Population Genetic Analyses of Botrytis cinerea Isolates From Michigan Vineyards Using a High-Throughput Marker System Approach.

Rachel P Naegele,Jeff Delong,Safa A Alzohairy,Timothy D Miles,Noor Abdelsamad,Seiya Saito

doi:10.3389/fmicb.2021.660874

Abstract

As sequencing costs continue to decrease, new tools are being developed for assessing pathogen diversity and population structure. Traditional marker types, such as microsatellites, are often more cost effective than single-nucleotide polymorphism (SNP) panels when working with small numbers of individuals, but may not allow for fine scale evaluation of low or moderate structure in populations. Botrytis cinerea is a necrotrophic plant pathogen with high genetic variability that can infect more than 200 plant species worldwide. A panel of 52 amplicons were sequenced for 82 isolates collected from four Michigan vineyards representing 2 years of collection and varying fungicide resistance. A panel of nine microsatellite markers previously described was also tested across 74 isolates from the same population. A microsatellite and SNP marker analysis of B. cinerea populations was performed to assess the genetic diversity and population structure of Michigan vineyards, and the results from both marker types were compared. Both methods were able to detect population structure associated with resistance to the individual fungicides thiabendazole and boscalid, and multiple fungicide resistance (MFR). Microsatellites were also able to differentiate population structure associated with another fungicide, fluopyram, while SNPs were able to additionally differentiate structure based on year. For both methods, AMOVA results were similar, with microsatellite results explaining a smaller portion of the variation compared with the SNP results. The SNP-based markers presented here were able to successfully differentiate population structure similar to microsatellite results. These SNP markers represent new tools to discriminate B. cinerea isolates within closely related populations using multiple targeted sequences.

Highlights

Botrytis cinerea is a necrotrophic pathogenic fungus that infects hundreds of plant species (Alfonso et al, 2000; Ma and Michailides, 2005; Williamson et al, 2007; Fillinger, 2016) including economically important crops such as fruits, ornamentals, and vegetables (Elad et al, 2004; Leroux, 2007)
Our objectives were to study the population structure of MI isolates of B. cinerea related to year, location, fungicide resistance, and to compare the use of microsatellite and amplicon-based sequencing single-nucleotide polymorphism (SNP) strategies to quantify genetic diversity and population structure
SNP distribution across the genome ranged from three SNPs on chromosome 11 to 101 SNPs on chromosome 1 with a total of 496 SNPs detected post filtering used for subsequent analyses (Table 1)

Summary

Introduction

Botrytis cinerea is a necrotrophic pathogenic fungus that infects hundreds of plant species (Alfonso et al, 2000; Ma and Michailides, 2005; Williamson et al, 2007; Fillinger, 2016) including economically important crops such as fruits, ornamentals, and vegetables (Elad et al, 2004; Leroux, 2007). B. cinerea infects all grapevine plant parts, though fruit rot, known as botrytis bunch rot (preharvest) and gray mold (postharvest), is the most common (Gabler et al, 2003; Elmer and Michailides, 2007; Saito et al, 2019). Fungicide resistance has been reported in different countries worldwide (Leroch et al, 2011; Angelini et al, 2014; Panebianco et al, 2015; Yin et al, 2015) and in the US including Michigan (Alzohairy et al, 2020) and California (Saito et al, 2019; Avenot et al, 2020; Delong et al, 2020). Fungicide resistance frequencies have been shown to differ between years, crop hosts, and locations (Fernández-Ortuño et al, 2015; Delong et al, 2020; Kozhar et al, 2020)

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