The Development Of Brassica Napus EST-SSRs And QTL Mapping Of 1000-seed Weight And Quality-related Traits

We loaded down 64,566 EST sequences from NCBI.Searching with SSRPrimer,we found 1200 EST which the length of SSR in it was more than 18bp and the motif of SSR was from dinucleotide to pentanucleotide.Alignment these sequence with CAP3,we obtained 130 contigs and 821 singlets.In total,we designed 995 SSR primer pairs using the 951 EST.These SSRs contained 1020 microsatellites,consisted of 0.3% mononucleotide,30.8%dinucleotide,55.9%trinucleotide,6.4%tetranucleotide and 6.6% pentanucleotide,respectively.AG/TC(25.4%)å’ŒAAG/TTC(21.5%) were the most rich motifs.Screening the 995 SSR makers with six Brassica napus lines,773(77.7%) of them showed clear and strong PCR products and 518(67.0%) performed polymorphism in at least two of the six Brassica napus lines.(AG) n and AT-rich trinucleotide had high polymorphism.The polymorphism increased with repeat length.Among the 951 EST,487 sequences had top blast hit regions of the Arabidopsis genome.150 microsatelites in B.napus have a corresponding microsatellite in the Arabidopsis genome.The corresponding microsatellites in Arabidopsis were usually much shorter than that in B.napus.The results suggested that SSR markers derived from the EST were a good marker resource for comparative mapping between the Brassica and Arabidopsis genomes.We chose 23 pair primers,which had successful amplified in the six lines,to do transferable amplification among Cruciferae.The EST-SSRs showed good transferability among Cruciferae.Three EST-SSRs were found to be genome-specifie in Brassica: BnEMS56 produced the PCR product in only A- type genome species,BnEMS22 had no PCR product only in B genome and BnEMS630 could not amplify in only C genome. These makers could use to identify genome in Brassica.186 F₂ population derived from a crossed between large seed B.napus parent S1 and small seed parent S2 were used to identify quantitative trait loci(QTLs) for 1000-seed weight and quality-related traits.A total of 213 SSRs which contained 233 loci were used to construct linkage map.It consisted of 19 linkage groups.The total map length was 1440.4 cM with average distance of 6.2cM.By using complex interval mapping of WinQTLCart2.5,a total of 28 QTLs were found for 7 traits.4 QTLs were for 1000-seed weight,distributing on N9 and N11,explaining the phenotypic variation ranged from 4.41～9.08%;2 QTLs for erucic acid,on N8 and N13,explaining 50.28%and 19.77%, respectively;5 QTLs for oleic acid,on LG5,N8,N11and N13,explaining 3.84%～46.81%; 5 QTLs for linoleic acid,on N2,N8,N13,N18 and LG14,explaining 1.47%～38.12%; 4 QTLs for glucosinolate,on N8 and N9,explaining 5.48%～8.45%;2 QTLs for eicosenoic acid,on N8 and N13,explaining 21.92%and 18.11%;6 QTLs for palmitic acid,on LG5,N8 and N13,explaining 5.40%～20.37%.QTLs of quality traits were cluster at the same or similar position in N8 and N13,there were significant correlations between the corresponding traits.Condition QTL analysis showed that the pleiotropism was the important genetic base for trait correlations.In this study,the molecular marker linked closely with main QTLs of 1000-seed weight and quality-related traits could be used for MAS in B.napus breeding program.