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ABSTRACT Recent advancements in plant regeneration and synthetic polyploid creation have been documented in _Gossypium arboreum_ ZB-1. These developments make ZB-1 a potential model within

the _Gossypium_ genus for investigating gene function and polyploidy. This work generated the sequence and annotation of the ZB-1 genome. The contig-level genome was constructed using the

PacBio high-fidelity reads, encompassing 81 contigs with an N50 length of 112.12 Mb. The Hi-C data assisted the construction of the chromosome-level genome, which consists of 13

_pseudo_-chromosomes and 39 un-anchored contigs, with a total length of about 1.67 Gb. Repetitive sequences accounted for about 69.7% of the genome in length. Based on _ab initio_ and

evidence-based prediction, we have identified 48,021 protein-coding genes in the ZB-1 genome. Comparative genomics analysis revealed conserved gene content and arrangement between ZB-1 and

_G. arboreum_ SXY1. The single nucleotide polymorphism occurrence rate between ZB-1 and SXY1 was about 0.54 per 1,000 nucleotides. This study enriched the genomic resources for further

exploration into cotton regeneration and polyploidy mechanisms. SIMILAR CONTENT BEING VIEWED BY OTHERS CHROMOSOME-LEVEL GENOME ASSEMBLY AND ANNOTATION OF THE PRICKLY NIGHTSHADE _SOLANUM

ROSTRATUM_ DUNAL Article Open access 01 June 2023 CHROMOSOME-LEVEL GENOME ASSEMBLY OF COTTON THRIPS _THRIPS TABACI_ (THYSANOPTERA: THRIPIDAE) Article Open access 16 September 2024 A

CHROMOSOME-LEVEL GENOME ASSEMBLY OF THE _KNOXIA ROXBURGHII_ (RUBIACEAE) Article Open access 15 November 2023 BACKGROUND & SUMMARY Cotton (_Gossypium spp_.) is one of the most crucial

fiber and oilseed crops worldwide. The _Gossypium_ genus comprises about 50 species, which include 45 diploids categorized into eight genome groups (A-G and K) and seven tetraploids denoted

as AD1-AD71,2. The tetraploids originated from a single hybridization event between the A- and D-genome progenitors, followed by polyploidization3. Historically, four of these species have

undergone independent domestication, _i.e_., the diploids _G. arboreum_ (A2) and _G. herbaceum_ (A1)4, as well as the tetraploids _G. hirsutum_ (AD1) and _G. barbadence_ (AD2)5. Notably, AD1

has emerged as the predominant species in contemporary cotton cultivation. A2 is critical for investigating the mechanisms underlying natural polyploidy in the genus _Gossypium_. For an

extended period, one of the major concerns in cotton biology is the origin of the A-genome in the natural tetraploids, with the controversy over whether it is from A1 or A2. In the last

decade, the genome sequences of A1, A2, and several tetraploids have been successively generated6. With the insights gained from comparative genomics, researchers are now inclined to

conclude that the ancestor (termed A0) of A1 and A2 is the A-genome donor7,8,9. However, as A0 no longer exists, A1 and A2 are still necessary substitutes for assessing the evolution of the

natural cotton polyploids upon genome merge and doubling. Due to its broader distribution and, more significantly, the more profound understanding of its biology in comparison to A1,

researchers often opt for A2 as the substitute progenitor when studying the evolution of natural tetraploids. Besides, A2 has also frequently been used to generate synthetic cotton

polyploids to investigate issues such as gene expression modulation in the early stage of polyploidy. For example, hybridization between A2 and _G. thurberi_ (D1), _G. raimondii_ (D5), and

