Study On Strain-Specific Molecular Markers In Ganoderma Strains

Ganoderma lucidum was used to treat different diseases in China for a long time.In recent years,the cultivation of Ganoderma was developed quickly,with the germplasm resource becoming more and more plentiful.But the isolates cultivated in China are presently in chaos seriously.Different strains were assigned with the same name or the same strain has different names.It brought too much difficulty for fundamental research and development of the Ganoderma lucidum industry.The purpose of this study was to establish Ganoderma strain-specific SCAR markers, which would be used in the Ganoderma strains fast examination and identification.The method of the strain-specific SCAR marker should be benefit to protect Ganoderma species in Chinese.On the genetic relationship at the level of 0.850,forty-eight DNA pools were prepared using standardized DNA extracted from 152 Ganoderma isolates(128 Chinese cultivars and 24 non-Chinese isolates) delineated on the basis of ERIC-PCR data.SRAP and ISSR methodologies were used to amplify DNA from the different pools, and four specific marker bands(one SRAP and three ISSR) were selected,cloned and sequenced.Then four pairs of SCAR primers,C4A/C4S,C38A/C38S,C39A/C39S and C45A/C45S,were designed.Then SRAP markers and ISSR markers were converted into more stable and more highly specific SCAR markers.Four pairs of SCAR primers were used to amplify targeted bands on 152 strains that made up of 48 pools.The strain with specific SCAR marker was used as positive control. Four pairs of SCAR primers,C4A/C4S,C38A/C38S,C39A/C39S and C45A/C45S,were confirmed to be strain-specific SCAR markers,specific only for G0014,G0121,G0142 and G0126 respectively.The feasibility and reliability of adopting strain-specific SCAR markers for the rapid identification of Ganoderma strains were confirmed.Multiplex PCR products,C4(777bp) and 38(774bp),differing from each other by only 3bp in length,could not be conveniently separated on gels of commonly used agaroses. Two multiplex PCR systems were developed and optimized using the corresponding highly specific SCAR primers.The primers were successfully used in a multiplex test for the reliable detection of three mixed species,each represented by a single individual respectively,G0126,G0014,G0142 and G0126,G0121,G0142.By simultaneously amplifying more than one locus in the same reaction,multiplex PCR would result the reduction in labor from one or two weeks to less than one day,and the lower reagent costs in research laboratory and management practices.