The chloroplasts genomic analyses of Caragana arborescens and Caragana opulens

doi:10.21203/rs.3.rs-3036665/v1

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Research Article

The chloroplasts genomic analyses of Caragana arborescens and Caragana opulens

https://doi.org/10.21203/rs.3.rs-3036665/v1

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published 09 Feb, 2024

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Background

Numerous species within the genus Caragana have high ecological and medicinal value. In this genus, however, species identification based on morphological characteristics is quite complicated; this issue can be resolved by analyzing the complete plastid genomes.

Results

We obtained the chloroplast genomes of two species using lllumina sequencing technology: Caragana arborescens and Caragana opulens, with lengths of 129,473 bp and 132,815 bp, respectively. The absence of inverted repeat sequences in the two species allowed them to be ascribed to the inverted repeat-lacking clade (IRLC). They comprise a total of 111 distinct genes (4 rRNA, 31 tRNA, and 76 protein-coding genes). In addition, 16 genes containing introns were identified in two genomes, the majority of which contained a single intron. C. arborescens and C. opulens were found to contain 129 and 229 repeats, as well as 277 and 265 simple repeats, respectively. The codon usage bias analysis revealed that the two Caragana species exhibit similar codon usage patterns. rpoC2-rps2, accD-cemA, rps18-clpP, rpoA-rpl36, and rpl2-rpl23 were identified as the five regions most likely to be mutated based on analysis of nucleotide diversity (Pi). Analysis of sequence divergence revealed that certain intergenic regions (matK-rbcL, psbM-petN, atpA-psbI, petA-psbL, psbE-petL, and rps7-rps12) are highly variable. Phylogenetic analysis showed that C. arborescens and C. opulens were related and clustered together as the other four Caragana species. And the genus Astragalus and Caragana were relatively closely related.

Conclusions

In our research, the chloroplast genomes of C. arborescens and C. opulens were sequenced and their genomic structural characteristics were compared. We have also confirmed that both plants lack IR regions, which resulted in unclear boundary analysis, and that two plants could be classified as IRLC. This study provides a foundation for future phylogenetic research and the development of molecular markers for Caragana plants.

Caragana arborescens

Caragana opulens

plastid genomes

comparative genomics

phylogenetic

Approximately 100 species of the genus Caragana, which belongs to the subfamily Papilionoideae of the family Fabaceae, are primarily found in arid and semiarid regions of Asia and Europe. The majority of plants in this genus can withstand adverse environmental conditions, including sterile soil, drought, cold, high temperatures, strong winds, and insect and disease damage[1]. China was home to a total of 66 species, 32 of which were endemic. In China, they were primarily found in areas of high altitude and harsh environments, such as shady and semi-shady terrain in the northwest, southwest, northeast, and north[2]. Caragana is a deciduous undershrub with extensive adaptability and strong stress resistance, and it is known to resist wind and fix sand[3]. In addition, the majority of them can fix nitrogen via nodules, thereby enhancing soil fertility, preventing dust cyclones, and preventing land desertification[4]. Caragana arborescens, also known as Siberian pea shrub, is typically found in Northeast China, North China, and Northwest China[5, 6]. It has a height of 4–5 meters. The species, which blooms in May with yellow flowers and pods that mature in midsummer, is typically used for foliage and garden decoration[7]. Caragana opulens is a shrub with a yellow corolla that thrives in the hills up to 3400 meters above sea level in North China, Northwest China, and Southwest China and is distributed throughout these regions[8]. In addition, previous research has demonstrated that numerous species of this genus possess outstanding pharmacological properties, including anti-cancer, anti-HIV, anti-rheumatoid arthritis, and hypertension[1, 9, 10]. Caragana arborescens has been documented in traditional Chinese medicine and is a significant Mongolian medicine used to treat pulmonary hemorrhage and rheumatism[1].

Current studies have revealed that the CP genome of Caragana plants contains only 14 reports, that the quantity of data available for analysis is extremely limited, that the phylogenetic relationship of Caragana plants is unclear, and that there are also questions regarding the classification of medicinal plants[2, 11–13]. Therefore, it is crucial to discover a precise and convenient method for identifying Caragana plants.

