Development and characterization of microsatellite markers for the oomyceta Aphanomyces euteiches

Abstract

Aphanomyces euteiches Drechsler is a serious pathogen of leguminous crops that causes devastating root rot of pea worldwide. Given that A. euteiches is a diploid organism, robust, codominant markers are needed for population genetics studies. We have developed and screened a microsatellite-enriched small-insert genomic library for identification of A. euteiches SSR containing sequences. Fourteen out of the 48 primer pairs designed to amplify SSR, produced unambiguous polymorphic products in our test population of 94 isolates. The number of alleles at each locus ranged from one to four. The identification of new markers would enhance the ability to evaluate the genetic structure of A. euteiches populations, and pathogen evolution.

Genetic and Pathogenicity Diversity of Aphanomyces euteiches Populations From Pea-Growing Regions in France

“…A low level of genetic diversity in A. euteiches populations was observed between the different locations in France, as shown by the moderate number of multilocus genotypes identified (13 different MLGs within 205 isolates) including a predominant MLG (MLG1) shared by 54% of the isolates studied. This result is consistent with previous studies conducted on French and American A. euteiches populations, using codominant or dominant markers (Malvick and Percich, 1998 ; Wicker, 2001 ; Grünwald and Hoheisel, 2006 ; Mieuzet et al, 2016 ; Le May et al, 2018 ). In this study, the similar level of differentiation observed within and among populations also confirmed the absence of a clear genetic structure, as previously reported (Malvick and Percich, 1998 ; Wicker et al, 2001 ; Grünwald and Hoheisel, 2006 ).…”

“…Twenty SSR primers, previously developed to amplify A. euteiches DNA (Mieuzet et al, 2016 ), were used in this study. The PCR reaction in simplex was carried out in 10 μL containing 1X Green GoTaq®Flexi Buffer, 2 mM MgCl2, 0.2 mM each dNTP (Promega, France), 1 μM each forward and reverse primer and 0.25 μM fluorescent-labeled M13 primer (FAM, Applied Biosystem), 1 U of GoTaq Flexi DNA polymerase (final concentration) (Promega, France) and 20 ng template DNA.…”

“…Amplifications were conducted on a S1000 Thermal Cycler (Bio-Rad). The cycling conditions for PCR amplification were as described in Mieuzet et al ( 2016 ). The amplified products were separated on 1.5% agarose (Lonza) gels, stained in 1X Tris-Borate EDTA (TBE) buffer containing 1X Sybr® Safe (Invitrogen) and visualized under UV light.…”

“…In France, a preliminary study of the genetic diversity using AFLP markers found unstructured populations among 56 pea A. euteiches isolates collected in infested fields (unpublished data). Recently, Mieuzet et al ( 2016 ) and Le May et al ( 2018 ) used SSR and Sequence Related Amplified Polymorphisms (SRAP) markers, respectively, to show the absence of genetic structure among French pea isolates sampled from research disease nurseries. However, in the same study, Le May also found significant genetic structure among American isolates sampled from four American nurseries.…”

Genetic diversity of the pea root pathogen Aphanomyces euteiches in Europe

“…Here, we confirm the pattern with an overall low level of genetic diversity found in previous studies of French and North American A . euteiches populations (Grünwald & Hoheisel, 2006; Le May et al, 2018; Malvick et al, 1998; Mieuzet et al, 2016; Quillévéré‐Hamard et al, 2018; Wicker et al, 2001). In the Grünwald and Hoheisel (2006) study, it was thought that a relatively higher diversity between populations was due to the limited spread of the soilborne pathogen.…”