Abstract
Gardenia jasminoides, belonging to the Rubiaceae family, is widely distributed and planted in China. It has traditionally been used as an ornamental and medicinal plant in several Asian countries. The rapid development of high-throughput sequencing technology makes it feasible to obtain complete chloroplast (cp) genome sequences and will deepen our understanding of evolution of G. jasminoides. In this study, we sequenced the complete cp genomes of two botanical varieties of G. jasminoides. The complete cp genomes of both botanical varieties of G. jasminoides showed highly conserved structures and the length was 154,954 base pairs (bp) for G. jasminoides var. radicans (GJR) and 155,098 bp for G. jasminoides var. grandiflora (GJG). A total of 132 and 133 genes were identified in GJR and GJG, respectively. The cp genomes of two newly sequenced G. jasminoides were further compared with two published G. jasminoides cp genomes. Multiple repeats and simple sequence repeats (SSRs) were detected among different genotypes of G. jasminoides. The intron sequences of rps16 and rpl16 genes were slightly divergent among four genotypes of G. jasminoides. Phylogenetic analyses based on the complete cp genome sequences showed that G. jasminoides was closely associated with Fosbergia shweliensis, with Coffea as their close relative. Taken together, the complete cp genomes of GJG and GJR provided significant insights and important information that can be used to identify related species and reconstruct their phylogeny.
Gardenia jasminoides is an evergreen shrub, belonging to the Rubiaceae. It originates from the middle part of China, and it has traditionally been used as an ornamental and medicinal plant in several Asian countries (Debnath et al., 2011). The highly fragrant white flowers of gardenia are commonly borne singly in the leaf axils. The organs of this plant, such as flower, fruit, leaf, and root are scientifically proven to have numerous medicinal applications (Choi et al., 2007; Miyasita, 1976; Tseng et al., 1995). Due to the long history of their planting and broad covering areas, diverse germplasm resources of G. jasminoides have been developed, which provided bright prospects of application.
The Gardenia genus includes more than 200 species, with the most common being G. jasminoides. Two botanical varieties of G. jasminoides, G. jasminoides var. radicans (GJR) and G. jasminoides var. grandiflora (GJG), have been widely cultivated. GJG is a plant ≈2 m high, with 8-cm-wide leaves and double-petalled blossom. In contrast, GJR is typically a 0.5-m-high plant, with narrow leaves (1 cm wide) and small flowers. In comparison with GJR, GJG is more tolerant to cold stress. However, GJR possesses a large number of advantages, including long flowering period, easy to culture, and being appropriate for potting, thus, it is highly significant for standard production and transportation.
Because of its great medicinal and ornamental value, previous studies on G. jasminoides have mainly concentrated on characterizing properties of extracts from leaves and fruit. The information on phylogeny and adaptive evolution of GJR and GJG is limited. It is well known that cps in green plants are responsible for photosynthesis and providing energy (Douglas, 1998), and they are also involved in the biosynthesis of amino acids, fatty acids, vitamins, and pigments (Prabhudas et al., 2016). In recent years, it has been reported that the cp genome has several advantages in the study of the evolutionary biology of plants, owing to its genetic stability, genome structure, and a higher evolutionary rate in comparison with mitochondria (Downie and Jansen, 2015; Saina et al., 2018). With the rapid development of high-throughput sequencing technology, it has become feasible to accurately obtain complete cp genome data (Huang et al., 2014). These data proved to be valuable sources for studying phylogeny and adaptive evolution within diverse genera, such as Urophysa (Xie et al., 2018), Camellia (Huang et al., 2014), and Amomum (Cui et al., 2019).
G. jasminoides is adapted to divergent habitats, and it may form different genotypes due to environmental factors. Sequence-characterized amplified region markers, including ZZH11, ZZH31, ZZH41, and ZZH51, have been developed to specifically identify cultivars of G. jasminoides at the DNA level. Furthermore, ZZH31 could be used to distinguish GJG from other G. jasminoides botanical varieties. Recently, Zhao and Zhou (2020) and Wang et al. (2021) reported the complete cp genome sequences of wild-type G. jasminoides based on the materials collected from different regions of China. However, both studied only one G. jasminoides genotype without considering the botanical variety differences; the information on different botanical varieties and genotypes of G. jasminoides is still limited. In the present study, the complete cp genomes of two widely planted botanical varieties in middle China were sequenced. The objectives of this study were to 1) establish and characterize the organization of the complete cp genomes from GJG and GJR, 2) conduct comparative genomic studies by combining the whole cp genomes of other species of Rubiaceae from GenBank [National Center for Biotechnology Information (NCBI), Bethesda, MD], and 3) provide a theoretical basis for genetic breeding, as well as determining phylogenetic relationships of G. jasminoides with related species.
