High genome heterozygosity and endemic genetic recombination in the wheat stripe rust fungus - PubMed (original) (raw)

Lili Huang, Jinqun Huang, Xiaojie Wang, Xianming Chen, Jie Zhao, Jun Guo, Hua Zhuang, Chuangzhao Qiu, Jie Liu, Huiquan Liu, Xueling Huang, Guoliang Pei, Gangming Zhan, Chunlei Tang, Yulin Cheng, Minjie Liu, Jinshan Zhang, Zhongtao Zhao, Shijie Zhang, Qingmei Han, Dejun Han, Hongchang Zhang, Jing Zhao, Xiaoning Gao, Jianfeng Wang, Peixiang Ni, Wei Dong, Linfeng Yang, Huanming Yang, Jin-Rong Xu, Gengyun Zhang, Zhensheng Kang

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High genome heterozygosity and endemic genetic recombination in the wheat stripe rust fungus

Wenming Zheng et al. Nat Commun. 2013.

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Abstract

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat. Here we report a 110-Mb draft sequence of Pst isolate CY32, obtained using a 'fosmid-to-fosmid' strategy, to better understand its race evolution and pathogenesis. The Pst genome is highly heterozygous and contains 25,288 protein-coding genes. Compared with non-obligate fungal pathogens, Pst has a more diverse gene composition and more genes encoding secreted proteins. Re-sequencing analysis indicates significant genetic variation among six isolates collected from different continents. Approximately 35% of SNPs are in the coding sequence regions, and half of them are non-synonymous. High genetic diversity in Pst suggests that sexual reproduction has an important role in the origin of different regional races. Our results show the effectiveness of the 'fosmid-to-fosmid' strategy for sequencing dikaryotic genomes and the feasibility of genome analysis to understand race evolution in Pst and other obligate pathogens.

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Figures

Figure 1

Figure 1. Life cycle of Puccinia striiformis Westend f. sp. tritici.

In most of the wheat-growing regions worldwide, urediniopores are the only inoculum for the initial and recurrent infection of wheat plants, which is known as the mini cycle of Pst. Although the alternate host was identified recently, the role of sexual reproduction in genetic variation and race evolution of Pst is largely unknown.

Figure 2

Figure 2. The K-mer distribution and contig frequency analysis.

(a) Seventeen k-mer depth distribution of whole-genome Illumina reads. Two peaks at 16 and 30 were identified. (b) Two peaks of Cvg 7 and 17 were identified in the distribution of contig length and depth. These results indicated that the two haploid nuclei of Pst urediniospore are highly heterozygous. The haploid genome size can be estimated using a modified Lander–Waterman algorithm based on K-mer analysis (Supplementary Notes 1 and 2). K-mer, a unique sequence of k nucleotides long; Cvg, coverage.

Figure 3

Figure 3. Evaluation of assembly integrity.

Scaffold 475 of the _Pst_-CY32 assembly has only one PE supported gap, and it matches to one region of fosmid clone_Txjkx that was fully sequenced by Sanger sequencing. The same region consists of 11 contigs in the _Pst_-130 assembly generated solely by WGS Illumina sequencing. Therefore, the fosmid-to-fosmid strategy greatly improved assembly integrity. High PE (paired end) support density indicates high assembly confidence. PE supported gaps are regions that may have SVs derived from genome heterozygosity. Green, yellow and orange curves show paired-end supports from sequences of 6-, 2- and <1-kb insert libraries, respectively.

Figure 4

Figure 4. Reciprocal Blast analysis of Pst coding sequences against Pgt and Mlp.

Venn diagram showing reciprocal Blast search results for orthologous coding sequences of Pst, Pgt and Mlp (_e_-value of 1e−7). For genes with multiple alternative transcripts, the transcript with the best alignment was selected.

Figure 5

Figure 5. Heterozygous regions in the Pst genome.

Heterozygous regions were identified by aligning the sequence of Txjex, a fosmid fully sequenced by Sanger sequencing with c19720 and c18230, two highly similar scaffolds of the _Pst_-CY32 assembly generated with Illumina sequences from fosmid pools 7PB_Index52_Index26/20PB_Index47_Index8 and 14PB_Index44_Index29_Index6_Index35, respectively. SNPs between the reference _Pst_-CY32 genome and re-sequenced strains Hu09-2, _Pst_-78, CY23, PK-CDRD or 104E137A- are marked with blue vertical lines. SNV, heterozygous SNV in the dikaryotic _Pst_-CY32 genome.

Figure 6

Figure 6. Functional categories and comparative analysis of predicted Pst genes.

(a) Functional categories and distribution of predicted Pst genes based on the PAMGO database. Among the 2,067 Pst genes categorized with the PAMGO database, major groups with 5 genes or more are presented. The most abundant functional groups belong to metabolic process, transport and DNA recombination. (b) Venn diagram showing orthologous gene clusters among the Pst, Pgt and Mlp genomes. For genes with multiple alternative transcripts, the transcript with the best alignment was selected. Orthologous gene families were merged into multiple species orthologous groups by MultiParanoid. (c) The result showed the percentage of _Pst_-specific genes in the total number of genes belonging to each gene category.

Figure 7

Figure 7. Assays for three putative Pst SP genes specifically upregulated during infection of Berberis leaves.

Pst genes striiformis_9106, 9305, and 1374 are putative SP genes that are specifically upregulated at the pycnial stage in the infected Berberis leaves. RNA samples were isolated from urediniospores (US), germ tubes (GT), infected wheat leaves at 24 h post inoculation (h.p.i.) or 120 h.p.i. and infected berberis (Berberis shensiana Ahrendt) leaves (BE) at 11 days post inoculation (11 d.p.i.), respectively. The relative expression levels were calculated by the comparative Ct method with the elongation factor-1 gene of Pst as the internal standard. Means and s.e. were calculated from three replicates. Error bars represent the s.d. of the mean.

Figure 8

Figure 8. Relationship among seven Puccinia striiformis f. sp. tritici isolates.

(a) Heat map and race relationship of 7 Pst isolates according to their virulence on 17 differential cultivars. The susceptible and resistant types were recorded as 1 and 0, respectively. Heatmap.2 was used for clustering. (b) Phylogenic tree of 7 Pst isolates constructed with their cSNPs using Mega5-nj. The origin and isolation date of each isolate were marked.

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