Optimization of DNA extraction and amplification of Kikuyu (Pennisetum clandestinum Hochst. ex Chiov) for molecular identification

Kikuyu (Pennisetum clandestinum Hochst. ex Chiov) is an important forage containing high crude protein for livestock. Molecular analysis of kikuyu relies on high yields of pure DNA and suitable PCR conditions. This research aimed to extract DNA from kikuyu based on weight of the sample and amplify the DNA of Burangrang accession using specific primers. 100 grams and 200 grams leaves of 3 accessions of kikuyu from Burangrang, Bukit Tunggul, and Tangkuban Perahu were extracted by Qiagen Mini Kit Plant. Concentration and purity of DNA were analyzed by NanoDrop Spectrophotometer 2000. DNA from Burangrang accession was amplified using six specific primers at different annealing temperatures. The result showed that the yield of DNA ranged 2.2 µg/µl to 21.4 µg/µl and the purity (ratio) were 1.08 to 2.01. Bukit Tunggul and Burangrang accession showed the same interaction pattern on the sample weight for concentration and purity. One hundred grams of leaves from Burangrang accession produce the highest concentration and the best purity of DNA, but no difference between other weight and accession. Reproducible amplifiable products were observed in all PCR reactions except primer K2. These results indicated that optimized protocol is suitable for further work on molecular identification of kikuyu.

Development of a Panel of Genotyping-in-Thousands by Sequencing in Capsicum

Genotyping by sequencing (GBS) enables genotyping of multiple loci at low cost. However, the single nucleotide polymorphisms (SNPs) revealed by GBS tend to be randomly distributed between individuals, limiting their direct comparisons without applying the various filter options to obtain a comparable dataset of SNPs. Here, we developed a panel of a multiplex targeted sequencing method, genotyping-in-thousands by sequencing (GT-seq), to genotype SNPs in Capsicum spp. Previously developed Fluidigm® SNP markers were converted to GT-seq markers and combined with new GT-seq markers developed using SNP information obtained through GBS. We then optimized multiplex PCR conditions: we obtained the highest genotyping rate when the first PCR consisted of 25 cycles. In addition, we determined that 101 primer pairs performed best when amplifying target sequences of 79 bp. We minimized interference of multiplex PCR by primer dimer formation using the PrimerPooler program. Using our GT-seq pipeline on Illumina Miseq and Nextseq platforms, we genotyped up to 1,500 (Miseq) and 1,300 (Nextseq) samples for the optimum panel size of 100 loci. To allow the genotyping of Capsicum species, we designed 332 informative GT-seq markers from Fluidigm SNP markers and GBS-derived SNPs. This study illustrates the first application of GT-seq in crop plants. The GT-seq marker set developed here will be a useful tool for molecular breeding of peppers in the future.

DNA technologies (molecular marking) in tomato breeding for resistance to Tobacco Mosaic Virus

Relevance. The purpose of this work is to create new discrete competitive tomato genotypes based on modern biotechnological approaches with increased resistance to the tobacco mosaic virus, economically valuable traits and adapted to the soil and climatic conditions of cultivation in the southern regions of the Russian Federation.Methods. At the first stage of the study, SSR markers taken from literature sources were tested [1;2] and PCR conditions were optimized for the identification of target Tm genes (Tm2a, Tm22, providing resistance to tobacco mosaic virus at the tissue level) in the breeding material available in the department of vegetable and potato growing.Results. Two informative molecular markers Tms 37 and UMD 2060 were selected, which reveal the allelic difference between resistant and susceptible samples. Hybridization of FMS tomato lines with samples having the desired genes of interest in the genotype for obtaining tomato lines resistant to TMV was carried out. Seeds of paternal forms - donors of target genes were obtained for their further use in breeding work. The scientific novelty lies in the study of the discrete genetic material of Solanumlycopersicum, used in the breeding process, and preserving its functions in a new genetic environment.

