Plant Pathology and Plant Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, NY, United States of America.
The Sainsbury Laboratory, University of East Anglia, Norwich, United Kingdom.
PLoS Genet. 2020 Jan 27;16(1):e1008571. doi: 10.1371/journal.pgen.1008571. eCollection 2020 Jan.
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.
长读测序有助于复杂基因组区域的组装。在植物中，含有核苷酸结合、富含亮氨酸重复（NLR）抗病基因的基因座是此类区域的一个重要例子。NLR 基因是植物中最大的基因家族之一，通常通过复制、收缩和转位进行聚类进化。我们最近将美国传统品种卡罗莱纳黄金选择（Carolina Gold Select）中抗细菌性叶斑病和细菌性条斑病的 Xo1 基因座定位到一个在 Nipponbare 参考基因组中富含 NLR 基因的区域。在这里，为了鉴定 Xo1 基因，我们将纳米孔和 Illumina 读取数据相结合，生成了高质量的卡罗莱纳黄金选择基因组组装。我们鉴定了 529 个完整或部分 NLR 基因，并发现相对于 Nipponbare，Xo1 基因座的 NLR 基因扩张。其中一个与克隆的、功能相似的 Xa1 基因具有高度的序列相似性。两者都含有一个整合的 zfBED 结构域，并且每个蛋白质中的重复几乎是完美的。在不同的 Oryzeae 中，我们鉴定了具有这些特征的两个 NLR 基因亚家族，其差异在于 zfBED 结构域的存在和重复的数量。卡罗莱纳黄金选择基因组组装还在 Xo1 基因座上发现了一个稻瘟病抗性基因和一个编码多酚氧化酶（PPO）的基因。在某些品种中，该基因座上的 PPO 活性已被用作抗稻瘟病的标记；然而，卡罗莱纳黄金选择序列揭示了 PPO 基因的功能丧失突变，打破了这种关联。我们的研究结果表明，将纳米孔和 Illumina 读取数据相结合的全基因组测序有效地解决了 NLR 基因座问题。我们鉴定的 Xo1 候选基因是对其进行机制特征分析（包括 zfBED 结构域的作用）的重要步骤。最后，卡罗莱纳黄金选择基因组组装将有助于鉴定该历史重要品种中的其他有用性状。
