Chromosomal-scale genome assembly and annotation of the land slug (meghimatium bilineatum)

ABSTRACT _Meghimatium bilineatum_ is a notorious pest land slug used as a medicinal resource to treat ailments in China. Although this no-model species is unique in terms of their ecological

security and medicinal value, the genome resource of this slug is lacking to date. Here, we used the Illumina, PacBio, and Hi-C sequencing techniques to construct a chromosomal-level genome

of _M. bilineatum_. With the Hi-C correction, the sequencing data from PacBio system generated a 1.61 Gb assembly with a scaffold N50 of 68.08 Mb, and anchored to 25 chromosomes. The

estimated assembly completeness at 91.70% was obtained using BUSCO methods. The repeat sequence content in the assembled genome was 72.51%, which mainly comprises 34.08% long interspersed

elements. We further identified 18631 protein-coding genes in the assembled genome. A total of 15569 protein-coding genes were successfully annotated. This genome assembly becomes an

important resource for studying the ecological adaptation and potential medicinal molecular basis of _M. bilineatum_. SIMILAR CONTENT BEING VIEWED BY OTHERS HAPLOTYPE-RESOLVED

CHROMOSOMAL-LEVEL GENOME ASSEMBLY OF BUZHAYE (MICROCOS PANICULATA) Article Open access 15 December 2023 A CHROMOSOMAL-LEVEL GENOME ASSEMBLY OF _BEGONIA FIMBRISTIPULA_ (BEGONIACEAE) Article

Open access 12 March 2025 CHROMOSOME-LEVEL GENOME ASSEMBLY OF THE TRADITIONAL MEDICINAL PLANT _LINDERA AGGREGATA_ Article Open access 03 April 2025 BACKGROUND & SUMMARY The _Meghimatium

bilineatum_ (_syn. Philomycus bilineatus_ Benson, 1842) is a member of the Philomycidae family and is a notorious quarantine pest land slug that can cause enormous damage to commercial

crops, horticultural crops, grasslands, and forests in East Asia1,2,3,4,5. It has a strong ecological adaptation to terrestrial environments and has been widely distributed in various

regions of China6. It does not only feed on stems, leaves, fruits, or juices of plants causing direct economic losses but also secretes mucus and excretes feces contaminating fruits and

vegetables. This contamination results in a reduction in the market value of products and transmits diseases. Thus, it poses great harm to local agricultural productivity and ecological

security, resulting in substantial economic and ecosystem losses7. However, from another perspective, _M. bilineatum_ also exhibits medicinal properties. For example, its crude extracts are

used in the treatment of bacterial-induced infectious diseases, the polysaccharides in slug cell are used as natural antioxidants to prevent cancer, and the antimicrobial peptide derived

from the slug is utilized to combat skin infections caused by _Candida albicans_8,9,10. At present, some researchers have carried out in-depth studies on the pharmacological effects of slug

extract, indicating that slugs can be used as a valuable medicinal resource with development and application value9,10. Thus, the study of slug species is very meaningful. In addition to its

ecological threat and medicinal value, _M. bilineatum_, as a member of 30000 described terrestrial gastropod mollusks with shell-less, has completed the transition from aquatic to

terrestrial. Similar to other slug species, they have developed many various robust features, including a pulmonate for breathing air, a sophisticated neural-immune system, and the ability

to produce mucus to adapt to the terrestrial environments11,12,13. However, compared with land snails, land slugs display unique life strategy for terrestrial environments, such as defense

by secreting mucus including specific chemical compounds and better mobility under predation, because they have no protective shell1,14. Furthermore, shell-less land slugs do not expend

energy ingesting large amounts of calcium, enabling them to grow faster. Although land slugs have strong adaptation mechanism, their evolutionary history remains unclear. In recent years,

molecular phylogenetics analysis of land slugs of the genus _Meghimatium_ based on the mitogenome and nuclear loci has offered new perspectives into the taxonomic revisions and evolution of

these species15,16,17. However, these studies cannot fully explain the molecular mechanism of wide ecological adaptation information and the potential genetic basis of medicinal resource

