靶向测序基因型检测（GBTS）技术及其应用

doi:10.3864/j.issn.0578-1752.2020.15.001

中国农业科学 ›› 2020, Vol. 53 ›› Issue (15): 2983-3004.doi: 10.3864/j.issn.0578-1752.2020.15.001

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

靶向测序基因型检测（GBTS）技术及其应用

徐云碧^1,^3,^4,⁷(),杨泉女³,郑洪建⁴,许彦芬²,桑志勤⁵,郭子锋¹,彭海⁶,张丛²,蓝昊发²,王蕴波³,吴坤生²,陶家军²,张嘉楠²()

¹中国农业科学院作物科学研究所,北京 100081
²石家庄博瑞迪生物技术有限公司,石家庄 050035
³佛山科学技术学院食品科学与工程学院/CIMMYT-中国热带玉米研究中心,广东佛山 528225
⁴上海市农业科学院作物研究所/CIMMYT-中国特用玉米研究中心,上海 201403
⁵新疆农垦科学院作物研究所,新疆石河子 832000
⁶江汉大学,武汉 430056
⁷国际玉米小麦改良中心,墨西哥特斯科科 56130

收稿日期:2020-05-06 接受日期:2020-06-16 出版日期:2020-08-01 发布日期:2020-08-06
通讯作者: 徐云碧,张嘉楠
基金资助:
国家重点研发计划(2016YFD0101803);石家庄市科技孵化计划(191540089A);河北省创新能力提升计划项目新型研发机构建设专项(19962911D);中央级公益性科研院所基本科研业务费专项(Y2020PT20);武汉市科技局应用基础前沿专项(2018020401011298);中国农业科学院农业科技创新计划;中国农业科学院作物科学研究所中央非公益类基础研究项目;比尔盖茨基金会、CGIAR MAIZE项目

Genotyping by Target Sequencing (GBTS) and Its Applications

XU Yunbi^1,^3,^4,⁷(),YANG QuanNü³,ZHENG HongJian⁴,XU YanFen²,SANG ZhiQin⁵,GUO ZiFeng¹,PENG Hai⁶,ZHANG Cong²,LAN HaoFa²,WANG YunBo³,WU KunSheng²,TAO JiaJun²,ZHANG JiaNan²()

¹Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081
²MolBreeding Biotechnology Co., Ltd., Shijiazhuang 050035
³School of Food Science and Engineering, Foshan University/CIMMYT-China Tropical Maize Research Center, Foshan 528225, Guangdong
⁴Institute of Crops, Shanghai Academy of Agricultural Sciences/CIMMYT-China Specialty Maize Research Center, Shanghai 201403
⁵Xinjiang Academy of Agricultural Reclamation, Shihezi 832000, Xinjiang
⁶Jianghan University, Wuhan 430056
⁷International Maize and Wheat Improvement Center (CIMMYT), El Batan Texcoco 56130, Mexico

Received:2020-05-06 Accepted:2020-06-16 Online:2020-08-01 Published:2020-08-06
Contact: Yunbi XU,JiaNan ZHANG

摘要/Abstract

摘要：

借助于分子标记进行基因型检测的技术在生物遗传改良等领域发挥着重要的作用。国际跨国种业公司凭借其高通量、自动化、大规模的共享检测平台,基因型检测技术得到广泛应用。随着从3G时代的高成本固相芯片和随机测序式基因型检测（genotyping by sequencing,GBS）发展到成本低、对检测平台要求较低、基于靶向测序基因型检测（genotyping by target sequencing,GBTS）的液相芯片,基因型检测技术完成了向4G时代的转变。在本文中首先介绍了两项最新的GBTS技术（基于多重PCR的GenoPlexs和基于液相探针捕获的GenoBaits）及其原理。同时,发展了可以在单个扩增子内检测多个SNP,称之为多聚单核苷酸多态性（multiple single-nucleotide-polymorphism cluster,mSNP或multiple dispersed nucleotide polymorphism,MNP）的技术,极大地提高了目标位点（扩增子）内变异的检测效率。与GBS和固相芯片相比,GBTS技术具有平台广适性、标记灵活性、检测高效性、信息可加性、支撑便捷性和应用广谱性。同一款标记集（例如玉米40K mSNP）,可以获得3种不同的标记形式（40K mSNP、260K SNP和754K单倍型）;并可以根据应用场景的需求,通过控制测序深度获得多种不同的标记密度（1—40K mSNP）。GenoPlexs和GenoBaits 2种技术相结合,可广泛应用于生物进化、遗传图谱构建、基因定位克隆、标记性状关联检测（全基因组关联分析——GWAS和混合样本分析——BSA）、后裔鉴定、基因渐渗、基因累加、品种权保护、品种质量监测、转基因成分/基因编辑/伴生生物检测等领域。目前,已经在20余种主要农作物、蔬菜以及部分动物和微生物中开发了GBTS标记50余套,并已广泛应用于上述领域。最后,展望了与未来GBTS应用相关的几个问题,包括便携式、自动化、高通量、智能化检测平台;根据用户需求定制的可变密度、多功能分子检测;GBTS与其他技术（KASP、高密度芯片、BSA策略等）的整合;基于资源共享的开源育种等。这些将推动GBTS技术在动物、植物和微生物遗传改良等领域的广泛应用。

