Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. dnia ngn otn n ie(ahrg 03 ahrge l,21) aiu ee (including genes Various 2014). al., nearly et or Bachtrog similar and 2013; are ZW) (Bachtrog, pairs complex size Z/W or and or genotypic amhr2 with content X/Y ZW fishes gene where Z0, In homomorphic, genotypic in than 2006). often identical prevalent Avise, hermaphroditism, are & more chromosomes sequential Promislow, sex being Mank, including 2014; sex-determination, XY Sex- al., variable, complex et species. (Bachtrog highly or dependency fish environmental XY are monomorphic 2013). X0, many al., fish in (with et teleost Pavlova studies maintenance sex-determination 2014; such in and al., hampers evolution systems et markers driving (Amos genetic determining management processes sex-specific conservation of of developing lack understanding in However, assist our and enhance biodiversity, biology of sex-specific both for of assays Studies sexing multi-locus facilitate will here resources Introduction The more. homozy- or are Similarly, 2 females which at biology. Percichthyid development. in heterozygous on system assay are research XX/XY scaffolds. males in predominantly and non-homologous a and used species across in 22, populations perch loci all Macquarie sex-linked the at in different for gous SNPs revealed sex-linked only SNPs 22 Macquarie reliability of identified DArTseq within we sequencing 100% and genome-wide Overall, this Amplicon interspecifically, approached perch tested differ species. golden We test positions confamilial SNP. and sex-linked sexing other one few Macquarie- its three of the of that that allele panels revealed Y-linked such smaller region the perch 146-bp and targeting the perch, assay SNPs, encompassing Macquarie sexing sex-linked bp known-sex 5 PCR-RFLP 400 of containing a panel haplotypes developed a 146-bp females, and Y-linked in in SOX, and assay homozygous comparisons X- of loci- pool-seq reconstructed upstream gametologous we In-silico for Kb scaffold enriched ˜38 this perch. was Within scaffold Macquarie SOX-containing males. known-sex identify 275-Kb To in 100 a heterozygous ambigua. on but M. annota- differences, perch resequencing golden sex and genome the few sequences whole species, revealed genome sister conducted whole facilitate its we To and report australasica loci, we systems. Macquaria sex-linked biota, sex-determination perch freshwater Macquarie diverse endangered Australian their the the by of challenged in tions monomor- predominant is of assays fish, differences Percichthyid sexing sex on molecular genomic research developing understanding and But species management. fish conservation phic aid can biology sex-specific Understanding Abstract 2021 23, February 7 6 5 4 3 2 1 male Ingram a be Pavlova to Alexandra ways Many Australian species: in fish Percichthyid sex-determination freshwater on light shed data Genomic eeE d Bhd Sdn GeneSEQ Industries Primary of Department Authority Fisheries Campus Waterfront Victorian Geelong - University Deakin University National Australian University Trobe La University Monash , 5 bcar1 enGilligan Dean , , dmrt1/dmY/dm-W 1 ahrn Harrisson Katherine , 6 alSunnucks Paul , , sf,sdY gsdfY, 1 n a igGan Ming Han and , 2 n ebr fthe of members and utmTurakulov Rustam , 1 O:SRY SOX: 7 3 i egLee Peng Yin , -like HMG 4 -box-containing Brett , amhy , Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. nunesxdtriaina nerysaeo eeomn nsm ecctyd Iga,H,Turchini, Ho, (Ingram, Percichthyids some in may development conditions of environmental that stage suggests early work an (Devlin Hatchery at temperature 2008). determination especially Piferrer, sex factors, & environmental influence by Penman influenced 2002; be young Nagahama, homozy- have can & may fishes always sex-chromosomes many and in perch males determination Macquarie Sex in in that heterozygous suggests always This i.e. 2020). homology, al., X/Y strata. et strict (Lutz females or in only) gous a males morphological 2014), in low (Lavou´e al., with present perch et Consistent Macquarie (i.e. 2019). sister-species al., its in et of chromosomes, (Shams set sex sex-chromosomes perch perch in golden golden detected in of was homomorphism differentiation Heteromorphism perch but 48. = cod, golden 2n Murray number species, of chromosome Percichthyid diploid two with males) for les/XY karyotypes examined sex and cod the (2019) biology Murray determine al. species to et of ways understanding and Shams dimorphism better sexual hinders of conservation. seasons lack efficient breeding gametes, more outside produces non-invasively it individuals when of ascribed be ( can Asia adult eastern in domi- ( family occur perch this America genera Macquarie from three South Species and Percichthyidae. and fauna, family fish ), the freshwater of Australian fish variability the in nate sex-specific understood well be and in not will depth is expected loci Sex-determination read is loci. Z-linked density on sex-linked and SNP based detecting X- higher purifying in approaches degeneration, and weaker assist W combining depth), can or Accordingly, and read Y sex. recombination (halved substantial homogametic sex reduced with heterogametic (Palmer the strata to sex the older homogametic due in than in hemizygous heterogametic Z contrast, the effectively In or in expected 2019). X is al., strata than et young in faster an density strata sex-chromosomes, mutations SNP young of higher will accumulating selection, regions in depth younger W But read heterogametic these in sex. the or For the W, homogametic in regions. reflected Y in and autosomal the differences be Z of reads in on that or will absent many based to Y be as approach pattern and and will sexes half X This between loci between show chromosome. similar similarity W- will be high or sex Z having Y- each sex-chromosomes, or (e.g. and homomorphic of X sex one, or copy heterogametic on homogametic whereas the strata one chromosome, as old have sex sex example, same will for the females) coverage: of ZW copies read-depth two or have approach males will first males) XY The ZZ from maps. or obtainable linkage & females sequence build Dean, XX to DNA Rogers, breeding use Palmer, captive they different require in because uses not (reviewed wildlife, do chromosomes to and sex applicable samples detect non-lethally-collected particularly to are used Two 2019). be Wright, in can evolutionary approaches resulting different genomic at gene, Several stopped sex-determining recombination often 2005). where a suppression evolve chromosome al., the from et This distinct, of (Charlesworth 2005). starting regions highly times i.e. Marais, chromosomes, ages, and & along different and diverged Charlesworth, of stepwise chromosomes strata (Charlesworth, are sex and W W, between progressively or recombination and occurs Y contrast, of Z In of suppression 2014). or degradation through al., subsequent chromosomes Y, et (Bachtrog sex and determination homomorphic sex X from for where used pair sex-chromosomes, result chromosome may heteromorphic the and recently, in relatively switches recombined recent have reliable, from gametologs of where Meril challenging. frequently, identification occur be Natri, and sex-chromosomes Thus, can 2014; Homomorphic 2019). rapidly fish al., al., in relatively to et Mart´ınez markers et evolve (Natri sex-linked contribute 2010; can sex-determination applicable all Peichel, in systems generally variation & can sex-determination intra-specific Graves loci, new in 2019; multiple Moreover, resulting al., 2019). or et Shikano, inversions, (Bao & (SNPs), fish polymorphisms in nucleotide sex-determination single family), gene > 20gnm-ierdcdrpeetto N oiddntrva akr ossetwt Y-linkage with consistent markers reveal not did loci SNP reduced-representation genome-wide 1200 ac peelii Macc. ploidy aqai australasica Macquaria fsxcrmsmsdvre hog rWdgnrto:tehmgmtcsx(e.g. sex homogametic the degeneration: W or Y through diverged chromosomes sex of Percichthys n o ohrpre aehtrgmtcsxcrmsm ytm X fema- (XX systems sex-chromosome heterogametic male reported both for and , e-pcfi N density SNP sex-specific .Mn utainseisaetraee,icuigteendangered the including threatened, are species Australian Many ). n ru cod trout and 2 aclohlamacquariensis Maccullochella cosgnmcrgosi oeaporae With appropriate. more is regions genomic across Coreoperca lhuhsxo an of sex Although . aq ambigua Macq. and Siniperca and ¨ a, Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eeapoe yrlvn nmlcr n tiscmite.Fedsml olcinwsapoe by of approval approved under was conducted were and collection sampling P01/0059(A)-3.0 Snobs sample and Permit employed work at Collection Field Hatchery procedures Scientific determined RP827. committees. 15/02, All sex Permit AEC ethics their Research S1). and Fisheries with and 14/04 Material their Victorian AEC cod, Supplementary care and DELWP males trout 14/10, two in animal project, and ACEC from sexing relevant NSW another cod clips and by Murray Fin for sampling approved of gonads. lethally each of were mature sampled for (details or used were maturing hatchery also of 66 Creek were presence populations, females on two three based and from dissection during perch ethanol identified absolute golden sex individuals in 68 two preserved and the but and all Creek Of non-lethally For Parrot collected season. were King breeding samples inland the -20 fin-clip during was from at section), The gametes Sex fish next mature populations. 2017). in sampled-and-released of al., coastal (exceptions were observation 21 et five by 25 and Beheregaray, in Creek (Pavlova, and program), one broodstock Holland’s distinguishable inland coastal hatchery breeding morphologically the four for from the are from hatchery-produced diverged two identified of populations) Creek sampled the Abercrombie details Snobs the Pleistocene; adults and a for the Dartmouth from were Yarra, was 2020 62 (including broodstock one lineage al., hatchery remaining River, (Supplementary inland et Creek the Abercrombie cod trout Snobs the Lutz and 4 were from juvenile, (see and 173 broodstock Dartmouth cod hatchery individuals, Murray Lake Narrandera 4 perch and perch, Macquarie River golden 261 68 Yarra Of perch, Macquarie S1). 