traits of this slug. Furthermore, the Philomycidae slug genomics have yet to be published. Therefore, assembling a genome of this slug species should be urgently assembled. The study of

genomes in certain terrestrial mollusks, has shown advancements, including the release of genomic data for two land snails, _Achatina fulica_ and _Pomacea canaliculata_. However, thorough

investigations into the evolutionary mechanisms associated with terrestrial adaptation remain scant18,19. Recently, one genome study of _Achatina immaculata_, namely giant African snail has

verified that some genes related to respiratory system, dormancy system, and immune system have undergone great expansion to adapt to the terrestrial environments20. However, to date,

high-quality genomic resources for land slugs are rarely reported. The land slugs and snails, as terrestrial gastropod mollusks with or without shell protection, have different biological

processes related to their terrestrial lifestyle. Hence, assembling a genome of the land slug species would facilitate intensive study of this species’ adaptive evolution. Herein, we

assembled the genome of _M. bilineatum_ by uniting the sequencing techniques of Illumina, PacBio, and Hi-C. Three methods, including _ab initio_ gene prediction, homolog and RNA-Seq-based

prediction, were used to perform genomic annotation. In addition, the comparative genomics analysis of _M. bilineatum_ and 11 other distantly related species were performed. This study

offers insights for the effective management and utilization of slug populations and provides valuable genome information into the evolutionary history and genetic mechanisms of this

important gastropod group. METHODS LAND SLUG COLLECTING AND SEQUENCING Adult land slugs _M. bilineatum_ were collected from a wild area in Zhoushan, Zhejiang, China (122.212 E, 29.979 N).

Total DNA was extracted from whole body of the land slug _M. bilineatum_ using the SDS-based extraction method. Then, the DNA samples were purified using QIAGEN® Genomic kit (QIAGEN,

Germany) for genome sequencing. First, Illumina short-read library with insert sizes of 300–350 bp was generated, and was sequenced using the Illumina Novaseq. 6000 platform. Second, PacBio

HiFi-read library with insert sizes of 10–40 kb was generated using SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, USA) and sequenced using the PacBio Sequel II platform.

Finally, Hi-C short-read library was generated using the purified DNA from the whole body of _M. bilineatum_ according to the previously performed protocol by Belton _et al_. with given

adjustments; it was sequenced using the Illumina Novaseq. 6000 platform21. A total of 250.12 Gb of clean Illumina short-reads, 71.33 Gb HiFi CCS reads and 140.69 Gb clean Hi-C reads were

obtained (Table 1). Total RNA was isolated from whole body of the land slug using TRIzol reagent (Invitrogen, MA, USA) for transcriptome sequencing. The RNA-seq library was generated using

NEBNext® Ultra™ RNA Library Prep Kit (NEB, USA) and sequenced using the Illumina Novaseq. 6000 platform. The RNA-seq reads were used for genome annotation. A total of 21.79 Gb of clean data

was obtained (Table 1). GENOME SIZE ESTIMATION Based on 250.12 Gb clean Illumina short-reads, the genome size, heterozygosity and repetitive sequence content was determined using the k-mer

analysis with GCE (1.0.0) following the default parameter22. A total of 223,346,880,670 k-mers with a depth of 144 was obtained (Fig. 1). In addition, the genome size of _M. bilineatum_ was

approximately 1.5 Gb, with a heterozygosity of 1.05% and proportion of repeat sequences at 43.69%. CHROMOSOMAL-LEVEL GENOME ASSEMBLY In the initial genome assembly, HiFiasm (v0.16.0) method

was used for _ab initio_ to assemble the genome using the HiFi reads from PacBio23. This preliminary assembly yielded a genome size of 1.80 Gb (Table 2). Subsequently, the redundant

sequences were filtered out using Purge_Haplotigs (v1.0.4) software with the parameter of cutoff ‘-a 70 -j 80 -d 200’24. Based on PacBio sequencing data, a 1.63 Gb contig-level genome

assembly of _M. bilineatum_ was obtained, and 2526 contigs displayed contig N50 and N90 sizes of 1.37 and 320.449 Mb, respectively (Table 2). The chromosome-level assembly of _M. bilineatum_

