香京julia种子在线播放

Fresh leaf samples from three species of Edgeworthia namely, E. albiflora, E. chrysantha, and E. gardneri, were collected from plants in their natural habitat and were stored in Ziplock bags filled with silica gel beads prior to transportation to the laboratory. Voucher specimens of the three species were deposited in the Herbarium of Yunnan Normal University (YNUB) (Table 1). Total genomic DNA was extracted from the silica-dried leaves using the modified cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987) and was further purified using Wizard^® DNA Clean-Up System (Promega, United States).

Next-generation sequencing was conducted on the Illumina HiSeq 2500 platform and a 350-bp paired-end library was prepared. The raw reads were filtered to obtain high-quality clean reads using NGS QC Toolkit v2.3.3 with default parameters (Patel and Jain, 2012). The cp genome was assembled using NOVOPlasty (Dierckxsens et al., 2017) with the rbcL gene of Daphne kiusiana Miq. extracted from the complete cp genome sequence (GenBank accession KY991380) as the seed sequence. The complete cp genome was annotated using Geneious v10.1.3 (Kearse et al., 2012) by referring to the cp genome sequence of D. kiusiana. Annotations on the protein-coding (CDS) sequences present in the genome were manually checked using the open reading frame (ORF) and the tRNA genes were verified using the online tRNAscan-SE web server with default settings (Lowe and Chan, 2016). The complete cp genome was visualized using OGDRAW v1.3.1 (Greiner et al., 2019); all cp genome sequences were deposited in the NCBI GenBank database under the accession numbers MW246180 (E. albiflora), MN511715 (E. chrysantha), MW246181 (E. gardneri) for future reference.

Simple sequence repeats (SSRs) were identified using the MISA-web (Beier et al., 2017). The minimum number of repeats was set at 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta-, and hexanucleotides, respectively. SSRs were manually checked for redundancy. Identification of the four different types (forward, palindromic, reverse, and complement) of large repeats were conducted with REPuter (Kurtz et al., 2001). The size and identity of the large repeats were limited to not less than 30 bp and 90%, respectively; while the Hamming distance was set at 3.0.

The junctions and borders of the inverted repeat (IR) regions were visualized using IRscope (Amiryousefi et al., 2018) and further edited using Adobe Photoshop CS6 (Adobe, United States). For comparative analysis, the three sequences of the Edgeworthia cp genomes were compared using mVISTA (Mayor et al., 2000) using Shuffle-LAGAN mode. The cp genome sequence of E. albiflora was selected as the reference genome. The output image was manually edited using Adobe Illustrator 2020 (Adobe, United States). All three genome sequences of the Edgeworthia cp were aligned using MAFFT v7.409 (Katoh and Standley, 2013). Highly divergent regions between the species were identified using DnaSP v5.10 (Librado and Rozas, 2009). The nucleotide divergence values of the cp genome sequence alignment were analyzed using the sliding window method, with a window length of 1000 bp and a 500-bp step size.

All the CDS gene sequences were manually extracted from the chloroplast genome. The codon usage frequency in each of the three species of Edgeworthia was analyzed for all the PCGs using MEGA5 (Kumar et al., 2008). The relative synonymous codon usage (RSCU) was conducted to determine if the plastid genes were under selection.

Polymerase chain reaction (PCR) amplification was conducted on a final reaction volume, with a 20 μL volume reaction consisting of 10 μL of 2× Taq PCR StarMix with loading dye (Genstar Biosolutions, China), 1 μL of each primer, 6 μL of distilled water, and 2 μL 5 ng genomic DNA as a template. The ITS universal primer set: ITS1, 5′-TCC GTA GGT GAA CCT GCG G-3′ (forward) and ITS4, 5′-TCC TCC GCT TAT TGA TAT GC-3′ (reverse), was used to obtain the ITS region (White et al., 1990). PCR amplification was programmed with thermal settings of an initial denaturation at 94°C for 5 min; denaturation at 94°C for 60 s, annealing at 55°C for 60 s, extension at 72°C for 60 s; and a final extension at 72°C for 7 min. Upon verification via electrophoresis on a 1.0% agarose gel and documented under the UV machine, the PCR products were sent for direct Sanger sequencing at both ends using an ABI 3730 DNA Analyzer (Applied Biosystems, United States). Results acquired from the Sanger sequencing were aligned and manually edited to obtain the clean sequences of the three species of Edgeworthia. The ITS sequences for E. albiflora (MW255615), E. chrysantha (MW255616), and E. gardneri (MW255617) were deposited in the NCBI GenBank database for future reference.

