Department of Plant and Soil Sciences, Texas Tech University, Lubbock, TX 79409, USA.
Plant Stress and Germplasm Development Unit, United States Department of Agriculture-Agricultural Research Service, Lubbock, TX 79415, USA.
Genes (Basel). 2020 Oct 18;11(10):1220. doi: 10.3390/genes11101220.
The use of molecular markers in plant breeding has become a routine practice, but the cost per accession can be a hindrance to the routine use of Quantitative Trait Loci (QTL) identification in breeding programs. In this study, we demonstrate the use of targeted re-sequencing as a proof of concept of a cost-effective approach to retrieve highly informative allele information, as well as develop a bioinformatics strategy to capture the genome-specific information of a polyploid species. SNPs were identified from alignment of raw transcriptome reads (2 × 50 bp) to a synthetic tetraploid genome using BWA followed by a GATK pipeline. Regions containing high polymorphic SNPs in both A genome and B genomes were selected as targets for the resequencing study. Targets were amplified using multiplex PCR followed by sequencing on an Illumina HiSeq. Eighty-one percent of the SNP calls in diploids and 68% of the SNP calls in tetraploids were confirmed. These results were also confirmed by KASP validation. Based on this study, we find that targeted resequencing technologies have potential for obtaining maximum allele information in allopolyploids at reduced cost.
在植物育种中，使用分子标记已经成为一种常规做法，但每个品系的成本可能会阻碍定量性状基因座（QTL）鉴定在育种计划中的常规使用。在这项研究中，我们展示了靶向重测序作为一种经济高效的方法的概念验证，用于获取高度信息丰富的等位基因信息，并开发了一种生物信息学策略来捕获多倍体物种的基因组特异性信息。使用 BWA 对原始转录组读数（2×50 bp）进行比对，然后使用 GATK 管道对合成的四倍体基因组进行比对，从原始转录组读数（2×50 bp）中鉴定出 SNPs。选择在 A 基因组和 B 基因组中都含有高多态性 SNP 的区域作为重测序研究的目标。使用多重 PCR 扩增目标，然后在 Illumina HiSeq 上进行测序。二倍体中 81%的 SNP 调用和四倍体中 68%的 SNP 调用得到了确认。这些结果也通过 KASP 验证得到了确认。基于这项研究，我们发现靶向重测序技术具有在降低成本的情况下在异源多倍体中获取最大等位基因信息的潜力。
