T H E C R O P J O U R N A L 3 ( 2 0 1 5 ) 1 – 9

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Divergence in homoeolog expression of the grain

length-associated gene GASR7 during wheatallohexaploidization

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, ChinabTriticeae Research Institute, Sichuan Agricultural University, Chengdu 611130, ChinacThe 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

⁎ Corresponding authors.E-mail addresses: maolong@caas.cn (L. MPeer review under responsibility of Crop S

1 These authors contributed equally to this

http://dx.doi.org/10.1016/j.cj.2014.08.0052214-5141/© 2014 Crop Science Society of Chreserved. This is an open access article unde

A B S T R A C T

Article history:Received 18 June 2014Received in revised form 29 July 2014Accepted 29 August 2014Available online 5 October 2014

Hexaploid wheat has triplicated homoeologs for most of the genes that are located insubgenomesA, B, andD.GASR7, amember of theSnakin/GASAgene family, has beenassociatedwith grain length development in wheat. However, little is known about divergence of itshomoeolog expression in wheat polyploids. We studied the expression patterns of the GASR7homoeologs in immature seeds in a synthetic hexaploid wheat line whose kernels are slenderlike those of itsmaternal parent (Triticum turgidum, AABB, PI 94655) in contrast to the round seedshapeof its paternal progenitor (Aegilops tauschii, DD,AS2404).We found that the BhomoeologofGASR7 was the main contributor to the total expression level of this gene in both the maternaltetraploid progenitor and the hexaploid progeny, whereas the expression levels of the A and Dhomoeologsweremuch lower. To understand possiblemechanisms regulating differentGASR7homoeologs, we firstly analyzed the promoter sequences of three homoeologous genes andfound that all of them contained gibberellic acid (GA) response elements, with the TaGASR7Bpromoter (pTaGASR7B) uniquely characterized by an additional predicted transcriptionalenhancer. This was confirmed by the GA treatment of spikes where all three homoeologswere induced,with amuch stronger response for TaGASR7B. McrBC enzymeassays showed thatthe methylation status at pTaGASR7D was increased during allohexaploidization, consistentwith the repressed expression of TaGASR7D. For pTaGASR7A, the distribution of repetitivesequence-derived 24-nucleotide (nt) small interferingRNAs (siRNAs)were foundwhich suggestspossible epigenetic regulation because 24-nt siRNAs are known to mediate RNA-dependentDNAmethylation. Our results thus indicate that both genetic and epigenetic mechanismsmaybe 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 CCBY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Keywords:TaGASR7Gibberellic acidTriticum aestivumPolyploidy

ao), liaili@caas.cn (A. Li).cience Society of China and Institute of Crop Science, CAAS.work.

ina and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. All rightsr the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

2 T H E C R O P J O U R N A L 3 ( 2 0 1 5 ) 1 – 9

1. Introduction

Polyploidy leads to whole-genome duplication and iswidespread in the plant kingdom [1,2], providing opportu-nities for duplicated genes to diverge in several evolutionaryways such as subfunctionalization, neofunctionalization, andnonfunctionalization (deletion or pseudogenization) [3,4], as wellas genetic and epigenetic interactions between homoeoalleles[1,5]. Bread wheat or common wheat (Triticum aestivum) is ahexaploid (2n = 6x = 42, AABBDD) that arose 7000–12,000 yearsago following the hybridization of the early-domesticatedallotetraploid T. turgidum ssp. dicoccon (AABB) and the diploidgoat grass Aegilops tauschii (2n = 2x = 14, DD) [6–9]. As one of themost important staple crops, common wheat is also a goodmodel for studying genetic interactions between multiplerelated genomes. New allohexaploid synthetic wheat can bereadily synthesized in the laboratory by interspecific hybridiza-tion [10] and chromosome doubling, facilitating the study ofgenetic, functional, and epigenetic changes during this process.Previously, a series of allohexaploid wheat lines were generatedby hybridizing T. turgidum ssp. dicoccon accession PI 94655 withthe diploid Ae. tauschii ssp. strangulata accession AS2404 as apaternal parent [11]. The synthetic predominantly resembledT. turgidum in grain length (Fig. 1), implying, as far as the graindevelopment is concerned, the genetic architecture of hexaploidprogeny 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 tissueand temporal specificity. Most Snakin/GASA genes are regu-lated by hormones and participate in signaling pathwaysthat modulate hormonal responses and levels. In Arabidopsis,GASA4 plays a role in regulating floral meristem identityand promotes the development of seed size and seed weight[14]. In rice, OsGASR7 (Os06g0266800), which is highly similarto Arabidopsis GASA4, was identified as a candidate genedetermining grain length [15]. Inwheat, a homolog ofArabidopsisGASA4 and rice OsGASR7 was also involved in grain lengthdevelopment [16,17]. In Triticum urartu (2n = 2x = 14, AA), theputative A genome donor of common wheat and twohaplotypes (H1 and H2) of TuGASR7 were identified among92 diverse accessions collected from different regions,among which H1 was found to be significantly associatedwith greater values of grain length and grain weight. Incommon wheat, TaGASR7-A1 was shown to be a majorgenetic determinant of grain length, with pleiotropic effectson grain weight and yield [17].

Fig. 1 – The slender seeds of synthetic allohexaploid wheat andtetraploid maternal parent; AS2404, Ae. tauschii (DD), paternal prBar = 1 cm.

Despite triplication of many genes in hexaploid wheat,not all homoeoalleles are equally transcribed. Transcriptionaldivergence of homoeologous genes has been reported. Ourprevious work showed that the A homoeolog of wheatcalcium-dependent protein kinase (CDPK) gene TaCPK2-Awas inducible by Blumeria graminis f. sp. tritici (Bgt) infectionwhereas TaCPK2-D mainly responded to cold treatment, sug-gesting subfunctionalization of these homoeologs [18]. Similar-ly, the homoeologs of methyl-binding domain protein genesTaMBD2-5B and TaMBD2-5D were highly responsive to saltstress, but in the seedling leaves only TaMBD2-5B was specifi-cally up-regulated by low temperature [19]. Although all threehomoeologs of a wheat expansin gene (TaEXPA1) were silencedin wheat seedling roots, TaEXPA1-A and TaEXPA1-D wereexpressed in seedling leaves, indicating tissue specificity ofhomoeolog expression [20]. On the other hand, among thethreehomoeologs of thewheat SEPALLATA-likeMADS-boxgeneWLHS1, only WLHS1-D was functional. The protein structureof WLHS1-A is interrupted by a DNA insertion whereasWLHS1-B is predominantly silenced by cytosine methylation,suggesting different evolutionary effects that lead to divergencein homoeologous gene expressions [21].

We are interested in understandinghowexpressions ofwheatGASR7 homoeologs are regulated in polyploid wheat and theirdifferential regulation during wheat polyploidization becausehomoeologs of GASR7 are associated with grain length develop-ment and hexaploid synthetic wheat has similar grain shape toits tetraploid progenitor. Towards this end, we examined theexpression patterns of wheat GASR7 homoeologs in immatureseeds of several generations of a synthetic wheat line and itsprogenitors and studied the promoter sequences of all threehomoeologs. We found that both genetic and epigenetic mech-anisms may be involved in the expression divergence of GASR7homoeologs in polyploid wheat.

2. Materials and methods

2.1. Plant materials and GA treatment

A set of newly synthesized hexaploid wheat lines and theirprogenitors were used, including the third and the fourthgeneration plants of self-pollinated synthetic wheat (S3and S4) and their progenitors the T. turgidum ssp. dicocconaccession 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,

PI94655

AS2404

S3

its tetraploid progenitor. PI 94655, T. turgidum (AABB),ogenitor; third generation (S3) synthetic plants (AABBDD).

3T H E C R O P J O U R N A L 3 ( 2 0 1 5 ) 1 – 9

transplanted into soil, and grew in a controlled environment[24]. Immature seeds were collected from a pool of 10–12plants for each genotype at 6, 11, 14, 17, 21, and 25 days afterflowering (DAF) and were stored in liquid nitrogen until RNAextraction. Samples were prepared for two consecutive yearsin the same experimental field as two biological replicates.For gibberellic acid (GA) treatment, young spikes of S3 andS4 plants were sprayed with 100 mg L−1 GA3 once a day for10 days from 2 to 11 DAF. Spikes sprayed with water wereused as controls.

