DNA Molecular Markers Research Articles

Detection of mRNA Expression and Copy Number Variations Within the Goat Fec B Gene Associated With Litter Size.

The Booroola fecundity (FecB) gene, as the first major fecundity gene identified in Booroola sheep, has attracted careful attention. So far, previous research have uncovered the FecB mutation (Q249R) as the main mutation by virtue of which sheep exhibits multiple lambing phenomena. This mutation is now being intensively studied and widely used. However, such effect of the FecB mutation has not been applied to goats, and similar types of the FecB gene in goats still need to be studied. Thus, the current study attempted to verify potential mutations in the goat FecB gene as well as investigate their functions related to fecundity. First, FecB expression was investigated in six different goat tissues, and we found that FecB expression was highest in the mammary gland, followed by the ovary. Next, the influence of the FecB gene was analyzed from a new perspective, where five potential copy number variations (CNVs) (CNV1–5) within the FecB gene were identified for the first time, and then their effects on litter size were measured. Our results point out that CNV3 (P = 3.44E-4) and CNV5 (P = 0.034) could significantly influence the litter size of goats. Identically, the combination genotype of CNV3 and CNV5 which consisted of their dominant genotypes was also significantly associated with goat litter size (P = 7.80E-5). Hence, CNV3 and CNV5 could serve as potential DNA molecular markers applied to DNA editing and DNA microarray. Additionally, the abovementioned study has laid a theoretical foundation for the detection of potential fertility-related quantitative trait loci within the goat FecB gene.

Estimation of the Genetic Diversity among Twenty Guar (Cyamopsis tetragonoloba L. Taub) Genotypes Using Molecular Markers

The aim of this study was to determine the genetic diversity and relationships among twenty newly developed genotypes of Cyamopsis tetragonoloba using Randomly Amplified Polymorphic DNA (RAPD) molecular technique. Data obtained from application of ten molecular markers were subjected to the procedure of clusters analysis. RandomAmplified Polymorphic DNA (RAPD) molecular markers indicated the presence of a wide genetic diversity among the studied twenty genotypes of guar. The constructed dendrogram, based on Jaccard’s similarity coefficients,differentiated the genotypes into seven main clusters. The closest genotypes were genotypes Gm34 and L53 with (98 % similarity), while the most diverse ones were genotype L14 and genotype Gm16 with 29% genetic similarity. It was concluded that use of RAPD markers can be used for efficient estimation of genetic diversity in guar. Hence, the current investigation can contribute toenhancement of molecular breeding approach for the development of improved guar genotypes.

Anti-trypanosomal activity and DNA fingerprinting of fifteen Euphorbia species using ISSR and SCoT markers

BackgroundEuphorbia is the largest genus in family Euphorbiaceae with a great biological and genetic diversity. The anti-trypanosomal activity of the crude extract of 15 Euphorbia species against Trypanosoma brucei brucei was carried out. Furthermore, DNA fingerprinting of the tested species using ISSR and SCoT markers was also investigated.ResultsThe anti-trypanosomal activity of the 15 Euphorbia species revealed the highest activity of E. officinarum L. and E. milli Des Moul. against Trypanosoma brucei brucei with IC50 values < 10 μg/mL after 48- and 72-h incubation. Moreover, the assessment of the genetic diversity among the 15 tested species showed similar correlation coefficients of 0.76–0.98 which classified Euphorbia species into two main groups, one contained two species and the other contained 13 species.ConclusionsSome Euphorbia species exhibited significant growth inhibitory activity toward Trypanosoma brucei strain TC221. Results also indicated the suitability of both markers for genetic fingerprinting of the tested Euphorbia species. To our knowledge, this is the first detailed comparison of the performance of two targeted DNA molecular markers (SCoT and ISSR) on the tested 15 Euphorbia species. The results guide future efficient use of these molecular markers in the genetic analysis of Euphorbia.

