Limits...
Rapid genotyping by low-coverage resequencing to construct genetic linkage maps of fungi: a case study in Lentinula edodes.

Au CH, Cheung MK, Wong MC, Chu AK, Law PT, Kwan HS - BMC Res Notes (2013)

Bottom Line: Here, we developed a rapid genotyping method based on low-coverage (~0.5 to 1.5-fold) whole-genome resequencing.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.Two hundred sequence-based markers were placed on the map, with an average marker spacing of 3.4 cM.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong.

ABSTRACT

Background: Genetic 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.

Findings: Shiitake 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.

Conclusions: 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.

Show MeSH

Related in: MedlinePlus

Flowchart of the proposed genotyping approach. Low-coverage (~0.5 to 1-fold) resequencing of a selected mapping population, here single-spore isolates (SSIs) I1 to In, is first performed on a next-generation sequencing (NGS) platform such as Roche 454, generating a library of short shotgun reads R1 to Rn for each isolate. Genotyping of the SSIs is then carried out by mapping the reads of each SSI to the draft parental reference genomes, represented in scaffolds S1 to Sn here. On each scaffold, identical mapped reads are assigned the same genotype of the parental strain being mapped whereas reads with high-quality single-nucleotide polymorphisms (SNPs) that are present in the other parent are assigned the opposite genotype. For each SSI, the final genotype (GT) of each scaffold, serving as a genetic marker, is called by a simple majority vote. All the scaffolds are genotyped in this manner. A genetic linkage map, on which linkage information of the markers on a set of linkage groups (LGs) is displayed, is then built based on the genotype segregation ratio of every marker examined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750829&req=5

Figure 2: Flowchart of the proposed genotyping approach. Low-coverage (~0.5 to 1-fold) resequencing of a selected mapping population, here single-spore isolates (SSIs) I1 to In, is first performed on a next-generation sequencing (NGS) platform such as Roche 454, generating a library of short shotgun reads R1 to Rn for each isolate. Genotyping of the SSIs is then carried out by mapping the reads of each SSI to the draft parental reference genomes, represented in scaffolds S1 to Sn here. On each scaffold, identical mapped reads are assigned the same genotype of the parental strain being mapped whereas reads with high-quality single-nucleotide polymorphisms (SNPs) that are present in the other parent are assigned the opposite genotype. For each SSI, the final genotype (GT) of each scaffold, serving as a genetic marker, is called by a simple majority vote. All the scaffolds are genotyped in this manner. A genetic linkage map, on which linkage information of the markers on a set of linkage groups (LGs) is displayed, is then built based on the genotype segregation ratio of every marker examined.

Mentions: Genotyping with various types of traditional molecular markers has been laborious and time-consuming. Sequencing simultaneously a large number of samples in multiplex on NGS platforms allows rapid and more cost-effective genotyping of whole mapping populations. Huang et al. [3] genotyped 150 recombinant inbred lines (RILs) of rice using low-coverage (~0.02-fold) resequencing on an Illumina Genome Analyzer platform. Rapid genotyping of 244 sorghum RILs was also achieved using ~0.07-fold resequencing [5]. Here, we developed a method to rapidly genotype basidiospore-derived progenies of L. edodes by low-coverage resequencing in ~0.5 to 1.5-fold (FigureĀ 2). The approach could very likely be applied on other fungi. However, as the current approach requires genome data from the parental line, for fungal species lacking such information, the parent-independent genotyping approach developed by Xie et al. [2] should be employed instead.


Rapid genotyping by low-coverage resequencing to construct genetic linkage maps of fungi: a case study in Lentinula edodes.

Au CH, Cheung MK, Wong MC, Chu AK, Law PT, Kwan HS - BMC Res Notes (2013)

Flowchart of the proposed genotyping approach. Low-coverage (~0.5 to 1-fold) resequencing of a selected mapping population, here single-spore isolates (SSIs) I1 to In, is first performed on a next-generation sequencing (NGS) platform such as Roche 454, generating a library of short shotgun reads R1 to Rn for each isolate. Genotyping of the SSIs is then carried out by mapping the reads of each SSI to the draft parental reference genomes, represented in scaffolds S1 to Sn here. On each scaffold, identical mapped reads are assigned the same genotype of the parental strain being mapped whereas reads with high-quality single-nucleotide polymorphisms (SNPs) that are present in the other parent are assigned the opposite genotype. For each SSI, the final genotype (GT) of each scaffold, serving as a genetic marker, is called by a simple majority vote. All the scaffolds are genotyped in this manner. A genetic linkage map, on which linkage information of the markers on a set of linkage groups (LGs) is displayed, is then built based on the genotype segregation ratio of every marker examined.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750829&req=5

Figure 2: Flowchart of the proposed genotyping approach. Low-coverage (~0.5 to 1-fold) resequencing of a selected mapping population, here single-spore isolates (SSIs) I1 to In, is first performed on a next-generation sequencing (NGS) platform such as Roche 454, generating a library of short shotgun reads R1 to Rn for each isolate. Genotyping of the SSIs is then carried out by mapping the reads of each SSI to the draft parental reference genomes, represented in scaffolds S1 to Sn here. On each scaffold, identical mapped reads are assigned the same genotype of the parental strain being mapped whereas reads with high-quality single-nucleotide polymorphisms (SNPs) that are present in the other parent are assigned the opposite genotype. For each SSI, the final genotype (GT) of each scaffold, serving as a genetic marker, is called by a simple majority vote. All the scaffolds are genotyped in this manner. A genetic linkage map, on which linkage information of the markers on a set of linkage groups (LGs) is displayed, is then built based on the genotype segregation ratio of every marker examined.
Mentions: Genotyping with various types of traditional molecular markers has been laborious and time-consuming. Sequencing simultaneously a large number of samples in multiplex on NGS platforms allows rapid and more cost-effective genotyping of whole mapping populations. Huang et al. [3] genotyped 150 recombinant inbred lines (RILs) of rice using low-coverage (~0.02-fold) resequencing on an Illumina Genome Analyzer platform. Rapid genotyping of 244 sorghum RILs was also achieved using ~0.07-fold resequencing [5]. Here, we developed a method to rapidly genotype basidiospore-derived progenies of L. edodes by low-coverage resequencing in ~0.5 to 1.5-fold (FigureĀ 2). The approach could very likely be applied on other fungi. However, as the current approach requires genome data from the parental line, for fungal species lacking such information, the parent-independent genotyping approach developed by Xie et al. [2] should be employed instead.

Bottom Line: Here, we developed a rapid genotyping method based on low-coverage (~0.5 to 1.5-fold) whole-genome resequencing.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.Two hundred sequence-based markers were placed on the map, with an average marker spacing of 3.4 cM.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong.

ABSTRACT

Background: Genetic 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.

Findings: Shiitake 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus