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.

Figure 1

Allelic ranges of the 106 SSRs analysed in this study by fluorescence labelling (A–D). The name of the multiplex PCRs in which each marker is included is indicated between brackets. The asterisk indicates that the marker was selected to be included in the supermultiplex PCRs.

Figure 2

Allelic ranges of the 40 SSRs selected for the supermultiplex (SM) PCRs. The markers are shown by SM (A–D).

Figure 3

Cumulative success rate for parentage assignment based on exclusion with markers ranked on PIC value. The grey area indicates the loci required to reach more than 99% probability of assigning a correct parent–offspring relationship. SMA n = 92 parents; SMB, n = 15 parents; SMC, n = 15; SMD, n = 15.

Figure 4

Integrated SSR genetic map of Senegalese sole (S. senegalensis). SseLG refer to the linkage groups according the high-density SNP genetic map. Genetic distance is indicated on the left. SSRs of this study are indicate in black and those from Molina-Luzón et al., 2015 in blue. The LGs previously assigned to these markers are shaded and indicated on the left. (a) SseLG1- SseLG10; (b) SseLG11- SseLG21.