was conducted using Hi-C technology. Initially, Bowtie2 (v2.3.4.3) following the default parameters was used to match the 140.69 Gb clean Hi-C reads to the contig-level genome to obtain

unique mapped paired-end reads25. A total of 185.36 million paired-end reads were uniquely mapped (Table S1), of which 88.02% represented valid pairs (Table S2). Subsequently, contigs were

assembled into the chromosome-level scaffolds using the 3D-DNA processes (v180922) (parameters: -r 0) with all valid pairs, and the JuiceBox (v1.11.08) was used to correct the errors in the

genome assembly26,27. We anchored and obtained 25 pseudo-chromosomes with seven unanchored scaffolds. The 25 pseudo-chromosomes covering ~99.95% of the final genome with size ranging from

25.66 Mb to 135.71 Mb (Fig. 2; Table 3). Ultimately, we obtained a 1.61 Gb chromosomal-level genome assembly of _M. bilineatum_ with contig N50 size and scaffold N50 size of 1.36 Mb and

68.08 Mb, respectively. Genome assembly results showed that the genome size of _M. bilineatum_ is similar to that of the Spanish slug _Arion vulgaris_ (1.54 Gb) in the previous study28.

REPEAT-CONTENT IDENTIFICATION AND CLASSIFICATION Repetitive sequences, including tandem repeats and interspersed repeats, in _M. bilineatum_ genome were determined using the _de novo_

prediction and homolog-based methods. Based on homology comparison, RepeatMasker (open-4.0.9) (parameters: default) and RepeatProteinMask (parameters: default) software were utilized to find

the interspersed repeats against the RepBase database (http://www.girinst.org/repbase)29. On the basis of _de novo_ prediction, TRF (v4.09) software (parameters: default) was used to

identify the tandem repeats30. In addition, a repetitive sequence library was constructed using the RepeatModeler (open-1.0.11) with default parameters and LTR-FINDER_parallel (v1.0.7) with

default parameters31,32. Then, the RepeatMasker (open-4.0.9) with default parameters was used to identify the repeat element against this repeat library31. After combining the results from

_de novo_ prediction and homolog-based methods, we identified and classified 1.18 Gb of repetitive sequences, taking up 72.51% of the assembled genome, mainly including 7.99% DNA elements,

34.08% long interspersed elements (LINE), and 16.35% unknown sequences (Tables 4 & 5). The repeat-content in the _M. bilineatum_ genome is similar to the Spanish slug _A. vulgaris_

(75.09%), and is higher than other studied gastropod species28,33. These results further validate the accuracy of our genome assembly. IDENTIFICATION AND ANNOTATION OF PROTEIN-CODING GENES

First, we used repeat-masked genome sequences to perform _ab initio_ gene prediction, and then used AUGUSTUS (v3.3.2), Genscan (v1.0) and GlimmerHMM (v3.0.4) software to detect the

protein-coding genes34,35,36. Second, to conduct homology-based prediction, protein sequences from _Candidula unifasciata_ (GCA_905116865.2), _Elysia chlorotica_ (GCA_003991915.1), _Haliotis

rubra_ (GCA_003918875.1), _Haliotis rufescens_ (GCA_023055435.1), _Lottia gigantea_ (GCA_000327385.1), _Pakobranchus ocellatus_ (GCA_019648995.1), and _Pomacea canaliculate_

(GCA_003073045.1) were compared with the _M. bilineatum_ genome utilizing TBLASTN (v2.2.29) (e-value ≤ 1e-5) to determine candidate regions, and further used GenWise (v2.4.1) software to

accurately map the screened proteins to the _M. bilineatum_ genome to obtain splice sites37. Third, to perform transcriptome sequencing-based prediction, the RNA-seq reads from Illumina were

mapped to the _M. bilineatum_ genome by using the TopHat (v2.1.1) software following default arguments, and the transcripts were assembled using Cufflinks (v2.2.1) software with the “-e 100

