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Comprehensive comparative analysis and development of molecular markers for Lasianthus species based on complete chloroplast genome sequences

BMC Plant Biology volume 24, Article number: 867 (2024) Cite this article

Abstract

Background

Lasianthus species are widely used in traditional Chinese folk medicine with high medicinal value. However, source materials and herbarium specimens are often misidentified due to morphological characteristics and commonly used DNA barcode fragments are not sufficient for accurately identifying Lasianthus species. To improve the molecular methods for distinguishing among Lasianthus species, we report the complete chloroplast (CP) genomes of Lasianthus attenuatus, Lasianthus henryi, Lasianthus hookeri, Lasianthus sikkimensis, obtained via high-throughput Illumina sequencing.

Results

These showed CP genomes size of 160164-160246 bp and a typical quadripartite structure, including a large single-copy region (86675–86848 bp), a small single-copy region (17177–17326 bp), and a pair of inverted repeats (28089–28135 bp). As a whole, the gene order, GC content and IR/SC boundary structure were remarkably similar among of the four Lasianthus CP genomes, the partial gene length and IR, LSC and SSC regions length are still different. The average GC content of the CP genomes was 36.71–36.75%, and a total of 129 genes were detected, including 83 different protein-coding genes, 8 different rRNA genes and 38 different tRNA genes. Furthermore, we compared our 4 complete CP genomes data with publicly available CP genome data from six other Lasianthus species, and we initially screened eleven highly variable region fragments were initially screened. We then evaluated the identification efficiency of eleven highly variable region fragments and 5 regular barcode fragments. Ultimately, we found that the optimal combination fragment' ITS2 + psaI-ycf4' could authenticated the Lasianthus species well. Additionally, the results of genome comparison of Rubiaceae species showed that the coding region is more conservative than the non-coding region, and the ycf1 gene shows the most significant variation. Finally, 49 species of CP genome sequences belonging to 16 genera of the Rubiaceae family were used to construct phylogenetic trees.

Conclusions

Our research is the first to analyze the chloroplast genomes of four species of Lasianthus in detail and we ultimately determined that the combination fragment' ITS2 + psaI-ycf4' is the optimal barcode combination for identifying the genus of Lasianthus. Meanwhile, we gathered the available CP genome sequences from the Rubiaceae and used them to construct the most comprehensive phylogenetic tree for the Rubiaceae family. These investigations provide an important reference point for further studies in the species identification, genetic diversity, and phylogenetic analyses of Rubiaceae species.

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Introduction

Lasianthus is a large genus with more than 200 species in family Rubiaceae [1]. These plants have the effect of promoting blood circulation and alleviating pain, and they are used in several traditional Chinese folk medicines to treat conditions such as fever, blood loss and bone pain with L. lucidus [2], and L. hookeri can be used as a food for promoting blood circulation [3]. The root decoction of L. oblongus is applied orally to hasten constriction of the organs for postpartum mothers [4]. L. acuminatissimus is used in traditional Chinese folk medicine for the treatment of rheumatoid arthritis [5]. In addition, researchers used various chromatographic methods to isolate and identify secondary metabolites of the Lasianthus species. These included antitumor anthraquinone glycosides isolated from L. acuminatidis, five new iridoid glycosides isolated from L. verticillatus, and iridoid terpenoids isolated from L. attenuatus [5,6,7]. However, source materials and herbarium specimens are often not well explored due to the similarities in morphological characters among Lasianthus species and their medicinal parts. This has further led to a chaotic situation in the Chinese folk medicinal market, characterized by the cross mixing of different original medicinal materials. These problems have severely hindered the clinical use of scientific research related to medicinal Lasianthus species.

Most of the Lasianthus species are shrubs, including a few small trees [8]. At present, the classification of Lasianthus species is mainly focused on microscopic and macroscopic morphological identification [8, 9]. However, with the change in growth environment, the microscopic morphology of plants also changes slightly, so morphological classification is difficult to identify the species of Lasianthus accurately. Studies on molecular identification of Lasianthus species are scarce, only Arshed et al. [10] reported evaluating the feasibility of five candidate DNA barcoding loci for Philippine Lasianthus Jack., and the results indicate that ITS, matK, rbcL, rps16 and trnT-F markers could not accurately identify all Lasianthus species. These results indicate that commonly used DNA barcoding sequences are not sufficient for accurately identifying the Lasianthus species.

