新开发的禾本科通用性分子标记的检测与评估  

Universal genetic markers for the Poaceae family

作　　者：李兵娟 肖国辉[1] 许在恩 郭小勤[1] 

机构地区：[1]浙江农林大学亚热带森林培育国家重点实验室培育基地,浙江临安311300

出　　处：《浙江农林大学学报》2014年第4期508-514,共7页Journal of Zhejiang A&F University

基　　金：国家自然科学基金资助项目(30901155);浙江省自然科学基金资助项目(Y307499)

摘　　要：为开发更多的适用于禾本科Poaceae植物通用性分子标记,丰富竹类植物的遗传信息,依据禾本科单拷贝同源基因(COSⅡ)已知序列的保守区域设计了14对引物,以此引物对刚竹属Phyllostachys植物进行聚合酶链式反应(PCR)扩增,并对部分扩增产物进行测序。结果所有引物可以在至少1个材料中得到特异性扩增,其中7对引物在5个供试材料中均能扩增到产物,13对引物在5个样本中表现出多态性,占引物总数的92.9%。对部分扩增产物进行测序,所测序列均为特异性扩增序列。同一引物在不同样本中的扩增片段间存在单核苷酸多态性(SNP)位点,部分SNP位点会导致氨基酸序列提前终止或酶切位点变化。对有合适内切酶存在的变异位点进行PCR-RFLP验证,验证结果与测序结果一致。利用NTsys 2.10e软件,对5个供试竹种材料进行了亲缘关系分析,毛竹Phyllostachys edulis与绿粉竹Phyllostachys virdiglaucescens亲缘关系最近,紫竹Phyllostachys nigra与其他4个竹种亲缘关系最远。所开发的14个COSⅡ引物在刚竹属植物中具一定通用性,同时挖掘出更多竹类植物中的遗传信息。To develop more universal molecular markers for grasses, rich genetic information of bamboo plants and 14 pairs of primers were designed according to the conserved regions of the single-copy orthologous （COS Ⅱ） genes of the Poaceae （grass） family. Then, polymerase chain reaction （PCR） was used to test five bamboo accessions. Also, Primer 1 and Primer 2 were used to sequence parts of the amplified products, and single nu- cleotide polymorphisms （SNP） sites were validated with the PCR-restriction fragment length polymorphism （RFLP） test. Based on different band styles amplified with the 14 primer pairs, genetic distance was calculated with NT sys 2.10e software and a cluster analysis of five species： Phyllostachys edulis, Ph. viridiglaucescens, Ph. nigra, Ph. violascens, and Ph. vivax f. aureocaulis were conducted. Results showed that all primers exhibit- ed specific PCR products in at least one sample. Seven pairs of primers were able to amplify the products in tested samples with 13 （92.9%） pairs of primers exhibiting polymorphic PCR products in all samples. Sequencing showed that Primer 1 and Primer 2 were homologous. These sequences were amplified with the same primer and existed at SNP sites. Some SNP sites led to early termination of the amino acid sequence, and some result- ed in changes of restriction endonuclease sites. Results of the PCR-RFLP test corresponded with the sequencing. The cluster analysis indicated that Ph. edulis and Ph. viridiglaucescens were closely clustered, and Ph. nigra was the most isolated. Overall, this classification coincided with morphological classification; so, thesemolecular markers were suitable for application in genetic analyses and other related research with Phyllostachys.

关 键 词：植物学 竹类植物 通用性分子标记 单拷贝同源基因(COSⅡ) 单核苷酸多态(SNP) 

分 类 号：S718.46[农业科学—林学]