基于SLAF-seq技术的古茶树SNP位点开发
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摘要
为给古茶树遗传图谱构建、分子辅助育种和物种进化等研究提供参考,以48份贵州古茶树种质为研究对象,利用SLAF-seq技术,获取大量多态性SLAF标签,进而开发一批特异性强、稳定性高的单核苷酸多态性(SNP)位点。结果表明,利用猕猴桃参考基因组进行电子酶切预测,选择HaeIII酶进行酶切,得到237 773个SLAF标签,其双端比对效率为91.40%,酶切效率为96.41%,说明SLAF建库正常。测序后各样品所获得的读长数为1 279 534～8 460 233,测序质量值Q30范围为92.61%～94.88%,均值为93.84%,测序获得的GC比例为43.52%～47.18%。共开发1 656 258个古茶树SLAF标签,样品的平均测序深度为24.21×,其中多态性SLAF标签有462897个,根据所获得的多态性SLAF标签来统计SNP位点信息,共得到2 690 638个群体SNP标记,根据完整度大于0.8且次要基因频率(MAF)大于0.05的标准进行过滤,得到283 376个高一致性的群体SNP标记。
In order to provide some references for the researches on genetic map construction, molecular assisted breeding and species evolution in old Camellia sinensis, using 48 samples of old C. sinensis germplasm in Guizhou as research objects, a large number of polymorphic SLAF tags were obtained by using SLAF-seq technology, and a large number of single nucleotide polymorphism(SNPs) sites were developed, which had strong specificity and high stability.The results showed that kiwifruit reference genome was used for electronic digestion prediction, HaeIII endonuclease was selected, and 237 773 SLAF tags were predicted. Double-end alignment efficiency was 91.40%, and enzyme digestion efficiency was 96.41%, indicating that SLAF library was normal. Number of sequencing reads was at the range of 1 279 534-8 460 233. Average quality of sequencing Q30 was 93.84%, ranging from 92.61% to 94.88%. Based on sequencing results, GC percentage was 43.52%-47.18%. Average sequencing depth of samples was 24.21×, and total1 656 258 SLAF tags were developed, including 462 897 polymorphic SLAF tags. Based on obtained polymorphic SLAF tags, information of SNP sites was counted, and 2 690 638 population SNP markers were obtained. Total 283 376 highly consistent population SNP markers were obtained through filtering according to integrity greater than 0.8 and minor allele frequency(MAF) greater than 0.05.
In order to provide some references for the researches on genetic map construction, molecular assisted breeding and species evolution in old Camellia sinensis, using 48 samples of old C. sinensis germplasm in Guizhou as research objects, a large number of polymorphic SLAF tags were obtained by using SLAF-seq technology, and a large number of single nucleotide polymorphism(SNPs) sites were developed, which had strong specificity and high stability.The results showed that kiwifruit reference genome was used for electronic digestion prediction, HaeIII endonuclease was selected, and 237 773 SLAF tags were predicted. Double-end alignment efficiency was 91.40%, and enzyme digestion efficiency was 96.41%, indicating that SLAF library was normal. Number of sequencing reads was at the range of 1 279 534-8 460 233. Average quality of sequencing Q30 was 93.84%, ranging from 92.61% to 94.88%. Based on sequencing results, GC percentage was 43.52%-47.18%. Average sequencing depth of samples was 24.21×, and total1 656 258 SLAF tags were developed, including 462 897 polymorphic SLAF tags. Based on obtained polymorphic SLAF tags, information of SNP sites was counted, and 2 690 638 population SNP markers were obtained. Total 283 376 highly consistent population SNP markers were obtained through filtering according to integrity greater than 0.8 and minor allele frequency(MAF) greater than 0.05.
引文
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[22]LI B,TIAN L,ZHANG J Y,et al.Construction of a highdensity genetic map based on large-scale markers developed by specific length amplified fragment sequencing(SLAF-seq)and its application to QTL analysis for isoflavone content in Glycine max[J].BMC Genomics,2014,15:1086.
[23]陶红霞.基于SLAF标记的苹果遗传连锁图谱构建[D].杨凌:西北农林科技大学,2015.
[2]陈亮.茶组植物的分子系统学研究:遗传多样性与分类[D].杭州:浙江大学,2002.
