Few public sector rice breeders have the capacity to use NGS-derived markers in their breeding programmes despite rapidly expanding repositories of rice genome sequence data. They rely on > 18,000 mapped microsatellites (SSRs) for marker-assisted selection (MAS) using gel analysis. Lack of knowledge about target SNP and InDel variant loci has hampered the uptake by many breeders of Kompetitive allele-specific PCR (KASP), a proprietary technology of LGC genomics that can distinguish alleles at variant loci. KASP is a cost-effective single-step genotyping technology, cheaper than SSRs and more flexible than genotyping by sequencing (GBS) or array-based genotyping when used in selection programmes. Before this study, there were 2015 rice KASP marker loci in the public domain, mainly identified by array-based screening, leaving large proportions of the rice genome with no KASP coverage. Here we have addressed the urgent need for a wide choice of appropriate rice KASP assays and demonstrated that NGS can detect many more KASP to give full genome coverage. Through re-sequencing of nine indica rice breeding lines or released varieties, this study has identified 2.5 million variant sites. Stringent filtering of variants generated 1.3 million potential KASP assay designs, including 92,500 potential functional markers. This strategy delivers a 650-fold increase in potential selectable KASP markers at a density of 3.1 per 1 kb in the indica crosses analysed and 377,178 polymorphic KASP design sites on average per cross. This knowledge is available to breeders and has been utilised to improve the efficiency of public sector breeding in Nepal, enabling identification of polymorphic KASP at any region or quantitative trait loci in relevant crosses. Validation of 39 new KASP was carried out by genotyping progeny from a range of crosses to show that they detected segregating alleles. The new KASP have replaced SSRs to aid trait selection during marker-assisted backcrossing in these crosses, where target traits include rice blast and BLB resistance loci. Furthermore, we provide the software for plant breeders to generate KASP designs from their own datasets.

中文翻译：

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利用源自rice稻全基因组测序的高密度KASP标记促进公共部门稻米育种。

尽管迅速扩大了水稻基因组序列数据的存储库，但很少有公共部门的水稻育种者能够在其育种计划中使用NGS衍生的标记。他们使用凝胶分析依靠> 18,000个映射的微卫星（SSR）进行标记辅助选择（MAS）。缺乏对靶SNP和InDel变异基因座的了解，阻碍了许多育种者对竞争性等位基因特异性PCR（KASP）的吸收，这是LGC基因组学的专有技术，可以区分变异基因座上的等位基因。当用于选择程序时，KASP是一种经济高效的单步基因分型技术，比SSR便宜，并且比测序（GBS）或基于阵列的基因分型更灵活。在此研究之前，公共领域有2015年水稻KASP标记基因座，主要通过基于阵列的筛选来鉴定，剩下的水稻基因组大部分没有KASP覆盖。在这里，我们已经满足了对多种合适的水稻KASP分析的广泛选择的迫切需求，并证明NGS可以检测更多KASP来提供完整的基因组覆盖。通过对9个in稻育种系或已发布的品种进行重测序，本研究确定了250万个变异位点。严格过滤变体产生了130万种潜在的KASP分析设计，包括92,500种潜在的功能标记。在所分析的cross杂交中，该策略以每1 kb 3.1的密度将潜在的可选KASP标记增加了650倍，每个杂交平均增加了377,178个多态KASP设计位点。这些知识可供育种者使用，并已被用来提高尼泊尔公共部门育种的效率，可以在相关杂交的任何区域或定量性状基因座处鉴定多态性KASP。对39个新的KASP进行了验证，方法是对一系列杂交的后代进行基因分型，以表明它们检测到分离的等位基因。新的KASP取代了SSR，以帮助在这些杂交的标记辅助回交中选择性状，这些杂交的目标性状包括稻瘟病和BLB抗性基因座。此外，我们为植物育种者提供了从他们自己的数据集生成KASP设计的软件。目标特征包括稻瘟病和BLB抗性基因座。此外，我们为植物育种者提供了从他们自己的数据集生成KASP设计的软件。目标特征包括稻瘟病和BLB抗性基因座。此外，我们为植物育种者提供了从他们自己的数据集生成KASP设计的软件。