Nucleotide diversity and molecular evolution of the WAG-2 gene in common wheat (Triticum aestivum L) and its relatives

In this work, we examined the genetic diversity and evolution of the WAG-2 gene based on new WAG-2 alleles isolated from wheat and its relatives. Only single nucleotide polymorphisms (SNP) and no insertions and deletions (indels) were found in exon sequences of WAG-2 from different species. More SNPs and indels occurred in introns than in exons. For exons, exons+introns and introns, the nucleotide polymorphism π decreased from diploid and tetraploid genotypes to hexaploid genotypes. This finding indicated that the diversity of WAG-2 in diploids was greater than in hexaploids because of the strong selection pressure on the latter. All dn/ds ratios were < 1.0, indicating that WAG-2 belongs to a conserved gene affected by negative selection. Thirty-nine of the 57 particular SNPs and eight of the 10 indels were detected in diploid species. The degree of divergence in intron length among WAG-2 clones and phylogenetic tree topology suggested the existence of three homoeologs in the A, B or D genome of common wheat. Wheat AG-like genes were divided into WAG-1 and WAG-2 clades. The latter clade contained WAG-2, OsMADS3 and ZMM2 genes, indicating functional homoeology among them.

The success of polyploid plants is often attributed to their genomic characteristics, including genomic stability, chromosomal rearrangement, genome size and differential gene expression (Soltics et al., 2003). In addition, nucleotide diversity is always generated in the course of polyploidy. Nucleotide diversity in wheat reflects the rich history of human selection and migration combined with a high level of recombination and out-breeding characteristic of this species. In the past decades, DNA markers such as RAPD, AFLP, and SSR have been extensively used for fingerprinting and exploring the genetic diversity and evolutionary relationships of wheat genetic resources. Although these molecular techniques are constantly being improved, in recent years several highly efficient and economic protocols have been introduced to differentiate closely related wheat species and their relatives.
Single nucleotide polymorphisms (SNP) are the most abundant type of variation in DNA sequences in all genomes studied to date (Brookes, 1999) and are considered last-generation molecular markers. SNPs are the most valuable molecular markers for research and application, including the detection of risk-associated alleles linked to human diseases, the study of evolutionary conservation among species, gene mapping and cloning, and crop breeding. The discovery of SNPs usually relies on re-sequencing alleles from different individuals or comparing the sequences to a reference sequence.
Wheat is the most important food crop in the world. The increase in the global human population and the very limited availability of suitable farmland make it essential to improve wheat yields. Wheat floral organs provide the ba-sis for grain formation such that wheat yield and quality are directly influenced by floral organ development. Hence, understanding wheat floral development is important for improving wheat yield. Past research has been limited to the morphological description and physiology of flowering, whereas recent developments in molecular genetics have led to studies of flower development at a molecular level. Some MADS-box cDNA sequences, such as WAP-1, WAP-2, WAP-3, WPI1, WPI2, WAG-1, WAG-2 and TaMADS1, have been identified (Murai et al., 1998(Murai et al., , 2002Hama et al., 2004;Zhao et al., 2006;Mizumoto et al., 2009;Shimada et al., 2009). Ciaffi et al. (2005) described the cloning and expression analysis of eight MADS-box cDNA sequences, and Zhao et al. (2006b) reported the characterization and expression of 42 wheat MADS-box gene sequences. Shitsukawa et al. (2007) undertook a detailed structural, expression and functional analysis of the homoeologous sequences of two wheat SEP-like genes. Paolacci et al. (2007) cloned 45 full-length MIKC-type MADS-box cDNA sequences of common wheat and studied their expression and function.
WAG -2, a class C MADS-box gene, has been isolated from the wheat expressed sequence tag (EST) database . Compared to other MADS-box genes, few studies have compared the sequences and investigated the evolutionary dynamics of WAG-2. In this study, we partially cloned the WAG-2 gene from wheat and its relatives and used the data (1) to analyze the sequence characteristics and investigate the inter-specific variability of the WAG-2 gene, and (2) to examine the relationship between the WAG-2 gene in common wheat and related species and other AG-like genes from different species.

