Expression of the Arabidopsis redox-related LEA protein, SAG21 is regulated by ERF, NAC and WRKY transcription factors.

SAG21/LEA5 is an unusual late embryogenesis abundant protein in Arabidopsis thaliana, that is primarily mitochondrially located and may be important in regulating translation in both chloroplasts and mitochondria. SAG21 expression is regulated by a plethora of abiotic and biotic stresses and plant growth regulators indicating a complex regulatory network. To identify key transcription factors regulating SAG21 expression, yeast-1-hybrid screens were used to identify transcription factors that bind the 1685 bp upstream of the SAG21 translational start site. Thirty-three transcription factors from nine different families bound to the SAG21 promoter, including members of the ERF, WRKY and NAC families. Key binding sites for both NAC and WRKY transcription factors were tested through site directed mutagenesis indicating the presence of cryptic binding sites for both these transcription factor families. Co-expression in protoplasts confirmed the activation of SAG21 by WRKY63/ABO3, and SAG21 upregulation elicited by oligogalacturonide elicitors was partially dependent on WRKY63, indicating its role in SAG21 pathogen responses. SAG21 upregulation by ethylene was abolished in the erf1 mutant, while wound-induced SAG21 expression was abolished in anac71 mutants, indicating SAG21 expression can be regulated by several distinct transcription factors depending on the stress condition.

Genetic diversity and forensic statistical support for the 12 X-STR markers in the Malaysian Indian population using Qiagen Investigator® Argus X-12 QS kit.

X-chromosome short tandem repeats (X-STRs) are useful for human identification, especially in complex kinship scenarios. Since forensic statistical parameters vary among populations and the X-STRs population data for the diverse population of Peninsular Malaysia's are unavailable, this attempt for Indians (n = 201) appears forensically relevant to support the 12 X-STRs markers' evidential value for human identification in Malaysia. The Qiagen Investigator® Argus X-12 QS kit showed that DXS10135 was the most polymorphic locus with high genetic diversity, polymorphism information richness, heterozygosity, and exclusion power. Based on allele frequencies, the strength of discrimination and mean exclusion chance (MECKrüger, MECKishida, MECDesmarais, and MECDesmaraisDuo) values for the Malaysian Indians were ≥0.999997790686228. As for haplotype frequencies, the overall discrimination power and mean exclusion probability (MECKrüger, MECKishida, MECDesmarais, and MECDesmaraisDuo) were ≥0.9999984801951. The genetic distance, neighbor-joining phylogenetic tree, and principal component analysis also supported the evidential value of the 12 X-STRs markers for forensic practical caseworks in Malaysia.

Complete genome sequences of Lactococcus lactis D1_2, a bacterium with antimicrobial properties isolated from peat soil.

Lactococcus lactis is a beneficial lactic acid bacterium commonly studied for its probiotic properties and role in dairy production. Here, we present a complete genome of Lactococcus lactis D1_2, isolated from peat swamp forests. To discover the potential antimicrobial properties, the complete genome of the strain was sequenced and analyzed.

Internal validation of reduced PCR reaction volume of the Qiagen Investigator® Argus X-12 QS Kit from blood samples on FTA cards.

The onus of proof in criminal cases is beyond any reasonable doubt, and the issue on the lack of complete internal validation data can be manipulated when it comes to justifying the validity and reliability of the X-chromosomal short tandem repeats analysis for court representation. Therefore, this research evaluated the efficiency of the optimized 60% reduced volumes for polymerase chain reaction (PCR) amplification using the Qiagen Investigator® Argus X-12 QS Kit, as well as the capillary electrophoresis (CE) sample preparation for blood samples on Flinder's Technology Associates (FTA) cards. Good-quality DNA profile (3000-12,000 RFU) from the purified blood sample on FTA card (1.2 mm) were obtained using the optimized PCR (10.0 µL of PCR reaction volume and 21 cycles) and CE (9.0 µL Hi-Di™ Formamide and 0.3 µL DNA Size Standard 550 [BTO] and 27 s injection time) conditions. The analytical and stochastic thresholds were 100 and 200 RFU, respectively. Hence, the internal validation data supported the use of the optimized 60% reduced PCR amplification reaction volume of the Qiagen Investigator® Argus X-12 QS Kit as well as the CE sample preparation for producing reliable DNA profiles that comply with the quality assurance standards for forensic DNA testing laboratories, while optimizing the analytical cost.

