Legume Research

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Legume Research, volume 44 issue 2 (february 2021) : 145-151

Comparative Chloroplast Genome Analysis and Evolutionary Relationships in Some Species of Asclepiadeae, Apocynaceae

Abidina Abba1,2,*, Dhafer Alzahrani1, Samaila Yaradua1, Enas Albokhari1,3
1Deparment of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, KSA.
2Deparment of Biological Sciences, Faculty of Science, Federal University Lokoja, Kogi State, Nigeria.
3Department of Biological Sciences, Faculty of Applied Sciences, Umm Al-Qura University, Makkah, KSA.
  • Submitted03-05-2020|

  • Accepted26-11-2020|

  • First Online 26-01-2021|

  • doi 10.18805/LR-565

Cite article:- Abba Abidina, Alzahrani Dhafer, Yaradua Samaila, Albokhari Enas (2021). Comparative Chloroplast Genome Analysis and Evolutionary Relationships in Some Species of Asclepiadeae, Apocynaceae . Legume Research. 44(2): 145-151. doi: 10.18805/LR-565.
Background: Comparative study of the complete chloroplast genomes of some species in the Subtribe Asclepiadeae was conducted to evaluate the variations and similarities between the species and to resolve the phylogenetic relationship within the subtribe. P. tomentosa has been used for medicinal uses in Saudi Arabia, Middle East, Africa and Brazil. It is used often in cosmetics and tanning industries, although it’s very well utilized as a traditional medicine in many civilizations.

Methods: The genomes were compared using Mvista Bioinformatics tools to evaluate the inverted repeats (IR), large single copy (LSC) and small single copy (SSC) regions and also the border junctions were visualized with IR scope to express the expansion and contraction of the circular genome structure. While SSR markers were determined using the Reputer program, the genome map was done using OGDRAW (OrganellarGenomeDRAW).

