Genotyping-by-Sequencing (GBS) of large amphibian genomes: a comparative study of two non-model species endemic to Italy

in Animal Biology
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Abstract

Next Generation Sequencing (NGS) and related technologies have revolutionized the field of conservation and population genetics, providing novel tools and the capacity to discover thousands of new Single Nucleotide Polymorphisms (SNPs) for the analysis of population parameters. However, gathering NGS data for organisms with very large genomes, such as amphibians, remains challenging because it is still unclear how the current methods perform. Here, we use the Genotyping-by-Sequencing (GBS) approach to generate SNP data for the genotyping of two amphibian species that are of conservation concern, the Sardinian brook salamander (Euproctus platycephalus) and the Italian stream frog (Rana italica). Both E. platycephalus and R. italica have very large genomes (5.53 Gb and >20 Gb, respectively) so genomic data are not available for either of them. We used 95 individual samples and one Illumina lane for each species, with an additional lane for E. platycephalus. After filtering, we obtained 961 and 854 high-coverage SNPs for E. platycephalus and R. italica, respectively. Our results suggest that GBS can serve as a reliable and cost-effective method for genotyping large amphibian genomes, including non-model species.

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Figures
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    Fragment size distribution of GBS libraries made with a single DNA sample (left: E. platycephalus; right: R. italica) using three restriction enzymes (top: ApeKI; middle: EcoT22I; bottom: PstI). The x-axis represents elution time and the y-axis shows fluorescence units. Numbers below hatch marks on the x-axis indicate fragment size (bp). Tall peaks at 15 and 1500 bp are size standards.

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    Summary of the number of tags and SNPs obtained with the restriction enzymes EcoT22I and SbfI during the optimization process.

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    Summary of statistical results of sequencing for E. platycephalus and R. italica, divided by locus, individual and plex level.

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    Distribution of the number of sequence reads. (A) Number of good barcoded reads in 95 DNA samples in one Illumina lane for E. platycephalus. (B) Number of good barcoded reads in 95 DNA samples in two combined Illumina lanes for E. platycephalus. (C) Number of good barcoded reads in 95 DNA samples in one Illumina lane for R. italica.

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    Comparison of the number of SNPs obtained for E. platycephalus and R. italica with different sequencing depth. For E. platycephalus, results for one lane (EP 1 lane) and two lanes (EP 2 lanes) are shown. For R. italica we have only one lane (RI 1 lane). White bars represent the number of SNPs obtained from the application of the UNEAK pipeline without filter application, gray bars represent the number of SNPs retained after having filtered for Call Rate < 0.80 and black bars represent the final number of SNPs obtained after having filtered for MAF < 0.05. Note the y-axis break between 2500 and 20 000.

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    Estimates of population genetic parameters for each of the E. platycephalus and R. italica populations.

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    Unrooted neighbor-joining trees, based on pairwise genetic distance, representing the basic population genetic structure of (A) E. platycephalus and (B) R. italica.

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