PCR amplification and sequencing
The gene sequence data of WAG-2 (AB465688) were obtained from NCBI (http://www.ncbi.nlm.nih.gov). Specific primers used for partial amplification of the WAG-2 gene were designed with Primer Premier 5.0 (Clarke and Gorley 2001). The forward primer was WAG-2F (5'-CATTGAGAGGTACAAAAAGGCC-3') and the reverse primer was WAG-2R (5'-CCGTATGATTCCAGAA ACTTCC-3'). Figure 1 schematically shows the partial sequence of the WAG-2 gene and the location of the primers. Using the primers WAG-2F and WAG-2R, the region from exon II to exon VII of the WAG-2 gene was amplified in different species. PCR amplification was done in a thermocycler (My-Cycler; Bio-Rad, San Diego, CA, USA) in a volume of 50 mL containing approximately 100 ng of template DNA, 100 mM each of dNTP, 1.5 mM Mg 2+ , 1 U of Taq DNA polymerase, 0.4 mM of each primer and 1 x PCR buffer. The PCR cycling included pre-denaturation at 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 60°C for 1 min and 72°C for 2 min, and a final extension at 72°C for 5 min. The amplified products were visualized by gel electrophoresis in 1% agarose gels and then documented with a Gel Doc 2000TM system (Bio-Rad). The target DNA bands were recovered and purified from the gels using Qiaquick Gel extraction kits (QIAGEN, Shanghai, China). The purified PCR products were cloned in the p MD 19T vector according to the manufacturers instructions (Takata, Dalian, China). Transformants were plated on LB agar containing ampicillin. Clones with inserts were identified us- Wei et al. 607  ing blue/white colony selection. Positive clones were then screened and sequenced by Taihe Biotechnology Co. Ltd. (Beijing, China). In total, 56 clones of 11 accessions belonging to eight species were sequenced using the universal M13 forward and reverse primers. The partial nucleotide sequences of 15 WAG-2 gene haplotypes were submitted to GenBank at NCBI. The GenBank accession number of each nucleotide sequence is listed in Table 1.

Nucleotide diversity estimate
Fifteen partial WAG-2 gene sequences were verified and corrected. The complementary strands were assembled using DNAman 5.2.2 software (Lynnon Biosoft Company). To confirm whether the sequences were WAG-2 homoeologs, the coding regions were compared with WAG-2 gene sequences available in GenBank using BLAST searches (Altschul et al., 1990). Nucleotide sequence alignments were done using ClustalW (Thompson et al., 1994) and then optimized using SeaView version 4.1 (Galtier et al., 1996). The alignments were refined manually using the GeneDoc program (Nicholas and Nicholas, 1997) in order to maximize the positional homoeology. To reduce the possible effect of PCR artifacts, unique substitutions in single clones were ignored. Several sequences were represented by a single original sequence in alignments.
Exon+intron, exon, and intron sequences from diploid, tetraploid and hexaploid species were used to estimate nucleotide diversity based on the calculation of p as described by Tajima (1989). The value of p quantifies the mean percentage of nucleotide difference among all pairwise comparisons for a set of sequences. The p values of non-synonymous (dn) or synonymous (ds) differences and the mean dn/ds ratios were also estimated (Nielsen and Yang, 1998). These parameters were calculated using DnaSP 4.10.9 (Rozas et al., 2003).

Phylogenetic analysis
The 15 partial WAG-2 gene sequences were also used to construct phylogenetic trees. Neighbor-joining and maximum likelihood trees were generated with MEGA software version 5.0 (Saitou and Nei, 1987;Tamura et al., 2007) with 1000 bootstrap replicates. Gaps were treated as missing values.

Results
Characteristics of the partial WAG-2 gene sequences and genomic structure Partial sequences of the WAG-2 gene were amplified and sequenced from different species using the primers WAG-2F and WAG-2R. The sequences were deposited in GenBank under accession numbers JF330184 to JF330198. BLAST searches, which explored the coding region of each sequence, confirmed the cloned sequences to be homoeologs of the WAG-2 orthologs found in GenBank. Sequence comparisons showed that the 15 partial sequences of the species studied had similar structures, including six exons and five introns ( Figure 1). The exon+intron lengths ranged from 1824 to 1899 bp among species. The total length of the six exons studied was 425 bp. The five introns fulfilled the GT-AG rule, but they varied markedly in length and sequence composition. The length of intron II was 938-964 bp, which was longer than the other four introns. The lengths of intron III were 98, 95 and 94 bp for genomes AA, BB and DD, respectively. For AABB tetraploids (T. dicoccoides and T. turgidum ssp durum cv.) and the AABBDD hexaploid (T. aestivum) the length of intron III was 98 and 95 bp, respectively. Introns IV, V and VI also showed marked variations in length. The lengths of intron IV were 151, 193 or 206, and 231 bp for the AA, SS and DD genome diploids, respectively; two of these lengths (151 and 193 bp) were also observed for intron IV in AABB tetraploids (T. dicoccoides and T. turgidum ssp durum cv.) and the AABBDD hexaploid (T. aestivum), respectively.
