| Objective: We identify and verify the SNP may be present in starch synthesis genes and elucidate the causes of wheat varieties(lines) starch content differ from a single base mutation.This will give us the subsequent development of SNP markers and assisted selection basis.Method: In this study, the SS IIa 、SBE IIa and SBE II b genes were cloned from wheat varieties(lines)with different contents of starch. By comparison and analysis of polymorphisms,we discover many different single base site, and design specific PCR amplification primers.Then we use matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS) to detect 162 colony samples,to explore significantly correlated sites with starch content by SPSS statistical analysis.The main results and conclusions:1、SSⅡa gene sequences are highly conserved,compared with the sequence published in Gen Bank similarity of more than 98.19%.It was found 40 single nucleotide mutation,resulting in 20 amino acids mutated. Protein domain analysis found that these mutation sites are located outside of the α- amylase enzyme N- terminal, and in structural domain analysis was not found any differences in alpha starch catalytic enzyme. In addition, we also found a deletion fragment of 249 bp in Xin Chun 12,which including part of the glycosyltransferase locus.2 、 SSⅡa gene nucleotide variation is richful, and the second exon is mutation enrichment Area.SBEⅡa gene SNP variation occurs mainly in the 3rd, 15 th and 18 th exon Area. SBEⅡb gene mutation sites are concentrated in the third exon. For SBEⅡb and SBEⅡa are similar to the sequences which published in Gene Bank more than 99.04% and 98.64%.3、These three gene single nucleotide polymorphism analysis showed that, π value between 0.01637-0.01884, the scope of Watterson’s θw value is 0.01668- 0.02523, suggesting that these genes have a high degree of nucleotide diversity. The three genes have the same haplotype number, but it is not consistent haplotype diversity. Tajima’s D test D value not significant, in line with the neutral theory of molecular.4、In the haplotype analysis, these three genes were divided into different haplotypes, but there is good correspondence between different haplotypes and amylopectin content, higher levels of amylopectin varieties(lines) often was in the same cluster, the low amylopectin content cultivars(lines) are also classified in the same category, explanatory material similar amylopectin content may have similar single nucleotide sequence variations.5、 Detected by 90 SNP loci we found that all these sites were no significant correlation between amylose content, there is a significant correlation between ss2a-rs31 site and sbe2a-rs10 sites and amylopectin content. Correlation analysis found that the differences between ss2a-rs31 locus genotype CT and TT genotypes with high amylopectin was not significant(P> 0.05), while in the medium starch and low-branched amylopectin content of the difference significant(P <0.05); CC genotype individual branched starch content was significantly higher than CT genotype and TT genotype. In the high amylopectin, sbe2a-rs10 locus GG genotype was significantly lower than AA and AG genotypes(P <0.05);while in the middle amylopectin content and a low content of amylopectin, AG genotype was significantly higher than that AA genotype and GG genotype.It concluded that the two sites have a larger contribution on the content of amylopectin,and it may be a candidate locus for SNP marker development.In this study,we study three starch synthesis related enzymes SS II a, SBE II a and SBE II b from wheat varieties(lines) with different contents of resistant starch,and explore the SNP locus information which may be present inside the gene. Combined with matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS) to detect the SNP, using SPSS statistical analysis, we found ss2a-rs31 site(C?T) and sbe2a-rs10 site(A? G) may be effective sites cause differences amylopectin content. |
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