基于新一代高通量测序技术开发巨人半边莲Lobelia deckenii的SSR标记  

Development of SSR Markers in Giant Lobelia(Lobelia deckenii)Based on Next-generation High-throughput Sequencing

作　　者：米奇 龙志成[1,2] Muchuku John Kamau 陈进明[1] 王青锋[1] 

机构地区：[1]中国科学院武汉植物园,中国科学院水生植物与流域生态重点实验室,武汉430074 [2]中国科学院大学,北京100049

出　　处：《植物科学学报》2015年第6期847-854,共8页Plant Science Journal

基　　金：国家自然科学基金项目(31570220);中国科学院海外拓展工程项目(23Y323771W0777)

摘　　要：为了开发东非半边莲属特有植物的微卫星分子标记（SSR）,本研究基于Illumina-HiSeq 2000测序平台对巨人半边莲Lobelia deckenii的基因组进行高通量测序。利用MISA软件对获得的基因组数据库进行搜索与分析,共鉴别出58 966个SSR位点,并利用Primer软件成功设计出3558对特异性的SSR引物。利用L.deckenii 3个居群的6个样品对随机挑选的40对SSR引物进行扩增效率检验,发现有32对重复性好且可扩增出清晰条带。利用筛选出的32对SSR引物对来自肯尼亚山居群的24株个体进行PCR扩增并采用荧光分型技术检测多态性,结果显示有14对可扩增出稳定的多态性条带,共有86个等位基因,各SSR位点的等位基因数（NA）为4~9个,观测杂合度（Ho）和期望杂合度（He）分别为0.000~1.000和0.625~0.854。本研究结果表明,通过高通量测序技术开发东非特有植物巨人半边莲的SSR标记是一种简单而高效的途径,这些新的SSR分子标记为巨人半边莲的居群遗传多样性、遗传结构以及对其开展保护生物学研究提供了工具。To develop co-dominant microsatellite molecular markers（SSRs）for studying conservation genetics of the giant lobelia endemic to east Africa,we sequenced the genome of the giant lobelia,Lobelia deckenii,using next-generation high-throughput sequencing technology.Using the MISA program,we acquired a total of 58 966 SSRs,from which we designed 3558 SSR primer pairs using Primer software.We selected 40 primer pairs at random to evaluate their application across six individuals from three L.deckenii populations（two individuals per population）.Thirtytwo markers were successfully amplified,yielding clear and discernible bands.Using24 L.deckenii individuals from the Mountain Kenyapopulation,we tested the polymorphism of the 32 SSR markers and found that 14 were polymorphic.Using these 14 polymorphic SSR markers,we detected a total of 86 alleles.The number of alleles per locus ranged from four to nine.Observed heterozygosity（Ho）and expected value（He）per locus varied from 0.000 to 1.000 and 0.625 to 0.854,respectively.Our results indicated that development of SSR markers from genomic data by high-throughput sequencing in the giant lobelia was valuable and effective.These newly generated SSR markers will provide novel tools for studying genetic diversity,population genetic structure and conservation biology of giant lobelias in east Africa.

关 键 词：东非特有植物 半边莲属 高通量测序 SSR标记 

分 类 号：Q346[生物学—遗传学]