Primer design of D-loop region for wild population genetics of Rusa timorensis in Indonesia

Rusa timorensis (Javan deer) is endemic wildlife in Indonesia and is estimated at less than 10.000 individuals with continuously declining populations due to habitat loss and illegal hunting in the wild. This declining low population indicates a greater risk of extinction. Unfortunately, the genetic information of the wild Javan deer population for conservation management strategies still lacks data due to challenging sampling in the wild. Most recent studies were analysing the breeding populations outside Indonesia. Here, we propose the primer design of the D-loop genetic marker to determine the genetic population of wild Javan deer. We used metadata analysis of genetic sequences and new samples from five wild populations to design the specific primer of the D-loop region of the wild Javan deer in Indonesia. We used software, i.e.., Primer3 to design the primers, BLAST for specificity and Oligo Analyzer™ Tool for efficiency of the primer. The Annealing temperature optimisation started with pre-denaturation at 94 °C followed by 35 cycles of denaturation at 95°C; 51-56°C annealing for each one degree’s different per PCR treatment; and 72°C extensions. We successfully designed a specific primer (RL-3.1a) to amplify 235 bp of the D-loop region at 52°C annealing’s temperature.

The presence of Austropuccinia psidii and the threat to Myrtaceae plantations in Indonesia

Austropuccinia psidii is an invasive pathogenic rust that infects the Myrtaceae family. This rust is a threat to Myrtaceae plantations around the world due to its widespread distribution. In this study, we observed the presence of A. psidii in three species of Myrtaceae, i.e. Melaleuca cajuputi, Syzygium myrtifolium, and Syzygium polyanthum planted in Yogyakarta and Sukabumi. The symptoms of infection were yellow-reddish spot in young leaves, presence of urediniospores in infected spot, foliage, and branch dieback. To confirm the presence of A. psidii on those trees, a molecular detection was performed using specific primer for A. psidii (Ppsi1/Ppsi6) on DNA samples extracted from diseased leaves. The presence of A. psidii was proved by the presence of DNA amplicon sized around 500bp in all samples collected from three different hosts. In this study, S. myrtifolium was firstly reported to be infected by this rust in Indonesia. Further study about the presence and the economic impact of this pathogen in Indonesia should be conducted. Indonesia has many species numbers of Myrtaceae and some species are important for medicines, herbs, foods, and as industrial plants. A strategy to control this pathogen should be established to avoid large economic losses in Myrtaceae plantations in Indonesia.

Occurrence of the Lance Nematode Hoplolaimus stephanus Infecting Bentgrass Agrostis stolonifera in Georgia, USA

A high population of lance nematodes Hoplolaimus spp. were found associated with creeping bentgrass (Agrostis stolonifera L.) in May 2019 in Georgia, USA. The nematode was pathogenic to bentgrass as its population increased by over 3-fold 180 days after inoculation under greenhouse conditions. Morphological measurements of body and stylet lengths of both mature females and males were similar to a grass population of H. stephanus from South Carolina. DNA sequence analyses of the D1-D3 expansion segments of the 28s gene identified the nematode as H. stephanus. The DNA sequence of the nematode was 99.7% identical to a H. stephanus isolate from South Carolina. Also, the PCR method using a species-specific primer set confirmed the identity of H. stephanus. To the best of our knowledge, this is the first report of H. stephanus Sher, 1963, infecting creeping bentgrass in Georgia.

Study on milky haemolymph diseases infection in wild and cultured of spiny lobster, Panulirus homarus in Indonesia

Milky Haemolymph Disease in Spiny Lobster (MHD-SL) is the most pathogenic diseases in spiny lobster (Panulirus homarus). Research on MHD-SL infection has not been undertaken in Indonesia. Therefore, present study aims to determine the infection of MHD-SL lobster. In 2016 a total of 240 lobsters for 30 each both from wild and cultured sample were collected from four locations (Candi Kusuma Bay of Bali Island, Gerupuk, Awang, and Telong-Elong Bays of Lombok Island) and in 2019, 50 lobster samples were collected for artificial infection study. While in January 2020, another 40 lobsters were collected from 2 different sites of culture (coastal and offshore cages) within Telong Elong Bay to determine infection of MHD-SL and for transmission study. The MHD-SL diseased was first check by clinical sign and confirmed by PCR-DNA molecular with specific primer of 254 bp. An experimental infection of MHD-SL was carried out by injection and cohabitation. The result showed that infected MHD-SL lobster shows inactive, loose appetite to eat, reddish and white colour of abdomen then moribund and all positive by PCR test. MHD-SL was found only in cultured lobster on the cages located at coastal water and no in the cages located at offshore within Telong-Elong Bay. In the experiment of artificial infection, either by injection or cohabitation, shows clinical sign of MHD-SL appeared at 8 days and all died after 14 days for both treatments. The present study approved that MHD-SL is pathogenic agent belonged to Rickettsia-like bacterium and infection occurred by horizontal transmission.

