DNA-based Markers Research Articles

Rapid Diagnostic PCR Assay Method for Species Identification of Mantidis Ootheca (Sangpiaoxiao) Based on Cytochrom C Oxidase I (COI) Barcode Analysis.

Mantidis Ootheca (sangpiaoxiao), the egg case of the mantis, is a type of insect-derived traditional medicine widely used in East Asia. However, species identification based on egg morphology is challenging, leading to the distribution of counterfeit and adulterated products. The use of inauthentic ingredients can pose serious health risks to consumers. This study aimed to develop PCR markers that can rapidly and accurately differentiate between authentic and counterfeit Mantidis Ootheca. The mitochondrial cytochrome c oxidase I (COI) region was sequenced in thirteen samples from four mantis species: Tenodera angustipennis, Statilia maculata, Hierodula patellifera, and T. sinensis. Four sets of SCAR primers were designed based on species-specific nucleotide polymorphisms, and a multiplex SCAR assay was developed by combining all sets of the primers. The sequence-characterized amplified region (SCAR) primers successfully produced amplicons for each target species, even with low-DNA templates or templates containing DNA from multiple samples. No amplification was observed for nontarget species. This study presents a novel approach for identifying authentic Mantidis Ootheca species using DNA-based diagnostic marker assays, which enable rapid and precise species identification. The SCAR assays developed in this study will aid in maintaining quality control and promoting the standardization of commercial Mantidis Ootheca products.

Comparison of multi-parallel quantitative real-time PCRs targeting different DNA regions and detecting soil-transmitted helminths in stool

BackgroundSoil-transmitted helminths infect an estimated 18% of the world’s population, causing a significant health burden. Microscopy has been the primary tool for diagnosing eggs from fecal samples, but its sensitivity drops in low-prevalence settings. Quantitative real-time polymerase chain reaction (qPCR) is slowly increasing in research and clinical settings. However, there is still no consensus on preferred qPCR targets.MethodsWe aimed to compare soil-transmitted helminth (STH) DNA detection methods by testing naïve stool samples spiked with known quantities of STH eggs and larvae. DNA extracts from spiked samples were tested using independent quantitative realtime PCR (qPCR) assays targeting ribosomal or putative non-protein coding satellite sequences.ResultsFor Trichuris trichiura, there was a strong correlation between egg/larvae counts and qPCR results using either qPCR method (0.86 and 0.87, respectively). Strong correlations also existed for A. lumbricoides (0.60 and 0.63, respectively), but weaker correlations were found for Ancylostoma duodenale (0.41 for both assays) and Strongyloides stercoralis (0.48 and 0.65, respectively). No correlation for Necator americanus was observed when testing with either qPCR assay. Both assays had fair-to-moderate agreement across targets when using field-collected stool samples (0.28–0.45, for all STHs), except for S. stercoralis (0.12) with slight agreement.ConclusionsThere is a strong correlation between qPCR results and egg/larvae counts. Our study confirms that qPCR is an effective diagnostic tool, even with low-intensity infections, regardless of the DNA-based diagnostic marker used. However, the moderate agreement between the two different qPCR assays when testing field samples highlights the need to understand the role of these targets in the genome so that the parasite burden can be quantified more accurately and consistently by qPCR.Graphical abstract

Effect of Chromosomal Localization of NGS-Based Markers on Their Applicability for Analyzing Genetic Variation and Population Structure of Hexaploid Triticale.

This study aimed to determine whether using DNA-based markers assigned to individual chromosomes would detect the genetic structures of 446 winter triticale forms originating from two breeding companies more effectively than using the entire pool of markers. After filtering for quality control parameters, 6380 codominant single nucleotide polymorphisms (SNPs) markers and 17,490 dominant diversity array technology (silicoDArT) markers were considered for analysis. The mean polymorphic information content (PIC) values varied depending on the chromosomes and ranged from 0.30 (2R) to 0.43 (7A) for the SNPs and from 0.28 (2A) to 0.35 (6R) for the silicoDArTs. The highest correlation of genetic distance (GD) matrices based on SNP markers was observed among the 5B-5R (0.642), 5B-7B (0.626), and 5A-5R (0.605) chromosomes. When silicoDArTs were used for the analysis, the strongest correlations were found between 5B-5R (0.732) and 2B-5B (0.718). A Bayesian analysis showed that SNPs (total marker pool) allowed for the identification of a more complex structure (K = 4, ΔK = 2460.2) than the analysis based on silicoDArTs (K = 2, ΔK = 128). Triticale lines formed into groups, ranging from two (most of the chromosomes) to four (7A) groups depending on the analyzed chromosome when SNP markers were used for analysis. Linkage disequilibrium (LD) varied among individual chromosomes, ranging from 0.031 for 1A to 0.228 for 7R.

