RFLP Map of Cotton, Gossypium hirsutum X Chromosome Organization

Copyright 0 1994 by the Genetics Society of America

A Detailed RFLP Map of Cotton, Gossypium hirsutum X Gossypium barbadense: Chromosome Organization and

Evolution in a Disomic Polyploid Genome

~

Alesia J. Reinisch," Jian-mjn Dong,* Curt L. Brubaker,t David M. Stelly," Jonathan F. Wendelt and Andrew H. Paterson"

*Department of Soil and Crop Sciences, Texas A&M University, College Station Texas 77843-2474, and +Department of Botany, Iowa State University, Ames, Iowa 50011

Manuscript received January 6, 1994 Accepted for publication July 12, 1994

ABSTRACT We employ a detailed restriction fragment length polymorphism (RFLP) map to investigate chromo-

some organization and evolution in cotton, a disomic polyploid. About 46.2% of nuclear DNA probes detect RFLPs distinguishing Gossypium hirsutum and Gossypium barbadense; and 705 RFLP loci are assembled into 41 linkage groups and 4675 cM. The subgenomic origin (A us. D) of most, and chromosomal identity of 14 (of 26), linkage groups is shown. The A and D subgenomes show similar recombinational length, suggesting that repetitive DNA in the physically larger A subgenome is recombinationally inert. RFLPs are somewhat more abundant in the D subgenome. Linkage among duplicated RFLPs reveals 11 pairs of homoeologous chromosomal regions-two appear homosequential, most differ by inversions, and at least one differs by a translocation. Most homoeologies involve chromosomes from different subgenomes, putatively reflecting the n = 13 to n = 26 polyploidization event of 1.1-1.9 million years ago. Several observations suggest that another, earlier, polyploidization event spawned n = 13 cottons, at least 25 million years ago. The cotton genome contains about 400-kb DNA per cM, hence mapbased gene cloning is feasible. The cotton map affords new opportunities to study chromosome evolution, and to exploit Gossypium genetic resources for improvement of the world's leading natural fiber.

T HE genus Gossypium L. has long been a focus of genetic, systematic and breeding research. Gos-

sypium comprises about 50 diploid and tetraploid species indigenous to Africa, Central and South America, Asia, Australia, the Galapagos, and Hawaii (FRYXELL 1979, 1992). Cultivated types derived from four species, namely G. hirsutum L. ( n = 2x = 26), G. barbadense L. ( n = 2x = 26), G. arboreum L. ( n = x = 13), and G. herbaceum L. ( n = x = 13), provide the world's leading natural fiber, cotton, and are also a major oilseed crop. Cotton was among the first species to which the Men- delian principles were applied (BALLS 1906), and has a long history of improvement through breeding, with sus- tained long-term yield gains of 7-10 kg lint/ha/yr (MEREDITH and BRIDGE 1984). The annual world cotton crop of ca. 65 million bales (of 218 kg/bale) , has a value of ca. US$15-20 billion/yr.

Diploid species of the genus Gossypium are all n = 13, and fall into 7 different "genome types," designated A-G based on chromosome pairing relationships (BEASLEY 1942; ENDRIZZI et al. 1984). A total of 5 tetraploid ( n = 2x = 26) species are recognized. All tetraploid species exhibit disomic chromosome pairing (KIMBER

1961). Chromosome pairing in interspecific crosses between diploid and tetraploid cottons suggests that tet-

raploids contain two distinct genomes, which resemble the extant A genome of G. herbaceum ( n = 13) and D genome of G. raimondii Ulbrich ( n = 13), respectively. The A and D genome species diverged from a common ancestor about 6-1 1 million years ago (WENDEL 1989). The putative A X D polyploidization event occurred in the New World, about 1.1-1.9 million years ago, and required transoceanic migration of the maternal A genome ancestor (WENDEL 1989, WENDEL and ALBERT

1992), which is indigenous to the Old World (FRYXELL 1979). Polyploidization was followed by radiation and divergence, with distinct n = 26 AD genome species now indigenous to Central America (G. hirsutum), South America ( G. barbadense, G. mustelinum Miers ex Watt), the Hawaiian Islands (G. tomentosum Nuttall ex See- mann), and the Galapagos Islands ( G. danuinii Watt) (FREXELL 1979).

Variation in ploidy among Gossypium spp., together with tolerance of aneuploidy in tetraploid species of Gos- sypium, has facilitated use of cytogenetic techniques to explore cotton genetics and evolution. Among 198 morphological mutants described in cotton, 61 mutant loci have been assembled into 16 linkage groups, through the collective results of many investigators. Using nul- lisomic, monosomic, and monotelodisomic stocks, l l of

Genetics 138 829-847 (November, 1994)

830 A. J. Reinisch et al.

these linkage groups have been associated with chromosomes (ENDRIZZI et al. 1985).

Our objectives were to use low copy DNA markers to investigate cotton genome and chromosome organization at the molecular level, in a cross of G. hinuturn X G. barbadense. We have established a detailed restriction fragment length polymorphism (RFLP) map of cotton, identifymg genomic origins of, and homoeologies among, most of the linkage groups in tetraploid ( n = 2% = 26) cotton, and characterizing the nature and frequency of rearrangements which distinguish homoeologs. Identification of homoeologous chromosomal regions reveals the approximate locus of DNA probes monomorphic in one subgenome but polymorphic in the other, and represents a means to greatly increase the informativeness of genetic maps in polyploids such as cotton. Characterization of the comparative organization of homoeologous chromosomes promises to increase the marker density of molecular maps in disomic polyploids, facilitating both genetic and physical mapping applications.

MATERIALS AND METHODS

Genetic stocks: The genetic mapping population com- prised 57 F2 individuals from a cross between single individuals of G. hirsutum race “palmeri” (see BRUBAKER and WENDEL 1993) and G. barbadense acc. “K101.” These accessions were selected because they are largely homozygous, and relatively primitive, thus free from the interspecific introgression that characterizes some populations of these two species from sym- patric portions of their indigenous ranges (PERCY and WENDEL 1990; WENDEL and ALBERT 1992; BRUBAKER et al. 1993), as well as cultivated types (PERCY and WENDEL 1990; G. WANC and A. H. PATERSON, unpublished data). Self-pollinated progeny from the “palmeri“ and “K101” parents were used for preliminary surveys of DNA polymorphism, prior to mapping.

Genomic identity of RFLP alleles (see Figures 2 and 3) were inferred based upon comparison of G. barbadense and G. hirsutum parents to accessions representing the diploid progeni- tor genomes of tetraploid Gossypium, i.e., the A genome d ip loids G. arboreum (accession 447) and G. herbaceum (accession 4 A,97), and the D genome diploids G. triEobum (Mocino & Sese ex DeCandolle) Skovsted (un-named accession) and G. raimondii (un-named accession).

Genetic stocks monosomic for G. barbadense chromosomes 1, 2, 4, 6, 9, 10, 17, 20 and 25, and stocks mono-tele disomic for G. barbadense chromosome arms >Lo, 14L0,15Lo, l8L0, 22Sh and 26Sh were used to identlfy linkage groups corresponding to these chromosomes. A monosomic substitution stock has a single chromosome from a donor genotype (in our case, G. barbadense acc. “Pima 3-79”) substituted for the corresponding pair of chromosomes of the recipient gene type (in our case, G. hirsutum acc. “TM-1”). A mono-telodisomic stock is similar, except that a single chromosome arm from the donor is substituted for a chromosome pair in the recipient. In the series of monosomic and mono-telodisomic substitution stocks, an individual genetic stock exhibits the G. barbadense allele only for loci falling on the substituted chromosome (or arm). hsignment of chromosome numbers in tetraploid cotton is based on pairing relationships in diploid X tetraploid crosses, with chromosomes 1-1 3 corresponding to the A subgenome, and chromosomes 14-26 corresponding to the D subgenome (UMBER 1961).

DNA probes: Mapped DNA probes included PstI-genomic fragments from G. raimondii (prefix G: 75 probes), G. herbaceum subsp. africanum Watt (Mauer) (accession A,-73; prefix A 192 probes), and G. hirsutum accession “TM-1” (pre- fured M: 21 probes; and P 78 probes), low copy genomic restriction fragments selected from a libraly of G. barbadense cv. “Pima S6” DNA cut with a mixture of five different blunt- cutting fourcutters (prefmed LXP: 1 probe and PXP: 40 probes; to be described elsewhere; X. Zhao and AHP, in prepa- ration), genomic probes derived from G. hirsutum cv. “Tam- cot GNCH” and containing Not1 sites (prefixed pVNC), and cDNAs (143 probes) from a library prepared from drought- stressed tissue of G. hirsutum accession ”T25.”All probes were prepared by PCR amplification of plasmid or phage DNA, using M13 primers (SP010 and SP030, Operon, Alameda, Cali- fornia), followed by chromatography through homemade Sephadex G50 (Sigma) spuncolumns (SAMBROOK et al. 1989).