Chloroplasts (CP) are important organelles of photosynthesis in green plants. The chloroplast gene is a closed circular DNA molecule composed of a typical quadripartite structure: a large single-copy region (LSC), a small single-copy region (SSC) and a pair of mirrored inverted repeat sequences (IRa and IRb) [11,12,13]. Chloroplast genomes are often used for species identification, systematics research, and the development of molecular markers because of their stable structure, maternal clonal inheritance, and low genetic recombination rate [14, 15]. Many chloroplast gene fragments such as trnH-psbA, matK and rbcL are used as DNA barcodes for species identification. However, it is difficult to identify related species by common fragments alone [16]. With the rapid development of high-throughput sequencing technology in recent years, the complete CP genome sequence becomes easy to obtain. The whole CP genome as a super-barcode has been widely used in plant phylogenetic relationship evaluation or species identification, and the sequences selected from the highly-variation regions of the whole CP genome have been used for species identification [17, 18]. For example, Yang et al. [18] conducted plant phylogenetic analysis and molecular marker development based on chloroplast whole genome sequencing of five medicinal plants in the genus Alpinia. Zhang et al. [19] developed barcode markers by comparing the complete CP genome sequences of Dracaena species to aid in the accurate identification of the origin of Dragon’s blood (Dracaena) medicinal herbs. However, there is limited publicly available data for Lasianthus. Although L. attenuatus, L. hookeri, L. sikkimensis, L. chrysoneurus, L. japonicus, L. rigidus, L. verticillatus have published the fasta format sequences of the chloroplast genome in NCBI, but these sequences have not been properly annotated. Relative to other families and genera in the plant kingdom, the CP genome data of Lasianthus plants are very limited. Therefore, it is necessary to obtain more CP genome data to solve the small intraspecific and interspecific differences among species of Lasianthus, to support the effective utilization of medicinal plant resources.

Here, we sequenced the complete CP genomes of L. attenuatus sampled from Guangxi and L. henryi, L. hookeri, L. sikkimensis sampled from Yunnan, using the Illumina HiSeq4000 sequencing platform. We also investigated their basic characteristics: including molecular structure analysis, simple sequence repeats (SSRs) and long repeat sequence analysis. Next, we compared the chloroplast genomes of Lasianthus species, analyzed nucleotide diversity, and identified hypervariable regions to develop DNA markers. Then, we collected 35 samples from 7 Lasianthus species to verify the identification efficiency of molecular markers and found the optimal identification fragments. Finally, we collected 49 species CP genome sequences from the Rubiaceae family and used them as a super-barcode to identify the species in this group and analyzed their phylogenetic relationships. This study provides important genetic information for species identification and phylogenetic analysis of Lasianthus. At the same time, it is also helpful to alleviate the problem of accurate identification of Lasianthus plants in the medicinal material market.

Materials and methods

Sample collection and DNA extraction

Fresh young leaves were collected from the L. attenuatus growing in Guangxi and L. henryi, L. hookeri, L. sikkimensis growing in Yunnan. The voucher specimens were deposited in the herbarium, Yunnan branch of the Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences herbarium (voucher numbers: IMDY2022051002, IMDY2022091311, IMDY2021102605, IMDY2021110615) and identified by Zhonglian Zhang. The collected leaves were cleaned with 75% ethanol, transported in dry ice, and preserved at -80 ° C for plant DNA extraction. Using the TIANGEN plant Genomic DNA kit (Tianjin Biotech, Beijing, Co., Ltd.) to extract total genomic DNA from frozen leaves according to a standard protocol. The concentration and quality of total DNA were evaluated using electrophoresis in 1% (w/v) agarose gel and Nanodrop 2000 instrument (Thermo Fisher Scientific Inc., Waltham, MA, USA). The OD260/280 value ranges from 1.8 to 2.2, and ≥ 2 µg of was equally pooled from individuals of the four species could be used to construct the library.

Chloroplast genome sequencing, assembly and annotation

DNA was broken into 300–500 bp fragments using the Covaris M220 focused ultrasonicator (Covaris, Woburn, MA, United States), and fragments of 500 bp size were screened for library construction. The DNA library was constructed using the Illumina TruSeq™ Nano DNA Sample Prep Kit (Illumina, San Diego, CA, United States). The library enrichment was performed by eight cycles of polymerase chain reaction (PCR) amplification, and the target band was recovered from 2% agarose gel (Certified Low Range Ultra agarose). The library was sequenced using the Illumina HiSeq4000 sequencing platform at Biozeron Company (Shanghai, China), and 2 × 150 bp paired-end reads were obtained. Raw reads were checked(Q ≥ 25) using the FastQC Toolkit [20]. Low-quality reads were filtered out from the raw data, reads containing 10% N were removed, and small fragments of < 75 bp were discarded after high-quality pruning to obtain high-quality data (clean reads) for subsequent analysis. Then, the above data was uploaded to the server with FileZilla 3.51.0, and the chloroplast genome was De novo assembled using Get Organelle [21]. The filtered ‘gfa’ file was visualized in Bandage v.0.8.1 [22]. Next, Bowite 2 in Geneious v.8.0.2 [23] was used to align the raw sequence to the assembled chloroplast genome to verify the assembly results. Finally, the reference genome was used to correct the starting position of the CP assembly sequence, and determine the position and direction of the four CP regions (LSC, IRa, SSC, and IRb) to obtain the assembled CP genome sequence.

