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

Abstract

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

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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Figures/Tables 8

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Fig. 3

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Table 1

Platforms and advantages of genotyping by target sequencing (GBTS)"

	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						●

Genotyping by Target Sequencing (GBTS) and Its Applications

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