A report on the chloroplast genome of Melia dubia Cav. (Sapindales: Meliaceae), a potential agroforestry species

doi:10.21203/rs.3.rs-1503416/v1

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A report on the chloroplast genome of Melia dubia Cav. (Sapindales: Meliaceae), a potential agroforestry species

https://doi.org/10.21203/rs.3.rs-1503416/v1

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published 30 Dec, 2022

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Background: Melia dubia Cav. is a fast-growing plywood species gaining popularity due to high economic returns. This study aimed to assemble and annotate the chloroplast (cp) genome of M.dubia and compare it with previously published cp genomes within the Meliaceae family.

Methods and Results: The chloroplast genome was constructed by the de novo and reference-based assembly of paired-end reads generated by long-read sequencing of genomic DNA. The cp genome, sized 171,956 bp, comprised a typical angiosperm quadripartite structure. The large single-copy (LSC) region of 76,055 bp and a small single-copy (SSC) region of 18,693 bp, covers 55% of the genome. The pair of inverted repeats (IRA and IRB) of 38,604 bp each (covering 45% of the genome). We identified unique genes (112), including protein-coding genes (79), tRNA (29) and 4 rRNA genes. Phylogenetic analysis using complete cp genomes of 11 species from Meliaceae revealed that M. dubia and M. azedarach hared a sister clade. Comparative analysis using cp genome of M.dubia , M.azedarach and Azadirachta indica revealed a high sequence similarity ( >70%). Five intergenic regions were highly conserved among the three cp genomes. The gene trnG-UCC at LSC region was found to be more divergent in M.dubia and M.azedarach while it shows complete conservation within M.dubia and A.indica.

Conclusions: The available levels of taxonomic expertise and clarity in species delineation within the Melia genus is low. The information generated provides scope for identifying new barcodes which increases the discriminatory power of the species within the genus beyond morphological identification.

short rotation

Meliaceae

cp genome

Melia dubia

phylogeny

conservation strategies

This is the first report on chloroplast genome sequencing and annotation of Melia dubia. There are many confusions related to the taxonomy of the species, especially with M. azedarach, of the same genus. Phylogenetic analysis and comparative cp genomic analysis reveal species-specific markers that could be used to delineate the species. Analysis of the whole chloroplast genome also provides opportunities for tree improvement, skimming the genetic variability in the natural populations of this species.

Chloroplasts (cp) are plastids characterised by a double-layered membrane and having independent DNA and thylakoid structures[1]. They evolved through endosymbiosis between a photosynthetic bacterium and a non-photosynthetic host, retaining their distinct genome information [2]. This intracellular organelle is the site of photosynthesis, providing energy for plants and algae and supporting biosynthesis of primary metabolites. A typical angiosperm cp genome is quadripartite comprising a large and small single-copy region (LSC and SSC) separated by a pair of inverted repeats (IRA and IRB) [3]. The number of cp genomes reported and deposited in the National Center for Biotechnology Information (NCBI) database is rising.

The plastid is non-recombinative and displays uniparental inheritance [4]. Compared with nuclear and mitochondrial genomes, cp genomes are the most conserved in DNA sequences, organisation, and structure. Hence they are widely used in phylogenetic analysis and molecular taxonomy.

Mader et al. report complete cp genomes of four Meliaceae species (Order: Sapindales) and their comparison with neem (Azadirachta indica), the first published Meliaceae plastome sequence[5]. Presently cpDNA sequences are available for Melia azedarach, Carapa guianensis, Cedrela sp, Entandrophragma cylindricum, Khaya senegalensis, Khaya madagascariensis, Aglaia odorata, Aphanamixis polystachya, Swietenia mahagoni, Toona sp., Azadirachta indica in Meliaceae. However, there are no reports on the cpDNA of Melia dubia at the Organellar Genome Resource at NCBI [6].

