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采用正交设计方法,对Mg2+、dNTPs和引物浓度、TaqDNA聚合酶及模板DNA用量等5个因素进行筛选,得到适于菊属植物的SCoT标记PCR反应体系,25μL体系中含有:Mg2+1.2 mmol·L-1、dNTPs 0.15 mmol·L-1、引物0.8μmol·L-1、TaqDNA聚合酶1 U、模板DNA 50 ng。优化的最适退火温度为49.4℃。改进电泳方法,采用8%非变性聚丙烯酰胺凝胶电泳,银染法染色。运用不同倍性菊属材料基因组DNA对优化的SCoT-PCR反应体系进行验证,获得了多态性丰富、条带清晰的扩增图谱,表明确立的菊属植物SCoT分子标记技术体系稳定可靠。利用该体系及筛选出的12个SCoT引物,对18份菊花近缘种属材料的遗传多样性及亲缘关系进行分析,共检测到209个位点,其中多态性位点数189个,多态性比率为90.43%。应用NTSYS-pc2.1软件,计算菊花近缘种属材料之间的相似性系数并采用不加权成对算术平均法(UPGMA)进行聚类分析,结果 18份菊花近缘种属材料的遗传相似性系数范围为0.4516～0.7035,平均遗传相似性系数为0.58。聚类分析显示在相似系数0.530处可以将18份材料分成Ⅰ、Ⅱ两个大组,Ⅱ组包括芙蓉菊和绢毛蒿,其余16份材料属于Ⅰ组。Ⅰ组在相似系数0.615水平又分为6个亚组。结果表明SCoT分子标记体系适用于菊属及其近缘属种遗传多样性及亲缘关系的研究。
Orthogonal design method was used to screen SCoT-labeled PCR reaction system for Mg (superscript 2 +), dNTPs and primer concentration, Taq DNA polymerase and template DNA dosage. The 25μL system contained Mg2 + 1.2 mmol L-1, dNTPs 0.15 mmol·L-1, primer 0.8 μmol·L-1, Taq DNA polymerase 1 U, template DNA 50 ng. The optimal annealing temperature is 49.4 ℃. Improve the electrophoresis method, using 8% non-denaturing polyacrylamide gel electrophoresis, silver staining staining. The optimized SCoT-PCR reaction system was verified by genomic DNA of different Pleurotus species, and the amplified polymorphic bands with clear bands were obtained. The established SCoT molecular marker technology system was stable and reliable. Using this system and 12 SCoT primers screened, genetic diversity and genetic relationship among 18 accessions were analyzed. A total of 209 loci were detected, of which 189 polymorphic loci were polymorphic Sex ratio was 90.43%. NTSYS-pc2.1 software was used to calculate the similarity coefficient between the relative species of chrysanthemum and the unweighted pairwise arithmetic average method (UPGMA) was used for cluster analysis. Results The genetic similarity of 18 chrysanthemum germplasm resources was similar The range of sex coefficient was 0.4516 ~ 0.7035, and the average genetic similarity coefficient was 0.58. Cluster analysis showed that 18 materials could be divided into two groups Ⅰ and Ⅱ at similarity coefficient of 0.530. Group Ⅱ consisted of Hibiscus chinensis and Artemisia sphaerocephala and the other 16 belong to group Ⅰ. Group Ⅰ was divided into 6 subgroups at the 0.615 level of similarity coefficient. The results showed that the SCoT molecular marker system is suitable for the study of genetic diversity and genetic relationship of Gerntia and its related genera.