Silencing of a metaphase I-specific gene resultsin a phenotype similar to that of the Pairinghomeologous 1 (Ph1) gene mutationsRamanjot Bhullara, Ragupathi Nagarajana, Harvinder Bennypaula,b, Gaganpreet K. Sidhua,c, Gaganjot Sidhua,Sachin Rustgia, Diter von Wettsteina,d,e,1, and Kulvinder S. Gilla,1

aDepartment of Crop and Soil Sciences, dSchool of Molecular Biosciences, and eCenter for Reproductive Biology, Washington State University, Pullman, WA99164; bCanadian Food Inspection Agency, Charlottetown, PE, Canada C1A 5T1; and cInstitute of Cancer Genetics, Columbia University, New York, NY 10032

Contributed by Diter von Wettstein, August 26, 2014 (sent for review May 30, 2014)

Although studied extensively since 1958, the molecular mode ofaction of the Pairing homeologous 1 (Ph1) gene is still unknown. Inpolyploid wheat, the diploid-like chromosome pairing is principallycontrolled by the Ph1 gene via preventing homeologous chromo-some pairing (HECP). Here, we report a candidate Ph1 gene (C-Ph1)present in the Ph1 locus, transient as well as stable silencing ofwhich resulted in a phenotype characteristic of the Ph1 genemutants, including HECP, multivalent formation, and disruptedchromosome alignment on the metaphase I (MI) plate. Despite ahighly conserved DNA sequence, the C-Ph1 gene homeologuesshowed a dramatically different structure and expression pattern,with only the 5B copy showing MI-specific expression, further sup-porting our claim for the Ph1 gene. In agreement with the previousreports about the Ph1 gene, the predicted protein of the 5A copy ofthe C-Ph1 gene is truncated, and thus perhaps less effective. The 5Dcopy is expressed around the onset ofmeiosis; thus, it may functionduring the earlier stages of chromosome pairing. Along with alter-nate splicing, the predicted protein of the 5B copy is different fromthe protein of the other two copies because of an insertion. Thesestructural and expression differences among the homeologues con-curred with the previous observations about Ph1 gene function.Stable RNAi silencing of the wheat gene in Arabidopsis showedmultivalents and centromere clustering during meiosis I.

neofunctionalization | VIGS | recombination | orthologs |centromere–microtubule interaction

The Pairing homeologous 1 (Ph1) gene was discovered in 1958based on the observation that plants lacking wheat chromo-

some 5B exhibit homeologous pairing (1, 2). Lack of the generesults in multivalents during metaphase I (MI) of meiosis, re-sulting in partial sterility. Conversely, six doses of the gene in thetriisosomic line of chromosome 5BL resulted in interlocking of thebivalents and reduced chiasmata frequency even among homologs,along with rare multivalents (3). Several other genes promotingor suppressing homeologous chromosome pairing (HECP) havealso been reported (4, 5), although their effect is difficult tomeasure in the presence of the Ph1 gene (6). Ph1-like genes werealso reported in other sexually propagating polyploids, includingAvena sativa, Festuca arundinacea, Brassica napus, Gossypiumhirsutum, andGossypium barbadense, as well as in some diploids,including Lolium perenne, Lolium multiflorum, and Loliumrigidum (7–11).Ph1 gene mutants in tetraploid (ph1c) (12, 13) and hexaploid

(ph1b) (14) wheat were shown to be interstitial deletions involvingan ∼0.84-μm region and an ∼1.05-μm region around the gene,respectively (15, 16) (SI Appendix, Fig. S1). Physical mapping lo-calized the gene to an ∼2.5-Mb chromosomal region referred to as“Ph1 gene region,” bracketed by the distal breakpoint of ph1c de-letion on the distal end and the breakpoint of deletion line 5BL-1on the proximal end (16) (SI Appendix, Fig. S1). Various markerenrichment efforts identified nine markers for the region (17).Detailedmicrosynteny analyses and comparativemapping identified

a 450-kb region of rice chromosome 9 (17). The corresponding riceregion contained 91 genes. The major objective of the present studyis to identify the gene(s) responsible for the Ph1 gene-like functionusing the available mapping information.

