Topographically Distinguished Microbiome Taxonomy and Stress-Response Genes of Royal Belum Rainforest and Raja Muda Musa Peat Swamp Revealed through Metagenomic Inquisition.

Soil ecosystems are home to a diverse range of microorganisms, but they are only partially understood because no single-cell sequencing or whole-community sequencing provides a complete picture of these complex communities. Using one of such metagenomics approaches, we succeeded in monitoring the microbial diversity and stress-response gene in the soil samples. This study aims to test whether known differences in taxonomic diversity and composition are reflected in functional gene profiles by implementing whole gene sequencing (WGS) metagenomic analysis of geographically dispersed soils from two distinct pristine forests. The study was commenced by sequencing three rainforest soil samples and three peat swamp soil samples. Soil richness effects were assessed by exploring the changes in specific functional gene abundances to elucidate physiological constraints acting on different soil systems and identify variance in functional pathways relevant to soil biogeochemical cycling. Proteobacteria shows abundances of microbial diversity for 52.15% in Royal Belum Reserved Forest and 48.28% in Raja Musa; 177 out of 1,391,841 and 449 out of 3,586,577 protein coding represent acidic stress-response genes for Royal Belum and Raja Musa, respectively. Raja Musa indicates pH 2.5, which is extremely acidic. The analysis of the taxonomic community showed that Royal Belum soils are dominated by bacteria (98% in Sungai Kooi (SK), 98% in Sungai Papan (SP), and 98% in Sungai Ruok (SR), Archaea (0.9% in SK, 0.9% in SP, and 1% in SR), and the remaining were classed under Eukaryota and viruses. Likewise, the soils of Raja Muda Musa are also dominated by bacteria (95% in Raja Musa 1 (RM1), 98% in Raja Musa 2 (RM2), and 96% in Raja Musa 3 (RM3)), followed by Archaea (4% in RM1, 1% in RM2, and 3% in RM3), and the remaining were classed under Eukaryota and viruses. This study revealed that RBFR (Royal Belum Foresr Reserve) and RMFR (Raja Musa Forest Reserve) metagenomes contained abundant stress-related genes assigned to various stress-response pathways, many of which did not show any difference among samples from both sites. Our findings indicate that the structure and functional potential of the microbial community will be altered by future environmental potential as the first glimpse of both the taxonomic and functional composition of soil microbial communities.

Transcriptome analysis of Rafflesia cantleyi flower stages reveals insights into the regulation of senescence.

Rafflesia is a unique plant species existing as a single flower and produces the largest flower in the world. While Rafflesia buds take up to 21 months to develop, its flowers bloom and wither within about a week. In this study, transcriptome analysis was carried out to shed light on the molecular mechanism of senescence in Rafflesia. A total of 53.3 million high quality reads were obtained from two Rafflesia cantleyi flower developmental stages and assembled to generate 64,152 unigenes. Analysis of this dataset showed that 5,166 unigenes were differentially expressed, in which 1,073 unigenes were identified as genes involved in flower senescence. Results revealed that as the flowers progress to senescence, more genes related to flower senescence were significantly over-represented compared to those related to plant growth and development. Senescence of the R. cantleyi flower activates senescence-associated genes in the transcription activity (members of the transcription factor families MYB, bHLH, NAC, and WRKY), nutrient remobilization (autophagy-related protein and transporter genes), and redox regulation (CATALASE). Most of the senescence-related genes were found to be differentially regulated, perhaps for the fine-tuning of various responses in the senescing R. cantleyi flower. Additionally, pathway analysis showed the activation of genes such as ETHYLENE RECEPTOR, ETHYLENE-INSENSITIVE 2, ETHYLENE-INSENSITIVE 3, and ETHYLENE-RESPONSIVE TRANSCRIPTION FACTOR, indicating the possible involvement of the ethylene hormone response pathway in the regulation of R. cantleyi senescence. Our results provide a model of the molecular mechanism underlying R. cantleyi flower senescence, and contribute essential information towards further understanding the biology of the Rafflesiaceae family.

New Insights on the Evolution of the Sweet Taste Receptor of Primates Adapted to Harsh Environments.