Complete cp genome sequences of 15 taxa from the family Thymelaeaceae were included for phylogenetic analyses using maximum likelihood (ML) methods and Bayesian inference (BI). Multiple sequence alignment was carried out using MAFFT v7.409 (Katoh and Standley, 2013). Based on the Akaike information criterion calculated from the Modeltest 3.7 (Posada and Crandall, 1998), the generalized-time-reversible (GTR) model with gamma (+G) and invariant sites included (+I) (=GTR + G + I) was the best-fitting substitution model for both the ML and BI analyses. The ML tree was constructed using RAxML 8.2.11, under 1,000 bootstrap replicates (Stamatakis, 2014). BI analysis was conducted using MrBayes 3.2.5 (Drummond and Rambaut, 2007), in which the Markov Chain Monte Carlo analysis was performed under 1,000,000 generations and four Markov chains. Samplings were conducted at every 1,000 generations. The first 25% of the trees was discarded as burn-in; the remaining trees were estimated using the 50% majority-rule consensus tree and Bayesian posterior probabilities. Two closely related species, Hibiscus hamabo (Malvaceae; KR259988) and Eugenia uniflora (Myrtaceae; KR867678) were included as outgroups.

A total of 23 ITS sequences from the members of Thymelaeaceae, representing 21 taxa from eight genera in the Daphne group of tribe Daphneae, two taxa from tribe Aquilarieae and one taxon from the subfamily Octolepidoideae, were retrieved from the NCBI GenBank database. The latter three taxa were then selected as outgroups. Along with the ITS sequences of the three Edgeworthia species, the sequences were MUSCLE-aligned using MEGA 5 (Kumar et al., 2008) and trimmed using trimAL v1.2 (Capella-Gutiérrez et al., 2009) with the gappyout method in order to reduce the systematic errors produced by poor alignment. Phylogenetic analyses were carried out using both the ML and BI method. For ML analysis, the optimal DNA substitution model for the ML analysis calculated using the “Find Best DNA/Protein Model (ML)” function embedded in MEGA 5 (Kumar et al., 2008) was the Kimura two-parameter (K2P) with discrete Gamma model (+G) and invariant included (+I) (=K2P + G + I). Calculation was conducted with 1,000 bootstrap replicates on each branch node and all gaps and missing data were included in the analysis. For BI analysis, calculation was performed using MrBayes v3.2.5 (Drummond and Rambaut, 2007) following the same parameters and settings as mentioned above.

A total of 23,660,708 raw reads were obtained and the raw reads were directly fed into the assembly pipeline to obtained the maximum amount of useful data. Prior to genome assembly, a total of 374,342 aligned reads were acquired, and 222,318 assembled reads of an average coverage depth of 217 times per site were incorporated in the genome assembly. Three contigs representing three species of Edgeworthia were obtained at the end of the assembly process.

The cp genomes of the species of Edgeworthia were typical quadripartite structures that ranged in size from 172,708 bp (E. chrysantha) to 173,621 bp (E. albiflora) (Figure 1). All genomes contained a pair of IRs (41,952–42,039 bp), separated by a large single-copy (LSC) region (85,824–86,862 bp) and a small single-copy (SSC) region (2,681–3,017 bp) (Table 1). A total of 139 genes were predicted for the cp genomes of E. albiflora and E. chrysantha, including 93 CDS genes, 38 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. However, only 138 genes were recorded in the cp genome of E. gardneri, in which only 92 CDS genes, 38 tRNA genes, and eight rRNA genes were predicted. In the LSC region, E. albiflora and E. chrysantha each had 61 CDS genes, but only 60 CDS genes were reported in E. gardneri. The CDS gene, cemA, was presumably a pseudogene in the cp genome of E. gardneri; while only two CDS genes were recorded in all three species of Edgeworthia. All three cp genomes of Edgeworthia had 27 genes replicated in both IR regions, including 15 CDS genes (ccsA, ndhA, ndhB, ndhD, ndhE, ndhG, ndhH, ndhI, rpl2, rpl23, rps12, rps15, psaC, ycf1, and ycf2), eight tRNA genes (trnA-UGC, trnI-CAU, trnI-GAU, trnL-CAA, trnL-UAG, trnN-GUU, trnR-ACG, and trnV-GAC), and four rRNA genes (rrn4.5, rrn5, rrn16, and rrn23) (Table 2). Of the 19 intron-containing genes, 17 of them (atpF, ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, rps16, trnA-UGC, trnG-UCC, trnI-GAU, trnK-UUU, trnL-UAA, and trnV-UAC) contained one intron, whereas two of them (rps12 and ycf3) contained two introns (Table 3). The GC content of E. chrysantha and E. albiflora was 36.5%, while the GC content of E. chrysantha was 36.7%.