ResultsIdentification of the Candidate Gene. The following criteria wereused to select the potential Ph1 gene candidates from the 91 genespresent in the 450-kb rice region: (i) genes expressed duringmeiosis and (ii) genes involved in chromatin reorganization,microtubule attachment, DNA binding, as well as acetyl- andmethyltransferase activity. This detailed bioinformatics anal-ysis identified 26 genes for further characterization. Virus-induced gene silencing (VIGS) was optimized for wheat meiosisusing the disrupted meiotic cDNA1 (DMC1) gene, lack of whichresults in mostly univalents at MI. VIGS of TaDMC1 (Triticumaestivum DMC1) with an antisense construct resulted in an av-erage of 37.2 univalents and 2.4 bivalents (18).Except for about 4–6% of the MI cells that usually show ab-

errant chromosome pairing including multivalents, 21 bivalentswere observed in the WT wheat cultivars (cv.) Chinese Spring(CS) and Bobwhite (BW) (SI Appendix, Tables S1 and S2). In theph1b mutant, about 60% of the cells showed the aberrantchromosome pairing, with an average of 1.29 multivalents and1.93 univalents per cell (SI Appendix, Table S1). The highernumber of univalents observed in the ph1b mutant may be due tothe combined effect of other genes present in the ∼1.05-μmchromosomal region deleted in the mutant line. At least two

Significance

Maintaining diploid-like pairing behavior is essential for a poly-ploid to establish as a new species. The Pairing homeologous 1(Ph1) gene, regulating such behavior in polyploid wheat, wasidentified in 1958, but its molecular function remained elusive.The present communication reports identification of the candi-date Ph1 (C-Ph1) gene that is expressed exclusively during mei-otic metaphase I, whose silencing resulted in formation ofmultivalents like the Ph1 gene mutations. Although the C-Ph1gene has three homoeologous copies, the 5B copy has divergedin sequence from the other two copies. Heterologous gene si-lencing of the Arabidopsis homologue of the C-Ph1 gene alsoconfirmed its function. Molecular characterization of this genewill make it possible to develop precise alien introgressionstrategies.

Author contributions: R.B., D.v.W., and K.S.G. designed research; R.B., R.N., G.K.S., andS.R. performed research; H.B. contributed new reagents/analytic tools; R.B., R.N., G.S., andS.R. analyzed data; and R.B. and K.S.G. wrote the paper.

The authors declare no conflict of interest.

Freely available online through the PNAS open access option.1To whom correspondence may be addressed. Email: diter@wsu.edu or ksgill@wsu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1416241111/-/DCSupplemental.

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genes (LOC_Os09g30310 and LOC_ Os09g31310) have beenidentified in the deleted part of ph1b, silencing of which resultedin two to eight univalents. About 8% of the ph1b cells showedbivalent interlocking. The ph1b (SI Appendix, Fig. S2) and otherPh1 mutant and deletion lines show relatively normal chromo-some alignment on the MI plate, and chromosome clumping isusually not observed.VIGS screening of the 26 candidates (17) identified a gene that

we designated as candidate Ph1 (C-Ph1; LOC_Os9g30320, wheatexpressed sequence tag (EST) homolog BE498862) (SI Appendix,Fig. S2), silencing of which showed chromosome pairing behaviorcharacteristic of the Ph1mutant and deletion lines. Compared withalmost all bivalents in 91% of the MI cells of the negative control[VIGS with pγ.MCS, carrying the sequence-matching multiplecloning site (MCS) of a plasmid], two of the five plants inoculatedwith the hairpin construct pγ.C-Ph1hp2 (Fig. 1 and SI Appendix,Table S3) showed multivalents and higher order pairing in 70.3%of the cells. In addition to multivalents, the MI chromosomesshowed severe clustering and disrupted alignment on the MIplate. In comparison, only about 9% of the MI cells of MCS-inoculated plants showed misalignment and multivalents (Fig. 1).Replicated VIGS experiments with the hairpin and antisense

constructs showed a similar phenotype in the silenced plants. Theconstruct pγ.C-Ph1hp1 showed the silencing phenotype in twoof the 20 plants compared with seven of 20 plants for the pγ.C-Ph1as construct. The two plants with the pγ.C-Ph1hp2 con-struct showed multivalents and chromosome clustering in 72.7%(plant VIGS-7; Fig. 1) and 68% (plant VIGS-5; Fig. 1) of thecells, respectively, with an average number of only 6.93 and 9.08bivalents, respectively. Only plant VIGS-7 showed bivalent in-terlocking in 5% of the cells, whereas no bivalent interlockingwas observed in any of the control plants. Measured by quanti-tative real-time PCR analysis, expression of the gene in plantVIGS-7 was only 21.83% of expression of the gene in controlplants compared with 54.56% in plant VIGS-5 (SI Appendix, Fig.S3). The expression of the gene in the remaining three plantsthat did not show any aberration in chromosome pairing rangedfrom 89–92% of the control plants (SI Appendix, Fig. S3).Stable RNAi silencing of the C-Ph1 gene was accomplished by

