Complete chloroplast genome sequence of Buchanania latifolia: genome structure, and phylogeny of basal Anacardiaceae family relationships

doi:10.21203/rs.3.rs-4668030/v1

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Complete chloroplast genome sequence of Buchanania latifolia: genome structure, and phylogeny of basal Anacardiaceae family relationships

https://doi.org/10.21203/rs.3.rs-4668030/v1

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The chloroplast (cp) genomes are valuable resource with multiple applications, encompassing species identification, phylogenetic reconstruction, and evolutionary investigations. In this study, the complete chloroplast genome sequence of Buchanania latifolia was de novo sequenced, assembled and annotated. The chloroplast genome of B. latifolia exhibits a typical quadripartite structure, with a total length of 160,088 bp, containing 88 protein-coding sequences (CDS), 37 tRNA genes, and 8 rRNA genes, with an overall GC content of 37.7%. A total of 99 SSR loci and 63 repeat sequences were identified, which can be utilized for marker development, phylogenetic and population studies of B. latifolia. Codon usage analysis revealed a preference for Leu codons ending with A/U. Additionally, the study investigated IR boundaries, DNA polymorphism, positive selection suites, and phylogenetic position. Comparative analysis with five other species from the Anacardiaceae family confirmed the nearly identical and highly conserved chloroplast genome features of B. latifolia, which can be valuable for understanding the plastid evolution and evolutionary relationships within Anacardiaceae. Phylogenetic analysis reveals that B. latifolia is positioned at the base of Anacardiaceae, sister to Choerospondias axillaris, Lannea coromandelica, and Sclerocarya birrea. These findings could provide important genetic information for further research into breeding of Anacardiaceae, phylogeny, and evolution of B. latifolia.

Buchanania latifolia Roxb.

Anacardiaceae

Chloroplast genome

SSR loci

Phylogeny

Buchanania latifolia Roxb. also known as Chironji, is a tree species of Anacardiaceae (Weber, 2018), native to the eastern Himalayas, Southwest China, and part of Southeast Asia. It's valued for its edible fruits and oil. As a pioneer species, it is commonly found in hot-dry valleys and forests in Yunnan, China. Initially named Coniogeton arborescens, it was reclassified under the genus Buchanania by Blume in 1850 (Parveen and Ilyas, 2021). The genus Buchanania, with B. latifolia as a notable species, is considered to occupy a basal position in the second ancestral clade of the Anacardiaceae family. This genus comprises approximately 25 species predominantly found in the tropical regions of Asia and Oceania. However, prior to this study, no plastid genome data of the genus Buchanania were available for phylogenetic reconstruction, which could significantly enhance our understanding of phylogenetic relationship, divergence time and biogeographic patterns of these basal clades within the Anacardiaceae family.

The next-generation sequencing (NGS) technology is generating vast amounts DNA data including both nuclear and organelle genomes. The chloroplast genome (cp genome), with its self-replication mechanism, relatively independent evolution, appropriate natural evolutionary rate, and unique maternal inheritance is typically highly conserved in terms of genome size, structure, gene content, and gene types in most terrestrial plants (Park and Lee, 2016, Li et al., 2019a). These conserved and divergent gene features provide crucial information for identifying, classifying, and reconstructing phylogenetic relationships between species and families (Jansen et al., 2007). In this study, the chloroplast genome of B. latifolia was sequenced, assembled, annotated and uploaded to NCBI database. The key features of the chloroplast genome were analyzed, including simple sequence repeat (SSR) loci and optimal codon usage boundaries.A plastid phylogenomic analysis was conducted to examine the phylogenetic relationships among the basal Anacardiaceae taxa. The publication of this plastome will widen our edge of knowledge for the applications both of DNA barcoding and for the biogeographic investigations of this tropical distributed woody clade of Angiosperm.

