Progress - University of California, Davisgrcp.ucdavis.edu/publications/doc10/Report10.pdf · or low copy clones, to chromosome arms in wheat using aneuploids so that these clones

Progress in Genome Mapping of Wheat

and Related Species

Proceedings of the 2nd Public Workshop of the International Triticeae Mapping Initiative

27-29 September 1991 Manhattan, Kansas

Edited by Bikram S. Gill, W. John Raupp, and Harold Corke

Published by Genetic Resources Conservation Program Division of Agriculture and Natural Resources

ClNlVERSlTY OF CALIFORNIA

Genetic Resources Conservation Program Report No. 10, September 1992

This report is published by the University of Califomia Genetic Resources Conservation Program as part of the public information function of the Program. The Program sponsors projects in the collection, inventory. maintenance, preservation, and utilization of genetic resources important for the State of Califomia as well as research and education in conservation biology. Further information about the program may be obtained from:

Genetic Resources Conservation Program University of Califomia Davis, CA 95616 USA (916) 757-8920 FAX (916) 757-8755

- ~ -

In accordance with applicable Federal laws and University policy, the University of California does not discriminate in any of its policies, procedures, or practices on the basis of race, religion, color, national origin, sex, marital status, sexual orientation, age, veteran status, medical condition, or handicap. Inquiries regarding this policy may be addressed to the Affirmative Action Director. University of California. Agriculture and Natural Resources, 300 Lakeside Drive, 6th Floor, Oakland, CA 94612-3650. (415) 967-0097.

Olin D. Anderson, USDA-ARS, Western Regional Research Center, Albany, CA USA Rudi Appels. Division of Plant Industry, CSIRO, Canberra City, ACT AUSTRALIA Jan Dvorak. Agronomy and Range Science, University of California. Davis. CA USA Michael D. Gale. Cambridge Laboratory, Nolwich, Norfolk, UNITED KINGDOM Bikram S. Gill, Plant Pathology, Kansas State University, Manhattan, KS USA J. Perry Gustafson. USDA-ARS. University of Missouri, Columbia, MO USA Gary E. Hart. Soil and Crop Sciences, Texas A8M University, College Station, TX USA David Hoisington, Applied Molecular Biology. CIMMYT, Mexico DF. MEXICO Andris Kleinhofs. Agronomy and Soils. Washington State University, Pullman. WA USA Peter J. Langridge, Ken W. Shepherd, A.K.M.R. Islam.

Waite Agricultural Research Institute, Glen Osmond, SA. AUSTRALIA Calvin 0. Qualset. Genetic Resources Consewation Program, Univenity of California. Davis, CA USA Peter J. Sharp, Plant Breeding Institute, Cobbitty, NSW, AUSTRALIA Mark E. Sorrello, Plant Breeding and Biometry, Comell University. Ithaca, NY USA Steven D. Tanksley, Plant Breeding and Biometry, Comell University, Ithaca. NY USA

CIMMM. MBxico ICARDA, Syria Sogetal, Inc., Hayward. CA USA

CITATION: Gill, B.S., W.J. Raupp, and H. Corke. (eds.) 1992. Progress in genome mapping of wheat and related species: Proceedings of the 2nd public workshop of the International Triticeae Mapping Initiative, Manhattan, Kansas, 1991. Repotl No. 10, University of California Genetic Resources Conservation Program. Davis. C A ~

Table of Contents

Introduction ............................... .. .... . . . . . . . . . . 1 B.S. GIN

Summaries of Presentations

Development and application of a .......... 3 chromosomal arm map for wheat based on RFLP markers

M.E. Sorrells*, J.A. Anderson, Y. Ogihara, and S.D. Tanksley

Genetic llnkage mapping of Triticum ................. 18 tauschii chromosomes using RFLP probes

E.S. Lagudah, D. Schachfman, and R. Appels*

The North American barley genome ................. 20 mapping project progress report

A. Kleinhofs

The physical mapping of RFLP markers ............ 26 in rice

J.P. Gustafson

An updated genetic linkage map of Triticum ...... 27 tauschii, the D-genome progenitor of wheat

KS. Gill*, D.S Hassawi, W.J. Raupp, A.K. Fritz, B.S. Gill, TS. Cox, and R. G. Sears

The relationship between physical and .............. 30 genetic maps of wheat chromosomes

A.J. Luknszouski* and CA. Curtis

Chromosome maps of wheat based on .............. 33 RFLP markers

G. E. Hart

Deletion mapping of wheat chromosomes ......... 34 R.S. Kota*, J.E. Werner*, D.E. Delaney*, K.S. Gill, T.R. Endo, and B.S. Gill

Fine physical mapping ofPhl , a chromosome .... 37 pairing regulator gene in polyploid wheat

KS. Gill*, T.R. Endo, andB.S. GIN

Chemiluminescent methods for RFLP analysis .... 39 in the Triticeae

D. Hoisington

RFLP mapping of wheat-lye recombinants ....... 41 and the use of PCR in cereal mapping

P. Rogawski

Genetic and molecular approaches to studying .... 44 a disease resistance gene in maize

S H Hulbert

A 'zebra' chromosome arising from multiple ...... 46 translocations between wheat chromosome 5A and Elymus trachycaulus chromosome 1Ht

J Jiang* and B.S. GIN

Mapping Initiatives

ITMl- The International Triticeae Mapping ...... 47 Initiative

C. 0. Qualset

ATMI - The Australian Triticeae Mapping ....... 49 Initiative

P. J. Sharp

Canada Wheat Genome Mapping Group ........... 50 W.K. Kim* and T.F. Townley-Smith

Japan Wheat Genome Mapping Initiative ........... 54 K. Tsunewaki

French Triticeae Mapping Initiative ................... 58 P. Leroy *. M. Bernard, S. Bernard, G. Branlard, M. Rousset, P. Nicalas, H. Thiellement, G. Doussinault, J. Jahier, P. Joudrier, M.F. Gautier, F. Quktier, and C Hartmann

Chromosome Croup Reports

..... Group2 P. J. Sharp

Group 3 ......... M.E. Sorrells

Group 5 .......................................................... 62 B.S. Gill

Cytogenetic Stocks

The E.R. Sears collection of wheat .................. 66 aneuploids: present status

A.J. Lukasrewski

Cytogenetic stocks maintained by the Wheat .... 68 Genetics Resource Center

B. Friebe*, W.J. Raupp, andB.S Gill

The production of homozygous recombinant ...... 73 lines from the Langdon d u r n - T. dicoccoides substitution lines

L.R. Joppa

Data Management

Wheat Database Meeting ................................ 75 0. Anderson and H. Corke

Attendance List ....................................... 79

* Presented the Paper

With this rather cursory look at worldwide small grain cereal genetics efforts, I want to make a few comments about the ITMI workshop program in Man- hattan. We focused the program on genome mapping (Imkage and physical mapping), mapping using non- conventional approaches, such as chemiluminescence and PCR, and on a survey of national genome initiatives, cytogenetic stocks, data management software demonstration, and poster sessions. About 100 scientists from eleven countries participated. Based on the comments from the participants, the program was quite successhl. The intent of the program was to present exciting science and separate the business sessions from scientific presentations. I hope this strategy will be followed in future meetings.

Of course, the organization of such a meeting entails a lot of hard work. One single individual who did more than anyone else to make this meeting such a success is John Raupp. John deserves our heartfelt thanks. I want to thank Duane Wilson and the rest of my laboratory staff for their tremendous efforts. We received considerable help from the ITMI office in Davis, CA, particularly from Cal Qualset, Pat

McGuire, and Harold Corke. I express my appreciation to them.

I shall close by offering a few suggestions for future meetings. First and foremost, the invited speak- ers must present cutting-edge science, new data, new protocols, innovative research. It is imperative that laboratory leaders encourage participation from their colleagues, bright postdoctoral fellows, and graduate students; the hands-on researchers who have much to contribute. We must encourage participation from all over the world. We must constantly encourage comparative mapping in wheat, rye, barley, oats, and beyond. We need communal library resources such as YAC libraries and cDNA libraries that are beyond the resources of individual laboratories. We need techniques for transformation, chromosome walking, and so on, so that small grain and polyploid genetics research remains an exciting and competitive enterprise.

Bikram S. Gill Professor, Wheat Genetics Resource Center, Kansas State University

2 I T M I Manhattan. Kansas 1991

Development and Application of a Chromosomal Arm Map for Wheat Based on RFLP Markers

M.E. Sorrellsl, LA. Anderson', Y . Ogiharaz, and S.D. Tanksleyl

'Department of Plant Breeding and Biometry, Coroell University, Ithaca, NY 14853 USA and "stitute for Biological Research, Yokohama City University, Yokohama, 232, JAPAN

I n cultivated bread wheat (Triticurn aesrivum L. em. Thell.), an allohexaploid (2n=6x=42), an RFLP map will be particularly useful as homoeoloci of each genome can be visualized. The development and use of RFLP markers in wheat has been slow because of limited polymorphism (Chao et al., 1989; Kam-Mor- gan et al., 1989; Liu et al., 1990; unpublished data) likely due to its relatively recent origin (Bell, 1987). Bread wheat has three genomes, and theoretically would require three times the number of polymorphic restriction fragments as a diploid to wnstmct an RFLP l~nkage map of equal density. One approach to facilitate the development and application of RFLPs in wheat is the construction of a chromosomal arm map. Wheat is uniquely suited for such a mapping strategy because of an abundance of aneuploid stocks (Sears, 1954). Since the 19501s, breeders and geneticists have made use of these aneuploids to determine the chro- mosonlal location of many genes (for review see Hart and Gale, 1990; Milne and McIntosh, 1990). The objective of this research was to locate a large number of DNA restriction fragments, corresponding to single or low copy clones, to chromosome arms in wheat using aneuploids so that these clones may be applied to gene tagging, linkage and mapping of quantitative trait loci (QTL), cytogenetic manipulations, estimation of genetic distance, and genetic studics. This study also adds to our knowledge of the comparative organization of homoeologous chromosomes in wheat.

Materials and Methods

Genetic stocks and Southern hybridizations Seeds of nullisomic-tetrasomic (NT) lines of Chi-

nese Spring wheat (Sears 1966) (complete except for 2A and 4B) and ditelosornics (DT) of Chinese Spring (Sears and Sears, 1978) (complete except for 2AL, 4AS, SAS, ZBS, 4BL, 5BS, 5DS, and 7DL) were kindly provided by E.R. Sears, University of Missouri. An independently derived set of DTs of Chinese

Spring, provided by Y. Ogihara, Kihara Institute, was also used for some probings. The designations of 4A and 4B used here are those agreed upon at the Seventh International Wheat Genetics Symposium held in 1988 at Cambridge, UK.

Leaf DNA was extracted from 3 to 4 weck old seedlings (bulk of 4 or 5) as described by Anderson et al. (1992). Digested DNA was loaded into gels (approximately 20 to 25 pg per lane), electrophoresed, and transferred to Hybond N+ membranes (Amersham Int., Arlington Heights, IL). Prehybridization of membranes, hybridization, washing, and exposure were as described by Anderson et a[. (1992).

Probe selection and chromosome arm assignment The clones used were from barley cDNA (BCD),

oat cDNA (CDO), and wheat genomic (WG) libraries. Wheat chromosome locations of the clones BG 131 and DG F15, from barley and Triticum tauschii genomic libraries, respectively, used previously in the wnstruction of a barley RFLP map (Heun et al. 1991) are also reported. Clones from each library were pre- screened by hybridization with membranes containing DNA of Chinese Spring (the genetic background of the aneuploid stocks) digested with the restriction enzymes Eco RI, Eco RV, and Dra I. Low copy clones (those hybridizing to 6 or fewer discernible fragments) were probed onto membranes containing DNA of NTs and DTs digested with one of the three restriction enzymes. Preference was given to clone/wqme combinations that yielded three fragments (presumably one for each genome) of approximately equal hybridization intensity. Resulting films of the NTDT probing were visually scored to identify fragments absent in any of the stocks. If a fragment was absent in a particular NT stock, we inferred its location on the chromosome in the nullisomic condition. Concomitant presence of a double-dose fragment in the stocks tetrasomic for a particular chromosome was used as additional evidence for the proper localization of fragments except in the

1 T M 1 Manhattan, Kansas 1931 3

Introduction Pe rhaps the third workshop of ITMI represents the coming of age of global cereal genetics research. ITMl was born at a time of despau in 1989. The privatiza- tion of the Cambridge Plant Breeding Institute was a traumatic event for the world cereal genetics community. Cambridge under Dr. Riley, Missouri under Dr. Sears, and Kyoto under Dr. Kihara had traditionally provided world leadership in wheat genetics research. It was a public research enterprise! With the retire- ments of these father figures from wheat genetics, and the death of Dr. Kihara in 1986, the turmoil at PBI was particularly jarring. The news (in 1988) of a private genetic map from the Cambridge group further fueled the fires of despair.

In this background, when the picture of wheat genetics looked so dismal, the first ITMI meeting was held in Davis, California in 1989. The details of events leading up to that meeting were summarized by Dr. Qualset in the introduction to the 1990 ITMl Public Workshop Proceedings. The only thing I might add is that ITMI's forerunners were two 'North American Wheat Genetic Mapping and Cytogenetic Stocks' workshops held in 1986 and 1988 in Columbia, Mis- souri'. These workshops were sponsored by the National Association of Wheat Growers Foundation. It should be noted that Dr. Sears personally blessed the birth of ITMI by actively participating in all its meetings until his death on February 14, 1991. I hope his dedication to wheat genetics, and his unselfishness in sharing genetic stocks and knowledge will continue to inspire us in turn.

If the 1991 ITMI workshop in Manhattan, Kansas was any indication, wheat genetics is flourishing. The transformed Cambridge Laboratory at John Innes Insti- tute is again setting the pace in wheat genetics research. Dr. Gale and his group have been very con- scientious in sharing their probes with colleagues else-

'Qualset, C.O. and P.E. McGuire (Eds.). 1986. Ernest R. Sears 50-year celebration symposium and North American wheat genetic mapping and cytogenetic stocks workshop, April 1986. Nat. As%. Wheat Growers Foundation, Washington, DC. 54 p. Kimber, G.E. (Ed.). 1988. The second North American wheat genetic mapping and cytogenetic stocks workshop, Nov.. 1988. Nat. Assoc. Wheat Growers Foundation, Washington, DC. 42 p.

where. Professor Tsunewaki's research group at Kyoto has made tremendous strides in wheat RFLP mapping, and we hope Japan will participate more actively in ITMI efforts.

The Australian group's contributions under the leadership of Drs. Appels, Sha~p, McIntosh, and Lan- gridge have been exemplary. Dr. Moshe Feldman has set up a good team at the Weizmann Institute of Sci- ence in Israel, and we would ask them to participate more actively in ITMI. Canada is well along the way. Other countries, notably Italy, Spain, France, and India, are beginning to get organized for active participation. There are perhaps other active groups of which I am unaware.

The setting-up of a molecular genetics laboratory at CIMMYT, under the dynamic leadership of Dave Hoisington, is a key to bridging the gap between developing and developed countries. In the US, the large CIMMYT commitment to the Comell wheat genetics program has been very positive. Drs. Sorrells and Tanksley at Comell have been generous in the sharing of probes and information.

In California, Adam Lukaszewski is finding that Riverside may be the best place to regenerate wheat aneuploid stocks. Drs. Qualset, Dvorak, and McGuire in Davis are providing leadership for the wheat genetic mapping effort. Kathleen Ross, with Dr. Gustafson in Missouri, has begun distributing the wheat aneuploid stocks. Wheat genetics got a considerable boost with the establishment of the Wheat Genetic Resource Cen- ter at Kansas State University. Hart and Tuleen's group in Texas, and Joppa and Maan's group in North Dakota are making solid contributions in wheat genetics and cytogenetic stocks research.

The impact of USDA's crop plant genome mapping initiative on wheat genetics has as yet been minimal. However, Olin Anderson's wheat database project in Albany, California holds great promise.

The contributions of the North American Barley Genetic Mapping project, under the leadership of Andy Kleinhofs, have nicely complemented wheat genetic efforts. We need to stay in touch with other barley genome mapping groups, too. We should also not be hesitant about making bridges to other cytogenetics groups. I want to mention the Phillips and Rines's oat cytogenetics group in Minnesota, in particular.

1 T M I Manhattan, Kansas 1991

case of 2A and 4B. For these two chromosomes, clone assignment was based on the presence of doubledose merits in the tetrasomic stock since nullisomics for these two chromosomes were not available. In the analysis of the DTs, a fragment absent in a stock indicated its presence on the opposing arm of that chromosome (i.e., a fragment absent in DT IAL would indicate that that fragment originated from the short arm of chromosome one). In those cases where a complete ditelosomic set was not available, the assignment of restriction fragments to chromosome arms was inferred based upon the presence of the fragment in the ditelosomic stock available. For each clone, fragments were classified as being major or minor according to their relative intensities. Minor fragments were those with ca. 50% weaker autoradiographic signal compared to major fragments produced by the same clone. For most of the selected clonelenzyme combinations, three major fragments were observed, a varying number of minor fragments, and also faint fragments at moderate stringency of 0.5 X SSC at 65'C. Fragments that accounted for less than ca. 5% of the total hybridization signal were not analyzed. Twenty-eight of the clones were mapped using more than one enzyme to confirm fragment locations when there were ambiguities. In those cases, only data from one enzyme was included when totaling the number of restriction fragments located.

Results and Discussion

A total of 804 restriction fragments corresponding to 210 clones were assigned to wheat chromosome arms (Appendix 1). Overall, 88% of all hybridizing fragments considered reproducible were located to a chromosome or chromosome arm. The fragments not disappearing in any of the aneuploid stocks are likely the result of comigrating restriction fragments. All fragments from 116 out of the 210 clones were assigned to a chromosome arm. An example of a probing in which three fragments were assigned to each of the three homoeologous long arms of group 3 chromosomes is shown in Figure 1.

The mapped loci from randomly chosen clones (198 out of 210) are not uniformly distributed on all chromosome arms (Figure 2). Homoeologous group 6 chromosomes are the least populated having only 61 loci (8.2% of the 745 fragments located from random clones) compared to 14.8%, 16.5%, 16.1%, 12.8%, 18.3%, and 13.4% for homoeologous groups 1, 2, 3,4,

5, and 7 chromosomes, respectively. Heterogeneity of the BCD and CDO libraries for the total number of restriction fragments located to chromosomes was significant and greatest for groups 3 and 7 @<0.05) (Table 1). There were not enough wheat genomic clones mapped to make a valid comparison.

Table 1. x2 tests for heterogeneity between BCD and CM3 libraries for distribution of single copy restriction fragments to chromosomes.

Chromosomes Degrees of compared freedom - -

ALL 6 1 1 2 1 3 1 4 1 5 1 6 1 7 1

x2 Probability

23.7 ,001 1.86 .50 0.90 .90 7.74 .01 1.63 .50 2.34 .SO 2.52 .50 3.85 .05

Arm homoeolog~es With the exception of three clones, the arm

homoeologies revealed by probings of the aneuploid stocks agree with those previously deduced. Within chromosome groups, long arms were homoeologous to long anns, and consequently short arms to short arms, with the exception of group 4 chromosomes in which 4 A L 4 B S 4 D S and 4AS=4BL=4DL. These results are consistent with those reported by Hart (1973) based on the locations of isozymes of alcohol dehydrogenase and acid phosphatase, and later supported by the locations of isozymes of lipoxygenase (Hart and Langston, 1977). The group 4 homoeologies can be explained by an inversion in chromosome 4A during the evolution of wheat.

The clones yielding exceptions to expected arm homoeologies were BCD 446 (fragments assigned to IAL, IBS, and IDS) and two chromosome group 4 clones (BCD 1262 and CDO 669). The BCD 446 result may be due to the presence of a small pencentric inversion in 1A. Mistakes due to mislabeling or gel loading can be ruled out because of the results of CDO 580 (fragments assigned to IAS, IBS, and IDS) using the same membrane. One group 4 exception, CDO 669 (fragments assigned to 4AL, 4AS, 4BL, and 4DS), could be explained by polymorphism among the

I T M I Manhattan, Kansas 1991

aneuploid stocks since one of the two 4A-specific fragments is abscnt in DT 4AL while the other is at about twice the relative intensity of the same fragment in other stocks. The results of clone BCD 1262 (fragments assigned to 4AL, 4BL. 4DL) indicate that the pencentric inversion believed to have occurred in 4A did not involve the entire long arm.

Duplications Thirty-four cloncs hybridized to multiple frag-

ments on the same chromosome. This result might be attributed to intrachromosomal duplication of loci, andlor the presence of restriction sites within the chromosomal segment hybridizing to the clone. Forty clones hybridized to fragments on non-homoeologous chromosomes and may represent interchromosomal duplications. For 35 of these 40 clones, only two chromosome groups were involved. In the majority of these cases, three or more major fragments were assigned to chromosomes in one homoeologous group and minor fragments were assigned to the other group. All fragments from all enzymes tested were assigned to a chromosome from 18 of the 40 clones revealing interchromosomal duplications. For the remainder of the clones, it is likely that additional fragments could be assigned to a chromosome if more enzymes were used. Group I chromosomes were involved in the most duplications (17 of 35 clones ) compared to 6, 9, 7, 14, 6, and 1 l for homoeologous groups 2, 3,4, 5, 6, and 7 chromosomes, respectively (Table 2). The three genomes (A, B, or D) were involved in about the same number of duplications.

Evidence for homoeologous recombination in NSBTSD

The 42 chromosomes of hexaploid wheat pair as 21 bivalents due largely to the effect of the phi gent-, located on chromosome 5BL, which suppresses homoeologous recombination (Okamato 1957; Riley and Chapman 1958). We have detected homoeologous recombination between the short arms of chromosomes 2A and 2D in the nullisomic 5B, tetrasomic 5D (NSBTSD) stock. This putative recombination event($ is evidenced by six clones (BCD 348, CDO 418, CDO 426, CDO 783, CDO 666, and CDO 981) assigned to homoeologous group 2 chromosomes. For these clones, the same molecular weight fragment(s) was absent from N2DT2A and N5BT5D and was compensated by the presence of the corresponding doubledose fragment@) (Figure 3). Mistakes due to mislabeling of

stocks or loading of gels were ruled out since other clones probed to the same membranes had fragments missing in only the homoeologous group 2 or 5 stocks (data not shown).

A single fragmcnt was absent in more than one lane witbin a set on NTs or DTs for fourteen other clones as well; however, no compensating doubledose fragment was observed. This could be the result of homoeologous recombination due to the action of other pairing inhibitorsfenhancers, insertion, deletion, or loss of a restriction site in which the size of the 'new' fragment is less than lkb or more than 25kb, the typical size range that can bc detected in Southern hybridizations. For all cases in which the same fragment was absent in more than one lane with a set of NTs or DTs, the fiagment(s) were assigned to only one chromosome arm based on the location of other fragments and assuming homoeology.

Table 2. Number of clones detecting interchromosomal group duplication of loci for different combinations of chromosomes.

