Development Of Wheat Glutenin Subunits Near Isogenetic Lines And Identification Of Wheat Glutenin Subunits In Ningxia

Wheat is one of the most important food crops in China,and quality improvement is a significant objective in wheat breeding.The type and composition of the gluten subunits have been proved to be closely related to wheat processing quality.Further analysis for the quality effects of different gluten subunits and their composition is the basis for wheat processing quality improvement.Near isogenic lines?NILs?differ in glutenin subunits had been recognized as the ideal materials to clarity the quality contributions of different subunits under common genetic background.In this study,a series of HMW-GS and LMW-GS NILs under the genetic backgrounds of some main cultivars from Huanghuai and Xinjiang wheat regions were developed,in which some advanced lines were preliminary evaluated to provide valuable genetic materials for wheat quality improvement.The identification of novel glutenin subunits in wheat could enrich the genetic resources for breeding specific wheat cultivars meeting quality requirements of different end-products.As so,the HMW-GS composition of a Ningxia durum wheat named DQM were detected and their genes were cloned.Then the gene structure and evolutional relationship of these novel subunits genes were characterized using the bioinformatics tools.Finally,the quality contributions of the identified subunits were predicted respectively based on their secondary structure features deduced from the amono acic sequence.Ningxia is one of the main producing areas for spring wheat in China,clarifying the LMW-GS composition and distribution in this area is very important for wheat quality improvement.Therefore,the LMW-GS composition and distribution of 98 Ningxia cultivars were characterized based on the allelic variations of LMW-GSs at Glu-A3 and Glu-B3 loci detected by related STS markers.The results are as follows:1.Basing on the previous research in our lab,158 HMW-GS NILs under 5 genetic backgrounds were obtained as identified by the optimized methods used for the target subunits tracing and detecting.The evaluation of the NILs showed that,differences in HMW-GS composition among the NILs and their recipient parents were detected although consistent agronomic character was observed.Compared with 1Dx2+1Dy12 lines,the protein content,wet gluten content and sedimentation value of 1Dx5+1Dy10 lines were significantly increased by 3.0%⁷.9%in Xiaoyan 22,Xinong 2208 and Huayu 8 genetic background?P<0.05?or?P<0.01?.But there was no significant difference in the genetic background of Xiaoyan 6,and the sedimentation value was significantly increased by 8.4%?P<0.05?under the genetic background of Xinong 1718.There have obtained 633 heterozygous lines under 8 genetic backgrounds.In development of LMW-GS NILs,there have obtained108 BC₆ lines under 3 genetic backgrounds.2.The HMW-GS composition in DQM was detected by SDS-PAGE.DQM contains three expressed HMW-GS,their electrophoretic mobility are different from control subunits.Therefore,it is speculated that DQM may contain new subunit combinations.The DQM-encoding HMW-GS gene was cloned by homologous cloning.obtained 3 amplification products,and the size of 2352 bp,2109 bp and 1827 bp,respectively,the similarity of1Bx14*,1Bx15*and 1Ay gene were 99%,99%and 97%,the corresponding subunits were temporarily named 1Bx14.1,1By15.1 and 1Ay.1,respectively.The results of amino acid sequence prediction showed that 1Bx14.1,1By15.1 and 1Ay.1 were composed of 783,702and 606 amino acids,respectively.The primary structure consisted of a signal peptide,the conserved N-terminal,a rich variation of central repetitive domain and conserved C-terminal four structural units,indicating that they have typical characteristics of HMW-GS.Genetic evolution analysis showed that 1Bx14.1,1By15.1 and 1Ay.1 were closest to 1Bx14*,1By15*and 1Ay,respectively.Using MALDI-TOF-MS to validate the correctness of gene cloning,and the results showed that subunit 1Bx14.1 and 1By15.1 had the highest similarity to1Bx14*and 1By15*.The sequence coverage was 24%?185aa?and 15%?101aa?,respectively.More than half of the identified peptides were distributed in the conserved N-terminal and C-terminal.The secondary structure of 1Bx14.1 and 1By15.1 was predicted by SSpro8 online tool.The results showed that both of them were similar to those of high quality subunits1Bx14 and 1By15,they had high Hydrogen bonded turn and?-helix content,indicating that it may be a high quality subunit.3.STS markers were used to detect the allelic variations of LMW-GSs at Glu-A3 and Glu-B3 loci in 98 wheat cultivars from Ningxia.The results showed that Glu-A3a,Glu-A3b,Glu-A3c,Glu-A3d and Glu-A3e were found in Glu-A3 locus in Ningxia wheat,where the frequency of Glu-A3c is 55.1%.Glu-B3a,Glu-B3b,Glu-B3c,Glu-B3d,Glu-B3f,Glu-B3g,Glu-B3h,Glu-B3i and Glu-B3j were found in Glu-B3 locus,and Glu-B3j accounts for about one quarter?25.5%?.At the same time,there were also differences in the types and distributions of alleles between different regions and different cultivars in the same area.