The mitogenome data of Holothuria (Mertensiothuria) leucospilota (Brandt,1835) from Malaysia.

White threads fish Holothuria (Mertensiothuria) leucospilota (Brandt, 1835) or locally known as bat puntil is a neritic marine organism, and it is widely distributed in Indo Pacific. They serve many important roles in ecosystem services and were discovered to contain many bioactive compounds that are useful for medicinal value. However, despite its abundance in Malaysian seawater, there is still a lack of records on H. leucospilota mitochondrial genome (mitogenome) from Malaysia. The mitogenome of H. leucospilota originating from Sedili Kechil, Kota Tinggi, Johor, Malaysia, is presented here. Whole genome sequencing was successfully sequenced using Illumina NovaSEQ6000 sequencing system and the mitochondrial-derived contigs were assembled using de novo approach. The size of the mitogenome is 15,982 bp which consists of 13 protein-coding genes (PCGs), 21 transfer RNAs, and 2 ribosomal RNAs. The overall composition of nucleotide bases was estimated to be 25.8% for T, 25.9% for C, 31.8% for A and 16.5% for G (with A + T content of 57.6%). Maximum likelihood phylogenetic tree analysis revealed that the mitochondrial Protein-Coding Genes (PCGs) sequence data from our H. leucospilota is closely related to H. leucospilota from accession number MK940237 and H. leucospilota from accession number MN594790, followed by H. leucospilota from accession number MN276190, forming sister group with H. hilla (MN163001), known as Tiger tail sea cucumber. The mitogenome of H. leucospilota will be valuable for genetic research, mitogenome reference and future conservation management of sea cucumber in Malaysia. The mitogenome data of H. leucospilota from Sedili Kechil, Kota Tinggi, Johor, Malaysia is available in the GenBank database repository with accession number ON584426.

DNA Barcoding, Phylogenetic Analysis and Secondary Structure Predictions of Nepenthes ampullaria, Nepenthes gracilis and Nepenthes rafflesiana.

Nepentheceae, the most prominent carnivorous family in the Caryophyllales order, comprises the Nepenthes genus, which has modified leaf trap characteristics. Although most Nepenthes species have unique morphologies, their vegetative stages are identical, making identification based on morphology difficult. DNA barcoding is seen as a potential tool for plant identification, with small DNA segments amplified for species identification. In this study, three barcode loci; ribulose-bisphosphate carboxylase (rbcL), intergenic spacer 1 (ITS1) and intergenic spacer 2 (ITS2) and the usefulness of the ITS1 and ITS2 secondary structure for the molecular identification of Nepenthes species were investigated. An analysis of barcodes was conducted using BLASTn, pairwise genetic distance and diversity, followed by secondary structure prediction. The findings reveal that PCR and sequencing were both 100% successful. The present study showed the successful amplification of all targeted DNA barcodes at different sizes. Among the three barcodes, rbcL was the least efficient as a DNA barcode compared to ITS1 and ITS2. The ITS1 nucleotide analysis revealed that the ITS1 barcode had more variations compared to ITS2. The mean genetic distance (K2P) between them was higher for interspecies compared to intraspecies. The results showed that the DNA barcoding gap existed among Nepenthes species, and differences in the secondary structure distinguish the Nepenthes. The secondary structure generated in this study was found to successfully discriminate between the Nepenthes species, leading to enhanced resolutions.

