Draft Genome of the Medicinal Tea Tree Melaleuca Alternifolia (Maiden & Betche) Cheel (Myrtaceae)

Draft Genome of the Medicinal Tea Tree Melaleuca Alternifolia (Maiden & Betche) Cheel (Myrtaceae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Draft Genome of the Medicinal Tea Tree Melaleuca Alternifolia (Maiden & Betche) Cheel (Myrtaceae) Xiaoning Zhang, Silin Chen, Ye Zhang, Yufei Xiao, Yufeng Qin, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1293742/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Background Melaleuca alternifolia (Maiden & Betche) Cheel is a commercially important medicinal tea tree native to Australia. Tea tree oil (TTO), the essential oil distilled from its branches and leaves, has broad-spectrum germicidal activity and is highly valued in the pharmaceutical and cosmetic industries. Genome size is a key biology index, the study of which provides reference for genome engineering and sequencing work; besides, it also provides direct data for evolution biology and so on. Methods and results In the present study, the next-generation sequencing (NGS) was used to investigate the whole genome of Melaleuca alternifolia. 114 Gb high quality sequence data were obtained and assembled into 1,838,159 scafolds with an N50 length of 1021 bp. The assembled genome size is about 595 Mb, twice of that predicted by flow cytometer (300 Mb) and k-mer analysis (345 Mb). Benchmarking Universal Single-Copy Orthologs analyses indicated that only 11.3% of the conserved single-copy genes were miss. Repetitive regions cover over 40.43% of the genome. M. alternifolia maybe a diploid with highly heterozygous (heterozygosity is 0.8%). A total of 44,369 protein-coding genes were predicted in the assembled genome and 32,909 genes of these were functionally annotated, of which 8,436 genes were annotated in KEGG. Additionally, 457,661 simple sequence repeats (SSRs) and 1,109 transcription factors (TFs) form 67 TF families were identified in the assembled genome. Conclusion This draft genome may provide a reference for the deep sequencing strategies and be useful for future functional or comparative genomics analyses. Genome sequencing Melaleuca alternifolia. Tea tree oil. Whole-genome shotgun sequencing (WGS) Figures Figure 1 Figure 2 Figure 3 Introduction The Myrtaceae family includes about 130 genera and 5,000 species, many of which accumulate essential oils in their foliage and branchlets [1]. Melaleuca alternifolia , native to Australia, is thought to be a diploid species with a chromosome number of 2n = 22 and is the best-known member of the Melaleuca genus planted in China [2]. The leaves and branches of M. alternifolia produce tea tree oil (TTO), which has broad-spectrum germicidal, antioxidant, anti-inflammatory, and anticancer activities [3] [4], and is highly valued in the pharmaceutical, cosmetic, and food industries [5, 6]. Terpenoid compounds contained in TTO also have important adaptive roles in plant defense against pests and abiotic stress. M. alternifolia were introduced into China in the 1980s. After decades of efforts, we have selected superior individuals with high-quality TTO, established a mature tissue culture technology system, and realized large-scale tissue culture production. TTO consists of over 100 components, and terpinen-4-ol accounts for about one-third of the TTO in the terpinen-4-ol chemotype, which plays a critical role in the antimicrobial activity [7, 8]. Improved productivity of TTO will yield significant profit income, and might be a major target of breeders at present and for a long time [9]. Next-generation sequencing (NGS) can be used to generate and assemble high-quality genomes, based on its high throughput, high resolution, high precision, and low cost. Short-read sequencing is usually performed to estimate genome size, heterozygosity, repetitive rate, and evaluate the simple sequencing repeat (SSR) markers at the genome level, in advance of long-read sequencing due to its cost-effective [10]. M. alternifolia genome had been sequenced recently [11], as well as several other Myrtaceae trees such as Eucalyptus pauciflora [12], Eucalyptus grandis [13], Metrosideros polymorpha [14], and Leptospermum scoparium [15]. These genome sequences not only provide foundational information for genetic engineering of better trees, but also contribute to a deeper understanding of their genomic evolution, while supplying a reference for genome sequencing projects of other tree species. Here, we employed whole-genome sequencing (WGS) and de novo assembly to produce a draft genome of M. alternifolia (terpinen-4-ol chemotype with high terpinen-4-ol content more than 30% and low 1,8-cineole content less than 5%) with its in-vitro aseptic plantlets. The size of the M. alternifolia genome was estimated using both the k-mer frequency distribution and flow cytometry (FCM). Moreover, the heterozygous and duplication rates of M. alternifolia genome were deduced using the JELLYFISH tool [16]. Our data provides valuable information for precise and in-depth sequencing and pave the way for further investigation of the genes involved in terpinen-4-ol biosynthesis. Materials And Methods Plant materials and DNA extraction Melaleuca alternifolia (terpinen-4-ol chemotype, with high terpinen-4-ol and low 1,8-cineole) plantlets of tissue culture were grown at 25 ± 2℃ with 16 h/8 h (light/dark) photoperiod and 2,500 lx illumination intensity for 1 month. Leaf tissues of M. alternifolia were collected and ground in liquid nitrogen for DNA extraction. Genomic DNA was then extracted using the DNA extraction kit DP350(Tiangen biotech LTD, Beijing, China). Flow cytometry analysis Twenty milligram plant tissues were collected and immediately chopped in 1 mL cold nuclei isolation buffer (45 mM MgCl 2 ∙6H 2 O, 20 mM MOPS, 30 mM Na-Citrate, 1% (w/v) PVP 40, 0.2% (v/v) Triton X-100, 10 mM Na 2 EDTA, 20 µL/mL β-mercaptoethanol, pH 7.0). Homogenate was filtered using 42 µm nylon mesh, and nuclei were collected in new sample tube. Then 50 mg/mL DNA fluorochrome propidium iodide (PI) simultaneously with RNase at 50 mg/mL were added and gently mixed with samples. The nuclei were incubated on ice for 1 h with occasional shaking before analysis. Maize ( Zea mays ) B37 was selected as the internal reference, and the relative fluorescence of the stained nuclei was measured using BD FACScalibur (Becton Dickinson, NY, USA). Data processing Two genomic DNA libraries with an insert size of 270 bp and 500 bp were constructed, respectively. Paired-end sequencing of both libraries was performed using the Hiseq X-ten platform (Illumina, NY, USA). To reduce the effect of sequencing errors on assembly, all raw sequencing reads were then filtered with the SOAPnuke1.5.5 software [17]. To estimate the size and heterozygosity of the M. alternifolia genome, the filtered reads were used for k-mer (K = 17) determination within subsequent assembly steps [16]. Moreover, the JELLYFISH tool was used to simulate the heterozygosity and repetition rates of 15-Gb and 60-Gb data sets, which were randomly extracted from the filtered data used to calculate the k-mer. De novo assembly and quality evaluation SOAPdenovo v2.04 software [18] with default parameters were used to assemble the filtered reads for each library (270 bp and 500 bp inserts). Post-assembly processing included removal of contaminating bacterial and viral DNA sequences by aligning all assembled sequences to viral and bacterial genome sequences. The genome sequences we used were obtained from previous local (Basic Local Alignment Search Tool) BLASTn alignments and NCBI (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/ ) upload filter. Aligned sequences which shared >90% identity and >200 bp were removed from the final assembly. BUSCO v5.0.0 [19] with the viridiplantae_odb10 dataset which consisting of 425 Benchmarking Universal Single-Copy Orthologs (BUSCOs) from 57 species were used to evaluate the assembly completeness of gene content. GC content analysis was then performed and five sequences of each GC cluster were randomly selected for nucleotide (nt) library comparison, using Megablast software and the parameter was set with ‘-evalue 1e-5’. Genome annotation To annotate the genome, BALSTp searches were performed against with NCBI NR database using Diamond v0.9.14.115 [20] with parameter ‘--evalue 1e-5’. Moreover, genes in the