Researchers have improved their understanding of chloroplasts over the past decade, including their origin, structure, evolution, and genetic engineering[14–16]. The chloroplast (cp) is derived from the hypothesis of bacterial endosymbiosis in eukaryotes; it is the site of photosynthesis in plants and has its own DNA[17]. Cp possesses its own DNA (cpDNA) and genetic system, which exist as covalent double-stranded circular DNA[18, 19]. With the rapid advancement of sequencing technology, scientists have discovered that the chloroplast genome contains more effective molecular markers, which facilitates the precise identification of species. The chloroplast genome is optimal for molecular identification, phylogenetic, and species conservation research[20]. Unlike the nuclear genome, the mitochondrial genome is characterized by unisexual inheritance, a simple structure, and more gene copies[19, 21]. Typically, the chloroplast genome is maternally inherited in angiosperms[22]. Its structure is comparatively stable and consists of a large single copy (LSC) and a small single copy (SSC) region separated by two inverted repeats (IRs)[23]. It has been reported that the phenomenon of inverted repeat-lacking clade (IRLC) occurs in leguminous plants[24–28]. There have been reports of eight species of Caragana plants with IRLC[2, 12, 13, 29]. With the refinement of Caragana chloroplast genome data, Caragana will presumably represent a broad IRLC spectral system for scientific investigation. Moreover, the chloroplast genome sequence offers more secure information for the study of genetic relationships, phylogeny, and population genomics among closely related species[29–31].

In this study, the complete chloroplast genomes of C. arborescens and C. opulens were obtained using lllumina sequencing technology, and their structural properties and phylogenetic relationships were elucidated. The completion of this endeavor has enriched the chloroplast genomic database of Caragana, which is anticipated to serve as a foundation for systematic evolution research and the protection and utilization of Caragana's germplasm.

Chloroplast genome assembly and features

C. arborescens and C. opulens chloroplast genomes were sequenced using the Illumina Novaseq platform. According to the sequencing results, the chloroplast whole genome sequence was assembled at 129,473 (Fig. 1A)and 132,815 base pairs (Fig. 1B). Due to the loss of the IR region, neither of their chloroplast genomes have the typical tetrad structure of most angiosperm chloroplast genomes, and their length has been shortened accordingly. Nonetheless, their genetic structures are extremely comparable.

In the chloroplast genomes of C. arborescens and C. opulens, there were 111 unique genes, including 76 protein-coding genes, 31 tRNA genes, and 4 rRNA genes, and their respective GC contents were 34.30% and 34.71% (Table 1), indicating that the GC content between the two species was extremely similar. This paper compares and analyzes the chloroplast genome sequences of six species of Caragana plants lacking the IR region. According to the results, the total length of their sequences varied between 129,331 and 133,122 base pairs. Due to the absence of the IR region, the chloroplast genome length of C. korshinskii was the shortest, at only 129,311 bp, and that of C. rosea was the longest, at a total length of 133,122 bp. In addition, the number of genes in C. arborescens and C. opulens was one gene greater than that of other species (tRNA encoded by the trnN-GUU gene), whereas the number of protein-coding genes and rRNA genes was consistent among the six plants. In terms of gene content, the number of protein-coding genes was the highest among the six species, comprising approximately half of the full-length genome, followed by the number of tRNA genes, whose length was shorter than that of other genes. C. rosea has the highest GC content in its chloroplast genome, at 34.84 percent, followed by C. kozlowii (34.5 percent), and C. microphylla, which has the lowest GC content, at 34.2 percent. We also examined the variations in GC concentration between the three gene types. The GC concentration of rRNA was over 50%, which was high and stable, followed by tRNA, and the GC content of protein-coding genes was approximately 37%. In conclusion, the sequence length and gene number of the chloroplast genomes of the six Caragana species were generally consistent, and the average GC content of the species was approximately 34%, which suggests that the evolution of the Caragana genus was relatively conservative.

