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.

References 47

[1]	SHARMA L,ALI S,SIVA C,et al.Genetic diversity and population structure of the threatened chocolate mahseer (Neolissochilus hexagonolepis McClelland 1839) based on SSR markers:implications for conservation management in Northeast India[J].Mol Biol Rep,2019,46(5):5237-5249.
[2]	ARIEDE R B,FREITAS M V,HATA M E,et al.Development of microsatellite markers using next-generation sequencing for the fish Colossoma macropomum[J].Mol Biol Rep,2018,45(1):9-18.
[3]	TAUTZ D.Hypervariability of simple sequences as a general source for polymorphic DNA markers[J].Nucleic Acids Res,1989, 17(16):6463-6471.
[4]	SCHLÖTTERER C,TAUTZ D.Slippage synthesis of simple sequence DNA[J].Nucleic Acids Res,1992,20(2):211-215.
[5]	LORENZINI R,FANELLI R,TANCREDI F,et al.Matching STR and SNP genotyping to discriminate between wild boar,domestic pigs and their recent hybrids for forensic purposes[J].Sci Rep,2020,10(1):3188.
[6]	YADAV R,LONE S A,GAIKWAD K,et al.Transcriptome sequence analysis and mining of SSRs in Jhar Ber (Ziziphus nummularia (Burm.f.) Wight & Arn) under drought stress[J].Sci Rep,2018,8:2406.
[7]	WANG H Y,FU Y,GU P,et al.Genome-wide characterization and comparative analyses of simple sequence repeats among four miniature pig breeds[J].Animals (Basel),2020,10(10):1792.
[8]	李军成,景志忠,路彩霞,等.微卫星标记对高原蕨麻猪的遗传多样性研究[J].中国实验动物学报,2007,15(5):355-360.LI J C,JING Z Z,LU C X,et al.Microsatellite marker analysis of genetic diversity in Plateau Juema pigs[J].Acta Laboratorium Animalis Scientia Sinica,2007,15(5):355-360.(in Chinese)
[9]	牛荣,商海涛,魏泓,等.版纳小耳猪近交系5家系35个微卫星座位的遗传分析[J].遗传学报,2001,28(6):518-526.NIU R,SHANG H T,WEI H,et al.Genetic analysis of 35 microsatellite loci in 5 lineages of Xishuangbanna Miniature pig inbred line[J].Acta Genetica Sinica,2001,28(6):518-526.(in Chinese)
[10]	RBAŁA K,RABTSAVA A A,KOTOVA S A,et al.STR profiling for discrimination between wild and domestic swine specimens and between main breeds of domestic pigs reared in Belarus[J].PLoS One,2016,11(11):e0166563.
[11]	LIN Y C,HSIEH H M,LEE J C I,et al.Establishing a DNA identification system for pigs (Sus scrofa) using a multiplex STR amplification[J].Forensic Sci Int Genet,2014,9:12-19.
[12]	GROENEN P M A,BUNSCHOTEN A E,VAN SOOLINGEN D,et al.Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis;application for strain differentiation by a novel typing method[J].Mol Microbiol,1993, 10(5):1057-1065.
[13]	TIAN R Z,ZHANG C H,HUANG Y X,et al.A novel software and method for the efficient development of polymorphic SSR loci based on transcriptome data[J].Genes (Basel),2019,10(11):917.
[14]	QI W H,LU T,ZHENG C L,et al.Distribution patterns of microsatellites and development of its marker in different genomic regions of forest musk deer genome based on high throughput sequencing[J].Aging (Albany NY),2020,12(5):4445-4462.
[15]	LI T T,FANG Z W,PENG H,et al.Application of high-throughput amplicon sequencing-based SSR genotyping in genetic background screening[J].BMC Genomics,2019,20(1):444.
[16]	GUO A H,YUAN J L,LIAN Q P,et al.Isolation and characterization of 20 polymorphic microsatellites loci for Xenocypris davidi based on high-throughput sequencing[J].Mol Biol Rep,2020,47(10):8305-8310.
[17]	LIU C C,LIU Y,ZHANG X Y,et al.Characterization of porcine simple sequence repeat variation on a population scale with genome resequencing data[J].Sci Rep,2017,7(1):2376.