M. alternifolia genome were also annotated by KEGG Automatic Annotation Server (KAAS) [21]. To generate the species-specific transposable elements (TEs) and repeats library of M. alternifolia , RepeatModeler v2.0.1 [22] was used in our research. The soft-mask repetitive elements in the M. alternifolia genome were identified using RepeatMasker v4.0.7 [23] running with NCBI/RMBLAST v2.6.0+ as search engine, and gff3 file was then generated based on the masked genome using AUGUSTUS v3.3 [24]. SSRs and TFs identification MISA-microsatellite searching tool [25] was used to identify the SSRs in the M. alternifolia genome. Transcription factors (TFs) of genome-wide were identified and classified by the software iTAK [26]. Results Genome sequencing The young shoots of the Melaleuca alternifolia superior individuals (Figure 1 a) were used as explants donor of in vitro culture. Induced sterile buds was grown in proliferation medium for 1 month with about 2-3 cm shoot height and 0.5 cm leaf length. They were cut into about 2 cm stem segments then transferred into rooting medium for another 1 month when developing more than 10 roots per shoot (Figure 1 b). Well-rooted plantlets were transplanted into greenhouse in a red mud cup for three months (Figure 1 c) then used for afforestation (Figure 1 d). The fresh leaves of these well-rooted plantlets were used as test materials to extract DNA for genome sequencing (Figure 1 b). We generated a total of 402 and 465 million reads from the paired-end sequencing of our two M. alternifolia genomic libraries. After filtering and corrections, we retained 355 and 408 million reads for each library, corresponding to 53 Gb and 61 Gb of clean data, high-quality sequences for the 270-bp and 500-bp genomic libraries, with the clean data rate of 88.3% and 88.4%, respectively. When combined, the two genomic libraries therefore include 114 Gb data (Supplement Table S1). Genome size estimation and heterozygous simulation A k-mer analysis was performed to estimate the genome size. We determined the k-mer distribution in our sequencing data with K = 17. The distribution showed two peaks: a major peak at depth = 37, and a minor peak at depth = 18, the height of the main peak (depth = 37) was about twice of the minor peak (depth = 18). The value of the main peak was also about twice that of the minor peak (Figure 2 ). The total number of 17-mers amounted to 12,767 Mb of sequence, resulting in a genome size estimation of 345 Mb when taking the major peak (depth = 37) and using the formula: Genome size = k-mer num/peak depth. The presence of the minor peak suggests that the M. alternifolia genome is highly heterozygous. We therefore simulated the heterozygosity and repetition rate of the M. alternifolia genome with JELLYFISH software [16],when using a depth of 37 as the main peak, the duplication rate was 41.95% and the heterozygosity rate was 0.80%, which is considered highly heterozygous [27], the read coverage is about 42 (Supplement Table S2). Fluorescence cell sorting of purified nuclei was used to estimate the DNA content of the M. alternifolia genome. M. alternifolia cell nuclei were stained with the fluorescent dye propidium iodide (PI), which could be uniformly embedded into DNA double chains, and then flow cytometry was performed to obtain the fluorescence signal. As the number of nuclei labeled with PI is proportional to the DNA content, the relative DNA content in the sample could be quantified based on the observed fluorescence signal intensity. The mean fluorescence intensity for the reference genome (maize inbred line B73) was 215.13, whereas that for M. alternifolia was only 28.31 (Supplement Table S3 & Figure S1). Based on the known genome size of maize B73 of 2.3 Gb [28], we therefore estimate that the genome size of M. alternifolia is approximately 300 Mb and hypothesize that multiple peaks may have been caused by endoreduplication. Sequence assembly and GC content analysis SOAPdenovo software was then used for genome de novo assembly and 63 mers were selected. A total of 1,838,159 scaffolds were assembled into a final genome sequence of 595 Mb with an N50 length of 1,021 bp (Table 1 ). This de novo assembly produced a sequence twice as large as the estimated genome size by k-mer analysis (345 Mb) and flow cytometry (300 Mb), which may indicate that the assembled genome is a highly heterozygous diploid. We calculated the GC content of the M. alternifolia genome in 20-kbp sliding windows without overlap (Supplement Figure S2). Our data fell into three clusters. Clusters A and B correspond to M. alternifolia , and the much smaller cluster C is presumed to represent DNA contaminants from exogenous sources such as bacteria, fungi, or insects. Mean GC content was 41.9%, as shown in Supplement Figure S1. The genome survey data represent 192 coverage of the M. alternifolia genome, and the splicing coverage was 198% (Table 1 ), both indicators of the high quality of our M. alternifolia sequence. Splicing coverage refers to the proportion of the assembled genome sequence to the whole genome. A splicing coverage of >95% is generally considered to be a robust genome assembly. Occasionally, the coverage is >100%, which may be due to the existence of repeat regions in the genome [29]. Table 1 Summary for the de novo assembly of the M. alternifolia genome. Statistical item Contig Scaffold N10 length (bp) 15,009 28,042 Number of N10 2,291 1,151 N50 length (bp) 796 1,021 Number of N50 100,342 71,431 N90 length (bp) 118 119 Number of N90 1,362,130 1,294,949 Maximum length (bp) 136,294 332,689 Total size (bp) 588,879,542 595,305,086 Total number (≥100 bp) 1,900,016 1,838,159 GC content (%) 42.4 41.9 Depth of sequencing (×) 192 Splicing coverage (%) 198 Genome completeness evaluation The level of genome completeness for the assembled sequences was evaluated by using BUSCO v5.0.0 (BUSCO, RRID:SCR_015008), which quantitatively assesses genome completeness using evolutionarily-informed expectations of gene content from near-universal single-copy orthologs selected from viridiplantae_odb10 (viridiplantae odb, RRID:SCR_011980; http://busco.ezlab.org/ ). BUSCO analysis showed that separate 67.5% and 21.2% of the 425 expected plant genes were identified as complete and fragmented genes respectively, while 11.3% of genes were considered to be missing from the assembled M. alternifolia genome sequence (Supplement Table S4). Repeat element annotation The total repeat length of the M. alternifolia genome (total length 595,305,086 bp) was estimated to be 240,657,903 bp, around 40.43% (Supplement Figure S3). 354,647,183 bp of non-redundant repetitive elements were also identified, accounting for 59.57%. About 6.93% of the genome can be attributed to TEs: DNA transposons, retrotransposons such as long interspersed nuclear elements (LINEs), short interspersed nuclear elements (SINEs), long terminal repeat (LTR); 1.78% of the repeats were predicted to be small RNAs, satellites, simple and low-complexity repeats (Supplement Figure S3 & Table S2), among which 191,238 simple sequence repeats were annotated, which will provide valuable genetic markers to assist M. alternifolia breeding programs. The GC content was 42.39% across the genome. The final assembly contains 44,369 predicted protein-coding genes (Supplementary Files S1), around 74.2% (32,909 genes) were functionally annotated in NCBI nonredundant protein (NR) database (Supplementary Files S2). Further, 8436 genes were annotated in KEGG metabolic pathways (Supplementary Files S3). Table 2 Major types of repeat elements identified in the M. alternifolia genome assembly. Repeat class No. of elements Total length, bp % of genome LINE 23,544 6,784,958 1.14 SINE 1,090 93,904 0.02 LTR 116,833 30,788,551 5.17 DNA transposons 14,545 3,588,626 0.60 Unclassified 1,457,484 186,499,107 31.33 Rolling-circles 5,871 2354517 0.40 Small RNA 29,702 2,262,577 0.38 Satellites 127 38,343 0.01 Simple repeats 191,238 7,015,736 1.18 Low complexity 25,911 1,231,584 0.21 SSR identification The scaffolds were utilized for SSR identification to ensure higher reliability. A total of 457,661 SSRs, including 255,359 (55.8%) mono-, 176,264 (38.51%) di-, 21,775 (4.76%) tri-, 2839 (0.62%) tetra-, 810 (0.18%) penta-, and 614 (0.13%) hexanucleotide repeats, were identified in 355,921 scaffolds (Supplement Figure S4). The mononucleotide repeat was overwhelming, and the 20 most frequent SSR types were exhibited in Supplement Figure S5. The A/T repeat was the most abundant type, accounting for 51.09% of all SSRs, followed by AG/CT (32.95%) and C/G (4.71%). Other dinucleotides and trinucleotides repeat types, such as AT/AT, AAG/CTT also made up an outstanding proportion. TF Identification There are 1109 transcription factors (TFs) coding genes form 67 TF families were identified and classified using the online tools Itak ( http://itak.feilab.net/cgibin/ itak/online_itak.cgi) in M. alternifolia . 