Characterization of the nearly complete mitochondrial genome of ochraceous darkies, Euphaea ochracea Selys, 1859 (Odonata: Zygoptera: Euphaeidae) and phylogenetic analysis.

In the present study, the nearly complete mitochondrial genome of Euphaea ochracea was described and its phylogenetic position in the family Euphaeidae was analyzed. Here, we recovered 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs and a partial control region, resulting in a mitogenome length of 15,545bp. All protein-coding genes were initiated by the typical ATN codon except nad3 and nad1, which utilizes the TTG codon. Four protein-coding genes (cox1, cox2, cox3 and nad5) are terminated by an incomplete stop codon T, while others end with either a TAA or TAG codon. The intergenic spacer region, S5, is absent in this mitogenome, supporting the lack of this region as a specific character in damselflies. Phylogenetic analysis showed that the newly sequenced E. ochracea is phylogenetically closer to E. ornata with a high support value.

Complete mitochondrial genome data and phylogenetic analysis of the Great Marquis, Bassarona dunya (Doubleday, 1848) (Lepidoptera: Nymphalidae: Limenitidinae) from Malaysia.

The Great Marquis or Bassarona dunya is a butterfly species commonly found in the tropical regions of Asia, America, and Africa. This butterfly is a member of the subfamily Limenitidinae and the classification within this subfamily has been unstable. Here, we report the first complete mitochondrial genome (mitogenome) of B. dunya sampled from Malaysia. The mitogenome is 15,242 bp long, comprising a set of 13 protein-coding genes (PCGs), 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and an A + T rich region. All PCGs were initiated by the typical ATN codon, except for COX1 which started with a CGA start codon. Nine PCGs were terminated with a TAA or TAG stop codon, while COX1, COX2, NAD4, and NAD5 ended with an incomplete T. The 12S and 16S rRNAs were 716 bp and 1269 bp in length, respectively. Phylogenetic analysis supported the placement of B. dunya within Limenitidinae with a high support value.

Integrated Approach for Species Identification and Quality Analysis for Labisia pumila Using DNA Barcoding and HPLC.

Labisia pumila is a precious herb in Southeast Asia that is traditionally used as a health supplement and has been extensively commercialized due to its claimed therapeutic properties in boosting a healthy female reproductive system. Indigenous people used these plants by boiling the leaves; however, in recent years it has been marketed as powdered or capsuled products. Accordingly, accuracy in determination of the authenticity of these modern herbal products has faced great challenges. Lack of authenticity is a public health risk because incorrectly used herbal species can cause adverse effects. Hence, any measures that may aid product authentication would be beneficial. Given the widespread use of Labisia herbal products, the current study focuses on authenticity testing via an integral approach of DNA barcoding and qualitative analysis using HPLC. This study successfully generated DNA reference barcodes (ITS2 and rbcL) for L. pumila var. alata and pumila. The DNA barcode that was generated was then used to identify species of Labisia pumila in herbal medicinal products, while HPLC was utilized to determine their quality. The findings through the synergistic approach (DNA barcode and HPLC) implemented in this study indicate the importance of both methods in providing the strong evidence required for the identification of true species and to examine the authenticity of such herbal medicinal products.

Characterization of the mitogenomes of long-tailed giant rat, Leopoldamys sabanus and a comparative analysis with other Leopoldamys species.

Two mitogenomes of long-tailed giant rat, Leopoldamys sabanus (Thomas, 1887), which belongs to the family Muridae were sequenced and assembled in this study. Both mitogenomes have a length of 15,973 bp and encode 13 protein-coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes and one control region. The circular molecule of L. sabanus has a typical vertebrate gene arrangement. Phylogenetic and BLASTn analysis using 10 Leopoldamys species mitogenomes revealed sequence variation occurred within species from different time zones. Along with the taxonomic issues, this suggests a landscape change might influence genetic connectivity.

