, The sex locus of E. lucius is located in the sub-telomeric region of LG24

, To identify the 213 sex chromosome in the genome of E. lucius, we generated RAD-Seq data from a single full-214 sibling family of E. lucius with two parents, 37 phenotypic male offspring, and 41 phenotypic 215 female offspring. In total, 6,512 polymorphic markers were aligned to the 25 nearly 216 chromosome-length linkage groups and 741 polymorphic markers were aligned to, The pool-seq results located the sex locus on unplaced scaffold 1067

, 025) had a higher average F st between males and females 220 than the genome average, and only markers on LG24 showed genome-wide significant 221 association with sex phenotype (Fig 3A), indicating that LG24 is the sex chromosome of E. (Fig 3B), and the strongest association with sex was also identified for markers 225 aligned to this region (Fig 3C), pinpointing the location of the sex locus to the sub-telomeric 226 region of LG24. In addition, with our parameters, 32 non-polymorphic RAD markers were 227 found only in the father and all male offspring, 15 of which could be aligned to the reference 228 XX genome (Table S3): five (33%) to a region located distal to 22, LG2 (F st = 0.006) and LG24 (F st = 0

, Moreover, two of the other 17 male-specific markers which could not be aligned to the reference 231 genome aligned perfectly to the sequence of amhby

, mg/ml) for 30 minutes at room temperature, and incubated 594 in post-fix solution (4% paraformaldehyde and 0.2% glutaraldehyde) for 20 minutes. Then, 595 samples were incubated for 1 h at 65 °C in a hybridization buffer containing 50% formamide, 596 5% SCC, 0.1% Tween 20, 0.01% tRNA (0.1 mg/ml), and 0.005% heparin. RNA probes were 597 added and samples were left to hybridize at 65 °C for 16 h. Afterwards, samples were washed 598 three times with decreasing percentages of hybridization buffer, 2000.

D. Corp and I. ). Indianapolis, After visual inspection of coloration, samples were 604 dehydrated and embedded in plastic molds containing paraffin with a HistoEmbedder

. Medite, ) instrument. Imaging 607 of the slides was performed with an automated microscope (Eclipse 90i

. Huang, For each subunit, the target-specific TALE DNA binding domain consisted 613 of 16 RVD repeats obtained from single RVD repeat plasmids kindly provided by Bo Zhang 614 (Peking University, China), vol.81

, At 624 two months post fertilization, fin clips were collected from 36 surviving animals for genotyping 625 with primers flanking the TALENs targets. Amplification of primers flanking the TALENs 626 targets on amhby was also used for sex genotyping. Only genetic males with a disrupted amhby 627 sequence were raised until the following reproductive season. The sperm of three one-year old 628 G0 mosaic phenotypic males with a disrupted amhby sequence was collected and then used in 629 in vitro fertilization with wild-type eggs

, G1 amhby mutants were then euthanized and dissected, and their gonads were subjected to

, 100 ng/L at the one-cell stage. Two months post fertilization, fin 637 clips were collected and used for genotyping with primer pairs in which one primer was located 638 on the fosmid vector sequence, which does not come from the Northern pike genome, and the with Ovaprim (Syndel, Ferndale, 643 fosmid was collected and used for in vitro fertilization with wild-type eggs, vol.636

, Gonads to be processed for histology were fixed immediately after dissection in Bouin's 651 fixative solution for 24 hours. Samples were dehydrated with a Spin Tissue Processor Microm 652 (STP 120, ) and embedded in plastic molds in 653 paraffin with a HistoEmbedder

. Medite, Embedded samples were 654 cut serially into slices of 7 µm using a MICRO HM355

, Germany) and stained with Hematoxylin. Imaging was performed with an automated 656 microscope (Eclipse 90i

, were by histological analysis of the gonads at 8-month post fertilization. The library was 667 sequenced in one lane of Illumina HiSeq 2500. Raw reads were analyzed with the Stacks 668 (Catchen et al., 2011) program version 1.44. Quality control and demultiplexing of the 669 169,717,410 reads were performed using the process_radtags.pl script with all settings set to 670 default, All statistical analyses, including Wilcoxon signed rank test and Chi-squared test, vol.128, p.481

