COMMENTARY. For the article ‘‘Confusing cortical columns,’’ byPasko Rakic, which appeared in issue 34, August 26, 2008, ofProc Natl Acad Sci USA (105:12099–12100; first publishedAugust 20, 2008; 10.1073�pnas.0807271105), the authors notethat, due to printer’s errors, three references were omitted anda phrase appeared incorrectly. On page 12099, left column,second paragraph, line 11, ‘‘(e.g., ref. 2)’’ should appear as ‘‘(e.g.,refs. 2, 22, and 23).’’ Also on page 12099, right column, line 6,‘‘single receptive field’’ should instead read: ‘‘single modalities inthe receptive field.’’ Finally, on page 12100, center column, line21, ‘‘(15, 19)’’ should appear as ‘‘(15, 24)’’; and in line 25,‘‘(19–21)’’ should appear as ‘‘(21–24).’’ In addition to theseerrors, the authors note that in ref. 3, the author names‘‘Herculano-Housel S, Collins CE, Wang P, Kaas J’’ should haveappeared as ‘‘Herculano-Houzel S, Collins CE, Wong P, KaasJH, Lent R.’’ The corrected and omitted references appearbelow.

3. Herculano-Houzel S, Collins CE, Wong P, Kaas JH, Lent R (2008) The basic nonuniformityof the cerebral cortex. Proc Natl Acad Sci USA 105:12593–12598.

22. Purves D, Riddle DR, LaMantia AS (1992) Iterated patterns of brain circuitry (or how thecortex gets its spots). Trends Neurosci 15:362–368.

23. Horton JC, Adams DL (2005) The cortical column: A structure without a function. PhilosTrans R Soc London Ser B 360:837–862.

24. Rakic P (1995) A small step for the cell, a giant leap for mankind: A hypothesis ofneocortical expansion during evolution. Trends Neurosci 18:383–388.

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CELL BIOLOGY. For the article ‘‘Dual-color superresolution imag-ing of genetically expressed probes within individual adhesioncomplexes,’’ by Hari Shroff, Catherine G. Galbraith, James A.Galbraith, Helen White, Jennifer Gillette, Scott Olenych, Mi-chael W. Davidson, and Eric Betzig, which appeared in issue 51,December 18, 2007, of Proc Natl Acad Sci USA (104:20308–20313; first published December 12, 2007; 10.1073�pnas.0710517105), the authors note that on page 20312, rightcolumn, in Materials and Methods, under Sample Preparation,line 4, ‘‘Cell Line Nucleofector Kit SF’’ should have appeared as‘‘Cell Line Nucleofector Kit SE.’’

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GENETICS. For the article ‘‘Fine structure mapping of a gene-richregion of wheat carrying Ph1, a suppressor of crossing overbetween homoeologous chromosomes,’’ by Gaganpreet K.Sidhu, Sachin Rustgi, Mustafa N. Shafqat, Diter von Wettstein,and Kulvinder S. Gill, which appeared in issue 15, April 15, 2008,of Proc Natl Acad Sci USA (105:5815–5820; first published April8, 2008; 10.1073�pnas.0800931105), the authors note that ‘‘a partof our Fig. 1 was reproduced from figure 1 of the paper by SimonGriffiths et al. [Griffiths S, et al. (2006) Nature 439:749–752]. Inour paper we neglected to reference this figure as adapted fromthe original work.’’

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Fine structure mapping of a gene-rich region ofwheat carrying Ph1, a suppressor of crossing overbetween homoeologous chromosomesGaganpreet K. Sidhu*, Sachin Rustgi*, Mustafa N. Shafqat*†, Diter von Wettstein*‡§¶, and Kulvinder S. Gill*¶

*Department of Crop and Soil Sciences and ‡School of Molecular Biosciences, Washington State University, Pullman, WA 99164; †Department ofEnvironmental Sciences, Commission on Science and Technology for Sustainable Development in the South Institute of Information Technology,Abbottabad, Pakistan; and §Institute of Phytopathology and Applied Zoology, Justus-Liebig-Universitat, D-35392 Giessen, Germany

Contributed by Diter von Wettstein, February 8, 2008 (sent for review December 5, 2007)