2.2. RNA extraction and cDNA 3′ RACE

Total RNA was isolated from seeds using a Trizol-based RNAisolation protocol (Life Technologies, NY, USA). First-strandcDNAwas synthesized using 2 μgDNase-treated total RNAwitholigo (dT) primers (Promega, WI, USA). Full-length cDNAs wereobtained by PCR using primers TaGASR7-F and TaGASR7-Rdesigned according to EST sequences (see text and Table S1).PCR products were subcloned and sequenced. The 3′ UTR ofGASR7 homoeologous genes were cloned using a SMART-rapidamplification of cDNA end kit (SMART-RACE; Clontech, Moun-tain View, CA, USA) following the manufacturer's protocol.Primers for 3′ RACE and homoeolog-specific PCR are also listedin 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 nucleotidepolymorphisms of cDNA sequences of the three TaGASR7homoeologs (Table S1). Amplification efficiency of primerswas detected according to the method described by Hu et al.[20]. For homoeolog-specific expression analysis, tubulin andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) geneswere used as endogenous controls for semi-quantitative andreal time PCR, respectively.

2.4. BAC library screening to obtain promoter sequences ofGASR7 homoeologs

To isolate thepromoter sequences ofGASR7B andDhomoeologs,a BAC library from T. aestivum cv. Renan was screened by PCRusing a BAC clone pooling approach [25]. Two BAC clones wereobtained using primers designed from the coding regions ofTaGASR7B and TaGASR7D. Promoter sequences of TaGASR7B/Dwere obtained by PCR from the BAC clones using primersdesigned from the 5× Chinese Spring genome sequences (TableS1) [26] and sequenced.

2.5. cis-element prediction and siRNA distribution analysis

DNA fragments encompassing the 1.5 kb upstream the startcodon and part of the coding region were amplified using PCR.By comparing the coding sequences, three promoters werediscriminated into pTaGASR7A, pTaGASR7B, and pTaGASR7D.cis-acting regulatory elements were predicted from the PLACEonline 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 distribu-tion was identified by BlastN [28] search against the smallRNA dataset [12] using promoter sequences as queries.

2.6. McrBC enzyme assay

Total genomic DNAwas extracted using a DNeasy PlantMini kit(Qiagen) following the manufacturer's instructions. PCR-basedDNA methylation assays were conducted as described [29]. Atleast three independent biological replicateswere performedonpTaGASR7D using primers listed in Table S1.

3. Results

3.1. Acquirement of TaGASR7 homoeolog-specific primers

ABlastN search using the T. urartu GASR7 gene (TuGASR7) [16] asa query against the PlantGDB Chinese Spring EST assemblyretrieved three ESTs, PUT-163b-Triticum_aestivum-2301165442,2301165443, and 147657 that contained open reading frames(ORF) of 306, 300, and 285 bp, respectively. A comparison ofT. urartu and Ae. tauschii GASR7 sequences [30] showed thatthe 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′ UTRsequences that were 216 (A), 233 (B), and 259 bp (D) and werequite polymorphic (Fig. 2-A). Homoeolog-specific reverse primerswere 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, thesehomoeolog-specific reverse primers amplified only two of thethree bands in Chinese Spring (CS) nulli-tetrasomic lines thatlacked the one on corresponding missing chromosomes. Asshown in Fig. 2-B, the primer pair TaGASR7-4F/A-4R amplified aband from all nulli-tetrasomic lines except N7AT7B andN7AT7Din which chromosome 7A was missing. The other two primercombinations (TaGASR7-4F/B-4R and TaGASR7-4 F/D-5R) alsogave expected amplification patterns (Fig. 2-B). These resultsindicate that the primers used here were indeed specific to eachhomoeolog.