Plant material, sequencing, and genome assembly

Fresh leaves of the Buchanania latifolia were collected from Xinping County, Yunnan Province, China. (102°7′41.75″E, 24°1′54.84′N). The voucher specimen (yue2021006) was deposited in Wetland College of Southwest Forestry University, Kunming, China (email: [email protected]). Total DNA was extracted from fresh leaves using the E. Z. N. A ® HP Plant DNA Kit (Omega Bio-tek, GA, United States) following the manufacturer’s protocol. Shotgun libraries (350 bp) were constructed using a TruSeq DNA Sample Prep Kit (Illumina, United States). Next-generation sequencing (NGS) was conducted on an Illumina nova-seq 6000 sequencing platform (Illumina, CA, United States), from which approximately 5 Gb of raw NGS data was obtained. Raw reads were filtered using the NGS QC Toolkit (Patel and Jain, 2012) to remove low quality reads and to trim off the sequence adaptors. The filtered reads were then fed into the GetOrganelle (Jin et al., 2020) pipeline for de novo genome assembly. The Chloroplast genome of B. latifolia was annotated using Plastid Genome Annotator (PGA) (Qu et al., 2019), using the platome of Mangifera indica (GenBank accession number: NC_035239) as a reference. As for the annotated results, the start & stop codons were checked manually, and the intron/exon boundaries of protein-coding genes were checked by using the Geneious Prime (Kearse et al., 2012). The chloroplast genome maps were drawn using CPGview (http://www.1kmpg.cn/cpgview/). Finally, the annotated chloroplast genome sequence was submitted to GenBank with an accession number of OM000214.

Genome structure analysis

The Geneious software was used to export the types and numbers of annotated genes in the cp genome into a CSV file (Kearse et al., 2012). Geneious was also used to identify the LSC, SSC, and IR regions in each cp genome sequence and to calculate the AT and GC content.

Codon preference analysis

The CDS coding sequences of the B. latifolia cp genome were extracted using Geneious (Kearse et al., 2012). The number of codons and the relative usage of synonymous codons were analyzed using CodonW 1.4.4 (Peden and John, 2000). Relative Synonymous Codon Usage (RSCU) refers to the relative probability of using a synonymous codon when encoding a specific amino acid. An RSCU > 1 indicates that the codon is used frequently, an RSCU = 1 indicates no preference, and an RSCU < 1 indicates low usage.

Repeat sequence analysis

Simple sequence repeats (SSRs) were identified and counted using MISA-web (Beier et al., 2017), in which the parameters for SSR identification were set at minimal repeat numbers of 10, 5, 4, 3, 3, and 3 for mono-, di-, tri-, tetra-, penta- and hexa- nucleotide repeats, respectively. For large repeats, REPuter (Kurtz et al., 2001) was used to identify forward, palindrome, reverse, and complementary long repeats. The hamming distance was set as 3, while the minimal repeat size was 30 bp.

Comparative analysis of chloroplast genome

IRscope was used to compare IR/SSC and IR/LSC boundaries and junctions among the six Anacardiaceae species (https://irscope.shinyapps.io/irapp/) (Amiryousefi et al., 2018). The mVISTA (https://genome.lbl.gov/vista/mvista/submit.shtml) (Frazer et al., 2004) was used to compare the complete chloroplast genome of the six Anacardiaceae species. Before nucleotide divergence analysis, the cp genome were aligned using MAFFT v7 (Katoh and Standley, 2013). The nucleotide variability of the six cp genomes were calculated using DnaSP v5.10 (Librado and Rozas, 2009), in which highly mutational hotspot regions were identified through a sliding window analysis. The length of the window frame was set at 4400 bp and a 200 bp step size was selected.

Nucleotide substitution rate (Ka/Ks)

A total of 79 protein-coding genes shared by the B. latifolia cp genome were extracted using Geneious. After aligning them with MAFFT, the online program ALTER (https://www.sing-group.org/ALTER/) was used to convert the file from ‘.fas’ format to ‘.aln’ format. Subsequently, the ‘.aln’ files were converted to ‘.axt’ format using AXTConvertor from the KaKs-Calculator in a Linux system (Zhang et al., 2006). Finally, the ratio of non-synonymous (Ka) to synonymous (Ks) substitutions was calculated using the KaKs-Calculator, selecting the NG model and setting the genetic code type to 11(bacterial and plant plastid codon). When Ka = 0 and Ks = 0, the value of Ka/Ks was represented by NA.

Phylogenetic analysis

We conducted a phylogenetic analysis of 37 chloroplast genomes in the family Anacardiaceae, using three species from the family Burseraceae, Commiphora gileadensis, Commiphora wightii, and Bursera fagaroides, as outgroups(The accession numbers can be found in Supporting Table 1). The Phylogenetic trees of complete chloroplast genomes were constructed using the maximum likelihood (ML) method. ML analysis was conducted using IQ-TREE (Nguyen et al., 2015), during which a substitution model of GTR + F + I were automatically calculated and applied with a bootstrap replicates of 1,000 times.

