基于RAD-seq技术的鹅掌楸基因组SNP标记开发

doi:10.3969/j.issn.1000-2006.201806010

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南京林业大学学报（自然科学版） ›› 2019, Vol. 62 ›› Issue (04): 1-7.doi: 10.3969/j.issn.1000-2006.201806010

基于RAD-seq技术的鹅掌楸基因组SNP标记开发

陆叶^1,2,龙晓飞^1,2,王鹏凯³,陈金慧^1,2,施季森^1,2*

(1. 南京林业大学林木遗传与生物技术省部共建教育部重点实验室,江苏南京 210037; 2.南京林业大学,南方现代林业协同创新中心,江苏南京 210037; 3.苏州农业职业技术学院,江苏苏州 215008)

出版日期:2019-07-22 发布日期:2019-07-22
基金资助:
收稿日期:2018-06-08 修回日期:2019-03-11 基金项目:江苏省高校“青蓝工程”创新团队项目(2016); 江苏省林业科技创新与推广项目(LYKJ[2017]42); 江苏高校优势学科建设工程资助项目(RAPD); 江苏省高等职业院校专业带头人高端研修资助项目(2018GRGDYX070)。第一作者:陆叶(luye@njfu.edu.cn)。*通信作者:施季森(jshi@njfu.edu.cn),教授,ORCID(0000-0002-3376-9233)。

Development of genomic SNP markers based on RAD-seq and genome data in Liriodendron

LU Ye ^1,2,LONG Xiaofei ^1,2,WANG Pengkai ³,CHEN Jinhui ^1,2,SHI Jisen ^1,2*

(1. Key Laboratory of Forest Genetics and Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, China; 2. Co-Innovation Center for the Sustainable Forestry in Southern China,Nanjing Forestry University,Nanjing 210037,China; 3. Suzhou Polytechnic Institute of Agriculture, Suzhou 215008,China)

Online:2019-07-22 Published:2019-07-22

摘要/Abstract

摘要： 【目的】开发大量可靠的SNP标记,为鹅掌楸高密度遗传连锁图谱的构建和基于基因组的林木选择育种提供分子基础。【方法】从北美鹅掌楸NK基因型为母本、鹅掌楸LS基因型为父本的F1代杂交群体中,选取198株个体为作图群体。用限制性内切酶EcoR I对包括2个亲本和198个子代在内的200个单株的基因组DNA进行酶切,构建RAD(restriction-site associated DNA)文库并进行RAD-seq测序。采用读长为91 bp的双末端测序。2个亲本的平均测序深度为2×,198个子代的平均测序深度为0.8×,平均产量为1.94 Gb,共获得约387.21 Gb数据。用Stacks软件将每个样品的RAD-reads作生物信息学分析,对候选位点进行卡方检验和缺失率检验,再将符合孟德尔遗传的标记及与之相对应的RAD-tag序列和鹅掌楸参考基因组序列进行比对。最后,从本研究开发的SNP标记中选取27个候选SNP位点,设计引物,对随机挑选的16个F1代进行PCR扩增并将结果进行测序,同时验证SNP的有效性。【结果】从候选群体中共鉴定到22 019个SNP位点,符合孟德尔遗传规律的标记为4 233个,最终获得3 501个候选SNP标记。SNP验证中,共有293个SNP标记完成测序并能判读结果,所有位点都为SNP位点,共有194(66.2%)个SNP变异类型得到了验证。【结论】基于RAD-seq技术和鹅掌楸参考基因组序列为基础的策略,能够作为一种快速有效的手段,实现大规模的分子标记开发,可用于鹅掌楸等林木的高密度遗传图谱的构建。

Abstract: 【Objective】 The large-scale development of reliable SNP markers is the key to obtaining molecular markers for the construction of high-density genetic linkage maps and for innovative selection and breeding strategies based on genomic information in Liriodendron. 【Method】One hundred and ninety-eight F1 trees were selected as the mapping population, which was obtained by intraspecific cross between the NK(♀)(Liriodendron tulipifera Linn.)and LS(♂)[L. chinense(Hemsl.)Sargent] genotypes. Genomic DNA of 200 individuals, including 2 parents and 198 offspring, was extracted and digested using the restriction endonuclease EcoR I. RAD libraries were constructed using genomic DNA from both parents and F1 trees. The read length of paired-end sequencing is 91 bp. In total, 387.21 Gb of data were obtained, with an average sequencing depth of 2× in the parents RAD libraries and 0.8× in their progeny RAD libraries, and the average production was 1.94 Gb. RAD-reads from each sample were analyzed using Stacks software. The distribution of candidate loci was tested by chi-squared test and the deletion rate was evaluated. Then each RAD-tag sequence containing genetic markers,whose segregation ratios were consistent with the expected Mendelian inheritance rations, was aligned to the reference genome of L. chinense. In the end, we selected 27 pairs of SNP markers then designed primers for amplification within random 16 F1 by PCR in order to validate the accuracy of the candidate SNPs.【Result】A total of 22 019 SNPs were observed in the candidate population, and 4 233 markers conformed to Mendelian inheritance. Finally, 3 501 candidate SNP markers were identified. In the validation experiment, 293 SNPs were successfully sequenced and all of these were identified as SNP loci. A total of 194(66.2%)SNPs were validated in variation types.【Conclusion】RAD-seq is a fast and effective technique for the large-scale development of SNPs and construction of genetic linkage maps in Liriodendron and other taxa.