关键词: 靶向测序基因型检测（GBTS）, 多重PCR, 液相探针, 多聚单核苷酸多态性（mSNP）, 多个分散型核苷酸多态性（MNP）, 单倍型, 遗传改良, 开源育种

Abstract:

Genotyping technology, via molecular markers, has been playing a key role in many biological fields including genetic improvement. Genotyping has been widely applied in multi-national seed companies due to their high-throughput, automatic, large-scale and shared genotyping platforms. Genotyping has moved from its third generation (G3), dominated by expensive DNA chips and random genotyping by sequencing (GBS), to the fourth generation (G4), characterized by low-cost, less facility-demanding and performed via genotyping by target sequencing (GBTS). In this article, we first introduced two GBTS protocols, GenoPlexs, based on multiplexing PCR, and GenoBaits, based on sequence capture in-solution (also called liquid chip). For both protocols, multiple single-nucleotide-polymorphisms (mSNP) or multiple dispersed nucleotide polymorphisms (MNP) can be generated to reveal the genetic variation hidden within each amplified locus (amplicon). Compared to DNA chips and GBS, GBTS has several advantages, including wide applicability to genotyping facilities, very flexible marker types, highly efficient genotyping, sharable and accumulative marker data, less required information management and support, and wide suitability in biological applications. With the same marker panel (for example, 40K maize mSNPs), three types of genotyping (40K mSNPs, 260K SNPs, and 754K haplotypes) can be achieved, and multiple panels with various marker densities (1K to 40K mSNPs) can be generated by sequencing at different depths. Applications of GenoPlexs and GenoBaits in biology were then reviewed, including biological evolution, germplasm evaluation, genetic map construction, gene mapping and cloning, marker-trait association (genome-wide association study and bulked sample analysis, BSA), progeny testing, gene introgression, gene pyramiding, variety right protection, variety quality monitoring, transgenic event and gene editing detection, and bioassay. More 50 marker panels have been developed so far for more than 20 plant, animal and microorganism species and applied in some of the fields described above. Lastly, we prospected for future GBTS by looking insights into carry-on, automatic, high-throughput and intelligent genotyping platforms, multi-functional marker panels with various marker densities designed to meet specific requirements, integration with other technologies such as KASP, high-density DNA chips and BSA strategies, and open-source breeding by sharing germplasm and breeding materials and information. The development in these fields will greatly facilitate the applications of GBTS in genetic improvement and other fields of animals, plants and microorganisms.

Key words: genotyping by target sequencing (GBTS), multiplexing PCR, sequence capture in-solution (liquid chip), multiple single-nucleotide-polymorphisms (mSNP), multiple dispersed nucleotide polymorphisms (MNP), haplotypes, genetic improvement, open-source breeding

徐云碧,杨泉女,郑洪建,许彦芬,桑志勤,郭子锋,彭海,张丛,蓝昊发,王蕴波,吴坤生,陶家军,张嘉楠. 靶向测序基因型检测（GBTS）技术及其应用[J]. 中国农业科学, 2020, 53(15): 2983-3004.

XU Yunbi,YANG QuanNü,ZHENG HongJian,XU YanFen,SANG ZhiQin,GUO ZiFeng,PENG Hai,ZHANG Cong,LAN HaoFa,WANG YunBo,WU KunSheng,TAO JiaJun,ZHANG JiaNan. Genotyping by Target Sequencing (GBTS) and Its Applications[J]. Scientia Agricultura Sinica, 2020, 53(15): 2983-3004.