Applications of Marker-Assisted Gene Pyramiding in Rice Crop Improvement

In order to give wide-ranging and long-lasting disease resistance that reduces rice output, breeding techniques are used such as the importance of selection assisted by markers and gene pyramiding. Bacterial leaf blight (BLB), which is carried on by the pathogen Xanthomonas oryzae pv. oryzae, is one such disease that significantly reduces rice yield. Numerous molecular marker techniques have already been developed because molecular markers are crucial for both marker-assisted selection and gene pyramiding. Currently, DNA-based markers, sometimes referred to as molecular markers, are the most frequently employed ones. Based on the methods used to find them, the molecular markers are divided into two main types. These markers are based on hybridization and polymerase chain reaction. The morphological (traditionally based) and biochemical (enzyme-based) markers are two more types of markers that are available. The best method for preventing rice BLB disease is host plant/varietal resistance. Gene pyramiding with the help of markers offers the potential to speed up breeding operations and ensure that the host plant’s acquired resistance will last. This research explores the use, economic significance, constraints, and potential of marker-assisted selection and gene pyramiding for rice BLB disease resistance.

Exploring Genetic Variability, Path Analysis and Divergence in Sorghum (Sorghum bicolor (L.) Moench)

The grain crop known as sorghum [Sorghum bicolor (L.) Moench], which belongs in the Poaceae family, is said to have evolved from Ethiopia or Sudan. This is a crucial crop for food security, especially within the tropical and semi-arid regions in African nations. By terms of long-term domestication and genetic diversity, this crop was native from Ethiopia. showed differences between the crop's cultivated and wild ancestors that gathered within the nation. Due to its enormous diversity of sorghum, which shows innate biological resistant to disease, dryness, and insects in addition to having large lysine content and excellent quality of grain, Ethiopia has become the world's top contributor of germplasm for the sorghum breeding programmes. Ethiopian landraces referred to as "zera zera" sorghum and the generations that sprang of those were used in ICRISAT along with other nations' contemporary sorghum breeding programmes for hybrids creation. Sorghum is at present Ethiopia's second most significant cereal crop after the tef with regard to of its combined area covered and productivity, which means it's used to make injera. Sorghum genotype from Ethiopia is varied and has responded to a variety of rainfall conditions and elevations. Plant breeding starts with the characterisation and recognition of sorghum seeds that offer desired features for improved genetics. When contrast with field trial evaluation, DNA-based molecular markers and PCR-based methods were most effective in characterising and identifying sorghum genotypes which offer desired features since they are unaffected by duration or climate. The most economical method of increasing sorghum production for many applications is genetic enhancement. Gaining more knowledge about sorghum's genetic diversity could be extremely beneficial for improving the crop in terms of quality of food along with other crucial agronomic characteristics. Sorghums from the Ethiopian Centre of Crop Diversity have a vast amount of genetic variety. To effectively gather and conserve this genetic variety before it is overrun and lost, some serious work is required.