DNA extraction, electrophoresis, blotting, and hybridization: DNA extractions followed PATEMON et al. (1993). Replica blots were prepared using each of the restriction enzymes used for polymorphism screening (below). DNA electrophoresis, blotting, probe labeling, Southern hybridization, and autoradiography were as described by CHITTENDEN et al. (1994). except that after liquid hybridization, filters were washed at 2X, l X , and 0.5X SSC (instead of three washes at 0.1X).

Individual DNA probes were screened for polymorphisms distinguishing the grandparents ( G . hirsutum race “palmeri” and G. barbadense acc. “K101”) using 4-6 restriction enzymes (EcoRI, EcoRV, HindIII, XbaI in all cases, and BamHI, and CfoI in a subset of cases), and filters were washed for 20 min each in 2X, lX, and 0.5X SSC, 0.1% SDS at 65” prior to autoradiography.

Data analysis Linkage maps and related statistics were determined using MapMaker 1.0 (LANDER et al . 1987; provided by L. PROCTOR, DuPont Co.) running on a Macintosh Quadra 700 under System ’7.01. A LOD (base 10-log of the ratio between odds of linkage and odds of non-linkage) score of 4.0 was used to infer linkage in two-point analyses: since the cotton RFLP map spans nearly 5000 cM, the standard LOD 3.0 used for smaller genetic maps is not sufiiciently stringent. lnitial frameworks of markers were evaluated using the “compare” function, and only orders preferred by a LOD of at least 2.0 (e.g. , a 100-fold difference in likelihood) over alternate orders were accepted. New markers were added to the frameworks using the “try” function, and the maximum likelihood orders of each linkage group ultimately verified by the “ripp1e”function. Segregation ratios were calculated using SAS (SM Institute, 1989). Deviation of segregation ratios from the Mendelian expectation was assessed by calculating chiquared values.

Since the mapping population is tetraploid, determination of allelism between genomic restriction fragments unique to the respective grandparents is more complicated than in diploids, or largely diploidized paieopolyploids. Basically, all DNA polymorphisms fit one of the following three scenarios: (1) Only one of the two homozygous grandparents had a unique fragment. This was commonly observed, as many fragments were presumably masked by co-migrating duplicated fragments. Consequently, mapping was based on presence or absence of an individual fragment, because heterozygotes could not be reliably (e.g. , qualitatively) distinguished from homozygotes for the variant fragment. (2) Each of the two homozygous grandparents had one unique fragment. In principle, the two fragments could be allelic, or could represent polymorphisms at two different loci. These alternatives were readily distinguished in most cases by the following logic: If the two fragments are allelic, all individuals in the mapping population must have at least one of the two. On the other hand, if the

831 Organization of Cotton Chromosomes

TABLE 1

Hybridization of homologous and homoeologous cotton DNA probes to EcoRIdigested genomic DNA from A, D and AD genome cottons

A. Number of restriction fragments B. Fraction of restriction fragments hybridizing to genomic DNA strongly hybridizing to genomic

from different genomes. DNA from different genomes. Source of genomic DNA Source of genomic DNA

Source of DNA probe’ A D AD Total A D AD Average

A genome PstI fragments 157 155 187 499 0.64 0.62 0.69 0.65 D genome PstI fragments 178 161 232 571 0.53 0.52 0.60 0.56 AD genome PstI fragments 1 49 126 226 50 1 0.55 0.63 0.60 0.59 AD genome cDNAs 167 154 262 583 0.51 0.57 0.53 0.54 Total 65 1 596 907 2154 Average per DNA probe 2.96 2.71 4.12 0.56 0.58 0.60 0.58

‘A total of 55 DNA probes from each of the four classes were used. Only 55 D genome probes were available which provided the necessary data; probes from the other three classes were chosen randomly from candidates for which equivalent data was available.

two fragments represent polymorphisms at two unlinked loci, an average of 1/16 of the individuals in the mapping population should not show either fragment (assuming Mendelian segregation ratios), and in a population of 57 individuals, there is approximately 95% confidence of seeing such an individual. In cases where one or more such individuals were observed, the fragments were mapped as independent dominant/recessive loci, as in case #1, above. If no such individual was observed, the fragments were tentatively mapped as alleles. This algorithm should be reliable for all cases except those rare cases where the two fragments represent linked duplications in “trans” configuration. This final possibility is difficult to exclude with certainty in any genetic mapping experiment, although the number of cases fitting this scenario is small. Should the alternative fragments represent different loci separated by an appreciable recombinational distance, at- tempting to map them as alleles would result in conflicting information, and either imprecise mapping or an inordinately large expansion of the most likely map interval (relative to its recombinational length without this “locus”). A few such cases were found, and the fragments were re-mapped as independent loci. (3) Each of the two homozygous grandparents showed multiple unique fragments. This is similar to scenario (2), except that the possibility of allelism must be evaluated between all possible pairs of loci derived from alternate grandparents. In most cases, allelism could be inferred not only by complementarity between one pair of genomic fragments, but also by lack of complementarity to other genomic fragments.

Nomenclature: Genetic loci detected by DNA markers have been designated by the name of the DNA probe detecting the locus. If a probe detected RFLPs at multiple loci, letters (a, b, c, . . . ) were arbitrarily assigned to these loci. If a locus could be assigned to a particular chromosome (by analysis of aneuploid stocks as described above), the locus name was followed by an indication of chromosome number (e.g., pAR042.05, for chromosome 5 ) .

Linkage groups not yet assigned to chromosomes are des ignated by the subgenome from whence they appear to be derived (A, D or U if unknown) based upon alloallelic evidence (see below), and numerically by decreasing recombinational length within a subgenome.

RESULTS

Sequence duplication in diploid and tetraploid Gos sypium genomes: Genomic hybridization patterns of cDNAs and hypomethylated (PstIdigested) genomic sequences revealed considerable duplication in the ge-

nomes of both diploid and tetraploid cottons (Table l , Figure la). Based on a sample of 220 cotton DNA probes for which equivalent data were available [55 G. hirsutum cDNAs, and 55 PstI-digested genomic sequences derived from each of three species, G. herbaceum (A genome), G. raimondii (D genome) and G. hirsutum (AD genome)], the average “low copy” cotton genomic probe hybridizes to 2.71 genomic EcoRI fragments in the ( n = 13) D genome, 2.97 genomic EcoRI fragments in the ( n = 13) A genome and 4.12 genomic EcoRI fragments in the ( n = 26) AD genome. Since DNA probe lengths averaged ca. 1 kb (ranging from ca. 0.5 to 2.0 kb), while EcoRI genomic fragments averaged ca. 4 kb, only about one-fourth of the genomic probes should contain internal EcoRI sites, giving a mean for true “single copy” probes of 1.25 fragments per probe. Even in contemporary diploid ( n = 13) cottons, most DNA probes detect more than twice this number of fragments, with an even larger number of fragments found in tetraploid ( n = 2x = 26) cottons.

Most probes did not hybridize equallywell to different genomic restriction fragments. EcoRI genomic fragments denoted as “weakly hybridizing” (less than 10% of signal in the lane, based on visual assessment), accounted for 44% of fragments in the A genome diploids, 42% of fragments in the D genome diploids, and 40% of fragments in the tetraploids (Table 1). In each of the three genomes, DNA probes hybridizing to multiple fragments detected similarly increasing proportions of “weakly hybridizing” fragments (Figure 1, b-d). While we acknowledge that our visual classification of “strong” us. “weak” bands may be somewhat confounded with the number of bands per lane (e .g . , multicopy probes are more likely to include copies which explain less than 10% of signal), it remains clear that most low copy probes detect both “strong” and “weak” bands.