Inter-specific variation in the partial sequences of the WAG-2 gene Table 3 summarizes the WAG-2 sequence variation identified in this study. The nucleotide variation in exons was generally less than in introns because of strong constraints.
In four haplotypes from two accessions of the hexaploid, 288 SNPs and 68 indels were identified from 7228 bp sequences (average of 1 SNP/25 bp and 1 indel/106 bp). A total of 288 SNPs were shared by 146 loci of the WAG-2 sequences cloned. Only eight SNPs (polymorphic rate of 1 SNP/213 bp) were detected in the exon region (Table 3). Seven SNPs (87.5% of total) were in exon IV. The intron region contained 280 SNPs (1 SNP/20 bp). All 68 indels were detected in the intron region. Forty indels were found in intron II and were involved in 18 sites (1 indel site/53 bp). Two indels located in intron III belonged to one site (1 indel site/96 bp). In intron IV, there were 24 indels shared by eight sites (1 indel site/21 bp). Finally, two indels in intron V were shared by one site (1 indel site/96 bp). The indel mutation of a single nucleotide in intron VI was detected only at one site; indel mutations at the other seven sites were mainly large fragment aberrations involving at least two nucleotides ( Figure S1). The high polymorphic rates of SNPs and large fragment aberrations meant that exon IV and intron IV were two hotspots for the WAG-2 gene in hexaploid genotypes.
496 SNPs and 113 indels were identified from 10,964 bp sequences in the six haplotypes from four tetraploid accessions, which yielded an average of 1 SNP/22 bp and 1 indel/97 bp, respectively (Table 3). Among the 33 SNPs in the exon region, 18 SNPs (54.5%) were in exon VII. 463 SNPs were detected in the intron region (1 SNP/18 bp). The intron region had 113 indels associated with 31 sites; intron IV had 35 indels with a higher frequency (1 indel site /21 bp) than the others. Consequently, highly polymorphic SNP was detected in exon VII. Intron IV also had a high frequency of indels ( Figure S1).
389 SNPs and 80 indels were identified in 9,236 bp sequences in five haplotypes from the five diploid accessions (1 SNP /24 bp and 1 indel /115 bp, respectively) (Table 3). The highest frequency of SNPs occurred in exon VII, which had 64.4% (18 out of 28) SNPs. The indel frequency in intron IV (1 indel site/24 bp) was higher than in the other four introns. Apparently, exon VII and intron IV are two mutation hotspots for the WAG-2 gene in diploid genotypes. Table 3 shows the nucleotide polymorphism p for synonymous differences, non-synonymous differences and Wei et al. 609   mean dn/ds in exons+introns, exons and introns. The nucleotide polymorphism p was lowest in hexaploids and highest in diploids. The values of p for synonymous differences were lowest in hexaploids (0.04245) and highest in diploids (0.06048). The values of p for non-synonymous differences were 0.00301, 0.00803 and 0.00663. The mean dn/ds for all coding regions was < 1. Point mutations involve transitions between purines (A and G) or pyrimidines (C and T), but may also involve transversions or changes between purines and pyrimidines. Table 4 shows the types of base substitutions in SNPs in different species. The exon+intron Ti/Tv ratio was 0.84:1, 0.88:1 and 0.96:1 for hexaploid, tetraploid and diploid genotypes, respectively. In exons, the main point mutations involved transitions, with Ti/Tv ratios of 3, 5.6 and 4.6. The ratio of A « G vs. C « T mutations was approximately 1 in hexaploid genotypes. In contrast, in tetraploid and diploid genotypes the main transition was A « G, with the ratio of A « G vs. C « T transitions being 2.1 and 1.3, respectively. In introns, the ratio of A « G vs. C « T transitions was 0.95 for hexaploid genotypes. The difference between tetraploid and diploid genotypes was that their ratios of A « G vs. C « T transitions were 1.14 and 1.07, respectively.
