Development Of Single-Locus SSR Markers And Fine Mapping Of A Dwarf Gene BnaA3.DWF In Brassica Napus

B.napus is an allotetraploid Brassica species that was formed by recent hybridization of diploid B.rapa and B.oleracea.Due to this high polyploidization,most of the SSR markers usually display two or more loci that locate in homoeologous chromosomal regions in B.napus,although numerous SSR markers had been developed in B.napus.These multi-locus markers make it hard to use in B.napus.However,single-locus markers could be used conveniently in genetic studies and breeding programs.Moreover,plant height is one of the important trait in B.napus.However,the study about plant height and dwarfing breeding was very less in B.napus because of the lack of dwarf mutants.In this study,a set of single-locus SSR markers were obtained in B.napus based on the markers newly developed from available Brassica genome sequences and published markers in previous studies.Also,a dwarf mutant was identified from the EMS mutant library and performed comprehensively for the inheritance analysis and genetic mapping.1.Development and evaluation of single-locus SSR markers in B.napus.5,968SSR markers were newly developed from genomic sequences of B.rapa,B.oleracea and B.napus and 3,890 SSR markers were also collected from previous studies.2,701markers that displayed single amplicons were obtained from the polymorphism screening in six B.napus inbred lines.A total of 283 high-quality putative single-locus SSR markers were selected from the 1,087 markers with single amplicon that showed polymorphism between No.2127 and ZY821,the two parents of the Bna NZDH population.Finally,230 high-quality single-locus markers were assigned to the Bna NZDH linkage map using Bna NZDH population with 154 DH lines.These markers were distributed randomly all over the genome.Furthermore,78 single-locus SSRs across the A and C genomes were selected to genotype B.napus 45 inbred lines and 45 hybrids.These markers displayed single and double allele in most of inbred lines and hybrids,which is consistent with the genome characteristics of inbred lines and hybrids,respectively.The results revealed that these single-locus SSR marker should be the universal ones and could be used for various aspects of genetic studies in rapeseed gene pools.Meanwhile,these markers displayed different levels of polymorphism and were proved to be effective in cultivar identification and fingerprinting.And the universality of single-locus SSR markers were investigated in other cultivated species of the U’s triangle.These 230 single-locus markers displayed single,two and three or more amplicons.Of these,118（51.3%）,125（54.4%）,85（37.0%）,134（58.3%）and 121（52.6%）markers produced a single fragment in B.rapa,B.oleracea,B.nigra,B.juncea and B.carinata,respectively.These SSR markers producing single amplicon in each species could be identified as putative single-locus.It is further found that 27 and 15 markers might be A-genome and C-genome specific.2.Identification and fine mapping of a dwarf mutant in B.napus.To get a mutant library,the seeds of Huashuang 5 were induced mutagenesis by ethyl methyl sulfone（EMS）and a total of 104 mutants were obtained.And a dwarf mutant named as10e41-3 was selected to be studied.There were obvious difference of hypocotyl and leaves between 10e41-3 and wild-type at seedling stage.At the adult stage,it decreased significantly that the plant height,first branch height and average internode length except main inflorescence length.Meanwhile,it also reduced significantly that the main yield-related traits including effective main branch numbe,silique number per plant,silique length and seed number per silique,except 1000-seed weight.The mutant 10e41-3was crossed to four B.napus cultivars,Huashuang 5,018A1,ZY821 and Zhongshuang 11,with normal plant height.These results demonstrated that the there was significantly reduce with plant height,which was almost similar to that of median of corresponding parents,but no obviously affect on yield per plant and flowering time for F₁plants.The inheritance of dwarf was studied based on F₁ lines,F₂ and BC₁ poplution derived from mutant 10e41-3 and tall cultivar 018A.it was revealed that plant height of F₁ was between that of two parents and a goodness-of-fit test indicated that the segregation of plant height fits the expected Mendelian ratios of 1:2:1 and 1:1 in F₂ and BC₁ population,respectively.These results indicated that the dwarf was controlled by a single semi-dominant nuclear gene.The morphology of seedling in the dark was almost same between 10e41-3 and wild-type,which indicated that the dwarf might not be directly related to Brassinosteroid.Also,the 10e41-3 was sensentive but could not restore a normal hypocotyl length same to wild-type under GA₃ treatment,which indicated there was not relationship between the dwarf and GA synthesis and signal transduction.Based on the SSR linkage map in B.napus,8 SSR markers linking to the target gene were sorted with tall and dwarf Bulk derived from 10e41-3 and cultivar ZY821.The target gene was mapped to a 17.28 c M region on A3 chromosome of B.napus and named as Bna A3.DWF.Then the Bna A3.DWF gene was finely mapped based on F₂ population derived from 10e41-3 and cultivar 018A.5 linked SSR makers（SSR81,SSR90,BrGMS4583,BrGMS4741 and BrGMS4752）were developed from B.rapa A3chromosome sequence,and 4 high-quality linked InDel markers（BnInDel18,BnInDel26,BnInDel28 and BnInDel32）were also developed successfully from the resequencing data of two parents using next-generation sequencing technology.Lastly,the Bna A3.DWF gene was narrowed between BnInDel28 and SSR81 using 2,534 tall individuals and 9linkage markers,which is corresponding to 85.6 kb region of B.napus ’Darmor-bzh’ reference genome.