DOI: 10.5061/dryad.cvdncjtc5
title: Mitochondrial epigenetics brings new perspectives on doubly uniparental inheritance in bivalves

description: Mitochondrial epigenetics, particularly mtDNA methylation, is a
 flourishing field of research. MtDNA methylation appears to play multiple
 roles, including regulating mitochondrial transcription, cell metabolism
 and mitochondrial inheritance. In animals, bivalves with doubly
 uniparental inheritance (DUI) of mitochondria are the exception to the
 rule of maternal mitochondrial inheritance since DUI also involve a
 paternal mtDNA transmitted from the father to sons. The mechanisms
 underlying DUI are still unknown, but mtDNA methylation could play a role
 in its regulation. Here, we investigated mtDNA methylation levels and
 machinery in gonads of the mussel Mytilus edulis using
 methods based on antibodies, enzymatic cleavage and methylome sequencing.
 Our results confirm the presence in mitochondria of methylated cytosines
 and adenines and methyltransferases and unveil a more variable cytosine
 methylation state among males than females. Also, spermatid mtDNA is
 always methylated, while only few spermatozoa present methylated mtDNA
 suggesting a relation between cytosine methylation and development stage
 of male gametes. We propose that mtDNA methylation could play a role in
 the different fates of the parental mtDNAs in male and female embryos
 in M. edulis. Our study provides novel insights into the
 epigenetic landscape of bivalve mtDNA and highlights the multiple roles of
 mtDNA methylation in animals.
description: <strong>DNA extractions</strong> : DNA extractions
 used for enzymatic digestions were performed on 9 male and 9 female of
 <em>Mytilus edulis</em> gonad samples with phenol-chloroform
 following a protocol adapted to molluscan tissues (Sokolov, 2000). DNA
 extractions used for methylome sequencing were performed on gonad tissues
 of 5 males and 3 females of <em>M. edulis </em>following the
 DNeasy blood & tissue<em> </em>kit (QIAGEN) protocol.
 DNA samples were then digested to linearize mtDNA using
 <em>Swa</em>I restriction enzyme (New England BioLabs),
 following the manufacturer’s protocol. Linearized mtDNA (and digested
 nuclear DNA) was then purified with a QIAquick PCR purification kit
 (QIAGEN) according to the manufacturer’s protocol.
 <strong>Enzymatic digestions : </strong>DNA samples
 were digested with the cytosine methyl-sensitive restriction enzyme (MSRE)
 <em>Hpa</em>II and its insensitive isoschizomer restriction
 enzyme <em>Msp</em>I, as well as with an adenine MSRE,
 <em>Dpn</em>I, and its methyl-insensitive isoschizomer
 <em>Dpn</em>II. Digestions were performed separately for each
 restriction enzymes in a 50 µl reaction mix containing 1 µg of total DNA,
 10 to 20 U of restriction enzymes, <em>i.e. </em>FastDigest
 <em>Msp</em>I, FastDigest <em>Hpa</em>II
 (Thermofisher Scientific), <em>Dpn</em>I or
 <em>Dpn</em>II (New England Biolabs) and 1X of their
 appropriate buffer. Digestions were carried out at 37°C for 10, 20, 30 or
 60 min (<em>Msp</em>I and <em>Hpa</em>II) or 15
 min (<em>Dpn</em>I and <em>Dpn</em>II).
 Adenine-specific enzymes were inactivated by heat treatment at 80°C
 (<em>Dnp</em>I) or 65°C (<em>Dpn</em>II) for 20
 min. <strong>Methylome sequencing</strong>
 : DNA samples were submitted to mitochondrial methylome sequencing.
 Libraries were performed at the Génome Québec Innovation Centre (Montreal,
 QC, Canada) using 200 ng of linearized and purified DNA samples and a
 lambda phage DNA (negative control) with the NEBNext® Enzymatic Methyl-Seq
 kit (New England BioLabs), following the manufacturer’s protocol.
 Libraries were subjected to 150 bp paired-end sequencing with Illumina
 NovaSeq 6000, using the NovaSeq Xp protocol. Finally, all reads from the
 libraries were generated with the Illumina bcl2fastq2 (v2.20)
 program.
description: # The epigenetic hypothesis of doubly uniparental inheritance of
 mitochondria: unveiling the mtDNA methylation toolkit in the blue mussel
 Mytilus edulis
 [https://doi.org/10.5061/dryad.cvdncjtc5](https://doi.org/10.5061/dryad.cvdncjtc5) ## Description of the data and file structure The dataset comprises text files of enzymatic digestion levels (labelled "ratios.merged.MF.txt", "ratios.enzymes.txt" and "ratios_enzymes_diff.txt.") of *Mytilus edulis* DNA samples (**available on Zenodo**), which can be used in the R code (see below). Digestion levels were calculated using ImageJ software program following the DNA band quantification protocol from Davarinejad (2015). The dataset also comprises fastqsanger files of paired-end sequencing data (R1 and R2) from *M. edulis* DNA samples (**available on Dryad)**, as well as text files of the processed reads (**available on Zenodo**), labelled "*SampleNumber_RefGenome*_seq.txt" (Sample number = M1 to M5 male samples and F3, F5, F6 for female samples), which are also used in the R code. Ref genome = mitotype M, mitotype F, nDNA or Lambda DNA; see below for genome accession numbers in Genbank). Reads processing includes quality control of reads using FastQC 0.11.8 (Andrews, n.d.) and trimming using Fastp 0.23.2 (Chen *et al.*, 2018) as well as Trim Galore! 0.6.7 (Krueger, 2021). Alignment to *M. edulis* reference genomes was done using Bismark Mapper with the relaxing stringency function L,0,-0.4 (Krueger and Andrews, 2011). Methylation calls were obtained using Bismark Meth. Extractor 0.22.1 (Krueger and Andrews, 2011). ## R Code The code file ("Script_Lerouxetal.R") can be used on R software (Ihaka and Gentleman, 1996) and includes data calling and formatting, figures build-up and statistical analysis necessary for the article. The R code and the datasets are available in Zenodo. **Statistical analysis** : * Enzymatic digestion analysis : Statistical analysis for CCGG methylation determination was made with Student’s T-tests to detect any significant difference in mean digestion level by *Msp*I and *Hpa*II. To assess whether the amount of methylated/unmethylated CCGG or GATC was uniform among males and females, the difference in variability of digestion levels among males and females was statistically evaluated by Fisher’s F-tests. * Methylome sequencing : Statistical analysis for strand-bias coverage was made with a series of Wilcoxon tests. ## Reference genomes ***Mytilus edulis*** **genomes accession numbers (Genbank):** * M mtDNA (Mitotype M): AY823624 * F mtDNA (Mitotype F): AY484747 * Nuclear DNA (nDNA) contig: CAJPWZ010000084 **Lambda spike-in genome accession number (Genbank)**: J02459