Based on the different species studied, 57 particular SNP mutations were identified, with seven (12.3%), 11 (19.3%) and 39 (68.4%) in hexaploid, tetraploid and diploid genotypes, respectively. Of the 39 SNPs, six SNPs were found in species of genome AA whereas 13 belonged to species of genome BB. Twenty SNPs were specific in species of genome DD. A common feature of these findings was that the main type of base substitution involved transitions among species. In exons, only transitions were detected among the species, and in introns the main substitutions in particular SNPs were also transitions. Seven SNPs from hexaploid genotypes were all transitions. Seven out of 11 SNPs were transitions from tetraploid genotypes. Diploid genotypes also had 24 SNPs, of which 66.7% were transitions. In addition, several specific SNPs shared by four sites were detected in groups A, B and D genomes.

Phylogenetic analyses of the partial sequences
The first series of phylogenetic analyses, which was done using the 15 WAG-2 gene sequences and the MEGA program, resulted in two phylogenetic trees generated by different algorithms. The topologies of the two phylogenetic trees were very similar to each other. A neighborjoining tree (Figure 2) indicated that the WAG-2 genes formed three major clades (I, II and III). Clade I, referred to as the A genome group of WAG-2 (although it may include the B genome), contained seven sequences from the AA diploid (T. monococcum, T. urartu), AABB tetraploid (T. turgidum, T. dicoccoides) and AABBDD hexaploid (T. aestivum) genomes. This clade was called WAG-2A because all of the species belonging to the clade contained the A genome. This clade also contained seven sequences with identical lengths in introns III (98 bp), IV (151 bp) and V (102 bp and 100 bp in T. aestivum). These seven sequences showed high sequence identity with 81 SNPs. Only 19 SNPs were shared by 12 sites in exons. Clade II, referred to as WAG-2B, contained seven sequences from the BB diploid (A. speltoides), AABB tetraploid (T. turgidum, T. dicoccoides) and AABBDD hexaploid (T. aestivum) genomes. In this clade, the lengths of introns III, IV and V in the seven sequences were 95, 193 and 92 bp, respectively. High sequence identities were also detected in the seven sequences with 45 SNPs, in which only two SNPs were shared by two sites in the exons. Lastly, clade III, re-610 WAG-2 gene diversity in wheat   Table 1. Bootstrap values are shown as percentages for 1000 replicates.
ferred to as WAG-2D, contained only one sequence from the DD diploid genome (A. tauschii).
To examine the relationships among wheat AG-like genes, the 15 WAG-2 genes and other AG group genes were analyzed phylogenetically. The neighbor-joining and maximum likelihood trees were reconstructed using amino acid sequences. The neighbor-joining tree is shown in Figure 3. Both phylogenetic trees were very similar to each other and differed only in the positions of ZAG1 (L18924) and OsMADS58 (FJ750942). The neighbor-joining tree indicated that the AG group was classified in dicot and monocot clades. Monocot genes were subdivided into WAG-1 and WAG-2 clades. The WAG-1 clade consisted of barley HvAG2, rice OsMADS58 and maize ZAG1, while the WAG-2 clade consisted of barley HvAG1, rice OsMADS3 and maize ZMM2. The WAG-2 clade was divided into three subclades: OsMADS3 and ZMM2 were included in subclade 1, WAG-2 (JF330184, AB465688), WM29A and TaAGL39 were clustered in subclade 2, and the other WAG-2 genes that were amplified in this study, along with WM29B and HvAG1, were clustered in subclade 3.

Discussion
Nucleotide diversity of WAG-2 in different species Most protein-coding nuclear genes of plants have exons and introns. Exons, which are usually under strong selection pressure that eliminates deleterious mutations, have a relatively slow rate of nucleotide substitution whereas, unlike chloroplast genes, the introns of nuclear genes have a higher rate of nucleotide substitution (Gaut, 1998). As shown here, all exons of the WAG-2 gene shared high sequence similarities. Only SNPs were found in the exons of different species; there were no indels. Compared with exons, the introns showed greater inter-specific polymorphism. In all species, there were more SNPs in introns than in exons because of the stronger selection pressure in the latter compared to the former.