A deletion mutation within the goat AKAP13 gene is significantly associated with litter size

A-kinase anchoring protein 13 (AKAP13) is one of the AKAP protein family members, which is correlated with estrogen receptors (ERs) and progesterone receptor (PR) activity. Consequently, the AKAP13 gene is considered to be one of the candidate genes for regulating female fertility. Hence, the objectives of this study were to discover the potential insertion/deletion (indel) variants within the AKAP13 gene and evaluate their associations with litter size of Shaanbei white cashmere goats (SBWC) to screen candidate genes for the molecular marker-assisted selection (MAS). Ultimately, we found the 16-bp deletion of AKAP13 gene which displayed three genotypes (II, ID and DD). However, it was not confirmed to Hardy-Weinberg equilibrium (HWE) in the tested population. Statistical analysis demonstrated that this 16-bp indel locus was significantly associated with litter size in goats (p < 0.05), in which the ID genotype was a key genotype for increasing litter size in goats. Besides, independent χ 2 tests between different genotypes and litter size showed that high-prolific groups had higher frequency of the ‘D’ allele (p < 0.05). Briefly, AKAP13 gene is a candidate gene for improving fertility, and its 16-bp indel locus can be used as a valid DNA molecular marker for the MAS in goat breeding.

Evaluating heat stress tolerance and molecular relationship among inbred lines of maize during early generations

Heat stress is one of abiotic stress that has deleterious effects on crops yield. Therefore, this study aims are to evaluate heat-tolerant maize lines and characterizing them by DNA molecular markers. Inbred lines were generated for two generations by selfing pollination. They were evaluated in each generation for the performance of some agro-morphological traits under normal and heat stress conditions. All the traits of the S1 and S2 inbred lines varied significantly among them under both conditions, except plant height was not significant under heat stress for the S1 lines. The L6 inbred line had the highest yield under the stress conditions in both S1 andS2 generations. Moreover, the heat susceptible index showed that the lines, L6 and L40 were the highest tolerant in the both generations. Furthermore, cluster analysis based on morphological traits for the 5 selected S2 inbred lines could be able to isolate the worst S2 inbred line under heat stress conditions in an independent cluster. In addition, they were characterized by ISSR and SRAP molecular markers. The ISSR detected higher polymorphism (79.79%) than SRAP marker (58.46%). The ISSR clustering patterns managed to classify the highest yield line (L6) under the heat stress in a separated cluster, but both the SRAP and combined isolated the worst line (L32) in one cluster. The Mantel’s test showed a positive correlation among all the studied markers. Additionally, the correlation was significant and highly strong (r=0.915) between morphological traits under normal conditions and SRAP marker. However, the identified S2 inbred lines with resistance to heat tolerance could be a beneficial source in the development of heat-tolerant maize hybrids.

Tightly Linked Molecular DNA Markers with High Predictive Trait Value: A Current Increasing Demand in the Breeding Program of Upland Cotton (Gossypium hirsutum L.)

Cotton (Gossypium spp.) is a major cash crop of India and is the second-largest cotton producer in the world after China. Gossypium hirsutum and Gossypium barbadense are two tetraploid species that are majorly cultivated besides two diploid species (desi cotton). In cotton, fiber quality, drought tolerance, boll weight, boll number, and yield are essential quantitative traits with many components that are controlled by several genes present at different loci. Identifying such genes from different genomic resources of cotton using various molecular markers is necessary to accelerate the Quantitative Trait Loci (QTL) analysis. In the public domain of cotton, there is a vast number of molecular markers. However, not all are very useful for trait mapping, as most markers are away from the QTL region. Thereby, cotton improvement programs pay more attention to tightly linked markers with high predictive trait values. The present review provides an overview and updates on the comparative studies and the application of various molecular markers, i.e., RFLP, AFLP, RAPD, SSR, EST-SSR, and SNP in the cotton-breeding program. Insights gained from the study may help in successful cotton breeding and improvement.

