杏高通量荧光SSR检测体系及分子身份证的构建

The Establishment of Fluorescent SSR Markers for High-throughput Detection and Construction of Molecular Identity on Apricot

本研究以杏(Prunus armeniaca L.)为试材,根据M13荧光引物和巢式PCR理论,通过调整巢式PCR内两部分循环数、M13荧光引物与正向引物比例和4种荧光(FAM,VIC,NED和PET)产物混合比例3个主要因素,建立了高通量荧光标记SSR检测体系。结果显示,当循环数比设为15∶25、M13荧光引物与正向引物比例为4∶1时,巢式PCR反应体系达到最佳。4种荧光产物按照1μL∶1μL∶1μL∶2μL混合稀释30倍上机检测,所得荧光信号较强、峰高均匀、稳定性好。在建立的体系基础上,筛选了12对多态性良好的李属(Prunus)SSR引物,构建了50份中国杏种质资源的分子身份证。各材料的遗传分子身份证具有唯一性、易读性,可以将种质资源精确的区分开,有利于中国杏种质资源鉴定和保护、品种权益保护和品种创新的激励。

In this study,apricot(Prunus armeniaca L.) was selected as the test material.Based on M13fluorescent primer and nested PCR theory,by adjusting the number of two-part cycles in nested PCR,the ratio of M13fluorescent primers to forward primers and the mixing ratio of four fluorescent products(FAM,VIC,NED and PET),a high-throughput fluorescent marker SSR detection system was established.The results showed that the nested PCR system was optimal when the cycle ratio was 15:25 and the ratio of M13fluorescent primers to forward primers was4∶1.Four kinds of fluorescent product mixed by diluting 30 times in accordance with 1 ∶1∶1∶2(μL) was detected with strong,uniformity and stabilize fluorescence signal.Twelve pairs of primers with well polymorphism were screened,and 50 molecular identity of apricot germplasm resources were conducted.The unique molecular ID of various quality resources can accurately distinguish the germplasm resources,which is conducive to the identification and protection of apricot germplasm resources,the protection of variety rights and the incentive of variety innovation in China.