261 Material of samples used We identification species sex fish collection, other Sample sex to tests Methods molecular and More developing de- Materials sex-determination. for resources of used and chromosomes. evolution be sex results the could monomorphic The including here with Percichthyidae, scenarios. described across on workflow potentially sex-determination research and the complex species, broadly, facilitate across more will variation across for sex-determination, chromosomes, here variation Y XY calls veloped and sequence supports regions, X be Overall, generally revealed both genomic populations developed. could results on perch was other region furthermore Our Macquarie test sexing and two in the species. in deletions species-specific which region four but in is 633 the those scaffold perch for small to Macquarie amplicon perch one related region used in Macquarie populations We this known-sex region to cod. in of genomic trout specific variation and panel XY-homologous cod sequence a the Murray in explore perch, that assay golden further PCR-RFLP this of a a to panels targeting tested equivalent developed individu- sequencing We assay smaller explored and sexing allele. on We region, molecular (male-specific) and 633. polymorphism) this individuals, Y-linked scaffold length for a perch fragment with haplotypes restriction Macquarie SNP Y-linked - on and reaction males) chain X- in (polymerase identified two variation, predominantly females, showing sequence in region, genomic al allele small a (one genotypes of for patterns using identification sources enabled sexes, XY as This between used pools. differentiated assembled populations 50 population-by-sex significantly large four newly for SNPs relatively of the data two for (WGS) of representing searched We equally resequencing some interventions. perch whole-genome perch on conservation Macquarie obtained golden clustered male we and are 50 Next, and perch loci obtained genomes. female sex-linked Macquarie SNPs reference putative for of respective whether sex-linkage 2012) of examined for al., scaffolds tested and and et We sex, affordable (Kilian individuals. known non-lethal, DArTseq of fully of a representation sex determination develop genomic genetic sex to the reduced XY and determine whether using species, to test these test to in molecular variation aimed we rapid genetic sex-linked particular, resolution, of different In for patterns species. conservation targeted explains these Per- datasets in benefit sexually-monomorphic genomic sex-linkage related using would explored closely Then, two perch. we fish of golden genomes and of perch the trout Macquarie sex annotated cichthyids, and of genetic assembled population recovery sequenced, determine a we for Here, in to implications have males able species. fish as Percichthyid being stocked females threatened in many of stocking, biases management as through sex-ratio times Because populations 2.5 fish. reported of hatchery-bred (2012) from al. stocked et cod Lyon 2012). Holland, & ° C. 3 Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ec rmteDrmuhadYrappltos n 1fml n 5ml odnprhfo Macquarie, from Macquarie perch male golden male 78 25 and and female female 93 41 se- using populations. and genotyped markers Murrumbidgee restriction-associated We populations, and SNP Yarra S1). double-digest Murray and genome-wide Material Dartmouth to obtained Supplementary the we similar from in sex perch samples method known of sequencing of (list representation perch quencing golden reduced 66 a and perch DArTseq, golden perch and Macquarie perch Macquarie 171 for For al., genotypes reduced-representation DArT et in (Jones loci 5 sex-linked InterProScan for Searching using Borodovsky, completed & was Lomsadze, proteomes Stanke, Hoff, predicted (Bruna, the the 2014). v2.1.2 as of BRAKER used et annotation were in (Dobin Functional genome prediction repeat-masked v2.7.1a 2020). gene and STAR coding Green, file) using protein & bam for Hubley, genome (single-species input (Smit, repeat-masked alignment 4.0.9 transcriptome the v The to RepeatMasker 2013). aligned al., with were genome reads the Transcriptome (soft-mask) with 2013-2015). repeat-mask 2019) to used constructed Zdobnov, and was & 2020), library Manni, repeat (Seppey, were A v4 then actinopterygii which the BUSCO approach, on using graph based Overlap-Layout-Consensus assessed for setting, Bruijn contigs was default de read” “mega the completeness generating by long reads, Genome contigs long assembly. Nanopore Nanopore construct and the to error-correct 2018), to used Gu, used and & error-corrected Chen, first Zhou, were (Chen, assembled v0.19.5 hybrid perch were fastp golden reads using and adapter-trimmed perch reads, Macquarie NEBNext Illumina for using genomes NovaSeq6000. beads of Illumina annotation poly-T Plus an Universal and via on TapeStation using Assembly mRNA sequenced processed a for was and mRNA enriched Kits using enriched Preparation was quantified The Library This (NEB). Research), mRNA-Seq Module tissue. (Zymo Isolation per Magnetic Kits pooled mRNA Poly(A) Quick-RNA ng 440 using and samples (Agilent) Shield tissue DNA/RNA in individual -80 preserved and at from brain, collected stored of were and Samples samples Research) RNA 2018. killed, (Zymo April collected. humanely in were were fish Victoria, muscle analysis), after River, cheek otolith Immediately Ovens by and the years liver in 3 kidney, captured as gonads, (aged male, heart, GPTT01 putatively female perch gills, as adult golden field adult used the used We in We sexed species. collected. were both muscle for and kidney sequencing signal mRNA Nanopore MP performed the perch we of Macquarie annotations, Base-calling genome flowcell. facilitate To R9.4 UK). Nanopore Nanopore, UK). (Oxford a Nanopore, v2.0.1 (Oxford on instructions Albacore sequenced manufacturer’s used subsequently the to was according library kit gDNA The preparation and of library Kit 2 LSK108 ng Preparation 1 a using Library 100 data, with Centre Illumina sequencing, long-read Genomics Ultra Deakin obtain Illumina NEB To the For a A). at with kits. Novaseq6000 processed Tissue a QSonica, on & on using preserved sequenced bp Blood based were 350 DNeasy years samples to Qiagen fragmented DNA 3+ was using species, as extracted both (aged For was sex 2017. DNA -20 unknown May genome, at of in perch kept Victoria, GOP001 golden and MP For River, adult ethanol 2012. Broken of from in November the tissues in non-lethally in muscle born collected captured origin, and size), tissue River fin Yarra bone of used tail sex we unknown fin, of used juvenile we produced perch perch, golden and Macquarie perch 05/06. For Macquarie committee for ACEC sequencing Fisheries transcriptome NSW and and Genome 0067 Jul09 AEC Fish DPI VIC 2,cpue oebr21 rmLk atot.Smlsfo ie,oay brain, ovary, liver, from Samples Dartmouth. Lake from 2017 November captured 527, ° C. enovo de μ fgN a rgetdt buigaCvrsGTb n processed and G-Tube Covaris a using kb 8 to fragmented was gDNA of g enovo de ° Mcureprh r-20 or perch) (Macquarie C sn auC 324(ii ta. 07.TesotIlmn reads Illumina short The 2017). al., et (Zimin v3.2.4 MaSuRCA using o h sebe eoewt eetoee2(ln tal., et (Flynn RepeatModeler2 with genome assembled the for d1 database. odb10 4 ° gle ec) oa N a extracted was RNA Total perch). (golden C × 5 prncngrto (Appendix configuration run bp 151 C1:a2mnhodhatchery- 2-month-old a SCH12: ® Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. oi ordc h ubro as oiie u osalsml ie nylc ucsflysoe in scored successfully loci only size, sample small t.hom to of due values positives in false the individuals of of of number ( None 90% the to reduce homozygous. up To allowed be loci. settings to these t.hom heterozygotes respectively; have and is homozygous. systems perch, to be t.hom ‘zw’ to expected and and allowed sex ’xy’ heterozygous, males) under the (XY be used heterozygous to were be allowed 0.9 to = females) expected (XX sex ( the homozygous find of To be individuals to of expected proportion the sex the of individuals ( gametologs females. ( in absent and sex-determination XY males, with in consistent izygous) strata of types four ( of one 2019): to al., belonging et for [?]5e- (Shams Project locus e-value SNP WGS with each BLAST, (NCBI tested sex-linked using We genomes finding perch) reference [?]90%. golden of identity for newly-assembled chance sequence VMKM01 the respective a and and reproducibility maximize their with perch 5 a to Macquarie to loci stage, with for aligned pairs: this SEMN01 then SNPs were was accessions: at allelic and calls loci performed the SNP replicates, was DArT for of technical filtering loci. were Reproducibility counts other between rejected. bases, read No were consistency 3 removed. allele of score each were than balance for on secondary more counts the a based no read ensuring used in by assessed and while difference differing low-quality template, higher loci tags corrected or a SNP sequence, comprising 5-fold as the separate Clusters, members of into multiple calling. rest with SNP parsed the tags for than collapsed (DArTsoft14) using region 2012). pipeline tags barcode al., singleton included the et from to Kilian lane bases criteria 2001; each stringent Kilian, control, loci more & DArT quality Feinstein, applying 69-bp using Peng, For short performed assembles (Jaccoud, were A). genotyping method pipeline enzymes and Appendix The analytical discovery restriction in SNP proprietary of libraries. P/L’s (details independent combination DArT from regions a replicates using genomic technical of digested low-copy following ˜25% were Australia) target samples (Canberra, that DNA Ltd Pty I (2012). Technology al. Arrays Diversity et at Kilian prepared were libraries Sequencing oa f2 ilo ardedraswr admysbape rmec niiulsWSdt and data WGS individual’s each from subsampled randomly were reads S1). Material paired-end Supplementary ( million in depth 25 coverage 10X were read-depth of least individual these at was total and to library of A Centre samples Novaseq (all Genome of Illumina population Deakin list an at library; per individual, sequenced each per sex were For data each Libraries above. of above. as 100 individuals as extracted for constructed, (WGS) 25 was data Yarra; DNA resequencing and DArT-sequenced). genome also whole Dartmouth perch obtained from Macquarie we perch genome, for draft Macquarie data the (WGS) across sequencing sex-linkage explore genome To whole in loci sex-linked for species. Searching both in females for than smaller function the ’xy’- = system 0, = t.hom 0, = t.het ( settings- with default consistent Under of patterns 2020). depth for Team, read Core looks (R the R half in the have 2018) used (males we males tests these in For hemizygous always but females can in females). Y-gametologs heterozygous recombination. on or X-X Variation mozygous than males. X-Y lower in to heterozygous ( due or X-gametologs homozygous on either than and faster accumulate females all in mozygous > iii ii i iv -nysqecso l taa(eefe,“ (hereafter, strata old on sequences Y-only ) 8 fml aqai ec and perch Macquarie male of 58) ieetallsfrnnrcmiigX n -gmtlg nmdrtl l taa(eefe “ (hereafter strata old moderately on gametologs Y- and X- non-recombining for alleles Different ) -iie aito nodsrt o oimsigo -aeoos(eefe “ (hereafter Y-gametologs on missing loci for strata old on variation X-limited ) -iie aito nyugsrt hratr“ (hereafter strata young on variation Y-limited ) iii ) ooyosi l eae n eeoyosi l males. all in heterozygous and females all in homozygous ”): n ( and ) < . ngle ec eundsxlne oi niaigvr o aiblt o sex-linked for variability low very indicating loci, sex-linked returned perch golden in 0.7 iv ,dfie eea oihtrzgu in heterozygous loci as here defined ), gl.sexlinkage > i 5 ( 95% n ( and ) ucino h at akg Gue,Umc,Bry Georges, & Berry, Unmack, (Gruber, package dartR the of function > 3 aegle ec eecniee,ml apeszsbeing sizes sample male considered, were perch golden male 23) ii enovo de ;hr,tetlrneprmtrthti h rprinof proportion the is t.het parameter tolerance the here, ); Y-linked 5 h ATppln eoe orqaiysequences, poor-quality removed pipeline DArT The . oibaigrcn--pcfi polymorphism recent-Y-specific bearing loci ) lasaprnl ooyos(culyhem- (actually