The assembly results were imported into Geneious v.8.0.2 [23] for annotation, and then the positions of start codon, stop codon and intron of protein-coding genes were manually adjusted in Geneious v.8.0.2 [23]. The tRNA gene was validated online using the tRNAscan SE service [24]. The chloroplast genome map was drawn using the online website (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html)) [25]. Finally, we obtained the sqn file and submitted our report to NCBI. The complete CP genome sequences of L. attenuatus, L. henryi, L. hookeri and L. sikkimensis were deposited in GenBank with accession numbers of OR490208, OR490209, OR490210 and OR490211, respectively.

Codon usage and repeat sequence analysis

CodonW software (University of Texas, Houston, TX, usa) was used to obtain relative synonymous codon usage (RSCU) and investigate the codon distribution [26]. Molecular Evolutionary Genetics Analysis Version X was used to analyze guanine-cytosine (GC) content [27]. Simple sequence repeats were detected using the MISA Perl Script (http://pgrc.ipk-gatersleben.de/misa/). The minimum number of repeat units was set as follows: 10 repeat units for mononucleotide repeats, 5 for di-nucleotide repeats, 4 for tri-nucleotide repeats, and 3 for tetra-, penta-, and hexanucleotide repeats. REPuter was used to detect L. attenuatus, L. henryi, L. hookeri and L. sikkimensis of long repeats, including forward, palindromic, reverse, and complementary repeats [28].

Genome comparison analyses and marker development

The whole CP genomes were initially aligned using the online MAFFT software [29]. Conserved sequences between the CP genomes of L. attenuatus, L. henryi, L. hookeri and L. sikkimensis were identified using BLASTN with an E-value cutoff of 1e-10. The mVISTA [30] program in Shuffle-LAGAN mode was used to compare the four Lasianthus CP genomes using the L. henryi CP genome as a reference. Then, we used DnaSP [31] software to determine the nucleotide diversity (Pi) with a 200 bp step size and a 600 bp window length.

We used the primer design tool Primer-BLAST to design labeled primers for the highly variable regions (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). This enabled verification of interspecies polymorphisms in the CP genomes and the development of DNA markers to identify Lasianthus species via genomic comparisons and analyses. Next, we obtained seven species of Lasianthus (L. attenuatus, L. henryi, L. hookeri, L. sikkimensis, L. fordii var. trichocladus, L. hookeri var. dunnianus and L. verticillatus) to validate the efficiency of DNA barcoding based on the selected highly variable regions. The sample number and location information are listed in Table S1. Total genomic DNA was extracted using the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit with a standard protocol (TaKaRa) and 1% agarose gel electrophoresis. We used an ultra-micro ultraviolet spectrophotometer to assess the purity and concentration of the extracted genomic DNA. The PCR reactions were conducted in a total reaction volume of 25 µL, which contained DNA (15 ng), 10× PCR buffer (2.5 µL), dNTPs (10 mM, 2 µL), primers (0.5 µL each), Taq DNA polymerase (5 U/µL, 0.5 µL; TaKaRa), and double-distilled water (18.5 µL). For each reaction, we used the following program: an initial 5 min of denaturation at 94℃; 35 cycles of 30 s at 94℃, 30 s of annealing at Tm with different primers, and 15 s of extension at 72℃; and a final extension for 7 min at 72℃. The PCR products were visualized using 1.5% agarose gels, and the successfully amplified PCR products were sent to Sangon Biotech (Shanghai, China) for bidirectional sequencing.

Phylogenetic analysis

To determine the phylogenetic positions of L. attenuatus, L. henryi, L. hookeri and L. sikkimensis, we downloaded 63 complete CP genomes of Rubiaceae from the NCBI database. The sequences were initially compared using MAFFT [29]. We also used the CP genomes of Lonicera gynochlamydea (NC_064373), L. similis (NC_060471), and Sambucus williamsii (MW788534) as outgroups. We constructed phylogenetic trees of CP genomes sequences of Rubiaceae family species using the Neighbor-Joining (NJ), Maximum Parsimony (MP) and Maximum Likelihood (ML) methods with MEGA X [27] software and 1000 bootstrap replicates, and the best-fit substitution models were selected by ModelTest-NG [32].