_G. bickii_ (G1) gave rise to several hybrids and synthetic tetraploids10,11. Gene expression analysis of these synthetic hybrids or polyploids revealed that the subgenome expression pattern

was rapidly established in the early generations of the progenies, which would then be reconciled during long-term evolution. Likewise, Ke _et al_. generated a novel cotton tetraploid

between A2 (accession ZB-1) and _G. stocksii_ (E1) by somatic hybridization12. As meiosis does not occur during somatic hybridization, the novel polyploid 2(A2E1) might be an ideal model for

further exploring the transcriptional and epigenomic alteration upon genome doubling. In addition, A2-derived new cotton polyploids provide valuable germplasm resources for improving

agronomically important traits in cotton cultivars. For example, A2 and G1-derived tetraploids have glandular vegetative tissue and glandless seeds (without toxic gossypol)13, which provide

novel germplasm to breed cultivars with the seeds could be more widely utilized in industrial production14. Likewise, by hybridizing A2 with E1 and following genome doubling, Nie and

colleagues generated novel tetraploids 2(A2E1), of which the high generations produce high-strength fiber15. Moreover, Chen _et al_. generated a novel cotton hexaploid by hybridization

between A2 (SXY1 or Shixiya 1) and AD1 (TM-1), which could be further utilized to develop the _G. arboreum_-introgressed lines to transfer the drought and _Verticillium_ resistance to

cultivars16. In addition to serving as the progenitor for synthetic cotton polyploids in polyploidy research and breeding, A2 is also a potential model organism for gene function exploration

in cotton species. Currently, the creation of transgenic cotton plants predominantly relies on AD16,17. Nevertheless, ascribed to the doubled genomes in AD1, each allele is associated with

four haplotypes that are similar in sequence. The complexity of the genetic background poses a challenge in gene manipulation in AD1, such as the risk of off-target effects in

CRISPR/Cas9-mediated genome editing. Furthermore, the gene duplication resulting from polyploidy also adds to the complexity of evaluating gene functions in AD1. For instance, several

studies have highlighted that subfunctionalization and neofunctionalization of duplicated genes frequently occur after cotton polyploidization11,18,19. Consequently, diploids such as A2 are

better models for characterizing gene functions in cotton species. However, the method of regenerating plants by somatic embryogenesis has yet to be established for most cotton species,

which significantly hindered the transgenic study in cotton cells. Recently, we have achieved plant regeneration using A2 (ZB-1) with alternative solid-liquid culture method20. The callus

was able to be induced from different explants (hypocotyl, root, and cotyledon), and the regenerated plants could be regularly grown to maturity in soil or be grafted onto A2 seedlings. Upon

further optimization, ZB-1 will become a readily available tool for the functional analysis of cotton genes. Taken together, the accession ZB-1 with A2 genome is an essential material for

the studies on cotton polyploidy and gene function exploration (Fig. 1). At the same time, we have noticed that although some genome assemblies of A2 are available now and tens of A2

accessions have been resequenced, the chromosome-level A2 genomes were almost exclusively generated from the accession SXY17,21,22,23,24. Therefore, a high-quality genome assembly of ZB-1 is

necessary to provide additional genomic information for studies on polyploidy, gene manipulation, and the mechanisms underlying the regeneration of cotton plants. In this work, we

constructed a chromosome-level genome assembly of _G. arboreum_ ZB-1. The contig-level genome assembly was generated using PacBio HiFi reads. The Hi-C data was used to scaffold the contigs

into _pseudo_-chromosomes. Four transcriptomes of ZB-1 (boll, flower, sepal, and stem) were generated to assist the gene identification. The final gene set was generated by integrating the

results from _ab initio_ prediction, homologs-based gene annotation, and transcripts-derived gene identification using the PASA pipeline. The completeness of the genome assembly was

evaluated by BUSCO assessment and the mapping back of the short reads, which showed the high quality of the provided genome assembly of ZB-1. Moreover, a comparative analysis was carried out

between the assemblies of ZB-1 and SXY1, revealing a large number of single nucleotide polymorphisms (SNPs), suggesting this genome assembly is necessary for further studies using ZB-1 as a

test material. METHODS PLANT MATERIAL COLLECTION _G. arboreum_ ZB-1 was planted in the experimental field on the campus of Zhejiang Sci-Tech University in Hangzhou, Zhejiang Province,

China, in May 2022. The tender leaves (the third to fifth), bolls (10 days post anthesis), flowers (bloom day, including petals, stamens and pistils), sepals, and stems were harvested from

at least five adult plants and pooled. All the tissues were snap-frozen in liquid nitrogen and stored at −80 °C until use. DNA LIBRARY CONSTRUCTION AND GENOME SEQUENCING The genomic DNA