中图分类号:

Q755

陆叶,龙晓飞,王鹏凯,等. 基于RAD-seq技术的鹅掌楸基因组SNP标记开发[J]. 南京林业大学学报（自然科学版）, 2019, 62(04): 1-7.

LU Ye ^1,2,LONG Xiaofei ^1,2,WANG Pengkai ³,CHEN Jinhui ^1,2,SHI Jisen ^1,2*. Development of genomic SNP markers based on RAD-seq and genome data in Liriodendron [J].Journal of Nanjing Forestry University (Natural Science Edition), 2019, 62(04): 1-7.DOI: 10.3969/j.issn.1000-2006.201806010.

参考文献

[1] 方炎明. 中国鹅掌楸的地理分布与空间格局[J]. 南京林业大学学报, 1994, 18(2): 13-18. DOI:10.3969/j.jssn.1000-2006.1994.02.003. FANG Y M. Geographical distribution and spatial pattern of Liriodendron chinese(Hemsl.)Sarg[J]. Journal of Nanjing Forestry University, 1994, 18(2): 13-18.
[2] 季孔庶, 王章荣. 鹅掌楸属植物研究进展及其繁育策略[J]. 世界林业研究, 2001, 14(1): 8-14. DOI:10.13348/j.cnki.sjlyyj.2001.01.002. JI K S, WANG Z R. Current advances on Liriodendron and their breeding strategies[J]. World Forestry Research, 2001, 14(1): 8-14.
[3] 王章荣. 鹅掌楸属树种杂交育种与利用[M]. 北京:中国林业出版社,2016. WANG Z R. Utilization and species hybridization in Liriodendron[M]. Beijing: China Forestry Publishing House, 2016.
[4] 李周岐,王章荣. 鹅掌楸属种间杂交可配性与杂种优势的早期表现[J].南京林业大学学报(自然科学版),2001,25(2):34-38. DOI:10.3969/j.jssn.1000-2006.2001.02.009. LI Z Q, WANG Z R. Cross ability and heterosis for seed-traits in Liriodendron[J].Journal of Nanjing Forestry University(Natural Sciences Edition),2001,25(2):34-38.
[5] 叶金山,王章荣.杂种马褂木杂种优势的遗传分析[J].林业科学,2002,38(4):67-71. DOI:10.11707/j.1001-7488.20020411. YE J S, WANG Z R. Genetic analysis of heterosis for hybrid tulip tree[J]. Scientia Silvae Sinicae, 2002,38(4):67-71.
[6] 王晓阳,李火根.鹅掌楸苗期生长杂种优势的SSR分析[J].林业科学,2011,47(4):57-62. WANG X Y, LI H G. Possible mechanism analysis for heterosis of hybrid Liriodendron based on seedling growth and SSR markers[J]. Scientia Silvae Sinicae, 2011,47(4):57-62.
[7] 陈金慧,施季森,诸葛强,等. 杂交鹅掌楸体细胞胚胎发生研究[J]. 林业科学,2003,39(4): 49-53. DOI:10.11707/j.1001-7488.2003408. CHEN J H, SHI, J S, ZHUGE Q, et al. Studies on the somatic embryogenesis of Liriodendron hybrids(L. chinese×L. tulipifera)[J]. Scientia Silvae Sinicae, 2003,39(4): 49-53.
[8] 陈金慧,张艳娟,李婷婷,等. 杂交鹅掌楸体胚发生过程的起源及发育过程[J]. 南京林业大学学报(自然科学版),2012,36(1):16-20. DOI:10.3969/j.jssn.1000-2006.2012.01.004. CHEN J H, ZHANG Y J, LI T T, et al. Study on origin and development of somatic embryos of Liriodendron hybrids[J].Journal of Nanjing Forestry University(Natural Sciences Edition), 2012, 36(1): 16-20.
[9] 成铁龙,孟岩,陈金慧,等. 茉莉酸甲酯对杂交鹅掌楸体胚发育的影响[J]. 南京林业大学学报(自然科学版),2017,41(6):41-46. DOI:10.3969/j.issn.1000-2006.201709011. CHENG T L, MENG Y, CHEN J H, et al. Effects of methyl jasmonic acid on somatic embryogenesis of Liriodendron hybrid[J]. Journal of Nanjing Forestry University(Natural Sciences Edition),2017,41(6):41-46.
[10] 鲁路,陆叶,盛宇,等.不同活性炭对杂交鹅掌楸体胚发生的影响[J].南京林业大学学报(自然科学版),2016,40(2):59-64. DOI:10.3969/j.issn.1000-2006.2016.02.010. LU L, LU Y, SHENG Y, et al. Effects of different activated carbon on somatic embryogenesis of Liriodendron hybrids[J].Journal of Nanjing Forestry University(Natural Sciences Edition), 2016, 40(2):59-64.