Genome structure of B. latifolia chloroplast genome

The chloroplast (cp) genome of B. latifolia exhibits a quadripartite structure, characterized by a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of inverted repeats (IRa and IRb), measuring 87,689 bp, 17,941 bp, and 27,217 bp, respectively (refer to Fig. 1; Table 1). The overall nucleotide composition reveals a predominance of A (30.8%) and T (31.5%), with C (19.2%) and G (18.5%) constituting the remaining nucleotides, resulting in a total AT content of 62.3% and GC content of 37.7%. A comprehensive annotation identified a total of 133 genes within the cp genome of B. latifolia, including 88 protein-coding genes (CDS), 37 transfer RNA genes (tRNAs), and 8 ribosomal RNA genes (rRNAs). Notably, the AT content is higher than the GC content (62.3% vs. 37.7%). Moreover, the GC content varies across different regions, with the LSC, SSC, and IR regions exhibiting values of 35.8%, 32%, and 42.6%, respectively, with the IR regions demonstrating higher GC content compared to the LSC and SSC regions. Functional classification of the genes in the cp genome of B. latifolia categorizes them into four classes: photosynthesis-related genes (44), genes involved in self-replication (74), other annotated genes (6), and genes with unknown functions (7) (see Table 2).

Table 1

Characteristics of the cp genome of *B. latifolia.*
Category	Item	Describe
Chloroplast genome structure	Cp gene/bp	160088
	LSC/bp	87713
	SSC/bp	17941
	IRA/IRB/bp	27217
Gene composition	Cp gene	133
	CDS	88
	tRNA	37
	rRNA	8
GC Content (%)	Cp gene	37.7
	LSC	35.8
	SSC	32
	IRA/IRB	42.6

A total of 19 duplicated genes including one NADH dehydrogenase subunit gene (ndhB), five self-replication genes (rpl2, rpl23, rps12, rps19, rps7), four rRNA genes (rrn16, rrn23, rrn4.5, rrn5), seven tRNA genes (trnA-UGC, trnI-CAU, trnI-GAU, trnL-CAA, trnN-GUU, trnR-ACG, trnV-GAC), and two unknown function protein genes (ycf15, ycf2) were illustrated. Most genes are non-coding genes, and a few genes contain one or two introns.Among them, ndhA, ndhB, petB, petD, atpF, rpl16, rpl2, rps12, rps16, rpoC1, trnA-UGC, trnI-GAU, trnK-UUU, trnL-UAA, trnT-CGU, and trnV-UAC contain one intron, while rps12, clpP and ycf3 contain three introns. It is worth noting that further research using advanced molecular techniques to determine the functions of the 7 genes with unknown functions were necessary (Table 2).

Table 2

Genes present in chloroplast genome of *B. latifolia*
Category	Gene group	Gene name
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(2), 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
	Subunits photochlorophyllide reductase	-
Self-replication	Proteins of large ribosomal subunit	rpl14, rpl16, rpl2(2), rpl20, rpl22, rpl23(2), rpl32, rpl33, rpl36
	Proteins of small ribosomal subunit	rps11, rps12(2), rps14, rps15, rps16, rps18, rps19(2), rps2, rps3, rps4, rps7(2), rps8*
	Subunits of RNA polymerase	rpoA, rpoB, rpoC1, rpoC2*
	Ribosomal RNAs	rrn16(2), rrn23(2), rrn4.5(2), rrn5(2)
	Transfer RNAs	trnA-UGC(2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnG-GCC, trnH-GUG, trnI-CAU(2), trnI-GAU(2), trnK-UUU, trnL-CAA(2), trnL-UAA, trnL-UAG, trnM-CAU, trnN-GUU(2), trnP-UGG, trnQ-UUG, trnR-ACG(2), trnR-UCU, trnS-GCU, trnS-GGA, trnS-UGA, trnT-CGU, trnT-GGU, trnT-UGU, trnV-GAC(2), trnV-UAC, trnW-CCA, trnY-GUA, trnfM-CAU
Other genes	Maturase	matK
	Protease	clpP**
	Envelope membrane protein	cemA
	Acetyl-CoA carboxylase	accD
	c-type cytochrome synthesis gene	ccsA
	Translation initiation factor	infA
	other	-
Genes of unknown function	Conserved hypothetical chloroplast ORF	ycf1, ycf15(2), ycf2(2), ycf3*, ycf4*
Notes: Gene: Gene with one introns; Gene*: Gene with two introns; #Gene: Pseudo gene; Gene (2): Number of copies of multi-copy genes.