Low copy DNA sequences in the A, D and AD genomes have not diverged extensively, as PstIdigested genomic probes from each source showed only small differences in detection of genomic fragments across


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FIGURE 1.-Copy number and hybridization affinity (signal intensity) of DNA probes in A, D, and AD genome cottons. (a) Histogram of the number of DNA probes detecting 1, 2, 3, or up to 14 (the maximum observed) genomic EcoRI fragments, for the A, D and AD genomes. The average “low copy” cotton genomic probe hybridized to 2.71 genomic EcoRI fragments in the ( n = 13) D genome (represented by G. raimon- d i i ) , 2.97 genomic EcoRI fragments in the ( n = 13) A genome (G. fmhxmm), and 4.12 genomic E d fragments in the ( n = 26) AD genome ( G. himLcum). The most highly duplicated probes hybrid- izedtoll (D),12(AD),and14(A)fhg- ments, respectively. (b-d) Fraction of ‘’weakly hybridizing” restriction fragments detected, us number of total restriction fragments detected, in the A, D and AD genomes, respectively. Most probes did not hybridize equaUy well to Merent genomic restriction fragments, but detecting an increasing proportion of “weakly hybridizing” hgments with increasing total number of fragments.

the different genomes (Table 1). Cloned genes (cDNAs), and PstI sequences derived from the smallest (D) genome ( c = 0.68 pg; GEEVER et al. 1989), detected appreciably more genomic fragments than PstI sequences from the A ( c = 1.05 pg; GEEVER et al. 1989) or AD genomes ( c = 1.8-2.5 pg; ARUMUNGANATHAN and EAIUE 1991; MICHAELSON et al. 1991).

The present results regarding sequence duplication differ somewhat from previously published results which suggested that most Lea genes are represented in only one copy in diploid Gossypium, and two copies in tetraploid Gossypium (GALAU et al. 1988). The higher stringency we employed in our final wash (0.5 X SSC at 65” herein, us. 1 X SSC at 68”; GALAU et al. 1988) would

suggest that we should detect a lower level of duplication, all other factors being equal. One difference which may have afforded us greater power to detect weakly homologous genomic DNA fragments is the fact that we used -2 X as much DNA on genomic Southern blots as did GALAU et al. (1988) ; specifically, 2-5 pg us. 1-3 pg (stoi- chiometrically adjusted in proportion to the cvalue of the respective genomes, in both experiments). The effect of differences in blotting protocol, and other conditions, cannot be evaluated based upon present information.

Frequency of DNA polymorphisms between G. hirsutum and G. barbadense: Averaged across all classes of probes, 46.2% of DNA probes detected one or more DNA polymorphisms between G. hirsutum “palmeri”

Organization of Cotton Chromosomes 833

TABLE 2

Observed and expected frequencies of RFLPs between G. hirsutum race ‘‘palmm”’ and G. barbadense acc. “KlOl with different numbers of restriction enzymes

A. Average frequency of B. No. of enzymes per probe detecting RFLPs

Enzyme probes detecting RFLPS Obs. Exp. 7‘ (1 d.f.)’

BamHI 0.10 0 594 350 170

EcoFU 0.22 2 147 235 33 EcoRV 0.20 3 103 65 22 HzndIII 0.17 4 58 10 234 XbaI 0.16 5 15 1 257

6 12 0 5681 Total 1108 1108 6559

CfOI 0.19 1 179 447 161

Only probes for which data from all six restriction enzymes was available were used in the analysis. Expected values were calculated from the average frequencies of RFLF’s for each restriction enzyme or combination of restriction enzymes, using the binomial expansion, and summed across the Dossible combinations of restriction enzvmes. ExDected values are rounded to the nearest integer in this table, however exact values were used to calculate chi-square statistics.

All values had a deviation significant at 0.001 or less.

and G. barbadense “K101,” based on evaluation of 782 PstI-genomic probes and 326 anonymous cDNAs screened with the same six restriction enzymes. The frequency of RFLPs for genomic clones (47.0%) was not significantly different from that for cDNAs (44.2%).

DNA probes showing no polymorphism, or showing polymorphism with each of several enzymes are found far more frequently than expected, while probes showing RFLPs with only one or two enzymes were found far less frequently than expected (Table 2). The tendency for restriction enzymes with different target sites to reveal RFLPs with common DNA probes suggests that an appreciable number of RFLPs between G. hirsutum and G. barbadense may be due to genomic rearrangements (e.g., insertions/deletions or localized duplications and slippage at repeat units) which occur between the resmc- tion sites of several Werent enzymes. GALAU et al. (1988) previously suggested this, based on similar observations.

Segregation: Among more than 1200 DNA probes examined with four to six restriction enzymes, 563 DNA probes revealed RFLPs at 705 loci, including 455 loci (average 1.25 per probe) detected by PstI-genomic sequences, 187 loci (average 1.31 per probe) by cDNAs, and 63 loci (average 1.17 per probe) by other cotton genomic sequences. Among the 705 mapped RFLP loci, 452 (64.1 %) were scored as co-dominant alleles at single loci, 122 (17.3%) were scored for presence or absence of the G. hirsutum fragment, and 131 (18.6%) were scored for presence or absence of the G. barbadense fragment. This high proportion of “dominant-recessive” markers presumably results from polyploidy, whereby the alternate allele is masked by monomorphic co-migrating duplicated DNA fragments. In many cases, this was evident from stoichiometric diminution of signal from the “monomorphic” restriction fragment (filter zone) in the parent exhibiting the unique fragment; however, quan- titative discrimination of genotypes in these cases was

not attempted. Our results underestimate the true frequency of such RFLPs, as we preferentially mapped DNA probe X restriction enzyme combinations showing the maximum number of bands differing between parents. [Since determination of allelism in a tetraploid requires genetic analysis, simple observation of apparent differences between parents also underes- timates the frequency of “dominant” polymorphisms- from our initial screen of grandparental genotypes, only 27.5% of RFLPs exhibited a single unique band, while genetic mapping showed that 36% of RFLPs segregated as “dominant-recessive” markers.]

Deviations from Mendelian segregation ratios were widespread, as is commonplace in interspecific crosses. Averaged across the entire genome, segregation fit closely with the Mendelian expectation for an F, population, with 25.5% of loci homozygous for the G. hirsutum allele, 22.8% homozygous for the G. barbadense allele, and 51.7% heterozygous. However, specific regions of the genome showed marked deviations from the average. A total of 42 distinct regions, on 26 of the 41 linkage groups, showed significant deviation from the Mendelian expectation (at the nominal 0.05 level: regions depicted in Figure 2). Heterozygote excess was found in 16 regions, while a deficiency of heterozygotes was found in one region. Nine regions showed a deficiency of G. hirsutum homozygotes, including three regions showing an overall deficiency of the G. hirsutum allele. Sixteen regions showed a deficiency of the G. barbadense homozygote, including six regions showing an overall deficiency of the G. barbadense allele.

There appears to be no strong bias in gross nuclear genome composition, despite the fact that the cytoplasm was derived from G. hirsutum. Further, no expected single-locus genotype was completely absent from the population. However, the knowledge that segregation distortion occurs in crosses between these species (which segregate for a number of agriculturally impor-


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FIGURE 2.-RFLP map of the cotton genome. The cotton map presently includes 41 linkage groups, and spans 4675 cM, using the KOSAMBI (1944) cM function. A total of 683 loci link to this map; an additional 22 loci (not shown) were mapped but do not show statistically significant linkage yet. The identity, and genomic affinity of 15 linkage groups assigned to chromosomes is indicated. Nomenclature for loci, and for linkage groups not yet assigned to chromosomes, is described in MATEW AND METHODS. Genomic affinities of linkage groups represent a consensus of data from several markers (inferred as described in RESULTS, and Figure 3). Evidence for genomic affinity of individual markers (denoted as shown in Figure 3) is presented in bold. Solid lines connecting probes on different linkage groups indicate putative evidence of homoeology, based on duplication of three or more loci on common linkage groups. Dotted lines connecting LG DO1 and A05, and LG A02 and DO3 reflect probes which show two loci on one homoeolog, and a single locus on another, thus the orthologous loci are unknown. Dotted lines connecting


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PXP4-49 -

(D)pAR138bJ

4.5 5.1 11.2 0.0 4.1 0.0 4.0 3.7 21.0 9.8 3.9

chromosome 4 to LG DO1 and A05 reveal putative evidence of "paleo-homoeology." The criteria for inferring this, as well as a description of three additional cases which could not be shown directly in the figure, are described in RESULTS. Regions of segregation distortion are indicated by filled rectangles which span the marker loci showing significant deviations from the Mendelian expectation, and end halfway between the most distal locus showing distortion and the nearest locus for which segregation does not deviate significantly (0.05 level) from the Mendelian expectation. Shading of the rectangles indicates the nature of segregation distortion, as shown in legend.


