Identification of Cotton F1 Hybrids and their Parents through Molecular Marker under Salinity Stress

An experiment was carried out at Main Cotton Research Station, NAU, Surat, Gujarat, India during 2018–2020 to identify F1 hybrids and their parents through SSR marker for salinity tolerance in cotton. The four cotton parents (two salt tolerant and two salt sensitive) were crossed in a diallel fashion to obtain twelve cotton hybrids and subjected to DNA isolation and PCR amplification with SSR markers. In the present study, six SSR markers (TMB0409, DPL0094, BNL686, JESPR153, CM45 and MGHES006) were identified to be polymorphic between parents and the hybrids. The SSR primer TMB0409, DPL0094, JESPR153 and CM45 identified two fragments each from different parents in two, two, four and eight cotton hybrids, respectively, which confirmed true hybrids. Hence, the SSR molecular marker, individually or in combination can be used to distinguish and confirm the hybrid and parents in cotton with special reference to salinity. The PCA analysis revealed that BNL686–1 (248 bp) allele contributed significantly to the quantum of variation as explained by PC1. Hence, this allele is able to serve as a benchmark for ascertaining the efficient pattern of grouping between genotypes. Further, the marker CM45 amplified a fragment specific to the saline tolerant parents which was absent in sensitive parents as well as a fragment produced in sensitive parent which was absent in the tolerant parents, hence the molecular marker CM45 may associate with the salinity tolerance in cotton and can be used for salinity tolerant breeding program after confirming in a large population.

Identification of Saltol QTL in the breeding rice samples

One of the main problems in most of the world rice-growing regions is soil salinity. Rice is considered a saline sensitive crop, especially at the early stages of development and in the period of maturity. In the Rostov region, rice is grown in the south-eastern parts, where there are currently difficulties with the operation of the existing reclamation facilities. The problem of saline soils for this region is especially urgent, since a significant part of the arable lands has alkali complexes. In order to return the saline lands into exploitation, it is necessary to develop salt tolerant varieties, which, under crop rotation and maintenance, can contribute to soil desalinization. Due to the difficulty of determining salt tolerance only by estimating the phenotype, it is necessary to use molecular markers associated with this trait. Thus, the purpose of the current work was to identify one of the main Saltol QTL in breeding rice samples of the eighth generation (F8) obtained from hybridizing the donor variety NSYC Rc106 with Russian varieties. For that purpose, there have been used such marker-assisted selection methods as DNA isolation, polymerase chain reaction (PCR), electrophoresis on 2% agarose gels, gels’ coloring in ethidium bromide solution, photography in ultraviolet light and evaluation of the obtained electrophoregrams. As a result of the study of 398 breeding rice samples, there have been identified 67 samples with the functional allele of Saltol QTL (6865/3, 6874/2, Don 7343/4, Don 7343/5, Don 7343/6, Don 7343/7, Don 7343/8, Don 7343/9, Don 7343/10, Don 7337/1, Don 7337/3, Don 7337/4, Don 7337/5, Don 7337/6, Don 7337/7, Don 7337/8, etc.). There have been recommended to use these samples in the further breeding process in order to develop new salinity resistant rice varieties.

Comparison of culture-dependent and culture-independent techniques in the detection of lactic acid bacteria biodiversity and dynamics throughout the ripening process: The case of Turkish artisanal Tulum cheese produced in the Anamur region

Our objective was to analyze the diversity of the microbiota over 180 d of ripening of eight batches of artisanal goatskin Tulum cheeses by culture-dependent and culture-independent (PCR-DGGE) methods. V3 region of the bacterial 16S rRNA gene was amplified with the PCR after direct DNA isolation from the cheese samples. Nine different species and five genera were determined by culturing, while 11 species were identified in the PCR-DGGE technique. This diversity revealed the uniqueness of artisanal cheese varieties. The dominant genera in all the cheese samples were composed of Enterococcus species. The culture-dependent method revealed five genera (Enterococcus,Bacillus,Lactococcus,Lactobacillus, Sphingomonas) while three genera (Enterococcus, Streptococcus, Lactococcus) were detected in the culture-independent method. It was concluded that combining the two methods is important for characterizing the whole microbiota of the Tulum cheese varieties produced in the Anamur region.

EFFICIENT METHOD OF DNA ISOLATION FROM BULKING SAMPLES OF SEEDLINGS

Forage annual and perennial grasses are the difficult subject for molecular and genetic studies because of the problem with obtaining qualitative genomic DNA for PCR, due of high content of proteins, polysaccharides and polyphenols. The known methods of DNA extraction or the numerous commercial kits allow isolating purified nucleic acids from the leaf tissue, but characterized by low efficiency at seedlings using. The modified method of DNA isolation, based on the SDS-extraction buffer (sodium dodecil sulfate), is presented in this study. Significant modifications were introduced in the reagents compound and the steps of procedure accordingly to used type of plant tissue and the result was positive at usage on the bulking samples, as well as on the individual genotypes (the only seedling). Reliability of this method and the functionality of the obtained DNA samples were tested in PCR with different molecular markers (SSR, SRAP and PawS) in researches on revealing of forage legume grasses DNA polymorphism. The general advantages of the proposed method are simplicity and effectiveness, the possibility to isolate qualitative DNA without toxic reagents application, as well as relatively low cost and availability of reagents. This method can be useful for studying the genetic biodiversity and for decision the different tasks, required the rapid analysis of large plant populations.