Cp genome is usually constructed by direct isolation of the cp DNA or cp genome amplification using PCR from total DNA. However, neither methods provide sufficient yield and quantity for all the plant species [7]. Since whole cellular DNA consists of chloroplast, mitochondrial and chromosomal DNA, Whole Genome Sequence (WGS) data is used to construct the cp genome directly.

Since the first genome sequencing in 1986 [8,9], different strategies and various bioinformatics tools have been developed to assemble cp genomes [7]. Deducing a cp genome from the WGS data requires extracting cp reads from the WGS followed by assembly and resolution of the unique circular structure, including the inverted repeats. In the absence of a reference cp to map the reads, a k-mer analysis is used to extract the most frequent reads. GetOrganelle (https://github.com/Kinggerm/GetOrganelle) is one such toolkit available in the public domain to extract cp sequence data. This toolkit applies the “baiting and iterative mapping” approach to recruit organelle-associated reads[10].

CPGAVAS 2.0 is an integrated web server for annotating, visualising, analysis and GenBank submission of cp genome sequence. The software is freely accessible from http://www.herbalgenomics.org/cpgavas2/. Based on the identification and mapping of the similar protein, CPGAVAS predicts protein-coding, rRNA and cDNA sequences by integrating results from Blastx, Blastn, protein2genome, est2genome programs. Further, tRNA genes and inverted repeats are identified using tRNAscan, ARAGORN and vmatch. It also generates a circular map [11].

Melia dubia Cav. (Family-Meliaceae) is a deciduous fast-growing multipurpose species distributed in India, Sri Lanka, Myanmar, Malaysia, Java, China and Australia. It has high-quality termite and fungus-resistant timber, making it a money-spinning species for farmers. It is widely used in agro and farm forestry plantation programmes [12]. Melia has the potential to be developed into biofuel because of its high photosynthesis efficiency and high biomass yield. The species has received considerable attention from researchers due to its short rotation, high coppicing ability and better economic returns [12].

The species is often confused with Melia azedarach, belonging to the same family[13]. Though the Plants Of The World Online (https://powo.science.kew.org/) identifies them as separate species, the Plantlist (http://www.theplantlist.org/), reports M. dubia as a synonym of M. azedarach [14]. Reports are available on the use of barcodes to delineate them. However, there is little genomic information reported. Hence in this present study, we assembled the complete cp sequence of Melia dubia using whole-genome data to develop new markers for delineation. This is the first report on the cp genome information of the species.

Further annotations revealed its gene content and a circular map was constructed using CPGAVAS2 [16]. The results also provide insights into the systematic classification. Secondly, a comparative analysis of Meliaceae species was conducted. Finally, highly variable and conserved genes of the Melia cp genome were identified compared to the M. azedarach and A. indica. The complete cp genome sequence would help to elucidate evolutionary and phylogenetic relationships in the other members of Meliaceae. The information would also provide comprehensive information relevant to the identification, and scope for genetic improvement in the species.

Whole genome data: The whole genome sequence raw data of Melia dubia was downloaded from NCBI SRA database (GenBank accession: SRR5576232) using fastq-dump tool of SRA-toolkit in two forward and reverse reads. The reads were then quality checked using fastqc tool and used for cp genome generation and assembly.

Chloroplast genome assembly and annotation: The de novo assembly of Melia dubia cp genome from the whole genome sequence of Melia dubia was carried out using GetOrganelle v1.7.5 toolkit. The generated complete cp sequence was annotated using CPGAVAS2- an integrated plastome annotator and analyser http://www.herbalgenomics.org/cpgavas2/, followed by manual correction using BLAST[17]. The complete cp sequence generated was submitted to NCBI Third Party Annotation database under the accession number TPA: BK059531. A circular map was generated by CPGAVAS using the annotated genome.