Benchmarking laboratory processes to characterise low-biomass respiratory microbiota

The low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available laboratory protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community profile, we amplified the 16S rRNA gene of respiratory samples using various bacterial loads and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 or V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community profile of low biomass samples. Purification methods and MiSeq reagent kits provided nearly similar microbiota profiles (paired Bray–Curtis dissimilarity median: 0.03 and 0.05, respectively). While profiles of positive controls were significantly influenced by the type of dilution solvent, the theoretical profile of the Zymo mock was most accurately analysed when the Zymo mock was diluted in elution buffer (difference compared to the theoretical Zymo mock: 21.6% for elution buffer, 29.2% for Milli-Q, and 79.6% for DNA/RNA shield). Microbiota profiles of DNA blanks formed a distinct cluster compared to low biomass samples, demonstrating that low biomass samples can accurately be distinguished from DNA blanks. In summary, to accurately characterise the microbial community composition we recommend 1. amplification of the obtained microbial DNA with 30 PCR cycles, 2. purifying amplicon pools by two consecutive AMPure XP steps and 3. sequence the pooled amplicons by V3 MiSeq reagent kit. The benchmarked standardized laboratory workflow presented here ensures comparability of results within and between low biomass microbiome studies.

Detection and phylogeny of Wolbachia in field-collected Aedes albopictus and Aedes aegypti from Manila City, Philippines

Wolbachia is the most common bacterial endosymbiont of arthropods, such as the medically important Aedes albopictus and Aedes aegypti. Both Wolbachia and mitochondrial DNA are maternally inherited. This study collected 12 adult Ae. albopictus and 359 Ae. aegypti from 183 households in a dengue-prone area, Manila, Philippines, between June and September 2017. Aedes larvae (n = 509) were also collected from 17 water containers from 11 households. The DNA of the Aedes larvae and adults were screened for the presence of Wolbachia using the wsp and 16S markers, following optimized polymerase chain reaction (PCR) conditions, and sequenced. We also performed PCR using the mitochondrial DNA cox1 marker and sequenced the Wolbachia-positive samples. Our results showed that 3 out of 359 (0.84%) adult Ae. aegypti and 12 out of 12 (100%) adult Ae. albopictus were Wolbachia positive, whereas all larvae tested negative for Wolbachia (0/509; 0%). The wsp marker revealed six Wolbachia-positive Ae. albopictus, whereas the 16S marker showed Wolbachia in three Ae. aegypti and ten Ae. albopictus. Three Wolbachia-positive Ae. albopictus from one household were found to have a single haplotype for all Wolbachia (wsp and 16S) and mitochondrial cox1 markers. This result can be used to infer that maternal transmission of Wolbachia in the household because siblings of a Wolbachia-infected mother mosquito share the Wolbachia and mitochondrial genomes. Our results suggest that the combined sequencing analyses of Wolbachia (wsp and 16S) and mitochondrial DNA can infer maternal transmission in field-collected mosquitoes.

Screening of miRNAs in plasma as a diagnostic biomarker for cardiac disease based on optimization of extraction and qRT-PCR condition assay through amplification efficiency

Background Although quantitative real-time PCR (qRT-PCR) is a common and sensitive method for miRNAs analysis, it is necessary to optimize conditions and minimize qRT-PCR inhibitors to achieve reliable results. The aim of this study was to minimize interference by contaminants in qRT-PCR, maximize product yields for miRNA analyses, and optimize PCR conditions for the reliable screening of miRNAs in plasma. Methods The annealing temperature was first optimized by assessing amplification efficiencies. The effects of extraction conditions on levels of inhibitors that interfere with PCR were evaluated. The tested extraction conditions were the volume of the upper layer taken, number of chloroform extractions, and the inclusion of ethanol washing, a process that reduces PCR interference during RNA extraction using TRIzol. Results An acceptable amplification efficiency of RT-qPCR was achieved by the optimization of the annealing temperature of the tested miRNAs and by the collection a supernatant volume corresponding to about 50% of the volume of TRIzol with triple chloroform extraction. These optimal extraction and PCR conditions were successfully applied to plasma miRNA screening to detect biomarker candidates for the diagnosis of acute myocardial infarction. Conclusion This is the first study to optimize extraction and qRT-PCR conditions, while improving miRNA yields and minimizing the loss of extracted miRNA by evaluations of the amplification efficiency.

