EcoTILLING Reveals Allelic Variations In The Starch Synthesis Gene TaSSIV And Its Haplotypes Associated With Grain Weight In Wheat(Triticum Aestivum L.)

Wheat is a staple food commodity grown worldwide,and wheat starch is a valuable source of energy and carbon that constitutes 70% of the grain weight.Starch has great utility in several industries as an inexpensive,adaptable and recyclable polymer,thus the demand for starch is always increasing.Starch synthase is controlled by different gene classes such as granule bound starch synthase,starch synthase I,II,III and IV.Manipulation of genes involved in starch synthesis significantly affect wheat grain weight and yield.Ta SSIV plays an important role in starch synthesis and its main function is granule formation.To mine and stack more favourable alleles,single nucleotide polymorphisms(SNPs)of Ta SSIV-A,B,and D were investigated across 362 wheat accessions by Eco TILLING.These 362 wheat accessions were collected from diverse geographical areas of China and Pakistan to identify nucleotide diversity.The SNPs were converted into specific KASP(kompetitive allele specific PCR)markers to find the haplotypes of the focus gene.Association analysis was done to examine the correlations between Ta SSIV haplotypes and agronomic traits.The core results of current work are as follows.i)A total of 38 SNPs in the amplified regions of three Ta SSIV genes were identified through Eco TILLING,of which 10,15,and 13 were in Ta SSIV-A,B,and D,respectively.Among the identified sequence polymorphisms,two missense polymorphisms were identified in Ta SSIV-A and Ta SSIV-B each,while Ta SSIV-D possessed three missense polymorphisms.ii)KASP markers were developed on the identified sequence polymorphism.Among the developed KASP markers,four markers(A1673T,C5952 T,A2403C and C2436T)in Ta SSIV-A,while two markers(C1560T,C6107T)in Ta SSIV-B showed diversity more than 5%,whereas the rest was less than 5% which were considered to be minor alleles.Similarly,no any SNPs of Ta SSIV-D showed allelic frequency more than 5%,so they were excluded for further analysis.iii)Three haplotypes of Ta SSIV-A,i.e.,Hap-1-1A(AATC),Hap-2-1A(ACTT)and Hap-3-1A(ACTC)were identified in Chinese mini core collection(MCC),which showed frequency more than 5%.Similarly,Ta SSIV-B three haplotypes i.e.,Hap-1-1B(CC),Hap-2-1B(TC)and Hap-3-1B(CT)in MCC,which showed frequency more than 5% The results of an association study in the Chinese mini-core collection with thousand grain weight(TGW)showed that Hap-2-1A(ACTT)was significantly associated with TGW.iv)In landraces,the frequency of the favoured haplotype Hap-2-1A for TGW was low while in modern cultivars,the favoured haplotype Hap-2-1A frequency was high(more than 50%)in regions I-IV.Allelic frequency and geographic distribution indicated that the favoured haplotype(Hap-2-1A)has been positively selected in Chinese wheat breeding.v)In Pakistani wheat accessions,geographic distribution was investigated in Ta SSIV-A haplotypes.The frequency of Hap-2-1A among five different regions of Pakistan was higher in the Punjab irrigated region(78%)and the Punjab rainfed region(52%).These results suggested that the Eco TILLING can be applied to mine the natural allelic variations and KASP markers can be applied in starch improvement which ultimately improve wheat yield by marker assisted selection in wheat breeding.This study identified 38 unique alleles in Ta SSIV among 362 wheat accessions collected.Association analysis showed that one haplotype in Ta SSIV were significantly associated with TGW and this haplotype can be used in future studies to assess their usefulness as selection criteria for improving these yield-related traits.