Repeat structure and simple sequence analysis: REputer (https://bibiserv.cebitec.uni-bielefeld.de/reputer/) was used to assess the forward, palindrome and reverse repeats[18], whereas TRF version 4.09 was used to identify long tandem repeats with size of repeat units greater than or equal to 6 and the parameters set to alignment weights for match (2) mismatch(7) and indels (7), match probability (P_M=80), indel probability (P_I = 10), minimum pattern size (50) and minimum alignment score of 500 [19]. The tuple sizes were 0,4,5 and7 while the tuple distances were fixed at 0, 29, 159 and 500. In REputer, the minimum repeat size was set as 30 bp and hamming distance 3 with a maximum repeat size set to 50bp. SSRs in Melia dubia complete cp genome were detected using MISA webserver http://misaweb.ipk-gatersleben.de/ [20]. The parameters were set by unit size (x) and minimum number of repeats (y): 1/10, 2/6, 3/5, 4/5, 5/5, 6/5 (x/y). Maximal number of interrupting base pairs in a compound microsatellite was set to 100.

Relative synonymous codon usage analysis : RSCU for M.dubia cp genome was calculated using protein coding gene sequence. Compute codon usage bias from MEGA X software was employed for RSCU and frequency analysis [21, 22]

Phylogenetic analysis: The complete cp genome sequence of Melia dubia generated in the study was used. Nine other species of the Meliaceae (Order Sapindales) and one species of adjacent order Geraniales were downloaded from NCBI database (Supplementary Table 1). The ten complete cp sequences were aligned using MAFFT v7.4.0.9 (https://mafft.cbrc.jp/alignment/software/index.html)with the parameters set to FFT-NS-2 [23]. The aligned sequences were further trimmed to equal length using BioEdit v7.2.5. Maximum likelihood method was followed to infer the phylogenetic relationship with 1000 bootstrap replicated in MEGA 10.2.4 and a phylogenetic tree was generated [24].

Comparative genomics of M.dubia cp genome: The newly sequenced cp genome of M.dubia was compared with two other closely related species namely M.azedarach and A.indica respectively (NC_050650.1, NC_023792.1). Multiple alignments were carried out using mVISTA programme and sequence identities were plotted using shuffle LAGAN mode[25]. The annotation of M.dubia was set as reference.

General features of Melia dubia cp

The complete cp sequence of Melia dubia retrieved from WGS data (GenBank accession: SRR5576232) through de novo assembly using GetOrganelle toolkit was identified and submitted to third party annotation section of the GenBank databases under the accession number TPA: BK059531.

The cp genome of M.dubia is a small, double-stranded circular structure with a genome size of 171,956 bp. It exhibits a typical angiosperm quadripartite structure with a pair of inverted repeats (IRA and IRB) of the same size of 38,604 bp; accounting for 45% of the genome separated by a large (LSC) and a small (SSC) single copy regions of 76,055 bp and 18693 bp with coverage of 44% and 11% respectively. The frequencies of all the four bases were 53,837, 54,192, 32,596 and 31,331 (Supplementary Figure 1). G+C content of all land plants cp genome plays a significant role in its own structural evolution. The universal GC content of M.dubia cp was found to be 37.18%, with uneven distribution along the single copy and inverted repeat regions. GC content was low at SSC region (31.19%), moderate at LSC region (35.23%) and high at IRs (40.55%) (Table 1).

Table 1 Summary of the Md cp genome characteristics

Characteristics	Length in bp	Genome coverage (%)	GC content(%)
Whole cp genome	171,956	100	37.18
LSC Region	76055	44	35.23
SSC Region	18693	11	31.19
IR Regions	38604	45	40.55

The GC content accounted for 40.55% in the IR regions, while in the LSC and SSC regions, GC contents accounted for 35.23 and 31.19 %, respectively. In contrast with the LSC and SSC regions, IR regions had higher percents of GC content (Table 2).