3.2. Differential expression of TaGASR7 homoeologsin immature seeds

To ensure that homoeolog-specific primers have comparableamplification efficiencies, Ct values were checked using geno-mic DNA of S3 plants and their progenitors. As shown in Fig. S2the above homoeolog-specific primer pairs gave very similarthreshold cycle numbers or Ct values on various samples withthe same DNA concentrations in either diploid or polyploidsamples, indicating that these primers were suitable forsubsequent expressionanalysis.We then comparedhomoeologexpression levels in immature seeds of different species (S3 andits progenitors PI 94655 and AS2404). To do this, seeds at 6, 11,14, 17, 21, and 25 days after flowering (DAF) were collected froma pool of 10–12 plants for each species according to its floweringtime. We found that GASR7B was highly expressed from 6 DAFuntil 14 DAF in developing seeds of the tetraploid accessionPI94655 and began to decrease at 17 DAF. In comparison, the

A

B

TaGASR7A-4F/A-4R

TaGASR7B-4F/B-4R

TaGASR7D-4F/D-5R

Fig. 2 –Design of GASR7 homoeolog-specific primers. A: Alignment of 3′UTR sequences of GASR7A, B, and D homoeologs. Greenboxes indicate homoeolog-specific reverse primers corresponding to the homoeologs with their names at the beginning of thealignment. 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 ofhomoeolog-specific primer pairs are indicated on the right side and their sequences are listed in Table S1.

4 T H E C R O P J O U R N A L 3 ( 2 0 1 5 ) 1 – 9

expression of GASR7A started to decrease from 11 DAF, while inAS2404, the expression of GASR7D was repressed immediatelyafter 6 DAF (Fig. 3-A, B, C). Similar expression patterns werefound for each GASR7 homoeolog in the S3 (Fig. 3-D, E, F). Thesedata indicate that the three homoeologs of GASR7 weredifferentially regulated during seed development.

3.3. GASR7B dominates GASR7 expression in synthetic wheat

To study homoeolog expression regulation during wheathexaploidization we measured the expression levels ofGASR7 homoeologs in S3 seeds at 6 DAF and compared theresults with those of the parents PI 94655 and AS2404. GASR7Bmaintained high expression in the S3 synthetic similar to thematernal parent PI 94655, whereas GASR7A and GASR7D werefurther repressed in newly synthesized hexaploid wheat(Fig. 4-A, B, C). These data indicate that the three GASR7homoeologs were differentially regulated during wheatpolyploidization. This was confirmed in S3 11 DAF seeds bysemi-quantitative RT-PCR where GASR7B was found to havehigher expression, similar to that in its tetraploid progenitor(Fig. 4-D), whereas expressions of GASR7A and GASR7D weremuch lower in S3 as in the progenitors PI 94655 and AS2404(Fig. 4-D). Since GASR7 belongs to the Snakin/GASA genefamily, we wondered whether the homoeologs were inducibleby gibberellic acid (GA) and which one might dominate GASR7

expression under this condition. We treated young spikesof S3 and S4 plants daily using 100 mg L−1 GA3 over 10consecutive days from 2 to 11 DAF and collected seeds at 11DAF for RNA extraction. Spikes sprayed with water at thesame time were used as controls. As shown in Fig. 4-E, allthree GASR7 homoeologs were induced by GA treatment. Theexpression of GASR7B however became even more dominantin both S3 and S4 plants, indicating that GASR7B is moreresponsive to GA treatment than the other two homoeologs.

To gain further insight into the differential response ofGASR7 homoeologs to GA, we isolated the promoter se-quences. About 1500 bp of the GASR7A promoter regions wasPCR amplified using primers designed from the genomicsequence 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 toamplify them using genomic DNA as templates. Two BACclones were obtained and the promoter sequences for GASR7Band GASR7D were amplified through PCR and sequenced.The three promoter sequences were named pTaGASR7A,pTaGASR7B, and pTaGASR7D. We found that pTaGASR7Bwas more similar to pTaGASR7D (60.2%) than to pTaGASR7A(38.7%). The promoter sequences were then subjected tocis-acting regulatory element prediction using the PLACEonline service (http://www.dna.affrc.go.jp/PLACE/signalscan.html) [27]. As shown in Fig. 5 and Fig. S3, at least one GA-related

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TtGASR7A

D7RSAGaTB7RSAGaTA7RSAGaT

TtGASR7B AeGASR7Re

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pres

sion

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l

Days a�er flowering (DAF)

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6 11 14 17 21 25

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

5T H E C R O P J O U R N A L 3 ( 2 0 1 5 ) 1 – 9

cis-element was found for each promoter (pTaGASR7A, “Pyrimi-dine box”, PYRIMIDINEBOXOSRAMY1A; pTaGASR7B, “TATCCACbox”; TATCCACHVAL21; pTaGASR7D, “RY repeat motif”,RYREPEATVFLEB4), consistent with the GA responsive pat-terns of GASR7 homoeologs. Two additional GA-response cis-elements, including a transcriptional enhancer, were found atpTaGASR7B (“TATCCAY” motif, TATCCAYMOTIFOSRAMY3Dand “TATCCA”, TATCCAOSAMY), supporting the markedlyincreased expression of this homoeolog under GA treat-ment. These data suggest that genetic regulation plays a role inGASR7 homoeolog expression in polyploid wheat.