The first mitochondrial genome data of an old world fruit bat, Cynopterus sphinx from Malaysia.

We assembled the complete mitogenome of Cynopterus sphinx (Vahl, 1797) of the family Pteropodidae originating from Malaysia. The total mitogenome size was 16,710bp which consists of 37 genes (13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one control region). A phylogenetic and BLASTn result showed the mitogenome sequence in this study varies by nearly 7% (93.48% similarity) from the same species in Cambodia. The next closest match of BLASTn was at 92% similarity to the C. brachyotis. This suggests the species-complex in Cynopterus sp. has given rise to the genetic variability.

The first complete mitochondrial genome data of Hippocampus kuda originating from Malaysia.

The spotted seahorse, Hippocampus kuda population is exponentially decreasing globally due to habitat loss contributed by massive coastal urbanization as well as its large exploitation for Chinese herbal medicine. Genomic data would be highly useful to improve biomonitoring of seahorse populations in Malaysia via the usage of non-invasive approaches such as water environmental DNA. Here we report the first complete mitogenome of two H. kuda individuals originating from Malaysia, generated using BGISEQ-500RS sequencer. The lengths of both mitogenomes are 16,529bp, consisting of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. The overall base composition was 32.46% for A, 29.40% for T, 14.73% for G and 23.41% for C with AT rich features (61.86%). The gene organization of Malaysian H. kuda were similar to that of most teleost species. A phylogenetic analysis of the genome against mtDNA data from other Hippocampus species showed that Malaysian H. kuda samples clustered with H. capensis, H. reidi and H. kuda. Notably however, analysis of the data using BLASTn revealed they had 99.18% similarity to H. capensis, and only 97.66% to H. kuda and H. reidi, which are all part of the unresolved H. kuda complex. The mitogenomes are deposited in Genbank under the accession number MT221436 (HK1) and MT221436 (HK2).

Molecular identification and phylogenetic analysis of a Callosciurus notatus complete mitogenome from Peninsular Malaysia.

The mitogenome of a plantain squirrel, Callosciurus notatus, collected from Bukit Tarek Forest Reserve (Extension), Selangor, Malaysia was sequenced using BGISEQ-500RS technology. The 16,582 bp mitogenome consists of 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and 1 control region. A phylogenetic and BLASTn analysis against other available datasets showed that the mitogenome matched with 99.49% similarity to a previously published C. notatus mitogenome from Peninsular Malaysia. However, it also diverged by nearly 8% (92.24% match) from a second previously published mitogenome for the same species, sampled in East Kalimantan, Indonesia. This suggests a difference in landscape features between both localities might affect its genetic connectivity.

The first complete mitochondrial genome data of Geoffroy's rousette, Rousettus amplexicaudatus originating from Malaysia.

The increasing interest in understanding the evolutionary relationship between members of the Pteropodidae family has been greatly aided by genomic data from the Old World fruit bats. Here we present the complete mitogenome of Geoffroy's rousette, Rousettus amplexicaudatus found in Peninsular Malaysia . The mitogenome constructed is 16,511bp in length containing 37 genes; 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and a D-loop region. The overall base composition is estimated to be 32.28% for A, 25.64% for T, 14.09% for G and 27.98% for C, indicating a slightly AT rich feature (57.93%). A phylogenetic and BLASTn analysis against other available mitogenomes showed Malaysian R. amplexicaudatus matched 98% similarity to the same species in Cambodia and Vietnam. However, it differed considerably (92.53% similarity) with the same species in the Philippines. This suggests flexibility in Rousettus sp. with regards to adapting to mesic and dry habitats, ability for long-distance dispersal and remarkably precise lingual echolocation thus supporting its wide-range distribution and colonization. Further taxonomical and mitogenomic comparatives are required in resolving the evolutionary relationship between Rousettus spp.

Authenticity Testing and Detection of Eurycoma longifolia in Commercial Herbal Products Using Bar-High Resolution Melting Analysis.