Materials and Methods
Plant materials and DNA extraction.
Fresh leaves of one plant of GJR and GJG were collected from the germplasm nursery of Xinyang Agriculture and Forestry University, Henan, China (lat. 32.117°N, long. 114.058°E). The total genomic DNA was extracted from mature fresh leaves (>1.0 g) through a modified ceyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle, 1987). Briefly, CTAB was augmented with 3% polyvinylpyrrolidone and 3% beta-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). Organic phase separation was repeated by centrifuging at 16,200 gn for 10 min until the aqueous fraction was clear. DNA pellets were resuspended in 200 μL DNase-free water, following by treating with RNase A (Thermo Fisher Scientific, Waltham, MA). The samples were then subjected to phase separation with chloroform, and DNA was recovered by ethanol precipitation. Finally, samples were resuspended in DNase-free water, and DNA quality was measured through electrophoresis in 1% agarose gel using a spectrophotometer (NanoPhotometer; IMPLEN, Duren, Germany).
Chloroplast genome sequencing and assembling.
A total amount of 1 μg DNA (per sample) was used as an input material for the DNA sample preparation. Sequencing libraries were generated using a library prep kit (NEBNext Ultra DNA Library Prep Kit; New England BioLabs Inc., Ipswich, MA) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Briefly, the DNA sample was fragmented to 300 bp by sonication; then, the inherited DNA fragments were end-polished, A-tailed, and ligated with the full-length adaptor for Illumina sequencing with further polymerase chain reaction (PCR) amplification. Last, PCR products were purified by magnetic beads (AMPure XP; Beckman Coulter Inc., Brea, CA), library-generated (for size distribution) by a bioanalyzer (model 2100; Agilent Technologies, Santa Clara, CA), and quantified by real-time PCR. The clustering of the index-coded samples was performed on a cluster generation system (cBot Cluster Generation System; Illumina Inc., San Diego, CA), according to the manufacturer’s instructions. After clustering, the prepared libraries were sequenced on an on an Illumina platform (NovasEq. 6000; Illumina Inc.) for sequencing and paired-end 150-bp reads were generated.
The quality of the raw paired-end reads was evaluated by FastQC v0.11.7 software (Anders and Huber, 2010). After quality assessment, the data were assembled into optimal contigs by NovoPlasty v2.7.0 (Dierckxsens et al., 2017) after multiple iterations with a k-mer of 33. Zea mays chloroplast gene for the large subunit of RUBP (GenBank No.: V00171.1) was used as the seed sequence. After that, cp genomes were annotated by PGA software (Qu et al., 2019) under default parameters (-i: 1000, -p: 40, -q: 0.5,2), and were corrected manually. The gene map was plotted using the OGDraw v1.2 online software (Lohse et al., 2007). Two complete cp genomes were deposited to the GenBank [GJR (accession no. MZ151501) and GJG (accession no. MZ151502)].
PCR primer design, amplification, and Sanger sequencing.
The sequences of ycf1 and psbC of GJG and GJR were amplified by PCR and sequenced by Sanger sequencing. The following primers were used: ycf1-F, 5'-CGGGCCGAGAAGATCTTAGA-3'; ycf1-R, 5'-CGACTGCCGTTATTGGTATCA-3'; psbC-F, 5'- CAATTTGACCCTTAGCCCAGG-3'; psbC-R, 5'-TCCCTTTTCAAATCCTGCTGC-3'. Multiple gene alignments were conducted by the program of DNAMAN 6.0 (Lynnon Biosoft, Pointe-Claire, QC, Canada).
Repeat sequences and SSRs.
