PCR-RFLP Analysis Research Articles

Outbreak of toxoplasmosis associated with muscular lesions in finishing pigs caused by an atypical Toxoplasma gondii genotype.

Toxoplasma gondii infections are usually asymptomatic in pigs, and an acute clinical disease is rare in this host. This study aimed to determine the pathological and molecular aspects of an outbreak of fatal systemic toxoplasmosis in finishing pigs in Brazil. The outbreak occurred on a commercial finishing pig farm in the state of Santa Catarina in southern Brazil. The farm had 1500 pigs and 3.8% of mortality rate during the outbreak. The pigs had fever, anorexia, apathy, and locomotor deficits. Seven pigs were necropsied. Gross findings included multifocal to coalescent pale areas in skeletal muscles, lymphadenomegaly, hepatosplenomegaly, and non-colapsed lungs. The histological findings included granulomatous lymphadenitis, hepatitis and splenitis, necrotizing myositis, and lymphoplasmacytic interstitial pneumonia. Lung and liver lesions were occasionally accompanied by T. gondii parasitic structures. Positive immunolabeling for T. gondii tachyzoites and encysted bradyzoites was detected in all examined pigs. PCR-RFLP (11 markers) and microsatellite analysis (15 markers) identified the non-archetypal genotype #278 in pigs. This is the first report of systemic toxoplasmosis in pigs with muscle lesions and additionally shows the diversity of disease-causing T. gondii genotypes circulating in animals in Brazil.

ГЕНЕТИЧЕСКАЯ СТРУКТУРА ОВЕЦ ЗАПАДНО-СИБИРСКОЙ МЯСНОЙ И КУЛУНДИНСКОЙ ТОНКОРУННОЙ ПОРОД ПО ГЕНАМ CAST, GDF9 И KRT1.2

The purpose of the study is to analyze the genotypic structure and population-genetic parameters of the West Siberian meat and Kulunda fine-wool breed of sheep by the CAST, GDF9 and KRT1.2 genes and blood groups. Molecularnogenetic and immunogenetic studies were carried out in the SRIAH (Siberian Research Institute of Animal Husbandry SFSCA RAS) biotechnology laboratory. PCR-RFLP analysis to determine genotypes was performed according to the described and approved methods on a C1000 “BioRad” amplifier; the results were visualized using an E-Box-CX5.TS-20.M gel documentation system. It was found that the frequency of genotypes and alleles of the CAST gene of West Siberian meat and Kulunda fine-wool breeds have no significant differences. In both breeds, CASTMM was predominant, with a frequency of 69.0–75.0%, CASTNN carriers being only 1.6–5.1%. The Kulunda fine-wool breed is characterized by a very high frequency of the GDF9GG genotype 92.7%, which is higher than that of the West Siberian meat sheep by 26.3%. GDF9AA genotype is extremely rare in breeds (0–5%). The GDF9G allele in the compared breeds was found to range from 0.811–0.960 and the GDF9A allele from 0.189–0.040. There are differences in the ratio of genotypes of the KRT1.2 gene in the compared breeds. In the Kulunda fine-wool breed, the vast majority of animals have homozygous genotype KRT1.2MM (95.5%), whereas in the West Siberian meat breed such sheep constitute 32.8%. Consequently, the frequency of the KRT1.2M allele is 0.565–0.978 and the KRT1.2N allele is 0.435–0.022. The genetic balance in the studied genes is not disturbed, χ2 = 0,033–1,025. The index of genetic similarity between breeds, calculated on the basis of the frequencies of genotypes and blood groups is 0.901 ± 0.028 and 0.833 ± 0.024. Population and genetic characteristics are practically identical in the compared breeds, except for gene homozygosity (Ca), which is higher in the Kulunda fine-wool sheep breed by 24.3% for the GDF9 gene and KRT1.2 by 29.1%, compared to the West Siberian meat sheep breed. The number of effective alleles is insignificant (1.04–1.46). The genetic variability (V) by individual genes in the breeds varies from 5.0 to 49.6%. The negative value of the Fis coefficient indicates the absence of inbreeding in sheep the studied breeds.

