National Key Laboratory of Crop Genetic Improvement, Key Laboratory of Rapeseed Genetic Improvement (Ministry of Agriculture), Huazhong Agricultural University, Wuhan, China.
Ann Bot. 2013 Feb;111(2):305-15. doi: 10.1093/aob/mcs260. Epub 2012 Dec 13.
Spontaneous male sterility is an advantageous trait for both constructing efficient pollination control systems and for understanding the developmental process of the male reproductive unit in many crops. A triallelic genetic male-sterile locus (BnMs5) has been identified in Brassica napus; however, its complicated genome structure has greatly hampered the isolation of this locus. The aim of this study was to physically map BnMs5 through an integrated map-based cloning strategy and analyse the local chromosomal evolution around BnMs5.
A large F(2) population was used to integrate the existing genetic maps around BnMs5. A map-based cloning strategy in combination with comparative mapping among B. napus, Arabidopsis, Brassica rapa and Brassica oleracea was employed to facilitate the identification of a target bacterial artificial chromosome (BAC) clone covering the BnMs5 locus. The genomic sequences from the Brassica species were analysed to reveal the regional chromosomal evolution around BnMs5.
BnMs5 was finally delimited to a 0·3-cM genetic fragment from an integrated local genetic map, and was anchored on the B. napus A8 chromosome. Screening of a B. napus BAC clone library and identification of the positive clones validated that JBnB034L06 was the target BAC clone. The closest flanking markers restrict BnMs5 to a 21-kb region on JBnB034L06 containing six predicted functional genes. Good collinearity relationship around BnMs5 between several Brassica species was observed, while violent chromosomal evolutionary events including insertions/deletions, duplications and single nucleotide mutations were also found to have extensively occurred during their divergence.
This work represents major progress towards the molecular cloning of BnMs5, as well as presenting a powerful, integrative method to mapping loci in plants with complex genomic architecture, such as the amphidiploid B. napus.
自发雄性不育是构建高效授粉控制系统和理解许多作物雄性生殖单位发育过程的有利特征。已经在甘蓝型油菜中鉴定出一个三等位基因遗传雄性不育位点（BnMs5）；然而，其复杂的基因组结构极大地阻碍了该位点的分离。本研究旨在通过整合图谱基于克隆策略对 BnMs5 进行物理作图，并分析 BnMs5 周围局部染色体的进化。
利用一个大的 F2 群体整合 BnMs5 周围现有的遗传图谱。采用图谱基于克隆策略与甘蓝型油菜、拟南芥、白菜和甘蓝的比较作图相结合，有助于鉴定覆盖 BnMs5 位点的目标细菌人工染色体（BAC）克隆。对来自 Brassica 物种的基因组序列进行分析，揭示 BnMs5 周围的区域染色体进化。
BnMs5 最终被限定在一个整合的局部遗传图谱 0.3-cM 的遗传片段内，并锚定在油菜 A8 染色体上。筛选油菜 BAC 克隆文库并鉴定阳性克隆证实了 JBnB034L06 是目标 BAC 克隆。最接近的侧翼标记将 BnMs5 限制在 JBnB034L06 上包含六个预测功能基因的 21-kb 区域内。在几个 Brassica 物种中，BnMs5 周围观察到很好的共线性关系，然而在它们的分化过程中，也发现了广泛发生的染色体进化事件，包括插入/缺失、重复和单核苷酸突变。
这项工作代表了朝着油菜 BnMs5 分子克隆的重大进展，同时也为在基因组结构复杂的植物（如异源四倍体油菜）中定位基因提供了一种强大的、综合的方法。
