与大白菜霜霉病抗性主效QTL连锁的分子标记开发

Development of molecular markers linked to the resistant QTL for downy mildew in Brassica rapa L. ssp. pekinensis

霜霉病是危害大白菜的三大病害之一,该病的发生会严重影响大白菜的产量及品质,因而研究与霜霉病抗性QTL紧密连锁的分子标记对大白菜抗病新品种培育具有重要意义。该研究在前期工作的基础上,选用高感霜霉病株系91-112、高抗霜霉病株系T12-19以及由二者为双亲构建的DH群体为实验材料,针对大白菜霜霉病抗性主效QTL——BrDW所在的标记区间,利用已有的大白菜基因组信息发展与抗性QTL紧密连锁的分子标记,通过Blast和IMap分析,将与BrDW连锁的RAPD标记K14-1030定位于大白菜KBrB058M10上(位于Contig214上),根据KBrB058M10附近的BAC及BAC-end序列设计引物,结合限制性内切酶酶切及HRM分析方法,筛选得到5个与BrDW连锁的分子标记,包括1个Indel标记Brb062-Indel230,3个CAPS标记Brb094-DraⅠ787、Brb094-AatⅡ666和Brb043-BglⅡ715,1个SNP标记Brh019-SNP137;同时,通过筛选与目标区域具有同源性的Unigene序列得到了1个与BrDW紧密连锁的SSR标记bru1209。标记Brb062-Indel230、Brb094-DraⅠ787、Brb094-AatⅡ666、Brb043-BglⅡ715、Brh019-SNP137和bru1209与RAPD标记K14-1030之间的遗传距离分别为4.3 cM、1.7 cM、5.9 cM、5.9 cM、4.6 cM和0.8 cM,在对DH群体中的抗性株系选择上准确率分别为69.7%、70.9%、72.4%、72.4%、58.3%和74.2%,可应用于分子标记辅助选择,为霜霉病抗性分子育种奠定了良好基础。

Downy mildew,caused by the oomycete Hyaloperonospora parasitica Constant.（Pers.ex Fr.）,is one of the most severe diseases in Chinese cabbage,leading to reduction of yield and quality of the harvested products.Therefore,identifying molecular markers linked to the major QTL for downy mildew resistance will be helpful in breeding resistant varieties of Chinese cabbage.Here,one highly susceptible line 91-112,one highly resistant line T12-19,and the derived DH population were employed to develop linked molecular markers for the major QTL,BrDW,for downy mildew.With BLAST and IMap analysis,the RAPD marker K14-1030 linked to BrDW was anchored on KBrB058M10（on Contig214）.On the basis of the BAC and BAC-end sequences around KBrB058M10,a set of PCR primers were designed,and the methods of restriction analysis and HRM analysis were used to develop molecular makers.Finally,five polymorphism markers were developed,containing one Indel marker named Brb062-Indel230,three CAPS markers named Brb094-DraⅠ787,Brb094-AatⅡ666 and Brb043-BglⅡ715,and one SNP marker named Brh019-SNP137.In addition,one SSR marker from Unigene sequence homologous with KBrB058M10（known as bru1209） was developed.The map distances between the six markers and RAPD marker K14-1030 were 4.3 cM,1.7 cM,5.9 cM,5.9 cM,4.6 cM,and 0.8 cM,respectively.The percentage of accuracy in selecting for downy mildew-resistant lines from the DH population were 69.7%,70.9%,72.4%,72.4%,58.3%,and 74.2%.These markers could be used in marker assisted selection to improve downy mildew resistance in Chinese cabbage.