Analysis of Sequence Variability and Transcriptional Profile of Cannabinoid synthase Genes in Cannabis sativa L. Chemotypes with a Focus on Cannabichromenic acid synthase

Cannabis sativa L. has been long cultivated for its narcotic potential due to the accumulation of tetrahydrocannabinolic acid (THCA) in female inflorescences, but nowadays its production for fiber, seeds, edible oil and bioactive compounds has spread throughout the world. However, some hemp varieties still accumulate traces of residual THCA close to the 0.20% limit set by European Union, despite the functional gene encoding for THCA synthase (THCAS) is lacking. Even if some hypotheses have been produced, studies are often in disagreement especially on the role of the cannabichromenic acid synthase (CBCAS). In this work a set of European Cannabis genotypes, representative of all chemotypes, were investigated from a chemical and molecular point of view. Highly specific primer pairs were developed to allow an accurate distinction of different cannabinoid synthases genes. In addition to their use as markers to detect the presence of CBCAS at genomic level, they allowed the analysis of transcriptional profiles in hemp or marijuana plants. While the high level of transcription of THCAS and cannabidiolic acid synthase (CBDAS) clearly reflects the chemical phenotype of the plants, the low but stable transcriptional level of CBCAS in all genotypes suggests that these genes are active and might contribute to the final amount of cannabinoids.

Characterisation of the Prevailing Multidrug Pseudomonas aeruginosa Strains from Surgical Wound Using 16S rRNA Sequencing Technique

Background: Pseudomonas aeruginosa (P. aeruginosa) is prevalent in hospital-acquired surgical wound infections. It exhibits both innate and acquired resistance to a broad range of antimicrobials and remains a principal problem in clinical practice. Methods: In total, 284 sterile surgical wound swabs (142 each) were collected from two government hospitals: Central Hospital Benin (CHB) and University of Benin Teaching Hospital (UBTH) in Benin City, Nigeria. Pseudomonas spp. isolated from both hospitals were screened with eight different antibiotics by way of disk diffusion method. Polymerase chain reaction (PCR) amplification of 34 multiple drug-resistant isolates was carried out using genus-specific primer set on extracted genomic DNA for the identification of Pseudomonas spp. and substituent 16S rRNA sequencing to determine the prevailing strains in the two locations. Results: Sixty-two Pseudomonas spp. were isolated from the two locations (27 isolates from CHB and 35 isolates from the UBTH). Surgical wound infections screened with regularly used antibiotics revealed that 17 (62.9%) isolates from CHB and 20 (57.1%) isolates from UBTH were multiple drug resistant Pseudomonas spp. PCR identification using Pseudomonas spp. specific primer showed that 16 (94.1%) isolates from CHB and 18 (90%) isolates from UBTH were confirmed. 16S DNA sequencing revealed that P. aeruginosa strain H25883 was dominant in both locations. Conclusion: High antibiotic resistance among P. aeruginosa isolates was established in our study. PCR technique revealed a more reliable method of bacterial identification. H25883 strain of P. aeruginosa is the prevalent strain in both locations and it should be given attention in nosocomial surgical wound infections.

Application of DNA Molecular Identification Method to Distinguish Ejiao and its Adulterants

To identify Ejiao and its adulterants at the DNA level by using DNA molecula marker. Ejiao (Asini Corii Colla) is a commonly used medicinal material. However, its adulteration is a serious concern. Due to the morphological characteristics of Asini Corii Colla and its adulterants, traditional identification techniques often complex and professional, which is not conducive to the circulation management and safety of the medicinal materials. To improve the distinction between Asini Corii Colla and its adulterants accurately, this study identified and its adulterant samples based on the CytB sequence. Sequence characteristics, Basic Local Alignment Search Tool (BLAST) application, genetic distance, construction of phylogenetic tree showed the CytB sequence to accurately identify Asini Corii Colla from its adulterants. Furthermore, in this study, we designed a specific primer, based on the CytB sequence, and established a PCR detection system for rapid, sensitive, and specific identification of Asini Corii Colla. Compared to DNA barcoding technology, this method has shorter detection time, stronger specificity, and higher sensitivity, which lays the foundation for the rapid identification of Asini Corii Colla.

Contribution of HLA Class II Genes, DRB4*01:01, DRB1*07:01, and DQB1*03:03:2 to Clinical Features of Vitiligo Disease in Iranian Population

Background: Vitiligo is a multifactorial depigmentation condition, which is due to skin melanocyte destruction. The increased expression of HLA class II genes in patients with pre-lesions of Vitiligo suggests an important role for the participation of immune response in the Vitiligo development. Recent studies progressively focused on HLA-DRB1 and DQB1 genes. In this study, we have evaluated the association and role of HLA-DRB4*01:01, -DRB1*07:01, and -DQB1*03:03:2 genes in different clinical subtypes of Vitiligo in the Iranian population.Methods: First, Genomic DNA from peripheral blood of 125 unrelated Vitiligo patients and 100 unrelated healthy controls were extracted through salting-out method. Then, HLA CLASS II genotyping were performed using sequence-specific primer PCR method. Finally, the clinical relevance of the testing for these genotypes were evaluated by applying the PcPPV (prevalence-corrected positive predictive value) formula.Results: Our results indicated the positive associations of DRB4*01:01 and DRB1*07:01 allelic genes with early-onset Vitiligo (P= 0.024 and 0.022, respectively). The DRB4*01:01 also showed a strong protection against late-onset Vitiligo (P= 0.0016, RR=0.360). Moreover, our data revealed that the DRB1*07:01 increases the susceptibility to Sporadic Vitiligo (P=0.030, RR=1.702). Furthermore, our findings proposed that elevated vulnerability of Vitiligo patients due to DRB4*01:01 and DRB1*07:01 alleles may be is correlated with the presence of amino acid Arginine at position 71 at pocket 4 on the antigen-binding site of the HLA-DRB1 receptor.Conclusion: Our findings on different subtypes of Vitiligo suggest that, despite a more apparent autoimmune involvement, a non-autoimmune nature for the etiology of Vitiligo could also be considered.