Cetyltrimethylammonium Bromide Method Research Articles

Whole-Genome Resource of Lasiodiplodia pseudotheobromae BaA, the Causative Agent of Black Root Rot Morinda officinalis.

Whole-Genome Resource of Lasiodiplodia pseudotheobromae BaA, the Causative Agent of Black Root Rot Morinda officinalis.

First Report of Bacterial Wilt Disease Caused by Ralstonia solanacearum on Southern Highbush Blueberries (Vaccinium corymbosum interspecific hybrids) in Georgia U.S.A.

Members of the Ralstonia solanacearum (Rs) species complex have recently been reported to cause bacterial wilt on southern highbush (SHB) blueberries in Florida (Norman et al. 2018), a disease first reported on blueberry (northern highbush; Vaccinium corymbosum) in New Jersey (Patel et al. 2013). SHB blueberries are widely grown in the southern United States, and SHB cultivars represent the majority of the blueberry acreage in Georgia - the U.S. state with the largest blueberry acreage (NASS 2022). In Fall 2020, three-year old SHB plants (cv. 'Indigocrisp') showing leaf bronzing, wilting, and dieback were collected from two field sites in Clinch County, GA. At these locations, numerous plants were rapidly dying, with symptoms appearing to have spread down rows. Plant material tested positive using the ImmunoStrip® for Rs (Agdia, Inc., Elkhart, IN). From one location, the remaining sample was submitted to USDA-APHIS Select Agent Services who determined that Rs was present but a select agent (Rs Race 3, Biovar 2) was not. Following this, six adjacent, symptomatic SHB plants were collected from the same field location. These plants tested positive using the ImmunoStrip® for Rs, and red-pigmented mucoid colonies typical of Rs formed within 48 h at 28°C on triphenyltetrazolium chloroide (TZC) isolation medium (Kelman 1954). DNA was extracted from pure Rs cultures using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1987) and tested using polymerase chain reaction (PCR). Primers pairs AMB013/AMB014 (Fegan and Prior 2005) and ENDO-F/ENDO-R (Ji et al. 2007) were used to amplify 558 nt and 843 nt portions of the 16S rRNA region and Rs endoglucanase gene, respectively. Resulting amplicons were purified using an E.Z.N.A.® Cycle Pure Kit (Omega Bio-Tek, Norcross, GA), Sanger sequenced in both directions (Eurofins Genomics, Louisville, KY), and compared to publicly available Rs sequences in Genbank. The 16 rRNA sequence from all obtained isolates (accession ON938207) had 100% identity to Rs strain CFBP2957 (FP885897), while the endoglucase sequence (ON938206) had 100% identity to phylotype IIa, sequevar 5 Rs strain CIP-426 (MF461810) and phylotype IIa, sequevar 39 Rs strain 19-058 (MT314067), among others. To fulfill Koch's postulates, an isolate ('Ral21-1') was grown on TZC medium for 48 h at 28°C and suspended in 8.5 g/L NaCl at 1 x 108 CFU/ml. Five young, tissue cultured SHB plants (cv. 'Kestrel') in 25 cm pots were drenched with 50 ml of Rs suspension. For six weeks, plants were maintained in the greenhouse at 21-32°C. Typical bacterial wilt symptoms (leaf bronzing/scorching) developed in all inoculated plants, and infections were confirmed using Immunostrip®. Rs was reisolated and confirmed via PCR and sequencing as previously described. While Ralstonia has been known to cause disease on numerous crops in Georgia, this represents a first report of bacterial wilt in Georgia blueberries. Relative to rabbiteye blueberries (V. virgatum), recent reports suggest that SHB are much more susceptible to bacterial wilt (Conner et al. 2022). Accordingly, given the transition from rabbiteye to SHB within Georgia's blueberry production region over the past two decades and the ability of Rs to spread easily in water, soil, or via infected plant material, the presence of this disease within the state represents a significant threat to blueberry production. Additional characterization of Ralstonia isolates from Georgia may help assess the risk of future outbreaks.

First Report of Flower Stalk Wilting Caused by Rhizopus oryzae on Chinese cabbage in China.

