Divergence in homoeolog expression of the grain length-associated gene GASR7 during wheat allohexaploidization

Dongdong Zhng，Bingnn Wng，Junmin Zho，Xuo Zho，Linqun Zhng，Dengi Liu，Lingli Dong，Dowen Wng，Long Mo，*，Aili Li，*

aNational Key Facility for Crop Gene Resources and Genetic Improvement，Institute of Crop Science，Chinese Academy of Agricultural Sciences，Beijing 100081，China

bTriticeae Research Institute，Sichuan Agricultural University，Chengdu 611130，China

cThe State Key Laboratory of Plant Cell and Chromosome Engineering，Institute of Genetics and Developmental Biology，

Chinese Academy of Sciences，Beijing 100101，China

Divergence in homoeolog expression of the grain length-associated gene GASR7 during wheat allohexaploidization

Dongdong Zhanga，1，Bingnan Wanga，1，Junmin Zhaoa，1，Xubo Zhaoa，Lianquan Zhangb，Dengcai Liub，Lingli Dongc，Daowen Wangc，Long Maoa，*，Aili Lia，*

aNational Key Facility for Crop Gene Resources and Genetic Improvement，Institute of Crop Science，Chinese Academy of Agricultural Sciences，Beijing 100081，China

bTriticeae Research Institute，Sichuan Agricultural University，Chengdu 611130，China

cThe State Key Laboratory of Plant Cell and Chromosome Engineering，Institute of Genetics and Developmental Biology，

Chinese Academy of Sciences，Beijing 100101，China

A R T I C L E I N F O

Article history：

Accepted 29 August 2014

Available online 5 October 2014

TaGASR7

Gibberellic acid

Triticum aestivum

Polyploidy

Hexaploid wheat has triplicated homoeologs for most of the genes that are located in subgenomesA，B，andD.GASR7，amemberoftheSnakin/GASAgenefamily，hasbeenassociated with grain length development in wheat.However，little is known about divergence of its homoeolog expression in wheat polyploids.We studied the expression patterns of the GASR7 homoeologs in immature seeds in a synthetic hexaploid wheat line whose kernels are slender likethoseofitsmaternal parent（Triticumturgidum，AABB，PI 94655）incontrastto theround seed shapeofitspaternalprogenitor（Aegilopstauschii，DD，AS2404）.WefoundthattheBhomoeologof GASR7 was the main contributor to the total expression level of this gene in both the maternal tetraploid progenitor and the hexaploid progeny，whereas the expression levels of the A and D homoeologs were much lower.To understand possible mechanisms regulating different GASR7 homoeologs，we firstly analyzed the promoter sequences of three homoeologous genes and found that all of them contained gibberellic acid（GA）response elements，with the TaGASR7B promoter（pTaGASR7B）uniquely characterized by an additional predicted transcriptional enhancer.This was confirmed by the GA treatment of spikes where all three homoeologs wereinduced，withamuchstrongerresponseforTaGASR7B.McrBCenzymeassaysshowedthat the methylation status at pTaGASR7D was increased during allohexaploidization，consistent with the repressed expression of TaGASR7D.For pTaGASR7A，the distribution of repetitive sequence-derived24-nucleotide（nt）smallinterferingRNAs（siRNAs）werefoundwhichsuggests possible epigenetic regulation because 24-nt siRNAs are known to mediate RNA-dependent DNA methylation.Our results thus indicate that both genetic and epigenetic mechanisms may be involved in the divergence of GASR7 homoeolog expression in polyploid wheat.

?2014 Crop Science Society of China and Institute of Crop Science，CAAS.Production and hosting by Elsevier B.V.All rights reserved.This is an open access article under the CC BY-NC-ND license（http：//creativecommons.org/licenses/by-nc-nd/3.0/）.

