A novel intragenic marker targeting the ectodomain of bacterial blight-resistance gene <i>Xa21</i> for marker-assisted selection in rice

The rice (<i>Oryza sativa</i> L.) <i>Xa21</i> gene is known to confer resistance against <i>Xanthomonas oryzae</i> pv. <i>oryzae</i>, the causal organism of bacterial blight (BB) through its involvement in pathogen recognition and immunity responses. The closely linked sequence-tagged site marker <i>pTA248</i> is frequently used for marker-assisted selection (MAS) of <i>Xa21</i>. However, lower precision and linkage drag of linked-markers hinders its reliability and effectiveness in MAS compared to flanking/intergenic markers. In the current study, a diagnostic intragenic marker was developed for MAS of <i>Xa21</i> in rice. The new marker <i>ABUOP0001</i> targets a 19-bp insertion/deletion on the ectodomain of <i>Xa21</i> and amplifies a 200-bp amplicon from rice line IRBB 62 (known to carry the “resistance allele”), and a 181-bp amplicon from IRBB 7 (known to carry the “susceptible allele”). The <i>ABUOP0001</i> marker assay identified the resistance allele in 15 rice lines/accessions. In a field study, 14 of these lines/accessions had highly resistant BB responses, and one had an intermediate response. Further, <i>Xa21</i> resistance allele was identified in 1,675 rice accessions through an <i>in-silico</i> genomic sequence analysis. The marker <i>ABUOP0001</i> performs better than the previously used linked-marker <i>pTA248</i> (anchored to a gene 225 kbp upstream of <i>Xa21</i>) as <i>ABUOP0001</i> is an intragenic marker targeting the functionally important ectodomain of <i>Xa21</i>. Further, <i>ABUOP0001</i> is compatible for high-throughput screening with high-resolution melting and can be multiplexed effectively with an intragenic marker of BB resistance gene <i>Xa4</i>. Therefore, <i>ABUOP0001</i> can be recommended as a high-throughput diagnostic intragenic marker for MAS of <i>Xa21</i> in rice.