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the main biennial vegetables in China and its flowers can be produced in the second year. In May 2021, approximately 50% of the flower stalks of Chinese cabbage wilted in a field in Laizhou, China. Water-soaked lesions were first observed on the lateral shoots of flower stalks, leading to wilting at a later stage. Small diseased tissues were excised from the margin of lesions, surface disinfected in 75% alcohol, rinsed in distilled water twice, and transferred onto potato dextrose agar (PDA) medium for incubation at 28 ℃. Five fungal isolates were obtained using single spore isolation method. The fungal colonies were initially white and became gray or black within 5 days. The columella was globose to subglobose and 82.86±5.25 μm (n=5) in diameter; sporangiophores were smooth-walled, simple or branched; the globose sporangia were 86.06±15.37 μm (n=5) in diameter and black; the sporangiospores were subglobose and abundant and 5.23±0.98 μm (n=5) in diameter; and the rhizoids were dark brown and 5.69±1.82 μm (n=5) wide. A cetyl tri-methyl ammonium bromide method was used to extract DNA from 3-day-old hyphae (Ausubel et al. 1987). PCR was performed for ITS (White et al. 1990), the RNA polymerase II large subunit (RPB1) gene (Voigt et al. 2000) and the actin (ACT) gene (Stiller et al. 1997). The DNA sequences of the five isolates were identical, therefore, the sequence of Isolate RO21 was submitted to GenBank. According to BLAST search, the ITS (MZ452687), RPB1 (OK431470), and ACT (OK431471) sequences showed 99.66% similarity to Rhizopus oryzae Strain CBS 112.07 (NR103595), 100% to Strain CBS 127.08 (KJ566325) and 100% to Strain CBS 102660 (KJ551423), respectively. A neighbor-joining phylogenetic tree was reconstructed based on the ITS of Isolate RO21 and 14 other Rhizopus species sequences obtained from GenBank. Isolate RO21 was found to be most closely related to R. oryzae and far from other species. Based on morphological and phylogenetic characteristics, Isolate RO21 was identified as R. oryzae (Dolatabadi et al. 2014, Kwon et al. 2015, Palemón-Alberto et al. 2020). Sporangiospores were harvested from 5-day-old PDA cultures, suspended in sterilized distilled water, adjusted to 106 spores/ml and amended with 0.1% Tween-80. Chinese cabbage inbred line "A54-1" was inoculated near the middle of the flower stalk by applying 20 μl of spore suspension (106 spores/ml) to each of three sites wounded using a sterilized knife or to the unwounded site. Sterilized distilled water was used as the control. Forty flower stalks (20 for the inoculation treatment and the rest for the control) selected from ten plants were used for pathogenicity test. All plants were incubated in a growth chamber at 28/22 °C (day/night), with 80 to 90% of relative humidity. Wilting symptoms similar to those in the field were observed in the wounded flower stalks after 5 days and in the non-wounded flower stalks after 15 days. All control flower stalks remained asymptomatic. The fungus was re-isolated from the artificially infected flower stalks and identified as R. oryzae by morphological characteristics and sequencing to fulfill the Koch's postulates. To our knowledge, this is the first report that R. oryzae causes flower stalk wilting on Chinese cabbage in China. The results can provide the basis for future studies on the occurrence, prevention and management of this disease.

Genetic Diversity of Durian (Durio Zibethinus Murr.) in Nias Island, North Sumatera Use Random Amplified Polymorphic Dna (Rapd) Markers

Durian (Durio zibethinus Murr.) is one of tropic fruits that has varian taste depending on the places of their production. Nias Island, well known as durian production place in North Sumatera. The information about it’s varian was less studied and now day we need that information to analyze variant of durian especially in Nias Island. The aim of this research was to analyze genetic diversity of durian in Nias Island with Random Amplified Polymorphic DNA (RAPD). Ten shoots of durian was collected from each 5 regencies, which were: Nias Selatan, Nias Barat, Nias Utara, Nias Kota, dan Nias Induk. Durian’s DNA has been isolated by using Cetyl Trimethyl Ammonium Bromide (CTAB) method and amplified with six RAPD primers namely: OPA 01, OPA 03, OPA 07, OPA 10, RAPD 05, and OPN 06. The amplified band were scored and translated to biner data, then analyzed using Numerical Taxonomy and Multivariate System (NTSys) and clustered by Unweighted Pair Group Method With Arithme Average (UPGMA) method. Isolated DNA showed a clear and unsmeared band. The size of amplified band was around 221-2855bp with 100% of polymorphism. The dendogram was constructed by 50 accession of Nias’s durian with 0.51-0.59 of similarity value in each population. All of Nias’s durian accession has similiar value of 67% and B2 (Nias Barat) as a farthest genetic distance from all accession.