Exploring crop genomics: role of molecular markers in identifying genetic diversity and characterization

and facilitating germplasm characterization for crop improvement. Understanding and harnessing crop genetic diversity are paramount with the escalating demand for sustainable agricultural practices and the need to address global food security challenges. Molecular markers have emerged as powerful tools enabling precise identification, characterization, and utilization of genetic resources for crop enhancement. The paper provides an in-depth analysis of various molecular marker techniques, encompassing DNA-based markers such as single nucleotide polymorphisms (SNPs), simple sequence repeats (SSRs), and amplified fragment length polymorphisms (AFLPs), as well as RNA-based markers like expression sequence tags (ESTs) and microRNAs. By elucidating genetic relationships, population structure, and phylogenetic analysis, these markers facilitate the conservation and utilization of crop germplasm. Moreover, the review highlights recent advancements in high-throughput genotyping technologies and bioinformatics tools, which have revolutionized crop genomic research. These advancements enable comprehensive genome-wide analyses, accelerating breeding efforts for the development of improved crop varieties with enhanced traits such as yield, quality, and stress tolerance. Integration of molecular marker-assisted selection (MAS) into breeding programs further enhances the efficiency and precision of crop improvement strategies. Despite the significant contributions of molecular markers to crop genomics, challenges persist. The review addresses these challenges and discusses potential solutions, emphasizing the importance of collaborative efforts, data sharing, and interdisciplinary research endeavors. By overcoming these hurdles, the agricultural community can fully harness the potential of genomics for sustainable crop improvement, thus addressing global food security challenges in the face of changing environmental conditions and population growth. In conclusion, understanding the role of molecular markers in crop genomics is crucial for optimizing breeding strategies, conserving genetic resources, and developing resilient crop varieties to meet the demands of a growing population and ensure food security in a changing world.

Advancing Hemidesmus indicus propagation and conservation: Somatic embryogenesis, histology, metabolite assessment and genetic stability

Hemidesmus indicus L. R. Br. is a commonly traded ethnomedicinal plant, and a method has been developed to produce somatic embryos (SEs) from calli grown from the leaves of the plant. With a maximum callus induction frequency of 91.66 %, pink-red friable proembryogenic calli were generated on Murashige and Skoog (MS) and 5.0 µM 2, 4-dichlorophenoxyacetic acid (2, 4-D) from the leaf explant (LE). With the aim of achieving a high success rate of 95.00 % for the induction of SEs, with a yield of 91.33 SEs per 100 mg of callus, the proembryogenic calli were transferred to fresh culture medium containing MS and 5.0 µM 6-benzylaminopurine (BA). This induction medium allowed the embryos to develop to the torpedo stage, but their further growth into emblings required a different culture medium. ½ MS with 5.0 µM BA and 1.0 µM gibberellic acid (GA3) resulted in 99.16 % maturation and 39.66 emblings regeneration out of 40 immature embryoids, respectively. With a maximum survival rate of 91.67 %, healthy emblings were successfully acclimatized using Soilrite. DNA-based molecular markers (RAPD and ISSR) were used to confirm the genetic similarities of the in vitro grown plants. Histological examinations were performed to determine the ontogeny, morphology, and development of somatic embryoids. Gas chromatography and mass spectrometry (GCMS–) were performed for both the mother and the in vitro generated plant to analyse the root for secondary metabolites. In the physiological and biochemical studies, enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), and glutathione reductase (GR) were found to increase in vitro derived H. indicus plants during different acclimatization periods (0, 7, 14, 21 and 28 days). With increasing acclimatization time, net photosynthetic rate (PN) and total chlorophyll show a decrease at first, then an increase. The protocol provides high callus induction rates, efficient somatic embryogenesis, successful maturation and regeneration, robust acclimatization, genetic fidelity, detailed insight into development, and screening of secondary metabolites.

An improved and effective DNA extraction protocol for Pyracantha crenulata with optimal PCR reliability

Abstract There are many challenges involved in obtaining high-quality DNA from tissues/cells, especially when using plants as the raw material. Three different DNA extraction protocols were employed in order to isolate high quality of genomic DNA from Pyracantha crenulata leaves. P. crenulata is a complex, versatile, and evergreen shrub species in Rosaceae family which is ecologically, economically and culturally important. This species produces valuable antioxidants like polyphenols, polysaccharides and secondary metabolites which interfere with DNA extraction thus making molecular marker based studies difficult in this species. This study aimed to develop a simple, rapid, cost-effective and highly efficient protocol for P. crenulata leaves rich in salts and phenols. The results demonstrated that modified CTAB (double buffer) protocol is very effective in overcoming the challenges that could impede next-generation sequencing analysis. The yield of the extracted DNA was excellent ranging from 368 to 410 ng/μL DNA with A260/280 ratio ranging from 1.80 to 1.84. The extracted DNA was amenable to PCR amplification making it suitable for DNA-based molecular marker studies. None procedures have been published for the purpose up this species, therefore, double buffer based extraction protocol is reported for the first time in P. crenulata and might prove useful in other species of family Rosa-ceae. This study describes an effective DNA isolation method that is appropriate for frequent population genetic screening. In conclusion, the double buffer protocol showed the most efficient and effective approach for extracting DNA from phenol rich, fresh, mature, and dry leaves.

