A high throughput molecular marker assay for bacterial leaf blight resistance gene Xa21 in rice

Abstract

Molecular markers are used as tools for precise and efficient screening of desirable genotypes in modern day plant breeding. One of the attractive attributes of a molecular marker is its compatibility to high throughput screening. The Xa21 gene is a major resistance gene against bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae. The closely-linked sequence-tagged site marker pTA248 has been frequently used for the screening of Xa21, however, compared to a flanking or intergenic marker, a linked-marker may have low reliability and effectiveness in marker-assisted selection (MAS). In the current study, a diagnostic intragenic marker ABUOP0001 was developed for MAS of Xa21, which flanks over a 19-bp insertion/deletion on the ectodomain of the Xa21. The marker amplifies a 200-bp and a 181-bp amplicon from the rice lines IRBB 62 and IRBB 7, known-to-carry the resistance and susceptible alleles for Xa21, respectively. The marker ABUOP0001 is compatible for high-throughput screening with high resolution melting technology, where the genotypic scoring could be effectively carried out based on the normalized melt curves. Further, the marker ABUOP0001 was successfully assayed as a single-tube multiplexed PCR with another molecular marker assaying for the BLB resistance gene Xa4. The products of the two markers can be effectively binned together to facilitate the detection of the presence of resistance and/or susceptible alleles in a single PCR reaction. The marker ABUOP0001 performed equally or better than the linked-marker pTA248 in detecting BLB resistant phenotypes in a field trial involving 63 rice accessions. Therefore, the marker ABUOP0001 can be recommended as a diagnostic intragenic marker for marker-assisted high throughput screening of Xa21 in rice.