基于转录组测序的枫香EST-SSR引物开发及有效性评价

李 辉<sup>1; 2</sup>; 冯源恒<sup>2</sup>; 唐生森<sup>2</sup>; 杨章旗<sup>1; 2*</sup>

引用本文:	李辉, 冯源恒, 唐生森, 杨章旗.基于转录组测序的枫香EST-SSR引物开发及有效性评价[J].广西植物,2023,43(2):327-335.[点击复制]
	LI Hui, FENG Yuanheng, TANG Shengsen, YANG Zhangqi.Development and validity evaluation of Liquidambar formosana EST-SSR primers based on transcriptome sequencing[J].Guihaia,2023,43(2):327-335.[点击复制]

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基于转录组测序的枫香EST-SSR引物开发及有效性评价
李辉^1,2, 冯源恒², 唐生森², 杨章旗^1,2*
1. 广西师范大学生命科学学院, 广西桂林541006;2. 广西壮族自治区林业科学研究院, 国家林业和草原局马尾松工程技术研究中心, 广西马尾松工程技术研究中心, 广西优良用材林资源培育重点实验室, 南宁 530002

摘要:

枫香是广西重要的乡土树种之一,具有较高的用材、观赏及药用价值。为验证枫香SSR引物的实际应用效果,该研究基于转录组测序技术,检测枫香SSR位点并设计引物,通过PCR扩增和聚丙烯酰胺凝胶电泳筛选出具有较高多态性的枫香EST-SSR引物,并对1个枫香天然群体进行遗传多样性分析。结果表明:(1)共发掘到23 777个SSR位点,单核苷酸重复类型SSR位点占总位点比例最高(46.54%),在重复次数上5～12次之间的SSR位点占比最高(72.36%)。(2)共开发出262对SSR引物,有效扩增率为53.1%,最终筛选出扩增稳定、条带清晰的引物18对。(3)多态性检测结果显示所有位点均具有多态性,天然群体遗传多样性结果显示该天然群体中等位基因数量(Na)、有效等位基因数量(Ne)、Shonnon信息指数(I)、观测杂合度(Ho)变化范围分别为2～4、1.112 8～2.609 6、0.208 9～1.112 7和0.275 9～1.000 0,平均值分别为2.333 3、1.957 4、0.708 5和0.722 6。综上认为,枫香中占优势的SSR位点重复类型和重复基序与其他物种基本相同,所开发的18对枫香EST-SSR引物可以满足开展枫香群体遗传学研究的需要。该研究结果为后续枫香种群遗传多样性研究提供了丰富的标记引物,对该树种的保护、开发和利用具有重要意义。

关键词: 枫香, 转录组, SSR分子标记, 引物开发, 遗传多样性

DOI：10.11931/guihaia.gxzw202202046

分类号:Q943

文章编号:1000-3142(2023)02-0327-09

基金项目:广西科技基地和人才专项(桂科AD19254004); 八桂学者专项(2019A26)。

Development and validity evaluation of Liquidambar formosana EST-SSR primers based on transcriptome sequencing

LI Hui^1,2, FENG Yuanheng², TANG Shengsen², YANG Zhangqi^1,2*

1. College of Life Sciences, Guangxi Normal University, Guilin 541006, Guangxi, China;2. Guangxi Forestry Research Institute, Engineering Research Center of Masson Pine of State Forestry Administration, Engineering Research Center of Masson Pine of Guangxi, Guangxi Key Laboratory of Superior Timber Trees Resource Cultivation, Nanning 530002, China

Abstract:

Liquidambar formosana is one of the important native tree species in Guangxi, which has high timber, ornamental and medicinal value. This study designs and develops EST-SSR markers of L. formosana based on data from the transcriptome sequencing. Primers were developed, and screened out by PCR amplification and polyacrylamide gel electrophoresis for high polymorphism, and the efficiency was tested by using 30 individuals from a wild L. formosana population to verify the practical application of these SSR primers. The results were as follows:(1)A total of 23 777 SSR loci were obtained by searching SSR Unigenes from transcriptome data. The repeat type of the SSR loci in L. formosana was dominated by mononucleotide repeat type(46.54%). The highest proportion of SSR loci(72.36%)was between 5 and 12 times.(2)A total of 262 pairs of SSR primers were developed, and among them, 139 pairs of effective amplifications with a success rate of 53.1%, and 18 pairs of them that could be used to steadily obtain clear bands were finally identified.(3)The polymorphism detection showed that all sites had a high degree of polymorphism. The number of alleles(Na), effective alleles(Ne), Shannon's diversity index(I), observed heterozygosity(Ho)of the L. formosana population ranged in 2-4, 1.112 8-2.609 6, 0.208 9-1.112 7 and 0.275 9-1.000 0, the average values were 2.333 3, 1.957 4, 0.708 5 and 0.722 6, respectively. In conclusion, the dominant SSR repeat type and repeat motif in L. formosana are basically the same as those in other species, the developed 18 pairs of EST-SSR markers can meet the needs of population genetic studies of L. formosana, which provides abundant primers for the study of genetic diversity of L. formosana. It is of important significance to the protection, development and utilization of L. formosana.

Key words: Liquidambar formosana, transcriptome, SSR molecular markers, primer development, genetic diversity