Abstract
The Senegalese sole (Solea senegalensis) is an economically important flatfish species. In this study, a genome draft was analyzed to identify microsatellite (SSR) markers for whole-genome genotyping. A subset of 224 contigs containing SSRs were preselected and validated by using a de novo female hybrid assembly. Overall, the SSR density in the genome was 886.7 markers per megabase of genomic sequences and the dinucleotide motif was the most abundant (52.4%). In silico comparison identified a set of 108 SSRs (with di-, tetra- or pentanucleotide motifs) widely distributed in the genome and suitable for primer design. A total of 106 markers were structured in thirteen multiplex PCR assays (with up to 10-plex) and the amplification conditions were optimized with a high-quality score. Main genetic diversity statistics and genotyping reliability were assessed. A subset of 40 high polymorphic markers were selected to optimize four supermultiplex PCRs (with up to 11-plex) for pedigree analysis. Theoretical exclusion probabilities and real parentage allocation tests using parent–offspring information confirmed their robustness and effectiveness for parental assignment. These new SSR markers were combined with previously published SSRs (in total 229 makers) to construct a new and improved integrated genetic map containing 21 linkage groups that matched with the expected number of chromosomes. Synteny analysis with respect to C. semilaevis provided new clues on chromosome evolution in flatfish and the formation of metacentric and submetacentric chromosomes in Senegalese sole.
Highlights
Genomes are an essential source of markers required for ecological studies, breeding programs, traceability or functional studies
The aim of this study was to: (1) provide de novo improved assembly of a female Senegalese sole based on long and short reads; (2) identify tetra- or pentanucleotide SSRs in silico and carry out a flatfish cross-species comparison to design whole-genome Multiplex PCRs; (3) validate all SSR loci, structure in multiplex PCRs according to allelic ranges and optimize amplification conditions for whole genome mapping; (4) design supermultiplex PCRs containing the most polymorphic loci to sustain breeding genetic programs in this species in which offspring is communally reared; and (5) integrate SSR markers available in Senegalese sole in a genetic linkage map and carry out a synteny analysis with the flatfish C. semilaevis to understand chromosome evolution
The final set of SSRs selected for primer design included 103 tetranucleotides, 5 pentanucleotides and 5 compound markers containing at least two tetranucleotide SSRs separated by a spacer (Supplementary Table S2 tab "InitialMultiplexDesign")
Summary
Genomes are an essential source of markers required for ecological studies, breeding programs, traceability or functional studies. Genome analysis provides enough information for in silico analysis to select and combine high polymorphic SSR markers while they maintain an reliable and robust scoring for multiplex PCRs. The aim of this study was to: (1) provide de novo improved assembly of a female Senegalese sole based on long and short reads; (2) identify tetra- or pentanucleotide SSRs in silico and carry out a flatfish cross-species comparison to design whole-genome Multiplex PCRs; (3) validate all SSR loci, structure in multiplex PCRs according to allelic ranges (with up to 11-plex amplification) and optimize amplification conditions for whole genome mapping; (4) design supermultiplex PCRs containing the most polymorphic loci to sustain breeding genetic programs in this species in which offspring is communally reared; and (5) integrate SSR markers available in Senegalese sole in a genetic linkage map and carry out a synteny analysis with the flatfish C. semilaevis to understand chromosome evolution
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