Table 2. Nucleotide composition and AT, GC content (%) in the four regions of the M.dubia cp genome

Region	A(%)	T(U)(%)	C(%)	G(%)	C + G (%)	A + T (%)
LSC	45.41	45.79	42.61	41.19	35.23	64.77
SSC	11.96	11.85	9.37	8.86	31.19	68.81
IRA	22.07	20.43	24.75	24.21	40.55	59.45
IRB	20.56	21.93	23.27	25.75	40.55	59.45
Total	31.31	31.52	18.96	18.22	37.18	62.82

Chloroplast genome annotation and contents

A total of 146 genes were annotated using a web-based programme called CPGAVAS2- an integrated Plastome Annotator and Analyzer and validated using BLAST X. M.dubia cp encodes 146 genes - 112 are unique; they include 79 CDS, 29 tRNAs and 4 rRNAs. 34 genes were in duplicate condition, thereby giving a total of 146 genes altogether that code for 101 CDS, 37 tRNAs and 8 rRNAs. For the 34 genes in duplicates, 22 were CDS, 8 were tRNAs and 4 were rRNAs (Figure 2).

The protein coding genes of this cp was mainly classified into 3 categories

Genes for photosynthesis that contain 52 genes which include subunits of ATP synthase genes, photosystem I genes, photosystem II genes, NADH-dehydrogenase genes, cytochrome b/f complex genes, RUBISCO gene(rbcL).
Genes for self-replication that enclose 38 genes including large subunit of ribosome, DNA dependent RNA polymerase,small subunit of ribosome.
Other genes like subunit of acetyl-CoA-carboxylase (accD), c-type cytochrome synthesis gene (ccsA), Envelope membrane protein gene(cemA), protease gene(clpP) and maturase gene(matK) along with ycf genes having unknown conserved open reading frames (Table 3).

Table 3. List of protein-coding genes present in M.dubia cp genome

Category of genes	Group of genes	Name of genes
Genes for photosynthesis	Subunits of ATP synthase	atpA, atpB, atpE, atpF, atpH, atpI
	Subunits of photosystem II	psbA, psbB, psbC, psbD, psbE, psbF,psbH, psbI, psbJ, psbK, psbL, psbM, psbN, psbT, psbZ, ycf3
	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 photosystem I	psaA, psaB, psaC, psaI, psaJ
	Subunit of rubisco	rbcL
Self replication	Large subunit of ribosome	rpl14, rpl16, rpl2, rpl20, rpl22, rpl23, rpl32, rpl33, rpl36
	DNA dependent RNA polymerase	rpoA*, rpoB, rpoC1, rpoC2
	Small subunit of ribosome	rps11, rps12#, rps14, rps15, rps16, rps18, rps19, rps2, rps3, rps4, rps7, rps8
Other genes	Subunit of Acetyl-CoA-carboxylase	accD
	c-type cytochrom synthesis gene	ccsA
	Envelop membrane protein	cemA
	Protease	clpP
	Maturase	matK
Unknown	Conserved open reading frames	ycf1, ycf15, ycf2, ycf4

* Genes found in duplicates; #trans splicing gene

16 genes were in duplicates from CDS genes and found in inverted repeat regions. As with other land plants rps 12 in Melia is also a trans-splicing gene with its 5′ end located in LSC and two replicative 3′ ends at IRs. Fifty-one coding genes were in single copy and the remaining 16 were split genes. Twenty four genes in Melia Cp harbours introns of which 2 genes (ycf3, clpP) harbour a second intron. The intron size ranged from 2544 bp for trnK-UUU to 527 bp for trnL-UAA (Supplementary table 2). Among the 37 tRNAs, 6 were duplicate genes, 17 genes in single copy and 8 were split genes. Moreover, there were 8 ribosomal RNA genes in duplicate conditions located at IR regions.

Codon Usage Patterns

Melia cp uses 64 different codons that code for 21 different types of amino acids. The number of codons ranged from 84 to 2257. Methionine and tryptophan amino acids possess only one codon while the remaining amino acids hold 2-6 codons (Figure 3).

