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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。
Functional markers based on gene sequences are ideally markers for genotyping, mapping, and marker-assisted selection of related traits. In this study, pears hybrids and nine pear cultivars from ’Dwarf Pear’ × ’Pear’ were used as experimental materials, and the key enzyme genes of kanamycin synthase pathway - the kaolinite oxidase gene (PpKO) Based on the cDNA sequence, five pairs of PCR primers were designed and used to screen a pair of primers which could be well genotyped by high resolution melting (HRM) analysis. The sequencing analysis of the hybrid progeny and the tested varieties showed that the amplicon of the primer was a 200 bp-long sequence located on the exon 8 of PpKO. Two SNPs were detected from the CCP single nucleotide polymorphism (SNP) changes, and are non-synonymous cSNP. Both SNP markers can be used as genetic markers for high-throughput analysis in genetic mapping, gene mapping or resource identification.