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Abstract Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi.

Molecular studies of fungi within the order Laboulbeniales (Ascomycota, Pezizomycotina) have been hampered for years because of their minute size, inability to grow in axenic culture, and lack of reliable and cost-efficient DNA extraction protocols. In particular, the genus Laboulbenia is notorious for low success with DNA extraction and polymerase chain reaction (PCR) amplification. This is attributed to the presence of melanin, a molecule known to inhibit PCR, in the cells. We evaluated the efficacy of a standard single cell-based DNA extraction protocol by halving the recommended amount of reagents to reduce the cost per extraction and adding bovine serum albumin (BSA) during the multiple displacement amplification step to reverse the effect of melanin. A total of 196 extractions were made, 111 of which were successful. We found that halving the reagents used in the single cell-based extraction kit did not significantly affect the probability of successful DNA extraction. Using the halved protocol reduces cost and resource consumption. Moreover, there was no significant difference in the probability of successfully extracting DNA based on whether BSA was added or not, suggesting that the amount of melanin present in cells of the thallus has no major inhibitory effect on PCR. We generated 277 sequences from five loci, but amplification of the internal transcribed spacer region, the mitochondrial small subunit rDNA, and protein-coding genes remains challenging. The probability of successfully extracting DNA from Laboulbeniales was also impacted by specimen storage methods, with material preserved in > 95% ethanol yielding higher success rates compared to material stored in 70% ethanol and dried material. We emphasize the importance of proper preservation of material and propose the design of Laboulbeniales-specific primers to overcome the problems of primer mismatches and contaminants. Our new insights apply not only to the genus Laboulbenia; Laboulbeniales generally are understudied, and the vast majority of species remain unsequenced. New and approachable molecular developments will benefit the study of Laboulbeniales, helping to elucidate the true diversity and evolutionary relationships of these peculiar microfungi. [ABSTRACT FROM AUTHOR]

A total of 19 fungal species were isolated from the seeds of selected rice varieties (BRRI dhan 90 to BRRI dhan 99) following Tissue planting method and Blotter method. The isolated fungi were Aspergillus niger, A. ochraceus, A. oryzae, A. tamarii, A. terreus, Chaetomium globosum, Cladosporium oxysporum, Colletotrichum gloeosporioides, Corynespora cassiicola, Curvularia lunata, Curvularia soli, Daldinia eschscholtzii, Fusarium solani, Penicillium oxalicum,Penicillium sclerotiorum, Pestalotiopsis guepinii, Pyricularia oryzae and Rhizopus stolonifer. Fourteen fungi were selected for molecular identification. Out of the 19 fungal isolates, 14 were confirmed up to species level through ITS sequence based molecular analysis. Among the isolated fungi Pnicillium sclerotiorum and Curvularia soli are the new record for Bangladesh. Association of Daldinia eschscholtzii with rice seeds is also recorded first time from world. [ABSTRACT FROM AUTHOR]

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DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a variant of CE7 (Colicin E7 DNase) from Escherichia coli. The resulting novel recombinant open reading frame was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 105 nM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required. Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics. [ABSTRACT FROM AUTHOR]

Mutations in the SARS‑CoV‑2 genome make it possible to effectively escape defense mechanisms of the host, which explains the spread of infection among vaccinated or previously affected by the virus individuals.The aim of the study was to investigate the dynamics of mutations in the SARS‑CoV‑2 virus genome during the rise of the seasonal incidence in the Chuvash Republic.Materials and methods. Under conditions of the clinical diagnostic laboratory of the Federal Center for Traumatology, Orthopedics and Endoprosthetics of the Ministry of Health of Russia (Cheboksary), samples, containing SARS‑CoV‑2 RNA, taken in January-February and July-October, 2022 were tested using reverse transcription PCR. The “MBS-Test SARS‑CoV‑2 RNA” (Technical Specifications 21.20.23-068-26329720-2021, Russia) and “AmpliTest SARS‑CoV‑2 VOC v.3” (Series V017, Certificate of Registration No. 2022/16307, Russia) were utilized in compliance with the manufacturer’s instructions.Results and discussion. Variations in the sets of SARS‑CoV‑2 S gene mutations have been revealed in the studied samples obtained during different periods of the spread of SARS‑CoV‑2 coronavirus. Timely detection of various mutations in the virus genome at the beginning of the epidemiological season and the alleged rise in the incidence of coronavirus infection is valuable information for forecasting the rate of virus transmission. It can also be used to create vaccines (taking into account changes in the virus genome) and to choose the adequate tactics for treating coronavirus infection.

At the Maritsa Vegetable Crops Research Institute, species X. euvesicatoria and X. vesicatoria were isolated from pepper and molecularly identified. The isolates belonging to X. euvesicatoria refer to pathotypes P (P6 with 2 strains and P4 with 2 strains) and PT (P4T2 with 5 strains and P2T2 with 2 strains). The predominant races of X. vesicatoria were PT (P1T2 with 3 strains and P2 with 2 strains). From all examined isolates, it was established that X. euvesicatoria was more often isolated from pepper. Of the available pepper gene pool, 13 breeding lines and 3 accessions have been studied for their reaction to X. euvesicatoria P6, P4T2, and X. vesicatoria P2 and P1T2. Immune to X. euvesicatoria P6, P4T2, and X. vesicatoria P2, P1T2 were of pepper genotypes K915, K917, K925, SOL-300 and SOL-361, the rest were classified as resistant. The studied genotypes varied by fruit characteristics-size, shape, orientation, color, and taste. Breeding line K915 has been identified as immune to all studied races of bacterial spot (X. euvesicatoria P6 and P4T2 and X. vesicatoria P2 and P1T2), which combined with its fruit traits, makes it extremely valuable for breeding activity in this direction. [ABSTRACT FROM AUTHOR]

