Genetic Properties Of An Integrated Y-type HMW-GS Genes In Common Wheat From Wild Emmer And Its Potential Value To Quality Improvement

High molecular weight glutenin subunits?HMW-GSs?are important components of storage proteins in wheat.The quality and quantity of HMW-GSs are closely correlated with the the processing quality of wheat flour.Wild emmer wheat?Triticum dicoccoides,AABB,2n=4x=28?is a secondary gene source of common wheat,and harbors abundant HMW-GS allelic variations.Therefore,the characterization and utilization of HMW-GS genes from wild emmer to broaden the genetic diversity of HMW-GS genes in common wheat is possible and such researches can also provide excellent alien gene resources for wheat quality improvement.In bread wheat,the 1Ax and 1By subunits are not usually expressed and 1Ay subunit is frequently silenced.In our former works,three HMW-GS genes including silenct 1Bx,active 1By,and active 1Ax were cloned and characterized from wild emmer wheat.Based on these results,we preformed further studies on characterization and utilization of the wild emmer 1Ay.The wild emmer wheat accession D97 with active y-type HMW-GS genes were distantly crossed as the male parent with the high yielding,low-gluten wheat cultivar Chuannong16?CN16?as the female parent,and the pentaploid F₁?AABBD?was obtained.An advanced generation hybrid line TaAy7-40with common wheat genetic background was obtained via the repeated self-fertilization of the pentaploid hybrid and breeding selection on the basis of ideal agronomic and yield characters.The y-type HMW-GS genes in advanced hybrids were characterized to evaluate the feasibility of introducing active y-type genes from wild emmer into common wheat and its potential utilization for improving the processing quality of wheat.In addition,the inheritability and expression characteristics of the 1Ay active gene in common wheat background and the potential value of 1Ay on enriching the wheat Glu-A1 locus and wheat processing quality improvement were further investigated.The main results are as follows:1.In this study,an active 1Ay gene from wild emmer wheat D97 was cloned and prokaryotic expressed,and its structural characteristics and phylogenetic relationship with other known 1Ay genes were analyzed.SDS-PAGE analysis showed that the offspring line expressed six HMW-GSs including the 1Ay subunit.We further identified the 1Ay genes in the parents and offspring line.Molecular cloning and sequence analysis revealed that the1Ay open reading frames?ORFs?of both D97 and offspring line TaAy7-40 were 1830 bp in length,which encoding 608 amino acid residues,and the similarity of two coding sequences was 99.2%.The authenticity of the functional 1Ay alleles was further confirmed by prokaryotic expression system.The ORF of 1Ay gene from female parent CN16 is only1791 bp.A premature stop codon?TGA?at the position 1000-1002 bp was found that suggested it was a pseudogene without express 1Ay subunit.Further comparison of the nucleotide sequences revealed that there were 14 single nucleotide polymorphisms?SNPs?in the line TaAy7-40 compared with the male parent D97,including 13 transitions and one transversion.In addition,30 SNPs were identified between TaAy7-40 and CN16,including25 transitions and 5 transversions.Similarly,the amino acid primary structure prediction of the three groups also showed that there were three deletions and a premature stop codon in CN16 compared with male parent D97 and TaAy7-40.Comparative cluster analyses of the1Ay genes in this study with the 1Ay allele sequences in the GenBank database were performed.We found that the 1Ay genes of TaAy7-40 and wild emmer D97 were clustered together and both were estranged from the 1Ay of CN16.These results demonstrated that the active 1Ay in TaAy7-40 was derived from wild emmer D97 and this wild emmer allele has been successfully integrated and stably expressed in common wheat.The obtained line TaAy7-40 is a genetic resource with active 1Ay gene that can be used for wheat quality improvement.2.The 1By genes in D97,CN16 and offspring line TaAy7-40 were identified.Molecular cloning and sequence analysis showed that the 1By ORFs of D97 and TaAy7-40were each of length 2166 bp and encode for 720 amino acid residues.The similarity of the two sequences was 99.2%.The ORF of 1By gene from female parent CN16 is only 2157bp in length.Nucleotide sequences alignment of three 1By genes