Development of whole-genome multiplex assays and construction of an integrated genetic map using SSR markers in Senegalese sole

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.

Chromosome anchoring in Senegalese sole (Solea senegalensis) reveals sex-associated markers and genome rearrangements in flatfish

“…The estimated gene integrity, as determined by BUSCO analysis, revealed 97.0% completeness. For comparison purposes, the assembly statistics for a recent female genome draft of S. senegalensis 20 , 20 are also shown in Supplementary Table S4 . Both genome assemblies had a similar size (608–610 Mb) although the newly assembled male genome had longer contigs with higher N50 values.…”

“…A genome for a female sole was previously reported 17 although it was highly fragmented (N50 = 85 kb, 600.3 MB long). Later, this assembly was improved by integrating Nanopore and Illumina reads, resulting in 5,748 contigs with N50 = 339.9 kb and 608 Mb long 20 (Supplementary Table S4 ). In this study, the newly obtained male assembly has a lower number of contigs (3,403) and higher N50 (512.7 kb) and confirmed that the genome size of sole is around 609 Mb.…”

“…Most importantly, markers were distributed into 21 SseLGs that match with the haploid karyotype (2n = 42) of the species 51 . Until now, two genetic maps with 129–229 microsatellites were reported in Senegalese sole 13 , 20 Moreover, a cytogenetic map was also published although the number of BACs did not still cover all chromosomes 14 , 16 . This new high-density SNP map (Fig.…”

“…Soles used for the preparation of ddRAD libraries and sequencing were selected from the genetic breeding program carried out by the IFAPA in collaboration with a commercial aquaculture company (CUPIMAR S.A.). Production of families used in this study, genotyping and parentage assignment were previously published 19 , 20 . Five families (three full-sib and two maternal half-sib families) containing between 48 and 96 individuals per family (total n = 356) were selected to construct the genetic linkage map (Table 1 ).…”

Genetic Estimates for Growth and Shape-Related Traits in the Flatfish Senegalese Sole

“…DNA was quantified using a Nanodrop ND-8000 and quality was evaluated by agarose gene electrophoresis. Genotyping of breeders and offspring was carried out using an 11-loci supermultiplex PCR [ 20 ] on an ABI3130 sequencer (Applied Biosystems, Foster City, CA, USA) and genotypes were collected using Genemapperv3.8 (Applied Biosystems, Foster City, CA, USA). Finally, parentage assignment was performed with Vitassign v8.2.1 [ 21 ] following the allelic exclusion method.…”