homozygous apparently always ”): > 0 fmlso eae,tht= n t.hom and =0 t.het females, or males of 10% old-X-linked < . nMacquarie in 0.8 Pst > and I bof Gb 7 ) ho- ”): ) ho- ”): > < XY- 75% 95% Sph Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. efis sdBat osac h odnprhgnm o einhmlgu o60b fMcureperch Macquarie of develop bp 600 To to homologous region males. a for species, sequenced genome other perch most possibly all golden and the in in populations search allele perch to present Macquarie Blastn reference all allele used (bases (X-linked) in first 633 (Y-linked) amplify we the to scaffold alternative for intended perch primers homozygous the Macquarie PCR conservative loci on with region SNP females, 146-bp sex-linked a sequenced 5 targeted contained we which assay, sexing 93182-93327), molecular sex the develop perch process: To Macquarie (biological for GO:0007530 assay sexing term molecular Ontology PCR-RFLP Gene (https://www.ebi.ac.uk/QuickGO/term/GO:0007530). terms and child 2011) its al., and et determination) Rodriguez-Mari location 2013; and Graves, presence for output the 1, InterProScan5 for searching tor an- looked fish, has also teleost We test 633 from brca2 known scaffold to sex-determination. genes perch perch sex-determining in Macquarie of golden role amh whether genomes and a and perch perch play the regions, might in Macquarie gene that in for genes located annotations notated were functional SNPs and genomes sex-linked reference organization in whether genes genome sex-determining used candidate seeking We variants and structural SNPs of sex-linked classes of score different annotations of to Exploring counts used compare samples. to sex-by-population was Y-specific used and libraries 2016) split-read of were R sexes, and in respectively al., between accumulation implemented paired 2012) tests et from Jimenez, supporting beta-binomial (Chen & on and expected than Fisher (Pham based Manta males be parameters. repeats) default in and would recombination. using inversions indels evidence, as in deletions, more (insertions, reduction clustered, has variants recent are structural loci to indels sex-linked due with these polymorphisms enriched for whether depth 633 and read scaffold all calculated females, whether We of investigated size. number we genome the for sex- by Finally, as four depth divided separately. and calculated the Read alleles length) was across R. alternate (read averaged individual in and bp each and 151 analysed reference for by depth, and multiplied average read merged reads average genome-wide were mapped genome-wide The files its output samples. by files, by-population normalized alignment was with enriched individual explored individual region from also each the 633 We scaffold using 2011) the collected 2018b). al., of was and base al., et et depth each female- et (Milne for Read resolved coverage Kijas Tablet read-depth libraries loci. in investigating in sex-linked sequenced by approach males loci the (similar these for of of inspection architecture alignments ploidy manual mate-pair XY-gametologs, individual via the being visualized haplotypes whether with we male-specific investigate consistency 633, to for scaffold 2012) genotypes examined on al., Individual were ( loci 2018). tests polymorphism SNP al., CMH recent-Y-specific et differentiated paired-end by bearing (Kim calling sex-linked trimmed or variant v2.9.10 strelka by be loci the using followed old-X-linked to aligning individuals, v0.7.17-r1188, inferred all by bwa-mem loci from using performed, files for genome also BAM reference merged was the the to data on individual (p read other. each levels WGS the for significance all in reads test monomorphic using loci, CMH but genotyping differentiated lower sex most Individual with one the in loci (scaffold of polymorphic other scaffold set were one had a loci Because it (p contained some whether region. significantly SEMN01000633) genomic examined highly accession same also GenBank frequencies the we length, in allele sexes bp clustered between with 274905 being differences loci for 633, frequency the examined allele SNP were onto consistent sexes and mapped replicates. between significant (Li in were biological detect v0.7.17 implemented pools two to bwa-mem test used four in using (CMH) was the SEMN01) Cochran-Mantel-Haenszel 2011) from Dartmouth Schlotterer, A accessions: DF, Reads & Project females, 2009). YM. WGS Dartmouth Durbin, males, (NCBI Yarra & population): genome and per reference sex YF, perch each Macquarie females, of Yarra individuals DM, (25 males, pools four into combined niMleinhormone, anti-Mullerian , ratcne ye2ssetblt protein susceptibility 2 type cancer breast , m,d-,dW gsdfY, dmW, dm-W, dmY, amhr, oaa oadrvdgot factor growth derived soma gonadal niMleinhroereceptor, hormone anti-Mullerian dmrt1 , CollectAllelicCounts 6 , doublesex- ii-iv bv) o h e fms togysex- strongly most of set the For above). and bcar1 ucino AKv.. (DePristo v4.1.0 GATK of function sdY , mab-3 ratcne anti-resistance, cancer breast , PoPoolation2 and rltdtasrpinfac- transcription -related SOX < e2)differentiated 1e-20) < e5 n whether and 1e-5) Boe l,2019; al., et (Bao Kflr Pandey, (Kofler, R-core and ibb Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ihol 4(.% u f360BSOgop isn ihnteAtnprgiob0dtbs.The of genes annotation. database. protein-coding GO odb10 predicted having Actinoperygii 33,422 16,264 complete, with with the 96.9% InterProScan5 repetitive within by be 27.67% missing annotated to be functionally groups estimated 675.98 to were BUSCO was of 29,940 found 3,640 and which size was of 1) a genome Table out had perch (2.0%) SEMN01) Kb; Macquarie accession 74 N50=845 Project (scaffold only WGS scaffolds with (NCBI 2,962 perch comprising Macquarie perchMb for golden genome and assembled perch The Macquarie for annotation and assembly Genome examined also were haplotypes. reads sex-specific Raw for of Results 16365-16988 presence perch). bases the Macquarie VMKM01003747 for for cod, Pro 93001-93600 trout Geneious bases and in SEMN01000633 cod and and Murray perch containerized for golden 2013), 83361 al., 82756- et bases Auwera tag: with (LKNJ01002546 der (Van repository docker protocol the using v4.1.0 poly-G on GATK saved system) of PCR-RFLP best-practice trimmed Illumina the using were NextSeq500/NovaSEQ6000/ISeq100 region on files newer amplicon fastq based the sex the in coastal each in Reads two of artefact cutadapt with S1). one along sequencing Material be sequenced, Supplementary (a using to were in sequences species appearing region samples in four of (details but bp (list bp the sex ˜400 assay 300 of unknown sexing x each a fragments of 1 of amplified individuals amplified of females then configuration perch two ISeq100 perch, the read We and Macquarie Illumina unidirectional an golden of males on the B7). Two the length it using A). sequenced (Malaysia) of in and Fig. Appendix Bhd B1) variation Table males Sdn B, R3, GeneSEQ two (Appendix sex-specific and by (F2 system site and any genomes three cut reveal females the across male-specific two not conservative primers a on did of (above) cod presence assay perch trout or sexing Macquarie and sex-linked PCR-RFLP cod Murray putatively the the Murray four B1). sex-linked of from other missing perch (Fig. tests the was genomes Macquarie Initial which to three the of all corresponding of part in Positions one microsatellite, present that a were B1). revealed SNPs in species located (Fig. was three sequence 82756- 93181) all cod (LKNJ01002546; base 2546 of 633 scaffold scaffolds species cod (scaffold Murray of SNPs other (VMKM01003747; of alignment 3747 section three scaffold An a 93182- perch with in (SEMN01000633; golden gaps) 83361). of 633 (7% sex-linked part identity scaffold corresponding 88% also a perch and with 16365-16989) is Macquarie gaps) (6% of region identity fragment 91% sex-linked had sex-linked 93327) 600-bp perch The Macquarie Percichthyids. the of other whether in investigated region sex-linked We perch Macquarie the at variation sex-specific unknown species coastal). for of six perch test F1-R1 and Macquarie to 41 with sequencing inland Amplicon-based and (two Abercrombie, PCRs Creek, populations and ran Holland’s Yarra we other and Dartmouth, eight sample, Creek from from larger Parrot sex individuals King a from hatchery-sexed on primer individuals perch assay on field-sexed Macquarie final 21 dependent the 45 length For using (of shorter tested males. two band was Ap- XY and long band for (see X by enzyme) one perch long followed the a produce Macquarie primers (including by to male bands cut three two expected ones and and was Y homozygous) (always assay female females two This XX 93229). in for combination) details). at initially base for trialled (T B were sequence pendix combinations and male-specific primer respectively) the four in alleles. 93474, only All Y-specific and R2 occurs with 93380, that F2, SNPs ATTAAT 93107, perch F1, recognizes 93027, Macquarie primers it (https://www.bioinformatics.org/sms2/rest targeting bases because B1, sites (Table tool 633 cut species online scaffold endonuclease two SMS on restriction the designed the starting We in 5’-end used identical B1). the then regions Fig. to in with B, aligned primers (Appendix R1, we Pro reverse and which Geneious two 16365-16988), in and search (bases haplotypes forward This perch VMKM01003747 two Macquarie scaffold 93001-93600). Y-linked perch bases and golden (SEMN01000633, X- on putative 633 region scaffold 624-bp of a region yielded 146-bp the encompassing sequence Mri,21)vateoption the via 2011) (Martin, Ase ieto Apni ;ls fsmlsi upeetr aeilS) hsassay This S1). Material Supplementary in samples of list B; (Appendix digestion I trust1/gatk:version0.3.1 –nextseq-trim 7 ahidvda a hngntpdfrte400-bp the for genotyped then was individual Each . h 60b einwsue sareference a as used was region bp ˜600 The . a.tl ofidpstosof positions find to map.html) Ase a chosen was I Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eepoe niiulsrlagntpsfr5 eae n 0mlsfra5K eino cffl 633 scaffold of region Kb 5 a for males 50 and females 50 for haplotypes Y-linked genotypes multiple strelka suggests individual polymorphisms explored Y-specific 90152-94017 We of region inheritance 633 of scaffold patterns on Complex loci WGS-derived a. ˜6.3 sex-linked the for within (p genotypes clustered 633 perch loci loci scaffold Macquarie most 27 on C1). Individual (Table females, the loci and in Two pools had male- allele female C1). bases), in alternative (Table in allele 274,905 an 633 two for one (length scaffold and count had of 633 single 87668-94017 scaffolds (p scaffold a region strongly 678 scaffolds, had Kb three in they of with scaffolds loci because together set 644 1,715 list 36, former in this these and the Of loci pools, C). Of 1,711 male were (Appendix these, in (36) SNPs pools. Of two female these and S3). in of female- Material two in Five Supplementary allele in one 3). (results had Table pools p perch populations; to lowered Macquarie both was female ‘ and threshold ˜3.8-Kb in the test populations), a CMH (hereafter, two both within the 93182-93327 in same When located fragment allele (the 146-bp all same short alleles SNPs, (the a present two these