3.4. Possible epigenetic regulation of GASR7 homoeologsduring wheat polyploidization

Although the relative expression of GASR7D was lower thanthat of GASR7B in the synthetic, GASR7Dwas further repressedduring hexaploidization (Fig. 4-C, D). Since sequences of theGASR7D promoter and coding region from the diploid progen-itor AS2404 are identical to that in S3 synthetic, repression ofGASR7D should contribute to epigenetic modification. To testthis hypothesis, we studied the methylation status at theGASR7D promoter using the methylation-dependent restric-tion enzyme McrBC. PCR on DNAs pre-incubated with McrBCenzymes showed that two regions represented by P1 (−1392to −887) and P2 (−928 to −446) were more digested in S3than that in its progenitor AS2404, indicating increased DNAmethylation in the GASR7D promoter after hexaploidization

(Fig. 6-A). Such an increase in promoter DNA methylationmay account for the repression of TaGASR7D during wheathexaploidization. In other words, epigenetic modificationmay be indeed involved in the regulation of GASR7 expression.

However, we were not able to design pGASR7A- andpGASR7B-specific primers for the other two promoters, due tothe lack of appropriate polymorphisms between them andtherefore could not conduct PCR-based methylation assays onthem. We then studied the presence of 24-nt siRNAs on thesetwo promoters because siRNAs are indicators of DNA methyl-ation status by mediating RNA-dependent DNA methylation(RdDM). SiRNAs are also known to be important for the main-tenance of chromatin stability and regulation of geneexpression in interspecific hybrids and polyploids [31–33].We searched the three promoter sequences against our smallRNA dataset previously generated from immature seeds at 11DAF [12]. A total of 152 24-nt siRNA (8.2 transcripts per million,or TPM, 152/18,441,717) from S3 plants were mapped topGASR7A (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 BLASTsearch against the wheat repeat sequence dataset showedthat the region on pTaGASR7A that generated siRNAs (from−1250 nt to −1160 nt) corresponded to an unclassifiedrepetitive sequence (TREP1579; Fig. 6-B). In light of the role oftransposon- or repeat-derived 24-nt siRNA in DNAmethylation[31], it is possible that TaGASR7A is also regulated by means ofepigenetic modification during wheat polyploidization.

D7RSAGaTB7RSAGaTA7RSAGaT

Rela

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TaGASR7A

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TaGASR7D

Tubulin

GA - + +S3 S3 S4 S4

GASR7A

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Tubulin

D E-

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 6DAF seeds from the diploid paternal parent Ae. tauschii (AS2404) compared to S3 synthetic plants. All values representmeans ± SD of n = 3 independent determinations with two biological replicates. D: Semi-quantitative RT-PCR confirmation ofGASR7 homoeolog expression patterns in seeds at 11 DAF. The tubulin gene was used as the endogenous control. E: Inductionof TaGAR7 homoeolog expression by GA3 treatment of S3 and S4 seeds at 11 DAF. −, H2O treatment; +, GA3 treatment.

6 T H E C R O P J O U R N A L 3 ( 2 0 1 5 ) 1 – 9

4. Discussion

Common wheat is a hexaploid species with genome com-position of AABBDD originating from three putative diploidancestral species: T. urartu, Aegilops speltoides and Ae.tauschii [34]. Allopolyploidization leads to the generation ofduplicated homoeologous genes, whose expression needsto be re-balanced in a polyploid genome [20]. Evidence is

100 bp

-433

-1025

CCTTTT

TATCCAYTATCCACTATCCA

Fig. 5 – Distribution of GA related cis-elements in the GASR7A, B,PYRIMIDINEBOXOSRAMY1A; red triangle, “TATCCAY” motif, TATATCCACHVAL21, and “TATCCA”, TATCCAOSAMY; purple triancodon.