A family screening of CD19 gene mutation by PCR-RFLP

Introduction and aim. Mutation(s) in the gene encoding the CD19 molecule affect CD19 protein expression and primary immunodeficiency (PID) occurs. The PCR-RFLP method, which is faster and cheaper than other mutation detection methods, is rarely used in the diagnosis of PID. The study aimed to genetically identify CD19 deficiency, which is a PID, using the PCR-RFLP method. Material and methods. A total of 8 patients and two healthy controls were included in the study and the relevant region genotypes in the CD19 gene were determined by performing PCR-RFLP analysis. Results. The index case, newborn baby and mother were also included in the study. It was determined that the index case (P6) was homozygous mutant, the newborn baby (P7) and mother (P8) had heterozygous genotype. Based on this situation, one child (P1) was found to be homozygous mutant, mother (P2), father (P3) and other children (P4 and P5) had heterozygous genotype in the family, which was determined to be related to the first case. Conclusion. In our study, it has been shown that PCR-RFLP is a method that can be used in the diagnosis of PID by determining genotypes using PCR-RFLP, and especially in terms of rapid genetic testing of family screenings.

Распространение дирофилярий (Spirurida, Onchocercidae) в природных популяциях малярийных комаров (Diptera, Culicidae) Томского Приобья

Dirofilariasis is a dangerous transmissible disease of carnivores and humans caused by two species of parasitic nematodes - Dirofilaria repens Railliet et Henry and D. immitis Leidy. The intermediate hosts of Dirofilariae are blood-sucking mosquitoes. Both species are typical of the mild climate of southern Europe, but in recent decades the parasites have spread to the north and northeast due to the global climate change. They have been registered in Western Siberia since 1989, and diro-filariasis cases in both animals and humans cause concern. To assess prospects for the spread of dirofilariasis and develop strategies to combat this disease, one has to determine the main mosquito vectors. Malaria mosquitos are the significant dirofilaria vectors in Europe, but there is a shortage of information on potential dirofilariasis vectors in Siberia. This paper is meant to estimate how extensive the invasion of different species of the malaria mosquitos D. repens and D. immitis is in the territory of Tomsk Region due to the global climate change. 64 samples of mosquitos from 36 localities of Tomsk Region were taken during the summer seasons of 2018 to 2020. Mosquitos were caught in stables, dissected to determine invasion via light microscopy. The species of malaria mosquitos and dirofilariae were identified through an ITS2 PCR-RFLP analysis (Artemov et al., 2021) and COI site-specific PCR (Rishniw et al., 1998), respectively. In total, the authors analyzed 7,591 female malaria mosquitos and 233 female non-malaria mosquitos, where 159 and 1 of them were infected with dirofilariae, respectively. They registered invasion in 44 samples from 21 localities, including the northernmost one - Strezhevoy town (60°44N, see Figure 1). Dirofilariasis is a common helminthiasis in Tomsk Region. The average invasion extensiveness is 2.1+0.2% in the region. In the natural populations of malaria mosquitos on the right bank of the River Ob, the invasion extensiveness is 3.7 times higher vs. the left bank of the Ob, despite the high transport accessibility of this bank (see Figure 2). Invasion extensiveness decreases from the south to the north, but the local conditions might disrupt this trend (see Table 3). Three species of malaria mosquitos - Anopheles daciae, An. messeae and An. beklemishevi (but not An. claviger) - are dirofilariasis vectors in Tomsk Region (see Table 1). South of 57° N, only D. repens and one mixed invasion case were found in An. daciae being the dominant species in this area. However, all the three species mainly invaded with D. repens were detected as vectors north of 57° N. One case of An. messeae invasion with the nematode D. immitis and mixed invasions of An. daciae and An. beklemishevi were found in Kolpashevo agglomeration. All the invasions with D. immitis (including mixed ones) were observed on the right bank of the Ob (see Table 2). The extensiveness of An. messeae and An. daciae invasion were significantly higher vs. An. beklemishevi, despite its domination north of 57° N. The northernmost location where an infective female of An. beklemishevi invaded with D. repens was found is Bolshaya Griva village at 59° N. Approximately 6.5% of the infected mosquitos had superinvasion - re-invasion with dirofilariae (see Figure 3). Thus, malaria mosquitos play a significant role in the circulation and, probably, the spread of dirofilariasis in Tomsk Region. Conclusion: 1. Dirofilariae are widespread within Tomsk Region. The northernmost border of the Dirofilaria repens spread is 60° 44' and that of D. immitis - 58° 32' N. The northernmost border of the D. repens transmission is 59° N. 2. The contribution made by malaria mosquitos to the spread of dirofilariasis in Tomsk Region is as high as that of other blood-sucking mosquitos. 3. Anopheles daciae, An. messeae and An. beklemishevi are the vectors of Dirofilaria repens and D. immitis. Anopheles beklemishevi get infected less often than the two remaining species. 4. The invasion extensiveness of malaria mosquitos is higher on the right bank of the River Ob. 5. Mixed invasions and superinvasions of malaria mosquitos with dirofilariae are frequent phenomena for Tomsk Region. The article contains 4 Figures, 3 Tables, 55 References. The Authors declare no conflict of interest.