Results and discussion

Chloroplast genome features of Lasianthus species

We analyzed and compared the basic characteristics of four Lasianthus species. The results showed that the CP genomes of L. attenuatus, L. henryi, L. hookeri and L. sikkimensis have the typical quadripartite structures [13, 16, 18] with a genome size of 160,164 bp, 160,240 bp, 160,246 bp and 160,203 bp, respectively (Fig. 1). Among them, the chloroplast genome of L. hookeri is the longest and L. attenuatus is the shortest, with a difference of only 82 bp. The chloroplast genome of Lasianthus species has a typical four-region structure like other higher plants [13, 33]. It contained a LSC region (86,675–86,848 bp), an SSC region (17,177–17,326 bp) and a pair of IRs (28,089–28,135 bp). The CP genomes of four Lasianthus species encodes a total of 129 genes, including 83 different protein-coding genes, 8 different rRNA genes and 38 different tRNA genes. Similar results have been reported in other angiosperms. The GC content of CP genome of the four Lasianthus species is very similar, ranging from 36.71 to 36.75% (Table 1).

Fig. 1
figure 1

Gene map of four Lasianthus complete chloroplast genomes. Genes on the inside of the outer circle are transcribed in a clockwise direction, while genes on the outside of the outer circle are transcribed in a counterclockwise direction. Genes belonging to different functional categories are different color-coded. The inner circle indicates the range of the LSC, SSC, and IRs. Also, the darker gray area in the inner circle corresponds to the GC content, whereas the lighter gray area corresponds to the AT content

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Table 1 Summary of chloroplast genome characteristics of four Lasianthus chloroplast genomes
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Codon usage

Codon usage bias plays an important role in CP genome evolution [34]. Some researchers pointed out that natural selection, mutation, phylogenetic relationship and other factors may lead to different codon use preferences [34,35,36]. The relative synonymous codon usage (RSCU) ratio is used to measure the usage of synonymous and non-synonymous codons in coding sequences. When the RSCU ratio < 1.00, the frequency of codon usage is lower than expected, and when the RSCU ratio > 1.00, the frequency of codon usage is higher than expected [26, 37]. We analyzed the codon usage levels of the shared protein-coding genes in the four Lasianthus species CP genomes (Fig. 2, Table S2). In total, the genes in the L. attenuatus, L. henryi, L. hookeri and L. sikkimensis CP genomes contain 25,698, 25,697, 25,700 and 25,647 codons, respectively. The codon for leucine is the most common in the four Lasianthus species CP genomes, accounting for 10.8% of the total number of codons on average. In the CP genomes of these Lasianthus species, usage of the codons AUG and UGG (encoding methionine and tryptophan, respectively) is not biased (RSCU ratio = 1.00). The AUG is also the initiator codon used by most protein-coding genes in the CP genome of terrestrial plants. Most amino acids were coded by more than one synonymous codon, such as leucine and arginine, which encode six codons. Only methionine and tryptophan do not have alternative codons. In the CP genome of higher terrestrial plants, the preference of the third base of the codon for A / T(U) is generally higher than C / G [38, 39]. In this study, codons ending in A and/or U accounted for 69.29–69.37% of all protein-coding genes in the CP genomes of four Lasianthus species. Moreover, these codons typically have high RSCU ratios in the four CP genomes, such as UUA (1.81–1.82) encoding leucine, GCU (1.71–1.73) encoding alanine. These codon usage results are similar to with those previously reported for Saxifraga species, Cardamine hupingshanensis, Alpinia galanga and Alpinia kwangsiensis [34, 37, 40]. Our results also showed that all types of RSCU ratio > 1.00 in the four Lasianthus species end with A or U except Ile-AUA and Leu-CUA. The high RSCU ratio may be related to the function of amino acids or the structure of peptides required to avoid transcription errors during the evolution of the CP genome [41, 42]. Therefore, stable CP genome evolution helps to reduce harmful mutations while improving the adaptability of important CP genes to selection pressure [37, 43, 44].