The frequencies of SNPs and indels were analyzed in hexaploid, tetraploid and diploid species. In exons+introns, the frequency of SNPs among hexaploids (1 SNP/25 bp), tetraploids (1 SNP/22 bp) and diploids (1 SNP/24 bp) was similar. Likewise, there were no marked differences in the frequency of indels among hexaploids (1 indel/106 bp), tetraploids (1 indel /97 bp) and diploids (1 indel /117 bp). SNPs are the most abundant form of variation in all genomes studied to date. Plant SNPs in genomic regions are at least as abundant as in humans. Preliminary estimates range from 1 SNP/60 bp in outbreeding maize (Ching et al., 2002) to 1 SNP/300 bp for inbreeding rice and Arabidopsis (Schmid et al., 2003). Some plant species also display a large number of indels (Bhattramakki et al., 2002;Rafalski, 2002). In the present study, the frequencies of SNPs and indels among different species were higher than those reported in most other plants. This may reflect the fact that the exon+intron region investigated here corresponded to the most polymorphic region of WAG-2 genome DNA. This conclusion agrees with the detection of hotspot regions, such as exon IV in hexaploid species and exon VII in tetraploid and diploid species (high rate of SNP variation), and intron IV, which had a high indel mutation rate in different species.
Strong selection pressure is important in decreasing the nucleotide diversity of some plant species. Most studies on the effect of selection pressure on nucleotide diversity have focused on domesticated crops and on comparing the diversity between wild relatives and cultivars. During the selection of advantageous phenotypes, some crops appear to pass through bottlenecks that substantially reduce diversity (Doebley, 1992). In contrast, many grass domesticates have undergone moderate decreases in diversity compared with their wild relatives (Buckler et al., 2001). In the present study, the nucleotide diversity of the WAG-2 gene during the evolution of common wheat was estimated by Wei et al. 611 Figure 3 -Neighbor-joining phylogenetic tree of AG group MADS-box genes. Predicted amino acid sequences of the partial coding region were obtained from the NCBI: X53579 (AG) from Arabidopsis, (S53900) PLE from Antirrhinum, (L18924) ZAG1 and L81162 (ZMM2) from maize, L37528 (OsMADS3) and FJ750942 (OsMADS58) from rice, AF465688 (HvAG1) and AF486649 (HvAG2) from barley, AB084577 (WAG-1), AB465688 (WAG-2), AM502898 (WM29A), AM502898 (WM29B) and DQ (TaAGL39) from wheat. The GenBank accession numbers (JF330184 to JF330198) for the cloned WAG-2 sequences are shown in Table 1. Bootstrap values are shown as percentages for 1000 replicates.
calculating p using the method employed by Tajima (1989). For exons, p was 0.01812, 0.01788 and 0.01137, indicating that polymorphism was 1 SNP/55 bp, 1 SNP/56 bp and 1 SNP/88 bp between two homoeologous chromosomes in diploid, tetraploid and hexaploid species, respectively. For the exon+intron and intron regions, there was a common trend in that nucleotide polymorphism p decreased from diploid and tetraploid to hexaploid genotypes.
These results indicate that the nucleotide diversity of the WAG-2 gene in hexaploid species was lower than in diploid species. Hexaploid species probably suffered from long, strong artificial selection pressure that reduced nucleotide diversity. The dn/ds ratio represents the selection effect and its direction. A dn/ds ratio < 1.0 indicates negative selection and a conserved gene, whereas a dn/ds ratio > 1.0 indicates positive selection and a gene susceptible to rapid evolutionary change. All of the dn/ds ratios calculated here were < 1.0, indicating that WAG-2 is a conserved gene affected by negative selection (Nielsen, 2005). Furthermore, the dn/ds ratio was lowest among hexaploids and highest among diploids. This result implies that the WAG-2 gene experienced rapid changes in its genome during the evolution of common wheat. The selection pressure was weak among diploid and tetraploid species, whereas natural and artificial selection pressure were stronger among hexaploid species.