Homoeologous Group Homoeologous 2 3 4 5 6 7

Group 1 2 5 4 2 1 4 2 1 1 3 0 0 3 0 2 1 0 4 1 0 1 5 2 4

Evidencefor translocations Based on the pairing frequencies of chromosomes

in 5B- or 3D- deficient l i e s (enhanced frequency of homoeologous rccombination) of Chinese Spring wheat, a double translocation of 4AL to 5AL, 5AL to 7BS, and 7BS to 4AL has been proposed by Naranjo et al. (1987). This proposal is supported by the location of structural genes for the isozymes of P-amylase on chromosome arms 4BL, 4DL, and 5AL (Ainsworth el al. 1983) and of endosperm peroxidase on 4AL, 7AS, and 7DS (Kobrehel and Feillet, 1975; Kobrehel, 1978; and Benito and Perez de la Vega, 1979).

Six clones (BCD 87, BCD 93, BCD 1302, CDO 780, CDO 1312, and WG 114) revealed putative inter- homoeologous translocations supporting the above proposed translocations (Appendix 1). For four of these clones, two of the three major fragments were


5 10 ..

E B * P a SHORT ARM

30 LONG ARM

35

I A B D A B D A B D A B D A B D A B D A B D

1 2 3 4 5 6 7

CHROMOSOME

Figure 2. Numbers of DNA restriction fragments from randomly chosen barley cDNA, oat cDNA, and wheat genomic clones located to individual chromosome arms using aneuploids of 'Chinese Spring' wheat. Fragments assigned to a chromosome group, but not an arm are not included.

assigned to their respective homoeologous chromosomes in two of the genomes, whereas the third was assigned to a different chromosomal group in the remaining genome. In agreement with the 4AL>5AL>7BS>4AL translocations proposed by Naranjo et al. (1987). we detected 4AL-specific hag- ments on 5AL (clones BCD 1302, CDO 13 12, and WG 114); a SALspecific fragment on 7BS (clone BCD 87); and 7BS-specific fragments on 4AL (clones BCD 93 and CDO 780). In addition, we detected a 5AL-specific fragment on 4AL (clonc CDO 484). One possible explanation of these results that expands upon Figure 4 of Naranjo et al. (1987) is illustrated in Figure 4. In the A genome progenitor of hexaploid wheat a reciprocal translocation occurred between knninal segments of the long arms of chromosomes 4 and 5. In the tetraploid (AABB), the rearranged 4AL exchanged tenninal segments with 7BS via a reciprocal translocation. The reasons for the occurrence of the translocations in the diploid and tetraploid, respectively were discussed by Naranjo et al. (1987). Our proposal adds to that of Naranjo et al. (1987) in that a segment of 5AL was transferred to 4AL. This segment is most likely interstitial, thus allowing for the 4AL-7BS reciprocal translocation to involve terminal portions of the chromosomes.

Polymorphism among stocks and other anomalies Polymorphism (an observable change in the

molecular weight of one or more fragments compared with Chinese Spring) was observed with 21 different clones on the aneuploid stocks. Ditelosomic stocks 4AL and IBL were polymorphic for 12 and 5 clones, respectively, with other aneuploids involved in 0, 1, or 2 each. A portion of the 14 clones with fragments absent in more than one lane (excluding group 2 homoeologous recombination in N5BT5D) may fall in this category also. Clone CDO 395, located on chromosome 3S, produced more intense 3DS fragments (ca. 10X) in the DT 6BL stock with respcct to the same molecular weight fragment in other stocks. This suggests some form of localized duplication of this locus on chromosome 3DS in the DT 6BL stock.

Twenty-eight of the clones were mapped using more than one enzyme to confirm fragment locations when there were ambiguities. As listed in Appendix 1, the different enzymes often resulted in a different num- her of fragments localized within a chromosomal group. This was expected since only clones giving ambiguous or incomplete results were probed with additional enzymes. Only four clones (BCD 348, CDO 484, CDO 836, and CDO 1400) yielded fragments assigned to different homoeologous groups from the

I T M 1 Manhattan, Kansas 1991 7

Figure 1. Autoradiogram from clone BCD 1127 probed onto Chinese Spring wheat aneuploids digested with EcoRV. h indicates lambda DNA digested with Hind III and used as a size marker. a) Result of probing onto nullisomic (N)- tetrasomics Q. Each ofthe three fragments corresponds to one of the genomes of the group 3 chromosomes. b) Result of simultaneous probing onto ditelosomic stocks. The fragments were assigned to the long arms of group 3 chromosomes based on their absence in 3BS, 3AS, and 3DS.


Figure 3. Evidence for the occurrence of homoeologous recombination from autoradiogram of clone BCD 348 probed onto 'Chinese Spring' wheat anenploids digested with Eco RV. ?. indicates lambda DNA digested with Hind III and used as a size marker. a) Result of probing onto nnllisomic 0-tebasomics (T). Note the concomitant absence of restriction fragments in N2DT2A and N5BT5D and the presence of doubledose fragments in N2DT2A (indicative of their location on 2D). b) Result of simultaneous probing onto ditelosomic stocks. Fragments assigned to group 2 chromosomes are further located tn 2S based on their absence in 2BL and 2DL. Fragments absent in 4AL, 6AL, and 6BS may also be the result of homoeologous recombination or polymorphism.

use of the different enzymes. The results from BCD 348 and CDO 1400 may be largely due to polymorphism since these two clones revealed the highest level of polymorphism compared to 40 other clones probed onto 18 hexaploid wheat genotypes (Anderson et al. in preparation).

The anomalies detected (translocations, homoeologous chromosome pairing, and polymorphism among DNAs) in these aneuploid stocks using molecular markers dictate that caution should be exer- cised in interpreting the results from genetic studies utilizing aneuploid stocks.

Applications of arm specific map We view the development of this chromosomal

arm map as a complement to, rather than a substitute for, a conventional RFLP map in wheat. The conservation of gene synteny among the three genomes of wheat and relatives such as T. tauschii (DD) (Kam-Morgan et al., 1989; Gill et al., 1991), barley, and rye (Hart, 1987) means that linkage maps produced in these diploids may be suitable for use on hexaploid wheat. Linkage maps should be more efficiently produced using diploid relatives of wheat such as T. tauschii (Kam-Morgan et al., 1989; Gill et al., 1991); T.

1 T M I Manhattan. Kansas 1991

monococcum (A genome) (M. RGder, pers. comm.), or barley (Heun et al., 1991) since higher levels of polymorphism can be found and only a single genome needs to be mapped.

I ) Reclprocal t ranslocat ton i n A genome dlDiold

4 A 5 A 4A 5 A

2) Reclprocal translocation I n AB genome t e t r a D l o l d

4A 7 0 4 A 7 0

R e s u l t a1 t ranslocat lons

Figure 4. Sequence of translocations during the evolution of Chinese Spring wheat. The approximate location of centromeres, based on somatic ann ratios, are indicated by ovals. The actual sizes of the translocations are not known. From Naranjo et al. (1987).


The efficiency and usefulness of a chromosomal arm map versus a linkage map in wheat will vary depending on the application. Since polymorphisms in cultivated bread wheat are relatively rare for any given pair of lines (Chao et al., 1989; Kam-Morgan et al., 1989; Liu el al., 1990; unpublished data), a large pool of clones will be required for selecting those informative on specific populations. The initial construction of a chromosomal arm map is relatively rapid since all low copy clones that are polymorphic among the A, B, and D genomes can be mapped using aneuploids. A disadvantage of conventional RFLP linkage mapping is that only those clones polymorphic for at least one fragment are mapped on the specific mapping population, thus eliminating clones that may be informative in other populations (Anderson et al., in preparation). In addition, linkage mapping populations are often constructed from a cross of distantly related genotypes within the primary gene pool or from different gene pools. The more distantly related the parents, the more polymorphism can be expected, but there may be a greater risk of cytological abnormalities such as reduced recombination and the presence of translocations or other chromosome rearrangements. A chromosomal arm map should be especially useful in deci- phering genetic relatedness of varieties and species accessions based on RFLPs since one could choose clones that represent all chromosome arms. Clones from this arm map may also find immediate application in the field of wheat cytogenetics as previous outlined (Gale et al., 1989). A subset of clones may be useful in following the introgression of alien chromosome segments, reducing or eliminating linkage drag (Young and Tanksley, 1989), detecting changes in cytogenetics stocks, constructing addition or substitution lines, and detecting other cytological abnormalities.

The tagging of qualitatively inherited traits should be enhanced by knowing the chromosomal arm location of clones. Not only does this make the search for unmapped genes more efficient, but in the case of wheat many genes of economic importance have already been located to chromosome arms of particular genomes (Hart and Gale, 1990; Milne and Mclntosh, 1990). Putative linkages to genes of economic importance have been identified in our lab using clones from the chromosomal arm map. These include linkages to genes for resistance to Hessian fly (Mayetiola destruc- for) (2. Ma, pers. comm.), leaf rust (Puccinia recondita f. sp. tritici), and stem rust (Puccinia graminis f. sp. tritici) (E. Autrique, pers. comm.).

The identification of quantitative trait loci (QTL) is best facilitated by a l i e map because of the need for uniform genome coverage to ensure detection of as many QTLs as possible (Paterson e t al., 1988). This can be accomplished in the case of wheat by selecting clones from l i a g e maps of related diploids and sup- plementing those with clones on the arm map in order to adequately cover the chromosomes of the hexaploid with polymorphic markers.

One of the drawbacks of a chromosomal arm map is that the lmear arrangement and genetic l i e of clones is not known. This is not as critical for wheat as for other crops since many genes of interest have been mapped only to a chromosome or chromosome arm using aneuploids. Therefore, tagging genes of interest will initially require the use of clones located throughout a chromosome arm rather than just a portion of an arm. In addition, the benefit of knowing the relative position of a clone on a chromosome may be inconsequential since only a portion of all clones on

any kind of map will be polymorphic on the population of interest. A second drawback of a chromosomal arm map is that the clones have been selected for intergenomic versus intragenomic polymorphism. As a result, the clones should be most useful for introgression studies, hut only a subset will be informative (with today's technology) in crosses between cultivated wheat varieties.

The complementation of RFLP mapping with a chromosomal map is likely to be efficient for other species in which the level of polymorphism is low, and aneuploid stocks are available that allow the placement of clones to chromosome arms.

Acknowledgments

Financial support was provided by the Australian and Netherlands governments, CIMMYT, and Hatch projects 418 and419.

References Cited

Ainsworth, C.C., M.D. Gale, and S. Baird. 1983. The relationship to the D genome of Triticum aestivum genetics of P-amylase isozymes in wheat. I. Allelic van- (AABBDD). Genome 34:362-373. ation among hexaploid varieties and intrachromosomal gene locations. Theor. Appl. Genet. 66:39-49. Hart, G.E. 1973. Homoeologous gene evolution in hexap-

loid wheat. In: Proc 4th Int Wheat Genet. Symp. (Sears, Anderson, J.A., Y. Ogihara, M.E. Sorrells, and S.D. E.R. and L.M.S. Sears eds.) Agr. Expt. Stn., College of Tanksley. 1992. Development of a chromosomal arm map Agr., Univ. Missouri, Columbia, pp 805-810. for wheat based on RFLP markers. Theor. Appl. Genet., submitted. Hart, G.E. 1987. Genetic and biochemical studies of

enzymes. In: Wheat and Wheat Improvement (Heyne, E.G. Bell, G.D.H. 1987. The history of wheat cultivation. In: ed.j. American Society of ~ ~ r o n o m ~ , Madison WI, pp 19- Wheat breedine. Its scientific basis. nuoton. F. G. H. Ed) 214 . . . Chapman and k l l Ltd., London, pp 31-49.

Hart. G.E. and M.D. Gale. 1990. BiochemicaVmolecular Benit0, C. and M. Perez de la Vega. 1979. The chromo- loci of hexaploid wheat (Triticum aestivum 2n=42 genomes soma1 location of peroxidase isozymes of the wheat kernel. AABBDD). In: Genetic Maps (O'Brien, S.J. Ed). Cold Theor. Appl. Genet. 5573-76. Spring Harbor Laboratory, Cold Spring Harbor NY.

Chao, S., P.J. Sharp. A.J. Worland, E.J. Warham, RM.D. Hart, G.E. and P.J. Langston. 1977. Chromosomal location Koebner, and M.D. Gale. 1989. RFLP-based genetic maps and evolution of isozyme structural genes in hexaploid of wheat homoeologous moup 7 chromosomes. Theor. wheat. Heredity 39:263-277. - Appl. Genet. 78:495-504.

Heun, M., A.E. Kennedy, J.A. Anderson N.L.V. Lapitan, Gale, M.D., P.J. Sharp, S. Chao, and C.N. Law. 1989. M.E. Sorrells, and S.D. Tanksley. 1991. Construction of an Applications of genetic markers in cytogenetic manipula- RFLP map for barley (Hordeum vulgare L). Genome in tion of the wheat genomes. Genome 31:137-142. press.

Gill, K.S., E.L. Lubbers, B.S. Gill, W.J. Raupp, and T.S. Kam-Morgan, L.N.W., B.S. Gill, and S. Mulhukrishnan. Cox. 1991. Linkage map of Triticum tauschi @D) and its 1989. DNA restriction fragment length polymorphisms: a

10 I T M I Manhattan, Kansas 1991

strategy for genetic mapping of D genome of wheat. Genome 32:724-732.

Kobrehel, K. 1978. Identification of chromosome segment controlling the synthesis of peroxidase in wheat seeds and in transfer lines with A g r o w n elongaturn. Can. J. Bot. 56:1091-1094.

Kobrehel, K. and P. Feillet. 1975. Identifidon of genomes and chromosomes involved in peroxidase synthesis of wheat seeds. Can. J. Bot. 53:2326-2344.

Liu, Y.G., N. Mori, and K. Tsunewaki. 1990. Resuiction Pdgment length polymorphism W P ) analysis in wheat. I. Genomic DNA library construction and RFLP analysis in common wheat. Jpn. J. Genet. 65:367-380.

Milne, D.L. and L.A. Mclntosh. 1990. Triticum aestivum (common wheat). In: Genetic Maps (O'Brien, S.J. Ed). Cold Spring Harbor Laboratoly, Cold Spring Harbor NY.

Naranjo, T., A. Raq P.C. Coicoechca, and R. Giraldez. 1987. Arm homoeology of wheat and rye chromosomes. Genome 29:873-882.

Okamato, M. 1957. Asynaptic effect of chromosome V. WheatInJ .W. 5:6.

Paterson, A.H., E.S. Lander, J.D. Hewitt, S. Peterron, S.E. Lincoln, and S.D. Tanksley. 1988. Resolution of quantitative traits into Mendelian factors by using a complete linkage map of restriction fragment length polymorphisms. Nature 339721-726.

Riley, R., and V. Chapman. 1958. Genetic control of the cytologically diploid behaviour of hexaploid wheat. Nature 182:713-715.

Sears, E.R. 1954. The aneuploids of common wheat. Mo. Agric. Exp. Stn. Res. BUN. 572:l-58.

Sears, E.R. 1966. Nullisomic-tetrasomic combinations in hexaploid wheat. In: Chromosome manipulation and plant genetics @ley, R. and K.K. Lewis, Eds). Oliver and Boyd, Edinburgh. pp 29-45.

Sears, E.R and L.M.S. Sears. 1978. The telocentric chromosomes of common wheat. In: Proc. 5th lnt. Wheat Genet. Symp. (Ramanujam, S. Ed) Indian Soc. Genet Plant Breed, New Delhi, pp 389-407.

Young, N.D. and S.D. Tanksley. 1989. Restriction fragment length polymorphism maps and the concept of graphical genotypes. Theor. Appl. Genet. 77:95-101

I T M I Manhattan. Kansas 1991 11

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Genetic Linkage Mapping of Triticum tauschii Chromosomes Using RFLP Probes

E.S. Lagudah, D. Schachtman, and R. Appels CSIRO Division of Plant Industry, P.O. Box 1600, Canberra ACT 2601, AUSTRALIA

T h e detailed molecuIar/genetic characterization of Trificum tauschii accessions has been camed out in Kansas State University in the US (Gill et al . , 1991; Lubbers et al., 1991) and in CSIRO, Australia (Lagudah er al. , 1991a,b), and is continuing as new disease resistance genes are incorporated into the map. An update of the map produced in CSIRO, Canberra is shown below (Figure 1). In terms of the ITMI collaboration two main issues will be addressed in the following 12 months:

Improve linkage of DNA markers to disease-

resistance and salt-exclusion genes, to allow these characters to be mapped in wheat breeding programs that incorporate these genes from T tauschii.

Combine the Kansas and Canberra maps by exchanging markers and mapping them in the F, progeny used to produce the maps. This will allow a consensus, or working, map to be produced. A similar exercise has recently been carried out using the maps for chromosome 1R produced in Canberra and Cambridge (UK) and, except for one discrepancy, provides a consistent map (Baum and Appels, 1991).

References Cited

Baom, M.B. and R. Appels. 1991. The cytogenetic and molecular architecture of chromosome 1R - one of the most widely used sources of alien chromatin in wheat varieties. Chromosoma 101:l-10.

Gill, K.S., E.L. Lubbers, B.S. Gill, W.J. Raupp, and T.S. Cox. 1991. A genetic linkage map of Triticum tauschii @D) and its relationship to the D genome of bread wheat (AABBDD). Genome 34:362-374.

Lagudah, E.S., R Appels, A.H.D. Brown, and D. McNeil. 1991a. The molecular genetic analysis of Triticurn tauschii

-the D genome donor to hexaploid wheat. Genome 34:375- 386.

Lagudah E.S., R. Appels, and D. McNeil. 1991b. The Nor-D3 locus of Triticum tauschii: natural variation and linkage to chromosome 5 markers. Genome 34:387-395.

Lubbers, E.L., K.S. Gill, B.S. Gill, W.J. Raupp, and T.S. Cox. 1991. Variation of molecular markers among geographically diverse accessions of Triticum tauschii. Genome 34:354-361.


Chromosome 10 Chromosome 2 0

Chromosome 4 0 Chromosome 5D Chromosome 60 Chromosome 7D

The North American Barley Genome Mapping Project Progress Report

A. Kleinhofs Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164 USA

T h e North American Barley Genome Mapping Pro- and leaf pubescence (Pub). To differentiate these ject (NABGMP) is organized to develop a 10 centi- markers from the RFLP markers they are designated Morgan (cM) map of the barley genome and to use that with the prefix 'm'. The isozymes are esterase 5 and 9 map to identify quantitative trait loci (QTL) for all of (Est5; Estg), phospbogluconate dehydrogenase 2 the economically important characteristics of barley. (PgdZ), alkaline P-galactosidase (Bgl), aconitate The Project consists of 49 scientists in 26 laboratories hydratase 2 (AcoZ), and an unknown isozyme desig- in the United States and Canada. It is directed by a nated ABII51 (American Barley Isozyme 151. The project coordinator: Dr. R.A. Nilan (Washington State isozyme markers are prefixed with an 'i' to differentiate University), the Canadian co-director, Dr. K.J. Kasha them from the RFLP markers. (University of Guelph), a five-member steering corn- Currently there are four RAPD markers on the mittee, and map construction and economic trait map. These are designated ABR (American Barley analysis coordinating committees. RAPD) plus an arbitrary number and are described in

Map construction is based on 150 doubled haploid Table 1. The RAPD markers have turned out to be (DH) lines derived from each of two crosses, Morex by more difficult than originally expected. There are Steptoe (6-rowed) and Harrington by TR306 (2- problems with segregation distortion and reproduci- rowed). The parents were selected to represent a broad bility. We have found that it is extremely important to sample of genetic variation and quantitative trait map only major bands and to maintain high quality of expression, as well as adequate DNA polymorphism. the template DNA. RAPD markers that show unusual Morex is the malting industry standard cultivar rec- segregation distortion are discarded. ommended by the American Malting Barley Associa- The RFLP markers are from many sources and tion (AMBA). Steptoe is a hi& yielding, broadly are either cDNA or genomic DNA probes. The cDNA adapted barley with poor malting and feed quality. probes are from the NABGMP libraries (Steptoe leaf; Morex is a derivative of the Manchurian group while Steptoe malted seed), from Cornell University libraries Steptoe is a Coast-type barley. Harrington is the stan- (barley cDNA, oat cDNA), from Plant Science dard 2-rowed malting barley in Canada while TR306 is Research Institute, Cambridge Laboratory library a feed barley with better adaptation, yield, and disease (Chinese Spring wheat cDNA) and various known resistance than Harrington. This report presents data function probes obtained from the individuals who on mapping of the Steptoe/Morex cross. Major con- cloned them (mostly cDNAs). The NABGMP group tributors to the work described, in alphabetical order, cDNA probes are designated ABC (American Barley are: J. Franckowiak (North Dakota State University), cDNA) plus an arbitrary number. The Cornell probes P. Hayes (Oregon State University), D. H o b are designated as in Heun et al. (1991); BCD for bar- (USDA, Aberdeen, ID), A. Kilian (Silesian University, ley cDNA and CDO for oat cDNA plus an arbitrary Katowice, Poland), A. Kleinhofs (Washington State number, except that for markers where the map loca- University), S. Knapp (Oregon State University), N. tion of the marker is different in our (SteptoeIMorex) Lapitan (Colorado State University), M.A. Saghai cross from the Cornell (Proctor/Nudinka) cross, we use Maroof (Virginia Polytechnic Institute), R. Skadsen a different capital letter designation following the arbi- (USDA, Madison, WI), M. Sorrells (Cornell Univer- trary number. The Plant Science Research Institute sity). probes are designated PSR plus a number (Chao et al.,

The current map consists of 2 morphological. 6 1988; Sharp et al., 1988). The known function gene isozyme, 4 RAPD, and 175 RFLP markers. The two probes are given a locus designation consistent with morphological markers are rachilla hair length (Srh) previous designations, or, if none, a symbol is assigned


Table 1. Random amplified polymorphic DNA (RAPD) marker description and primer sequence

Number Designation Primer Sequence Chr Probe* Source and Description**

84 ABP.315 CTGCTTAGGG 4 G 'Operon' primer J-16; S-; band 500 bp 140 ABR3 13 CG TA CG CG 'L'I' 7s G 'Genosys' prbttcr CS43; S i ; band 500 bp 180 ABR303 CC GC CTAGTC 1 G 'Operon' primer 1-04.> S-; band 600 bp 181 ABR329 TGTTCCACGG 1 G 'Operou' primer 1-05: S+; band 600 bp

* Primer quality rating: E - excellent; major band, easy to read G - good; strong band, fairly easy to read F - fair,, fGly strong band, but some diOiculty in reading P - minor band, not recommended

** Source was either Operon Technologies lnc., 1000 Atlantic Ave., Suite 108, Aliuneda, CA 94501 or Ccnosys Biotechnologies Inc., 8701A New Trails Drive, The Woodlands, TX 773814244. S+/- refers to the presence or absence of the scored band in the cultivar Steptoe. Approximate band size is indicated in base pairs.

following established guidelines (Barley Genetics Newsletter 11:l-16, 1981). In cases where it was not possible to determine which previously used locus designation corresponded to the locus uncovered by the RFLP probe, a previously unused locus number is used. For example, we mapped five alcohol dehydrogenase loci using the alcohol dehydrogenase cDNA probe (Good et al., 1988), but there are three Adh loci previously identified by isozyme analyses (Sogaard and von Wettstein-Knowles, 1987). Since we were not able to resolve which one was Adh 1, 2 or 3, we designated the RFLP detected loci as Adh 4, 5, 6, 7, and 8. Simi- lar problems arise with the peroxidase gene loci. Here we have chosen to use the gene symbol Prx for the RFLP identified loci to differentiate them from the more commonly used isoqme symbol Per. The known function gene designations are outlined in Appendix 1.

