Microsatellite loci are increasingly used as markers in the human, animal and plant genomes. Being highly mutable, microsatellite regions are able to differentiate between related taxa, even at the level of individual isolates in a single species. Studies on mushroom population structure, gene flow and dispersal between natural and cultivated species have become central in breeding programmes and the knowledge of new polymorphic, codominant markers will be a promising avenue to exploit wild genetic resources. The molecular phylogeny in 50 different commercial cultivated strains of Pleurotus eryngii using PCR amplification with URP primers and mitochondrial microsatellite primer was studed. The sizes of the polymorphic fragments obtained were in the range of 200 to 2000 bp. RAPD analysis techniques were able to detect genetic variation among the tested strains. With these isolated PCR amplification with URP primers we intend to analyse the population structure of the P. eryngii species complex and investigate the structure of the basidiomycete genome which deserves. A few single-locus microsatellite markers have been isolated in Pleurotus eryngii and Pleurotus ferulae. This technique is useful in those species where microsatellite loci are rare in the mitochondria.

The mating-type genes control formation of the dikaryon from two haploid strains. These genes are now used in mating-type-assisted breeding programs for economically important mushrooms, especially the oyster mushroom, Pleurotus ostreatus, aiming at high-yield and high-quality standard mushroom production. However, it improves the breeding program when the breeder is able to quickly identify compatible strains in a given set of progeny. The two mating factors with their mating-type loci are used as markers for breeding and have been incorporated in a chromosome mapping investigation. The linkage maps include not only genetic markers such as the mating types that can be cored, but also molecular markers such as PCR-assisted approaches, e.g. RAPD analyses, or RFLP markers. Once mating-type genes within progeny may be more easily identified by the use of PCR-directed cloning of partial mating-type genes. We analyzed homeodomain (HD1 and HD2) and pheromone receptor(rcb1, 2 and 3) genes as molecular markers for breeding using mating type A and B of Pleurotus eryngii and Pleurotus ferulae by direct PCR.

[n order to obtain the trlle expected DNA prod uct from PCR and RT-PCR using genornic DNA or cDNA reversely transcribed from mRNA. the PCR should be done in an appropriated condition. Sometimes the PCR was repeatedly fail ed. and cventllally the PCR product was turned out to be nonspecific and rudimentary . And more‘ t he PCR prodllctwas not reproducible even though careflll repeat of experiments. As the PCR was based on the exact primel hybridization. the condition of primer hybridization should be properly controlled by a nnealing temperatllre. But the selection of primer seqllences for targeting a specific gene is mostly important. A new method of primer eval uation is now available llsing DNA base pair polarity program. This study presents an example of PCR targeting to human Bax gene using genomic DNA. The DNA base pair polarity theory can di vide the genetic cord into propel DNA segments and calclllaLe their DNA base pair hybridization energy. Thus. mathematically the degree 0(' exact primer hybridization can be expected for the t r1l8 targeting of PCR. However, the DNA base pair polal'ityanalysis demonstrates that the more frequent number of DNA segment incl'eased the specificity of PCR. but decreased its sensitivity . While the greater polarity of DNA segment composed of increased nllmber of polarized DNA base pairs showed increased sensitivi ty 0 1' PCR. bllt relati vely decreased specificity of PCR. With the mllltiple analysis of PCR. especially for PCR cloning from the gDNA and cDNA, we found that the primers themselves showed secondary strllcture of partial hybridization between sameprimers or each pair primers. The DNA base pail‘ polarity signal can directly demonstrated symmetric sequences 0 1' each primer. and also can distinguish the dimmer formation from each pair primers. At least the symmetric seqllence of fOlll‘ base pairs dramatically showed the dimrner formation. On the other hand. in addi tion Lo the statlls of DNA base pair polarity the three-dimensional strllctllre of DNA dOllble helix targeted by the primer seqllences may affect the sensitivity and specificity of PCR detection. The present study introduced a new method of primer evalllation and selection in order to obtain abundant and exacL! y-trlle DNA product for genomic ffilltation analysis and gene expression profï le

Transforming growth factor (TGF) family is well known to induce the chondrogenic differentiation of mesenchymal stem cells (MSC). However, the precise signal transduction pathways and underlying factors are not well known. Thus the present study aims to evaluate the possible role of C2 domain in the chondrogenic differentiation of human mesenchymal stem cells. To this end, 145 C2 domains in the adenovirus were individually transfected to hMSC, and morphological changes were examined. Among 145 C2 domains, C2 domain of protein kinase C eta (PKCη) was selected as a possible chondrogenic differentiation factor for hMSC. To confirm this possibility, we treated TGFβ3, a well known chondrogenic differentiation factor of hMSC, and examined the increased-expression of glycosaminoglycan (GAG), collagen type II (COL II) as well as PKCη using PT-PCR, immunocytochemistry and Western blot analysis. To further evaluation of C2 domain of PKCη, we examined morphological changes, expressions of GAG and COL II after transfection of PKCη -C2 domain in hMSC. Overexpression of PKCη-C2 domain induced morphological change and increased GAG and COL II expressions. The present results demonstrate that PKCη involves in the TGF-β3-induced chondrogenic differentiation of hMSC, and C2 domain of PKCη has important role in this process.