1109 TFs belonged to 67 TF family were identified in M. alternifolia genome (Supplemental Files S4). Top 10 TFs including C2H2, MYB-related, bHLH, AP2/ERF-ERF, zn-clus, NAC, bZIP, C3H, WRKY, MYB accounts for 56.45% of the total TFs (Figure 3 ). Discussion The results of flow cytometer revealed that the genome size of M. alternifolia is approximately 300 Mb (Supplement Table S3), which is similar to the result obtained by k-mer analysis, i.e., 345 Mb. The genome size of our results are consistent with the previous research (356Mb) of Calvert’s (2017). And similar to that of Metrosideros polymorpha at 347 Mb [14] and Leptospermum scoparium at 297 Mb [15], but less than Eucalyptus pauciflora at 595 Mb and Eucalyptus grandis at 640 Mb [12, 13], all of which belong to the Myrtaceae family. The genome size of M. alternifolia is considered “very small” according to the definition of genome size [30]. Such a small genome will facilitate genome research and molecular manipulation, and provide insights into genome size variation in tree genomes. A GC content that is too high (>65%) or too low (<25%) may result in sequence bias during whole-genome sequencing, and may negatively affect genome assembly. The mean GC content of our result was 41.9%, quite consistent with the result of Voelker’s, 42%. According to the GC content and depth analysis, two of three detected clusters were derived from M. alternifolia . However, the de novo-assembled genome was 595 Mb (Table 1 ), which is twice about the genome sizes estimated using flow cytometry (300 Mb) and k-mer frequency distribution analyze (345 Mb). This larger assembly length was also performed in Metrosideros polymorpha [31]. This notable larger assembly length in M. alternifolia may suggest more than one haplotype were assembled. Based on these observations, we speculate that this species may be a diploid with a highly heterozygous genome. Genome heterozygosity reflects the reproductive lifestyle of the species, such as self-incompatibility or outcrossing, but may confound the quality of the genome assembly. Our k-mer analysis identified two depth peaks: the height of the main peak (depth = 37) was about twice that of the minor peak (depth = 17), suggesting that the M. alternifolia genome is highly heterozygous, and the heterozygosity determined using JELLYFISH was 0.8% (Supplement Table S2) smaller than that of Eucalyptus grandis (1.0%) [12] and Eucalyptus pauciflora (1.5%) [13]. However, the M. alternifolia genome is on the threshold for being considered a highly heterozygous genome with heterozygosity ≥0.8% [27] . Flowers of M. alternifolia are hermaphroditic, protandrous, pollinated by insects and predominantly outcrossing by nature, with occasional geitonogamy (self-pollination among flowers within the inflorescence) [32, 33]. The high heterozygosity of the M. alternifolia genome is likely the result of outcrossing [34] and highly confounds genome sequencing and assembly [35]. Contig N50 and BUSCO (BUSCO, RRID: SCR 015008) scores are usually used to assess and compare genome assemblies. A higher N50 length suggests an assembled genome with fewer and larger contigs. The length of contig N50 assembled by SOAPdenovo of M. alternifolia genome is 1,021 bp, lower than that of the previous research of this species(8,778bp) [11]. May be related to its short-read assembly method. However, it is much higher than that of Mikania cordata (312∼352 bp) which used the same SOAPdenovo software [10]. Genome completeness, estimated by BUSCO using the viridiplantae dataset, was 88.7%, indicating a relatively high geonome completeness. However, these two mentods of short-read assembly were much lower than Pacific Biosciences using MaSuRCA assembly. Repetitive regions cover over 40.43% of the genome, which similar with that in Eucalyptus pauciflora (44.77%) and Eucalyptus grandis (41.22%) [12]. A total of 44,369 protein-coding genes were predicted in the M. alternifolia assembled genome, more than those encoded by Metrosideros polymorpha (39,305) [14], Eucalyptus grandis (36,376) [13] and Leptospermum scoparium (31,220) [15]. 1109 TFs were annotated in the whole genome. C2H2, Myb related, bHLH were the top 3 TFs, accounted for 17.5%, 14.2%, 12.7%, respectively. Analyse of SSRs was conventional performed in genome survey, 457,661 SSRs have been identified, A/T, AG/CT, AAG/CTT are the most common motifs of mono-, di, and trinucleotide, respectively. This result showed the same patten with that in the Camellia sinensis [36]. The genomic SSRs identified here would benefit assessing the genetic diversity of M. alternifolia . In conclusion, we report a draft genome sequencing of M. alternifolia . This cost-effective strategy based on short-read data helped us to reveal its basic genomic parameters with small genome size, mid GC content, high heterozygosity rate. Its genome annotation has also been characterized, including repeat element annotation, the SSR identification, and TFs prediction at the genome level. This M. alternifolia draft genome will be useful for functional and comparative genomics research in the future. Conclusions We have sequenced the Melaleuca alternifolia genome and obtained 129 Gb of sequencing data from two genomic DNA libraries (270 bp and 500 bp), resulting in 114 Gb of clean data after quality control (Supplement Table S1). A total of 1,838,159 scaffolds were assembled for a total sequence length of 595 Mb, with an N50 length of 1,021 bp (Table 1 ). We estimated the genome size to be 345 Mb by k-mer frequency distribution and 300 Mb with FCM (Supplement Table S3). These numbers are only half of the total assembled sequence of 595 Mb. GC content and average depth analysis further confirmed that M. alternifolia may be a highly heterozygous diploid. The draft genome assembly of M. alternifolia lays the foundation for further genome engineering and sequencing work, which can be used to accelerate the molecular breeding of M. alternifolia with improved tea tree oil yield and quality. Declarations Funding This study was funded by the Department of Human Resources and Social Security of Guangxi Zhuang Autonomous Region, China (GuiCaiSheHan [2018]112); Guangxi Science and Technology Project (Gui Ke AD18281083 & Gui Ke AB18221058); Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards Open Project [202002]. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Conceptualization, Hong Yang and Hailong Liu; Data curation, Xiaoning Zhang and Silin Chen; Formal analysis, Ye Zhang, Yufei Xiao and Yufeng Qin; Funding acquisition, Hailong Liu; Investigation, Hong Yang; Methodology, Ye Zhang, Yufei Xiao and Yufeng Qin; Resources, Qing Li, Buming Liu and Ling Chai; Supervision, Hailong Liu; Visualization, Qing Li, Buming Liu and Ling Chai; Writing-original draft, Xiaoning Zhang and Silin Chen; Writing-review and editing, Li Liu. Consent to participate Informed consent was obtained from all individual participants included in the study. Consent to publish The authors affirm to grant the Publisher an exclusive licence to publish the article or to transfer copyright of the article to the Publisher. Data Availability The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2021), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences, under accession number CRA004893 that are publicly accessible at ( https://ngdc.cncb.ac.cn/gsa ) or at (https://ngdc.cncb.ac.cn/gsa/browse/CRA004893). Supplemental Information Supplemental information for this article can be found in the submission system. References Wu ZY, Raven PH (2013) Flora of China. Science Press: Beijing, China & Missouri Botanical Garden Press: St. Louis, MO, USA 321 Zhang X, Liang G, Yan Y, Yu Y, Yang G, Yang T (2000) Rapid propagation and polyploid induction in Melaleuca alternifolia . 