Modern approaches to the genetic identification of farm animal breeds (review)

To improve the breed characters of farm animals, there is the need for breeding based on reliable information about the particular breed of animals, which is the key to high-quality breeding work. In this regard, there is a need to search for new methods of reliable identification of the breed of animals. The article presents an analysis of methods based on the achievements of modern genetics, which are used to obtain more accurate information when determining the breed of ani-mals. A brief description of methods for determining the species and breed identification of animals based on molecular DNA markers is given. Methods based on the study of polymorphism of mitochondrial DNA and microsatellites are analyzed. Spe-cial attention in this review is paid to the latest and most promising achievements in the field of high-performance genotyping technologies based on single nucleotide polymorphism (SNP). The analysis of the literature sources has shown that each breed of farm animals is characterized by its unique genetic structure, which should provide an objective result when deter-mining the breed of the animal.

Evaluation of a Novel Molecular Marker Associated with the Tan Spot Disease Response in Wheat

After nearly 40 years of DNA molecular marker development in plant breeding, the wheat research community has amassed an extensive collection of molecular markers which have been widely and successfully used for selection of agronomic, physiological and disease resistance traits in wheat breeding programs. Tan spot is a major fungal disease of wheat and a significant global economic challenge and is caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr). Here, the potential for using a PCR-based marker (Ta1AS3422) present on the short arm of wheat chromosome 1A, was evaluated for effectiveness in distinguishing tan spot disease susceptibility. The marker was initially screened against 40 commercial Australian hexaploid wheat varieties, and those that amplified the marker had an overall lower disease score (2.8 ± 0.7 for seedlings and 2.4 ± 0.4 for plants at the tillering stage), compared to those lacking the marker which exhibited a higher disease score (3.6 ± 0.8 for both growth stages). The potential of Ta1AS3422 as a marker for the tan spot disease response was further assessed against a panel of 100 commercial Australian hexaploid wheat varieties. A significant association was observed between marker absence/presence and tan spot disease rating (Pearson’s chi-squared test, χ2 (6) = 20.53, p = 0.002), with absence of Ta1AS3422 associated with susceptibility. This simple and cost-effective PCR-based marker may be useful for varietal improvement against tan spot, although further work is required to validate its effectiveness.

Isolation and HPLC assisted quantification of two iridoid glycoside compounds and molecular DNA fingerprinting in critically endangered medicinal Picrorhiza kurroa Royle ex Benth: implications for conservation.

The online version contains supplementary material available at 10.1007/s12298-021-00972-w.

Effects of magnetite nanoparticles on physiological processes to alleviate salinity induced oxidative damage in wheat.

One of the major abiotic stressors that have a serious effect on plant growth and productivity worldwide is the salinity of soil or irrigation water. The effect of foliar application of magnetite nanoparticles (size=22.05 nm) at different concentrations (0, 0.25, 0.5, and 1.0 ppm) was investigated to improve salinity tolerance in two wheat cultivars, namely, Misr1 (Tolerant) and Gimmeza11 (Sensitive). Moreover, toxicological investigations of magnetite oxide nanoparticle in Wistar albino rats were estimated. The magnetite nanoparticles positively affected growth, chlorophyll, and enzymatic antioxidants such as superoxide dismutase (SOD), stimulating reduced glutathione and improving the aggregation of several polypeptide chains that may be linked to the tolerance of saline stress. In contrast, magnetite nanoparticles reduced malondialdehyde (MDA). Inverse sequence-tagged repeat (ISTR) assay of DNA molecular marker showed the change in band numbers with the highest polymorphic bands with 90% polymorphism at primer F3, B5 and 20 positive bands in Gimmeza11 with 0.5 ppm magnetite nanoparticles. In the median lethal dose (LD50 ) study, no rats died after the oral administration of magnetite nanoparticle at different doses. Therefore, the iron oxide nanoparticle was nontoxic when administered orally by gavage. Magnetite nanoparticles partially helped to alleviate the effects of salt stress by activating growth, chlorophyll content, SOD, glutathione, and soluble proteins in two wheat cultivars (Misr1 and Gimmeza11) and decreasing MDA content. © 2021 Society of Chemical Industry.

Mitochondrial DNA Analysis Clarifies Taxonomic Status of the Northernmost Snow Sheep (Ovis nivicola) Population.

