Characterization Of X-type HMW-GS Of Wild Emmer And Its Potential Improvement For Processing Quality Of Common Wheat

Wild emmer(Triticum dicoccoides,AABB,2n=4x=28)possesses abundant HMW-GS allelic variations.Therefore,the identification and utilization of novel HMW-GS of wild emmer can not noly enrich the genetic diversity of HMW-GS genes,but also provide genetic resources for improving the quality of common wheat.In this study,we will identify the x-type HMW-GS and its coding genes of wild emmer D97,especially study the Glu-Ax genes in advanced generations(>F9)of hybrids between D97 and "Chuannong 16" with high grain yield but weak gluten for resolving the low genetic diversity on Glu-1Ax locus in common wheat,and further test the processing quality of these hybrids.The objective is to investigate the potential of using wild emmer for enriching HMW-GS especially Glu-1Ax locus genetic basis,and improving processing quality of common wheat.The main results are as follows:1.SDS-PAGE analyses showed,the band of "Bx subunit" of D97 was weak,and designated "likely 1 Bx6.2 subunit”according electrophoretic mobility.Through mass spectrometry detect,the "likely 1Bx6.2 subuni" should be degradation product of lAx subunit.Sequence and phylogenetic analyses showed,the open reading frame(OFR)of 1Bx gene of D97 was 2354 bp,and it was most similar with 1Bx-null(JQ007588)and 1Bx14(KF733216)genes in GenBank,with the identities 98.69%and 98.57,respectively.But,IBx gene of D97 had 12 and 15 single nucleotide polymorphisms(SNP),respectively,and two deletion fragments,compared with the two genes.Nucleotide and amino acid sequence analyses revealed that the frameshift mutation occurred by the deletion of "C base",which resulted the premature stop codon(TAG)at position 515 of amino acid sequence.In addition,the prokaryotic expression analysis did not detect the expression products of lBx gene of D97.Therefore,the IBx gene of D97 was silent.This study enriched the silencing mechanism of 1Bx subunit.2.The electrophoretic mobility of lAx subunit of D97 was faster than 1Dx2 subunit of "Chinese Spring" and 1Ax2a*subunit of "Longfumai 1”in SDS-PAGE,designated 1Ax2.2.The results of gene cloning,sequence analysis and phylogenetic analysis showed,the open reading frame(OFR)of 1Ax2.2 gene of D97 was 2496 bp,encoding 830 amino acid(aa),and its activity was proved by prokaryotic expression.In comparision with other lAx alleles in GenBank,1Ax2.2 gene had 17 SNPs,including 11 non-synonymous mutations.And it had the highest similarity(98.8%)with 1Ax1(KJ531446,X61009)and 1Ax-null(HQ613179)genes.Hovewer,1x2.2 gene was estranged from the two similar genes according to the result of phylogenetic analysis,indicating the IAx2.2 gene was a novel allele.3.The hybrids(>F9)were the generations(2n=6x=42)resulting from bagging selfing over nine times since the pentaploid F1 with the AABBD genome between female CN16 and male D97 in combination with the continuously directional selection of single plant having desirable agronomic properties.Cytological observation showed the chromosome number in root-tip cells of hybrids were 2n=42,indicating they have already been hexaploid wheat.SDS-PAGE showed the hybrids had 1Dx5+1Dy10 at Glu-D1 locus of female CN16,of which,the hybrids 1-6?5-1?54-2?BAd7-210?11-1 and 12-1 had the 1Ax2.2 subunit of D97,while 21-6?17-5 and 48-1 had the 1Axl subunit of CN16.However,the 1Ax subunit of BAd7-209 was different from 1 Ax2.2 and 1 Axl of parents.In addition,at Glu-B1 locus there was both the 1By8 subunit from wild emmer and the 1Bx7 subunit different their parents.Therefore,HMW-GSs of wild emmer D97 could be transferred into common wheat,also produce novel HMW-GS allelic variation through wide hybridization.The hybrids 1-6,11-1 and 12-1(1Ax2.2,1Bx20,1Dx5+1Dy10)were better than female CN16(1Axl,1Bx20,1Dx5+1Dy10)in quality parameters.It suggested 1Ax2.2 subunit had better quality potential than 1 Ax1 subunit.Apparently,the introduced HMW-GSs from wild emmer could be stably inherited and expressed as well as effectively improve quality of common wheat.4.The two sister lines of hybrid offspring BAd7-209 and BAd7-210 had the the similar agronomic traits of common wheat.The lAxl.2 of BAd7-209 and 1Ax2.2 of BAd7-210 were identified by SDS-PAGE.The ORF of 1Ax2.2 gene of BAd7-210 was 2496 bp,encoding 830 aa.The 1Ax2.2 of BAd7-210 was highly similar to the 1Ax2.2 of D97 with identity for 99.8%,and a total of only 4 SNPs.Comparatively,it had lower identity of 98.7%with the 1Ax1 gene of CN16,and a total of 33 SNPs.Meanwhile,the phylogenetic analysis showed the 1Ax2.2 of BAd7-210 and D97 were clustered closcly together,but it was estranged from the I Ax I of CN16.Therefore,the I Ax 1.2 gene of BAd7-210 was inherited from D97.The OFR of 1Ax1.2 gene of BAd7-209 was 2514 bp,encoding 836 aa.In comparision with other 1Ax alleles in GenBank,1Ax1.2 had six SNPs including two non-synonymous mutations.The lAx1.2 of BAd7-209 had low identities of 94.5%and 94.8%with the 1Ax2.2 of D97 and the 1Ax1 gene of CN16,respectively.It had 31 and 22 SNPs with IAx2.2 of D97 and 1Axl of CN16,respectively.Compared with the 1Ax alleles of parents,the 1Ax1.2 had identical one insertion and two deletions.The phylogenetic analysis showed 1Ax1.2 was estranged from the 1Ax alleles of parents.Obviously,the 1Ax1.2 of BAd7-209 was different from those of parents,it was a novel allelic variation,and its amino acid sequence had higher variations per unit than those of 1Ax2.2 and 1Ax1.The results of quality measurement showed that BAd7-209 and BAd7-210 were much better than CN16,and BAd7-210 was better than medium gluten wheat cultivar "Mianmai 37”,and BAd7-209 was far better than moderate to strong gluten wheat cultivar "Shumai 482".And,BAd7-209(lAxl.2,1Bx7+1By8,1Dx5+1Dy10)had better processing quality than BAd7-210(lAx1.2,1Bx7+1By8,1Dx5 + 1Dy10),implying 1Axl.2 had better quality potential than 1Ax2.2,which may be associated with the more glutamine(Gln)or longer polypeptide of lAx1.2.Therefore,wild emmer could be effectively utilized to enrich the basis of the HMW-GS genes of common wheat through direct cross transferring,even further generating novel allele variation,which could effectively improve weak gluten wheat into medium or medium to strong gluten wheat.These results fully demonstrated the practivity and effectiveness using wild emmer for improving processing quality of common wheat.