(gDNA) was extracted from the tender leaves using the CTAB (cetyltrimethylammonium bromide) method. After chilling in liquid nitrogen, 20 mg leaves were ground with steel balls in a 2 mL

polypropylene tube. 600 μL pre-heated (65 °C) CTAB buffer (2% w/v CTAB, 100 mM Tris-HCl, 20 mM EDTA, 1.5 M NaCl, pH 8) was added into the tube and thoroughly vortexed. The tube was placed in

a 65 °C water bath for 1 h. The homogenate was then centrifuged for 10 min at 12,000 rpm. The supernatant was transferred to a new tube, and 5 μL of RNase A solution was added. After

incubating at 37 °C for 20 min, an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added and mixed. The sample was centrifuged for 10 min at 12,000 rpm. The upper aqueous

phase was transferred to a new tube, and repeat this extraction once. Cold isopropanol was added with a volume of about two-thirds of the upper phase. The sample was gently mixed and

incubated at −20 °C for 30 min. Next, the sample was centrifuged for 10 min at 12,000 rpm at room temperature, and the pellet was washed using 500 μL ice-cold 70% ethanol. Decant the ethanol

and naturally dry the pellet to remove the ethanol residual. Finally, about 20 uL TE buffer (10 mM Tris, pH 8, 1 mM EDTA) was added to dissolve the DNA pellet. ILLUMINA SEQUENCING For

Illumina short-read sequencing, the purified gDNA was randomly sheared into fragments by a Covaris M220 Ultrasonicator (Covaris, USA). Subsequently, a library with an average length of about

350 bp for the inserted fragments was constructed according to the recommended protocols of the Illumina TruSeq DNA Sample Prep Kits. Qubit 2.0 Fluorometer (ThermoFisher Scientific, USA)

was used to quantify the dsDNA of the library, which was then diluted to 1 ng/μL. Agilent 2100 Bioanalyzer (Agilent, USA) was used to assess the integrity and size of the inserted fragments.

A final concentration of the library was determined with QuantStudio 3 Real-Time PCR Instrument (Thermo Fisher Scientific, USA) (>2 nM). The library was sequenced using the Novaseq 6000

platform (Illumina, USA) and a program of pair-end 150 bp. Quality control of the raw data was performed using Fastp (v0.21.0)25. The read pairs were removed if the ambiguous base (N)

exceeds 10% or the low-quality bases (Q < 5) exceed 20% of a single read in length. The generated raw data contained about 101.03 Gb paired short reads, with 99.91% retained after quality

control (Table 1). PACBIO SEQUENCING A HiFi library was constructed using the SMRTbell Template Prep Kit (Pacific Biosciences, USA). The high molecular weight DNA fragments (_ca_. 20 Kb)

were isolated and purified from the g-TUBE (Covaris, USA) sheared gDNA sample using the AMPure beads (Beckman Coulter, USA), followed by damage repair, end-repair/A-tailing and hairpin

adapter ligation according to the manufacturer’s instructions. The SMRTbell library was sequenced on the Pacbio Sequel II platform with the Circular Consensus Sequencing (CCS) model. The

HiFi reads were generated using SMRT Link software (v12.0) with default settings. About 4.01 M HiFi reads were finally acquired, with a total length and average length of about 71 Gb and

17.7 kb, respectively (Table 1). HI-C LIBRARY CONSTRUCTION AND SEQUENCING The Hi-C library was constructed according to the method previously described by Belton _et al_. with certain

modifications26. Tender leaves of ZB-1 were ground with liquid nitrogen and crosslinked by 4% formaldehyde at room temperature for 30 mins. 2. 5 M glycine was added to quench the

crosslinking reaction. The pellet was centrifuged at 2,500 rpm at 4 °C and resuspended with the lysis buffer (1 M Tris-HCl, pH 8, 1 M NaCl, 10% CA-630, and 13 units protease inhibitor). The

supernatant was centrifuged at 5,000 rpm at room temperature. The pellet was washed twice using the ice-cold NEB buffer and centrifuged again. Then, the nuclei were resuspended by the NEB