The cp genome sequences of two reported botanical varieties of G. jasminoides (MW160432 and MN735462) and Fosbergia shweliensis (NC_050962) downloaded from the GenBank, together with two newly sequenced cp genomes, were used for repeat sequences and SSRs. Repeat sequences, including palindromic, forward, reverse, and complement repeats, were identified by the REPuter program (Kurtz et al., 2001). The following conditions for identification of repeat sequences within the cp genome were considered: 1) hamming distance equal to 3; 2) a minimal size of 30 bp; and 3) a sequence identity ≥ 90%. The SSRs in the complete cp genome sequences of four genotypes of G. jasminoides and F. shweliensis, which belongs to the Gardenieae tribe (Li et al., 2006), were identified using a Perl script called MISA (Thiel et al., 2003). For different lengths of SSRs, including mononucleotides, dinucleotides, trinucleotides, tetranucleotides, pentanucleotides, and hexanucleotides, minimum numbers (thresholds) were 10, 5, 4, 3, 3, and 3, respectively.
Sequence variation map and variations of inverted repeat sequences.
The sequence variation map was plotted by the mVISTA comparative genomics server (Frazer et al., 2004), with the annotation of GJR (MZ151501) as the reference. Variations of inverted repeat (IR) sequences, including expansion and contraction, were analyzed by the IRscope online program (Amiryousefi et al., 2018).
Phylogenomic reconstruction based on cp genomes.
The phylogenomic analysis was conducted based on two newly sequenced cp genomes of G. jasminoides, together with 15-cp genomes of the Rubiaceae family downloaded from the GenBank, including two botanical varieties of G. jasminoides, two species of Coffea, two species of Neolamarckia, two species of Ophiorrhiza, Fosbergia shweliensis, Ixora chinensis, Antirhea chinensis, Scyphiphora hydrophyllacea, Mussaenda hirsutula, Cinchona officinalis, and Uncaria rhynchophylla. The MAFFT (for multiple alignment using fast Fourier transform) program (Katoh et al., 2002) was used to align the sequences, and after that, the datasets were trimmed by the trimAl online tool (Capella-Gutiérrez et al., 2009). The best substitution model, GTR+G, was chosen in the jModelTest v2.1.7 software (Darriba et al., 2012), and the maximum-likelihood method was used to infer the phylogenetic relationship with 1000 bootstrap replicates in the MEGA 6.0 software (Kumar et al., 2008). Bayesian inference was performed using the MrBayes 3.2 software (Ronquist et al., 2012). Markov chain Monte Carlo analysis was conducted using 1 × 108 generations. Samples were taken every 1000 generations, and the first 25% were discarded as burn-in. When the average standard deviation of the splitting frequency is kept below 0.01, it is considered that the stationarity is achieved.
Results
The overall features of complete cp genomes.
The complete cp genomes of both G. jasminoides botanical varieties showed a single circular molecule with a quadripartite structure. The size of GJR and GJG cp genomes was 154,954 and 155,098 bp, respectively. The cp genomes consisted of a large single copy [LSC (85,270–85,411 bp)] region, and a small single copy [SSC (18,096–18,103 bp)] region, separated by a pair of IRs (IRa and IRb, with the length of 25,792–25,794 bp) (Table 1). The guanosine and cytosine (GC) content in the complete cp genome of GJR and GJG was less than 37.49%, which was similar in different genera of Rubiaceae (Table 1). In each species, the GC content of the IR region was higher than that in other regions, which was ascribed to the high GC content of four ribosomal RNA (rRNA) genes (Table 2) located in the IR regions (Fig. 1).
Gene map of the chloroplast genomes of Gardenia jasminoides var. radicans (GJR) and Gardenia jasminoides var. grandiflora (GJG). Genes shown outside the outer circle are transcribed clockwise, and those shown inside are transcribed counterclockwise. Genes belonging to different functional groups are color-coded. The darker gray color in the inner circle indicates the guanosine and cytosine (GC) content of the chloroplast genome, and the lighter gray color corresponds to the adenine and thymine (AT) content. SSU = small subunit; LSU = large subunit.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Gene map of the chloroplast genomes of Gardenia jasminoides var. radicans (GJR) and Gardenia jasminoides var. grandiflora (GJG). Genes shown outside the outer circle are transcribed clockwise, and those shown inside are transcribed counterclockwise. Genes belonging to different functional groups are color-coded. The darker gray color in the inner circle indicates the guanosine and cytosine (GC) content of the chloroplast genome, and the lighter gray color corresponds to the adenine and thymine (AT) content. SSU = small subunit; LSU = large subunit.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Gene map of the chloroplast genomes of Gardenia jasminoides var. radicans (GJR) and Gardenia jasminoides var. grandiflora (GJG). Genes shown outside the outer circle are transcribed clockwise, and those shown inside are transcribed counterclockwise. Genes belonging to different functional groups are color-coded. The darker gray color in the inner circle indicates the guanosine and cytosine (GC) content of the chloroplast genome, and the lighter gray color corresponds to the adenine and thymine (AT) content. SSU = small subunit; LSU = large subunit.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Statistics on the basic features of the chloroplast genomes of two Gardenia jasminoides botanical varieties and four Rubiaceae species.