[11] 李周岐,王章荣.杂种马褂木无性系随机扩增多态DNA指纹图谱的构建[J].东北林业大学学报,2001,29(4):5-8. DOI:10.13759/j.cnki.dlxb.2001.04.002. LI Z Q, WANG Z R. RAPD fingerprints of interspecific hybrid clones in Liriodendron[J]. Journal of Northeast Forestry University, 2001,29(4):5-8.
[12] 施季森,童春发.林木遗传图谱构建和QTL定位统计分析[M].北京:科学出版社,2006. SHI J S, TONG C F. Establishment of tree genetic map and statistical analysis of QTL location[M]. Beijing: Science Press, 2006.
[12] LANDER E S. The new genomics: global views of biology[J]. Science, 1996, 274(5287):536-539. DOI: 10.1126/science.274.5287.536.
[14] 李小白, 崔海瑞, 张明龙. EST分子标记开发及在比较基因组学中的应用[J]. 生物多样性, 2006, 14(6): 541-547. DOI: 10.3321/j.issn:1005-0094.2006.06.010. LI X B, CUI H R, ZHANG M L. Molecular markers derived from EST: their development and applications in comparative genomics[J]. Biodiversity Science, 2006, 14(6): 541-547.
[15] 贺道华,邢宏宜,赵俊兴,等. 多倍体植物中单核苷酸多态性(SNPs)的开发[J]. 浙江大学学报(农业与生命科学版), 2011, 37(5): 485-492. DOI: 10.3785/j.issn.1008-9209.2011.05.003. HE D H, XING H Y, ZHAO J X, et al. Single nucleotide polymorphism(SNP)discovery in polyploid plants[J]. Journal of Zhejiang University(Agriculture & Life Sciences), 2011, 37(5): 485-492.
[16] 唐立群, 肖层林, 王伟平. SNP分子标记的研究及其应用进展[J]. 中国农学通报, 2012, 28(12): 154-158. DOI: 10.3969/j.issn.1000-6850.2012.12.028. TANG L Q, XIAO C L, WANG W P. Research and application progress of SNP markers[J]. Chinese Agricultural Science Bulletin, 2012, 28(12): 154-158.
[17] 王振玉, 李威, 周晓箭, 等. 棉花单核苷酸多态性标记研究进展[J]. 棉花学报, 2016, 28(4):399-406. DOI: 10.11963/issn.1002-7807.201604012. WANG Z Y, LI W, ZHOU X J, et al. Review of single nucleotide polymorphism markers in cotton[J]. Cotton Science, 2016, 28(4):399-406.
[18] 王洋坤, 胡艳, 张天真. RAD-seq技术在基因组研究中的现状及展望[J]. 遗传, 2014, 36(1):41-49. DOI: 10.3724/SP.J.1005.2014.0050. WANG Y K, HU Y, ZHANG T Z. Current status and perspective of RAD-seq in genomic research[J]. Hereditas, 2014, 36(1):41-49.
[19] MILLER M R, DUNHAM J P, AMORES A, et al. Rapid and cost-effective polymorphism identification and genotyping using restriction site associated DNA(RAD)markers[J]. Genome Research, 2007, 17(2): 240-248. DOI: 10.1101/gr.5681207.
[20] BARCHI L, LANTERI S, PORTIS E, et al. Identification of SNP and SSR markers in eggplant using RAD tag sequencing[J]. BMC Geno-mics, 2011, 12(1): 304. DOI: 10.1186/1471-2164-12-304.
[21] PFENDER W F, SAHA M C, JOHNSON E A, et al. Mapping with RAD(restriction-site associated DNA)markers to rapidly identify QTL for stem rust resistance in Lolium perenne[J]. Theoretical and Applied Genetics, 2011, 122(8): 1467-1480. DOI: 10.1007/s00122-011-1546-3.
[22] CHUTIMANITSAKUN Y, NIPPER R W, CUESTA-MARCOS A, et al. Construction and application for QTL analysis of a restriction site associated DNA(RAD)linkage map in barley[J]. BMC Genomics, 2011, 12(1): 4. DOI: 10.1186/1471-2164-12-4.
