Improving Broad Spectrum Blast Resistance by Introduction of the Pita2 Gene: Encoding the NB-ARC Domain of Blast-Resistant Proteins into Upland Rice Breeding Programs

Abstract

Blast disease is generally more important in upland rather than lowland rice cultivation, representing one of the biotic obstacles in the development of upland rice. The objective of this study was to detect broad-spectrum blast resistance gene Pita2 encoding the NB-ARC (nucleotide-binding adaptor common in APAF-1, R proteins, and CED-4) domain of blast-resistant proteins in new upland rice lines from the breeding program for landrace rice varieties, with the goal of providing a novel source of blast-resistant germplasm for application in future upland rice breeding programs. In this study, we screened 19 inbred lines of landrace rice varieties challenged using local virulent isolates in greenhouse conditions and performed field evaluations to confirm blast resistance. Molecular analysis was conducted using six specific primers to detect broad-spectrum blast resistance, and sequence analysis was performed to detect the NB-ARC domain of blast-resistant proteins in the lines. Consistent results were observed between greenhouse screening and field evaluations, although there was variance in the level of resistance. The PCR assay showed that there were eight positive lines (G7, G8, G9, G11, G13, G14, G15, and G18) containing the Pita2 gene. Conserved domain analysis revealed that eight blast-resistant rice lines encode NB-ARC at sequence lengths ranging between 300 and 870 (450 bp). Using these sequences in BLASTX searching revealed 15 gene homologs of the eight rice lines, which were detected as Pita2 genes, with a similarity level of 81–99%. Further comprehensive studies should be performed to confirm the performance and resistance of candidate lines in field trials in various blast-endemic areas before being released as new upland rice varieties able to overcome the problem of blast disease in the field. In addition, the lines can also be used as a novel genetic resource in the blast-resistant upland rice breeding program on various rice cultivars.