PCR-RFLP analysis of rbcL chloroplast gene

To identify intergeneric, interspecific, and intraspecific polymorphisms, PCR-RFLP studies of the chloroplast rbcL gene profiles were carried out using five restriction endonucleases (AluI, HpaII, BsuRI, BstHHI, BstMBI) in 14 species belonging to 6 different genera and 7 Thuja occidentalis cultivars. For all the genera studied (Pinus sp., Abies sp., Picea sp., Microbiota sp., Syringa sp., Thuja sp.), A genus-specific restriction profile characterized by a unique combination of restriction DNA fragments was revealed. The combination of enzymes used in the study did not reveal the interspecies and intraspecific variability of the rbcL gene. Species belonging to the same genus and all varieties of western thuja were characterized by identical PCR-RFLP profiles. It is assumed that further research, with the inclusion of a larger number of species and enzymes, will reveal polymorphism at the interspecies and intraspecific levels.

Prevalence and genotype of Toxoplasma gondii in stranded Hawaiian cetaceans.

Toxoplasma gondii is a significant threat to endangered Hawaiian wildlife including birds and marine mammals. To estimate the prevalence of T. gondii in stranded cetaceans from 1997 to 2021 in Hawai'i, we tested tissues from 37 stranded spinner dolphins Stenella longirostris and 51 stranded individuals that represented 18 other cetacean species. DNA from cetacean tissue extracts were screened using a nested polymerase chain reaction (PCR) assay targeting the Toxoplasmatinae internal transcribed spacer 1 of the nuclear ribosomal DNA. A positive result was obtained in 9 tissues examined for each of 2 spinner dolphins out of 525 tissue samples analyzed by PCR. The PCR-positive spinner dolphins had disseminated acute toxoplasmosis with necrosis, inflammation, and intralesional protozoal cysts and tachyzoites in multiple organs. Discrete positive immunostaining for T. gondii was observed in all tissues tested including the adrenal gland, brain, liver, and lung. Both positive spinner dolphins were negative for cetacean morbillivirus. The T. gondii genotyping was performed by restriction fragment length polymorphism (PCR-RFLP) based on 10 genetic markers. The PCR-RFLP analysis revealed the T. gondii belonged to PCR-RFLP-ToxoDB genotype #24, previously detected in wild pig Sus scrofa in O'ahu, bobcats Lynx rufus from Mississippi, USA, and chickens Gallus gallus from Costa Rica and Brazil. These cases represent the first report of this genotype in aquatic mammals and the second and third reports of fatal disseminated T. gondii infection in stranded spinner dolphins from Hawai'i. Nearshore species, like spinner dolphins, may be at increased risk of mortality from this parasite in marine coastal waterways via sewage systems, storm water drainage, and freshwater runoff.