buffer, solubilized with dilute sodium dodecyl sulphate (SDS), and incubated at 65 °C for 10 mins. Triton X-100 quenched SDS, and the crosslinked chromatin was digested overnight by the

restriction enzyme _DPN II_ (400 units, 37 °C). The 5’ overhangs were filled in with biotinylated residues (biotin-14-dCTP), and the proximate blunted ends were ligated. Biotin was removed

from the un-ligated ends using T4 DNA polymerase. The ligated DNA was then sheared with the Covaris instrument, and the fragments with an average size of 350 bp were purified via agarose gel

electrophoresis and gel elution. After end repairing by the mixture of T4 DNA polymerase, T4 polynucleotide kinase, and Klenow DNA polymerase, the biotin-labelled DNA fragments were pulled

down using the streptavidin C1 magnetic beads. The library was then constructed with a standard protocol and sequenced on the Illumina Novaseq 6000 platform with 2 × 150 bp paired-end reads

(PE150). About 3.80 Gb short reads were generated from Hi-C library sequencing, with 3.78 Gb (99.6%) retained after removing the low-quality reads (Table 1). Further quality control was

mainly carried out with HICUP27. The truncated sequences were identified and mapped with hicup_truncater and hicup_mapper. The re-ligated and same circularised sequences were removed by

hicup_filter, and the PCR-raised duplication was filtered using hicup_deduplicator. As a result, 2,494,732 unique di-Tags were obtained, with the effect rate of Hi-C sequencing data about

23.32%. RNA LIBRARY CONSTRUCTION AND SEQUENCING Total RNA was isolated from the tissue samples (boll, flower, sepal, and stem) using the RNAprep Pure Plant Plus Kit (DP441, TIANGEN) and

purified with RNase-free DNase I. The sequencing libraries were constructed using NEB Next Ultra RNA Library Prep Kit for Illumina (New England Biolabs, USA). The mRNAs were enriched using

the oligo(dT) attached beads, and the cDNAs were synthesized using Superscript II reverse transcriptase. Fragmented cDNAs with a length of around 350 bp were used for sequencing library

construction with standard protocol. The constructed libraries were sequenced on Novaseq 6000 with the PE150 strategy. About 6.75 Gb, 6.16 Gb, 6.82 Gb, and 6.36 Gb raw data were generated

from sequencing of the libraries of the boll, flower, sepal, and stem, respectively. After quality control, about 6.1 to 6.7 Gb clean data were retained for further analysis, with their

effective ratio ranging from 98.21% to 98.93% (Table 1). GENOME ASSEMBLY The Pacbio sequencing generated HiFi reads were used to construct the contig-level genome assembly with Hifiasm

(v0.13.0-R307)28. 81 contigs were generated with a total length of 1,667.63 Mb. The length of the contigs ranged from 3.8 kb to 149.26 Mb, with the N50 and N90 lengths of 112.12 Mb and 41.97

 Mb, respectively. The Hi-C data was then used to construct the chromosome-level assembly with the ALLHiC pipeline29. The tool juicebox (v2.20.00) was used for a manual check and accuracy of

the generated assembly (Fig. 2)30. 52 scaffolds were finally acquired, with the N50 and N90 lengths of 137.59 Mb and 102.94 Mb, respectively (Table 2). The total length of 13

_pseudo_-chromosomes was 1,663.66 Mb, accounting for 99.77% of the entire assembly in length (Table 3). GENOME ANNOTATION AND GENE FUNCTION PREDICTION ANNOTATION OF REPETITIVE SEQUENCES A

hybrid strategy was used to identify the repetitive sequences in the ZB-1 genome. The tandem repeats were predicted using TRF (v4.09) (Match = 2, Mismatch = 7, Delta = 7, PM = 80, PI = 10,

MaxPeriod = 2000)31. The homology-based prediction was carried out using the Repbase database (v202101) and the software RepeatMasker (v4.1.0) (-a -nolow -no_is -norna) and RepeatProteinmask

(v4.1.0) (-noLowSimple -pvalue 0.0001 -engine ncbi) (http://www.repeatmasker.org/)32. The _de novo_ repetitive elements database was built with LTR_FINDER (v1.06) (-C -w 2)33, RepeatScout