Currently, the intraspecific taxonomy of snow sheep (Ovis nivicola) is controversial and needs to be specified using DNA molecular genetic markers. In our previous work using whole-genome single nucleotide polymorphism (SNP) analysis, we found that the population inhabiting Kharaulakh Ridge was genetically different from the other populations of Yakut subspecies to which it was usually referred. Here, our study was aimed at the clarification of taxonomic status of Kharaulakh snow sheep using mitochondrial cytochrome b gene. A total of 87 specimens from five different geographic locations of Yakut snow sheep as well as 20 specimens of other recognized subspecies were included in this study. We identified 19 haplotypes, two of which belonged to the population from Kharaulakh Ridge. Median-joining network and Bayesian tree analyses revealed that Kharaulakh population clustered separately from all the other Yakut snow sheep. The divergence time between Kharaulakh population and Yakut snow sheep was estimated as 0.48 ± 0.19 MYA. Thus, the study of the mtDNA cytb sequences confirmed the results of genome-wide SNP analysis. Taking into account the high degree of divergence of Kharaulakh snow sheep from other groups, identified by both nuclear and mitochondrial DNA markers, we propose to classify the Kharaulakh population as a separate subspecies.

Keragaman genetik Anggrek Grammatophyllum scriptum asal biji dari hasil Kultur In Vitro berdasarkan penanda RAPD

Propagation through tissue culture by using orchid seed as explants will produce a lot of orchid plants. This study aims to measure the genetic character of orchid plantlets that were regenerated from seeds which have been resulted from in vitro culture. The genetic character of the original orchid plants produced from in vitro culture was determined using Random Amplified Polymorphic DNA (RAPD) molecular markers. The results showed that the primers used in the RAPD analysis showed a polymorphic band pattern of 14 DNA bands, with sizes between 500 bp - 8000 bp. The genetic distance of Grammatophyllum scriptum orchids that was regenerated from seeds is between 0.229 and 0.649. The progenis produced from in vitro culture were clustered into seven groups at a dissimilarity coefficient of 45%.

Meta-topolin and liquid medium enhanced in vitro regeneration in Scaevola taccada (Gaertn.) Roxb

Scaevola taccada (Gaertn.) Roxb. is a hemi-sclerophyllous littoral shrub of the family Goodeniaceae. It is a salt-tolerant plant and used in soil reclamation and coastal landscaping programs to control beach erosion. The present article reports a reproducible in vitro regeneration system using meta-topolin (mT) through liquid medium employing nodal segment cultures. The highest number of axillary shoots (5.8 ± 0.29) was differentiated on Murashige and Skoog’s (MS) medium containing meta-topolin (1.5 mg L−1). The amplification of shoots (49.8 ± 0.37) and fresh growth was achieved by recurrent subcultures on liquid MS medium supplemented with 0.25 mg L−1 mT and 0.15 mg L−1 indole-3-acetic acid (IAA) in the presence of additives (50 mg L−1 ascorbic acid + 25 mg L−1 citric acid, adenine sulphate and l-arginine). Rooting and acclimatization was simultaneously achieved using concurrent ex vitro rooting and acclimatization (CEVRA) technique by pulse treating the shoot base with 300 mg L−1 IBA for 5 min. The in vitro developed shoots represented a maximum percentage (100%) of rooting in autoclaved soilrite in a greenhouse. After 2 mo, the plantlets were established in the field with cent percentage survival success. The genetic fidelity analysis using inter simple sequence repeats (ISSR) DNA molecular markers developed maximum of 55 monomorphic bands and revealed the genetic homogeneity of in vitro regenerated plantlets with the mother plant.

Application Techniques of Molecular Marker and Achievement of Marker Assisted Selection (MAS) in Three Major Crops Rice, Wheat and Maize

With the discovery of new genetic technology, the researcher focuses on using DNA molecular markers to improve new varieties worldwide. Such as resistance to biotic and abiotic stresses and enhancing quality and quantity at different plant breeding fields. Conventional breeding selection is based on phenotype data selection, time-consuming, and has a high chance of linkage drag. Thus, DNA molecular marker method usage is faster, easy, and not expensive than conventional breeding programs. This review focused on applying molecular markers such as genetic diversity analysis, the genotype of identification and fingerprinting, gene tagging and mapping, QTL analysis, and marker-assisted selection. In another part of this review, we focused on MAS's achievements related to improving agronomic traits, quality traits, and biotic/abiotic stresses for three major cereal crops like Wheat, Rice, and Maize.

