Roselle (Hibiscus sabdariffa L.) is recognized for its phytochemical compounds such as anthocyanins, which possess pharmacological potentials in the treatments of hypertension, diabetes, cancer, hyperlipidaemia and hyperglycaemia. The calyx is the most commercially valuable part of the roselle and usually harvested at maturation. However, genetic study to understand the transcriptome changes in the calyx during maturation has yet to be explored. In this study, we sequenced the transcriptomes of roselle calyces at maturation stages III and IV using Illumina NextSeq 500 platform. These are the two most critical maturation stages in roselle, as these stages are often associated with the quality of the calyx. Over 200 million good quality paired-end reads were generated and de novo assembled into a reference transcriptome consisting of 221,334 transcripts with N50 score of 491bp. Among these transcripts, 92,974 transcripts (42%) were successfully annotated. The total number of significantly differentially expressed genes (DEGs) and the top five most significantly regulated genes in each of the maturation stage were presented. Twenty-one genes implicated in the biosynthesis of anthocyanins and their relative expressions in the calyx tissues at the two maturation stages were reported. Two secondary metabolites biosynthesis pathways that attained a relatively higher number of DEG mappings compared to other pathways were also reported. The findings from this work provide novel insights to better understand the transcriptional changes in roselle during calyx maturation, and the data made available here is intended for continued genetic study on roselle. The work is registered under NCBI Bioproject PRJNA664826. The raw sequencing reads are available in Short Read Archive with the accession numbers SRX9171161, SRX9171162, SRX9171163, SRX9171164, SRX9171165 and SRX9171166.
Stevia rebaudiana, a perennial herb native to northeastern Paraguay, has gained immense attention globally over the recent decades due to the natural sweetness of its leaves. Like in most plants, this particular species contains high amount of secondary metabolites, thus rendering the isolation of high quality and quantity RNA extract for molecular applications rather challenging. An effective, high-yield and high-quality RNA isolation protocol for this economically important plant species was devised here based on the cetyltrimethylammonium bromide (CTAB) extraction method, with an additional genomic DNA (gDNA) removal step. DNA and other contaminants that may affect downstream applications were effectively removed. Our results exhibited that RNA samples isolated from the leaves and stems of Stevia rebaudiana using this improvised method are high in integrity and quality with RNA integrity number (RIN) of more than 8 and low in contaminants.
Macaca fascicularis, also known as the cynomolgus macaque, is an important non-human primate animal model used in biomedical research. It is an Old-World primate widely distributed in Southeast Asia and is one of the most abundant macaque species in Malaysia. However, the genetic structure of wild cynomolgus macaque populations in Malaysia has not been thoroughly elucidated. In this study, we developed genic-simple sequence repeat (genic-SSR) markers from an in-house transcriptome dataset generated from the Malaysian cynomolgus macaque via RNA sequencing, and applied these markers on 26 cynomolgus macaque individuals. A collection of 14,751 genic-SSRs were identified, where 13,709 were perfect SSRs. Dinucleotide repeats were the most common repeat motifs with a frequency of 65.05%, followed by trinucleotide repeats (20.55%). Subsequently, we designed 300 pairs of primers based on perfect di- and trinucleotide SSRs, in which 105 SSRs were associated with functional genes. A subset of 30 SSR markers were randomly selected and validated, yielding 19 polymorphic markers with an average polymorphism information content value of 0.431. The development of genic-SSR markers in this study is indeed timely to provide useful markers for functional and population genetic studies of the cynomolgus macaque and other related non-human primate species.
A molecular sexing method by polymerase chain reaction (PCR) amplification of a portion of the sex-determining region Y (SRY) and the zinc finger (ZF) gene, as well as six equine Y-chromosome-specific microsatellite markers, were tested in the Malayan tapir (Tapirus indicus). While the microsatellite markers did not yield any male-specific amplicons for sex-typing, the SRY/ZF marker system produced reliable molecular sexing results by accurately sex-typing 31 reference Malayan tapirs, using whole blood, dried blood spot (DBS), or tissue samples as materials for DNA extraction. The marker system was also tested on 16 faecal samples, and the results were in general consistent with the pre-determined sexes of the animals, despite some amplification failures. A preliminary estimation of wild Malayan tapir population sex ratio was estimated from the Wildlife Genomic Resource Bank (WGRB) database of the Malaysian Department of Wildlife and National Parks (PERHILITAN), zoos, and the Sungai Dusun Wildlife Conservation Centre (WCC), as well as from the results of molecular sexing 12 samples of unknown sex. The overall sex ratio favoured females, but the deviation from parity was statistically not significant when tested using the binomial test (p > 0.05), which may be due to reduced statistical power caused by small sample sizes.
The cynomolgus macaque (Macaca fascicularis) is an extensively utilised nonhuman primate model for biomedical research due to its biological, behavioural, and genetic similarities to humans. Genomic information of cynomolgus macaque is vital for research in various fields; however, there is presently a shortage of genomic information on the Malaysian cynomolgus macaque. This study aimed to sequence, assemble, annotate, and profile the Peninsular Malaysian cynomolgus macaque transcriptome derived from three tissues (lymph node, spleen, and thymus) using RNA sequencing (RNA-Seq) technology. A total of 174,208,078 paired end 70 base pair sequencing reads were obtained from the Illumina Hi-Seq 2500 sequencer. The overall mapping percentage of the sequencing reads to the M. fascicularis reference genome ranged from 53-63%. Categorisation of expressed genes to Gene Ontology (GO) and KEGG pathway categories revealed that GO terms with the highest number of associated expressed genes include Cellular process, Catalytic activity, and Cell part, while for pathway categorisation, the majority of expressed genes in lymph node, spleen, and thymus fall under the Global overview and maps pathway category, while 266, 221, and 138 genes from lymph node, spleen, and thymus were respectively enriched in the Immune system category. Enriched Immune system pathways include Platelet activation pathway, Antigen processing and presentation, B cell receptor signalling pathway, and Intestinal immune network for IgA production. Differential gene expression analysis among the three tissues revealed 574 differentially expressed genes (DEG) between lymph and spleen, 5402 DEGs between lymph and thymus, and 7008 DEGs between spleen and thymus. Venn diagram analysis of expressed genes revealed a total of 2,630, 253, and 279 tissue-specific genes respectively for lymph node, spleen, and thymus tissues. This is the first time the lymph node, spleen, and thymus transcriptome of the Peninsular Malaysian cynomolgus macaque have been sequenced via RNA-Seq. Novel transcriptomic data will further enrich the present M. fascicularis genomic database and provide future research potentials, including novel transcript discovery, comparative studies, and molecular markers development.