Chr. 22(D) 305.3 cM

G1074

A 1310 15.1

L G. DO5 132.1 cM

11.8 8.8

13.1 9.5

4.9 0.0 2.2 9.0

16.8 3.6 0.0

13.5

n

pA R051 A 1524

20.0 pA ROO6

12.5 4.1 0.0 4.3 4.3 4.6 9.4

14.3 3.5 3.6

13.6 7.8

14.5

~~

A 1252 26.8

6.2 7.0 0.9 2.7 8.9 7.9 0.0 5.6 2.0 0.0 3.6 0.0 12.1 3.1 12.4 11.4 0.0 3.5 1.5 9.1 14.4 0.0 0.4 4.9 9.3 22.4 7.3 1.3 21.2

A 1740 A 1264

A 1719bMA 171ga PXP2-78 P9-3a A 1557 pA R 144h A 1780 pA R218a A 1807 G1005 A 1685a- A 16856 ~ 1 7 7 6 G1045(A 1575)d

DAR177a-

L. G. U03 50.1 cM

M16-41 pA R 188 pA R057

A 1625

pA R078a A 1826a

Pll-72 14.8 10.6

L G. A06 98.2 cM

n

Chr. 77(D) A1608 suppl. A A 17076 1194'$ ili 4'8 cM A 1482.17

(D) G1071

Chr. 77(D) A1583 121.38 cM

FIGURE 2.Pontinued

tant traits) suggests that subsequent QTL mapping stud- Recombination: Among the 705 loci at which segre- ies, or phenotype-based DNA pooling experiments gation has been determined, 683 (96.8%) have been to isolate diagnostic markers (MICHELMORE et al. 1991; assembled into 41 linkage groups of two or more loci, GIOVANNONI et al. 1991) should compensate for segre- with the remaining 22 not yet linked to the map (Figure gation distortion with increased population size (WANC 2). The map presently spans 4675 centimorgans (cM), and PATEMON 1994). with DNA marker loci distributed at average intervals of


LG. DO3 Chr. 7(A) 169.3 cM

Chr. 75(D) 128.6 cM

L.G. A02 205.2 cM

11.9 P1-8 3.8 G1114

6.4 A 1698 0.0

12.8 2.0 6.4

14.8 3.4 6.0

10.4 10.3 5.3 4.6 0.9 0.0 0.0 0.0 0.0 3.6 4.3

16.4 7.0

17.9 4.1 5.6

155.7 cM

P10-56a-)&4.4 A 1590e

-E - p2-58$1 ~~~ ~ 5.0

(D)pA R019.15 20.1

11.9 - 4 A 1549.07

pAR099a -

12.1

10.7 8.2 2.9

11.9 2.7 4.9

16.9 10.1 4.2 0.1 3.6 5.0

31.2 5.9

16.2 6.4 7.9 1.3 0.0 7.4

A 1216 A 1706 A 1731 A 1679 M 16-88 A 1412fAA)

I I 1-38.2 6.8 5.8 2.7 0.0 0.0 3.2 8.1

10.3 7.5 3.6 0.0 0.0

14.4 3.6 2.1 5.4 5.3

12.8

A 1204.0 1 M 16-78 pAR297.OqA) (D)P5-39 A 1686a.OqAA) pA R226\(DIDY\ 16866

G1171 pA ROE8

61097 A 1720 A 1204.0 1 pA R o l l pA R132 A 1553

pA R07 A 1225

pAR121 A 1588 A 1109 A 1340

A 1643 (D DID) P5-32

A 1155 A 1257 P9-54 A

pA R077a A 1643

A 1097 pA R245

P5-3 1

A 17g4Aw pxp3-42

1197 1658 1010

6.9 8.6 6.5 0.1 3.4 4.6 1.5 1.0 0.0 0.0 0.0 0.0 3.9 22.0 13.0 26.0

A 1691c A 1348 A 1 108 (D/D pAR309

VNC1646 P5-24

A 1590a -( A 1168

A 140 1 61013

pAR118 A 15906

M 16-200 A 1667a

P 1-26 pAR118a P5-9 A 1632 P5- 18 A 1562 61018 A1107 P1-46a P5-37 A 1658 AA) P5-4 G l lO l pA R248 G1021 G 1078(A) pA R 123 G1276a(D/D) 4 1214d

e3.1 12.3 ' J

J 20.8

LG. A01 205.75 cM

A 1552a - A 1417qD\ A 1520A \

A 1835 p A R238

LG. U06 19.14 cM

A 15526 A 1676

A 14176 ' A 15208

P10-7 3.4 P1-19

0.0 20.6

1.5 12.1 12.5 4.4

14.9

pA R265c A 1208a

P11-16 pA R274a

PXP2-25

P5- l la

10.6

22.2

26.5

~

LEGEND FOR SEGREGAl lON DlSTORl lON

Excess of heterozygotes

Deficiency of hetenzygotes

bficiency of G. hilsutum homozygotes = Deficiency of G. hilsutum allele

0 bficiency of G. babadense allele

Deficiency of G. babadense homozygotes

Chr. 78( D) 24.6 cM 15.7

7.6 0.0 5.5

19.4 14.5

A 1713a(AA)-A 17136

G11256(Ak(DD)G1125a PXP 1-69 PXP3-25

pA R046 A 1364

1258a(A) (A ) A 164 7.18 A 1428a

17.7 - pA R282

FIGURE 2 . 4 o n t i n u e d

7.1 cM along the chromosomes. Estimates of recom- 26 chromosomes of cotton are presently represented by bination in cotton based upon chiasmata counts indi- 41 linkage groups, and that we can detect linkage at up cate a minimum overall map length of 4660 cM (STELLY to 30 cM, filling the gaps between existing linkage 1993), suggesting that most regions of the genome are groups should add at least 450 cM (=30 cM X 15 gaps) already covered by the present map, although gaps in to the map, for a minimum overall length of 5125 cM. some linkage groups remain to be filled. Given that the Once the map reaches a density of about one marker per


L. G. UO1 200.34 cM

L G. Dl0 6.86 cM

A 1619a 6.8 PI-246

P12-1qD)

34.7

3.3 7.0

18.1

23.5

L. G. A04 L. G. U02 Chr: 2(A) 120.22 cM 63.82 cM 51.9 cM

A 1826b

G I185b pA R027 A 16914A) pA R257a

23.7 -

0.0 29.2 -

A 1719c

p BA M325b

P9-6 1 P2-356

pA R260c

10.6 1.7 6.8 4.3 2.1

14.0 12.4

G 1148

A 1325 P2-35c pAR151.OqD)

pA R318.04AA) pA R316

A I 146.02

A 1436

1 r pAR278b

L G. Dl1 L G. DO9 33.7 - 3.3 cM 7.05 cM

L G. U04 L. G. A07 L G. A08 28.4 cM 27.65 cM 21.77 cM

0.1 M 16-85 M16-45c

L. G. U05 L G. U07 L G. UO9 26.1 0 cM 16.21 cM 6.27 cM

FIGURE 2.-Continued

5 cM, or a total of about 1025 markers, there should be fewer than 1% of intervals between markers measuring >25 cM (TANKSLEY et al. 1988), and the map should “link up” into 26 linkage groups corresponding to the 26 gametic chromosomes of cotton.

Assignment of linkage groups to chromosomes: A subset of the mapped DNA probes were hybridized to genomic digests of a series of monosomic and monotelodisomic substitution stocks, with a single G. bu&xknse chromosome substituted for one pair of G. himturn chre mosomes. This method is conceptually similar to use of chromosomedeficient lines to determine chromosomal location of DNA probes (HELENTJARIS et al. 1986; GALAU et al. 1988), except that assignment of probes to chromosomes is based on detection of an RFLP, rather than on dosage. Based on multiple genetically linked loci which correspond to common aneuploid substitution stocks, we have tentatively determined the linkage groups which correspond to chromosomes 1, 2, 4, 6, 9, 10, 1 7, 22 and 25 (Figure 2). The identity of chromo-

somes 5, 14, 15, 18 and 20 is suggested by single loci, which are neither corroborated nor contradicted by any other locus on the linkage group. These determinations were based upon single plants derived from isoline development projects for each monosome and telosome. Abnormal meioses occasionally generate new forms of aneuploidy from existing aneuploids, thus independent corroboration of these results with newly isolated stocks is underway. Further, additional aneuploid stocks are being characterized to identtfy chromosomes and chromosome arms corresponding to additional linkage groups.

Three pairs of (tentatively) identified chromosomes showed homoeology in our map, chromosomes 1 and 15, chromosomes 5 and 20, and chromosomes 6and 25. Homoeology between chromosomes 1 and 15 is supported by the observation that duplicated mutations in three morphological traits map to this pair of chromosomes (ENDRIZZI et al. 1984), with one inversion in order. Homoeology between chromosomes 6 and 25 has been suggested previously (ENDRIZZI and RAMSAY 19’79), based


upon similar phenotypes of plants monosomic for each of these chromosomes. Although several mutant phenotypes have been assigned to chromosome 5, no evidence is available regarding chromosome 20. Classi- cal evidence for homoeology between chromosomes 7 and 16, and chromosomes 12 and 26 (ENDRIZZI et al. 1984), cannot yet be corroborated by molecular evidence as the linkage groups corresponding to these chromosomes have not yet been determined.

