Background & Summary

Lithocarpus is the second largest genus of the Fagaceae family, comprising more than 320 species1. Species belonging to the genus Lithocarpus, commonly known as stone oaks, are dominant canopy trees in the tropical and subtropical forests of East Asia2. Lithocarpus polystachyus Rehder (2n = 2x = 24; syn. Lithocarpus litseifolius [Hance] Chun) is an evergreen plant widely distributed in south China and the adjacent southeast Asian countries3. The L. polystachyus is commonly known as “sweet tea”, because its leaves have a sweet taste when brewed. The leaves of L. polystachyus also have medicinal properties and have long been used as herbal tea to prevent and manage diabetes.

The leaves of L. polystachyus contain high concentration of dihydrochalcones (DHCs), which is the main source of its sweet taste. DHCs is a class of minor flavonoids (e.g. trilobatin and neohesperidin), which have been reported to act as flavor sweeteners and bitterness blockers4,5,6. DHCs such as phloretin, phlorizin, and sieboldin have also been demonstrated to play important roles in human health by providing a wide range of beneficial effects against diabetes, cardiovascular, cancer, and free radical-involving diseases7,8,9,10. In addition, phloretin exhibits strong broad-range bactericidal and fungicidal activities, and sieboldin is a powerful multipotent antioxidant9,11. DHCs have been isolated from many medicinal plants belonging to different families, but their contents vary significantly both among and within species12,13,14,15. Four DHCs (phloretin, phlorizin, trilobatin, and sieboldin) have been reported in sweet tea, and the biosynthesis pathway of the first three DHCs have been proposed14,16. However, our knowledge of DHC biosynthesis and regulatory mechanisms is limited in sweet tea. Specifically, candidate genes and transcription factors involved in the DHCs biosynthesis pathway remain to be investigated.

Here, we assembled a high-quality chromosome-scale genome of sweet tea, the first ever in the genus Lithocarpus, using PacBio HiFi and Hi-C data. The assembled sweet tea genome had a total length of 952 Mb, with a contig N50 of 21.4 Mb and a complete BUSCO score of 98.6%. A total of 922.6 Mb (96.9%) of the sequences were anchored to the 12 chromosomes. Genome annotation predicted 37,396 protein-coding genes and 597.5 Mb repetitive sequences. The high-quality sweet tea genome provides a valuable resource for exploring key genes and molecular regulatory mechanisms involved in the biosynthesis of important compounds, including DHCs, and will further serve as a strong foundation for trait improvement in this species.

Methods

Plant material and sequencing

A healthy sweet tea tree growing in the South China National Botanical Garden (accession number: 19940074) was selected for de novo genome assembly (Fig. 1). Young leaves were collected from the selected individual for whole-genome sequencing. The leave, stem, and root were collected for RNA-sequencing (RNA-seq) for the transcriptome assembly. All samples were immediately flash-frozen in liquid nitrogen after harvest, and stored at −80 °C for subsequent nucleic acid extraction.

Fig. 1
figure 1

The sweet tea tree sequenced in this study. (a) The sequenced tree planted in South China National Botanical Garden (accession number: 19940074) (b) The fruits of sequenced tree.

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Genomic DNA and total RNA were isolated from the leaves using DNeasy Plant MiniKit (Qiagen, Germany) and RNAprep Pure Plus Kit (Tiangen, China), respectively. The mRNA was purified from total RNA using poly-T oligo-attached magnetic beads for subsequent sequencing. To perform short-read sequencing for genome survey, transcriptome assembly, and transcriptomic profiling, libraries with an insert size of ~350 bp was constructed and sequenced on the Illumina NovaSeq 6000 platform to generate 150-bp paired-end reads. To perform de novo genome assembly, a 15–20-kb PacBio HiFi library was prepared using the SMRTbell Express Template Preparation Kit 2.0 (Pacific Biosciences, USA) and sequenced on the PacBio Sequel IIe platform to produce PacBio HiFi long reads. To generate the high-throughput chromatin conformation capture (Hi-C) data, DNA was isolated from the leaves and fixed with paraformaldehyde. The genomic DNA was then enzymatically digested with DnpII, generating fragments with sticky ends. These sticky ends were repaired by ‘A’ or ‘C’ deoxynucleotides with biotin by using DNA polymerase. Subsequently, the DNA fragments were ligated together to form chimeric circles using DNA ligase. The ligated DNAs were then uncrosslinked, purified, and sheared into 300–500 bp in size. Finally, the Hi-C sequencing library was sequenced on the Illumina NovaSeq 6000 platform, generating 150-bp paired-end reads.