The wheat gene-rich region (GRR) 5L0.5 contains many importantgenes, including Ph1, the principal regulator of chromosome pair-ing. Comparative marker analysis identified 32 genes for the GRRcontrolling important agronomic traits. Detailed characterizationof this region was accomplished by first physically localizing 213wheat group 5L-specific markers, using group 5 nulli-tetrasomics,three Ph1 gene deletion/insertion mutants, and nine terminaldeletion lines with their breakpoints around the 5L0.5 region. ThePh1 gene was localized to a much smaller region within the GRR(Ph1 gene region). Of the 61 markers that mapped in the foursubregions of the GRR, 9 mapped in the Ph1 gene region. Highstringency sequence comparison (e < 1 �10�25) of 157 group5L-specific wheat ESTs identified orthologs for 80% sequences inrice and 71% in Arabidopsis. Rice orthologs were present on all ricechromosomes, although most (34%) were on rice chromosome 9(R9). No single collinear region was identified in Arabidopsis evenfor a smaller region, such as the Ph1 gene region. Seven of the ninePh1 gene region markers mapped within a 450-kb region on R9with the same gene order. Detailed domain/motif analysis of the91 putative genes present in the 450-kb region identified 26candidates for the Ph1 gene, including genes involved in chromatinreorganization, microtubule attachment, acetyltransferases, methyl-transferases, DNA binding, and meiosis/anther specific proteins. Fiveof these genes shared common domains/motifs with the meiosisspecific genes Zip1, Scp1, Cor1, RAD50, RAD51, and RAD57. Wheatand Arabidopsis homologs for these rice genes were identified.

Triticum aestivum � rice and wheat BAC contigs

Bread wheat (Triticum aestivum L., 2n � 6x � 42) possesses16 billion base pairs per haploid genome, of which only 1–5%

are expected to represent genes (1). Physical mapping of 3,025gene loci on 334 single-break deletion lines identified 18 majorand 30 minor gene-rich regions (GRRs), accounting for 85% ofknown genes and encompassing only �29% of the genome (2).One of the major GRRs is present around fraction length 0.5 ofthe long arm of group 5 chromosomes (5L0.5 region) and is bestlocalized on chromosome 5BL (Fig. 1), where it is bracketed bythe breakpoints of deletions 5BL-1 (FL0.55) and 5BL-11(FL0.59) (2–4). The region physically spans �2.6% of chromo-some 5B that translates to �21 Mb of DNA (5). Comparisons ofthe physical map with consensus genetic linkage map revealedthat �20% of chromosome 5B recombination occurs in thisregion.

In addition to agronomically important genes, the 5L0.5region contains Ph1, a principal suppressor of crossing overbetween homoeologous chromosomes (6). Although breadwheat contains three genomes with considerable homology, itbehaves as a normal diploid with disomic inheritance: 21bivalents are present at diakinesis-metaphase I of meiosis, and,in euhaploids, chiasma formation is virtually absent (7, 8).Chromosome pairing leading to exclusive bivalent formationstarts with multivalent formation with shifts of pairing partners

between homoeologous chromosomes at zygotene (9–11), asillustrated by reconstruction of a hexavalent at late zygotene inFig. 2. These multivalents are corrected into bivalents throughdissolution and reassembly of the synaptonemal complex—nolonger requiring DNA homology resulting in 21 bivalents atpachytene. This correction mechanism is in agreement with thatobserved in other polyploids and translocation heterozygotes(12). A review of information on meiosis in wheat is provided assupporting information (SI) Text.

By telocentric analysis, the Ph1 gene in hexaploid wheat wasgenetically mapped 1–5 cM from the centromere on 5BL (13, 14).In tetraploid wheat, the gene was physically mapped between theC-bands 5BL1.5 and 5BL2.1 (5, 15). Two mutants ph1b (5, 13, 14)and ph1c (16, 17) were generated for the Ph1 gene in hexaploid andtetraploid wheat, respectively. Both mutants are a result of inter-stitial deletions of �1.05 �m and 0.84 �m, respectively (3, 4, 14).Another mutant line, dup.Ph1, carries four doses of Ph1 gene. It isspeculated that the duplication in this line corresponds to the regiondeleted in ph1c. Fine mapping has localized the Ph1 gene to asubregion within the 5L0.5 region (3, 5). The breakpoints ofterminal deletion 5BL-1 and the distal breakpoints of the ph1b andph1c mutants, all map between C-bands 5BL1.7 and 5BL2.1 (18).The Ph1 gene maps between the breakpoints of 5BL-1 and ph1c,which is referred to as the Ph1 gene region (Fig. 1). A DNA markerwas identified for the Ph1 gene region by screening 602 randommarkers present on the Aegilops tauschii map (3, 19). Threeadditional markers were identified for the region by RNA finger-printing-deletion mapping analysis of the ph1b and its wild type,Chinese Spring (3, 20).