The genomic DNA probes are from a NABGMP barley Psr I library (preparcd by N. Lapitan), Comell University barley and wheat libraries (Heun et al., 1991), and a Kansas State University Triticum tauschii library (Gill et al., 1991). The NABGMP probes are designated ABG (American Barley Genomic DNA) plus an arbitrary number. The Comell U~versity probes are designated BG barley genomic) or WG (wheat genomic) plus an arbitrary number. The Kansas State University probes are designated ksu followed by a capital letter and an arbitrary number. If a probe detects more than one Locus, an additional number or capital letter is added to the designation in an alternating order, i.e., if the designation ends in a letter add a number, and if it ends in a number add a capital letter.

The 187 markers mapped to the SteptoeMorex cross constitute 7 linkage groups spanning 1,344 cM. The distribution of the markers to the seven chromosomes and the genetic length of each are shown in Table 2 Chromosomes 6 and 7 are the shortest and longest, respectively, in recombination distance. Chro- mosomes I and 4 have the most and the least number of markcrs mapped, respectively. We suspect that in many cases we have not yet reached the ends of the chromosomes. However, we do not expect the map to expand dramatically. In fact, in many cases the map has actually decreased in length as we have discovered and corrected marker classification errors. The maps are presented in Fig. 1.

In summary, much has been accomplished and much remains to be done. Although we have identified seven linkage groups for the seven barley chromosomes, there are still several major gaps to be filled. Ceutromeres and telomeres need to be identified and the genetic map needs to be related to physical locations on the metaphase chromosomes. Perhaps most important, tecluiques necd to bc developed to facilitate rapid, simple, and inexpensive application of the map and markers by breeders.

The QTL analyses are in progress. In 1991 the Steptoe x Morex doubled haploids were grown at 10 locations, in Oregon, Washington, Idaho, Montana, North Dakota, Minnesota, New York, and Saskatche- wan. Agronomic, pest resistance, nutritional quality, and malting quality traits are being evaluated.

In order to facilitate communication, we have developed an electronic bulletin board for the barley genome mapping effort. This bulletin board is located

at Washington State University, the address is WSUVMS 1 .CSC.WSU.EDU, the user-name is BARMAPBB, and the current password is STEPTOE. Eventually the bulletin board will contain up-to-date barley mapping information, standard techniques, and messages of general interest. Outside users will be able to leave messages on the bulletin board and down-load data to their personal computers.

In addition to NABGMP, there are several other groups mapping the barley genome. Two such groups are those headed by Dr. Andreas Granor, Biologische Bundesanstalt f i r Land-und Forshvirtschaft, Institut fiir Resistenzgenetik, Graf-Seinsheim Str. 23, D8059 Griinbach, Germany, and Dr. Henriette Giese, Rise National Laboratory, Postboks 49, DK-400 Roskilde, Denmark.

References Cited

Chao, S., P.J. Sharp, andM.D. Gale. 1988. A linkage map of wheat homoeologous group 7 chromosomes using RFLP markers. In: T.E. Miller and RM.D. Koebner, eds., Proc. Seventh lnternational Wheat Genetics Symposium, Vol. 1 (Cambridge, England, 1988). Institute of Plant Science Research, Cambridge. pp. 493-498.

Gill, K., E. Lubbers, B.S. Gill, W.J. Raupp, and T.S. Cox. 1991. A genetic linkage map of Triticum tauschii @D) and its relationship to thc D genome of bread wheat (AABBDD). Genome 34:362-374.

Good, A.G., L.E. Pelcher, and W.L. Crosby. 1988. Nuclwtide sequence of a complete barley alcohol dehydrogenase 1 cDNA. Nucleic AcidsResearch 16:7182.

Heun, M., A.E. Kennedy, J.A. Anderson, N.L.V. Lapian, M.E. Sorrells, and S.D. Tanksley. 1991. Construction of a restriction fragment length polymorphism map for barley (Hordeum vulgare). Genome 34:437-447.

genetic linkage maps of experimental and natural populations. Genomics 1:185-199.

Liu, B.H. and S.J. Knapp. 1990. GMENDEL: A program for Mendelian segregation and linkage analysis of individual or multiple progeny populations using log- likelihood ratios. J. Hered. 81:407.

Proctor, L., A. Rafalski, S. Tingey, S. Lincoln, and E. Lander. 1987. Mapmaker Macintosh V1.O for the constluction of genetic linkage maps. Copyright 1987 WIBR; copyright 1990 E.I. DuPont de Nemours and Co.

Sharp, P.J., S. Desai, S. Chao, and M.D. Gale. 1988. Isolation and use of a set of fourteen RFLP probes for the identitication of each homoeologous chromosome arm in the Triticeae. In: T.E. Miller and R.M.D. Koebner, eds., Proc. Seventh International Wheat Genetics Symposium, Vol. 1 (Cambridge, England, 1988). Institute of Plant Science Research, Cambridge. pp. 639-646.

Lander, E.S., P. Green, J. Abrahamson, A. Barlow, M.J. Segaard, B. and P. von Wettstein-Knowles. 1987. Barley: Daly, S.E. Lincoln, and L. Nehurg. 1987. MAPMAKER: genes and chromosomes. CarlsbergRes. Commun. 52:123- an interactive computer package for constructing primary 196.

i2 1 T M I Manhatlan, Kansas 1991

Dill MU*.. Dmt Mah.r D bl Mufar Db l Mart., CM Nun. CM Nmma cM Nam. cM Nun.

Di.1 Marker sM Name

1 4

6.4 ksuF2A

5.1 A8C1528

2.9 pcr2

0.7 BCD351C

3.3 WG7898

8.8 A80452

0.0 Glbl

9.7 OoG?l160)

14.6 ABC164

1.5 HisU

9.4 A80184

15.8 ABW07A 1Pgd2120)

-

Dirt Marker c U Name

Figure 1. The 187 markers mapped to the Steptoe/Morex barley cross constitute seven Linkage groups spanning 1,344 cM,

1 T M I Manhattan, Kansas 1991 23

Table 2. Distribution of markers to and genetic length of the seven barley chromosomes

Chromosome cM cDNA Genomic RAPD Other Total 1 229 25 8 2 2 37 2 208 24 9 0 0 33 3 170 9 9 0 2 20 4 182 10 7 1 1 19 5 168 17 5 0 1 23 6 134 12 11 0 0 23 7 253 17 12 1 2 32

Total 1.344 114 61 4 8 187

Appendix 1. Known function gene loci designation and description

Number Designation Description Chr Probe Source and Reference

Narl Ale Dhn6 Nar7 Glx Prx2 Brz RbcS Bmy 1 Bmy2 Amy 1 Amy 1 Lth Horl HorZ Ubi 1 Ical cxp Ips PrxlA Cab 1 Cab2 Gln2 ChslA ChslB Chs3 His3 Pgkl pgk2 Pcrl Pcr2

NADH nitrate reductase 6S E aleurain 7 E dehydrin 4 E NAD(P)H nitrate reductase 6L E starch synthetase 1 G leaf peroxidase 2L G UDPgluaflav3-0-glucosylsyl trans I G ribulose bis P carboxylase; SS 2 G p- amylase 4 G p- amylase 2 G a- amylase 6L G a- amylase I G leaf thionins 6S G C-hordeins 5S G B-hordeins 5S G Ubiquitin 1 P chemotlypsin inhibitor 1 5 G carboxypeptidase 6S E amylase/proteinase inhibitor (PAPI) 7 G seed peroxidase; B 1 1 G chlorophyll ah bindmg protein 7 F chlorophyll ah biding protein 5L F glutamine spthase, chloroplast 2 E chalcone synthase 2s P chalcoue synthase 2s P chalcone synthase 5 P histone 3 1 F phosphoglycerate kinase 4 G phosphoglycerate kinase 1 G protcchlorophylide reductase 2L G protochlorophylide reductase 5 F

A. Kleinhofs; MGG 227:411,91 1. Rogers; PNAS 82:6512,85 T. Close; Amt.JPP 17:333,90 A. Kleinhofs; MGG 228:329,91 W. Rhode; NAR 16:7185,88 SK Rasmussen; unpublished R. Wise; PMB 14:277,90 K. Gausing; DevGen 8:495,87 M. Kreis; GenResCamb 51:13,88

I. Rogers; JBC 263:18953,88

K. Apel; MGG 207:446,87 PR Sheuly; EMBO J 4:9,85

G. Gaming; EnrJBio 158:57,86 PR Shewry; PMB 10:521,88 DC Baulcombe; JBC 262:13726,87 J. Rogers; Planta 169:51,86 SK Rasrnussen; PMB 16:3 17,9 1 K. Gausing; DevGen 8:497,87

BG Forde; PMB 14:297,90 W. Rhode; PMB 16:1103,91

A. Brandt; CarResCom 51:211,86 S. Chao; MGG 218:423,89

K. Apel; JCellBiol 23: 18 1.83

1 T h! I Manhattan, Kansas 1991

Appendix 1 (continued).

Number Designation

83 Rml I14 Rml 126 Chi 127 Glb3 128 Glb4 133 Adh4 134 Adh5 135 Adh6 136 Adh7 137 Adh8 138 Hor5 141 Glbl 142 Tha 143 Pox 153 PIC 156 His4A 157 Dor4A I57 Dor4B 160 Dor2 174 Rpll7A 178 Nir 179 Aga7

Pmbe quality rating:

Description Chr Probe

ribosomal RNA spacer DNA 6S E ribosomal RNA spacer DNA 7S P chitinase; 26 kDa 1 F (1-3)-p-glucana~e 3L G (1-3)-p-glucanase 3L G alcohol dehydrogenase 4 P alcohol dehydrogenase 3 P alcohol dehydrogenase 7 P alcohol dehydrogenase 1 P alcohol dehydrogenase 2 P gamma hordeins 5 F (1-3; 1-4)-P-glucanase 5 G thaumatin-like protein 1 E pathogen-induced peroxidase 2S E plastocyanin 1 F histone 4 5 P dormin group 4 3 P dormin group 4 1 F dormin group 2 5 G ribosomal protein 7 G nitrate reductase 6 G ADP-glucose pyophosphate, endo 5 G

E - excellen< major band, easy to use G - god, easy to use F - fair, can be used but some difficulty P - poor, not recommended

Source and Reference

M. O'Dell; NAR 7:1869,79

R Leah; PMB 12:673,89 R Leah; JBC 266: 1564,91

A. Good; NAR 16:7182,88

A. Brandt; PMB 11:449,88 J. Litts; PNAS 83:2081,86 R. Dudler; PMB 17:283,91 R Dudler; PMB 16:329,91 K. Gausing; FEBS L 225:159,87 M. Iwabuchi; NAR 11:5865,83 K. Simmons; PlantPbysiol 95:814,91

K. Sinunons; PlantPhysiol 95:814,91 K. Gausing; CurrGen 19:417,91 S. Rothstein; PlantPhysiol 88:741,88 MR Olive; PMB 12:525,89

I T M I Manhattan, Kansas 1991 25

The Physical Mapping of RFLP Markers in Rice

J. P. Gustafson USDA-ARS, Plant Genetics Research Unit and Plant Science Unit,

University of Missouri, Columbia, MO 65211 USA

Physical mapping of lowcopy and unique-sequence DNA probes to plant chromosomes using a non- radioactive biotin-labeling in situ hybridization (ISH) technique is possible. The detection of unique-sequence probes has been improved by the utilization of root-tip protoplasts and enzyme-conjugated reporters. The production of protoplasts involved the removal of cellulose and allowed for improvement in detection levels of unique-sequence DNA probes.

With the creation of restriction fragment length polymorphism (RFLP) l i g e maps in cereals and the use of non-radioactive ISH techniques, studies analyzing the extent to which linkage maps covered the physical length of a genome were started. There are indications that tremendous differences occur between physical and genetic distances as determined by recombinational map units.

The available physical mapping data on plant linkage maps is very limited and at present most of the data involves some rice ( O v a sativa L.) l i e . The large linkage groups mapped to chromosomes 1 and 5 were the only ones that spanned a centromere. All of the other l i e groups were either completely on the long arm or the short arm of the chromosome to which they were mapped. The largest l i e group, consist- ing of 21 RFLPs covering 183 cM, was located on the short arm of chromosome 2. The chromosome 9 linkage group contained six RFLPs covering 66 cM, while the chromosome 10 linkage group contained only three RFLPs covering 43 cM. It was interesting to note that the physical distance covered by both linkage groups was quite similar in size. The chromosome 2 linkage group showed a significant difference when comparing the physical distances between the linked RFLPs RG322 and RG139 (56 cM) vs. RG139 and RG83

(127 cM). The physical distance between RG322 and RG139 was significantly larger than the physical distance between RG139 and RG83. This indicated that the largest cM distance was definitely not the largest in terms of the physical distance covered on the chromosome.

These differences could be due to first, the presence or absence of blocks of heterochromatin within one of the l i e groups that have not yet been detected. A second possibility could be the presence of a 'cold spot' of recombination within one of the linkage groups. Third, it has been shown that during mitosis, rice chromosomes appear to condense differently from one part of the chromosome to another. Maybe different levels of recombination exist because of the condensation differences. However, the real reason 1s unknown. These two examples of the differences that can exist when comparing genetic and physical distances illustrate the problems that can arise even within a single chromosome, let alone between chromosomes of a species.

It would be a mistake to conclude that the ISH technique is the ultimate solution to physical mapping. The physical location on a chromosome utilizing ISH techniques is at best a fair estimate of the actual distance. Considerable chromosome walking and DNA sequencing is needed.

Analysis of several rice RFLP linkage groups have shown that only a limited portion of the genome's physical length could be involved. This was not unex- pected as several reports have suggested that the recombinational location of genes may vary consider- ably from their physical location in both animals and plants.

26 1 T M I Manhattan, Kansas 1991

An Updated Genetic Linkage Map of Triticum tauschii, the D-genome Progenitor of Wheat

K.S. Gill, D. Hassawi, KJ. Raupp, A.K. Fritz, B.S. Gill, T.S. Cox, and R.G. Sears Departments of Plant Pathology and Agronomy and the USDAIARS,

Kansas State University, Manhattan, KS 66506-5502 USA

T h e current map of ~ri t icum tauschii consists of 196 loci, 182 of which are present as llnkage groups (Fig. Table 1. Number of loci and average chromosome lcngth 1). An F, population of 60 plants, derived from a cross for each of the 7 D-genome chromosomes in Tritjcum between two accessions of T. tauschii (TA1691 / tauschii. TA1704), was used for the mapping. Triticum tauschii was used to make the map because it is diploid, highly Chromosome Number Length polymorphic, and its D genome is almost identical to of loci (cM)

that of polyploid wheat. The clones used as RFLP ID 23 27 1 (restriction fragment length polymorphism) markers 2D 27 342 were isolated from a Pst I genomic library of T. 3D 36 390

tauschii accession TA1642 (Gill et al. , 1991). One 4D 19 221 5D 24 281

hundred and seventy-six loci exist as seven linkage 6D 34 462 groups corresponding to the seven chromosomes of T. 7D 13 185 tauschii (ID, 2D, . . . , 7D). Six loci are present in two Total 176 2152 linkage groups not assigned to any chromosome. The number of loci and the genetic length for each chromosome is given in Table I . There is an average of 25 loci pcr chromosome. All the markers are RFLP loci except Reference Cited for eight protein loci and a leaf rust resistance gene. The map covers all seven D-genome chromosomes Gill, K.S., E.L. Lubbers, B.S. Gill, W.J. Raupp, and T.S.

with an average genetic length of 307 cM per chromo- Cox. 1991. A genetic linkage map of Tr~ticum tauschii (DD) and its relationship to the D genome of bread wheat some. Ninety per cent of the genome is covered and (AABBDD), Genome 34:362-374,

most of the probes are also mapped in wheat.

-M LOCUS -..I LOCUS CM LOCUS

RUST

Q U I

B ' f D o l l

10 .

Figure 1. Genetic linkage map of Triticum tauschiz OD)

CM LOCUS

CM Locus


- 96

16 - 11 - 0 - 5- 7

W G Y I

--Mom

- ADKf

- M 1 4 m

- EoOO - .,,*A

,I

5- 7 - 9

1- '- 11 - 4

- r)

- 2)

- 43

- ~ m 6 - PSRl63

Golo -urn - MOT1

- WQtj4

- MMlB

- MPP2

- M147

- G W A

ell LOCUS cM LOCUS dl LOCUS


The Relationship Between Physical and Genetic Maps of Wheat Chromosomes

A.J. Lukaszewski and C.A. Curtis Department of Botany and Plant Sciences, University of California, Riverside, CA 92521 USA

T h e use of polymorphism for C-bands or other and was concentrated in the distal (ca. 20%) of the physical attributes of chromosomes in genetic mapping arms. There were no statistically significant differences allows the integration of genetic and physical maps and in this pattern of recombination between different the study of the distribution of recombination along the chromosome arms. However, significant differences in chromosome. Such attempts have been made in wheat recombination frequencies were observed for the same (Jampates and Dvorak, 1986; Curtis and Lukaszewski, segments in different hybrid combinations. This may 1991), barley (Linde-Laursen, 1982), and rye be the result of different pairing affinities of chromo- (Lukaszewski, 1992), showing that, in all three species, somes or differences in transmission rates, particularly recombiation was concentrated in the distal regions of of univalents following pairing failure. As a rule, chromosomes and was rare or absent in the proximal reduced pairing leads to overestimation of genetic dis- regions. As a result of this uneven distribution of tances. recombination, genes that mapped close to the cen- When the genetic distances from the centromere tromere genetically, were physically localized in more were plotted against the physical lengths of the chro- distal regions of chromosome arms (for review, see mosome arms (Fig. I), it became apparent that the Curtis and Lukaszewski, 1991). Disparity between proportion of distal recombination was higher and of physical and genetic distances could create problems proximal recombination was lower in the physically when l i e d markers are used in the isolation of genes short arms (IBS, 2BS, 3BS, 6BS, 6BL, and 7BS) than of interest since the genetic distance between them it was in the physically long arms (IBL, 2BL, 3BL, would correspond to Merent physical lengths (number 5BL, and 7BL). This suggests chiasma interference. of base pairs) depending on their locations with respect Because pairing initiation in wheat is distal, the forma- to the centromere. tion of distal chiasmata could be favored. Once a distal

Extensive polymorphism for C-banding patterns, chiasma is formed, interference may prevent the for- particularly in the B-genome chromosomes, was found mation of a second chiasma, thus limiting the physi- among different accessions of Triticum dicoccoides cally short arms to a single exchange. There would be and between T. dicoccoides and T. durum. This poly- less space limitation in the physically long arms to the morphism involved either the presence or absence, or formation of a second, interstitial chiasma. Average clear differences in size, of certain C-bands. It allowed interference calculated for all pairs of adjacent seg- us to study the distribution of recombination in 90 ments involved in recombination was 81.3%. Interfer- segments in chromosome arms lBS, IBL, ZBS, 2BL, ence tested in segments pooled to cover the entire por- 3BS, 3BL, 5BL, 6BS, 6BL, 7BS, and 7BL. The tions of chromosome arms in which any recombination analysis was performed in both F2 and backcross was detected averaged 56.6%. The lengths of genetic progenies and up to three chromosomes were studied in maps of the B-genome chromosomes generated in this any single hybrid combination. The segments varied in study corresponded closely to the map lengths calcu- length from 3 to 70% of the relative arm length. The lated on the basis of chiasma frequencies observed total number of chromosomes studied was 916. cytologically by Sallee and Kimber (1978). Similar

The results demonstrated that the frequency of distribution of chiasmata in metaphase I and of recombination in wheat chromosomes increased expo- recombiation along chromosome arms indicate no nentially with distance from the centromere. Among the chiasma terminaliration. analyzed sample of chromosomes, the most proximal It is apparent that for all practical purposes the recombination event was detected at 37.4% of the proximal halves of the B-genome chromosomes do not relative arm length away from the centromere; recom- recombine. There are good indications that the same bination was infrequent in the middle part of the arms holds true for the A- and D-genome chromosomes.


However, there are no reasons to believe that these proximal segments are incapable of recombination. Chromosomes deficient for up to 49% of their relative arm lengths showed regular pairing in meiosis (Curtis et al., 1991) indicating that the proximal regions are fully capable of pairing initiation and normal synapsis. Premeiotic applications of wlchicine, which are believed to disturb the telomere alignment at the nuclear membrane and thus prevent normal telomeric initiation of synapsis, significantly increased the ratio of proximal to distal recombination (Curtis, unpublished).

Using the data from Flavell et al. (1987), on the DNA content and arm ratios of individual wheat chro-

mosomes, and the data on genetic distances between the telomeric and sub-telomeric C-bands on the IB satellite (Curtis and Lukaszewski, 1991) and between the centromere and the breakpoint in 5B chromosome deficient for 49% of the long arm (Curtis et al., 1991) we calculated that a genetic distance of 1 cM may vary from about 1,530 kb in the distal chromosome regions to about 234,000 kb in the regions proximal to the centromere, a 153-fold difference. This difference, in conjunction with 354% average range in estimates for genetic distances between the same markers, implies that chromosome walking studies that use linked markers in the isolation of genes may be difficult in wheat.

Physically short arms 0,

a, 70 o m Physically long arms d

E ,, " . / 0

0 5 10 15 20 25 30 Physical distance from centromere (a.u.)

Figure 1. Relationship behveen genetic and physical distances in physically short (IBS, 2BS. 3BS, 6BS, 6BL, 7BS) and physically long (IBL. ZBL, 3BL, 5BL, 7BL) chromosome arms in wheat. Physical length expressed in arbitrary units (am).

References Cited

Curtis, C.A. and A.J. Lukaszewski. 1991. Genetic linkage between C-bands and storage protein genes in chromosome 1B of tetraploid wheat. Theor. Appl. Genet. 81:245-252.

Curtis, C.A., A.J. Lukaszewski, and M. Chrzastek. 1991. Metaphare I pairing of deficient chromosomes and genetic mapping of deficiency breakpoints in common wheat. Genome 34553-560.

Flavell, R.B., M.D. Bennett, A.G. Seal, and J. Hutchinson. 1987. Chromosome structure and organization. In: Wheat Breeding: Its Scientific Basis (Lupton, F.G.H., ed.) Chapman and Hall, pp 21 1-268.

Jampates R. and J. Dvorak. 1986. Location of the Ph1 locus in the metaphase chromosome map and the linkage

map of the 5Bq arm of wheat. Can. J. Genet. Cytol. 28:511-519.

Linde-Laursen, I. 1982. Linkage map of the long arm of barley chromosome 3 using C-bands and marker genes. Herediw 49:27-35.

