Ziarsolo Peio, Hasing Tomas, Hilario Rebeca, Garcia-Carpintero Victor, Blanca Jose, Bombarely Aureliano, Cañizares Joaquin
COMAV, Universitat Politècnica de València, 46022, Valencia, Spain.
Colección española de cultivos tipo (CECT), Universitat de València, 46980, Paterna, Spain.
Plant Methods. 2021 Mar 25;17(1):30. doi: 10.1186/s13007-021-00733-6.
K-seq, a new genotyping methodology based on the amplification of genomic regions using two steps of Klenow amplification with short oligonucleotides, followed by standard PCR and Illumina sequencing, is presented. The protocol was accompanied by software developed to aid with primer set design.
As the first examples, K-seq in species as diverse as tomato, dog and wheat was developed. K-seq provided genetic distances similar to those based on WGS in dogs. Experiments comparing K-seq and GBS in tomato showed similar genetic results, although K-seq had the advantage of finding more SNPs for the same number of Illumina reads. The technology reproducibility was tested with two independent runs of the tomato samples, and the correlation coefficient of the SNP coverages between samples was 0.8 and the genotype match was above 94%. K-seq also proved to be useful in polyploid species. The wheat samples generated specific markers for all subgenomes, and the SNPs generated from the diploid ancestors were located in the expected subgenome with accuracies greater than 80%.
K-seq is an open, patent-unencumbered, easy-to-set-up, cost-effective and reliable technology ready to be used by any molecular biology laboratory without special equipment in many genetic studies.
介绍了K-seq，一种新的基因分型方法，该方法基于使用短寡核苷酸通过两步Klenow扩增来扩增基因组区域，随后进行标准PCR和Illumina测序。该方案还附带了为辅助引物组设计而开发的软件。
作为首批示例，在番茄、狗和小麦等多种物种中开发了K-seq。K-seq在狗中提供的遗传距离与基于全基因组测序（WGS）的遗传距离相似。在番茄中比较K-seq和GBS的实验显示了相似的遗传结果，尽管在相同数量的Illumina读数下，K-seq在发现更多单核苷酸多态性（SNP）方面具有优势。使用番茄样本的两次独立运行测试了该技术的可重复性，样本间SNP覆盖率的相关系数为0.8，基因型匹配率高于94%。K-seq在多倍体物种中也被证明是有用的。小麦样本为所有亚基因组生成了特异性标记，从二倍体祖先产生的SNP位于预期的亚基因组中，准确率大于80%。
K-seq是一种开放、无专利限制、易于设置、经济高效且可靠的技术，任何分子生物学实验室无需特殊设备即可在许多遗传研究中使用。
