Abstract
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.
Highlights
Recent advances in sequencing technology enable the assembly of complex genomic loci by generating read lengths long enough to resolve repetitive regions [1]
We found that assembly by Flye [46] using only Nanopore data yielded long contigs but a high consensus error rate
Whole genome sequencing using Nanopore long reads along with Illumina short reads delineated a complex, NLR gene-rich region of interest, the Xo1 locus for resistance to bacterial leaf streak (BLS) and bacterial blight (BB), in the American heirloom rice variety Carolina Gold Select. This revealed an expansion at the locus relative to the reference (Nipponbare) genome and allowed identification of an Xo1 gene candidate based on sequence similarity to the functionally similar, cloned Xa1 gene, including an intergrated zfBED domain and nearly identical repeats
Summary
Recent advances in sequencing technology enable the assembly of complex genomic loci by generating read lengths long enough to resolve repetitive regions [1]. Repetitive regions are often hotspots of recombination and other genomic changes, but difficulties assembling them mean that they often remain as incomplete gaps for many years after a genome’s initial draft assembly. The most straightforward way to span lengthy or complex repeats is to generate single reads that are longer than the repeats themselves, so that repeats can be placed in the correct genomic location. One of the most promising current technologies for resolving complex repeats is nanopore-based sequencing from Oxford Nanopore Technologies ("Nanopore"), for which validated reads as long as 2,272,580 bases have been reported [2], and improvements in base calling software are increasingly improving fidelity [3]. Nanopore sequencing has been used for various applications, including genome sequencing of Arabidopsis and a wild tomato relative [4,5], resolving complex T-DNA insertions [6], and disease resistance gene enrichment sequencing [7]
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