Lukaszewski, A.J. 1992. A comparison of physical distribution of recombination in chromosome 1R in diploid rye and in hexaploid triticale. Theor. Appl. Genet. (In press).

Sallee, P.J. and G. Kimber. 1978. An analysis of the pairing of wheat telocentric chromosomes. In: Proc. 5th Int. Wheat Genet. Symp., New Delhi, India (Ramanujam, S., ed.) 408-419.


Chromosome Maps of Wheat Based on RFLP Markers

Gary E. Hart

Soil a n d C r o p Sciences Department, Texas A&M University, College Station, T X 77843 USA

I n research being conducted at Texas A&M Univer- sity, compensating nullisomic-tetrasomic strains and ditelosomic strains of Triticum aestivum cv. Chinese Spring are being used to assign DNA restriction fragments to chromosomes and chromosome anns; and derivatives of T. turgidum var. durum cv. Langdon - T turgidum var. dicoccoides disomic chromosome substitution lines are being used to develop RFLP maps of selected chromosomes.

Sixty-six genomic DNA (gDNA) clones in pUC8 and nine cDNA clones in pBR322 have been used to determine the chromosomal locations of about 200 RFLP loci. The number of probes isolated that identify loci in one or more chromosomes in the seven homoeologous groups of hexaploid wheat ranges from four for group 4 to 22 for group 6. The number of loci detected per chromosome ranges from only one for both 4B and 4D to 16 in each of the chromosomes of hornoeologous group 6. Forty-eight probes (64%) detect one group, 18 (24%) detect duplicate loci in one homoeologous group, four (5%) detect one locus only, five (7%) detect two or more loci in different homoeologous groups and one detects loci in more than two non-homoeologues.

Dr. Leonard Joppa has developed the 14 possible T. turgidurn var. durum cv. Langdon-T. turgidum var. dicoccoides chromosome substitution lines and is also deriving recombinant substitution lines (RSLs) from the substitution lies. Using 30 probes on DNA from the substitution lines and of Langdon cut with from four to six restriction enzymes, we have found 70% RFLP for 6A and 73% for 6B. Eighteen probes detected RFLPs between Langdon and dicoccoides for both 6A and 6B, five for neither chromosome, and seven for one of the two chromosomes only (three for 6A and four for 6B). Little difference was found among restriction enzymes in the level of RFLP detected, the range being from 29% for Bgl II to 38% for Hind III.

Dr. Joppa has supplied us with 66 chromosome 6A RSLs and 85 chromosome 6B RSLs. We have produced 100 or more F2 seeds from crosses of both the 6A and 6B substitution lines to Langdon and in turn have produced F, populations from 80 to 100 F2 individuals for both of these chromosomes. We are presently mapping 6A and 6B using RSLs and bulked F, populations.


Deletion Mapping of Wheat Chromosomes

R.S. Kota, J.E. Werner, D.E. Delaney, K.S. Gill, T.R. Endo, and B.S. Gill Department of Plant Pathology, Kanses State University, Manhattan, KS 66506-5502 USA

A l i e n chromosome transfers to wheat reveal that certain chromosomes of Aegilops (=Triticum) cause extensive breakage of wheat chromosomes. The chromosomal instability in wheat is caused either by gametocidal action (Endo, 1988) or nongametocidal action (Kota and Dvorak, 1988). The gametocidal chromosomes of Aegilops in a single dose (monosomic addition, 2n = 43) in wheat ensure preferential transmission by causing extensive chromosome breakage in the gametes that do not contain the gametocidal chromosome. No chromosome structural changes are detected among the gametes that contain the gametocidal chromosome (Maan, 1975; Miller et al., 1982; Finch et al., 1984; Endo, 1985; 1988). Progeny from the crosses of Chinese Spring with monosomic addition 4S of Triticum sharonense (Finch et al., 1984) and T. longissimum (Endo, 1985) were sterile. However, progeny from the cross of Chinese Spring with monosomic addition 3C of T. cylindricum are fertile.

The nongametocidal chromosomes include chromosome 6S of T. speltoides ( s p . Aegilops speltoides, Kota and Dvorak, 1988) and 6S1 of T. longissimum (Kota and Dvorak, 1986). The presence of a nongametocidal chromosome like 6S in the wheat genetic background affects only certain chromosomes. As a result, only a limited number of deletion stocks for certain chromosomes were recovered (Kota, unpublished results). Whereas, in a cross of Chinese Spring with monosomic addition 3C of T. cylindricum, about half of the plants lacking chromosome 3C have extensive deletion, translocation, ring, and acentric chromosomes.

Isolation of chromosome deletion stocks Utilizing this system, Endo and Gill (unpublished results) isolated a total of 226 independent deletion stocks for the 21 chromosomes of Chinese Spring wheat. Among these, 145 deletions are homozygous and 81 deletions are heterozygous. Once a deletion was identified in heterozygous condition by C-banding, ten plants from the progeny were screened for homozygous deletion. If none were recovered, then heterozygous plants were crossed with corresponding nulli-tetra-

somic or opposite arm deletion stocks. Five plants, analyzed from these crosses to identify monosomic deletion chromosomes were then selfed to isolate homozygous deletion stocks. The list of deletion stocks for each chromosome arm is provided in Table 1.

Table 1. The list of deletion stocks, in Chinese Spring wheat, for each chromosome arm.

Chromo- L S L S L S TOTAL some

1 - 1 1 2 7 5 - 25

TOTAL 22 50 39 60 24 31 226

Deletion mapping The cytologically-based physical maps of the molecular markers of wheat are being ~ 0 n ~ t ~ c t e . d by locating breakpoint positions, indicated as a fraction length from the centromere, and mapping probes to chromosome regions defined by deletion breakpoints. Initially, for most of the chromosomes, we are using the genomic probes that were generated from Triticum tauschii at Kansas State University. These probes are readily available and will allow comparison of the hexaploid wheat and I: tauschii genetic maps (Gill et al., 1991). Other probes, provided by Cornell Uni- versity and IPSR, Cambridge will be added later. The group 7 deletions were mapped using mainly Cam- bridge probes since very few T. tauschii probes are available for this group.

The deletion mapping is carried out in two steps. To determine if any possible chromosome rearrangements have occurred between hexaploid wheat and T. tauschii, we first hybridized our probes to control blots containing restriction enzyme digested DNA from all the available nulli-tetrasomic and ditelosomic lines. This data indicated which probes were specific for


which wheat chromosomes. Most of the probes (87%) that mapped to a specific chromosome in T. fauschii could be assigned to the same group in hexaploid wheat. Exceptions were probe GO34 on chromosome 1D of T. tauschii that is not present on 1B in wheat; probes E003,G049, and F036 on chromosome 2D that map to chromosome group 6 in wheat; probes DO18, H005, and E009 on chromosome 4D that map to group 7 in wheat; and probes F043, F019, and F037 on chromosome 6D that map to group 2 in wheat. It would be premature at this point to make a general conclusion from these data that chromosomal rearrangements had occurred in the evolution of hexaploid wheat since the discrepancies may be isolated to the particular accessions of T. tauschii (var. meyeri and fypica) used to construct the map.

The second step was hybridization of the probes, specific for a particular chromosome group, to blots containing DNA from the deletion lines for that group. From autoradiography of these hybridizations we were able to determine two things: 1) the linear order of the probes on the chromosomes in hexaploid wheat that we could compare to T. tauschii; and 2) the physical location of the probes on each chromosome when we have all the breakpoint measurements for the deletion l ies . Most of the probes (95%) were in the same linear order in both hexaploid wheat and T. tauschii. Exceptions were on chromosome 3D where the probes on the short arm appear to be in the reverse order in hexaploid wheat, and on chromosome 5 where probe KSU024 maps to the short arm in T. tauschii but is on the long a m in wheat. Probes are placed on the physical map in the chromosome region between the breakpoint of the largest deletion showing the band of interest and the next larger deletion where the band is not expressed.

The comparison between physical and genetic maps (Chao er al., 1989) using the same probes for chromosomes 7B and 7D are indicated in Figure 1. The gene order on the physical map of chromosomes 7A, 7B, and 7D corresponds very well to the genetic map except for loci 72 and 117. These two loci genetically map in the distance from 0 to 5 cM, depending on the recombination population u s 4 and appeared in re-

verse order on the physical map. General agreement in the gene order between the two maps confirms the cytological observation that most deletions arise from a single breakage with concomitant loss of the distal segment. Massive chromosome rearrangements would have resulted in gene order disparities between the two maps.

The discrepancy between the physical and the genetic maps was observed for the distances of loci in relation to the centromere. Loci that genetically map close to the centromere are physically localized to more distal chromosome regions. Loci 152 and 165, on opposite sides of the centromere on 7B, are 7 cM apart physically but span about 60% of the total chromosome length. The mid-arm region spanning 0.4 to 0.8 of 7BL has a genetic length of 24 cM. The distal 15% of the 7BL chromosome has a high rate of recombination with a genetic length exceeding 100 cM. A similar physical distribution of recombination was observed for the 7D chromosome. The fact that recombination is suppressed in the proximal regions of the chromosomes, limits the power of recombination mapping in ordering proximal loci. Loci 105, 165, and 169, that were not resolved genetically, were easily ordered physically. Therefore, physical maps are more powerful for resolving gene order for the proximal loci while genetic mapping is more precise for distally located genes.

Cytogenetically-based physical maps of molecular markers provide:

a simple and rapid method to construct maps; genetic analysis is not required, and any probe can be utilized without the necessity of identifying polymorphism;

a direct estimation of the physical size of a region;

the order of DNA markers spanning millions of base pairs;

the translation of genetic distances into physical distances;

the integration of information from different mapping strategies, leading to the ultimate goal of gene cloning.


Figure 1. Comparison between physical and genetic maps using the same probes for chromosomes 79 and 7D.

References Cited

Chao, S., P.1 Sharp, A.J. Worland, E. J. Warham, R.M.D. Koebner, and M.D. Gale. 1989. RFLP-based genetic maps of wheat homoeologous group 7 chromosomes. Theor. Appl. Genet. 78:495-504.

Endo, T.R. 1985. An Aegilops longissima chromosome causing chromosome aberrations in common wheat. Wheat InJ Sen. 60:29.

Endo, T.R. 1988. Induction of chromosomal structural changes by a chromosome of Aegilops cylrndrica L. in common wheat. .I Hered. 79:366-370.

Finch, RA., T.E. Miller, and M.D. Bennett. 1984. 'Cuckoo' Aegilops addition chromosome in wheat ensures its transmission by causing chromosome breaks in meiospores lacking it. Chromosoma 90:84-88.

Gill, K.S., E.L. Lubbers, B.S. Gill, W.J. Raupp, and T.S. COX. 1991. A genetic linkage map of Triticum tauschii

(DD) and its relationship to the D genome of bread wheat (AABBDD). Genome 34:362-373.

Kota, RS. and J. Dvorak. 1986. Mapping of a chromosome pairing gene and 5 s rDNA genes in Triticum aestiwrm L. by spontaneous deletion in chromosome arm 5Bp. Can. J. Genet. Cytol. 28:266-271.

Kota, R.S. and 1. Dvorak. 1988. Genomic instability in wheat induced by chromosome 6BS of Triticum speltoides. Genetics 120:1085-1094.

Maan, S.S. 1975. Exclusive preferential transmission of an alien chromosome in common wheat. Crop Sci. 15:287- 292.

Miller, T.E., I. Hutchinson, and V. Chapman. 1982. Investigation of a preferentially transmitted Aegilops sharonensis chromosome in wheat. Theor. Appl. Genet. 6127-33.

36 I * T M I Manhattan, Kansas 1991

Fine Physical Mapping of Phl, a Chromosome Pairing Regulator Gene in Polyploid Wheat

KS. Gill, T.R. Endo, and B.S. Gill Department of Plant Pathology, Kansas State University, Manhattan, KS 665065502 USA

I n polyploid wheat, the diploid-like chromosome pairing mechanism is principally controlled by the Ph l (pairing homoeologous) gene that is located on the long arm of chromosome 5B (Riley and Chapman, 1958; Sears and Okamoto, 1958). Here we report that in hexaploid wheat, the Ph l gene is located in the chromosome region between fraction length (FL) 0.51 and 0.59 of chromosome 5BL. We identified three (XksuS1-5, Xpsr128-5 and Xksu75-5) DNA probes that map in the deleted region of chromosome 5BL of thephlb mutant. The probes XksuS1-5 and Xksu75-5 map in the same chromosome region that the Phl gene maps, XksuS 1-5 being closer to the gene than Xksu75- 5. The probe Xpsr128-5, however, maps proximal to FL 0.55.

The chromosome deletion lines used for the physical mapping of the Ph l gene were generated using gametocidal chromosome 3C of Triticum cylindricurn (Endo, 1988). The two mutants of the Ph l gene @hlb andphlc), used in the study, were generated independently using X-ray irradiation (Sears, 1977; Giorgi and Barbera, 1981). Both mutants, most probably, are interstitial deletions (Sears, 1977; Gill and Gill, 1991; Dvorak et al., 1984). The duplication line used has a duplicated interstitial region, encompassing the Phl gene of chromosome 5BL. The mutant phlb is in hexaploid wheat cultivar Chinese Spring (CS) whereas the mutant ph l c and the duplication line are in tetraploid wheat cultivar CappeUi. Wheat homoeologous group 5 nullisomic-tetrasomic and ditelosomic lines of CS were used to map DNA fragments to their respective arms of group 5 chromosomes.

The presence of the Ph l gene in the deletion lines was scored by analyzing MI (metaphase I) chromosome pairing of the F, from a cross of each deletion line with Triticum peregrinum (syn. Aegilops van- abilis). The deletion l i e s 5BL-9 and I I, at MI, have an average of 3.2 I1 + 29.8 I and 1.5 I1 + 32.5 I, respectively, thus possessing the Ph l gene. The deletion lines 5BL-1 and 3, however, have an average of 0.4 IV + 1.5 III+ 8.9 II+ 14.1 Iand 0.4 IV + 2.1 111 +

8.9 I1 + 9.3 I, respectively, indicating the absence of the Phl gene. The breakpoints of the two deletion lines which bracket the Ph l gene (5BL-1 and 5BL-11) are at FL 0.55 and 0.59, respectively. Therefore, the Ph l gene is present in the chromosome region between FL 0.55 and 0.59.

We analyzed the P h l mutant and chromosome 5BL deletion lines by 'Southern' analysis, using chromosome group 5 long-arm-specific probes. Chromo- some 5BL specific fragments (from Southern analysis) were identified using chromosome group 5 nullisomic- tetrasomic and ditelosomic lines. We identified three (out of 17) probes (XksuS1-5, Xpsr128-5, and Xksu75-5) each of which detected a missing chromosome 5BL fragment in ph lb mutant indicating that these three probes map within the deletion(s) of chromosome 5BL of thephlb mutant. The probes XksuS1- 5 and Xksu75-5 mapped in the chromosome region between FL 0.55 and 0.59, whereas Xpsr128-5 mapped proximal to FL 0.55. The probe XksuS 1-5 also detects a missing 5BL band in the mutant phlc, however, Xksu75-5 detects a normal band. In the duplication line, that possesses an interstitial duplication encompassing the Phl gene, only the probe XksuS1-5 detects the 5BL band, at twice the intensity of the normal Cappelli band. Another DNA probe (Xksu24-5), that maps in the same chromosome region as the Ph l gene maps (between FL 0.55 and 0.59), detects a normal 5BL band in the mutant phlb and phlc.

According to the deletion mapping data, the Ph l gene maps in the chromosome region distal to the breakpoint of deletion 5BL-I (FL 0.55) and proximal to deletion 5BL-11 (FL 0.59). The probes Xksu75-5, Xksu24-5, and XksuS1-5 also map in the same chromosome region. Among these probes, XksuSl-5 is present closest to the Ph l gene a s it detects a missing 5BL band in both the mutants, and in double the intensity in the duplication line. The probe Xksu24-5 is present in the chromosome region that is not deleted in either mutant, most probably the region marked by the C-

I - T M I Manhattan, Kansas 1991 37

band 5BL2. I. The breakpoint of the deletion in chromosome 5BL of the phlc mutant separates Phl and Xksu75-5.

The breakpoiits of the deletions 5BL-1 and 5BL- 11 are at FL 0.55 and 0.59, respectively, just surrounding the C-band 5BL2.1. The mutant phlb possesses a deletion proximal to the C-band 5BL2.1 and the probes Xpsr128-5 maps in the deleted region. Since the chromosome region marked by the C-band 5BL2.1 is not deleted in either of the mutants, the Phl gene is present in the region just proximal or distal to the C-band. The proximal location of the gene would suggest the presence of only one deletion in the mutant phlb, whereas the distal location would indicate that there are two deletions in chromosome 5BL of the mutant l i e .

On the genetic linkage map of T. tauschii (a progenitor species of wheat), the probes Xksu75-5 and XksuS1-5 map 3 cM apart, XksuS1-5 beiig distal to Xksu75-5 (Gill, K.S. et al . , 1991). The probe order should also be. the same in wheat, assuming that the gene synteny between wheat and T. tauschii is wn- sewed for these markers. The probe order is the same between wheat and T. tauschii for 11 of the other 13 chromosome-group-5-specific DNA markers (K.S. Gill et al. , unpublished results). Therefore, probe Xksu75-5

Referenc

Dvorak, J., K.-C. Chen, and B. Giorgi. 1984. The C- banding pattern of a Ph-mutant of durum wheat. Can. J. Genet. 26: 360-363.

Endo, T.R 1988. Induction of chromosomal structural changes by a chromosome of Aegilops cylindrica L. in common wheat. J. Hered. 79: 366-370.

Giorgi, B. and F. Barbera. 1981. Increase of homoeologous pairing in hybrids between a Ph murant of T. turgidum L. var. Arum and two tetraploid species of Aegilops: Aeg- ilops kotschyi and Ae. cylindrica. Cereal Res. Comm. 9: 205-211.

Gill. K.S. and B.S. Gill. 1991. A DNA fragment mapped within the submicroscopic deletion of Phl, a chromosome pairing regulator gene in polyploid wheat. Genetics 129: 257-259.

Gill, B.S., B. Friebe, and T.R. Endo. 1991. Standard

is probably proximal to XksuS1-5. Since the two probes are 3 cM apart and the breakpoint of deletion 5BL-1 is just above the C-band 5BL2.1 (FL 0.5 I), it is likely that the two marken are spaced by the C-band 5BL2.1, the probe Xksu75-5 being proximal and XksuS 1-5 being distal. In that case the probe XksuS1- 5, along with the Phl gene, maps in the chromosome region between C-bands 5BL2.1 and 5BL2.5. The FL of the chromosome region between the C-bands 5BL2.1 and 2.5 is 0.15 and 0.14 in CS and phlb mutant, respectively, with a confidence interval value ranging from H.01 to 0.02. Any deletion in this region, therefore, has to be very small (submicroscopic).

In wnclusion, the Phl gene is located in the chromosome region between FL 0.55 and 0.59 of chromosome 5BL of hexaploid wheat. The probes XksuS1-5 and Xksu75-5 map in the same chromosome region as the Phl gene, the probe XksuS1-5 King closer to the gene wmpared to Xksu75-5. Although there is no proof yet, the current data suggest the possibility of two independent deletions in chromosome 5BL of the phlb mutant, the larger being proximal to C-band 5BL2.1 and the submicroswpic, encompassing the Phl gene, being distal.

:es Cited

karyotype and nomenclature system for description of chromosome bands and structural aberrations in wheat (Triticum aestiwm). Genome 34:830-839.

Gill, K.S., E.L. Lubbers, B.S. Gill, W.J. Raupp, and T.S. Cox. 1991. A genetic linkage map of Trificum tauschii @D) and its relationship to the D genome of bread wheat (AABBDD). Genome 34: 362-374.

Riley, R. and V. Chapman. 1958. Genetic control of the cytologically diploid behavior of hexaploid wheat. Nature 182:713-715.

Sears, E.R 1977. An induced mutant with homoeologous pairing in common wheat. Can. J. Genet. Cytol. 19: 585- 593.

Sears, E.R and M. Okamoto. 1958. Intergenomic chromosome relationship in hexaploid wheat. Proc. Tenth lnt. Cong. Genet. 2:258-259.

- 38 1 T M I Manhattan, Kansas 1991

Chemiluminescent Methods for RFLP Analysis in the Triticeae

David Hoisington Applied Molecular Genetics Laboratory, CIMMYT, Mkuco DF, MEXICO

O n e of the major limitations for the widespread use of molecular techniques in developing countries is the requirement to use radionuclides for the detection of nucleic acid hybridizations. This is particularly limiting in the case of RFLP analyses given the large quantity of 32P that must be used. Since one of the goals of the new Applied Molecular Genetics Laboratory at CIMMYT is to provide training and, ultimately, technology transfer to developing country laboratories, we decided to attempt RFLP analyses in maize, wheat, and barley without using radioactivity. Recent reports demonstrated that the digoxigenin anti-digoxigenin- alkaline phosphatase system could be used to detect single-copy DNA-DNA hybrids (Krieke et al., 1990). In addition, with the use of the chemical AMPPD as the substrate for the alkaline phosphatase, the probes wuld be removed and the memhranes rehybridized with additional probes.

Our early attempts at non-radioactive hybridizations involved the use of biotin and strepavidin-alkaline phosphatase (we were using the Photogene kit from BRL). We were successful in detecting single-copy sequences in maize although there was a large problem in repeatability of the system. At times, the same probe did not hybridize or resulted in tremendous background upon detection. We also had difficulty in applying the detection reagent uniformly over the membranes (particularly when a large number of membranes were being detected).

At the lTMI meetings held in Sacramento, Cali- fornia last year, Tim Helentjaris informed me that the digoxigenin-based system of Boehringer-Mannheim was superior to that of biotin. Once the appropriate reagents were purchased, we quickly determined that it was both easier to use and apparently better in detecting single-copy sequences in maize and wheat. In our early experiments, we were able to detect a single-copy sequence after loading 1 mg of digested genomic DNA on a gel (on first-use membranes). The protocols employed were essentially those of Kreike e! al. (1990). Since then, we have refined the protocols to

utilize minimal quantities of chemicals (for wst sav- ings) and to allow for overnight exposures (for ease of use). The protocol currently in use in the laboratory is outlined below.