within allele had of located single nine pools a For had male alleles 3). pools two both (Table female had 90152-96798 both SNPs (p 90152-94017, region 20 p-values region ˜6.6-Kb other test CMH the a lowest and within the SNP), 633, with sexes. females one loci between in at SNP frequencies one allele eleven allele only The second different but on a with males (nine of but in SNPs count sex alleles 10 single each two these, a in had Of of SEMN01000251) exception 3). (p on (Table the strongly one comparisons (with loci and population-by-sex SNP four SEMN01000633, 30 all 633, detected comprising in scaffold individuals) (each females 25 and samples of coverage. each males population-by-sex 92X between in pooled of reads these four outlier paired-end 20X; run, million one to to first 25 with applied the 11X 57X, tests during from to (CMH) yield 32X ranged Cochran-Mantel-Haenszel insufficient from coverage to coverage samples, due of of re-sequenced 11.3X- range (range were (75) a sample individuals majority had per Dartmouth 16.3X the for was For perch libraries Macquarie 25 S1). male 50 Material and pools Supplementary female population-by-sex 50 93.0X; only using for coverage of SNPs depth WGS-derived heterozygotes WGS perch Median had Macquarie 2) one in Table in loci in same sex-linked present population. (indicated for the were single Tests species on a SNPs per in were sex-linked only SNPs of loci but two perch Heterozygotes sex except golden one other. populations, two and each multiple except aligned males in from genomes, loci sex Kb in reference sex-linked 226 homozygous respective bearing putative VMKM01000203, with their were All consistent scaffold of males, and perch S2). of scaffolds perch golden Material 10-30% Macquarie different in Supplementary in in to heterozygous 2, loci Macquarie and 34 (Table two females to and and in polymorphism compared homozygous old-X-linked, recent-Y-specific perch were perch being perch Macquarie and with golden golden consistent Materials in in in females, in 14 found loci of defined were 10-30% as Two loci in gametologs heterozygosity/homozygosity. sex-linked heterozygous ZW-) of follows. more (or patterns magnitude as XY- sex-linked or of for perch, linkage order perch Y- An 5,997 golden with and consistent male perch, Methods. loci 25 Macquarie male had and 78 species female and Neither female 41 93 for for golden scored scored loci and loci SNP DArT-derived perch biallelic 6,939 Macquarie tested We for be SNPs to DArT-derived found annotated in 95.9% was functionally perch patterns genome were be perch 31,895 homozygosity/heterozygosity to golden which annotation. less Sex-linked estimated GO The of was having was genes above. 16,792 VMKM01) protein-coding and groups with accession predicted InterProScan5 BUSCO 1) by 36,108 Project the Table with of WGS Kb; repetitive out (NCBI N50=252 25.83% (2.3%) perch 85 (scaffold golden missing scaffolds complete, for 7,165 genome comprising assembled contiguous, Mb 661.43 The < e5 hr ee6,5 oidffrnitdbtenml and male between differentiated loci 61,452 were there 1e-5, 8 < e2)wr oae nMcureprhscaffold perch Macquarie on located were 1e-24) < e2)dffrnitdlc htddntmake not did that loci differentiated 1e-20) 4-psxn region sexing 146-bp < e5 a n leei male- in allele one had 1e-5) < e2)differentiated 1e-20) ’). Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ftesrcua ainswr togysxlne Fg 1.I ohppltos ae a oeindels more had males populations, None both bp). In 8 Appendix of D1). cutoff 274267; size to (Fig. default 8693 sex-linked minimum despite (from strongly bp, were 633 50-425 variants scaffold (range structural bp on males, 98 the (47 positions was individuals of size 28 90 indel in through detected deletions) evenly Mean 179 D). and relatively insertions spread (42 variants females) last structural 221 43 The of total 2). a detected 633 (Fig. Manta being genome-wide scaffold males the allele perch to some Macquarie alternative close in on the was 1). depth Y-allele variation for Fig. where Structural of depth region 4; deletion (Table a read in drop suggesting sexes male was both allele, a 94017, with for reference region, with 2). 1), average this the associated Fig. in (Fig. for Y0; was locus sex-linked that females SNP (i.e. polymorphism) XY-gametologous for of of too recent-Y-specific half males that males a a some to in (bearing in just compared alleles haploid the 93879 depth alternative thus being locus male to chromosomes, region SNP of X compared the or the the supports alleles some of 93327 contrast, reference that one and that In of suggesting on 93315 than depth 93299, deletion region, lower Lower 93229, (through sexing consistently SNPs region 146-bp was X0). this the females for of being for hemizygous majority region depth be containing region Average could ˜93600, in the females to 1). same in most ˜93250 apparent the from (Fig. were was males region sex-differences depth for But sexing average shown). 146-bp 90152-92914 (not the region average containing in genome-wide approaching polymorphism sexes, 28 recent-Y-specific deletions both and recovered bearing sex-linked females depth loci segregating 31 the the suggests For coverage in that depth reads except read (zero in ˜93800, ˜93400 Sex-differences by at average c. males. zero genome-wide Dartmouth 93250, almost for the each position to ˜93650) to for Near (at samples) dropped returned normalized earlier sex-by-population but 1). gradually four and average, the then 93000-94200 genome (Fig. across in males), region the region averaged a the to being this 93327) revealed before close of in (93182- also depth was average base 633 It genome-wide genome each average scaffold the for by Y-haplotype). of individual to (calculated (a compared coverage region SNPs depth read-depth sexing male-specific average read 146-bp of of the linkage drop physical for strong confirmed reads males mate-pair heterozygous mapped indicating issues these, sequencing of of Illumina Examination more short-read or was indicates two coverage it at read-depth reference suggesting heterozygous Depressed the were males, for b. males other homozygous and most were alleles, Y-haplotypes. were females sex-specific multiple Overall, with as of (MP loci mixed-up. presence locus, 22 sample male this all or at one at individual were allele heterozygous except mis-sexed females was a heterozygous, all region, simply were MP not sexing the male XY-gametologous: males 146-bp for Notably, being all the homozygous were for Y-allele. with and females the consistent X-allele all for was and reference homozygous males 94017 the 12 SNP-locus for other Lastly, the homozygous heterozygous, were and males allele, the allele. 37 alternative for polymorphism, reference and the recent-Y-specific homozygous (MP locus bearing one and for more 93879 and homozygous loci or SNP-locus loci, two one the one three For at for for Y-allele Y-allele loci. heterozygous seven the the and all for for locus homozygous homozygous alleles one 11 three (Y-) for included alternative were ones, in X-allele males and remaining females inherited this 15 the All 93278) for remaining for at heterozygous C1). The heterozygous were insertion other Fig. loci. males and The seven S4; 35/50 93184 all and Material 93182- C1). at at alleles Supplementary region (X-) deletion (Fig. sexing 4; reference (Table 146-bp males male-specific for The XY-gametology of (a homozygous with loci. sets indels consistent these different two at fashion in and allele a present SNPs reference generally the five for loci had homozygous of 93327 were sets females two all (90152 and the two males with these, males, Of in of C1). p heterozygous 90152- 14-32% test (Fig. were region polymorphism (CMH the 91773) recent-Y-specific loci in bearing and differentiated alleles SNP-loci significantly 13 sex-specific most contained with 90152-92914 nine loci the on containing focussing 94017 S4), Material Supplementary (90,000-95,000; > 4 fmls(4ad2,rsetvl)ad1 eehtrzgu in heterozygous were 11 and respectively) 27, and (34 males of 54% B7 oe bv sdslyn eaelk genotype female-like a displaying as above noted CBR72 9 B7)hmzgu o h leefor allele X the for homozygous CBR72) < e2;Fg 1.Teregion The C1). Fig. 1e-20; CBR101) Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eae hwdasnl ad xetdfo ooyoe o h -ikdCall t929(pedxB, (Appendix 93229 at allele Parrot C remaining The X-linked King the 93229. of at for 20% allele homozygotes did T from the so expected of but presence band, patterns, the single indicating two 3-band a ones, showed these or Abercrombie females of 2- in 60% Abercrombie yielded and promising and Ck consistently females Creek/Holland’s appears Creek males Parrot King assay of that from showed sexing perch out Macquarie populations the known-sex male in single tested 93229, assay a same at The showing X-allele WGS putative and reflecting a variation, likely bands, this for C/T populations. all product Despite showed homozygous for variation three PCR being length number. these digested revealed 50 of repeat for also in assay and genotype males, microsatellite band PCR-RFLP data Y0 in at The X-specific WGS displayed variation Y region). had five uncut from sexing of males all the bp males five fragments 146 (although 10 of because the cut cut-site and lack unexpected, short the females The was two for 10 males heterozygotes only of B3). these at but sample of cut (Fig. larger some females, Y-allele A genotype for in of with Y0 fragments band B2). two consistent with single and females, Fig. consistent a allele in B; X showed in band (Appendix uncut homozygote also single expected with (C/C Dartmouth/Yarra a consistent as genotypes showed SNP, males, known data) in Y-specific with WGS bands females the on three two and and based with males genotype, males digested two XX in in primers heterozygote tested F1-R1 C/T 93229) by base females, at bounded T product allele (PCR 633 assay sexing PCR-RFLP The using perch Macquarie in containing sexing scaffolds Molecular the in E5). were (Table genome genes these the to mapped SNPs annotations: and perch VMKM01000340, golden in VMKM01002618, observed ones were sex-determining genes known VMKM01002284, regions. than SNPs sex-determining annotated other sex-linked in putative protein genes not perch 146-bp Forty-five the annotated were Macquarie the (of of SNPs genome-wide containing region introns other other 52003-131250, the coding in the E4); were region a (Table seven Of 633 in 35783- and was scaffold gene. bases SEMN01000396), one The on a 3), (g10285; to gene. and Table gene annotated this 43200; 2 a (base (Tables not of DArT was was 5 by region intron region, found 93182- guanyl-nucleotide SNP sexing within sexing and region sex-linked 146-bp located (GO:0005515) 633 sexing function scaffold was the 146-bp The binding 2) of protein the (GO:0005085). upstream activity with (including of factor Kb annotated downstream exchange loci domains bases 41 Kb sex-linked several (g10286; About ˜38 with strongly which 52002) located 11 3). of was containing Table one gene region 93327; This E3), 633 (Table (GO:0006355). scaffold factor genes transcription transcription the eight gene DNA-templated contain the regulating to to of annotated found was was 132300) E1). 