马鹿特异性SNP分子标记的验证

doi:10.16431/j.cnki.1671-7236.2021.04.018

摘要/Abstract

摘要： 试验旨在对基于基因分型测序（genotyping by sequencing，GBS）技术筛选出的马鹿特异性SNPs位点的准确性进行验证，为梅花鹿、马鹿及其杂交后代的鉴别提供可靠的分子遗传标记。随机选取30个马鹿特异性SNPs位点，根据SNPs位点前后各200 bp的序列，利用Primer Premier 6.0软件设计特异性引物，以随机选取的验证样本DNA作为模板进行PCR扩增，并进行Sanger测序，对测序结果利用BioEdit软件进行峰图的观察，利用Mega 6.0软件对测序得到的序列进行比对分析并观察每个特异性SNP位点在不同验证群体中的基因型，对每一个马鹿特异性位点的峰图和比对信息进行统计分析。结果表明，30个马鹿特异性位点中有28个和前期研究结果一致，其中1个SNP位点（SNP3）中G等位基因在梅花鹿中的基因频率为0.05，而G等位基因在马鹿中的基因频率为1，G等位基因在马鹿个体中的基因频率比在梅花鹿个体中高，同时，利用SPSS 22.0进行统计分析发现，该位点在马鹿和梅花鹿中基因型分布表现出显著性差异（P<0.05）；另外1个SNP位点（SNP5）中T等位基因在梅花鹿的基因频率为0.15，而在马鹿中的基因频率为1，且这个SNP位点在梅花鹿和马鹿中基因型分布差异显著（P<0.05），所以这2个位点仍然可以作为马鹿的特异性SNPs位点。研究结果说明了GBS测序筛选出的马鹿特异性SNPs可以作为鉴定的分子标记，对梅花鹿、马鹿及其杂交后代的鉴别奠定了理论基础。

关键词: 马鹿; 特异性SNP; Sanger测序; 验证

Abstract: The purpose of this study was to verify the accuracy of specific SNPs of red deer based on genotyping sequencing (GBS) technology,and provide a reliable basis for the identification of sika deer,red deer,and their hybrid offspring.Thirty specific SNPs of red deer were randomly selected,based on the sequences of 200 bp each before and after the SNPs,Primer Premier 6.0 software was used to design specific primers,and the randomly selected DNA of the validation samples was used as a template for amplification,PCR was performed,and the amplified products were sequenced in one generation.The sequences obtained by sequencing were analyzed by Mega 6.0 software,and the genotypes of each SNP site in different validation populations were observed.The results showed that 28 out of 30 specific loci were consistent with the results of the previous study,and the gene frequency of the G allele at one of SNPs (SNP3) was 0.05 in sika deer,while the gene frequency of the G allele was 1 in red deer.The loci showed significant differences in genotype distribution between red deer and sika deer (P<0.05).The gene frequency of the T allele in the other SNP (SNP5) was 0.15 in sika deer,while the gene frequency of the T allele was 1 in red deer,and the genotype distribution of this SNP in sika deer and red deer was significantly different (P<0.05).So these two loci could still be used as specific SNPs of red deer.The results showed that the specific SNPs of red deer selected by GBS sequencing could be used as molecular markers for identification,and laid a theoretical foundation for the identification of sika deer,red deer and their hybrid offspring.

Key words: red; deer:specific; SNPs:Sanger; sequencing:validation

中图分类号:

S813.1

范欢欢, 王天骄, 董依萌, 李洋, 刘汇涛, 邢秀梅. 马鹿特异性SNP分子标记的验证[J]. 中国畜牧兽医, 2021, 48(4): 1313-1322.

FAN Huanhuan, WANG Tianjiao, DONG Yimeng, LI Yang, LIU Huitao, XING Xiumei. Verification of Red Deer Specific Molecular Marker SNP[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(4): 1313-1322.

