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Genetic characterization of Plasmopara halstedii populations in Argentina using simple sequence repeats (SSR) and effector-based markers Materials and Methods

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Abstract

a) Race determination of P. halstedii isolates Fig. 1. A= recovery of inoculum at field B= detail of leaf sporulation C= spore solution D= reinfection of differential set of sunflower inbred lines E= phenotyping and race determination. b) SSR analysis The isolates were analyzed by eight loci SSR. The number of alleles per locus (APL), the expected (HE) and observed heterozygosity (HO) were determined. SSR data was analyzed with regions (2), collection years (4) and races (5) as variation sources (AMOVA) and the population structure was defined (Bayesian method). • The races of the isolates were 330, 710, 711, 730 and 770. • Based on AMOVA, genetic variation was observed among regions (7%) and years (32%), but not between races. • Average number of alleles= 2.625; Average Observed Heterocigosity= 0; He Average expected heterocigosity= 0.369; Average Inbreeding coefficient F=1
Genetic characterization of Plasmopara halstedii populations in Argentina
using simple sequence repeats (SSR) and effector-based markers
Materials and Methods
a) Race determination of P. halstedii isolates
Fig. 1. A= recovery of inoculum at field B= detail of leaf
sporulation C= spore solution D= reinfection of differential set of
sunflower inbred lines E= phenotyping and race determination.
b) SSR analysis
The isolates were analyzed by eight loci SSR. The number of
alleles per locus (APL), the expected (HE) and observed
heterozygosity (HO) were determined. SSR data was analyzed
with regions (2), collection years (4) and races (5) as variation
sources (AMOVA) and the population structure was defined
(Bayesian method).
The races of the isolates were 330,710,711,730 and 770.
Based on AMOVA, genetic variation was observed among regions (7%) and years
(32%), but not between races.
Average number of alleles= 2.625; Average Observed Heterocigosity= 0; He
Average expected heterocigosity= 0.369; Average Inbreeding coefficient F=1
Introduction
Sunflower downy mildew, caused by the pathogen Plasmopara
halstedii (Farl.) Berl. et de Toni is an economically important
disease of sunflower. Since 2012, P. halstedii epiphytotics were
identified in several production regions of Argentina. However,
the genetic diversity of the pathogen in Argentina remains
largely unknown.
Results
Ana Laura Martínez 1, Antonio Garayalde 2, Macarena Petruccelli 3, Ignacio Erreguerena 4 , Facundo Quiroz 4, Alicia Carrera 5
almartinez@cerzos-conicet.gob.ar
Fig. 2. PCO analysis based on the SSR matrix of genetic distance. Shapes
indicate isolates collection years, and black or grey colors indicate
Northern and Central regions, respectively.
Fig. 3. Histogram of
Bayesian clustering. Each
individual is represented by
a bar proportional to the
inferred clusters. In the
upper part of the figure are
indicated the races of
isolates.
Year N Number of
alleles detected
Novel alleles
detected
Races determined
1991 1 8 --- 770
2013 13 13 5nd
2016 814 3710
2017 16 17 5 330, 710, 711, 730
2018 410 0 710, 770
Table 1. Preexistent and novel alleles in different years of isolates collection.
N= number of analyzed isolates . Nd= non determined.
42 isolates were collected in years 1991, and 2013 to 2018 in
fields located in Central (Buenos Aires) and Northern (Chaco y
Santa Fe) Argentina. The races of the isolates were determined
by pathogenicity assays (Figure 1).
c) Polymorphisms analysis on sequence data
Fig. 4. Polymorphisms in the nucleotide sequences of the effector CRN and RXLR. The races
of P. halstedii from Argentina (ARG) and France (FR) are shown in color circles
Conclusions
The race spectrum in Argentina is increasing in the last years. A novel race was detected for the first time (711).
The heterozygotes deficit found is coincident with the homothallic reproduction of P. halstedii.
The increased genetic variability observed in recent years seems to parallel with the emergence of novel races reducing
fungicides and/or R genes efficacy.
The examined sites from the effector sequences resulted homogeneous among the Argentinean races.
Race determinations and effector CRN/RXLR polymorphisms were concordant in the pathotypes shared by Argentina and
France, except for 330, that showed alternative RXLR haplotypes in each country.
The study based on two effector genes suggests that the races evolution depends on country specific factors.
DNA sequences of two effector genes type CRN and RXLR were obtained from two isolates per race and were analyzed along
with the polymorphisms reported by Gascuel et al, 2016 on 14 French races. The polymorphisms were analyzed by multiple
alignment (ClustalW).
Gascuel, Quentin, et al. Effector polymorphisms of the sunflower downy mildew pathogen Plasmopara halstedii and their use to identify pathotypes from field isolates. PLoS One 11.2 (2016).
1) Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET),
CERZOS, Bahía Blanca, Argentina; 2) Dto. de Matemática, Universidad
Nacional del Sur (UNS), Bahía Blanca; 3) FCA, Universidad de Mar del Plata,
Balcarce; 4) Instituto Nacional de Tecnología Agropecuaria (INTA); 5) Dto.de
Agronomía, UNS, Bahía Blanca.
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