Developing New Markers And Populations For Fine Mapping Of Major QTL Controlling Sclerotinia Disease Resistance And Flowering Time In Brassica Napus

Rapeseed is an important oil crop and one of the major sources of vegetable oil in the world.Sclerotinia stem rot,is a major disease and constrain of rapeseed production,which causes billions of economic loss.At present,resources only showed lighter sclerotinia disease or avoiding disease immune were found in rapeseed,and resistant or complete immune varieties haven't been found,which led to the big difficulty in disease-resistant breeding.On the other hand,obtaining early flowering and precocious rapeseed varieties is the breeding objective in rapeseed in China.In our previous study,quantitative trait locus(QTL)mapping was performed using a population of 244 recombinant inbred lines(RILs)which were derived from a cross between 888-5(a line with susceptible to Sclerotinia sclerotiorum,early flowering,dwarf and more branch number line)and M083(a line with resistance to Sclerotinia sclerotiorum,later flowering,tall and less branch number line),through phenotyping data from 9 environments or experiments and genotyping data from 9278 single nucleotide polymorphisms(SNPs)array.The results showed that overlapped or linked resistance and flowering major QTL were located on the interval(26.8 cM,from 5257 kb to 7494 kb)on chromosome A02.In order to explore the linkage degree of stem rot resistance and flowering time,break down the linkage between the two traits,and provide effective molecular markers for breeding disease resistant and early flowering varieties,based on the preliminary QTL mapping,a large-scale segregation population for fine mapping was constructed in this study.New SNP and insertion-deletion(InDel)markers were developed through genome re-sequencing,which lays a solid foundation for the fine mapping and cloning of associated genes.The results are showed as follows:1.Molecular markers development.1662616 SNPs and 106622 InDels were developed basing on the biparental re-sequencing data which covered five-fold of Brassica napus genome information,and the size of InDels varied from 1 bp to 67 bp.90053 SNPs and 6809 InDels were developed on the A02 chromosome,and the size of InDels varied from 1 bp to 43 bp with an average density of one InDel per 3.64 kb.In the SNP variation type,the frequence of A/G type is higher,which accounted for 29%of total makers;while the frequence of A/T and C/G type is lower,which accounted for 12%and 8%of total SNPs respectively.In total,11540 SNPs and 939 InDels were developed near the target QTL interval.2.Markers selection of the target QTL interval.257 InDels were selected from target QTL interval on A02 chromosome.Seven pairs of primers from the 257 InDels showed polymorphism by agarose gel electrophoresis and 6%denatured polypropylene amide gel electrophoresis.The locations of the seven InDels were verified by genetic map in RIL population,and the physical positions were verified by PCR product sequencing.Among which,two InDels(markers D2 and D3)were used for foreground selection in each generating,which produced fragments near the target QTL interval only 543 kb and 346 kb respectively.Five pairs of InDels(markers D6-D10)were used for densing the target QTL interval and distributed intensively from 6532 kb to 7447 kb.3.Markers screening for background selection.Forty-two pairs of primers were showed polymorphism between parents from 835 pairs of SSR primers,and used for further background screening.42 SSR markers were distributed over seventeen chromosomes except the C03 and C08 chromosome in Brassica napus;Among which,the A01,A07,A09,A10,C05 and C09 chromosome have the least number of SSR markers;the A03 chromosome had the most number of SSR markers.4.Fine mapping population construction.BC3F2 and BC4F1 populations were constructed by backcrossing female parent 12CA02-233 and recurrent parent 888-5(the female parents were chosen by phenotyping combined with the markers of the target QTL interval).QTL-NILs population(BC3F2)was constructed by self-crossing the hybrid individuals,which had the target QTL interval of BC3F1.115 exchanging plants had been detected using foreground markers(markers D2 and D3)from the BC3F2 population(12000 plants)in the field.