Comparison of the size of chloroplast genes among two Gardenia botanical varieties and four Rubiaceae species.
The complete cp genomes of both GJR and GJG contained 132 and 133 genes, respectively (Table 3). The cp genome of GJR contained 87 protein coding sequences (CDSs), 37 transfer RNA (tRNA) genes, and 8 rRNA genes. Among them, 94 were unique genes and 19 were duplicated genes. The 94 unique genes contained 73 CDSs and 21 tRNA genes, and 8 tRNA genes, 4 rRNA genes, and 7 CDSs were duplicated. In comparison with GJR, two genes, trnP-GGG and ycf68, were unique in GJG, and rps19 was only found in GJR. In addition, 19 repeat genes (including all rRNA genes) were located in the IR region.
List of genes in the chloroplast genome of Gardenia jasminoides var. radicans and G. jasminoides var. grandiflora.
Repeat analysis.
Large repeats and SSRs were investigated in the cp genomes of two botanical varieties of G. jasminoides, together with F. shweliensis, which also belongs to the Gardenieae tribe. In the cp genomes of GJR, repeat analysis revealed 25 palindrome repeats, 15 forward repeats, nine reverse repeats, and one complement repeat. Among them, eight palindromic, five forward, and nine reverse repeats were found in a length of 10 to 20 bp. Moreover, 14 palindromic, nine forward, and one reverse repeats were detected in a length of 21 to 40 bp, and three palindromic and one forward repeats were noted in a length of more than 40 bp (Fig. 2). Similarly, 25, 23, 24 palindromic repeats; 15, 16, 17 forward repeats; 8, 10, 6 reverse repeats; and 2, 0, 0 complement repeats were detected in GJG, MN735462, and MW160432, respectively, and the details of length distribution of repeats are shown in Fig. 2. Although the number of each repeat type of F. shweliensis cp genomes was similar to that of G. jasminoides cp genomes, the length distribution of each repeat in NC_050962 cp genomes was distinct from that of G. jasminoides cp genomes. Importantly, the number of both palindromic and forward repeats in a length of 21 to 30 bp was more abundant in F. shweliensis cp genomes than that in cp genome of G. jasminoides. In contrast, lower palindromic and forward repeats in a length of 10 to 20 bp were detected in F. shweliensis cp genomes.
Analysis of repeated sequences in the chloroplast genomes of Gardenia jasminoides [G. jasminoides var. radicans (GJR) and G. jasminoides var. grandiflora (GJG)] and Fosbergia shweliensis. (A) A total of four types of repeats. (B) Frequency of the palindromic repeat by length. (C) Frequency of the forward repeat by length. (D) Frequency of the reverse repeat by length. The chloroplast genome sequences of F. shweliensis (NC_050962) and G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Analysis of repeated sequences in the chloroplast genomes of Gardenia jasminoides [G. jasminoides var. radicans (GJR) and G. jasminoides var. grandiflora (GJG)] and Fosbergia shweliensis. (A) A total of four types of repeats. (B) Frequency of the palindromic repeat by length. (C) Frequency of the forward repeat by length. (D) Frequency of the reverse repeat by length. The chloroplast genome sequences of F. shweliensis (NC_050962) and G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Analysis of repeated sequences in the chloroplast genomes of Gardenia jasminoides [G. jasminoides var. radicans (GJR) and G. jasminoides var. grandiflora (GJG)] and Fosbergia shweliensis. (A) A total of four types of repeats. (B) Frequency of the palindromic repeat by length. (C) Frequency of the forward repeat by length. (D) Frequency of the reverse repeat by length. The chloroplast genome sequences of F. shweliensis (NC_050962) and G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Four categories of SSRs (mono, di-, tri, and tetra-nucleotide repeats) were identified in the cp genomes of four genotypes of G. jasminoides. Penta- and hexa-nucleotide repeats were only detected in the cp genomes of F. shweliensis (NC_050962). The number of SSRs ranged from 31 (GJR) to 42 (GJG, MW160432, and NC_050962) (Fig. 3A). The most abundant SSRs were mononucleotide repeats, which accounted for 69.05% (NC_050962) to 78.57% (GJG and MW160432) of the total SSRs. Trinucleotide repeats were the least abundant in G. jasminoides, whereas penta- and hexa-nucleotide repeats were the least abundant in F. shweliensis. In GJR, GJG, and MW160432, all the mono- and dinucleotides were composed of adenine/thymine (A/T), and most mononucleotides (88.00% and 96.55%) were composed of A/T in MN735462 and NC_050962, respectively (Fig. 3B).
