Integrative GWAS and Haplotype-Based Dissection Identify Additive Genomic Hotspots conferring Bacterial Leaf Streak Resistance in the Barley Interspecific Cytonuclear Multi-Parent Population (CMPP)

<title>Abstract</title> <p> Bacterial leaf streak (BLS), caused by <italic>Xanthomonas translucens</italic> , is an emerging threat to barley ( <italic>Hordeum vulgare</italic> L.) production in the Great Plains. Due to the lack of information on resistant cultivars and limited chemical or biological control, it is essential to identify and deploy resistance in current high-yielding barley varieties. To discover the genomic architecture of BLS resistance, we performed an integrative genome-wide association study (GWAS) and haplotype-based analysis in the barley doubled haploid interspecific Cytonuclear Multi-Parent Population (CMPP) to discover marker-trait associations (MTAs). The mapping population was genotyped using an Illumina Infinium 50K iSelect SNP array, resulting in 13,209 high-quality SNP markers. The BLS disease screening using a scale of 1 (highly resistant) to 9 (highly susceptible), revealed substantial phenotypic variation in the 700 lines, with only 2% highly resistant. Several GWAS models identified 28 significant MTAs, of which 13, were classified as high-confidence (HC) MTAs. LD-based clustering identified two major additive genomic hotspot Quantitative Trait Loci (QTL) regions: <italic>QHvBLS_1H_GAT</italic> (2.18 Mb) on chromosome 1H, harboring seven HC MTAs, and <italic>QHvBLS_2H_GAT</italic> (1.72 Mb) on chromosome 2H, harboring two HC MTAs. Resistant allele stacking exhibited a significant cumulative effect, confirming polygenic additive inheritance. Haplotype dissection revealed distinct resistant (R1, R2) and susceptible (S1, S2) haplotypes within both QTLs, and haplotype stacking effects demonstrated that <italic>QHvBLS_1H_GAT</italic> acts as the primary resistance locus, while <italic>QHvBLS_2H_GAT</italic> enhances resistance additively. Together, these findings define the genomic architecture of BLS resistance in barley and highlight deployable haplotypes for genomics-assisted breeding of durable BLS-resistant barley varieties. </p>