Construction Of Genetic Linkage Map Of Flammulina Velutipes With SCAR Markers

Random Amplified Polymorphic DNA (RAPD),Inter Simple Sequence Repeat (ISSR),and Sequence-related Amplified Polymorphism (SRAP) molecular makers were used to obtain polymorphic bands,which were converted into more stable and more reliable markers SCAR.A haploid progeny of 120 monokaryons were used as mapping population to construct a genetic linkage map of Flammulina velutipes.A hybrid strain of Flammulina velutipes 8801 and FL19 was produced with protoplast monokaryogenesis crossbreeding technic.Basidiospores were isolated from fresh fruiting bodies by holding the cap,120 monokaryons were selected randomly to be mapping population.8801 and its protoplast monokaryotic strain W23 ,FL19 and its protoplast monokaryotic strain L11,as well as the hybrid strain 1123 were used as templates in PCR to select primers which can produce polymorphic bands between two parental monokaryons,the polymorphic bands were converted into SCAR molecular maker.41 polymorphic bands were produced by 150 selected RAPD primers,and 29 polymorphic bands were produced by 102 pairs of SRAP primers which combined by 17 upstream primers and 6 downstream primers,while only 4 polymorphic bands were produced by 20 ISSR primers.As a result, 13 polymorphic bands of RAPD molecular maker,and 3 of SRAP marker were converted into SCAR molecular marker successfully.The ratio of that is 31.7% and 10.3% separately.In this research,SCAR marker S500-1500 correlated with the gene of yellow fruitbody.The gene encoding MIP(mitochrondrial )was accomplished using a pair of degenerate oligonucleotide primers. Based on the sequence of MIP,three nested primers was designed.TAIL-PCR was performed with five arbitary degenerate(AD) primers paring with three nested primers.A flanking sequence less than 500bp was generated,which will be confirmed whether the A mating-type gene was contained ,and at last the A mating-type gene will be used in the genetic linkage map.Combined with the SCAR marker linked to the color gene of white fruitbody, 17 SCAR markers were analysed and used to construct linkage map of Flammulina velutipe by Mapmaker software. Only 4 SCAR makers were from parental strain W23,and 13 SCAR markers was from parental strain L11.Chi-square analysis indicated 3 of all were distorted segregation markers,other 14 were in agreement with 1:1 Medelian ration,which were used to construct genetic linkage map.7 out of the 14 markes could not be assigned to any linkage group,and the remaining 7 markers were assigned to 3 linkage group,which span a total of 76.6 cM.