Verification of Maternal Transmission of Wolbachia in Field-collected Aedes Albopictus and Aedes Aegypti Using a Combination of Wolbachia and Mitochondrial DNA Sequencing

Wolbachia is the most common bacterial endosymbiont of arthropods, such as the medically important Aedes albopictus and Aedes aegypti. Both Wolbachia and mitochondrial DNA are maternally inherited. This study collected 12 adult Aedes albopictus and 359 Aedes aegypti from 183 households in a dengue-prone area, Manila, Philippines, between June and September 2017. Aedes larvae (n = 509) were also collected from 17 water containers from 11 households. The DNA of the Aedes larvae and adults were screened for the presence of Wolbachia using the wsp and 16S markers, following optimized polymerase chain reaction (PCR) conditions, and sequenced. We also performed PCR using the mitochondrial DNA cox1 marker and sequenced the Wolbachia-positive samples. Our results showed that three out of 359 (0.84%) adult Ae. aegypti and 12 out of 12 (100%) adult Ae. albopictus were Wolbachia positive, while all of the larvae tested negative for Wolbachia (0/509; 0%). The wsp marker revealed six Wolbachia-positive Ae. albopictus, while the 16S marker showed Wolbachia in three Ae. aegypti and 10 Ae. albopictus. Three Wolbachia-positive Ae. albopictus from one household were found to have a single haplotype for all Wolbachia (wsp and 16S) and mitochondrial cox1 markers. This result indicates maternal transmission of Wolbachia in the household because siblings of a Wolbachia-infected mother mosquito share the Wolbachia and mitochondrial genomes. Our results showed the feasibility of combined sequencing analyses of Wolbachia (wsp and 16S) and mitochondrial DNA to test maternal transmission in field-collected mosquitoes.

Whole genome survey of big cats (Genus: Panthera) identifies novel microsatellites of utility in conservation genetic study

Big cats (Genus: Panthera) are among the most threatened mammal groups of the world, owing to hunting, habitat loss, and illegal transnational trade. Conservation genetic studies and effective curbs on poaching are important for the conservation of these charismatic apex predators. A limited number of microsatellite markers exists for Panthera species and researchers often cross-amplify domestic cat microsatellites to study these species. We conducted data mining of seven Panthera genome sequences to discover microsatellites for conservation genetic studies of four threatened big cat species. A total of 32 polymorphic microsatellite loci were identified in silico and tested with 152 big cats, and were found polymorphic in most of the tested species. We propose a set of 12 novel microsatellite markers for use in conservation genetics and wildlife forensic investigations of big cat species. Cumulatively, these markers have a high discriminatory power of one in a million for unrelated individuals and one in a thousand for siblings. Similar PCR conditions of these markers increase the prospects of achieving efficient multiplex PCR assays. This study is a pioneering attempt to synthesise genome wide microsatellite markers for big cats.

STANDARDIZATION AND APPLICATION OF PCR TARGETING CHLORELLA SPECIES ISOLATED FROM ENVIRONMENTAL SAMPLES

Objective: Identification of Chlorella species from the environment through 18s ribosomal RNA sequencing. This study was aimed to design primer targeting Chlorella and other closely related algal species targeting 18s ribosomal RNA, ITS1 region. Methods: Sanger sequencing was carried out for the identification of algae up to the genus and species level using an in-house designed primer and optimized PCR conditions. Results: Out of 2 algae samples identified phenotypically, one isolate identified as Chlorella vulgaris and other one identified as Chlorella sorokiniana based on the results of Basic Alignment Search Tool (BLAST). Conclusion: To conclude, this study provided primers with PCR conditions to characterize algal samples through molecular identification with 100% accuracy than the phenotypic method.

Benchmarking laboratory processes to characterise low-biomass respiratory microbiota

The low biomass of respiratory samples makes it difficult to accurately characterise the microbial community composition. PCR conditions and contaminating microbial DNA can alter the biological profile. The objective of this study was to benchmark the currently available protocols to accurately analyse the microbial community of low biomass samples. To study the effect of PCR conditions on the microbial community composition, we amplified the 16S rRNA gene of respiratory samples using various DNA input and different number of PCR cycles. Libraries were purified by gel electrophoresis or AMPure XP and sequenced by V2 and V3 MiSeq reagent kits by Illumina sequencing. The positive control was diluted in different solvents. PCR conditions had no significant influence on the microbial community composition of low biomass samples. Purification methods and MiSeq reagent kits had only a modest impact on microbiota profiles, while profiles of positive controls were significantly influenced by type of dilution solvent. Microbiota profiles of low biomass samples can be accurately distinguished from DNA blanks. Microbiota profiling of low biomass samples is stable under several PCR conditions, purification methods and MiSeq reagent kits. We recommend to use amplification with 30 PCR cycles. The amplicon pools can best be purified by two consecutive AMPure XP steps and sequenced by V3 MiSeq reagent kit. The benchmarked standardized workflow presented here ensures comparability of results within and between low biomass microbiome studies.