Simple sequence repeat analysis detected 127, 121, and 115 SSRs in E. albiflora, E. chrysantha, and E. gardneri, respectively (Figure 2A). Most of the SSRs were in the LSC regions when compared to the SSC and IR regions (Figure 2B). The SSRs were more abundant in the intergenic spacer region when compared to both the intronic and exon regions; more than 80 SSRs were detected in the intergenic spacer regions of the three species of Edgeworthia, and the number of SSRs in the intronic and exon regions were only recorded between 16 and 25 (Figure 2C). Cp genomes of all three Edgeworthia species contained mono-, di-, tri-, and tetranucleotide SSRs; while E. albiflora and E. chrysantha had one and three pentanucleotide SSRs, respectively, none were recorded in E. gardneri. Yet, E. gardneri was recorded with one hexanucleotide SSR, which was not present in the other two species. Considering sequence complementary, eight classified repeat types were found present in all three species of Edgeworthia (data not shown). The repeat type C/G were only detected in E. albiflora and E. chrysantha; while the repeat type AATT/AATT were only detected in E. chrysantha and E. gardneri. The repeat types AAG/GTT and ACAT/ATGT were exclusive to E. chrysantha, and E. gardneri was the only species recorded with the repeat types AATTC/AATTG and ACCCC/GGGGT.

For large repeats, forward repeats were recorded most abundant in the cp genome of the three Edgeworthia species, ranging from 44 to 47, followed by the palindromic repeats that ranged from 35 to 38 (Figure 2D). However, there was no records for reverse repeats in E. albiflora and E. chrysantha, but one in E. gardneri. The large repeats were recorded mostly in sequence length of 30–40 bp (Figure 2E). Edgeworthia gardneri was recorded with one large repeat with sequence length over 70 bp, but only large repeats with length from 60 to 70 bp were recorded longest for E. albiflora and E. chrysantha. The large repeats were recorded mostly distributed in the IR region, followed by the LSC region; at least one large repeat was recorded in the SSC region in E. gardneri (Figure 2F).

Chloroplast genome structure and the junction positions between IR regions were well-conserved among the three species of Edgeworthia, but structural variation was present in the IRs/SC borders (Figure 3). The ndhF gene extended to the IRB region in the cp genome of E. chrysantha, but not for E. albiflora and E. gardneri. The ndhF gene in the latter two species was located in the SSC region. The rps19 gene that was located in the LSC region in E. chrysantha extended into the IRB region in E. albiflora and E. gardneri. When compared to other seven closely related genera in the family Thymelaeaceae, the placement of genes adjacent to the IR junctions were identical to those in the cp genome of Gonystylus affinis, D. kiusiana, Phaleria macrocarpa, Stellera chamaejasme, and Wikstroemia chamaedaphne.

Based on the genome sequence alignment of the three species of Edgeworthia, distinct sequence variation was detected in three gene regions; petN-psbM, trnL-UAG-rpl32, and rps16-trnQ-UUG (Figure 4). With a nucleotide diversity (Pi) cut-off point set at Pi ≥ 0.04, the sliding window analysis detected three highly variable regions in the genome sequence alignment of the three species of Edgeworthia (Figure 5). The highly variable regions were all located within the protein-coding genes, ndhF and mainly manifested in the SSC region, between ndhF and rpl32 of the cp genome.

A total of 29,529–30,093 codons of the CDS genes were recorded in the three cp genomes of Edgeworthia. The RSCU value for each species exhibited similar codon preference in the 64 codons in the CDS genes (Supplementary Table 1). As a result, 30 of them exhibited greater preference (RSCU > 1); 32 of them were least preferred (RSCU < 1); two of them displayed no preferences (RSCU = 1). The isoleucine (Ile)-encoded codon AUU exhibited the greatest occurrence (n = 1,269); while the Ile-encoded codon UGA exhibited the least occurrence (n = 20). Among the preferred codons, 27 of them were A/U-ended. Among the three stop codons, UAA was recorded to be more abundant than UAA and UGA, thus displaying higher preferences. There were no rare codons (RSCU < 0.1) found in the CDS genes of the three cp genomes of Edgeworthia.

Phylogenetic analyses using the complete cp genome sequence for both ML and BI methods revealed similar topological structure between the two phylogenetic trees (Figure 6A). Strong bootstrap support and high posterior probabilities were recorded at all branch nodes. All taxa included in this study displayed monophyletic relationships. In the Daphneae, Edgeworthia diverged before Daphne, Stellera, and Wikstroemia. The three species of Edgeworthia formed a monophyletic clade, with E. albiflora diverging before E. chrysantha and E. gardneri.

For the ITS-based ML and BI analyses, both phylogenetic trees exhibited identical tree structure and species placement (Figure 6B). The Daphne group displayed a paraphyletic relationship, in which the genera Daphnopsis, Dirca, and Ovidia formed a cluster; while Edgeworthia was placed at the base of the latter cluster, with strong bootstrap support and Bayesian posterior probability (ML ≥ 75, BI ≥ 0.95) that also consisted of Daphne, Diarthron, Stellera, Thymelaea, and Wikstroemia. In the Edgeworthia clade, the three Edgeworthia species formed a monophyletic clade, with E. albiflora diverging before E. chrysantha and E. gardneri under strong bootstrap support and Bayesian posterior probability.