| In this paper,systemically genetic analyses were carried out for some quantitative traits closely related to yield and quality and their relationship in Lentinula edodes. Moreover,using combined RAPD,ISSR,SRAP and SSR molecular markers,a genetic linkage map of L.edodes was constructed based on a population of 131 basidiospores derived from HW21,a wild strain of this basidiomycete,and the most quantitative trait loci of dikaryons and monokaryons were mapped.The main results are summarized as following:1.The mating-types of 238 basidiospores,derived from HW21,a wild strain of L. edodes,collected from hinterland of Shennongjia National Nature Reserve in Hubei Province,China,were exactly identified through normal mating test,OWE-SOJ technique and nuclear migration tests.In order to construct a molecular genetic map of L.edodes,a comparably lager scale and higher genetic diversity mapping population was created with 131 individuals,including 113 spores,belonging to 4 normal mating-types,A1B1,A2B2, A1B2 and A2B1,and 18 ones pertaining to the putative secondary recombination mating types,A2B3,A3B4 and A1B3,out of the mentioned 238 isolates.For genetic analysis of quantitative traits and mapping of quantitative traits loci,four series of tested dikaryons were produced by crossed each basidiospore of mapping population with 4 sporulated or protoplasted monokaryons respectively derived form 2 wild and 2 cultivated strains of Lentinula edodes.2.The correlation and principal component analyses of 17 quantitative traits of dikaryons were made in present study.The results of genetic correlation analyses showed that,fresh weight of single fruitbody(FWS) is positively correlated with other traits of single fruitbody,but negatively correlated with amount of fruitbodies(AF),CMCase activity and xylanase activity at significant or highly significant level.The genetic correlations between fresh yield(FY) and primordium forming period(PFP) and fruiting period(FP) are highly significantly negative.The genetic correlations between FY and AF, growth rates of two kinds of mycelia and pileus thickness are highly significantly positive. These results suggested that,the traits of mycelial growth rate and enzyme activities can be used as the indirect indexes for selection of single fruitbody and fresh yield in edible fungi crossbreeding.In addition,six principal components(PCs) were obtained from seventeen tested traits through the principal component analysis.According to their variance contributive percentage and the cumulative variance contributive percentage,i.e. 80.23%,these PCs were designated as single fruitbody,development,yield,enzyme activity,primordium factor and color-changing factors respectively.These PCs or their subcomponents could be selected independently to improve selective efficiency and objective pertinency for different breeding target.The correlation analyses of 11 quantitative traits of monokaryons were also made in this paper.The positively phenotypic and genetic correlation ship at significant or highly significant level were found among two kinds of mycelial growth rates of monokaryons and the resistance against Trichoderma,and between CMCase activity and xylanase activity,laccase activity and guaiacol oxidase activity.Two kinds of mycelial growth rates were negatively correlated with laccase activity and guaiacol oxidase activity in phenotypic and genetic pattern at significant or highly significant level.The positive correlation ship at highly significant level were also found among mycelial density(MD), regularity degree of mycelia(RDM) and mycelial growth vigor(MDV),while negative pattern in present or absent of pigment against RDM and MDV.The correlation between present or absent of pigment and MD was not significant.3.11,150 ESTs(including 10 cDNAs),about 8.1×106 bp in length,were downloaded from ESTs databases of L.edodes in Fungal Genomics Project(FGP) website and NCBI website.By searching with SSRhunter 1.3 software and handwork,469 SSRs were identified in 316 ESTs,approximately 2.83%of total ESTs.The average distance between SSRs was about 17.3kb.Trinucleotide and hexanucleotide ESR-SSRs were found to be the two most abundant repeats,accounting for 38.00%and 20.00%of the total ESR-SSRs respectively,and(A)n,(T)n,(GA)n,(AG)n,(TGA)n,(GAT)n and(TCTTT)n to be the most familiar motifs,which comprised about 35.94%of all EST-SSRs.Based on the sequence of EST-SSR searched from ESTs databases of L.edodes,87 SSR primer pairs were designed,of which 67(77.0%) primer pairs amplified 90 distinct and denumerable DNA bands,with the average of 1.34 bands per primer pairs.There were 41 polymorphic bands produced by 36 SSR primer pairs in parental protoplast monokaryons and their sporulated progeny.4.A genetic linkage map of L.edodes was constructed on the basis of the 1:1 Mendelian segregation of 94 RAPD markers,53 ISSR markers,104 SRAP markers,16 SSR markers and the A and B mating-type loci among 131 random basidiospore progeny from a single fruitbody of a wild strain HW21.Fourteen linkage groups,including eight major and six minor,covering a total of 2944.1cM,about 210.3cM per linkage group, were identified with the well-proportioned markers distributing on it.The average marker interval was 10.7cM,varying from the maximum(36.8cM between S471290 and S421400 in LG8) and minimum(0.4cM between F107400 and F33170 in LG10) intervals.A total of 16 gaps spanning more than 20cM were observed in this map.This map covers nearly the whole genome of L.edodes.5.There were 78 putative QTLs controlling 28 traits of L.edodes identified in this just constructed linkage map when scanned from the whole genome for 30 traits by means of composite interval mapping(CIM) method,using Winqtlcart 2.5 software.The threshold LOD value of each trait was determined with 1000-times permutations test(P＜0.05).In addition,the LOD scores of another 4 putative QTLs were equal to or higher than 2.5,in despite of no significance when respectively compared to the corresponding LOD criterion.The number of QTLs of most traits ranged between 1 to 4,in despite of one extreme example of 8 QTLs detected controlling mycelial growth rate of monokaryons in PDA medium(MoMGRP).More than one QTL were mapped in all linkage groups except LG9,LG12,LG13 and LG14.The distribution of QTLs were not even resulting in 59 QTLs,about 72.0%were clustered in LG1,LG2,LG3 and LG8.Most of traits controlled by clustered QTLs were closely correlated with each other.The phenotypic variation explained by a single QTL ranged between 6.59%and 63.66%,with the average of 17.29%.A total of 23 QTLs,about 28.05%of all QTLs,with more than 20% contributions were identified and could be considered to be major QTLs.The length of a single QTL,i.e.95%confidence intervals of QTL position,varied from 2.8cM to 28.7cM, with the average of 11.02cM.When performed repetitive mapping of QTLs for 30 traits by Bayesian-MCMC method with QTLnetwork 2.0 software,26 QTLs controlling 17 traits were identified, many of them except few QTLs were found too by CIM,sharing the same marker interval and position on LG,and the similar length,contribution to phenotypic variation and additive effect of certain QTL.Furthermore,6 pairs of epistatic QTL controlling four traits were also mapped,explaining 3.58%to 36.84%of the phenotypic variation,with the average of 18.65%.As a whole,the constructed genetic linkage map of Lentinula edodes in this study had more mapping population,more molecular markers,lengger map distance and new type markers(ISSR,SRAP and SSR) compared with orther genetic maps of this species. The mapping QTLs about some important quantitative traits based on this map was the first time in the world.
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