Based on comparisons of genetic linkage maps, wheat and ricegenomes were reported to be significantly collinear, but analysesat micro level show many exceptions (21). Comparison of 4,485physically mapped wheat ESTs confirmed that wheat-rice col-linearity is frequently interrupted by segments from other chro-mosomes (22, 23). The objective of this study was to identify Ph1gene candidates in rice and Arabidopsis by first mapping wheatgenes to the Ph1 gene-containing region and then identifying theorthologous Ph1 gene region in rice and Arabidopsis. Addition-ally, we wanted to line-up our maps with those published inref. 24.

ResultsMarker Enrichment of the 5L0.5 Region. A set of 213 DNA markersfor the 5L0.5 region was identified by comparative analysis of 36

Author contributions: G.K.S., S.R., D.v.W., and K.S.G. designed research; G.K.S., S.R., andM.N.S. performed research; G.K.S., S.R., D.v.W., and K.S.G. analyzed data; and G.K.S., S.R.,D.v.W., and K.S.G. wrote the paper.

The authors declare no conflict of interest.

¶To whom correspondence may be addressed. E-mail: diter@wsu.edu or ksgill@wsu.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0800931105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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Poaceae maps and by screening of physically mapped wheatESTs as follows. Initially nulli-tetrasomics, ditelocentrics andsingle-break deletion lines for homoeologous group 5 chromo-somes identified the location of DNA restriction fragmentscomprising 90 RFLPs from genomic DNA and cDNA clones, sixSSRs, and 10 markers isolated by RNA fingerprinting deletionmapping (20). Additional markers and useful genes in the targetregion were retrieved with two successive rounds of selectingmarkers flanked by two previously identified anchors in thePoaceae maps. The EST probes for physical mapping wereobtained from http://wheat.pw.usda.gov/wEST/binmaps. Map-ping was performed on relevant deletion lines with gel-blot DNAanalyses, using three restriction enzymes (3).

Location of 238 5BL specific loci (representing 207 DNAmarkers) is shown in Fig. 1 A. The 5L0.5 region contained 113 ofthese loci. For 68 marker loci, the corresponding 5B-band wasmissing in both 5BL-1 and 5BL-11 deletions and are thereforelocalized distal to the breakpoint of 5BL-11. Similarly, 57 markerloci are localized proximal to the 5L0.5 region, because thecorresponding 5B-bands were present in both 5BL-1 and 5BL-11deletions. The probes were derived from A. tauschii genomicclones or from barley/wheat genomic or cDNA clones. A list ofthe genes and symbols is presented in Table S1, Table S2, andDataset S1. Many probes detected more than one locus. Theseloci are designated with a letter (a, b, or c) at the end of thesymbol. Some of the second or third loci mapped within the

Fig. 1. Physical map of 238 marker loci on the long arm of chromosome 5B (5BL) covering region 5L0.5 spanning proximal deletion breakpoint PB,dup.Ph1 anddistal deletion breakpoints DB,dup.Ph1 and 5BL-11. (A) Major C-bands, dark blue; minor C-bands, light blue. The markers are color-coded according tohomologues identified in rice and Arabidopsis. (B) Mapped wheat markers color coded for homology in 12 rice and 5 Arabidopsis chromosomes. (C) BAC contigof R9 with location of markers from the physical map. (D) Location of marker genes in BAC clones mapped on wheat chromosome 5B between breakpoints 5BL-1and ph1c (24).

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5L0.5 region; others were proximal or distal from this region.The fragment band for all of the Ph1 gene region markers waspresent in double intensity in dup.Ph1, suggesting that the Ph1gene region is duplicated in this line. Eighty-three markersmapped in the 5BL region, which is duplicated in the dup.Ph1line, because the intensity of the 5BL specific bands for thesemarkers was approximately twice that in the normal CS line. Thismarker data accurately demarcated the duplicated region in thedup.Ph1 line (Fig. 1 A) and showed that, contrary to previousbelief, the duplicated region is not the same as that deleted in theph1c mutant.