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Science 326(5956):1112-5 Desai A, Marwah VS, Yadav A, Jha V, Dhaygude K, Bangar U, et al (2013) Identification of optimum sequencing depth especially for de novo genome assembly of small genomes using next generation sequencing data. PLoS One 8(4):e60204 Camillo J, Leao AP, Alves AA, Formighieri EF, Azevedo AL, Nunes JD, et al (2014) Reassessment of the Genome Size in Elaeis guineensis and Elaeis oleifera , and Its Interspecific Hybrid. Genom Insights 7:13-22 Izuno A, Hatakeyama M, Nishiyama T, Tamaki I, Shimizu‑Inatsugi R, Sasaki R, et al (2016) Genome sequencing of Metrosideros polymorpha (Myrtaceae), a dominant species in various habitats in the Hawaiian Islands with remarkable phenotypic variations. J Plant Res 129(4):727-736 Baskorowati L, Moncur MW, Doran JC, Kanowski PJ (2010) Reproductive biology of Melaleuca alternifolia (Myrtaceae) 1. Floral biology. Aust J Bot 58:373-83 Baskorowati L, Moncur MW, Cunningham SA, Doran JC, KanowskiA PJ (2010). Reproductive biology of Melaleuca alternifolia (Myrtaceae) 2. Incompatibility and pollen transfer in relation to the breeding system. Aust J Bot 58:384-91 Butcher P, JC B, GF M (1992) Patterns of Genetic Diversity and Nature of the Breeding System in Melaleuca alternifolia (Myrtaceae). Aust J Bot 40:365-75 Wei C, Yang H, Wang S, Zhao J, Liu C, Gao L, et al (2018) Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality. Proc Natl Acad Sci U S A. 115(18):E4151-E8 Wei Y, Jing W, hua D, Youxiang Z, Mingming Z, Dingjin. H (2013) Characteristic Analysis and Application of Microsatellites from EST Sequence of Camellia sinensis (in chinese). Hubei Agricultural Sciences 52(24):6178-81 Supplementary Files Supplementaryfiguresandtables.pdf supplementaryfiles.rar Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major Revisions Needed 23 Mar, 2022 Reviews received at journal 01 Mar, 2022 Reviewers invited by journal 11 Feb, 2022 Editor assigned by journal 08 Feb, 2022 First submitted to journal 24 Jan, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1293742","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":83100249,"identity":"7a465dbb-8255-40a2-9617-b37aeb6b8146","order_by":0,"name":"Xiaoning Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIiWNgGAWjYDAC5sMNQNKGh5+/gVgtbIkgpWkykjMOkKblsI1BQwKROuTbGJs/8+44z2PAcIDxw8ccIrQYHGNsMJx55jaPOXMDs+TMbcRokW9sSPjYdpvHsuEAGzMvMVqADms4kNh2jsfgQAKRWhiOMTY2fGw7QIIWoF+aGWe2JfNIzjjYTJxf5NuYD3/mbbOz5+dvPvjhI1EOQwDGBtLUj4JRMApGwSjADQAuAzWbokilcQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-2105-0560","institution":"Guangxi Forestry Research Institute","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiaoning","middleName":"","lastName":"Zhang","suffix":""},{"id":83100250,"identity":"265af366-d505-4ec7-b14e-9eef41d16661","order_by":1,"name":"Silin Chen","email":"","orcid":"","institution":"Kunming Institute of Botany Chinese Academy of Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Silin","middleName":"","lastName":"Chen","suffix":""},{"id":83100251,"identity":"5beaf6e8-d167-491d-9ec6-7fd322e5c106","order_by":2,"name":"Ye Zhang","email":"","orcid":"","institution":"Guangxi Forestry Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ye","middleName":"","lastName":"Zhang","suffix":""},{"id":83100252,"identity":"0dfe74c7-b757-4f89-9ac1-f62c7ade54cc","order_by":3,"name":"Yufei Xiao","email":"","orcid":"","institution":"Guangxi Forestry Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yufei","middleName":"","lastName":"Xiao","suffix":""},{"id":83100253,"identity":"cd924ff6-6d8c-47c0-89a2-b3d71c82be05","order_by":4,"name":"Yufeng Qin","email":"","orcid":"","institution":"Guangxi Forestry Research 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Standards","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Buming","middleName":"","lastName":"Liu","suffix":""},{"id":83100257,"identity":"4deca3b1-a6b1-4638-99d0-a23e6aee7c7a","order_by":8,"name":"Ling Chai","email":"","orcid":"","institution":"Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Chai","suffix":""},{"id":83100258,"identity":"467828af-91fc-4d1a-b890-6f2c5614f456","order_by":9,"name":"Hong Yang","email":"","orcid":"","institution":"Hubei University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Yang","suffix":""},{"id":83100259,"identity":"c7d57dc6-a231-441a-9992-c51ef565cdea","order_by":10,"name":"Hailong Liu","email":"","orcid":"https://orcid.org/0000-0002-4983-8555","institution":"Guangxi Forestry Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hailong","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2022-01-25 03:26:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1293742/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1293742/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":18213594,"identity":"c0856803-3d97-4d75-b09f-0db813a8a05d","added_by":"auto","created_at":"2022-02-15 00:16:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":902334,"visible":true,"origin":"","legend":"\u003cp\u003eAfforestation with tissue culture plantlets of M. alternifolia. Adult superior strains (a); Single bud of about 2 cm height grown in rooting medium for 1month (b); Well-rooted plantlets were transplanted into greenhouse in a red mud cup for 3 months (c); M. alternifolia stands (d).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1293742/v1/03a96b43fca0ff3b23408fc2.png"},{"id":18213692,"identity":"31a4ef8f-894e-4b21-aac2-2a4ec5ad4653","added_by":"auto","created_at":"2022-02-15 00:19:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":291214,"visible":true,"origin":"","legend":"\u003cp\u003eK-mer (k = 17) analysis for \u003cem\u003eM. alternifolia \u003c/em\u003egenome size estimation. The x-axis indicates the coverage depth (×); the y-axis indicates the frequency of k-mers at a given depth divided by the total frequency at all depths.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1293742/v1/ecfa628e0fe9233be22df89f.png"},{"id":18213592,"identity":"f2eb413b-753c-4d37-81d3-63e22eeab006","added_by":"auto","created_at":"2022-02-15 00:16:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":225520,"visible":true,"origin":"","legend":"\u003cp\u003eTop 20 TF families of \u003cem\u003eM. alternifolia \u003c/em\u003egenome.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1293742/v1/593bbbab6c277c2011854d6e.png"},{"id":18213693,"identity":"c12668bf-3bcf-47d3-8663-297c3bfa6b9d","added_by":"auto","created_at":"2022-02-15 00:19:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":514979,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1293742/v1/8b53cee1-d1cc-47d3-b577-43feaf07255d.pdf"},{"id":18213595,"identity":"8a5be27c-9dc9-49f4-9b76-b3a421def9c0","added_by":"auto","created_at":"2022-02-15 00:16:41","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":479250,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiguresandtables.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1293742/v1/f0cc3a1b61bf6793d15d7b5d.pdf"},{"id":18213596,"identity":"06d29974-cc81-471d-860c-a49fb0cf7bb4","added_by":"auto","created_at":"2022-02-15 00:16:41","extension":"rar","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":6953159,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfiles.rar","url":"https://assets-eu.researchsquare.com/files/rs-1293742/v1/7b4da7f4f7be50f436134a91.rar"}],"financialInterests":"","formattedTitle":"\u003cp\u003eDraft Genome of the Medicinal Tea Tree \u003cem\u003eMelaleuca Alternifolia\u003c/em\u003e (Maiden \u0026amp; Betche) Cheel (Myrtaceae)\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe Myrtaceae family includes about 130 genera and 5,000 species, many of which accumulate essential oils in their foliage and branchlets [1]. \u003cem\u003eMelaleuca alternifolia\u003c/em\u003e, native to Australia, is thought to be a diploid species with a chromosome number of 2n = 22 and is the best-known member of the \u003cem\u003eMelaleuca\u003c/em\u003e genus planted in China [2]. The leaves and branches of \u003cem\u003eM. alternifolia\u003c/em\u003e produce tea tree oil (TTO), which has broad-spectrum germicidal, antioxidant, anti-inflammatory, and anticancer activities [3] [4], and is highly valued in the pharmaceutical, cosmetic, and food industries [5, 6]. Terpenoid compounds contained in TTO also have important adaptive roles in plant defense against pests and abiotic stress.