Analysis of codon preference

There are significant differences in codon usage patterns among different species. Each amino acid is encoded by at least one codon and up to six codons. This unequal usage of synonymous codons is known as codon bias. Natural selection and base mutations are considered the main factors influencing codon bias. Based on 88 coding sequences (CDS), the codon usage frequency and Relative Synonymous Codon Usage (RSCU) of the cp genome were calculated. These CDS consist of 22,855 codons each, encoding 20 amino acids in the chloroplast genome. Among these, six codons encode arginine (Arg), leucine (Leu), and serine (Ser), while only one codon encodes methionine (Met) and tryptophan (Trp). Among them, leucine (Leu: 10.53%) is the most frequently utilized amino acid, while cysteine (Cys: 1.12%) is the least utilized amino acid in the cp genome. According to RSCU analysis, except for methionine (Met) and tryptophan (Trp), almost all amino acids are encoded by 2–6 synonymous codons. The relative synonymous codon usage for methionine (Met) and tryptophan (Trp) is 1. There were 30 high-frequency codons with RSCU > 1, among which 13 and 16 high-frequency codons ended with A and U, accounting for 93.55% of the total. Only one high-frequency codons ended with G, and no high-frequency codons ending with C were found. A preference of A or U to end codons was indicated for cp genome of the B. latifolia chloroplast.

Repeat sequences analysis

In this study, 99 SSRs were identified in the cp genome of B. latifolia, including 77 mononucleotides (Mono-), 8 dinucleotides (Di-), 5 trinucleotides (Tri-), 7 tetranucleotides (Tetra-), and 2 hexanucleotides (Hexan-) (Fig. 3A). 16 SSRs were located in the IR region, 22 SSRs in the SSC region, and the LSC region contained the highest number of SSRs, with 61 (Fig. 3B). The repeat units were primarily composed of A or T, with B. latifolia cp genome being A/T types rather than G/C types. Furthermore, dinucleotides are primarily composed of AT/AT, with only 1 or 2 occurrences of other nucleotide types.(Fig. 3C).

Within this cp genome, a total of 63 repeat sequences were identified. Among these, forward repeat sequences constituted 39.68% (25 repeats) of the total repeats, while reverse repeat sequences accounted for 3.17% (2 repeats). The remaining 57.14% (36 repeats) were attributed to palindromic repeat sequences. Notably, no complementary repeat sequences were detected (refer to Fig. 4A). Furthermore, the analysis revealed that repetitive sequences ≤ 40 bp in length were the most prevalent, with 37 occurrences. Subsequently, there were 10 occurrences in the 41–50 bp range, and 16 occurrences in the > 50 bp range (Fig. 4B).

IR expansion and contraction

The chloroplast genome of B. latifolia was compared with those of five reported chloroplast genomes from the Anacardiaceae family (Fig. 5). The main chloroplast marker genes, rpl22, ndhF, ycf1, and trnH, were found at the boundaries of LSC/IRA, IRA/SSC, SSC/IRB, and IRB/LSC, respectively. Among these genes, rpl22 primarily located in the LSC region or crossed the LSC/IRA boundary. In B. latifolia, Dobinea delavayi, Lannea coromandelica, and Pegia nitida, the rpl22 gene was partly located within the IRb region, the IRb part of the rpl22 genes ranged from 39 to 69 bp, while in Searsia paniculata and Semecarpus reticulatus, the entire rpl22 gene was within the IRb region. The ndhF gene was mainly located in the LSC region or across the IRb/SSC boundary, showing positions ranging from 2208 to 2259 within the SSC region. Notably, among all the compared six Anacardiaceae species, the ycf1 gene crossed the IRa/SSC boundary, with positions ranging from 952 to 1479 bp within the IRa region and from 3996 to 4568 bp within the SSC region. The ndhF gene was located within the SSC region, with distances to the IRb/SSC junction of 5, 56, 61, 64, 143, and 154 bp. The trnN gene was entirely located within the IRa region and contracted by 971 to 1479 bp. The trnH gene is located within the LSC region, positioned 74–75 bp away from the IRa/LSC boundary.

