Allelic variation at a hypervariable compound microsatellite locus in the ascomycete Ascochyta rabiei

Abstract

The genome of the fungal chickpea pathogen Ascochyta rabiei was screened for polymorphisms by microsatellite-primed PCR. While ethidium-bromide staining of electrophoretically separated amplification products showed only limited polymorphism among 24 Tunisian A. rabiei isolates, Southern hybridization of purified PCR fragments to restriction digests of fungal DNA revealed polymorphic DNA fingerprints. One particular probe that gave rise to a hypervariable single-locus hybridization signal was cloned from the Syrian isolate AA6 and sequenced. It contained a large compound microsatellite harbouring the penta- and decameric repeat units (CATTT)n, (CATTA)n, (CATATC-ATTT)n and (TATTT)n. We call this locus ArMS1 (Ascochyta rabiei microsatellite 1). Unique flanking sequences were used to design primer pairs for locus-specific microsatellite amplification and direct sequencing of additional ArMS1 alleles from Tunisian and Pakistani isolates. A high level of sequence variation was observed, suggesting that multiple mutational mechanisms have contribute to polymorphism. Hybridization and PCR analyses were performed on the parents and 62 monoascosporic F1 progeny derived from a cross between two different mating types of the fungus. Progeny alleles could be traced back to the parents, with one notable exception, where a longer than expected fragment was observed. Direct sequencing of this new length allele revealed an alteration in the copy number of the TATTT repeat [(TATTT)53 to (TATTT)65], while the remainder of the sequence was unchanged.

Simple Sequence Repeat Markers Distinguish among Morphotypes of Sphaeropsis sapinea

“…ISSR-PCR of S. sapinea isolate CMW 5977 (belonging to the A morphotype) was conducted with seven primers, 5ЈDDB (CCA) 5 , 5ЈDHB(CGA) 5 , 5ЈYHY(GT) 5 G, 5ЈHVH(GTG) 5 , 5ЈNDB(CA) 7 C, 5ЈNDV(CT) 8 , and 5ЈHBDB(GACA) 4 , as previously described (34) except that an annealing temperature of 49°C was used for all of the primers. The amplification products from each ISSR primer were purified with the High Pure PCR product purification kit (Roche Diagnostics, Mannheim, Germany).…”

“…During the last decade, SSR markers have been extensively used in population studies of many plants and animals (31), although to date there have been few studies of fungi (16). Fungal studies have generally used only a few markers, predominantly for genotyping (7,8,14,17). Recently, however, Epichloë endophytes have been identified in planta using 11 polymorphic microsatellite markers (16).…”

Genetic diversity analysis of a world-wide collection of Ascochyta rabiei isolates using sequence tagged microsatellite markers

“…Polymorphism detected among fungal isolates collected from different chickpea growing localities of Syria and Tunisia by microsatellite primers in microsatellite primed PCR (MP-PCR) was rather low, and most of the isolates could not be differentiated from each other (Geistlinger et al 1997b). However, a PCR amplification fragment obtained in A. rabiei with a combination of primers (GTTTGG) 3 and (GGAT) 4 revealed the presence of a compound microsatellile (ARMS1).…”

“…However, a PCR amplification fragment obtained in A. rabiei with a combination of primers (GTTTGG) 3 and (GGAT) 4 revealed the presence of a compound microsatellile (ARMS1). The locus composed of arrays of four continuous penta-and decanucleotide motifs surrounded by extremely AT-rich DNA combined typical features of microsatellites (compound repeats of pentaand decameric motifs), minisatellites (total length of 200-500 bp) and satellites (occurrence at a few genomic loci only) (Geistlinger et al 1997b). The ARMS1 locus was reported to have a high mutation rate and direct sequencing revealed one newly mutated allele in the progeny of a cross (Geistlinger et al 1997b).…”

Population Biology of Fungal Plant Pathogens

“…Originally described by Hamada et al ( 68 ) , the method was rapidly established in various eukaryotic systems as it exhibited great variability useful for genotyping at the subspecies level (69)(70)(71)(72) . Subsequently, such SSR were identifi ed in fungi (73)(74)(75) and oomycetes ( 76,77 ) . Use of SSRs for interspecifi c analyses is commonly considered to be less effective than for subspecies population analyses ( 78,79 ) .…”