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Genetic variation of single nucleotide polymorphisms identified at the mating type locus correlates with form-specific disease phenotype in the barley net blotch fungus Pyrenophora teres

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Abstract

Mating-type (MAT) locus-specific single nucleotide polymorphisms (SNPs) have been shown to be sufficient for conventional PCR-based differentiation of Pyrenophora teres f. teres (Ptt) and P. teres f. maculata (Ptm), the cause of the net and spot form, respectively, of barley net blotch. Here, we report the cloning and characterization of the MAT locus from 10 California isolates that cause atypical blotch symptoms on barley. Analysis of the full-length nucleotide sequences of one MAT1-1 (1,993 bp) and nine MAT1-2 (2,149 or 2,161 bp) idiomorphs revealed high (98–99 %) similarity to those of Ptt isolates. However, distinct SNP patterns were identified in the newly cloned MAT idiomorphs. Two new MAT1-2-specific SNPs were found to be conserved in one Australia and eight California isolates that all cause similar atypical blotch symptoms. Phylogenetic analysis indicated that all 10 California isolates form a separate branch (or clade) within the Ptt group, except for one that appears to be ancestral to both Ptt and Ptm. PCR primers designed based on the identified SNP patterns were used successfully to differentiate each atypical isolate from highly virulent forms. This study extends our previous work and, taken together, the results demonstrate that the genetic variation at the MAT locus correlates with variation in the form-specific disease phenotype, and that MAT-specific SNPs can serve as reliable and convenient markers for subspecies level differentiation in P. teres, an economically important plant-pathogenic ascomycete (http://en.wikipedia.org/wiki/Single_nucleotide_polymorphism).

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Acknowledgments

We thank Kelsey Dunnell and Danielle Holmes for technical assistance. This study was supported by USDA-ARS CRIS projects 5442-21000-033-00D and 5442-22000-043-00D and the NIFA-AFRI Coordinated Agricultural Project (CAP) Competitive Grant no. 2011-68002-30029.

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  1. USDA-ARS, Cereal Crops Research Unit, Northern Crop Science Laboratory, Fargo, ND, 58102-2765, USA

    Shunwen Lu, Michael C. Edwards & Timothy L. Friesen

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  1. Shunwen Lu
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  2. Michael C. Edwards
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  3. Timothy L. Friesen
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Correspondence to Shunwen Lu.

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Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Nucleotide sequence data reported are available in the GenBank databases under the accession numbers JN980080 (for MAT1-1 idiomorph of isolate CAWB05-Pt-5), JN980081 to JN980088 (for MAT1-2 idiomorphs of CAWB1 to 5, CAWB05-Pt-2 to Pt-4, respectively) and JN980089 (for MAT1-2 idiomorph of CAWB05-Pt-1).

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Supplemental Figure 1

Nucleotide sequence alignments of internal transcribed spacer (ITS) regions of the 10 California isolates identified in this study and representative isolates of P. teres f. teres (Ptt), P. teres f. maculata (Ptm) and P. graminea (Pg) reported in previous studies (Andrie et al. 2008; Liu and Friesen 2010). The ITS regions of the 10 California isolates that include 18S ribosomal RNA gene (partial sequence), internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, and 28S ribosomal RNA gene (partial sequence) were PCR-amplified with primers 5′-TCCGTAGGTGAACCTGCGG-3′ and 5′-TCCTCCGCTTATTGATATGC-3′. Shown here are the sequences corresponding to nucleotide positions 7 to 476 of GenBank accession number GU014819 (Ptt isolate 0-1, Liu and Friesen 2010). Note that only one P. graminea-specific single nucleotide polymorphism (SNP) site was identified (triangle). The variations found in the two short stretches of T-rich sequences (asterisks) are not form- or species-specific and are likely due to PCR or sequencing errors. (DOCX 662 kb)

Supplemental Figure 2

Phylogenetic relationships of the glyceraldehyde-3-phosphate dehydrogenase-like (GPD) genes of P. teres isolates. A 570-bp genomic sequence of the GPD gene was PCR-amplified from the 10 California isolates, the Australia isolate SNB172i, and 16 other P. teres f. teres (Ptt) and nine P. teres f. maculata (Ptm) isolates (Lu et al. 2010). PCR primers (5′-CGTATCGTCTTCCGCAAC -3′ and 5′-TTGGAGAGCACCTCAATGT -3′) were designed to GenBank accession number EF513236 (Ptt isolate ND, Andrie et al. 2008). The amplified sequences were aligned using the CLUSTALX program (Thompson et al. 1997) and the neighbor joining tree was constructed with the PHYLIP 3.61 package (http://evolution.genetics.washington.edu/phylip.html; Felsenstein 1989) and drawn using TreeView software (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). Bootstrapping values (>50 %, from 1,000 replicates) are indicated on the branches. A part of the sequence alignment was given below to show the identified polymorphic sites (triangles). Nucleotide sequences of the PCR-amplified GPD genes have been deposited into the GenBank database under accession numbers JQ837841 to JQ837876. (DOCX 198 kb)

Supplemental Figure 3

Nucleotide sequence alignments of MAT1-2 idiomorphs of the nine California isolates and other P. teres f. teres isolates identified from previous studies. Gray bars on the top indicate the highly conserved sequences flanking the MAT idiomorphs. Triangles indicate the positions of conserved single nucleotide polymorphisms (SNPs) with the corresponding nucleotide and the positions indicated at the top. Black triangles indicate the six SNPs used to differentiate typical isolates of P. teres f. teres from P. teres f. maculata. White triangles indicate the two newly identified SNPs conserved among the eight California isolates and the Australia isolate SNB172i (brackets). Gray triangle indicates a less conserved SNP which is found in six California isolates (CAWB3 to 5 and CAWB05-Pt-2 to Pt-4). Asterisk indicates the positions of a 12-bp deletion in CAWB05-Pt-1. Start and stop codons of the MAT1-2-1 open reading frames are underlined. Intron sequences are boxed. GenBank accession numbers: CAWB1 to 5, JN980081 to JN980085; CAWB05-Pt-2 to Pt-4, JN980086 to JN980088; CAWB05-Pt-1, JN980089; 0-1, AY950585 (Ellwood et al. 2010); BrPteres, HM121999; Jpt0101, HM121998; LDNH04Pt1, HM121998; NB077, HM122000; SNB172i, HM122001 (Lu et al. 2010). (DOCX 1.08 kb)

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Lu, S., Edwards, M.C. & Friesen, T.L. Genetic variation of single nucleotide polymorphisms identified at the mating type locus correlates with form-specific disease phenotype in the barley net blotch fungus Pyrenophora teres . Eur J Plant Pathol 135, 49–65 (2013). https://doi.org/10.1007/s10658-012-0064-8

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