The amount of pathogenic bacteria in feces is an indicator characterizing the health status of animals. This is important to protect the environment and the health of farm workers. The aim of the present study is to determine the effect of probiotic Zoovit on the microbiome of the faecal mass of Holstein cattle. In the experiment, two groups were formed - experimental and control, 30 each. A probiotic was added to the combined feed of the experimental group, but not to the control group. The food ration with the participation of probiotic is placed twice a day. To determine the microbiome in fecal matter in cattle, PCR analyzes were used for the detection of Lactobacillus delbrueckii subsp. bulgaricus, a species-specific PCR assay for the presence of total DNA in L. delbrueckii and L. delbrueckii subsp. bulgaricus and methods for determination of sulfite-reducing colonies. No representatives of Staphulococccua aureus and Clostridium sp were isolated in the faecal mass of the animals from both groups. For Escherichia coli and Coliforms, a significant decrease in the faecal mass of cows fed with feed containing probiotics was found. The presence of L. delbrueckii subsp. bulgaricus in animals receiving a probiotic. Colonies with a colonial characteristic typical of L. delbrueckii subsp. bulgaricus were isolated in the animals receiving probiotic Zoovit and in the control group, no colonies typical of Lactobacillus bulgaricus were found. [ABSTRACT FROM AUTHOR]

Most Dalbergia species are economically valuable and have been over-exploited, which has raised concerns. The regulation and protection of this genus require accurate and rapid authentication and identification processes. To address the issue of high residual inhibitors in extracted DNA from the Dalbergia xylem, an optimized DNA extraction experiment was performed on 10 species of Dalbergia wood stored for 1–5 years; in particular, no gene sequence for D. tsoi can be found in the NCBI database. Additionally, universal primers ITS2 were used for PCR amplification and sequencing to confirm the effectiveness of DNA extraction. The results revealed that rinsing the wood with 0.25 M ammonium acetate buffer produced DNA with a high purity, without a significant decrease in the DNA yield. To achieve an optimal DNA yield, the wood DNA should be rinsed with ammonium acetate fewer than three times. All the wood DNA obtained using the kit method and treated with the ammonium acetate buffer rinsing solution one to four times was successfully amplified. The NJ phylogenetic tree constructed based on ITS2 can distinguish D. tsoi from other Dalbergia spp., and the predicted ITS2 secondary structure showed the difference between species. This experiment extracted high-quality DNA from wood, without the need for purification kits, thereby improving the efficiency of the extraction process. The extracted DNA was directly used for follow-up molecular experiments. [ABSTRACT FROM AUTHOR]

The approach based on molecular modeling was developed to study dNTP derivatives characterized by new polymerase-specific properties. For this purpose, the relative efficiency of PCR amplification with modified dUTPs was studied using Taq, Tth, Pfu, Vent, Deep Vent, Vent (exo-), and Deep Vent (exo-) DNA polymerases. The efficiency of PCR amplification with modified dUTPs was compared with the results of molecular modeling using the known 3D structures of KlenTaq polymerase–DNA–dNTP complexes. The dUTPs were C5-modified with bulky functional groups (the Cy5 dye analogs) or lighter aromatic groups. Comparing the experimental data and the results of molecular modeling revealed the decrease in PCR efficiency in the presence of modified dUTPs with an increase in the number of non-covalent bonds between the substituents and the DNA polymerase (about 15% decrease per one extra non-covalent bond). Generalization of the revealed patterns to all the studied polymerases of the A and B families is discussed herein. The number of non-covalent bonds between the substituents and polymerase amino acid residues is proposed to be a potentially variable parameter for regulating enzyme activity. [ABSTRACT FROM AUTHOR]

DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a variant of CE7 (Colicin E7 DNase) from Escherichia coli. The resulting novel recombinant open reading frame was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 105 nM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required. Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics.

Copyright of Mycosystema is the property of Mycosystema Editorial Board and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The recombinant protein of heterologous proteins in Escherichia coli strains K12 has various and different systems tested and demands a detailed insight into the multiple factors affecting the encoded protein. One of the crucial factors is the acceptable quality of the DNA copies inserted inside the bacteria. Firstly, the amplification procedure needed to be performed well; thus, designing the primer and selecting the optimum annealing temperature are the focus indicators in this study. This study obtained a reference gene from the NCBI data bank with Reference Sequence: NM_174187.2. Two types of primers (SPP1FSPP1R and OPN1F - OPN1R) with different targeted bands were designed and selected after being reconstructed using the software. Online software such as addgene.org is also used to identify the right restriction site. The annealing temperature distinguished the PCR system used to amplify each primer. The result of this study revealed the best annealing at 65ºC successfully amplified 820 bp of the targeted band. The phenomenon not following the theory of blue-white screening is the empty plasmid control, where not a single colony grows on the media. Competent cells inserted with empty plasmids should still be able to expand on LB-Amp agar media because the presence of these plasmids is capable of providing resistance to antibiotics (in this case, ampicillin). This discrepancy is thought to have been caused by the improper insertion of the empty plasmid so that the plasmid did not enter the competent cell. [ABSTRACT FROM AUTHOR]

Background: The bacterial speck disease of tomato caused by a bacterial pathogen Pseudomonas syringae pv. tomato is a most important disease causing severe crop losses. Methods and results: Present study was conducted to investigate and characterize the population diversity of P. syringae pv. tomato pathogen isolated from infected tomato plants from various regions of Egypt. Significant variation among the isolates was observed which demonstrated considerable virulence. All isolates were pathogenic and the CFU population recovered from inoculate tomato leaves by isolate Pst-2 was higher than other isolates. Genetic disparity among the isolates was investigated by PCR analysis by amplifying hrpZ gene using random amplified polymorphic DNA (RAPD), sequence-related amplified polymorphism (SRAP), and inter-simple sequence repeats (ISSR) markers. The amplified products for ITS1 were found to have 810 bp length whereas 536 bp length was observed for hrpZ gene using primer pairs (1406-f/23S-r) and (MM5-F, MM5-R) respectively. The restriction analysis of amplified regions "ITS" and hrpZ by using 5 and 4 endonucleases respectively demonstrated slight variation among the bacterial isolates. The results of RAPD, ISSR and SRAP showed higher polymorphism (60.52%) within the isolates which may assist for successful characterization by unique and specific markers based on geographical distribution, origin and virulence intensity. Conclusion: The results of present study suggested that the use of molecular approach may provide successful and valuable information to differentiate and classify P. syringae pv. tomato strains in future for the detection and confirmation of pathogenicity. [ABSTRACT FROM AUTHOR]

