基于非变性聚丙烯酰胺凝胶电泳的菜心SCoT分析

以菜心(Brassica rapa var. parachinensis)抗小菜蛾品种Caixin65和感小菜蛾品种Caixin69及6份F2株系为材料，利用非变性聚丙烯酰胺凝胶电泳检测菜心抗虫株系的SCoT多态性，同时进行SCoT多态性的非变性聚丙烯酰胺凝胶电泳检测效率、遗传多样性分析和差异条带克隆分析。结果表明，非变性聚丙烯酰胺凝胶能检测出亲本与子代间的SCoT多态性和遗传多样性变化，条带数量较多且清晰，提高了SCoT标记检测效率。选取亲本的10条非变性聚丙烯酰胺凝胶差异片段克隆测序，获得感虫序列4条、抗虫序列6条，GENSCAN预测其中8条具有启动子、终止子、阅读框等基因结构序列。同源性检索分析表明，感虫序列分别与泛素羧基末端水解酶mRNA序列、大白菜克隆序列、白菜线粒体丙酮酸载体蛋白序列、甘蓝基因组编码未知蛋白的HDEM序列同源，抗虫序列分别与白菜RNA假尿苷合酶4线粒体mRNA序列、京水菜线粒体DNA序列、白菜未知蛋白mRNA序列、白菜4-香豆酸：辅酶A连接酶mRNA序列、大白菜克隆序列、哈茨木霉mRNA序列同源。本研究提高了SCoT标记清晰度、遗传多样性检测水平和差异片段克隆的精准性，使得SCoT成为批量克隆差异片段的高效工具，有助于挖掘SCoT功能性标记信息，开展初步的功能基因组学研究，提高优异株系筛选鉴定效率，加快育种进程，为菜心抗虫性机制的进一步研究提供理论基础。

SCoT Analysis of Flowering Chinese Cabbage (Brassica rapa var. parachinensis) Based on Native Polyacrylamide Gel Electrophoresis

In order to provide theoretical basis for the screening and identification of excellent materials and the preliminary study of resistance mechanisms in flowering Chinese cabbage(Brassica rapa var. parachinensis) cultivars, the SCoT polymorphism was used to analyze by native polyacrylamide gel electrophoresis with Caixin65, Caixin69 and F2 segregating lines. The detection efficiency, genetic diversity and differential cloning bands of native polyacrylamide gel electrophoresis were analyzed. The results showed that the native polyacrylamide gel could detect the SCoT polymorphism and genetic diversity between parents and offspring, and SCoT bands were more and clearer on native polyacrylamide gel than agarose gel, which elevated the detection efficiency of SCoT. Ten native polyacrylamide gel differential fragments were selected for cloning and sequencing, and four insect-susceptible sequences and six insect-resistant sequences were obtained. GENSCAN predicted that 8 of 10 SCoT sequences had promoter, terminal and open reading frame of genes. Homology search showed that function from 4 SCoT sequences obtained from Caixin69 included Brassica napus ubiquitin carboxyl-terminal hydrolase MINDY-1-like (LOC106367017) mRNA, B. rapa subsp. pekinensis clone KBrB026J02 complete sequence, B. rapa mitochondrial pyruvate carrier 2 (LOC103839865) mRNA, B. oleracea HDEM genome scaffold C3. Function from 6 SCoT sequences obtained from Caixin65 included B. rapa RNA pseudouridine synthase 4 mitochondrial (LOC103869454) mRNA, Trichodermaharzianum CBS 226.95 hypothetical protein (M431DRAFT_397944) mRNA, B. rapa subsp. nipposinica mitochondrial DNA complete sequence cultivar: Oushou hakusai, B. napus uncharacterized LOC106451263 (LOC106451263) mRNA, B. rapa subsp. pekinensis clone KBrB017P15 complete sequence. The analysis based on native polyacrylamide gel electrophoresis elevated the clarity and resolution SCoT marker, the detection level of genetic diversity level and accuracy of clone sequences. SCoT analysis based on native polyacrylamide gel electrophoresis was not only an efficiency method to detect genetic level, but also a simple and efficiency method to clone genes which could be used in genomics. The study improved the screening and identification efficiency of excellent strains, speeded up the breeding process, and provided a theoretical basis for further research on the mechanism of the insect-resistant breeding of flowering Chinese cabbage.