MARKER-ASSISTED SELECTION FOR GENOTYPING HERDS OF HYBRID GILTS BASED ON POPULATION-GENETIC VARIABILITY

In the study, the allelic effect of single nucleotide polymorphisms of SNPs was determined to estimate fattening productivity in a herd of hybrid gilts (n=101) (Large White × Landrace) × Maxgro. The studied herd of hybrid gilts was obtained as a result of direct (Large White × Landrace) and reciprocal crossing (Landrace × Large White) with boars of the Maxgro terminal line. We conducted a population analysis of polymorphisms by melanocortin genes 4 MC4R (c.1426 A>G), cathepsin D CTSD (g.70 G>A), and ryanodine receptor 1 RYR1 (g.1843 C>T) using software GenAlEX6. To genotyping a herd of hybrid gilts, 2 experimental groups were selected: control (uncastrated) in quantity (n=56) and experimental (immunologically castrated) (n=45) grown in conditions of LLC SPE «Globinsky Pig Farm». Laboratory tests are carried out in the laboratory of genetics in the Institute of Pig Breeding and Agro-Industrial Production of the NAAS of Ukraine. Due to the fact that information about polymorphism on the above SNPs in the population uncastrated and immunologically castrated herd of hybrid pigs which are breed in Ukraine is absent, this requires breeding work involving marker-associated selection on the inside of the breed base, to find out which alleles will be determined as desirable in the marker selection of hybrid pigs. Marker breeding is an effective tool in improving fattening qualities for the pig industry - modern commercial lines of pigs. Therefore, we decided to carry out the typing of an experimental herd of hybrid gilts. The results of genotyping will provide useful information for the selection of the commodity population of hybrid pigs for fixing the desired useful signs of the genotype. Polymorphism of the SNPs studied MC4R (c.1426 A>G), CTSD (g.70 G>A), and RYR1 (g.1843 C>T) is determined by the method of PCR-RFLP analysis. Thus, a breed character was established distribution of frequencies of encounter ability of alleles of investigated SNPs.

Karyotype and plasmotype characteristics in wheat-rye hybrids with the different structural organization of the nuclear genome

Aim. Genome structure analysis and plasmotype identification in wheat-rye hybrids of various types (triti- cale, secalotriticum) and ploidy level. Mcth0ds. Cytological and molecular-genetic analysis. Rcsults. The karyotype and plasmotype analysis was carried out in 11 stable lines of secondary recombinant hexaploid triticale with the introgression of D-genome chromosomes of the wheat (A/B/DRR, 2n = 6x = 42), 14 stable and highly productive secalotriticum lines of F6–16 generations (Secalotriticum, S/RRAABB, 2n = 6x = 42), 9 stable lines of tetraploid triticale (A/BRR, 2n = 4x = 28). By means of differential chromosome staining, the chromosomal composition of the experimental material was characterized and the intergenomic substitution and translocation of chromosomes were detected. The PCR-RFLP analysis of the 18S/5S mitochondrial (mt) repeat and the ndhH-region of chloroplast DNA showed that these organ- elle DNA regions are in the homoplasmic state and belong to rye-type cytoplasm in secalotriticum lines and wheat-type cytoplasm in tetraploid and secondary recombinant hexaploid triticale lines. C0nclusi0ns. Cytological and molecular genetic analysis revealed significant genetic diversity of the created gene pool of wheat-rye hybrids by nuclear-cytoplasmic structure. The synthesized linear material of wheat-rye hybrids may be used in cytogenetic research and practical breeding.

Lactoferrin gene polymorphism of exons 8 and 13 in Murrah buffalo

Lactoferrin is one of the important candidate genes for mastitis resistance in dairy animals. The gene is located on chromosome BBU21 and consists of 17 exons spanning over 32.95 kb of genomic DNA. The present study was undertaken with the objectives to identify allelic variants in exon 8 and 13 of lactoferrin gene and to analyze their association with incidence of clinical mastitis in Murrah buffalo. A total of 200 lactating Murrah buffaloes, grouped as mastitis affected and non-affected, were included in the study. Genomic DNA was isolated from the whole blood sample of each animal. Two primer sets were used to amplify exon 8 and 13 of lactoferrin gene, which yielded respective amplicons of 216 bp and 211 bp. The PCR-RFLP analysis of lactoferrin gene revealed monomorphic pattern in exon 8 and polymorphic pattern in exon 13. Hpy 188I -RFLP for exon 13 exhibited polymorphism with three genotypes: AA, AB and BB with respective frequencies of 0.20, 0.58 and 0.22 whereas, frequencies for A and B alleles were estimated as 0.49 and 0.51. Comparison of nucleotide sequence of exonic region of lactoferrin gene in Murrah buffalo with that of Bos taurus cattle revealed a total of 5 mutations out of which 2 were transition and 3 were transversion. The SNPs in exon 8 were found to be non synonymous and revealed two amino acid changes in exon 8 of Murrah buffalo as compared to Bos taurus cattle. Chi-square (χ2) analysis indicated non significant association between genetic variants of exon 13 and incidence of clinical mastitis.