Fig. 2
figure 2

Heatmap of codon distribution of common protein-coding genes of four Lasianthus species

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Analyses of simple sequence repeats and long repeats

Simple sequence repeats (SSRs) are tandem repeat sequences composed of 1–6 nucleotide repeat units, widely distributed in the protein-coding genes [15, 45]. We analyzed the distribution and types of SSRs in CP genomes of four Lasianthus species. A total of 68,63,60,63 SSRs were found in the L. attenuatus, L. henryi, L. hookeri and L. sikkimensis CP genomes using MISA software, respectively (Table 2). Among these repeats, the mononucleotide SSRs were the most abundant, which were found (25–32) times in the four Lasianthus species. Followed by trinucleotide (9–15), tetranucleotide (8–9), pentanucleotide (7–8) and dinucleotide (6–7) repeats. Furthermore, the main repeats were constituted by A/T (21–27) in the four Lasianthus species, followed by AAT/ATT (8–14) trinucleotide repeats, AT/AT (5–6) dinucleotide repeats and AAAT/ATTT (5–6) tetranucleotide repeats. Our results are consistent with previous studies reporting that the Chloroplast genome SSRs are mostly composed of polyadenine ( Poly-A ) or polythymine ( Poly-T ) repeat, and the contents of C and G repeat are rare, which is consistent with the general characteristics of chloroplast genome SSRs in many plants [46,47,48].Due to the high substitution rate of CP SSRs, SSRs markers are widely used in genetic diversity and population structure evaluation, marker-assisted selection breeding, genetic map development and germplasm resources of plant populations [37, 49, 50].

Table 2 The simple sequence repeats (SSRs) types of the four CP genomes of Lasianthus species
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Long Repeats sequences include four types: complementary, forward, reverse, and palindromic repeat [28, 37]. These repetitive structures help facilitate the molecular recombination and diversity analysis of the CP genome in the population [51]. In this paper, we detected complementary, forward, reverse, and palindromic repeats in four Lasianthus species CP genomes using REPuter software tools. Results of the Long repeat-sequence analysis is shown in Fig. 3. The results showed that L. attenuatus has the least number of repeats, including 19 forward, 19 palindromic and 10 reverse repeats. The number of forward repeats (19–26) was the most abundant, followed by palindromes (17–23) and reverse repeats (10–20), with complementary repeats (0–2) being the least abundant. L. attenuatus does not have any complementary repeat types. Among the four Lasianthus species, the length of these repeat sequences is mostly between 30 and 39 and 40–49 bp, with none exceeding 70 bp. Repeat sequences play an important role in genome rearrangement and recombination [52, 53]. The repeat sequences identified in this study provide useful resources for species identification, genetic diversity and population structure of Lasianthus.

Fig. 3
figure 3

Long repeat sequence analysis of four Lasianthus complete chloroplast (CP) genomes. F, P, R, and C indicate the forward, palindromic, reverse, and complementary repeat types, respectively. Repeats with different lengths are indicated by different colors

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Contraction and expansion of IRs

The chloroplast genome of angiosperms is highly conserved. Some researchers believe that the contraction and expansion of the boundary between the IR and LSC/SSC regions are the main reasons for the size change of the chloroplast genome [48, 54]. In this study, we compared the IR / LSC and IR / SSC boundary structures of four Lasianthus species. The expansion and contraction of the IR regions are shown in Fig. 4. The results showed that there was no significant difference among the four Lasianthus species in terms of the length range of IR regions, which was 28,089–28,135 bp. The psbA gene of all Lasianthus species was identical in location, as it was completely located in the LSC region and was 92 bp away from the IRa / LSC boundary. The ndhF encoding gene located at the IRB-SSC boundary, and the ndhF gene has a length of 25–53 bp in the IRb region. Furthermore, all the IRa regions expand 3246–3250 bp into ycf1 and form a pseudogene ycf1 with a length of 3245–3249 bp in the IRb region. This also resulted in a 25–53 bp overlap between the pseudogene ycf1 and the gene ndhF in the IRb region. The pseudogenization of ycf1 and the location of ycf1 copies were also frequently found in other plants [55, 56]. In summary, although the chloroplast genomes of the four species are well conserved including gene number and genomic structures, the partial gene length and IR, LSC and SSC regions length are still different. This phenomenon indicates suggested expansions and contractions of the IR regions, as contraction and expansion of the IR/ SC boundary are considered to be the main reason for the length change of the chloroplast genome [38, 54, 57]. This is also a driving force in plant CP genome variation [58].

