大白菜核雄性不育基因Ms的精细定位及BAC文库筛选

发布时间：2018-06-13 04:59

本文选题：大白菜 + 核基因雄性不育　；参考：《沈阳农业大学》2016年硕士论文

【摘要】：大白菜核复等位基因遗传的雄性不育材料的发现,为核不育系的选育和利用开辟了新途径。本实验室前期已将大白菜核不育复等位基因中的Ms基因,初步定位在A07染色体上。本研究以大白菜核雄性不育“两用系”AB01中的不育植株为母本,与大白菜小孢子DH系FT杂交,杂交后代的不育株与FT回交,构建大规模BC1精细定位群体,在A07染色体上继续开发与不育基因Ms连锁更紧密的SSR标记。同时,构建大白菜基因组BAC文库,筛选定位区间所在的染色体片段,为克隆Ms基因奠定基础。主要研究结果如下：1.利用构建的BC1分离群体,筛选到了与Ms基因紧密连锁的SSR分子标记LZY6和RF37,其与目的基因Ms的遗传距离分别为0.45cM和0.62cM。RF37与LZY6之间的遗传距离为0.17cM,确定Ms基因在A07染色体上的方向。2.构建了含有51840个克隆的BAC文库,其单个孔的原始克隆数为540个,文库的菌株为大肠杆菌DH10B,采用的载体是plndigoBAC-5。对BAC文库质量进行鉴定,随机选取文库中的9个单克隆,经过摇菌、酶切等检测到单克隆的平均插入片段大小为160Kb。基因组覆盖率为15x,能筛到阳性单克隆的几率在99.9%以上。随机选取的9个单克隆都含有插入片段,空载率为0%。3.设计引物SKRF1,利用PCR对BAC文库进行阳性混合池、阳性孔、阳性单克隆的逐级筛选,获得了阳性单克隆SRF,其片段大小约为140Kb。对SRF进行第二代测序,拼接成5个scaffold,预测编码37个基因。对37个预测基因在大白菜数据库中搜索,其中11个基因来自数据库中的已知基因。对37个预测基因在拟南芥中进行同源搜索(Blastx, E-10)其中18个基因可以找到同源基因,包括功能已知基因12个,功能未知基因6个。其余的19个基因在Nr数据库中搜索,能匹配到基因序列的有18个基因,包括功能已知基因1个,功能未知基因17个。
[Abstract]:The discovery of male sterile materials inherited by nuclear multiple alleles in Chinese cabbage opens up a new way for breeding and utilization of GMS lines. In our laboratory, the Ms gene of gene-sterile complex allele in Chinese cabbage was preliminarily located on chromosome A07. In this study, the sterile plants in "dual-purpose line" AB01 of Chinese cabbage were used as female parent, and FT hybridization with microspore DH line of Chinese cabbage was carried out. The sterile plants of hybrid progenies were backcrossed with FT, and a large scale BC1 fine localizing population was constructed. SSR markers linked to sterility gene Ms continued to be developed on chromosome A07. At the same time, the genomic BAC library of Chinese cabbage was constructed, and the chromosomal fragments with the location interval were screened, which laid the foundation for the cloning of Ms gene. The main results are as follows: 1. The SSR markers LZY6 and RF37, which were closely linked to Ms gene, were screened by using the constructed BC1 segregated population. The genetic distance between LZY6 and target gene Ms was 0.45cM and 0.62cM.RF37, respectively, and the genetic distance between LZY6 and LZY6 was 0.17cM. The BAC library containing 51840 clones was constructed. The original clone number of 51840 clones was 540, the strain of the library was E. coli DH10B, and the vector was plndigo BAC-5. The quality of the BAC library was identified. Nine monoclonal clones were randomly selected. The average size of the inserted fragment was 160 kb by shaking bacteria and enzyme digestion. The genome coverage was 15 x, and the probability of screening positive monoclonal was over 99.9%. The randomly selected nine monoclonal clones all contained inserted fragments with a no-load rate of 0. 3. The primers SKRF1 were designed, and the positive mixed pool, positive pore and positive monoclonal were screened step by PCR, and the positive monoclonal SRFs were obtained, the size of which was about 140 kb. The SRF was sequenced in the second generation and spliced into 5 scaffolds and encoded 37 genes. Thirty-seven predictive genes were searched in Chinese cabbage database, 11 of which were from known genes in the database. In Arabidopsis thaliana, 18 of the 37 predicted genes were homologous to Blastx (E-10), including 12 genes with known function and 6 genes with unknown function. The remaining 19 genes were searched in Nr database and 18 genes were found to match the gene sequence, including 1 gene with known function and 17 genes with unknown function.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S634.1

【相似文献】