Table_3_Long-Read–Based de novo Genome Assembly and Comparative Genomics of the Wheat Leaf Rust Pathogen Puccinia triticina Identifies Candidates for Three Avirulence Genes.XLSX

Leaf rust, caused by Puccinia triticina (Pt), is one of the most devastating diseases of wheat, affecting production in nearly all wheat-growing regions worldwide. Despite its economic importance, genomic resources for Pt are very limited. In the present study, we have used long-read sequencing (LRS) and the pipeline of FALCON and FALCON-Unzip (v4.1.0) to carry out the first LRS-based de novo genome assembly for Pt. Using 22.4-Gb data with an average read length of 11.6 kb and average coverage of 150-fold, we generated a genome assembly for Pt104 [strain 104-2,3,(6),(7),11; isolate S423], considered to be the founding isolate of a clonal lineage of Pt in Australia. The Pt104 genome contains 162 contigs with a total length of 140.5 Mb and N50 of 2 Mb, with the associated haplotigs providing haplotype information for 91% of the genome. This represents the best quality of Pt genome assembly to date, which reduces the contig number by 91-fold and improves the N50 by 4-fold as compared to the previous Pt race1 assembly. An annotation pipeline that combined multiple lines of evidence including the transcriptome assemblies derived from RNA-Seq, previously identified expressed sequence tags and Pt race 1 protein sequences predicted 29,043 genes for Pt104 genome. Based on the presence of a signal peptide, no transmembrane segment, and no target location to mitochondria, 2,178 genes were identified as secreted proteins (SPs). Whole-genome sequencing (Illumina paired-end) was performed for Pt104 and six additional strains with differential virulence profile on the wheat leaf rust resistance genes Lr26, Lr2a, and Lr3ka. To identify candidates for the corresponding avirulence genes AvrLr26, AvrLr2a, and AvrLr3ka, genetic variation within each strain was first identified by mapping to the Pt104 genome. Variants within predicted SP genes between the strains were then correlated to the virulence profiles, identifying 38, 31, and 37 candidates for AvrLr26, AvrLr2a, and AvrLr3ka, respectively. The identification of these candidate genes lays a good foundation for future studies on isolating these avirulence genes, investigating the molecular mechanisms underlying host–pathogen interactions, and the development of new diagnostic tools for pathogen monitoring.

由小麦叶锈菌（Puccinia triticina，简称Pt）引起的小麦叶锈病是小麦最具破坏性的病害之一，几乎在全球所有小麦种植区均对生产造成严重影响。尽管该病害的经济重要性突出，但目前针对Pt的基因组学资源仍极为有限。本研究采用长读长测序（long-read sequencing，LRS）技术结合FALCON与FALCON-Unzip（v4.1.0）分析流程，完成了首个基于LRS的Pt从头基因组组装。本研究利用平均读长11.6 kb、平均覆盖度150倍的22.4 Gb测序数据，对Pt104[菌株104-2,3,(6),(7),11；分离株S423]进行了基因组组装，该分离株被认为是澳大利亚Pt无性繁殖谱系的奠基性分离株。Pt104基因组包含162个重叠群，总长度为140.5 Mb，N50值为2 Mb；关联的单倍型重叠群可为91%的基因组提供单倍型信息。这是目前已发布的最高质量的Pt基因组组装结果：与此前公开的Pt race1组装相比，重叠群数量减少91倍，N50值提升4倍。本研究结合多维度证据构建了基因注释流程，包括基于RNA测序（RNA-Seq）得到的转录组组装结果、已报道的表达序列标签以及Pt race1的蛋白序列，最终为Pt104基因组预测得到29043个编码基因。基于信号肽存在、无跨膜结构域且无线粒体靶向序列的筛选标准，共鉴定出2178个分泌蛋白（secreted proteins，SPs）。研究团队对Pt104及另外6个针对小麦叶锈病抗性基因Lr26、Lr2a与Lr3ka表现出差异化毒力的菌株进行了全基因组测序（采用Illumina双端测序技术）。为鉴定对应无毒基因AvrLr26、AvrLr2a与AvrLr3ka的候选基因，本研究首先将各菌株的测序数据比对至Pt104参考基因组以获取菌株间的遗传变异信息；随后将预测的分泌蛋白基因中的菌株间变异与毒力表型进行关联分析，分别鉴定得到38、31和37个AvrLr26、AvrLr2a与AvrLr3ka的候选基因。上述候选基因的成功鉴定，为后续分离上述无毒基因、解析宿主与病原体互作的分子机制以及开发新型病原菌监测诊断工具奠定了坚实的研究基础。