Hybridize 15 to 18 hrs. at 65°C Wash 3x 15' in 0.15X SSC, 0.1% SDS at 65°C Rinse in Buffer 1 110 mM Tris, pH7.5; 150 mM NaCI] Incubate 30' in Buffer 2 [lo mM Tris, pH 7.5; 150 mM NaCI; 0.1% blocking] Incubate 30' in Anti-Digoxigenin-AP [1:15000 in Buffer 21

a Wash 3x 10' in Buffer 2 Wash 3x 10' in Buffer 1 Wash lx 5' in Buffer 3 [lo mM Tris, pH 9.5; 100 mM NaCI; 50 mh4 MgCIZ] Incubate 20' in AMPPD 110 d m 1 in Butrer 31 Expose 15 to 18 hrs. to XAR-5 X-ray film

DigoxigenindUTP can be incorporated into DNA by nick translation, random priming, or PCR. We routinely use PCR amplification, starting with 1 to 50 ng of plasmid DNA, 2.5 to 5.0% digoxigenindUTP and 20 to 25 cycles of amplification. Probes which are difficult to amplify can be labeled by either of the other two methods. No purification of the labeled probe has been found to be necessary. Hybridizations are routinely performed in glass tubes in a hybriduation oven, although seal-a-meal bags have been used successfully. Prehybridizing is performed only long enough to wet the memhranes - essentially the time necessary to denature the probe (95°C for 10'). The hybridization solution contains 5X SSC, 0.1% blocking agent (Boehringer-Mannheim), 0.1% lauroylsarcosine, and 0.02% SDS. If the signal on the developed film is weak, a second, stronger exposure can be made by incubating the membrane in a more concentrated solution of AMPPD. For re-use, the membranes are first rinsed in 2X SSC for 5' at RT; followed by washing in O.2N NaOH, 0.1% SDS for 5' at 3%. After stripping, the membranes are rinsed for 5' in TE. The membranes can be immediately placed in tubes for


rehybridization, stored in TE at 4"C, or dried and stored at RT. It should be noted that the stripping protocol is specific for our membranes and the time of the NaOH treabnent should be determined for the specific membranes and conditions in use.

In maize, very little difference is observed behveen 32P and chemiluminescent detection. Mem- branes can be re-used 10 to 15 times (with no change in exposure times, as the substrate is essentially de- pleted in 15 to 18 hours). Unfortunately in wheat and barley, reusability is somewhat of a problem. Under the best of conditions, 3 to 5 re-uses are possible. We are currently investigating whether improvement in DNA isolations, probe removal, or probe labeling can increase the number of times a membrane can be re- probed.

A recent spreadsheet analysis by Michel Ragot in our laboratory has allowed us to determine the total

wst and per-data-point cost of the RFLP protocols. Surprisingly, the cost of RFLP analyses using the chemiluminescent protocol is cheaper than those using 32P, at least within MBxico (and probably within most developing wuntries). When the price of jZP in the US is used, the costs of the two methods are approximately equal. Given the hazards of radioactivity, we feel that the chemiluminescent protocol has a definite advan- tage.

If you would like our current protocols, please write and we would be happy to send them.

Reference Cited

Kreike, C.M., IRA. de Koning, and F.A. Krens. 1990. Non-radioactive detection of single-copy DNA-DNA hybrids. PlantMol. Biol. Rep. 8: 172-179.

40 I T M i Manhattan, Kansas 1991

RFLP Mapping of Wheat-Rye Recombinants and the Use of PCR in Cereal Mapping P.M. Rogowsky, S. Weining, K. W. Shepherd and P. Langridge

Centre for Cereal Biotechnology, Waite Agricultural Research Institute, Glen Osmond, SA 5064, AUSTRALIA

T h e introgression of genetic material from alien species has become an important tool in modem wheat breeding. For example, cereal rye (Secale cereale) has been successfully used as a source of disease resistance genes and yield-benefit traits. Using wheat-rye translocation l i e s as the starting material, rare recombination between wheat and rye chromatin could be achieved in chromosome pairing control mutants (like the phlb mutant) leading to rye segments smaller than chromosome arms present in wheat. Wheat-rye recombinants involving both the short (Rogowsky et al., 1991) and long arms (Koebner and Shepherd, 1985) of chromosomes 1R and 1D were isolated. A total of five recombinants with proximal and four recombinants with distal rye chromatin were isolated for the short arm, while three recombinants with proximal and eight recombinants with distal rye chromatin were isolated for the long arm (see Figure 1). Homologous recombination between recombinants 82- 180 and 193 yielded the derived recombinant DRA-1, in which only a small internal piece of rye chromatin containing the stem rust resistance gene Sfl is present.

While isozyme markers were used to isolate these four groups of recombinants, these markers were not numerous enough to allow a further classification of individual isolates within groups or to order their breakpoints along the chromosome arm. In contrast, over 30 RFLP markers had been mapped to chromosome group 1 of wheat by various labs around the world. Therefore, we screened all the available RFLP probes from Cornell University, Texas A&M, CSIRO Canberra, PBI Cambridge, and from our own collection on the recombinants. Using wheat-rye translocation l i e s as controls, all group 1 bands could be assigned to the A, B, D, or R genome. RFLPs between the wheat and rye chromatin were found with 13 short- arm and 18 longarm probes. While in most cases the lack of one or more 1D bands was accompanied by the appearance of a 1R band, five markers on the short

arm and five markers on the long arm were mapped by the lack of ID bands or the presence of 1R bands only.

The RFLP probes clearly demonstrated at least three different breakpoints for the four short-arm recombinants with distal rye chromatin. With probes BCD98, M69, and SSDNA, the recombinant I93 showed the rye pattern, while recombinants WR-1, WD-1, and WD-2 showed the wheat pattern. Conse-

. quently the 193 breakpoint is located proximal to these markers and to the other three breakpoints. Probe Secl.5, on the other hand, showed a rye band with I93 and WR-1 but not with WD-1 or WD-2, placing the WD-1 and WD-2 breakpoint(s) distal to this marker. In contrast to the results with this group of recombinants, no differences wuld be found between the five short- arm recombinants with proximal rye or within the two groups of long-arm recombinants.

Since only a few of the RFLP probes have been linkage mapped, it is not known how evenly the corresponding markers are distributed along the chromosome arms. However, assuming a relatively random distribution, the results would indicate that there are hot spots for recombination between wheat and rye chromatin. In combination with the low frequency of recombination, this would limit the chances of intro- gressing a rye segment of interest and rule out the development of the recombinants as a mapping tool. On the other hand, four RFLP markers were identified, which map to the rye segment present in DRA-1. This segment also carries the Sfl stem rust resistance gene. These markers will be useful for fine mapping of the region and may ultimately help to isolate the resistance gene.

The PCR reaction in cereal mapping Two types of genetic markers are used to detect

polymorphisms in the DNA sequence of organisms: RFLP markers are usually based on sequence changes in the recognition site of a given restriction endonucle-


Het Glu Tri Sec Gli I

K 1 ( 1D

Figure 1. Wheat chromosome 1D (top) and nine wheat-rye recombinants

ase, while PCR markers are based on polymorphism in the length of an amplied sequence between the annealing sites of two synthetic DNA primers. The DNA sequences of the PCR primers can be completely arbitrary as in the case of the random amplified polymorphic DNA (RAPD) markers (Williams et al., 1990) or they can be defined by the known sequence of a gene of choice (D'Ovidio et al., 1990). In a combination of both, we have taken a semi-random approach where the first primer corresponds to the consensus sequence of intron splice junctions in plants (ISJ primer), while the second one has a random sequence (Weining and Langridge, 1991). Using the junction between the generally rather conserved exons and the often quite variable introns, we target a region in the genome which is likely to be present in a large number of cultivars and at the same time exhibits a high degree of variability.

To be useful in mapping programs, markers have to be polymorphic between varieties of a given species. Therefore, we tested the ISJ primers on a whole range of barley and wheat varieties. This also offered a simple tool for varietal identification. Using a maximum of only three primer pairs, we were able to definitively identify every single one of 30 barley varieties, which included all major commercially grown barley varieties in Australia. The only exceptions were WI2736 and WI2737, two known sister lines. In the case of wheat, five groups could be established among 60 varieties with the same primer pairs. The different degree of polymorphism between barley and wheat varieties cor- roborates earlier results with RFLP markers.

While polymorphism are sought between varieties, uniformity is desired within varieties. Screening 50 individual plants of the barley variety 'Clipper' with two primer pairs, uniformity was found in 49/50 plants. This result is consistent with a rate of 1% cross pollination typically found in barley. Similar consistency was found with rye cultivar 'Imperial' (30130 plants) with one primer pair; in contrast, the second primer pair led to two groups of uniform plants. In the case of South Australian rye, a high degree of polymorphism between individuals was found with both primer pairs. The markers generated with these primer pairs are therefore useful for mapping in inbr&g species like barley but not in outcrossing species like rye. In the case of inbred lines of outcrossing species (such as the rye cultivar Imperial) some of the markers may be useful. PCR markers generated with the ISJ primers have been successfully assigned to cbromosome groups using wheatmarley addition lines, and linkage analysis is in progress.

That linkage mapping of PCR markers can be successfully done in cereals was shown with a primer pair from the border of an R173 element. With this primer pair, two rye cultivars, 'King 11' and 'Petkus', showed a polymorphism on IRS to each other and to cultivar Imperial. In a mapping population the corresponding 180bp King 11 band and the 300bp Petkus band segregated in a Mendelian fashion. They were not allelic, although both mapped relatively close to each other between the centromere and a secalin marker. The fact that the two PCR products were not allelic seems to be a frequent finding associated especially

. 42 I T M I Manhattan, Kansas 1991

with random PCR markers. It is a major drawback to lished with two standard cultivars are difficult to trans- the use of PCR as a mapping tool because maps estab- fer to a cultivar of interest.

References Cited

D'Ovidio. R.. O.A. Tanzarella. and E. Porceddu. 1990. some arm IDS of wheat. Theor. A P P ~ . Genet. in Dress . . . . Rapid and efficient detection of genetic polymorphism in Weinin& S, and P, Langridge, 1991, ldentifi&on and wheat through amplification of polymerase chain reaction. mapping of in cereals based on the Plant Mol. Biol. 15: 169-171. oolvmerase chain reaction. Theor. ADD/. Genet. 82209- . , , , Koebner, RM.D. and K.W. Shepherd. 1985. Induction of 216. recombination rye chromosome IRL and wheat Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski, chromosomes. Theor. Appl. Genet. 71:208-215. and S.V. Tingey. 1990. DNA polymorphisms amplified by Rogowsky, P.M., F. Lo, Y. Guidet, P. Langridge, K.W. arbitrary primers are useful as genetic markers. Nucleic Shepherd, and R.M.D. Koebner. 1991. Isolation and char- AcidsRes. 18:65314535. acterization of wheat-rye recombinants involving chromo-


Genetic and Molecular Approaches to Studying Rust Resistance Genes in Maize

S. H. Hulbert Department of Plant Pathology, Kansas State University, Manhattan, KS 66506-5502 USA

M o s t of the 25 genes which control resistance to common rust in maize map to two complex loci, Rpl and Rp3. Wheat also has complex rust resistance loci, since genes exhibiting different spec5cities often map to the same genomic location. Complex loci in wheat sometimes include genes conferring resistance to two or three species of Puccinia rusts. Similarly, resistance to two different Puccinia rust species map to the Rpl area of maize. Since the rust resistance genes of maize and wheat have similar phenotypes and control resistance to closely related pathogens, it seems likely these genes may belong to orthologous families in the two species.

The maize genes have several advantages for experimental analysis. Most genes arc very easy to score at the seedliig stage, and provide high levels of resistance. Most of the preliminaq mapping work was done by Hooker and co-workers in the 1960s (Hooker and Russell, 1962; Lee et al., 1963; Hagan and Hooker, 1965; Saxena and Hooker, 1968; Wilkinson and Hooker, 1968). In addition, a large series of near- isogenic lines was made in the highly susceptible inbred R168. The ability to make large test cross populations in maize makes it an ideal system for studying the structure of complex rust resistance loci.

Another aspect of the maize rust system that makes it suitable for the genetic analysis of rust resistance are the abundance of characterized genetic markers in maize. Genetic markers are a prerequisite for studying the genetic fine structure of complex rust resistance loci. Unless the lines carrying the resistance genes are well marked, it is impossible to tell rare recombinants from seed or pollen contaminants. Markers that flank the locus are particularly useful since they can be used to distinguish recombination from events such as mutation, and can also distinguish certain types of recombination events. Crossing-over results in flanking marker exchange. Flanking marker exchange can also result &om unequal crossing-over if duplicated sequences are present that can mispair. Possible types of recombination events that result in

parentally marked chromosomes are intrachromatid exchange, unequal sister chromatid exchange and gene conversion. The Rpl area is flanked by the RFLP loci bn13.04 (about one cM distal) and by npi422 (about one cM proximal). These markers are ideal for analysis of Rpl recombination since they are multiallelic and are frequently heterozygous in Rpl heterozygotes. We have not yet positioned the Rp3 locus on the maize RFLP map, and have therefore not yet identified flanking RFLP markers for Rp3.

The multiple specificities expressed by a complex disease resistance locus could be controlled either by a cluster of closely linked genes or an allelic series at a single locus. The Rpl complex appears to be a combination of these two possibilities. The Rpl area includes fourteen genes (RplA - RplN) which were originally considered to be allelic, and RpS and Rp6 which are closely linked to Rpl (Saxena and Hooker, 1968). We have mapped RpS approximately two map units distal to most of the Rpl genes. RplG, however, maps about 0.2 map units distal to Rp5. Eight other Rpl genes that we have mapped to date lie within about 0.4 map units of each other and belong to a complex locus (Hulbert and Bemetzen, 1991); recombination analyses have indicated that this complex locus wries two or more Rpl genes in at least some maize lines. RplA, RplB, Rpl', and RplL all recombine very rarely, at a frequency of about one susceptible in 4,000 or more test cross progeny. We believe they are allelic, since intragenic recombination has been reported to be this frequent in other maize loci in which this has been analyzed @ooner, 1986). RplD, RplJ, and RplF map distally to these genes. They recombine with each other at a fairly high frequency; roughly one recombinant for every 1,000 test cross progeny or possibly higher in the RplF/RplJ heterozygotes. We are able to obtain susceptible recombinants and those with the combined resistance of both parents. For example, we have constructed homozygous maize lines that carry RplJ with Rp ID in the cis arrangement. These recombinants show both non-parental combinations of flanking

P

44 1 T td I Manhattan, Kansas 1991

markers, indicating they arise by unequal crossing- over. Since unequal crossing-over generates variable numbers of duplicated sequences, this implies that different maize lines carry different numbers of Rpl genes in the wmplex Rpl locus. RplA and RplA' are pheno- typically identical but map to two different spots in this complex locus. Analysis of flanking RFLP markers in recombinant test cross progeny indicates that RplA' maps just distally to RplJ, while RplA, RplB, Rpll, and RplL map just proximally.

We are currently trying to place the remaining Rpl genes on our fine structure map and delimit the extent of the Rpl area. We have not yet been able to map the Rp6 locus because we do not have a rust isolate that detects (is avimlent on) Rp6. Wilkinson and Hooker (1968) estimated its map position to be 1 map unit from Rpl and three map units from Rp5. This suggests that Rp6 is very close to our proximal RFLP marker.

While identifying the limits of the Rpl area, we are also trying to determine which of the Rp genes maps closest to one of our RFLP markers. We would like to determine the feasibility of using a map based cloning approach to clone an Rp gene. We are cur-

rently tlying two approaches to target RFLPs to the Rpl area. The first involves the use of RAPD markers on our Rpl and Rp3 isogenic lines. The second approach involves a subtractive hybridization approach and is dependent on our isolation of a homozygous Rpl deletion. We have recently generated several Rpl derivatives using X-ray irradiated pollen, but we do not yet know which of these are due to deletions or which of the putative deletions we can make homozygous.

We are also interested in the possibility of using sorghum DNA libraries to chromosome walk in maize. Most cloned maize genes and RFLP markers cross- hybridize to sorghum and their genetic maps are somewhat collinear (Hulbert et al., 1990). The sorghum genome, however, is three to four times smaller and carries much less repetitive DNA. We have recently constructed a wsmid library of sorghum genomic DNA and have isolated !NO clones homologous to our flanking RFLP markers. We have not yet determined the usefulness of these clones for initiating chromosome walks in sorghum or their ability to provide probes which rehybridize to maize. The utility of this approach has therefore not yet been determined.

References Cited

Dooner, H.K. 1986. Genetic fine structure of the Bronze locus in maize. Genetics 113:1021-1036.

H a g q W.L. and A.L. Hooker. 1965. Genetics of reaction to Puccinia sorghi in eleven wrn inbred lines from Central and South America. Phytopathology 55193-197.

Hooker, A.L. and W.A. Russell. 1962. Inheritance of resistance to Puccinia sorghi in six wrn inbred lines. Phytopa- thologv 52122-128.

ghum and related crops using maize RFLP probes. Proc. Natl. Acad. Sci. U U 87:4251-4255.

Lee, B.H.. A.L. Hooker, W.A. Russell, J.G. Dickson, and A.L. Flangas. 1963. Genetic relationships of alleles on chromosome LO for resistance to Puccinia sorghi in 11 corn lines. Crop Sci. 3:24-26.

Saxena, K.M.S. and A.L. Hooker. 1968. On the structure of a gene for disease resistance in maize. Proc. Natl. Acad.

Hulbert, S.H. and J.L. Bennetzen. 1991. Recombination at Sci. USA 68:1300-1305.

theRpl locus of maize. Mol. Gen. Genet. 226:377-382. Wilkinson, D.R and A.L. Hooker. 1968. Genetics of

Hulbert, S.H.. T.D. Richter, J.D. Axtell, and J.L. Bennet- reaction to Puccinia sorghi in ten corn inbred lines from

zen. 1990. Genetic mapping and characterization of sor- Africa and Europe. Phytopathologv 58:605-608.

= T M I Manhattan, Kansas 1991 45

A 'Zebra' Chromosome Arising From Multiple Translocations Between Wheat Chromosome 5A and

EZymus trachycaulus Chromosome 1 Ht

J. Jiang and B.S. Gill Department of Plant Pathology, Kansas State Univeraity, Manhattan, KS 66506-5502 USA

An aUoplasmic wheat-Elymus trachycaulus chromosome translocation line 5A-IHt was isolated from the derivatives of an E. trachycaulus x 'Chinese Spring' wheat hybrid. N-banding analysis showed that a modified 5A (5A-IHt) chromosome substituted for the normal 5A in this line. Chromosome 5A-IHt paired with the telocentric chromosome lHtS from E. trachycaulus and normal 5A of wheat at metaphase I in pollen mother cells, and carried several genetic markers, including fertility restoration gene Rf-Etl, storage protein gene Gli-Htl, repeated DNA sequence probe pCb4.14, and a leaf-rust-resistance gene from 1HtS. Genomic in sifu hybridization, using total DNA of E.

trachycaulus as a probe, revealed that chromosome 5A-IHt actually consists of four chromosome segments derived from E. trachycaulus and five chromosome segments, including the centromere, from wheat. The chromosome was named as 'zebra' because of its striped genomic in situ hybridization pattern. We analyzed the chromosome constitution of the sib plants of the previous generation from which the zebra chromosome was recovered. These results show that the zebra chromosome probably originated from spontaneous multiple translocations between chromosomes 5A and 1Ht.

46 I T h! I Manhattan, Kansas 1991

The International Triticeae Mapping Initiative

C. 0. Qualset ITMI Management Off~ce, Genetic Resources Conservation Program,

University of California, Davis, CA 95616 USA

Goals and objectives ITMI was developed as an informal, unincorporated

effort that had as its specific objective the development of comprehensive genetic maps of the genomes of the Triticeae crop plants and their relatives. The value of such maps has been increasingly appreciated for use in plant breeding and basic biological research. The objectives for ITMI were enumerated in the Prospectus (ITMI, 1989) as follows:

Progress is being made and it appears that ITMI is serving a role considering the success of the 1990 (McGuire et al., 1991) and 1991 international workshops.

Organizational aspects The founders of ITMI conceived a rather ambitious

scheme for coordinating genetic mapping activities (ITMI, 1989). It was decided that a coordinating sci-

1. Develop linkage and metaphase chromosome maps entist should be designated for each homoeo~~~ous

utilizing RFLP markers of the chromosomes of durum chromosome group in Triticeae species, and addition-

wheat (Trificum furgidum) and common wheat (T. ally, coordinators for specific genomes, especially of

aestivum). important diploid species - barley, rye, and selected wild relatives. These coordinators were called 'ITMI

2. Develop a comparative map of barley (Hordeum vulgare) utilizing RFLP markers.

3. Develop a comparative map of rye (Secale cereale) utilizing RFLP markers.

4. Develop comparative maps of representative diploid species of the genera in Triticeae.

5. Construct comparative linkage maps of the diploid ancestors of the wheat A, B, and D genomes.

6. Determine l i e between RFLP markers and genes controlling specific agronomically important traits.

These are ambitious objectives, but not unrealistic for the world community of Triticeae geneticists. ITMI was formed as a strategic means to develop cooperation and direct collaboration among scientists in developing genetic maps. It meant to provide a forum for interchange of ideas, and, of utmost importance, the free exchange of genetic stocks and DNA probes among scientists in developing the maps and among those who would use the mapping information in plant breeding or other research. The ITMI strategy also was meant to assist in developing research funding by influencing donors and research administrators. The advancement of genetic knowledge about the Triticeae was promoted as critical to any nation that depended upon wheat, barley, rye, or triticale in its economy.

Investigators', and their roles were to assemble information about the chromosome included in their assignment and to reconcile data from different laboratories by experimental verification if necessary. It was not intended that the coordinators be solely responsible for mapping of their assigned chromosomes, but they would exchange stocks and data from their own research with coordinators. Thus, ITMI encourages any group to proceed with developing genetic maps for any species of Triticeae, and the coordinators can provide useful information and probes to help these efforts.

'Affiliate Member' was a second category of collaborators designated in the original formulation of ITMI. The Affiliate Members were envisioned to be organizations which could benefit from using genetic maps in their research and development programs, but may not be able to develop comprehensive maps by their own resources. The intention was to include public and private research organizations and corporations whose business was largely dependent on Triticeae crops. The Affiliate Members were invited to support ITMI financially for five years at $10,000 per year. This was viewed as a means to provide research funds to the major mapping laboratories. This concept has, up to now, proven to be only marginally successful, in part due to the difficulty in identifying and contacting


individuals in relevant organizations, but also because received from CIMMYT, Mexico, and from the Cam- public research organizations are reluctant to provide bridge Laboratory, International Plant Sciences research funds to other public research organizations. Research, Nonvich. The invitation from CIMMYT

These issues were discussed during this workshop, was accepted, with David Hoisington to confirm local and by consensus the roles and definitions of ITMI arrangements for the meeting to be held at CIMMYT participants were redefined as follows: Headauarters at El Batan near Mexico Citv in

I M Coordinator - an individual who has accepted a role in assembling, interpreting, reconciling, and reporting information for a particular chromosome group or a whole genome. One of these coordinators will be responsible for administrative aspects of ITMI.

ITMI Investigator - an individual who is active in Triticeae genetics research, including the development of genetic stocks useful in mapping, andlor contributes directly to genetic maps.

A#liate Member - Organizations that make financial contributions directly to ITMI, or provide scientific expertise to the ITMI Coordinators. The provision of expertise may be the assignment of scientists or students to ITMl Coordinator laboratories with support for one or more years.

These definitions were designed to provide the broadest possible opportunities for scientists and organizations to be integral contributors to the advancement of genetic mapping in Triticeae. It is an immediate goal of ITMl to invite participation in the above categories. Anyone interested should contact the Management Office or any of the current ITMI Coordinators (listed in the front of these proceedings).

