Sequencing, assembly and annotation of the mitochondrial and plastid genomes of Gelidium pristoides (Turner) Kützing from Kenton-on-Sea, South Africa
- Authors: Mangali, Sandisiwe
- Date: 2019
- Subjects: Gelidium -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/19109 , 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.
- Full Text:
- Date Issued: 2019
- Authors: Mangali, Sandisiwe
- Date: 2019
- Subjects: Gelidium -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10353/19109 , 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.
- Full Text:
- Date Issued: 2019
Phylogeography and epifauna of two intertidal seaweeds on the coast of South Africa
- Authors: Mmonwa, Lucas Kolobe
- Date: 2009
- Subjects: Phylogeography -- South Africa , Marine algae -- South Africa , Red algae -- South Africa , Gelidium -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5738 , http://hdl.handle.net/10962/d1005424 , Phylogeography -- South Africa , Marine algae -- South Africa , Red algae -- South Africa , Gelidium -- South Africa
- Description: Southern African biogeographic boundaries delimit the phylogeographic distribution of some coastal and estuarine invertebrates. This study investigated the impact of these boundaries on the phylogeographic distribution of two intertidal red seaweeds, Gelidium pristoides and Hypnea spicifera using the mitochondrial Cox2-3 spacer and the nuclear ITS1 regions. G. pristoides spores have short distance-dispersal, while long distance-dispersal is more likely in H. spicifera via spores and drifting fertile thallus fragments. Both markers revealed a south-western and south-eastern lineage within G. pristoides but the breaks between lineages do not coincide with any recognised biogeographic limits. The Cox2-3 spacer revealed a boundary between the two lineages at the Alexandria Coastal Dunefield (ACD) and ITS1 at the Gamtoos-Van Stadens Dunefields (GVD) which is approximately 80km west of the ACD. The minor difference between the two markers regarding location of the phylogeographic boundary is probably due to the dating differences between the two dunefields. The ACD as developed currently is superimposed on the ancient dunefields which formed during the Pleistocene, coinciding with the Cox2-3 spacer sequences divergence which dates back 500,000 - 580,000 years. The GVD formed during the Holocene (6,500 - 4,000 years ago), coinciding with the ITS1 sequences divergence which dates 4,224 - 4,928 years ago. Thus, these phylogeographic boundaries probably appeared without the influence of biogeographic boundaries, but rather due to the lack of suitable habitat in the dunefields, coupled with short dispersal-distances of the spores. Analysis of the ITS1 and Cox2-3 spacer regions in H. spicifera revealed that the species is characterized by uniform genetic structure along the coastline. This reflects the species`s potential for long range expansion as it inhabits both the intertidal and subtidal zones; and this presumably leads to high gene flow among populations. The ITS1 sequences showed minimal genetic variation of one substitution between the gametophyte and tetrasporophyte generations within H. spicifera. This suggests the predominance of asexual reproduction, which reduces gene flow and fixes alleles between generations. ANOSIM and Bray-Curtis cluster analyses showed scale-dependant variation in the abundances of epifauna (mainly amphipod, isopod, mollusc and polychaete species) on G. pristoides. At small local (within site) and large (among sites) scales, there were weak and no structure in epifaunal abundances respectively. However, at larger, biogeographic scales, samples from the same biogeographic region tended to be clustered together. Thus, there was a group containing predominantly south coast samples and a group containing east coast samples mixed with the remaining south coast samples. Such scale-dependant variation in epifaunal abundances is probably due to the effects of factors driving species richness at small local (within site) scales (e.g. wave exposure, seaweed biomass) and at larger, biogeographic scales (e.g. surface sea temperature). Moreover, at very small (individual samples) scales; there was no correlation between epifauna composition and genotype of the seaweed. Seaweed samples characterized by distinct ITS1 or Cox2-3 spacer sequences did not show any significant differences in epifaunal composition. Although the distributional pattern of the epifaunal community observed at large biogeographic scale is not clear, it seems to be associated with the biogeographic regions. However, phylogeographic distribution of Gelidium pristoides is not connected to biogeographic regions. Thus, at larger, biogeographic scales, there is no correlation between phylogeographic distribution of G. pristoides and distribution of the associated fauna
- Full Text:
- Date Issued: 2009
- Authors: Mmonwa, Lucas Kolobe
- Date: 2009
- Subjects: Phylogeography -- South Africa , Marine algae -- South Africa , Red algae -- South Africa , Gelidium -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5738 , http://hdl.handle.net/10962/d1005424 , Phylogeography -- South Africa , Marine algae -- South Africa , Red algae -- South Africa , Gelidium -- South Africa
- Description: Southern African biogeographic boundaries delimit the phylogeographic distribution of some coastal and estuarine invertebrates. This study investigated the impact of these boundaries on the phylogeographic distribution of two intertidal red seaweeds, Gelidium pristoides and Hypnea spicifera using the mitochondrial Cox2-3 spacer and the nuclear ITS1 regions. G. pristoides spores have short distance-dispersal, while long distance-dispersal is more likely in H. spicifera via spores and drifting fertile thallus fragments. Both markers revealed a south-western and south-eastern lineage within G. pristoides but the breaks between lineages do not coincide with any recognised biogeographic limits. The Cox2-3 spacer revealed a boundary between the two lineages at the Alexandria Coastal Dunefield (ACD) and ITS1 at the Gamtoos-Van Stadens Dunefields (GVD) which is approximately 80km west of the ACD. The minor difference between the two markers regarding location of the phylogeographic boundary is probably due to the dating differences between the two dunefields. The ACD as developed currently is superimposed on the ancient dunefields which formed during the Pleistocene, coinciding with the Cox2-3 spacer sequences divergence which dates back 500,000 - 580,000 years. The GVD formed during the Holocene (6,500 - 4,000 years ago), coinciding