Taste perception is an essential function that provides valuable dietary and sensory information, which is crucial for the survival of animals. Studies into the evolution of the sweet taste receptor gene (TAS1R2) are scarce, especially for Bornean endemic primates such as Nasalis larvatus (proboscis monkey), Pongo pygmaeus (Bornean orangutan), and Hylobates muelleri (Muller's Bornean gibbon). Primates are the perfect taxa to study as they are diverse dietary feeders, comprising specialist folivores, frugivores, gummivores, herbivores, and omnivores. We constructed phylogenetic trees of the TAS1R2 gene for 20 species of anthropoid primates using four different methods (neighbor-joining, maximum parsimony, maximum-likelihood, and Bayesian) and also established the time divergence of the phylogeny. The phylogeny successfully separated the primates into their taxonomic groups as well as by their dietary preferences. Of note, the reviewed time of divergence estimation for the primate speciation pattern in this study was more recent than the previously published estimates. It is believed that this difference may be due to environmental changes, such as food scarcity and climate change, during the late Miocene epoch, which forced primates to change their dietary preferences. These findings provide a starting point for further investigation.

Transcriptome landscape of Rafflesia cantleyi floral buds reveals insights into the roles of transcription factors and phytohormones in flower development.

Rafflesia possesses unique biological features and known primarily for producing the world's largest and existing as a single flower. However, to date, little is known about key regulators participating in Rafflesia flower development. In order to further understand the molecular mechanism that regulates Rafflesia cantleyi flower development, RNA-seq data from three developmental stages of floral bud, representing the floral organ primordia initiation, floral organ differentiation, and floral bud outgrowth, were analysed. A total of 89,890 transcripts were assembled of which up to 35% could be annotated based on homology search. Advanced transcriptome analysis using K-mean clustering on the differentially expressed genes (DEGs) was able to identify 12 expression clusters that reflect major trends and key transitional states, which correlate to specific developmental stages. Through this, comparative gene expression analysis of different floral bud stages identified various transcription factors related to flower development. The members of WRKY, NAC, bHLH, and MYB families are the most represented among the DEGs, suggesting their important function in flower development. Furthermore, pathway enrichment analysis also revealed DEGs that are involved in various phytohormone signal transduction events such as auxin and auxin transport, cytokinin and gibberellin biosynthesis. Results of this study imply that transcription factors and phytohormone signalling pathways play major role in Rafflesia floral bud development. This study provides an invaluable resource for molecular studies of the flower development process in Rafflesia and other plant species.

Unravelling the adaptation strategies employed by Glaciozyma antarctica PI12 on Antarctic sea ice.

Glaciozyma antarctica PI12, is a psychrophilic yeast isolated from Antarctic sea. In this work, Expressed Sequence Tags (EST) from cells exposed to three different temperatures; 15 °C, 0 °C and -12 °C were generated to identify genes associated with cold adaptation. A total of 5376 clones from each library were randomly picked and sequenced. Comparative analyses from the resulting ESTs in each condition identified several groups of genes required for cold adaptation. Additionally, 319 unique transcripts that encoded uncharacterised functions were identified in the -12 °C library and are currently unique to G. antarctica. Gene expression analysis using RT-qPCR revealed two of the unknown genes to be up-regulated at -12 °C compared to 0 °C and 15 °C. These findings further contribute to the collective knowledge into G. antarctica cold adaptation and as a resource for understanding the ecological and physiological tolerance of psychrophilic microbes in general.

The Glaciozyma antarctica genome reveals an array of systems that provide sustained responses towards temperature variations in a persistently cold habitat.

Extremely low temperatures present various challenges to life that include ice formation and effects on metabolic capacity. Psyhcrophilic microorganisms typically have an array of mechanisms to enable survival in cold temperatures. In this study, we sequenced and analysed the genome of a psychrophilic yeast isolated in the Antarctic region, Glaciozyma antarctica. The genome annotation identified 7857 protein coding sequences. From the genome sequence analysis we were able to identify genes that encoded for proteins known to be associated with cold survival, in addition to annotating genes that are unique to G. antarctica. For genes that are known to be involved in cold adaptation such as anti-freeze proteins (AFPs), our gene expression analysis revealed that they were differentially transcribed over time and in response to different temperatures. This indicated the presence of an array of adaptation systems that can respond to a changing but persistent cold environment. We were also able to validate the activity of all the AFPs annotated where the recombinant AFPs demonstrated anti-freeze capacity. This work is an important foundation for further collective exploration into psychrophilic microbiology where among other potential, the genes unique to this species may represent a pool of novel mechanisms for cold survival.