Fig. 4
figure 4

Comparison of the borders of the LSC, SSC, IRs regions among four CP genomes of Lasianthus. JLB: boundary of the LSC and the IRb. JSB: boundary of the SSC and the IRb. JSA: boundary of the SSC and the IRa. JLA: boundary of the LSC and the IRa

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Comparative genomic analysis within Lasianthus

The structure of the plant CP genome is highly conserved, highly mutated regions can be easily identified by comparative analyses [16]. These highly variable regions help to elucidate the genetic structure and evolutionary relationships of plants in different environments [59, 60]. In order to evaluate the CP genome differences of the Lasianthus species, we downloaded the complete CP genome (.fas) format sequences of six Lasianthus species from the NCBI database. Then, we combined the CP genome information of L. attenuatus, L. henryi, L. hookeri and L. sikkimensis with six CP genomes (L. chrysoneurus, L. hookeri var. dunnianus, L. japonicus, L. rigidus, L. verticillatus, L. sp.) of Lasianthus species published in the NCBI database. We performed comparisons and analyses using mVISTA software with L. attenuatus as the reference sequence (Fig. 5). These analyses revealed that, except for L. sp., the Lasianthus species CP genome sequences had little difference. The sequence differences were mainly concentrated in the non-coding region, while the exon and untranslated region (UTR) had only slight differences between the genomes. The most differentiated non-coding regions include accD-psaI, psaI-ycf4, rbcL-accD, ycf4-cemA, ndhC-trnV-UAC, petA-psbJ-psbL, trnE-UUC- trnT-GGU and trnT-trnL. Furthermore, we found that the most of the sequence variation was in the LSC and SSC regions, with the smallest sequence variation in the IR region. This result further supports the idea that the coding regions are more conservative than the non-coding regions, and that the IR regions are more conserved than the LSC and SSC regions in higher plants [14, 61, 62]. This phenomenon may be due to gene conversion correcting mutations in the IR sequence [63].

Fig. 5
figure 5

The alignment and comparative analysis of the whole CP genome for ten Lasianthus species using mVISTA, and using L. attenuatus as a reference. Gray arrows and thick black lines above the alignments indicate gene orientations. White peaks represent differences among CP genomes. Exons, introns, and conserved noncoding sequences (CNSs) were displayed as different colors. A similarity cut-off value of 70% was used for the plots, and the Y-axis represents the percentage similarity (50–100%)

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Next, the nucleotide diversity (Pi) and the highly variable regions of whole CP genome sequence in Lasianthus were detected by using DnaSP [31] software (Fig. 6). The IR region exhibits lower variability than the LSC and SSC regions. The test results showed that the Pi average value was 0.002029 (Table S3). Additionally, the Pi values of two highly variable regions in the LSC and SSC regions with Pi value greater than 0.015, were 0.0266 (psaI-ycf4-cemA) and 0.0156 (ndhF), respectively. The relatively high Pi values in the LSC and SSC regions indicate that rapid nucleotide substitution may occur during genome evolution, which plays an important role in species identification and phylogenetic analysis.

Fig. 6
figure 6

Nucleotide diversity (Pi) values analysis based on the complete chloroplast (CP) genomes of ten Lasianthus species. Window length: 600 bp; step size: 200 bp. X-axis: position of the midpoint of a window. Y-axis: nucleotide diversity of each window

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Molecular marker development and polymorphism verification

Previous studies on the molecular identification of Panax, Zanthoxylum and Alpinia species showed that CP genetic markers had high identification efficiency [18, 64, 65]. Compared with the whole CP genome, CP barcode fragments have the advantages of low sequencing costs and easy and fast analysis. Therefore, based on the alignment of complete CP genome sequences, 11 highly variable sites were selected as candidate DNA markers for identifying Lasianthus species. A total of 35 samples of seven species of Lasianthus (L. attenuatus, L. henryi, L. hookeri, L. sikkimensis, L. fordii var. trichocladus, L. hookeri var. dunnianus and L. verticillatus) were collected to verify the identification efficiency of the candidate barcode fragments. The sample number and location information are listed in Table S1. To develop identification markers for species authentication of Lasianthus, specific primers for the conserved regions of eleven highly variable sites (rps16-trnQ, psbK-psbI, psbM-trnD, trnE-trnT, ndhC-trnV, accD-psaI, psaI-ycf4, cemA, petA-psbJ, ycf1, ndhF) were designed. Then, five barcode markers were successfully amplified into fragments of the expected sizes, and their PCR products were sent to the Sangon Laboratory for sequencing. Ultimately, sequences for three markers (psaI-ycf4, psbM-trnD, cemA) were successfully obtained. At the same time, five conventional DNA barcodes ITS, ITS2, psbA-trnH, rbcL and matK were used to amplify seven Lasianthus species to evaluate their identification efficiency. The information of conventional barcodes and selected chloroplast molecular marker are shown in Table S4.