【Objective】The study aims to explore the composition, sequence number and diversity of microbial community in Zhuhai Wailingding Marine Ranch, analyze the sensitivity and reducing power of eDNA technology, with a view to providing references for the management and maintenance of ecosystem in Zhuhai Wailingding Marine Ranch.【Method】In September 2021, 9 stations were set up in Zhuhai Wailingding Marine Ranch to sample at 2 m below the surface water for pretreatment, DNA extraction, PCR amplification, and high-throughput sequencing to analyze the composition of microbial species, the density and abundance of microbial resources, and determine the dominant species.【Result】A total of 30 species of microorganisms in Zhuhai Wailingding Marine Ranch were detected by eDNA technology, and 5 phyla, 7 classes, 14 orders, 19 families and 26 genera were identified, and a total of 779 706 Operational Taxonomic Units (OTUS) were detected. The Proteobacteria was the absolute dominant phylum accounting for 75.06%, followed by Bacteroidetes (11.35%), Firmicutes (5.28%), Planctomycetes (1.91%), and Actinobacteria (1.06%). There were 7 dominant species, which were Pseudoalteromonas, Marinobacter, Moraxella, Psychrobacter, Vibrio alginolyticus, Halomonas and Sphingobacterium. The dominance of Vibrio alginolyticus reached 0.19, which was the absolute dominant species, followed by Moraxella and Pseudoalteromonas reaching 0.134 and 0.127, respectively, which were the main dominant species.【Conclusion】The study reveals the microbial community information of the Zhuhai Wailingding Marine Ranch, explores the application of eDNA technology in marine ranch, and verifies the efficiency and sensitivity of this technology, providing basic data and scientific reference for future research on marine ranching microorganisms.

Highlight Research • The genetic diversity of fish and shrimp from mangrove habitats was relatively substantial, with a heterozygosity of 0.926 and the exception of Langsa samples. • AMOVA revealed that the diversity among individuals in the populations were higher compared to those within individuals. • DNA barcode in mangroves is useful in developing mangrove conservation and restoration initiatives Abstract Fish and shrimp are among the species that are vulnerable to high level of disturbance in mangrove ecosystem. This study aimed to investigate the implementation of 16S rRNA gene for fish and shrimp barcoding in mangrove ecosystems in North Sumatra and Aceh, Indonesia. 50 samples fresh fish and shrimp were collected from five mangrove ecosystems in North Sumatra, namely Belawan, Lubuk Kertang, Percut Sei Tuan, and Pulau Sembilan and Langsa of Aceh for DNA extraction process. The research was divided into four activities: sample collection, DNA isolation, PCR amplification, and data analysis. DNA was extracted using kit extraction (kit Reliaprep gDNA Tissue Miniprep System) and with PCR amplification. Genetic parameters were analyzed using Gen Alex 6.51 software and the relationship between sites was examined using the MVSP 3.22. The genetic diversity of fish and shrimp from mangrove habitats was relatively substantial, according to the findings, with a heterozygosity of 0.926, with the exception of Langsa samples, which were only identified in subclass A1. Genetic relationship between sites revealed that all samples clustered into two branches and were randomly dispersed within each site. This work confirmed the 16S rRNA gene worked for fish and shrimp barcoding in mangrove ecosystems, North Sumatra and Aceh, Indonesia and our findings are expected to be useful in developing mangrove conservation and restoration initiatives.

Pediatric COVID-19 determines a mild clinical picture, but few data have been published about the correlation between disease severity and PCR amplification cycles of SARS-CoV-2 from respiratory samples. This correlation is clinically important because it permits the stratification of patients in relation to their risk of developing a serious disease. Therefore, the primary endpoint of this study was to establish whether disease severity at the onset, when evaluated with a LqSOFA score, correlated with the gene amplification of SARS-CoV-2. LqSOFA score, also named the Liverpool quick Sequential Organ Failure Assessment, is a pediatric score that indicates the severity of illness with a range from 0 to 4 that incorporates age-adjusted heart rate, respiratory rate, capillary refill and consciousness level (AVPU). The secondary endpoint was to determine if this score could predict the days of duration for symptoms and positive swabs. Our study included 124 patients aged between 0 and 18 years. The LqSOFA score was negatively correlated with the number of PCR amplification cycles, but this was not significant (Pearson's index −0.14, p-value 0.13). Instead, the correlation between the LqSOFA score and the duration of symptoms was positively related and statistically significant (Pearson's index 0.20, p-value 0.02), such as the correlation between the LqSOFA score and the duration of a positive swab (Pearson's index 0.40, p-value < 0.01). So, the LqSOFA score upon admission may predict the duration of symptoms and positive swabs; the PCR amplification of SARS-CoV-2 appears not to play a key role at onset in the prediction of disease severity. [ABSTRACT FROM AUTHOR]

DNA barcoding of herbs allows accurate species authentication. However, the DNA of herbs are often not easily PCR amplified due to co-extraction of inhibitors. Methods have been developed to improve DNA extraction to reduce contaminants. These methods usually require toxic chemical treatments or expensive commercial kits and are labor intensive. In this report, we collected the air passed from the herbs and directly amplified the DNA obtained. Results showed that DNA could be obtained, and it was PCR amplifiable. Sequencing of the amplified DNA allowed species authentication. This DNA collection method is applicable to herbs from different plant tissues. It has the advantages of reducing the use of toxic substances and more economical.

[Objective] The study aims to explore the composition, sequence number and diversity of microbial community in Zhuhai Wailingding Marine Ranch, analyze the sensitivity and reducing power of eDNA technology, with a view to providing references for the management and maintenance of ecosystem in Zhuhai Wailingding Marine Ranch. [Method] In September 2021, 9 stations were set up in Zhuhai Wailingding Marine Ranch to sample at 2 m below the surface water for pretreatment, DNA extraction, PCR amplification, and high-throughput sequencing to analyze the composition of microbial species, the density and abundance of microbial resources, and determine the dominant species. [Result] A total of 30 species of microorganisms in Zhuhai Wailingding Marine Ranch were detected by eDNA technology, and 5 phyla, 7 classes, 14 orders, 19 families and 26 genera were identified, and a total of 779 706 Operational Taxonomic Units (OTUS) were detected. The Proteobacteria was the absolute dominant phylum accounting for 75.06%, followed by Bacteroidetes (11.35%), Firmicutes (5.28%), Planctomycetes (1.91%), and Actinobacteria (1.06%). There were 7 dominant species, which were Pseudoalteromonas, Marinobacter, Moraxella, Psychrobacter, Vibrio alginolyticus, Halomonas and Sphingobacterium. The dominance of Vibrio alginolyticus reached 0.19, which was the absolute dominant species, followed by Moraxella and Pseudoalteromonas reaching 0.134 and 0.127, respectively, which were the main dominant species. [Conclusion] The study reveals the microbial community information of the Zhuhai Wailingding Marine Ranch, explores the application of eDNA technology in marine ranch, and verifies the efficiency and sensitivity of this technology, providing basic data and scientific reference for future research on marine ranching microorganisms. [ABSTRACT FROM AUTHOR]