Occurrence of Leaf Spot Caused by Diaporthe phaseolorum on Bletilla striata in China.

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is a traditional Chinese medicinal plant widely distributed in eastern and southern Asia. In April of 2020, a leaf spot disease on B. striata was observed in plant nurseries in Guilin, Guangxi Province, China. Disease incidence was estimated at approximately 20% (n = 150 plants) across the survey area (~ 0.3 ha). The initial symptoms were small, reddish to brown spots, circular or irregular in shape. Subsequently, they developed into large dark brown, irregular lesions. As the lesions coalesced, leaves withered and defoliated. To isolate the causal agent, eighteen small pieces (~ 5 mm2) were collected from the margin of the necrotic lesions on Chinese ground orchid, surface disinfected (2 min in 1% NaOCl, and rinsed three times in sterile water), and placed on potato dextrose agar (PDA) at 26°C for 3 days. Hyphal tips were transferred to PDA to obtain pure cultures. Twelve isolates were obtained, of which eight isolates had similar morphological characteristics. After 7 days growth on PDA, colonies were grayish-white, fluffy, with white aerial mycelium. After 3 weeks, colonies formed white aerial mycelial mats, and pycnidia developed. The α-conidia were abundant, hyaline, aseptate, ellipsoidal to fusiform, measuring 4.6 to 6.7 μm × 2.1 to 3.0 μm (n = 55), whereas the β-conidia were hyaline, long, slender, straight or curved, measuring 10.3 to 17.2 μm × 0.9 to 1.8 μm (n = 59). Morphological features were similar to Diaporthe sp. (Santos et al. 2011, Udayanga et al. 2015). For further molecular identification, DNA was extracted from the mycelia of the representative isolate BJ26.3 following the CTAB (cetyltrimethylammonium bromide) method (Guo et al. 2000). The internal transcribed spacer (ITS) region, partial translational elongation factor subunit 1-α (EF-1α), calmodulin (CAL) , histone H3 (HIS3), β-tubulin (TUB) genes were amplified and sequenced using the primer pairs ITS1/ITS4, EF1-728F/EF1-986R, CAL-228F/CAL-737R, CYLH3F/H3-1b, and Bt2a/Bt2b, respectively (White et al. 1990, Guarnaccia et al. 2018). The obtained sequences were deposited in NCBI GenBank under the following accession numbers: OK560457, OK539595, OK539592, OK506726, OK539598. BLAST analysis of the deposited sequences showed 99 to 100% identity with accession numbers KC343177 (563/566 bp), KC343903 (521/523 bp), KC343419 (423/427 bp), KC343661 (340/340 bp), KC344145 (658/662 bp) of D. phaseolorum CBS 127465 (Guarnaccia et al. 2018). In addition, a phylogenetic analysis using concatenated sequences confirmed BJ26.3 as D. phaseolorum. Furthermore, pathogenicity tests were carried out on 1.5-year-old B. striata plants. Healthy leaves on three plants (1 leaf per plant) were inoculated with 5 × 5 mm mycelial discs of strain BJ-26.3 from 3-day-old PDA cultures. Another three plants treated with sterile water served as the control. All plants were covered with transparent plastic bags and maintained in a greenhouse at 26°C with a 12 h photoperiod. Nine days post-inoculation, the inoculated leaves showed leaf spot symptoms, while the control plants remained healthy. The experiments repeated three times showed similar results. Finally, D. phaseolorum was consistently re-isolated from the infected leaves and confirmed by morphology and sequencing, fulfilling Koch's postulates. To our knowledge, this is the first report of D. phaseolorum causing leaf spot of B. striata worldwide. This study might provide important information for growers to manage this disease.