\u003c/p\u003e \u003cp\u003e \u003cem\u003eM. alternifolia\u003c/em\u003e were introduced into China in the 1980s. After decades of efforts, we have selected superior individuals with high-quality TTO, established a mature tissue culture technology system, and realized large-scale tissue culture production. TTO consists of over 100 components, and terpinen-4-ol accounts for about one-third of the TTO in the terpinen-4-ol chemotype, which plays a critical role in the antimicrobial activity [7, 8]. Improved productivity of TTO will yield significant profit income, and might be a major target of breeders at present and for a long time [9].\u003c/p\u003e \u003cp\u003eNext-generation sequencing (NGS) can be used to generate and assemble high-quality genomes, based on its high throughput, high resolution, high precision, and low cost. Short-read sequencing is usually performed to estimate genome size, heterozygosity, repetitive rate, and evaluate the simple sequencing repeat (SSR) markers at the genome level, in advance of long-read sequencing due to its cost-effective [10].\u003c/p\u003e \u003cp\u003e \u003cem\u003eM. alternifolia\u003c/em\u003e genome had been sequenced recently [11], as well as several other Myrtaceae trees such as \u003cem\u003eEucalyptus pauciflora\u003c/em\u003e [12], \u003cem\u003eEucalyptus grandis\u003c/em\u003e [13], \u003cem\u003eMetrosideros polymorpha\u003c/em\u003e [14], and \u003cem\u003eLeptospermum scoparium\u003c/em\u003e [15]. These genome sequences not only provide foundational information for genetic engineering of better trees, but also contribute to a deeper understanding of their genomic evolution, while supplying a reference for genome sequencing projects of other tree species. Here, we employed whole-genome sequencing (WGS) and de novo assembly to produce a draft genome of \u003cem\u003eM. alternifolia\u003c/em\u003e (terpinen-4-ol chemotype with high terpinen-4-ol content more than 30% and low 1,8-cineole content less than 5%) with its in-vitro aseptic plantlets. The size of the \u003cem\u003eM. alternifolia\u003c/em\u003e genome was estimated using both the k-mer frequency distribution and flow cytometry (FCM). Moreover, the heterozygous and duplication rates of \u003cem\u003eM. alternifolia\u003c/em\u003e genome were deduced using the JELLYFISH tool [16]. Our data provides valuable information for precise and in-depth sequencing and pave the way for further investigation of the genes involved in terpinen-4-ol biosynthesis.\u003c/p\u003e "},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and DNA extraction\u003c/h2\u003e \u003cp\u003e \u003cem\u003eMelaleuca alternifolia\u003c/em\u003e (terpinen-4-ol chemotype, with high terpinen-4-ol and low 1,8-cineole) plantlets of tissue culture were grown at 25 \u0026plusmn; 2℃ with 16 h/8 h (light/dark) photoperiod and 2,500 lx illumination intensity for 1 month. Leaf tissues of \u003cem\u003eM. alternifolia\u003c/em\u003e were collected and ground in liquid nitrogen for DNA extraction. Genomic DNA was then extracted using the DNA extraction kit DP350(Tiangen biotech LTD, Beijing, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry analysis\u003c/h2\u003e \u003cp\u003eTwenty milligram plant tissues were collected and immediately chopped in 1 mL cold nuclei isolation buffer (45 mM MgCl\u003csub\u003e2\u003c/sub\u003e∙6H\u003csub\u003e2\u003c/sub\u003eO, 20 mM MOPS, 30 mM Na-Citrate, 1% (w/v) PVP 40, 0.2% (v/v) Triton X-100, 10 mM Na\u003csub\u003e2\u003c/sub\u003eEDTA, 20 \u0026micro;L/mL β-mercaptoethanol, pH 7.0). Homogenate was filtered using 42 \u0026micro;m nylon mesh, and nuclei were collected in new sample tube. Then 50 mg/mL DNA fluorochrome propidium iodide (PI) simultaneously with RNase at 50 mg/mL were added and gently mixed with samples. The nuclei were incubated on ice for 1 h with occasional shaking before analysis. Maize (\u003cem\u003eZea mays\u003c/em\u003e) B37 was selected as the internal reference, and the relative fluorescence of the stained nuclei was measured using BD FACScalibur (Becton Dickinson, NY, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData processing\u003c/h2\u003e \u003cp\u003eTwo genomic DNA libraries with an insert size of 270 bp and 500 bp were constructed, respectively. Paired-end sequencing of both libraries was performed using the Hiseq X-ten platform (Illumina, NY, USA). To reduce the effect of sequencing errors on assembly, all raw sequencing reads were then filtered with the SOAPnuke1.5.5 software [17]. To estimate the size and heterozygosity of the \u003cem\u003eM. alternifolia\u003c/em\u003e genome, the filtered reads were used for k-mer (K = 17) determination within subsequent assembly steps [16]. Moreover, the JELLYFISH tool was used to simulate the heterozygosity and repetition rates of 15-Gb and 60-Gb data sets, which were randomly extracted from the filtered data used to calculate the k-mer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDe novo assembly and quality evaluation\u003c/h2\u003e \u003cp\u003eSOAPdenovo v2.04 software [18] with default parameters were used to assemble the filtered reads for each library (270 bp and 500 bp inserts). Post-assembly processing included removal of contaminating bacterial and viral DNA sequences by aligning all assembled sequences to viral and bacterial genome sequences. The genome sequences we used were obtained from previous local (Basic Local Alignment Search Tool) BLASTn alignments and NCBI (National Center for Biotechnology Information, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003c/span\u003e) upload filter. Aligned sequences which shared \u0026gt;90% identity and \u0026gt;200 bp were removed from the final assembly. BUSCO v5.0.0 [19] with the viridiplantae_odb10 dataset which consisting of 425 Benchmarking Universal Single-Copy Orthologs (BUSCOs) from 57 species were used to evaluate the assembly completeness of gene content. GC content analysis was then performed and five sequences of each GC cluster were randomly selected for nucleotide (nt) library comparison, using Megablast software and the parameter was set with \u0026lsquo;-evalue 1e-5\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eGenome annotation\u003c/h2\u003e \u003cp\u003eTo annotate the genome, BALSTp searches were performed against with NCBI NR database using Diamond v0.9.14.115 [20] with parameter \u0026lsquo;--evalue 1e-5\u0026rsquo;. Moreover, genes in the \u003cem\u003eM. alternifolia\u003c/em\u003e genome were also annotated by KEGG Automatic Annotation Server (KAAS) [21]. To generate the species-specific transposable elements (TEs) and repeats library of \u003cem\u003eM. alternifolia\u003c/em\u003e, RepeatModeler v2.0.1 [22] was used in our research. The soft-mask repetitive elements in the \u003cem\u003eM. alternifolia\u003c/em\u003e genome were identified using RepeatMasker v4.0.7 [23] running with NCBI/RMBLAST v2.6.0+ as search engine, and gff3 file was then generated based on the masked genome using AUGUSTUS v3.3 [24].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSSRs and TFs identification\u003c/h2\u003e \u003cp\u003eMISA-microsatellite searching tool [25] was used to identify the SSRs in the \u003cem\u003eM. alternifolia\u003c/em\u003e genome. Transcription factors (TFs) of genome-wide were identified and classified by the software iTAK [26].