The techniques and the principles used in DNA isolation play a vital role in the obtaining of a purified genetic material. In present study, we investigated the efficiency of seven genomic DNA isolation techniques in terms of isolated DNA concentration, yield and purity from internal organs, gills and muscle tissues of Notopterus notopterus. Isolated DNA quality was analysed through Nanodrod and PCR using mitochondrial COI genetic markers. Results showed that GeneJET Genomic DNA Purification Kit was found significantly higher in terms of isolated DNA concentration (1200-1288 ng.ul-1), yield (257.6 ng.ul-1) and purity (1.91-2.00), and also successful in PCR amplification as compared to other evaluated six traditional DNA isolation methods. The nucleotide minimum bands range was observed 200 base pairs in heart sample and maximum 600 base pair was observed in intestine. There is no data on description of parameters analysed in this work to date, neither the evaluation of isolated DNA using PCR amplification of mitochondrial COI gene for the species N. notopterus. Present study also revealed that the traditional DNA isolation methods are the secondary choice for isolation of DNA. The data of present study also indicated that the GeneJET Genomic DNA Purification Kit is useful for DNA isolation and can be used best in genetic applications for fishes. [ABSTRACT FROM AUTHOR]

A total of 14 varieties (CB1-CB14) of cotton (Gossypium hirsutum L.) seeds were collected from Cotton Research, Training and Seed multiplication Farm, Sreepur, Gazipur to detect and identify the seed borne fungi by morphological and molecular techniques. The sequence results obtained using the ITS1 and ITS4 primers were compared with NCBI GenBank and BOL database using BLAST analysis. In the present investigation, a total of 29 fungal isolates were morphologically identified from different varieties of cotton seeds, of which 19 fungal isolates were identified by molecular techniques. Among the isolated fungi, Aspergillus subramanianii, A. toxicarius, A. wentii, Penicillium aculeatum, P. citrinum, Rhizomucor sp. and Meyerozyma guilliermondii have been reported as new records for Bangladesh. [ABSTRACT FROM AUTHOR]

Self-incompatibility is considered to be a growth-limiting factor in fruit plants. In species with hermaphrodite flowers, S-locus (S-allele) has been accepted to control self-incompatibility, and the genetic control of this locus is provided by multiple genes (alleles). Pear (Pyrus communis L.) belongs to the Pomoideae from the Rosaceae family and is found to have great genetic potential in terms of ecological features in Turkey. To protect these cultivation features, national garden collections have been established across the country and Atatürk Horticultural Central Research Institute–Yalova collection is considered as genes bank. Identification of the different features of this collection (fruit quality, stress tolerance, self-incompatibility, grafting incompatibility, etc.) is of great importance for its utilization in pear breeding and cultivation. However, to our knowledge, this collection has not been characterized for self-incompatibility trait. In the current study, PCR (Polymerase Chain Reaction)-based amplification of the S-allele regions (S1, S6, S7, S8) causing the self-incompatibility in 180 pear genotypes obtained from the national pear germplasm was investigated by molecular biological methods based on the comparison of amplified products. In 180 pear genotypes, the S6 allele was the most prevalent one with 63% frequency, while the S8 allele was the least common allele with a rate of 4%. In allele combinations, the S1-S6 allele combination was the most common allele combination with a rate of 18%, and trilateral allele combinations (S1-S6-S7 and S1-S6-S8) were observed at a rate of 1%. Findings of the current research will enable the classification of the materials and the analysed material is likely to be used in breeding studies as well as pear cultivation.

Background: Recent years, multi drug resistant pathogens and their pathogenicity were increased worldwide due to unauthorized consumption of antibiotics. In addition, correlation between multi drug resistant bacteria and biofilm formation is heightened due to the production of more virulence behavior. There is no better identification methods are available for detection of biofilm producing gram negative bacteria. Materials and methods: In this research work, multi drug resistant strains of Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) were identified based on the specific antibiotics and third generation cephalosporin discs by disc diffusion assay. Subsequently, biofilm forming ability of selected pathogens were identified tissue culture plate and tube test. Based on the multi-drug resistant ability and biofilm production, the molecular identification of P. aeruginosa and K. pneumoniae were confirmed by PCR using universal primers. Results and conclusions: No zone of inhibition present around the discs of muller hinton agar plates were confirm, selected P. aeruginosa and K. pneumoniae strains were multi drug resistant pathogens. Performed third generation cephalosporin antibiotics were also highly sensitive to selected pathogens of P. aeruginosa and K. pneumoniae. Further, biofilm forming ability of selected P. aeruginosa and K. pneumoniae was confirmed by tissue culture plate and tube methods. Finally, molecular identification of P. aeruginosa and K. pneumoniae was named as P. aeruginosa and K. pneumoniae. Our result was conclude, selected P. aeruginosa and K. pneumoniae as biofilm producing pathogens and also highly resistant to current antibiotics.