Synteny of Wheat Markers to the 12 Rice (R) and 5 Arabidopsis (A)Chromosomes. Translated nucleotide sequences of 157 wheatchromosome 5BL-specific ESTs or genes were compared withrice nucleotide sequences. Among orthologs for 126 (80%) of thesequences, 43 (34%) mapped on R9 and 82 on the other 11 ricechromosomes, with a range of 19 orthologs on R3 to two eachon R6 and R10 (Fig. 1B, Table S3, and Dataset S1). Significantdifferences were observed among various wheat regions both forthe proportion of sequences detecting rice orthologs and forsynteny conservation. Sequences from the proximal regionbracketed by the centromere and the proximal breakpoint ofph1b mutant (C-PB, ph1b) detected 30 rice orthologs, and thedistal region (5BL-11 to telomere) detected 41. Orthologs for theproximal region (C-PB,dup.Ph1) were present on eight differentrice chromosomes, with the highest number of 12 on R9,compared with the distal region, in which 38 orthologs weredistributed over nine rice chromosomes, with R3 carrying thehighest number (18).

The C-PB, ph1b region has maximum homology with R9 (12sequences) and R1 (6 sequences). The region Ph1b-5BL-1(0.55)is syntenic to R9, R1, R8 and R12. We confirm the synteny ofthe 5L0.5 region to R9 but also identify syntenic sequences onseven other chromosomes. The region from 5BL-11-telomere ishighly similar to R3, but significant orthologous sequences arealso detected on R1, R9, and R5. Seven of the nine Ph1 generegion markers have orthologous sequences on R9 and one onR7 (Fig. S1 and Table S3). Five of the eight markers for thePh1c-ph1b region, which is another very small region present just

distal to the Ph1 gene region (Fig. 1), have five orthologsequences on R7 and three on R9. Seven of the Ph1 gene regionorthologs were present within a 450-kb region present betweenbasepairs 18162398 and 18615877 on R9 (Fig. S1). The geneticorder of these markers was the same between wheat and rice(Figs. 1 and 3). The three R9 orthologs for the Ph1c-ph1b regionare located adjacent to the 450-kb region. The other fiveorthologs for the Ph1c-ph1b region identified an �2.7-Mb R7region that also contained one orthologue from the Ph1 generegion (Fig. 1 and Fig. S1). The wheat locus of cdo412 maps inthe region proximal to the Ph1 gene region but has a duplicatedcopy in the Ph1 gene region. The orthologous rice locus is locatedproximal to the 450-kb region and is present �1.2-Mb away fromthe 450-kb region. If the cdo412 orthologue is included, the totalrice Ph1 region is �1.6 Mb containing 337 genes. Because themajor locus (based on the hybridization intensity) for cdo412 inwheat maps in the proximal region, the rice region containing thissequence was not included in the rice Ph1 region. To further test theextent of rice-wheat homology for the Ph1 gene region, wheat ESThomologs for the rice 1.6 Mb region sequence were identified fromthe Gramene database. Deletion line-based physical mapping in-formation for 21 of the wheat genes was available, and 19 genesmapped between the deletion breakpoints of 5BL-1 and 5BL-11,which corresponds to the 5L0.5 region containing the Ph1 generegion (Fig. 1). These results further validate homology of the1.6-Mb rice region with the Ph1 gene region of wheat.

Of the 157 mapped wheat sequences, 112 identified ortholo-gous sequences on various Arabidopsis chromosomes (Fig. 1B

Fig. 2. Reconstruction of synaptonemal complexes from electron micro-graphs of serial sections of a hexavalent at late zygotene from microsporemeiocyte of wheat cv. Chinese Spring. S, short arm; L, long arm; bar, attach-ment to the nuclear envelope; red dots, recombination nodules [modifiedfrom Hobolth (9)].

Fig. 3. Genetic map of wheat chromosome 5BL spanning abc706 andmwg914. (A) Wheat genes, which have been placed on the map by theirphysical map position, and their orthologous sequence in the rice chromo-some are indicated by dashed lines. An asterisk indicates that the map positionis not verified. (B) Deduced protein functions of wheat orthologues in the450-kb region of the rice genome sequence of chromosome 9. Blue dotsdesignate location of mapped wheat genes; red dots indicate rice genes.Proteins shown in blue letters are high-priority candidates for the Ph1 protein.(C) Wheat EST homologues identified for rice genes.

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and Table S3). Arabidopsis chromosome A1 had 36 orthologsfollowed by 35 for A5, 19 for A3, 13 for A4, and 9 for A2 (Fig.1B). Comparison of the rice Ph1 gene region with that ofArabidopsis showed orthologs of A1 to be present on R1, R3, andR7 and orthologs of A5 to be present on R7 and R9 (Table S4).The greatest similarity between these two model species wasfound for R9 that has 18 wheat orthologs common with A5region. Another significant homology was between regions of R3and A1.