Structural comparison and divergence hotspot identification analysis

Using the annotation of B. latifolia as reference, the chloroplast genome sequences of the five Anacardiaceae species were compared by mVISTA (Fig. 6). The sequence divergences remarkably differed among regions. The alignment result indicates that the chloroplast genome is extremely conserved with only few variations detected. The data revealed that the non-coding region was more divergent than coding counterparts. IR regions of all cp genomes were less diverged than the LSC and SSC regions. The rRNA genes were highly conserved comparing to other genes. The exons of the ycf1 gene were regions representing the highest polymorphism.

The diversity of nucleic acids can reveal variations in nucleic acid sequences among different species. Regions with high variability can serve as potential molecular markers for population genetics. The nucleotide diversity (Pi) value for the six cp genomes ranged from 0 to 0.14438, with an average of 0.02078. At a cut-off point set at Pi ≥ 0.1, four hypervariable regions, including rps16 (exonl)-trnQ (0.12183), atpF (exon1)-atpH (0.11283), ndhF-rpl32(0.1098), rpl32-trnL (0.10633), and ycf1 (0.14433), were identified. Among the four hypervariable regions, two were in the LSC region, the other two were in the SSC region. None was detected in the IR region (Fig. 7).

Adaptive evolution analyses

Taking B. latifolia as a reference, alterations in 5 Anacardiaceae cp genomes were examined to uncover patterns of selection among protein coding genes (Table 3). Overall, the Ka/Ks ratios of 79 protein-coding genes shared by the cp genome of B. latifolia were compared. The results indicate that only the psbT (1.61) gene in L. coromandelica has a Ka/Ks ratio > 1. Additionally, in S. paniculata, the rpl22 (1.09), rpl32 (1.23), rps16 (1.64) and ycf2 (2.04) genes have Ka/Ks ratios greater than 1, while all other genes have Ka/Ks ratios < 1.

