Genomic DNA blot hybridization showing <i>P. sojae</i> strain-specific deletions of <i>Avr1a</i> and <i>Avr1c</i>.

<p>Selected strains of <i>P. sojae</i> genomic DNA were digested with <i>Pml</i>I and separated by electrophoresis prior to blotting and hybridization. Virulence phenotype of the <i>P. sojae</i> strains on <i>Rps</i>1c or <i>Rps</i>1a plants is shown as virulent (V) or avirulent (A). The positions and sizes of the <i>Pml</i>I segments of <i>Avr1a</i>, <i>Avr1c</i>, and <i>Avh72</i> genes are indicated on the right. The sizes of DNA markers are shown on the left; kb, kilo base pair. The <i>Avr1c</i> gene is deleted from parental strain P7076.</p

Genome Re-Sequencing and Functional Analysis Places the <i>Phytophthora sojae</i> Avirulence Genes <i>Avr1c</i> and <i>Avr1a</i> in a Tandem Repeat at a Single Locus

<div><p>The aim of this work was to map and identify the <i>Phytophthora sojae Avr1c</i> gene. Progeny from a cross of <i>P. sojae</i> strains ACR10×P7076 were tested for virulence on plants carrying <i>Rps</i>1c. Results indicate that avirulence segregates as a dominant trait. We mapped the <i>Avr1c</i> locus by performing whole genome re-sequencing of composite libraries created from pooled samples. Sequence reads from avirulent (Pool1) and virulent (Pool2) samples were aligned to the reference genome and single nucleotide polymorphisms (SNP) were identified for each pool. High quality SNPs were filtered to select for positions where SNP frequency was close to expected values for each pool. Only three SNP positions fit all requirements, and these occurred in close proximity. Additional DNA markers were developed and scored in the F₂ progeny, producing a fine genetic map that places <i>Avr1c</i> within the <i>Avr1a</i> gene cluster. Transient expression of <i>Avr1c</i> or <i>Avr1a</i> triggers cell death on <i>Rps</i>1c plants, but <i>Avr1c</i> does not trigger cell death on <i>Rps</i>1a plants. Sequence comparisons show that the RXLR effector genes <i>Avr1c</i> and <i>Avr1a</i> are closely related paralogs. Gain of virulence on <i>Rps</i>1c in <i>P. sojae</i> strain P7076 is achieved by gene deletion, but in most other strains this is accomplished by gene silencing. This work provides practical tools for crop breeding and diagnostics, as the <i>Rps</i>1c gene is widely deployed in commercial soybean cultivars.</p></div

Identification of SNPs linked to <i>Avr1c</i> by bulked segregant analysis and deep sequencing.

<p><b>A,</b> The procedure for discovery of candidate SNPs linked to <i>Avr1c</i> is shown. Selected F₂ progeny from a cross of <i>P. sojae</i> strains ACR10×P7076 were pooled according to their virulence phenotype. The avirulent (A) Pool1 and virulent (V) Pool2 composite DNA samples were deeply sequenced. Sequence reads were aligned against the reference genome, and SNPs were identified and filtered based upon quality scores. High quality SNPs were further filtered according to the predicted SNP frequencies for Pool1 and Pool2. After processing, only three SNPs passed all requirements. <b>B,</b> Genome location of three candidate SNPs. These three candidate SNPs occur in close physical proximity in the reference genome assembly. All three sites fall within a 92 kb segment on Scaffold_7 (V5.0). Reference (Ref) allele and alternate (Alt) alleles for three SNPs are shown. The SNP frequencies (Freq) in each pool are also shown.</p

Genetic and physical mapping of <i>Avr1c</i> region.

<p><b>A,</b> Physical map of <i>Avr1c</i> region. The position of the three identified SNP markers, <i>Avr1a</i>, and 25 predicted <i>Avh</i> genes are shown. <b>B,</b> Genetic analysis of the <i>Avr1c</i> region. The position of DNA markers and their recombination frequency (RF%) with <i>Avr1c</i> in a segregating F₂ population (n = 28) is indicated; Mbp, mega base pair. <b>C,</b> Predicted arrangement of the <i>Avr1a</i> locus in <i>P. sojae</i> reference strain P6497.</p

Analysis of alleles of <i>Avr1c</i>, and transcripts of <i>Avr1c</i> and <i>Avr1a</i> in <i>P. sojae</i> strains.

<p>Shown at the top of the figure are the <i>Avr1c</i> alleles present in each of the selected <i>P. sojae</i> strains. Virulence phenotype of the <i>P. sojae</i> strains on <i>Rps</i>1c or <i>Rps</i>1a plants is shown as virulent (V) or avirulent (A). Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed using mRNA from mycelia cultures, and <i>Avr1a</i> and <i>Avr1c</i> specific primers, to test for transcripts of these two genes. Results from RT-PCR of the control gene <i>Actin</i> are also shown.</p

Whole genome re-sequencing of composite and strain specific <i>P. sojae</i> libraries.

1<p>Illumina HiSeq 2000, read length 100 bp.</p>2<p>Sequence reads with quality score >30.</p>3<p>Sequence reads that map to the 82 Mb <i>P. sojae</i> reference genome assembly.</p>4<p>Average depth of sequence read coverage.</p

Sequence read coverage of the <i>Avr1a/Avr1c</i> region from re-sequencing of <i>P. sojae</i> strains ACR10 and P7076.

<p>An assembly of the <i>Avr1a</i> region from reference strain P6497 was used to align sequence reads from parental strains ACR10 and P7076. The DNA segment corresponding to the <i>Avr1c</i> gene lacks sequence reads in parental strain P7076. The longest segment of identity between the <i>Avr1a</i> and <i>Avr1c</i> DNA sequences is 98 nucleotides, which is shorter than the read length of 100 nucleotides.</p

Transient expression of <i>Avr1c</i> and <i>Avr1a</i> triggers cell death in <i>Rps</i>1c soybean plants.

<p>Measurement of cell death in soybean leaves by co-bombardment and transient expression of a GUS reporter together with a test gene. Test genes that cause cell death reduce GUS expression and blue staining. <b>A,</b> Leaves of Williams (<i>rps</i>) and the isoline L75-3735 (<i>Rps</i>1c) tested with each of the three <i>Avr1c</i> alleles, and <i>Avr1a</i>. <b>B,</b> Leaves of Williams (<i>rps</i>) and the isoline L75-6141 (<i>Rps</i>1a) tested with each of the three <i>Avr1c</i> alleles. Control test gene in each experiment corresponds to a synthetic <i>Avr1a</i> sequence with a frame-shift mutation. Results show means and standard errors of three independent biological replicates, with a minimum of three leaves per treatment, per replicate.</p

Photographs of leaves of Williams (<i>rps</i>) and the isoline L75-3735 (<i>Rps1c</i>) tested with each of the three <i>Avr1c</i> alleles, and <i>Avr1a</i>.

<p>Results from a representative co-bombardment experiment are shown, after staining for GUS expression.</p

Amino acid sequence alignment of the predicted proteins for Avr1a and each of the three alleles of Avr1c.

<p>The residues are colored according to their physicochemical properties. Signal peptide, RXLR and dEER motifs, and WY-domain are shown, and polymorphic residues among the three Avr1c alleles are underlined. An asterisk (*) indicates positions which have a single, fully conserved residue; a colon (:) indicates conservation between groups of strongly similar properties; a period (.) indicates conservation between groups of weakly similar properties.</p