Construction Of A Molecular Linkage Map In Lentinula Edodes

In this study, the single spores L6 and W26, derived from two genetic-differentiated cultivated mushroom strains L205 and WuXiang NO.1 respectively, used as parents, the hybrid L6XW26 was cultivated from which the spore monokaryons were obtained. Their mating-types were identified accurately by the clamp connection appearing or not during hybrid pairing, nuclear transfer experiments and SOJ-OWE experiments. we constructed a genetic segregation population with 148 spores monokaryons as map population of Lentinula edodes.SSR, SRAP, TRAP, and a pheromone receptor gene were used to analyse the map population for the construction of a molecular genetic linkage map in Lentinula edodes. Two polymorphic primers were screened from 77 tested SSR primers.25 out of 56 SRAP primer combinations were selected which had high polymorphic, PCR amplification results showed that the number of polymorphic bands was from 4 to 17 and the total polymorphic bands was 225, on tne average of 9 bands amplified by each primer combination. Through the analysis of 36 genes that associated with the fruit body development in Lentinula edodes, fixed primers for TRAP were designed, and 43 TRAP primer combinations were obtained. PCR amplification results showed that total of 474 polymorphic bands was obtained, on the average of 11.1 bands amplified by each TRAP primer combination. SRAP and TRAP molecular markers have shown high amplification efficiency, which was very beneficial to constructing high-density molecular genetic linkage map. According to the previous study about the Mat-B factor gene segments which also belonged to a pheromone receptor in our laboratory, we designed a specific primer and fortunately, a 371bp polymorphic DNA bands was obtained in our genetic segregation population, which aslo consistent with the theoretical size.In molecule experiments, we got total of 702 polymorphic bands (two SSR markers, one pheromone receptor gene,225 SRAP and 474 TRAP markers), and two mating type sites (MAT-A and MAT-B, total of 706 polymorphic bands and sites were analysed by JoinMap(?) 3.0 software for graphical representation of the genetic linkage map. JoinMap(?)3.0 generated a framework map consisting of 581 molecular marker and two mating type sties which distributed over 11 linkage groups. The 11 linkage groups was 963.2 cM with an average distance of 1.70 cM between two markers, the groups ranged in size from 21 cM～151 cM, and the largest distance between markers was 20.45 cM. Two mating type factors were linked in LG 3 and LG 10 groups, which was 111 cM and 39 cM respectively. Two markers zipl-4-295 and exg2-3-180 were closely linkaged to the MAT-A stie to which the distance was 6.6 cM and 7.3 cM respectively (LG 3), two markers zipl-4-390 and expl-4-190 were closely linkaged to MAT-B stie to which the distance was 2.48 cM and 6.55 cM respectively (LG 10). Pheromone receptor gene fragment "ste-1-371"(STE3) was linked in LG 10 too, but not closely with the MAT-B locus, the distance between two locus was 6.93 cM, which may be due to some transposition or gene insertion in B mating type system. The coverage of markers within 10 cM,5 cM and 2 cM were 99.9%,98.9% and 83.7% respectively.In our experiments, we constructed a high-density molecular genetic linkage map in Lentinula edodes. With the shortesr average distance 1.70 cM in recent research. The coverage of markers within 5 cM was 98.9%, which owed to the high amplification efficiency SRAP and TRAP molecular markers, this also lay a good foundation for future positional cloning, quantitative trait position, and other modern molecular genetic breeding in Lentinula edodes.