参考文献

[1] 周盼伊.利用Y染色体基因分析家养梅花鹿的父系类型[D].北京:中国农业科学院,2016. ZHOU P Y.Using the Y chromosome gene analysis the pater type of domestic sika deer[D].Beijing:Chinese Academy of Agricultural Sciences,2016.(in Chinese)
[2] 郑兴涛.梅花鹿,马鹿及其杂种茸毛遗传方式的研究[J].特产研究,1990,1:17-18. ZHENG X T.Study on the genetic pattern of antler hair in the sika deer,red deer and their hybrids[J].Special Wild Economic Animal and Plant Research,1990,1:17-18.(in Chinese)
[3] 王会,刘佳,付丽,等.单核苷酸多态性在多倍体作物油菜中的研究进展[J].中国油料作物学报,2014,36(3):422-429. WANG H,LIU J,FU L,et al.Review on single nucleotide polymorphisms in polyploid crop Brassica napus[J].Chinese Journal of Oil Crop Sciences,2014,36(3):422-429.(in Chinese)
[4] KIM S W,JANG H K,KIM K S,et al.Establishment of genetic characteristics and individual identification system using microsatellite loci in domestic beef cattle[J].Journal of Animal Science & Technology,2009,51(4):273-282.
[5] ALLEN A R,TAYLOR M,MCKEOWN B,et al.Compilation of a panel of informative single nucleotide polymorphisms for bovine identification in the Northern Irish cattle population[J].BMC Genetics,2010,11(1):1-8.
[6] 董世武,王天骄,刘华淼,等.基于GBS技术对梅花鹿、马鹿及其杂交后代基因组SNP特征的分析[J].畜牧兽医学报,2019,50(12):2422-2430. DONG S W,WANG T J,LIU H M,et al.Analysis of genomic SNP characteristics of sika deer,red deer and their hybrid progenies based on GBS technology[J].Chinese Journal of Animal and Veterinary Sciences,2019,50(12):2422-2430.(in Chinese)
[7] PATEL R K,JAIN M.NGS QC Toolkit:A toolkit for quality control of next generation sequencing data[J].PLoS One,2012,7(2):e30619.
[8] LI H,DURBIN R.Fast and accurate short read alignment with Burrows-Wheeler transform[J].Bioinformatics,2009,25(14):1754-1760.
[9] BHAKTA M S,JONES V A,EDUARDO V C,et al.Punctuated distribution of recombination hotspots and demarcation of pericentromeric regions in Phaseolus vul-garis L.[J].PLoS One,2015,10(1):e0116822.
[10] BOGATKEVICH G S,NIETERT P J,SILVER R M,et al.Rationale for anticoagulant therapy of pulmonary fibrosis[J].American Journal of Respiratory & Critical Care Medicine,2014,189(3):362-363.
[11] CANET L,CÁLIZ,R,LUPIANEZ C B,et al.THU0002 Estrogen-related polymorphisms and risk of rheumatoid arthritis:A multicenter study[J].Annals of the Rheumatic Diseases,2015,74(Suppl 2):191-193.
[12] HAND D,WALLIS C,COLYER A,et al.Pyrosequencing the Canine faecal microbiota:Breadth and depth of biodiversity[J].PLoS One, 2013,8(1):e53115.
[13] 刘刚.序列捕获高通量测序技术建立citrin缺乏症基因检测平台及其应用[D].广州:华南理工大学,2015. LIU G.A study about rstablishing the genetic test platform for citrin deficiency based on targeted sequence capture and high-throughput sequencing and its clinical applications[D].Guangzhou:South China University of Technology,2015.(in Chinese)
[14] 朱涛,谷洪昌,张泽宾,等.鸭不同品种特异性分子标记SNP的筛选与鉴定[J].中国家禽,2018,40(15):7-10. ZHU T,GU H C,ZHANG Z B,et al.SNP identificationfor specific molecular markers of different duck breeds[J].China Poultry,2018,40(15):7-10.(in Chinese)
[15] 聂红毅,许叔鹏,王雪妍,等.意大利蜜蜂染色体DNA非编码区抗白垩病相关的SNP的筛选与验证[J].昆虫学报,2019,62(10):1162-1171. NIE H Y,XU S P,WANG X Y,et al.Screening and validation of SNPs associated with chalkbrood resistance in non-coding regions of DNA of chromosomes of Apis mellifera ligustica[J].Acta Entomologica Sinica,2019,62(10):1162-1171.(in Chinese)
[16] 林晓煜,肖世俊,李完波,等.大黄鱼性别特异SNP标记的开发与验证[J].水产学报,2018,42(9):1329-1337. LIN X Y,XIAO S J,LI W B,et al.Development and validation of sex-specific SNP markers in Yellow croaker[J].Journal of Fisheries of China,2018,42(9):1329-1337.(in Chinese)
[17] 岳阳,张哲,徐忠.基于单核苷酸多态性鉴别小梅山猪及其制品[J].农业生物技术学报,2018,26(12):2168-2175. YUE Y,ZHANG Z,XU Z.Identification of Small Meishan pig (Sus scrofa) and its products based on single nucleotide polymorphism[J].Journal of Agricultural Biotechnology,2018,26(12):2168-2175.(in Chinese)
[18] 王晓歌,阴祖军,王俊娟,等.陆地棉转录组耐盐相关SNP挖掘及分析[J].分子植物育种,2016,14(6):1524-1532. WANG X G,YIN Z J,WANG J J,et al.Mining and analyzing of SNP related to salinity stress in transcriptome of upload cotton(Gossypium hirsutum L.)[J].Molecular Plant Breeding,2016,14(6):1524-1532.(in Chinese)
[19] 赵列平,韩欢胜,赵广华.东北梅花鹿与天山马鹿种间杂交效果[J].经济动物学报,2011,15(3):153. ZHAO L P,HAN H S,ZHAO G H.Effect of interspecific hybridization between Tianshan Wapiti and Northeast sika[J].Journal of Economic Animal, 2011,15(3):153.(in Chinese)