-C” parameter38,39, and the protein-coding genes were determined using the PASA (v2.3.2)40. Fourth, using the MAKER2 (v2.31.10) and HiFAP software following default parameters, we combined

the three predictions to construct a complete and nonredundant reference gene database41. Finally, in the _M. bilineatum_ genome, 18631 identified protein-coding genes were found. The length

of the average gene, including CDS, exon, and intron, is presented in Table 6. These predicted gene structures were also compared with the seven other homologous species (Fig. 3). We

annotated these protein-coding genes functions through the alignment of gene sequences to the InterPro, GO, KEGG, SwissProt, TrEMBL, TF, Pfam, NR, and KOG database by using BLAST + (2.11.0)

software (e-value ≤ 1e-5)42,43,44,45,46,47. In addition, based on InterPro database and Pfam database, the conserved protein domain and motif associated with the function annotated was

determined using the InterProScan tool (v5.61-93.0) with the “-seqtype p -formats TSV -goterms -pathways -dp” parameter48. Ultimately, a total of 15569 genes (83.57%) were successfully

annotated (Table 7). IDENTIFICATION OF NON-CODING GENES The tRNA, rRNA, miRNA, and snRNA non-coding RNAs are not translated into proteins. In the annotation process of non-coding RNAs,

tRNAscan-SE (v1.3.1) software following the default parameters was used to find the tRNA sequences in the assembled genome according to the structural characteristics of tRNA49. BLASTN was

applied to identify rRNA genes in the assembled genome according to the highly conserved characteristics of rRNA. In addition, according to the covariance model of Rfam database (v14.8), we

used the INFERNAL program with default arguments to predict the miRNA and snRNA sequences50. Finally, 1424 rRNAs, 941 tRNAs, 588 snRNAs, and 49 miRNAs were annotated (Table 8). COMPARATIVE

GENOMIC ANALYSIS The single-copy ortholog genes of _M. bilineatum_ and 11 other molluscan species (Table S3), including _Nautilus pompilius_, _Octopus minor_, _Bathymodiolus platifrons_,

_Chrysomallon squamiferum_, _Elysia chlorotica_, _Biomphalaria glabrata_, _Candidula unifasciata_, _Pomacea canaliculate_, _Haliotis rubra_, _Gigantopelta aegis_ and _Lottia gigantea_, were

determined using the “-l 1.5” parameter of hcluster_sq software from OrthoMCL (v2.0.9) to validate the phylogenetic relationships among the 12 molluscan species51. A total of 29157 gene

families were determined, including 671 common orthologous gene families and 135 single-copy gene families, in the 12 molluscan species (Fig. 4; Table S4). The MAFFT (v7.487) software with

default parameters was used to compare the single-copy genes52. All conserved sequences in the single-copy genes were extracted using Gblock (v0.91b) software with the “-t = c” parameter53.

Subsequently, the ML phylogenetic tree was constructed using the “-f a -N 100 -m GTRGAMMA” parameter of RAxML (v8.2.12)54, with _N. pompilius_ and _O. minor_ as the outgroup. Moreover, the

divergence time of the 12 mollusks were estimated using the MCMCtree (v4.4) program in software PAML (v4.9) with “clock = 3; model = 0” parameter according to the calibration times of _N.

pompilius_-_B. platifrons_ (619.1–527.6 MYA), _B. platifrons_-_P. canaliculata_ (541.7–463.4 MYA), _N. pompilius_-_O. minor_ (452.6–364.2 MYA), _B. glabrata_-_P. canaliculata_ (496.0–310.0

MYA) and _G. aegis_-_C. squamiferum_ (100.0–42.4 MYA) from the Timetree database55. The evolutionary tree showed that _M. bilineatum_ and _C. unifasciata_ were clustered together, and

diverged ~231.4 MYA (Fig. 5). We also identified the expanded genes and contracted gene families in the 12 mollusks using CAFE (v5.0.0) with the “-p 0.05 -t 4 -r 10000” parameter56. The

result showed that there were 879 expanded gene families and 1385 contracted gene families in the _M. bilineatum_ (Fig. 5). DATA RECORDS All sequencing data from three sequencing platforms

have been uploaded to the NCBI SRA database (transcriptomic sequencing data: SRR2586702857, genomic Illumina sequencing data: SRR2590398958, genomic PacBio sequencing data: SRR2591904459 and