Analysis of repeat sequences in Melia dubia chloroplast

Identifying the diversity in species using molecular markers and its development can facilitate analysis of population genetics, species identification and polymorphism studies in M.dubia . Using MISA, we detected 81 SSR loci in M.dubia cp genome, which includes 97.5% mononucleotides and 2.5% dinucleotides (Supplementary Figure 2). The predominant SSR motifs were T and A with an average frequency of 51 and 44 %. This abundance contributes to the bias in AT rich sequences than GC in cp genome. Dinucleotides were completely composed of either AT or TA. No other nucleotides were present in M.dubia .

Repeat Structure and inversions

Repeat motifs play crucial role in genome rearrangement and phylogenetic analysis. The REPuter results for the cp genome of length 171,956 bp with maximum distance 3 and minimum repeat size 30 bp revealed 3 categories: forward (23), reverse (1), palindromic (26) repeats. The TRF tool identified sixty-five long tandem repeats with repeat unit size > 6 bp (Supplementary Table 3 and 4).

Phylogenetic relationship analysis

The complete cp genome of 10 Meliaceae members and Melianthus villosus outgroup species belonging to Geraniales order were downloaded from NCBI database and aligned along with the constructed cp genome of M.dubia . Phylogeny using cp genome was resolved and determined with 100% bootstrap in almost all nodes (Figure 3). Cp genome of Melia dubia and M. azedarach occurred as sister clades in the phylogenetic tree with a common internal node showing that they share a common ancestor and have a closer relationship than A.odarata, A.indica, A.polystachya, K.madagascariensis, S.mahagoni, C.odorata, C. guianensis, T.ciliata and a lower relationship with Melianthus villosus outgroup species.

Comparative genomics analysis

Comparative analysis using cp genome of M.dubia , M.azedarach and A.indica was performed using mVISTA. The cp genomes had a relatively high degree of conservation in exon regions than the CNS and introns. The sequence similarity was 72%. Trans splicing gene rps 12 was found to be conserved in 3 plastomes. trnG-UCC gene at LSC region was found to be more divergent in M.dubia and M.azedarach whereas shows complete conservation within M.dubia and A.indica. rpoC2-rpoC1, petL-petG, psbN-psbH, rpl23-trnI-CAU, psbH-petB were the intergenic regions which are highly conserved among 3 cp genomes (Figure 4) .

In this study, the complete cp genome of Melia dubia was sequenced and annotated (Figure 1, Table 1) and compared with previously published Meliaceae cp sequences, using the sequences of nine other species. Across eight other families within the Sapindales, more than 50 complete cpDNA sequences are available at the Organellar Genome Resource at NCBI [6]. There is a need to enrich the database with members from Sapindales to better understand the evolution of different families in this Order.

The GC content accounted for 40.55% in the IR regions. In contrast, in the LSC and SSC regions, GC contents accounted for 35.23 and 31.19 %, respectively. In contrast with the LSC and SSC regions, IR regions had a higher percent of GC content. The high GC contents in the IR regions could be attributed to DNA leading and lagging strands, origin of replication and termination, which are always highly conserved regions[26].

The genes of chloroplast mainly work for photosynthesis and self-replication. In addition, there are six genes with other functions and 4 with unknown functions (Table 2). We found clpP and ycf3 in duplicate, with two introns, similar to reports in cp genomes of four Meliaceae species [5].

Codon Usage Bias

Using the coding sequence, we calculated the RSCU frequency of the cp genome (Figure 3). The total protein-coding genes had 54,251 codons. Isoleucine had 4592 codons, making up 8.46 % of the total, as the most abundant amino acid in the M.dubia cp genome. Cysteine accounted for only 0.09%. In addition, almost all RSCU values greater than one were in the A/U-ending codons, while all RSCU values less than one were in the C/G-ending codons. Met and Trp were encoded by only one codon, so they showed no codon preference.

SSRs and long Repeat Analysis

Our study analysed the SSRs in the cp genome of M.dubia (Supplementary Fig. 2). We detected only 81 SSR loci. The mono-nucleotide A and T repeats accounted for 95%, the highest proportion in all SSRs identified. These markers have high intraspecific variability and could help resolve species delineation issues. The general observation is that plastid SSRs are usually made up of A and T and rarely contain G or C repeats. Xie et al. [27] attribute this that A-T transformation is simpler than G-C transformation in cp genomes.