Several tentatively identified chromosomes show homoeology to as yet unassigned linkage groups. For example, chromosome 9 appears homoeologous to a linkage group (LG) initially named D06. Based on independent evidence of homoeology between chromosomes 9 and 23 (CRANE et al. 1994), we have tentatively changed the name of LG DO6 to chromosome 23. Un- assigned linkage groups showing partial or complete homoeology to chromosomes 10, 14, 1 7 and 22 have been identified; however, the identities of the homoeologs to these chromosomes remain unknown.

Deducing the genomic origin of linkage groups in allotetraploid cotton: Some DNA probes detected genomic fragments in tetraploid cottons which were shared with either A or D genome ancestors, but not both. In a subset of these cases, polymorphism between G. hirsutum and G. barbadense permitted mapping of one or both of the homoeologous genomic fragments. Such “alloalleles” (e.g. , orthologous diploidgenome- specific DNA fragments) have been suggested to be a means of deducing the diploid origin of particular linkage groups (or chromosome segments) in tetraploid cotton (GALAU et al. 1988).

The degree of support for inferences of subgenomic origin of alloalleles varied among RFLP loci and linkage groups (logic summarized in Figure 3). Among 276 mapped loci for which the A, D, and AD genomes could be compared, 115 yielded information suggesting the genomic origin of particular restriction fragments. These 115 loci yielded a total of 159 comparisons (in some cases, a locus yielded more than one piece of evidence of genomic origin; see Figure 3). Among these, 138 (86.8%) fit with the most parsimonious genomic origin of their respective linkage group. Based on alloallelic loci, the genomic origin of 33 of the present 41 linkage groups, including all of the known chromosomes, has been determined. One linkage group (LG UO1) showed conflicting information (2 A, 2 D), and seven small linkage groups (totaling 224.8 cM and 34 loci) harbored no alloallelic loci.

To evaluate the reliability of our subgenomic inferences, we calculated the frequency at which alloallelic information coincided with the classical assignment of cotton chromosomes to subgenomes based upon pairing relationships in diploid X tetraploid hybrids (A = chromosomes 1 - 1 3 D = chromosomes 14-26). Based on the 14 chromosomes we have identified, the majority

FIGURE 3.-Inference of genomic affinity for individual DNA marker loci. Genomic restriction fragments which revealed RFLPs between G. hirsutum and G. barbadense were evaluated for genomic affinity by comparison to corresponding genomic digests from G. raimondii and G. trilobum (D genome) and G. arboreum and G. herbaceum (A genome). Six basic scenarios were found. (1) A and D genomes have different restriction fragments, one polymorphic restriction fragment in AD genome co-migrates with one (or both, in this case) fragments in a diploid genome (D, in this case). In the case shown, the locus would be denoted “DD,” because it co- migrated with fragments unique to the D genome but present in both D genome types. This was deemed to represent two “units” of information in support of a D genome affinity of the chromosomal region near this locus. (2) A case similar to # 1 would be denoted “D,” for co-migration with a fragment unique to just one of the two D genome types studied. This was deemed to represent only one “unit” of information in support of D genome affinity. (3) If each of two allelic polymorphic restriction fragments in the AD genome cottons correspond to a polymorphic restriction fragment in one of the two D genome types, the locus is denoted “D/D,” and two “units” of information in support of D genome finitywere inferred. (4) This case exemplifies the risk of artifacts due to convergent gain/loss of restriction sites. The available information war- rants the same designation as case #2 (‘ID”). The most parsimonious explanation of this scenario is that one D genome ancestor and one of the tetraploid subgenomes have indepen- dently incurred mutations resulting in new co-migrating fragments. However, there is no a priori way of determining which of the AD subgenomes was involved in the mutation. An alternative explanation would be polyphyletic evolution of the two tetraploid species, involving each of the two diploid species. No information is inferred. (5) In this case, one AD genome fragment corresponds to a fragment unique to one A genome type. Another AD genome fragment corresponds to a fragment unique to a D genome type. Consequently, there is conflicting information of equal weight. Such cases were considered ambiguous, and no information is inferred. (6) In this case, the polymorphic band(s) is/are unique to the tetraploid, and no information is inferred.

of alloallelic data for a chromosome agreed with classical subgenomic assignment of chromosomes in all 14 (100%) cases. Individual pieces of information from alloallelic probes on known chromosomes were correct in 63 (86.3%) of 73 cases. We conclude that alloallelic information is a reliable, although not infallible, indicator of genomic origin of a chromosome or linkage group. For cotton, agreement among three independent pieces of alloallelic information affords ca. 99% confidence (1 - [ 1 - 0.8631’) of correct subgenomic assignment of a chromosomal region.

Six of the 10 loci where alloallelic information dis- agreed with classical subgenomic assignment of chromosomes occurred as linked pairs on three linkage groups.


In each of these three cases (chromosome 14, LG A03, LG UOl), the two deviant loci are consecutive along the linkage group (with reference to alloallelic probes). This suggests the possibility that exchanges of chromatin between homoeologous chromosomes may have occurred during the evolution of tetraploid cottons. How- ever, by the criteria we suggest in the preceding para- graph, three pieces of information are necessary to make this inference, thus more data is needed to s u b stantiate the possibility of homoeologous exchanges.

Determination of homoeology from map positions of duplicated lock A total of 106 (18.8%) DNA probes detected RFLPs at two loci, 13 (2.3%) probes detected RFLPs at three loci, 2 (0.36%) at four loci and 1 (0.18%) at six loci. We preferentially mapped probe X enzyme combinations which showed multiple polymorphic fragments; hence, these data overestimate the frequency at which DNA clones detect multiple RFLPs.

In many cases, loci linked to one another had duplicated sites also linked to one another. This could occur by at least three mechanisms: (1) chance associations between loci generated by retrotransposon-like or other duplication mechanisms, (2) recent polyploidy and (3) paleopolyploidy. If duplicated loci are randomly distributed and linkage groups are of equal size, synteny between n duplicated DNA markers in a map of 41 linkage groups would occur with a likelihood of (1/41)(”-’). [We recog- nize that each of these assumptions probably are violated to some degree, and that this calculation represents only a crude estimate of the likelihood of the underlying event.] Thus, synteny of two duplicated loci by chance is somewhat improbable ( ca. 2.4%), but synteny between three or more loci by chance is highlyimprobable (ut. 0.06%), andclearly suggests that duplication of a chromosome (or segment), rather than independent local duplications of short sequences, is the most likely explanation. We have considered synteny of three or more duplicated loci as evidence of homoeology.

By evaluating the distribution of linked duplicated loci across the map, we find evidence for duplication of chromosomes or segments of 23 of our 41 linkage groups, covering 1668 cM (36% of the genome). These homoeologous relationships are based upon association among 62 (51%) of the 122 duplicated probes we mapped. In all cases, any one region of a linkage group was associated with only one region of another linkage group, when associations based upon three or more duplicated loci are considered.

Homoeologous relationships between linkage groups are described below (and shown in Figure 2). In all cases where structural mutations were inferred, homosequential order was rejected at LOD scores of 2.0 or greater, unless otherwise specified.

Chromosome 6 (A genome) and chromosome 25 (0 p o m e ) : These are deemed homoeologous based on six duplicated loci, spanning 150.4 cM (76.8%) of chromosome

6 and 141.9 cM (92.2%) of chromosome 25. The duplicated loci are homosequential. Three additional DNA probes mapping to these chromosomes show duplicated loci which fall on different linkage groups. Alloallelic evidence indicates that chromosome 6 is A genomederived, while chromosome 25 is D genomederived.

Linkagegroups DO1 and A05r These are deemed homoeologous based upon seven duplicated loci, spanning 152.7cM (87.8%) ofLGD01 and 108.7 cM (92.3%) of LG A05. At least two inversions are necessary to account for differences in locus order. Four additional duplicated DNA probes mapping to these chromosomes show duplicated loci. Two of these are on different linkage groups, but the other two, PAR1 97 (on LG Dol) and G1045 (on LG A05), each show a duplicated locus on chromosome 4 (an A genome chromosome), possibly indicating “paleo-homoeology” (see below).

Chromosome 10 (A genome) and LG 0 0 4 : These are deemed homoeologous based upon 4 DNA probes, spanning 75.2 cM (35.0%) of chromosome 10 and 66 cM (45.9%) of LG D04. A single inversion can account for the difference in order between loci. Eleven other duplicated loci are found on these two groups. Two of these ( A 1 18?a, b on chromosome 10) represent a proximal duplication, while three show duplicated sites widely distributed throughout the genome. Several additional loci show evidence consistent with paleopolyploidy (see below).