transforming wheat cultivar BW with the hairpin RNAi constructpHellsgate8 1-1 involving 200 bp of the gene segment. Seven ofthe 54 confirmed T0 (plants after regeneration) transgenic plants(Materials and Methods) were randomly selected for meioticchromosome pairing analysis. Compared with the negative con-trol (BW), reduction in the gene expression among these sevenplants ranged from 7–83% (SI Appendix, Fig. S4). Transcriptionalsuppression between 22% and 44% appears to be necessary toinduce HECP. Two of the seven plants (RNAi-7 and RNAi-3) thatexhibited 7% and 22% transcript suppression, respectively, showedrelatively normal chromosome pairing, with only 6.2% and 14.2%of the MI cells, respectively, showing aberrant chromosome pair-ing. RNAi-3 showed slight, although nonsignificant, multivalents,along with misalignment. Multivalents were not observed inRNAi-7, although slight misalignment was observed similar tothat of the negative control, which showed misalignment in only6% of the cells (Fig. 2). These two plants were fully fertile.RNAi-5 with a 44% reduction in gene expression was the most

interesting finding because it showed a chromosome pairing

phenotype similar to that seen in the ph1b mutant (Fig. 2). Mul-tivalents were observed in 22% of the cells (Fig. 2). The numberof bivalents in this plant was 12, compared with 16 in the ph1bmutant (SI Appendix, Tables S2 and S4). As is the case for theph1 mutants, RNAi-5 was partially fertile. Misalignment andchromosome clustering were not commonly seen in this plant. Thereduction in gene expression in the remaining four plants rangedfrom 50–83% (SI Appendix, Tables S2 and S3). The number ofbivalents in these four plants ranged from 10.6 to 14.3, comparedwith 19.7 in the negative control. The number of MI cells showingmultivalents, chromosome clustering, and misalignment rangedfrom 71.7–90% (Fig. 1 and SI Appendix, Table S3). The plant(RNAi-6) showing a 50% reduction in gene expression exhibiteda more severe phenotype than RNAi-5, with additional chromo-some clumping and disrupted alignment on the MI plate. Thisplant exhibited aberrant chromosome pairing in 71.7% of the cells(SI Appendix, Table S3). There was a significant increase in thelevels of chromosome clumping and misalignment along the MIplate with a further 31.64% reduction in gene expression. RNAi-4showed the maximum level of silencing (Figs. 1 and 2). Bivalentinterlocking, which was not observed in the negative control, waspresent in 31.2% of the MI cells of these four transgenic plants.Overall, chromosome pairing aberration was very severe in thesefour plants compared with that seen in the ph1b mutant (Figs. 1and 2). These four plants were completely sterile but exhibited noother phenotypic abnormality.

Structure of the C-Ph1 Gene. Detailed bioinformatics and sequenceanalyses revealed three genomic and cDNA copies of the gene,one on each of the three wheat group 5 chromosomes (Fig. 3). Atthe DNA level, the three homeologues from cv. CS were 90%similar and the differences were due to several structural changes,including deletions and insertions (Fig. 3). The 5B copy of the genesequence showed two insertions of 46 bp and 14 bp present 5 bpapart in exon II (referred to as a 60-bp insertion) (Fig. 3). A de-letion of 29 bp was observed 80 bp upstream of the two insertions.There were two 5D-specific deletions of 12 bp and 15 bp presentin exon II. Additional differences among the homeologues werea 13-bp deletion in the 5D copy present 11 bp downstream of theexon–intron junction and two insertions in the 5A copy: a 7-bpinsertion in the intron and a 6-bp insertion in exon II (Fig. 3). The5′ UTR of the 5B copy was 96.8% similar to that of the 5D copyand 94.3% similar to that of the 5A copy. A similar comparisonbetween the 5A and 5D copies showed 92.6% similarity. The 3′UTRs of the 5B and 5D copies were 92.6% similar, and the dif-ferences were mainly due to an 11-bp insertion in the 5B copy,alongwith six homeologous sequence variants (HSVs). The 3′UTRof the 5A copy did not match with either the 5B or the 5D copymainly due to a major deletion/insertion. Of the HSVs among thethree gene copies, 26.7% were synonymous and 73.3% were non-synonymous. Overall, the percentage of nonsynonymous bases inthe gene was 77.8%, with the remaining 22.3% being synonymous.The genomic copy of 5B was the largest (954 bp) compared with

the 5D (883 bp) and 5A (539 bp) copies. Excluding insertions anddeletions, the 5B genomic copy is 95% similar to that of the 5Dcopy. The similar proportions are 94.4% for the comparison be-tween 5B and 5A and 85.9% for the comparison between 5A and

A BFig. 1. Chromosomal pairing (CP) and C-Ph1 geneexpression analysis in the VIGS and RNAi-silencedplants. (A) Chromosome spreads of PMCs at MI fromFES and MCS controls and C-Ph1 silenced plantsVIGS-5 and VIGS-7. CP analysis shows (i) the per-centage of cells exhibiting aberrant pairing and thetotal number of cells analyzed (given in parenthe-sis) and (ii) the average number of multivalentsper cell, with the range given in parentheses. EXPdenotes the transcript expression levels (%) in the C-Ph1 silenced plants relative to the FES control, as observed by quantitative real-time PCR analysis. (B)Chromosome spreads of PMCs of control (BW) and C-Ph1 silenced RNAi plants indicating CP and EXP. (Scale bar, 5 μm.) (SI Appendix, Tables S2 and S3.)