633 Table 131251- SEMN01000434) E, scaffold on Appendix perch scaffolds; g32091 Macquarie 34 and on genes SEMN01000023 37 on family; g21038 HMG-box protein; susceptibility ( 2 genes/gene type to cancer InterProScan5 SEMN01000098), by including on annotated species, fish were other SEMN01000046), in BRAKER2 on genes by sex-determining sex as found act one that genes of families individuals perch of annotations Macquarie genome proportion Forty-four perch large golden a and in perch sexes Macquarie haploid the P Exploring are between populations: that difference pooled the other. regions for the but large test not respectively) show beta-binomial but 35, not P=0.38, vs did test 46 Manta Fisher Yarra: population Overall, 58, (per vs significant 82 not was (Dartmouth: females did than doublesex- and mab-3 brca2: amhr: mr ( amhr2 bcar1 SOX rltdtasrpinfco ;g53o EN1010,and SEMN01000160), on g4593 1; factor transcription -related 152o MM1001adg64 nVMKM01000026 on g16441 and VMKM01000091 on g12532 319o MM10408adg23o VMKM01004095, on g6203 and VMKM0100047038 on g34189 bes acrat-eitnegn;g46o SEMN01000123), on g8496 gene; anti-resistance cancer (breast ee n3 cffls al 2.Treo 4gle ec e-ikdDArT- sex-linked perch golden 44 of Three E2). Table scaffolds; 36 on genes niMleinhroercpo g96o EN1041adg28883 and SEMN01000461 on (g4926 2 receptor hormone anti-Mullerian 10 DUF3504 SOX SOX ee,a itneo 765K from Kb 67-635 of distance a at genes, IR207,wihhstefunction the has which (IPR022097), amh fukonfnto;gn g30509 gene function; unknown of > Ase at-ulra omn;g1995 hormone; (anti-Mullerian fohrml-pcfi oiin loci male-specific other of 3 tteYseicscaffold Y-specific the at I dmrt1: , SOX amh: bcar1: ( brca2 Sry 258on g25528 146on g13446 97 on g9675 dmrt1 , -related > (breast 0.62). Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. a ossetylwrdphta ae Fg ) n iia et o -lee sec fteX n Y- and X- the of each as X-alleles for females depth for similar region: sexes, and this both for XY-gametologous 1), in (X0) loci this (Fig. females SNP haploid these of and males appeared of males than females part diploidy for most depth implies for depth 93327 lower alleles 94017) read haploid and and consistently sex-linked similar (93182 93315 had be eight for region Whereas 93299, this these might separately X-variants). 93229, in of the males loci and loci seven intervening of last and loci to one and females sex-linked first on up the deletion for some for for a alleles that depth through Y- suggested read (i.e. or female 2) XY-gametologous region X- this and for 1, within homozygous male recombination being (Figs. of and ongoing males homozygous or Comparisons of be past equally 30% to approximately Rare loci. two by females loci. had all suggested and these showed pools was genotypes 93182-93327, at male region WGS and heterozygous region guided Individual allele males XY-gametologous sexing single tests of SNPs. 146-bp including a six 70% CMH had the loci, these with pools in for sex-linked Female alleles perch indels with 94017. represented two Macquarie enriched at and SNP for 90152-94017, SNPs additional region data an five 633 WGS encompassing scaffold of 93182-94017, on region pools focus 2021). population to al., female et us the (Nguyen and on loci male based female-linked inferred Exploring moderately were and catfish coastal sex-determination male- in ZW the has moderately including and DArTseq from systems, of XY sex-determination 2017). presence both Basin infer al., where Darling et and sex-chromosomes, Gan, loci homomorphic Murray (Pavlova, sex-linked with isolation the find population to of and variation used contraction colonization sex-linked been range lost in Macquarie the recent perch have during involved in with Macquarie could or bottleneck perch than systems, to range Macquarie Pleistocene perch sex-determination homologous Alternatively, independently-evolved golden the VMKM01003747 recent. have more in scaffold during being species perch loci one these golden DArT perch that sex-linked Macquarie the indicates more on species, were 633, these magnitude of which scaffold of sex-chromosomes re- of on order that present none an suggests if in perch, This recombination), Finding only of scaffolds. polymorphic lack short. different long-term loci with through are (with found consistent scattered strata but old SNPs females), perch, to with DArTseq sometimes Macquarie karyology gions uncover males, or by not (sometimes perch inferred did golden sex We is XY either one have in perch, 2019). to XY-gametology al., golden cod or Murray et species, Y-linkage confamilial identify (Shams two the these chromosomes and to in of chromosomes, heteromorphic aimed markers One sex and we homomorphic sexing and species. sequencing, molecular sex-determination confamilial amplicon XY test have two and and with sex, develop along known re- and species of whole-genome regions individuals and genomic of (DArTseq) sex-linked panels sequencing for reduced-representation (WGS) genomes, sequencing assembled newly two F2), (Table Using perch golden in to variation homologous sex-linked F3). Discussion (Table clear fragment cod reveal consistently amplicon-sequenced trout not be or the might did F1) cod assay) for region (Table Dartmouth Murray sexing sex-linked Genotypes populations PCR-RFLP from perch coastal the males Macquarie for from individuals. two the chosen individuals sequenced site and two (the across females the 93229 two sex-linked for at variation the sequences only Amplicon for that using genotypes showed sequencing F1). amplicon WGS Table by with F, captured amplicon 633 consistent (Appendix scaffold by were of revealed region primers ˜400-bp relatives, the F2-R3 three for genotypes its perch, Macquarie not For but perch, three or Macquarie two and in sequencing individuals, B5). polymorphism perch of Fig. Macquarie 34% sequence B, in in Sex-specific (Appendix band tested single individuals assay of a of PCR-RFLP rate yielded were 66% same fertility populations that in The eggs produced additional bands an that eggs six fertilized. showed females from successfully produced one six sex were fertilized, two unknown Of which of not bands), single fertilized. eggs were be had a yielded that to one eggs produced that failed and produced that that ten four eggs females (male-like), of have had four bands two to (out two Of and appear assay hatchery, males) in 1). PCR-RFLP fertilized feminized Material the successfully potentially (Supplementary on (i.e. rate band allele fertilization female-like male-specific lower a with with eggs females had Abercrombie B4). Fig. 11 Macquaria lra gariepinus Clarias ec sister- perch < 1%, Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eotdfrgle ec n urycdsxcrmsms(hm ta. 09.Gvnishg read-depth high its Given 2019). al., et (Shams sex-chromosomes cod Murray and perch golden for reported hslwrdphmgtb ikdt iclyo eunigtruhalwcmlxt ein nldn the 1). including region, (Fig. zero low-complexity depth a including sequence through genome-average, overall sequencing the their of to of difficulty compared to regardless linked region individuals be sex-linked (GT) of might the 59% depth for lower for 2014). This depth 93400 al., obscured read position et was in 633 around (Wilson scaffold coverage W-alleles drop of of region the XY-gametologous loss by the after at natural here Bradley de-novo homo/hemizygosity evolved example, 2012; of which nature For al., the 2012) et Understanding al., drift. (Anderson et inferred by Liew been sex-chromosomes have 2011; of strains al., loss laboratory et different population- to of response and systems in sex-determination shared- ( rapidly species evolve zebrafish sex, can same of systems on the populations sex-determination Different of temperature between 2018). differentiation populations Patrelle, environmental Wapstra, sex-chromosome in & of Merila, of Ezaz, degree Rodrigues, different Burridge, influence a (Hill, skink 2015; suggesting of populations detected, al., snow were degree et loci spotted sex-linked their Lubieniecki specific the sex-determining in 2018a; of In al., perch differ locations Macquarie et chromosomal which 2014). in (Kijas in variation variation vertebrates Perrin, sequencing sex-specific Intraspecific amplicon other & that in in populations. loci suggests occurs some these further at loci to sex variation specific reference unknown Unique the be of male-specific populations. for may could individuals the some homozygosity males had coastal to female of perch, Abercrombie limited two Macquarie males, ˜2% from be for of (egg-producing) except might 60% populations feminized band), 93299 and other are one at in Creek well individuals on allele Parrot work (based these King individuals to females Unless from Yarra expected or females and not allele. putative gel) Dartmouth is of identify test a in same correctly 20% on present The should because bands was more test females. Y-allele three as in the This or region missexed hence two and species. be sexing region tested, on the perch, sexing 50 of (based 146-bp this golden subset of males the a that out in as to showed male in limited perch, cod) one be 93229 trout Macquarie but even and all could of of cod and Y-allele populations (Murray perch-specific, Percichthyidae Macquarie other the family is in the targeted from 633, that of relatives scaffold mechanisms distant protocol, King perch other sexing of for Macquarie 36% molecular call of in model our and 93229 suggested Dartmouth of at the the Tests Y-allele from on the deviation based phenotype. of single mechanism female presence this This a Y-alleles However, with of B4). producing consistent individuals phenotype. presence not (Fig. whereas male only is phenotype, populations which a 14%- females female Yarra in to in Abercrombie a and sex-determination leads develop and 93315, of loci Creek loci and these Parrot hypothesis the 93278 of working of 93184, 22 100% a 93182, some all for in suggests at at of present X-alleles This 96% were the 93327, at loci. Y-alleles and homozygous other and 93299 the X-alleles, 93229, individuals for for for 2016), 90152-94017 100 98% homozygous 76% region in al., 94017, were screened 633 for females et C1), scaffold males all in Fig. of (Chen Yarra, heterozygosity polymorphism; Manta of and recent-Y-specific patterns Dartmouth bearing of sex-biased from loci with tests 14 loci and would original 22 XY-gametologs that of (8 in depth variants panel read used the structural our in ˜50x any however Overall, D1), detect the Y-specific not (Fig. than analysis. suggested did patterns our lower females these analysis underpowering to much with Manta potentially compared consistent was 2). males polymorphism (˜10-20x) and in deletion coverage Y- 93879 1 or for Y (Figs. X- depth and support locus read X this lower between region at recombination The multiple XY-gametologous occasional deletions that The C1). suggesting suggest 93879, perch. C1) (Fig. SNP haplotypes Macquarie SNPs recent-Y-specific (Fig. in another sex-difference. males 13 segregating included of in containing itself are region polymorphisms emerging 90152-92914 Y-haplotypes recent-Y-specific recently recombining region these more occasionally of the a appearance males be was of some might 