DOI: 10.5061/dryad.kwh70rzc3
title: Data from: High-resolution chromosome-level genome of Scylla paramamosain provides molecular insights into adaptive evolution in crab

description: Crabs exhibit adaptability across diverse ecosystems, encompassing shallow
 coral reefs, hydrothermal vents, as well as freshwater, seawater, and
 terrestrial habitats. Our study presents a thorough genomic analysis of
 the mud crab leveraging ultralong sequencing technologies. We achieved a
 high-quality chromosome-level assembly, covering 1.21 Gb (scaffold N50
 23.61 Mb) with 33,662 protein-coding genes. Anchoring contigs into 49
 pseudochromosomes revealed Chromosome 06 as a significant sex chromosome.
 Notably, the Hox genes are implicated in abdomen limb morphology, with the
 Abd-A gene identified as a linchpin in pleopod development, shedding light
 on brachyurization. fru gene, negatively regulated by novel-miR35, plays
 critical role in ovarian maturation. Additionally, we elucidated
 neo-functionalization of the elovl6 gene and identified tissue-specific
 splice variants, suggesting a potential role for Elovl6 in long-chain
 polyunsaturated fatty acid biosynthesis. These findings contribute
 significantly to our understanding of crab adaptability and evolutionary
 dynamics, offering a robust foundation for future investigations.
description: <strong>Sample preparation and genomic DNA
 isolation</strong> Genomic DNA for sequencing was
 extracted from the testis of a wild adult male green mud crab <em>S.
 paramamosain</em> with stage III testis, which was caught off the
 coast of Shantou, China. A Grandomics Genomic BAC-long DNA Kit was used to
 isolate ONT ultralong DNA according to the manufacturer's guidelines.
 The total DNA quantity and quality were evaluated using a NanoDrop One
 UV-Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and a
 Qubit 3.0 Fluorometer (Invitrogen Life Technologies, Carlsbad, CA). Large
 DNA fragments were obtained through gel cutting with the Blue Pippin
 system (Sage Science, Beverly, MA). Qualification of DNA involved visual
 inspection for foreign matter, assessment of degradation and size
 <em>via</em> 0.75 % agarose gel electrophoresis, check on
 purity (OD260/280 between 1.8-2.0; OD260/230 between 2.0-2.2) using
 Nanodrop, and precise quantification with Qubit 3.0 Fluorometer
 (Invitrogen, USA). <strong>Library construction
 and sequencing</strong> Approximately 8-10 µg of
 genomic DNA (&gt; 50 kb) was selected using the SageHLS HMW library
 system (Sage Science, Beverley MA, USA) and processed with the Ligation
 Sequencing 1D Kit (Oxford Nanopore Technologies, Shanghai, China)
 following the manufacturer's instructions. Library construction and
 sequencing were conducted on the PromethION (Oxford Nanopore Technologies)
 at the Genome Center of Grandomics (Wuhan, China). After quality
 inspection, large DNA fragments were recovered using the BluePippin
 automatic nucleic acid recovery instrument. Terminal repair, A-tailing,
 and ligation were performed using the LSK109 connection kit. Qubit was
 employed to assess the constructed DNA library precisely. The library was
 loaded into a flow cell, transferred to the PromethION sequencer, and
 subjected to real-time single-molecule sequencing. In the ONT sequencing
 platform, base calling, the conversion of nanopore-generated signals to
 base sequences (<em>69</em>), was executed using the Guppy
 toolkit (Oxford Nanopore Technologies). Pass reads with a mean
 qscore_template value greater than or equal to 7 were obtained and
 directly used for subsequent assembly
 (https://github.com/nanoporetech/taiyaki).
 <strong>Genome assembly, evaluation and
 correction</strong> Filtered reads post quality
 control were employed for a pure three-generation assembly using
 NextDenovo software (reads_cutoff:1k, seed_cutoff:28k)
 (https://github.com/Nextomics/NextDenovo.git). The NextCorrect module was
 employed to correct the original data, yielding a consistency sequence
 (CNS sequence) after 13 Gb of error correction. <em>De
 novo</em> assembly using the NextGraph module produced a preliminary
 assembly of the genome. ONT three-generation data and Pb HiFi
 three-generation data were utilized with Nextpolish software
 (https://github.com/Nextomics/NextPolish.git) for genome correction. The
 corrected genome (Polish Genome) was obtained after three rounds of
 correction for both ONT and Pb HiFi data. Bwa mem default parameters were
 used to compare next-generation data to the genome, and Pilon was
 iteratively calibrated three times to derive the final genomic sequence.