DNA-based molecular identification of Urnula mediterranea (Ascomycota, Pezizales) collected in Central Serbia

Molecular systematics is a branch of systematics that analyzes genetic markers for the classification of organisms. Genetic markers include target sequences of molecules DNA, RNA, and amino acids. The most often used genetic markers are DNA markers, localized on DNA molecules. The study aim was DNA-based molecular identification of species Urnula mediterranea (M. Carbone, Agnello &amp; Baglivo) M. Carbone, Agnello &amp; P. Alvarado (2013), by using molecular systematics methods. This species was recently discovered for the first time in Central Serbia and determined only based on morphological characteristics. For the first time, U. mediterranea sampled in Serbia, was identified with DNA molecular markers. Extraction of DNA molecules was performed from the fruiting body of U. mediterranea. ITS region and the part of the gene for 28S rRNA were amplified and sequenced by the Sanger method. Comparing the analyzed sequences with the sequences from the database, a match of 99.72% was found for the ITS region and 100% for the part of the gene for 28S rRNA. By molecular identification, it has been established that the sample belongs to the species U. mediterranea. Both analyzed DNA sequences of U. mediterranea were deposited into the NCBI database.

English

Production of extended-spectrum beta-lactamases (ESBLs) can lead to treatment failures when the antibiotics are used. This study detected ESBLs genes on Multidrug Resistant Escherichia coli from HIV-infected individuals attending some hospitals in Jos. Eighty (80) isolates of multidrug resistant (MDR) E. coli were screened for plasmids. ESBLs genes including bla-CTX, bla-TEM and bla-SHV were detected on the plasmids using polymerase chain reaction (PCR) with 100 bp DNA ladder as DNA molecular weight marker. Out of the total 38 plasmids tested, ESBLs genes occurred in 13 (34.21%), with the bla-TEM dominating [7 (53.85%)] over the bla-CTX [4 (30.77%)].&nbsp; Two (15.38%) of the isolates carried both genes. None of the isolates carried the bla-SHV gene in our study. All the strains showed resistance to SXT, AMC and CTX. Resistance was most frequently observed against SXT (13), AMC (13), CTX (13), CFM (12), F (8), NA (6), GN (5), CRO (4), OFX (2) and CIP (1). None of the ESBLs-bearing strains showed resistance to IPM. Result showed 34.21% prevalence of ESBLs and suggested the need to be more cautious with the clinical use of third generation Cephalosporins especially for the treatment of acute infections caused by E. coli due to the high resistance recorded. &nbsp; Key words:&nbsp;Extended spectrum beta-lactamase, resistance genes, antimicrobial, plasmids, Escherichia coli, HIV-infected, Jos. &nbsp

Detection of InDel and CNV of SPAG17 gene and their associations with bovine growth traits

Sperm-associated antigen 17 (SPAG17) gene encodes a central pair protein, which is involved in flagellar motility, male fertility and skeletal growth in ruminants. The insertions/deletions (indels) and copy number variations (CNVs) influence phenotypic traits by altering the sequences and copy numbers of functional genes, respectively. This study identified a novel 8-bp indel of SPAG17 gene in 1520 individuals from eight different cattle breeds, as well as a novel CNV region in 355 animals. The correlation analysis of indel showed that the individuals of ID genotype had superior performance traits such as body height (p = 0.038) and body slanting length (p = 0.041) as compared to other genotypes in Xianan cattle. For the CNV, different copy numbers were closely related to the body height in Qinchuan (p = 0.045) and body weight in Xianan (p = 0.036) breeds. Importantly, significant difference was observed between the 8-bp indel and the copy number loss in Xianan breed (p < 0.01). These findings indicated that the variations within the bovine SPAG17 gene can be considered as an effective DNA molecular marker for beef cattle breeding.