Analysis of simple sequence repeats (SSRs) in the chloroplast genomes of Gardenia jasminoides [G. jasminoides var. radicans (GJR) and G. jasminoides var. grandiflora (GJG)] and Fosbergia shweliensis. (A) The number of different SSRs detected in each species. (B) Type and frequency of each identified SSR. The chloroplast genome sequences of F. shweliensis (NC_050962) and G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Analysis of simple sequence repeats (SSRs) in the chloroplast genomes of Gardenia jasminoides [G. jasminoides var. radicans (GJR) and G. jasminoides var. grandiflora (GJG)] and Fosbergia shweliensis. (A) The number of different SSRs detected in each species. (B) Type and frequency of each identified SSR. The chloroplast genome sequences of F. shweliensis (NC_050962) and G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Analysis of simple sequence repeats (SSRs) in the chloroplast genomes of Gardenia jasminoides [G. jasminoides var. radicans (GJR) and G. jasminoides var. grandiflora (GJG)] and Fosbergia shweliensis. (A) The number of different SSRs detected in each species. (B) Type and frequency of each identified SSR. The chloroplast genome sequences of F. shweliensis (NC_050962) and G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
IR expansion and contraction.
The IR/LSC and IR/SSC junction regions of G. jasminoides and F. shweliensis were compared to indicate whether they have expansion or contraction. At the junction of LSC/IRb, SSC/IRa, and IRa/LSC regions, rps19, ycf1, and trnH genes were located respectively (Fig. 4). No gene was found at the junction of IRb/SSC, whereas the ndhF gene was located close to the line between IRb and SSC (JSB line). Although the length of IR regions in the four Gardenia genotypes was quite similar, ranging from 25,792 to 25,794 bp, some expansion and contraction was observed. The line between LSC and IRb (JLB line) intersected the rps19 gene. In four Gardenia genotypes, 245 bp of the rps19 gene located in LSC and 31 bp located in IRa with an exception of MN735462, whose rps19 gene locating in LSC and IRb were in the length of 248 and 31 bp, respectively. The distance between ndhF gene and JSB line was either 12 bp (GJG and MW160432) or 13 bp (GJR, MN735462, and NC_050962). The line between SSC and IRa (JSA line) transected the ycf1 gene, and the transection location was variable among species. Ycf1 gene located in SSC ranged from 4479 to 4520 bp, while it ranged from 1111 to 1152 bp in IRa. In comparison with MW160432 and MN735462, trnH gene located in LSC had 3 bp extending to the IRa region, resulting in an overlap of trnH gene and JLA line in GJR and GJG. The line between IRa and LSC (JLA line) intersected trnH gene in GJR, GJG, and NC_050962, and IRa only got 1 bp of trnH for GJR and GJG and got 3 bp for NC_050962. In addition, the distance between psbA gene and the JLA line ranged from 305 to 310 bp in the four cp genomes of Gardenia, whereas it was 285 bp in NC_050962.
Comparison of large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regional boundaries of chloroplast genomes between Gardenia jasminoides [G. jasminoides var. radicans (GJR) and G. jasminoides var. grandiflora (GJG)] and Fosbergia shweliensis. The chloroplast genome sequences of F. shweliensis (NC_050962) and G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD). JLB = junction line between LSC and IRb; JSB = junction line between IRb and SSC; JSA = junction line between SSC and IRa; JLA = junction line between IRa and LSC.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Comparison of large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regional boundaries of chloroplast genomes between Gardenia jasminoides [G. jasminoides var. radicans (GJR) and G. jasminoides var. grandiflora (GJG)] and Fosbergia shweliensis. The chloroplast genome sequences of F. shweliensis (NC_050962) and G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD). JLB = junction line between LSC and IRb; JSB = junction line between IRb and SSC; JSA = junction line between SSC and IRa; JLA = junction line between IRa and LSC.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Comparison of large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regional boundaries of chloroplast genomes between Gardenia jasminoides [G. jasminoides var. radicans (GJR) and G. jasminoides var. grandiflora (GJG)] and Fosbergia shweliensis. The chloroplast genome sequences of F. shweliensis (NC_050962) and G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD). JLB = junction line between LSC and IRb; JSB = junction line between IRb and SSC; JSA = junction line between SSC and IRa; JLA = junction line between IRa and LSC.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Variations of the cp genomes.
