摘要
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 was applied to optimize SCoT-PCR amplification system of Chrysanthemum in five factors such as Mg2＋, dNTPs, primer, Taq DNApolymerase and template DNA. An optimal reaction system of species from Chrysanthemum was completed： 25 gL PCR reaction mixtures contained Mg2＋ 1.2 mmol · L-1, dNTPs 0.15 mmol · L-l, primers 0.8 μmol · L-1, Taq polymerase 1 U, template DNA 50 ng. The most suitable annealing temperature of primers was 49.4 ℃. The improvedmethod for separating PCR products was 8% polyacrylamide gel electrophoresis followed by silver staining. Three materials from Chrysanthemum with different ploidy levels （2x, 4x, 6x） were used to test the optimized reaction system. Ideal results with clear polymorphic bands showed that the stable and reliable reaction system was obtained. SCoT markers were applied to study the genetic diversity and relationship of 18 species of Chrysanthemum and its related genera. Using 12 selected SCoT primers 209 bands were generate, of which 189 （90.43%） were polymorphic. The Jaccard genetic similarity coefficients among the species were calculated with NTSYS-pc2.10e software and the values were between 0.4516 and 0.7035 with an average of 0.58. These coefficients were utilized to construct a dendrogram using the unweight pair-group method with arithmetic averages （UPGMA） . The results showed that 18 species of Chrysanthemum and its related genera could be grouped into two distinct clusters based on similarity of 0.530. The others except Crossostephium chinense and Artemisia sericea were in one cluster, which was further divided into six groups when the similarity coefficient value was 0.615. The results demonstrate that the SCoT marker system is useful for studying genetic diversity and relationships among species of Chrysanthemum and its related genera.
出处
《园艺学报》
CAS
CSCD
北大核心
2013年第10期2015-2025,共11页
Acta Horticulturae Sinica
基金
国家自然科学基金项目(31272196)
‘948’计划项目(2003-S13)
江苏省科技支撑计划项目(BE2011325
BE2012350)
江苏省农业科技自主创新资金项目[CX(11)1034
CX(12)2020]
教育部新世纪优秀人才支持计划项目(NCET-10-0492)