Estimation of C-derived introgression into A. m. mellifera colonies in the Russian Urals using microsatellite genotyping.

Marker-assisted selection is well established in animal breeding method of selecting individuals with desirable traits in a breeding scheme based on DNA molecular marker patterns. Genetic diversity and C-derived admixture into local purebred gene pool of A. m. mellifera colonies was assessed using polymorphism of nine microsatellite loci in order to provide further marker-assisted selection of desired honey bee colonies. The genetic diversity and the level of C-derived introgression into A. m. mellifera colonies in the Shulgan-Tash Nature Reserve (Russia) was assessed based on nine microsatellite loci (ap243, 4a110, A24, A8, A43, A113, A88, Ap049, A28), which were analized using the fragment analysis of the PCR products in Applied Biosystems 3130 DNA Analyzer. Phylogenetic relationship of colonies was evaluated using Neighbor-Joining methods with Cavalli-Sforza and Edwards genetic distance using the PHYLIP 3.68. The model-based Bayesian clustering algorithm implemented in STRUCTURE2.3.3 was employed to infer membership and introgression proportions (Q-value). In the Shulgan-Tash Nature Reserve colonies of A. m. mellifera subdivided into four groups by level of C-derived introgression. Only five colonies of A. m. mellifera had C-derived introgression which varied from 0.5 to 2%. The genetic diversity in colonies of A. m. mellifera varied from 0.12 to 0.40. The Neighbor-Joining tree demonstrates the genetic relationship of A. m. mellifera colonies, which subdivided into three groups with different levels of C-derived introgression. Group 1 combined five honey bee colonies Bort_1, Bort_2, Bort_3, Baisalyan_1, and Kush_7 with a fraction of introgression close to 0.000 and genetic diversity from 0.20 to 0.25. The results showed the excellence of nine microsatellite loci genotyping in estimation of genetic diversity, distinguishing the two European evolutionary lineages M and C and estimating C-derived introgression. These genetic parameters can be applied further to perform the marker-assisted selection of purebred dark European honey bees.

Quality assurance of Chinese Herbal Medicines (CHMs)

As more and more people worldwide are using Chinese Herbal Medicines (CHMs), the safety of their use has raised international concern. Authentication and standardization of CHMs are needed. Authentication of a CHM includes both identifying its botanical origin and evaluating its pharmaceutical quality. In this paper, the important parameters for quality assurance are discussed. Quality control methods such as the examination of taxonomic, morphological and/or microscopic characters, fingerprint chromatography, DNA molecular marker are described and compared. Some topics related to herbal quality control such as contamination, processing as well as the new method MALDI-TOFMS are also introduced. Finally, modernization of Chinese medicine information was brought up.

Chemical Markers to Distinguish the Homo- and Heterozygous Bitter Genotype in Sweet Almond Kernels.

Bitterness in almonds is controlled by a single gene (Sk dominant for sweet kernel, sk recessive for bitter kernel) and the proportions of the offspring genotypes (SkSk, Sksk, sksk) depend on the progenitors’ genotype. Currently, the latter is deduced after crossing by recording the phenotype of their descendants through kernel tasting. Chemical markers to early identify parental genotypes related to bitter traits can significantly enhance the efficiency of almond breeding programs. On this basis, volatile metabolites related to almond bitterness were investigated by Solid Phase Microextraction-Gas Chromatography-Mass Spectrometry coupled to univariate and multivariate statistics on 244 homo- and heterozygous samples from 42 different cultivars. This study evidenced the association between sweet almonds’ genotype and some volatile metabolites, in particular benzaldehyde, and provided for the first time chemical markers to discriminate between homo- and heterozygous sweet almond genotypes. Furthermore, a multivariate approach based on independent variables was developed to increase the reliability of almond classification. The Partial Least Square-Discriminant Analysis classification model built with selected volatile metabolites that showed discrimination capacity allowed a 98.0% correct classification. The metabolites identified, in particular benzaldehyde, become suitable markers for the early genotype identification in almonds, while a DNA molecular marker is not yet available.