Genome assembly and characterization of a complex zfBED-NLR gene-containing disease resistance locus in Carolina Gold Select rice with Nanopore sequencing

Long-read sequencing facilitates assembly of complex genomic regions. In plants, loci containing nucleotide-binding, leucine-rich repeat (NLR) disease resistance genes are an important example of such regions. NLR genes constitute one of the largest gene families in plants and are often clustered, evolving via duplication, contraction, and transposition. We recently mapped the Xo1 locus for resistance to bacterial blight and bacterial leaf streak, found in the American heirloom rice variety Carolina Gold Select, to a region that in the Nipponbare reference genome is NLR gene-rich. Here, toward identification of the Xo1 gene, we combined Nanopore and Illumina reads and generated a high-quality Carolina Gold Select genome assembly. We identified 529 complete or partial NLR genes and discovered, relative to Nipponbare, an expansion of NLR genes at the Xo1 locus. One of these has high sequence similarity to the cloned, functionally similar Xa1 gene. Both harbor an integrated zfBED domain, and the repeats within each protein are nearly perfect. Across diverse Oryzeae, we identified two sub-clades of NLR genes with these features, varying in the presence of the zfBED domain and the number of repeats. The Carolina Gold Select genome assembly also uncovered at the Xo1 locus a rice blast resistance gene and a gene encoding a polyphenol oxidase (PPO). PPO activity has been used as a marker for blast resistance at the locus in some varieties; however, the Carolina Gold Select sequence revealed a loss-of-function mutation in the PPO gene that breaks this association. Our results demonstrate that whole genome sequencing combining Nanopore and Illumina reads effectively resolves NLR gene loci. Our identification of an Xo1 candidate is an important step toward mechanistic characterization, including the role(s) of the zfBED domain. Finally, the Carolina Gold Select genome assembly will facilitate identification of other useful traits in this historically important variety. Author summary Plants lack adaptive immunity, and instead contain repeat-rich, disease resistance genes that evolve rapidly through duplication, recombination, and transposition. The number, variation, and often clustered arrangement of these genes make them challenging to sequence and catalog. The US heirloom rice variety Carolina Gold Select has resistance to two important bacterial diseases. Toward identifying the responsible gene(s), we combined long- and short-read sequencing technologies to assemble the whole genome and identify the resistance gene repertoire. We previously narrowed the location of the gene(s) to a region on chromosome four. The region in Carolina Gold Select is larger than in the rice reference genome (Nipponbare) and contains twice as many resistance genes. One shares unusual features with a known bacterial disease resistance gene, suggesting that it confers the resistance. Across diverse varieties and related species, we identified two widely-distributed groups of such genes. The results are an important step toward mechanistic characterization and deployment of the bacterial disease resistance. The genome assembly also identified a resistance gene for a fungal disease and predicted a marker phenotype used in breeding for resistance. Thus, the Carolina Gold Select genome assembly can be expected to aid in the identification and deployment of other valuable traits.