SRR2591904360, Hi-C sequencing data: SRR2591915561 and SRR2591915462). The final chromosome-level assembled genome file has been uploaded to the GenBank database under the accession

JAXGFX00000000063. Genome annotation files (including repeat-content annotation, gene structure annotation, gene functional annotation and non-coding genes annotation) have been uploaded to

the Figshare database64. TECHNICAL VALIDATION EVALUATING QUALITY OF THE DNA AND RNA Prior to the genome sequencing, we used the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, San

Jose, CA, USA) and Qubit 3.0 Fluorometer (Thermo Fisher Scientific, San Jose, CA, USA) to determine the quality (OD260/280 and OD260/230) and concentration of the DNA and RNA samples to

ensure the accuracy of sequencing data. We also used the agarose gel electrophoresis and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California, USA) to determine the

integrity of the DNA and RNA samples. EVALUATING QUALITY OF THE GENOME ASSEMBLY To evaluate the sequence consistency and assembly quality, the BWA (v0.7.17-r1188) and Minimap2

(v2.24_x64-linux) software were used to map the short reads from Illumina and HiFi reads from PacBio to the assembled genome, respectively65,66. After these processes, 99.35% of the short

reads from Illumina and 99.62% of the HiFi reads from PacBio were aligned, covering 99.81% and 99.99% of the assembled genome, respectively (Table S5 & S6). Moreover, BUSCO (v5.4.3)

analysis was conducted to evaluate the assembly quality based on the mollusca_odb10 database67. A total of 91.70% of the 5295 single-copy orthologs in the assembled genome were determined as

complete, including 4015 single-copy (75.80%) and 842 duplicated (15.90%), 0.89% and 7.46% of the total single-copy orthologs were fragmented and missing, respectively (Table 9). EVALUATING

QUALITY OF THE GENOME ANNOTATION BUSCO (v5.4.3) analysis was conducted to evaluate the genome annotation quality based on the mollusca_odb10 database67. A total of 91.60% of the 5295

single-copy ortholog genes in the assembled genome were determined as complete, including 3912 single-copy genes (73.90%) and 939 duplicated genes (17.70%), 1.30% and 7.10% of the total

genes were fragmented and missing, respectively (Table 9). CODE AVAILABILITY No specific code was used in this study. The standard bioinformatic tools were used for data analysis.

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single-copy orthologs. _Bioinformatics_ 31, 3210–3212 (2015). Article  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the Zhejiang Provincial Natural

Science Foundation of China (LR21D060003) and the Introduction of Talent Research Start-up Fund of Zhejiang Ocean University (JX6311031923). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS *

Fishery College, Zhejiang Ocean University, Zhoushan, Zhejiang, 316022, China Shaolei Sun, Xiaolu Han & Zhiqiang Han * Wuhan Onemore-tech Co., Ltd, Wuhan, Hubei, 430076, China Qi Liu

Authors * Shaolei Sun View author publications You can also search for this author inPubMed Google Scholar * Xiaolu Han View author publications You can also search for this author inPubMed 

Google Scholar * Zhiqiang Han View author publications You can also search for this author inPubMed Google Scholar * Qi Liu View author publications You can also search for this author

inPubMed Google Scholar CONTRIBUTIONS Z.Q.H. designed the project. S.L.S., X.L.H. and Q.L. collected the samples and analyzed the data. S.L.S. and Z.Q.H. wrote the manuscript. S.L.S., Z.Q.H.

and Q.L. revised the manuscript. All authors read and approved the final version of the manuscript. CORRESPONDING AUTHORS Correspondence to Zhiqiang Han or Qi Liu. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps

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Han, Z. _et al._ Chromosomal-scale genome assembly and annotation of the land slug (_Meghimatium bilineatum_). _Sci Data_ 11, 35 (2024). https://doi.org/10.1038/s41597-023-02893-7 Download

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