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5D. Overall, the genomic copies of the three homeologues shared41% DNA sequence similarity.Among the three gene copies, 5A produced the smallest

transcript (excluding 5′ and 3′ UTRs) of 420 bp by splicing anintron of 120 bp, whereas 5D produced a transcript of 783 bp bysplicing an intron of 100 bp (Fig. 3). The 5B copy of the geneshowed signs of alternate splicing to produce transcripts of 954bp and 763 bp. The difference between the two variants was twointrons of 113 bp and 78 bp, which were spliced to generate the763-bp version and were retained to generate what turned out tobe the largest transcript among all gene copies (Fig. 3). The 5Bcopy with the larger transcript generated a smaller protein thanits alternate form due to retention of the intron around the 60-bpaddition, which contained an in-frame stop codon (Fig. 3). Pre-dicted proteins from either of the two splice variants from the 5Bcopy were smaller than proteins produced by the 5D copy butlarger than proteins produced by the 5A copy.Predicted proteins from the 5B copy of the gene were 204 aa

and 221 aa in length compared with 174 aa from the 5A copy and260 aa from the 5D copy. The most conserved part of the genecorresponding to exon I was not present in either of the predicted

proteins from the 5B copy but was present in the predicted pro-teins from the 5A and 5D copies. The two proteins representingthe 5B copy of the gene were 91% similar, with the only differencebeing an additional 17 aa on the N terminus in the larger version.Not counting the 86-aa deletion in 5A created by a premature stopcodon, the predicted proteins of the three copies were 82% sim-ilar. Considering all deletions and insertions however, the 5A copyprotein is only 25–30% and 46.4% similar to the 5B copy and 5Dcopy proteins, respectively. Similarly, the 5D copy and the 5B copyproteins were 68–74% similar. Likewise, the predicted 3D structuresof the four proteins were significantly different, indicating functionaldivergence of the homoeologues after allopolyploidization (SIAppendix, Fig. S5).

C-Ph1 Expression Pattern. Quantitative real-time expression analysisrepresenting the cumulative expression of all homeologues showedthat the gene is primarily expressed during the postflower initiationstages, although significant expression was observed in the roots aswell (Fig. 4). Maximum expression of the gene was during meiosis I;however, the flag leaf of the plants undergoing meiosis also showedsignificant expression. Essentially no expression was observed inmature pollen grains or the subsequent seed development stages.To “pinpoint” the gene expression pattern during various mei-

otic stages, one of the three anthers from each floret was used forthe meiotic chromosome analysis and the remaining two were usedfor real-time gene expression analyses (primer details are providedin SI Appendix, SI Text and Table S4). All three anthers from asingle wheat floret are known to be at a developmentally identicalstage (19). Chromosome spreads of pollen mother cells (PMCs)fromwheat cv. CS denoting the various stages ofmeiosis are shownin Fig. 4A. Expression of the gene increased 13-fold in the transi-tion from prophase I to late prophase I, followed by a further in-crease of about 26-fold during MI (Fig. 4C). Relative to MI,expression dropped by 34-fold during anaphase I, followed by afurther drop of 6.4-fold during the dyad stage (Fig. 4C). Maximumexpression of the gene was observed duringMI. Surprisingly, therewas a 16.5-fold increase in gene expression during the tetrad stage,suggesting additional functions of the gene.Expression analysis of each of the gene copies individually by

single-strand conformation polymorphism analysis revealed thatthe three copies of the gene have dramatically different expres-sion patterns. With the exception of roots, where almost allcopies showed expression, the 5B copy specifically expressed in a3- to 5-cm long spike, which, in CS, contains meiotically dividingcells (Fig. 4D). Expression of the 5B copy dropped significantlyin the 6- to 8-cm spike that contains cells at the meiosis II stage.Essentially no expression was observed for the copy at the mature

Fig. 2. Cytogenetic analysis showing different levels of C-Ph1 gene silencing in RNAi plants compared with ph1b. Chromosome spreads of PMCs from ph1b,BW (negative control), and four RNAi plants showing different levels of gene silencing. Expression denotes the normalized transcript expression levels (%)relative to BW, using the delta-delta threshold cycle (Ct) method, observed by quantitative real-time PCR analysis. Aberrant pairing (%) denotes the per-centage of cells exhibiting aberrant chromosome pairing. “Multivalents/cell” denotes the average number of multivalents per cell, and the range is given inparentheses. Chromosome (Chr.) clustering and misalignment phenotype are represented by “+” and “−,” where (+) indicates increased severity levels and(−) indicates decreased severity levels. (Scale bar, 5 μm.)