A sequence variation map of G. jasminoides and F. shweliensis was plotted by the mVISTA comparative genomics server, with the annotation of GJR as the reference (Fig. 5). Overall, the IR regions were less variable compared with LSC and SSC regions. The protein coding regions in relative species were highly conserved, thus, only rps16 and rpl16 genes were relatively divergent among four genotypes of G. jasminoides because of the intron regions, which could be used as potential molecular markers for plant identification in Rubiaceae. Single nucleotide polymorphisms (SNPs) were observed at coding regions of psbC and ycf1 genes among G. jasminoides. These SNPs were further confirmed by PCR of both genes and Sanger sequencing (Supplemental Fig. 1). The highly variable regions of Gardenia in cp genomes mainly appeared in intergenic spacers (IGSs), such as accD-psaI, ndhF-rpl32, and rpl32-ccsA. There was no significant difference in coding regions of rrn16, rrn23, rrn4.5, and rrn5 between G. jasminoides and F. shweliensis.
Comparison of chloroplast genomes via annotation of Gardenia jasminoides var. radicans (GJR) as a reference. The vertical scale indicates the percentage of identity, ranging from 50% to 100%. The horizontal axis indicates the coordinates within the chloroplast genome. Genome regions are color-coded as exons, introns, untranslated regions (UTRs), and conserved noncoding sequences (CNSs). The chloroplast genome sequences of Fosbergia shweliensis (NC_050962) and G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD). GJG = G. jasminoides var. grandiflora; k = kilobps.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Comparison of chloroplast genomes via annotation of Gardenia jasminoides var. radicans (GJR) as a reference. The vertical scale indicates the percentage of identity, ranging from 50% to 100%. The horizontal axis indicates the coordinates within the chloroplast genome. Genome regions are color-coded as exons, introns, untranslated regions (UTRs), and conserved noncoding sequences (CNSs). The chloroplast genome sequences of Fosbergia shweliensis (NC_050962) and G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD). GJG = G. jasminoides var. grandiflora; k = kilobps.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Comparison of chloroplast genomes via annotation of Gardenia jasminoides var. radicans (GJR) as a reference. The vertical scale indicates the percentage of identity, ranging from 50% to 100%. The horizontal axis indicates the coordinates within the chloroplast genome. Genome regions are color-coded as exons, introns, untranslated regions (UTRs), and conserved noncoding sequences (CNSs). The chloroplast genome sequences of Fosbergia shweliensis (NC_050962) and G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD). GJG = G. jasminoides var. grandiflora; k = kilobps.
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Phylogenetic analysis.
To achieve more clear phylogenetic information of Gardenia, we collected other 15 cp genomes of the Rubiaceae family from the NCBI database. GJR showed a close relationship with G. jasminoides (MN735462.1), whereas GJG was closely correlated with another genotype of G. jasminoides (MW160432.1). A new phylogenetic relationship was proposed, in which the cp genes of G. jasminoides showed the closest relationship with F. shweliensis (Fig. 6). The cp genes of G. jasminoides also showed a close relationship with Coffea, which included both C. canephora and C. arabica within the Rubiaceae. Another clade consisted of eight species of the Rubiaceae, and two species, S. hydrophyllacea and I. chinensis, did not gather together with other species of the Rubiaceae.