Rhizobium sophorae is the dominant rhizobial symbiont of Vicia faba L. In North China

Faba bean (Vicia faba L.) is a major introduced grain-legume crop cultivated in China. In this study, rhizobia that nodulated faba bean grown in soils from three sites in North China (Hebei Province) were isolated and characterized. Firstly, isolates were categorized into genotypes by ribosomal IGS PCR-RFLP analysis, then representatives of the different IGS genotypes were further identified by phylogenetic analyses of 16S rRNA, housekeeping (atpD, recA) and nodulation (nodC) gene sequences. Rhizobial distribution based on the IGS genotype was related to the different soil physicochemical features by redundancy analysis. IGS typing and phylogenetic analyses of 16S rRNA and concatenated housekeeping gene sequences affiliated the 103 rhizobial strains isolated into four Rhizobium species/genospecies. A total of 69 strains of 3 IGS types were assigned to R. sophorae, 20 isolates of 5 IGS types to R. changzhiense and 9 isolates of 3 IGS types to R. indicum. The representative strain of the five remaining isolates (1 IGS type) was clearly separated from all Rhizobium type strains and was most closely related to defined genospecies according to the recently described R. leguminosarum species complex. Rhizobium sophorae strains (67% of total isolates) were common in all sites and shared an identical nodC sequence typical of faba bean symbionts belonging to symbiovar viciae. In this first study of rhizobia nodulating faba bean in Hebei Province, China, R. sophorae was found to be the dominant symbiont in contrast to other countries.

Genetic Diversity and Distribution of Vitamin D Receptor (VDR) Genotypes in Breast Cancer Cases from Pakistan

Background: Breast carcinoma is one of the most commonly diagnosed invasive malignancies in females. In Pakistan, it is more commonly detected in women at a young age as compared to the West. Among all women, the risk of developing breast cancer is equal irrespective of their ethnic or racial basis. The aim of the study was to determine vitamin D receptor gene polymorphisms (FokI and TaqI) and allele frequency distribution in Pakistani women with newly diagnosed breast cancer. This study also aimed to find and compare genetic diversity of VDR polymorphisms among breast cancer cases in different population groups. Methods: Newly diagnosed women having breast cancer (n=300) were selected for the study. Blood samples of all the participants were analyzed for vitamin D levels and isolated DNA was subjected to PCR-RFLP analysis. Results: Results revealed that allelic frequency of FokI and TaqI was ‘F’; ‘f’ 50.67 and 49.33% and ‘T’ and ‘t’ was 46.67 and 53.33 respectively in Pakistani women with breast cancer. The genotypic frequency is significantly (P<0.05) distributed.Conclusion: The current study concluded significant difference in genotypes and allele frequency of VDR gene polymorphism in Pakistani population suffering from breast cancer when compared with other population.

Interaction between rs6446482 polymorphisms in the WFS1 gene in type 2 diabetes patients

BackgroundType 2 diabetes mellitus (T2DM) is a chronic metabolic condition characterized by high blood glucose levels. Mutations in the WFS1 gene cause β-cell death, leading to Wolfram syndrome and diabetes. There have been few studies that have been linked to T2DM and the rs6446282 polymorphism in the WFS1 gene, and there are no studies from Saudi Arabia. As a result, the purpose of this study was to investigate the association between the rs6446482 polymorphism in the WFS1 gene and T2DM in the Saudi population. MethodsOne hundred T2DM patients and 100 healthy controls were recruited based on inclusion and exclusion criteria. Genomic DNA was obtained from collected blood, and using oligonucleotides, PCR was performed followed by RFLP analysis. Statistical analysis was performed with SPSS software to compare the numerical and categorical data. ResultsThis case-control study compared T2DM patients to healthy controls. Age, weight, BMI, FBG, HDL-c, TC, TGs, and family history (p < 0.05). The effect of the rs6446482 polymorphism was performed with PCR-RFLP analysis, and the study results confirmed the positive association with HWE (p = 0.01), genotypes (GG vs. CC: OR-3.29 [95% CI: 1.33–8.13]; p = 0.007), dominance (CC vs. CG + GG: OR-2.12 [95% CI: 1.24–3.92]; p = 0.006), and allele frequencies (G vs. C: OR-2.15 [95% CI: 1.37–3.38]; p = 0.0007), revealing a positive association. ANOVA results showed a negative association between the rs6446482 polymorphism and T2DM risk parameters such as age, weight, height, BMI and FBG. ConclusionThe results of this study confirmed that the rs6446482 polymorphism is associated with T2DM in the Saudi population. This study recommends updating the meta-analysis studies in T2DM with the rs6446482 polymorphism and should also be performed in different modes of diabetes.