Communications ITMI has a major role in communicating informa-

tion and facilitating the exchange of genetic stocks and probes among Coordinators and Investigators. The initiation of a medium for rapid communication of information was discussed at the workshop. A newsletter patterned after Maize Genetics Cooperation Newsletter was proposed, and the concept of a 'Triticeae Genomes' newsletter will be investigated and proposed at the next ITMI Workshop. Since rapid communication is essential it was recommended that an electronic bulletin board and FAX transmission be used.

International Worhhops The venue of future annual workshops was dis-

cussed. Invitations for the 1992 Workshop were

Septe;ber, 1992. The ITMI Coordinators expressed interest in join-

ing the International Wheat Genetics Symposium in July, 1993. An inquiry to the organizing committee will be made to explore this possibility. Finally, a European site for a hture workshop is desired.

Acknowledgments The present Workshop was ably organized by Bik-

ram Gill and his colleagues at Kansas State University. Their efforts were greatly appreciated. The ITMI concept received financial support from Sogetal, Inc., and we regret that that company is no longer available to continue support. CIMMYT and ICARDA both joined in providing financial support, and we look forward to mutually valuable collaboration with these important international research centers. The US National Plant Genome Program, through the efforts of O l i Anderson and the Wheatbase project, provided financial support for publication of these proceedings, and for other ITMI Coordinators' activities. Jeny M i c h e , Director of the US National Plant Genome Program, has continually offered encouragement for ITMI. Their efforts are greatly valued. Richard Stuckey of the US National Association of Wheat Growers Foundation has assisted in informing the US wheat industry of the importance of the ITMI program. We are very pleased that research groups from several countries, including Australia, France, and Italy, are formalizing their genetic mapping programs and are integral participants in ITMI. We look forward to additional groups in 1992.

References Cited

McGuire, P.E., H. Corke, and C.O. Qualset, eds. 1991. Genome Mapping of Wheat and Related Species: Proceed- ings of a Public Workshop. Report No. 7, University of California Genetic Resources Conservation Program.

ITMI. 1989. ITMI Prospectus: Comparative Maps of Triticeae Based on RFLP Markers. November, 1989, 30 p.

9


ATMI - The Australian Triticeae Mapping Initiative

P. Sharp University of Sydney, Plant Breeding Institute, Cobbitty, NSW, AUSTRALIA

ATMI wnsists of three cooperating centers:

(1) CSIRO Division of Plant Industry Canberra ACT (R A P P ~

(2) University of Sydney Plant Breeding Institute Cobbitty NSW (P.J. Sharp)

(3) Center for Cereal Biotechnology Waite Agricultural Researcb Institute Adelaide S.A. (I? Langi&e)

The Australian Triticeae Mapping Initiative was set up with funding initially from the Wheat and Barley Research Committees; this has now been assumed by the Grains Research and Development Corporation. The funding provides a half-time research assistant and maintenance and travel expenses.

ATMI has two principal aims: to provide a center for the storage and distribution of Triticeae RFLP probes, and to run an educational program on molecular marker techniques and approaches for cereal breeders and researchers.

The probe center activity is collecting, checking, storing, and distributing cloned probes. Probes are being stored in three ways: as stabs, frozen stocks at - 70°C, and as isolated plasmid DNA at -20°C. A wm- puter database of information about the probes is also being assembled.

The educational program is being organized as short, intensive courses. These are of five days dura- tion and are attended by eight to ten students, two to three teachers and one to two teachers' assistants. Students have ranged from postgraduates at the start of their projects through to senior wheat breeders, and have wme from universities, government research stations, and private industry. The teachers are the heads of the cooperating laboratories. Two courses have been held so far, one in Canberra and one in Adelaide. Instruction has been both theoretical and practical, covering the full range of molecular marker technology from DNA isolation (witb plant and p h - mid) through library construction, PCR, to genetic mapping, and computer analysis. The course is provided free to eligible people, those working with the Triticeae in Australia (and New Zealand).


Canada Wheat Genome Mapping Group WK. Kim and T.F. Townley-Smith

Agriculture Canada Research Station, Winnipeg, Manitoba R3T 2M9, CANADA

T h e ~ a n a d a wheat Genome Mapping Group was formed in Winnipeg in February 1990, when the RFLP Workshop-Agriculture Canada BioCrop Network was held in conjunction with the Canada Expert Committee on Plant Breeding and Plant Disease. Emphasis was placed on the establishment of a network among wbeat workers for the purpose of establishing stronger corn- munication. It was decided at the Discussion Session (chaired by Dr. K.J. Kasha, University of Guelph) of thc Cereals RFLP Syndicate that the coordination would be headed by Drs. Won K. Kim and N.K. Howes of the Agriculture Canada Winnipeg Research Station. A mailing list of members was sent out in Feb- ruary 1991 at the time of the Expert Committee meeting. The Group adopted the concept that rather than initiate wheat RFLP mapping immediately, the focus should be placed on 1) the application of technology such as PCR and barley probes to wheat, 2) support of the barley initiatives (North America Barley Genome Mapping Project), 3) use of available probes to tag smut and bunt resistance and high protein genes.

The group has 34 individual members with the following interests: RFLP mapping - 6; cytogenetics (including in situ hybridization and development of double-haploids) - 5; genetic analysis and field testing (agronomic and disease evaluation) - 19; molecular biology - 2; quality testing - 2. Research establishments which have cereal biotechnology programs on- site are involved in the mapping effort and some members of these establishments would serve on the Steer- ing Committee to be formed in February 1992. The participating institutions are: Plant Research Centre, Agriculture Canada, Ottawa; W i p e g Research Station; Lethbridge Research Station; University of Guelph; and University of Saskatchewan. More research establishments would be added once the Group's mandate and genetic resources are identified for the mapping effort. A Genome Mapping Workshop is planned tentatively in Februav 1992 in Saskatoon at the Canada Expert Committee meeting.

RFZP analysis of wheat and barley genomes at the Winnipeg Research Station

This project is coordinated by Dr. W.K. Kim and Dr. T.F. Townley-Smith. The objective is to identify the genes for rust and smut resistance and sprouting resistance. These are the integral components of the cereal cultivar development program at the Winnipeg Research Station. Recently, RAPD (random amplified polymorphic DNA) primers (arbitmy nucleotide sequence) have been widely used to amplify genomic DNA by PCR and detect polymorphism among cereal cultivars, and can thus be used as genetic markers and for construction of genetic maps. We are screening 400 RAPD primers that were synthesized by the University of British Columbia Biotechnology Centre. Of 150 screened, we found 41 polymorphic primers in barley and 35 in wbeat. The following cereal cultivars are being used for genetic analysis with selected RAPD markers:

Barley (in collaboration with Dr. P.L. Thomas). We use Hanncben (2 row) with covered smut resistance gene and Plush (6 row) with covered smut resistance gene (these two genes are not l i e d ) . Nineteen RAPD markers are being analyzed, using F6 recombinant inbred lines. Sixty of 100 single seed descent lines were screened for disease reaction.

Wheat (in collaboration with Dr. N.K. Howes and Mr. R. Knox). We are interested in tagging a bunt resistance gene and sprouting resistance gene. A wheat germplasm RL 4555 (soft white, sprouting resistance gene) x Biggar BSR (bunt resistance gene bt-10 derived from BW553) and smut resistance gene (derived from Glenlea). We have F3 single seed descent lines for analysis, using 15 RAPD markers.

Wheat (in collaboration with Mr. E. Czarnecki). We are interested in the mapping of bunt resistance genes and finding DNA markers that would detect polymor-

50 I T M 1 Manhattan. Kansas 1991

phism. Wheat cultivar Roblin x BW553, BC, F2 and BC, F, are now available for RAPD analysis. We also found polymorphism between these materials when ribosomal DNA repeat unit was restricted with Barn HI and probed with pMF2 (ribosomal RNA genes of Neurospora crassa).

For cereal DNA analysis at the Station, we identified several useful parental lines: RL 4555 (soft, white, sprouting resistance gene); Biggar BSR (bt 10, bunt resistance gene and smut resistance gene); Chinese Spring Sr 6 (stem rust resistance gene); BW 121 (streak mosaic virus resistance gene); Tigalen (Australian germplasm, stem st resistance genes, Sr 5, Sr 6, Sr 8 and Sr Tt and Sr T (Australian wheat cultivars by CSIRO, 1975). The mapping populations

would include single seed descent and double-haploids. Other related research Ribosomal DNA repeat unit polymorphism in 27 accessions of six species of Aegilops (in collaboration with Dr. E.R. Kerber and Mr. B. Innes). Species of the genus Aegilops are an important source of useful alien genes in developing wheat cultivars with disease resistance, insect resistance, and cold hardiness. In order to understand the genetic diversity among several species, the ribosomal RNA genes were double-digested with Barn HI and Eco RI and it was found that two accessions of Ae. squarrosa var. strangulata collected from Turkmenia and Azerbaijan, USSR had two additional Barn HI sites in the non-transcribed spacer region, in addition to two sites in coded regions.

For further information, please contoct Dr. Won K. Kim Agriculture Canada Research Station W i p e g , Manitoba CANADA, R3T 2M9

Tel. (204) 983-5533 or 983-2340 FAX (204) 983-4604


Canada Wheat Genome Mapping Group Interested Members as at February 5,1991

Armstrong, KC. PRC, Agriculture Canada Ottawa, Ontario. KIA OC6 (613) 995-3700

Cytogmetics, DNA markers

Ballance, G.M. Department of Plant Science University of Manitoba Winnipeg. Manitoh R3T 2N2 (204) 474-6086

Gene expresrion in cereals

Baker, R J . Crop Science Department University of Saskatchewan Saskatoon, Saskatchewan, S7N OW0 (306) 966-4969

Fuld testing, plant breeding

Brule-Babel, A. L Department of Plant Science University of Manitoba Winnipeg, Manitoba, R3T 2N2 (204) 474-6062

CuItivar improvement, double-kaploids

Clarke, J. Agriculture Canada Research Station Swift Current Saskatchewan, S9H 3x2 (306) 773-4621

Field testing, pplant breeding

Clarke, P. Agriculture Canada Research Station Beaverlodge, Alberta, TOH OCO (403) 354-2212

Fierd testing, plant breeding

Czaroecki, E. Agriculture Canada Research Station Winnipeg, Manitoba, R3T 2M9 (204) 983-0573

Fieiii testing, plant breeding

Dc Pauw, RM. Agriculture Canada Research Station Swift Current, Saskatchewan, S9H 3x2 (306) 773-4621

FieW testing, plant breeding

Fcdak, G. PRC, Agriculture Canada Ottawa, Ontario, KIA OC6 (613) 995-3700

RFLP, genetic analysb

Graf, RJ. Saskatchewan Wheat Pool Saskatoon, Saskatchewan, S7K 3W (306) 975-3794

PuId testing, plant breeding

Howes, N. K. Agriculture Canada Research Station Winnipeg, Manitoba, R3T 2M9 (204) 983-2385

Immunoassay, qualify testing

Hucl, P. Crop Science Department University of Saskatchewan Saskatoon, Saskatchewan, S7N OW0 (306) 966-8667

Field testing, plant breeding

Hughes, G.R Crop Science Department University of Saskatchewan Saskatoon, Saskatchewan, S7N OW0 (306) 966-8667


Kasha, K.J. Crop Science Department University of Guelph Guelph, Ontario, NlG 2W1 (519) 824-4120

Cytogenefics, RFLP, genetic analyst

Kerber, E.R Agriculture Canada Research Station Winnipeg, Manitoba, R3T 2M9 (204) 983-0476

Cytogenetics

Kim, W.K. Agriculture Canada Research Station Winnipeg, Manitoba, R3T 2M9 (204) 983-2340

RFLP, molecular bwlogv

52 1 T M 1 Manhattan, Kansas 1991

Knon, D.R Crop Science Department University of Saskatchewan Saskatoon, Saskatchewan, S7N OW0 (306) 966-8890

Genetic analysis, pplant breeding

Knox, R Agriculture Canada Research Station Swift Current, Saskatchewan, S9H 3x2 (306) 7734621

Disease testing, plant breeding

Lamari, L. Department of Plant Science University of Manitoba Winnipeg, Manitoba, R3T 2N2 (204) 474-607 1

RFLP, disease tedng

Leisle, D. Agriculture Canada Research Station Winnipeg, Manitoba, R3T 2M9 (204) 983-0487

Field testing, genetic analysis

Laroehe, A. Agriculture Canada Research Station Lethbridge, Alberta, T1J 4B1 (403) 3274561

RFLP, molecular biology

Lukow, 0. Agriculture Canada Research Station Winnipeg, Manitoba, R3T 2M9 (204) 983-1629

Qua& tesfing, plant breeding

M c h d , J.G. Agriculture Canada Research Station Swift Current, Saskatchewan, S9H 3x2 (306) 773-4621


McKenzie, RLH. Agriculture Canada Research Station Winnipeg, Manitoba, R3T 2M9 (204) 983-0628

Genetic analysis, plant breeding

Sadasivaiab, RS. Agriculture Canada Research Station Lethbridge, Alberta, TI J 4B1 (403) 327-4561


Salmon, D.F. Alberta Agriculture Lacombe, Albem, TOC IS0 (403) 7824641

Disease tem'ng, genetic analysis

Shugar, LP. W. G. Thompson Seed Ltd. Blenheim, Ontario, NOP 1AO (519) 2324341


Simmonds, J. PRC, Agriculture Canada Ottawa, Ontario, KIA OC6 (613) 995-3700

RFLP, genetis analysis

Smith, J.A. U.G.G. Berthoud Colorado, USA (303) 532-3721

Field testing

Townley-Smith, F. Agriculture Canada Research Station W i p e g , Manitoba, R3T 2M9 (204) 983-0610

Genetis analysis, plant breeding

Thomns, J. Agriculture Canada Research Station Lethbridge, Alberta, T1J 48 1 (403) 327-4561

Field testing, genetic analysis

Whelm, E.D.P. Agriculture Canada Research Station Lethbridge, Alberta, TIJ 4B1 (403) 3274561



Japan Wheat Genome Mapping Initiative

K. Tsunewaki Laboratory of Genetics, Kyoto University, Sakyo-ku, Kyoto 606, JAPAN

I n Japan, genetic mapping of RFLP sites in common wheat has been canied out only in my laboratory. The results so far obtained are briefly summarized below.

DNA clones prepared for use as probes (1) Genomic clones of Chinese Spring. Using pUC119, the following numbers of low-copy clones have been tested:

Source Number of clones Number of clones screened with RFLP

EcoRI fragments 232 45 Hindlll 188 36 PstI 1560 361 Total 1980 422

Results

In total, 198 RFLP loci have been grouped into 32 l i e groups (Table 1). The total map size for all chromosomes is about 1800 cM at present. By nulli- tetrasomic analysis, 105 RFLP loci and 226 monomorphic loci have been located on 21 chromosomes (Figure 1). By ditelosomic analysis, 115 RFLP loci have been located on either the short or long arm of most chromosomes. From this result, the position of the centromere of 12 chromosomes has been determined (Figure 2).

Future plans Two other groups will join this program. Their and our coverage of RFLP studies have been agreed as follows:

(2) cDNA clones prepared from mRNA of Chinese Tsunewaki's group at Kyoto University - Spring; about 100 clones. homoeologous groups 1,2, and 3.

Methods employed for RFLP analyses Ogihara's group at Yokohama City

Linkage analysis, using 66 F2 plants (F, progenies) of University - a cross. Chinese Sorine x Triticum soelra. nulli-tetra- homoeologous groups 4 and 5. . - somic analysis for both RFLP and monomorphic loci, Furuta's group at Gifu University - and ditelosomic analysis for RFLP loci. homoeologous groups 6 and 7.

- 54 1 T M 1 Manhattan, Kansas 1991

Table 1. a) Numbers of RFLP loci mapped by linkage analysis, b) chromosome location determined by nulli-tetrasomic analysis, c) chromosome arm location determined by ditelosomic analysis, d) chromosome location of monomorphic loci determined by nulli-tetrasomic analysis. Map size is also given. Results of Y.-G. Liu, N. Mori, and S. Nasuda in the Labora- tory of Genetics, Faculty of Agriculture, Kyoto University, Japan.

Chromosome 1A 2A 3A 4A 5A 6A 7A Total a) Mapped 2 13 8 17 17 16 10 83 b) Chromosome 2 6 2 6 8 9 7 40 c) ~ r m location (short) 0 1 0 1 1 4 4 11 c) Arm location (long) 0 4 5 5 9 6 4 33 d) Monomorphic 1 11 11 9 6 8 16 62 Map size (cM) 6 103 55 147 198 136 47 692 Centromere located (0) x ? x o ? o o

Chromosome 1A 2A 3A 4A 5A 6A 7A Total a) Mapped 9 29 11 7 8 11 12 87 b) ~Gdmosome 4 15 7 4 4 5 5 44 c) Arm location (short) 2 6 3 2 2 2 2 19 c) Arm location (long) 5 9 4 4 3 4 3 32 d) Monomorphic 7 6 10 14 19 16 7 79 Map size (cM) 51 161 126 76 115 74 100 703 Centromere located (0) o o o o o o o

Chromosome 1A 2A 3A 4A 5A 6A 7A Total a) Maowd 3 9 2 2 5 3 4 28 bj ~h;dmosome 2 6 2 2 4 2 3 21 c) Arm location (short) 0 3 I 1 0 0 4 9 c) Arm location (long) 0 3 1 1 4 2 0 11 d) Monomorphic 2 20 11 7 17 16 12 85 Map size (cM) 35 142 62 71 59 21 16 406 Centromere located (0) x ? o o x x x

Total 1A 2A 3A 4A 5A 6A 7A Total (Homeologous group)

a) Mapped 14 51 21 26 30 30 26 198 b) ~hromosome 8 27 11 12 16 16 15 105 c) Arm location (short) 2 10 4 4 3 6 10 39 c) Arm location (long) 5 16 10 10 16 12 7 76 d) Monomomhic 10 37 32 30 42 40 35 226 Map size (CM) 92 406 243 294 372 231 163 1801 Centromere located 1 1 2 3 1 2 2 12


6A - M? UI

l2 11

Em'=]

a a

sm=l Il7d u rns1 U7W

14 - -- gp(cle f I:.

Figure 1. Linkage maps of RFLP loci corresponding to 21 chromosomes. The underlined loci are the ones for which canier chromosomes have been determined by nulli-tetrasomic analysis.


Figure 2. Linkage maps of RFLP loci of which camer chromosome arms (indicated by S or L) have been determined by ditelosomic analysis. The chromosome nomenclature follows the recommendation of the 7th IWGS. S and L: loci localized on the long and short arms, respectively; Z - loci specilic to T. spelto; 8 - centromere; Q - Q locus; # - located on both arms; 4 - could not be assigned to either arm.


French Triticeae Mapping Initiative P. Leroy, M. Bernard, S. Bernard, G. Branlard, M. Rousset, (1)

P. Nicolm, H. Thiellement (2)

G. Doussinault, J. Jahier, (3) P. Joudrier, M.I? Gautier, (4 I? Quitier, C. Hartmann, (5)

(I) INRA, Domaine de Crouelle, 63039 Clermont-Ferrand ddex, FRANCE @) Universitb Blaise Pascal (Clermont II), 4 rue Ledru, 63038 Clermont-Ferrand cbdex 1, FRANCE 0 INRA, Domaine de la Motte BP 29 35650 Le Rheu, FRANCE (4) INRA, 2 place Viala 34060 Montpellier cbder 1, FRANCE (VUniversitC de Paris Sud, 91405 Orsay ceder, FRANCE

A research project on wheat molecular mapping has recently been started in France. Five French public institutions are participating in the program, funded by the French Ministry of Agriculture. The INRA (Institut National de la Recherche Agronomique) Plant Breed- ing Station at Clermont-Femd is the major development center for the whole project. The main objectives are the following :

Establishing genetic maps using nucleic acid (RFLP) and protein gene markers of hexaploid wheat (Triticum aestivum).

Using molecular markers: to monitor the introgression of useful genes from

related wild species into wheat to detect correlations between markers and quali-

tative or quantitative characters of interest to evaluate the degree of genetic dissimilarity

between wheat varieties, local and exotic germplasm, and related species.

I. RFLP analysis Clones A partial Psf I genomic library, and a partial genomic library enriched with unique sequences at a given Cot value (>loo), both from etiolated seedlings; and three cDNA libraries, respectively from etiolated seedlimgs, embryogenic calli, and tissue culture, are being developed from wheat (I? Quefier - Orsay). Moreover, a cDNA library has been developed from mid-maturation seeds of T. durum (P. Joudrier- INRA Montpellier). We also have a cDNA library from lye mid-maturation seeds developed by M. Delseny (CNRS - Perpignan).

We are starting to work with three of these libraries: Pst I, cDNA 'embryogenic calli' and cDNA 'mid- maturation seeds'. The cDNA 'embryogenic calli' library has been developed using lambda gtlO vector (101 1 PFUIml) and the cDNA 'mid-maturation seeds' library has been developed using pUC18 at the Pst I restriction enzyme cloning site. All the clones will first be tested by PCR (polymerase chain reaction) and then the cDNA libraries will be tested for redundancy against the whole library using the non-radioactive system ECL from Amersham. Then, the amplified clones will be evaluated by Southern hybridization with total DNA of the French wheat variety 'Courtot', cut with four restriction enzymes: Eco RI, Hind III, Eco RV, and Dra I. Only clones giving a good hybridization pattern will be kept for the characterization step.

A clone database will be developed on SUN-UNIX workstation using the database software ORACLE. We will follow as much as possible the structure of the database already developed by Olin D. Anderson and collaborators in the US.

Characterization Each of the non-redundant and PCR-amplified clones will be characterized by Southern hybridization with total DNA of several species: barley, rye, Triticum monococcum (A genome), T. tauschii @ genome), T. dunm (AB genome), T. speltoides (S genome), Dasypynm villosum (V genome) and oats with the restriction enzyme EcoRI. Two mapping populations will be developed: Chinese Spring (CS) x Courtot (CT) and CS x AW, an amphiploid wheat from T. durum x T. tauschii. The parents will be analyzed for polymorphism in the same Southern blot. In parallel, the degree

P p


of polymorphism for each clone will be tested using 15 commercial wheat varieties with the same four restriction enzymes previously described.

Mapping Each clone showing polymorphism will be chromoso- mally located using CS nullisomic-tetrasomic and ditelosomic lines (E.R. Sears) and the same four restriction enzymes previously described. The segregating wheat families used for mapping will be two populations of doubled haploids @H) obtained from the crosses CS x AW and CS x CT, the first through androgenesis (male meiosis), the second from an inter- specific cross with maize (female meiosis). Morcover, we will also use recombinant inbred lines generated by single seed descent (F7) h m the cross CS x AW.

Segregation analysis and estimation of painvise recombination frequencies between loci sill be performed using several softwares such as MAPMAKER (E. Lander), LINKAGE (M. Lathrop), and G-MEN- DEL (S. Knapp) installed on a SUN-UNTX workstation. For QTL analysis we will use essentially MAP- MAKER QTL (E. Lander).

11. Protein Markers The same plant materials (parents, hybrids and segre-

gants) will be examined by two-dimensional gel elec- trophoresis (2D) of total proteins extracted from at l a s t two different stagcs or organs. The 2D will be performed on a large dimension gel using immobilized pH gradient in the first dimension. As previously demonstrated, e.g. on barley, genetic maps can also be constructed from such abundant protein markers. We therefore plan to add as many protein markers as possible to the RFLP genetic map.