Ulmus villosa Brandis ex Gamble, an economically and ecologically important forest tree, is native to the western Himalayas of Pakistan. The long pressure imposed by unsustainable utilization and market demands has resulted in the rapid decline of the U. villosa population in the wild. To date, very limited information on the genomic diversity of U. villosa is available and this can tremendously limit our understanding of distribution and future conservation of U. villosa. Therefore, the current study aimed to assess genetic diversity within U. villosa wild populations of the Hazara Division using four barcoding markers (i.e., rbcL, matK XF + 5R, matK 390F + 1326R and trnH-psbA). A total of six high quality sequences were obtained with rbcL, four with matK XF + 5R, four with matK 390F + 1326R, and fourteen with trnH-psbA. The sequenced regions contained insertions and deletions as well as several SNPs across the length of sequences, and PCR-based markers may be developed from these novel SNPs markers. The phylogenetic results supported the polyphyletic origin of the genus Ulmus, and the data indicated that multiple speciation events may have led to the evolution of U. villosa in this region. For deeper understanding of the origin and evolution of U. villosa, sequencing of the complete nuclear and chloroplast genomes will be pivotal. The results herein provide novel insights into the genomic diversity as well as phylogenetic relationships of U. villosa with other species, and may facilitate both in situ and ex situ conservation efforts for this endangered species. [ABSTRACT FROM AUTHOR]

Museum collections harbor millions of samples, largely unutilized for long-read sequencing. Here, we use ethanol-preserved samples containing kilobase-sized DNA to show that amplification-free protocols can yield contiguous genome assemblies. Additionally, using a modified amplification-based protocol, employing an alternative polymerase to overcome PCR bias, we assembled the 3.1 Gb maned sloth genome, surpassing the previous 500 Mb protocol size limit. Our protocol also improves assemblies of other difficult-to-sequence molluscs and arthropods, including millimeter-sized organisms. By highlighting collections as valuable sample resources and facilitating genome assembly of tiny and challenging organisms, our study advances efforts to obtain reference genomes of all eukaryotes., Competing Interests: Competing interests The authors have no competing interests.

Indonesia is a country with high variability of microorganisms, including bacteria, yeast, and fungi. Yeast isolates could be isolated from the honeycomb of Apis dorsata. Molecular approaches were used to identify yeast using ribosomal DNA gene sequences, called the ITS gene. The optimum condition for DNA extractions and amplifications are needed for the successfully of molecular identification. Therefore, it is necessary to optimize the DNA extraction and amplification of several protocols to obtain good identification results. This study aimed to compare the effects of DNA extraction with various temperatures and different amplification protocols. LIPI reference DNA extraction protocol with the boiling method and variations in incubation time of 10, 15, and 20 minutes at a temperature of 98° C. Meanwhile, for the amplification of yeast DNA using a variety of different amplification protocols. The results showed the optimal time of incubation was 10 minutes in K1 isolates with DNA purity of 1.896. meanwhile, for isolates K2, K3, and K4 each with a purity of 2.246, 2.335, and 1.748. optimal DNA amplification results were indicated by the presense of DNA bands for each sample K1, K2, K3, and K4, namely 503, 542, 492, and 526 bp. In this study, it can be concluded that the optimal incubation time for the extraction process is 10 minutes. In addition, the optimal amplification protocol was shown in the DNA bands in all sample.

Abstract Background Phalaris species (Poaceae) occupy diverse environments throughout all continents except Antarctica. Phalaris arundinacea is an important forage, ornamental, wetland restoration and biofuel crop grown globally as well as being a wetland invasive. The nuclear ribosomal internal transcribed spacer (ITS) region has been used for Phalaris barcoding as a DNA region with high nucleotide diversity for Phalaris species identification. Recent findings that P. arundinacea populations in Minnesota USA are most likely native and not European prompted this analysis to determine whether Eurasian vs. native North American P. arundinacea differed in ITS regions. Our objectives were to amplify and compare ITS regions (ITS1 and ITS2) of historic herbaria (1882–2001) and extant (fresh) Phalaris specimens; analyze ITS regions for species-specific polymorphisms (diagnostic SNPs) and compare ITS regions of historic Phalaris specimens with known, extant Phalaris species. Results We obtained complete ITS1 and ITS2 sequences from 31 Phalaris historic (herbaria samples, 1908 to 2001) and five extant (fresh) specimens. Herbaria Phalaris specimens did not produce new SNPs (single nucleotide polymorphisms) not present in extant specimens. Diagnostic SNPs were identified in 8/12 (66.6%) Phalaris species. This study demonstrates the use of herbaria tissue for barcoding as a means for improved species identification of Phalaris herbaria specimens. No significant correlation between specimen age and genomic DNA concentration was found. Phalaris arundinacea showed high SNP variation within its clade, with the North American being distinctly different than other USA and most Eurasian types, potentially allowing for future identification of specific SNPs to geographic origin. Conclusions While not as efficient as extant specimens to obtain DNA, Phalaris herbaria specimens can produce high quality ITS sequences to evaluate historic genetic resources and facilitate identification of new species-specific barcodes. No correlation between DNA concentration and age of historic samples (119 year range) occurred. Considerable polymorphism was exhibited in the P. arundinacea clade with several N. American accessions being distinct from Eurasian types. Further development of within species- and genus-specific barcodes could contribute to designing PCR primers for efficient and accurate identification of N. American P. arundinacea. Our finding of misidentified Phalaris species indicates the need to exercise stringent quality control measures on newly generated sequence data and to approach public sequence databases in a critical way.

The success of polymerase chain reaction (PCR) assay depends on template deoxyribonucleic acid (DNA) being sufficient with respect to both quantity and quality. Some biological materials contain compounds which inhibit the functioning of DNA polymerase and thus need to be removed as part of the DNA extraction procedure. The aim of the present experiments was to optimise the process of DNA isolation from various types of black, fruit and herbal teas. A comparison was made between two cetyltrimethylammonium bromide (CTAB)-based protocols and two commercially available DNA purification kits. The yield and integrity of the extracted DNA were monitored both spectrophotometrically and using agarose gel electrophoresis. The presence/absence of inhibitors in the DNA preparations was checked by running quantitative real-time PCRs. The optimal protocol was deemed to be the CTAB method described in ISO 21571:2005, so this method is recommended for the routine sample analysis of tea products.