[18]	DENG T X,PANG C Y,LU X R,et al.De novo transcriptome assembly of the Chinese swamp buffalo by RNA Sequencing and SSR marker discovery[J].PLoS One,2016,11(1):e0147132.
[19]	DING N,HAN Q,LI Q,et al.Comprehensive analysis of Sichuan white geese (Anser cygnoides) transcriptome[J].Anim Sci J,2014,85(6):650-659.
[20]	HAN Z F,XIAO S J,LI W B,et al.The identification of growth,immune related genes and marker discovery through transcriptome in the yellow drum (Nibea albiflora)[J].Genes Genomics,2018,40(8):881-891.
[21]	JIANG J P,YUAN X,QIU Q Q,et al.Comparative transcriptome analysis of gonads for the identification of sex-related genes in giant freshwater prawns (Macrobrachium Rosenbergii) using RNA sequencing[J].Genes (Basel),2019,10(12):1035.
[22]	IP J C H,MU H W,CHEN Q C,et al.AmpuBase:a transcriptome database for eight species of apple snails (Gastropoda:Ampullariidae)[J]. BMC Genomics,2018,19:179.
[23]	SONG W,LI L Z,HUANG H L,et al.Tissue-based transcriptomics of Chionodraco hamatus:sequencing,de novo assembly, annotation and marker discovery[J].J Fish Biol,2019,94(2):251-260.
[24]	FENG B,YI S V,ZHANG M M,et al.Development of novel EST-SSR markers for ploidy identification based on de novo transcriptome assembly for Misgurnus anguillicaudatus[J].PLoS One,2018,13(4):e0195829.
[25]	郭欢,王刚,张树田,等.基于RNA-seq数据的窄足真蚋SSR分子标记开发[J].昆虫学报,2018,61(7):815-824.GUO H,WANG G,ZHANG S T,et al.Development of SSR primers for Simulium (Eusimulium) angustipes (Diptera:Simuliidae) based on RNA-seq dataset[J].Acta Entomologica Sinica,2018,61(7):815-824.(in Chinese)
[26]	CAI C B,LI M,ZHANG Y W,et al.Comparative transcriptome analyses of Longissimus thoracis between pig breeds differing in muscle characteristics[J].Front Genet,2020,11:526309.
[27]	成志敏,张宁芳,王媛媛,等.基于RNA-Seq技术筛选影响猪肌纤维性状的候选基因[J].畜牧兽医学报,2019,50(5):918-929.CHENG Z M,ZHANG N F,WANG Y Y,et al.Screening of candidate genes for muscle fiber characteristics in pig using RNA-seq[J].Acta Veterinaria et Zootechnica Sinica,2019,50(5):918-929.(in Chinese)
[28]	郭晓红,王效京,张敏,等.晋汾白猪生长发育性状的选育研究[J].中国畜牧兽医,2013,40(9):228-231.GUO X H,WANG X J,ZHANG M,et al.Study on selection of growth and development performance of Jinfen White pig[J]. China Animal Husbandry & Veterinary Medicine,2013,40(9):228-231.(in Chinese)
[29]	李超,侯吉伦,王桂兴,等.基于牙鲆RNA-seq数据中SSR标记的信息分析[J].海洋渔业,2015,37(2):122-127.LI C,HOU J L,WANG G X,et al.Bioinformatic analysis of SSR markers in transcriptomic sequenceing Paralichthys olivaceus[J]. Marine Fisheries,2015,37(2):122-127.(in Chinese)
[30]	熊翠玲,张璐,付中民,等.基于RNA-seq数据大规模开发中华蜜蜂幼虫的SSR分子标记[J].环境昆虫学报,2017, 39(1):68-74.XIONG C L,ZHANG L,FU Z M,et al.Large-scale development of SSR primers for Apis cerana cerana larvae based on its RNA-seq datasets[J].Journal of Environmental Entomology,2017,39(1):68-74.(in Chinese)
[31]	XIAO S J,HAN Z F,WANG P P,et al.Functional marker detection and analysis on a comprehensive transcriptome of large yellow croaker by next generation sequencing[J].PLoS One,2015,10(4):e0124432.
[32]	CHEN W,LIU Y X,JIANG G F.De novo assembly and characterization of the testis transcriptome and development of EST-SSR markers in the cockroach Periplaneta americana[J].Sci Rep,2015,5:11144.