Our hope is to receive a maximum of available RFLP markers which are already well mapped on the wheat genome and could help us as guidelines for mapping our own RFLP markers. This research will be carried out in cooperation with another EEC member (M. Gale), and with international institutions, including CIMMYT (D. Hoisington), and other collaborators in lTMI. We wish to be in contact vcly rapidly with a large number of laboratories involved in wheat mapping.

To contribute from our side to the ITMI project we plan to work on the A genome, probably using a cross between T. monocconrm and T. boeticum. We are preparing the genetic materials. Several T. monococcum and i? boetinrm accessions will be analyzed using RFLP probes developed from our previous program (T. urarfu) will also be investigated.

For further information, plense contact M. Bernard, P. Leroy INRA Station dlAmelioration des Plantes Domaine de Crouelle 63039 Clermont-Ferrand FRANCE

Tel: (33) 73 62 43 37 Fax: (33) 73 62 44 53 E-mail: [email protected]


Chromosome Group Reports

Group 2 P.J. Sharp, University of Sydney, Plant reedi in^ Institute, Cobbitty, NSW AUSTRALIA

T a b l e I lists the current progress in mapping the TWO sorts of data are given: homoeologous group 2 chromosomes in Triticum . n, .,,,,,&, of fragments or probes located by aestivum and T. tauschii. The data from Kyoto analysis of various meuploid stocks to particular University are from a T. aestivum x T. spelta cross. chromosomes and chromosome arms. Laboratories

progress has in differ in fragments or probes located, mapping the group 2 chromosomes of Secale cereale and Hordeum vulgare (see separate reports for these The mmber of loci mapped to the GOUP linkage species), e,g., the North American Barley Genome groups by genetic recombination analysis. Not all data

Mapping Project has mapped 39 markers on 2H (A. available from all Sources

Kleinhofs, pers. comm.).

Table 1. Current progress in mapping the homoeologous group 2 chromosomes in Triticum aestivum and T. tauschii. Data from Kvoto Universitv are from a T. aestivum x T. s~e l ta cross.

T. aestivum T. tauschii Reporting A m 2A 2B 2D 2D laboratory

Number of probedfragments S 24 23 28 IF'SR, Norwichl located by aneuploids 13 13 16 Cornel13

Not arm-located 17 21 27 Kyoto2 l l * 18 Kansas State4

13 C S I W 10 9 9 Texas A&M6

Total 107 114 138 31 Number of loci detected S 9 10 10 IF'SR, Norwichl and linkage mapped 10 Kyoto2

L 9 12 11 Kansas State4 19 Kyoto2

Not arm-located 13 9 Kyoto2 27 Kansas State4 15 CSIROS

Total 31 51 30 42 * Many of these detect other hgments, often located on 2A andlor 2B in T. aesfiwm; only D-genome aneuploids were examined by (4)

1. Institute of Plant Science Research, Norwich, UK; (M.D. Gale, pers. w m . ) 2. Laboratory of Genetics, Kyoto University, JAPAN., Liu, Y.G. and K. Tsunewaki. Jpn. J. Genet. 1991. 66:617-633. 3. w e n t of Plant Breeding and Biometry, Cornell University, USA; Anderson J.A., Y. Ogihara, M.E. Sorrells, and S.D. Tanksley. Theor. Appl. Genet. in press, (M. Sorrells, pers. comm.). 4. Deparhnent of Plant Pathology, Kansas State University, USA; Gill, K.S., E.L Lubbers, B.S. Gill, W.J. Ranpp, and T.S. Cox. 1991. Genome 34:362-374. (B.S. Gill, pers. comm.). 5. CSIRO, Division of Plant Industry, AUSTRALIA; Lagudah, E.S., R. Appels, A.H.D. Brown, and D. McNeil. 1991. Genome 34:375- 386, (R. Appels, pers. corn.). 6. Department of Soil and Crop Sciences, Texas A&M University; (G.E. Hart, pen. comm.)


Group 3

M.E. Sorrells, Department of Plant Breeding and Biometry, Cornell University, Ithaca, NY 14853 USA

Table 1. RFLP probes on wheat homoeologous group 3, September 1991

Chromosome Mapping Number of Number of markers By genome arm population probes or fhpents located A B D

3L Aneuploidsl 32 110 35 37 38 3 s 8 31 12 10 9 3 1 3 0 2 1 3 T. aestivumZ 30 80 - - -

3 T. tauschii3 12 21 - - 21 3 T. tauschii4 6 8 - - 8 3L BarleyS 11 11 - - - 3 s 2 2 - - - 3 1 1 - - -

3 Barley6 12 12 - - -

3 Barley7 40 47 - - -

Total (not lncl. duplications) 147 317

1. Anderson, J.A., Y. O-a, M.E. Sorrells, and S.D. Tanksley. 1992. Development of a chromosomal a m map for wheat based on RFLP markers. Theor. Appl. Genet., in press.

2. Gale, M.D. e ta / . , Institute for Plant Science Research, Nonvich, p r s . c o r n

3. Gill. K.S., E.L. Lubbers, B.S. Gill, W.J. Raupp, and T.S. Cox. 1991. A genetic linkage map of Triticum tauschii @D) and its relationship to the D genome of bread wheat (AABBDD). Genome 34:362-374.

4. Lagudah, E.S., R. Appels, A.H.D. Brown, and D. McNeil. 1991. The molecular-genetic analysis of Triticum tauschii, the D-genome donor to hexaploid wheat. Genome 34:375-386.

5. Hue% M., A.E. Tanksley, J.A. Anderson, N.L.V. Lapitan, M.E. Sorrells, and S.D. Tanksley. 1991. Construction of a restriction fragment length polymorphism map for barley (Hordeum vulgare). Genome 34:437-447.

6. Granor, A. e t a / . , Biologische Bundesanstalt f i r Land- und Forstwirtschaft, Institut f ir Resistenzgenetik, pets. c o r n ,


Group 5

B.S. Gill, Department of Plant Pathology, Kansas State University, Manhattan, KS 66506 USA

I n preparing the group 5 report, I have summarized pertinent references are cited. The present report information for each clone in terms of restriction en- includes data from three labs (Kansas State University, zyme used, number of bands observed, and the number Cornell University, and Kyoto University). I thank W. of bands mapped on a chromosome or chromosome John Raupp, K.S. a l l , Mark Sorrells, Jim Anderson, arm (S or L). The 'others' category includes location on K. Tsunewaki, and S. Nasuda for their contributions to additional homoeologous group(s) andlor arm(s). The the preparation of this report.

Bands Restriction Chmmosome location Clone (located/total) enzyme Arm A B D Others

Kansas State University Robes DG A003 518 DGD016 314 DG GO07 5/14 DG GO44 313 DG GO57 313 DG HOOl 313 KSU024 718 KSU026 313 KSU029 414 KSU058 616 KSU075 13/14

Cornell University Probes BCD1130 313 CD0 2 313 CDO 677 314 CDO 687 3 I5

315 CDO 749 317 CDO 948 313 CDO 1335 313 WG 184 118 WG 341 417 WG 363 414 BCD 21 316 BCD 87 213

213 BCD 157 313 BCD 204 516 BCD 450 313 BCD 508 314 BCD 603 313

Eco RI Hind ZZZ Hind Ill Hind Ill Hind 111 Eco RI Hind Ill Hind Ill Hind 111 Hind Ill Hind ZZZ

Dra 1 Eco RI Eco RV Eco RI Eco RV Eco RV Dra I Dra I Eco Rl Eco RI Dra I Eco RI Dra 1 Eco RV Eco RI Eco RI Eco RV Eco RV Eco RV


Bands Restriction

Clone Oocated/total) enzyme

Chromosome location

A B D

Cornell University Probes (continued) BCD 926 313 Eco RV BCD 1087 313 Eco RV BCD 1088 313 Eco RV BCD 1032 113 Dra I BCD 1381 313 Eco Rl CDO 57 316 Eco RV CDO 213 3 I3 Eco RI CDO 388 315 Dra I CDO 400 415 Dra 1 CDO 412 313 Dra I CDO 484 214 Eco RI

213 Eco RV CDO 548 415 Eco Rl CDO 662 215 Dra 1

216 Eco RI 114 Eco RV

CDO 666 318 Eco Rl CDO 735 616 Eco RV CDO 786 315 Dra I CDO 836 115 Eco RI

117 Eco RV CDO 981 317 Dra 1 CDO 1049 313 Dra 1 CDO 1168 818 Eco RI CDO 1189 313 Dra 1 CDO 1312 115 Eco RI

215 Eco RV CDO 1333 213 Eco RI CDO 1338 114 Eco RI

216 Eco RV CDO 1400 115 Dra 1

115 Eco RV WG 114 113 Dra 1

113 Eco RI WG 405 115 Dra 1 WG 419 313 Eco RI WG 583 313 Eco RI WG 876 114 Eco RI WG 889 4/10 Eco RI

316 Eco RV WG 909 315 Eco RI WG 1026 416 Dra 1

317 Eco RI

Others


Bands Restriction Chmmosome location Clone (locnted/totnn enzvme Arm A B D Olhers

Plant Science Research Robes PSR940 414 PSR945 313 PSR118 313 PSRl7O 819 PSR326 515 PSR618 414 PSR628 313 PSR929 313 PSR1204 313 PSR79 316 PSR94 7/8 PSRlOO 818 PSRI I5 313 PSR120 618 PSR128 313 PSR145 818 PSR15O 819 PSR164 313 PSR360 515 PSR370 414 PSR426 3/3 PSR574 313 PSR580 515 PSR637 3 I3 PSR906 313 PSR9 1 1 313 PSR912 515 PSR9 18 111 PSR1094 314 PSRllOl 414 PSR1202 515 PSR1206 518 PSR1316 313

pACPI1 (XACII)~ 313 pACPl (XAC13)2 414 pGC19 (XEmbp)3 717 pcPC51 (Xp-Amy-1)' 516

Eco RI Hind III Hind Ill Dra 1 Eco RI Dra I Dra I Hind III Dra 1 Dra I Eco RV Eco RZ Eco RV Eco RV Hind Ill Eco RI Eco RI Eco RV Dra I Dra I Dra I Eco RI Eco RI Eco RI Eco RI Eco RV Eco RV Dra I Eco RV Eco RI Eco RV Eco RV Dra I

Eco RI Dra l Eco RV Eco RV

1. Sharp, P.J., S. Chao, S. Desai, and M.D. Gale. 1989. I h e isolation, characterization and application in the Triticeae of a set of wheat RFLP probes identifying each homomlogous chromosome m. Theor. Appl. Genet. 78:342-348.

2. Devos, K.M.. C.N. Chinoy, M.D. Atkinson, L. Hansen, P. von Wettstein-Knowles. and M.D. Gale. 1991. Chmmosomal location in wheat of the genes ccdimg for the acyl carrier proteins I and m. Theor. Appl. Golet. 823-5.

3. Devos, K.M., M.D. Atkinson, C.N. Chinoy, M.J. Guiltinan, R.S. Qnatrano, and M.D. Gale. 1991. Chromosomal loeation and variability in wheat, barley and lye of a wheat gene encoding a bZIP protein (EmBP-1). Theor. Appl. Genet. 82:665667.


Bands Clone Ilocatedltotal\

Restriction e n m e Arm

Chromosome location A B D Others

Kyoto University Probes pTag 165 516 pTag 222 111 pTag 25 1 212 pTag 259 5/14 pTag 301 2/14 pTag 354 316 pTag 5 10 414 pTag 520 216 pTag 546 216 pTag 558 111 1 pTag 562 113 pTag 587 313 pTag 644 214 pTag 65 1 213 pTag 655 416 pTag 724 3/14 pTag 754 718 pTag 756 211 1 pTag 317 112 pTag 3 19 111 pTag 554 118 pTag 614 113 pTag 62 1 111 pTag 695 113

Texas A&M University pTaTAM37 313 pTaTAM3 8 519 pTaTAM40 214 pTaTAM43 315 pTaTAM68 314 pTaTAM70 114 pTaTAM75 ? pTaTAM4 I 315 pTaTAM53 415 pTaTAM54 616 pTaTAM0 l 313 pTaTAM 16 313 pTaTAM29 313

University of Adelaide pWJLl l 515 pAWJL26 6/12 pAWBMl pAWBM13 8/10 pAWBM22 416 pAWGM3 1 315 pWJL3 14/21

Dra I Barn HI Hind Ill Hind Ill Hind Ill Dra I Dra I Dra I Barn HI Hind III Eco RI Barn HI Barn HI Hind III Barn HI Dra 1 Barn HI Eco RV Hind Ill S Eco RI S Eco RV L Eco RI L Hind lll L Dra I L

Hind Ill Bgl III Sac I Barn HI Sac1 Hind III Barn HI Bgl II Barn HI Sac I Hind III L Hind III L Eco RI

Dra 1 Dra I Barn HI Barn III/ Hind Ill Hind Ill Barn HI Hind III S

ZA 6 4 IB, 4B. 6B, ZD, 4D. I ? 4 4 6 4 7.4s. IB, 3B,6B,7D, Z?

3A, 4A

IBS, 2B, 3BL, 4B 6A7ALZBS.3BS.68L68.70.40.5D

ID?, ID, 2D, 3D, 3? 6AS, 4B

2? 7a5 IA, I? 3BS, 4B, 6B. 6BL, 2D, 6 7B? 2A, 3A, 6AL, IB, 3BL, 17 7D, 2? 6AS, 4B 2AL, 7A, 2BL, 3B, 3? 6B, 6D


The E.R. Sears Collection of Wheat Aneuploids: Present Status

Department of Botany and Plant Sciences, University of California, Riverside, CA 92521 USA

T h e collection of Triticum aestivum L. em. %ell. cv. Chinese Spring aneuploids developed by Dr. E.R. Sears is being regrown and multiplied for preservation and distribution. During the last two years, nullisomics, monosomics, tetrasomics, nulli-tetrasomics, ditelosomics, double ditelosomics, and disomic additions of 'Imperial' rye were analyzed cytologically, proper plants selected, grown, and seed placed in storage at -20°C. Cytological screening is based on C-banding of root-tip squashes; in monosomics, tetrasomics, and nulli-tetrasomics. MI pairing was also observed.

Considerable somatic instability was observed in all sets analyzed. It may be related to seed age and storage conditions. It was rare in samples less than 10 years old and frequent in samples over 10 years old, and was particularly high among the monosomics which, because of the demand, were stored only under refrigeration with frequent removal to room tempera- ture (E.R. Sears, pers. comm.). Probably as a result of the somatic instability, deficient and translocated chromosomes were present in the analyzed stocks. These are being eliminated from the affected stocks by crosses to normal Chinese Spring and selection.

Nullisomics (N): lA, lB, ID. 3A, 3B, 6 4 7A, 7B, and 7D were increased and sufficient amounts of seed were produced. The single N4D plant obtained set only about a dozen seeds; samples will be available after another seed increase.

Monosomics (M): 1A (P87-1.1-2) was homozygous deficient for about 30% of 2BL. Monosomic 3B (P75-13.4-4) was the most somatically unstable of all accessions analyzed with up to three ring and five dicentric chromosomes per mitotic root-tip cell. Cor- rect 3B monosomics were identified in the second generation of accession P75-13.4-1. Monosomic 7A (P80- 7.3-6) was homozygous for a nonreciprocal translocation of 5BL to 7BL.

Terrasomics (T): No tetrasomics in homoeologous group 3 were found among the accessions of T3A (P84-203-2), T3B (P84-213.2), and T3D (P80-223-3).

Dr. Sears suspected that T3D was in fact trisomic 3D, but did not anticipate problems with T3A and T3B, and had no alternative seed sources. Tetrasomic 5A (P78-205-2) was segregating for a translocation 2AS.5AL; after one round of selection, a correct T5A was selected.

Nullisomic-tetrasomics (N-T): N1D-TlB (P87- 321.11-1) was in fact another NIB-TlD. A correct NlD-T1B was supplied by Dr. B.S. Gill and increased. Among the progeny of Mono2A-T2B (P83- 302.12b-3), several N2A-T2B plants were identified which set seed under Riverside conditions. Therefore, this combination will be maintained as a true N-T line. The line listed by Dr. Sears as N2A-T2D (P90-302.22- 1) must have been Mono2A-T2D; no correct N2A- T2D plants have so E?r been identified. N5B-T5A (P88-315.5-4) segregated for the same 2AS.5AL translocation as T5A. A correct N-T line has not yet been selected. N7D-T7B (P80-327.17-4) segregated for two C-banding patterns on 7B, one of which was that of Chinese Spring. The origin of such polymorphism is not clear.

Because of severe necrosis associated with the presence of four doses of 4A, Dr. Sears maintained only the Mi4BS-Tri4A and N4D-Tri4A lines. How- ever, T4A plants are reasonably viable and fertile under Riverside conditions and true N-T lines can be maintained. I would appreciate receiving seed samples of Mi4BS-T4A and N4D-T4A, if available.

Ditelosomics (Dt): This set now includes 35 true Dt lines and seven compensating dimonotelosomics (2AS'+2ALW, 4ASN+4AL', 5ASW+5AL', 2BSn+2BL', 4BS1+4BL", 5BSV+5BL', and 5DSU+5DL').

The Dt3DL (P79-23.1-1) line obtained from Dr. Sears has white s d , suggesting a mix-up. A Dt3DL line obtained from Dr. G. Kimher tested correct and was included in the set. Dt2BS-Mt2BL (P78-12.5-1) and Dt2BL (P86-12.4-1) lines were homozygous deficient for about 8% of 4AL.

The 4AS chromosome present in the Dt4AS-


Mt4AL lines was an acrocentric. A true 4AS telocentric was isolated and new Dt4AS-Mt4AL and DDt4A lines were produced and need to be increased.

Double ditelosomics (DM): The full set of 42 lines was checked and seed supply increased. The only problem is with the DDt2B line (F'74-12.5-1) which is homozygous deficient for about 8% of 4AL.

CS-Imperial rye addition lines: Addition 3R was homozygous deficient for about 50% of one of the 3R arms. AAer screening several sources of the line, a heterozygote was identified. After two rounds of selection among its progeny, a correct disomic addition 3R was identified. Another round of seed increase is in order before the line is available for distribution.

The nomenclature of chromosomes 4R and 7R used by Dr. Sears was reversed to that accepted as standard for rye. Although it better reflected the homoeology relationship to wheat, for the sake of

consistency, the designation of chromosome 4R and 7R additions in the set was changed to wnform to the standard. T.E. Miller has been using the standard nomenclature for several years and there is some wn- fusion as to the chromosomal location of some genes on rye chromosomes 4 and 7.

The remaining sets in the collection will be checked cytologically where necessary and grown in the next two to three years; time, space, and funding permitting. Priority will be given to correcting the problems encountered so far and to double monosomics (1 9"+ 1'+1'), monotelosomics, monoisosomics, and single chromosome substitution lines. By agreement with Dr. Sears, the remaining 35 dimonotelosomics will be given low priority. The demand for this set has been low and it can be re-created by crosses between Dt and DM lines when needed.


Cytogenetic Stocks Maintained by the Wheat Genetics Resource Center

B. Friebe, W.J. Raupp, and B.S. Gill Department of Plant Pathology, Kansas State University, Manhattan, KS 66506-5502 USA

DESCRIPTION NUMBER OF SOURCE ACCESSIONS

ALIEN ADDITIONS 1 1 1

in Alcedo Disomic Additions

from T. dichasianc

in Chinese Spring Disomic Additions

from D. villosum from T. cylindricurn from T. Iongissimum

from T. searsii from T. umbellulatum from T. timopheevii from Th. eelngafum from Th. intermedium from 7h. ponticum from Betzes Barley from Imperial Rye Ditelosomic Additions from T. longissimum from Th. elongatum from Betzes Barley from Imperial Rye

in (;brtot Disomic Addition

from Th. intermedium

in Lungdon Ditelosomic Addition

from T. longissimum

in Moisson Disomic, Ditelosomic Addition

from T. ventricosum

Lukaszewski Endo Endo, Hart, Maan, Waines Tuleen Sears Sears Dvorak Sears Sears Islam Miller, Sears

Tuleen Dvorak Islam Miller

Cauderon

Chen

Dosba


in Norin 26 Disomic Addition

from T. triunciale

in Novi Sad 60 Disomic Additions

from Th. intermedium in Panoija

Disomic Additions from Th. intermedium

in PBWII4 Durum wheat Monosomic Additions

from T. fauschii

in Selkirk Disomic Additions

from T. Iongissimum

in Vilmorin 27 Disomic Additions

from Th. intermedium Ditelosomic Additions

from Th. intermedium

ALLOPLASMZC

AMPHZPLOID

ANEVPLOZD

in Cheyenne Monosomic

in Chinese Spring Di-isosomic Ditelo monotelosomic Ditelosomic Double Ditelosomic Double Monosomic Duplication Nullisomic Monoisosomic Monosomic Monotelosomic Mono-Tetrasornic Nulli-Tetraromic

in Federation Monosomic

Cauderon

Cauderon

Dhaliwal

Cauderon

Cauderon

Maan, Nakata

Badaev, Cauderon, Chen Dewey, Dhaliwal, Dvorak, Kerber, Maan, Metzger, Sears, Sharma

Morris

Sears Sears Sears Sears Sears Lukaszewski Sears Sears Sears Sears, WGRC Sears Sears

P


in Langdon Durum Ditelo monotelosomic Double Ditelosomic

Nishikawa J ~ P P ~

in Shin Ebisu Telotrisomic Trisomic

Tsuchiya Tsuchiya

in Spica Monosomic McIntosh

in T. monococcum Trisomic Kuspira

in Wichita Ditelosomic Monosomic

Morris Moms

Miscellaneous lines Kerber

DELETION Endo, Lukaszewski, Tsujimoto, WGRC

GERMPLASM Harvey, Hatchett, Sears, WGRC

MARKER /MUTANT Anthocyanin pigmentation Allan, Knott, McIntosh,

Schmidt Sears Sears McIntosh Maan Gale Metzger Sears Sears McIntosh Metzger Gale McIntosh

Darvey, Maan, Metzger, Sears, Williams

Georgi, Sears Moseman

Acosta, Knott, Loegering, McIntosh, Sears Dyck, Kerber, Knott

Line

Chlorina Club spike shape Corroded Restorer for cytoplasmic male sterility Gibberellic acid insensitivity Grass-clump dwarfness Hairy glume Hairy peduncle Hairy node Hybrid necrosis Isozyme Liguleless Male sterility

Pairing homoeozygous mutant Reaction to Etysiphe graminis Reaction to Puccit~ia graminis

Reaction to Puccinia recondita Reaction to Puccinia striiformis Reduced height Spelt Factor Sphaerococcum Factor Storage Protein Virescent

Allan, Gale, Metzger Sears Schmidt, Sears Gale Sears

-- 1 T M I Manhattan, Kansas 1991

SUBSTITUTIONS

in Chinese Spring Disomic Substitution

from Axminster from Cheyenne from Hope from Indian from Kenya Farmer from Marquis from Red Egyptian from Sapporo from T. boeoticum from T. dicoccoides from T. longrssimum from T. timopheevii from Th. elongatum from Thatcher from Tistein from White Federation

Ditelosomic Substitution from Th. elongatrrm

in Hobbit Disomic Substitution

in Lungdon Disomic Substitution Monosomic Substitution

in NE66536 Disomic Substitution from Th. elongatum

in Novosibirskq from T. timopheevii

in Pirotrix from T, timopheevii

in Saratovskqa from T. timopheevii

in Selkirk Disomic Substitution

from T. Iongissimum

in Wichita Disomic Substitution

from Cheyenne from Chinese Spring

Sears Moms Moms Sears McIntosh McIntosh Sears Sears WGRC Sears WGRC Sears Dvorak Sears Moms Sears

Sears

Snape

Moms

Badaeva

Badaeva

Badaeva

Moms Moms


SUBSTITUTIONS (continued)

in 56-1 Durum wheat Disomic Substitution

from T. boeoticum

in unknown cultivars Disomic Substitution

from Th. elongatum from Th. intermedium

TRANSLOCATIONS

in Cappelli in Chinese Spring in Selkirk in Thatcher in Viking in unknown cultivars

TOTAL

Sebesta Wells

Georgi Sears Maan Sears Schlegel

Liang, Maan, Wells

72 I T M 1 Manhattan, Kansas 1991

The Production of Homozygous Recombinant Lines from the Langdon Durum - Triticum

dicoccoides Substitution Lines

Leonard R. Joppa Northern Crop Science Laboratory, State University Station, Fargo, North Dakota 58105, USA

T h e Langdon d u r n - T. dicoccoides chromosome large number of polymorphic loci. substitution lines can be used to produce homozygous recombinant lines (HRLs) by crossing each line to *The lines should be relatively homozygous for loci on Langdon dumm. Crossing over in the F, is expected all of the chromosomes, except for the chromosome between the Langdon chromosome and the substituted that is recombined. dicoccoides chromosome, and each F, gamete will be different. The various products of recombination can Once a line has been obtained, it can be increased be sampled and fixed by crossing the F, plants to the and used in replicated field experiments to assess dif- respective Langdon D-genome disomic substitution ferences in quantitative traits. These traits can then be line. The advantages of using these lines are that: studied to determine if there are associations between

the quantitative trait loci (QTLs) and restriction frag- Previous studies have shown that the dumm wheat ment length polymorphisms (RFLPs) or other qualita-

cultivar Langdon differs from T. dicoccoides by a tive trait loci.