Table 1

Summary of complete chloroplast genomes for six *Caragana* species.
Plastome Characteristics		Caragana arborescens	Caragana opulens	Caragana kozlowii	Caragana rosea	Caragana microphylla	Caragana korshinskii
Protein Coding gennes	Length(bp)	66,222	66,333	66,234	66,243	66,231	66,231
	GC(%)	36.89	37.01	37.03	37.13	36.88	36.88
	Length(%)	51.15	50.0	50.45	49.76	50.94	51.21
	Number	76	76	76	76	76	76
tRNA	Length(bp)	2,379	2,370	2,285	2,359	2,370	2,379
	GC(%)	52.74	52.83	53.15	52.73	53.14	53.05
	Length(%)	1.83	1.80	1.74	1.77	1.82	1.83
	Number	31	31	30	30	30	30
rRNA	Length(bp)	4,522	4,520	4,521	4,537	4,520	4,520
	GC(%)	54.8	54.56	54.75	54.77	54.82	54.82
	Length(%)	3.49	3.40	3.44	3.4	3.48	3.49
	Number	4	4	4	4	4	4
Total	Length(bp)	129473	132815	131274	133122	130029	129331
	Number Of genes	111	111	110	110	110	110
	GC(%)	34.3	34.71	34.5	34.84	34.26	34.36

Comparable to other species, the chloroplast genomes of C. arborescens and C. opulens encode three categories of genes (Table 2). Self-replication was associated with 57 genes. 3 subunits (large, small, and DNA-dependent RNA polymerase), including ribosomal RNA genes, transporting RNA genes, and encoding chloroplast RNA polymerase; 44 photosynthesis-related genes; other genes and unknown genes. In the chloroplast genomes of C. arborescens and C. opulens, 16 genes with introns were detected, of which one gene, ycf3, had two introns, and the remaining 15 genes (trnK-UUU, trnV-UAC, trnL-CAA, rpoC1, atpF, trnG-UCC, clpP, petB, petD, rpl16, rpl2, ndhB, trnI-GAU, trnA-UGC, ndhA) had only one intron (Table 3). Among these 16 intron-containing genes, the intron lengths of the two genes were remarkably similar.

Table 2

Genes in the chloroplast genome of *Caragana* species.
Category	Group of genes	Name of genes
Self-replication	Proteins of large ribosomal subunit	rpl14, rpl16, rpl2*, rpl20, rpl23, rpl32, rpl33, rpl36*
	Proteins of small ribosomal subunit	rps11, rps12, rps14, rps15, rps18, rps19, rps2, rps3, rps4, rps7, rps8*
	Subunits of RNA polymerase	rpoA, rpoB, rpoC1, rpoC2*
	Ribosomal RNAs	rrn16, rrn23, rrn4.5, rrn5
	Transfer RNAs	trnA-UGC, trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnG-UCC, trnH-GUG, trnI-CAU, trnI-GAU, trnK-UUU, trnL-CAA, trnL-UAA, trnL-UAG, trnM-CAU, trnN-GUU(2), trnP-UGG, trnQ-UUG, trnR-ACG, trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-GGU, trnT-UGU, trnV-GAC, trnV-UAC, trnW-CCA, trnY-GUA, trnfM-CAU
Photosynthesis	Subunits of photosystem I	psaA, psaB, psaC, psaI, psaJ
	Subunits of photosystem II	psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ
	Subunits of NADH dehydrogenase	ndhA, ndhB, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK
	Subunits of cytochrome b/f complex	petA, petB, petD*, petG, petL, petN*
	Subunits of ATP synthase	atpA, atpB, atpE, atpF, atpH, atpI*
	Large subunit of rubisco	rbcL
Other genes	Maturase	matK
	Protease	clpP
	Envelope membrane protein	cemA
	Acetyl-CoA carboxylase	accD
	c-type cytochrome synthesis gene	ccsA
Unknown	Conserved hypothetical chloroplast ORF	ycf1, ycf2, ycf3*, ycf4*
Notes: Gene:Gene with one introns; Gene*:Gene with two introns; Gene(2):Number of copies of multi-copy genes.

