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.
[1] Mahajan V, Gill HS, More TA. Inheritance of downy mildew resistance in Indian cauliflower (group III). Euphytica, 1995, 86(1): 1-3.
[2] Farnham MW, Wang M, Thomas CE. A single dominant gene for downy mildew resistance in broccoli. Euphytica, 2002, 128(3): 405-407.
[3] Coelho PS, Monteiro AA. Inheritance of downy mildew resistance in mature broccoli plants. Euphytica, 2003, 131(1): 65-69.
[4] 冷月强, 侯喜林, 史公军. 白菜抗霜霉病基因的RAPD标记. 园艺学报, 2007, 34(3): 763-766.
[5] Farinhó M, Coelho P, Carlier J, Svetleva D, Monteiro A, Leitão J. Mapping of a locus for adult plant resistance to downy mildew in broccoli (Brassica oleracea convar. italica). Theor Appl Genet, 2004, 109(7): 1392-1398.
[6] Farinhó M, Coelho P, Monteiro A, Leitão J. SCAR and CAPS markers flanking the Brassica oleracea L. Pp523 downy mildew resistance locus demarcate a genomic region syntenic to the top arm end of Arabidopsis thaliana L. chromosome 1. Euphytica, 2007, 157(1-2): 215-221.
[7] Giovannelli JL, Farnham MW, Wang M, Strand AE. Development of sequence characterized amplified region markers linked to downy mildew resistance in broccoli. J Am Soc Hortic Sci, 2002, 127(4): 597-601.
[8] Yu SC, Zhang FL, Yu RB, Zou YM, Qi JN, Zhao XY, Yu YJ, Zhang DS, Li L. Genetic mapping and localization of a major QTL for seedling resistance to downy mildew in Chinese cabbage (Brassica rapa ssp. pekinensis). Mol Breed, 2009, 23(4): 573-590.
[9] 虞慧芳, 钟新民, 李必元, 顾宏辉. 与大白菜抗霜霉病基因连锁的分子标记研究. 中国农学通报, 2010, 26(15): 66-70.
[10] Marek LF, Shoemaker RC. BAC contig development by fin-gerprint analysis in soybean. Genome, 1997, 40(4): 420-427.
[11] Biber A, Kaufmann H, Linde M, Spiller M, Terefe D, Debener T. Molecular markers from a BAC contig spanning the Rdr1 locus: a tool for marker-assisted selection in roses. Theor Appl Genet, 2010, 120(4): 765-773.
[12] Kilian A, Chen J, Han F, Steffenson B, Kleinhofs A. To-wards map-based cloning of the barley stem rust resistance genes Rpg1 and rpg4 using rice as an intergenomic cloning vehicle. Plant Mol Biol, 1997, 35(1-2): 187-195.
[13] Quint M, Dußle CM, Melchinger AE, Lübberstedt T. Identification of genetically linked RGAs by BAC screening in maize and implications for gene cloning, mapping and MAS. Theor Appl Genet, 2003, 106(7): 1171-1177.
[14] Lehmensiek A, Sutherland MW, McNamara RB. The use of high resolution melting (HRM) to map single nucleotide polymorphism markers linked to a covered smut resistance gene in barley. Theor Appl Genet, 2008, 117(5): 721-728.
[15] Yang HB, Liu WY, Kang WH, Jahn M, Kang BC. Development of SNP markers linked to the L locus in Capsicum spp. by a comparative genetic analysis. Mol Breed, 2009, 24(4): 433-446.
[16] Van KJ, Hwang EY, Kim MY, Kim YH, Cho YI, Cregan PB, Lee SH. Discovery of single nucleotide polymorphisms in soybean using primers designed from ESTs. Euphytica, 2004, 139(2): 147-157.
[17] Mackay JF, Wright CD, Bonfiglioli RG. A new approach to varietal identification in plants by microsatellite high resolution melting analysis: application to the verification of grape-vine and olive cultivars. Plant Methods, 2008, 4(1): 8.
[18] 刘之熙, 刘云开, 詹庆才. 水稻孕穗期耐冷基因的CAPS标记辅助选择. 湖南农业大学学报 (自然科学版), 2008, 34(2): 127-131.
[19] 朱莎, 宋燕, 刘磊, Atanassova B, 徐和金, 周国龙, 李君明. 番茄ps-2基因的SSR及CAPS标记开发. 园艺学报, 2010, 37(2): 235-240.
[20] 束永俊, 李勇, 朱振雷, 朱延明. 大豆CAPS标记快速开发方法的建立与优化. 东北农业大学学报, 2009, 40(12): 62-65.
[21] 潘存红, 王子斌, 马玉银, 殷跃军, 张亚芳, 左示敏, 陈宗祥, 潘学彪. InDel和SNP标记在水稻图位克隆中的应用. 中国水稻科学, 2007, 21(5): 447-453.
[22] Hayashi K, Yoshida H, Ashikawa I. Development of PCR-based allele-specific and InDel marker sets for nine rice blast resistance genes. Theor Appl Genet, 2006, 113(2): 251-260.
[23] Harsh R, Rosy R, Rachel W, Peter