RNA-seq data from different developmental stages of Rafflesia cantleyi floral buds.

Rafflesia cantleyi, known as one of the world's largest flowers, is a specialised holoparasite due to dramatic morphological modifications. It possesses highly reduced vegetative structure and only appears as a flower for sexual reproduction. Moreover, it has an unusual life cycle in that its floral bud development takes up to nine months. In order to fully understand the highly modified floral organ structure and long life cycle of R. cantleyi, we used Illumina sequencing technology (HiSeq) for sequence generation followed by de novo assembly of sequence reads. We obtained the RNA-seq data from three different stages of floral bud, representing the early, mid and advanced developmental stages. These data are available via BioProject accession number PRJNA378435. More than 10.3 Gb raw sequence data were generated, corresponding to 102,203,042 raw reads. Following removal of low-quality reads and trimming of adapter sequences, a total of 91,638,836 reads were obtained. De novo assembly of these sequences using Trinity resulted in 89,690 unique transcripts with an N50 of 1653 bp. The obtained transcriptomic data will be useful for further study to understand the molecular interactions that result in R. cantleyi floral development.

Draft genome sequences of Helicobacter pylori isolates from Malaysia, cultured from patients with functional dyspepsia and gastric cancer.

Helicobacter pylori is the main bacterial causative agent of gastroduodenal disorders and a risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. The draft genomes of 10 closely related H. pylori isolates from the multiracial Malaysian population will provide an insight into the genetic diversity of isolates in Southeast Asia. These isolates were cultured from gastric biopsy samples from patients with functional dyspepsia and gastric cancer. The availability of this genomic information will provide an opportunity for examining the evolution and population structure of H. pylori isolates from Southeast Asia, where the East meets the West.

Insights into the genome structure and copy-number variation of Eimeria tenella.

BACKGROUND: Eimeria is a genus of parasites in the same phylum (Apicomplexa) as human parasites such as Toxoplasma, Cryptosporidium and the malaria parasite Plasmodium. As an apicomplexan whose life-cycle involves a single host, Eimeria is a convenient model for understanding this group of organisms. Although the genomes of the Apicomplexa are diverse, that of Eimeria is unique in being composed of large alternating blocks of sequence with very different characteristics - an arrangement seen in no other organism. This arrangement has impeded efforts to fully sequence the genome of Eimeria, which remains the last of the major apicomplexans to be fully analyzed. In order to increase the value of the genome sequence data and aid in the effort to gain a better understanding of the Eimeria tenella genome, we constructed a whole genome map for the parasite. RESULTS: A total of 1245 contigs representing 70.0% of the whole genome assembly sequences (Wellcome Trust Sanger Institute) were selected and subjected to marker selection. Subsequently, 2482 HAPPY markers were developed and typed. Of these, 795 were considered as usable markers, and utilized in the construction of a HAPPY map. Markers developed from chromosomally-assigned genes were then integrated into the HAPPY map and this aided the assignment of a number of linkage groups to their respective chromosomes. BAC-end sequences and contigs from whole genome sequencing were also integrated to improve and validate the HAPPY map. This resulted in an integrated HAPPY map consisting of 60 linkage groups that covers approximately half of the estimated 60 Mb genome. Further analysis suggests that the segmental organization first seen in Chromosome 1 is present throughout the genome, with repeat-poor (P) regions alternating with repeat-rich (R) regions. Evidence of copy-number variation between strains was also uncovered. CONCLUSIONS: This paper describes the application of a whole genome mapping method to improve the assembly of the genome of E. tenella from shotgun data, and to help reveal its overall structure. A preliminary assessment of copy-number variation (extra or missing copies of genomic segments) between strains of E. tenella was also carried out. The emerging picture is of a very unusual genome architecture displaying inter-strain copy-number variation. We suggest that these features may be related to the known ability of this parasite to rapidly develop drug resistance.