 GC depth analysis and BUSCO prediction (https://busco.ezlab.org/) were
 used to assess genome quality and completeness.
 <strong>Chromosome anchoring by Hi-C
 sequencing</strong> To anchor hybrid scaffolds
 onto chromosomes, genomic DNA for Hi-C library construction was extracted
 from green mud crab testes. Cell samples from the tissue used for genomic
 DNA sequencing were employed for Hi-C library preparation. The process
 involved cross-linking cells with formaldehyde, lysing cells, resuspending
 nuclei, and subsequent steps leading to proximity ligation. After
 overnight ligation, cross-linking was reversed, and chromatin DNA
 manipulations were performed. DNA purification and shearing to 400 bp
 lengths were followed by Hi-C library preparation using the NEBNext Ultra
 II DNA library Prep Kit for Illumina. Sequencing on the Illumina
 NovaSeq/MGI-2000 platform completed the Hi-C procedure. Briefly, cell
 samples were fixed with formaldehyde and subjected to lysis and extraction
 for sample quality assessment. After passing the quality test, the Hi-C
 fragment preparation process involved chromatin digestion, biotin
 labeling, end ligation, DNA purification, and library construction. The
 library was sequenced on the MGI-2000 platform, and data were processed to
 extract high-quality reads. The analysis included filtering for adapters,
 removing low-quality reads, and eliminating reads with an N content
 exceeding 5. Reads were aligned using Bowtie2 (<em>70</em>),
 and contig clustering was performed using LACHESIS software
 (<em>71</em>). <strong>Gene
 annotation</strong> Tandem repeats were annotated
 using GMATA (https://sourceforge.net/projects/gmata/?source=navbar) and
 Tandem Repeats Finder (TRF) (http://tandem.bu.edu/trf/trf.html),
 identifying simple repeat sequences (SSRs) and all tandem repeat elements.
 Transposable elements (TEs) were identified through an <em>ab
 initio</em> and homology-based approach, with RepeatMasker
 (https://github.com/rmhubley/RepeatMasker) used for searching known and
 novel TEs. Gene prediction employed three methods: GeMoMa
 (http://www.jstacs.de/index.php/GeMoMa) for homolog prediction, PASA
 (https://github.com/PASApipeline/PASApipeline) for RNAseq-based
 prediction, and Augustus (https://github.com/Gaius-Augustus/Augustus) for
 <em>de novo</em> prediction. EVidenceModeler (EVM)
 (http://evidencemodeler.github.io/) integrated gene sets, which underwent
 further filtering for transposons and erroneous genes. UTRs and
 alternative splicing regions were determined using PASA based on RNA-seq
 assemblies. Functional annotation involved comparisons with public
 databases, including SwissProt, NR, KEGG, KOG, and Gene Ontology.
 InterProScan identified putative domains and GO terms. BLASTp
 (https://blast.ncbi.nlm.nih.gov/Blast.cgi) against public protein
 databases was used to assess gene function information. The noncoding RNA
 (ncRNA) prediction entailed using tRNAscan-SE
 (http://lowelab.ucsc.edu/tRNAscan-SE/) and Infernal cmscan
 (http://eddylab.org/infernal/) for tRNAs and other noncoding RNAs. BUSCO
 was employed for gene prediction evaluation, aligning annotated protein
 sequences to evolution-specific BUSCO databases.