1.Introduction

Polyploidyleadstowhole-genomeduplicationandis widespread in the plant kingdom［1，2］，providing opportunities for duplicated genes to diverge in several evolutionary ways such as subfunctionalization，neofunctionalization，and nonfunctionalization（deletion or pseudogenization）［3，4］，as well as genetic and epigenetic interactions between homoeoalleles［1，5］.Bread wheat or common wheat（Triticum aestivum）is a hexaploid（2n=6x=42，AABBDD）that arose 7000-12，000 years ago following the hybridization of the early-domesticated allotetraploid T.turgidum ssp.dicoccon（AABB）and the diploid goat grass Aegilops tauschii（2n=2x=14，DD）［6-9］.As one of the most important staple crops，common wheat is also a good model for studying genetic interactions between multiple related genomes.New allohexaploid synthetic wheat can be readily synthesized in the laboratory by interspecific hybridization［10］and chromosome doubling，facilitating the study of genetic，functional，and epigenetic changes during this process. Previously，a series of allohexaploid wheat lines were generated by hybridizing T.turgidum ssp.dicoccon accession PI 94655 with the diploid Ae.tauschii ssp.strangulata accession AS2404 as a paternal parent［11］.The synthetic predominantly resembled T.turgidum in grain length（Fig.1），implying，as far as the grain developmentisconcerned，thegeneticarchitectureofhexaploid progeny may be more similar to the maternal progenitor［12］.

Snakin/GASA proteins are widely distributed in plants［13］. They are expressed in different plant organs with high tissue and temporal specificity.Most Snakin/GASA genes are regulated by hormones and participate in signaling pathways that modulate hormonal responses and levels.In Arabidopsis，GASA4 plays a role in regulating floral meristem identity and promotes the development of seed size and seed weight［14］.In rice，OsGASR7（Os06g0266800），which is highly similar to Arabidopsis GASA4，was identified as a candidate gene determininggrainlength［15］.Inwheat，ahomologofArabidopsis GASA4 and rice OsGASR7 was also involved in grain length development［16，17］.In Triticum urartu（2n=2x=14，AA），the putative A genome donor of common wheat and two haplotypes（H1 and H2）of TuGASR7 were identified among 92 diverse accessions collected from different regions，among which H1 was found to be significantly associated with greater values of grain length and grain weight.In common wheat，TaGASR7-A1 was shown to be a major genetic determinant of grain length，with pleiotropic effects on grain weight and yield［17］.

Despite triplication of many genes in hexaploid wheat，notallhomoeoalleles are equally transcribed.Transcriptional divergence of homoeologous genes has been reported.Our previous work showed that the A homoeolog of wheat calcium-dependent protein kinase（CDPK）gene TaCPK2-A was inducible by Blumeria graminis f.sp.tritici（Bgt）infection whereas TaCPK2-D mainly responded to cold treatment，suggesting subfunctionalization of these homoeologs［18］.Similarly，the homoeologs of methyl-binding domain protein genes TaMBD2-5B and TaMBD2-5D were highly responsive to salt stress，but in the seedling leaves only TaMBD2-5B was specifically up-regulated by low temperature［19］.Although all three homoeologs of a wheat expansin gene（TaEXPA1）were silenced in wheat seedling roots，TaEXPA1-A and TaEXPA1-D were expressed in seedling leaves，indicating tissue specificity of homoeolog expression［20］.On the other hand，among the threehomoeologsofthewheatSEPALLATA-likeMADS-boxgene WLHS1，only WLHS1-D was functional.The protein structure of WLHS1-A is interrupted by a DNA insertion whereas WLHS1-B is predominantly silenced by cytosine methylation，suggesting different evolutionary effects that lead to divergence in homoeologous gene expressions［21］.

Weareinterestedinunderstandinghowexpressionsofwheat GASR7 homoeologs are regulated in polyploid wheat and their differential regulation during wheat polyploidization because homoeologs of GASR7 are associated with grain length development and hexaploid synthetic wheat has similar grain shape to its tetraploid progenitor.Towards this end，we examined the expression patterns of wheat GASR7 homoeologs in immature seeds of several generations of a synthetic wheat line and its progenitors and studied the promoter sequences of all three homoeologs.We found that both genetic and epigenetic mechanisms may be involved in the expression divergence of GASR7 homoeologs in polyploid wheat.