Fig. 3. Structural differences among the C-Ph1 gene homeologues inhexaploid wheat. Nucleotide sequences of cloned CS C-Ph1-5B, its splicevariant (C-Ph1-5Balt), and C-Ph1-5D and C-Ph1-5A copies were aligned toeach other; the differences are drawn to scale (1 = 1 nucleotide). The sym-bols ▲ and ▼ represent deletions and insertions in the sequences, re-spectively. Insertions and deletions were determined by majority consensusrule. The shaded region in C-Ph1-5B and C-Ph1-5Balt represents a corre-sponding region similar to the C-Ph1-5D and C-Ph1-5A sequences; the nu-cleotide sequence is not translated as a protein (predicted) but forms a partof the UTR. The colored bars below C-Ph1-5B represent VIGS and RNAi oli-gos, denoted by as (antisense), hp1 (hairpin 1), hp2 (hairpin 2), and RNAi,respectively. The gray dots at the end of C-Ph1-5A exon II represent deletion/insertion not present in the C-Ph1-5B and C-Ph1-5D sequences.

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anther stage (Fig. 4D). The expression pattern of the 5D copy wasvery different from that of the 5B copy. Unlike 5B, the 5D copyshowed a low level of expression in the leaves and the 3- to 5-cmspike as well as during anthesis or 5 d postanthesis (Fig. 4D). The5A copy was expressed predominantly during meiosis II andshowed very little expression in the 3- to 5-cm spike, suggestingits role in cytokinesis and/or gametophyte development.In the substaged meiotic anthers, the 5B copy was specifically

expressed during MI and a low level of expression was also seenduring anaphase I. No expression was observed for the copy in thesubsequent meiotic stages (Fig. 4E). Unlike 5B, the 5A copy wasexpressed during the anaphase I, dyad, and tetrad stages. Comparedwith the other two copies, the 5D copy showed significantly higherexpression during interphase. A significant amount of expression ofthe 5D copy was also observed during prophase I and MI (Fig. 4E).Additionally, expression of the 5B copy was analyzed in the

wheat homeologous group 5 nullisomic-tetrasomic (NT) lines,ph1 mutants, and a series of 5BL deletion lines during meiosis(SI Appendix, Fig. S6). The results provide an additional line ofevidence to confirm that the newly identified C-Ph1 gene in thisstudy corresponds to the Ph1 locus.

Gene Orthologs in Other Plants. Structurally conserved orthologsof the C-Ph1 gene were observed in all studied monocots, includ-ing rice, barley, maize, and Brachypodium. The rice ortholog(LOC_Os09g30320) maps on chromosome 9, the Brachypodiumortholog (Bradi4g33300) maps on chromosome 4, and the maizeortholog (GRMZM2G078779)maps on chromosome 7. The geneshowed a typical pattern of three exons and two introns in all thesespecies except maize, which had two additional splice variants. Inaddition to the conserved BURP domain (named based on fourtypical members, BNM2, USP, RD22, and PG1-β), a 45-bp firstexon was observed, followed by a 94- to 138-bp intron and a 114-to 138-bp second exon. The size of second intron in rice was 599bp, which is comparatively larger than 97 bp, 119 bp, and 122 bp in

maize,Brachypodium, and barley, respectively. The third exon wasbetween 596 and 644 bp in size and contained BURP domain-related sequences. The transcribed part of the barley copy showed90% similarity with the 5B copy of the C-Ph1 gene, whereas theother species had 71–72% similarity. Structurally, the putativeC-Ph1 ortholog from several diploids, including barley, maize,Brachypodium, and rice, resembled the 5D copy more closely,suggesting it to be the conserved and ancestral version of the gene.Studied using the available microarray-based expression data,

the maize ortholog of the C-Ph1 gene showed the highest ex-pression in the meiotic tassel and anthers containing the PMCmeiotic stages (20). The developing endosperm and kernel alsoshowed a significant level of expression, but no other develop-mental stage showed any expression of the gene. Similarly, the riceortholog of the gene, LOC_Os09g30320, also showed the highestexpression in the heading panicle and stamens containing meiotictissues, and no expression was observed in any other develop-mental stages (21). The barley ortholog was identified from ESTsderived from immature male inflorescences (22), although a de-tailed expression pattern of the gene in barley is not yet known.Initially, DNA sequence analysis and a domain/motif search

identified At5g25610 as the putative Arabidopsis ortholog ofOs09g30320 (17). At5g25610 encoded a BURP domain con-taining protein with a putative function in the dehydration stressresponse, because it encodes dehydration-induced protein RD22(17), thus making it a less likely candidate inArabidopsis. Sequencecomparison at the DNA or protein level identified another ortho-log of the wheat gene in Arabidopsis, although with poor conser-vation. At1g78100 mRNA matched perfectly with the 22 bp of thewheat RNAi fragment. The gene expresses during anthesis andvarious other developmental stages, thus making it a likely candi-date for the gene ortholog. Poor sequence conservation amongorthologs has been well documented for many other meiotic genes.

