Restriction Fragment Length Polymorphism Sequence Research Articles

Screening of Type 2 Diabetes Patients of Khyber Pakhtunkhwa for CDKAL1 Variant (rs10946398)

Diabetes Mellitus Type 2 (T2DM) is a metabolic disorder influenced by environmental and genetic factors, with varied genetic predispositions across populations. Research on T2DM's genetic risk factors in the Pakistani community is limited. Objective: To explore the association between the CDKAL1 gene variant (rs10946398) and T2DM in the Khyber Pakhtunkhwa population in Pakistan. Methods: The study involved 100 T2DM patients and 100 controls, matched by age and gender, following specific inclusion and exclusion criteria. Sociodemographic data were collected alongside 3ml of whole blood for DNA extraction. The CDKAL1 gene was analyzed using PCR-based Sequence Restriction Fragment Length Polymorphism (RFLP), and the data were processed with SPSS version 26.0s. Results: Findings showed that 47% of cases were aged 39 to 59, with 94% having a family history of T2DM and 85% leading a sedentary lifestyle. A significant association was observed between the CDKAL1 rs10946398 variant and T2DM. The GC variant was linked to a fourfold increase in risk (OR: 4.833, CI: 1.197-19.509, P=0.027), and the CC variant had a twofold association (OR: 2.788, CI: 1.545-5.033, P=0.001). These associations persisted after adjusting for family history, age, socioeconomic factors, exercise, and diet. Conclusions: The study identified a significant correlation between the CDKAL1 rs10946398 variants and T2DM susceptibility in the Khyber Pakhtunkhwa population, emphasizing the role of genetic factors in the disease's prevalence within this community.

Full genome sequence analysis of a 1-7-4-like PRRSV strain in Fujian Province, China.

PRRS virus (PRRSV) has undergone rapid evolution and resulted in immense economic losses worldwide. In the present study, a PRRSV strain named FJ0908 causing high abortion rate (25%) and mortality (40%) was detected in a swine herd in China. To determine if a new PRRSV genotype had emerged, we characterized the genetic characteristics of FJ0908. Phylogenetic analysis indicated that FJ0908 was related to 1-7-4-like strains circulating in the United States since 2014. Furthermore, the ORF5 sequence restriction fragment length polymorphism (RFLP) pattern of FJ0908 was 1-7-4. Additionally, FJ0908 had a 100 aa deletion (aa329–428) within nsp2, as compared to VR-2332, and the deletion pattern was consistent with most of 1-7-4 PRRSVs. Collectively, the data of this study contribute to the understanding of 1-7-4-like PRRSV molecular epidemiology in China.

IDENTIFICATION OF GYMNEMA SPECIES BY RANDOM AMPLIFIED POLYMORPHIC DNA TECHNIQUE AND CHLOROPLAST trnK GENE

Gymnema is one of the important anti-diabetic medicinal plants used from ancient times and is commonly known as ‘sugar killer’. Most of its species have been used in many applications in Indian traditional medicine. Nevertheless, their efficiency is critically dependent on the use of the correct material. The sharing of similar vernacular name and morphological features make confusion in the usage of Gymnema species. In the present study, Gymnema sp. were identified through random amplified polymorphic DNA (RAPD) technique and species specific markers were generated for easy identification of G. elegans, G. montanum and G. sylvestre. Using the RAPD techniques of 3 species specific markers for G. sylvestre, 7 markers for G. elegans and 4 markers for G. montanum had been generated. Highest genetic identity was found between G. sylvestre and G. montanum and highest genetic distance was found between G. sylvestre and G. elegans. Further, DNA barcode was developed by sequencing chloroplast partial trnK DNA of these three species. No significant variation was found in partial trnK gene sequences between Gymnema species. But these sequences can efficiently differentiate the Gymnema and Mandevilla species. In-silico sequence–restriction fragment length polymorphism (RFLP) analysis revealed three fragments measuring G. sylvestre - 204, G. elegans - 174, and G. montanum - 168 bp Gymnema species. The present study concluded that RAPD markers were highly efficient for species detection than the partial trnK gene sequences. This could be used to confirm the Gymnema sp. identities and to ensure their safe application in pharmaceuticals.

