Open Access
Isolation, molecular characterization and location of telomeric sequences of the basidiomycete Pleurotus ostreatus var. florida
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Pérez Garrido, María Gumersinda; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
The white rot fungus Pleurotus ostreatus is an edible basidiomycete of increasing biotechnological interest due to its ability to degrade both wood and chemicals related to lignin degradation products. Telomeres are specialized structures at the end of all eukaryotic chromosomes. Ensure chromosome stability and protect the ends from degradation and from fusing with other chromosomes. Telomeres sequences are extraordinary highly conserved in evolution. The loss of telomeric repeats triggers replicative senescence in cells. For identification of restriction telomeric fragments in a previously described linkage map of Pleurotus ostreatus var. florida (Larraya et al., 2000), dikaryotic and eighty monokaryotic genomic DNAs were digested with diferents restriction enzymes (BamHI, BglII, HindIII, EcoRI, PstI, SalI, XbaI and XhoI) electrophoresed and transferred to nylon membranes. Numerous polymorphic bands were observed when membranes were hibridized with human telomericd probe (TTAGGG)132 (heterologous probe). Telomeric restriction fragments were genetically mapped to a previously described linkage map of Pleurotus ostreatus var.florida, using RFLPs identified by a human telomeric probe (tandemly repeating TTAGGG hexanucleotide). Segregation of each telomeric restriction fragment was recorded as the presence vs. absence of a hibridizing band. Segregation data for seventy three telomeric restriction fragments was used as an input table to be analysed as described by Ritter et al. (1990) and by Ritter and Salamini (1996) by using the MAPRF program software. Seventeen out of twenty two telomeres were identified. Telomere and telomere-associated (TA) DNA sequences of the basidiomycete Pleurotus ostreatus were isolated by using a modified version of single- specific-primer polymerase chain reaction (SSP-PCR) technique (Sohapal et al., 2000). Telomeres of Pleurotus ostreatus contain at least twenty five copies of non-coding tandemly repeated sequence (TTAGGG).
Open Access
Identification and functional characterisation of ctr1, a Pleurotus ostreatus gene coding for a copper transporter
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Peñas Parrila, María Manuela; Azparren Larraya, María Goretti; Domínguez, A.; Sommer, H.; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Copper homeostasis is primordial for life maintenance and especially relevant for ligning-degrading fungi whose phenol-oxidase enzymes depend on this micronutrient for their activity. In this paper we report the identification of a gene (ctr1), coding for a copper transporter in the white rot fungus Pleurotus ostreatus, in a cDNA library constructed from four-days old vegetative mycelium growing in submerged culture. The results presented here indicate that: (1) ctr1 functionally complements the respiratory deficiency of a yeast mutant defective in copper transport supporting the transport activity of the Ctr1 protein; (2) ctr1 transcription is detected in all P. ostreatus developmental stages (with exception of lamellae) and is negatively regulated by the presence of copper in the culture media; (3) ctr1 is a single copy gene that maps to P. ostreatus linkage group III; and (4) the regulatory sequence elements found in the promoter of ctr1 agree with those found in other copper related genes described in other systems. These results provide the first description of a copper transporter in this white rot fungus and open the possibility of further studies on copper metabolism in higher basidiomyetes.
Open Access
Genetic linkage map of the edible basidiomycete Pleurotus ostreatus
(American Society for Microbiology, 2000) Larraya Reta, Luis María; Pérez Garrido, María Gumersinda; Ritter, Enrique; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
We have constructed a genetic linkage map of the edible basidiomycete Pleurotus ostreatus (var. Florida). The map is based on the segregation of 178 random amplified polymorphic DNA and 23 restriction fragment length polymorphism markers; four hydrophobin, two laccase, and two manganese peroxidase genes; both mating type loci; one isozyme locus (est1); the rRNA gene sequence; and a repetitive DNA sequence in a population of 80 sibling monokaryons. The map identifies 11 linkage groups corresponding to the chromosomes of P. ostreatus, and it has a total length of 1,000.7 centimorgans (cM) with an average of 35.1 kbp/cM. The map shows a high correlation (0.76) between physical and genetic chromosome sizes. The number of crossovers observed per chromosome per individual cell is 0.89. This map covers nearly the whole genome of P. ostreatus.
Open Access
Nutritional value of protein from vegetative mycelia of edible mushroom Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Parada Albarracín, Julián Andrés; Urdaneta, Elena; Marzo Pérez, Florencio; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena; Ciencias del Medio Natural; Natura Ingurunearen Zientziak
The present work was designed to study the effects of supplementation a control diet with P. ostreatus mycelium for evaluation a nutritional value of mycoprotein and possible cholesterol lowering.
