Characterization of the Complete Chloroplast Genome of Blepharis Ciliaris (Acanthoideae, Acanthaceae)

Volume 13, Supplementary Issue, December 2020 ISSN 1995-6673 JJBS Pages 597 - 606 Jordan Journal of Biological Sciences Characterization of the Complete Chloroplast Genome of Blepharis ciliaris (Acanthoideae, Acanthaceae) Samaila S. Yaradua1, 2,*, Dhafer A. Alzahrani 1, Abidina Abba 1 and Enas J. 1 Albokhary 1Department of Biology, King Abdulaziz University, Jeddah, Saudi Arabia; 2Centre for Biodiversity and Conservation, Department of Biology, Umaru Musa Yaradua University, Katsina, Nigeria Received January 24, 2020; Revised February 17, 2020; Accepted: February 22, 2020 Abstract The complete chloroplast genome of Blepharis ciliaris, medicinal and endangered plant in Saudi Arabia was sequenced and characterized. NOVOPlasty was used to assemble the complete chloroplast genome from the whole genome data. The cp genome of B. ciliaris is 149, 717 bp in length with GC content of 38.5% and has a circular and quadripartite structure; the genome harbors one pair of inverted repeat (IRa and IRb 25, 331bp each) separated by large single copy (LSC, 87, 073 bp) and small single copy (SSC, 16, 998 bp). There are 131 genes in the genome, which include 79 protein-coding genes, 30 tRNA and 4 rRNA; 113 are unique while the remaining 18 are duplicated in IR regions. The repeat analysis indicates that the genome contained all types of repeats with palindromic occurring more frequently; the analysis also identified a total number of 91 simple sequence repeats (SSR) of which the majority are mononucleotides A/T and are found in the intergenic spacer. This study reported the first cp genome of the genus Blepharis and provides resources for studying the genetic diversity of B. ciliris as well as resolving phylogenetic relationship within the core Acanthaceae. Keywords: Acanthaceae; Blepharis ciliaris; chloroplast genome; SSR Walker et al., 2015; Sun et al., 2017). These 1. Introduction rearrangements occur as a result of contractions, expansions and inversions in the single copy regions (large Blepharis ciliaris (L.) B. L. Burtt. is a member of the single copy and small single copy) and the inverted repeats tribe Acantheae (Acanthaceae). The species is known to be (Palmer et al., 1987; Tangphatsornruang et al., 2009). The distributed in Saudi Arabia, East Africa, East Pakistan and rearrangements of the genes and inversion in the Egypt (Kamal and Abdul, 1956). The plant is used as chloroplast genome are reported to be useful in fodder for ruminant animals particularly camel and sheep. phylogenetic analyses to solve taxonomic problems at The plant seeds (powdered) are used as an antibacterial on various taxonomic levels because they do not occur often boils, wounds and sores. The seed of the plant is being and estimation of their homology and inversion event used to treat cough and has diuretic, aphrodisiac and polarity is simple (Johansson, 1999; Lee et al., 2007; antibacterial activity (Deshpande, 2006). In addition, the Jansen et al., 2008; Yan et al., 2017). With the importance charcoal from the root is used to improve eye vision and of complete chloroplast genome in evolutionary studies, treat sore eyes (Boulos, 1981; Tackholm, 1974). Despite resolving phylogenetic relationship issues and the large the endangered nature and uses in traditional medicine of number of genera and species in Acanthaceae, only the species, the complete chloroplast genome of the complete chloroplast genome of 8 genera have been so far species was not characterized until this study. reported (Andrographis paniculata (Burm.f.) Nees, Comparison of complete chloroplast genome provides NC_022451; Ruellia breedlovei T. F. Daniel, very informative information for the reconstruction of KP300014; Strobilanthes cusia (Nees) O. Kuntze, phylogeny and resolving evolutionary relationship issues MG874806 and four species Echinacanthus at various taxonomic levels (Shaw et al., 2007; Mardanov MF490441, MH045155, MH045156, and MH045157). et al.,2008; Moore et al., 2010; Park et al., 2017; Sun et Seven genera belong to cystolith clade, and only 1 genus al.,2017). This is as a result of the conservative nature of belongs to non cystolith clade the chloroplast genome (Wolfe et al., 1987); this In this study, we reported the characteristics of the conservative nature is because the plastome evolves about complete chloroplast genome of Blepharis ciliaris, the half the rate of other genomes like the nuclear genome second cp genome to be sequenced in Acantheae lineage. (Jansen et al., 2005; Walker et al., 2014). However, Moreover, its simple sequence repeats were reported to rearrangements in the sequence of chloroplast genome provide the tools for genetic diversity and identification of were reported by various plant chloroplast genome studies the species and lastly to resolve the status of Acantheae (Doyle et al., 1996; Tangphatsornruang et al., 2011; * Corresponding author e-mail: [email protected]. 