Abstract
BackgroundGenetic linkage maps are important tools in breeding programmes and quantitative trait analyses. Traditional molecular markers used for genotyping are limited in throughput and efficiency. The advent of next-generation sequencing technologies has facilitated progeny genotyping and genetic linkage map construction in the major grains. However, the applicability of the approach remains untested in the fungal system.FindingsShiitake mushroom, Lentinula edodes, is a basidiomycetous fungus that represents one of the most popular cultivated edible mushrooms. Here, we developed a rapid genotyping method based on low-coverage (~0.5 to 1.5-fold) whole-genome resequencing. We used the approach to genotype 20 single-spore isolates derived from L. edodes strain L54 and constructed the first high-density sequence-based genetic linkage map of L. edodes. The accuracy of the proposed genotyping method was verified experimentally with results from mating compatibility tests and PCR-single-strand conformation polymorphism on a few known genes. The linkage map spanned a total genetic distance of 637.1 cM and contained 13 linkage groups. Two hundred sequence-based markers were placed on the map, with an average marker spacing of 3.4 cM. The accuracy of the map was confirmed by comparing with previous maps the locations of known genes such as matA and matB.ConclusionsWe used the shiitake mushroom as an example to provide a proof-of-principle that low-coverage resequencing could allow rapid genotyping of basidiospore-derived progenies, which could in turn facilitate the construction of high-density genetic linkage maps of basidiomycetous fungi for quantitative trait analyses and improvement of genome assembly.
Highlights
MethodsSingle-spore isolate (SSI) cultivation and DNA extraction Twenty basidiospores were randomly isolated from fresh-fruiting bodies of the L. edodes dikaryotic L54 strain
Genetic linkage maps are important tools in breeding programmes and quantitative trait analyses
We used the shiitake mushroom as an example to provide a proof-of-principle that low-coverage resequencing could allow rapid genotyping of basidiospore-derived progenies, which could in turn facilitate the construction of high-density genetic linkage maps of basidiomycetous fungi for quantitative trait analyses and improvement of genome assembly
Summary
SSI cultivation and DNA extraction Twenty basidiospores were randomly isolated from fresh-fruiting bodies of the L. edodes dikaryotic L54 strain. Shotgun sequencing and genotyping Shotgun sequencing of the 20 SSIs was performed in multiplex with multiplex identifiers (MIDs) on a GS FLXTitanium sequencer (454 Life Sciences Corporation) in 1.5 plates run. Genotyping was performed by mapping the sequencing reads of each SSI to the parental L54A reference genome using the GS Reference Mapper version 2.6 (454 Life Sciences Corporation) with default parameters. Each scaffold sequence (genotype A or B) served as a genetic marker for linkage map construction. On a certain scaffold, if a particular SSI contained more mapped reads with genotype “A” than “B”, the marker was genotyped as “A”, and vice versa. A “X” was assigned in cases where there were an equal number of “A” and “B”, and a “-“was assigned when no reads were mapped on the scaffold.
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