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Bacterial pustule (BP), caused by Xanthomonas citri pv. glycines, is an important disease that, under favorable conditions, can drastically affect soybean production. We performed a genome-wide association study (GWAS) with a panel containing Brazilian and American cultivars, which were screened qualitatively and quantitatively against two Brazilian X. citri isolates (IBS 333 and IBS 327). The panel was genotyped using a genotyping by sequencing (GBS) approach, and we identified two main new regions in soybeans associated with X. citri resistance on chromosomes 6 (IBS 333) and 18 (IBS 327), different from the traditional rxp gene located on chromosome 17. The region on chromosome 6 was also detected by QTL mapping using a biparental cross between Williams 82 (R) and PI 416937 (S), showing that Williams 82 has another recessive resistance gene besides rxp, which was also detected in nine BP-resistant ancestors of the Brazilian cultivars (including CNS, S-100), based on haplotype analysis. Furthermore, we identified additional SNPs in strong LD (0.8) with peak SNPs by exploring variation available in WGS (whole genome sequencing) data among 31 soybean accessions. In these regions in strong LD, two candidate resistance genes were identified (Glyma.06g311000 and Glyma.18g025100) for chromosomes 6 and 18, respectively. Therefore, our results allowed the identification of new chromosomal regions in soybeans associated with BP disease, which could be useful for marker-assisted selection and will enable a reduction in time and cost for the development of resistant cultivars. [ABSTRACT FROM AUTHOR]