, M. retained sequences from the father, ~ 0.9 M. retained sequences from the mother, and

, GenBank assembly accession: GCA_000721915.3) using BWA (version 0.7.15-r1140, vol.86

, Results of ref_map.pl were 677 analyzed with populations using the --fstats setting to obtain population genetic statistics 678 between sexes. Fisher's exact test was performed on all polymorphic sites using Plink (version 679 1.90b4.6 64-bit, [87]) to estimate association between variants and phenotypic sex. A Manhattan 680 plot was constructed in R with homemade scripts showing -log 10

, The genome of one local phenotypic male Northern pike (Esox lucius) was assembled 685 using Oxford Nanopore long reads and polished with Illumina reads

, FC-687 121-4001) following the manufacturer's instructions. First, 200 ng of gDNA was briefly 688 sonicated using a Bioruptor sonication device (Diagenode, Liege, Belgium), end-repaired and 689 size-selected on beads to retain fragments of size around 550 bp, and these fragments were A-690 tailed and ligated to Illumina's adapter, Illumina short reads libraries were built using the Truseq nano kit (Illumina, ref

, Libraries were checked with a Fragment Analyzer (AATI) and quantified by qPCR using the

, Kapa Library quantification kit. Libraries were sequenced on one lane of a HiSeq2500 using

, DNA purity was checked using a NanoDrop 699 ND2000 spectrophotometer (Thermo scientific, Wilmington, DE) and size distribution and 700 degradation were assessed using a Fragment analyzer (AATI) High Sensitivity DNA Fragment 701 Analysis Kit

, A DNA damage 704 repair step was performed on 5 µg of DNA, followed by an END-repair and dA tail of double 705 stranded DNA fragments, and adapters were then ligated to the library, Libraries were loaded 706 on R9.4.1 flowcells and sequenced on a GridION DNA sequencer

, 346 nucleotides were 709 used in the assembly, Adapters were removed using Porechop, vol.17

, 711 using standard parameters, with genomeSize set to 1.1g to match theoretical expectations, vol.89

, Two rounds of polishing were performed with racon version 1.3.1 713 using standard parameters. For this step

, The 718 assembly's completeness was assessed with BUSCO [93] using the Actinopterygii gene set 719 (4,584 genes) and the default gene model for Augustus. The same analysis was performed on 720 the Elu_V3 reference genome

, ). separately. Libraries were constructed using a Truseq nano kit (Illumina, ref. FC-121-731 4001) following the manufacturer's instructions. DNAseq shorts reads sequencing was 732 performed at the GeT-PlaGe core facility of INRA Toulouse, DNA from 30 males and 30 females from the fish production unit of the fishing 725 federation of Ille-et-Vilaine (Pisciculture du Boulet, vol.728

T. Nano, D. Ht-library-prep-kit, ;. Illumina, and S. Diego, CA) following the 735 manufacturer's protocol. First, 200ng of DNA from each sample (male pool and female pool) 736 was briefly sonicated using a Bioruptor sonication device (Diagenode, Liege, Belgium), and 737 then end-repaired and size-selected on beads to retain fragments of size around 550 bp, and 738 these fragments were A-tailed and ligated to indexes and Illumina's adapter. Libraries were 739 checked with a Fragment Analyzer, ) and 740 quantified by qPCR using the Kapa Library Quantification Kit

I. ). Indianapolis, CA) 742 using paired-end 2x150 nt mode with Illumina NovaSeq Reagent Kits following the 743 manufacturer's instruction. The run produced 129 millions of read pairs for the male pool

, picard) with default 751 parameters. Then, a pileup file combining both BAM files was created using samtools mpileup 752 version 1.8 [94] with per-base alignment quality disabled (-B). A sync file containing the 753 nucleotide composition in each pool for each position in the reference was generated from the 754 pileup file using popoolation mpileup2sync version 1, Reads from the male and female pools were aligned separately to the reference genome, vol.748