with the ITS1 sequences divergence which dates 4,224 - 4,928 years ago. Thus, these phylogeographic boundaries probably appeared without the influence of biogeographic boundaries, but rather due to the lack of suitable habitat in the dunefields, coupled with short dispersal-distances of the spores. Analysis of the ITS1 and Cox2-3 spacer regions in H. spicifera revealed that the species is characterized by uniform genetic structure along the coastline. This reflects the species`s potential for long range expansion as it inhabits both the intertidal and subtidal zones; and this presumably leads to high gene flow among populations. The ITS1 sequences showed minimal genetic variation of one substitution between the gametophyte and tetrasporophyte generations within H. spicifera. This suggests the predominance of asexual reproduction, which reduces gene flow and fixes alleles between generations. ANOSIM and Bray-Curtis cluster analyses showed scale-dependant variation in the abundances of epifauna (mainly amphipod, isopod, mollusc and polychaete species) on G. pristoides. At small local (within site) and large (among sites) scales, there were weak and no structure in epifaunal abundances respectively. However, at larger, biogeographic scales, samples from the same biogeographic region tended to be clustered together. Thus, there was a group containing predominantly south coast samples and a group containing east coast samples mixed with the remaining south coast samples. Such scale-dependant variation in epifaunal abundances is probably due to the effects of factors driving species richness at small local (within site) scales (e.g. wave exposure, seaweed biomass) and at larger, biogeographic scales (e.g. surface sea temperature). Moreover, at very small (individual samples) scales; there was no correlation between epifauna composition and genotype of the seaweed. Seaweed samples characterized by distinct ITS1 or Cox2-3 spacer sequences did not show any significant differences in epifaunal composition. Although the distributional pattern of the epifaunal community observed at large biogeographic scale is not clear, it seems to be associated with the biogeographic regions. However, phylogeographic distribution of Gelidium pristoides is not connected to biogeographic regions. Thus, at larger, biogeographic scales, there is no correlation between phylogeographic distribution of G. pristoides and distribution of the associated fauna
- Full Text:
- Date Issued: 2009
The ecophysiology of Gelidium Pristoides (Turner) Kuetzing : towards commercial cultivation
- Authors: Steyn, Paul-Pierre
- Date: 2009
- Subjects: Marine algae -- South Africa , Marine algae -- Ecophysiology , Red algae -- South Africa , Gelidium -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10617 , http://hdl.handle.net/10948/1117 , Marine algae -- South Africa , Marine algae -- Ecophysiology , Red algae -- South Africa , Gelidium -- South Africa
- Description: The ecophysiology of the red alga Gelidium pristoides (Turner) Kuetzing was investigated in an effort to establish a technique for commercial cultivation. The seaweed is of commercial importance in South Africa where it is harvested from the intertidal zone rocky shores along the coast. It is dried and exported abroad for the extraction of agar. Yields and quality could be improved by cultivation in commercial systems. However, attempts at growing the seaweed in experimental systems have all ended in failure. This study aimed to describe the conditions in which the seaweed grows naturally; and investigate its physiological response to selected physical conditions in the laboratory in order to determine suitable conditions for mariculture. Ecological studies showed that G. pristoides grew above the spring low tide water level. The upper limit of the seaweed’s vertical distribution range, as well as its abundance, was largely dependent on wave exposure. The zone normally inhabited by G. pristoides was dominated by coralline turf in sheltered areas, while the abundance of G. pristoides increased towards more exposed rocky shore sites. The seaweed occurred among species such as Pattelid limpets and barnacles, but was usually the dominant macroalga in this zone, with coralline turf and encrusting algae being the only others. Physical conditions in the part of the intertidal zone inhabited by G. pristoides were highly variable. During low tide temperatures could vary by as much as 10°C within the three hours between tidal inundation of the seaweed population, while salinity varied by up to 9 ppt, and light intensity by as much as 800 μmol m-2 s-1. During these exposure periods the seaweed suffered up to 20% moisture loss. Laboratory experiments on the seaweed’s response to these conditions indicated that it was well adapted to such fluctuations. It had a broad salinity (20 and 40 ppt), and temperature tolerance range (18 to 24°C), with an o ptimum of temperature of 21°C for photosynthesis, while there was no difference in the photosynthetic rate of the alga within the 20 to 40 ppt salinity range. The alga had a low saturating irradiance (ca. 45 – 80 μmol m-2 s-1) equipping it well for photosynthesis in turbulent environments, with high light attenuation, but poorly to unattenuated light conditions. Exposure resulted in an initial increase in photosynthetic rate followed by a gradual decrease thereafter. pH drift experiments showed that low seawater pH, and associated increased carbon dioxide availability, resulted in an increase in photosynthetic rate. This response suggests that the seaweed has a high affinity for carbon dioxide, while the reduction in photosynthetic rate in response to bicarbonate use inhibition indicates that it also has the capacity for bicarbonate use. The high affinity of Gelidium pristoides for carbon dioxide as an inorganic carbon source appears to be the primary reason for the low abundance of the alga on sheltered rocky shore areas, and also explains the failure of the alga to grow in tank or open-water mariculture systems. Exposed rocky shores have experience heavy wave action, and the resultant aeration and mixing of nearshore waters increases the availability of carbon dioxide, which is considered a limiting resource. The absence of such mixing and aeration at sheltered site makes this less suitable habitat for G. pristoides. Periodic exposure also makes high levels of atmospheric carbon dioxide available from which the seaweed benefits. The traditional mariculture systems in which attempts have been made to cultivate the seaweed failed to satisfy either of the above conditions.