Chromosome 9 ( A genome) and chromosome 23: These are deemed homoeologous based upon 5 DNA probes, spanning 76.3 cM (40.9%) of chromosome 9 and 78.1 cM (63.9%) of chromosome 2?. A single terminal inversion can account for the difference in order between markers on these linkage groups. Six additional probes show unlinked duplications. We note that chromosome 23 has been identified based on prior evidence of homoeology to chromosome 9 (CRANE et al. 1994), as no aneuploid genetic stocks were available. The inversion we detected is consistent with the physical linkage of 5s and 18-28s rDNA sites in the same arm of chromosome 23, and opposite arms of chromosome 9 (18- 28s on short arm; CRANE et al. 1994).

Linkage groups A03 and 002: These are deemed homoeologous based upon 4 DNA probes, spanning 26 cM (15.4%) of LG DO2 and 54 cM (31.8%) of LG A03. The duplicated loci map in the same order on the two chromosomes. Nine loci falling outside the region of homoeology map to unlinked sites, several on very small linkage groups. Only one other duplicated locus occurs in the region of homoeology, with five sites on other chromosomes.

Chromosome 5 (A genome)/chromosome 20 (D genome) + LG 007: These are deemed homoeologous based upon 13 DNA probes (9 on chromosome 20,4 on LG D07). One region of 122.7 cM (50.2%) of chromosome 5 corresponds to a region of 127.6 CM (48.5%) of chromosome 20, while another region of 31.4 cM (12.8%)


of chromosome 5 corresponds to 19.3 cM (28.6%) of LG D07. A single terminal inversion can explain the difference in marker orders between chromosome 5 and chromosome 20, while another terminal inversion d is tinguishes chromosome 5 and LG D07. This association of chromosome 5, an A genome chromosome, to parts of two different D genome linkage groups could indicate that the relevant A and D genome chromosomes differ by a translocation, or that the two D genome linkage groups are parts of the same chromosome. We found no evidence of linkage between chromosome 20 and LG D07; however, LG DO7 is relatively small (67.4 cM) to be an entire chromosome.

Linkage groups A02 and 003: These are deemed homoeologous based upon three DNA probes, spanning 32 cM (16%) of LG A02 and 26 cM (17%) of LG D03. At least one inversion is necessary to explain the differences in order among the duplicated loci. The location of the breakpoint is unclear, as one probe ( A 1 5 9 0 ) de- tects two loci on LG DO3 at similar hybridization intensity, and it is unclear which of these is orthologous to the locus on LG A02. Seven other loci on the two chromosomes map to unlinked sites.

Chromosomes 1 and 15: These are deemed homoe ologous based upon two duplicated DNA probes, spanning 45 cM (26%) of chromosome 1 and 58 cM (45%) of chromosome 15. As described above, we have not inferred homoeology in other cases unless three duplicated loci were found- however, this case is supported by classical evidence that mutations in three morphological traits map to these two chromosomes (ENDRIZZI et al. 1984), with one inversion in order. The duplicated loci we have mapped do not detect the inversion, but span less than half of each chromosome. Three other duplicated loci on the two chromosomes map to unlinked sites.

Linkage groups A01 and U06: These are deemed homoeologous based on 3 markers, spanning 20.6 cM (10.0%) of LG A01 and 15.6 cM (81.5%) of LG U06. The duplicated loci are homosequential. At the opposite end of LG AO1, over 120 cM away, an association of two markers is found between LG A01 and chromosome 18. Four other duplicated loci on LG A01 map to unlinked sites. One, pAR274b, has not yet linked to the map, and the (putatively homoeologous) pAR274a locus corresponds to the center of the gap between LG U06 and chromosome 18.

Chromosome 22 (D genome) and LG DO5 + LG U03: In the upper region of chromosome 22,s loci spanning 93.5 cM (30.6%) correspond to a region of 86 cM (66.7%) on LG D05. The homoeologous regions are largely homosequential, with the most likely order of markers suggesting a small terminal inversion, although an alternate marker order with no inversion is only LOD 1.59 less likely (thus cannot be ruled out at the standard LOD 2.0 threshold).

In the lower region of chromosome 22, three loci spanning 41.8 cM (13.7%) correspond to a region of 45.1 cM

(22.4%) on LG U03, a linkage group of unknown sub genomic origin. The loci occur in homosequential order on the two linkage groups. The regions of homoeology to the different linkage groups do not overlap.

Homoeology of chromosome 2 2 to two other linkage groups may be the result of a translocation-or, the two other groups (DO5 + U03) could be different parts of the same chromosome. New markerswill be necessary to distinguish between these hypotheses.

Finally, we note that this is the only instance where two linkage groups inferred to be of the same subgenome (D) show evidence of homoeology. However, the subgenomic identity of LG DO5 is based on sharing of only a single restriction fragment between the D and AD genomes (cf. Figure 3, case 2), an observation which could easily have explanations other than homoeology.

Linkage group A06 and chromosome I4 (D genome) + chromosome 17 (D genome): In the lower region of LG A06, three loci spanning 55.8 cM (56.8%) correspond to 26 cM (17.4%) of chromosome 14. The difference in order of loci on the two groups can be explained by a single inversion. The upper region of L.G. A06 shows association to chromosome 17, based upon only two loci. Although this is not sufficient (by our criteria described above) to claim homoeology, we note that the corresponding regions of LG A06 and chromosome 1 7 do not show homoeology to any other genomic regions. The apparent homoeology of LG A06 to two different D genome chromosomes strongly suggests a translocation or Robertsonian fusion distinguishing the A and D genome chromosomes. Two other markers on chromosome 17 detect unlinked duplicated loci.

Possible evidence of segmental paleo-homoeology: In at least five cases, we found “nested” associations, between (segments of) different homoeologous pairs of linkage groups. While any one such association of two duplicated loci might well be seen by chance in a genome with as many chromosomes and duplicated loci as cotton (see likelihood calculation, above), the occurrence of five such events suggests paleopolyploidy. These associations are described below, and one example is illustrated (Figure 2; chromosome 4 with LG DO1 and A05):

Chromosome 4//LGDOI-LGAOS: Probes GI 045 and PAR197 map to loci 55.9 cM apart on chromosome 4, and duplicated sites map to loci on LG DO1 and A05, respectively, which are an estimated 40 cM apart (estimated from distances separating nearby duplicated markers).

Chromosome IO-LG D 0 4 / / L G A 0 3 - L G 0 0 2 : Probes A I 7 5 9 and P6-57 map to loci near the upper ends of chromosome IO-LG D04, and duplicated sites map about 40 cM apart in a region homosequential between LG A03 and LG D02.

Chromosome IO-LG D04//chromosome 5-chromosome 20: Probes AI 751 and P5-61 map to loci 1.3 cM apart


on chromosome 5, in a region which is homoeologous to chromosome 20. Duplicated loci map to homeolo- gous regions of chromosome 10 and LG D04, separated by about 36 cM (estimated from distances separating nearby duplicated markers).

Chromosome 5-chromosome 20//LG UO1: Probes A1 432 and P P I - 9 map to loci 51.7 cM apart on LG UO1, and are estimated to be more than 100 cM apart on chromosome khromsome 20 (estimated from distances separating nearby duplicated markers).

Chromosome 20 + LG D07//chromosome 4: Probes PAR138 and PAR21 9 map about 21 cM apart on chromosome 4, and are unlinked on chromosome 20 and LG D07. Chromosome 20and LG DO7 each show homoeology to Merent parts of chromosome 5, and it is unclear whether they are part of the same linkage group or not.

Several other “nested” associations were detected, but all involved one DNA probe that mapped to six loci, suggesting that these are due to mechanisms other than polyploidy.

DISCUSSION

Molecular mapping of disomic polyploids can be con- ducted with similar efficacy to molecular mapping of diploids, although polyploidy necessitates a more rigor- ous examination of allelism between polymorphic DNA fragments (see above). Further, polyploidy tends to increase the frequency of polymorphic bands masked by monomorphic co-migrating duplicated fragments, precluding qualitative identification of heterozygotes at such loci. Fortuitously, however, one occasionally finds polymorphism at each of two homoeologous loci, affording unique opportunities to study chromosome evolution, and to economize in genetic mapping.

Divergence of homoeologous chromosomes in n = 26 cotton: Mapping of multiple RFLPs derived from individual cotton DNA probes reveals the consequences of genome-wide duplication in “tetraploid” ( n = 2x = 26) cotton. We tacitly assume that most such associations between linkage groups are a result of the New World A X D hybridization event responsible for the occurrence of “tetraploid” ( n = 2x = 26) cottons.

