Identification Of Secondary Recombinants And Linked Molecular Markers For Mating-type Factors In Lentinula Edodes

The edible fungus Lentinula edodes is a heterothallic homobasidiomycete whose matingis controlled by a bifactorial incompatibility mating system determined by two unlinkedfactors (the A and B mating type factors). In this study, 13 L. edodes strains were used andmating types of monokaryons derived from each of these strains were exactly identifiedby OWE-SOJ (Oak Wood Extract Agar-Squeezed Orange Juice Agar) technique and bynuclear migration testing. Further analysis and confirmation were carried out upon HL01strain whose progeny may occur recombinants. In addition, taking advantage ofRAPD/BSA, representational difference analysis(RDA) and degenerate PCR, wescreened and identified DNA markers linked to mating type factors of L. edodes, andmade preliminary investigation of the sequences structure and basic properties of themarkers obtained. The main results are as follows.Based on the regular mating types analysis of the 13 L. edodes strains, OWE-SOJtechnique and nuclear migration testing were used for identification of mating typefactors of monkaryons derived from each of these strains. The result showed that, usingOWE-SOJ technique, three kinds of typical colony could be observed, e.g. fluffy colonywith clamp connection formed by A≠B≠pairs, barrage colony without clamp connectionformed by A≠B=pairs, and fluffy colony without clamp connection formed by A=B≠pairs. Furthermore, nuclear migration test demonstrated that nuclear migration occurredonly in A=B≠but not in A≠B=. Therefore both of the two methods can discriminatedefinitely mating reactions between A=B≠and A≠B=, and it is effective to use them toexactly identify mating type factors of L. edodes.In the process of mating type analysis of the 13 L. edodes strains, one strain HL01appeared exceptive phenotype: 132 random pairs in monokaryons derived from onesporocarp of HL01 were made and the proportion of incompatibility to compatibility was82:50, of which the chi-square value was 11.00, extremely distorted the theoreticalchi-square value that consists with 3:1. Using the four standard tester strains, weidentified the mating type of 189 spore monokaryons derived from HL01 and found that161 out of them could be classified into four normal mating types (A₁B₁, A₂B₂, A₁B₂ andA₂B₁) excepting the other 28 monokaryons that probably occurred secondaryrecombination. By crossing in all pairwise combinations, the 28 monokaryons werefurther analysed and found six mating types(A₁B₃, A₁B₄, A₂B₃, A₂B₄, A₃B₂ and A₃B₅),indicating that secondary recombination occurred in both A factor and B factor. And Afactor is composed of at least two subloci with 8.47% recombination frequency; B factor is composed of more than two subloci with 11.64% recombination frequency. Thesubsequent fruiting test revealed that all the compatible pairs contained at least one of therecombinants had the ability to produce fruiting body.To screen and identify the molecular markers related to mating type factors of L.edodes, several DNA marker techniques, such as RAPD-BSA, RDA and degenerate PCRwere employed. Among these methods, by means of degenerate PCR we obtained one773bp DNA fragment cosegregating with B₂ mating-type factor in L. edodes strain HL01.Sequencing analysis revealed it belongs to pheromone receptor, suggesting the geneticbasis for B factor in L. edodes is the same as in the two model mushroom species,Schizophyllum commune and Coprinus cinereus, structure and function of whose Bincompatibility factors have been studied in detail. Also, by degenerate PCR we identifiedpartial DNA fragment (419bp) of MIP gene coding mitochondrial intermediate peptidasethat has been shown tightly linked to A mating type loci (＜1kb) in most other mushroomspecies, including two model organisms. Thus the MIP fragment obtained from HL01establishes preliminary basis for further verification of the linked relationship between Amating loci and MIP gene in L. edodes.Finally, we made chromosome walking using TAIL-PCR and inverse PCR based onpartial DNA fragments of B factor and MIP gene obtained above. It walking from 773bpto 3119bp for B2 factor, 449bp to 2134bp for MIP gene. BLAST searches demonstratedthat nearly entirely sequences of B factor of L. edodes are obtained and that the MIP geneare most likely linked to A mating loci, which provides appropriate basis for cloning andsequencing of A factor in L. edodes...