Novel amplified fragment length polymorphism (AFLP) markers for typing medically relevant Fusarium and allied fusarioid genera

Fusariosis is an emerging mycosis caused by diverse Fusarium and allied fusarioid genera that are characterized by spindle-shaped macroconidia. These fungi possess a broad ecological distribution, causing infections in a wide diversity of hosts, spanning the animal and plant kingdoms. The spectrum of human fusariosis encompasses superficial lesions like keratitis and onychomycosis to invasive fungal diseases. Notable genera within the medically relevant fusarioid group include Neocosmospora, Fusarium s. str., and Bisifusarium. While Neocosmospora species (formerly F. solani species complex) are primary causative agents of human fusariosis, instances involving Fusarium s. str. and Bisifusarium (formerly F. dimerum species complex) have been reported. There is an urgent need for DNA-based markers to explore the epidemiology of these emerging fusarioid pathogens using molecular methods. We took advantage of fusarioid genomes available in NCBI (n = 20) to optimize the development of novel amplified fragment length polymorphism (AFLP) markers by conducting in-depth in silico analyses to refine their applicability for studies on these pathogens’ genetic epidemiology. In-silico screening highlighted eight primer pair combinations (C1–C8) to be tested in vitro. The AFLP protocol was used for genotyping 40 medically relevant fusarioid fungi. Based on the overall scored AFLP markers (77–93 fragments), the values of polymorphism information content (PIC = 0.3474–0.3725), marker index (MI = 0.0038–0.0056), effective multiplex ratio (E = 26.3750–40.4750), resolving power (Rp = 40.1500–54.6000), discriminating power (D = 0.7978–0.8857), expected heterozygosity (H = 0.4476–0.4949), and mean heterozygosity (Havp = 0.0001) demonstrated the utility of these primer combinations for discriminating Neocosmospora, Fusarium s. str., and Bisifusarium species. Of relevance, some AFLP panels were better than others at studying genetic trends in Neocosmospora (#2 EcoRI-AT/MseI-TA, #3 EcoRI-AA/MseI-TT, and #5 EcoRI-AT/MseI-AG) or Fusarium s. str. (mainly #2 EcoRI-AT/MseI-TA and #6 EcoRI-GA/MseI-TT) and Bisifusarium (#1 EcoRI-GA/MseI-AG and #6 EcoRI-GA/MseI-TT), and these combinations will better resolve disease transmission routes. Our DNA fingerprint assay has proven effective by exhibiting rapidity, reproducibility, and high discriminatory capabilities, which represents a valuable asset in the ongoing efforts to combat fusariosis and enhance our scientific understanding of medically relevant Fusarium and allied fusarioid genera.

Advances in Physalis molecular research: applications in authentication, genetic diversity, phylogenetics, functional genes, and omics.

The plants of the genus Physalis L. have been extensively utilized in traditional and indigenous Chinese medicinal practices for treating a variety of ailments, including dermatitis, malaria, asthma, hepatitis, and liver disorders. The present review aims to achieve a comprehensive and up-to-date investigation of the genus Physalis, a new model crop, to understand plant diversity and fruit development. Several chloroplast DNA-, nuclear ribosomal DNA-, and genomic DNA-based markers, such as psbA-trnH, internal-transcribed spacer (ITS), simple sequence repeat (SSR), random amplified microsatellites (RAMS), sequence-characterized amplified region (SCAR), and single nucleotide polymorphism (SNP), were developed for molecular identification, genetic diversity, and phylogenetic studies of Physalis species. A large number of functional genes involved in inflated calyx syndrome development (AP2-L, MPF2, MPF3, and MAGO), organ growth (AG1, AG2, POS1, and CNR1), and active ingredient metabolism (24ISO, DHCRT, P450-CPL, SR, DUF538, TAS14, and 3β-HSB) were identified contributing to the breeding of novel Physalis varieties. Various omic studies revealed and functionally identified a series of reproductive organ development-related factors, environmental stress-responsive genes, and active component biosynthesis-related enzymes. The chromosome-level genomes of Physalis floridana Rydb., Physalis grisea (Waterf.) M. Martínez, and Physalis pruinosa L. have been recently published providing a valuable resource for genome editing in Physalis crops. Our review summarizes the recent progress in genetic diversity, molecular identification, phylogenetics, functional genes, and the application of omics in the genus Physalis and accelerates efficient utilization of this traditional herb.