Translated DNA sequence comparison of the rice region or-thologous to the Ph1 gene region did not identify any significantsynteny with Arabodopsis. Orthologs to the rice sequences werepresent on all five Arabidopsis chromosomes, with the highestnumber of 10 present on A5 (4 on 5S and 6 on 5L) followed by 8for A3 (3 on 3S and 5 on 3L). Three were present on A2 and twoeach were present on A1 and A4. The orthologs were scattered onthe chromosomes interspersed by large chromosomal segments.The only significant homology was for a 222-kb region of A5 thatcontain four genes, At5g55730, At5g55820, At5g56220, andAt5g56270, which are orthologs of genes encoding rice methyl-transferase, bzip transcription factor, a meiosis specific protein, anda WRKY DNA binding protein, respectively.

Genetic Map of the Ph1 Gene Region and Its Alignment with the BACScaffolds of Rice Chromosome 9 and Wheat Chromosome 5B. The Ph1gene is present in the wheat region flanked by 5BL-1 and thedistal breakpoint of ph1c mutant. Now we have a total of ninemarkers for the region. A linkage map of this region flanked bymarkers abc706 and mwg914 is presented in Fig. 3A, and acomparison of the wheat Ph1 gene region between basepairs18162398 and 18748622 of R9 is shown in Fig. 3B. Blue dotsidentify wheat genes, and red dots annotated rice genes. Ad-dresses for wheat EST homologs of rice genes are shown in Fig.3C. This wheat-rice comparison allowed us to align the Ph1 generegion to the BAC scaffold of rice R9 and in turn to the BACscaffold of wheat chromosome 5B (Fig. 1 C and D) as publishedby Moore and coworkers (24).

It has been suggested that 5BL contains a subtelomericinsertion from wheat chromosome 3A, comprising two BACclones with the genes stk1and vmp1 and the genes hyp3 and bna(24). Because we did not find these genes in our map, wedesigned primers, using Primer 3 (http://frodo.wi.mit.edu), forthese four genes and for the cdc2–4 gene (Table S5), usinggenomic DNA sequences [supplemental table 2 in Griffiths etal.(24)]. Specificity of the primers to the target gene was ensuredby comparing the primer sequences against wheat genomic DNAand EST sequence database. Chromosomal localization of these

genes was revealed by amplifying cDNA and genomic DNA ofwheat nulli-tetrasomic lines along with cultivar Chinese Spring ascontrol. All primers amplified a fragment of expected size alongwith a few other fragments ranging from 2 to 10 fragments withgenomic DNA and 2 to 5 fragments with cDNA. None of theexpected sized fragments could be localized to chromosome 5Bfor any of these genes. However, fragments with unexpected sizeamplified from stk1 (size �400 bp) and vmp1 (size �120 bp)localized to 5B (Table S6). Surprisingly, a fragment of expectedsize (�215 bp) amplified with the hyp3 primers localized tochromosome 3A (Fig. S2 A and Table S6). An �150-bp fragmentamplified with the cdc2-4 primers that we designed localized tothe long arm of chromosome 2D (Fig. S2B and Table S6). Theexpected sized band amplified by the primers kindly provided byG. Moore (John Innes Centre, Colney, Norwich, U.K.), however,localized to chromosome 5B (Fig. S2C and Table S6). The reasonfor this discrepancy will have to be elucidated, but the results donot support the subtelomeric insertion from wheat chromosome3A that was suggested in ref. 24.

Among the genes encoding proteins involved in meioticprocesses the DNA strand exchange enzyme DMC1 and HOP1/Asy1 have been assigned to chromosome 5B. Primers for thesegenes amplified 5–10 fragments from wheat genomic DNA, andthe expected sized bands mapped to 5B when tested on a set of21 nulli-tetrasomic lines (Fig. S2D and Table S6).