Table 3

The intron-containing genes and the length of exons and introns in the chloroplast genomes of two *Caragana* species
Species	Gene	Exon I(bp)	Intron I(bp)	Exon II(bp)	Intron II(bp)	Exon III(bp)
C.arborescens	trnK-UUU	37	2488	29
	trnV-UAC	39	574	37
	trnL-CAA	37	550	50
	ycf3	126	713	228	877	153
	rpoC1	432	789	1623
	atpF	168	660	411
	trnG-UCC	23	682	49
	clpP	368	701	229
	petB	6	818	642
	petD	8	717	475
	rpl16	9	1054	399
	rpl2	396	685	435
	ndhB	723	685	762
	trnI-GAU	38	951	35
	trnA-UGC	38	812	35
	ndhA	552	1170	540
C.opulens	trnK-UUU	37	2485	29
	trnV-UAC	39	574	37
	trnL-CAA	37	534	50
	ycf3	126	702	228	875	153
	rpoC1	432	790	1623
	atpF	168	679	411
	trnG-UCC	23	682	49
	clpP	368	1159	223
	petB	6	826	642
	petD	8	720	475
	rpl16	9	1108	399
	rpl2	396	692	435
	ndhB	723	685	762
	trnI-GAU	38	953	35
	trnA-UGC	38	810	35
	ndhA	552	1169	540

Analyses of repetitive sequences and SSRs

Repeat sites are important in genomic evolution, such as in structural rearrangement and size-based evolution [32, 33]. In this study, we identified the repetitive sequences in the chloroplast genomes of C. arborescens and C. opulens and analyzed their content. The results indicated that the chloroplast genome with a repeat length greater than or equal to 30 bp contained four categories of repeats: forward (F), palindromic (P), reverse (R), and complementary (C) repeats. In the two plants, 129 (length range: 30–249 bp) and 229 (length range: 30–472 bp) repeats, respectively, were identified (Table S1). The length range of 30–49 bp sequences had the highest frequency among all classes of repetitive sequences (former: 68.22%, latter: 52.40%).

Structural analysis of the repetitive sequences showed that C. arborescens was composed of 85 forward repeats (65.89%), 36 palindromic repeats (27.91%), 7 reverse repeats (5.43%), and 1 complementary repeat (0.78%) (Fig. 2A, Fig. 2C), while there are no complementary repeats in the repeat sequence of C. opulens, which consists of three repeat types, including 165 forward repeats (72.05%), 62 palindromic repeats (27.07%), and 2 reverse repeats (0.87%) (Fig. 2B, Fig. 2C). The majority of repeat sequences exist in the IGS region, and the majority of them are forward repeats.

Numerous simple sequence repeats (SSRs) are present in the chloroplast genome of plants. This form of sequence is transmitted from parents to offspring. It has a relatively basic structure and low variability. SSRs are therefore more efficient molecular markers [34]. Using the software MISA v1.0, we identified a total of 18 varieties in the two Caragana plants. The chloroplast genomes of C. arborescens and C. opulens contain 277 and 265 SSR loci, respectively (Table S2). The proportion of mononucleotide in the two Caragana plants with the highest concentration were 57.04 and 63.40 percent, respectively. While dinucleotide and trinucleotide repeat sequences comprised 7.58 and 29.24 percent of the former, tetranucleotide repeat sequences comprised the smallest proportion (6.14 percent). In the latter, the proportions of dinucleotide, trinucleotide, and tetranucleotide repeat sequences were 4.91 percent, 28.68 percent, and 2.64 percent, respectively, while pentanucleotide represented the smallest proportion, 0.38 percent.

C. arborescens has the longest SSR on the ycf1 gene of the chloroplast genome, which is a single nucleotide repeat sequence (A) with a length of 46 bp, whereas C. opulens has the longest SSR, which is a mononucleotide (T) with a length of 26 bp (Table S3). In addition, the distribution of SSRs in coding and noncoding regions was analyzed. Figure 3A displays that the number of SSRs in the protein-coding region was significantly lower than in the non-coding region. The majority of these SSRs were A/T single nucleotide repeats; 158 and 167 of the two Caragana species contained A/T, while only one contained C/G(Fig. 3B, Fig. 3C). Similarly, the majority of dinucleotide repeats consist of AT/AT, resulting in a deviation in base composition, which is consistent with the finding that the overall AT content of plastids is greater than the GC content[35].