To detect molecular marker polymorphism and determine the most effective Lasianthus species identification barcode marker, we analyzed conventional barcodes and selected chloroplast molecular marker parameters, such as average differences length (bp), PCR success rate (%), intraspecific and interspecific differences (%), and average sequence differences between each marker and different markers (refer to Table 3). We constructed an NJ phylogenetic tree using conventional barcodes and screened chloroplast molecular markers. Our analysis revealed that no single fragment among these markers provided sufficient information to distinguish the seven species of Lasianthus. Ultimately, we discovered that the ideal combination fragment' ITS2 + psaI-ycf4 ‘, effectively identifies seven species within the genus (Fig. 7). Additionally, both fragments demonstrated high amplification and sequencing success rates, and the phylogenetic tree constructed using the combined fragments had anticipatively support rates. Based on our results, we suggest that more highly variable regions should be selected as candidate molecular markers, and the combination of two or more markers should be considered for the reliable identification of different species within some specifically genus which could not be authenticated efficiently in future studies. In recent years, numerous studies have utilized chloroplast genomes to detect highly variable regions as molecular markers for species identification, however, this method is still limited to a few taxa and limited samples [18, 66]. Therefore, we suggest that the combination of barcode fragments can be used for species identification for different taxa.

Table 3 Characteristics of the different barcode marker loci of seven Lasianthus species
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Fig. 7
figure 7

Neighbor-joining bootstrap trees (based on Kimura-2-Parameter) illustrating the resolution of the seven Lasianthus species for the “ITS2 + psaI-ycf4” barcoding

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Comparative genomic in Rubiaceae

To detect divergence in the CP genome of the Lasianthus species of Rubiaceae, we downloaded 45 species complete CP genome (.fas) format sequences of Rubiaceae from NCBI database (Table S5), and comparative analysis was conducted by aligning the CP genome sequences using L. attenuatus as a reference genome (Fig. S1, Fig. 8). The results showed that the chloroplast genome sequences of species in below the genus level are highly similar, but there are great differences among different genera. Thereinto, the most significant variation lies in the ycf1 gene, indicating that it was active in the evolution process of chloroplast genomes. Some researchers believe that ycf1 is the most variable plastid genome region and can serve as a core barcode of land plants [67]. Kikuchi et al. [68] provided evidence that ycf1 is indeed TIC 214, a crucial component of the protein translocon on the inner chloroplast membrane. Meanwhile, an interesting phenomenon was discovered in the comparison of chloroplast genomes Rubiaceae species, with the length of pseudogene ycf1 at the IRa / LSC boundary in the chloroplast genome of Lasianthus species is significantly longer than that in other genera of Rubiaceae species. Pseudogenes are classically believed to be insignificant and considered as ‘genomic junk’, were reported by Anand et al. [69] to undergo repair of pseudogene efeU under a designed selection pressure during adaptive laboratory evolution. This result indicates that some pseudogenes can recover their functions under certain pressures, emphasizing their importance for genome adaptive evolution. In addition, the intergenic region trnF-ndhJ, ycf1-trnN, rpl32-trnL, trnE-trnT and psaJ-rpl33 of Lasianthus species were significantly different from other groups in Rubiaceae. Due to the fact that intergenic regions are not directly involved in protein coding, their functional research has rarely reported. In recent years, some researchers have found that intergenic regions drive gene expression, indicating that intergenic regions are closely related to gene transcription regulation [70, 71]. The chloroplast intergenic regions also have great potential in species identification and phylogenetic evolution, which has been verified among species of Lilium, Dracaena and Alpinia [18, 19, 72]. On the whole, the coding region is more conservative than the non-coding region, and IRs are also more conservative than LSC and SSC. We speculate that during genome evolution, the LSC and SSC regions of Rubiaceae species undergo rapid nucleotide substitution. These variation regions are of great significance for species identification and genome adaptive evolution within the Rubiaceae family.

Fig. 8
figure 8

Nucleotide polymorphism (Pi) values analysis based on the complete chloroplast (CP) genomes of 49 Rubiaceae species. Window length: 600 bp; step size: 200 bp. X-axis: position of the midpoint of a window. Y-axis: nucleotide diversity of each window

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Phylogenetic relationships in Rubiaceae