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靶向测序基因型检测（GBTS）技术的平台与比较优势"

	优势 Advantages	具体特征 Description of advantages	展望 Prospects
平台 Platform	广适性 Wide suitability	适合所有二代和三代测序系统,包括Illumina、Ion Torrent和MGI Applicable to the second and third generations of sequencing facilities, including Illumina, Ion Torrent and MGI	与下一代测序或其他检测设备兼容 Also applicable to the next generation of sequencing and other genotyping facilities
标记 Markers	灵活性 Flexibility	适合各种标记类型（SNP、短SSR、长/短InDel、已知融合基因、甲基化位点）;不同的标记密度;一款多用 Suitable for various marker types (SNPs, short SSRs, long/short InDels, known fusion genes and methylated loci), densities and applications	广泛利用单倍型、LD区段和其他标记衍生物 Wide applications with haplotypes, LD blocks and other marker-derivatives
检测 Genotyping	高效性 High efficiency	样本多重化、多重PCR;开发和升级简便;设计、测试和检测成本低 Sample- and PCR-multiplexing, readily development and upgrade of marker panels, and low cost in design, test and genotyping	随检测技术进步推动检测的自动化、智能化、超高通量 More robotic, intelligent, and high-throughput with advanced genotyping facilities
信息 Information	可加性 Accumulativity	数据重复率高,缺失数据少;不同时间、地点、项目间的数据可比和累加;整合度高 High duplication rate; less missing; accumulative across times, locations and projects; integrative	可加性程度随技术进步而增强 Increasingly accumulative with technical advancement
支撑 Support systems	便捷性 Less demanding	不依赖检测技术或专业化的生物信息团队;通用而简化的实验室信息管理系统;通用的信息整合、处理、分析流程 Independent of professional genotyping and informatics supports, manageable through regular LIMS and data integration, treatment and analytical protocols	随技术进步而更加便捷、快速、智能化 Less demanding, much quicker but more intelligent, with technical development
应用 Applications	广谱性 Wide application	广泛应用于动物、植物、微生物及其互作群体的进化、遗传、育种、知识产权保护等领域 Wide application in the fields of evolution, genetics, breeding and variety right protection in animals, plants and microorganisms	随着海量信息的累计,将拓展在群体生物学、生态学等领域的应用 Applications extended to population biology and ecology as huge data accumulated

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应用领域 Applications	40K+	30K	20K	10K	5K	1K	<200
生物进化Biological evolution	+++	+++	++	++	+
种质资源评价Germplasm evaluation	+++	+++	++	++	+	+
分类Classification	+++	+++	+++	+++	++	++
图谱构建Linkage map construction	+++	+++	+++	++	+	+
基因定位和克隆Gene mapping/cloning	+++	+++	+++	+++	++	+
标记-性状关联Marker-trait association	+++	++	++	+
后裔测验Progeny testing	+++	+++	+++	+++	+++	+++	+++
基因渐渗Gene introgression	+++	+++	+++	+++	+++	++	+
基因累加Gene pyramiding	+++	+++	+++	+++	++	+	+
品种权保护Variety right protection	+++	+++	+++	+++	++	++	+
质量控制Quality control	+++	+++	+++	+++	++	++	+
生物检测Bioassay	+++	+++	+++	+++	++	+	+

物种名称 Species names	GenoBaits 标记集GenoBaits panels			GenoPlexs 标记集GenoPlexs panels
物种名称 Species names	40K	20K	10K	500-1K	<100	功能标记 Functional markers
玉米 Zea mays L.	●	●	●	●		○
水稻Oryza sativa L.	●	●	●	●		○
棉花Gossypium spp				●		○
大豆Glycine max (Linn.) Merr.	●	●	●	●
花生 Arachis hypogaea L.	●	●	●		●
小麦Triticum aestivum L.	○	○	●			●
谷子 Setaria italica				●
大麦 Hordeum vulgare L.	●	●	●
番茄Solanum lycopersicum				●	●	○
黄瓜 Cucumis sativus L.				●	●	○
辣椒 Capsicum annuum L.				●	●	○
西瓜Citrullus lanatus (Thunb.) Matsum. et Nakai				●	●
白菜Brassica pekinensis (Lour.) Rupr.				●	●
荔枝Litchi chinensis Sonn.				●
猕猴桃Actinidia spp.				●
甘蓝Brassica oleracea L.					●
胡萝卜Daucus carota L. var. sativa Hoffm.					●
西葫芦Cucurbita pepo L.					●
白萝卜 Raphanus sativus					●
苹果Malus domestica						●
牛Bos holsatiae						●

靶向测序基因型检测（GBTS）技术及其应用

Genotyping by Target Sequencing (GBTS) and Its Applications

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