A total of 32.8 Gb PacBio HiFi long reads (~37.6 × coverage), 118.8 Gb Hi-C reads (~135.9 × coverage), 44.3 Gb (~51 × coverage) paired-end Illumina reads, and 21.5 Gb RNAseq reads were generated for the genome assembly, genome survey, and transcriptome assembly.

Genome survey

The 21-bp K-mers with Illumina reads were counted using Jellyfish v1.1.1117, with default parameters. The genome size, the level of heterozygosity, and repeat content were estimated using GenomeScope v1.018. The estimated genome was 874 Mb in length and a heterozygosity of 1.5% (Fig. 2).

Fig. 2
figure 2

K-mer (21-mer)-based estimation of genome characters of sweet tea.

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De novo genome assembly

The HiFi long reads were initially assembled into contigs using hifiasm v0.18.6-r51319 with default parameter to generate one primary haplotype-collapsed assembly and a pair of partially phased assemblies. The primary assembly was selected for further scaffolding because it was much more contiguous than the partially phased assemblies. To anchor the contigs onto scaffolds, the Hi-C reads were mapped on to the contigs using Juicer v2.020 with DpnII (GATC) as the restriction enzyme site, and scaffolds were generated using the 3D-DNA (v201008) pipeline21 with the following parameters: “-m haploid -i 150000 -r 0--editor-repeat-coverage 5”. Based on the chromosome number of sweet tea (n = 12) determined previously22,23 and the interaction information of Hi-C reads generated with the 3D-DNA pipeline, the chromosome segmentation boundaries and assembly errors were manually checked and adjusted using Juicebox v1.11.0824.

The total length of the final sweet tea genome assembly was 952.3 Mb, which is slightly larger than the genome size estimated by K-mer analysis (Fig. 2 and Table 1), and smaller than the size measured by flow cytometry (ca. 1,149 Mb)22. The contig and scaffold N50 values of the sweet tea genome were 21.4 and 78.6 Mb, respectively, which are comparable with those of recently published Fagaceae genome assemblies (Table 2). A total of 922.6 Mb (96.9%) of the sequences were anchored to the 12 chromosomes (Table 1). The Hi-C interaction map showed a strong intrachromosomal interactive signal along the diagonal (Fig. 3).

Table 1 Statistics of the sweet tea genome assembly and annotation.
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Table 2 Summary of the genomic features of 12 Fagaceae species.
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Fig. 3
figure 3

Genome-wide chromatin interaction heatmap of sweet tea based on Hi-C data.

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Identification and characterization of repetitive elements

Tandem duplications were identified using TRF 4.0925, and both structurally intact and fragmented transposable elements (TEs) were annotated using EDTA (Extensive de-novo TE Annotator) v1.9.626. Overlapping regions of each class of repetitive elements were counted only once when calculating their total size. The divergence (K) of intact LTRs identified was estimated by Kimura two-parameter distance (K2P)27. The insertion time was calculated by the formula: T = K/(2 × r), where r refers to a substitution rate of 1.3 × 10–8 per site per year28.

A total of 597.5 Mb (62.74%) of the assembled sequences were annotated as TEs, with LTR (33.49%), TIR (14.27%), and Helitron (10.82%) being the three most abundant TE superfamilies (Fig. 4 and Table 3). We found most of the LTRs have been accumulated recently over a short time span with the peak of 0.3 million years ago (Ma), suggesting an expansion event (Fig. 5). In addition, TEs were unevenly distributed along the genome, with greater accumulation in regions with low gene density (Fig. 4).

