The stripe rust pathogen, Puccinia striiformis f. sp. tritici (Pst), threats world wheat production. Resistance to Pst is often overcome by pathogen virulence changes, but the mechanisms of variation are not clearly understood. To determine the role of mutation in Pst virulence changes, in previous studies 30 mutant isolates were developed from a least virulent isolate using ethyl methanesulfonate (EMS) mutagenesis and phenotyped for virulence changes. The progenitor isolate was sequenced, assembled and annotated for establishing a high-quality reference genome. In the present study, the 30 mutant isolates were sequenced and compared to the wide-type isolate to determine the genomic variation and identify candidates for avirulence (Avr) genes. The sequence reads of the 30 mutant isolates were mapped to the wild-type reference genome to identify genomic changes. After selecting EMS preferred mutations, 264,630 and 118,913 single nucleotide polymorphism (SNP) sites and 89,078 and 72,513 Indels (Insertion/deletion) were detected among the 30 mutant isolates compared to the primary scaffolds and haplotigs of the wild-type isolate, respectively. Deleterious variants including SNPs and Indels occurred in 1866 genes. Genome wide association analysis identified 754 genes associated with avirulence phenotypes. A total of 62 genes were found significantly associated to 16 avirulence genes after selection through six criteria for putative effectors and degree of association, including 48 genes encoding secreted proteins (SPs) and 14 non-SP genes but with high levels of association (P ≤ 0.001) to avirulence phenotypes. Eight of the SP genes were identified as avirulence-associated effectors with high-confidence as they met five or six criteria used to determine effectors. Genome sequence comparison of the mutant isolates with the progenitor isolate unraveled a large number of mutation sites along the genome and identified high-confidence effector genes as candidates for avirulence genes in Pst. Since the avirulence gene candidates were identified from associated SNPs and Indels caused by artificial mutagenesis, these avirulence gene candidates are valuable resources for elucidating the mechanisms of the pathogen pathogenicity, and will be studied to determine their functions in the interactions between the wheat host and the Pst pathogen.

中文翻译：

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小麦条锈菌的全基因组测序f。sp。小麦突变体分离株鉴定无毒力基因候选物

条锈病病原体，Puccinia striiformis f。sp。Tritici（Pst），威胁世界小麦产量。对Pst的抗药性通常可以通过病原体毒力的变化来克服，但其变化机理尚不清楚。为了确定突变在Pst毒力变化中的作用，在先前的研究中，使用甲磺酸乙酯（EMS）诱变从最低毒性的分离物中开发了30个突变株，并对毒力变化进行了表型分析。对祖先分离物进行测序，组装和注释，以建立高质量的参考基因组。在本研究中，对30个突变株进行了测序，并与宽型分离株进行比较，以确定基因组变异并确定无毒力（Avr）基因的候选对象。将30个突变体分离株的序列读数定位到野生型参考基因组，以鉴定基因组变化。在选择EMS首选突变后，与野生型分离株的主要支架和单倍体相比，在30个突变株中分别检测到264,630和118,913个单核苷酸多态性（SNP）位点以及89,078和72,513个Indels（插入/缺失）。包括SNP和Indel在内的有害变体出现在1866个基因中。全基因组关联分析确定了754个与无毒性表型相关的基因。通过六种推定效应子和关联度的标准选择后，发现总共有62个基因与16个无毒力基因显着相关，包括48个编码分泌蛋白（SPs）的基因和14个非SP基因，但具有很高的关联度（P≤ 0。001）为无毒力表型。SP基因中有8个符合5或6个确定效应子的标准，因此被高度确定为与毒力相关的效应子。突变株与祖先分离株的基因组序列比较揭示了沿基因组的大量突变位点，并确定了高信度效应基因作为Pst中无毒力基因的候选者。由于无毒力基因候选物是通过人工诱变从相关的SNP和Indels中鉴定出来的，因此这些无毒力基因候选物是阐明病原体致病性机制的宝贵资源，并将对其进行研究以确定其在小麦宿主与小麦之间的相互作用中的功能。 Pst病原体。SP基因中有8个符合5或6个确定效应子的标准，因此被高度确定为与毒力相关的效应子。突变株与祖先分离株的基因组序列比较揭示了沿基因组的大量突变位点，并确定了高信度效应基因作为Pst中无毒力基因的候选者。由于无毒力基因候选物是通过人工诱变从相关的SNP和Indels中鉴定出来的，因此这些无毒力基因候选物是阐明病原体致病性机制的宝贵资源，并将对其进行研究以确定其在小麦宿主与小麦之间的相互作用中的功能。 Pst病原体。SP基因中有8个符合5或6个确定效应子的标准，因此被高度确定为与毒力相关的效应子。突变株与祖先分离株的基因组序列比较揭示了沿基因组的大量突变位点，并确定了高信度效应基因作为Pst中无毒力基因的候选者。由于无毒力基因候选物是通过人工诱变从相关的SNP和Indels中鉴定出来的，因此这些无毒力基因候选物是阐明病原体致病性机制的宝贵资源，并将对其进行研究以确定其在小麦宿主与小麦之间的相互作用中的功能。 Pst病原体。突变株与祖先分离株的基因组序列比较揭示了沿基因组的大量突变位点，并确定了高信度效应基因作为Pst中无毒力基因的候选者。由于无毒力基因候选物是通过人工诱变从相关的SNP和Indels中鉴定出来的，因此这些无毒力基因候选物是阐明病原体致病性机制的宝贵资源，并将对其进行研究以确定其在小麦宿主与小麦之间的相互作用中的功能。 Pst病原体。突变株与祖先分离株的基因组序列比较揭示了沿基因组的大量突变位点，并确定了高信度效应基因作为Pst中无毒力基因的候选者。由于无毒力基因候选物是通过人工诱变从相关的SNP和Indels中鉴定出来的，因此这些无毒力基因候选物是阐明病原体致病性机制的宝贵资源，并将对其进行研究以确定其在小麦宿主与小麦之间的相互作用中的功能。 Pst病原体。