Development Of GSS-SSR Markers And Identification Of Quantivitive Trait Loci (QTL) For Oil Content In Brasscia Napus

Rapeseed is an important oil crop.Increasing oil content is always one of the major objectives in rapeseed genetics and breeding research.Oil content is a complex quantitative trait and controlled by complex environmental factors and multiple genes. With the advent of molecular marker technology,great efforts have been made to map QTLs controlling oil content.However,the number of currently available mapped SSR markers is about 1,000 and insufficient for genetic analysis and map-based gene cloning in Brassica.Therefore,a great demand exists for many more SSR markers to construct high-density linkage maps for Brassica genome research and breeding.In this study,SSR markers were developed based on publicly available genome survey sequences（GSS） of B.napus and a SSR linkage map was constructed.F₂ population and RILs F₅ population were generated for mapping genes controlling oil content in seeds.Two NIL populations have been developed for major QTLs fine mapping.The main results are as follows:1) A total of 13,794 GSSs downloaded from the GenBank database were searched for SSR-containing sequences.Of which,2,113 GSSs containing 2,578 SSRs with repeat lengths of at least 12 nucleotides were identified.A total of 627 new SSR markers （designated BnGMS） were developed based on these GSSs and used to survey polymorphisms among six B.napus cultivars that serve as parents for established populations.Among these SSR markers,591（94.3%） successfully amplified at least one fragment and 434（73.4%） detected polymorphism among the six B.napus cultivars.No correlation was observed between SSR motifs,repeat number or repeat length with polymorphism levels.A linkage map was constructed using 163 newly developed BnGMS marker loci and anchored with 164 public SSRs in a doubled haploid population, which covered a total of 1,821.33 cM with an average interval of 5.07 cM.2) M201 and M202 have high and low seed oil content,respectively.With these two Brassica napus lines,an F₂ population and a RILsF₅ population consisting of 162 and 157 lines respectively were generated for mapping genes controlling oil content in seeds.The genetic linkage map of F₂ population was constructed based on 221 SSR loci,which covered a tatal of 1,374.87 cM with an average interval of 6.22 cM;and the genetic linkage map of RILsF₅ population was constructed based on 239 SSR loci,which covered a total of 1,945.21 cM with an average interval of 8.14 cM.3) In the F_2:3 families,7 QTLs（qOCO7F3-C2,qOCO7F3-A9-1,qOC07F3-A9-2, qOC07F3-A9-3,qOC07F3-A10,qOC07F3-A7-2 and qOC07F3-C4） and 4 QTLs （qOC08F3-C1,qOC08F3-A8,qOC08F3-A9-3 and qOC08F3-A10） were detected based on composite interval mapping in both the years 2007 and 2008,respectively.Of which, qOC07F3-A9-3 and qOC08F3-A9-3,qOC08F3-A10 and qOC08F3-A10 were detected in the same marker intervals,respectively,indicating that qOC07F3-A9-3 and qOC08F3-A9-3,qOC08F3-A10 and qOC0SF3-A10 are the same QTL,respectively.Then they are renamed qOCF3-A9-3 and qOCF3-A10.No epistatic QTL was detected using QTLNetwork 2.0 soft,and only 2 major QTLs qOCF3-A9-3 and qOCF3-A10 were detected in F_2:3 population,indicating that additive effects are the main factors contributing to variation in oil content.In the RILsF₅ population,5 QTLs （qOC08F5-A9-1,qOC08F5-A9-2,qOC0SF5-A10,qOC08F5-A7 and qOC08F5-C6） were detected in 2008.qOC08F5-A9-2 and qOCF3-A9-3,qOC08FS-A10 and qOCF3-A10 were mapped in the same marker intervals,respectively,indicating that qOC08F5-A9-2 and qOCF3-A9-3,qOC08F5-A10 and qOCF3-A10 are the same QTL,respectively.Then they are renamed qOC-A9-3 and qOC-A104) After normalized with the protein content data of each line,the oil contents in F_2:3 population and F₅ population were re-analyszed using conditional QTL analysis approach. The QTLs coltrolling oil content can be differentiated two types:typeⅠQTLs not noly control oil content,but also protein content,such as qOC08F3-A8;and typeⅡQTLs only control oil content,such as qOC-A010.The 2 major QTLs,qOC-A9c and qOC-A10, belong to typeⅡQTL and only control oil content.5) Based on performance of phenotype and genotype,two NIL populations have been isolated to fine map the two major QTLs from recombinant inbred lines.