[33]	JI P F,LIU G M,XU J,et al.Characterization of common carp transcriptome:sequencing,de novo assembly,annotation and comparative genomics[J].PLoS One,2012,7(4):e35152.
[34]	GAO Z X,LUO W,LIU H,et al.Transcriptome analysis and SSR/SNP markers information of the blunt snout bream (Megalobrama amblycephala)[J].PLoS One,2012,7(8):e42637.
[35]	ZHANG Y P,ZHENG Y,LI D S,et al.Transcriptomics and identification of the chemoreceptor superfamily of the pupal parasitoid of the oriental fruit fly,Spalangia endius Walker (Hymenoptera:Pteromalidae)[J].PLoS One,2014,9(2):e87800.
[36]	LUO H,XIAO S J,YE H,et al.Identification of immune-related genes and development of SSR/SNP markers from the spleen transcriptome of Schizothorax prenanti[J].PLoS One,2016,11(3):e0152572.
[37]	李庆伟,华一杉,冯少姝,等.基于RNA-Seq技术的日本七鳃鳗(Lampetra japonica)肝脏转录组从头组装及分析[J].辽宁师范大学学报:自然科学版,2018,41(1):68-78.LI Q W,HUA Y S,FENG S S,et al.De novo assembly and analysis of Lampetra japonica liver transcriptome by RNA-Seq[J]. Journal of Liaoning Normal University:Natural Science Edition,2018,41(1):68-78.(in Chinese)
[38]	DU M,LI N,NIU B Z,et al.De novo transcriptome analysis of Bagarius yarrelli (Siluriformes:Sisoridae) and the search for potential SSR markers using RNA-Seq[J].PLoS One,2018,13(2):e0190343.
[39]	TEMNYKH S,DECLERCK G,LUKASHOVA A,et al.Computational and experimental analysis of microsatellites in rice (Oryza sativa L.):frequency,length variation,transposon associations,and genetic marker potential[J].Genome Res,2001,11(8):1441-1452.
[40]	BRENIG B,SCHÜTZ E.Recent development of allele frequencies and exclusion probabilities of microsatellites used for parentage control in the German Holstein Friesian cattle population[J].BMC Genet,2016,17:18.
[41]	LIU Y,CHEN Y Y,GONG Q,et al.Paternity assignment in the polyploid Acipenser dabryanus based on a novel microsatellite marker system[J].PLoS One,2017,12(9):e0185280.
[42]	GAO X G,HAN J B,LU Z C,et al.RETRACTED:characterization of the spotted seal Phoca largha transcriptome using Illumina paired-end sequencing and development of SSR markers[J].Comp Biochem Physiol Part D:Genomics Proteomics,2012, 7(3):277-284.
[43]	LU T F,SUN Y J,MA Q,et al.De novo transcriptomic analysis and development of EST-SSR markers in the Siberian tiger (Panthera tigris altaica)[J].Mol Genet Genomics,2016,291(6):2145-2157.
[44]	ZHANG J,MA W G,SONG X M,et al.Characterization and development of EST-SSR markers derived from transcriptome of Yellow Catfish[J].Molecules,2014,19(10):16402-16415.
[45]	贺希文,高彩霞,姜骞,等.加系SPF大白猪和长白猪群体遗传学分析[J].中国实验动物学报,2015,23(6):551-556.HE X W,GAO C X,JIANG Q,et al.Analysis of the pedigreed population genetics of SPF Yorkshire and Landrace pigs imported from Canada[J].Acta Laboratorium Animalis Scientia Sinica,2015,23(6):551-556.(in Chinese)
[46]	李桢,储明星,曹红鹤,等.中外11个猪种A-FABP基因微卫星遗传变异的研究[J].遗传,2004,26(4):473-477.LI Z,CHU M X,CAO H H,et al.Genetic variation of A-FABP microsatellites in eleven pig breeds[J].Hereditas (Beijing), 2004,26(4):473-477.(in Chinese)
[47]	曹果清,李步高,石建中,等.应用21个微卫星标记监测马身猪遗传多样性变化趋势[J].畜牧兽医学报,2010,41(8):932-938.CAO G Q,LI B G,SHI J Z,et al.Genetic monitoring on genetic diversity of Mashen pig using 21 microsatellite markers[J].Acta Veterinaria et Zootechnica Sinica,2010,41(8):932-938.(in Chinese)

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

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