基于SSR荧光标记的MCID快速鉴定13个湖北茶树品种

Rapid identification of 13 Hubei tea cultivars by MCID method based on SSR fluorescent markers

【目的】通过人工绘制品种鉴别图(Manual cultivar identification diagram,MCID)快速区分鉴别湖北茶树品种,为湖北茶树种质资源评价、品种保护及苗木纯度早期鉴定提供技术支持。【方法】以湖北省13个优良茶树品种为材料,利用30对均匀分布于遗传连锁图谱上的SSR荧光引物进行PCR扩增,并用荧光标记毛细管电泳检测扩增产物以筛选出核心引物,利用MCID法构建CID图谱。【结果】30对SSR引物共扩增出145个等位基因,各引物等位基因数为3~9个,平均每对引物为4.83个;共检测到194个基因型,各引物检测出的基因型为3~12个,平均每对引物6.47个;多态性信息量(PIC)为0.29~0.85,平均为0.58。13个茶树品种的Nei’s遗传距离为0.19~0.44。基于Nei’s遗传距离,采用Neighbor-Joining(NJ)法可将13个品种分为三大类(Nei’s遗传距离为0.16),结果表明地理来源相同的品种可能因为具有相似的遗传背景而聚在一起,也可能因为亲本来源不同而存在明显的遗传差异。利用筛选出的3对核心引物(TM547、TM552和TM107)可快速鉴别所有参试品种,通过MCID法构建的CID图谱可直观看出各参试茶树品种鉴定所需要的引物及其对应的基因型。【结论】基于SSR荧光标记的茶树品种MCID鉴定方法具有快速、准确、高效的特点,可用于湖北茶树良种知识产权保护、苗期鉴定及品种区分。

【Objective】In order to provide theoretical basis for the evaluation of Hubei tea germplasm resources,proprotection of varieties and early identification of the purity of seedlings,manual cultivar identification diagram（MCID）was constructed to quickly distinguish and identify Hubei tea cultivars.【Method】Thirty pairs of fluorescent SSR markers distributed evenly on the genetic linkage group were used to carried out PCR amplification of thirteen Hubei fine tea cultivars. Fluorescence labeled capillary electrophoresis was used to detect the amplification products and screen the core primers. The cultivar identification diagram（CID）was drawn by MCID method.【Result】Thirty pairs of SSR primers amplified 145 alleles,ranging from 3 to 9 alleles for each primer,with an average of 4.83. A total of 194 genotypes were detected,and the genotypes detected by each primer were 3 to 12 with an average of 6.47. The range of polymorphism information content（PIC）was 0.29-0.85 with an average of 0.58. Relationship analysis showed that Nei’s genetic distance of the 13 tea cultivars was 0.19-0.44,and were classified into three groups by Neighbor-Joining（NJ）method based on Nei’s genetic distance（Nei’s genetic distance was 0.16）. It indicated that cultivars with the same geographical origin may grouped due to similar genetic backgrounds,or may also show great differences because of their different parental origin. Three pairs of SSR primers,TM547,TM552 and TM107,could rapidly identify all the tested cultivars. According to the CID map constructed by MCID method,the primers needed for the identification of various tea cultivars and their corresponding genotypes could be seen directly.【Conclusion】The MCID method based on fluorescent SSR markers is rapid,accurate and efficient in identification of tea cultivars. It can support the intellectual property protection,seedling appraisal and variety identification of Hubei fine tea cultivars.