Transitions generally occur more frequently than transversions such that the rate of transitions in DNA sequences differs from that of transversions. In mammalian nuclear DNA, transition mutations are approximately twice as frequent as transversions (Gojobori et al., 1982;Li et al., 1984;Cargill et al., 1999;Lindblad-Toh et al., 2000;Rosenberg et al., 2003). In contrast to the moderate transition bias observed in mammalian nuclear DNA, transitions are approximately 15 times as frequent as transversions in human mitochondrial DNA (Brown et al., 1982;Tamura and Nei, 1993). Transition bias also occurs in plant nuclear DNA. Transitions are about twice as frequent as transversions in rice (Hayashi et al., 2004) and maize (Batley et al., 2003). As shown here, the Ts/Tv ratio of the WAG-2 gene partial sequences was nearly 1:1. The characteristics of transition bias suggest that the transition frequency was probably higher in other regions of the WAG-2 genome DNA. These regions may not have been detected and should be studied in future research based on the overall WAG-2 genome DNA. There was also a gradual decrease in the Ts/Tv ratio of the WAG-2 gene partial sequences, from diploid and tetraploid to hexaploid species, especially in the exon region. Additional investigations on how transitions and transversions occur in the WAG-2 gene of hexaploid wheat should improve our understanding of the molecular evolution of this gene. The A « G to C « T ratio was larger in tetraploid and diploid genotypes (> 1) than in hexaploid genotypes (approximately 1 or < 1). This finding implies a higher level of monthycation in hexaploid geno-types than in tetraploid or diploid species since most cytosines in CpG can be monthycated and changed into thymine through deamination. 57 specific SNP mutations were identified, of which 39 belonged to diploid species. Eight of 10 indels were also detected in diploid species. These SNPs and indels can be used as specific molecular markers. Those specific SNPs and indels in diploid species may persist for longer than in tetraploid or hexaploid species and have a long evolutionary history, which could explain why the number of specific SNPs and indel mutations decreases with the increase of ploidy. There is also the possibility that the specific SNPs and indel mutations may occur later than common variants. As suggested by population genetics theory, in contrast to common variants, rare variants are more likely to be recently derived and therefore more likely to be population-specific (Stephens et al., 2001).

Copy number of the WAG-2 gene
The partial sequences of the WAG-2 gene cloned from different species were assigned to three genomes: AA, BB and DD. The WAG-2 gene of common wheat (T. aestivum L.) can also be copied to A, B and D genomes. The degree of divergence in the length of introns among WAG-2 clones suggests conclusively that more than one copy of WAG-2 is present in hexaploid or tetraploid species (Table 2). In the A or B genome group of WAG-2, sequences with high identities were clustered in WAG-2A, WAG-2B and WAG-2D. A previous study by Paolacci et al. (2007) revealed the possibility of three homoeologs in the A, B or D genome of common wheat. Most nuclear genes are considered members of multi-gene families (Vision et al., 2000;Martin and Burg, 2002). The differences between two products within a taxon could be attributed to differences in the introns alone, as indicated elsewhere (Grob et al., 2004). This finding proves that the number of copies of WAG-2 is an important consideration for future work. Functional diversification of the three homoeologs should also be examined in subsequent research.

Phylogenetic relationships among AG-like genes
The two phylogenetic trees using different algorithms had very similar topologies, thus confirming the validity of the phylogenetic analysis. Most MADS-box genes are organized into distinct gene groups that share similar functions in regulating plant development (Purugganan et al., 1995;Theissen et al., 1996). Wheat AG-like genes were grouped into WAG-1 and WAG-2 clades (Figure 3), indicating that wheat AG orthologs were duplicated in monocot wheat species and showed functional differentiation. WAG-1, ZAG1, OsMADS58 and HvAG2 were clustered into one subgroup, suggesting similar functions among them. A loss of functional analysis indicates that maize ZAG1 determines the floral meristem (Mena et al., 1996). Mutant and transgenic studies indicate that OsMADS58 has 612 WAG-2 gene diversity in wheat an important role in determining floral meristem and in regulating carpel morphogenesis (Yamaguchi et al., 2006). Wheat WAG-1 transcripts preferentially accumulate in developing spikes during the late stages, suggesting that WAG-1 is involved in the development of floral organs (Meguro et al., 2003).
WAG-2 genes, together with OsMADS3 and ZMM2, were clustered in the WAG-2 clade (Figure 3), indicating functional homoeology among them. OsMADS3 plays a more predominant role in inhibiting lodicule development and in specifying stamen identity (Kang et al., 1995). ZMM2 participates in regulating the formation of stamens and carpels (Mena et al., 1996). WAG-2 has been isolated from a wheat EST database , however its function remains unknown. WAG-2 transcripts have been preferentially detected in the central region of pistils but not in developing stamens during floral organ development (Mizumoto et al., 2009). Future work should examine the functional role of WAG-2 in the development of the floral organ in wheat. Apart from the WAG-2 gene (JF330184), other WAG-2 genes (JF330185-JF330198) clustered with WM29B (AM502899) and HvAG1 (AF486648)in a single subgroup, probably because of their functional similarity and the high similarity in their amino acid sequences. Likewise, the WAG-2 gene (JF330184, AB465688), WM29A (AM502898) and TaAGL39 were clustered in a single subgroup, probably also because of the similarities in their amino acid sequences and functions. The WAG-2 clade clearly contains several different wheat genes clustered into two subgroups in diploid, tetraploid and hexaploid species. Further study will show whether this sequence diversity involves different functions.