Species identification of ruminant milk by genotyping of the κ-casein gene

The development of molecular genetic and bioinformatic systems for identifying the species of milk and the raw material composition of dairy products is of great scientific and practical importance with the purpose of introducing developments in the system for controlling the turnover of falsified products. The aim of the research is to develop a method of PCR-RFLP analysis for species identification of milk and dairy products from agricultural ruminant animals by the κ-casein gene (CSN3) with the possibility of qualitative and relative quantitative assessment of species-specific DNA of the tested biomaterial. The objects of research were samples of raw milk and milk powder, pasteurized cream, and hard and semi-hard cheeses. The developed method of species identification of milk and dairy products includes sample preparation of the studied samples, nucleic acid extraction, combined PCR-RFLP technique, detection of obtained results by the method of horizontal electrophoresis in agarose gel and their analysis, including using the developed mathematical algorithms and software. The synergistic effect established in combined operation of 2 restriction enzymes ensured their application in a mix with increased performance in an ergonomic way in the context of DNA authentication of cow, goat, and sheep milk and dairy products based on them. The specificity and sensitivity of the proposed method is potentially suitable for implementing the development of a system to control the turnover of falsified and counterfeit goods.

Молекулярно-филогенетическое исследование рода Hypericum L. во флоре Азербайджана

Hypericum is one of the 100 largest flowering plant genera forming the family Hypericaceae Juss., which belongs to the clusioid clade of the Malpighiales. Hypericum is represented in Azerbaijan flora by 19 native species and 1 subspecies belonging to 7 taxonomic sections. The chloroplast DNA of 8 species from the genus was studied by PCR-RFLP analysis. Total genomic DNA was extracted from leaf tissue using the DNeasyPlantMini kit. (Qiagen Inc.; Valencia, CA, USA) following the supplied protocol and quanti field using a Nanodrop (Nanodrop Technologies; Wilmington, DE, USA) spectrophotometer. The article is part of an experimental study that comprises molecular-phylogenetic research of this genus in the flora of Azerbaijan.

UCP2 and CFH Gene Variants with Genetic Susceptibility to Schizophrenia in Turkish Population.

Schizophrenia (Sch) is a complex, multifactorial psychiatric disorder. Growing evidence shows that oxidative damage and immunological dysfunction exist in the Sch physiopathology. In the present study, we aimed to evaluate whether the Uncoupling protein 2 and Complement factor H gene variants play any role in susceptibility to Sch. This study was carried out on 200 individuals (100 Sch patients and 100 healthy controls). Genomic DNA was extracted from blood samples. UCP2-866G /A (rs659366) and CFHY402H variants were analyzed by PCR-RFLP analysis. The UCP2 -866G/A variant G/G genotype and G allele were associated significantly with increased risk of Sch (p=0.001, p=0.001, respectively). The subjects were carrying UCP2 -866G/A variant G/G genotype had 4.377-fold increased risk for Sch. There was no significant difference between the groups for the genotype and allele frequencies of the CFH Y402H variant (p>0.05). The observed genotype counts deviated significantly from those expected in Sch patients according to the HWE for UCP2 -866G/A variant (p=0.001). We present the first results investigating UCP2 -866G/A/ and CFH Y402H variants for susceptibility to Sch in a Turkish population. These results indicate that the UCP2 -866G/A, but not CFH Y402H variant, might play an important role in the development of Sch.