Pigeon pea Cajanus cajan (L.) Millsp. and cowpea Vigna unguiculata (L.) Walp. are two of the important seed legumes of Africa and are relatively more drought-tolerant than many other legumes. Nigeria is the largest producer of cowpea in Africa, while pigeon pea is yet to be commercially cultivated in Nigeria. The genomic information on cowpea is known but not for pigeon pea, and this has hindered the development of superior varieties. Most importantly, although various genetic markers have been used in both legumes to elucidate their genetic diversity, simple sequence repeats and single nucleotide polymorphism markers are preferred for genetic and plant breeding applications. We investigated the genetic diversity in the two legumes and traced the evolutionary relationship of the large subunit of the ribulose-bisphosphate carboxylase/oxygenase (RuBisCo) (rbcl) gene in selected organisms using cowpea and pigeon pea rbcl gene sequences as queries. The genetic diversity study analysed 25 pigeon pea and 22 cowpea accessions using rbcl gene region primers. A total of 432 polymorphic sites were detected from the obtained legume DNA sequences. The phylogeny of the two legumes was a location/origin of adoption to the germplasm. Analysis of molecular variance of the rbcl gene showed that among-population variance was 0% and within-population variance was 100%, suggesting a wide genetic diversity among pigeon pea and cowpea populations. This study provided a basis for the understanding of the evolutionary relatedness and the genetic diversity of cowpea and pigeon pea accessions. To substantiate the diversity of these legumes, wider regional sampling of seeds in Nigeria should be implemented. [ABSTRACT FROM AUTHOR]

The main culprits of catastrophic agricultural losses or crop mortality are phytopathogenic fungi and parasitic plants. To manage the plant pathogens, a simple and rapid disease diagnosis is needed. The aim of the experiment was to develop simple and rapid genomic DNA extraction and PCR amplification methods for fungi and parasitic plants. For the DNA extraction, mycelia from 16 fungi species and stems from two parasitic plants species were incubated in a lysis buffer and were homogenised using a sterilised wooden stick. The homogenates were incubated at 95°C temperature for 10 min. Crude extracts served as a template for the PCR amplification containing UKOD polymerase. The application of lysis buffer, mechanical and heat disruption resulted in a fast DNA extraction from fungi and parasitic plants. DNA amplification time is reduced when using Master Mix containing UKOD polymerase. The presented results confirm that these simple and rapid DNA extraction and PCR amplification methods are applicable to diverse fungi species and parasitic plants. [ABSTRACT FROM AUTHOR]

Hereditary properties of strong growth and huge accumulation of biomass in Oryza officinalis exhibit a great potential; however, the genes that code for its high photosynthesis performance are not established. This study screened eight hybrids, using biomass accumulation and photosynthesis analysis, based on the introgression lines constructed by analyzing distant hybridization patterns between Oryza officinalis and cultivars HY-8. We designed 23 primer pairs using transcriptome sequencing of Oryza officinalis and screened two types of photosynthetic enzymes: phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK), which are two related proteins of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco), its binding protein (rubis-subs-bind), and a small subunit (rbcS). Results showed that C4 photosynthetic pathway enzymes, PEPC and PPDK, were highly expressed in hybrids and the source plant, Oryza officinalis. Homology analysis also indicated that the sequences of those two genes were different from those of the C3 and C4 plants investigated. Therefore, a better understanding of the photosynthetic characteristics of Oryza officinalis would provide clues for further isolation of valuable genes from this plant.

A modified method for the direct extraction of DNA from alkaline-saline soils with minimum DNA fragmentation and a possible reduction in chimera formation during polymerase chain reaction (PCR) was developed. The commercial extraction kit Power Soil DNA (Mo Bio™ Laboratories, Inc.) was used as a reference technique. The method reported here was based on cell lysis employing ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), and cell disruption with mechanical force with FastPrep-24™ equip followed by one cycle of freezing at -40 °C for 60 min and thawing at 65 °C for 20 min. The extraction method was tested for allophonic soils with large concentrations of organic matter, fulvic and humic acids, electrolytic conductivity (EC) ranging between 2.6 dS m-1 and 39.9 dS m-1, and pH between 8.8 and 10.9. The yield of DNA extracted depended on soil type, i.e., DNA extracted from soil varied between 2.35 (Texcoco-2) to 3.66 (Texcoco-1) μg DNA g-1 soil. The proposed method in this study produced enough DNA with yield and quality for PCR amplification of 16S rDNA when bovine serum albumin (BSA) was added to the reaction buffer. The DNA obtained had sufficient quality and yield for later use for 16S sequencing or possible use in other sequencing technologies, e.g. whole metagenome shotgun sequencing.

Pediatric COVID-19 determines a mild clinical picture, but few data have been published about the correlation between disease severity and PCR amplification cycles of SARS-CoV-2 from respiratory samples. This correlation is clinically important because it permits the stratification of patients in relation to their risk of developing a serious disease. Therefore, the primary endpoint of this study was to establish whether disease severity at the onset, when evaluated with a LqSOFA score, correlated with the gene amplification of SARS-CoV-2. LqSOFA score, also named the Liverpool quick Sequential Organ Failure Assessment, is a pediatric score that indicates the severity of illness with a range from 0 to 4 that incorporates age-adjusted heart rate, respiratory rate, capillary refill and consciousness level (AVPU). The secondary endpoint was to determine if this score could predict the days of duration for symptoms and positive swabs. Our study included 124 patients aged between 0 and 18 years. The LqSOFA score was negatively correlated with the number of PCR amplification cycles, but this was not significant (Pearson’s index −0.14, p-value 0.13). Instead, the correlation between the LqSOFA score and the duration of symptoms was positively related and statistically significant (Pearson’s index 0.20, p-value 0.02), such as the correlation between the LqSOFA score and the duration of a positive swab (Pearson’s index 0.40, p-value < 0.01). So, the LqSOFA score upon admission may predict the duration of symptoms and positive swabs; the PCR amplification of SARS-CoV-2 appears not to play a key role at onset in the prediction of disease severity.

Self-incompatibility is considered to be a growth-limiting factor in fruit plants. In species with hermaphrodite flowers, S-locus (S-allele) has been accepted to control self-incompatibility, and the genetic control of this locus is provided by multiple genes (alleles). Pear (Pyrus communis L.) belongs to the Pomoideae from the Rosaceae family and is found to have great genetic potential in terms of ecological features in Turkey. To protect these cultivation features, national garden collections have been established across the country and Atatürk Horticultural Central Research Institute-Yalova collection is considered as genes bank. Identification of the different features of this collection (fruit quality, stress tolerance, self-incompatibility, grafting incompatibility, etc.) is of great importance for its utilization in pear breeding and cultivation. However, to our knowledge, this collection has not been characterized for self-incompatibility trait. In the current study, PCR (Polymerase Chain Reaction)-based amplification of the S-allele regions (S1, S6, S7, S8) causing the self-incompatibility in 180 pear genotypes obtained from the national pear germplasm was investigated by molecular biological methods based on the comparison of amplified products. In 180 pear genotypes, the S6 allele was the most prevalent one with 63% frequency, while the S8 allele was the least common allele with a rate of 4%. In allele combinations, the S1-S6 allele combination was the most common allele combination with a rate of 18%, and trilateral allele combinations (S1-S6-S7 and S1-S6-S8) were observed at a rate of 1%. Findings of the current research will enable the classification of the materials and the analysed material is likely to be used in breeding studies as well as pear cultivation. [ABSTRACT FROM AUTHOR]

