- Title
- Sequencing, assembly and annotation of the mitochondrial and plastid genomes of Gelidium pristoides (Turner) Kützing from Kenton-on-Sea, South Africa
- Creator
- Mangali, Sandisiwe
- Subject
- Gelidium -- South Africa
- Date
- 2019
- Type
- Thesis
- Type
- Masters
- Type
- MSc
- Identifier
- http://hdl.handle.net/10353/19109
- Identifier
- vital:39883
- Description
- The genome is the complete set of an organism's hereditary information that contains all the information necessary for the functioning of that organism. Complete nuclear, mitochondrial and plastid DNA constitute the three main types of genomes which play interconnected roles in an organism. Genome sequencing enables researchers to understand the regulation and expression of the various genes and the proteins they encode. It allows researchers to extract and analyse genes of interests for a variety of studies including molecular, biotechnological, bioinformatics and conservation and evolutionary studies. Genome sequencing of Rhodophyta has received little attention. To date, no published studies are focusing on both whole genome sequencing and sequencing of the organellar genomes of Rhodophyta species found in along the South African coastline. This study focused on genome sequencing, assembly and annotation mitochondrial and plastid genomes of Gelidium pristoides. Gelidium pristoides was collected from Kenton-on-Sea and was morphologically identified at Rhodes University. Its genomic DNA was extracted using the Nucleospin® Plant II kit and quantified using Qubit 2.0, Nanodrop and 1% agarose gel electrophoresis. The Ion Plus Fragment Library kit was used for the preparation of a 600 bp library, which was sequenced in two separate runs through the Ion S5 platform. The produced reads were quality-controlled through the Ion Torrent server version 5.6. and assessed using the FASTQC program. The SPAdes version 3.11.1 assembler was used to assemble the quality-controlled reads, and the resultant genome assembly was quality-assessed using the QUAST 4.1 software. The mitochondrial genome was selected from the produced Gelidium pristoides draft genome using mitochondrial genomes of other Gelidiales as search queries on the local BLAST algorithm of the BioEdit software. Contigs matching the organellar genomes were ordered according to the mitochondrial genomes of other Gelidiales using the trial version of Geneious R11.12 software. The plastid genome was also selected following the same approach but using plastid genomes of Gelidium elegans and Gelidium vagum as search queries. Gaps observed in the organellar genomes were closed by amplification of the relevant gap using polymerase chain reaction with newly designed primers and Sanger sequencing. Open reading frames for both organellar genomes were annotated using the NCBI ORF-Finder and alignments obtained from BlastN and BlastX searches from the NCBI database, while the tRNAs and rRNAs were identified using the tRNAscan-SE1.21 vi and the RNAmmer 1.2 servers. The circular physical map of the mitochondrial genome was constructed using the CGView server. Lastly, in silico analysis of cytochrome c oxidase 3 and Heat Shock Protein 70 was performed using the PRIMO and the SWISS-MODEL pipelines respectively. Their phylogenies were analysed through Clustal omega and the trees viewed on TreeView 1.6.6 software. Qubit and Nanodrop genomic DNA qualification revealed A260/A280 and A230/A260 ratios of 1.81 and 1.52 respectively. The 1% agarose gel electrophoresis further confirmed the good quality of the genomic DNA used for library preparation and sequencing. Pre-assembly quality control of reads resulted in a total of 30 792 074 high-quality reads which were assembled into a total of 94140 contigs, making up an estimated genome length of 217.06 Mb. The largest contig covered up to 13.17 kb of the draft genome, and an N50 statistic value of 3.17 kb was obtained. The G.pristoides mitochondrial genome mapped into a circular molecule of 25012 bp, with an overall GC content of 31.04% and a total of 45 genes distributed into 20 tRNA-coding, 2 rRNAcoding genes and 23 protein-coding genes, mostly adopting the modified genetic code of Rhodophyta. The SecY and rps12 genes overlapped by 41 bp. This study presents a partial plastid genome composed of 89 (38%) fully annotated genes, of which 71 are protein-coding, and 18 are distributed among 15 tRNA-coding, 2 rRNA-coding and 1 RNaseP RNA-coding genes. Sixty-one (26%) partial protein-coding genes were predicted, while approximately 84 (36%) genes are not yet predicted. In silico analysis of the cytochrome c oxidase and heat shock protein 70 showed that the gene sequences obtained in this study and the resultant transcribed protein have sequences and structures that are similar to those from several other different species, thus validating the integrity of the genome sequences. This study provides genomic data necessary for understanding the genomic constituent of G.pristoides and serve as a foundation for studies of individual genes and for resolving evolutionary relationships.
- Format
- 152 leaves
- Format
- Publisher
- University of Fort Hare
- Publisher
- Faculty of Science and Agriculture
- Language
- English
- Rights
- University of Fort Hare
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UFH Department of Biochemistry & Microbiology
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