Effects of fruits and vegetables on gut microbiota in a mouse model of metabolic syndrome induced by high-fat diet.

The aim of this study was to evaluate the effect of fruit and vegetable intake on gut microbiota using a mouse model of metabolic syndrome (MS) induced by a high-fat diet. Forty-eight male mice were randomly divided into four groups, control group (C), high-fat diet-fed model group (H), high fat plus low intake of fruits and vegetables diet-fed group (H.LFV), high fat plus high intake of fruits and vegetables diet-fed group (H.HFV), and each group were fed for 60 days. During the experiment, mouse body weights were recorded and fecal samples were collected. Cetyltrimethyl ammonium bromide (CTAB) method was used to extract fecal bacterial DNA, and the purity and concentration of the DNA were detected by electrophoresis. DNA samples underwent PCR amplification (primers in 16 S V4 (515F and 806R)). Raw sequencing data were processed, and sample complexity and multiple-sample comparisons were investigated. Mouse organ coefficient, serum lipid levels, fecal TC (total cholesterol) and TBA (total bile acid) levels, and hepatic glutathione and malondialdehyde levels were determined. Compared to the H group, the fecal TC and TBA levels decreased significantly in the H.HFV group (p < .05), and hepatic glutathione and malondialdehyde levels decreased significantly in both H.LFV and H.HFV groups (p < .05). Decreased abundance of Firmicutes, Burkholderiales, Syntrophomonas, and Pseudomonadales in gut microbiota was observed in H.LFV and H.HFV groups compared to the H group. The Anosim results showed significant differences in pairwise comparison between groups. The linear discriminant analysis effect size (LEfSe) results showed that k_bacteria not only exhibited statistically differences between H and C groups but also among H.LFV, H.LFV, and H groups, and hence, could be used as a biomarker between groups. To sum up, fruit and vegetable powder could increase the fecal excretion of TC and TBA, and the antioxidant capacity in C57BL/6N mice. Meanwhile, the mechanism that fruit and vegetable powder could prevent MS in C57BL/6N mice was related to the decreased abundance of gut microbiota, including Firmicutes, Syntrophomonadales, and Pseudomonadales. Hence, fruit and vegetable powder could be used as a recommended food to regulate gut microbiota and prevent the occurrence of MS-related diseases.

Detection of soy in food from the Czech market using ELISA and PCR methods

Soy is considered an allergen under Regulation No. 1169/2011 of the European Parliament and of the Council, which mandates the labelling of soy allergen on food packaging. This study is focused on detecting soy in food products using two kinds of methods. The first method, enzyme-linked immunosorbent assay (ELISA), was performed using two commercial kits (Veratox for Soy Allergen; Neogen, USA). The second method, polymerase chain reaction (PCR), was carried out with two different deoxyribonucleic acid (DNA)-isolating kits and DNA isolation via the cetyltrimethylammonium bromide (CTAB) method. A total of 57 samples of food were tested, including 45 samples of animal origin and 12 samples of plant origin. The results were compared with information on the packaging. From the group of samples that contained soy according to the packaging (12 pieces), 9 samples were found to be positive by ELISA method, 10 samples by the CTAB method and 11 samples by GeneSpin. On the other hand, from the group of samples that should not contain soy according to the packaging (30 pieces), the presence of soy in 2 samples was detected by ELISA. No significant difference was found between the examined methods. Results show that the situation on the market is satisfactory and that only products declared as containing traces of soy appear to be problematic.

Pencirian Genetik Pepper Yellow Leaf Curl Virus pada Tanaman Cabai Merah (Capsicum annuum) di Bengkulu

Chili is one of the horticultural commodities with increasing demand yearly. Rejang Lebong and Kepahiang regencies are the most significant chili-producing areas in Bengkulu. One of the crucial viruses in chili plants is Pepper yellow leaf curl virus (PYLCV). PYLCV virus infection in the vegetative phase can cause stunted plants and fail to bear fruit. This study aims to detect and genetically characterize PYLCV. Samples of chili plants were taken in these two districts using the purposive sampling method. Virus detection was carried out at the Genetics and Biotechnology Laboratory, Department of Biology. Extraction of viral DNA using the Cetyl Trimethyl Ammonium Bromide (CTAB) method. Rep and Trap gene amplification using universal primers of Bemovirus. SPG1 and SPG2. The PCR product was visualized on a 1.2% agarose gel in TAE.. Furthermore, the PCR products were sequenced in PT Genetics Science Indonesia. Virus gene sequences were aligned using Bioedit and MEGA X software. The results showed that of the 24 chili plants sampled in the study, 21 samples showed an amplicon with a size of ~ 900 bp, which follows the length of the amplicon based on the primer used. Of the 21 amplicon results, 6 of them were sequenced and genetically characterized. The results of the genetic characterization of the 6 samples showed the presence of 552 conservative sites (C) (75.6%), 178 variation sites (Vi) (24.4%), parsimony sites (Pi) (15.9%), and 70 singleton (S) sites (8.5%). The highest nucleotide base composition was thymine (T) with an average value of 30.1%, and the lowest was guanine (G) 21.9%, while the highest nucleotide combined composition was AT with an average value of 55%, the lowest was GC approximately 45%. The average genetic distance between samples was 0.13 (13%). Based on the phylogenetic tree, 6 samples were divided into two groups. Keywords: begomovirus, Bengkulu, Capsicum annuum

Expression of human papillomavirus 16 and 18 DNA in oral lichen planus using polymerase chain reaction.

Oral lichen planus (OLP) is a chronic inflammatory disease with cell-mediated immune dysregulation. The aetiology of OLP has been studied extensively for decades. Viruses like Hepatitis C Virus (HCV), human papillomavirus (HPV), herpes simplex virus (HSV), and stress have been hypothesized to play a role in the pathogenesis and malignant transformation of OLP. HPV has been proved to be an etiological agent in oropharyngeal cancers and non-tobacco-associated leukoplakia. The role of human papillomavirus in the pathogenesis of OLP has to be studied extensively. This study aims to detect the presence of HPV 16 and HPV 18 DNA in the biopsy samples of OLP and also to determine the role played by the virus in the pathogenesis and malignant transformation of OLP. Biopsy samples comprising 30 OLP tissues were collected. The DNA was extracted by the cetyltrimethylammonium bromide method. Polymerase chain reaction was performed by using general primers to amplify the HPV E6 gene. Twelve out of 30 (40%) OLP cases were positive for HPV DNA. A significant relation was found between HPV, site (buccal mucosa) and the type (reticular) of the lesion (P = 0.007). However, the difference between the percentage of HPV positive males and females was statistically insignificant (P = 0.852). This study confirmed the presence of high-risk HPV 16 and HPV 18 DNA in OLP. The study showed a significantly higher expression of HPV in erosive OLP when compared to reticular OLP, suggesting a possible role of HPV in the malignant transformation of OLP.

Visual Detection of Chicken Adulteration Based on a Lateral Flow Strip-PCR Strategy.

The aim of this study was to develop an accurate, easy-to-use, and cost-effective method for the detection of chicken adulteration based on polymerase chain reaction (PCR) and lateral flow strip (LFS). We compared six DNA extraction methods, namely the cetyltrimethylammonium bromide (CTAB) method, salt method, urea method, SDS method, guanidine isothiocyanate method, and commercial kit method. The chicken cytb gene was used as a target to design specific primers. The specificity and sensitivity of the PCR-LFS system were tested using a self-assembled lateral flow measurement sensor. The results showed that the DNA concentration obtained by salt methods is up to 533 ± 84 ng µL−1, is a suitable replacement for commercial kits. The PCR-LFS method exhibits high specificity at an annealing temperature of 62 °C and does not cross-react with other animal sources. This strategy is also highly sensitive, being able to detect 0.1% of chicken in artificial adulterated meat. The results of the test strips can be observed with the naked eye within 5 min, and this result is consistent with the electrophoresis result, demonstrating its high accuracy. Moreover, the detection system has already been successfully used to detect chicken in commercial samples. Hence, this PCR-LFS strategy provides a potential tool to verify the authenticity of chicken.

Molecular detection of human papillomavirus DNA in dysplastic and non-dysplastic oral lichen planus patients

Introduction: Oral Lichen Planus (OLP) is a chronic inflammatory disease of cell-mediated dysregulation characterized by relapses and remissions. The aetiology of OLP has been extensively studied for decades. The pathogenesis is unclear with a debatable malignant transformation. The pathogenesis and malignant transformation of OLP may be affected by viruses such as Hepatitis C Virus (HCV), Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), and stress. HPV has been proved to be an etiological agent in oropharyngeal cancers and non-tobacco-associated leukoplakia. The role of human papillomavirus in the pathogenesis of OLP and its malignant transformation has to be studied extensively. Aim: This study aims to detect the presence of HPV DNA in the biopsy samples of dysplastic and non-dysplastic OLP and thus determine the role the virus played in the malignant transformation of OLP. Materials and Methods: Biopsy samples comprising 250 OLP tissues were collected. The DNA was extracted from the fixed tissue by using the Cetyltrimethylammonium bromide (CTAB) method. Polymerase Chain Reaction (PCR) was performed using primers to amplify the HPV E6 gene. Results: Hundred and three out of 250 (41.2%) OLP cases were positive for HPV DNA.

Study on the Algae Contamination and Its Effects on the Properties of RTV-Coated Insulators

The surface of organic insulating materials such as room temperature vulcanized silicone rubber (RTV) coatings often has serious contamination deposition. In humid areas such as the Southwest region of China, algae contamination layers are present on the surface of the insulators. In this study, the geographical and creeping distribution of algae contamination on the surfaces of RTV insulators were studied by investigating and sampling various substations in the Southwest region. The main components of soluble salts in the contamination were studied by atomic absorption spectroscopy and ion chromatography. The algal genome was extracted by the cetyltrimethylammonium bromide method, and the species of algae and other microorganisms in the contamination layer were determined. The effects of algae or their secretions on the surface resistance and hydrophobicity were studied by quantitatively inoculating algae and smearing extracellular secretions on the RTV surface. The damage of the algae contamination layer to the microstructure of the silicone rubber was investigated by microscopic observation and thermogravimetric analysis. Results showed that the growth of algae was positively correlated with the surface contamination of RTV. The extracellular secretion of algae destroys the surface microstructure of RTV and causes the removal of alumina hydroxide, leading to the reduction of siloxane. Therefore, the resistance and hydrophobicity of the RTV surface were reduced. It is of great significance to study the characteristics and effects of the algae contamination layer for RTV maintenance.

A comparison of total RNA extraction methods for RT-PCR based differential expression of genes from Trichoderma atrobrunneum

The genus Trichoderma is ubiquitous in various niches and is currently used for biocontrol, biofertilizer, enzyme production and bioremediation. However, molecular mechanisms underlying its diverse biological functions are yet not fully elucidated. Extraction of high-quality RNA for downstream applications such as quantitative polymerase chain reaction is a prerequisite. The current study aims to optimize a total RNA extraction protocol for high-quality and quantity RNA from Trichoderma atrobrunneum. Seven RNA extraction protocols including Trizol, RiboEx PureLink RNA mini kit, high salt CTAB (cetyltrimethylammonium bromide), modified high salt CTAB, SDS (sodium dodecyl sulfate) and CTAB-PVP (polyvinylpyrrolidone) were performed separately, to extract RNA. Quality and quantity of extracted RNA samples were further analyzed by Nanodrop spectrophotometry, agarose gel electrophoresis and RT-PCR analysis. The results of quantitative and qualitative analysis of RNA samples showed that more intact, high-quality RNA was extracted using the modified high salt CTAB as compared to other methods. The RT-PCR results for the amplification of the genes encoding β-tubulin and Ubiquitin carrier protein also showed lowest threshold cycle (Ct) and coefficient of variation (CV) for RNA samples extracted with the modified high salt CTAB method as compared to RNA samples extracted with other protocols. Therefore, it is proposed that the modified high salt CTAB protocol is an excellent method to obtain high-quality RNA with good yield from T. atrobrunneum for its downstream applications. Moreover, the optimized protocol is very economical and can be used to extract total RNA from a large number of samples.