(v1.0.5)34, and RepeatModeler (v2.0.1) (-engine ncbi), then all repeat sequences with lengths greater than 100 bp and gap ‘N’ less than 5% constituted the raw transposable element (TE)

library. This TE library was combined with the results from the homology search against Repbase to construct the custom library using Uclust (-id 80)35, and supplied to RepeatMasker for

DNA-level repeat identification. A total of 1,161.62 Mb of repetitive sequences were identified in the ZB-1 genome, representing 69.7% of the entire assembly in length. Apart from the tandem

repeats (61.4 Mb, 3.68%), the other repetitive sequences were mainly classified into long terminal repeats (LTR), DNA, and long interspersed nuclear elements (LINE) (Table 4). ANNOTATION OF

CODING GENES The coding genes were identified by integrating the results from _ab initio_ prediction, homology-based prediction, and cDNA-assisted prediction. Augustus (v3.2.3) (--genemodel

 = complete,--noInFrameStop = true)36, GlimmerHMM (v3.0.4)37, SNAP (v2013.11.29)38, Geneid (v1.4), and Genscan (v1.0) were used for _ab initio_ prediction39. The predicted proteomes of

closely related species, _i.e_., A2 (‘SXY1’ genome WHU-updated v1)7, E1 (ZSTU_v1)40, D5 (JGI_v2_a2.1)41, AD1 (‘TM-1’ genome NBI_v1.1)42, and _Theobroma cacao_43, were downloaded from

cottongen (https://www.cottongen.org/) and Ensembl Genomes (http://ensemblgenomes.org/) and used for homology-based gene prediction. These protein sequences were aligned to the ZB-1 genome

assembly using TBLASTN (v2.2.26) (-F T -e 1e-5), and the matching ones were further aligned to the genome with GeneWise (v2.4.1) for an accurate prediction of the gene structures44,45. In

addition, RNA-seq data of the ZB-1 tissues (leaf, boll, flower, sepal, and stem) were used to assist the gene prediction. Notably, the RNA-seq data of ZB-1 seedling leaves were generated and

submitted to the public database by our previous work40, including SRR13933598, SRR13933601, and SRR13933602. Trinity (v2.1.1) (--normalize_reads --full_cleanup –min_glue 2 –min_kmer_cov 2

--KMER_SIZE 25) was used to generate _de novo_ transcripts46. In addition, the short reads from RNA-seq were aligned to the ZB-1 genome using Hisat2 (v2.2.1), and the resulting bam files

were used for re-constructing the gene models using Stringtie (v1.3.3)47,48. The non-redundant gene annotation was then generated by merging the results derived from three methods with

EvidenceModeler (EVM, v1.1.1) (--segmentSize 200000 --overlapSize 20000 --min_intron_length 20) and was improved by using PASA pipeline and manual check49. This study finally identified

50,058 loci of coding genes, including 48,236 genes of high confidence and 1,822 pseudogenes (Table 5). The majority of coding genes (48,021) were found within the constructed chromosomes,

in contrast to a small number (215) of genes in the unanchored contigs. The high confidential genes spanned an average of 2,620 bp in the genome, with an average exon number of 4.3 and an

average coding sequence (CDS) length of 1,047 bp. FUNCTIONAL ANNOTATION Functional annotation of the coding genes was performed by homology search against the non-redundant protein database

(NR) in NCBI using BLASTP (cutoff evalue 1e-4 and identity 25%). The domain architectures and Gene Ontology annotations of the predicted proteins were characterized by Interproscan

(v5.35–74.0) (-appl ProDom, SMART, ProSiteProfiles, PRINTS, Pfam, Panther -iprlookup -dp -goterms) and online service of eggNOG (http://eggnog-mapper.embl.de/)(--evalue 0.001 --score 60

