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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 publication of the reference genome sequence of Brassica rapa ssp. Pekinensis and the reduction of the sequencing cost have made it possible to develop the Insertion / Deletion (In Del) marker on a large scale. In this study, Chinese cabbage inbred lines He102 and 06-247 with significant differences in traits were used as parents to carry out genome-wide resequencing, marker development and validation. The two sequences were sequenced at depths of 10 × and 8 ×, respectively. A total of 330 218 In Dels loci were found by screening, with a frequency of 1.2 / kb, of which 11 238 were located in the coding region, including 5 184 differences Genes contain an average of 2.2 differential loci per gene. According to the polymorphism In Dels site screened from He102 and 06-247 sequencing data, 933 sites were randomly selected from 10 chromosomes for PCR amplification and electrophoresis. The results showed that the depth of sequencing had a direct effect on the false positive rate, and the difference of the screening sites in the deep sequencing materials served as a reference. The false positive rate of the validation results was also higher, and vice versa. In this study, the average false-positive rate of In Dels locus was 51.9% and 22.5% respectively based on He102 and 06-247. A total of 593 loci that actually showed polymorphisms among parents were screened out from 933 loci. 375 of these loci were located in the coding region and were potential functional markers. Using the above-mentioned difference sites, the population of F7 recombinant inbred lines derived from He102 and 06-247 was detected, and the pure / heterozygous status of each of the F7 lines at 593 loci was determined. In order to utilize the remaining hybrid lines, Analysis and precise positioning of Chinese cabbage resistant to bolting, antiviral, anti-heartburn and other important agronomic traits laid the foundation.