Computational protocol: ddRADseq reveals determinants for temperature-dependent sex reversal in Nile tilapia on LG23

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Protocol publication

[…] Flanking sequences to amplify SNP Oni23063 and Oni28137 were derived from Scaffold NC022199.1. The genomic sequence of the amh gene was derived from Scaffold GL831234.1 of the Nile tilapia genome sequence deposited in the Ensembl database (; Orenil1.0 GCA_000188235.1; location of amh: Scaffold GL831234.1, 1.688.687–1.691.779). Gene- and locus-specific primers were designed using the Primer3 software (see Additional file : Table S4). Primers were used to amplify a specific fragment of 561 bp harbouring SNP Oni23063 and Oni28137 as well as another 1252 bp long PCR fragment flanking exon 6 of amh. PCRs were carried out using 20 ng of genomic DNA, 1× PCR buffer containing MgCl2, 1× Q-solution, 10 pM of each primer, 10 mM dNTPs and 2 U FastStart Taq DNA polymerase in a final volume of 25 μl. All PCR components, except for primers (MWG-Biotech, Ebersberg, Germany) and the 1× Q-solution (Qiagen, Hilden, Germany) were purchased from Roche (Roche,Penzberg, Germany). PCR was performed using a Biometra T-3000 Thermocycler (Biometra,Goettingen, Germany) with an initial denaturation at 95 °C for 10 min, followed by 35 cycles of 92 °C for 30 s, 60 °C for 30s and 72 °C for 1 min with a final extension at 72 °C for 5 min. The fragment identity was controlled via gel-electrophoresis on 1.5% agarose gels. Primers for amh were specific for all three reported forms of amh, i.e. the X-linked amh, and the Y-specific amhy homolog as well as amhΔY []. First of all amh was screened for 120 genetically female individuals and the 10 YY supermales. AmhΔY features a 5 bp insertion in Exon VI (ATGTC), which contains a Taq αI specific recognition site. Therefore, all amplicons were Taq α I (NEB, Frankfurt, Germany) digested and separated on 2% agarose gels. The X-linked amh and the Y-linked amhY were represented by the amplicon of 1252 bp length, whereas, the Y-linked amhΔY was represented by cleaved fragments of 829 and 423 bp in length. In addition to the restriction digest, all results were further confirmed using Sanger sequencing. M13-tailed primers enabled direct bidirectional sequencing on an ABI-PRISM 3100® capillary analyser (Life Technologies, Darmstadt, Germany) using the Big Dye terminator Kit. PCR products were before purified with Exo-SAP-IT® (Thermo Fisher Scientific, Schwerte, Germany). The obtained sequences were trimmed, contigs were built, and SNPs were manually identified using the program software suite DNASTAR LasergeneTM6® (DNASTAR, Madison, WI, USA). In Nile tilapia populations with an LG1 sex chromosome, genetic females are supposed to carry genotypes G/G and T/T at loci Oni23063 and Oni28137, respectively []. The available genome reference sequence of O. niloticus exhibits G/G at locus Oni23063 and T/T at Oni28137, as it was derived from an individual of an XX homozygous isogenic line. Genetic males are supposed to be heterozygous showing genotypes A/G and G/T for loci Oni23063 and Oni28137, respectively []. [...] The resulting raw single-end reads were demultiplexed using a custom Perl script allowing 1 mismatch in the adapter sequence. Reads shorter than 95 bp were filled with ‘N’. All reads were then truncated to 95 base pairs, quality filtered, and merged into one file per individual. Reads were subsequently mapped onto the Tilapia genome version Orenil1.1.( using the programme package BWA []. All resulting BAM-files were further analysed using the Stacks program Version 1.34 []. The datasets generated and analysed during the current study are available in the NCBI Bioproject repository, (Accession: PRJNA354565; First, ddRAD tags were analysed using the script, with a minimum stack depth of 5 (−m 5), allowing 1 mismatch between loci when building the catalogue (−n 1). The file catalog.snps.tsv was screened for erroneous SNP alleles resulting from in silico elongation of reads to 95 bp. All loci containing the erroneous ‘N’-allele were blacklisted using the –B option in subsequent analyses.Secondly, the population script was applied in order to calculate population genetic parameters. As the present study explicitly aimed to further decipher determinants for temperature-dependent sex reversal, exclusion of autosomal genes or other sex-skewing modifiers was paramount. As only family 1 was devoid of males in the control group, initially this family was chosen for the ddRADseq approach. Subsequently families 2 and 3, which showed some sex-reversal in the controls, too, were additionally sequenced for a case-control approach. Independent runs of the populations script were performed comprising the following data sets: data set 1) comparison of 20 temperature-treated males with 20 females in family 1; 2) comparison of 60 temperature-treated males (affected cases) and 60 temperature-treated but non-masculinized females (unaffected controls) from families 1, 2, and 3. All data sets were filtered equally to a stack depth of >5 (−m 5), a minor allele frequency of >0.01 (--min_maf 0.01), and a minimum percentage of individuals in the population required to process the locus >70% (−r 0.7), ddRAD tags were requested to be present in all populations within a data set (−p 2). SNP and haplotype-based F-statistics were requested using the --fstats command, kernel smoothing of FST was enabled using the –k option applying a default window size of 3σ (150 Kbp). The fixation index FST was calculated with Stacks 1.34 [] using an adapted formula, accounting for unequal sample size among populations by weighting []:FST=1−∑inj2πjπ.∑inj2where n j is the number of alleles sampled in population j, p j is the nucleotide diversity within population j, and π is the total nucleotide diversity across the pooled populations. Expected and observed heterozygosity (H) as well as nucleotide diversity (π) were calculated using the populations script. […]

Pipeline specifications

Software tools Primer3, BWA
Applications Population genetic analysis, qPCR
Organisms Oreochromis niloticus, Homo sapiens, Danio rerio