--pident 35)50,51. The combined analysis assigned tentative functions for about 99% of the predicted coding genes. 17,700 genes were mapped to the KEGG pathways, and a similar number of

genes (17,774) were assigned GO terms. ANNOTATION OF NONCODING RNAS The tRNAs in the ZB-1 genome were identified using tRNAscan-SE (v2.0.12)52, and the rRNA genes were identified via

homology search with BLASTN (v2.2.26) (-e 1e-10). In addition, Rfam (v14.1) and INFERNAL (v1.1.3) (http://infernal.janelia.org/) were used to predict the miRNAs and snRNAs53,54. 274 miRNA

genes and 1,369 tRNA genes were identified, as well as 14,383 rRNA and 7,467 snRNA loci (Table 6). COMPARATIVE GENOMICS ANALYSES GENOMIC SYNTENY The predicted proteomes of ZB-1 and SXY1

(WHU-updated v1) were compared using BLASTP (-evalue 1e-5, cutoff identity 50%, coverage 50%). Based on the homology analysis, MCScanX (-s 10) was then used to identify the collinear

blocks55, with the result visualized using the R package RIdeogram (0.2.2)56. Extensive genomic synteny was observed between the genomes of these two A2 accessions (Fig. 3), i_.e_., 99.1% of

the coding genes (including pseudogenes) were encoded by the collinear blocks. SNP CALLING Discovery of the SNPs between ZB-1 and SXY1 was carried out using the MUMmer tools (v4.0.0)57. The

script _nucmer_ was used to align the two genome sequences, and the script _delta-filter_ was subsequently used to remove the redundancy in the alignments (−1 -q -r). The structural

variations were then called using the script _show-snaps_ (-C), and the result was displayed using the R package of CMplot (v4.5.0)58. The overall rate of the SNP occurrence was about 0.53

in every 1,000 nucleotides (Fig. 4). Specifically, 19,017 SNPs were identified in the coding regions (cSNPs), with the occurrence rate of about 0.38 in 1,000 nucleotides. DATA RECORDS All

the high-throughput sequencing data generated in this work were available through NCBI with the project PRJNA935667. The clean data generated by sequencing the genome libraries were

deposited in the Sequence Read Archive (SRA) under the accession numbers SRR27009933 (Illumina PE150) and SRR27009931 (Pacbio CCS)59,60. The data generated from sequencing of the Hi-C

library was deposited under SRR2700993261. The RNA-seq data of the boll, flower, sepal, and stem could be retrieved from the SRA with the accession numbers

SRR27009934-SRR2700993762,63,64,65. The assembled genome sequence of _G. arboreum_ ZB-1, together with the information on the coding gene structures, has been deposited in GenBank under the

accession number JARKNE00000000066. The annotation of noncoding genes and repeat sequence, as well as the gene function prediction, were available in the Figshare database

(https://doi.org/10.6084/m9.figshare.24736338)67. TECHNICAL VALIDATION EVALUATION OF THE COMPLETENESS AND QUALITY OF THE GENOME ASSEMBLY MAPPING THE SHORT READS TO THE GENOME The short reads

generated from Illumina sequencing of the genome library were aligned to the assembly using BWA (v0.7.17)68, which showed that 99.22% of the reads were mapped to 99.95% of the genome

sequence. The average depth of short reads mapping was about 50, with 99.73% of the genomic regions covered by at least four reads, suggesting high concordance and completeness of the ZB-1

genome assembly (Table 7). Based on the result of short reads alignment, SNP calling was performed using samtools (v1.7)69, which showed an extremely low rate of heterozygosis SNP

(0.000563%) and rare homology SNP (<10). In addition, the _k_-mer based method Merqury was also used to estimate the quality of the genome assembly70, which showed high base accuracy of

the genome assembly (Q-value about 50). MAPPING OF RNA-SEQ DATA The RNA-seq data generated by this work, _i.e_., for the ZB-1 samples of flower, sepal, stem, and boll, were aligned to the

genome assembly using HiSat2 (v2.2.1) with default settings71. According to the statistics of the alignment, 94.5% (boll), 96.2% (flower), 91.9% (sepal), and 94.4% (stem) of the properly

paired short reads from RNA-seq could be mapped to the _pseudo_-chromosomes. BUSCO ASSESSMENT The BUSCO (Benchmarking Universal Single-Copy Orthologs) method was also used to evaluate the

completeness of the genome assembly72,73. The ZB-1 genome assembly captured 99.4% of the BUSCO 1614 reference gene set (embryophyta_odb10), indicating the completeness of the ZB-1 genome

assembly (Table 8). EVALUATION OF THE GENOME ANNOTATION The genomic features of the coding genes were compared between _G. arboreum_ ZB-1 and closely related species within the genus