Background: The COBL genes encode a plant-specific glycosylphosphatidylinositol (GPI)-anchored protein. Recently identified COBRA genes are supposed as a key regulator of the orientation of cell expansion in the root indicating that COBRA gene family members are likely to be important players at the plasma membrane-cell wall interface. Methods and results: Five COBL gene namely, TaCOBL 1, TaCOBL 2, TaCOBL 3, TaCOBL 4 and TaCOBL 5 were identified using database search and domain predictions. Chromosomal location of each gene was mapped on karyotype. Structure of genes, promoter analysis and phylogenetic analysis were performed using different bioinformatics tools. Set of novel SNPs were also predicted. Gene ontologies were analyzed, and the processes and pathways were identified in which COBRA genes were involved. The molecular weight all the cobra proteins was in range of 50–75 KDa with 429–461 amino acid residues. The COBL genes were mapped on homeologous groups 2, 4, 5, 6 and 7. Gene ontology analysis revealed that TaCOBL genes were involved in cellulose microfibril organization, mucilage biosynthetic process involved in seed coat development, plant-type cell wall biogenesis plant-type cell wall cellulose biosynthetic process, seed coat development and seed development. Three drought responsive cis-elements (WRKY, ABRE and DRE) were found nearby COBL genes The qRT-PCR revealed TaCOBL genes are drought responsive and can be further explored to understand their role in drought tolerance in wheat. Conclusion: The comprehensive annotation and expression profiling of COBL genes revealed that all five COBL genes are drought response. The promoter cis-regulatory element analysis revealed that COBL genes had stress related WRKY, ABRE and DRE cis-regulatory elements. This evidence suggest that TaCOBL genes are involved in drought stress tolerance. [ABSTRACT FROM AUTHOR]

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Recent years, multi drug resistant pathogens and their pathogenicity were increased worldwide due to unauthorized consumption of antibiotics. In addition, correlation between multi drug resistant bacteria and biofilm formation is heightened due to the production of more virulence behavior. There is no better identification methods are available for detection of biofilm producing gram negative bacteria. In this research work, multi drug resistant strains of Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) were identified based on the specific antibiotics and third generation cephalosporin discs by disc diffusion assay. Subsequently, biofilm forming ability of selected pathogens were identified tissue culture plate and tube test. Based on the multi-drug resistant ability and biofilm production, the molecular identification of P. aeruginosa and K. pneumoniae were confirmed by PCR using universal primers. No zone of inhibition present around the discs of muller hinton agar plates were confirm, selected P. aeruginosa and K. pneumoniae strains were multi drug resistant pathogens. Performed third generation cephalosporin antibiotics were also highly sensitive to selected pathogens of P. aeruginosa and K. pneumoniae. Further, biofilm forming ability of selected P. aeruginosa and K. pneumoniae was confirmed by tissue culture plate and tube methods. Finally, molecular identification of P. aeruginosa and K. pneumoniae was named as P. aeruginosa and K. pneumoniae. Our result was conclude, selected P. aeruginosa and K. pneumoniae as biofilm producing pathogens and also highly resistant to current antibiotics. [ABSTRACT FROM AUTHOR]

Objective(s): Human gastrointestinal tract harbors a variety of bacteria with vital roles in human health. Bacteroides fragilis is considered one of the dominant constituents of gut microflora which can act as an opportunistic pathogen leading to various diseases, including colon cancer, diarrhea, uterine and intrathecal abscesses, septicemia, and pelvic inflammation. In this study, multiple locus variable number of tandem repeats analysis (MLVA) was performed to genetically differentiate 50 B. fragilis isolates.Materials and Methods: Eight suitable tandem repeats (TRs) were selected by bioinformatics tools and were then subjected to PCR amplification using specific primers. Finally, MLVA profiles were clustered using BioNumerics 7.6 software package. Results: All VNTR loci were detected in all isolates using the PCR method. Overall, B. fragilis isolates were differentiated into 27 distinct MLVA types. The highest diversity index was allocated to TR1, TR2, TR5, TR6, and TR8; with this taken into account, strain type 14 was the most prevalent with 12 strains belonging to this type. Clustering revealed three major clusters of A, B, and C. With regards to the pathogenicity of B. fragilis and the outcomes of infections related to this microorganism, it is imperative to study this microorganism isolated from both patients and healthy individuals.Conclusion: This study aimed at evaluating the efficiency of MLVA for the genetic differentiation of B. fragilis. The results of this study indicate the promising efficiency of MLVA typing for cluster detection of this bacterium.

Background: Phalaris species (Poaceae) occupy diverse environments throughout all continents except Antarctica. Phalaris arundinacea is an important forage, ornamental, wetland restoration and biofuel crop grown globally as well as being a wetland invasive. The nuclear ribosomal internal transcribed spacer (ITS) region has been used for Phalaris barcoding as a DNA region with high nucleotide diversity for Phalaris species identification. Recent findings that P. arundinacea populations in Minnesota USA are most likely native and not European prompted this analysis to determine whether Eurasian vs. native North American P. arundinacea differed in ITS regions. Our objectives were to amplify and compare ITS regions (ITS1 and ITS2) of historic herbaria (1882–2001) and extant (fresh) Phalaris specimens; analyze ITS regions for species-specific polymorphisms (diagnostic SNPs) and compare ITS regions of historic Phalaris specimens with known, extant Phalaris species. Results: We obtained complete ITS1 and ITS2 sequences from 31 Phalaris historic (herbaria samples, 1908 to 2001) and five extant (fresh) specimens. Herbaria Phalaris specimens did not produce new SNPs (single nucleotide polymorphisms) not present in extant specimens. Diagnostic SNPs were identified in 8/12 (66.6%) Phalaris species. This study demonstrates the use of herbaria tissue for barcoding as a means for improved species identification of Phalaris herbaria specimens. No significant correlation between specimen age and genomic DNA concentration was found. Phalaris arundinacea showed high SNP variation within its clade, with the North American being distinctly different than other USA and most Eurasian types, potentially allowing for future identification of specific SNPs to geographic origin. Conclusions: While not as efficient as extant specimens to obtain DNA, Phalaris herbaria specimens can produce high quality ITS sequences to evaluate historic genetic resources and facilitate identification of new species-specific barcodes. No correlation between DNA concentration and age of historic samples (119 year range) occurred. Considerable polymorphism was exhibited in the P. arundinacea clade with several N. American accessions being distinct from Eurasian types. Further development of within species- and genus-specific barcodes could contribute to designing PCR primers for efficient and accurate identification of N. American P. arundinacea. Our finding of misidentified Phalaris species indicates the need to exercise stringent quality control measures on newly generated sequence data and to approach public sequence databases in a critical way. [ABSTRACT FROM AUTHOR]

