基于EST-SSR标记的沙棘品种鉴定及指纹图谱构建

doi:10.19741/j.issn.1673-4831.2023.0018

生态与农村环境学报 ›› 2024, Vol. 40 ›› Issue (3): 374-385.doi: 10.19741/j.issn.1673-4831.2023.0018

基于EST-SSR标记的沙棘品种鉴定及指纹图谱构建

赵雨欣¹, 张哲文¹, 考惠霞¹, 孙永江², 辛智鸣³, 赵喆¹, 董树斌¹, 程瑾¹

1. 北京林业大学生物科学与技术学院/林木育种与生态修复国家工程中心/花卉种质创新与分子育种北京市重点实验室, 北京 100083;
2. 北京林业大学林学院/森林培育与保护教育部重点实验室, 北京 100083;
3. 中国林业科学研究院沙漠林业实验中心, 内蒙古磴口 015200

收稿日期:2023-01-10 出版日期:2024-03-25 发布日期:2024-03-18
通讯作者: 董树斌,E-mail: dongshubin@bjfu.edu.cn; 程瑾,E-mail:chengjin@bjfu.edu.cn E-mail:dongshubin@bjfu.edu.cn;chengjin@bjfu.edu.cn
作者简介:赵雨欣(1998-),女,河北邯郸人,主要从事荒漠旱生林木种群遗传学和生理生态学研究。E-mail: zhaoyx98@163.com
基金资助:
国家重点研发计划政府间国际科技创新合作专项(2019YFE0116500);国家林业和草原局林业行业标准制修订项目(2020-LY-062);高等学校学科创新引智计划111项目(B13007)

Identification and Fingerprint Construction of Hippophae rhamnoides Varieties Based on EST-SSR Markers

ZHAO Yu-xin¹, ZHANG Zhe-wen¹, KAO Hui-xia¹, SUN Yong-jiang², XIN Zhi-ming³, ZHAO Zhe¹, DONG Shu-bin¹, CHENG Jin¹

1. College of Biological Sciences and Technology, Beijing Forestry University/National Engineering Research Center of Tree Breeding and Ecological Restoration/Beijing Key Laboratory of Ornamental Plants Germplasm Innovation and Molecular Breeding, Beijing 100083, China;
2. College of Forestry, Beijing Forestry University/Key Laboratory of the Ministry of Education on Forestry Silviculture and Conservation, Beijing 100083, China;
3. Experimental Center of Desert Forestry, Chinese Academy of Forestry, Dengkou 015200, China

Received:2023-01-10 Online:2024-03-25 Published:2024-03-18

摘要/Abstract

摘要： 以沙棘(Hippophae rhamnoides)优良品种"实优1号"为材料,对其叶片进行转录组测序,利用微卫星识别软件(microsatellite identification tool,MISA)和Primer 3(version 2.3.4)对获得的序列进行简单重复序列(simple sequence repeat,SSR)位点挖掘和引物设计,以收集的42份沙棘品种为研究材料,开展聚合酶链式反应(PCR)和毛细管电泳检测,旨在开发一套多态性高、稳定性好和通用性强的表达序列标签微卫星(express sequence tags from simple sequence repeat,EST-SSR)引物,构建沙棘指纹图谱,从而实现沙棘品种的快速准确鉴定,并对沙棘品种间亲缘关系进行分析。"实优1号"转录组测序共获得6 196个SSR位点,其中,重复基元类型为182种,SSR基序长度主要分布在10~21 bp区间,占全部SSR的81.58%,主要SSR重复类型为单核苷酸重复(48.72%)、二核苷酸重复(22.68%)和三核苷酸重复(18.85%)。利用筛选出的28对引物在42份沙棘品种中共检测出193个等位基因,等位基因数(Na)、有效等位基因数(Ne)、观测杂合度(Ho)、期望杂合度(He)、多态信息含量(PIC)和Shannon信息指数(I)等遗传多样性参数的均值分别为6.964、3.495、0.617、0.671、0.623和1.384。UPGMA聚类分析表明,42份沙棘品种间的遗传相似性系数为0.601~0.990,当遗传相似性系数为0.694时,供试品种可分为2组;当遗传相似性系数约为0.740 2时,供试品种可分为3组。优选6对引物构建指纹图谱,可以实现沙棘品种的快速准确鉴定。该研究可为沙棘的良种鉴定、指纹图谱构建以及遗传多样性和亲缘关系分析等提供分子水平的理论基础和数据支撑。

关键词: 沙棘, 表达序列标签微卫星, 指纹图谱, 遗传多样性

Abstract: To achieve swift and precise variety identification of Hippophae rhamnoides, a set of EST-SSR (express sequence tags from simple sequence repeat) markers was developed. These markers exhibit high polymorphism, stability and generality, and have allowed the construction of the fingerprint of H. rhamnoides. For the transcriptome sequencing data, the young and fresh leaves of 'Shiyou 1’ were selected for RNA extraction and cDNA library construction. Additionally, young and healthy leaves from 42 varieties of H. rhamnoides were collected. The EST-SSR markers were designed by using microsatellite identification tool (MISA) and Primer 3 (version 2.3.4). PCR and TP-M13-SSR combined capillary electrophoresis examined fluorescence signals and peak emergence for 42 varieties. Amplified product lengths and genetic information of EST-SSR markers were assessed by using GeneMarker (version 2.2.0), Popgene (version 1.32), Cervus (version 3.0.7), and Convert (version 1.31). Genetic relationship analysis of the varieties was conducted by using NTSYSpc (version 2.10e). As a result, a total of 6 196 SSR loci were obtained, with 182 motifs types among all the SSR loci. The dominant motif was (A/T) n. The lengths of the SSR motifs were 10-21 bp, accounting for 81.58% of all SSR sequences. The main types of SSR repeat motifs were mono-nucleotide (48.72%), di-nucleotide (22.68%) and tri-nucleotide (18.85%). 28 pairs of primers were developed and a total of 193 allele loci in the 42 varieties were detected. The genetic diversity indexes, including the mean number of observed alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC) and Shannon information index (I) were 6.964, 3.495, 0.617, 0.671, 0.623 and 1.384, respectively. The genetic similarity coefficients between varieties ranged from 0.601 to 0.990, as determined by UPGMA clustering. At a coefficient of 0.694, varieties formed two groups, while at coefficient to be approximately 0.740 2, they grouped into three. Using six selected primers, the fingerprint of H. rhamnoides was constructed for rapid and accurate variety identification. This study provides the theoretical basis and molecular-level data support for variety identification, fingerprint construction, genetic diversity and relationship analysis of H. rhamnoides.

Key words: Hippophae rhamnoides, EST-SSR (express sequence tags from simple sequence repeat), fingerprint, genetic diversity

中图分类号:

X176

赵雨欣, 张哲文, 考惠霞, 孙永江, 辛智鸣, 赵喆, 董树斌, 程瑾. 基于EST-SSR标记的沙棘品种鉴定及指纹图谱构建[J]. 生态与农村环境学报, 2024, 40(3): 374-385.

ZHAO Yu-xin, ZHANG Zhe-wen, KAO Hui-xia, SUN Yong-jiang, XIN Zhi-ming, ZHAO Zhe, DONG Shu-bin, CHENG Jin. Identification and Fingerprint Construction of Hippophae rhamnoides Varieties Based on EST-SSR Markers[J]. Journal of Ecology and Rural Environment, 2024, 40(3): 374-385.

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基于EST-SSR标记的沙棘品种鉴定及指纹图谱构建

Identification and Fingerprint Construction of Hippophae rhamnoides Varieties Based on EST-SSR Markers

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