Gene Copy Number Variation Analysis Of Glu-B3 And Glu-D3 Loci In Common Wheat

Low-molecular-weight glutenin subunit(LMW-GS)is the main component of wheat seed storage protein and plays an important role in determining the end-use quality of wheat(Triticum aestivum L.)by influencing the viscoelastic properties of dough.A considerable allelic variation of LMW-GS has been identified but the variation of LMW-GS gene copy number at Glu-B3 and Glu-D3 loci are less studied.Therefore,the present study was aimed to examine the variation of LMW-GS gene copy number at Glu-B3 and Glu-D3 loci in hexaploid(Triticum aestivum),tetraploid(Triticum turgidum)and diploid(Triticum monococcum)wheat genomes using micro-droplet digital PCR(ddPCR)method.The gene-specific primers and probes of Glu-B3 and Glu-D3 were designed on the basis of genomic sequences of wheat Glu-B3 and Glu-D3.The sequences of these genes were amplified by PCR and quantitative real-time PCR(qRT-PCR).A total of 231 wheat genotypes were used to analyze the variation in LMW-GS gene copy number at Glu-B3 and Glu-D3 loci by probe method using ddPCR.The plastid acetyl-CoA carboxylase gene(ACCase)was used as internal control in ddPCR.The number of mutations identified showed that the highest copy number of the two loci was 9 and the lowest was 3.The average copy number of Glu-B3 and Glu-D3 loci was 5.16 and 5.03,respectively,while the coefficient of variation of Glu-B3 and Glu-D3 was 30.90%and 31.37%.Whereas the coefficient of variation of Glu-B3 and Glu-D3 was 30.74%and 27.71%with a copy number of 4.These results showed that gene copy number of wheat Glu-B3 and Glu-D3 was negatively correlated with protein content,wet gluten content,formation time and stabilization time.The present study has potential applications for improving wheat quality through marker-assisted breeding.