2.Materials and methods

2.1.Plant materials and GA treatment

Fig.1-The slender seeds of synthetic allohexaploid wheat and its tetraploid progenitor.PI 94655，T.turgidum（AABB），tetraploid maternal parent；AS2404，Ae.tauschii（DD），paternal progenitor；third generation（S3）synthetic plants（AABBDD）. Bar=1 cm.

A set of newly synthesized hexaploid wheat lines and their progenitors were used，including the third and the fourth generation plants of self-pollinated synthetic wheat（S3 and S4）and their progenitors the T.turgidum ssp.dicoccon accession PI 94655（AABB，2n=4x=28）and the goat grass Ae. tauschii ssp.strangulata accession AS2404（DD，2n=2x=14）［11，22，23］.Seeds were germinated on moist filter papers，transplanted into soil，and grew in a controlled environment［24］.Immature seeds were collected from a pool of 10-12 plants for each genotype at 6，11，14，17，21，and 25 days after flowering（DAF）and were stored in liquid nitrogen until RNA extraction.Samples were prepared for two consecutive years in the same experimental field as two biological replicates. For gibberellic acid（GA）treatment，young spikes of S3 and S4 plants were sprayed with 100 mg L-1GA3once a day for 10 days from 2 to 11 DAF.Spikes sprayed with water were used as controls.

2.2.RNA extraction and cDNA 3′RACE

Total RNA was isolated from seeds using a Trizol-based RNA isolation protocol（Life Technologies，NY，USA）.First-strand cDNAwassynthesizedusing2 μgDNase-treatedtotalRNAwith oligo（dT）primers（Promega，WI，USA）.Full-length cDNAs were obtained by PCR using primers TaGASR7-F and TaGASR7-R designed according to EST sequences（see text and Table S1）. PCR products were subcloned and sequenced.The 3′UTR of GASR7 homoeologous genes were cloned using a SMART-rapid amplification of cDNA end kit（SMART-RACE；Clontech，Mountain View，CA，USA）following the manufacturer's protocol. Primers for 3′RACE and homoeolog-specific PCR are also listed in Table S1.

2.3.Semi-quantitative PCR and quantitative RT-PCR analysis

Gene-specific primers for quantitative PCR（Forward，TaGASR7-4 F；Reverse，TaGASR7A-4R，TaGASR7B-4R，and TaGASR7D-5R，respectively）were designed according to the nucleotide polymorphisms of cDNA sequences of the three TaGASR7 homoeologs（Table S1）.Amplification efficiency of primers was detected according to the method described by Hu et al.［20］.For homoeolog-specific expression analysis，tubulin and glyceraldehyde-3-phosphate dehydrogenase（GAPDH）genes were used as endogenous controls for semi-quantitative and real time PCR，respectively.

2.4.BAC library screening to obtain promoter sequences of GASR7 homoeologs

ToisolatethepromotersequencesofGASR7BandDhomoeologs，a BAC library from T.aestivum cv.Renan was screened by PCR using a BAC clone pooling approach［25］.Two BAC clones were obtained using primers designed from the coding regions of TaGASR7B and TaGASR7D.Promoter sequences of TaGASR7B/D were obtained by PCR from the BAC clones using primers designed from the 5×Chinese Spring genome sequences（Table S1）［26］and sequenced.

2.5.cis-element prediction and siRNA distribution analysis

DNA fragments encompassing the 1.5 kb upstream the start codon and part of the coding region were amplified using PCR. By comparing the coding sequences，three promoters were discriminated into pTaGASR7A，pTaGASR7B，and pTaGASR7D. cis-acting regulatory elements were predicted from the PLACE online service（http：//www.dna.affrc.go.jp/PLACE/signalscan. html；［27］）.Small RNAs were obtained from 11 DAF seeds and deep sequenced as previously described［12］.SiRNA distribution was identified by BlastN［28］search against the small RNA dataset［12］using promoter sequences as queries.