Gene Function in Arabidopsis. With the assumption of functionalconservation of the gene between Arabidopsis and wheat due toconserved catalytic motifs, we performed RNAi-based silencingof the Arabidopsis ortholog. The resistant transgenic lines, alongwith WT Columbia (Col-8), were analyzed for meiotic chromo-some pairing analysis, and the results are shown in Fig. 5 andsummarized in SI Appendix, Tables S5 and S6.Twenty-five cells were imaged and analyzed for chromosome

pairing defects during early stages of leptotene to pachytene. Inall of the cells analyzed, the WT Arabidopsis showed five bivalents(Fig. 5C) in contrast to an average of 3.05 bivalents in the silencedplants. On average, 0.9 quadrivalents and 0.05 hexavalents permeiotic cell were observed in the RNAi plants, whereas noquadrivalent or hexavalent was observed in the WT plants (SI Ap-pendix, Table S5). TheRNAi plants showedmultiple associations inthe form of centromere coupling in all of the analyzed cells at theleptotene stage (Fig. 5 and SI Appendix, Table S6), whereas no suchcentromere coupling was observed in the WT. The centromerecoupling led to the formation of multivalents during zygotene andpachytene stages. The silenced plants showed multivalent forma-tion in 90–95% of the analyzed cells in the zygotene and pachytenestages (Fig. 5 and SI Appendix, Table S6). The WT was normal andshowed no such chromosomal aberrations.

Cell Division Cycle 2 Gene Is Not a Good Candidate for the Ph1 Gene.The Cell division cycle 2 (Cdc2-4) gene is present in chromo-some deletion lines 5BL-1, 5BL-3, and 5BL-8 (SI Appendix, Fig.S7), which are known to lack the Ph1 gene, because their chro-mosome pairing matched with that of the ph1b and other Ph1gene mutants (16, 23). VIGS analysis with a 96-bp antisenseconstruct targeting the Cdc2-4 gene showed normal chromosomepairing at meiotic MI (SI Appendix, Fig. S8 and Table S7). TheVIGS plants showed an average of 20.9 bivalents compared with21 bivalents in the MCS and FES (abrasive agent used for in-oculation) control plants (SI Appendix, SI Text and Fig. S8).

Fig. 4. C-Ph1 gene expression pattern in various tissues and substagedmeiotic anthers. (A) Chromosome spreads of PMCs from CS denoting variousstages of meiosis. (Scale bar, 5 μm.) (B) Quantitative expression analysis usinggene-specific primers (SI Appendix, SI Text) in the root (R), leaf (L), flag leaf(FL), 3- to 5-cm spike (I to MI), and 6- to 8-cm spike (MII to T), as well as atanthesis (AN) and 5 d postanthesis (5DPA). (C) Quantitative expressionanalysis at interphase (I), prophase I (P), late prophase I and metaphase I (M),anaphase I (A), dyad (D), and tetrad (T). The y axis in B and C denotes thenormalized mRNA levels using the delta-delta Ct method. (D) Tissue- andstage-specific expression of homeologues analyzed by single-strand confor-mation polymorphism (SSCP) analysis (SI Appendix, SI Text). (E) Meioticstage-specific expression of homeologues in the different substages ofmeiosis, as mentioned in C.

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DiscussionSince its discovery in 1958, various studies have implicated the Ph1gene in many different meiotic processes. While studying the so-matic association of chromosomes in premeiotic cells, the Ph1gene was suggested to be involved in ensuring strict homologouspairing by suppressing premeiotic homeologous chromosome as-sociation (24). Careful analyses of the published data specificallyimplicates the 5D copy of the Ph1 gene in the initial chromosomepairing of both homologs and homeologues because asynapsis wasobserved in the absence of the 5D copy but not the 5A or the 5Bcopy (3, 25). The absence of chromosome 5A had essentially noeffect on HECP. Although the 5B copy of the Ph1 gene was shownspecifically to regulate diploid-like pairing (2, 26), various lineslacking either chromosome 5B, its long-arm, or the Ph1 locus allshowed increased HECP (14, 16, 27). The function of the 5B copyin differentiating homologs from homeologues was further sup-ported by the fact that four copies of either 5A or 5D were notable to restore normal chromosome pairing in the absence of 5B(3, 25). Multivalents and other types of higher order pairingobserved in the absence of the 5B copy were not observed in theabsence of the 5A copy and were not as robust in the absence ofthe 5D copy (3, 25). Along with asynapsis, lack of 5D exhibitedfrequent bivalent interlocking and rare multivalents. A minimumof four copies of the 5A copy were needed to compensate for theabsence of the 5D copy, suggesting that the two copies share acommon function, with the 5A copy having a weaker effect.The C-Ph1 gene that we have identified in the present study

