基于RNA-seq结果开发猪SSR标记

doi:10.11843/j.issn.0366-6964.2021.06.009

摘要/Abstract

摘要： 旨在开发猪SSR标记，为猪遗传多样性分析、亲缘关系鉴定、标记辅助选择等奠定基础。本研究基于前期对6月龄大白猪和马身猪背最长肌RNA-seq结果，根据SSR在染色体上的分布、碱基类型、重复次数等的差异随机筛选154个位点，设计合成SSR引物，进行PCR扩增、PAGE检测及克隆测序验证，开发多态性SSR标记。利用开发的SSR标记对马身猪、大白猪、晋汾白猪及山西黑猪等4个猪种各30头6月龄个体进行遗传多样性分析，以评价所开发SSR标记的应用效果。结果，从36 693条转录组Unigene序列中搜索到10 488个SSRs位点，纯合型SSR为9 424个，复合型SSR为1 064个，分布于6 953条Unigene序列，其中4 727条Unigene含有单个SSR，2 226条Unigene含有2个及以上SSRs，SSR发生频率为18.95%，出现频率为28.58%。优势SSR重复类型为单、三、二核苷酸重复，其重复次数主要集中于5~22次；优势重复基序序列类型为A/T、AC/GT、GCC/GGC，长度类型为10~20 bp。随机筛选154个位点进行验证，共有124对引物扩增出清晰、特异的条带，扩增效率为80.52%，其中25对SSR引物具有多态性，占比为16.23%。利用25对SSR引物对4个猪种共120个个体进行遗传多样性分析，共识别到131个等位基因，等位基因数为2~7个，平均等位基因数为5.24，平均有效等位基因数为3.487 1，平均PIC为0.646 7，呈高度多态，总体表现出较高的多样性。本研究结果表明，基于猪转录组测序结果开发SSR标记是可行的，结果丰富了猪可用SSR标记数据库，且开发的SSR标记准确可靠，多态性高，可用于猪的遗传多样性分析。

关键词: RNA-seq, SSR, 猪, 遗传多样性

Abstract: The aim of this study was to develop SSR markers, and lay the foundation for the applications in genetic diversity analysis, relationship identification, and marker assisted selection in the pig production. The previous RNA-seq results of longissimus dorsi of 6-month-old Large White pig and Mashen pig were used to predict SSR markers. A total of 154 SSRs were selected randomly based on the chromosome locations, base types, repeat numbers of SSR markers, and the amplification primers of the selected SSRs were designed and synthesized. The polymorphic SSR markers were developed by PCR, PAGE analysis and cloning sequencing verification, and used to investigate the genetic diversity of 6-month-old Mashen pig, Large White pig, Jinfen White pig, and Shanxi Black pig(30 individuals from each breed) for assessing the practicability of the developed SSR markers. The result showed that a total of 10 488 SSRs were identified from 36 693 Unigene of transcriptome sequences. There were 9 424 homozygous SSRs and 1 064 compound SSRs, which distributed in 6 953 Unigene sequences. A total of 4 727 Unigene contained the single SSR, and 2 226 Unigene contained two or more SSRs. The occurrence and appearance frequency of SSR were 18.95% and 28.58%, respectively. The dominant SSR repeats were mononucleotide, trinucleotide and dinucleotide, and the largest number of repeats mainly ranged from 5 to 22 times. The most repeated primitive sequences were A/T, AC/GT and GCC/GGC. The dominant repeat motif length type was 10-20 bp. A total of 154 sites were randomly screened for verification, and 124 pairs of SSR primers could amplify the bright and specific bands, the amplification efficiency was 80.52%, among them, 25 pairs of SSR primers were polymorphic, accounting for 16.23%. The polymorphism of 120 pigs from Mashen pig, Large White pig, Jinfen White pig and Shanxi Black pig was analyzed using 25 pairs of SSR primers. A total of 131 alleles were identified, and the range of allele number was 2-7, the average allele number and effective allele number was 5.24 and 3.487 1, respectively. The average PIC value was 0.646 7, which indicated the sites were highly polymorphic, and the genetic diversity of the tested populations was high. This study results indicated that it was feasible to develop SSR markers based on RNA-seq result in pig, and the developed SSR marker enriched the pig SSR database. The developed SSR markers in this study were reliable and highly polymorphic, and could be used to investigate pig genetic diversity.

Key words: RNA-seq, SSR, pig, genetic diversity

中图分类号:

S828.8

李文霞, 吴怡琦, 杨帅, 张燕伟, 路畅, 杨阳, 蔡春波, 高鹏飞, 郭晓红, 李步高, 曹果清. 基于RNA-seq结果开发猪SSR标记[J]. 畜牧兽医学报, 2021, 52(6): 1535-1549.

LI Wenxia, WU Yiqi, YANG Shuai, ZHANG Yanwei, LU Chang, YANG Yang, CAI Chunbo, GAO Pengfei, GUO Xiaohong, LI Bugao, CAO Guoqing. Development of SSR Markers Based on RNA-seq Result of Pig[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6): 1535-1549.

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