Domain–Motif Analysis of Meiotic Genes in the Highly SyntenousRegion of Rice Chromosome 9. To investigate whether homologs ofgenes required in other organisms for meiosis are located in therice 450-kb region of R9, the protein sequence of such genes wascompared against the rice genome in the Gramene database.Eleven potential rice meiosis genes present in this chromosomesegment were searched for conserved domains/motifs in theNational Center for Biotechnology Information conserved do-main database. The results are summarized in Table 1. Ofproteins encoded by the listed genes, Cor1 of hamster is ahelix–loop–helix (HLH) domain containing protein of the axialelement of the synaptonemal complex (25–27). Scp1 from mam-mals and Zip1 from yeast with a primary structure of 875–1,000aa, and a coil–coil domain forms the transverse filaments thatsynaps the axial elements into the synaptonemal complex. Nullmutants assemble only axial elements. RAD50 is a part of thetopoisomerase, and the Spo11 complex is required for theinduction and processing of double-strand breaks. Mutations inthis protein prevent the exonucleolytic 5�-3� digestion to yieldsingle stranded tails for invasion into a homologous DNA doublestrand. RAD55 and RAD51 encode DNA strand exchange

Table 1. Domain/motif analysis of 91 putative genes present in the 450-kb rice chromosome 9region syntenous with the wheat Ph1 gene region

Gene/domain Smc HLH RAD55 MAD Scp1 ERM SbcC Sms HOOK HHT1

RAD50 † — — ‡ — — † — — —Cor1 — † — — — — — — — —Scp1 § — — — † § — — — —Zip1 ‡ — — ‡ — — ‡ — — —RAD55 — — † — — — — § — —RAD51 — — ‡ — — — — § — —Os29970* ¶ — — — — — — — ¶ —Os29930* — † — — — — — — — —Os30380* — — ‡ — — — — § — —Os30310* — — — — — — — — — †Os31300* — † — — — — — — — —

Percent similarity to protein domains in meiosis specific genes is shown.*Rice Ph1 gene candidates: Os29970 represent Alignment: †, 75–100%; ‡, 50–75%; §, 25–50%; ¶, �25%; and �,no similarity.

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enzymes with homology to bacterial RecA enzymes. Among therice candidate proteins, LOC�Os09g30380 is an ATPase associ-ated with various cellular activities (AAA). Such proteins formhexameric ring structures involved in folding polypeptide chainsor unfolding coagulated proteins. LOC�Os09g30310 has a his-tones H3-and H4-like structure. The Hook motif in another ricecandidate protein might mediate attachment to microtubules. Ithas yet to be clarified whether any of the proteins with thesedomains are involved in the mentioned activities. All of thesegenes are our prime candidates for the Ph1 gene.

Discussion5L0.5 and the Ph1 Gene Region of Chromosome 5BL. The extensivephysical characterization of the 5L0.5 region of chromosome 5BLwas obtained by mapping 238 marker loci relative to eight deletionbreakpoints and two C-bands (Fig. 1). Two of these breakpoints anda C-band subdivide the 5L0.5 region into four subregions, includingthe Ph1 gene region, which is the smallest of the four and containsthe Ph1 gene. Comparative analysis physically localized 32 genesuseful to the 5L0.5 region and added 77 new markers to the region.Only �52% of the markers identified mapped in the 5L0.5 region.It may partly be due to a liberal approach in the selection ofmarkers, which we used because elimination of false positives wasrelatively easy. Second, two restriction enzymes may not be enoughto resolve all homoeologous loci, as observed for 12 probes that didnot detect a missing fragment band on group 5 nulli-tetrasomiclines. Third, 19 loci mapped in the chromosomal regions just outsidethe breakpoints of deletions 5BL-1 and 5BL-11. The breakpoints ofthe two deletions may bracket only a part of the GRR. It has beenreported earlier that both ph1b and ph1c mutants are a result ofinterstitial deletions (13, 14, 16). Contrary to earlier suggestions, theexact localization of the breakpoints of these deletions showed thebreakpoints for the duplicated 5B region in dup.Ph1 to be differentfrom that of ph1c (Fig. 1).

Wheat–Rice and Wheat–Arabidopsis Comparisons. Comparison ofrice and wheat genomes, using 4,485 ESTs (3,358 single-copy and1,127 multicopy genes) have been mapped in wheat chromosomebins with ordered rice BAC/PAC clones (28). Of these unigenes,375 matched at least one rice sequence, and 81% of the riceBAC/PAC clones were matched by a wheat EST. Despite theobserved sequence conservation, this comparison also revealeda complex chromosomal gene mosaic between the two genomeswith tremendous discontinuities between gene orders resultingfrom the evolution of these two species. The present study hasprovided confirmation of the syntenic relationships for markersin chromosome 5BL identified in refs. 21 and 22, but it is moreextensive, because physical and the genetic map is known for thewheat sequences used for the comparison (Figs. 1 and 3, Fig. S1,and Tables S3 and S4). A high percentage (71%) of wheat genesidentified orthologs in Arabidopsis, but their distribution washighly scattered (Fig. 1B and Table S3). Orthologous sequencesfor all 26 rice Ph1 gene candidates were identified in Arabidopsis,but they were scattered over five chromosomes, and a collinearregion between wheat and Arabidopsis for chromosome 5BL orthe Ph1 gene region could not be identified.