, PSASS outputs 1) all positions with sex-760 specific SNPs or high F ST , 2) the number of such positions in a sliding window over the genome, 761 3) average absolute and relative coverage for each sex in a sliding window over the genome, 762 and 4) number of sex-specific SNPs as well as coverage for each sex for all genes and CDS

, We used PSASS to identify non-overlapping 50 kb windows enriched in sex-specific

, allele 766 frequency for a heterozygous locus 0.5 ± 0.1 (--freq-het 0.5, --range-het 0.1), allele frequency 767 for a homozygous locus 1 (--freq-het 1, --range-het 0), --window-size 50000, and --output-768 resolution 50000. For scaffold1067, the number of sex-specific SNPs and coverage for each sex 769 were similarly computed in 2, SNPs in E. lucius, using the following parameters: minimum depth, vol.10

, A sync file containing the nucleotide composition in each pool for each position in the 774 reference was generated as described in the previous section, using the Nanopore assembly 775 (NCBI accession number SDAW00000000) to align the reads. This time, because we were 776 comparing reads coverage levels, the BAM files were filtered with samtools version 1.8 [94] to 777 only retain reads with a properly mapped pair and a mapping quality higher than 30 to reduce 778 the impact of false positive mapped reads. The resulting sync file was used as input for PSASS 779 to compute coverage for each sex in 1 kb non-overlapping windows along the genome using 780 the following parameters: --min-depth, vol.10

, To identify protein coding sequences, we performed alignments between the Y-specific 785 sequence and the teleostei (taxid:32443) non-redundant protein database using blastx 786

, 97]) was run on these Y-specific sequences with NCBI/RMBLAST (version 2.2.27+) 791 against the Master RepeatMasker Database (Complete Database: 20130422)

, Supplementary file 1, Figure and Text: Figure S1 to Figure S6 and additional results

, Tables: Table S1 to Table S8

, Sequencing data and assembly for the Esox lucius genome can be found under NCBI Bioproject 802 PRJNA514887. Sequencing data for identifying and charactering the sex locus, including RAD-803 seq, pool-seq reads

, We are grateful to the fish facility of INRA LPGP for support in experimental 808 installation and fish maintenance. We are grateful to the genotoul bioinformatics platform

, Bioinfo Genotoul) for providing help and/or computing and/or 810 storage resources. We also want to thank Nicolas Perrin, Susana Coelho and Eric Pailhoux for 811 feedbacks and helpful discussion on an earlier version of the manuscript. This project was 812 supported by funds from the Agence nationale de la recherché

, The GeT

(. Toulouse, . Cr, ). Jl, and . France, HP) core facilities were supported by France, LJ

, Investissement d'avenir" program 816 managed by Agence Nationale pour la Recherche (contract ANR-10-INBS-09). The funders 817 had no role in study design, data collection and analysis, decision to publish

, Writing -Original Draft Preparation, Writing -Review & Editing Amaury Herpin Conceptualization, Supervision, Writing -Review & Editing Yann Guiguen Conceptualization, Funding acquisition, Supervision, Writing -Original Draft Preparation, Qiaowei Pan Formal Analysis, Investigation, Visualization, Conceptualization

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, Sequence identity between amha and amhby and amplification in males and 1095 females. A) PCR amplification of amha (500 bp band) and amhby (1500 bp band) in male (n=4), vol.1

, Each lane corresponds to one 1097 individual. The number of tested animals of each sex positive for amha and amhby is indicated 1098 in the table on the right. B) Sequence identify of global pairwise alignment between amha and 1099 amhby genomic sequences with amhby sequence as the reference. C) Schematic representation 1100 of amha and amhby gene structure in E. lucius from the start to the stop codon. Exon1-Exon7 1101 are represented by green boxes with shared percentage identity indicated and introns are 1102 represented by white segment. The red segment in intron 1 represents the amhby specific 1103 insertion. TGF-? domains are indicated with diagonal lines. D) Global pairwise alignment 1104 between 5 kb upstream region of amha and amhby with amhby sequence as the reference. The 1105 start codon of, doi: bioRxiv preprint 1096 and female (n=4) genomic DNA samples from E. lucius