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eGenome sequencing\u003c/h2\u003e \u003cp\u003eThe young shoots of the \u003cem\u003eMelaleuca alternifolia\u003c/em\u003e superior individuals (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) were used as explants donor of in vitro culture. Induced sterile buds was grown in proliferation medium for 1 month with about 2-3 cm shoot height and 0.5 cm leaf length. They were cut into about 2 cm stem segments then transferred into rooting medium for another 1 month when developing more than 10 roots per shoot (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Well-rooted plantlets were transplanted into greenhouse in a red mud cup for three months (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec) then used for afforestation (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). The fresh leaves of these well-rooted plantlets were used as test materials to extract DNA for genome sequencing (Figure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). We generated a total of 402 and 465 million reads from the paired-end sequencing of our two \u003cem\u003eM. alternifolia\u003c/em\u003e genomic libraries. After filtering and corrections, we retained 355 and 408 million reads for each library, corresponding to 53 Gb and 61 Gb of clean data, high-quality sequences for the 270-bp and 500-bp genomic libraries, with the clean data rate of 88.3% and 88.4%, respectively. When combined, the two genomic libraries therefore include 114 Gb data (Supplement Table S1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGenome size estimation and heterozygous simulation\u003c/h2\u003e \u003cp\u003eA k-mer analysis was performed to estimate the genome size. We determined the k-mer distribution in our sequencing data with K = 17. The distribution showed two peaks: a major peak at depth = 37, and a minor peak at depth = 18, the height of the main peak (depth = 37) was about twice of the minor peak (depth = 18). The value of the main peak was also about twice that of the minor peak (Figure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The total number of 17-mers amounted to 12,767 Mb of sequence, resulting in a genome size estimation of 345 Mb when taking the major peak (depth = 37) and using the formula: Genome size = k-mer num/peak depth.\u003c/p\u003e \u003cp\u003eThe presence of the minor peak suggests that the \u003cem\u003eM. alternifolia\u003c/em\u003e genome is highly heterozygous. We therefore simulated the heterozygosity and repetition rate of the \u003cem\u003eM. alternifolia\u003c/em\u003e genome with JELLYFISH software [16],when using a depth of 37 as the main peak, the duplication rate was 41.95% and the heterozygosity rate was 0.80%, which is considered highly heterozygous [27], the read coverage is about 42 (Supplement Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFluorescence cell sorting of purified nuclei was used to estimate the DNA content of the \u003cem\u003eM. alternifolia\u003c/em\u003e genome. \u003cem\u003eM. alternifolia\u003c/em\u003e cell nuclei were stained with the fluorescent dye propidium iodide (PI), which could be uniformly embedded into DNA double chains, and then flow cytometry was performed to obtain the fluorescence signal. As the number of nuclei labeled with PI is proportional to the DNA content, the relative DNA content in the sample could be quantified based on the observed fluorescence signal intensity. The mean fluorescence intensity for the reference genome (maize inbred line B73) was 215.13, whereas that for \u003cem\u003eM. alternifolia\u003c/em\u003e was only 28.31 (Supplement Table S3 \u0026amp; Figure S1). Based on the known genome size of maize B73 of 2.3 Gb [28], we therefore estimate that the genome size of \u003cem\u003eM. alternifolia\u003c/em\u003e is approximately 300 Mb and hypothesize that multiple peaks may have been caused by endoreduplication.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSequence assembly and GC content analysis\u003c/h2\u003e \u003cp\u003eSOAPdenovo software was then used for genome de novo assembly and 63 mers were selected. A total of 1,838,159 scaffolds were assembled into a final genome sequence of 595 Mb with an N50 length of 1,021 bp (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This de novo assembly produced a sequence twice as large as the estimated genome size by k-mer analysis (345 Mb) and flow cytometry (300 Mb), which may indicate that the assembled genome is a highly heterozygous diploid.\u003c/p\u003e \u003cp\u003eWe calculated the GC content of the \u003cem\u003eM. alternifolia\u003c/em\u003e genome in 20-kbp sliding windows without overlap (Supplement Figure S2). Our data fell into three clusters. Clusters A and B correspond to \u003cem\u003eM. alternifolia\u003c/em\u003e, and the much smaller cluster C is presumed to represent DNA contaminants from exogenous sources such as bacteria, fungi, or insects. Mean GC content was 41.9%, as shown in Supplement Figure S1. The genome survey data represent 192 coverage of the \u003cem\u003eM. alternifolia\u003c/em\u003e genome, and the splicing coverage was 198% (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), both indicators of the high quality of our \u003cem\u003eM. alternifolia\u003c/em\u003e sequence. Splicing coverage refers to the proportion of the assembled genome sequence to the whole genome. A splicing coverage of \u0026gt;95% is generally considered to be a robust genome assembly. Occasionally, the coverage is \u0026gt;100%, which may be due to the existence of repeat regions in the genome [29].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary for the de novo assembly of the \u003cem\u003eM. alternifolia\u003c/em\u003e genome.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStatistical item\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eContig\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eScaffold\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN10 length (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15,009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28,042\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of N10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2,291\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,151\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN50 length (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e796\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of N50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100,342\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e71,431\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN90 length (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e119\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of N90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,362,130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,294,949\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaximum length (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e136,294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e332,689\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal size (bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e588,879,542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e595,305,086\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal number (\u0026ge;100 bp)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,900,016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,838,159\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGC content (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDepth of sequencing (\u0026times;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSplicing coverage (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGenome completeness evaluation\u003c/h2\u003e \u003cp\u003eThe level of genome completeness for the assembled sequences was evaluated by using BUSCO v5.0.0 (BUSCO, RRID:SCR_015008), which quantitatively assesses genome completeness using evolutionarily-informed expectations of gene content from near-universal single-copy orthologs selected from viridiplantae_odb10 (viridiplantae odb, RRID:SCR_011980; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://busco.ezlab.org/\u003c/span\u003e\u003c/span\u003e). BUSCO analysis showed that separate 67.5% and 21.2% of the 425 expected plant genes were identified as complete and fragmented genes respectively, while 11.3% of genes were considered to be missing from the assembled \u003cem\u003eM. alternifolia\u003c/em\u003e genome sequence (Supplement Table S4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRepeat element annotation\u003c/h2\u003e \u003cp\u003eThe total repeat length of the \u003cem\u003eM. alternifolia\u003c/em\u003e genome (total length 595,305,086 bp) was estimated to be 240,657,903 bp, around 40.43% (Supplement Figure S3). 354,647,183 bp of non-redundant repetitive elements were also identified, accounting for 59.57%. About 6.93% of the genome can be attributed to TEs: DNA transposons, retrotransposons such as long interspersed nuclear elements (LINEs), short interspersed nuclear elements (SINEs), long terminal repeat (LTR); 1.78% of the repeats were predicted to be small RNAs, satellites, simple and low-complexity repeats (Supplement Figure S3 \u0026amp; Table S2), among which 191,238 simple sequence repeats were annotated, which will provide valuable genetic markers to assist \u003cem\u003eM. alternifolia\u003c/em\u003e breeding programs. The GC content was 42.39% across the genome.