EmaxDB: Availability of a first draft genome sequence for the apicomplexan Eimeria maxima.

Apicomplexan parasites are serious pathogens of animals and man that cause diseases including coccidiosis, malaria and toxoplasmosis. The importance of these parasites has prompted the establishment of genomic resources in support of developing effective control strategies. For the Eimeria species resources have developed most rapidly for the reference Eimeria tenella Houghton strain (http://www.genedb.org/Homepage/Etenella). The value of these resources can be enhanced by comparison with related parasites. The well characterised immunogenicity and genetic diversity associated with Eimeria maxima promote its use in genetics-led studies on coccidiosis and recommended its selection for sequencing. Using a combination of sequencing technologies a first draft assembly and annotation has been produced for an E. maxima Houghton strain-derived clone (EmaxDB; http://www.genomemalaysia.gov.my/emaxdb/). The assembly of a draft genome sequence for E. maxima provides a resource for comparative studies with Eimeria and related parasites as demonstrated here through the identification of genes predicted to encode microneme proteins in E. maxima.

Metabolic routes affecting rubber biosynthesis in Hevea brasiliensis latex.

The cytosolic mevalonate (MVA) pathway in Hevea brasiliensis latex is the conventionally accepted pathway which provides isopentenyl diphosphate (IPP) for cis-polyisoprene (rubber) biosynthesis. However, the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway may be an alternative source of IPP since its more recent discovery in plants. Quantitative RT-PCR (qRT-PCR) expression profiles of genes from both pathways in latex showed that subcellular compartmentalization of IPP for cis-polyisoprene synthesis is related to the degree of plastidic carotenoid synthesis. From this, the occurrence of two schemes of IPP partitioning and utilization within one species is proposed whereby the supply of IPP for cis-polyisoprene from the MEP pathway is related to carotenoid production in latex. Subsequently, a set of latex unique gene transcripts was sequenced and assembled and they were then mapped to IPP-requiring pathways. Up to eight such pathways, including cis-polyisoprene biosynthesis, were identified. Our findings on pre- and post-IPP metabolic routes form an important aspect of a pathway knowledge-driven approach to enhancing cis-polyisoprene biosynthesis in transgenic rubber trees.

Oil palm (Elaeis guineensis Jacq.) tissue culture ESTs: identifying genes associated with callogenesis and embryogenesis.

BACKGROUND: Oil palm (Elaeis guineensis Jacq.) is one of the most important oil bearing crops in the world. However, genetic improvement of oil palm through conventional breeding is extremely slow and costly, as the breeding cycle can take up to 10 years. This has brought about interest in vegetative propagation of oil palm. Since the introduction of oil palm tissue culture in the 1970s, clonal propagation has proven to be useful, not only in producing uniform planting materials, but also in the development of the genetic engineering programme. Despite considerable progress in improving the tissue culture techniques, the callusing and embryogenesis rates from proliferating callus cultures remain very low. Thus, understanding the gene diversity and expression profiles in oil palm tissue culture is critical in increasing the efficiency of these processes. RESULTS: A total of 12 standard cDNA libraries, representing three main developmental stages in oil palm tissue culture, were generated in this study. Random sequencing of clones from these cDNA libraries generated 17,599 expressed sequence tags (ESTs). The ESTs were analysed, annotated and assembled to generate 9,584 putative unigenes distributed in 3,268 consensi and 6,316 singletons. These unigenes were assigned putative functions based on similarity and gene ontology annotations. Cluster analysis, which surveyed the relatedness of each library based on the abundance of ESTs in each consensus, revealed that lipid transfer proteins were highly expressed in embryogenic tissues. A glutathione S-transferase was found to be highly expressed in non-embryogenic callus. Further analysis of the unigenes identified 648 non-redundant simple sequence repeats and 211 putative full-length open reading frames. CONCLUSION: This study has provided an overview of genes expressed during oil palm tissue culture. Candidate genes with expression that are modulated during tissue culture were identified. However, in order to confirm whether these genes are suitable as early markers for embryogenesis, the genes need to be tested on earlier stages of tissue culture and a wider range of genotypes. This collection of ESTs is an important resource for genetic and genome analyses of the oil palm, particularly during tissue culture development.