[23] WANG N, FANG L, XIN H, et al. Construction of a high-density genetic map for grape using next generation restriction-site associated DNA sequencing[J]. BMC Plant Biology, 2012, 12(1): 148. DOI: 10.1186/1471-2229-12-148.
[24] WANG Y K, NING Z Y, HU Y, et al. Molecular mapping of restriction-site associated DNA markers in allotetraploid upland cotton[J]. PloS One, 2015, 10(4): e0124781. DOI:10.1371/journal.pone.0124781.
[25] DAVEY J W, BLAXTER M L. RADSeq: next-generation population genetics[J]. Briefings in Functional Genomics, 2010, 9(5-6):416-423. DOI: 10.1093/bfgp/elr007.
[26] 李文轲, 李丰余, 张思瑶, 等. 基因组二代测序数据的自动化分析流程[J]. 遗传, 2014, 36(6): 618-624. DOI: 10.3724/SP.J.1005.2014.0618. LI W K, LI F Y, ZHANG S Y, et al. Automatic analysis pipeline of next-generation sequencing data[J]. Hereditas, 2014, 36(6): 618-624.
[27] 王晓丽, 马祥庆. 遗传标记技术及其在林木遗传育种中的应用研究[J]. 世界林业研究, 2005, 18(5):37-41. DOI: 10.3969/j.issn.1001-4241.2005.05.008. WANG X L, MA X Q. Advances in genetic markers and its application in forest tree breeding[J]. World Forestry Research, 2005, 18(5):37-41.
[28] 罗光佐, 施季森. 利用RAPD标记分析北美鹅掌楸与鹅掌楸种间遗传多样性[J]. 植物资源与环境学报, 2000, 9(2):9-13. DOI: 10.3969/j.issn.1674-7895.2000.02.003. LUO G Z, SHI J S. Comparison of genetic diversity between Liriodendron tulipifera Linn. and Liriodendron chinense(Hemsl.)Sarg. by means of RAPD markers[J]. Journal of Plant Resources and Environment, 2000, 9(2):9-13.
[29] 骆鹏,曹玉婷,莫家兴,等.柳杉无性系指纹图谱的构建及遗传多样性分析[J].南京林业大学学报(自然科学版),2017,41(4):191-196.DOI: 10.3969/j.issn.1000-2006.201611006. LUO P, CAO Y T, MO J X, et al. Analysis of genetic diversity and construction of DNA fingerprinting of clones in Cryptomeria fortune[J].Journal of Nanjing Forestry University(Natural Sciences Edition),2017,41(4):191-196.
[30] BARCHI L, LANTERI S, PORTIS E, et al. A RAD tag derived marker based eggplant linkage map and the location of QTLs determining anthocyanin pigmentation[J]. PLoS One, 2012, 7(8): e43740. DOI: 10.1371/journal.pone.0043740.
[31] BAIRD N A, ETTER P D, ATWOOD T S, et al. Rapid SNP discovery and genetic mapping using sequenced RAD markers[J]. PloS One, 2008, 3(10): e3376. DOI: 10.1371/journal.pone.0003376.
[32] GUO F, YU H, TANG Z, et al. Construction of a SNP-based high-density genetic map for pummelo using RAD sequencing[J]. Tree Genetics & Genomes, 2015, 11(1): 1-11. DOI: 10.1007/s11295-014-0831-0.
[33] 王莹. 利用RAD-seq测序构建美洲黑杨×小叶杨高密度遗传连锁图谱[D]. 南京:南京林业大学, 2014. WANG Y. Construction of a high-density linkage map of P. deltoides&#215;P. simonii using restriction-site associated DNA sequencing[D]. Nanjing: Nanjing Forestry University.

基于RAD-seq技术的鹅掌楸基因组SNP标记开发

Development of genomic SNP markers based on RAD-seq and genome data in Liriodendron

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