DNA Barcoding for Dipterocarpus Species in Vietnam Based on Chloroplast Gene Region matK

Objective: Dipterocarp identification is challenging, based on traditional methods of morphological characteristics for species conservation and management. In the present study, the matK region was used as a DNA bar-code to identify the Dipterocarpaceae species in Vietnam. Methods: All the samples were collected at studied localities and identified by morphological methods by the botanist. DNA samples were extracted using the modified Cetyltrimethylammonium bromide method and amplified the matK gene by PCR. Sequencing was performed on an Avant 3100 Automated DNA sequencer system. Results: Thirty-three trees were analyzed, representing 11 species (eight in the Dipterocarpus genus, one in Hopea and one in Vativa) and showing low intraspecific divergence and interspecific divergence within and among Dipterocarpus species, with an average of 0.08% and 0.19%, respectively. However, there was zero distance between D. costatus, D. dyeri, D. intricatus and D. obtusifolius. Three Dipterocarpus species (D. alatus, D. condorensis and D. tuberculatus) were discriminated against, while others (D. costatus, D. dyeri and D. intricatus) showed too low divergent intraspecific lineages and suggested hybridization may be affected them. These results also indicated a low barcoding gap for Dipterocarpus species. The highest gene distance was found between Vatica and Dipterocarpus, with an average of 2.12%. Conclusion: In this study, we used the matK region as a tool to identify some Dipterocarpaceae species in Vietnam. The accurate identification of species may manage and conserve species effectively. Our results showed little sequence differentiation among most Dipterocarpus species. The lack of differentiation may be due to incomplete lineage sort and hybridization between sympatric species. In order to identify species, a combination of two or three chloroplast gene regions should be utilized in further studies. DNA barcoding gap should be analyzed within different genera in Dipterocarpaceae.

Genetic diversity of Sterculia quadrifida from Kupang based on ISSR profiles, stomatal density, and chlorophyll content

Abstract. Jannah N, Pharmawati M, Uslan. 2022. Genetic diversity of Sterculia quadrifida from Kupang based on ISSR profiles, stomatal density, and chlorophyll content. Biodiversitas 23: 2690-2698. This study investigated the genetic diversity of Sterculia quadrifida R.Br.or faloak based on Inter Simple Sequence Repeats (ISSR) molecular markers and determined stomatal density and chlorophyll content from several localities in Kupang District, East Nusa Tenggara, Indonesia. For genetic diversity analysis, samples were collected from the Oelsonbai forest area of Maulafa Sub-district, Kupang City, whereas for stomatal density and chlorophyll content analysis, samples were collected from areas with various degrees of anthropogenic activity from Kupang city. DNA was isolated using Cetyl Trimethyl Ammonium Bromide (CTAB) method and amplified by three ISSR primers (UBC 811, UBC 812, and UBC 815), whereas stomatal density was observed using a microscope and chlorophyll content was determined using a spectrophotometer. Genetic data were analyzed using the unweighted pair group method with arithmetic mean (UPGMA) method and Nei &amp; Li coefficient similarity in the MVSP software. A total of 78 ISSR bands ranging from 320-3200 bp were obtained, where populations were divided into four main clusters and six sub-clusters, similarly having moderate heterozygosity (He=0.50) not clustered based on their geographical distribution. There was a difference in stomatal density and chlorophyll content, probably influenced by anthropogenic disturbances. A conservation action plan is required to ensure the S. quadrifida's survival in the future.

Genome Resource of Colletotrichum spaethianum, the Causal Agent of Leaf Anthracnose in Polygonatum falcatum

Genome Resource of <i>Colletotrichum spaethianum</i>, the Causal Agent of Leaf Anthracnose in <i>Polygonatum falcatum</i>

An optimized method to\xa0obtain high-quality RNA from different tissues in Lilium davidii var. unicolor