[3]KAUNDUN S S,MATSUMOTO S.Heterologous nuclear and chloroplast microsatellite amplification and variation in tea,Camellia sinensis[J].Genome,2002,45(6):1041-1048.
[4]KAUNDUN S S.Genetic structure of six Korean tea populations as revealed by RAPD-PCR markers[J].Crop Science,2002,42(2):594-601.
[5]谢荟,祝文娟,张应中,等.基于SRAP分子标记的广宁红花油茶种质资源遗传多样性研究[J].中南林业科技大学学报,2017,37(8):93-97.
[6]MATSUMOTO S,KIRIIWA Y,TAKEDA Y.Differentiation of Japanese green tea cultivars as revealed by RFLP analysis of phenylalanine ammonialyase DNA[J].Theoretical and Applied Genetics,2002,104(6):998-1002.
[7]PAUL S,WACHIRA F N,POWELL W,et al.Diversity and genetic differentiation among populations of Indian and Kenyan tea(Camellia sinensis(L)O Kuntze)revealed by AFLP markers[J].Theoretical and Applied Genetics,1997,94(2):255-263.
[8]MISHRA R K,CHAUDHURY S,AHMAD A,et al.Molecular analysis of tea clones(Camellia sinensis)using AFLP markers[J].International Journal of Integrative Biology,2009,5(2):130-135.
[9]张汉卿,蔡秀兰,王海,等.山茶属种质资源亲缘关系的SSR分析[J].经济林研究,2018,36(4):130-134.
[10]FANG W,CHENG H,DUAN Y,et al.Genetic diversity and relationship of clonal tea(Camellia sinensis)cultivars in China as revealed by SSR markers[J].Plant Systematics and Evolution,2012,298(2):469-483.
[11]王亨洪,索化夷,杨坚,等.川渝地区重要野生大茶树遗传多样性的ISSR分析[J].茶叶科学,2009,29(2):168-172.
[12]牛素贞,刘玉倩,杨锦标,等.贵州茶树地方品种的EST-SSR遗传多样性分析[J].浙江农业学报,2012,24(5):836-841.
[13]ZHANG Y X,WANG L H,XIN H G,et al.Construction of a high-density genetic map for sesame based on large scale marker development by specific length amplified fragment(SLAF)sequencing[J].BMC Plant Biology,2013,13(1):1-12.
[14]SUN X W,LIU D Y,ZHANG X F,et al.SLAF-seq:An efficient method of large-scale de novo SNP discovery and genotyping using high-throughput sequencing[J].Plos One,2013,8(3):e58700.
[15]郑会敏.提莫菲维小麦和野燕麦远缘杂交后代的染色体遗传组成分析[D].贵阳:贵州大学,2013.
[16]KOZICH J J,WESTCOTTS L,BAXTERN T,e t a l.Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq illumina sequencing platform[J].Applied&environmental microbiology,2013,79(17):5112-5120.
[17]INITIATIVE A G.Analysis of the genome sequence of the flowering plant Arabidopsis thaliana[J].Nature,2000,408(6814):796-815.
[18]LI R,LI Y,KRISTIANSEN K,et al.SOAP:short oligonucleotide alignment program[J].Bioinformatics,2013,24(5):713-714.
[19]燕丽萍,吴德军,王因花,等.白蜡属优良种质资源亲缘关系的AFLP分析[J].中南林业科技大学学报,2018,38(7):89-95.
[20]郑丹琳,陈涛林,葛智文,等.柳州汪洞乡古茶树种质资源遗传多样性的ISSR分析[J].分子植物育种,2018,16(11):3629-3635.
[21]JAIN M,MOHARANA K C,SHANKAR R,et al.Genomewide discovery of DNA polymorphisms in rice cultivars with contrasting drought and salinity stress response and their functional relevance[J].Plant Biotechnol Journal,2014,12(2):253-264.
[22]LI B,TIAN L,ZHANG J Y,et al.Construction of a highdensity genetic map based on large-scale markers developed by specific length amplified fragment sequencing(SLAF-seq)and its application to QTL analysis for isoflavone content in Glycine max[J].BMC Genomics,2014,15:1086.
[23]陶红霞.基于SLAF标记的苹果遗传连锁图谱构建[D].杨凌:西北农林科技大学,2015.