应用SNaPshot技术对桉树SNP的检测

【目的】SNaPshot是一种单核苷酸多态性(SNP)检测的多重分析技术,具有检测速度快、准确性高、成本低廉等特点。利用多重PCR技术,结合SNaPshot分型方法,在桉树中构建SNP复合分型检测体系,促进SNP技术在桉树遗传图谱构建和无性系鉴定的应用。【方法】利用桉树已有的EST序列设计引物,通过PCR产物直接测序进行SNP标记位点的开发,利用多重PCR技术和SNaPshot分型方法建立SNP复合分型检测体系,并在尾叶桉和细叶桉作图群体的两亲本和6个F₁子代,以及16个国内常用的桉树无性系中进行分型检测。【结果】共设计合成12对SNP引物,分为3组(Ⅰ、Ⅱ、Ⅲ)建立了SNP复合分型检测体系,SNP分型情况与测序结果一致。对桉树作图群体的两个亲本和6个F₁子代的分型结果进行统计,发现12个SNP标记在6个子代中均发生了分离; 对桉树无性系进行多态性的分析,期望杂合度(H_e)为0.11～0.51、观测杂合度(H_o)为0.11～0.56,多态性信息含量(PIC)为0.10～0.37,表现为中度或低度多态性。【结论】利用多重PCR和SNaPshot技术可以快速地对桉树进行基因分型,其结果准确可靠,可以用于桉树遗传图谱的构建以及桉树无性系的鉴定等方面研究。

Application of SNaPshot to detect SNP markers in Eucalyptus

Abstract: 【Objective】The SNaPshot technique, a multiplex PCR method for genotyping SNP markers, is rapid, accurate and cost effective. In this paper, we used multiplex PCR and the SNaPshot technique to establish a multiplex primer assay for the SNaPshot reaction in Eucalyptus spp., in order to promote the application of SNPs in Eucalyptus spp. genetic linkage map construction and identification of the major commercial clones cultivated in China. 【Method】The primers were designed with Eucalyptus ESTs and the PCR products were sequenced using a PCR direct sequencing method to detect SNP loci. The selected SNP markers were used to establish the multiplex primer assay for the SNaPshot reaction. Finally, we applied this method to assay two parents and six F₁ mapping population sibs of E. urophylla × E. tereticornis, as well as 16 commercial Eucalyptus clones cultivated in China.【Result】Twelve SNP markers with three groups(I, II and III)were analyzed and the multiplex primer assay for the SNaPshot reaction in Eucalyptus was established. The SNaPshot assay genotyping results were in complete agreement with the PCR product direct sequencing results. The polymorphism diversity of these SNPs was also analyzed; all 12 loci were polymorphic among the six E. urophylla × E. tereticornis sibs and the commercial clones. The expected heterozygosity(H_e)ranged from 0.11 to 0.51, observed heterozygosity(H_o)ranged from 0.11 to 0.56, and polymorphic information content(PIC)ranged from 0.10 to 0.37, showing medium or low polymorphism.【Conclusion】The multiplex primer assay for the SNaPshot reaction established here is an efficient and accurate method for Eucalyptus genotyping, which will be useful for genetic linkage map construction and identification of Eucalyptus clones.