\u003c/p\u003e \u003cp\u003eThe final assembly contains 44,369 predicted protein-coding genes (Supplementary Files S1), around 74.2% (32,909 genes) were functionally annotated in NCBI nonredundant protein (NR) database (Supplementary Files S2). Further, 8436 genes were annotated in KEGG metabolic pathways (Supplementary Files S3).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMajor types of repeat elements identified in the \u003cem\u003eM. alternifolia\u003c/em\u003e genome assembly.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRepeat class\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of elements\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal length, bp\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e% of genome\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLINE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23,544\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6,784,958\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSINE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93,904\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLTR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e116,833\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30,788,551\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDNA transposons\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14,545\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3,588,626\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnclassified\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,457,484\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e186,499,107\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRolling-circles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5,871\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2354517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmall RNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29,702\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2,262,577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSatellites\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e127\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e38,343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSimple repeats\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e191,238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7,015,736\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow complexity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e25,911\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1,231,584\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSSR identification\u003c/h2\u003e \u003cp\u003eThe scaffolds were utilized for SSR identification to ensure higher reliability. A total of 457,661 SSRs, including 255,359 (55.8%) mono-, 176,264 (38.51%) di-, 21,775 (4.76%) tri-, 2839 (0.62%) tetra-, 810 (0.18%) penta-, and 614 (0.13%) hexanucleotide repeats, were identified in 355,921 scaffolds (Supplement Figure S4). The mononucleotide repeat was overwhelming, and the 20 most frequent SSR types were exhibited in Supplement Figure S5. The A/T repeat was the most abundant type, accounting for 51.09% of all SSRs, followed by AG/CT (32.95%) and C/G (4.71%). Other dinucleotides and trinucleotides repeat types, such as AT/AT, AAG/CTT also made up an outstanding proportion.\u003c/p\u003e \u003c/div\u003e\n\u003ch2\u003eTF Identification\u003c/h2\u003e\n\u003cp\u003eThere are 1109 transcription factors (TFs) coding genes form 67 TF families were identified and classified using the online tools Itak (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://itak.feilab.net/cgibin/\u003c/span\u003e\u003c/span\u003e itak/online_itak.cgi) in \u003cem\u003eM. alternifolia\u003c/em\u003e. 1109 TFs belonged to 67 TF family were identified in \u003cem\u003eM. alternifolia\u003c/em\u003e genome (Supplemental Files S4). Top 10 TFs including C2H2, MYB-related, bHLH, AP2/ERF-ERF, zn-clus, NAC, bZIP, C3H, WRKY, MYB accounts for 56.45% of the total TFs (Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of flow cytometer revealed that the genome size of \u003cem\u003eM. alternifolia\u003c/em\u003e is approximately 300 Mb (Supplement Table S3), which is similar to the result obtained by k-mer analysis, i.e., 345 Mb. The genome size of our results are consistent with the previous research (356Mb) of Calvert\u0026rsquo;s (2017). And similar to that of \u003cem\u003eMetrosideros polymorpha\u003c/em\u003e at 347 Mb [14] and \u003cem\u003eLeptospermum scoparium\u003c/em\u003e at 297 Mb [15], but less than \u003cem\u003eEucalyptus pauciflora\u003c/em\u003e at 595 Mb and \u003cem\u003eEucalyptus grandis\u003c/em\u003e at 640 Mb [12, 13], all of which belong to the Myrtaceae family. The genome size of \u003cem\u003eM. alternifolia\u003c/em\u003e is considered \u0026ldquo;very small\u0026rdquo; according to the definition of genome size [30]. Such a small genome will facilitate genome research and molecular manipulation, and provide insights into genome size variation in tree genomes.\u003c/p\u003e \u003cp\u003eA GC content that is too high (\u0026gt;65%) or too low (\u0026lt;25%) may result in sequence bias during whole-genome sequencing, and may negatively affect genome assembly. The mean GC content of our result was 41.9%, quite consistent with the result of Voelker\u0026rsquo;s, 42%. According to the GC content and depth analysis, two of three detected clusters were derived from \u003cem\u003eM. alternifolia\u003c/em\u003e. However, the de novo-assembled genome was 595 Mb (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), which is twice about the genome sizes estimated using flow cytometry (300 Mb) and k-mer frequency distribution analyze (345 Mb). This larger assembly length was also performed in \u003cem\u003eMetrosideros polymorpha\u003c/em\u003e [31]. This notable larger assembly length in \u003cem\u003eM. alternifolia\u003c/em\u003e may suggest more than one haplotype were assembled. Based on these observations, we speculate that this species may be a diploid with a highly heterozygous genome.\u003c/p\u003e \u003cp\u003eGenome heterozygosity reflects the reproductive lifestyle of the species, such as self-incompatibility or outcrossing, but may confound the quality of the genome assembly. Our k-mer analysis identified two depth peaks: the height of the main peak (depth = 37) was about twice that of the minor peak (depth = 17), suggesting that the \u003cem\u003eM. alternifolia\u003c/em\u003e genome is highly heterozygous, and the heterozygosity determined using JELLYFISH was 0.8% (Supplement Table S2) smaller than that of \u003cem\u003eEucalyptus grandis\u003c/em\u003e (1.0%) [12] and \u003cem\u003eEucalyptus pauciflora\u003c/em\u003e (1.5%) [13]. However, the \u003cem\u003eM. alternifolia\u003c/em\u003e genome is on the threshold for being considered a highly heterozygous genome with heterozygosity \u0026ge;0.8% [27] .\u003c/p\u003e \u003cp\u003eFlowers of \u003cem\u003eM. alternifolia\u003c/em\u003e are hermaphroditic, protandrous, pollinated by insects and predominantly outcrossing by nature, with occasional geitonogamy (self-pollination among flowers within the inflorescence) [32, 33]. The high heterozygosity of the \u003cem\u003eM. alternifolia\u003c/em\u003e genome is likely the result of outcrossing [34] and highly confounds genome sequencing and assembly [35].\u003c/p\u003e \u003cp\u003eContig N50 and BUSCO (BUSCO, RRID: SCR 015008) scores are usually used to assess and compare genome assemblies. A higher N50 length suggests an assembled genome with fewer and larger contigs. The length of contig N50 assembled by SOAPdenovo of \u003cem\u003eM. alternifolia\u003c/em\u003e genome is 1,021 bp, lower than that of the previous research of this species(8,778bp) [11]. May be related to its short-read assembly method. However, it is much higher than that of \u003cem\u003eMikania cordata\u003c/em\u003e (312\u0026sim;352 bp) which used the same SOAPdenovo software [10]. Genome completeness, estimated by BUSCO using the viridiplantae dataset, was 88.7%, indicating a relatively high geonome completeness. However, these two mentods of short-read assembly were much lower than Pacific Biosciences using MaSuRCA assembly. Repetitive regions cover over 40.43% of the genome, which similar with that in \u003cem\u003eEucalyptus pauciflora\u003c/em\u003e (44.77%) and \u003cem\u003eEucalyptus grandis\u003c/em\u003e (41.22%) [12].