Table 3

The 79 protein-coding genes in cp genome of the B. latifolia and 5 Anacardiaceae species were used for ka/ks analysis
Gene	D. delavayi	L. coromandelica	P. nitida	S. paniculata	S. reticulatus	Gene	D. delavayi	L. coromandelica	P. nitida	S. paniculata	S. reticulatus
accD	0.40	0.40	0.42	0.33	0.45	psbI	0.00	0.00	0.00	0.00	0.00
atpA	0.07	0.04	0.03	0.03	0.04	psbJ	NA	NA	NA	NA	NA
atpB	0.06	0.07	0.05	0.02	0.10	psbK	0.36	0.68	0.82	0.55	0.18
atpE	0.20	0.00	0.00	0.00	0.00	psbL	NA	0.00	0.00	NA	0.00
atpF	0.18	0.21	0.26	0.18	0.35	psbM	0.20	0.20	0.20	0.15	NA
atpH	0.00	0.00	0.00	0.00	0.00	psbN	0.00	0.00	0.00	0.00	0.00
atpI	0.04	0.06	0.06	0.06	0.09	psbT	0.52	1.61	0.98	0.73	0.64
ccsA	0.32	0.35	0.27	0.37	0.29	psbZ	0.00	0.17	0.17	0.00	NA
cemA	0.22	0.11	0.13	0.65	0.40	rbcL	0.30	0.16	0.17	0.18	0.21
clpP	0.03	0.03	0.04	0.19	0.10	rpl14	0.17	0.25	0.18	0.06	0.08
matK	0.52	0.58	0.48	0.60	0.76	rpl16	0.05	0.05	0.04	0.05	0.10
ndhA	0.23	0.14	0.15	0.23	0.04	rpl2	0.16	0.13	0.11	0.11	0.22
ndhB	0.21	0.16	0.31	0.21	0.16	rpl20	0.63	0.40	0.73	0.22	0.31
ndhC	0.08	0.15	0.22	0.17	0.15	rpl22	0.53	0.50	0.54	1.09	0.83
ndhD	0.16	0.21	0.14	0.17	0.13	rpl23	NA	NA	NA	0.29	NA
ndhE	0.18	0.40	0.30	0.12	0.15	rpl32	0.31	0.56	0.70	1.23	0.78
ndhF	0.22	0.32	0.29	0.26	0.27	rpl33	0.36	0.37	0.28	0.09	0.00
ndhG	0.31	0.24	0.31	0.40	0.19	rpl36	0.13	0.04	0.05	0.20	0.17
ndhH	0.04	0.08	0.06	0.07	0.03	rpoA	0.20	0.14	0.14	0.15	0.25
ndhI	0.24	0.52	0.53	0.27	0.64	rpoB	0.10	0.07	0.11	0.17	0.11
ndhJ	0.11	0.21	0.12	0.56	0.28	rpoC1	0.11	0.18	0.12	0.20	0.05
ndhK	0.11	0.11	0.14	0.35	0.19	rpoC2	0.37	0.35	0.41	0.32	0.31
petA	0.11	0.13	0.17	0.19	0.05	rps11	0.06	0.09	0.10	0.06	0.05
petB	0.03	0.03	0.03	0.03	0.21	rps12	0.18	0.12	0.35	0.00	0.00
petD	0.00	0.00	0.00	0.08	0.00	rps14	0.14	0.20	0.19	0.19	0.07
petG	0.00	NA	NA	NA	0.00	rps15	0.22	0.47	0.18	0.20	0.86
petL	NA	NA	NA	0.18	0.74	rps16	0.21	0.97	0.65	1.64	0.64
petN	NA	0.00	0.32	NA	NA	rps18	0.10	0.30	0.15	0.15	0.30
psaA	0.06	0.06	0.05	0.08	0.06	rps19	0.00	0.05	0.11	0.00	0.00
psaB	0.02	0.02	0.03	0.01	0.00	rps2	0.09	0.09	0.08	0.16	0.12
psaC	0.00	0.00	0.00	0.00	0.00	rps3	0.25	0.16	0.15	0.14	0.14
psaI	0.15	0.61	0.61	NA	NA	rps4	0.11	0.17	0.15	0.35	0.24
psaJ	0.33	0.33	NA	0.33	0.33	rps7	0.32	NA	NA	NA	NA
psbA	0.02	0.02	0.02	0.02	0.06	rps8	0.16	0.08	0.07	0.07	0.05
psbB	0.03	0.05	0.01	0.08	0.07	ycf1	0.73	0.87	0.99	0.98	0.77
psbC	0.02	0.03	0.01	0.04	0.08	ycf15	0.00	0.00	0.00	0.00	NA
psbD	0.00	0.00	0.00	0.00	0.00	ycf2	0.81	0.88	0.83	2.04	0.97
psbE	0.00	0.00	0.00	0.00	0.00	ycf3	0.19	0.29	0.17	0.29	0.29
psbF	0.00	0.00	0.00	0.00	0.00	ycf4	0.21	0.16	0.14	0.42	0.23
psbH	0.04	0.00	0.00	0.00	0.17

Phylogenetic relationships of Anacardiaceae species

B. latifolia is positioned within the basal clade of the Anacardiaceae family, sister to the clade containing Choerospondias axillaris, Lannea coromandelica, and Sclerocarya birrea (Fig. 8). Buchanania is grouped on the same branch with Choerospondias, Lannea, and Sclerocarya of the Spondioideae. Anacardium and Mangifera are clustered with Semecarpus into the clade of Semecarpeae.

Homology: Feature of Chloroplast Genomes

In this study, the chloroplast genome features and phylogenetic relationships of Buchanania latifolia were comprehensively analyzed. The chloroplast genome of B. latifolia showed a high degree of homology compared to other reported chloroplast genomes of Anacardiaceae species, including Dracontomelon delavayi, Lannea coromandelica, Pistacia nitida, Spondias paniculata, and Swintonia reticulata. The genome lengths of these species ranged from 159,485 to 162,509 bp, with a maximum difference of 3,024 bp. The overall GC content is comparable to that of other species in the Anacardiaceae family, such as Rhus chinensis (37.79%), Pistacia weinmannifolia (37.84%), Toxicodendron vernicifluum (37.96%), and Cotinus species (37.9%-38.1%)

(Wang et al., 2020, Zheng et al., 2018, Liu et al., 2023). The number and types of genes were also very similar, reflecting the highly conserved characteristics of chloroplast genomes. The homology among closely related woody species is typically considered reasonable due to their long generation times and the relatively low number of substitutions occurring over a given period. Substitutions from parent plants can only be passed to offspring during the process of germination.