Open Access
Molecular characterization of A cellobiohydrolase gene family in the fungus Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Eizmendi Goikoetxea, María Arantzazu; Sannia, Giovanni; Ramírez Nasto, Lucía; Pisabarro de Lucas, Gerardo; Producción Agraria; Nekazaritza Ekoizpena
Cellulose is the most abundant biological polymer on Earth. Its chemical composition consists of D-glucose units linked by β-1,4- glycosidic bonds forming linear polymeric chains with a reducing and a non-reducing end. Cellulose chains may either adhere to each other, via hydrophobic and van der Waals interactions, forming crystalline structures or remain more loosely packaged (amorphous cellulose). Consequently, the physical structure and morphology of native cellulose is complex and not uniform. Biological degradation of cellulose depends on the action of three types of enzymes: endoglucanases (E.C.3.2.1.4), cellobiohydrolases (E.C.3.2.1.91) and β-glucosidases (E.C.3.2.1.21). All them hydrolyse β-1,4-glycosidic bonds but they differ on the substrate specificity. Endoglucanases hydrolyse the amorphous regions of the cellulose fibbers generating new reducing and non-reducing ends, cellobiohydrolases attack the molecule ends yielding cellobiose units, and β-glucosidases hydrolyse cellobiose molecules yielding glucose. Cellobiohydrolases can be classified into two groups: type I (CBHI) and type II (CBHII), each having opposite chain-end specificities. CBHI prefer the reducing ends while CBHII act at non-reducing ends. By the screening of a genomic library from the basidiomycete Pleurotus ostreatus var. florida, we have isolated five cbhI genes, named cbhI1, cbhI2, cbhI3, cbhI4 and cbhI5, proving the occurrence of a multigenic family coding for this enzymatic activity. Using this sequences as probe, it has been possible to know the conditions in which are expressed those genes. This has allowed the synthesis of the each gene cDNA and, by comparison of this sequence with the corresponding genomic sequence, the characterization of their structure. On the other hand, using the RFLP technique and a progeny of 80 monokaryons derived from the dikaryon N001, the five genes have been mapped on the linkage map of P. ostreatus var. florida mapping the cbhI1 to the chromosome IV and the others to the chromosome VI.
Open Access
Mapping of genomic regions (quantitative trait loci) controlling production and quality in industrial cultures of the edible basidiomycete Pleurotus ostreatus
(American Society for Microbiology, 2003) Larraya Reta, Luis María; Alfonso Esquíroz, Mikel; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua
Industrial production of the edible basidiomycete Pleurotus ostreatus (oyster mushroom) is based on a solid fermentation process in which a limited number of selected strains are used. Optimization of industrial mushroom production depends on improving the culture process and breeding new strains with higher yields and productivities. Traditionally, fungal breeding has been carried out by an empirical trial and error process. In this study, we used a different approach by mapping quantitative trait loci (QTLs) controlling culture production and quality within the framework of the genetic linkage map of P. ostreatus. Ten production traits and four quality traits were studied and mapped. The production QTLs identified explain nearly one-half of the production variation. More interestingly, a single QTL mapping to the highly polymorphic chromosome VII appears to be involved in control of all the productivity traits studied. Quality QTLs appear to be scattered across the genome and to have less effect on the variation of the corresponding traits. Moreover, some of the new hybrid strains constructed in the course of our experiments had production or quality values higher than those of the parents or other commercial strains. This approach opens the possibility of marker-assisted selection and breeding of new industrial strains of this fungus.
Open Access
Quantitative trait loci controlling vegetative growth rate in the edible basidiomycete Pleurotus ostreatus
(American Society for Microbiology, 2002) Larraya Reta, Luis María; Idareta Olagüe, Eneko; Arana, Dani; Ritter, Enrique; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena; Gobierno de Navarra / Nafarroako Gobernua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Mycelium growth rate is a quantitative characteristic that exhibits continuous variation. This trait has applied interest, as growth rate is correlated with production yield and increased advantage against competitors. In this work, we studied growth rate variation in the edible basidiomycete Pleurotus ostreatus growing as monokaryotic or dikaryotic mycelium on Eger medium or on wheat straw. Our analysis resulted in identification of several genomic regions (quantitative trait loci [QTLs]) involved in the control of growth rate that can be mapped on the genetic linkage map of this fungus. In some cases monokaryotic and dikaryotic QTLs clustered at the same map position, indicating that there are principal genomic areas responsible for growth rate control. The availability of this linkage map of growth rate QTLs can help in the design of rational strain breeding programs based on genomic information.