Codon usage bias analysis

In the evolution of biology, plastids exhibit a prevalent codon usage bias. By analyzing codon usage bias, which may penetrate the phylogenetic relationship between bionts and the molecular phylogeny of genes[36], it is possible to study the origin, mutation model, and evolution of species. We have analyzed the codon distribution conditions in all protein-coding genes in these two plants. The 76 protein-coding gene sequences of the two Caragana species were used to generate 12,812 codons in total. Leucine (Leu) was the amino acid with the highest content, accounting for 10.58% and 10.65%, respectively, followed by codons encoding isoleucine (Ile) (9% and 8.89%), while cysteine (Cys) had the lowest abundance among the two plants (Table S4).

In the meantime, we also independently calculated the relative synonymous codon usage (RSCU) values, using which we determined the codon usage bias of the two plants' chloroplast genomes (Fig. 4). When the RSCU value is greater than one, the codon is considered optimal. Among the 31 codons with RSCU values greater than 1, the AUG codon encoding methionine had the highest utilization bias (C. arborescens RSCU: 2.99 (Fig. 4A), C. opulens RSCU: 2.98 (Fig. 4B)). Tryptophane had no codon usage bias among these 31 codons (only one codon). Except for UUG, which encodes leucine, and AUG, which encodes methionine, the remaining codons terminated in A (12) or U (16) (Table S4).

Sequence divergence analysis

Previous research has demonstrated that highly variable loci in the plastid genome can be used to investigate molecular markers [13]. Therefore, the software DNAsp6 [37] was used to calculate the nucleotide diversi (Pi) in order to identify highly variable regions in the chloroplast genomes of C. arborescens and C. opulens. According to the results of sliding window analysis, the Pi values of the two plants ranged from 0 to 0.05516, with an average value of approximately 0.0067655 (Fig. 5), indicating that the chloroplast genome sequences of the same genus have few distinctions and a high degree of similarity. rpoC2-rps2, accD-cemA, rps18-clpP, rpoA-rpl36, and rpl2-rpl23 were determined to be the most probable highly variable regions based on the pi values of 111 different genes. Furthermore, the rpoA-rpl36 region has the highest pi value, followed by the rps18-clpP region.

To demonstrate the distinct chloroplast genome sequence levels in C. arborescens and C. opulens. Caragana, including C. arborescens, C. opulens, C. kozlowii, C. rosea, C. microphylla, and C. korshinskii, had their whole plastid genome sequences compared to that of C. jubata (Fig. 6). Extremely low sequence divergence among species suggests that the CP genome was more conservative. IGS (matK-rbcL), IGS (psbM-petN), IGS (atpA-psbI), IGS (petA-psbL), IGS (psbE-petL), and IGS (rps7-rps1 2) contain significant differences among Caragana plants. Additionally, the majority of protein-coding regions were highly conserved, with the exception of a few (accD, ycf2, and rps7). This indicates that IGS is responsible for the accelerated evolution of Caragana species.

Phylogenetic development analysis

For determining the phylogenetic position of Caragana in the family Fabaceae, we have made multiple sequence matches using 86 protein sequences commonly found in 23 plastids. Except for the genus Caragana, the remaining 8 genus included Wisteria(1), Glycyrrhiza(2), Astragalus(1), Calophaca(1), Cicer(1), Medicago(3), Trifolium(3), and Lathyrus(4). Numbers in brackets indicate the number of species in the relevant group.