598 © 2020 Jordan Journal of Biological Sciences. All rights reserved - Volume 13, Supplementary Issue 2. Materials and Methods 3. Results and Discussion 2.1. Plant material and DNA extraction 3.1. Characteristics of B.ciliaris chloroplast genome Plant material (vegetative and floral part) was collected The complete plastome sequence of B. ciliaris has a through field survey of B. ciliaris in North Jeddah, Saudi circular and quadripartite structure. The total length of the Arabia. Plant identification and DNA extraction were done genome is 149, 717 bp (Samaila et al., 2019). The according to Samaila et al., (2019). plastome has four distinct regions which are Small Single 2.2. Library construction, sequencing and assembly Copy (SSC), Large Single Copy (LSC), and a pair of Inverted repeats (IRa and IRb) which separate the small A total amount of 1.0µg DNA was used as input single copy and large single copy (Figure 1). The region material for the DNA sample preparations. Sequencing coding for genes is 87, 073bp in length which constitutes libraries were generated using NEBNext® DNA Library 58.15% of the genome, the remaining 62, 644 bp is the Prep Kit following the manufacturer's recommendations non-coding region which includes intron and intergenic and indices were added to each sample (Samaila et al., spacer (41.85%). The length of the SSC, LCS, IRa and IRb 2019). The raw reads were filtered to get the clean reads (5 is 16, 998 bp, 82, 057 bp, 25, 331 bp and 25, 331 bp, Gb) using PRINSEQ lite v0.20.4 (Schmieder and Edwards, respectively. The LSC and SSC regions possessed GC 2011) and were subjected to de novo assembly using content of 36.6 % and 32.4 %, respectively, while the NOVOPlasty2.7.2 (Dierckxsens et al., 2016) with kmer K- inverted repeats IRa and IRb have 43.6 % (Table 1). The mer= 31 (Samaila et al., 2019) architecture of the B. ciliaris cp genome is like other 2.3. Gene annotation reported Acanthoideae cp genomes (Chunming et al., The program DOGMA (Dual Organellar Genome 2019; Yongbin and Erin, 2017). The plastome sequence Annotator, University of Texas at Austin, Austin, TX, has GC of 38.5% and AT content of 61.5%; this is USA) (Wyman, et al., 2004) was used to annotate the consistent with the plastome data of Strobilanthes cusia genes in the assembled chloroplast genome. The positions (Chen et al., 2018). The percentage of GC in the inverted of start and stop codon were adjusted manually. trnAscan- repeat regions is found to be higher than the large single SE server (http://lowelab.ucsc.edu/tRNAscan- copy region and small single region. SE/)(Schattner et al., 2005) was used to verify the tRNA Table 1. Base composition in the B. ciliaris chloroplast genome. genes and finally, the plastome genome circular map was Region T(U) C A G Total drawn using OGDRAW (Organellar Genome DRAW) (Lohse et al., 2007). The sequence of the chloroplast cp genome 31.1 19.6 30.4 18.9 149717.0 genome of B. ciliaris was deposited in the GenBank LSC 32.4 18.8 31.0 17.8 82057.0 database with the accession number (MK548576). SSC 33.7 16.7 33.9 15.7 16998.0 2.4. Sequence Analysis IRA 28.3 22.6 28.1 21.0 25331.0 MEGA 6.0 was used to analyze the relative IRB 28.1 21.0 28.3 22.6 25331.0 synonymous codon usage values (RSCU), base 1st Position 31 19.5 30.4 18.7 49906.0 composition and codon usage. Possible RNA editing sites present in the protein coding genes of B. ciliaris cp 2nd Position 31 19.6 30.0 19.1 49906.0 genome were determined using PREP suite (Kurtz et al., 3rd Postion 31 19.7 30.7 19.0 49905.0 2001) with 0.8 as the cutoff value. 2.5. Repeat analysis in B. ciliaris chloroplast genome The complete chloroplast genome of B. ciliaris contained a total of 131 genes; 113 genes out of the 131 Simple sequence repeats (SSRs) were identified in the are unique and are present in the single copy regions (large B. ciliaris chloroplast genome using the online software single copy containing 83 genes and the small single copy MIcroSAtellite (MISA) (Thiel et al., 2003) with the contained 13 genes); 18 genes are duplicated in the pair of following parameters: eight, five, four and three repeats the inverted repeat region which include 8 protein-coding units for mononucleotides, dinucleotides, trinucleotides genes, 4 rRNAs and 7 tRNAs. There are 79 protein-coding and tetra, penta, hexa nucleotides SSR motifs respectively. genes, 4 rRNAs and 30 tRNAS in the plastome (Table 2 For analysis of long repeats (palindromic, forward, reverse and Figure 1). The structural organization and the number and complement) the program REPuter of genes in the plastome is consistent with that of the (https://bibiserv.cebitec.uni-bielefeld.de/reputer) (Kurtz sequenced Acanthoideae cp genome (Chunming et al., et al., 2001) with default parameters was used to identify 2019; Yongbin and Erin, 2017).

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