the ganoderma strains listed in table 1 were obtained from the korean collection for type culture ( ktct , jeongeup , korea ) , the american type culture collection ( atcc , rockville , md , usa ) , the korean agricultural culture collection ( kacc , suwon , korea ) , the mushroom division of the rural development administration ( eumseong , korea ) , the centraalbureau voor schimmelcultures ( cbs , utrecht , netherlands ) , incheon university ( incheon , korea ) , and konkuk university ( seoul , korea ) .
ganoderma species were cultured on potato dextrose broth ( difco , detroit , mi , usa ) and incubated at 30 for 2 wk .
cultured mycelia , filtered through 2 layers of miracloth ( calbiochem , la jolla , ca , usa ) , were ground in liquid nitrogen , and genomic dna was extracted using the cetyltrimethylammonium bromide method .
pcrs were performed using a premixed polymerase kit ( taq premix ; tnt research , anyang , korea ) in a 20 l reaction mixture containing 1 l of dna .
amplification of the its region was carried out using a thermal cycler ( takara , tokyo , japan ) at the following conditions : 5 min at 94 for initial denaturation , followed by 30 cycles of 30 sec at 94 for denaturation , 30 sec at 56 for primer annealing , and 1 min at 72 for extension , and 10 min at 72 for a final extension .
pcr products were detected by electrophoresis on 1.2% agarose gels in 0.5 tris - acetate ethylenediaminetetraacetic acid buffer .
pcr products were ligated into the pgem - t easy vector ( promega , madison , wi , usa ) according to the manufacturer 's instructions .
ligation products were transformed into the escherichia coli dh5 competent cell ( rbc , taiwan ) by heat shock .
plasmid dnas were extracted using the dna hybrid - qtm plasmid mini dna isolation kit ( geneall , seoul , korea ) .
recombinant clones were identified , and the presence of inserts was confirmed by ecori restriction enzyme digestion and sequencing ( using the sp6 and t7 promoters ; genotech , daejeon , korea ) .
nucleotide sequences were deposited in the national center for biotechnology information genbank database ( table 1 ) .
the sequences of the its rdna were aligned for phylogenetic analysis using the program bioedit ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) .
the phylogenetic trees were constructed by using the mega5 program   and the neighbor - joining method .
confidence levels for individual branches of the resulting tree were assessed using the bootstrap test   in which 1,000 replicate trees were generated from resampled data .
the size of basidiospores from the korean cultivated yeongji strains was determined using a fluorescence microscope ( axio observer a1 ; carl zeiss , jena , germany ) and a 5% koh solution as a mounting medium .
the size of each spore was calculated , and the mean value was used in the description .
the ganoderma strains listed in table 1 were obtained from the korean collection for type culture ( ktct , jeongeup , korea ) , the american type culture collection ( atcc , rockville , md , usa ) , the korean agricultural culture collection ( kacc , suwon , korea ) , the mushroom division of the rural development administration ( eumseong , korea ) , the centraalbureau voor schimmelcultures ( cbs , utrecht , netherlands ) , incheon university ( incheon , korea ) , and konkuk university ( seoul , korea ) .
ganoderma species were cultured on potato dextrose broth ( difco , detroit , mi , usa ) and incubated at 30 for 2 wk .
cultured mycelia , filtered through 2 layers of miracloth ( calbiochem , la jolla , ca , usa ) , were ground in liquid nitrogen , and genomic dna was extracted using the cetyltrimethylammonium bromide method .
pcrs were performed using a premixed polymerase kit ( taq premix ; tnt research , anyang , korea ) in a 20 l reaction mixture containing 1 l of dna .
amplification of the its region was carried out using a thermal cycler ( takara , tokyo , japan ) at the following conditions : 5 min at 94 for initial denaturation , followed by 30 cycles of 30 sec at 94 for denaturation , 30 sec at 56 for primer annealing , and 1 min at 72 for extension , and 10 min at 72 for a final extension .
pcr products were detected by electrophoresis on 1.2% agarose gels in 0.5 tris - acetate ethylenediaminetetraacetic acid buffer .
pcr products were ligated into the pgem - t easy vector ( promega , madison , wi , usa ) according to the manufacturer 's instructions .
ligation products were transformed into the escherichia coli dh5 competent cell ( rbc , taiwan ) by heat shock .
plasmid dnas were extracted using the dna hybrid - qtm plasmid mini dna isolation kit ( geneall , seoul , korea ) .
recombinant clones were identified , and the presence of inserts was confirmed by ecori restriction enzyme digestion and sequencing ( using the sp6 and t7 promoters ; genotech , daejeon , korea ) .
nucleotide sequences were deposited in the national center for biotechnology information genbank database ( table 1 ) .
the sequences of the its rdna were aligned for phylogenetic analysis using the program bioedit ( http://www.mbio.ncsu.edu/bioedit/bioedit.html ) .
the phylogenetic trees were constructed by using the mega5 program   and the neighbor - joining method .
confidence levels for individual branches of the resulting tree were assessed using the bootstrap test   in which 1,000 replicate trees were generated from resampled data .
the size of basidiospores from the korean cultivated yeongji strains was determined using a fluorescence microscope ( axio observer a1 ; carl zeiss , jena , germany ) and a 5% koh solution as a mounting medium .
the size of each spore was calculated , and the mean value was used in the description .
the phylogenetic analysis of ganoderma species in this study was generally consistent with the findings reported by the group from china .
the 62 ganoderma strains were divided into 7 groups , a to g ( fig .
in addition , ganoderma lucidum were largely divided into two groups ( groups a and g ) .
group a included all the korean g. lucidum strains , as well as the g. lucidum strains from bangladesh and japan , and chinese g. sichuanense and g. lingzhi .
