| The basidiomycete Lentinula edodes, well-known for its highly nutritional and medicinal values, is one of most cultivated edible mushrooms throughout the world. Screening and breeding of superior strains are crucial for sustainable development of L. edodes production and marketing. China has abundant natural germplasm resources of L. edodes due to a vast territory. These rich germplasm resources are the basis of breeding programme of L. edodes. Most of agronomic traits of L. edodes were quantitative traits controlled by multi-gene and easily impacted by environment. It is difficult to perform genetic improvement only based on the phenotypes of L. edodes. Marker-assisted selection (MAS), using markers associated with phenotype, may be integrated into regular breeding schemes to improve the breeding efficiency. Association studies based on linkage disequilibrium (LD) offers a new method to analyze the genetic basis of the complex traits.95 wild strains and one cultivated strain of L. edodes were used in this study. Then we determined the phenotypic data of 15 agronomic traits in those strains.95 InDel markers and two SSR markers that developed from the whole genome sequence of L. edodes were used to scan the genome of the tested strains. Our objective was to:(1) determine the genetic diversity, genetic differentiations and population structure of the tested strains; (2) to analyze the physical distance of LD decay in a single scaffold; (3) to perform association analysis between 15 agronomic traits and 97 gennetic loci using mixed linear model. The major results are as follows:1. Multivariate statistical methods were used to analyze fifteen important agronomic traits in 96 shiitake strains. Results indicated that the number of fruiting bodies was positively correlated with yield, but negatively correlated with the weight of single fruiting body; positive correlations were found between pairs of agronomic traits related to the basidiocarp; there were no significant correlations between mycelium growth rate in synthetic media and agronomic traits in fruiting trials; the fruiting bodies precocity was negatively correlated with yield and the weight of single fruiting body respectively.2.97 markers, including 78 candidate-gene markers and 19 markers from L. edodes genome, were employed to evaluate genetic diversity, genetic differentiations and population structure among the wild strains. A high genetic diversity was found in Chinese L. edodes strains, yet there was considerably genetic differentiation between populations (FST=0.282). Results from analysis of molecular variance suggested that majority of the genetic variation was contained within populations (71%). Analysis of population structure revealed two unambiguous genetic groups, which were highly consistent with two geographic regions.3.4656 pairwise LD values between the 97 loci were relatively low, raning from 0 to 0.20. By calculating the r2 value of pairwise markers in Scaffold Le_N7_S278 in L. edodes draft genome, we found that r value declined rapidly as the genetic distance increased. When the cut off value of r2 was setted to 0.20, the decay of the LD in this Scaffold is about 3100bp.4. Twenty markers were found to associate with ten agronomic traits in L. edodes (P<0.001), nine of which were associated with two or more than two agronomic traits. Among these markers, three were significant associated with the mycelium growth rate in MYG medium. The numbers of marker significantly associated with the mycelium growth rate in sawdust, primordium forming period, fruiting bodies precocity and pileus diameter were respectively one. Four markers were significant associated with the pileus thickness. Nine markers were significant associated with the pileus weight. Two markers were significant associated with the stipe weight. Seven markers were significant associated with the weight of single fruiting bodies. Four markers were significant associated with the yield. Six associated markers were designed form the candidate genes including laccase gene, alpha-ketoglutarate-dependent dioxygenase gene, isoamyl alcohol oxidase gene, polygalacturonase gene, S-adenosyl-L-methionine-dependent methyltransferase gene and cysteine proteinase gene. These candidate genes were associated with pileus thickness, pileus weight, stipe weight and weight of single fruiting bodies.5. We uncovered the excellent allelic variations at loci significantly associated with the fifteen agronomic traits in L. edodes and their typical carrier strains. In total,22 carrier strains harboured 42 excellent allelic variations. The marker-trait association developed here provides a reference for molecular marker assisted breeding in L. edodes. |
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