基于简化基因组数据开发岭南青冈微卫星引物

Development of Microsatellite Primers of Quercus championii with RAD-seq Data

微卫星标记(simple sequence repeat,SSR)在基因组中普遍存在,具有共显性、高多态性等特点,在群体空间遗传研究中应用广泛。随着测序技术的发展,SSR的开发方法也更加多样化。岭南青冈(Quercus championii Benth)是中国南方常绿阔叶林的珍贵材用树种,对其分子标记的开发可促进岭南青冈的育种和种质保护。本研究基于4个岭南青冈个体的简化基因组序列进行SSR引物的开发。使用pyRAD软件分析显示:①每个个体中包含微卫星重复片段的序列在46000~84000条;②个体内聚类后得到的位点数在5500~24000;③个体间聚类后获得1158个一致性位点。在1158个位点中获得186个侧翼序列没有变异且重复碱基位于序列中间位置的位点。使用Primer Premier 5.0设计了25对引物,在来自3个群体的36个岭南青冈个体中进行验证,结果表明17对引物能成功扩增,共获得106个等位位点,每个引物的等位基因数在2~12,平均为6.2。引物的期望杂合度和观测杂合度分别为0.19~0.88和0.11~0.76。本研究的引物开发方法具有速度快、效率高、成本低的特点,可应用于群体遗传学分子标记的开发。

Microsatellite(simple sequence repeat,SSR)is a co-dominant molecular marker commonly found in plant genome with high polymorphism.This genotyping method is widely used in the study of spatial genetic structure of populations.With the blooming of the new sequencing technologies,the development method of SSR is also more diverse.Quercus championii is a precious wood species in the evergreen broad-leaved forest of South China.The development of its molecular markers can promote the species breeding and germplasm conservation.We used the reads obtained from RAD-seq(restriction-site associated DNA sequence)of four individuals of Q.championii to developed SSR primers.The pyRAD analysis showed that:(1)the reads containings 46000-84000 repeats sequence in each individual;(2)within the individual ranged clustering,5500-24000 loci were detected;(3)1158 concordance loci were obtained after clustering between individuals.Finally,186 loci with flanking sequences were not mutated and repeating base was in the middle were obtained.A total of 25 primers pairs were designed using premier 5.0 and validated in 36 individuals from 3 populations.The results showed that 17 primer sets were successfully amplified and 106 alleleswere obtained.The number of alleles per primer between 2-12,and the average number is 6.2.The expected heterozygosity and observed heterozygosity of the primers were 0.19-0.88 and 0.11-0.76,respectively.The rapid,effective,and low cost in this method can be applied to the development of molecular markers of population genetics.