Based on criteria described above, we have tentatively identified (at least parts of) 11 of the 13 expected homoeologous pairs in n = 26 cottons based upon 62 duplicated DNAprobes, spanning 1668 cM or 35.6% of the genome. Among the 265 mapped duplicated loci, 124 (46.8%) are accounted for by homoeology, and 16 (6.0%) by “paleo-homoeology.” Additional loci may be accounted for by these mechanisms as the remaining small linkage groups coalesce. The occurrence of proximal duplications, and duplicated loci inconsistent with homoeologous relationships, together with independent evidence (VANDERWIEL et al. 1993) reflect the ac-

tivity of other sequence duplication mechanisms in cotton, and further support the need for determining homoeology based on several loci along a linkage group (as we have done; see above).

Most of the homoeologous chromosomes in n = 26 cottons are distinguished by one or more inversions. In only two cases are putative homoeologs homosequential over the entire region of the chromosome for which homoeology can be inferred, although large blocks of sequence conservation are evident in most cases. Four pairs clearly differ by at least one inversion, and two additional pairs differ by at least two inversions. At least one pair shows a translocation, while several additional pairs may represent either translocations or simply small linkage groups which have not yet linked up.

Detection of rearrangements among the homoeologs of cotton is not surprising, in light of the 6-11 million years of divergence of n = 13 cottons from a common ancestor (WENDEL 1989). The observation that aneuploid D X AD genome hybrid cottons form >12 bivalents per cell suggests that few if any translocations differen- tiate D genome diploid and tetraploid chromosomes (KIMBER 1961). However, A X AD hybrids show evidence of at least two translocations distinguishing n = 13 A genome chromosomes from their n = 26 A genome counterparts (GERSTEL 1953), suggesting that the A and D subgenomes also differ by translocations, which is consistent with our observations. A companion study ( C . L. BRUBAKER, A. H. PATERSON and J. F. WENDEL, unpublished data) will describe mapping of a subset of the DNA probes used here in F, populations of G. arboreum X G. herbaceum (Agenome) and G. raimondii X G. trilobum (D genome). This should permit discrimination of structural mutations that occurred during divergence of the n = 13 A and D genomes, from mutations that occurred after formation of the n = 26 AD genome.

Despite structural rearrangement and reliable biva- lent pairing in tetraploid cotton (KIMBER 1961), low copy DNA sequences on the homoeologous chromosomes of both A and D diploid ( n = 13) genomes, and the corresponding AD tetraploid (n = 26) genome retain considerable similarity. Low copy DNA probes derived from either the A or D genomes are of similar effectiveness at detecting homologous and heterologous copies at mod- erate stringency (0.5 X SSC, 65”). A high frequency of “dominant/recessive” RFLPs suggests that many homoeologous DNA sequences retain flanking 6-base restriction sites from common ancestors of A and D genome cottons-thus one polymorphic restriction fragment is masked by monomorphic co-migrating duplicated fragments.

Extent of structural rearrangement of cotton chromosomes relative to chromosomes of other taxa: The nature and extent of chromosomal rearrangements distinguishing taxa provides one means by which the relative “genetic distance” between disparate taxa can be


assessed. For example, the two homoeologous chromosome sets (A and D) of n = 26 Gossypium are much more extensively rearranged than chromosomes of the genera Lycopersicon and Solanum, which differ by only five inversions and no translocations (TANKSLEY et al . 1992). Cotton homoeologs also exhibit more extensive rearrangement than the chromosomes of Brassica oleracea ( n = 9) and Brassica campestris ( n = lo), which differ primarily by structural rearrangements accounting for the difference in chromosome number ( SLOCUM 1989). Extension of this comparison to other Gossypium genomes (B, C, E, F, G) awaits future results.

In contrast, homoeologous cotton chromosomes are more similar than the chromosomes of Zea and Oryza (AHN and TANKSLEY 1993) which are distinguished by several translocations. Finally, homoeologous cotton chromosomes are far more similar than the chromosomes of Lycopersicon and Capsicum (TANKSLEY et al. 1988) or Arabidopsis and Brassica (KOWALSKI et al. 1994), which are extensively rearranged and exhibit only small islands of conserved order.

The extent of rearrangement between homoeologous cotton chromosomes most closely parallels that found between Zea mays and Sorghum bicolor (HULBERT et al. 1990; WHITKUS et al. 1992), where long homosequential stretches of chromatin are occasionally interrupted by inversions, but few if any translocations are evident.

Evidence repding paleopolyploidy of n = 13 cottons: It is apparent that the majority of duplicated loci mapped in n = 26 cotton became duplicated as a con- sequence of the relatively recent hybridization event (1.1-1.9 million years ago) that led to the evolution of polyploid Gossypium. This is supported by the identification of homoeologous linkage groups covering 35.6% of the genome, as shown above. In addition to recent polyploidy, however, evidence implicates other mechanisms in the generation of duplicated sequences. For example, the observation that DNA probes hybridize to a larger number of genomic fragments in the n = 13 A genome than in the n = 13 D genome cannot be explained by recent polyploidy (Table 1). Similarly, the fact that individual DNA probes revealed up to six RFLP loci demonstrates sequence duplication that is not ex- plicable by recent polyploidy alone. Additional duplications may have occurred by a variety of mechanisms, including replicative transposition (VANDERWIEL et al. 1993), chromosome segment duplication, and, of particular relevance here, more ancient polyploidization events involving progenitors of present-day “diploid” ( n = 13) Gossypium species.

New evidence from genomic hybridization patterns and linkage relationships among duplicated RFLP loci supports the hypothesis that “diploid” ( n = 13) cottons may be paleopolyploids, derived from an event or events involving ancestors with fewer chromosomes. This hypothesis was suggested decades ago, based on cytoge-

netic observations and the relatively high gametic chromosome number (DAVE 1933; SKOVSTED 1933; ABRAHAM 1940), and more recently (SUITER 1988; WENDEL and PERCIVAL 1990) stemming from the observation that gene numbers for allozymeencoding loci exceed expec- tations for “true” diploids ( c f . GOTTLIEB 1982). In our results, hybridization patterns of low copy genomic sequences reveal an appreciable level of DNA sequence duplication even in present-day “diploid” ( n = 13) cottons, although we note that our results show a somewhat higher level of DNA sequence duplication than prior results (GALAU et al . 1988; discussed above). Further, in at least four cases, associations of two pairs of linked duplicated loci were observed within individual subgenomes of tetraploid Gossypium, possibly reflecting conserved linkage blocks from an earlier polyploidization. At least one homoeologous pair (chromosome IO-LG D04) shows the possibility of two different putative “paleo-homoeologs” in only a small part of its length. These different putative regions of paleo-homoeology are non-overlapping. The fact that we could only docu- ment such associations with two pairs of loci in each region suggests that n = 13 cottons may have undergone much structural rearrangement and/or sequence evolution subsequent to the presumed paleopolyploidization event.

In suggesting the possibility that n = 13 cottons are paleopolyploid, we need to account for the observation that more than 30% of the DNA probes examined hybridized to only one (size of) genomic restriction fragment in the A and D genomes (Table 1; Figure 1). Some of these apparent “single” fragments may represent two co-migrating duplicated sequences, which origi- nated via polyploidization, and retain the restriction sites present in the putative n = 6-7 ancestor. Alter- nately, duplicated sequences may have diverged to the extent that genomic probes no longer detect “paleo- homoeologs.” We view this latter alternative as more likely, i . e . , that the n = 6-7 to n = 13 transition is ancient, primarily because paleopolyploidization must have antedated the evolution of the genus Gossypium (which is estimated to be at least 25 million years old; WENDEL and ALBERT 1992), and indeed, must have antedated the entire tribe Gossypieae and the closely related tribe Hibisceae, where all genera have high gametic chromosome numbers (FRWELL 1979). Accordingly, it would not be surprising if many genomic probes can no longer detect paleehomoeologous DNA sequences.

Relationship between physical size and recombinational length of subgenomes: Identification of linkage groups corresponding to the A and D subgenomes of n = 26 cotton (Figure 2) permits comparison of prop erties of the two subgenomes. Despite considerable differences in DNA content (GEEVER et al. 1989), and chromosome size (KIMBER 1961), the A and D subgenomes of n = 26 cottons exhibit virtually identical recombinational length (2119 and 2140 cM, respectively; Table 3).


This suggests that the much of the DNA comprising the (physically larger) A genome may be recombinationally inactive. Much of the difference in DNA content between the A and D diploid genomes is attributable to moderately and highly repetitive elements (GEEVER et al. 1989; X. ZHAO, R. WING and A. H. PATERSON, manuscript submitted for publication), suggesting that the repetitive fraction may be recombinationally inert. This con- cept is supported in a general sense by the observation that recombinational length of the genome varies much less than DNA content, across a wide range of plant and animal species. However, in no prior case have recombination rates in two genomes differing in DNA content been compared so directly, e.g. , within the gametes pro- duced by a common F, plant.