, A: Number of MSS in a 50 kb non-overlapping window 1110 is plotted for each linkage group (LG) and all unplaced scaffolds in the reference genome 1111 (GenBank assembly accession: GCA_000721915.3). The two highest peaks of MMS density 1112 are located on unplaced scaffold1067. B: Number of male and female-specific SNPs in 50 kb 1113 non-overlapping windows is plotted along scaffold1067. The male data are represented in blue 1114 and female in red. The ~ 40 kb region located between ~ 143 kb to ~ 184 kb (11% of the 1115 scaffold, Regions enriched with male-specific SNPs (MMS) in the genome of E. lucius 1109 based on pooled sequencing analysis, vol.2

, A: Rad-Seq marker association with sex across the 25 linkage groups of E. lucius 1119 genome. The log 10 of p-value from the association test between each marker and sex phenotype, Figure, vol.3

, doi: bioRxiv preprint 1121 concentration of markers significantly associated with sex phenotype. The dotted horizontal 1122 lines are indications for genome-wide significance level from the association test, with the 1123 lower dotted line indicating p-value of 1*10 -5 and higher dotted line indicating p-value of 1*10 -1124 8 . B: Differentiation of polymorphic markers demonstrated by F ST between males and females 1125 and their association with sex phenotypes on LG24. Between male and female F ST value is 1126 plotted against the mapped position of each marker on the upper panel. Average F ST value of 1127 markers on LG24 is presented as the dotted line (higher panel). C: The log10 of p-value from 1128 the association test of each marker with sex phenotype is mapped against the mapped position 1129 on the lower panel

, A-B: Boxplots showing the first quantile, median, and the third quantile 1135 of the temporal expression of amha (A) and amhby (B) during early development of E. lucius 1136 measured by qPCR. Outliers are displayed as a dot. The mRNA expression of amha and amhby 1137 were measured at 54, 75, 100, and 125 days post fertilization (dpf) in male and female samples 1138 of E. lucius and the log 10 of their relative expression is presented on the graph. Significant P-1139 values (<0.05) for Wilcoxon signed rank test between male and female expression at each time 1140 point are indicated by * and 'ns' indicates 'non-significant P-values. Statistical tests were not 1141 performed on amhby expression between sexes because of the complete absence of amhby 1142 expression in females. C-F: In situ hybridization on histological sections revealed the 1143 localization of amha in both 80 dpf female (C) and male (D) gonads, with a stronger expression 1144 in male gonads, Temporal and spatial expression of amha and amhby mRNA in male and female 1134 developing gonads

, doi: bioRxiv preprint 1145 signal detected in female gonad (E). The red scale bars denote 20 m and the dashed lines 1146 outlines the gonadal sections

, The amhby KO XY male (C) developed ovaries with oocytes and an ovary cavity, 1152 indistinguishable from the ovary of the control females (A). The amhby transgenic XX mutant 1153 female (D) developed testis with clusters of spermatozoids and testicular lobules identical to 1154 that of the control males (B). PVO: previtellogenic oocytes, Gonadal phenotypes of E. lucius in amhby knockout (KO) and additive 1149 transgenesis experiments, vol.5

, Orthologs of each gene are shown in the same 1159 color and the direction of the arrow indicates the gene orientation. Ortholog names are listed 1160 below and the genomic location of the orthologs are listed on the right side. For Esox lucius, 1161 amha, which is located on LG08, is used in this analysis. B: Phylogenetic reconstruction of 1162 teleost Amh protein orthologs. The phylogenetic tree was constructed with the maximum 1163 likelihood method (bootstrap=1000). Numbers at tree nodes are bootstrap values, Evolution of Amh in teleosts. A: Synteny map of genomic regions around amh 1158 genes (highlighted by the red box) in teleosts, vol.6

, The copyright holder for this preprint (which was not peer-reviewed) is the