At present, chloroplast genome sequences are widely used in phylogenetic relationships, genetic structure analysis and species identification of higher plants [73,74,75]. To explore the phylogenetic relationship of Lasianthus and the phylogenetic position of Lasianthus in the Rubiaceae family, we obtained 67 complete CP genome sequences belonging to 16 genera of Rubiaceae, and constructed NJ (Fig. 9), ML (Fig. S2), MP (Fig. S3) phylogenetic trees were constructed using L. gynochlamydea, L. similis and Sambucus williamsii as outgroups. Three methods generated nearly identical topology, and all nodes were well supported. The phylogenetic trees showed that 10 species of Lasianthus were clustered into one branch, and each species was separated from each other. Thus, we believe that the CP genomes can be used to identify the Lasianthus species. The chloroplast genome sequence serves as a super-barcode providing a useful method for species identification of advanced plants. Meanwhile, the 16 genera of Rubiaceae were divided into two large branches: the Lasianthus, Morinda, Paederia, Psychotria, Galium, Rubia, Dunnia, Leptodermis and Damnacanthus were grouped together in one large branch, whereas the remaining species are included in another large branch. Then, the ten species of Lasianthus split off into a branch, and the remaining eight genera species split off into another large branch. Rubiaceae includes approximately 700 species of 97 genera in China, and Lasianthus includes over 200 species. Therefore, for a clearer understanding of the species relationships in Rubiaceae and Lasianthus, future phylogenetic analyses should include more CP genome samples.

Fig. 9
figure 9

Phylogenetic tree constructed using neighbor-joining based on the 49 species CP genomes of Rubiaceae. Numbers at branch nodes are the bootstrap support values

Full size image

Conclusion

In this study, we reveal the detailed characteristics of the complete CP genome of four Lasianthus species, and the gene order, SSRs, GC content and IR/SC boundary structure were highly similar. we then combined these data with publicly available CP genome data from six other Lasianthus species to compared the CP genome sequences. Three highly variable regions (psaI-ycf4, psbM-trnD, cemA) were identified as valuable molecular markers, we ultimately determined that the combination fragment' ITS2 + psaI-ycf4' is the optimal barcode combination for identifying the genus of Lasianthus. Comparative analysis of chloroplast genome of Rubiaceae showed that the coding region is more conservative than the non-coding region, and IRs are also more conservative than LSC and SSC. Finally, the most comprehensive phylogenetic tree to date has been constructed for the Rubiaceae family. These findings provide an important reference point to further studies in the species identification, genetic diversity, and phylogenetic analyses of Rubiaceae species.

Data availability

The complete CP genome sequences of L. attenuatus, L. henryi, L. hookeri and L. sikkimensis are available in the GenBank with accession numbers of OR490208, OR490209, OR490210 and OR490211, respectively.

Abbreviations

CP:

Chloroplasts

LSC:

Large single-copy region

SSC:

Small single-copy region

IRs:

Inverted repeat regions

SSRs:

Simple sequence repeats

RSCU:

Relative synonymous codon usage

GC:

Guanine-cytosine

Pi:

Nucleotide diversity

ML:

Maximum Likelihood

NJ:

Neighbor-Joining

MP:

Maximum Parsimony

CNSs:

Conserved noncoding sequences

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Funding

Yunnan “Xingdian Talent Support Program " young talents special project and CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1–032).

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  1. Yunnan Key Laboratory of Southern Medicine Utilization, Yunnan Branch of Institute of Medicinal Plant Development Chinese Academy of Medical Sciences, Peking Union Medical College, Jinghong, 666100, China

    Yue Zhang, Meifang Song, Deying Tang, Haitao Li, Lu Qu, Yunqiang Wang, Cuiyun Yin, Lixia Zhang & Zhonglian Zhang

  2. College of Pharmacy, Dali University, Dali, 671000, China

    Xianjing Li & Niaojiao Xu

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Contributions

ZLZ, YZ conceived and designed the paper. YZ, MFS, LXZ, LQ, XJ Lanalyzed the experiments data. YZ execute the manuscript. ZLZ, HTL, NJX revised the manuscript. ZLZ, YZ, DYT, YQW, CYY collected the samples. The final manuscript was approved by all authors.

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Correspondence to Zhonglian Zhang.

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Fresh young leaves were collected from the Lasianthus attenuatus growing in Guangxi and Lasianthus henryi, Lasianthus hookeri, Lasianthus sikkimensis growing in Yunnan. The voucher specimens were deposited in the herbarium, Yunnan branch of the Institute of Medicinal Plant Development (IMPLAD), Chinese Academy of Medical Sciences herbarium (voucher numbers: IMDY2022051002, IMDY2022091311, IMDY2021102605, IMDY2021110615) and identified by Zhonglian Zhang. This study complies with relevant institutional, national, and international guidelines and legislation.

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Zhang, Y., Song, M., Tang, D. et al. Comprehensive comparative analysis and development of molecular markers for Lasianthus species based on complete chloroplast genome sequences. BMC Plant Biol 24, 867 (2024). https://doi.org/10.1186/s12870-024-05383-z

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