ABSTRACT
BACKGROUND: Epicoccum nigrum Link (syn. E. purpurascens Ehrenb. ex Schlecht) is a saprophytic ascomycete distributed worldwide which colonizes a myriad of substrates. This fungus has been known as a biological control agent for plant pathogens and produces a variety of secondary metabolites with important biological activities as well as biotechnological application. E. nigrum produces darkly pigmented muriform conidia on short conidiophores on sporodochia and is a genotypically and phenotypically highly variable species. Since different isolates identified as E. nigrum have been evaluated as biological control agents and used for biocompound production, it is highly desirable that this species name refers to only one lineage. However, according to morphological and genetic variation, E. nigrum present two genotypes that may comprise more than one species. METHODOLOGY/PRINCIPAL FINDINGS: We report the application of combined molecular (ITS and ß-tubulin gene sequence analysis, PCR-RFLP and AFLP techniques), morphometric, physiological, genetic compatibility and recombination analysis to study the taxonomic relationships within an endophytic population that has been identified as E. nigrum. This combined analysis established two genotypes showing morphological, physiological and genetic divergence as well as genetic incompatibility characterized by colony inhibition, strongly indicating that these genotypes correspond to different species. Genotype 1 corresponds to E. nigrum while genotype 2 represents a new species, referred to in this study as Epicoccum sp. CONCLUSIONS/SIGNIFICANCE: This research contributes to the knowledge of the Epicoccum genus and asserts that the classification of E. nigrum as a single variable species should be reassessed. In fact, based on the polyphasic approach we suggest the occurrence of cryptic species within E. nigrum and also that many of the sequences deposited as E. nigrum in GenBank and culture collection of microbial strains should be reclassified, including the reference strain CBS 161.73 sequenced in this work. In addition, this study provides valuable tools for differentiation of Epicoccum species.
Subject(s)
Ascomycota/classification , Ascomycota/genetics , Genetic Variation , Amplified Fragment Length Polymorphism Analysis , Ascomycota/enzymology , Ascomycota/growth & development , Base Sequence , Bayes Theorem , Colony Count, Microbial , DNA Fingerprinting , DNA, Intergenic/genetics , Fungal Proteins/metabolism , Mutant Proteins/isolation & purification , Mutant Proteins/metabolism , Mycelium/metabolism , Phylogeny , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Recombination, Genetic/genetics , Species Specificity , Spores, Fungal/cytology , Tubulin/geneticsABSTRACT
The objective of this work was to analyze the effects of RAPD markers with skewed segregation on genetic linkage maps. Segregation data for 123 Citrus sinensis (L.) Osbeck cv. Pêra markers and 53 C. reticulata Blanco cv. Cravo markers in F1 progeny composed of 94 hybrids were used. Genetic linkage maps of the two varieties were constructed with non-skewed markers (p < 0.05 and p < 0.01) using the program MAPMAKER 3.0 and a pseudo-testcross strategy. The maps were compared to those constructed with all markers. Alterations in the genetic distances were observed based on the location of the skewed markers within the linkage groups. Generally, the skewed markers were located at the end of the linkage groups, sometimes forming entire linkage groups, without causing significant distance modifications. However, skewed markers located between non-skewed markers caused significant distance modifications and, in some cases, altered the order of the markers. Most of the skewed markers can be included in linkage maps, but in each case the degree of distance modification caused by each marker needs to be assessed.