Alignment of the Ph1 Gene Region to the BAC Scaffold of RiceChromosome 9 and Wheat Chromosome 5B. The detailed physicalmap of the long arm of chromosome 5B between the breakpoints5BL-1 and 5BL-11 allowed identification of mapped markers atthe corresponding BAC contig of rice R9 and in turn at the BACcontig of wheat between the distal breakpoint of ph1c and 5BL-1(Fig. 1). Among the nine markers that map together with the Ph1gene, marker bcd1949 was found on BAC clone 1567N6. Alter-natively cdk-like genes B1–7 on BAC clones F-102J1, F-563C24,2212, 67H7, and 1236H11 were considered as components of thePh1 gene complex (24, 29) (The cdk genes were previously

designated cdc genes). The amino acid sequences of these genesare homologous to the mammalian meiotic checkpoint Cdk2cyclin phosphokinase. This enzyme is involved in the initiationof DNA replication, but without Cdk action synaptonemalcomplex proteins fail to assemble on homologous chromosomepairs in mice resulting in desynapsis and nonhomologous syn-apsis (30). In wheat, the Cdk cluster in chromosome 5B has moremembers and is specifically transcribed in chromosome 5B,whereas mRNA formation from the fewer paralogous genes inthe A and D genomes was not detected. Deletion of the locusin 5B seems to activate transcription of the Cdc-like paraloguesin the A and D genomes. It will be of interest to sequence theBAC(s) containing the bcd1949 gene from chromosome 5B ofhexaploid wheat and determine its gene content followed bymolecular and functional characterization of the identifiedgenes. One of the genes (mic1) present next to bcd1949 is knownto show Cdk-like expression pattern. In general, the BAC contigsof the Ph1 gene region provides a highly valuable tool to studythe structure and function of the genes present in this region.

Perspective. Both increases and decreases in the copy number ofPh1 gene(s) containing regions increases homoeologous and non-homologous chromosome synapsis and leads to meiotic arrest ofsynapsis and crossing over. A synopsis of ultrastructural analyses ofchanges in the number of Ph1 gene regions is provided in SI Text.From these investigations, it is likely that the Ph1 gene encodes aprotein or is part of a protein complex that is required for correctionof multivalents, resolution of interlockings, and the processes oflimiting crossovers to a few per chromosome or chromosome arm.The dosage effect of the number of the genes by increasing thenumber of 5BL chromosome arms causes increase of meiotic arrestand might involve gene silencing.

Understanding the phenomena displayed by the Ph1 gene re-quires the answer to several fundamental questions high-lighted bythe following observations. A knockout mutant of the double-strand breaking transesterase SPO11 of the ascomycete Sordariamacrospora was constructed by changing the active site tyrosine aswas a transformant expressing green fluorescent protein (GFP)tagged enzyme (31). The enzyme appears on the chromosomesduring karyogamy of the two haploid nuclei and is present asnumerous foci on the leptotene chromosomes during presynapticalignment and during synapsis. Then, the number of foci decreases,and, at mid to late pachytene, the Spo11-GFP tagged protein isspread over the entire nucleus. Antibodies against the DNA strandexchange enzyme Rad51 mark 60–50 foci from late leptotene toearly zygotene, considered as markers for double-strand breaks.Only some of these are processed into crossing-overs seen as �21chiasmata at diplotene. This, then, is reminiscent of the transientappearance and random location of recombination nodules (RNs)at zygotene in Bombyx males and human meiocytes and theirreduction, relocation, and conversion into chiasmata frompachytene to diplotene (12). The Sordaria spo11 knockout deletiondisplays only a few Rad51 foci and an �500-fold reduction in thenumber of chiasmata and a reduction in crossing over from 44% to2%. The lateral elements of the synaptonemal complexes areformed normally, but presynaptic coalignment, so characteristic forSordaria homologs, and synaptonemal complex formation areabsent. Most remarkably, exposure of young fruiting bodies of theknockout mutant to 200 Gy of �-rays led to appearance of manyRad51 foci at leptotene, restoration of homologous synapsis, andformation of chiasmata. Thus, �-ray-induced double-strand breakscan substitute for the double-strand breaks formed by the transes-terification reaction of the Spo11 topoisomerase. It is likely thathomologous pairing and synaptonemal complex formation inpolyploid plants also is initiated at leptotene and zygotene bytopoisomerase formed double-strand breaks but that these organ-isms have evolved mechanisms to prevent the consequences ofmultivalent formation by untimely resolutions into reciprocal cross-