Rapid cell isolation in breastmilk in a non-clinical setting by a deterministic lateral displacement device and selective water and fat absorption.

Breastmilk is a reliable source of biomarker-containing, sloughed breast cells that have the potential to give valuable health insights to new mothers. Furthermore, known DNA-based markers for pregnancy-associated breast cancer are chemically stable and can be safely stored on a commercially available FTA® Elute Micro (EM) card, which can subsequently be mailed to a testing facility for the cost of a stamp. In theory, this archiving process can be performed by nonprofessionals in very low-resource settings as it simply requires placing a drop of breastmilk on an EM card. Although this level of convenience is paramount for new mothers, the low cell density of breastmilk complicates archiving on an EM card as such commercial products and associated protocols were designed for high-cell density physiological fluids such as blood. In this study, we present the use of a deterministic lateral displacement (DLD) device combined with porous superabsorbent polymers and hydrophobic sponges to achieve simple and low-cost cell enrichment in breastmilk. As the critical separation diameter in a DLD device is more heavily dependent on lithographically controlled pillar layout than fluid or flow properties, our use of DLD microfluidics allowed for the accommodation of both varying viscosities in human breastmilk samples and a varying pressure of actuation resulting from manual, syringe-driven operation. We demonstrate successful cell enrichment (>11×) and a corresponding increase in the DNA concentration of EM card elutions among breastmilk samples processed with our hybrid microfluidic system. As our device achieves sufficiently high cell enrichment in breastmilk samples while only requiring the user to push a syringe for 4 min with reasonable effort, we believe that it has high potential to expand EM card DNA archiving for diagnostic applications with low-cell density physiological fluids and in low-resource settings.

Wild grapes of Armenia: unexplored source of genetic diversity and disease resistance.

The present study is the first in-depth research evaluating the genetic diversity and potential resistance of Armenian wild grapes utilizing DNA-based markers to understand the genetic signature of this unexplored germplasm. In the proposed research, five geographical regions with known viticultural history were explored. A total of 148 unique wild genotypes were collected and included in the study with 48 wild individuals previously collected as seed. A total of 24 nSSR markers were utilized to establish a fingerprint database to infer information on the population genetic diversity and structure. Three nSSR markers linked to the Ren1 locus were analyzed to identify potential resistance against powdery mildew. According to molecular fingerprinting data, the Armenian V. sylvestris gene pool conserves a high genetic diversity, displaying 292 different alleles with 12.167 allele per loci. The clustering analyses and diversity parameters supported eight genetic groups with 5.6% admixed proportion. The study of genetic polymorphism at the Ren1 locus revealed that 28 wild genotypes carried three R-alleles and 34 wild genotypes carried two R-alleles associated with PM resistance among analyzed 107 wild individuals. This gene pool richness represents an immense reservoir of under-explored genetic diversity and breeding potential. Therefore, continued survey and research efforts are crucial for the conservation, sustainable management, and utilization of Armenian wild grape resources in the face of emerging challenges in viticulture.

An efficient direct organogenesis protocol for in vitro clonal propagation of Rubia cordifolia L.

