Development of microsatellite markers for the characterisation of Phaeoisariopsis griseola (bean angular leaf spot agent) populations in Central America

Abstract

Although several researches revealed an important diversity within Phaeoisariopsis griseola, the bean angular leaf spot (ALS) agent, no sexual recombination was already detected for this fungus. That apparent contradiction gave rise to the interest to develop codominant markers in order of a more precise analysis of the pathogen populations. Microsatellites were expected to allow characterising P. griseola populations in terms of allele frequencies. A genomic library was constructed by ligating DNA fragments, previously prepared by enzymatic restriction of total DNA of two pathogen strains, into a pZERO plasmid. After transformation of TOP 10F' strains of E. coli with the recombinant plasmid, a total of 448 colonies were selected for zeocin resistance. The probe mixture [(GT)15, (GA)15, (GATA)8, (GTG)10], previously labelled with 32P, was used to screen the genomic library for the presence of microsatellite sequences. The vector DNA was then extracted from the positive colonies and sequenced. Based on the sequences, a first group of 10 microsatellite loci was identified and the corresponding primers designed. A size analysis using an ALF express system exhibited 3 polymophic microsatellites among a total of 4 loci already considered. The identification of other polymorphic microsatellites is continuing before a large scale analysis of our pathogen collection by using this new molecular tool developed for P. griseola.

Keywords

• angular leaf spot
• phaeoisariopsis griseola
• phaseolus vulgaris
• microsatellite