Subgenomic distribution of RFLPs, and its implications for the origin of polyploid cotton: A long-standing question in Gossypium evolution is whether polyploidization occurred only once (“monophyletic origin”) or two or more times (“polyphyletic origin”). While most evidence supports a single hybridization event leading to the evolution of tetraploid Gossypium species (ENDRIZZI et al. 1984), polyphyly has been suggested based on cytogenetic information (DAVIE 1933, 1935; KA”ACHER 1959,1960), flavonoid profiles (PARKS et al. 1975), and seed protein electrophoretic patterns (JOHNSON 1975). Chloroplast DNA restriction site data clearly demonstrate that only a single parental A genome was involved in polyploid formation (WENDEL 1989), but few data critically address whether this maternal A genome parent hybridized with one, or more than one, D genome species.

Herein, we note that approximately 10% more RFLPs were detected in the D subgenome than the A subgenome (329 us. 295; Table 3). If only a single hybridization event led to polyploid formation, then both subgenomes would be equivalently aged and might be expected to reveal similar levels of RFLP diversity (assuming equal rates of sequence divergence). In contrast, if different D genome species were involved in the par- entage of G. barbadense and G. hirsutum, one might expect greater D subgenome than A subgenome RFLP diversity in modern tetraploids.

Evidence from alloallelic polymorphisms could, in principle, support or contradict a polyphyletic origin of n = 26 cotton. Specifically, scenario #3 in Figure 3 could be consistent with a polyphyletic origin of n = 26 cotton. Nine D subgenome loci fit scenario #3, while only three A subgenome loci do so. However, an un- usually high rate of polymorphism between the D genome parents raises the possibility that a larger portion of the D genome matches are due to convergence, rather than conservation.

While one might be tempted to speculate that our data support a polyphyletic origin of n = 26 Gossypium, we view the apparent differences in subgenome RFLP

diversity to be equivocal with respect to the issue of polyploid monophyly us. polyphyly. The magnitude of observed differences in RFLP diversity between subgenomes is small, the RFLPs remaining unassigned to a subgenome are of sufficient number to eliminate this difference, we have little evidence regarding the relative activity in different subgenomes of processes which have given rise to new RFLP diversity since polyploid formation, and evidence from alloallelic polymorphisms may be biased by convergence between D-genome and tetraploid restriction fragments.

Application of DNA markers to cotton improvement: The cotton RFLP map is a starting point in use of DNA markers to manipulate genetic determinants of agriculturally important traits, and characterize the basis of agricultural productivity of cotton. Numerous introgression events may be detectable using existing DNA markers, including transfer of numerous genomic regions from G. hirsuturn into cultivated G. barbadense, as well as introgression of specific traits such as Verticillium wilt resistance (STATEN 1971), bacterial blight resistance (STATEN 1971), nectariless leaves (TYLER 1908), restoration of cytoplasmic male-sterity (MEYER 1975; WEAVER and WEAVER 1977), and improved fiber quality (CULP and HARRELL 1974: CULP et al. 1979). As in many largely self-pollinated crops, the gene pools of each of the cultivated cotton species show only modest levels of DNA polymorphism. Routine application of DNA markers to cotton breeding may benefit from technologies such as microsatellite-based markers, which are being superim- posed on the existing RFLP map (ZHAO et aL 1994).

Prospects for high density mapping and mapbased cloning in cotton and other polyploids: Several poten- tial complications associated with mapbased cloning in disomic polyploids are partly compensated for by some unique advantages of polyploid genomes. Utilization of genetic information from homoeologous relationships will accelerate development of a high density map of DNA markers in polyploid genomes with many chromosomes. About 21.7% of DNA probes segregated for RFLPs at two or more loci, accounting for 265 (37.5%) of the mapped loci. These duplicated loci provide a skel- eton for inferring the approximate homoeologous locus of DNA probes monomorphic in one subgenome but polymorphic in the other, increasing the marker density of the map. As additional markers are mapped it should be possible to catalog homoeologous relationships over most of the genome, and identify breakpoints of rear- rangementswith higher resolution. Once the remaining homoeologous relationships are identified, and the density of duplicated loci becomes greater, formal algo- rithms for genetic map integration ( cf. BEAVI~ and GRANT 1991; STAM 1993) can be employed to infer “unified maps” of particular homoeologous chromosomal regions, greatly increasing the density of DNA markers in maps of disomic polyploids such as cotton. Ultimately,


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The physical size of a centimorgan in cotton is not pro- hibitive to mapbased cloning, but the lengthy genetic map will require a large number of markers in order to be suf- ficiently close to most genes for “chromosome walking.” With a genetic map length of ca 5000 cM (Figure 2), and physical size of 2246 Mb (ARUMUNGANATHAN and EARLE 1991), the average physical size of a centiMorgan in cotton is about 400 kb, only slightly larger than that of Ara- bidopsis (ca . 290 kb), and smaller than that of tomato (ca . 600 kb), both species in which map-based gene cloning has been accomplished (GIRAUDAT et al. 1992; ARONDEL et al. 1992; MARTIN et al. 1993). However, even with the advantage afforded by homoeologous information, the genetic map of 5000 cM will require c a 3000 DNA markers to map at average 1 cM density, and the physical genome of 2246 Mb will require ca 75,000 YACs/BACs of average size 150 kb for 5X coverage. The suggestion above that repetitive DNA is recombinationally inert will further im- prove prospects for mapbased cloning in cotton, as a siz- able fraction of the genome may contribute little or noth- ing to the 5000 cM genetic length. Finally, we note that the estimate of physical genome size we have employed (2246 Mb: ARUMUNGANATHAN and EARLE 1991) falls at the median of several estimates (GEEVER et al. 1989; MICHAEL~~N et al. 1991; GOMEZ et al. 1993), which ”uy by *30%, introducing a corresponding range into our figures.

Mapbased cloning in polyploids such as cotton in- troduces a new technical challenge not encountered in diploid (or highly diploidized) organisms, e.g. , that virtually all “single-copy” DNA probes occur at two or more unlinked loci. This makes it difficult to assign Y mega- base DVA clones to their site of origin. One possible approadh to this problem is the utilization of diploids in physical mapping and map-based cloning; for example, exploiting the relatively small genome of D genome d i p loid cottons (approximately 65% of the size of the A genome, and 39% of the tetraploid; GEEVER et al. 1989). However, since D genome cottons produce minimal fiber, and are not cultivated, they are of limited utility for dissecting the fundamental basis of cotton productivity. The relatively larger size of the A genome (GEEVER et al. 1989), despite low copy sequence complexity similar to that of the D genome (Table 1; also see GEEVER et al. 1989), suggests that the repetitive fraction may provide a means of characterizing the genomic identity of individual YACs from tetraploid cottons (X. ZHAO, R. WING and A. H. PATERSON, manuscript submitted for publication). Such an approach may prove generally applicable to mapbased cloning in other major crops, many of

which are disomic polyploids (e.g., soybean, wheat, oat, canola, tobacco, peanut and many others).

A detailed map of DNA markers, in conjunction with well developed cytogenetic tools, offers the opportunity to utilize the polymorphic genus Gosy@um and its diverse relatives such as Hibiscus (kenaf, roselle) and Aklnwschus (okra), to investigate plant chromosome evolution in ex- quisite detail, and to have a major impact on improvement of one of the world’s oldest and most important crops.

We appreciate a cDNA library from N. TROLINDER, genomic probes from I. KWON, J. PRICE, T. MCKNIGHT, V. RASTOGI, and X. Zmo, technical

SARMIENTO, R. VAYLAY, and C. VASQUEZ-ROSADO, and helpful com- ments from D. ALTMAN, A. H. D. BROWN, C. CRANE, R EL-ZIK, J. GANNAWAY, R. KOHEL, T. MCKNIGHT, E. PERCIVAL, J. PRICE, D. RAY, W. SMITH, P. THAXTON, N. TROLINDER, T. WIWNS, R. WING, and three anonymous reviewers. Aspects of this work were supported by the U.S. Department of Agriculture (91-37300-6570 (to A.H.P.), and 92-37300-7655 (to D.M.S.), Texas Agricultural Experiment Sta- tion (to A.H.P. and D.M.S.), Texas Higher Education Coordinating Board (999902-148 to A.H.P.), and National Science Foundation (8918041 to J.F.W.). Subsets of the mapped DNA probes are available for academic research from A.H.P. by written request.

support from L. CHITTENDEN, D. CZESCHIN, J. GARZA, D. RASKA, D.

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Communicating editor: A. H. D. BROWN

Documents

RFLP Map of Cotton, Gossypium hirsutum X Chromosome Organization