explains the observations made on the Ph1 gene function. Theexpression and silencing data clearly suggest that the C-Ph1 genehas multiple functions during meiosis, each controlled by one ormore copies of the gene. One of these functions is the initialpairing of both homologs and homeologues, as suggested by thehigher expression level of the 5D copy during interphase andthe gene silencing phenotype. Chromosome 5B was implicated inthe specific function to differentiate homologous pairing fromHECP as shown by the unique expression pattern of the 5B copyof the C-Ph1 gene, along with the gene silencing phenotype. The39.7-fold increase in expression of the 5B copy between lateprophase and MI coincided with the stages when this precisefunction takes place. Expression of the 5D copy during the MIstage suggests an additive function of the copy during MI. The5A copy was expressed predominantly during meiosis II, sug-gesting its role in cytokinesis and/or gametophyte development.

In accordance with the interpretation that the 5A and 5D copiesof the Ph1 gene share a common function, the predicted proteins ofthe 5A and 5D copies of the C-Ph1 gene are very similar, exceptthat the 5A copy produces a truncated, and thus perhaps less ef-fective, protein. The two proteins share a highly conserved motifcorresponding to exon I, which is almost identical in the twohomeologous gene copies but is absent in the 5B copy proteins. Thepresence of this highly conserved motif suggests unique function(s)for the two copies, including initial pairing of both homologs andhomeologues. Alternatively, the unique function of the 5B copymay be due to the lack of this conserved motif along with an in-sertion of 60 bp that contains an in-frame stop codon, thus resultingin smaller proteins. The unique function(s) of the 5B protein(s)may also be due to its very specific expression pattern (mentionedabove). The presence of the two 5B copy proteins resulting fromalternate splicing suggests multiple functions of the 5B copy. Thedifferences in structure and expression patterns among the threecopies of the gene suggest neofunctionalization of the 5B copy, withat least one of the functions being different from that of the 5Aor 5D copy. Sequence similarity with diploid species suggests the5D copy to be the ancestral copy.VIGS and RNAi-silenced plants showed all of the chromo-

some pairing aberrations observed in the Ph1 gene mutationsalong with some additional phenotypes, including chromosomeclumping and disrupted alignment on the MI plate. In both theVIGS and RNAi plants, the severity of the chromosome pairingphenotype correlated well with the level of gene silencing. Oneof the RNAi plants (RNAi-5) with a 44% reduction in geneexpression showed a chromosome pairing phenotype similar tothat seen in the ph1 mutants and the lines lacking the Ph1 gene(Fig. 2). Characteristic of the ph1 mutants, multivalents wereobserved in this plant without any disruption in chromosomealignment on the MI plate or chromosome clumping (Fig. 2).These results suggest that about a 44% reduction in expressionof the gene is needed to show the aberrant chromosome-pairingphenotype, similar to that observed in the Ph1 gene mutants.Previously, bivalent interlocking was observed in ph1b mutantand in the plants lacking the 5B or 5D copy, as well as in plantstriisosomic for chromosome 5BL (3). We also observed biva-lent interlocking in the C-Ph1–silenced plants. The bivalentinterlocking is probably caused by pairing between distanthomologs in the absence of the gene. Bivalent interlocking in theplants carrying a triple dose of chromosome 5BL is probably dueto the dosage effect of Ph1 on the relative separation of homologsbefore meiosis (28). Bivalent interlocking could also be due tosilencing of the gene triggered by the higher copy number (29).All RNAi and VIGS plants with gene silencing of more than 44%

resulted in chromosome clustering and misalignment of chromosomeson the MI plate, in addition to the expected multivalent formation.This phenotype was not observed in any of the NT lines, probablybecause multiple copies of the gene perform the same function andloss of a copy in NT lines is compensated for by the other copies.Our data suggest a plausible explanation of the above-mentioned

observations. Firstly, expression of the 5B copy increases betweenlate prophase and MI coinciding with the stages when centro-mere–microtubule interactions takes place. Secondly, transientVIGS as well as stable RNAi silencing of the C-Ph1 gene resultedin severe centromere clustering, along with disrupted alignmentof chromosomes on the MI plate, suggesting a plausible rolein centromere–mictrotubule interaction. This dramatic clusteringand misalignment was not observed in the absence of any one ofthe three gene copies during previous studies, suggesting that twoor more of the gene copies act in an additive manner to accom-plish this very important function. Also, expression pattern of the5B copy of the C-Ph1 gene closely coincides with that of the motorprotein CENP-E (30), a kinetochore-associated protein involvedin the sustained movement of chromosomes leading to properalignment on the MI plate (31). Taken together, these and theobservations of disrupted chromosome alignment on the MI platein the VIGS and the RNAi plants suggest that either the C-Ph1gene functions by regulating centromere–microtubule interaction

