Computational protocol: Construction of high-resolution recombination maps in Asian seabass

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Protocol publication

[…] Three full-sib families were used for construction of genetic maps. In detail, Fam1 was a backcross generated by a F2 male offspring crossing its F1 female parent [], while both Fam2 and Fam3 were independent F2 populations from two different parents set up as described in our previous study []. For each family, two parents and 192 progeny randomly selected and genotyped with microsatellites and genotyping-by-sequencing using the ddRADseq approach [].Genomic DNA was isolated from fin tissue using the salt precipitation method []. 149 microsatellites, almost evenly covering the genome, were genotyped according to a previous study []. GBS libraries were constructed according to our previous method []. In brief, 300 ng genomic DNA was digested with PstI-HF and MspI restriction enzymes (New England Biolabs, USA) and was then ligated with adaptors using T4 ligase (New England Biolabs, USA). The ligation products were pooled for size selection of 300–500 bp by running gels, after clean up with QIAquick PCR Purification Kit (Qiagen, Germany). The libraries were enriched using PCR with Phusion® High-Fidelity DNA Polymerase (New England Biolabs, USA). After a final clean up using QIAquick PCR Purification Kit (Qiagen, Germany), ddRADSeq libraries were sequenced using a NextSeq 500 platform (Illumina, USA) for either paired-end (2 × 150 bp) or single-end (1 × 150 bp).Raw reads processing and SNP genotyping were conducted using the software package Stacks v1.34 []. Reads were trimmed to 120 bp and those with any uncalled base were removed. QC filtered reads were aligned against the reference genome of Asian seabass [] using the program BWA with a maximum of two mismatches []. Alignments with multiple genome targets were excluded from further analysis. Reference aligned reads were used for stacks assembly using pstacks implemented in the package Stacks v1.34 []. The stacks assembled for the parents of the three families were used to construct a catalogue with the program cstacks Stacks v1.34 []. The catalogue of loci was then used as reference for SNP discovery and genotyping for each family with the program populations implemented in Stacks v1.34 []. SNP filtrations were conducted similarly to our previous study with some modifications []. In brief, RAD tags with any SNP of > 2 alleles and showing heterozygosity of > 0.5 were removed []. A minimum of 10 × sequence depth and a genotyping success of > 85% of the individuals in each family were used for SNP genotyping. In order to reduce the number of missing genotypes, samples with low sequencing depth were ruled out for each family. Only one SNP per tag was retained for map construction. [...] All mapped microsatellites with flanking sequences were aligned against the genome assembly, including all contigs and scaffolds, of Asian seabass [] using BLAST with a cutoff of 1E−10 and a minimal sequence identity of 95%. The syntenic relationships between genetic maps and the genome assembly were constructed and the integration was achieved by anchoring the contigs and scaffolds onto the genetic maps according to marker positions. The program Circos [] was used to visualize the genomic synteny and integration between genetic maps and genome assembly. The accuracy of genome assembly was examined by integration of both consensus and individual genetic maps with the genome assembly. […]

Pipeline specifications

Software tools BWA, Circos
Applications GBS analysis, Genome data visualization
Organisms Lates calcarifer