Data Sheet 2_Development of cleaved amplified polymorphic sequence marker for powdery mildew resistance in Korean malting barley using QTL-seq.docx

IntroductionPowdery mildew (PM) caused by Blumeria graminis f.sp. hordei is a major fungal disease affecting barley (Hordeum vulgare L.). The most effective approach to controlling this disease is the development of resistant cultivars. In this study, we investigated the genomic regions associated with PM resistance by performing quantitative loci sequencing (QTL-seq) twice using the parental lines ‘Hopum’ (susceptible) and ‘Jeonju 182’ (resistant) as reference genomes.
MethodsThis study was conducted from 2022 to 2024 at the National Institute of Agricultural Sciences in Wanju, Republic of Korea. We conducted artificial crossing, genomic DNA extraction, phenotypic evaluation, QTL-seq analysis, and cleaved amplified polymorphic sequence (CAPS) marker development. Candidate gene expression was analyzed using real-time quantitative reverse transcription PCR.
ResultsA total of 2,130 common variants were identified in two regions of chromosome 1H (6,940,595–18,008,713 bp and 19,363,700–20,551,018 bp). Twenty-one non-synonymous single nucleotide polymorphisms among these variants were used to develop CAPS markers, which were validated in an F2 population and malting barley cultivars. The PMC_75 marker, which is annotated as HORVU.MOREX.r3.1HG0005790, showed a strong association with resistance and was highly expressed in ‘Jeonju 182.’ This marker is associated with a Clathrin Assembly Protein, which is involved in vesicle formation and intracellular trafficking, processes essential for cellular signaling and defense responses.
ConclusionThe development of the CAPS marker (PMC_75) provides a valuable tool for marker-assisted selection in breeding PM-resistant malting barley, improving breeding efficiency, and accelerating the development of resistant cultivars.

引言
由大麦白粉菌（Blumeria graminis f.sp. hordei）引起的白粉病（Powdery Mildew, PM）是影响大麦（Hordeum vulgare L.）的主要真菌病害。防控该病害最有效的手段是培育抗病品种。本研究以感病亲本株系‘Hopum’和抗病亲本株系‘Jeonju 182’作为参考基因组，通过两次数量性状位点测序（QTL-seq），解析与白粉病抗性相关的基因组区域。

材料与方法
本研究于2022年至2024年在韩国完州郡国立农业科学研究院开展。实验内容包括人工杂交、基因组DNA提取、表型鉴定、QTL-seq分析以及裂解扩增片段长度多态性（cleaved amplified polymorphic sequence, CAPS）标记开发。候选基因的表达水平通过实时定量反转录聚合酶链式反应进行分析。

结果
在1H号染色体的两个区域（6,940,595–18,008,713 bp与19,363,700–20,551,018 bp）中共鉴定出2130个共有的变异位点。其中21个非同义单核苷酸多态性位点被用于开发CAPS标记，并在F2分离群体及啤酒大麦品种中完成验证。注释为HORVU.MOREX.r3.1HG0005790的PMC_75标记与白粉病抗性呈现显著关联，且在‘Jeonju 182’中高表达。该标记对应的基因为网格蛋白组装蛋白（Clathrin Assembly Protein），其参与囊泡形成与细胞内运输过程，而这两类过程对细胞信号转导与防卫反应至关重要。

结论
CAPS标记PMC_75的开发为抗白粉病啤酒大麦的标记辅助选择育种提供了实用工具，可提升育种效率并加速抗病品种的培育进程。