Wang Yonggang, Yang Jingyi, Gao Yujiao, Ma Haigang, Dai Yi, Ma Hongxiang
Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding, Zhongshan Biological Breeding Laboratory/Key Laboratory of Plant Functional Genomics of the Ministry of Education, Agricultural College of Yangzhou University, Yangzhou, 225009, China.
Jiangsu Key Laboratory of Crop Genetics and Physiology, Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, 225009, China.
BMC Plant Biol. 2025 Jul 26;25(1):963. doi: 10.1186/s12870-025-07005-8.
Wheat (Triticum aestivum L.) cultivation suffers from significant yield loss owing to diseases such as Fusarium head blight (FHB) and powdery mildew (PM). Utilization of host resistance is an effective strategy for controlling diseases. Fhb7 and Pm21 are derived from wild relatives of wheat, which confer broad-spectrum resistance to FHB and PM, respectively, and can be used in breeding through marker-assisted selection. Kompetitive Allele Specific polymerase chain reaction (KASP) is a homogeneous fluorescence-based technology, which identifies single nucleotide polymorphisms (SNP), and is suitable for marker-assisted selection (MAS) in large-scale breeding. However, KASP typically identifies the alleles of a single SNP, which limits the efficiency of pyramiding multiple genes during breeding.
In this study, we developed and applied a novel multiplex KASP (Multi-KASP) system that enabled concurrent detection of two distinct genes in a single reaction. The Multi-KASP system increased the flexibility of primer requirements and differentiated genes using only basic fluorescent cassettes, thereby significantly enhancing genotyping efficiency while ensuring high specificity and accuracy. This system accurately distinguished homozygous and heterozygous genotypes for both genes, which was validated by comparison with conventional marker detection. Phenotyping showed that polymerization of Fhb7 and Pm21 using the multi-KASP system enhanced resistance to FHB and PM in the wheat breeding program.
A cost-effective Multi-KASP genotyping system, capable of simultaneously detecting two distinct genes in a single reaction, is an efficient and convenient solution for molecular breeding. The system demonstrates high potential for enhancing the efficiency of MAS in wheat breeding for resistance to FHB and PM.
小麦（Triticum aestivum L.）种植因诸如赤霉病（FHB）和白粉病（PM）等病害而遭受显著的产量损失。利用寄主抗性是控制病害的有效策略。Fhb7和Pm21源自小麦的野生近缘种，分别赋予对FHB和PM的广谱抗性，并且可通过标记辅助选择用于育种。竞争性等位基因特异性聚合酶链反应（KASP）是一种基于均相荧光的技术，可识别单核苷酸多态性（SNP），适用于大规模育种中的标记辅助选择（MAS）。然而，KASP通常识别单个SNP的等位基因，这限制了育种过程中多个基因聚合的效率。
在本研究中，我们开发并应用了一种新型多重KASP（Multi-KASP）系统，该系统能够在单个反应中同时检测两个不同的基因。Multi-KASP系统提高了引物要求的灵活性，并仅使用基本荧光盒区分基因，从而在确保高特异性和准确性的同时显著提高基因分型效率。该系统准确地区分了两个基因的纯合和杂合基因型，这通过与传统标记检测进行比较得到了验证。表型分析表明，使用多KASP系统聚合Fhb7和Pm21可增强小麦育种计划中对FHB和PM的抗性。
一种具有成本效益的Multi-KASP基因分型系统，能够在单个反应中同时检测两个不同的基因，是分子育种的一种高效便捷的解决方案。该系统在提高小麦抗FHB和PM育种中MAS效率方面显示出巨大潜力。
