靶向测序基因型检测（GBTS）技术及其应用

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...

Genotyping by Target Sequencing (GBTS) and Its Applications

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.