Conditional QTL Mapping Of Grain Weight At Different Grain Filling Stages In Wheat And Construction Of Gene Knockout Vector

Wheat?Triticum aestivum L.?is a worldwide food crop,and growth of the yield and quality has always been the top priority of wheat breeders and researchers.Grain filling stage is the key period for wheat yield formation,while wheat grain weight is a multi-gene controlled quantitative trait.Different genes are expressed in different grain filling stages,which ultimately affects wheat yield.In the present study,we tried to map the grain-weight QTLs through linkage analysis using the RIL population consisting of 173 F_8:9:9 lines derived from Shannong 01-35×Gaocheng 9411 and association analysis using a nature population consisting of 205 wheat varieties.The candidate genes were predicted using bioinformatics methods,and the CRISPR-Cas9 knockout vector was constructed.The study laid the foundation for fine mapping and map cloning of grain genes.The main results were as follows:?1?A total of 35 unconditional QTLs were detected for grain weight at four different grain filling stages,located at 1A,1B,2A,2B,3A,3B,3D,4A,4B,5B,5D,7A and 7B chromosomes,could explain the phenotypic variation rate of 3.72%-12.97%.QTKW4B.4-17,QTKW4B.2-13 and QTKW3A.4-42 were simultaneously detected in the third and fourth periods of grain filling stages.Besides,located in the marker of TaTkw₄B-Rht1 range?TKWd4B.4-17?is also detected in different environments,indicating that they are the stable QTL controls the grain weight.?2?Based on the conditions of different grain filling stages,the genetic mechanism of QTL dynamics of grain weight was analyzed by using the conditional QTL method.22conditional QTLs controlling grain weight were detected.7 of them were co-located in conditional and unconditional QTL analysis.The analysis results shows that TKWb7A.1-69,TKWb1B.1-4,TKWc1A.3-83,TKWc3A.4-42,TKWc4B.2-13,TKWd4B.4-17 and TKWd2B.6-45are stably expressed genes locus.?3?Except for the 1D,3D,4D and 7D chromosomes,a total of 104 significant association sites of controlling 1000-grain weight were detected in three environments,its genetic contribution rate was 5.79%-12.55%.On the 1B,4B,and 7A chromosomes,26markers associated with 1000-grain weight were detected,explaining 5.98%-9.14%of the genetic contribution rate.The genetic distance of wsnp_Ex_rep_c103064₈8104690 on chromosome 2B and QTKWd2B.8-3 was only 3.8 cM.As with wsnp_Ex_c23363₃2607637on chromosome 4B,could detected in multiple periods and multiple environments,indicating that these two sites plays an important role in grain weight.?4?The QTL loci QTKW4B.4-17 was analyzed by bioinformatics.It was found that the genetic distance and physical distance of QTKW4B.4-17 were 2.3 cM and 1.91 Mb,respectively.Contained 9 high-confidence genes with annotation function in the interval.?5?The knockout vector Sg2300-001 for the high confidence gene TraesCS4B01G042300 in the target segment was successfully constructed,which provides a basis for knockout of the target genes in subsequent experiments,and provides a reference for the identification and screening of grain weight genes.