大白菜InDels标记开发及其在剩余杂合体鉴定中的应用  

Development of In Dels Markers and Their Usage in Detection of Residual Heterozygous Lines in Chinese Cabbage(Brassica rapa ssp. pekinensis)

作　　者：张志刚[1] 赵智中[1] 李巧云[1] 王晓[1] 刘栓桃[1] 王淑芬[1] 徐文玲[1] 刘贤娴[1] 刘辰[1] 

机构地区：[1]山东省农业科学院蔬菜花卉研究所,山东省设施蔬菜生物学重点实验室/国家蔬菜改良中心山东分中心,济南250100

出　　处：《农业生物技术学报》2016年第4期510-518,共9页Journal of Agricultural Biotechnology

基　　金：山东省自然科学基金(No.ZR2014CM021,No.ZR2015YL073);山东省农业良种工程2014项目“十字花科名产蔬菜新品种选育”(No.2014LNK-96-PZTP-10);山东省农业科学院青年基金(No.2014QNM11)

摘　　要：大白菜(Brassica rapa ssp.pekinensis)参考基因组序列的公布及测序成本的降低为大规模开发插入/缺失(Insertion/Deletion,In Del)标记提供了可能。本研究以性状差异明显的大白菜自交系He102与06-247为亲本,开展了全基因组重测序、标记开发和验证工作。二者测序深度分别为10×和8×,经筛选共获得330 218个In Dels差异位点,出现频率为1.2个/kb,其中有11 238个差异位点位于编码区,包括5 184个差异基因,每个基因平均包含2.2个差异位点。分别以He102与06-247测序数据中筛选出的多态性In Dels位点为参照,从10条染色体上随机选择933个位点进行了PCR扩增及电泳检测验证。结果表明,测序深度对假阳性率有直接影响,以测序较深的材料中筛选到的差异位点为参照,其验证结果的假阳性率亦较高,反之则较低。本研究中以He102与06-247为参照选择In Dels位点验证的平均假阳性率分别是51.9%和22.5%。从933个位点中共筛选出593个在亲本之间实际表现为多态性的位点,这些差异位点中有375个位于编码区,是潜在的功能性标记。利用上述差异位点,检测了He102与06-247衍生的F7代重组自交系群体,明确了F7代各株系在593个位点的纯/杂合状态,为利用剩余杂合株系精细解析和精准定位大白菜耐抽薹、抗病毒、抗干烧心等重要农艺性状奠定了基础。The completion of Chinese cabbage （Brassica rapa ssp. pekinensis） whole genome and the reduced cost of re- sequencing provide possibility for high- throughput development of Insertion/Deletion （InDel） markers. In the present study, two Chinese cabbage inbred lines He 102 and 06-247 with significant differences in phenotype such as bolting tolerance, virus resistance and tip-burn tolerance were re-sequenced for genome- wide InDels identification. The sequencing depth of these two lines was 10 × and 8 × respectively. A total of 330 218 polymorphic InDels loci were predicted based on the sequence data. The frequency of InDels occurrence was 1.2/kb, among which 11 238 loci were located in deduced coding region and represented for 5 184 genes. We randomly selected 933 predicted polymorphic InDels loci that distributed on the 10 chromosomes of Hel02 and 06-247, respectively for PCR validation. Results showed that the positive rate varied greatly with sequencing depth. When the choosed loci from a higher sequencing depth material Hel02, the false positive rate was higher either, and vise versa. In the present study, the average false positive rate was 51.9% and 22.5% for the selected loci from Hel02 and 06-247, respectively. In other words, higher false positive rate based on higher-depth material reflected that the false positive loci were missed in lower-depth material; contrarily, lower false positive rate based on lower-depth material reflected that the positive loci were detected in higher-depth material. A total of 593 verified loci including 375 loci located in coding sequence （CDS）, were identified. More than 90% of the polymorphism loci were co-dominant while about 5% of them were dominant. The rest couldn＇t produce clear band or multiple bands were generated. The detected polymorphic loci were not evenly distributed on ten chromosomes. Chromosome A01 contained the most number （86） of loci, while A08 contains only 29 loci. The co-dominant polymorphic loci were used to detect the residual het

关 键 词：大白菜 重测序 全基因组 InDels标记 剩余杂合体(RHL) 

分 类 号：S634.1[农业科学—蔬菜学]