Result: Observed variations of the Mvista alignments is mainly at the coding regions of the sequences, while IR borders were varied at the SSC region of A. nivea genome; with ycf1 and rps19 due to evolutionary events. The genome sizes of C. procera are 166,010 bp, P. tomentosa 164,213bp, A. nivea 161,592 bp and C. wilfordii 161,180 bp. GC contents of A. nivea, C. wilfordii and P. tomentosa are 38% respectively; while C. procera is the least with 37%.; total SSR markers as well as the circular genome map were presented in this study.
Recent usage of the complete chloroplast genome in solving evolutionary questions has been on the rise due to its unique nature and its capability to unravel valuable information worthy of adopting, as well as the utilized widely in resolving phylogenetic problems. The nature of chloroplast varied as reported by different authors even though are similar in most angiosperms; in terms of their coding regions, sizes, tRNA genes and other related genes (Raubeson et al., 2007; Daniell et al., 2016; Yaradua et al., 2019). Complete chloroplast genomes have been used as a vital tool in revealing information for phylogenetic reexamining of complex taxa; it is used vividly in resolving some unanswered questions in plant taxonomy recently (Ruhsam et al., 2015). The chloroplast is a major distinctive feature of plants that is used in photosynthesis and production of starch, fatty acids, pigment and amino acid (Wicke et al., 2011).
The majority of the land plant has a circular quadripartite shape with four different sizes; which is very well conserved in its genes contents and also partaking in translation, transcription and photosynthesis (Jansen et al., 2005). Despite the conservative nature of the genes in cp genomes yet they undergo expansions and contractions over time which might result in loss and gains of introns which causes compounded rearrangements thereby causing variations in their contents and shapes from species to species. In addition to the evolutionary perspective, the chloroplast genome has important implications in chloroplast transformation such as the improved transgenic expression and absence of transgenic escape through pollen (Daniell et al., 2016).
P. tomentosa has been used for medicinal uses in Saudi Arabia, Middle East, Africa and Brazil. It is used often in cosmetics and tanning industries, although it’s very well utilized as a traditional medicine in many civilizations, it is also used for the treatment of bronchitis, headache and tuberculosis among other uses. In the present study whole plastome genome sequence of Pergularia tomentosa was characterized and compared with previously reported cp genomes of Calotropis procera, Asclepias nivea and Cynanchum wilfordii from the Genbank database as such; the study is done to make comparative genomics analysis of complete chloroplast genome of P. tomentosa, C. procera, Asclepias nivea and Cynanchum wilfordii Asclepiadoideae, Apocynaceae and also to evaluate the evolution and characteristics of accD gene within the species.
Plants sample collection and extractions of DNA
P. tomentosa plant material was collected from the Taif at Al-Shafa mountains (22° 4.9’31” N. 40° 18.6’28” E) a town about 25km from the holy city of Makkah and identified at the herbarium of KAAU Jeddah. Extraction of DNA was done according to the manual of the manufacture in which the leaves of P. tomentosa were washed with ethanol (70%) and Silica, followed by DNA extraction with DNeasy Plant Mini Kit (Qiagen, Inc., Germany). The quality of the DNA was checked using 1% agrose gel electrophoresis and Multiskan (Thermo Scientific Inc., USA). The DNA samples were sequenced at the Novogene, Beijing for paired-end reads 150 bp short reads and 350 bp size with Hiseq2500 (Illumina, USA).
Preparation of library, DNA sequencing and assembly
1.0 µg of the leaf sample was measured for DNA preparation to serve as an input. NEBNext DNA Library Kit was used to generate the sequencing libraries following the manufacturer’s manual using the DNeasy Plant Mini Kit (Qiagen, Inc., Germany); with indices included in each sample. DNA was fragmented erratically to 350bp size by shearings; polished DNAs strand was then ligated with NBNext adapter for Illumina sequencing followed with P5, P7 indexing; also purification of PCR and later analyzed for the effective magnitude of the DNA by the Agilent 2100 and measured using RT-PCR. Raw reads obtained from the Illumina sequencers were filtered to obtain a (5 GB) with PRINSEQlite v0.20.4 (Schmieder and Edward, 2011). NOVOPlasty2.7.2 program (Dierckxsen et al., 2016) was used for Assembly of the FastQ files with (Kmer = 32) to get the whole chloroplast genome out of the whole genome reads as well as the ndhAx1 to ndhAx2 ITS (intergenic spacer) of the Cynanchum wilfordii (KX352467K) were used as a seed while the sequence of Calotropis procera (NC_041440) as a reference. A complete chloroplast sequence was generated afterward.
Annotation of genes
Dual Organellar Genome Annotator program, (Wyman et al., 2004) University of Texas was employed for annotation of P. tomentosa plastome  in which manual adjustment of the stop and start codons were done; we then use tRNAscan-SE program www.lowaelab.ucsc.edu/tRNAscan-Se/in annotation of tRNAs.; while for drawing of chloroplast circular structure, an  OGDRAW (Lohse et al., 2007) was employed. The complete annotation was achieved and later deposited to GenBank with the assigned accession number (MN548766).
Repeats analysis
Simple sequence repeats (SSRs) were identified in the P. tomentosa chloroplast genome and genome of other three species of Asclepiadeae using the online software MIcroSAtellite (MISA) (Mayor et al., 2000) with the following parameters: eight, five, four and three repeats units for mononucleotides, dinucleotides, trinucleotides and tetra, Penta, hexanucleotides SSR motifs respectively.
Long repeats identification
Reputer software (Kurtz et al., 2001) was employed in tandem identifications, forward and a palindromic repeat of the genomes were identified following a manual method of 90% and >15 base pairs as sequence capture values.
The result of the analysis which includes the complete chloroplast genomes features and map of P. tomentosa and comparison with C. procera, Cynanchum wilfordii and Asclepias nivea as well as the presentation of the evolution of accD gene in Asclepiadoideae, Apoceanaceae were discussed.
Genes with Introns
Table 1 is narrating features of genes with introns in P. tomentosa whole chloroplast genome with clpP, accD and ycf3 both from LSC section of the genome were located at intron 2 and exon 3 respectively. The position of accD and ycf3 gens agrees with the finding of Yaradua et al., (2019).

Table 1: Genes with introns of P. tomentosa chloroplast genome with lengh of exon and intron observed.

SSR Analysis
Table 2 shows the SSR markers in P. tomentosa with about 42 repeats sequences which are either palindromic or forward. The findings are similar to the studies by Huang et al., 2020. The cp genome has a similar variability with the previously reported cp genomes of Asclepiadoideae subfamily such as the C. wilfordii (KT220733) (Park, et al., 2018) and C. procera (NC041440). All the genomes had a quadripartite structure consisting of SSC, SSC regions and inverted repeats.

Table 2: Total SSR Sequences in P. tomentosa genome.

Genome features analysis of P. tomentosa, C. procera, C. wilfordii and A. nivea
Table 3 Indicates genome features of P. tomentosa, C. procera, C.wilfordii and A. nivea, in which the C. procera genome size is 166,010 bp, P. tomentosa 164,213 bp, A. nivea 161,592 bp and C. wilfordii 161,180 bp (Fig 1). The total number of genes was highest in C. wilfordii 168, C. procera 163, while P. tomentosa and A. nivea are the least number of genes with 161 each However, the GC content of the genomes showed highest in A. nivea, C. wilfordii and P. tomentosa with 38% while C. procera is the least with 37%. tRNA content is highest in A. nivea and P. tomentosa 46% and low in C. procera and C. wilfordii 42% each; total rRNA genes were same in all the plants 8 each; the size of the inverted repeats (IR) were highest in C. procera 26,219bp, followed by A. nivea 25,472bp, C. wilfordii 24,637bp and P. tomentosa 21411bp. For the size of large singe copy (LSC) C. procera is the highest 94,104bp, then C. wilfordii 91,976bp, A. niveae 91,971bp and P. tomentosa being the least with 80,102bp; Also the size of Small single copy (SSC) of the plants C. wilfordii has the highest size with 19,930bp, then C. procera 19,468bp, followed by A. niveae with 18,771 and P. tomentosa with 17,022bp.

