Delayed effect of low rearing temperature on gonadal DNA methylation in juvenile barramundi (Lates calcarifer)

Induction of precocious females in the protandrous barramundi (Lates calcarifer) through implants containing 17β-estradiol - effects on gonadal morphology, gene expression and DNA methylation of key sex genes

“…This result is also consistent with previous research on barramundi, where methylation of cyp19a1a, but not foxl2, is significantly different in adult male and female barramundi (Domingos et al, 2018). While the expression of esr1 is known to be epigenetically mediated in humans (Yoshida et al, 2000) and DNA methylation in esr1 is significantly different in adult male and female barramundi (Budd, 2020), no significant changes in methylation of esr1 were observed in response to the E 2 treatment applied here. Given the variation of esr1 expression patterns seen across taxa (Budd, 2020), the functional role of esr1 in barramundi is still not definitive.…”

“…Approximately 500 ng of extracted gDNA was subject to bisulphite treatment using EZ DNA Methylation-Gold™ (Zymo Research) following the manufacturer"s instructions. Primers were derived from previous studies (Table 1) (Budd, 2020;Domingos et al, 2018) and PCR amplification was carried out using Platinum® Taq DNA Polymerase (Thermo Fisher Scientific) following the manufacturer"s instructions with an annealing temperature of 57.5 o C. PCR products were purified using Sera-Mag SpeedBeads as described above and quantified using QuantiFluor (Promega) fluorometric nucleic acid quantitation on an EnSpire Multimode plate reader (PerkinElmer). Library preparation was adapted from a 16s metagenomic sequencing library preparation protocol (Illumina, 2013) with the modifications and analysis performed as described in Budd (2020).…”

“…Primers were derived from previous studies (Table 1) (Budd, 2020;Domingos et al, 2018) and PCR amplification was carried out using Platinum® Taq DNA Polymerase (Thermo Fisher Scientific) following the manufacturer"s instructions with an annealing temperature of 57.5 o C. PCR products were purified using Sera-Mag SpeedBeads as described above and quantified using QuantiFluor (Promega) fluorometric nucleic acid quantitation on an EnSpire Multimode plate reader (PerkinElmer). Library preparation was adapted from a 16s metagenomic sequencing library preparation protocol (Illumina, 2013) with the modifications and analysis performed as described in Budd (2020). Dmrt1 reactions were observed to amplify poorly during PCR amplification, generating an insufficient number of sequence reads for further analysis, and as such were excluded.…”