Wild Emmer Wheat Enriching The Genetic Basis Of LMW-GS In Common Wheat And Its Potential Value For Wheat Processing Quality Improvement

Wild emmer wheat（Triticum dicoccoides,AABB,2n=4x=28）is unique in storage proteins,and it possesses abundant LMW-GS allelic variations.Therefore,the identification and utilization of LMW-GS of wild emmer might not only enrich the genetic diversity of LMW-GS genes,but also provide genetic resources for improving the quality of common wheat.In this study,LMW-GS of wild emmer D97 and‘Chuannong 16’（CN16）with high grain yield but weak gluten and their advanced generation(F₁₂)hybrids BAd7-209 and BAd7-213 sister lines which have the same HMW-GS but different processing quality properties,were identified by SDS-PAGE and 2-DE protein analysis methods.Based on the rich genetic diversity of Glu-3 locus,molecular cloning and structural analysis of Glu-A3 genes of the tested materials were carried out and their processing quality properties were measured.The objective was to investigate the potential of wild emmer for enriching LMW-GS genetic basis,and improving processing quality of common wheat.The main results are as follows:1.The results of SDS-PAGE and 2-DE showed that the male wild emmer D97and the female common wheat cultivar CN16 were different in composition of LMW-GS,and D97 with higher expression and more abundant LMW-GS than CN16.The interspecific hybrids BAd7-209 and BAd7-213 not only contained the LMW-GS of the female CN16,but also possessed the C-type subunits C1,C5 and D-type subunits D1,D2 that were similar to D97 electrophoretic mobility and charge.Furthermore,BAd7-209 contained C2,C3 and C4 subunits that were similar to those of D97,and BAd7-213 with D4 subunit that was similar to that of D97.The results showed that some LMW-GS genes in wild emmer were successfully introduced into common wheat through distant hybridization and inherited stably in the offspring.In addition,BAd7-209 also contained the two new LMW-GS D8 and D9,which were different from both parents,indicating that wild emmer was beneficial to further broaden the LMW-GS genetic basis of common wheat.2.A total of 35 LMW-GS Glu-A3 genes were isolated and sequenced.Among them,8,8,10 and 9 sequences were isolated in D97,CN16,BAd7-209 and BAd7-213,respectively.Among the 35 obtained sequences,a total of 27 LMW-m and 2 LMW-i encoding proteins full-length LMW-GS gene sequences were identified（accession numbers MG574321-MG574349）,and the two coding LMW-i protein genes were only detected in D97.MG574323 was shared in CN16,D97 and their hybrid offspring BAd7-209 and BAd7-213,indicating that this gene was highly conserved.Wild emmer D97 had the two endemic sequences MG574329 and MG574330,of which,MG574329 was only inherited in the offspring BAd7-213,while the gene MG574330was inherited in the two offspring BAd7-209 and BAd7-213.3.Cluster analysis showed that the two LMW-GS i-type genes from wild emmer D97 were clustered together with i-type genes from GenBank in one branch,but all of the cloned m-type LMW-GS genes were clustered together with the majority of m-type genes from GenBank in another branch.Of these,only MG574328 from the female CN16 was clustered together with the known m-type LMW-GS gene from GenBank,while the rest cloned m-type LMW-GS genes were clustered with published EF190882 in a large sub-branch.Further cluster anasis using the cloned Glu-A3 genes in the present study discovered that the LMW-m genes MG574329,MG574330,MG574333,MG574335,MG574337,MG574339-MG574341,MG574343,MG574344,MG574347 in group 1 had the closer relationship to male those of D97,while the LMW-m genes MG574321-MG574325,MG574336,MG574338,MG574349 in group 2 were closer to those of female CN16.Obviously,the hybrids have integrated the LMW-GS genetic materials from the two cross parents.4.Based on the deduced amino acid sequences,the 26 LMW-m subunits（MG574321-MG574327,MG574329,G574330,MG574332,MG574333 and MG574335-MG574349）contained eight conserved cysteine residues,and the two LMW-i subunits MG574331 and MG574334 had seven cysteine residues.Of which,the 6 cysteine residues located at the C-?and the C-Шregions might participate in the intramolecular disulfide bond,and the other two or one cysteine residues which located at the N-terminus and C-Пmight involve in intermolecular disulfide bond to form protein multimer.The gene MG574328 was unique to the female CN16 only containing 6 cysteine residues that located at the C-?region and the C-Шand lacked the cysteine probably forming intermolecular disulfide bonds,which might influence the protein multimer formation,and further affecting the processing quality of CN16.5.The results of protein secondary structure prediction showed that the average percentage ofα-helix in D97 was higher than that of CN16.And the average percentages ofα-helix in the offspring BAd7-209 and BAd7-213 were generally higher than that of female CN16.These indicated that D97 and offspring BAd7-209and BAd7-213 might improve the quality of dough byα-helix content,which can be used to improve the dough quality of common wheat.This was consistent with the processing quality properties of the offspring BAd7-209 and BAd7-213 which were higher than those of CN16,such as protein content,settlement value,wet gluten content,development time,stability,farinograph quality number.Among all the obtained LMW-GS genes,only MG574335 in D97 had two beta chains,equal to AY263369 which was believed to have a positive effect on dough quality.6.The quality analysis showed that the quality parameters of the two distant offspring were significantly better than those of the female CN16,which indicated wild emmer LMW-GS might improve the processing quality of common wheat,to some extent.Meanwhile,the offspring line BAd7-209 was significantly higher than its sister line BAd7-213 in all of the determined quality parameters.The quality differences might be associated with their different LMW-GS compositions besides their LMW-GS and/or gliadin.These results provided the theoretical evidence for using wild emmer LMW-GS in the quality improvement program of common wheat.