\u003c/p\u003e \u003cp\u003eA total of 44,369 protein-coding genes were predicted in the \u003cem\u003eM. alternifolia\u003c/em\u003e assembled genome, more than those encoded by \u003cem\u003eMetrosideros polymorpha\u003c/em\u003e (39,305) [14], \u003cem\u003eEucalyptus grandis\u003c/em\u003e (36,376) [13] and \u003cem\u003eLeptospermum scoparium\u003c/em\u003e (31,220) [15]. 1109 TFs were annotated in the whole genome. C2H2, Myb related, bHLH were the top 3 TFs, accounted for 17.5%, 14.2%, 12.7%, respectively. Analyse of SSRs was conventional performed in genome survey, 457,661 SSRs have been identified, A/T, AG/CT, AAG/CTT are the most common motifs of mono-, di, and trinucleotide, respectively. This result showed the same patten with that in the \u003cem\u003eCamellia sinensis\u003c/em\u003e [36]. The genomic SSRs identified here would benefit assessing the genetic diversity of \u003cem\u003eM. alternifolia\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, we report a draft genome sequencing of \u003cem\u003eM. alternifolia\u003c/em\u003e. This cost-effective strategy based on short-read data helped us to reveal its basic genomic parameters with small genome size, mid GC content, high heterozygosity rate. Its genome annotation has also been characterized, including repeat element annotation, the SSR identification, and TFs prediction at the genome level. This \u003cem\u003eM. alternifolia\u003c/em\u003e draft genome will be useful for functional and comparative genomics research in the future.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe have sequenced the \u003cem\u003eMelaleuca alternifolia\u003c/em\u003e genome and obtained 129 Gb of sequencing data from two genomic DNA libraries (270 bp and 500 bp), resulting in 114 Gb of clean data after quality control (Supplement Table S1). A total of 1,838,159 scaffolds were assembled for a total sequence length of 595 Mb, with an N50 length of 1,021 bp (Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We estimated the genome size to be 345 Mb by k-mer frequency distribution and 300 Mb with FCM (Supplement Table S3). These numbers are only half of the total assembled sequence of 595 Mb. GC content and average depth analysis further confirmed that \u003cem\u003eM. alternifolia\u003c/em\u003e may be a highly heterozygous diploid.\u003c/p\u003e \u003cp\u003eThe draft genome assembly of \u003cem\u003eM. alternifolia\u003c/em\u003e lays the foundation for further genome engineering and sequencing work, which can be used to accelerate the molecular breeding of \u003cem\u003eM. alternifolia\u003c/em\u003e with improved tea tree oil yield and quality.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Department of Human Resources and Social Security of Guangxi Zhuang Autonomous Region, China (GuiCaiSheHan [2018]112); Guangxi Science and Technology Project (Gui Ke AD18281083 \u0026amp; Gui Ke AB18221058); Guangxi Key Laboratory of Traditional Chinese Medicine Quality Standards Open Project [202002].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, Hong Yang and Hailong Liu; Data curation, Xiaoning Zhang and Silin Chen; Formal analysis, Ye Zhang, Yufei Xiao and Yufeng Qin; Funding acquisition, Hailong Liu; Investigation, Hong Yang; Methodology, Ye Zhang, Yufei Xiao and Yufeng Qin; Resources, Qing Li, Buming Liu and Ling Chai; Supervision, Hailong Liu; Visualization, Qing Li, Buming Liu and Ling Chai; Writing-original draft, Xiaoning Zhang and Silin Chen; Writing-review and editing, Li Liu.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all individual participants included in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors affirm to grant the Publisher an exclusive licence to publish the article or to transfer copyright of the article to the Publisher.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics \u0026amp; Bioinformatics 2021) in National Genomics Data Center (Nucleic Acids Res 2021), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences, under accession number CRA004893 that are publicly accessible at (\u003ca href=\"https://ngdc.cncb.ac.cn/gsa\"\u003ehttps://ngdc.cncb.ac.cn/gsa\u003c/a\u003e) or at (https://ngdc.cncb.ac.cn/gsa/browse/CRA004893).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplemental information for this article can be found in the submission system.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWu ZY, Raven PH (2013) Flora of China. 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Aust J Bot 40:365-75\u003c/li\u003e\n\u003cli\u003eWei C, Yang H, Wang S, Zhao J, Liu C, Gao L, et al (2018) Draft genome sequence of \u003cem\u003eCamellia sinensis\u003c/em\u003e var. \u003cem\u003esinensis\u003c/em\u003e provides insights into the evolution of the tea genome and tea quality. Proc Natl Acad Sci U S A. 115(18):E4151-E8\u003c/li\u003e\n\u003cli\u003eWei Y, Jing W, hua D, Youxiang Z, Mingming Z, Dingjin. H (2013) Characteristic Analysis and Application of Microsatellites from EST Sequence of \u003cem\u003eCamellia sinensis\u003c/em\u003e(in chinese). Hubei Agricultural Sciences 52(24):6178-81\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Genome sequencing, Melaleuca alternifolia. Tea tree oil. Whole-genome shotgun sequencing (WGS)","lastPublishedDoi":"10.21203/rs.3.rs-1293742/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1293742/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003e\u003cem\u003eMelaleuca alternifolia\u003c/em\u003e (Maiden \u0026amp; Betche) Cheel is a commercially important medicinal tea tree native to Australia. Tea tree oil (TTO), the essential oil distilled from its branches and leaves, has broad-spectrum germicidal activity and is highly valued in the pharmaceutical and cosmetic industries. Genome size is a key biology index, the study of which provides reference for genome engineering and sequencing work; besides, it also provides direct data for evolution biology and so on. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods and results \u003c/strong\u003eIn the present study, the next-generation sequencing (NGS) was used to investigate the whole genome of \u003cem\u003eMelaleuca alternifolia. \u003c/em\u003e114 Gb high quality sequence data were obtained and assembled into 1,838,159 scafolds with an N50 length of 1021 bp.\u003cem\u003e \u003c/em\u003eThe assembled genome size is about 595 Mb, twice of that predicted by flow cytometer (300 Mb) and k-mer analysis (345 Mb). Benchmarking Universal Single-Copy Orthologs analyses indicated that only 11.3% of the conserved single-copy genes were miss. Repetitive regions cover over 40.43% of the genome. \u003cem\u003eM. alternifolia\u003c/em\u003e maybe a diploid\u003cem\u003e \u003c/em\u003ewith highly heterozygous (heterozygosity is 0.8%). A total of 44,369 protein-coding genes were predicted in the assembled genome and 32,909 genes of these were functionally annotated, of which 8,436 genes were annotated in KEGG. Additionally, 457,661 simple sequence repeats (SSRs) and 1,109 transcription factors (TFs) form 67 TF families were identified in the assembled genome. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eThis draft genome may provide a reference for the deep sequencing strategies and be useful for future functional or comparative genomics analyses.\u003c/p\u003e","manuscriptTitle":"Draft Genome of the Medicinal Tea Tree Melaleuca Alternifolia (Maiden \u0026amp; Betche) Cheel (Myrtaceae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-02-15 00:16:39","doi":"10.21203/rs.3.rs-1293742/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revisions Needed","date":"2022-03-23T10:48:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-03-02T02:47:42+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-02-11T08:31:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-02-08T16:07:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2022-01-24T22:26:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4ced3397-90f8-4d63-a6f9-3776fdbef0db","owner":[],"postedDate":"February 15th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-11-15T15:13:18+00:00","versionOfRecord":[],"versionCreatedAt":"2022-02-15 00:16:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1293742","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1293742","identity":"rs-1293742","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}