Based on the chloroplast genomes of 22 Fabaceae and Arabidopsis thaliana (outgroup), phylogenetic trees were constructed by Bayesian and maximum likelihood methods. The similar topology of the phylogenetic trees acquired by the two methods. Phylogenetic analysis revealed that all samples were classified into three main branches. Undoubtedly, C. arborescens and C. opulens were related and clustered together as the other four Caragana species, with a bootstrap value of 100%(Fig. 7). The two pairs showed a closer relationship: C. microphylla and C. korshinskii, and C. opulens and C. rosea. From the results, it is noteworthy that the genus Astragalus and Caragana were relatively closely related(bootstrap:100%) and categorized into Subtrib. Astragalinae. This result was consistent with previous studies[2].

Caragana is one of the superb forages native to northwest China and certain plateau regions, and it has significant application value for the enhancement of natural pastures and the establishment of forage bases. Due to their adaptability to drought conditions, Caragana plants are extensively cultivated due to their resistance to drought, aridity, cold, and heat. Using the Illumina platform, we sequenced the complete CP genomes of two Caragana plants for this study. By assembling and annotating these genomes, more detailed information was obtained. Two plastids ranged in size from 129,473 to 132,815 (bp)base pairs and were found in C. arborescens and C. opulens, respectively. Other Caragana have comparable gene structures. In some species, the chloroplast genome reportedly lacks ycf2, rpl23, and accD [38–40], whereas these genes were present in Caragana. Throughout plant evolution, several genes have been lost from the plastid genome. Previous research had demonstrated that the rpl22 and infA genes were lost in some or all legumes [41], that infA is an abnormally unstable flowering plant cp gene, and that rpl22 is a gene encoding ribosomal protein CL22 that was lost in cpDNA and relocated to the nucleus [42, 43]. Similarly, the infA and rpl22 loci in C. arborescens and C. opulens were also deleted in this study. Recent research has demonstrated, however, that the infA gene was present in the chloroplast genomes of C. jubata, C. erinacea, and C. bicolor[2]. This phenomenon suggests that infA genes may not be lost or transferred to organelles in some Caragana plants. Due to the limited quantity of chloroplast genome data in Caragana plants, this conclusion must be confirmed through an extensive number of experimental studies.

As with the majority of plant species, the plastids of two Caragana species are conserved and no rearrangements have occurred. Multiple Caragana species, such as C. microphylla, C. erinacea, and C. intermedia, have reportedly lost their IR region [2, 11, 12]. Similarly, the CP genomes of C. arborescens and C. opulens examined in this study lacked the IR region, and the two plants share a high degree of similarity in terms of genomic structure, gene deletion, genomic size, gene types, repeat sequence distribution, etc. Moreover, cpDNA G/C content is a key determinant of inter-specific affinity [2], and the DNA G/C content of the two Caragana species discussed in this paper is extremely similar. Numerous repetitive sequences were identified in the plastid genomes of two plants. These sequences are significant genetic markers and are intimately associated with the origin and evolution of species[44]. Four types of repeats were identified in this study: complement repeats (C), reverse repeats (R), forward repeats (F), and palindromic repeats (P). The dispersed repeats were longer than 30 base pairs, and the repeat sequence length of two Caragana plants varied between 30 and 472 base pairs. In addition, SSRs are regarded as essential molecular markers for population variation research and are widely employed to assess genetic diversity, phylogenetic relationships, and evolution [45]. In total, the CP genomes of two Caragana plants contain between 265 and 277 SSRs with a significant A/T bias. In this study, it was determined that the majority of SSR types are single nucleotide repeats and that non-coding regions (IGSs) contain the most SSRs. Similar circumstances have been observed in other Caragana species, including C. rosea, C. microphylla, C. korshinskii, and C. kozlowii [27]. These repeat sequences provide a crucial starting point for the development of genetic indicators for Caragana species and can be utilized for phylogenetic and ecological research.

It is known that the codon utilization preference mirrors the species of origin and the mutational model. The study of codon bias patterns in chloroplast genomes can shed light on plant phylogenetic relationships, gene expression mechanisms, and molecular evolution [36]. Leucine (Leu) is the most abundant amino acid in C. arborescens and C. opulens (mean of 1969), and the same trend has been observed in other Caragana species. In addition, our research revealed that the majority of synonymous codons preferred for RSCU values terminated in A/U, resulting in a high AT content in the gene. Based on the preceding analysis, we infer that natural selection and gene mutation may be to blame. Codon preference and utilization patterns reflect the evolutionary relationship between species to a limited extent [46], but additional research is required.