Open Access
Genetic breeding of edible mushrooms: from the genome to the production of new varieties of Pleurotus ostreatus
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Pisabarro de Lucas, Gerardo; Peñas Parrila, María Manuela; Pérez Garrido, María Gumersinda; Park, Sang-Kyu; Eizmendi Goikoetxea, María Arantzazu; Parada Albarracín, Julián Andrés; Palma Dovis, Leopoldo; Idareta Olagüe, Eneko; Jurado Cabanillas, Javier; Castellón Gadea, Jordi; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
The breeding of new varieties of industrially cultivated edible mushrooms must proceed in the framework defined by the breeding objectives, the biological characteristics of the material and the legal and cultural constraints imposed to the breeding technology to be used. This last aspect is of the greatest importance in the case of a food that is considered in European countries as high quality and closer to nature than other industrially produced foods. This fact prevents the use of genetic-engineering based technologies for breeding, as the consumers would hardly accept genetically modified mushrooms. Consequently, mushroom breeding should be based on time-consuming processes of classic breeding. Molecular biology, however, can offer to the breeders useful tools for speeding up the selection process, for evaluating the new bred lines and, last but not least, to identify and eventually protect legally the outcome of their breeding programs.
Open Access
Mapping the Pleurotus ostreatus genome
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Castellón Gadea, Jordi; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; Producción Agraria; Nekazaritza Ekoizpena
Pleurotus ostreatus is a commercially important edible mushroom commonly known as oyster mushroom which has also important biotechnical applications. Industrial production of P.ostreatus is based on a solid fermentation process in which a limited number of selected strains are used. Optimization of industrial mushroom production depends on improving the culture process and breeding new strains with higher yields and productivities. In a previous study a linkage map of P. ostreatus strain N001 was constructed, which provided a basis for performing an efficient QTL (Quantitative trait loci) analysis based in a population of 80 sibling monokaryons. The map is based on the segregation of RAPD markers, RFLP markers, phenotypic characters and cloned genes. Nevertheless the linkage map is just a first step towards the selection of the appropiate parentals for new breeds. In order to organize and improve the access to the data and information accumulated in the previous works mentioned above, a Microsoft® Excel Linkage Map Matrix (MELMM) was designed and created. On this linkage map matrix we could have an easy and functional view of the P. ostreatus linkage map data, such as, recombination frequencies, genotypes information and degree of similarity between monokaryons that will help us in the design of breeding crosses aimed at improving QTLs of agronomic interest of new commercial strains.
Open Access
Enzymatic characterization of a monokaryon population of the edible mushroom, Pleurotus ostreatus with a view to genetic improvement
(Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa, 2006) Terrón, María del Carmen; López, María; Carbajo, José M.; Pisabarro de Lucas, Gerardo; Ramírez Nasto, Lucía; González Aldo, E.; Producción Agraria; Nekazaritza Ekoizpena
In this work the lignocellulolytic enzymes produced by the edible mushroom Pleurotus ostreatus var. florida were studied. The objective was to know their relationship with the degradation of the biopolymers present in the cell wall of wheat-straw for the purpose of explaining their influence on the production and quality characters of the fruiting bodies. The following enzymatic activities were studied both in solid and submerged culture: Ligninases (Lignin Peroxidase, Manganese Peroxidase (MnP) and Laccase), Cellulases (Glucohydrolases, Glucosidases) and Hemicellulases from the group Arabinofuran- Xylanases (Xylanase, Xilosidase, Glucoronidase, Arabinofuran-Oxidase and Acetylesterase), cooperating enzymes (Glyoxal Oxidase) and feedback enzymes (Glucose Oxidase (GOD), Aryl Alcohol Oxidase (AAO), Tyrosinase (TYR), Veratryl Alcohol Oxydase (VAO), Cellobiose Dehydrogenase (CDH)). The first studies regarding all the mentioned enzymes were performed using the dikayon (N001) and the parental monokarion strains “fast” (PC9) and “slow” (PC15). The studies on all this whole group of enzymes, which are enough representative of the lignocellulolytic complex, let to conclude that (both in solid or submerged culture) the enzymes of major influence in colonizing the natural substrate and also those whose activity-determination better guarantees their further mapping were Laccases, MnP, AAO and TYR. Subsequently these four activities were measured in the monokaryon population being Laccases and MnP, those yielding the best levels in medium-7 (rich in nitrogen). In addition both enzymes allow the discrimination between “fast-” or “slow-” monokaryon strains both in solid medium with several dyes, or in liquid culture in agitation. The analysis of the enzymatic activities detected in the assayed conditions, in the population of “fast” or “slow” strains let to the observation that they map in different places where the loci corresponding to Laccase (pox) and mnp genes are located. These results open the possibility to design more precise studies that could help to establish a correlation between the contribution of the genes already described and the activity of the different ligninolytic enzymes. In addition the results will contribute to know whether in P. ostreatus genome there are new genes or if they correspond with locations that regulate these enzymatic activities, or it is a gene that has a role in the transport system or a kind of effector in the exportation machinery of the protein to the culture medium.