 <strong>Evolutionary analysis</strong>
 The evolutionary analysis entailed the examination of gene
 families, construction of phylogenetic trees, estimation of divergence
 times, exploration of gene expansion and contraction phenomena,
 identification of orthologous genes, scrutiny of positively selected
 genes, and investigation of whole-genome duplications. To ascertain
 homologous relationships between <em>S. paramamosain</em> and
 other animal species, protein sequences were acquired and aligned using
 OrthMCL (https://orthomcl.org/orthomcl/). Initially, protein sets were
 gathered from 14 sequenced animal species, and the longest transcripts for
 each gene were selected, excluding miscoded and prematurely terminated
 genes. Subsequently, pairwise alignment of these extracted protein
 sequences was conducted to identify conserved orthologs, employing Blastp
 with an E-value threshold of ≤ 1 × 10<sup>-5</sup>. Further
 identification of orthologous intergenome gene pairs, paralogous
 intragenome gene pairs, and single-copy gene pairs was achieved using
 OrthMCL. Species-specific genes, encompassing <em>S.
 paramamosain</em>-specific unique genes and unclustered genes, were
 extracted. Functional annotation and enrichment tests of species-specific
 genes were performed utilizing information from homologs in the online
 Gene Ontology (http://www.geneontology.org/) and KEGG (Kyoto Encyclopedia
 of Genes and Genomes) (https://www.genome.jp/kegg/) databases.
 Building upon the orthologous gene sets identified with OrthMCL,
 molecular phylogenetic analysis was executed using shared single-copy
 genes. Coding sequences were extracted from single-copy families, followed
 by multiple alignment of each ortholog group using MAFFT
 (https://mafft.cbrc.jp/alignment/software/). Gblocks were applied to
 eliminate poorly aligned sequences, and the GTRGAMMA substitution model of
 RAxML (https://cme.h-its.org/exelixis/web/software/raxml/hands_on.html)
 was employed for phylogenetic tree construction with 1000 bootstrap
 replicates. The resulting tree file was visualized using Figtree/MEGA
 (http://tree.bio.ed.ac.uk/software/figtree/). The RelTime tool
 (https://www.megasoftware.net/) of MEGA-CC was then utilized to compute
 mean substitution rates along each branch and estimate species divergence
 times, with three fossil calibration times obtained from the TimeTree
 (http://www.timetree.org/) database serving as temporal controls,
 including the divergence times of <em>S.
 paramamosain</em>. The detection of significant
 expansions or contractions in specific gene families, often indicative of
 adaptive divergence in closely related species, was carried out based on
 OrthoMCL results. CAFE (https://github.com/hahnlab/CAFE), employing a
 birth and death process to model gene gain and loss over a phylogeny, was
 used for this purpose. Furthermore, in accordance with the neutral theory
 of molecular evolution, the ratio of the nonsynonymous substitution rate
 (Ka) to the synonymous substitution rate (Ks) of protein-coding genes was
 calculated. The average Ka/Ks values were determined, and a branch-site
 likelihood ratio test using Codeml
 (http://abacus.gene.ucl.ac.uk/software/) from the PAML package was
 conducted to identify positively selected genes within the <em>S.
 paramamosain</em> lineage. Genes with a p value &lt; 0.05 under
 the branch-site model were considered positively selected.
 Whole-genome duplication events in the green mud crab genome were
 investigated using four-fold synonymous third-codon transversion (4DTv)
 and synonymous substitution rate (Ks) estimation. Initial steps involved
 extracting protein sequences and conducting all-vs.-all paralog analysis
 through self-Blastp in these plants. After filtering by identity and
 coverage, the Blastp results underwent analysis with MCScanX
 (<em>71</em>), and the respective collinear blocks were
 identified. Subsequently, Ks and 4DTV were calculated for the syntenic
 block gene pairs using KaKs-Calculator
 (https://sourceforge.net/projects/kakscalculator2/), and potential WGD
 events in each genome were evaluated based on their Ks and 4DTv
 distribution.
description: # High-resolution chromosome-level genome provides molecular insights into
 adaptive evolution in crab
 [https://doi.org/10.5061/dryad.kwh70rzc3](https://doi.org/10.5061/dryad.kwh70rzc3) The sequences and the annotation files of the green mud crab genome. The dataset consists of SP2022.Chr.bed, SP2022.fasta, SP2022.genome.gff, and SP2022.genome.pep.fasta files. ## Description of the data and file structure The sequences file was in fasta format and the annotation file was in gff including genome sequencing, assembly, and annotation of green mud crab. ## Sharing/Access information Links to other publicly accessible locations of the data: * [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1059155](https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1059155)