基于RNA-seq结果开发猪SSR标记

doi:10.11843/j.issn.0366-6964.2021.06.009

Abstract

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

CLC Number:

S828.8

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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Development of SSR Markers Based on RNA-seq Result of Pig

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