Steele Katherine, Quinton-Tulloch Mark, Vyas Darshna, Witcombe John
School of Environmental and Natural Science, Bangor University, Bangor, Gwynedd LL57 2UW, UK.
LGC BioSearch Technologies, Units 1 and 2, Trident Industrial Estate, Pindar Road, Hoddesdon, Herts EN11 0WZ, UK.
G3 (Bethesda). 2025 Jan 8;15(1). doi: 10.1093/g3journal/jkae251.
This study aimed to broaden applicability of KASP for Oryza sativa across diverse genotypes through incorporation of ambiguous (degenerate) bases into their primer designs and to validate 4,000 of them for genotyping applications. A bioinformatics pipeline was used to compare 129 rice genomes from 89 countries with the indica reference genome R498 and generate ∼1.6 million KASP designs for the more common variants between R498 and the other genomes. Of the designs, 98,238 were for predicted functional markers. Up to 5 KASP each for 1,024 breeder-selected loci were assayed in a panel of 178 diverse rice varieties, generating 3,366 validated KASP. The 84% success rate was within the normal range for KASP demonstrating that the ambiguous bases do not compromise efficacy. The 3,366-trait-specific marker panel was applied for population structure analysis in the diversity panel and resolved them into 4 expected groups. Target variations in 13 genomes used for designs were compared with the corresponding KASP genotypes in different accessions of the same 13 varieties in the diversity panel. There was agreement for 79% or more markers in 12 varieties; 10 having agreement >88%. One variety, a selection from a landrace, had only 46.5% marker agreement. Breeders can search for the validated KASP and more than a million so-far untested designs in three reference genomes (including Niponbare MSU7) with a search tool, that includes designs in proximity to previously published microsatellite markers, and retrieve target variations for 129 rice genomes plus their genomic locations with ±25 bp flanking sequences.
本研究旨在通过在引物设计中引入模糊（简并）碱基，拓宽KASP在不同基因型水稻中的适用性，并对其中4000个进行基因分型应用验证。利用生物信息学流程将来自89个国家的129个水稻基因组与籼稻参考基因组R498进行比较，针对R498与其他基因组之间较常见的变异生成约160万个KASP设计。在这些设计中，98238个是针对预测功能标记的。在178个不同水稻品种的群体中，对1024个育种者选择的位点各检测了多达5个KASP，产生了3366个经过验证的KASP。84%的成功率在KASP的正常范围内，表明模糊碱基不会影响效果。将3366个性状特异性标记面板应用于多样性面板的群体结构分析，并将它们解析为4个预期组。将用于设计的13个基因组中的目标变异与多样性面板中同一13个品种不同种质中的相应KASP基因型进行比较。12个品种中79%或更多的标记一致；10个品种的一致性>88%。一个从地方品种中选出的品种，标记一致性仅为46.5%。育种者可以使用搜索工具在三个参考基因组（包括日本晴MSU7）中搜索经过验证的KASP以及超过100万个迄今未测试的设计，该工具包括与先前发表的微卫星标记相邻的设计，并检索129个水稻基因组的目标变异及其两侧各有±25 bp侧翼序列的基因组位置。
