ACTIVITIES OF THE GENETICS AND MICROBIOLOGY RESEARCH GROUP

1. Molecular biology of Pleurotus ostreatus
2. Marker-assisted breeding
3. Bacteriocins

MOLECULAR BIOLOGY OF Pleurotus ostreatus

    The Genetics and Microbiology Research Group (GMRG) started in 1993 the research on different aspects of the molecular biology of higher fungi using the white rot fungus Pleurotus ostreatus as model organism. The initial research interest of the group deals with the identification of genes controlling the switching from the vegetative mycelial growth to the reproductive fruit body formation. This long distance objective, however, necessitated previous characterization of different aspects of P. ostreatus genetics and molecular biology.

Currently, the GMRG is undertaking the following projects:

1.- Construction of a genetic linkage map of P. ostreatus. For this purpose, P. ostreatus florida has been used. The linkage map is based on Randomly Amplified polymorphic DNA markers (RAPD) whose segregation across a collection of monokaryons derived from a dikaryon of the strain used has been analyzed. The map identifies 11 linkage groups. In order to facilitate the use of the linkage map for breeding or cloning programs, most of the RAPD markers are  converted into Restriction Fragment Length Polymorphic (RFLP) ones.

2.- Physical map of P. ostreatus. The chromosomes of P. ostreatus N001 have been resolved by Pulse Field Gel Electrophoresis. This strain is a dikaryon whose two chromosomal complements differ markedly. To analyze the two parental chromosomal complements the strain N001 has been de-dikaryotized by protoplasting and culture of single isolated monokaryons. These monokaryons are called proto-clones and they present significant differences in chromosome length. Selected probes corresponding to RFLP markers of the linkage map described above have been used to identify the pulse field resolved chromosomes. This allowed us to ascribe linkage groups to separate chromosomes.

3.- Analysis of incompatibility genes in P. ostreatus. The efficient use of P. ostreatus in breeding or cloning programs requires the understanding of the genetic bases of its mating system. P. ostreatus is an heterothallic fungus whose mating is controlled by two genes which segregate independently (A and B). Crosses between P. ostreatus strains can only be accomplished when a double heterozygote is formed (AxAy BxBy). The analysis of the incompatibility genes in P. ostreatus encompasses three different approaches:

3.1.- Identification of incompatibility genes in P. ostreatus strains. For this approach the incompatibility alleles present in different wild and commercial P. ostreatus strains have been studied. This study includes the isolation of tester monokaryotic strains that allow to classify new incompatibility alleles present in other P . ostreatus isolates. Table 1 summarizes the incompatibility alleles identified in our strains and for which testers are available in our collection.

Table 1

3.2.- Genetic analysis of the B-type incompatibility genes. The appearance of new B-type incompatibility alleles in the spore progeny of a given dikaryon of P. ostreatus florida (strain N001) prompted the study of the mechanism of formation of new incompatibility alleles in this fungus. The current hypothesis is that this locus is a complex one formed by two different genes (matBa and matBb ) and recombination between them can occur giving rise to new incompatibility alleles

In our system this scheme has been proven to be supported by genetic evidence derived from the analysis of the incompatibility factors B3 and B4. The genetic distance between the loci matBa and matBb is of 17.5 cM in the strain N001.

3.3.- Identification of molecular markers genetically linked to the incompatibility loci A and B. In the analysis of the linkage map of P. ostreatus several molecular markers linked to either one of the two incompatibility loci have been identified.Several markers for the incompatibility factor B have been identified which cosegregate with either one of the two loci which constitute the B factor. One of the RFLP markers identified seems to cosegregate simultaneously but in an opposite way with both B loci. This marker can be useful to undertake the cloning of the B incompatibility factor.

4.- Analysis and characterization of hydrophobins. Hydrophobins are small hydrophobic proteins secreted by fungi. They are involved in protection against water loss and pathogens attack, and in the adhesion of hyphae between them and to the substrate. Hydrophobins, on the other hand, have several biotechnological applications as surfactants, as agents to biocompatibilize surfaces and in changing the wettability of surfaces. Hydrophobin expression is developmentally regulated and different hydrophobins can be isolated from vegetative mycelium, fruit bodies and spores. Hence, hydrophobins are good examples for genes whose expression switches during the phase change from vegetative growth to fruit body formation. In our laboratory we have isolated five different hydrophobins from P. ostreatus.

5.- The GMRG has also set up the technology to use molecular markers such as RAPD in the identification and classification of fungal isolates (P. ostreatus, Agaricus bisporus, Alternaria alternata, for instance) or other biological material (Prosopis spp., Staphylococcus aureus, etc.). This technology is used in collaborative projects together with different public and private institutions.

MARKER ASSISTED BREEDING

BACTERIOCINS

In summary, the GMRG can offer:

1.- Expertise in the handling of P. ostreatus both in genetic and molecular aspects.

2.- A linkage map already developed which is the most complete for this species up to now and it is complemented by the physical map based on Pulse Field Gel Electrophoretic separation of the chromosomes.

3.- Incompatibility tester collections which allow to speed up the genetic manipulation of the material.

4.- Expertise in the isolation and characterization of gene products including in situ expression analysis and recombinant gene expression.

5.- Expertise in the use of molecular markers to identify and characterize fungal isolates.