Identification of Novel Alleles of the Rice Blast-Resistance Gene Pi9 through Sequence-Based Allele Mining

As rice (Oryza sativa) is the staple food of more than half the world's population, rice production contributes greatly to global food security. Rice blast caused by the fungus Magnaporthe oryzae (M. oryzae) is a devastating disease that affects rice yields and grain quality, resulting in substantial economic losses annually. Because the fungus evolves rapidly, the resistance conferred by most the single blast-resistance genes is broken after a few years of intensive agricultural use. Therefore, effective resistance breeding in rice requires continual enrichment of the reservoir of resistance genes, alleles, or QTLs. Seed banks represent a rich source of genetic diversity; however, they have not been extensively used to identify novel genes and alleles.

We carried out a large-scale screen for novel blast-resistance alleles in 1883 rice varieties from major rice-producing areas across China. Of these, 361 varieties showed at least moderate resistance to natural infection by rice blast at rice blast nurseries in Enshi and Yichang, Hubei Province. We used sequence-based allele mining to amplify and sequence the allelic variants of the major rice blast-resistance genes at the Pi2/Pi9 locus of chromosome 6 from the 361 blast-resistant varieties, and the full-length coding region of this gene could be amplified from 107 varieties. Thirteen novel Pi9 alleles (named Pi9-Type1 to Pi9-Type13) were identified in these 107 varieties based on comparison to the Pi9 referenced sequence. Based on the sequencing results, the Pi2/Pi9 locus of the 107 varieties was divided into 15 genotypes (including three different genotypes of Pi9-Type5). Fifteen varieties, each representing one genotype, were evaluated for resistance to 34 M. oryzae isolates. The alleles from seven varieties with the highest resistance and widest resistance spectra were selected for transformation into the susceptible variety J23B to construct near-isogenic lines (NILs). These NILs showed resistance in a field test in Enshi and Yichang, indicating that the seven novel rice blast-resistance tandem-repeat regions at the Pi2/Pi9 locus of chromosome 6 could potentially serve as a genetic resource for molecular breeding of resistance to rice blast.

The thirteen novel Pi9 alleles identified in this study expand the list of available of blast-resistance alleles. Seven tandem-repeat regions of the Pi2/Pi9 locus from different donors were characterized as broad-spectrum rice blast-resistance fragments; these donors enrich the genetic resources available for rice blast-resistance breeding programs.