This comprehensive study delves into the intricate process of in vitro clonal propagation of R. cordifolia, with a particular focus on optimizing a method of direct organogenesis. The choice of direct organogenesis over indirect organogenesis is driven by the propensity for somaclonal variations associated with the latter. The presented research explores the critical factors influencing direct organogenesis, including the selection of appropriate explants and the optimum use of plant growth regulators. Notably, nodal explants have demonstrated great potential for regeneration, and the choice of cytokinins such as kinetin (KIN) and 2-isopentenyladenine (2-iP) for shoot induction has proven highly effective, with 1.0 mg/L KIN supplementation to MS media emerging as the most favourable choice resulting in 12.14 ± 1.58 shoots and 29.78 ± 1.93 axillary buds. Subsequent adventitious rooting using indole-3 butyric acid (IBA) and indole-3-acetic acid (IAA) resulted in a 100% rooting response. Among the concentrations tested, 1.0 mg/L IBA was found most effective, yielding the best rooting response (22.9 ± 1.06 roots/ micro-shoot). Acclimatization of in vitro cultures was achieved by gradually exposing them to natural conditions, maintaining high humidity and minimizing photorespiration. The morphological transformations observed, including robust leaf development contributed to the high acclimatization success (100% survival). Additionally, the genetic stability was confirmed using DNA-based ISSR markers, with complete monomorphism indicating no variations among the regenerants. The efficient direct organogenesis protocol developed in this study may prove a valuable contribution towards the mass clonal propagation and conservation of R. cordifolia, an industrially and medicinally important plant.

Molecular Genetic Identification of Some Sweet Sorghum - Sorghum bicolor L. (Ankolib) Accessions – Sudan

This study is an attempt to identify some Sudanese Sweet Sorghum (Ankolib) [Sorghum bicolor (L.) Moench.] accessions using two types of DNA-based markers: RAPD and SSR. Seven (Ankolib) accessions were assayed, namely: Black Ankolib, Black White Ankolib, Dark Red Ankolib, Red Yellow Ankolib, White Ankolib, White Black Ankolib and Bengaga. All of the accessions were uniquely identified and fingerprinted. The levels of polymorphism among the accessions as revealed by (22) RAPD primers and (16) SSR primer pairs were (58%) and (76%) respectively, indicating that SSRs markers were highly polymorphic. The scored data of the two markers were analyzed using the Dice Coefficient to assess genetic relationships among the (7) (Ankolib) accessions. The results of the statistical analysis revealed that the accession Bengaga was distantly related to the other (6) accessions which all showed a close genetic similarity among them. UPGMA cluster analysis generated a dendrogram for each marker alone and for the combined data of the two markers. It was observed that in all of the dendrogram the accession Bengaga was found in a unique cluster thus indicating its uniqueness, since it is the only accession that has seeds that produce flour besides its juicy sweet stem.

Estimating the Genetic Diversity of Oranges Citrus spp. in South Sulawesi, Indonesia, Using RAPD Markers

Oranges hold significant economic importance, being cultivated extensively worldwide and having a large global market. Indonesia, ranked eighth globally as a producer of oranges, is one of the countries with high genetic diversity of oranges. This diversity is distributed across various regions of Indonesia, including South Sulawesi. Despite the advancements in DNA-based molecular marker techniques for assessing genetic diversity, information on orange diversity in South Sulawesi is currently unavailable and under-researched. In this study, random amplified polymorphic DNA (RAPD) markers were utilized to analyze the genetic diversity of oranges in five production centers in South Sulawesi. Leaf samples of 13 orange varieties were collected from the five production centers: Pangkep, Sidrap, Bantaeng, North Luwu, and Selayar in South Sulawesi, Indonesia. Genomic DNA extraction from the orange leaves followed the protocol of the DNA Mini Kit Geneaid. DNA amplification was carried out using the RAPD method with 14 primers: OPE-04, OPH-04, OPH-15, OPN-14, OPN-16, OPR-08, OPR-20, OPW-06, OPW-09, OPX-07, OPX-11, OPX-17, UBC-18, and UBC-51. The RAPD primers yielded 109 amplified fragments ranging in size from 200 to 2000 base pairs (bp), and all RAPD primers showed 100% polymorphism. The genetic diversity value (He) of oranges in South Sulawesi was moderate (0.236). Cluster analysis based on a similarity coefficient of 77% divided the 175 orange genotypes into five groups. The most closely related genotypes were SB6 and SB7, exhibiting 100% similarity, followed by genotypes JS8 and JS9 and JS13 and JS17, with genetic similarities exceeding 99% for each pair. Genotypes P9 and SI5 displayed the highest genetic distance, with a similarity coefficient of 57%. The dendrogram diagram can serve as a basis for selecting desired plant traits in the improvement of plant characteristics through both conventional breeding and genetic engineering activities.

