桂花EST-SSR引物开发及在品种鉴定中的应用

EST-SSR primers and their application in cultivar identification of Osmanthus fragrans

利用L16(45)正交实验设计,针对模板DNA,镁离子(Mg2+),d NTPs,Taq DNA酶和引物5个因素进行正交优化,建立适用于桂花Osmanthus fragrans表达序列标签-简单重复序列(EST-SSR)的最优扩增反应体系,将其用于桂花转录组EST-SSR多态性引物的筛选,最后利用筛选得到的多态性引物和聚类分析对14份四川省常见的栽培丹桂(SC01~SC14)样本进行品种鉴定。结果表明:桂花EST-SSR最优反应体系包含40 ng模板DNA,2.25 mmol·L-1Mg2+,0.3μmol·L-1引物,0.3 mmol·L-1d NTPs,1.25×16.67 nkat Taq DNA聚合酶,10×PCR Buffer缓冲液2μL,双蒸水补至20μL;从150对EST-SSR引物中筛选得到了19对稳定性好、多态性高的SSR引物,多态性从高到低依次为五核苷酸(25.00%),六核苷酸(19.44%)和三核苷酸(16.67%)重复类型。利用筛选得到的3对高度多态性SSR引物(OF8623-1677,OF24299-844和OF28774-2088)对四川省成都市周边常见栽培的14份丹桂(Aurantiacus group)样本进行了有效鉴定。结果表明:14份样本中,有12份可判定为‘朱砂丹桂’‘Zhusha Dangui’,SC06为‘堰红桂’‘Yanhong Gui’,SC08与‘满条红’‘Mantiao Hong’和‘状元红’‘Zhuangyuan Hong’亲缘关系较近。本研究也为今后利用EST-SSR标记对桂花指纹图谱构建、遗传多样性分析、分子育种及品种鉴定与保护等工作奠定基础。

In this study,expressed sequence tags(ESTs)were identified by EST-simple sequence repeat(SSR)markers.The optimal amplification reaction system for EST-SSR was established by orthogonal design of an L16(45)orthogonal experiment,and it was established for orthogonal design of template DNA,Mg2+,dNTPs,and TaqDNA enzyme and primer.Then,Osmanthus fragrans transcriptase EST-SSR polymorphism primers were screened.Finally,the polymorphic primers and cluster analysis were used to identify the cultivars cultivated in Sichuan Province.Results showed that the EST-SSR optimal reaction system of O.fragrans contained 40 ng template DNA,2.25 mmol·L-1 Mg2+,0.3μmol·L-1 primer,0.3 mmol·L-1 dNTPs,TaqDNA polymerase 1.25×16.67 nkat,10×PCR Buffer 2μL,and ddH2O to 20μL.A total of 19 SSR primers with high stability and high polymorphism were screened from 150 pairs of EST-SSR primers.The polymorphism was five nucleotides(25.00%),six nucleosides acid(19.44%),and trinucleotide(16.67%).Altogether a total of 14 samplings of O.fragrans cultivars cultivated in Chengdu,Sichuan Province were identified by high sensitivity polymorphic SSR primers.Analysis of genetic diversity,molecular breeding,and identification and protection of cultivars have laid a strong foundation for O.fragrans.