_Gossypium_, including _G. arboreum_ SXY1, _G. hirsutum_, _G. raimondii_, and _G. stocksii_, with the versions of their genome assemblies as aforementioned. According to the distribution of

the exon number and the length of CDS, exon, gene, and intron, the gene features of ZB-1 were highly consistent with other cotton species (Fig. 5), suggesting the overall high accuracy of

the gene annotation. The predicted proteome of ZB-1 was also subjected to a search against the BUSCO 1614 reference gene set (embryophyta_odb10) using HMMER (v3.4) (http://hmmer.org/). With

the recommended cutoff by BUSCO, this assessment showed that 98.4% of the reference genes have been annotated in this study. EVALUATION OF THE SNP CALLING About 95.1 Gb of short reads from

sequencing the genome library of SXY1 were downloaded from SRA with the accession number SRR1306194374. About 97.5% of the reads were retained after quality control using Trimmomatic (v0.33)

(SLIDINGWINDOW:5:20 LEADING:5 TRAILING:5 MINLEN:50)75. 97.1% of the clean data were aligned to the ZB-1 genome sequence using BWA (v0.7.17) with default settings. GATK (v4.0.5.1) tools

_MarkDuplicates_ and _HaplotypeCaller_ were used to remove the redundancy raised by PCR amplification and identify the structure variations (default parameters), respectively76. According to

the result, 88.1% (16,752/19,017) of the cSNPs identified from genome sequence alignment (by MUMMER) were validated by the short reads, suggesting high accuracy of the estimation of the SNP

occurrence rate between SXY1 and ZB-1 and also high quality of the ZB-1 genome assembly. CODE AVAILABILITY All the bioinformatics tools were used according to the users’ manuals. Otherwise

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ACKNOWLEDGEMENTS The authors would like to thank Dr. Wang Tingzhang and Dr. Hua Xuejun for their assistance during the gene annotation and manuscript writing processes. This work was

supported by the National the National Natural Science Foundation of China (32170623 and U190320) and the Fundamental Research Funds of Zhejiang Sci-Tech University (19042398-Y). AUTHOR

INFORMATION AUTHORS AND AFFILIATIONS * Plant Genomics & Molecular Improvement of Colored Fiber Laboratory, College of Life Sciences and Medicine, Zhejiang Sci-Tech University, Hangzhou,

310008, China Rongnan Sun, Yuqing Wu, Xinyu Zhang, Minghua Lv, Dongliang Yu & Yuqiang Sun Authors * Rongnan Sun View author publications You can also search for this author inPubMed 

Google Scholar * Yuqing Wu View author publications You can also search for this author inPubMed Google Scholar * Xinyu Zhang View author publications You can also search for this author

inPubMed Google Scholar * Minghua Lv View author publications You can also search for this author inPubMed Google Scholar * Dongliang Yu View author publications You can also search for this

author inPubMed Google Scholar * Yuqiang Sun View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS D.Y. and Y.S. conceived and designed the

study; R.S., Y.W. and D.Y. analysed the sequencing data; X.Z. and M.L. collected the samples; R.S. and D.Y. wrote the draft manuscript; Y.S. and D.Y. modified the manuscript. All authors

have read and approved the final version for submission. CORRESPONDING AUTHORS Correspondence to Dongliang Yu or Yuqiang Sun. 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 and institutional affiliations. RIGHTS AND

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from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Sun, R., Wu, Y.,

Zhang, X. _et al._ Chromosome-level genome assembly and annotation of a potential model organism _Gossypium arboreum_ ZB-1. _Sci Data_ 11, 620 (2024).

https://doi.org/10.1038/s41597-024-03481-z Download citation * Received: 15 December 2023 * Accepted: 06 June 2024 * Published: 12 June 2024 * DOI: https://doi.org/10.1038/s41597-024-03481-z

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