accumulating that both genetic and epigenetic modifica-tions may contribute to the divergence of expression of thethree homoeologs in hexaploid wheat [18,20,21]. Divergentcis-element combinations in the promoters of two homoeologsof the wheat CDPK gene TaCPK2 were shown to contribute tofunctional differentiation, with TaCPK2A responding to pow-derymildew infection and TaCPK2D response to cold stress [18].On the other hand, epigeneticmodificationswere considered tocontribute to the divergence of expression of three TaEXPA1

pTaGASR7B

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CATGCATG

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and D promoter regions. Blue triangle, “Pyrimidine box”,TCCAYMOTIFOSRAMY3D; “TATCCAC box”,gle, “RY repeat motif”, RYREPEATVFLEB4. ATG, the start

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0

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TaGASR7A24-nt siRNAs covering ~302 bp

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Fig. 6 – Epigenetic regulation of GASR7 homoeologs duringwheat allohexaploidization. A: Locations of DNA fragmentsin the GASR7D promoter used for testing the methylationstatus (top, P1 and P2) and McrBC enzyme-based PCR assaysof methylation status of genomic regions (bottom). CKs, DNAtemplates not pre-incubated with McrBC. S3, third genera-tion plants of the synthetic. B: Distribution of wheat repeatTREP159 (the red line) and S3-derived 24-nt siRNAs (shortlines below the region indicated by lines with left and rightarrows) on pGASR7A and pGASR7D.

7T H E C R O P J O U R N A L 3 ( 2 0 1 5 ) 1 – 9

homoeologs during wheat development [20]. An ability toproduce new wheat synthetics provides opportunities tofurther study these regulatory processes by tracing homoeologexpression patterns in polyploid wheat [11,12].

Similar to that in T. urartu, the A homoeolog of the hexaploidwheat GASR7 is also found to be associated with grain length

Table 1 – Distribution of 24-nt siRNAs in promoters ofGASR7 homoeologs in S3 synthetic plants.

Number of mapped 24-ntsiRNAs

Transcript parts permillion

pGASR7A 152 8.2pGASR7B 0 0pGASR7D 9 0.5

Total small RNA reads: 18,441,717.

development [17]. Since synthetic wheat line used in this workhas slender seeds similar to its maternal parent the regulationof homoeolog expression of this gene in polyploid wheatand during wheat allohexaploidization is of great interest.By studying the promoter sequences of GASR7 homoeologs,we found that genetic composition may play a role indifferential regulation of homoeolog expression. The presenceof GA-responsive elements in promoters may explain theinducibility of GASR7 homoeologs by GA. However, epigeneticmodification may provide alternative regulatory approachesas shown by changes of methylation status in the GASR7Dpromoter. Similar epigenetic modifications may also apply toGASR7A due to the presence of repeat-derived 24-nt siRNAs inits promoter. In light of the persistently dominant expression ofGASR7B, it is possible that TaGASR7Bmayalso be related to grainlength in common wheat.

Recent work has shown that many structural changesin allohexaploid wheat may have taken place duringallotetraploidization [10,35,36]. Experiments with newlyformed allotetraploid wheat revealed localized genomicchanges and rapid changes in the copy number of genehomologs [37]. More recently, differential modifications ofA and D subgenome transposable element (TE)-associatedgenes were found in the hexaploid wheat, indicating thatRNA-dependent DNA methylation may be another regulatorymechanism during wheat allohexaploidization [12]. Additionalmechanisms are expected when more information on geneexpression 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 polyploidplants [38]. Such a phenomenon may be due to the intrinsicmechanisms of basic genetic rules such as those highlighted bythe gene dosage balance hypothesis [39]. The rules governinghomoeolog interaction are pivotal for genetic manipulation inpolyploid crops such as wheat, cotton, and canola. More workon genetic and epigenetic interactions of homoeoalleles shouldreveal new information on the modes of gene regulation inpolyploids and should promote their application in cropbreeding.

Acknowledgments

This work was supported by the Chinese National NaturalScience Foundation (31271716), National High TechnologyResearch and Development Program (2012AA10A308), andNational Key Programon Transgenic Research (2013ZX009-001).

Supplementary material

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

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