Recent and Advanced DNA-Based Technologies for the Authentication of Probiotic, Protected Designation of Origin (PDO) and Protected Geographical Indication (PGI) Fermented Foods and Beverages.

The authenticity of probiotic products and fermented foods and beverages that have the status of protected designation of origin (PDO) or geographical indication (PGI) can be assessed via numerous methods. DNA-based technologies have emerged in recent decades as valuable tools to achieve food authentication, and advanced DNA-based methods and platforms are being developed. The present review focuses on the recent and advanced DNA-based techniques for the authentication of probiotic, PDO and PGI fermented foods and beverages. Moreover, the most promising DNA-based detection tools are presented. Strain- and species-specific DNA-based markers of microorganisms used as starter cultures or (probiotic) adjuncts for the production of probiotic and fermented food and beverages have been exploited for valuable authentication in several detection methods. Among the available technologies, propidium monoazide (PMA) real-time polymerase chain reaction (PCR)-based technologies allow for the on-time quantitative detection of viable microbes. DNA-based lab-on-a-chips are promising devices that can be used for the on-site and on-time quantitative detection of microorganisms. PCR-DGGE and metagenomics, even combined with the use of PMA, are valuable tools allowing for the fingerprinting of the microbial communities, which characterize PDO and PGI fermented foods and beverages, and they are necessary for authentication besides permitting the detection of extra or mislabeled species in probiotic products. These methods, in relation to the authentication of probiotic foods and beverages, need to be used in combination with PMA, culturomics or flow cytometry to allow for the enumeration of viable microorganisms.

A physical map of traits of agronomic importance based on potato and tomato genome sequences.

Potato, tomato, pepper, and eggplant are worldwide important crop and vegetable species of the Solanaceae family. Molecular linkage maps of these plants have been constructed and used to map qualitative and quantitative traits of agronomic importance. This research has been undertaken with the vision to identify the molecular basis of agronomic characters on the one hand, and on the other hand, to assist the selection of improved varieties in breeding programs by providing DNA-based markers that are diagnostic for specific agronomic characters. Since 2011, whole genome sequences of tomato and potato became available in public databases. They were used to combine the results of several hundred mapping and map-based cloning studies of phenotypic characters between 1988 and 2022 in physical maps of the twelve tomato and potato chromosomes. The traits evaluated were qualitative and quantitative resistance to pathogenic oomycetes, fungi, bacteria, viruses, nematodes, and insects. Furthermore, quantitative trait loci for yield and sugar content of tomato fruits and potato tubers and maturity or earliness were physically mapped. Cloned genes for pathogen resistance, a few genes underlying quantitative trait loci for yield, sugar content, and maturity, and several hundred candidate genes for these traits were included in the physical maps. The comparison between the physical chromosome maps revealed, in addition to known intrachromosomal inversions, several additional inversions and translocations between the otherwise highly collinear tomato and potato genomes. The integration of the positional information from independent mapping studies revealed the colocalization of qualitative and quantitative loci for resistance to different types of pathogens, called resistance hotspots, suggesting a similar molecular basis. Synteny between potato and tomato with respect to genomic positions of quantitative trait loci was frequently observed, indicating eventual similarity between the underlying genes.