2.6.McrBC enzyme assay

Total genomicDNA wasextractedusing a DNeasyPlantMini kit（Qiagen）following the manufacturer's instructions.PCR-based DNA methylation assays were conducted as described［29］.At leastthreeindependentbiologicalreplicateswereperformedon pTaGASR7D using primers listed in Table S1.

3.Results

3.1.Acquirement of TaGASR7 homoeolog-specific primers

ABlastNsearchusingtheT.urartuGASR7gene（TuGASR7）［16］as a query against the PlantGDB Chinese Spring EST assembly retrievedthreeESTs，PUT-163b-Triticum_aestivum-2301165442，2301165443，and 147657 that contained open reading frames（ORF）of306，300，and 285 bp，respectively.A comparison of T.urartu and Ae.tauschii GASR7 sequences［30］showed that the three ESTs corresponded to the A，B，and D homoeologs，respectively（Fig.S1）.Rapid amplification of cDNA end（RACE）experiments showed that the three homoeologs had 3′UTR sequences that were 216（A），233（B），and 259 bp（D）and were quitepolymorphic（Fig.2-A）.Homoeolog-specificreverseprimers were then designed using the 3′UTR polymorphic regions（TaGASR7A-4R，TaGASR7B-4R，and TaGASR7D-5R；Table S1）. Combining with the same forward primer TaGASR7-4F，these homoeolog-specific reverse primers amplified only two of the three bands in Chinese Spring（CS）nulli-tetrasomic lines that lacked the one on corresponding missing chromosomes.As shown in Fig.2-B，the primer pair TaGASR7-4F/A-4R amplified a band from all nulli-tetrasomic lines except N7AT7B and N7AT7D in which chromosome 7A was missing.The other two primer combinations（TaGASR7-4F/B-4R and TaGASR7-4 F/D-5R）also gave expected amplification patterns（Fig.2-B）.These results indicate that the primers used here were indeed specific to each homoeolog.

3.2.Differential expression of TaGASR7 homoeologs in immature seeds

To ensure that homoeolog-specific primers have comparable amplification efficiencies，Ct values were checked using genomic DNA of S3 plants and their progenitors.As shown in Fig.S2 the above homoeolog-specific primer pairs gave very similar threshold cycle numbers or Ct values on various samples with the same DNA concentrations in either diploid or polyploid samples，indicating that these primers were suitable for subsequentexpressionanalysis.Wethencomparedhomoeolog expression levelsinimmatureseedsofdifferentspecies（S3 and its progenitors PI 94655 and AS2404）.To do this，seeds at 6，11，14，17，21，and 25 days after flowering（DAF）were collected from a pool of 10-12 plants for eachspecies accordingto its flowering time.We found that GASR7B was highly expressed from 6 DAF until 14 DAF in developing seeds of the tetraploid accession PI94655 and began to decrease at 17 DAF.In comparison，theexpression of GASR7A started to decrease from 11 DAF，while in AS2404，the expression of GASR7D was repressed immediately after 6 DAF（Fig.3-A，B，C）.Similar expression patterns were found for each GASR7 homoeolog in the S3（Fig.3-D，E，F(xiàn)）.These data indicate that the three homoeologs of GASR7 were differentially regulated during seed development.

Fig.2-Design of GASR7 homoeolog-specific primers.A：Alignment of 3′UTR sequences of GASR7A，B，and D homoeologs.Green boxes indicate homoeolog-specific reverse primers corresponding to the homoeologs with their names at the beginning of the alignment.B：Specific amplification of homoeolog-specific primer pairs among Chinese Spring（CS）nulli-tetrasomic lines. N7AT7B refers to the nullisomic 7A-tetrasomic 7B line and similar naming rules apply to the remaining lines.The names of homoeolog-specific primer pairs are indicated on the right side and their sequences are listed in Table S1.