revealed that TaAy7-40had 18 SNPs compared with D97,including 11 transitions and 7 translocations.In addition,32 SNPs were identified between TaAy7-40 and CN16,including 27 transitions and 5transversions.Similarly,the amino acid primary structure prediction analyses of the three groups showed a deletion and insertion in the parent CN16 compared with D97 and TaAy7-40.Comparative analyses of the 1By genes in this study with the 1By allele sequences in the GenBank database were performed.The 1By genes of TaAy7-40 and D97were clustered together with a bootstrap of 99 and separated from the 1By sequence of CN16.These results demonstrated the transferring feasibility of 1By gene from wild emmer wheat to common wheat and the potential value of wile emmer 1By allele for enriching the genetic diversity of Glu-B1 locus in common wheat.3.The TaAy7-40 with a common wheat genetic background and expressed six HMW-GSs including active 1Ay and 1By from wild emmer were obtained.The morphological and agronomical analyses indicated that the TaAy7-40 resembled CN16with respect to flowering period,plant height,spike length,spikelet shape,spikelet number and leaf shape,but all were significant from those of CN16.The kernel length,kernel width,kernel thickness,1000-kernel weight,and grain weight per plant of TaAy7-40 were significant differences from D97,while slight differences were occurred between TaAy7-40 and CN16.These results suggested that the introgression line TaAy7-40 had the properties of the agronomic and yielding traits of common wheat and can be further used for wheat quality study.Therefore,we compared the main processing quality parameters including protein content,sedimentation value and content of wet gluten in the flour of TaAy7-40,CN16,and the commercial varieties Mianmai 37 and Neimai 9 as control.The results showed that the TaAy7-40 contained the active y-type genes from wild emmer had better flour processing characteristics than its female parent CN16 and those parameters were even superior to the medium gluten common wheat cultivars Mianmai 37 and Neimai9.These results implied that the y-type HMW-GSs of wild emmer are to a certain extent correlated with the wheat quality improvement.4.Based on the studies above,the TaAy7-40 as the female parent was crossed with the medium gluten wheat cultivar Neimai 9 as male parent.SDS-PAGE was used to screen and select the lines with active 1Ay subunit in generations from F₂ to F₅.We identified sib lines with and without 1Ay from F₄ hybrid generation.The selected sib lines namely Q1-F4-2-5-1and Q1-F4-2-5-5 showed HMW-GSs differences only at the 1Ay subunit.Molecular characterization of the 1Ay genes in the two sib lines and their parents found that the 1Ay ORF of Neimai 9?MF429890?was 1812 bp in length,while that of TaAy7-40was 18 bp shorter.A total of 38 SNPs were found between Neimai 9 and TaAy7-40.Two premature stop codons at positions of 340 and 355 sites were identified in the 1Ay of Neimai 9,implying this gene is a pseudogene.We further cloned the 1Ay genes from three offspring lines Q1-F4-1-6-1,Q1-F4-2-5-1,and Q1-F4-2-5-5 with accession numbers MF429891,MF429892,and MF429893,respectively.The sequences length of these three1Ay genes were each of length 1830 bp with the same length to the 1Ay of TaAy7-40.However,few SNPs were identified among these 1Ay genes.MF429891 and MF4229892had 608 amino acids the same as that of the female TaAy7-40 and no premature stop codon was found.However,four premature stop codons were identified in the deduced amino sequence of MF429893,which further confirmed that the MF429893 and MF429890 of Neimai 9 were pseudogenes.The flour processing quality parameters of the two parents and F₅ lines were investigated.The TaAy7-40 showed higher protein content,higher content of wet gluten in the flour,and higher sedimentation value than Neimai 9,implying the TaAy7-40 has flour processing quality and gluten characteristics superior to those of medium gluten common wheat cultivars Neimai9.The hybrid line Q1-F5-2-5-1 expressed six HMW-GSs showed higher protein content,and longer dough stability time than bi-parents and sib line Q1-F5-2-5-5.Our findings suggested that the expression of wild emmer active 1Ay gene in bread wheat might have positive effect on wheat quality,and the advanced hybrid line TaAy7-40 obtained in the current study was a potential valuable germplasm for wheat quality improvement.