Variation of prolactin and β-Lactoglobulin genes in the Indonesian FH Cattle

Prolactin is a polypeptide hormone, encoded by the prolactin (PRL) gene, synthesized and secreted by anterior pituitary, and affecting milk yield and composition. β-Lactoglobulin (BLG) is the major whey proteinin the milk of ruminants. This study was conducted to identify the PRL and LGB genes polymorphism in the Indonesian FH cattle. A total of 139 individual cattle blood samples from West Java were used to obtain DNA samples through the DNA extraction process. Identification of the PRL and LGB genes was performed using PCR-RFLP method with RsaI (PRL gene) and HaeIII (BLG gene) restriction enzymes. The PRL gene was amplified using forward primer 5’-ccaaatccactgaattatgctt-3’ and reverse primer 5’-acagaaatcacctctctcattca-3’. The BLG gene was amplified using forward primer 5’-tgtgctggacaccgactacaaaaag-3’ and reverse primer 5’-gctcccggtatatgaccaccctct-3’. The PRL and BLG genes in the Indonesia FH cattle were polymorphic based on the PCR-RFLP analysis but the heterozygosity value was low. There were two alleles (G and A) and three genotypes (GG, GA, and AA) identified in the PRL gene of the Indonesian FH cattle with genotype frequencies were 0.914, 0.079, and 0,007 for GG, GA, and AA genotypes respectively. There were two genotypes (CC and CG) identified in the BLG gene with genotype frequencies were 0.91 (CC), and 0.09 (CG). Information about the PRL and BLG genes polymorphism in this study can be considered for further study to analyse its association with milk yield trait.

Population genetic structure of pedunculate oak (Quercus robur L.) in Belarus according to the analysis of chloroplast DNA

Pedunculate oak (Quercus robur L.) is one of the main forest forming species in the Republic of Belarus. Its population genetic structure was formed under the influence of various migration processes. Six chloroplast DNA loci (µdt1, µdt3, µdt4, µcd4, µcd5 and µkk4) were used for the genogeographic study. The material for the analysis was collected in 100 oak forest stands (2325 samples); 18 allelic variants were identified, which are grouped into 17 different combinations (haplotypes). Five of them are widespread (the proportion of occurrence varies from 7 to 48 %, totalling 85 %). The remaining 12 are rare (the proportion of occurrence varies from 1 to 3 %, totalling 15 %). Phylogenetic trees constructed using the nearest neighbour and maximum likelihood methods show the presence of two groups (branches) of haplotypes. One of it comprises 8 variants including 2 dominant haplotypes and the other comprises 9 variants including 3 dominant haplotypes. PCR-RFLP analysis of chloroplast DNA showed that the pedunculate oak in Belarus originates from the Balkan refugium. Haplotype No. 1 (µdt189, µdt3123, µdt4142, µcd494, µcd574, µkk4109) is found almost everywhere in Belarus with the exception of the southwest and northeast, while haplotype No. 8 (µdt189, µdt3121, µdt4142, µcd494, µcd574, µkk4109) is mainly localised in the southwest and northeast. Haplotypes No. 3 (µdt189, µdt3120, µdt4141, µcd494, µcd575, µkk4109) and No. 7 (µdt189, µdt3122, µdt4142, µcd494, µcd574, µkk4109) predominantly found in the west of the country. Haplotype No. 2 (µdt190, µdt3120, µdt4141, µcd495, µcd574, µkk4109) is typical for the southeast.

Relationships of αs1-casein (CSN1S1) and β-casein (CSN2) genotypes and their association with milk quality and coagulation properties in Bulgarian Brown cattle