g. meredithae ( usa and unknown sources ) in group b was closely related to g. lucidum in group a as evidenced by a high bootstrap value of 98% .
g. multipileum ( china and taiwan ) was grouped within group c and g. tropicum ( china and taiwan ) within group d , with 96% and 99% bootstrap support , respectively .
group e includes three strains of g. resinaceum from the czech republic and uk ( two strains ) , and was supported by very high bootstrap values of 99% .
strains of g. weberianum ( australia , china , and philippines ) and two other chinese g. sichuanense strains ( cui 7691 and hmas 86597 ; holotype ) were grouped within group f with 99% bootstrap support . groups e and f were closely related with bootstrap values of 96% .
it is worth noting that other g. lucidum strains from europe ( france , finland , italy , sweden , and uk ) and canada could be clustered into group g with g. tsugae strains from north america ( canada and usa ) , supported by a very high bootstrap value of 99% .
the findings of the current study are consistent with those of park et al .   who reported that g. lucidum strains from europe and north america could be clustered into one group together with g. tsugae based on both analyses of the its rdna gene and partial -tubulin gene sequences .
interestingly , korean g. lucidum strains could be clustered into a group together with g. sichuanense and g. lingzhi strains from china .
reported that g. lucidum is incorrectly recorded in china , as well as in other countries , and suggested that the name ' g. lucidum ' as used for the chinese species should be corrected as g. sichuanense .
however , they did not obtain sequences from type specimens of g. sichuanense ( the holotype ; hmas 42798 ) .
reported that korean g. lucidum ( asi-7004 ) could be clustered into one group together with chinese g. lucidum and g. lingzhi based on the its2 sequences and rna secondary structures .
furthermore , cao et al .   reported that g. sichuanense ( included the holotype ; hmas 42798 ) was distantly phylogenetically related to g. lingzhi ( included the holotype ; wu 1006 - 38 ) , but it was closely related to g. resinaceum . with regard to the morphological characteristics , it has been reported that chinese g. lucidum ( g. lingzhi ) has a yellow pore surface , european g. lucidum has a white pore surface , and korean g. lucidum has pale brownish thread - like tissues in the middle of the context .
cao et al .   found that the morphological characteristics of ' g. lucidum ' from east asia were consistent with those of g. lingzhi .
thus , they concluded that g. sichuanense was a distinct species since they display obvious morphological differences from g. lingzhi and suggested that the name ' g. lucidum ' should be corrected as g. lingzhi not g. sichuanense .
it is commonly called " yeongji " in korea , " lingzhi " in china , and " reishi " or " mannentake " in japan .
however , the ganoderma species from various countries including africa , oceania , america , asia ( china , korea , and japan ) , and europe   have been incorrectly reported as g. lucidum .
in addition , moncalvo et al .   classified g. lucidum collections from different regions ( asia and europe ) into different species based on its and partial nuclear large subunit ribosomal dna sequences .
this is similar to the results of our its nucleotide sequence analysis of g. lucidum .
likewise , hong and jung   reported that the g. lucidum from korea and japan were monophyletic , and were distinguished from the g. lucidum from europe and north america based on sequence analysis of mitochondrial small - subunit ribosomal dna .
as described above , the taxonomy of g. lucidum has been reported by many researchers .
nevertheless , the name g. lucidum has been misapplied to various species around the world .
the results of this study also reveal that korean g. lucidum was clustered into one group together with chinese g. sichuanense and chinese g. lingzhi ( both formerly g. lucidum ) , and it was clearly separated from g. lucidum from europe and north america .
in addition , g. sichuanense were divided into two groups ( groups a and f ) .
g. sichuanense strains ( cui 7691 and hmas 86597 ; holotype ) , including the holotype , could be clustered into group f together with g. weberianum , and it was closely related to g. resinaceum .
cao et al .   also reported that the size of basidiospores of g. lingzhi [ ( 9.85  0.85 )  ( 6.4  0.6 ) m ] differs from that of g. sichuanense [ ( 8.3  0.9 )  ( 5.8  0.8 ) m ] .
interestingly , the average size of the basidiospores from the korean cultivated yeongji strains [ ( 10.65  0.65 )  ( 6.6  0.6 ) m for asi-7004 , ( 10  1 )  ( 6.4  0.3 ) m for asi-7071 ] were similar to that of g. lingzhi ( fig .
the comparison of the its rdna sequences and the estimation of the basidiospores size presented in this study confirm previous findings and contribute additional evidence that suggests the naming korean cultivated yeongji strains of ' g. lucidum ' should be renamed as g. lingzhi .
 ganoderma lucidum has a long history of use as a traditional medicine in asian countries . 
 however , the taxonomy of ganoderma species remains controversial , since they were initially classified on the basis of their morphological characteristics . 
 recently , it was proposed that g. lucidum from china be renamed as g. sichuanense or g. lingzhi . in the present study , 
 phylogenetic analysis using the internal transcribed spacer region rdna sequences of the ganoderma species indicated that all strains of the korean ' g. lucidum ' clustered into one group together with g. sichuanense and g. lingzhi from china . 
 however , strains from europe and north american , which were regarded as true g. lucidum , were positioned in a clearly different group . in addition , the average size of the basidiospores from the korean cultivated yeongji strains was similar to that of g. lingzhi . based on these results , 
 we propose that the korean cultivated yeongji strains of ' g. lucidum ' should be renamed as g. lingzhi . 
MATERIALS AND METHODS
None
Genomic DNA extraction and PCR amplification
Cloning and sequencing
Sequence analysis
Basidiospore observation
RESULTS AND DISCUSSION