芥菜型油菜A9染色体BAC物理图谱构建及比较基因组分析

摘要

To abtain a precise physical map of Brassica juncea chromosome A9,the markers located on the A9 chromosome were used in this study to screen Brassica juncea BAC library by three-dimensional mixed PCR screening. A total of 3200 positive BAC were screened out;constructing a physical map composed of 16 contigs and 4 singletons. According to the principle of the minimum path,435 BACs were chosen as seed BACs to represent the whole physical map and were subject to end sequencing,at last,862 qualified sequences were received. As the average insert size of BAC is 126 kb,the contigs accounted up to 52. 08 M,covering 1. 37 times of chromosome A9. By BLAST,the seed BAC end sequences were mapped to chromosome A9 pseudomolecule of Brassica rapa and Brassica napus respectively,results showed that the physical map covered 36. 4 Mb and 31. 0 Mb,equivalent to 93. 9%、91. 8% of the whole genome assemble length. Among the 862 BAC end sequences,702 sequences (81. 4%)were located on Brassica rapa A9 chromosome and 646 sequences(74. 9%)to Brassica napus A9 chromosome,which means Brassica juncea A9 chromosome have higher homology with Brassica rapa A9 chromosome than Brassica napus A9 chromosome. Compared with Brassica napus A9 pseudomolecule and BAC end se-quence of Brassica juncea A9 chromosome,there are two chromosome inversions about 1. 4 Mb、3. 6 Mb in length at the po-sition of 6413031-7827177 and 14856710-18458808 in Brassica rapa A9. While compared with BAC end sequence of Bras-sica juncea A9,there is a inserted mutation about 0. 98 Mb in length at the position of 15676847-16064302 of Brassica na-pus A9 pseudomolecule,which lie in one of the two chromosome inversions and was consist with the predicted position of centromere of Brassica napus A9 chromosome remained to assembled.%为获得高精确度的芥菜型油菜A9染色体物理图，本研究利用定位在A9染色体上的标记对芥菜型油菜BAC文库进行三维混合PCR筛选，共得到阳性BAC 3200个；构建了由16个重叠群、5个单拷贝组成的芥菜型油菜A9染色体物理图谱。根据最小路径原则挑选代表整个物理图的种子BAC 435个，并进行双末端测序，得到质量良好的序列862条；按照BAC平均插入片段为126 kb，可知所有重叠群累积达52.08 M，覆盖A9染色体的1.37倍。利用BLAST软件（按参数值E＜1e－10）将种子BAC末端序列比对到白菜和甘蓝型油菜A9染色体假分子上，结果显示该物理图谱分别覆盖了36.4、31.0 Mb的物理距离，相当于全基因组组装长度的93.9％、91.8％；在862条BESs中，分别有702条（81.40％）定位在白菜A9染色体上，646条（74.9％）定位在甘蓝型油菜A9染色体上，表明芥菜型油菜A9染色体同白菜A9染色体同源性高于甘蓝型油菜A9染色体；芥菜型油菜A9染色体同甘蓝型油菜A9染色体，相对白菜A9染色体6413031～7827177、14856710～18458808的位置发生了两处相同的分别长为1.4、3.6 Mb的倒位，白菜和芥菜型油菜A9染色体相对于甘蓝型油菜15676847～16064302的区域存在长为0.98 Mb的插入片段，该区域位于前述的倒位区域，而且与预测但未组装出的甘蓝型油菜着丝粒位置一致。