3.3.GASR7B dominates GASR7 expression in synthetic wheat

To study homoeolog expression regulation during wheat hexaploidizationwemeasuredtheexpressionlevelsof GASR7 homoeologs in S3 seeds at 6 DAF and compared the results with those of the parents PI 94655 and AS2404.GASR7B maintained high expression in the S3 synthetic similar to the maternal parent PI 94655，whereas GASR7A and GASR7D were further repressed in newly synthesized hexaploid wheat（Fig.4-A，B，C）.These data indicate that the three GASR7 homoeologsweredifferentiallyregulatedduringwheat polyploidization.This was confirmed in S3 11 DAF seeds by semi-quantitative RT-PCR where GASR7B was found to have higher expression，similar to that in its tetraploid progenitor（Fig.4-D），whereas expressions of GASR7A and GASR7D were much lower in S3 as in the progenitors PI 94655 and AS2404（Fig.4-D）.Since GASR7 belongs to the Snakin/GASA gene family，we wondered whether the homoeologs were inducible by gibberellic acid（GA）and which one might dominate GASR7 expression under this condition.We treated young spikes of S3 and S4 plants daily using 100 mg L-1GA3over 10 consecutive days from 2 to 11 DAF and collected seeds at 11 DAF for RNA extraction.Spikes sprayed with water at the same time were used as controls.As shown in Fig.4-E，all three GASR7 homoeologs were induced by GA treatment.The expression of GASR7B however became even more dominant in both S3 and S4 plants，indicating that GASR7B is more responsive to GA treatment than the other two homoeologs.

To gain further insight into the differential response of GASR7 homoeologs to GA，we isolated the promoter sequences.About 1500 bp of the GASR7A promoter regions was PCR amplified using primers designed from the genomic sequence of T.urartu［16］.To obtain the other two promoters，we screened a hexaploid wheat BAC library［25］using GASR7B-and GASR7D-specific primers because we were not able to amplify them using genomic DNA as templates.Two BAC clones were obtained and the promoter sequences for GASR7B and GASR7D were amplified through PCR and sequenced. The three promoter sequences were named pTaGASR7A，pTaGASR7B，and pTaGASR7D.We found that pTaGASR7B was more similar to pTaGASR7D（60.2%）than to pTaGASR7A（38.7%）.The promoter sequences were then subjected to cis-acting regulatory element prediction using the PLACE online service（http：//www.dna.affrc.go.jp/PLACE/signalscan. html）［27］.As shown in Fig.5 and Fig.S3，at least one GA-relatedcis-element was found for each promoter（pTaGASR7A，“Pyrimidine box”，PYRIMIDINEBOXOSRAMY1A；pTaGASR7B，“TATCCAC box”；TATCCACHVAL21；pTaGASR7D，“RY repeat motif”，RYREPEATVFLEB4），consistent with the GA responsive patterns of GASR7 homoeologs.Two additional GA-response ciselements，including a transcriptional enhancer，were found at pTaGASR7B（“TATCCAY”motif，TATCCAYMOTIFOSRAMY3D and“TATCCA”，TATCCAOSAMY），supporting the markedly increased expression of this homoeolog under GA treatment.These data suggest that genetic regulation plays a role in GASR7 homoeolog expression in polyploid wheat.

Fig.3-Expression patterns of three GASR7 homoeologs during seed development.A，B：TtGASR7A and TtGASR7B are the two homoeologs of T.turgidum accession PI94655.C：AeGASR7 expression pattern in diploid Ae.tauschii accession AS2404.D，E，F(xiàn)：Expression pattern of three TaGASR7 homoeologs in S3（AABBDD）.All values represent means±SD of n=3 independent determinations with two biological replicates.