93182-94017 Patterns which that suggests region polymorphism, Y-allele recent-Y-specific XY-gametologous the bearing the than males of Y0). in Upstream depth (i.e. X-allele Y average for Lower haploid be 2). could (Fig. males in alleles n irstlieta nsaon 39 Fg 1.Acmlto frpttv eune a previously was sequences repetitive of Accumulation B1). (Fig. 93193 around ends that microsatellite ai rerio Danio aeamngncZ e-eemnto ytm hra polygenic whereas system, sex-determination ZW monogenic a have ) iesicsocellatus Niveoscincus 12 ihadadlwadpopulations, lowland and highland , Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ms .N,Hrisn .A,Rdod .Q,Wie . eel . a al,R,...Pvoa A. Pavlova, . . . R., Nally, Mac G., Newell, M., White, Q., J. Radford, A., K. Harrisson, N., J. Amos, Research NeCTAR on Australian hosted References servers Office, eResearch. Water virtual Monash Environmental on Sam- of Commonwealth run courtesy the were Cloud, Australia). from analyses (Western intensive funding Attractions Computationally (ACT by Government. and Directorate supported Conservation Development was Biodiversity, Sustainable collection of ple & Diversity Department Monash Planning DELWP, to Canberra, and Eco- Environment, of LP160100482 Government), of Victoria, University and Department Organizations Zoos Water, Partner Technology, within Melbourne with Arrays (now University, and Trobe Victoria Resources) La Fisheries and and DELWP), University Transport Planning; Jobs, Department Development, & within was (now Water nomic Flinders work (Victoria) Corpo- University, Environment Land, This ACTEW and Environment, Monash Montana, Sustainability assistance. of of of to and University Department lab Water), Organizations Tonkin LP110200017 for Icon Partner (now Grants Zeb Polesskiy with ration Canberra, Linkage Anna Thiem, of and Council University Jason Lutz and Research University Stoessel, Maiko Luke Australian Daniel and O’Mahony, by collection, Smith, Justin supported sample McLay, Chris for Duncan Sharley, Wooden McDonald, Joanne Ian Graeme Alasdair Rehwinkel, Doyle, Jonathon Lintermans, Rohan Butterfield, Tom Mark Pearce, Beitzel, Hartwell, Matt Babbs, Dean Mark Hackett, Ayres, Renae Asmus, species. Marty within thank We including lineages, closely-related quite in this mechanisms Acknowledgements in different sex-determination and known-sex of regions platyfish for understanding genomic data in our or WGS improving interact sites Acquiring for 633 in and crucial phenotypes. scaffold Sex-determination be male locus perch will species. or Macquarie determination populations female on other sex of region from development sex-linked samples the some with the at at links in variation mechanistic segregate chromosomes sequence has whether W) different demonstrate and on to needed present Y, are (X, maculatus systems alleles Xiphophorus sex-determining Three ZW p 2010). and detected (at XY loci tests both Metriaclima many CMH Also, the the given by 2015). possible, male-linked bass is and sea perch female- it Macquarie putatively Thus, in as sex-determination development. polygenic testes Alternatively, inducing perch. Macquarie expression in their development gonadal with controls species, that different possible in is determination particular, in sex genes male Three and 2013). ˜39 2005) complex). Hochedlinger, & protein located (Koopman, (Sarkar or protein was development and during protein but activity cells any gene, factor progenitor with exchange known selectively guanyl-nucleotide a (interacting with to associated binding superfamily, annotated of domain not (DH) gene 146-bp was homology scaffolds. the of 90152-94017 the perch of upstream region search Macquarie for loci BLAST sex-linked Kb other depth XY-homologous in 633 in regions: some regions repetitive drop scaffold for similar to the The highly due genotypes that find mapping unlikely Y0 failed not is or to did It X0 due region was an phenomenon. sexing region biological Thus, for sexing a male 633 population- 2). represents a scaffold and likely sex-specific including (Fig. individuals detecting depth, coverage some preclude WGS not depth in Apparently did reasonable of were also techniques. with depth patterns genotypes Low males both specific WGS-based done. for with was WGS, even analysed sequencing observed in amplicon individuals which were four depth homozygotes contain the read that Y-allele for reduced regions real genotypes genomic locally amplicon-based genotyping the to of way identical Despite reliable more fragments. a provide low-complexity may sequencing amplicon coverage, ietacu labrax Dicentrarchus ihis iheitssbtengntpsa w oicnrligsx(e,Rbrs Kocher, & Roberts, (Ser, sex controlling loci two at genotypes between epistasis with cichlids, SOX eeg08,mdltdb h aito ihntesxlne ein90152-94017, region sex-linked the within variation the by modulated g10286, gene SOX3 Shlhi,Bhe cat,Vl,&GlaaAnu,20) uuesuisare studies Future 2009). Galiana-Arnoux, & Volff, Schartl, Bohne, (Schultheis, SOX Macquaria rgntro the of progenitor a , tlatfu oiaeascae ihsxdtriain(aaootse al., et (Palaiokostas determination sex with associated are loci four least at rncito atrfml,ad4 bdwsra fapoenwith protein a of downstream Kb 41 and family, factor transcription per ob ope n yai,ptnilyivligmultiple involving potentially dynamic, and complex be to appears SRY 13 eei aml Gae,21) aearl in role a have 2013), (Graves, mammals in gene < e5 upeetr aeilS) In S3). Material Supplementary 1e-5; SOX ee euaeft fse and stem of fate regulate genes SOX9 , SOX8 Dbl Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. il .L,Brig,C . zz . asr,E 21) osraino e-ikdmresamong markers sex-linked temperature- of and Conservation genetic (2018). exhibiting E. ocellatus, Wapstra, Niveoscincus & skink, determination. T., viviparous sex Ezaz, a dependent dartR. P., of shared to C. populations to Introduction Burridge, conspecific due (2018). determination L., A. P. sex Georges, vertebrate & Hill, O., in Berry, homologies P., Unmack, Are B., (2010). Gruber, L. options? C. limited Peichel, to genes. & or determining ancestry M., sex A. vertebrate J. new Graves, evolve to How (2013). Repeat- (2013). 242 M. (2020). R. A. F. T. A. families. J. Smit, Gingeras, Graves, element & C., transposable . Feschotte, 117 of G., Sciences, . A. discovery of Clark, genomic Academy J., . automated Rosen, S., C., for Jha, Goubert, Modeler2 R., C., Hubley, of Zaleski, M., overview J. J., Flynn, an Drenkow, F., aligner. fish: RNA-seq Schlesinger, in universal A., differentiation ultrafast C. sex STAR: Davis, and determination A., Sex Dobin, (2011). influences. (2002). M. environmental Y. Hanna, and data. Nagahama, physiological, . sequencing & genetic, . DNA H., . next-generation R. C., Devlin, using Hartl, C. R., genotyping sequencing Saunders, J. and cancer Maguire, discovery . V., and 43 K. variation Genetics, germline Garimella, . for for R., framework Poplin, indels . E., A and Banks, M., variants A., Kallberg, M. structural DePristo, F., of Schlesinger, detection B., rapid Barnes, Manta: preprocessor. applications. R., FASTQ all-in-one Shaw, (2016). ultra-fast O., T. an sex Schulz-Trieglaff, fastp: heteromorphic X., (2018). of Chen, J. evolution Gu, the & Y., in 34 Chen, formatics, Steps Y., Zhou, (2005). S., Chen, G. Marais, & B., chromosomes. Charlesworth, D., Charlesworth, Database. Eukaryotic Protein Automatic a by BRAKER2: An Supported (2020). AUGUSTUS (2011). M. https://doi.org/10.1101/2020.08.10.245134 and Borodovsky, R. & GeneMark-EP+ J. A., with Lomsadze, Annotation Smith, M., Genome & Stanke, K., W., Hoff, chromosome E. T., Y Bruna, Knapik, W., The loci. K. determination (2019). Broman, sex T. reveals B., zebrafish Zhou, 3-9. D. for Melville, map . P., linkage J. fish. SNP-based . Breyer, teleost M., in . K. mechanisms Z., Bradley, determination Yuan, sex A., novel Elaswad, suggests (2014). Y., R. catfish Zhang, Ming, 17 channel S., . the Liu, of . C., sequence . it? T.-L., Tian, Ashman, doing L., P., of S. Bao, ways Otto, many M., so Kirkpatrick, why L., C. determination: Peichel, Sex E., degener- J. Y-chromosome Mank, D., of Bachtrog, processes into insights emerging and evolution: mapping ation. Y-chromosome RAD by (2013). revealed D. (2012). H. Bachtrog, zebrafish J. in Postlethwait, chromosome & P., sex Batzel, putative P., genomics. Hohenlohe, a population A., and Amores, birds. regions I., woodland Braasch, sex-associated of R., Multiple A. suite Mari, a L., in J. Anderson, fragmentation habitat of effects connectivity 95 sex-specific Ecology, and Species- (2014). 1,6. (1), 4,354-359. (4), aueRvesGntc,14 Genetics, Reviews Nature 6,15–58 doi:http://dx.doi.org/10.1890/13-1328.1 1556–1568. (6), 5,491. (5), iifrais 32 Bioinformatics, 1) i884-i890. (17), eeiy 95 Heredity, LSOE 7 ONE, PLoS 1) 9451-9457. (17), 2,118-128. (2), eoeBooyadEouin 10 Evolution, and Biology Genome eoeBooy 11 Biology, Genome 8,1220-1222. (8), 2,113. (2), (7). iifrais 29 Bioinformatics, LSBo,12 Biol, PLoS 4,205. (4), 14 qautr,208 Aquaculture, 1,15-21. (1), 4,1079-1087. (4), 7,e1001899. (7), 3 ee,Gnms eeis 1 Genetics, Genomes, Genes, G3: 34,191-364. (3-4), rceig fteNational the of Proceedings eeomna Dynamics, Developmental m Biology, Bmc iRi,doi: bioRxiv, Nature Bioin- (1), Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. atnz . ia,A . ace,L,Da,N,Rbs . ierr .(04.Gntcacietr of architecture Genetic aquaculture. (2014). in F. control Piferrer, ratio & sex L., to Ribas, applications N., Diaz, doi:10.3389/fgene.2014.00340 fish: L., in Sanchez, determination M., sex A. Vinas, P., reads. Martinez, sequencing high-throughput from sequences mechanisms adapter 17 sex-determining removes A. alternative journal, Cutadapt J. of (2011). Evolution C. M. Bradshaw, (2006). Martin, C. . J. Avise, . & fishes. E., teleost . D. in A., Promislow, B. E., Ingram, J. Mank, D., Stoessel, ( Using cod A., trout reproduction. MacDonald, Australian (2020). and J., threatened growth A. of S. Pavlova, success Nicol, Reintroduction C., . (2012). Todd, P., . of J. reproduction Lyon, . and J., survival Sharley, benefits B., population Ingram, extinct 2020 G., locally fish. freshwater a Johnson, endangered of D., an reintroduction instability J. during Genomic Yen, sources (2015). Z., multiple S. Tonkin, W. sex salar). M., Davidson, Polygenic (Salmo Lutz, & Y., salmon (2012). Y. Atlantic L. Lai, in J., Orban, Li, locus 2513-2522. & I., sex-determining E. R., the Cabana, K. S., of Siegfried, Lin, P., R., transform. K. Sreenivasan, Lubieniecki, Burrows–Wheeler Z., with Lim, zebrafish. alignment in R., read system Bartfai, short determination C., accurate W. and (2014). Fast Liew, M. Miya, (2009). R. . 