Fig. 4
figure 4

Genomic features of sweet tea. The tracks from the outer to the inner circle represent the 12 chromosomes (Chr1–Chr12), gene density, trnasposable element (TE; Copia, Gypsy, TIR, and Helitron) density, tandem repeat density, and GC content. The connecting lines in the center of the Circos plot indicate the syntenic gene blocks.

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Table 3 Summary of the repetitive sequences in the sweet tea genome assembly.
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Fig. 5
figure 5

The distribution of insertion time of intact LTRs in sweet tea. Ma, million years ago.

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Gene prediction and functional annotation

Protein-coding genes in the sweet tea repeat-masked genome were predicted by applying MAKER v3.01.0429 to the combined results of RNA-seq-based prediction, protein-homology-based prediction, and ab initio prediction. Trinity v2.14.030 was used to de novo assemble the transcriptome for RNA-seq-based gene prediction. HiSat2 v2.2.131 was used to align the RNA-seq reads against the sweet tea genome, and then Trinity and StringTie v2.2.132 were used to assemble the genome-guided transcriptomes. Then, the de novo and genome-guided transcriptome assemblies were merged and refined using CD-HIT-EST v4.8.133, generating the transcript-based gene prediction. Plant protein sequences from the Swiss-Prot database (https://www.uniprot.org/downloads) and annotated proteins from Arabidopsis thaliana Araport1134 and Populus trichocarpa v4.135 were used for protein-homology-based prediction. AUGUSTUS v3.4.036,37 with BUSCO single-copy genes as training data, GeneMark-ES v4.69_lic38 with a self-training algorithm, and SNAP v2006-07-2839 with the output of MAKER as training data were used for ab initio prediction. MAKER was run for a total of three rounds to obtain high-quality gene models.

To further improve the gene prediction, genes predicted by MAKER were integrated into consensus gene models using EVidenceModeler v1.1.140 and then further polished using PASA v2.5.241. The longest transcript of each predicated gene model was considered as the representative gene models. The completeness of gene models was assessed by searching the gene content of the embryophyta_odb10 database using BUSCO v5.4.342.

Functional annotations were performed using different tools: InterProScan v5.56-89.043 for predicting potential protein domains; KofamKOALA v1.3.044 for determining KEGG ortholog assignments; and eggNOG-mapper v2.1.945 for inferring orthology assignments. In addition, BLASTP v2.13.0 + was used to search the homologous sequences in Nr, Swiss-Prot, and TrEMBL databases. The GO (Gene ontology) terms of the genes were extracted from the eggNOG and InterPro entries. Transcription factors were annotated using PlantTFDB v5.046 and PlanTFcat47.

A total of 37,396 protein-coding genes were predicted, with 97.1% of the complete BUSCO genes covered (Table 1). The average length of genic regions, coding sequences (CDSs), and intron sequences was 4,903 bp, 1,163 bp, and 948 bp, respectively. The average length of genes and introns in sweat tea was comparable to that of Fagaceae species (Table 2). Among the predicted protein-coding genes, 36,096 (96.5%) were annotated in functional databases (Tables 1), and 3,200 were identified as transcription factor (TF) genes belonging to 99 different families.

Comparative genomic analysis

To investigate the syntenic relationships between the protein-coding genes of sweet tea and those of the other Fagaceae species, collinear blocks between sweet tea and the representative species of each genus or section were identified based on protein sequences using MCScan implemented in jcvi v1.2.748. The synonymous substitution rate (Ks) was estimated using KaKs_Calculator v3.049 based on paralogous gene pairs extracted from the collinear blocks.