Fig. 5. Multivalent formation in the RNAi-silenced Arabidopsis plants. Eachimage is a flat projection across the entire nucleus. Chromosomes were coun-terstained with DAPI (red); the centromeric probe was labeled with cyanine-5(green). (A–C) Normal meiosis progression from leptotene to late pachytene,leading to formation of five bivalents in the WT. Centromere coupling duringleptotene (D) and multivalent formation in zygotene (E) (Inset shows quadri-valent formation) in the gene silenced plants. Centromere coupling in pachy-tene, leading to formation of two clusters of centromeres (F) instead of fivepairs in the WT (C). (Scale bar, 5 μm.) (SI Appendix, Tables S5 and S6.)

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as was previously suggested, or that this is one of the additionalfunctions of the gene where two or more copies of the gene havethe same function, in addition to regulating HECP.Studied in the root-tip cells, chromosomes in ph1b mutant

showed higher mitotic association of homeologues and hy-persensitivity to colchicine compared to those in the normalCS, and disrupted centromere-microtubule association wassuggested to be the cause (32–34). A low level of expression ofthe 5B copy of the C-Ph1 gene was observed in all mitoticallydividing tissue, including roots and leaves, suggesting its rolein mitotic cell division. This also supports the previous reportsthat Ph1 gene functions during mitotic and particularly pre-meiotic stages, affecting chromosomal movement towards thepoles and, consequently, their arrangement in the nucleus.This may effect premeiotic association of homologous chro-mosomes and relative separation of homeologues, thus de-termining exclusive homologous pairing in wheat alreadybefore the commencement of synapsis (35–37).

Materials and MethodsPlant Material. Plant material used in this study includedWT hexaploid wheat(Triticum aestivum cv. CS and cv. BW), a CSmutant lacking thePh1 locus (ph1b),wheat homeologous group 5 NT lines, and a series of 5BL deletion lines. Basedon the efficient utilization of the cv. CS for chromosome squash preparations,itwas selected as an ideal cultivar for VIGS. CS,NT, and deletion lineswere usedfor mapping and cloning experiments. BW was used for RNAi experimentsbecause it can be efficiently transformed usingAgrobacterium-mediated genetransfer (38). The detailed growth conditions are given in SI Appendix, SI Text.

VIGS. The preparation of vector constructs, transcription, and inoculation of viralRNAs has been described previously (18). On the basis of comparative sequenceanalysis, the unique gene region for the C-Ph1 homeologue on chromosome 5Band the Cdc2-4 gene was selected for silencing. The procedure is elaborated inSI Appendix, SI Text. FES buffer (abrasive agent used for inoculation) was usedas a negative control, and the plasmid pγ.MCS [containing a 121-bp antisensefragment of the MCS (pBluescript K/S; Stratagene)] was used as a “virus-only”control to differentiate the effect of the target gene from that of the virus. Forthe experiment using an antisense construct, 10 plants were inoculated withpγ.MCS and four plants were inoculated with FES. Four CS plants were also usedas a control. Similarly, for VIGS using a pγ.C-Ph1hp2 construct, one and threeplants were inoculated with FES and pγ.MCS, respectively. Likewise, for theCdc2-4 gene, five plants each were inoculated with pγ.MCS and FES.

To target the gene in PMCs, the flag leaf of the main tiller was inoculatedat the boot stage by rubbing. Inoculated plants were lightlymistedwithwaterand covered with plastic bags for 16–18 h.

RNAi Genetic Transformation. For RNAi-based silencing of the Arabidopsisortholog, a 200-bp wheat RNAi construct was cloned in the pANDA35HKvector, driven by the 35S promoter and carrying a gene for hygromycin re-sistance. Details of this procedure are provided in SI Appendix, SI Text.

The RNAi construct for the stable wheat transformation was developed byamplifying a 200-bp target sequence from the C-Ph1 gene. Details of thisprocedure are provided in SI Appendix, SI Text.

ACKNOWLEDGMENTS. We thank Dr. Neeraj Kumar for help with meioticanalysis and for providing other technical assistance. This work was supportedby the Vogel Endowment Fund.

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