Phylogenetic analysis

A phylogenetic tree based on a multiple alignment of ten complete Meliaceae cpDNA sequences (Supplementary Table 1; Figure 4) and Melianthus villosus as an outgroup, shows that the analysed Meliaceae form a monophyletic sister clade within the Meliaceae. Within this clade, M.dubia and M.azedarach group together as sister clades as with Cedrela odorata and Toona ciliata together, as do Khaya madagascarensis, Sweitenia mahogani and Carapa guianensis (Figure 6). This is in agreement with reports from other researchers who showed similar grouping amongst Meliaceae members [5].

Comparative analysis of chloroplast genomes and phylogenetic analyses

Complete cp sequences help decipher phylogenetic relationships, especially between closely related taxa, or in cases where limited sequence variation exists due to recent divergence, rapid speciation or slow genome evolution [28,29]. Considering that most species-level diversity of Meliaceae is contemporary [30], a whole-plastome phylogeny provides insights into the patterns of evolution and delineation. The phylogeny that was generated in this study (Figure 4) based on the whole-cp alignment for three Meliaceae species (Figure 5), is an initial step in this direction. Here, a clear grouping was also observed between M.dubia and M.azedarach which were grouped as sister clades, revealing that the two species are distinct. Azadirachta indica, though a member of the subfamily Melioidaeae, formed a separate clade from the genus Melia. This, again is in agreeement with previous studies in Meliaceae [31,32]. This study, therefore, helps in the differentiation at lower taxonomic levels. Whole-genome alignments are also very useful to develop cpDNA markers for the genetic identification of species or other taxonomic categories [33].

Based on pairwise alignments using mVISTA, large deletions were identified for Melia azedarach at the intergenic region between ndhB and trnL-CAA (Figure 5). This deletion is a size of about 1587 bp (Figure 5; at position 148322-149905 of the Melia azedarach sequence). These deletions mainly contribute to the smaller genome sizes of M.azedarach over M.dubia . Such deletions could be developed into effective species-specific markers, which must then be further validated with more species and individuals within the family. The SSRs and indels identified (Supplementary Table 4), after further validation, could serve as targets for future marker development to delineate M.dubia from M.azedarach. PCR-marker development based on indels in small intergenic regions might be particularly robust, because the primers could be placed into conserved genic regions adjacent to the indel. Similarly, the trnG-UCC could serve as an excellent marker.

Studies focusing on cp genome variation among the mahogany family have provided a useful taxonomic framework for this study. We have added a cp genome reference for M.dubia to the growing collection of Meliaceae cp sequences. This is the first report of cp genome of Melia dubia.

This genomic resource for Melia dubia and the Meliaceae family will enable further detailed genetic study of Melia, a genus that contains three species. This would form the base to generate resources to provide a level of resolution that can support delineation of the species within the genus and further development of genetic tools for breeding in tree improvement, considering its potential as an agroforestry species.

cp chloroplast

cpDNA chloroplast DNA

CNS Non Coding Sequences

CDS Coding Sequences

IRA Inverted Repeat A

IRB Inverted Repeat B

LSC Large Single-Copy Region

NGS Next-Generation Sequencing

SSC Small Single-Copy Region

Compliance with Ethical Standards

Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.

Funding: This work was supported by the Indian Council of Forestry Research and Education, Dehradun.

Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results.

Author Contribution: All authors contributed to the study conception and design. Aghila Samji and Komal Eashwarlal contributed equally to material preparation, data collection and analysis. Senthil Shanmugavel and K. Santhosh Kumar provided technical support during the initial phases of analysis. Rekha R Warrier involved in conceptualisation, resources, supervision, funding acquisition, writing original draft and editing. All authors read and approved the final manuscript.

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A report on the chloroplast genome of Melia dubia Cav. (Sapindales: Meliaceae), a potential agroforestry species

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