基于梨贝壳杉烯氧化酶基因PpKO序列的功能性SNP标记

Development of Functional SNP Markers Anchored PpKO Gene in Pear

基于基因序列的功能性标记是基因分型、图谱定位和相关性状标记辅助选择的理想标记。以'矮生梨'×'茌梨'的梨杂交分离群体和9个梨栽培品种为试材,以梨赤霉素合成代谢途径的关键酶基因--贝壳杉烯氧化酶基因(PpKO)的cDNA序列为基础,设计5对PCR引物,通过高通量熔解曲线(high resolution melting,HRM)分析,筛选到可对基因进行良好分型的1对引物。通过对杂种后代和测试品种的测序分析表明,此对引物的扩增子是一个位于PpKO第8外显子上的长度为200bp的序列,从中共检测到两处单核苷酸多态性(single nucleotide polymorphism,SNP)变化,且均为非同义cSNP。这两处SNP标记均可以作为适合高通量分析的遗传标记在遗传作图、基因定位或资源鉴定中加以利用。

The functional markers based on gene sequences are ideal markers for genotyping,gene mapping and marker assistant selection for associated traits. In this research,5 PCR primer pairs were designed according to the cDNA sequence of PpKO gene in pear which codes the protein of ent-kaurene oxidase（KO）,one of the key enzymes in the path of gibberellin synthesis. These primer pairs were tested in the population derived from the cross of‘Aishengli’בChili’by high resolution melting（HRM） ananlysis,and one of them was selected for genotyping. Furthermore,the fine genotyping function of this primer pair was confirmed by 9 cultivars. Sequencing and comparison of amplicons from the progenies and the 9 cultivars showed that the primer pair could amplified a fragment of 200 bp in length,which located in the eighth exon of PpKO gene,with 2 non-synonymous SNPs（single nucleotide polymor- phisms）detected. The two SNPs are genetic markers suitable for high resolution analysis,which are useful tools for genetic mapping,gene location and germplasm identification.