Table 3: Genome features of Pergularia tomentosa, Calotropis procera, Cynanchum wilfordii and Asclepias nivea.


Fig 1: Circular Complete Chloroplast genomes of P. tomentosa with genes which are residing within the circular shape are on clockwise direction while those outside are at the anti-clockwise.

Comparative genome analysis using Mvista alignment
Comparative genomic analysis in Fig 3 indicate that genes at non-coding regions are more conserved compared to the ones at the coding regions; such as ycf15, cemA, rpl16 and clpP genes were the most divergent regions observed. Genetic diversity in angiosperm chloroplast genomes has been reported in families such as the Angiosperms such as the Amaranthaceae, Caryophyllaceae, with some showing a character of loss of introns in coding regions (Daniell et al., 2008). The nature of chloroplast varied as reported by different authors even though are also similar in most angiosperms; in terms of the coding regions sizes, complete  gene number,  complete tRNA genes and complete protein genes (Raubeson and Jansen, 2005; Daniell et al., 2016; Yaradua et al., 2019). Distribution in the number of SSR Fig 2. revealed that non-coding region has higher SSR, followed by the coding region and the cp genome region.

Fig 2: Features of the non-protein genes, SSR types and coding genes in P. tomentosa genome.


Fig 3: Sequence alignment of four complete chloroplast of sub family Asclepiadoideae in Apoceanacee using mVISTA program.

Furthermore, the accD gene deals with the formation of fatty acids in plants programming the betacarboxyl transferase a derivative of acetyl coA (Sasaki and Nagano, 2004; Rousseau-Guetin et al., 2013; Kode et al., 2005). It was also reported that fatty acids contents and metabolic activities in plants are influenced by the accD gene expression (Lee et al., 2004; Nakkew et al., 2008); accD gene is a pseudo-gene in some angiosperm (Xiang et al., 2016) while exist as a complete gene in others (Ni et al., 2016b) the result of these study indicate about 6 highly conserved regions in the accD of P. tomentosa and A. nivea while two  and four highly conserved regions detected on accD gene of C. wilfordii and C. procera, respectively.

IR border junction analysis
Variations exist between the chloroplast genomes of P. tomentosa. C. procera, A. nivea and C. wilfordii as seen in Fig 4 where trnH, rpl22, ycf1, ndhF and rps19 genes are designated by the junction of IR andSSC regions. Variation was also observed at the SSC section of A. nivea genome; with ycf1 and rps19. Angiosperms chloroplast genome was generally known for its uniqueness and conservative features in structures and topology; even with many evolutionary events there exists  slight changes that lead to the variations among members of the same taxonomic groups due to contraction in the borders of the genomes. The comparison between plastome genomes of P. tomentosa, C. procera, A. nivea and C. wilfordii reveals some variations in their genome architecture.

Fig 4: Genome borders comparison of IR, SSC and LSC of four species of Asclepideae.

Phylogenetic analysis
Phylogenetic inference revealed that the genus Pergularia is a monophyletic genus (Fig 5) and a sister genus to Calotropis and Asclepias. This result agrees with the findings of Rapini et al., 2012 and Abba et al., 2020 based on complete chloroplast genome sequences of some species in the subfamily Asclepiadoideae. Therefore, this study highlighted the chloroplast genome architecture in Asclepiadoideae and the variations among the species studied. There is a close resemblance in the genome’s features; identified SSR markers (Fig 2) could be used in population genetics and Phylogenetic studies of the subfamily Asclepiadoideae.

Fig 5: Phylogenetic tree of Asclepiadoideae subfamily using complete Chloroplast genomes to infer Bayesian inference of the 9 species from Apocynaceae family.

The study involving the comparative genome sequence analysis of P. tomentosa and other species of Asclepideae was done in which the genome features such as the SSR markers and accD gene evolution were evaluated; as such that the information could be used for future decision making in Asclepiadoideae as well as the studies of evolutionary relationship, herb-genomics, plant breeding and conservation.
The project was financially supported by the Deanship of Scientific Research (DSR), King Abdulaziiz University, Jeddah, with grant No. (D144-260-131) and therefore authors appreciate the gesture.

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