Comparative genomics approach to build a genome-wide database of high-quality, informative microsatellite markers: application on Phytophthora sojae, a soybean pathogen

Guohong Cai,Tomara J Fleury,Ning Zhang

doi:10.1038/s41598-019-44411-z

Abstract

Microsatellites are a tract of repetitive, short DNA motifs (usually 1 to 6 bp) abundant in eukaryotic genomes. They are robust molecular markers in many areas of studies. Development of microsatellite markers usually involves three steps: (1) obtaining microsatellite-containing sequences, (2) primer design, and (3) screening microsatellite loci for polymorphism. The first and third steps require considerable resources. Next generation sequencing technologies have greatly alleviated the constraint of the first step. In this study, we leveraged the availability of genome assemblies of multiple individuals in many species and designed a comparative genomics approach to bioinformatically identify polymorphic loci. Our approach can eliminate or greatly reduce the need of experimental screening for polymorphism and ensure that the flanking regions do not have length difference that would confound interpretation of genotyping results using microsatellite markers. We applied this approach on Phytophthora sojae, a soybean pathogen, and identified 157 high-quality, informative microsatellite markers in this oomycete. Experimental validation of 20 loci supported bioinformatics predictions. Our approach can be readily applied to other organisms of which the genomes of multiple individuals have been sequenced.

Highlights

Microsatellites, known as simple sequence repeats (SSRs) or short tandem repeats (STRs), are a tract of repetitive, short DNA motifs
The development of microsatellite markers follows these steps: (1) genomic DNA is fragmented, microsatellite-containing fragments can be enriched, and the enriched microsatellite-containing DNA fragments are cloned and sequenced; (2) primer pairs are designed from the flanking regions; and (3) the primer pairs are screened using a small subset of individuals to identify polymorphic microsatellite loci — those having multiple alleles in the tested individuals
In Anisogramma anomala, a fungal pathogen of hazelnut, 23 loci were found to be polymorphic among 236 loci screened[9]; in Fusarium virguliforme, a fungal pathogen on soybean, 12 polymorphic loci were identified in a screening of 92 microsatellite loci with very high repeat numbers[8]; and in Phytophthora sojae, an oomycete pathogen on soybean, 21 out of 158 microsatellite loci with extremely high repeat numbers were found to be polymorphic among 33 isolates[12]

Summary

Introduction

Microsatellites, known as simple sequence repeats (SSRs) or short tandem repeats (STRs), are a tract of repetitive, short DNA motifs (usually 1 to 6 bp). It is usually assumed that the flanking regions of microsatellite loci are conserved and the length difference in PCR amplicons is due to varying repeat numbers of the microsatellite motifs.

Results

Conclusion