25 Durbin, Bioinformatics, . & . with H., N., analysis. comparison Li, simultaneous Suzuki, (Percomorphaceae): and N., Centrarchiformes studies Yagishita, gene-based and Y., Percichthyidae nuclear Yamanoue, recent the R., of D. between genes. phylogeny Jerry, Sox differentiation Mitogenomic two K., identifying Nakayama, of tale S., (2018). PoPoolation2: a Lavoue, P. determination: Krusche, Sex (2011). . (Pool-Seq). (2005). C. P. samples . Koopman, Schlotterer, DNA pooled . & of M., V., sequencing Kallberg, using R. E., variants.populations Pandey, somatic Noh, and R., A., germline M. Kofler, arrays of Bekritsky, Diversity calling L., accurate (2012). A. and C. fast Halpern, Hopper, Strelka2: K., . Scheffler, platforms. . S., open Kim, . on Protocols H., technology and Blois, Methods profiling L., Genomics: genome Xia, Evolution Population J., generic (2018b). K. Carling, a Verbyla, E., . Huttner, technology: P., . Wenzl, . A., B., salmon. Evans, Kilian, Atlantic H., Evolution in King, (2018a). P., loci K. Kube, determination Verbyla, N., sex . M. of Sanchez, . S., InterProScan . McWilliam, B., J., (2014). Salmon. Evans, Kijas, G. Atlantic H., Nuka, in King, P., Loci . Kube, Determination N., . Sex M. of for Sanchez, . S., technology C., McWilliam, McAnulla, state J., W., Kijas, solid Li, classification. a M., function Fraser, protein arrays: H.-Y., genome-scale Diversity Chang, 5: D., (2001). Binns, A. P., Jones, Kilian, & genotyping. D., independent Feinstein, information K., sequence (2012). Peng, M. D., Holland, Jaccoud, & M., G. Turchini, Industries. broodstock K., cod H. Murray Ho, tive A., B. Ingram, doi:10.1111/eva.13173 . 1,10-12. (1), ilgclJunlo h ina oit,87 Society, Linnean the of Journal Biological 1) 1754-1760. (14), aieadFehae eerh 63 Research, Freshwater and Marine ihre itraRsac eotSre o 8:Dprmn fPrimary of Department 58.: No. Series Report Research Victoria Fisheries . vltoayApiain,1.11ea113 ulse nie2 November 22 online published 10.1111/eva.13173, Applications, Evolutionary LSOE 7 ONE, PLoS cetfi eot,8 Reports, Scientific 67-89. , uli cd eerh 29 Research, Acids Nucleic iifrais 30 Bioinformatics, cetfi eot,8 Reports, Scientific (4). 15 ee 549 Gene, 1,83-93. (1), 7,58 doi:10.1071/mf12034 598. (7), rnsi eeis 21 Genetics, in Trends 3 ee,Gnms eeis 5 Genetics, Genomes, Genes, G3: iifrais 27 Bioinformatics, 1,1-11. (1), aclohlamacquariensis Maccullochella 9,1236-1240. (9), 1,5664. (1), aeeqaiyadsann ncap- in spawning and quality Gamete aueMtos 15 Methods, Nature 1,46-57. (1), aaPouto n nlssin Analysis and Production Data 4,e25-e25. (4), rnir nGntc,5 Genetics, in Frontiers 2) 3435-3436. (24), 7,367-370. (7), 8,591. (8), ae on based ) EMBnet. (340). (11), Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. e,J . oet,R . ohr .D 21) utpeitrciglc oto e eemnto in determination sex control loci interacting Multiple (2010). D. T. Kocher, & fish. B., cichlid R. Malawi lake Roberts, Annotation R., and J. Assembly Genome Ser, Assessing BUSCO: (2019). Humana. (Ed.), M. NY: Kollmar E. M. Zdobnov, diversity In & determination Completeness. M., Sex Manni, (2009). M., D. fish. Seppey, Galiana-Arnoux, poeciliid & in stem J., evolution Volff, of chromosome M., regulators sex Schartl, versatile and A., factors: Bohne, transcription C., of Schultheis, family M. sox E. fate. The genome Gray, cell and (2013). progenitor . tumors, K. and . Hochedlinger, gonad & gametogenesis, . A., Y.-L., architecture, Sarkar, Yan, nuclear A., oocyte R. in BreMiller, zebrafish. (fancd1) C., in brca2 Canestro, stability of A., Roles T. differ- Titus, sex-chromosome (2011). C., in variation Wilson, Geographic A., Rodriguez-Mari, (2014). ( N. frog Perrin, common & the C., in Patrelle, entiation J., Statistical for Merila, Foundation N., R Rodrigues, http://www.R-project.org/ computing. statistical from for data. Retrieved environment count and Austria. language for Vienna, A test R: Computing, sample (2020). paired Team. accurate Core R An (2012). R. of C. i596-i602. mechanisms Jimenez, environmental (18), & and V., genetic T. I: Part Pham, gonadogenesis. Fish (2008). F. determination. Piferrer, sex & Pu- J., endangered (2017). an D. P. in Penman, Sunnucks, genome & mitochondrial M., the Lintermans, of D., evolution fish. Gilligan, Pleistocene freshwater M., shaped Australian C. drift Australian Austin, genetic P., and Sunnucks, endangered Y. selection an Lee, . rifying M., of . H. flow. diversity Gan, . gene genetic A., A., Pavlova, assisted on B. for fragmentation Ingram, call A., (2013). habitat a K. P. of fish: Harrisson, Sunnucks, consequences freshwater D., Severe Gilligan, . R., (2017). Coleman, . P. B., L. . Beheregaray, S., A., Pavlova, J. the in Keogh, environment bird. J., Australian with an J. correlate in doi:10.5061/dryad.f5q32 DRYAD flow Austin, lineages gene mitochondrial K., nuclear divergent ongoing Loynes, their of crossroads: face and L., mito-nuclear chromosomes Joseph, the sex identify N., at to Perched J. How Amos, (2019). A., E. Pavlova, A. Wright, & R., Dean, Vandeputte, F., (Dicentrarchus . turnover. T. bass Rogers, sea . European H., . in D. . determination B., Palmer, sex Chatain, of J., . genetics B. the of McAndrew, . vision K., SNP-based labrax). Gharbi, W., new A B., Singchat, J. (2015). Taggart, M. E., a M., African Kraichak, Bekaert, with North C., in N., associated Palaiokostas, Systems Determination Laopichienpong, determination Sex XX/XY J., 1822). sex and Burchell, Ponjarat, gariepinus, ZZ/ZW of (Clarias of Investigation Catfish T., evolution An (2012). Panthum, The (2021). D. K. M., Marshall, Srikulnath, (2019). H. T. . D. Shikano, Nguyen, . & J., . L., Merila, inversion. chromosomal Cardle, M., L., H. Pritchard, data. Natri, A., sequencing J. second-generation doi:10.1093/bib/bbs012 P. of 193-202. Cock, exploration (2), visual M., for Bayer, Tablet G., Using Stephen, I., Milne, eeisSlcinEouin 47 Evolution, Selection Genetics oeua Ecology Molecular eiw nFseisSine 16 Science, Fisheries in Reviews lsGntc,7 Genetics, Plos vlto:ItrainlJunlo rai vlto,64 Evolution, Organic of Journal International Evolution: 12 cell, stem Cell aueCmuiain,10 Communications, Nature eeiy 118 Heredity, . aatemporaria Rana eePeito.Mtosi oeua ilg,vl1962 vol Biology, Molecular in Methods Prediction. Gene 3,e1001357. (3), 6–7.doi:doi:10.1038/hdy.2016.120 466–476. , 1,68. (1), 1,15-30. (1), r nir nGntc,11 Genetics, in Frontiers vltoayApiain,10 Applications, Evolutionary eulDvlpet 3 Development, Sexual ). oeua clg,23 Ecology, Molecular 16 S) 16-34. (S1), 1,1-13. (1), vlto,67 Evolution, 1) 4232.doi:10.1111/evo.12107; 3412–3428. (12), 11) doi:10.3389/fgene.2020.562856 (1719). 23,68-77. (2-3), 1) 3409-3418. (14), 531–550. , rensi iifrais 14 Bioinformatics, in Briefings 2,486-501. (2), iifrais 28 Bioinformatics, e York, New . Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. cffl 5 4 b22Kb 252 scaffolds 7,165 Gb Mb 3.1 661.43 Kb 845 scaffolds 2,962 Gb 264.5 Mb SRX6595904 675.98 Gb SAMN12340637 7.6 SRX6595905 PRJNA556086 Gb 237.1 SRX7207074 SAMN10817714 MP SRX7207073 PRJNA516983 N Contig scaffolds of Number N50 Scaffold size genome Assembled assembly: Genome Nanopore accession NCBI-SRA data 1D Nanopore DNAseq accession NCBI-SRA data DNAseq Illumina DNAseq BioSample genome ID Sample DNAseq: BioProjectID NCBI genomes. reference authors all 1 manuscript, Table the drafted AP sequenced, Figures assays, HMG KH and sexing and Tables data, tested RT AP, hatchery and MHG, collected designed data, writing. KH DG WGS to and obtained and contributed PS and BI AP, libraries manuscript, prepared data, YPL the analysed genome, conceived annotated KH and assembled and PS HMG, Repository the AP, Research under NCBI Monash in Contributions Bridges for Author deposited in https://doi.org/10.26180/13551713 been available and also are perch. perch have annota- golden de- genotypes Macquarie perch (repeat been DArT Macquarie for data whole have 100 https://doi.org/10.26180/5ea1736cc3553 from annotation output) the generated PRJNA598965. Genome InterProScan reads of BioProjectID Raw (WGS) and resequencing (https://doi.org/10.5281/zenodo.4315338). genomes perch. repository genome repeat-masked golden PRJNA516983 Zenodo BioprojectID genes, for in the posited protein-coding PRJNA556086 under NCBI predicted and in deposited tion, Hybrid perch been have (2017). Macquarie assemblies L. genome for S. with and Salzberg, reads wheat, raw bread . The of M. . progenitor Schartl, a . tauschii, Accessibility G., Aegilops . Data Marcais, of S., genome . Koren, algorithm. repetitive mega-reads T., highly . MaSuRCA Zhu, and strains. A., the M.-C., large domesticated T. Luo, the in of D., Titus, determinant assembly Puiu, Y.-l., sex V., Yan, natural A. A., the Zimin, Amores, of . loss M., B. . best zebrafish: 1291-1308. McCluskey, A., toolkit in (3), K., analysis sex Levy-Moonshine, genome S. Wild G., the High, Angel, (2014). calls: A., Del variant C. R., high-confidence Wilson, Poplin, to C., data FastQ Hartl, From O., pipeline. (2013). M. practices J. Carneiro, Thibault, A., (2019). . Open-4.0 G. RepeatMasker Auwera, T. (2013-2015). der Ezaz, P. Van Green, & & Z., R., Hubley, Majtanova, A., D., Smit, ( J. Perch Golden Thiem, Fishes, P., Freshwater 20 Native ences, R. Australian Duncan, Two in R., ambigua Chromosomes Thompson, Sex and F., Karyotypes Dyer, F., Shams, n uryCd( Cod Murray and ) 50 eoeasml n noainmtisadacsinfrMcureprhadgle perch golden and perch Macquarie for accession and metrics annotation and assembly Genome 1) 4244. (17), urn rtcl nbonomtc,43 bioinformatics, in protocols Current aclohlapeelii Maccullochella eoeRsac,27 Research, Genome )(Percichthyidae). 17 1,1.0 11.0 33. 11-11.10. 11.10. (1), 5,787-792. (5), nentoa ora fMlclrSci- Molecular of Journal International < http://www.repeatmasker.org 7 b24Kb 244 Kb 679 perch golden perch Macquarie C1 GOP001 SCH12 eeis 198 Genetics, Macquaria > . Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 81652-/ MM10907673 5000 0 0 0 24 0 25 0 0 0 1 1 0 0 0 0 24 0 25 0 0 0 1 1 0 0 0 0 0 0 0 0 8 13 0 hom alt M2 0 25 25 24 24 0 loci 25 Old-X-linked 0 24 perch Macquarie hom 52 alt M2 65 loci Old-X-linked 65 62 loci Old-X-linked 0 1 0 0 perch Macquarie 36 0 36 loci 0 Old-X-linked het M1 loci Old-X-linked 0 0 0 perch Macquarie 1 4 6 5 loci Old-X-linked 6 5 loci 7 Old-X-linked 5 hom ref M0 6 perch Macquarie 0 0 loci Old-X-linked 8 8 loci 36 Old-X-linked 34 34 35 hom alt F2 perch 0 Macquarie 34 0 35 loci Old-X-linked loci Old-X-linked 78 92 70 71667 18984 66 perch Macquarie 7572 135276 het 130180 loci F1 Old-X-linked 21759 157581 173435 loci Old-X-linked VMKM01001960 1691939 VMKM01001928 perch 43200 Macquarie VMKM01001533 VMKM01001374 59288 1395201 VMKM01001181 loci Old-X-linked VMKM01001101 VMKM01001081 hom ref F0 VMKM01001065 loci Old-X-linked polymorphism sequence recent-Y-specific bearing locus Loci the 28619655-25-A/T of position Scaffold polymorphism SEMN01000020 SEMN01000633 recent-Y-specific 28618539-6-C/T bearing Loci perch Macquarie 37096050-57-C/T polymorphism recent-Y-specific bearing Loci 28931872-6-A/G SEMN01000754 polymorphism recent-Y-specific 28618575-31-A/T bearing Loci SEMN01000004 loci ID Old-X-linked 33707197-25-A/G scaffold polymorphism genome recent-Y-specific bearing reference Loci NCBI 28618482-9-G/A polymorphism recent-Y-specific 28619580-14-C/T bearing Loci loci Old-X-linked polymorphism recent-Y-specific perch bearing Loci Golden polymorphism 17035690-65-T/A* recent-Y-specific bearing 16,792 Loci 28931412-11-C/T* polymorphism Gb recent-Y-specific bearing 11.6 Loci perch Macquarie 36,108 36356824-43-G/A SAMN14943551 SRX8356284 25.8% 36356960-12-G/A loci Old-X-linked indicate scaffold Asterisks perch by Macquarie sorted genome). italic. are reference bold 31,895 perch Loci in ID golden 16,264 are population. SAMN13380527 locus the single loci 95.9% Dart to VMKM01 a 203 align Gb MP of scaffold not 15.1 sex are did GP alternative one DArT-tags that two SRX7207075 the only contigs perch the females/males for in 7,352 golden bold; present of homozygous (four number were are in number ID = heterozygotes that are and = hom which females/males loci (F) ref for het of 33,422 633 hom/M0 loci female number het/M1 scaffold ref = in F1 27.7% MP F0 hom individuals allele, alt details): allele. and heterozygous hom/M2 reference for ID alt and a S2 scaffold F2 homozygous for Material abbreviation, heterozygous, Supplementary of homozygous species (see count genome, females/males VMKM00000000.1 given reference loci, 10% of S2 is a in all 29,940 Material more sample contigs SEMN01 to For heterozygous or (M) Supplementary 3,369 mapped but 10% male see locus given. in females 96.9% DArT males; heterozygous are all each but 25 in For coordinate males and homozygous males. all polymorphism- females more in recent-Y-specific or 41 homozygous SEMN00000000.1 bearing those (GP; GCA loci loci- perch Old-X-linked females; golden information). and more males) for 78 and 10.5281/zenodo.4315338 females 2. 