Cloning, Chromosomal Location And Differential Expression Of A Flower Development Gene WAG-2in Common Wheat And Its Relatives

Wheat is an important crop, providing staple food for about one-third population in the world. As the basis for grain formation, floral organ development directly controls wheat yield and quality. AGAMOUS gene plays a vital role in floral organ formation and differentiation. Study on AG gene structure, expression and function has a potential application prospect for seed plant breeding. Wheat AG-like gene WAG-2, a class C MADS-box gene acts to specify wheat floral organ development, especially the pistil, carpel, ovule. In this study, using molecular biology and bioinformatics technology, WAG-2genes structure, sequences diversity, evolutionary dynamic, chromosomal location, expression pattern were discussed. The research results will lay the foundation for further studying on WAG-2gene function in wheat floral organs, add new data for wheat flower development, also has guiding significance for wheat breeding. The main results are summarized as follows:1. The full-length cDNA sequence of WAG-2was cloned from five accessions or varieties by RACE and RT-PCR. Fifteen allelic types of WAG-2were obtained. A total of38SNPs loci result in20sites amino acid residue variations. In addition,2sites of Indel mutation were detected. Fifteen WAG-2allelic variations encode13proteins with different amino acid sequences. The molecular weight of13WAG-2protein is30.83-31.39kDa with theoretical PI=8.99-9.23. Most amino acids of13WAG-2proteins are hydrophilic amino acids, so they may be soluble protein. Protein post-translational phosphorylation sites modification is more in Ser than Thr and Tyr residues. Glycosylation is mainly N-glycosylation site, but no O-glycosylation site. Secondary structures of13WAG-2proteins are mainly composed of α-helices, random coils, extended band and β-turn. The phylogenetic tree indicates that monocot class C gene family is separated into two groups, WAG-1clade and WAG-2clade. The WAG-2clade insists of barley HvAG2, rice OsMADS3, which suggests that the genes in the WAG-2clade have more similar function to dicot class C genes.2. Fifty sequences of WAG-2gene were characterized from29wheat accessions using overlapping primers. Sequence comparisons show that WAG-2genes of different species have similar structures, including seven exons and six introns. The nucleotide polymorphism π in coding region (0.00771-0.01108) is lower than that in non-coding region (0.03795-0.05728), which suggests that genetic variation in coding region is smaller than that in non-coding region because of stronger selection pressure in the coding region. For all region and coding region, the nucleotide polymorphism π is higher in tetraploid and hexaploid species than that in diploid species. Homologous genes sequences from A, B or D genome have a certain degree of polymorphism, which causes higher nucleotide polymorphism in tetraploid and hexaploid species. For the cultivation species, when the SNPs and Indels are eventually survived under the effects of natural selection, artificial selection and domestication, they may help individuals successfully evolve in some way. Ka/Ks values (0.12-0.41) of WAG-2are all<1, which indicates that WAG-2is a conservative gene. The Ti/Tv are2.0(AA genome species),1.73(SS genome specie),1.5(DD genome species),1.34(tetraploid species) and1.27(hexaploid species), which suggests that WAG-2genes have different evolution level in different species. The results of cluster analysis show that50WAG-2gene sequences are clustered into three groups. Group I named A contain all sequences from AA genome diploids and sixteen sequences containing genome A from AABB tetraploid and AABBDD hexaploid. The WAG-2gene from T. urartu may be more primitive. Group II named B contain all sequences from the BB genome diploids and sixteen sequences containing genome B from AABB tetraploid and AABBDD hexaploid. The WAG-2genes of tetraploid and hexaploid wheat may be provided by the one in Ae. speltoides PI486262. The WAG-2genes from SS genome have closer genetic relationship with those from DD genome species, probably because they belong to the Aegilops. Group III named D contain four sequences from the DD genome diploids AS75, Y207, Y172and RM188.3. The lengths of intron4of WAG-2gene are significantly different among A, B, D genome. For diploid, tetraploid and hexaploid genome species, the lengths of intron4are151,193or206and231bp respectively in genome A, S/B and D. In this study, three accessions from T.urartu, Ae. speltoides, Ae.tauschii (considered to be the donor of common wheat) and wild emmer wheat (T. dicoccoides), Chinese spring were selected to amplify intron4using primers designed in both sides of the exon. The results demonstrate that only one band was detected in T.urartu, Ae. speltoides and Ae.tauschii. Two bands corresponding to the ones from T.urartu, Ae. speltoides were obtained in emmer wheat. Three bands corresponding to the ones from three diploid donor species were detected in Chinese spring. For group3Nulli-tetra (NT) lines and Ditelosomic (DT) lines Dt3AL, Dt3BL, Dt3DL, two bands were detected and one band respectively corresponding to A, B, D genome was missing. These results indicate that in the wheat genome there are three homologous WAG-2genes located on the group3chromosomes short arm.4. The results of WAG-2gDNA and cDNA sequences alignment show that WAG-2is an alternative splicing gene. The alternative splicing loci locates in boundary between intron3and exon4, and WAG-2belongs to5’alternative splicing gene. In each species, WAG-2transcripts are differentially expressed, which suggests they have certain differentiation in function. The expression level of WAG-2A and WAG-2F are higher in PI428181(T.urartu) and465(Ae.tauschii) respectively and all reach the peak at anther seperation stage. Both WAG-2A and WAG-2F are produce by the alternative splicing type1, which indicate alternative splicing type1is the main splice form in PI428181(T.urartu) and465(Ae.tauschii) and contributes more to formation, development of staments and pistils. The expression of WAG-2C and WAG-2D(E) show bias and respectively pay important role in different development stages of spikelet. WAG-2C mainly participates in formation, development of staments and pistils because of the highest expression level at anther connective stage. However, WAG-2D(E) has the highest expression level at pistil and stamen primordiuxn formation stage, which suggests WAG-2D(E) is responsible for differentiation of staments and pistils, WAG-2J(K) produced by alternative splicing type2, which is the main alternative form in D48(T. dicoccoides), also reaches the expression peak at anther connective stage. Thus WAG-2J(K) pays a vital role in formation, development of staments and pistils. The expression patterns of four allelic variations from three pistil mutant and Chinese spring are markedly different. The WAG-2M has the highest expression level at pistil and stamen primordiuxn formation, as well as anther connective stage in three pistil mutant wheat. But unlike three pistil mutant, WAG-2L’has higher expression level at pistil and stamen primordiuxn formation stage. The expression level of WAG-2N’ reaches the peak at anther connective stage. WAG-2M and WAG-2N’are produced respectively by alternative splicing type1and type2. Whether changed expression pattern is associated with the pistil development of three pistil mutant needs to further study.