The aim of the present investigation was to establish the relationships of αs1-casein (CSN1S1) and β-casein (CSN2) genotypes in Bulgarian Brown cattle and to evaluate their association with milk quality and milk coagulation properties. Milk proteins’ polymorphism was found out in 155 tissue samples from cows reared at 4 farms. Innovative protocols and techniques were used for determination of genetic polymorphism of αs1-casein (CSN1S1) and β-casein (CSN2) in Brown cattle. After collection, tissue samples were shipped to the University in Padova, Italy for PCR-RFLP analysis. The analysis of milk composition was done in the lab of the Agriculture Institute – Stara Zagora on Lactoscan ultrasound milk analyzer, whereas coagulation properties of individual milk samples were evaluated on a Computerized Renneting Metter – Polo Trade, Italy. During the study, the following parameters were studied: milk fat and protein contents (%), rennet coagulation time (RCT, min), curd firmness (а30, mm) and curd firming time (k20, min). Heterozygous cows for both studied milk proteins: CSN1S1 and CSN2, had higher milk fat and milk protein percentages, 4.71% fat and 3.45% protein in genotype ВС of CSN1S1 and 5.00% fat and 5.33% protein in genotype АВ of CSN2. The milk of cows from different CSN1S1 and CSN2 genotypes was outlined with moderate curd firmness and good coagulation time. Homozygous cows for both studied milk proteins had higher curd firmness (32.31 mm) for the ВВ genotype of CSN1S1 and 31.29 mm for the АА genotype of CSN2.

Vitamin D receptor gene polymorphism in women with genital endometriosis, type 1 diabetes mellitus, and in the population

AIM: The aim of this study was to analyze the association between the vitamin D receptor gene polymorphism and the risk of developing genital endometriosis and type 1 diabetes mellitus.&#x0D; MATERIALS AND METHODS: The frequency of allelic variants of the VDR gene was studied by PCR-RFLP analysis in 282 women, including 129 patients with genital endometriosis (stages IIV), 71 patients with type 1 diabetes mellitus, and 82 women of the control group represented by the population sample.&#x0D; RESULTS: It was found that the frequency of the allele G polymorphic variant of rs1544410 (BsmI) in the VDR gene was significantly higher in the group of patients with genital endometriosis compared to the population sample (p = 0.048). Significant differences for the G / G genotype in patients with genital endometriosis relative to the control group (p 0.05) and the group of patients with type 1 diabetes mellitus (p 0.05) were revealed. According to the odds ratio, the risk of developing genital endometriosis was 1.9 times higher for this genotype (OR = 1.93 CI = 1.0823.450; OR = 1.892 CI = 1.0223.430). The combination of the A / A and G / A genotypes was significantly more common in patients with type 1 diabetes mellitus (p = 0.040) and in the population (p = 0.025), when compared to the patients with genital endometriosis. A significant increase in the t allele of the rs731236 polymorphism (TaqI) of the VDR gene was found in the group of patients with type 1 diabetes mellitus (p 0.05). The combination of the T / t and t / t genotypes of the rs731236 polymorphism (TaqI) of the VDR gene in patients with type 1 diabetes mellitus were significantly more common than in the group of patients with genital endometriosis (p = 0.017).&#x0D; CONCLUSIONS: The data obtained may be important for risk assessment of genital endometriosis and type 1 diabetes mellitus development and for developing new strategies for the prevention and treatment of these diseases.

&lt;i&gt;NAT2&lt;/i&gt; and &lt;i&gt;CYP1B1&lt;/i&gt; genetic polymorphisms in patients with genital endometriosis depending on tolerability of melatonin

BACKGROUND: Genital endometriosis is one of the most pressing problems of modern gynecology. Melatonin is a promising drug with a potentially curative effect on endometriosis.&#x0D; AIM: The aim of this study was to conduct a comparative analysis of the genetic polymorphism of some genes encoding enzymes involved in melatonin metabolism.&#x0D; MATERIALS AND METHODS: The genetic polymorphism in the NAT2 and CYP1B1 genes encoding enzymes involved in melatonin metabolism in patients with different tolerance to this drug was analyzed by PCR-RFLP analysis.&#x0D; RESULTS: In patients with genital endometriosis, the presence of a wild-type allele (N) of the NAT2 gene was associated with poor tolerance of melatonin. The NAT2 (N / N) rapid acetylator phenotype combined with the low catalytic activity of CYP1B1 (C / C) occurred more frequently in endometriosis patients having poor melatonin tolerability compared to the group of patients who tolerated the therapy well.&#x0D; CONCLUSIONS: For patients with genital endometriosis with the wild-type (N) allele of the NAT2 gene, melatonin administration is inappropriate due to numerous side effects during the drug use.