The present study demonstrated High Resolution Melting (HRM) analysis combined with DNA barcode (Bar-HRM) as a fast and highly sensitive technique for detecting adulterants in Eurycoma longifolia commercial herbal products. Targeting the DNA barcoding of the chloroplastic region-ribulose biphosphate carboxylase large chain (rbcL) and the nuclear ribosomal region- internal transcribed spacer 2 (ITS2), PCR amplification and HRM analysis using saturated Eva green dye as the source of fluorescence signals, was accomplished by employing a real-time cycler. The results were further validated by sequencing to identify unknown sequence from Genbank database and to generate phylogenetic tree using neighbour joint (NJ) analysis. Both of the DNA markers exhibited a distinguishable melting temperature and shape of the normalised curve between the reference and the adulterants. In the case of species identification, ITS2 was more successful in differentiating between species. Additionally, detection of admixture sample containing small traces of targeted E. longifolia DNA (w/v) can be detected as low as 5% for rbcL and less than 1% for ITS2, proving the sensitivity and versatility of the HRM analysis. In conclusion, the Bar-HRM analysis is a fast and reliable technique that can effectively detect adulterants in herbal products. Therefore, this will be beneficial for regulatory agencies in order to regulate food safety issues.

An expanded mammal mitogenome dataset from Southeast Asia.

Southeast (SE) Asia is 1 of the most biodiverse regions in the world, and it holds approximately 20% of all mammal species. Despite this, the majority of SE Asia's genetic diversity is still poorly characterized. The growing interest in using environmental DNA to assess and monitor SE Asian species, in particular threatened mammals-has created the urgent need to expand the available reference database of mitochondrial barcode and complete mitogenome sequences. We have partially addressed this need by generating 72 new mitogenome sequences reconstructed from DNA isolated from a range of historical and modern tissue samples. Approximately 55 gigabases of raw sequence were generated. From this data, we assembled 72 complete mitogenome sequences, with an average depth of coverage of ×102.9 and ×55.2 for modern samples and historical samples, respectively. This dataset represents 52 species, of which 30 species had no previous mitogenome data available. The mitogenomes were geotagged to their sampling location, where known, to display a detailed geographical distribution of the species. Our new database of 52 taxa will strongly enhance the utility of environmental DNA approaches for monitoring mammals in SE Asia as it greatly increases the likelihoods that identification of metabarcoding sequencing reads can be assigned to reference sequences. This magnifies the confidence in species detections and thus allows more robust surveys and monitoring programmes of SE Asia's threatened mammal biodiversity. The extensive collections of historical samples from SE Asia in western and SE Asian museums should serve as additional valuable material to further enrich this reference database.

Review: DNA Barcoding and Chromatography Fingerprints for the Authentication of Botanicals in Herbal Medicinal Products.

In the last two decades, there has been a tremendous increase in the global use of herbal medicinal products (HMPs) due to their claimed health benefits. This has led to increase in their demand and consequently, also, resulted in massive adulteration. This is due to the fact that most of the traditional methods cannot identify closely related species in a process product form. Therefore the urgent need for simple and rapid identification methods resulted in the discovery of a novel technique. DNA barcoding is a process that uses short DNA sequence from the standard genome for species identification. This technique is reliable and is not affected by external factors such as climates, age, or plant part. The difficulties in isolation of DNA of high quality in addition to other factors are among the challenges encountered using the DNA barcoding in the authentication of HMP. These limitations indicated that using DNA barcoding alone may ineffectively authenticate the HMP. Therefore, the combination of DNA barcoding with chromatographic fingerprint, a popular and generally accepted technique for the assessment and quality control of HMP, will offer an efficient solution to effectively evaluate the authenticity and quality consistency of HMP. Detailed and quality information about the main composition of the HMPs will help to ascertain their efficacy and safety as these are very important for quality control.