BSA-seq Localization And KASP Molecular Marker Development Of Thousand-grain Weight Related Genes In Secale Cereale Subsp.segetale

Rye is a cross-pollinating plant of the wheat family of the grass family,with excellent agronomic traits and rich genetic diversity.Secale cereale subsp.segetale is a wild rye,with many excellent genes related to yield and seed quality.By mining these genes,we can provide germplasm resources for excellent variety selection of rye and breeding of wheat crops.In this study,CP hybrid population were constructed by artificial crosses in order to explore rye thousand-grain weight related loci,and 13 yield related traits were further investigated and statistically analyzed;the CP population between wild rye 90R13 and cultivated rye Z837 was used to screen individuals with extreme phenotypic differences in thousand grain weight,and together with the parents,the BSA analysis was performed to initially locate the thousand grain weight trait in rye;KASP primers were designed by locating SNP loci associated with thousand-grain weight traits,and marker screening was performed in two combinatorial CP populations in order to obtain KASP markers and candidate genes closely associated with thousandgrain weight traits.The results of the study were as follows.1.Thirteen yield-related traits from seven rye CP populations in two years were investigated and counted,and there was abundant genetic variation for the same trait among populations and between years for the same population traits.The correlation analysis of the material from the crosses C1 and C5 showed that most of the traits were highly significantly correlated with each other,such as the increase of thousand grain weight along with the increase of spike weight,grain length,grain width and grain weight per spike.2.Using the CP population between the maternal wild rye 90R13 and the paternal cultivated rye Z837,twenty samples of each of the extreme traits of thousand-grain weight were selected from 376 single plants of their progeny for the BSA analysis,and their distribution between the two offspring was calculated using the △(SNP-index)method with a window of 1 Mb and a step of 1 kb for the initial localization of thousandgrain weight shape,using Weining rye as the reference genome.Under the 95%threshold line,six QTL loci were localized on seven chromosomes,three on chromosome 1 and one on each of chromosomes 3,4 and 5.Among them,q TGW-5-1was located on the 99% threshold line and could be used as a target segment for further study.3.The micro-effective QTL loci identified in the BSA project were screened,and genes with important mutations such as upstream,stop loss,stop gain,nonsynonymous mutations,and variable splicing were selected as candidate genes for KASP primer design.The KASP primers were designed for 68 SNP loci after screening.The KASP primers were used for genotyping in C1 and C5 populations,and the phenotypic data were analyzed by t-test,gene annotation and tissue expression analysis to obtain five candidate genes for rye thousand-grain weight,Sc WN2R01G320500,Sc WN6R01G478100,Sc WN7R01G175200,Sc WN7R01G178400 and Sc WN7R01G304400.Among them,Sc WN7R01G304400 could be used as a key gene for the next step of research.The designed KASP primers could be used for early seedling screening for selecting high quality thousand-grain weight lines to support molecular marker-assisted breeding.