Meanwhile, we identified five intergenic spacer regions (IGSs) with relatively high differentiation values (pi > 0.01037): rpoC2-rps2, accD-cemA, rps18-clpP, rpoA-rpl36, and rpl2-rpl23. In addition, fragmentary sequences of the ycf1, rps3, and rps7 genes exhibited comparatively high nucleotide diversity. These variable regions could potentially function as DNA barcode labels for phylogenetic relationships, species recognition, and population genetics research [47–49]. The sequence variations of six assembled Caragana plants were then compared. Analysis of the comparative CP genomes revealed that the code regions were more conserved than the untranslated regions, corroborating findings from other Caragana species.

The phylogenetic analysis of 23 Fabaceae species simultaneously revealed the phylogenetic position of Caragana and the relationship between Caragana and closely related species. The evolution of the plastid genome (nucleotide changes and structural changes) has been elucidated by advances in phylogenetic analysis [50, 51], according to previous research. Caragana species were monophyletic, and C. arborescens, C. opulens, C. kozlowii, C. rosea, C. microphylla and C. korshinskii were distinguishable from other species. Our findings may serve as a guide for future research into the phylogenetic evolution of Caragana plants and the creation of novel molecular markers. Our findings augment the chloroplast genome database of the Caragana genus.

In this study, we constructed phylogenetic relationships based on the chloroplast genome sequences of C. arborescens, C. opulens, and 23 Legume species belonging to IRLC. The long repeats, SSRs loci, codon usage bias and five hypervariable regions identified in our paper are helpful for future works, such as the development of new molecular markers, population genetics and phylogenetic analysis. We deeply analyzed sequences and structure information of the chloroplast genomes of two Caragana plants, as well as the genetic evolution position and evolutionary relationship with other genus Caragana, which provided a data basis for more in-depth and comprehensive study of Caragana species identification, genetic diversity and phylogenetic research. At the same time, the chloroplast genome database of Caragana plants were also effectively enriched.

DNA Extraction, library construction, and sequencing

C. arborescens and C. opulens leaves were collected in Qinghai Province (China) at the following coordinates: C. arborescens: 36°43′24.80′′N, 101°44′54.11′′E; C. opulens: 37°36′53.34′′N, 10°19′18.63′′E. The improved cetyltrimethylammonium bromide (CTAB) method was used to extract whole genome DNA from fresh leaves of Caragana plants [52]. The sample's genomic DNA needs to be evaluated. When the test was successful, the mechanical damage method was utilized to ultrasonically fragment the sample DNA, purify the genomic DNA, and stop the repair process. The DNA fragment size was subsequently determined using agarose gel electrophoresis, and the sequencing library was generated using PCR amplification. The qualified library was sequenced utilizing the Illumina Novaseq platform, with 150 bp pair-end reads.

Gene annotation and sequence analyses

This study utilized Trimmomatic [53]to remove low-quality data from the original data. The chloroplast genome sequence was then assembled using SPAdes [54]software to obtain its seed sequence, Kmer analysis of the seed sequences to obtain the contigs, and SSPACE v2 [55] software to connect the contigs and obtain the scaffold sequences. Gapfiller v2.1.1 [56] software was used to supplement the GAP found in the scaffold sequence to assure the integrity of the pseudogenome sequence. After adjusting the corrected pseudogenome sequence, complete CP genome sequences of C. arborescens and C. opulens were ultimately obtained. Blast(https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to derive the CP genome annotation results of the two plants. Hmmer (http://www.hmmer.org) and ARAGORN [57] (http://ogdraw.mpimp-golm.mpg.de/index.shtml) were used to obtain the annotation information for rRNA and tRNA, respectively. OGDRAW [58] (http://ogdraw.mpimp-gol.m.mpg.de/index.html)was used to plot the CP genome maps of C. arborescens and C. opulens. MT211962, OQ656872 are the NCBI accession numbers for the whole chloroplast genomic data reported in this investigation.