Phylogenetic tree of Gardenia jasminoides [GJR = G. jasminoides var. radicans; GJG = G. jasminoides var. grandiflora] and its related taxa based on the complete chloroplast genomes. The chloroplast genome sequences of Coffea arabica (EF044213), Coffea canephora (NC_030053), Fosbergia shweliensis (NC_050962), G. jasminoides (MN735462 and MW160432), Antirhea chinensis (NC_044102), Cinchona officinalis (MZ151891), Ixora chinensis (MZ221832), Mussaenda hirsutula (MK203878), Neolamarckia cadamba (NC_041149), Neolamarckia macrophylla (MN877388), Ophiorrhiza densa (MW683127), Ophiorrhiza pumila (MW528277), Uncaria rhynchophylla (NC_053701), and Scyphiphora hydrophyllacea (NC_049078) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Phylogenetic tree of Gardenia jasminoides [GJR = G. jasminoides var. radicans; GJG = G. jasminoides var. grandiflora] and its related taxa based on the complete chloroplast genomes. The chloroplast genome sequences of Coffea arabica (EF044213), Coffea canephora (NC_030053), Fosbergia shweliensis (NC_050962), G. jasminoides (MN735462 and MW160432), Antirhea chinensis (NC_044102), Cinchona officinalis (MZ151891), Ixora chinensis (MZ221832), Mussaenda hirsutula (MK203878), Neolamarckia cadamba (NC_041149), Neolamarckia macrophylla (MN877388), Ophiorrhiza densa (MW683127), Ophiorrhiza pumila (MW528277), Uncaria rhynchophylla (NC_053701), and Scyphiphora hydrophyllacea (NC_049078) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Phylogenetic tree of Gardenia jasminoides [GJR = G. jasminoides var. radicans; GJG = G. jasminoides var. grandiflora] and its related taxa based on the complete chloroplast genomes. The chloroplast genome sequences of Coffea arabica (EF044213), Coffea canephora (NC_030053), Fosbergia shweliensis (NC_050962), G. jasminoides (MN735462 and MW160432), Antirhea chinensis (NC_044102), Cinchona officinalis (MZ151891), Ixora chinensis (MZ221832), Mussaenda hirsutula (MK203878), Neolamarckia cadamba (NC_041149), Neolamarckia macrophylla (MN877388), Ophiorrhiza densa (MW683127), Ophiorrhiza pumila (MW528277), Uncaria rhynchophylla (NC_053701), and Scyphiphora hydrophyllacea (NC_049078) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Discussion
Chloroplast genome sequences provide global information for plant phylogenetic analyses and DNA chloroplast barcoding for accurate identification of plant species (Dong et al., 2017). Obtaining cp genome sequences may facilitate chloroplast transgenes that can be engineered to modify plant agronomic traits (Daniell et al., 2016). The present study reported the cp genomes of two botanical varieties of Gardenia and compared them with other species at various genetic distances of the Rubiaceae to attain an insight to further study the molecular evolution. The analysis of cp genome sequences of these species can broaden the knowledge about the evolution of G. jasminoides.
The structure of complete cp genomes of both G. jasminoides is similar to most plants, containing a single circular quadripartite molecule (Asaf et al., 2016; Kuang et al., 2011; Wang et al., 2017) (Fig. 1). The size of cp genomes of GJR and GJG was within the range of most angiosperm plastid genomes (Jansen et al., 2007). Previous studies reported that GC content varied among different regions of cp genomes and the IR regions contained high GC content because of the presence of rRNAs (Mehmood et al., 2020; Shahzadi et al., 2019). Our results have also shown similarities in GC content of each region (LSC, SSC, and IR) among five species (Table 1).
Chloroplast repeats are potentially significant genetic resources to study evolutionary and population genetics (Huang et al., 2017). SSRs, consisting of tandemly repeated multiple copies of mono, di-, tri, or tetranucleotide motifs, are ubiquitous in eukaryotic genomes and are frequently used as genetic markers, taking advantage of the length polymorphism (Powell et al., 1995). The number and proportion of SSRs vary among four genotypes of G. jasminoides, which is in accordance with previous report (Wang et al., 2021) (Fig. 3). In the cp genomes of Forsythia, the number of di-nucleotide repeats is the highest (Wang et al., 2017). Trinucleotide SSRs are the most abundant in Nicotiana species, accounting for ≈43.03% (Asaf et al., 2016). These results suggest that different repeats may contribute to the genetic variations among different species. Thus, the variations of SSRs identified in our study can be important for understanding the genetic diversity status of G. jasminoides and its relatives. In both newly sequenced cp genomes of G. jasminoides, all the mono- and dinucleotides were composed of A/T. It has been reported that SSRs found in the cp genomes are generally composed of poly-thymine (polyT) or poly-adenine (polyA) repeats and infrequently contain tandem cytosine (C) and guanine (G) repeats (Kuang et al., 2011), which is consistent with our findings. Therefore, these SSRs contributed to the AT richness of the cp genomes, as previously reported for other species (Chen et al., 2015; Kuang et al., 2011). The SSRs of four genotypes of G. jasminoides have abundant variations and could be used to detect genetic polymorphisms at intraspecific levels.
