Development And Evaluation Of Near-isogenic Lines,and Fine Mapping Of QTL For Thousand Grain Weight On Chromosomes 4A And 4B In Common Wheat(Triticum Aestivum L.)

Wheat(Triticum aestivum L.)is one of the major food crops in the world,and is also the second-largest food crop in China.The development of new wheat varieties with high and stable yield is one of the important targets for wheat breeders worldwide.As grain weight is the key determinant for grain yield,cloning and characterization of the major genes controlling grain weight is the basis of genetic improvement of yield and the precondition for molecular design breeding in wheat.Previous studies from our lab have identified two major genomic regions on chromosomes 4A and 4B with stable loci for thousand grain weight(TGW)across different environments in the Nongda3338/Jingdong6 doubled haploid(DH)population.Here,the QTgw-4A and QTgw-4B were studied in more depth by dissection,validation,fine mapping and candidate gene prediction with near isogenic lines(NILs)using Nongda3338 as the recurrent parent through marker-assisted selection.The main results obtained are as follows:1.Sixteen co-dominant SSR markers were developed to construct saturated genetic linkage map of QTgw-4A region,and five gene markers(TaARGOS-4A,TaSnRK2.10,TaCWI-4A,TaTGW6 and TaGS3-4A)associated with grain weight previously reported were anchored on chromosome 4A.Using the Nongda3338/Jingdong6 DH population,QTgw-4A was validated and mapped into a 5.73 cM interval flanked by Xcau4A₁1 and Xcau4A₁3 by CIM and ICIM methods,suggesting that QTgw-4A is a novel QTL loci for TGW.2.To fine map QTgw.4A,five BC3F3:4 segregating populations from BC3F3 plants covering different heterozygote fragments of the QTgw.4A region were generated.Thus,the QTgw.4A was finely mapped into a physical interval smaller than 10 Mb.The Wheat660K SNP array was used to analyze genetic similarities of five homozygous BC3F5 NIL pairs(4A-/4A+).Compared with NIL(4A-),NIL(4A+)showed an increase in TGWand a decrease in GNS in field trials.It was also found that NIL(4A+)showed significantly higher rate of grain filling than NIL(4A-)during the latter stage of grain filling.Moreover,it is predicted that a total of 49 candidate genes were identified within the finely mapped interval of QTgw-4A,combining with the transcriptome analysis of the 6-DAP grains of NILs(4A-/4A+)and the dynamic expression profiles of spike and grain tissue-specific developmental timecourse in an expVIP expression database.The allelic distribution at the QTgw-4A locus showed that the frequencies of Jingdong6 positive allele accounted for approximately 13.66%in 688 hexaploid wheat accessions.In addition,another NILs with QTgw-4A in the background of cultured spring wheat Y3002 were developed with the SSR marker Xcau4A₁0 assisted selection using Jingdong6 as the donor parent,which resulted a significant increase in TGW.3.To construct the saturated genetic map of the QTgw-4B region,a total of 59 SSR markers were developed based on comparative genomic analysis.The QTgw-4B region was validated using the Nongda3338/Jingdong6 DH population by CIM and ICIM methods,and was dissected into 3 fragments,namely QTgw-4B.1,QTgw-4B.2,and QTgw-4B.3.Furthermore,two BC3F2 and three BC4F2 segregating populations were generated to validate the QTgw-4B.1,and five BC3F3:4 segregating populations from BC3F3 plants covering different heterozygote fragments were generated to dissect and validate the QTgw-4B.2 and QTgw-4B.3 regions.To evaluate the genetic effect of Rht1 in the background of Nongda3338,NILs(Rht-B1a/Rht-B1b)were developed in the QTgw-4B.2 region,and field trials showed that Rht1 gene had the pleiotropic effect on yield component traits.4.For the QTgw-4B.3 region,three BC3F4:5 segregating populations from the BC3F4 recombinants covering different heterozygote fragments were developed for precise mapping,and this grain weight effect was fine-mapped to a 7.81 cM interval flanked by Xcau4B₃7 and Xcau4B₄2.The Wheat660K SNP array was used to analyze genetic similarities of two homozygous BC3F5 NIL pairs(4B.3-/4B.3+),and it was found that polymorphic SNPs were located in the centromeric region of chromosomes 4B.Field trials showed that NIL(4B.3+)had obviously increased the grain size and TGW compared with NIL(4B.3-).Additionally,the transcriptome analysis of the 6-DAP grains of NILs(4B.3-/4B.3+)were conducted,and the functional annotation and enrichment analysis revealed that the differentially expressed genes were significantly enriched in categories of cell cycle regulation,cell division,and starch biosynthesis pathways,which may contribute to the observed differences in grain weight between NIL(4B.3+)and NIL(4B.3-).