The high quality, yield and purity total RNA samples are essential for molecular experiments. However, harvesting high quality RNA in Lilium davidii var. unicolor is a great challenge due to its polysaccharides, polyphenols and other secondary metabolites. In this study, different RNA extraction methods, namely TRIzol method, the modified TRIzol method, Kit method and cetyltrimethylammonium bromide (CTAB) method were employed to obtain total RNA from different tissues in L. davidii var. unicolor. A Nano drop spectrophotometer and 1% agarose gel electrophoresis were used to detect the RNA quality and integrity. Compared with TRIzol, Kit and CTAB methods, the modified TRIzol method obtained higher RNA concentrations from different tissues and the A260/A280 ratios of RNA samples were ranged from 1.97 to 2.27. Thus, the modified TRIzol method was shown to be the most effective RNA extraction protocol in acquiring RNA with high concentrations. Furthermore, the RNA samples isolated by the modified TRIzol and Kit methods were intact, whereas different degrees of degradation happened within RNA samples isolated by the TRIzol and CTAB methods. In addition, the modified TRIzol method could also isolate high-quality RNA from other edible lily bulbs. Taken together, the modified TRIzol method is an efficient method for total RNA isolation from L. davidii var. unicolor.

Effective strategies for isolating DNA from members of Asteraceae with high concentrations of secondary metabolites.

The Asteraceae are the largest plant familybut among the least studied at the genome level. Our work investigated practical methods to reduce the influence of secondary metabolites -specifically, phenolic compounds - on the extraction of DNA from Silphium spp. This genus is in the Heliantheae tribe of Asteraceae that also includes sunflower (Helianthus annuus). Three methods were attempted in Silphium, with varying condition and age of the leaf sample. A modified cetyl trimethylammonium bromide (CTAB) method on young leaves resulted in the best DNA yield, with sufficient sample purity. No perceptible difference was observed between fresh and lyophilized samples for any extraction method or leaf age. These results provide an excellent basis for DNA extraction of difficult plant samples.

Molecular Characterization and Phylogenetic Analysis of Earthworm Species Collected from Different Soil Habitats of Poonch Division Azad Jammu and Kashmir Pakistan.

This study aims to analyze molecular characterization and phylogenetic analysis of earthworm species collected from different soil habitats of Poonch division Azad Kashmir Pakistan by using CO1 gene partial sequencing methodology. Samples gathered randomly from 18 study sites (127 localities) by digging and hand sorting methods were preserved in pure ethanol at -20°C. The modified CTAB (Cetyltrimethyl ammonium bromide) method extracted high quality DNA from region of representative earthworm's caudal region. This extracted DNA was used to amplify the 700 bp partial region of the cytochrome oxidase I (COI) gene with LCO1490 and HCO2198 universal primers. All of the obtained amplified gene sequences were aligned, edited and analyzed using MEGA X software to characterize different species of earthworms. Thirty-eight (38) Barcoding sequences belonging to 11 different strains of earthworms were successfully generated. Their phylogenetic analysis revealed that 7 Barcoding sequences gave maximum similarity with the available online database, while the rest of the 4 sequences gave lower similarity than the maximum threshold level. The collected DNA barcode sequences were also clustered together by the maximum likelihood method and the resultant phylogenetic tree revealed they belong to different family lineages. Moreover the identified earthworm species have a close evolutionary link with the earthworm fauna of south and central Asia instead of Europe, which might be due to similar climate of both regions.

An optimized method for high-quality DNA extraction medicinal fungi Mycoleptodonoides aitchisonii for whole genome sequencing

The extraction of pure high molecular weight DNA and collecting genomic DNA from fungi is difficult. This is because filamentous fungi has a sturdy cell wall, high protein and polysaccharide levels resistant to the usual DNA extraction procedures. A low-cost and reliable DNA extraction method was designed from Mycoleptodonoides. aitchisonii fit for whole-genome sequencing for identification and mapping of the A mating-type gene. Mycoleptodonoides. aitchisonii belongs to the Climacodontaceae family has pharmaceutical activities. In the present study, the mycelia of M. aitchisonii, which grew from the different concentrations of malt extract and minimal liquid media, have been compared systematically to determine the DNA extraction procedure resulted in DNA concentration with excellent purity and quality. The best protocol that resulted in good quality DNA was further validated using polymerase chain reaction amplification with a specific primer to amplify the homeodomain protein (Mahd2-18) gene that encodes a transcription factor. The method proposed DNA extraction using CTAB (Cetyl Trimethyl Ammonium Bromide) method and purify by commercial kit from mycelium grown in the minimal liquid media give the best result with the high concentration of DNA for whole-genome sequencing by Next Generation Sequencing and other applications.