3.4.Possible epigenetic regulation of GASR7 homoeologs during wheat polyploidization

Although the relative expression of GASR7D was lower than that of GASR7B in the synthetic，GASR7D was further repressed during hexaploidization（Fig.4-C，D）.Since sequences of the GASR7D promoter and coding region from the diploid progenitor AS2404 are identical to that in S3 synthetic，repression of GASR7D should contribute to epigenetic modification.To test this hypothesis，we studied the methylation status at the GASR7D promoter using the methylation-dependent restriction enzyme McrBC.PCR on DNAs pre-incubated with McrBC enzymes showed that two regions represented by P1（-1392 to-887）and P2（-928 to-446）were more digested in S3 than that in its progenitor AS2404，indicating increased DNA methylation in the GASR7D promoter after hexaploidization（Fig.6-A）.Such an increase in promoter DNA methylation may account for the repression of TaGASR7D during wheat hexaploidization.In other words，epigenetic modification may be indeed involved in the regulation of GASR7 expression.

However，we were not able to design pGASR7A-and pGASR7B-specific primers for the other two promoters，due to the lack of appropriate polymorphisms between them and therefore could not conduct PCR-based methylation assays on them.We then studied the presence of 24-nt siRNAs on these two promoters because siRNAs are indicators of DNA methylation status by mediating RNA-dependent DNA methylation（RdDM）.SiRNAs are also known to be important for the maintenance of chromatin stability and regulation of gene expression in interspecific hybrids and polyploids［31-33］. We searched the three promoter sequences against our small RNA dataset previously generated from immature seeds at 11 DAF［12］.A total of 152 24-nt siRNA（8.2 transcripts per million，or TPM，152/18，441，717）from S3 plants were mapped to pGASR7A（Tables 1 and S2）and nine 24-nt siRNA（0.5 TPM，9/ 18，441，717）were mapped to pTaGASR7D（Tables 1 and S3）whereas no siRNA was found in pTaGASR7B（Table 1）.A BLAST search against the wheat repeat sequence dataset showed that the region on pTaGASR7A that generated siRNAs（from -1250 nt to-1160 nt）corresponded to an unclassified repetitive sequence（TREP1579；Fig.6-B）.In light of the role of transposon-or repeat-derived 24-nt siRNA in DNA methylation［31］，it is possible that TaGASR7A is also regulated by means of epigenetic modification during wheat polyploidization.

Fig.4-Expression divergence of GASR7 homoeologs in synthetic wheat.A，B：Expression regulation of GASR7A（A）and GASR7B（B）in the tetraploid maternal parent T.turgidum（PI 94655）and the S3 synthetic at 6 DAF.C：Expression pattern of GASR7D in 6 DAF seeds from the diploid paternal parent Ae.tauschii（AS2404）compared to S3 synthetic plants.All values represent means±SD of n=3 independent determinations with two biological replicates.D：Semi-quantitative RT-PCR confirmation of GASR7 homoeolog expression patterns in seeds at 11 DAF.The tubulin gene was used as the endogenous control.E：Induction of TaGAR7 homoeolog expression by GA3treatment of S3 and S4 seeds at 11 DAF.-，H2O treatment；+，GA3treatment.

4.Discussion

Common wheat is a hexaploid species with genome composition of AABBDD originating from three putative diploid ancestral species：T.urartu，Aegilops speltoides and Ae. tauschii［34］.Allopolyploidization leads to the generation of duplicated homoeologous genes，whose expression needs to be re-balanced in a polyploid genome［20］.Evidence is accumulating that both genetic and epigenetic modifications may contribute to the divergence of expression of the three homoeologs in hexaploid wheat［18，20，21］.Divergent cis-element combinations in the promoters of two homoeologs of the wheat CDPK gene TaCPK2 were shown to contribute to functional differentiation，with TaCPK2A responding to powdery mildew infection and TaCPK2D response to cold stress［18］. On theotherhand，epigenetic modificationswereconsideredto contribute to the divergence of expression of three TaEXPA1homoeologs during wheat development［20］.An ability to produce new wheat synthetics provides opportunities to further study these regulatory processes by tracing homoeolog expression patterns in polyploid wheat［11，12］.