Repeat structure, SSRs and codon usage analysis

Vmatch Web [59] identified repeats (forward repeats, palindromic repeats, reverse repeats, and complementary repeats). MISA [60] software was used to identify SSRs in two Caragana plants with the following search parameters: mononucleotides set to ≥ 10 repeat units, dinucleotides ≥ 8 repeat units, trinucleotides, tetranucleotides, pentanucleotides and hexanucleotides ≥ 3 repeat units. CodonW1.4.2 calculated the relative synonymous codon usage (RSCU) values of protein-coding genes using the default settings.

Comparative genome analysis

C. arborescens and C. opulens whole cp genome sequences were compared with those of C. kozlowii, C. rosea, C. microphylla, and C. korshinskii using mVISTA (shuffle-lagan mode). As a reference, C. jubata plastid was labeled. 111 gene sequences between C. arborescens and C. opulens were aligned using MEGA7 [61]. To calculate the nucleotide diversity (Pi) values with the software DnaSP6, the following parameter configurations are utilized: Normal parameters, 200-bp step size and 600-bp window length, appeared in the document [62].

Phylogenetic analysis

Phylogenic tree was established using plastome sequences of 20 species (pertaining to IRLC)downloaded from NCBI database and two species sequenced in this study, with Arabidopsis as an outgroup. All 23 complete chloroplast genomes were aligned using the tool MAFFT(default parameters), and the aligned sequences were optimized using MACSE. Bayesian inference (BI) and maximum likelihood (ML) methods were used to construct phylogenetic trees. MrBayes was used for BI analysis, with the model selected using Modelfinder [63] and the nucleotide substitution model set to GTR + F + I + G4. The MrBayes analysis was set to run for 1,000 cycles, and the first 25% of the cycles were removed as burn-in. The average standard deviation of splitfrequencies was set to > 0.01 [64]. IQ-TREE was used for ML analysis, with the automatic partitioning module and bootstrap analysis set to 1,000 repetitions to evaluate the confidence of the branches.

Acknowledgments

This research was funded by Innovation Platform for the Development and Construction of Special Project of Qinghai Province (Grant Number 2021-ZJ-T05) and National Natural Science Foundation (No. 32160386) of China.

Authors’ contributions

LiE Liu was the study's experimental designer and executor, completing data analysis and writing the first draft of the paper. HongYan Li, JiaXin Li, and XinJuan Li all contributed to the experimental design and analysis of the results. Na Hu and Honglun Wang assisted in sample collection and species identification. Wu Zhou was the project developer and leader, guiding the experimental design, data analysis, and paper writing and revision. The final text has been read and approved by all authors.

Funding

Innovation Platform for the Development and Construction of Special Project of Qinghai Province (Grant Number 2021-ZJ-T05) and National Natural Science Foundation (No. 32160386) of China.

Availability of data and materials

The original sequencing data have been submitted to the NCBI database and received GenBank accession numbers MT211962(C. arborescens), OQ656872(C. opulens). The data used in this study are already entirely in the public domain (https://www.ncbi.nl m .nih.gov).

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Author details

Address: ¹ School of Ecological and Environmental Engineering, Qinghai University, Xining, China, ² Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810008, China.

Email: LiE Liu - [email protected]; HongYan Li - [email protected]; [email protected]; XinJuan Li - [email protected]; Na Hu - [email protected]; Honglun Wang - [email protected]; Wu Zhou* - [email protected]

* Corresponding author.

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The chloroplasts genomic analyses of Caragana arborescens and Caragana opulens

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Abstract

Background

Results

Conclusions

Figures

Background

Results

Chloroplast genome assembly and features

Codon usage bias analysis

Sequence divergence analysis

Phylogenetic development analysis

Discussion

Conclusions

Materials and methods

DNA Extraction, library construction, and sequencing

Gene annotation and sequence analyses

Repeat structure, SSRs and codon usage analysis

Comparative genome analysis

Phylogenetic analysis

Declarations

References

Additional Declarations

Supplementary Files

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Journal Publication

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