Diversity Analysis Of Aegilops Tauschii And Triticum Dicoccoides And Gene Cloning Of Their ?-gliadins

Common wheat(Triticum aestivum,2n=6x=42)is an allohexaploid hexaploidy crop originated from diploid wheat(Aegilops tauchii,2n=14,DD)and tetraploid wild durum wheat(Triticum turgidum ssp.dicoccoides,2n=28,AABB).With the development of wheat breeding techniques such as hybridization,domestication and self crossbreeding,the genetic variation of common wheat is narrower and narrower than its wild ancestor species,which severely restricts the cultivation and production of new common wheat varieties.It is very important to expoit valuable genes and germplasm resources from its wild relatives for wheat improvement.Gliadin is one of the two major components of wheat gluten proteins.It is not only closely related to wheat processing quality,but also is the main external stimulus of celieac disease(CD),a chronic and life-long genetic disease.It is of great significance for wheat improvement and CD prevention to screen distinctive gliadin genes or germplasm resources.In this work,the genetic diversity of Aegilops tauchii and Triticum dicoccoides based on the gliadin composition and ISSR markers were studied,and based on the PCR strategy,the ?-gliadin genes were cloned from Aegilops tauchii accession T093 and Triticum turgidum ssp.dicoccoides TD93.The results were shown as belows:Firstly,based on the A-PAGE electrophoresis,the gliadin composition of 169 Aegilops tauchii accessions was analyzed.The results showed that there was a wide variety of genetic variation in the gliadin composition,11~34 gliadin bands were in each accession,90 kinds of gliadin bands and 28 polymorphic bands were obtained in 169 accessions.Genetic diversity based on ISSR markers of 76 accessions with different gliadin bands were further analyzed.18~37 bands could be amplified by each ISSR primer,251 bands were amplified by 9 ISSR primers,and 237 bands are polymorphic and the polymorphism ratio was 94.4%.The software of Ntsys 2.1 was used in cluster analysis and principal component analysis(PCoA)based on the results of gliadin components and ISSR markers.The results showed that there were obvious genetic differences and geographical differentiation.At about 0.56 of the similarity coefficient,all the accessions can be significantly divided into two groups,except for several accessions clustered into a small branch II,the majority of the other accessions were clustered into branch I and further could divided into several small clusters based on its geographical origin,such as the Middle East,Huang Huai and Xinjiang.In addition,based on the results of genetic diversity,it was suggested that some accessions native to China(T002,T006,T093,T102,SL002,SX009)and out of China(AY007,AY073,Y175,Y128,Y212)may be the gene and germplasm resources for wheat improvement.Secondly,based on the A-PAGE electrophoresis,the gliadin composition of 96 Triticum turgidum ssp.dicoccoides accessions was analyzed.The results showed that there was a wide variety of genetic variation in the gliadin composition,11-34 gliadin bands were in each accession,90 kinds of gliadin bands and 43 polymorphic bands were obtained in 96 accessions.Genetic diversity based on ISSR markers of 76 accessions with different gliadin bands were further analyzed.18~37 bands could be amplified by each ISSR primer,244 bands were amplified by 9 ISSR primers,and 231 bands are polymorphic and the polymorphism ratio was 94.67%.The software of Ntsys 2.1 were used in cluster analysis and principal component analysis(PCoA)based on the results of gliadin components and ISSR markers.The results showed that there were obvious genetic differences among the different accessions.All the accessions can be significantly divided into three branches(I,II,III),and most of them were clustered in the branch I and further clustered in several subgroups(A,B,C,D).Moreover,it was suggested that some accessions TD012,TD046,TD062,J129 and J214 may be the useful gene and germplasm resources for wheat improvement.Thirdly,based on PCR strategy,78 ?-gliadin genes with unique nucleotide sequences from the Aegilops tauchii accession T093 and Triticum turgidum ssp.dicoccoides accession TD93 were cloned with a pair of degenerate primer.Among them,36 genes with complete open reading frames(ORF)and 42 genes were pseudogenes due to the early emergence of the terminator.Cluster analysis on the deduced amino acid sequences and the four major immune dominant peptides combination of the 36 complete ORF genes showed that,except that T093-70 a had an additional cysteine residue in the C-terminal region,and T093-61 a and T093-88 a were clustered into the branch B which represent the genes originated from B genome,other than the branch of their true origin of D genome,the vast majority genes had the typical characteristics of ?-gliadin gene,which contains 6 conserved cysteine residues,have significant genomic specificity in whole sequences of deduced amino acids and the distribution of four major immune dominant peptides.