The root lesion nematode Pratylenchus penetrans parasitizes a wide range of economically important crops, including potato (Solanum tuberosum). Damage by P. penetrans impacts not only the potato yield but can also reduce the tuber quality. Detailed information on tuber infection by P. penetrans is scarce for most cultivars and molecular detection of nematodes from infected tubers is needed. The objective of this study was to assess tuber symptomatology due to P. penetrans infection in 10 potato cultivars and to provide an accurate molecular methodology for nematode detection using tuber peels. Sprouts of certified potato seed from cultivars Agata, Agria, Camel, Désirée, Dirosso, Kennebec, Laura, Picasso, Royata, and Stemster were planted in 2 L pots, and soil was inoculated with 4 P. penetrans/g of soil. Sixty days after inoculation, tubers were harvested, inspected for lesions, and the number of nematodes/g of potato peel assessed. Observations of tubers with symptoms showed the presence of P. penetrans in superficial layers of peels around the lenticels and injured necrotic tissue. Different nematode stages were detected in tubers of all inoculated cultivars, varying from 4 to 46 nematodes/g of potato peel. Species‐specific primers showed suitable sensitivity and reproducibility for the detection of P. penetrans in tuber potato peel samples. The molecular detection of P. penetrans directly from tuber peels can facilitate routine nematode inspections of potato seed tubers or cull potatoes for nematode detection, and prevent further dissemination of this species. [ABSTRACT FROM AUTHOR]

The success of polymerase chain reaction (PCR) assay depends on template deoxyribonucleic acid (DNA) being sufficient with respect to both quantity and quality. Some biological materials contain compounds which inhibit the functioning of DNA polymerase and thus need to be removed as part of the DNA extraction procedure. The aim of the present experiments was to optimise the process of DNA isolation from various types of black, fruit and herbal teas. A comparison was made between two cetyltrimethylammonium bromide (CTAB)-based protocols and two commercially available DNA purification kits. The yield and integrity of the extracted DNA were monitored both spectrophotometrically and using agarose gel electrophoresis. The presence/absence of inhibitors in the DNA preparations was checked by running quantitative real-time PCRs. The optimal protocol was deemed to be the CTAB method described in ISO 21571:2005, so this method is recommended for the routine sample analysis of tea products. [ABSTRACT FROM AUTHOR]

• Wood samples are potential sources of DNA information verified by two common isolation methods. • DNA isolation was partly recovered from dry and fresh wood samples across 20 tropical tree species. • Recovered DNA were successfully amplified based on ITS and rbcL loci for detecting DNA signals. • A pair method based on dry-fresh wood samples can be integrated to verify the protocol development. The successful isolation of DNA (Deoxyribonucleic Acid) is essential for the investigation process of forestry molecular genetics. Samples used are usually retrieved either from soft or juvenile plant organs because of their excellent DNA source. However, in certain cases, aforesaid samples are hard to obtain, as for forensic purposes. Alternatively, woods possess potential as alternative source of DNA whose extraction method has been developed with varying degrees of success. However, to date, effectiveness on tropical wood grown in Indonesia has not been widely reported. Therefore, objective of this study was to compare the results of DNA isolation of various dried and fresh wood samples by using two isolation methods: Cetyl Trimethyl Ammonium Bromide (CTAB) and Qiagen DNeasy Plant Mini Kit (QDPMK). Extraction results were visualized in agarose gels and quantified using Nanophotometer NP80 Implen which were then amplified using two universal primers: ITS and rbcL for detecting DNA signals. Extraction results from dried wood indicated no visualization in the gel, while fresh wood samples showed thick smeared bands on both extraction methods. Quantity test results denoted higher concentration in CTAB-extracted samples compared to samples extracted using QDPMK, in both types of samples, even though both resulted in optical density ratios outside the range of purity (λ260/280: 1,8–2,0 and λ260/230: 2,0, respectively). Success rates of ITS and rbcL primary amplification in dried wood samples were quite low yet outputs from the two methods did not differ significantly. Meanwhile, outcome of ITS and rbcL amplification on fresh wood samples had a fairly high success. [ABSTRACT FROM AUTHOR]

Ulmus villosa Brandis ex Gamble, an economically and ecologically important forest tree, is native to the western Himalayas of Pakistan. The long pressure imposed by unsustainable utilization and market demands has resulted in the rapid decline of the U. villosa population in the wild. To date, very limited information on the genomic diversity of U. villosa is available and this can tremendously limit our understanding of distribution and future conservation of U. villosa. Therefore, the current study aimed to assess genetic diversity within U. villosa wild populations of the Hazara Division using four barcoding markers (i.e., rbcL, matK XF + 5R, matK 390F + 1326R and trnH-psbA). A total of six high quality sequences were obtained with rbcL, four with matK XF + 5R, four with matK 390F + 1326R, and fourteen with trnH-psbA. The sequenced regions contained insertions and deletions as well as several SNPs across the length of sequences, and PCR-based markers may be developed from these novel SNPs markers. The phylogenetic results supported the polyphyletic origin of the genus Ulmus, and the data indicated that multiple speciation events may have led to the evolution of U. villosa in this region. For deeper understanding of the origin and evolution of U. villosa, sequencing of the complete nuclear and chloroplast genomes will be pivotal. The results herein provide novel insights into the genomic diversity as well as phylogenetic relationships of U. villosa with other species, and may facilitate both in situ and ex situ conservation efforts for this endangered species.