Molecular Genetic Identification of Some Sweet Sorghum - Sorghum bicolor L. (Ankolib) Accessions - Sudan

This study is an attempt to identify some Sudanese Sweet Sorghum (Ankolib) [Sorghum bicolor (L.) Moench.] accessions using two types of DNA-based markers: RAPD and SSR. Seven (Ankolib) accessions were assayed, namely: Black Ankolib, Black White Ankolib, Dark Red Ankolib, Red Yellow Ankolib, White Ankolib, White Black Ankolib and Bengaga. All of the accessions were uniquely identified and fingerprinted. The levels of polymorphism among the accessions as revealed by (22) RAPD primers and (16) SSR primer pairs were (58%) and (76%) respectively, indicating that SSRs markers were highly polymorphic. The scored data of the two markers were analyzed using the Dice Coefficient to assess genetic relationships among the (7) (Ankolib) accessions. The results of the statistical analysis revealed that the accession Bengaga was distantly related to the other (6) accessions which all showed a close genetic similarity among them. UPGMA cluster analysis generated a dendrogram for each marker alone and for the combined data of the two markers. It was observed that in all of the dendrogram the accession Bengaga was found in a unique cluster thus indicating its uniqueness, since it is the only accession that has seeds that produce flour besides its juicy sweet stem.

Investigation of genetic diversity of Iranian wild relatives of bread wheat using ISSR and SSR markers

BackgroundWild relatives of wheat are one of the most important genetic resources to use in wheat breeding programs. Therefore, identifying wild relatives of wheat and being aware of their diversity, is undeniably effective in expanding the richness of the gene pool and the genetic base of new cultivars and can be a useful tool for breeders in the future. The present study was performed to evaluate the molecular diversity among 49 accessions of the genera Aegilops and Triticum in the National Plant Gene Bank of Iran using two DNA-based markers, i.e., SSR and ISSR. Also, the present study aimed to examine the relationships among the accessions studied belonging to different genetic backgrounds. ResultsTen SSR and tan ISSR primers produced 2065 and 1524 polymorphism bands, respectively. The number of Polymorphic Bands (NPB), the Polymorphism Information Content (PIC), Marker Index (MI), and Resolving Power (Rp) in SSR marker was 162 to 317, 0.830 to 0.919, 1.326 to 3.167, and 3.169 to 5.692, respectively, and in the ISSR marker, it was from 103 to 185, 0.377 to 0.441, 0.660 to 1.151, and 3.169 to 5.693, respectively. This indicates the efficiency of both markers in detecting polymorphism among the accessions studied. The ISSR marker had a higher polymorphism rate, MI, and Rp than the SSR marker. Molecular analysis of variance for both DNA-based markers showed that the genetic variation within the species was more than the genetic diversity between them. The high level of genomic diversity discovered in the Aegilops and Triticum species proved to provide an ideal gene pool for discovering genes useful for wheat breeding. The accessions were classified into eight groups based on SSR and ISSR markers using the UPGMA method of cluster analysis. According to the cluster analysis results, despite similarities between the accessions of a given province, in most cases, the geographical pattern was not in accordance with that observed using the molecular clustering. Based on the coordinate analysis, neighboring groups showed the maximum similarities, and distant ones revealed the maximum genetic distance from each other. The genetic structure analysis successfully separated accessions for their ploidy levels. ConclusionsBoth markers provided a comprehensive model of genetic diversity between Iranian accessions of Aegilops and Triticum genera. Primers used in the present study were effective, informative, and genome-specific which could be used in genome explanatory experiments.

DNA Fingerprinting of Crops and Its Significance in Crop Improvement

One of the improvements made possible by the era of genomics is DNA fingerprinting. DNA fingerprinting can be used to detect a variety of genetic changes. In breeding programmes fingerprinting is used to improve productivity and performance. Variety differentiation was relied on morphological characteristics prior to the introduction of genomics and proteomics approaches. Although morphological markers have been used for decades, protein-based markers like isozymes were discovered in the middle of the twentieth century and used for fingerprinting and crop genetic variety assessments. But nowadays, the emergence of genome sequencing has led to the widespread use of genetic markers for crop fingerprinting. The discovery of DNA-based genetic markers has improved genetic research. For genetic diversity estimation, crop evolution, gene mapping and phylogeny, diploid/haploid crop appraisal, heterosis analysis, varietal identification by using marker assisted selection (MAS), highly polymorphic DNA markers are generated. The use of DNA markers for crop fingerprinting started with RFLPs that did not involve PCR and subsequently advanced to PCR amplifiable markers such as AFLPs, RAPDs, SSRs, ISSRs, SNPs, and GBS.This review will provide an outlook on several types of markers and their significance in DNA fingerprinting of crops and possible applications, as well as suggestions for further research.