The syntenic gene blocks showed 1:1 syntenic patterns between sweet tea and other Fagaceae species (Fig. 6a), suggesting a conserved genome structure in Fagaceae. The Ks distribution pattern of the sweet tea syntenic genes presented the same signature of Ks peaks only at ~1.2 as that observed in the other Fagaceae and Vitis vinifera genomes, indicating that the genome of sweet tea did not undergo WGD after the gamma (γ) hexaploidy event (Fig. 6b).

Fig. 6
figure 6

Syntenic anasis between sweet tea and other Fagaceae species. (a) Syntenic blocks between sweet tea and other Fagaceae species. (b) Distribution of the synonymous substitution (Ks) rates of paralogous gene pairs within syntenic blocks. The blue dashed line represents the 1.2 of Ks. (c) Phylogenetic tree of sweet tea and 11 other Fagaceae species. The numbers in square brackets indicate the 95% confidence intervals of the divergence time, and two fossil calibrations are indicated in blue. The red solid circle represents support lower than 100%, based on 1,000 bootstrap replicates.

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Phylogenetic analyses

We conducted phylogenetic analyses to infer the relationship between sweet tea and other Fagaceae species. To do that, we utilized OrthoFinder v2.5.450 to identify orthologous genes between 12 Fagaceae species (Table 2). A total of 3,498 single-copy orthologous genes identified by OrthoFinder were aligned using MAFFT v7.50551, trimmed using trimAl v1.4.rev1552, and then concatenated together. Based on the concatenated alignment, a maximum likelihood phylogenetic tree was constructed using IQ-TREE v2.0.553,54. The divergence times among Fagaceae species were estimated using MCMCTree in the PAML v4.9j package55, based on four-fold degenerate sites. Two fossil calibrations were used to constrain the age of nodes: (1) the split between the Fagus genus and the rest of the Fagaceae species at 82–81 Ma56, and (2) the divergence between the Castanopsis and Castanea genera at 53–52 Ma57. The phylogenetic analyses showed that sweet tea was sister to the genus Quercus, with strong bootstrap support (100%). Calibration of the phylogenetic tree showed that sweet tea diverged from the genus Quercus ~59 Ma (95% HPD: 41.05–77.38 Ma) (Fig. 6c).

Data Records

The genome assembly have been deposited in the GenBank database of NCBI with accession number JAWTZU00000000058. The raw sequence data have been deposited in the Genome Sequence Archive (GSA) in National Genomics Data Center (NGDC) database (https://ngdc.cncb.ac.cn/) under the accession number CRA01239759. The genome annotation files and synteny data were deposited in the Figshare database60.

Technical Validation

Genome completeness was assessed by searching the gene content of the embryophyta_odb10 database with Benchmarking Universal Single-Copy Orthologs (BUSCO) v5.4.342; the quality of repetitive genomic regions was assessed using the LAI vbeta3.2 program (Ou et al., 2018); per-base consensus accuracy (QV) and k-mer completeness was estimated with Merqury v1.361 using PacBio HiFi long reads with a K-mer value of 20-bp; and PacBio HiFi long reads were mapped on to the genome using minimap2 v2.24-r112262 to calculate the mapping rate. The telomeric sequences in the sweet tea genome assembly were identified using quartet v1.1.363 with “-c plant”.

The sweet tea genome assembly showed a high degree of completeness and accuracy at the chromosome scale as indicated by the following statistics (Table 1): (1) the complete BUSCO score was 98.6%, which suggests high-gene space completeness of the assembly; (2) the LTR Assembly Index (LAI) was 21.5 for the assembly, which suggested it was a high quality genome64; (3) the values of QV and k-mer completeness were estimated as 60.6 and 81.4, respectively, based on the analysis of 20-mer spectra from the PacBio HiFi long reads; (4) 99.88% of PacBio HiFi long reads were aligned to the sweet tea genome assembly, with an average coverage depth of ~32 × along the 12 chromosomes (Fig. 7); and (5) telomeric sequences were detected at both ends of two chromosomes (Chr5 and Chr10).

Fig. 7
figure 7

Coverage of HiFi long reads mapped across the 12 chromosomes of sweet tea.

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