The study of codon preference aids in understanding the evolutionary processes of plant species and optimizing the expression of exogenous genes in chloroplasts, enabling the prediction of gene function and expression levels (Li et al., 2019b). Consistent with previous findings, Buchanania latifolia demonstrates a preference for A or U bases in its chloroplast genome codons, a common trait in plant chloroplast genomes (Zhou et al., 2008). In B. latifolia, 30 high-frequency codons were identified, with 29 of them ending with an A or U base, likely influenced by natural selection and mutations (Necşulea and Lobry, 2007). Previous studies have also noted that in the chloroplast genomes of Anacardiaceae, high-frequency codons tend to utilize A or U bases as the third codon base (Liu et al., 2023, Xin et al., 2023, Wang et al., 2020).

The most prevalent SSRs in the chloroplast genomes of B. latifolia were mononucleotide repeats. Similar to other plants, chloroplast SSRs predominantly consist of short poly-A or poly-T repeats, with mononucleotide repeats being the most common forms (Tao et al., 2023, Vu et al., 2020, Djedid et al., 2021, Provan et al., 2001, Yang et al., 2020). Moreover, the majority of SSRs are located in the LSC and SSC regions, consistent with previous findings on chloroplast genomes (Alshegaihi, 2024, Liu et al., 2023, Wang et al., 2020). Palindrome sequences accounted for 39.68%, forward repeated sequences for 57.14%, and reverse sequences for 3.17%. No complementary sequences were found. Forward and palindromic repeats were the most common repeat types, with most dispersed repeats being less than 40 bp, as reported in previous studies (Liang et al., 2020, Kirov et al., 2020, Tian et al., 2021, Yuan et al., 2005).

The IR region of the chloroplast genome is thought to be the most conservative section. The expansion and contraction of the IR region are pivotal factors influencing the length variation observed in plant chloroplast genomes, typically categorized into two types (Yi et al., 2013, Zhang et al., 2013). Such expansions and contractions in the IR region across most species manifest as minor deviations in the IR/SC boundary within a few fixed genes, which may lead to pseudogenization of certain genes. The examination of the chloroplast genome of B. latifolia and the Anacardiaceae family in this study aligns with prior reports in angiosperms (Liu et al., 2023, Wang et al., 2020, Xin et al., 2023). Typically, the LSC/IRb boundary is situated on or near rps19, rpl2, or rpl22, while IRb/SSC is generally positioned on ycf1 or between ycf1 and ndhF. The SSC/IRa boundary typically lies on ycf1, and the IRa/LSC boundary usually falls on or near rps19, rpl2, rpl12, and trnH (Alshegaihi, 2024).

Intergenic spacers are more divergent than introns and protein-coding sequences (Meng et al., 2018). However, pseudogenes suffer the same fate as intergenic spacers due to a lack of functional importance, leading to less conservative strains. Pseudogenization was common in the evolution of chloroplast genomes, such as accD, ccsA, ycf1, rps19 and psbB pseudogenes (Krawczyk et al., 2018, Li et al., 2021). The most divergent genes among the six Anacardiaceae species were two pseudogenes accD and ycf1, an intron of the rps16 genes, and the intergenic spacer ndhF-rpl32. These genes or regions can be utilized for developing molecular markers for species identification and population studies (Magdy et al., 2019).,

The synonymous (Ks) and non-synonymous (Ka) nucleotide substitution pattern is a well-recognized marker for assessing genome evolution; and the Ka/Ks ratio reflects selection pressure on genes (Yang and Nielsen, 2000, Guo et al., 2017). Ka/Ks < 1, Ka/Ks = 1, and Ka/Ks > 1 indicate genes that underwent purifying, neutral, and positive selections, respectively (Yang and Nielsen, 2000). Generally, synonymous mutations occur more frequently than nonsynonymous mutations within genes, causing the Ka/Ks values to be below 1 (Makałowski and Boguski, 1998). Our results using B. latifolia and five species from the Anacardiaceae family indicate that only the psbT gene in L. coromandelica and the rpl22, rpl32, rps16 and ycf2 genes in S. paniculata have Ka/Ks values > 1, suggesting strong positive selection acting on these two genes. Ka/Ks values for all other detected genes are below 1, indicating widespread purifying selection on these chloroplast genomes.