Fig.5-Distribution of GA related cis-elements in the GASR7A，B，and D promoter regions.Blue triangle，“Pyrimidine box”，PYRIMIDINEBOXOSRAMY1A；red triangle，“TATCCAY”motif，TATCCAYMOTIFOSRAMY3D；“TATCCAC box”，TATCCACHVAL21，and“TATCCA”，TATCCAOSAMY；purple triangle，“RY repeat motif”，RYREPEATVFLEB4.ATG，the start codon.

Fig.6-Epigenetic regulation of GASR7 homoeologs during wheat allohexaploidization.A：Locations of DNA fragments in the GASR7D promoter used for testing the methylation status（top，P1 and P2）and McrBC enzyme-based PCR assays of methylation status of genomic regions（bottom）.CKs，DNA templates not pre-incubated with McrBC.S3，third generation plants of the synthetic.B：Distribution of wheat repeat TREP159（the red line）and S3-derived 24-nt siRNAs（short lines below the region indicated by lines with left and right arrows）on pGASR7A and pGASR7D.

Similarto that inT.urartu，the A homoeolog of thehexaploid wheat GASR7 is also found to be associated with grain lengthdevelopment［17］.Since synthetic wheat line used in this work has slender seeds similar to its maternal parent the regulation of homoeolog expression of this gene in polyploid wheat and during wheat allohexaploidization is of great interest. By studying the promoter sequences of GASR7 homoeologs，we found that genetic composition may play a role in differential regulation of homoeolog expression.The presence of GA-responsive elements in promoters may explain the inducibility of GASR7 homoeologs by GA.However，epigenetic modification may provide alternative regulatory approaches as shown by changes of methylation status in the GASR7D promoter.Similar epigenetic modifications may also apply to GASR7A due to the presence of repeat-derived 24-nt siRNAs in its promoter.Inlight ofthe persistentlydominant expression of GASR7B，itispossiblethatTaGASR7Bmayalsoberelatedtograin length in common wheat.

Table 1-Distribution of 24-nt siRNAs in promoters of GASR7 homoeologs in S3 synthetic plants.

Recent work has shown that many structural changes in allohexaploid wheat may have taken place during allotetraploidization ［10，35，36］.Experiments with newly formed allotetraploid wheat revealed localized genomic changes and rapid changes in the copy number of gene homologs［37］.More recently，differential modifications of A and D subgenome transposable element（TE）-associated genes were found in the hexaploid wheat，indicating that RNA-dependent DNA methylation may be another regulatory mechanism during wheat allohexaploidization［12］.Additional mechanisms are expected when more information on gene expression patterns and regulation are available in the future.

Despite the presence of multiple homoeologs for one gene，there is a preference in the use of one homoeolog in polyploid plants［38］.Such a phenomenon may be due to the intrinsic mechanisms of basic genetic rules such as those highlighted by the gene dosage balance hypothesis［39］.The rules governing homoeolog interaction are pivotal for genetic manipulation in polyploid crops such as wheat，cotton，and canola.More work on genetic and epigenetic interactions of homoeoalleles should reveal new information on the modes of gene regulation in polyploids and should promote their application in crop breeding.

Acknowledgments

This work was supported by the Chinese National Natural Science Foundation（31271716），National High Technology Research and Development Program （2012AA10A308），and National KeyProgramon TransgenicResearch（2013ZX009-001）.

Supplementary material

Supplementary figures to this article can be found online at http：//dx.doi.org/10.1016/j.cj.2014.08.005.

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18 June 2014

in revised form 29 July 2014

s.

E-mail addresses：maolong@caas.cn（L.Mao），liaili@caas.cn（A.Li）.

Peer review under responsibility of Crop Science Society of China and Institute of Crop Science，CAAS.

1These authors contributed equally to this work.

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