STEP 1: CROSS LDN(DIC-1~) BY LDN STEP 2: GROW F,

( STEP 3: CROSS LDN ID(1A) / P, MALE GAMETE 1

$TEP 4: SELF TllESE PROGENIES AND SELECT 1 A D I S O M E ~

Figure 1. Illustration of procedures for using the Langdon - T. dicoccoides chromosome substitution lines for the production of 'homozygous recombinant lines'. LDN@IC-1A) is used as an example.


The procedures for the production of HRLs are illustrated schematically in Figure 1. The materials required are: a set of Langdon - T. dicoccoides disomic substitution lioes (LDN(DIC)), Langdon dunun, and a set of Langdon D-genome disomic substitution lines. As indicated, the LDN(D1C) lime is crossed with the recurrent parent Langdon. This line is expected to be similar to LDN for all loci with the exception of those on the substituted chromosome. In the F, plants the LDN and dicoccoides chromosome will cross over. Crossing the F, plants to the LDN D- genome disomic substitution line that is nullisomic for that LDN chromosome produces a BC-FI that has a single copy of the recombinant chromosome. Selfing these plants produces some plants that received two recombinant chromosomes and these plants will show 14 chromosome pairs at metaphase I of meiosis. However, some plants with 14 chromosome pairs may have a pair of D-genome chromosomes. Therefore, it is necessary to test cross the 14" plants to the proper double ditelosomic. The test cross plants with 13" + tlt"' at metaphase I of meiosis must have a pair of recombinant chromosomes and that BCIF, plant is homozygous for all loci on the recombinant chromosome.

The disadvantage of this method of producing HRLs is that it is time consuming and requires careful monitoring using cytology. One alternative would be to use anther culture. We are investigating this possibility, but have had little success with the tetraploid wheats.

A number of HRLs have been produced for chromosomes IA, IB, 3B, 6A, and 6B (Table 1).

In addition, the 4A and 4B lines are being produced at Davis, California, by Dr. Dvorak. Additional lines for these chromosomes as well as lines of the remaining chromosomes will be produced as quickly as time, funding and facilities permit.

Table 1. Current list of Langdon d m - T. dicoccoides homozygous recombinant lines, as at September 29, 1991.

Cbromosome Number of number Lines

I A 118


Wheat Database Meeting Manhattan, Kansas, September 2627,1991

ine, soybean, and maize)

Those attending the meeting were:

Olin Anderson - USDA, ARS, Albany, CA Rudi Appels - CSIRO, Canberra, Australia Doug Bigwood - National Agricultural Library Bob Busch - USDA, ARS, University of Minnesota Harold Corke - University of California, Davis Stan Cox - Kansas State University Mike Gale - Cambridge Laboratory, Nonvich, England Bikram Gill - Kansas State University Bob Graybosch - USDA, ARS, University of Nebraska Perry Gustafson - USDA, ARS, University of Missouri Gary Hart - Texas A&M University Steve Jones - USDA, ARS, Pullman, WA John McCarthy - Lawrence Berkeley Laboratory Jerry Miksche - USDA Plant Genome Research Program David Porter - USDA, ARS, Stillwater, OK Cal Qualser - University of California, Davis Peter Sharp - University of Sydney, Australia Mark Sorrells - Comell University, Ithaca, NY

O l i n Anderson opened the meeting and outlined the agenda, shown in Table 1. Jerry M i c h e said that the legislature wants a National Genetic Resources Pro- gram, to include plant and animal genome research, plant and animal germplasm, and microorganisms, and headed by an appointee chosen by the Secretary for Agriculture1. The present ARS Plant Genome Database Project is funded at $3,674,000 for one year, including the wheat, corn, soybean, and forest tree projects, plus a joint DOEYNSFMIHKJSDA effort on Arabidopsis. Dave Bigwood is Database Manager of plant genome database efforts at the National

'On May 15, 1992, Dr. Henry L. Shands was appointed Associate Deputy Administrator to head the new U.S. National Genetic Resources Program.

Agricultural Library. The question was asked whether the different database designs of different crops would cause compatibility problems if one model were M l y chosen as the standard, and others had to change their formats. The answer (Bigwood, McCarthy, Anderson) was that the databases would be similar at a conceptual level, and coordiited as much as possible. If this is successful, then later modifications of the database could be made independently, and would be hidden from users who would interact with the programs through a standard interfafie.

Sorrells and Anderson emphasized the necessity of scientists in different crops but similar areas, e.g., cytogenetics, communicating directly at this early stage. The upcoming meeting in Tucson of representative of all four crops would discuss this further.

Anderson introduced McCarthy from Lawrence


Table 1: Discussion topics

I. Maps TYPB

Genetic, RFLP * Cytogenetic = Other?

construction Presentation Multiple Maps? Map UnZication Coordination

Berkeley Laboratories (LBL). LBL was chosen to design the wheat database because of their cutting edge position in database theory and development, wn- nection to the human genome project in informatics and other technologies, and their proximity to the ARS Western Regional Research Center, where the database will be located.

The goals of the wheat database, provisionally named Wheatbase', include: archiving and accessibility of data, integration of diverse data types, and making available information systems for coarse and h e structure mapping of the wheat and related genomes. The immediate objectives of the coordinator (Anderson) and the LBL staff are:

To obtain general guidance from researchers on database design and construction

To include representation of the international community in Wheatbase

To decide on the specifics of what information Wheatbase will contain

To decide on standardization, where feasible Some of the main data types include:

different types of genomic maps DNA fragment information germplasm characteristics pedigrees for populations and lines

11. Genetic Stocks - Germplasm - Pedigrees Genetic Stocks, Germplasm Pedigrees Pathology Quality Boteins Seed Repositories

111. All Other Nomenclature ' Inucging

Robe, Clone, Library Repositories Data Input Hardware Software

gel patterns and images additional topics, e.g. enzymes, gene products, pathogens, citations, software

Wheatbase itself will not be publicly available, but will download to NAL data systems for public access. It will be accessible to designated curators on an X- windows interface using passwords allowing the curator to change specific portions of Wheatbase. It is proposed to complete a prototype Wheatbase, with enough data to test the structure of the database design, by late spring of 1992.

Sofhare for analysis. Bigwood stated that NAL will not be supporting analysis software, but it would be possible to catalog existing software, and possibly allow downloading of analysis programs to researchers. A group consensus was reached that raw data, to the level of scores of the researcher, should be included in Wheatbase. This would allow users to use such data for new analyses. It is important to define the format of such data to be compatible with the most used analysis programs.

Misuse of data. Concern was raised about laboratories misusing data generated by others by unfairly using it for publications or grants. Anderson, McCarthy and Qualset answered that some of this may be inevitable, although Genbank has not had major problems, and it may well be a transitory problem because in a few years the mapping will be finished and using the maps


will predominate. We should be looking ahead five to ten years.

Utility of a stock center for probes. The group agreed that maintenance and distribution of probes is a serious problem. No clear agreement on the best method was reached, but there is a general mistrust of couections being kept by individuals and organizations not an active part of the cereal community. Miksche said that the Plant Genome Office is considering establishing a repository of probes at Ft. Collins, at a later date. Something needs to be done quickly for cereals, and a crop specific library was preferred. Anderson will start to organize a probe repository at the Western Regional Research Center in Albany, CA, which will involve one technician and part-time of a scientist. Anderson will coordinate with Sharp in Australia and Gale in England so that three duplicate collections will exist. The probes will be referenced in the database along with notations on availability. Individuals who generate probes will determine if their probes will be available from a repository or only from themselves.

Imaging. It was unclear whether or not to include an image capability in Wheatbase. While imaging was desirable, the utility versus cost and complexity balance was not clear. Original images may be most useful among collaborating laboratories. The database should make provision for standard banding patterns for both RFLP and protein data without storing images of the original gels. The exact format of such summary data is still under discussion. Since the technology is advancing quickly, and since there is agreement that images of original gels are desirable if feasible, Wheatbase will make provision for this feature, to be implemented as and when appropriate.

Nomenclamre. Hart and Gale are members of the international wheat nomenclature committee. Compatibility of the wheat nomenclature rules with those of other crops was discussed. It was agreed that no nomenclature changes were practical with so many different systems. The database could handle the problem with a synonym - thesaurus approach. Bob McIntosh (Australia) currently keeps catalogs of symbols, stocks and genes. It may be possible to aid him by including his catalogs in the wheat database; he could be given sole entry rights into this portion of the database.

Maps. How many maps and what type? It was agreed that the database will at least initially present sets of maps. Some unification will occur over time, but the user will have a choice of which maps to view and use. The maps will include all pertinent data; genetic, RFLP and cytogenetic. The original ITMI plan for coordinating the maps should be continued, i.e. seven laboratories will each coordinate one lmkage group, to include all types of data for that group. Those laboratories will have primary access to Wheatbase and will be the only authorized modifiers for their respective L i e groups. Each coordinator will also be responsible for data on relevant barley and rye chromosomes. The need for meetings among the coordinators should be minimized, with most communication occurring electronically or in conjunction with yearly ITMI meetings. Satellite Wheatbases will be run in Australia and England. Anderson has spoken with Alex Reisner, head of the Australian Genome Information Network and confirmed that the Australian network utilizes exactly the same hardware platform and database software as will Wheatbase, so there will be no compatibility problems. Gale also foresees no problem running the database in England. The coordinators will communicate with the most convenient version of Wheatbase, and the three computers will periodically exchange information so that all three versions are updated as often as practicable.

Potential participation by barley mapping group. It would be wasteful of resources for the what and barley groups to implement separate database models. Ideally a single database should contain information on all the Triticeae. The barley group should be invited to participate and collaborate. At the least, the software is being written and will be available to the barley group if they want to use it, although no resources are currently available from the wheat effort to divert to barley data input. Sorrells is also a coordinator for the homologous barley chromosome and will discuss this potential with the barley community.

GIUN. There was a lively discussion on the usefulness of GRIN and the intended relationship between Wheatbase and GRIN. There were widely varying opinions on GRIN, but a general agreement that the system is not user-friendly and does not contain much of the most useful information for the wheat


community, or have a mechanism to add such data. According to McCarthy, approaches to the Wheatbase- GRIN relationship are:

Include GRIN identifiers in Wheatbase Periodically download to Wheatbase a subset of GRIN information

Bigwood said that GRIN is beiig redesigned as a relational system, which may make inter-connection more simple. It was agreed that coordination with GRIN should be pursued as much as possible. Qualset stated that many important genetic stocks are not part of GRIN because they are not cultivars or closely cultivar-related. Wheatbase must include all genetically welldefined stocks.

Germplasm information and stocks. Support for the maintenance of genetic stocks is nearing a crisis at the same time willingness to share existing and new stocks shows signs of declining. Should stocks be included in the database even if the owner will not agree to distribute? It was agreed to include the information, with a database field giving availability.

What information should be entered, and who should enter it? Although it was suggested to settle on three broad categories (aneuploids, structural aberrations, and genetic stocks), McCarhy pointed out that Wheatbase doesn't have to be exclusive since the thesaums notion can present many different ways of

picking categories. The important initial point is to gather as complete as possible a selection of germplasm data for use in the Wheatbase prototype. Gill and Gustafson agreed to aid in the compilation of sample data.

It was not settled how much genetic stock information to include, e.g., the whole world's resources? How much trait data? While we may still be discussing for some time how much trait data to include, this type of data is essential to Wheatbase or it will be useful only to researchers not to breeders or germplasm enhancers.

On the advisability of having a genetic stock curator, the problem was how to support such a person, and how a well-qualified person in that position would get recognition for the effort. No clear solution was apparent, but this issue should be discussed further in future. A related issue is the current lack of support for the physical maintenance of important genetic stocks, particularly critical now with the passing of Ernie Sears.

Pedigrees will be recorded using the Purdey system (Weibe system) [Crop Science 8:405, 19681.

Availability of data in machine-readable form. Once the database format is settled, the easiest data to load is that which is already in machine-readable form, i.e. spreadsheets, etc. In the longer term there will be problems in bow to enter new information. Anderson, McCarthy and Sorrells are to discuss this further.

Reported by OIin D. Anderson and Harold Corke

- 78 I T M I Manhattan, Kansas 1991

Attendance List Olin Anderson USDNARS/WRRC 800 Buchanan Street. Albany, CA 94710

Rudi Appels CSIRO G.P.O. Box 1600 Canberra, ACT 2601 AUSTRALIA

Taing Aung Agriculture Canada Cytogeneticdl95 Dafoe Road Winnipeg, Manitoba 3RT 2M9 CANADA

Carol Carter Kansas State University Department of Plant Pathology Throckmorton Hall Manhattan, KS 66506-5502

Carla Ceoloni University of Tuscia-Viterbo Vis S. Carnillo De Lellis Viterbo, 01 100 ITALY

Pei du Chen Nanjing Agricultural University Department of Agronomy Nanjing, Jiangsv 210041 CHINA

Sally Clayshulte Cargill Hybrid Seed 2540 E. Drake Road Fort Collins, CO 80525

Harold Corke University of California Department of Agronomy & Range Science Davis, CA 95616

Stan Cox Kansas State University USDAIARSDepartment of Agronomy Throckmorton Hall Manhattan, KS 66506

Donna Delaney Kansas State University Department of Plant Pathology Throckmorton Hall Manhattan. KS 66506-5502

Jack DeWitt Washington Wheat Commission Route 5 Box 64A Walla Walla, WA 99362

Hai Sbui Dong Kansas State University Department of Agronomy Throdimolton Hall Manhaltan, KS 66506

lsmail Dweikat Fwdue University Agronomy Depanment Lilly Hall of Life Sciences West Lafavette. IN 47906 . .

Bend Friebe Kansas State University Department of Plant Pathology Thmckmorton Hall Manhattan. KS 66506-5502

Allan Fritz Kansas State University Department of Agronomy Throckmorton Hall Manhattan, KS 66506

Mike Gale IPSR Cambridge Laboratoty Colney Lane Nonvich, Norfolk NR4 7UJ UNITED KINGDOM

Bikram Gill Kansas State University Department of Plant Pathology Throckmorton Hall Manhattan, KS 66506-5502

Kulvinder Gill Kansas State University Department of Plant Pathology Throckmorton Hall Manhatran, KS 66506-5502

Bob Graybosch USDNARS U~versity of Nebraska 322 Keim Hall Lincoln, NE 68583


Aaron Gueozi Oklahoma Srate University Agronomy D e p m e n t 550 Agriculture Hall Stillwater, OK 74078

Jane Guo Kansas State University Department of Agronomy Throckmorton Hall Manhattan, KS 66506

P e n y Gustnfson USDAIARS University of Missouri Curtis Hall Columbia, MO 652 11

Gary Hart Texas A&M University Department of Soil & Crop Sciences College Station, TX 77801

Dbia Hassawi Kansas State University Department of Agronomy Throckmorton Hall Manhattan, KS 66506

Jim Hatchett USDAIARS Kansas State University Department of Entomology Waters Hall Manhattan, KS 66506

E. k Hockett Montana State University Plant & Soil Science Department Bozemq MT 597174W2

David Hoisington CIMMYT Applied Molecular Genetics Lisboa 27 Mexico DF 06600 MEXICO

David Hole Utah State University Department of Plants, Soils, & Biometeorology UMC 4820 L o g q UT 87322

Kate Eouchins USDAIARS University of Missouri Curtis Hall Columbia, MO 6521 1

Jie Hu Texas A&M University TAES - Amarillo 6500 Amarillo Amarillo, TX 79106

Scott Hnlbert Kansas State University Department of Plant Pathology Throckmo~ton Hall Manhattan, KS 66506-5502

Eric Jellen University of Minnesota Department of Agronomy &Plant Genetics St. Paul, MN 55108

Jiming Jiang Kansas State University D e p m e n t of Plant Pathology Throckmorton Hall Manhattan, KS 66506-5502

Dave Johnston Cargill Hybrid Seed 2540 E. Drake Road Fort Collins, Co 80525

Stephen Jones USDAIARS Washington State University Pullman, WA 99164

Leonard Joppa USDAIARS North Crop Science Laboratory Box 5677 Fargo, ND 58105

Ken Kasba University of Guelph Department of Crop Science Guelph, Ontario, NIA 2W CANADA

Won Kim Agriculture Canada Research Station 195 Dafoe Road Winnipeg, Manitoba, R3T 2M9 CANADA

Andy Kleinhofs Washington State University Crop & Soil Sciences Department Pullman, WA 99164-6420

-


Rama Kotn Kansas State University Department of Plant Pathologj Throckmorton Hall Manhattan, KS 66506-5502

Mark Lazar Texas A&M University TAES - Amarillo 6500 Amafill0 Amarillo, TX 79 106

Philippe LeRoy INRA Domaine de Crouelle 63039 Clermont-Ferrand &ex 1 FRANCE

Weiqi Li Kansas State University Department of Agronomy Throckmorton Hall Manhattan, KS 66506

George Liang Kansas State University Department of Agronomy Throckmorton Hall Manhattan, KS 66506

Adam Lukawewski University of California Department of Botany and Plant Science Bachelor Hall Riverside, CA 92521

Susan McCarthy National Agriculwe Library 10301 Baltimore Blvd. Beltsville, MD 20705

Patrick McGuire Genetic Resonrces Conservation Program University of California Davis, CA 95616

Leigh Mickelson Kansas State University Department of Agronomy Throckmorton Hall Manhattan, KS 66506-5502

Doug Miller Kansas State University Department of Plant Pathology Throckmorton Hall Manhattan, KS 66506-5502

Herbert Ohm Purdue University Agronomy Department Lily Hall of L i e Sciences West Lafayette, IN 47906

David Porter USDAIARS 1301 North Western Sweet Stillwater, OK 74074

Cal Qualset Genetic Resources Conservation Program University of California Davis, CA 95616

S. Ramagopal USDAI ARS P. 0. Box 307 Aberdeen, ID 83201

W. John Raupp Kansas State University Department of Plant Pathology Throckmorton Hall Manhattan, KS 66506-5502

Howard Rines USDAIARS University of Minnesota Department of Agronomy and Plant Genetics St. Paul, MN 55108

Carlos Riede Brigham Young University IAPAEIBRAZIC Department of Botany and Range Science Provo, UT 84602

Peter Rogowsky Waite Research Institute Department of Plant Science Glen Osmond, SA 5064 AUSTRALIA

Jackie Rudd Kansas State University Department of Agronomy Throckmorton Hall Manhattan, KS 66506

Fred Schwenk Kansas State University Department of Plant Pathology Throckmorton Hall Manhattan, KS 66506-5502


Rollie Sears Kansas State University Department of Agronomy Throckmorton Hall Manham KS 66506

Peter Sharp Plant Breeding Institute Cobbitly Road Cobbitly, NSW 2570 AUSTRALIA

Lesley Sitch Joint AEA /FA0 Division Wagramerstrase 5 Vienna AUSTRIA

Virgil Smail US Grain Marketing Research Laboratory USDAlARS 1515 College Avenue Manhattan, KS 66502

Rex Smith University of Florida Agronomy Department Building 935 Gainesville, FL 3261 1

Mark Sorrells Cornell University Department of Plant Breeding 252 Emerson Hall Ithaca, NY 14853

Richard Stnckey NAWGF 415 Second Street NE Suite 300 Washington, DC 20002

Yi Sun Kansas State University Department of Agronomy Throckmorton Hall Manhattan, KS 66506

0. A. Tnnzarella University of Tuscia Agrobiology and Agrochemistry Department Via. S. C. de Lellis Viterbo, 01 100 ITALY

Fred TownleySmith Agriculture Canada Research Station 195 Datk Road Winnipeg, Manitoba, 3RT 2M9 CANADA

Ned Tuleen Texas A&M University Department of Soil and Crop Science College Station, TX 77843

David Van Sanford University of Kentucky Agronomy Department N-106 A-S Building Lexington, KY 40546-0091

Jean-Marc Von Allmen Ciba-Geigy Seeds Postpach - Basel, 4002 SWlTZERLAND

Mike Wanous University of Missouri Deparlment of Agronomy Columbia, MO 6521 1

Joanna Werner Kansas State University Department of Plant Pathology Throckmorton Hall Manhattan KS 66506-5502

Duane Wilson Kansas State University Department of Agronomy Throckmorton Hall Manhattan, KS 66506-5502

Yang Yen University of Nebraska Agronomy Department 307 Keim Hall Lincoln, NE 685834915

Yaw-Ching Ynag Texas A&M University X-3-K Hensel Apt. College Station, TX 77840

Manfred Zom Lawence Berkeley Laboratory 1 Cyclotron Road MS 508-3238 Berkeley, CA 94720


Documents

Progress - University of California, Davisgrcp.ucdavis.edu/publications/doc10/Report10.pdf · or low copy clones, to chromosome arms in wheat using aneuploids so that these clones