Application on Phylogenetic Reconstruction

The informative sites provided by single genes or gene combinations are often limited. Differences in the evolutionary trajectories of various gene sequences can result in low resolution of the constructed phylogenetic tree, posing challenges in elucidating the evolutionary relationships within the Anacardiaceae family. To optimize phylogenetic outcomes, we conducted a phylogenomic analysis of Anacardiaceae based on complete chloroplast genomes using the maximum likelihood method. Overall, branch support at the generic level was generally high, with only relatively lower support observed for the branch of the genus Rhus and its sister groups (BS = 68), while support for all other branches was robust (BS = 100). This suggests that our analysis accurately reflects the phylogenetic relationships within Anacardiaceae. B. latifolia is sister to Choerospondias, Lannea, and Sclerocarya, consistent with previous phylogenetic trees constructed using sequences such as nuclear ribosomal external transcribed spacer (ETS), the chloroplast trnL intron and trnL-F intergenic spacer (trnL-F region), and the chloroplast rps16 intron (Weeks et al., 2014). However, Choerospondias, Lannea, and Sclerocarya all belong to the tribe/subfamily Spondiadeae/Spondioideae. Phylogenetically, they are distant from Anacardium and Mangifera, which are in the same tribe/subfamily Anacardieae/Anacardioideae as Buchanania. Although Buchanania is typically considered a member of the tribe/subfamily Anacardieae/Anacardioideae, it is here regarded as a sister group to those traditionally belonging to the tribe/subfamily Spondiadeae/Spondioideae, which aligns with Weeks' view (Weeks et al., 2014).

In this study, the complete chloroplast genome sequence of B. latifolia was assembled and annotated de novo. The chloroplast genome of B. latifolia exhibits a typical quadripartite structure, with a length of 160,088 bp, containing 88 protein-coding sequences (CDS), 37 tRNA genes, and 8 rRNA genes, with a GC content of 37.7%. A total of 99 SSR loci and 63 repeat sequences were identified, which can be utilized for marker development, phylogenetic and population studies of B. latifolia. Codon usage analysis revealed a preference for Leu codons ending with A/U. Additionally, the study investigated IR boundaries, DNA polymorphism, positive selection suites, and phylogenetic position. Comparative analysis with five other species from the Anacardiaceae family confirmed the nearly identical and highly conserved chloroplast genome features of B. latifolia, which can be valuable for understanding the plastid evolution and evolutionary relationships within Anacardiaceae. Phylogenetic analysis reveals that B. latifolia is positioned at the base of Anacardiaceae, sister to Choerospondias, Lannea, and Sclerocarya. These fndings could provide important genetic information for further research into breeding of Anacardiaceae, phylogeny, and evolution of B. latifolia. This study reveals the structural characteristics of the chloroplast genome, SSR loci, and phylogenetic analysis, providing valuable genetic information for understanding the origin and evolution of Anacardiaceae in the future.

Data Availability

The genome sequence data that support the fndings of this study are openly available in GenBank of NCBI at https://www.ncbi.nlm.nih.gov/ under the accession OM000214.1. The associated BioProject, SRA, and Bio-Sample numbers are PRJNA783515, SRR17036892, and SAMN23436132 respectively.

Funding

This research was funded by the National Natural Science Foundation of China (grant No. 42161015). The Key Project of Basic Research of Yunnan Province, China (202202AS07337) to Gao Chen. This work is supported by Yunnan Science and Technology Talent and platform Program (202305AM340008).

Acknowledgements

Thanks to Zhefei Zeng and Kaiwen Jiang for their support of field sampling.

Contributions

LLY designed the study. CMM performed statistical and bioinformatic analyzes and wrote the first draft. RR proofreads the image text and provides suggestions for revision. QWL sampling, data analysis. WTC Molecular Materials Management. CMM and RR contribute equally in this study. All-authors read and approved the final manuscript.

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Informed consent.

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Conflict of interest

The authors declare no competing interests.

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Complete chloroplast genome sequence of Buchanania latifolia: genome structure, and phylogeny of basal Anacardiaceae family relationships

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Abstract

Figures

Introduction

Materials and Methods

Plant material, sequencing, and genome assembly

Genome structure analysis

Codon preference analysis

Repeat sequence analysis

Comparative analysis of chloroplast genome

Nucleotide substitution rate (Ka/Ks)

Phylogenetic analysis

Results

Analysis of codon preference

Repeat sequences analysis

IR expansion and contraction

Structural comparison and divergence hotspot identification analysis

Adaptive evolution analyses

Phylogenetic relationships of Anacardiaceae species

Discussion

Homology: Feature of Chloroplast Genomes

Application on Phylogenetic Reconstruction

Conclusions

Declarations

References

Supplementary Files

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