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ing-overs. This is clearly demonstrated by the reorganization ofmultivalent associations with the synaptonemal complex into opti-mal bivalent formations at pachytene. The reorganization involvesdegradation and reformation of synaptonemal complexes, elimina-tion and reformation of RNs and their placement in specificchromosome segments. With the cloned genes available, it will nowbe possible to determine whether the repair processes like thatcarried out by the Ph1 involves reversal of the transesterificationreaction, if a suitable homologous DNA invasion partner forrecombination is not found for covalent linkage of the 5� termini tothe Spo11 topoisomerase (32). It is also possible that the suppres-sors operate by favouring cleavage between the two Hollidayjunctions leading to a noncrossover (gene conversion).

The increase and decrease of the number of Ph1 genes causecorrelated degrees of arrest of synapsis and crossing-over but noeffect on chromosome compaction and progress of later stagesof meiosis. This is reminiscent of meiotic arrest triggered bysynapsis checkpoints as identified in yeast and mammals (30, 33).In yeast, an allele of Zip1 was constructed in which four leucineresidues in the central coiled coil domain were replaced byalanines (34). Morphologically normal synaptonemal complexesassemble, but crossing over is delayed and decreased. Themutant elicits strong Pch2 check-point induced arrest in mid-meiotic prophase. Pch2 is a cyclin-dependent cell cycle control-ling phosphokinase. If the Zip1 mutant is combined with thePch2 checkpoint mutant, the crossing over is restored to wild-type levels, indicating that the defects in the genetically engi-neered zip1 mutant is due to the trigger of the crossing overdefect-sensing wild-type Pch2 protein.

Conclusion. The availability of the extended physical map of the Ph1gene region together with the corresponding wheat chromosome5B BAC contig provides excellent facilities to device experimentsfor elucidation of the mechanisms that suppress crossing over

between homoeologous chromosomes in wheat and the function ofmany other interesting genes of this gene-rich region.

Materials and MethodsGenetic and Aneuploid Stocks. Wheat homoeologous group 5 nullisomic-tetrasomic lines (35) were used to identify the chromosomal location of DNArestriction fragments. These; the Ph1 gene mutant lines (ph1b, ph1c, anddup.Ph1); and the single-break deletion lines 5A-3 (FL-0.56), 5A1–4 (FL-0.55),5AL-10 (FL-0.57), 5AL-12 (FL-0.35), 5BL-5 (FL-0.54), 5BL-1 (FL-0.55), 5BL-11(FL-0.59), and 5DL-11 (FL-0.52) (4) were obtained from the Wheat Genetic andGenomic Resources Center, Manhattan, KS.

DNA Sequence Analysis and Motif Search. For DNA sequence analysis and motifsearch, the nucleotide sequences of the wheat genes mapped to chromosome5BL were used as queries in tBLASTx (translated query vs. protein database)(36) searches against the GenBank nonredundant database (nr) to search forthe orthologous sequences in rice and Arabidopsis that exhibit significantsimilarity based on the alignment and e value (i.e., e value significance � l �10�25). The precise chromosomal location of these orthologous sequences wasidentified by screening Gramene database (www.gramene.org) for rice andthe TAIR database for Arabidopsis (www.arabidopsis.org). The domains andmotifs of the genes present in the orthologous Ph1 gene region on R9 wereanalyzed by the CD searches (37) on the National Center for BiotechnologyInformation database (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Theprotein sequences of the rice Ph1 candidates were used as queries in tBLASTnsearches against GenBank wheat EST database to identify ESTs exhibitingsignificant homology (based on the alignment and e values). The putativefunctions to the representative wheat ESTs were assigned by using the nucle-otide sequence of the ESTs in BLASTn (36) searches against GenBank pdb andBLASTx (36) searches against GenBank nr and SwissProt databases. Putativefunctions were assigned after analyzing all of the results.

ACKNOWLEDGMENTS. This work was supported by the United States Depart-ment of Agriculture National Research Initiative, the Vogel and the NilanEndowments, and the Washington Wheat Commission. We thank the WheatGenetic and Genomic Resources Center for providing seeds for various ane-uploid stocks. This is scientific paper CSS0202-08 from The College of Agricul-tural, Human, and Natural Resources Sciences.

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