- Full Text:
- Date Issued: 2009
- Authors: Steyn, Paul-Pierre
- Date: 2009
- Subjects: Marine algae -- South Africa , Marine algae -- Ecophysiology , Red algae -- South Africa , Gelidium -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:10617 , http://hdl.handle.net/10948/1117 , Marine algae -- South Africa , Marine algae -- Ecophysiology , Red algae -- South Africa , Gelidium -- South Africa
- Description: The ecophysiology of the red alga Gelidium pristoides (Turner) Kuetzing was investigated in an effort to establish a technique for commercial cultivation. The seaweed is of commercial importance in South Africa where it is harvested from the intertidal zone rocky shores along the coast. It is dried and exported abroad for the extraction of agar. Yields and quality could be improved by cultivation in commercial systems. However, attempts at growing the seaweed in experimental systems have all ended in failure. This study aimed to describe the conditions in which the seaweed grows naturally; and investigate its physiological response to selected physical conditions in the laboratory in order to determine suitable conditions for mariculture. Ecological studies showed that G. pristoides grew above the spring low tide water level. The upper limit of the seaweed’s vertical distribution range, as well as its abundance, was largely dependent on wave exposure. The zone normally inhabited by G. pristoides was dominated by coralline turf in sheltered areas, while the abundance of G. pristoides increased towards more exposed rocky shore sites. The seaweed occurred among species such as Pattelid limpets and barnacles, but was usually the dominant macroalga in this zone, with coralline turf and encrusting algae being the only others. Physical conditions in the part of the intertidal zone inhabited by G. pristoides were highly variable. During low tide temperatures could vary by as much as 10°C within the three hours between tidal inundation of the seaweed population, while salinity varied by up to 9 ppt, and light intensity by as much as 800 μmol m-2 s-1. During these exposure periods the seaweed suffered up to 20% moisture loss. Laboratory experiments on the seaweed’s response to these conditions indicated that it was well adapted to such fluctuations. It had a broad salinity (20 and 40 ppt), and temperature tolerance range (18 to 24°C), with an o ptimum of temperature of 21°C for photosynthesis, while there was no difference in the photosynthetic rate of the alga within the 20 to 40 ppt salinity range. The alga had a low saturating irradiance (ca. 45 – 80 μmol m-2 s-1) equipping it well for photosynthesis in turbulent environments, with high light attenuation, but poorly to unattenuated light conditions. Exposure resulted in an initial increase in photosynthetic rate followed by a gradual decrease thereafter. pH drift experiments showed that low seawater pH, and associated increased carbon dioxide availability, resulted in an increase in photosynthetic rate. This response suggests that the seaweed has a high affinity for carbon dioxide, while the reduction in photosynthetic rate in response to bicarbonate use inhibition indicates that it also has the capacity for bicarbonate use. The high affinity of Gelidium pristoides for carbon dioxide as an inorganic carbon source appears to be the primary reason for the low abundance of the alga on sheltered rocky shore areas, and also explains the failure of the alga to grow in tank or open-water mariculture systems. Exposed rocky shores have experience heavy wave action, and the resultant aeration and mixing of nearshore waters increases the availability of carbon dioxide, which is considered a limiting resource. The absence of such mixing and aeration at sheltered site makes this less suitable habitat for G. pristoides. Periodic exposure also makes high levels of atmospheric carbon dioxide available from which the seaweed benefits. The traditional mariculture systems in which attempts have been made to cultivate the seaweed failed to satisfy either of the above conditions.
- Full Text:
- Date Issued: 2009
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