Diversity and potential application of endophytic bacteria in ginger.

Here, 248 endophytic bacterial strains were isolated to assess the distribution and population diversity of endophytic bacteria in ginger plants. A total of 10.4 x 10(4) to 20.2 x 10(4) CFU/g fresh weight endophytic bacteria of different growth stages were isolated. Maximum bacterium numbers were obtained at the seedling stage. A total of 107 functional strains were screened, including 17 antibacterial strains and 90 indole acetic acid-producing strains. Based on 16S rDNA sequence restriction fragment length polymorphism and 16S rDNA sequences, these 107 strains were mapped and grouped into 16 genera. Bacillus and Pseudomonas were the dominant genera; however, the bacteria belonged to a tremendous range of genera, with the highest species richness being observed at the seedling stage. Sixteen strains exhibited antimicrobial activity against Pythium myriotylum Drechsler, while 7 strains exhibited antimicrobial activity against Phyllosticta zingiberi Hori. Bacillus was the dominant antibacterial strain. Pseudomonas fluorescens, B. megaterium, and Enterobacter ludwigii produced remarkably high levels of IAA. Only a few endophytic bacterial strains were inhibited in fresh ginger juice. Most of these strains were present during seedling stage, including Roseateles depolymerans, Chryseobacterium taiwanense, E. ludwigii, Agrobacterium larrymoorei, P. fluorescens, and Bacillus amyloliquefaciens. This study indicates that the community of endophytic bacteria in ginger changes with the synthesis of antibacterial substances.

Cotton genetic resources. A review

Since 6000 BC, cotton has been cultivated for lint fiber, which now dominates the natural textile industry worldwide. Common resources such as an integrated web database, a microsatellite database, and comparative quantitative trait loci (QTL) resources for Gossypium have accelerated the progress towards quantifying the impact of repeated human dispersals and selection regimes on various gene pools of the genus Gossypium. Out of 50 Gossypium species, four have been domesticated—two diploids and two tetraploids—for elimination of hard seed coat, improvement in lint percentage of about 40% and fiber length of 22%, larger boll size, and day-neutral reproductive habit. The major drawback of domestication is the lack of genetic diversity. This lack of genetic diversity is observed more in Gossypium hirsutum L. cultivars characterizing upland cotton than in Gossypium barbadense, typical of Pima and Egyptian cotton. Much of the genetic diversity among G. barbadense cultivars is attributed to the introgression of G. hirsutum alleles. This process highlights the importance of introgression of new alleles from accessions of all the Gossypium species into cultivated cotton species. Among the genomic resources, about 16,162 publicly available SSRs and 312 mapped cotton RFLP sequences containing simple sequence repeat (SSR), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), and random amplified polymorphic DNA (RAPD) markers have been surveyed on numerous mapping populations, and developed about 26 linkage maps (SSR, RFLP, AFLP, and RAPD). Reports show the identification of DNA markers associated with over 29 important traits or QTLs such as fiber quality and yield, leaf and flower morphology, trichome density and their distribution, and disease resistance. In comparative mapping studies, 432 QTLs mapped on 11 different mapping populations were aligned on a high-density reference map containing 3,475 loci. In a meta-analysis study of over 1,000 QTLs obtained from backcross population and recombinant inbred line populations derived from the same parents, most consistent meta-clusters were reported for fiber color, fineness, and length. For exploring the function of genes, the targeting induced local lesions in genomes (TILLING) approach—avoiding gene transfer process was used for identifying a brassino steroid receptor gene that is involved in fiber development. Lastly, cotton genome has been enriched with genes isolated from distantly related organisms using various transformation methods. For example, Cry1Ac, Cry1Ab, and herbicide-resistant genes were transformed in cotton that covered a vast majority of cotton acreage worldwide. Here the authors discuss investigations for improving the efficacy of transformation and regeneration systems, and for searching new genes or silencing the unwanted cotton genes using RNAi technology. We suggest initiating projects on sequencing the diploid and tetraploid genomes for exploring the extent of genetic variations, developing TILLING populations, initiating nested association mapping studies, and developing third generation genetically modified cotton, collectively setting the stage for sustaining cotton production under continually changing production conditions, climates, and human needs.