Expansion and contraction of boundaries of IR regions are the main factors for the size variations of the cp genomes and play a vital role in evolution of species (Raubeson et al., 2007; Wang et al., 2008). We observed certain similarities and differences in the junction of LSC, SSC, and IR among five compared species. Moreover, some genes, such as ycf1, rps19, and trnH, were shifted at the borders (Fig. 4). The expansion and contraction detected in the IR regions may act as the main mechanism in creating the length variation of the cp genomes in G. jasminoides and related species (Asaf et al., 2016; Yang et al., 2016).
The variations in cp genomes are important to understand the evolution and genome structure of the chloroplast (Kim et al., 2017). In the present study, the IR regions were less variable compared with LSC and SSC regions, which was consistent with the results of a previous report (Xie et al., 2018). As noncoding regions typically mutate faster than coding regions (Gielly and Taberlet, 1994), the highly variable regions of Gardenia in the cp genome mainly appear in IGSs, such as accD-psaI, ndhF-rpl32, and rpl32-ccsA. Several rRNA genes did not show a significant difference in coding regions between G. jasminoides and F. shweliensis (Fig. 5), which was consistent with the results of previous research, in which the sequences of rRNA genes were highly conserved (Shen et al., 2018). The nucleotide substitution in IGS and the intron region, as well as the pseudogenes that are not translated into proteins, are neutral or near-neutral (Akashi et al., 2012), and they are not influenced by natural selection. Therefore, a noncoding region acts as a good material for inferring the evolutionary history (Baker et al., 2000). Some coding regions, which have a relatively high sequence deviation, also have been shown to be good sources for the phylogenetic analysis of interspecies (Asaf et al., 2016; Chen et al., 2015). Highly variable regions in coding and noncoding regions together provide potential molecular markers of G. jasminoides for phylogenetic analysis and identification of botanical varieties.
Chloroplast genomes have been proven to be significant in reconstructing phylogenetic relationships and evolutionary history (Gitzendanner et al., 2018). In this study, we enriched the information on cp genomes of different botanical varieties and genotypes of G. jasminoides. GJR clustered closely to G. jasminoides (MN735462.1, Fig. 6), which was collected from Guangzhou province, located in the south of China. GJG showed a close relationship with another botanical variety of G. jasminoides (MW160432.1), which was grown in Zhejiang province, located in the middle east of China. The distribution of two botanical varieties was consistent with that reported previously (Fu et al., 2002). We reported for the first time that G. jasminoides showed the closest relationship with F. shweliensis (Fig. 6), which is understandable morphologically, as both species have yellow, white, and fragrant flowers. In terms of origin, both Gardenia botanical varieties, which we studied here, and F. shweliensis originated from China. The cp genomes of G. jasminoides also showed a close relationship with Coffea, which is consistent with the results of a previous study (Xu et al., 2020; Zhao and Zhou, 2020). Taken together, the phylogenetic analysis will increase our understanding of the evolutionary relationship among different species of the Rubiaceae.
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Supplemental Information
Alignments of ycf1 (A) and psbC (B) among four genotypes of Gardenia jasminoides. The ycf1 and psbC sequences of G. jasminoides var. grandiflora (GJG) and G. jasminoides var. radicans (GJR) were obtained by polymerase chain reaction and Sanger sequencing, whereas those of G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Alignments of ycf1 (A) and psbC (B) among four genotypes of Gardenia jasminoides. The ycf1 and psbC sequences of G. jasminoides var. grandiflora (GJG) and G. jasminoides var. radicans (GJR) were obtained by polymerase chain reaction and Sanger sequencing, whereas those of G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
Alignments of ycf1 (A) and psbC (B) among four genotypes of Gardenia jasminoides. The ycf1 and psbC sequences of G. jasminoides var. grandiflora (GJG) and G. jasminoides var. radicans (GJR) were obtained by polymerase chain reaction and Sanger sequencing, whereas those of G. jasminoides (MN735462 and MW160432) were downloaded from GenBank (National Center for Biotechnology Information, Bethesda, MD).
Citation: Journal of the American Society for Horticultural Science 147, 5; 10.21273/JASHS05225-22
