Porphyromonas gingivalis is among the major etiological pathogens of chronic periodontitis. The virulence mechanisms of P. gingivalis is yet to be identified as its activity is largely unknown in actual disease process. The purpose of this study is to identify antigens of P. gingivalis expressed only in patients with chronic periodontitis using a unique immunoscreening technique. Change Mediated Antigen Technology (CMAT), an antibody-based screening technique, was used to identify virulence-associated proteins of P. gingivalis that are expressed only during infection stage in patients having chronic periodontitis. Out of 13,000 recombinant clones screened, 22 tested positive for reproducible reactivity with rabbit hyperimmune anti-sera prepared against dental plaque samples acquired from periodontitis patients. The DNA sequences of these 18 genes were determined. CMAT-identified protein antigens of P. gingivalis included proteins involved in energy metabolism and biosynthesis, heme and iron binding, drug resistance, specific enzyme activities, and unknown functions. Further analysis of these genes could result in a novel insight into the virulence mechanisms of P. gingivalis.

Two strains one of 29 strains were picked out Hm1-1, and Hm3-10 as parental strains for breeding, such as Hm1-1 was taken 15 point as the highest grade. Because Hm3-10 was wild strain collected form Deogyu mountain. The result of RAPD has shown that 29 strains fell into 6 distinct groups while a wild strain collected from DeogYu Mt. did not belong to any of the groups. The 20 selected monokaryon mycelia were crossed by Hm3-10 × Hm1-1, and then we picked up dikaryotic mycelia which did form clamp connection. We acquired 343 dikaryons from Hm3-10 × Hm1-1 with the mating rates of 85.7%, respectively. Total 343 dikaryon strains were cultivated and then 58 strains with good morphological and cultivation characteristics were selected. The differences of the 58 strains were verified by morphology of fruiting bodies. Finally, we selected 3 new strains (Hm15-3, Hm15-4, Hm17-5). Lastly, in order to develop molecular markers that can identify developed 3 new cultivars, were subjected to the random amplified polymorphic DNA (RAPD) analysis using 3 commercially available random primer sets (OPS-1, OPS-10, and OPL-13). I selected 10 distinct DNA bands from the three RAPD gels which were amplified with OPS-1, OPS-10, or OPL-13 primers. Bands 1, 6, and 7 were unique for Hm1-1 and Hm1-6. Bands 2∼5 and 8∼10 were unique for Hm3-10. The sequences were deposited in GeneBank and were used to design the 15-base primer sets using their 5’- and 3’-ends. The primer sets P1∼P5 and P7 produced DNA bands with corresponding sizes from all H. marmoreus strains and presented no strain specificity. The P6 marker appeared only for Hm1-1 while the P8 marker appeared for most hybrid strains except Hm16-1 and the wild Hm3-10. This is interesting because the P6 marker showed broader specificity than the P8 marker in the identification of strains. The P9 marker appeared on Hm16-1, Hm16-2, Hm17-5, and Hm3-10 and the P10 marker appeared on Hm15-3, Hm15-4, Hm16-1, Hm16-2, and Hm3-10. The hybrid strain Hm15-3 was the only strain that did not contain either the P9 or P10 marker. The resulting DNA markers were applied for the identification of the strains and employed for the molecular breeding of H. marmoreus.

This study was carried to develop new white cultivars that are suitable export using back cross method of Hypsizygus marmoreus. At first, we did select mother strains(Hm3-8 as brown and Hm0-7 as white) that have excellent cultivational caracteristics and morphology. The two strains are collected spore and selected 150 monokaryon mycelia by dilution method, and then picked out, 10 monokaryon strains, that havn‘t clamp connection and different mycelial morphology on plate. The selected monokaryon mycelia of GPHm3-8 and GPHm0-7 was crossed in PDA plate and the, acquired 100 dikaryon strains as mating rate is 100%. The 100 dikaryon strains is cultivated by sawdust media. And, we are selected 5 strains (BW16, BW41, BW56, BW76, BW96). To practice back cross, we selected 10 monokaryon mycelia that didn't clamp connection from the 5 stains(BW16, BW41, BW56, BW76, BW96). And then, we were carried out back cross with Hm0-7. The mating rate were investigated 31%, 33%, 33%, 34%, and 47% within BW16, BW41, BW56, BW76, and BW96, each other. We obtained 15 hybrid strains as brown line, and 23 hybrid stains as white line among 178 hybrid strains, and picked up 23 white strains because 15 brown strains were not suitable our object to develop new white cultivars among 38 strains. To select the best white strain , cultivated by standard growing condition, and BW76W-13 strain has been selected because it had good quality and morphology, finally. We were carried RAPD to verify difference between mother strains. We confirmed that band pattern of BW76W-13 had different pattern compared with mother strains.