Climate and Tick Seasonality Are Predictors of Borrelia burgdorferi Genotype Distribution

The blacklegged tick, Ixodes scapularis, is of significant public health importance as a vector of Borrelia burgdorferi, the agent of Lyme borreliosis. The timing of seasonal activity of each immature I. scapularis life stage relative to the next is critical for the maintenance of B. burgdorferi because larvae must feed after an infected nymph to efficiently acquire the infection from reservoir hosts. Recent studies have shown that some strains of B. burgdorferi do not persist in the primary reservoir host for more than a few weeks, thereby shortening the window of opportunity between nymphal and larval feeding that sustains their enzootic maintenance. We tested the hypothesis that climate is predictive of geographic variation in the seasonal activity of I. scapularis, which in turn differentially influences the distribution of B. burgdorferi genotypes within the geographic range of I. scapularis. We analyzed the relationships between climate, seasonal activity of I. scapularis, and B. burgdorferi genotype frequency in 30 geographically diverse sites in the northeastern and midwestern United States. We found that the magnitude of the difference between summer and winter daily temperature maximums was positively correlated with the degree of seasonal synchrony of the two immature stages of I. scapularis. Genotyping revealed an enrichment of 16S-23S rRNA intergenic spacer restriction fragment length polymorphism sequence type 1 strains relative to others at sites with lower seasonal synchrony. We conclude that climate-associated variability in the timing of I. scapularis host seeking contributes to geographic heterogeneities in the frequencies of B. burgdorferi genotypes, with potential consequences for Lyme borreliosis morbidity.

Molecular markers linked to papaya ring spot virus resistance and Fusarium race 2 resistance in melon

In melon, the Fom-1 gene confers monogenic resistance against the soil-borne fungus Fusarium oxysporum f. sp. melonis, races 0 and 2, while the closely linked Prv gene specifies resistance against the papaya ring spot virus. Markers linked to these resistance (R) genes were identified using two recombinant inbred line populations, derived from crosses between Cucumis melo Vedrantais and C. melo PI 161375, and between C. melo Vedrantais and C. melo PI 414723, respectively. Using bulked segregant analysis, as well as systematic scoring of the mapping populations, we developed two amplified fragment length polymorphism markers, two random amplified polymorphic DNA markers and five restriction fragment length polymorphism (RFLP) markers linked to this locus. Four of the RFLP sequences bear homology to nucleotide-binding site-leucine-rich repeat R genes, indicating the presence of a significant R-gene cluster in this locus. Our study provides the most closely linked markers published so far for these important traits. It also improves the resolution of the whole linkage group IX, which was difficult to order in our previous studies. Two of the markers were converted to cleaved amplified polymorphic sequence markers to facilitate their application in marker-assisted selection. Testing these two markers in several melon lines revealed different marker haplotypes in the melon germplasm and supported multiple, independent origin of the Fusarium races 0 and 2 resistance trait.

"RFLP subtraction": a method for making libraries of polymorphic markers.

We developed a method, "RFLP subtraction," that isolates large numbers of unique sequence restriction fragment length polymorphisms (RFLPs) in a single experiment. The technique purifies small restriction fragments from one genome containing sequences that reside on large fragments in a related genome. We first isolate samples containing the small restriction fragments from two polymorphic strains. Subtractive hybridization then removes the fragments that are present in both samples. The remaining sequences are RFLPs: they occur on small fragments in one strain but not in the other. Here we use RFLP subtraction to make a library of hundreds of unique sequence RFLPs from two inbred mouse strains. We analyze and map a subset of the RFLPs and show that the genetic linkage of these markers can be rapidly determined by an efficient dot blot mapping technique. Several other potential applications of RFLP subtraction, including isolating region specific markers, are discussed.