10.5281/zenodo.4315338 Table InterProScan5 and BRAKER2 RepeatModeler2, by annotations for annotations DOI GO Zenodo with genes genes of protein-coding Number annotated functionally of genes Number protein-coding predicted of Number annotation: Genome RNAseq accession NCBI-SRA bases G RNAseq, Illumina RNAseq BioSample RNAseq ID Sample RNAseq: repetitiveness Genome completeness genome BUSCO3 accession Shotgun Genome Whole accession Project WGS accession assembly GenBank contigs of Number ATdrvdSPlc ihsxbae atrso eeoyoiyfrMcureprh(P 93 (MP; perch Macquarie for heterozygosity of patterns sex-biased with loci SNP DArT-derived 18 aqai ec odnperch golden perch Macquarie 2 GPTT01 527 0484. GCA 005408345.1 008360985.1 Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 30465-/ MM10914934 0500 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 25 0 1 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 25 5 0 0 4 1 25 0 3 3 0 1 0 3 3 0 1 0 4 0 0 3 0 0 4 0 3 2 3 0 0 3 3 0 0 0 25 20 perch Golden 0 21 0 0 21 0 0 22 0 0 0 22 22 0 1 21 0 0 22 0 0 0 21 0 22 0 22 0 0 22 22 perch 0 Golden 0 0 0 0 0 0 24 0 1 0 0 0 0 24 0 1 0 hom 25 alt 0 M2 25 0 0 25 0 24 0 0 perch Golden 25 0 23 1 25 25 2 0 25 25 25 0 0 25 25 0 0 hom alt 34 M2 24 0 0 25 0 25 perch 0 0 Golden 0 23 1 0 24 0 0 25 0 0 0 0 3 0 0 0 0 0 0 0 0 34 41 het perch M1 0 Golden 41 0 4 38 6 0 41 0 6 0 40 41 5 5 41 6 0 41 5 0 6 41 5 41 9 40 6 5 39 41 5 perch Golden hom ref M0 6 41923 5 99375 7 132713 6 268476 36 5 0 30436 4 2935 5 35 26259 6 36 sexing 50324 5 146-bp the 34 within 301933 located 5 SNP 36 five grey, 35 325681 6 shaded alleles two are 147539 but loci allowed) perch error 36 633 Golden VMKM01000991 (one Scaffold pools bold. female 1113651 in italic. 28 in VMKM01000475 are allele hom in 139620 93182-93327, one alt are just region F2 35 with pools 36 VMKM01000445 Loci male males. in in VMKM01000323 Yarra 25 test or Cochran-Mantel-Haenszel 36 females Yarra by detected 35 VMKM01003205 pools, p-value female VMKM01003059 and male 36 VMKM01002530 3. Table 0 VMKM01000212 3422 35 VMKM01000017 76143 36 312116 VMKM01000160 28619935-62-T/A 35 VMKM01001354 142832 33707406-50-T/C 36 polymorphism recent-Y-specific bearing 200871 Loci 35 VMKM01000010 20268621-32-A/C perch VMKM01000001 Golden 213128 polymorphism 26 recent-Y-specific 28619579-52-G/A bearing Loci 28036 20267873-5-G/C 36 polymorphism recent-Y-specific bearing 749908 Loci 100026247-65-T/A 35 174302 polymorphism recent-Y-specific 17038127-64-C/A bearing Loci het 168594 F1 28619198-20-C/A polymorphism recent-Y-specific bearing 1909 Loci 20267388-24-G/A VMKM01000987 18850 polymorphism recent-Y-specific 20265649-22-C/T bearing Loci VMKM01000979 15044 100025231-56-G/A VMKM01000097 polymorphism recent-Y-specific 112501 bearing Loci 37096713-34-G/T VMKM01000877 589065 polymorphism 100008933-5-C/A* recent-Y-specific bearing VMKM01000770 Loci 288497 37096522-21-G/A VMKM01000739 polymorphism recent-Y-specific 27593 bearing Loci VMKM01000738 perch Golden 29291 VMKM01000064 28618437-13-T/A 24594 VMKM01000526 52353 20268010-19-T/A VMKM01000489 64254 28619462-17-T/A VMKM01004724 251318 28618970-31-T/C VMKM01004054 476876 hom 28619737-22-G/A ref F0 VMKM01000394 37096210-58-C/T VMKM01000378 17038132-46-A/T VMKM01000035 17039139-40-T/C sequence VMKM01000328 locus the 28618085-27-G/A of VMKM01002906 position Scaffold 28618065-41-T/G VMKM01002812 28619004-10-A/C VMKM01002499 37095970-57-G/T VMKM01002345 37095883-61-C/T VMKM01002203 ID 28620041-44-A/G scaffold genome VMKM01000203 reference NCBI 37095925-14-T/A VMKM01000203 17036577-54-A/G 37096511-30-G/A 20266862-57-T/A 28618166-37-C/T 37096320-25-C/A 100010491-66-G/T 37096174-14-C/T* 33707200-37-T/C 28619537-13-C/A 37096215-12-T/A 37096062-11-G/A ID locus Dart . ahpo otis2 ilo ed rmec f2 atot eae,2 atot ae,25 males, Dartmouth 25 females, Dartmouth 25 of each from reads million 25 contains pool Each leecut o G-eie aqai ec oisrnl (P strongly loci perch Macquarie WGS-derived for counts Allele 19 PoPoolation2 < e2)dffrnitdbetween differentiated 1e-20) oisre ytest by sorted loci ; 1002 0 0 0 0 0 0 3 0 0 0 21 0 0 24 0 24 25 0 0 0 0 6 41 4 7 35 34 34 Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 38 00024 XY recent-Y recent-Y 1 0 0 47 27 34 Y on Deletion 2 23 16 4 0 0 0 44 0 0 0 2 50 50 50 pattern Sex-linkage 0 hom alt M2 A het T M1 A, 0 G hom ref M0 hom alt F2 het 50 F1 TGTGTGT hom T, ref F0 TGTGTGTGTGA T G C alleles Alt 93184 93182 91773 allele Ref 90152 deletion). position or allele 7.1893E-21 633 coverage Sex-linkage Y Scaffold low males scaffold. at to in homozygotes a (due Y0- of in not count XY-gametologs, number/position is the XY- X scaffold 4.7816E-21 is polymorphism, but by 39:0:55:0:0:0 hom recent-Y-specific present sorted alternative 3.0293E-21 alt is bearing the are M2 loci for Loci present, 2.6182E-21 recent-Y- homozygous was allele. are allele pattern: alternative that alternative 0:29:65:0:0:0 = common females/males one hom more of than are 2:0:97:0:0:0 0:54:0:54:0:0 ref the number more that hom/M0 = females/males 2.4059E-21 When ref of hom number allele. F0 alt 5:0:0:89:0:0 = SNP het hom/M2 bold). 0:54:30:0:0:0 het/M1 1.0428E-21 alt in F1 F2 shown allele, 33:0:70:0:0:0 0:15:0:89:0:0 reference heterozygous, is for region 0:29:43:0:0:0 homozygous (this females/males 1.0292E-21 of 93182-93327 34:0:0:46:0:0 8.6443E-22 number bases 0:26:66:0:0:0 42:0:0:52:0:0 (SEMN01000633) p 633 15:0:84:0:0:0 0:40:0:53:0:0 test 6.5797E-22 scaffold (CMH 0:80:14:0:0:0 alleles 5.3869E-22 male-specific 44:0:0:45:0:0 29:58:0:1:0:0 with 0:63:34:0:0:0 1:0:0:68:0:0 73:0:0:19:0:0 2.4506E-22 4. 0:15:0:78:0:0 Table 0:14:76:0:0:0 4:0:0:89:0:0 52:0:0:39:0:0 56:0:0:18:0:0 7.2449E-23 23:0:0:81:0:0 33:1:0:62:0:0 7:88:0:0:0:0 47:59:0:0:0:0 6.4122E-23 97477 0:31:50:0:0:0 22:0:0:92:0:0 9:0:0:69:0:0 85:0:0:5:0:0 56:0:0:33:0:0 SEMN01001079 300259 72:0:0:32:0:0 52:44:0:0:0:0 5.5955E-23 84:8:0:0:0:0 0:74:7:0:0:0 SEMN01000442 12203 1.5484E-23 8.3177E-24 65:0:0:40:0:0 19:0:0:77:0:0 64:0:0:32:0:0 12:0:0:93:0:0 SEMN01000713 477061 17:80:0:0:0:0 70:0:0:13:0:0 67:20:0:0:0:0 0:59:48:0:0:0 SEMN01000081 33:0:0:50:0:0 877605 4.9314E-24 0:42:33:0:0:0 69:0:0:30:0:0 14:0:0:60:0:0 SEMN01000055 657608 77:0:0:8:0:0 42:0:0:40:0:0 3.1558E-24 2.1701E-24 89:11:0:0:0:0 SEMN01000038 1931657 0:87:5:0:0:0 10:0:93:0:0:0 1:0:0:81:0:0 0:80:9:0:0:0 4.6027E-25 SEMN01000017 493881 7:0:0:75:0:0 47:0:0:36:0:0 15:0:79:0:0:0 43:0:0:47:0:0 8.8577E-27 SEMN01000338 11:0:0:65:0:0 1944824 0:53:24:0:0:0 54:0:51:0:0:0 17:0:0:35:0:0 0:41:28:0:0:0 3.4415E-27 SEMN01000020 280957 21:1:0:89:0:0 37:0:42:0:0:0 83:0:0:18:0:0 3.8445E-33 52:0:0:19:0:0 SEMN01000105 1.0476E-34 99134 18:0:88:0:0:0 0:0:0:87:0:0 41:0:0:58:0:0 0:0:0:78:0:0 SEMN01000248 305726 0:86:6:0:0:0 28:0:59:0:0:0 59:0:0:42:0:0 0:0:18:44:0:0 25:0:0:58:0:1 2.9651E-36 SEMN01000351 20:42:0:0:0:0 5.1401E-37 465844 40:0:61:0:0:0 14:0:0:35:0:0 0:0:0:64:0:0 SEMN01000282 7.5488E-39 722926 68:0:22:0:0:0 0:0:54:14:0:0 0:0:40:36:0:0 81:0:0:6:0:0 51:0:0:22:0:2 SEMN01000130 35:0:0:14:0:0 5.5909E-40 399756 0:82:0:0:0:0 18:0:0:73:0:0 0:1:0:81:0:0 SEMN01000251 0:33:0:15:0:0 5.156E-40 409936 0:0:25:49:0:0 10:0:0:42:0:0 1:0:88:0:0:0 SEMN01000396 0:15:33:0:0:0 836251 32:29:0:0:0:0 0:0:0:41:0:0 2.1971E-41 0:0:0:95:0:0 SEMN01000110 0:35:28:0:0:0 363800 0:0:0:45:0:0 0:0:45:22:0:0 0:0:56:8:0:0 SEMN01000409 32899 36:0:0:20:0:0 0:47:0:0:0:0 47:0:0:34:0:0 0:64:0:0:0:0 SEMN01000508 93879 0:36:0:20:0:0 0:0:71:0:0:0 0:0:74:0:0:0 SEMN01000633 96798 0:22:36:0:0:0 0:0:0:33:0:0 92:0:1:0:0:0 SEMN01000633 91773 0:34:28:0:0:0 0:0:0:38:0:0 SEMN01000633 93727 33:0:0:24:0:0 0:42:0:0:0:0 SEMN01000633 93182 0:0:46:0:0:0 SEMN01000633 90152 90:0:0:0:0:0 SEMN01000633 93327 SEMN01000633 93315 SEMN01000633 93299 SEMN01000633 93229 SEMN01000633 94017 SEMN01000633 ID Scaffold perch Macquarie umr fMcureprhidvda-ae tek eoye o ieWSdrvdSNPs WGS-derived nine for genotypes strelka individual-based perch Macquarie of Summary coordinate Scaffold :::::ne :::::ne :::::ne A:T:C:G:N:indel A:T:C:G:N:indel A:T:C:G:N:indel A:T:C:G:N:indel Females Dartmouth < e2) lstosxlne neswti h 4-psxn einof region sexing 146-bp the within indels sex-linked two plus 1e-20), 20 ae ar eae ar Males Yarra Females Yarra Males Dartmouth cutoff p-value CMH Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 41 00004 XY recent-Y recent-Y-specific bearing 1 XY locus a is 1 93879 Y except 93229. on at indicates XY-homologs, Insertion allele line 49 are alternative horizontal the loci alternative targets Dashed shade, assay All lighter YM. sexing (Ref)- PCR-RFLP 37 males- allele shade. reference Yarra polymorphism. male, darker 7 females- and blue- (Alt)- Dartmouth female, YF pink- allele samples: females- colours: population-by-sex Yarra Box four average. DM, genome-wide for males- 93182-94017 Dartmouth 6 region 42 DF, 633 0 0.72, scaffold 0.83, 0.92, perch SNP, 1.13, (yellow- Macquarie 12 1.15, region was this loci 2 in these Figure for respectively. located ratio 42 1.07, loci depth and sex-linked read 1.49 female:male nine 0.65, Mean of males- 0.68, Yarra 4). location (X-axis). Table blue), indicate (light see 93050-94150 DM lines indels; bases males- Vertical orange- Dartmouth (SEMN0100063, 1 0 blue). (orange), 633 YF (dark scaffold females- YM Yarra perch 0 (red), Macquarie DF of females- Dartmouth region sex-linked 2 along axis) 0 1 Figure 0 0 0 50 0 50 48 0 48 50 G 50 A pattern Sex-linkage A hom T alt M2 C het M1 CTA hom ref M0 present was allele reference hom but alt SNPs, F2 A these for § count read het G low F1 had females ++Two assay hom G sexing ref PCR-RFLP F0 for used G +Locus T C alleles 94017 Alt 93879 93327 93315 93299 93278 allele Ref 93229 position 633 Scaffold n aehdlwra on o hs Nsbtatraieall a present was allele alternative but SNPs these for count read low had male One + eddphcvrg o eeec n lentv lee tnn osctv e-ikdlc on loci sex-linked consecutive nine at alleles alternative and reference for coverage depth Read . omlzdWSra et oeaefrfu ouainb-e ape fidvdas(Y- individuals of samples population-by-sex four for coverage depth read WGS Normalized . 00014 XY 3 46 1 0 0 50 T C 21 ++ ++ 17 7 41 40 1 2 0 0 0 0 § § XY XY Posted on Authorea 23 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161410988.87857941/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 22