A metagenomic DNA sequencing assay that is robust against environmental DNA contamination

Metagenomic DNA sequencing is a powerful tool to characterize microbial communities but is sensitive to environmental DNA contamination, in particular when applied to samples with low microbial biomass. Here, we present contamination-free metagenomic DNA sequencing (Coffee-seq), a metagenomic sequencing assay that is robust against environmental contamination. The core idea of Coffee-seq is to tag the DNA in the sample prior to DNA isolation and library preparation with a label that can be recorded by DNA sequencing. Any contaminating DNA that is introduced in the sample after tagging can then be bioinformatically identified and removed. We applied Coffee-seq to screen for infections from microorganisms with low burden in blood and urine, to identify COVID-19 co-infection, to characterize the urinary microbiome, and to identify microbial DNA signatures of inflammatory bowel disease in blood.

Genetic diversity based on RAPD F2 generation of apple cactus (Cereus spp.)

Apple cactus consists of 2 types, long spines (Cereus jamacaru) and short spines (C. peruvianus); both are self-incompatible. So that only artificial crosses between species that produce fruit. But some fruits are found from natural crosses of long spines. The three results of these crosses, long spines with short spines; short spines with long spines; and natural crosses on long spines, have produced F2 seedlings. The purpose of this study was to study genetic diversity based on the RAPD of the F2 seedlings. DNA isolation was carried out using Doyle and Doyle method modified on the addition of Polivinilpirolidon. The RAPD technique uses 3 primers: OPD-11, OPM-10, and OPN-5. Analysis of genetic diversity using Simple Matching coefficient using NTSys 2.02 software. Based on the three primers, each F2 offspring had genetic diversity but was grouped according to the parent. Showed that the three RAPD primers were effective for markers of genetic diversity in apple cactus.

Isolation and amplification of mangrove plants using DNA barcode in Percut Sei Tuan, North Sumatra, Indonesia

Mangroves are a collection of several species of trees or shrubs that distribute around the coastline and can survive in high salinity environments. Around 60% of mangrove forests in North Sumatra are reported to have been damaged, the main factors of this damage being the mangrove forests conversion into ponds and the expansion of oil palm plantations. Identification of mangrove species is very important in protecting and applying the biodiversity of mangrove forests. Identification of living things has evolved from morphological charcetrization to molecular identification. This study aims to explain the DNA isolation and PCR methods to identify mangrove species in North Sumatra. The results suggested that the rbcL primer used can detect mangrove species that were visualized in the form of DNA bands. The length of DNA fragments of mangrove species Acrosticum aureum ranged 632.0-619.6 bp, species Rhizophora apiculata 619.6-585.8 bp, species Nypa fruticans 600- 592.9 bp, species Avicennia alba 549.1-533.5 bp, species Hibiscus tiliaceus was not detected, and mangrove species Acanthus ilicifolius 480.3 bp.

Genetic Polymorphisms of Chicken Antiviral Mx1 and TVB Genes in Indigenous and Giriraja Chicken

Background: Recent developments in molecular genetics lead to addressing certain poultry diseases via breeding for disease resistance. The present study was carried to identify and compare the genetic polymorphism in Chicken Mx1 and TVB genes among the indigenous and Giriraja chicken using PCR-RFLP technique. Methods: Blood samples were collected from 50 indigenous and 50 Giriraja birds and DNA isolation was done by Phenol: Chloroform: Isoamyl alcohol method. PCR amplification of Chicken Mx1 (exon 14) and Chicken TVB (exon 3) genes was carried out followed by RFLP analysis. Result: PCR product sizes of 301 bp and 303 bp of Mx1 and TVB genes, respectively were successfully amplified. RFLP analysis of Mx1 gene with Hyp8I restriction enzyme revealed three genotypes AA, AB and BB. In indigenous birds genotypic frequencies of AA, AB and BB were 0.314, 0.493 and 0.194, respectively and gene frequencies were 0.56 and 0.44 for alleles A and B, respectively. In Giriraja birds, genotypic frequencies for AA, AB and BB were 0.27, 0.499 and 0.23, respectively and gene frequencies were 0.52 and 0.48 for alleles A and B, respectively. RFLP analysis of TVB gene with NlaIII restriction enzyme revealed two genotypes viz., AA and AB. In indigenous birds genotypic frequencies of AA and AB were 0.81 and 0.18, respectively and gene frequencies were 0.9 and 0.1 for alleles A and B, respectively. In Giriraja birds genotypic frequencies for AA and AB were 0.774 and 0.211, respectively and gene frequencies were 0.88 and 0.12 for alleles A and B, respectively.

Recovery of Mycobacteria from Heavily Contaminated Environmental Matrices

For epidemiology studies, a decontamination method using a solution containing 4.0% NaOH and 0.5% tetradecyltrimethylammonium bromide (TDAB) represents a relatively simple and universal procedure for processing heavily microbially contaminated matrices together with increase of mycobacteria yield and elimination of gross contamination. A contamination rate only averaging 7.3% (2.4% in Cluster S; 6.9%% in Cluster R and 12.6%% in Cluster E) was found in 787 examined environmental samples. Mycobacteria were cultured from 28.5% of 274 soil and water sediments samples (Cluster S), 60.2% of 251 samples of raw and processed peat and other horticultural substrates (Cluster R), and 29.4% of 262 faecal samples along with other samples of animal origin (Cluster E). A total of 38 species of slow and rapidly growing mycobacteria were isolated. M. avium ssp. hominissuis, M. fortuitum, and M. malmoense were the species most often isolated. The parameters for the quantitative detection of mycobacteria by PCR can be significantly refined by treating the sample suspension before DNA isolation with PMA (propidium monoazide) solution. This effectively eliminates DNA residue from both dead mycobacterial cells and potentially interfering DNA segments present from other microbial flora. In terms of human exposure risk assessment, the potential exposure to live non-tuberculous mycobacteria can be more accurately determined.