Nucleotide Sequence Variation Research Articles

Copy number variation and elevated genetic diversity at immune trait loci in Atlantic and Pacific herring

BackgroundGenome-wide comparisons of populations are widely used to explore the patterns of nucleotide diversity and sequence divergence to provide knowledge on how natural selection and genetic drift affect the genome. In this study we have compared whole-genome sequencing data from Atlantic and Pacific herring, two sister species that diverged about 2 million years ago, to explore the pattern of genetic differentiation between the two species.ResultsThe genome comparison of the two species revealed high genome-wide differentiation but with islands of remarkably low genetic differentiation, as measured by an FST analysis. However, the low FST observed in these islands is not caused by low interspecies sequence divergence (dxy) but rather by exceptionally high estimated intraspecies nucleotide diversity (π). These regions of low differentiation and elevated nucleotide diversity, termed high-diversity regions in this study, are not enriched for repeats but are highly enriched for immune-related genes. This enrichment includes genes from both the adaptive immune system, such as immunoglobulin, T-cell receptor and major histocompatibility complex genes, as well as a substantial number of genes with a role in the innate immune system, e.g. novel immune-type receptor, tripartite motif and tumor necrosis factor receptor genes. Analysis of long-read based assemblies from two Atlantic herring individuals revealed extensive copy number variation in these genomic regions, indicating that the elevated intraspecies nucleotide diversities were partially due to the cross-mapping of short reads.ConclusionsThis study demonstrates that copy number variation is a characteristic feature of immune trait loci in herring. Another important implication is that these loci are blind spots in classical genome-wide screens for genetic differentiation using short-read data, not only in herring, likely also in other species harboring qualitatively similar variation at immune trait loci. These loci stood out in this study because of the relatively high genome-wide baseline for FST values between Atlantic and Pacific herring.

Prognostic significance of <I>TLR3</I> and <I>TLR9</I> gene polymorphism in assessing the severity of COVID-19

Objective: to study polymorphic variants of TLR3 (rs3775291) and TLR9 (rs352140) genes in patients with COVID-19 depending on the severity of the disease and their significance for determining the risk of severe course of COVID-19.Materials and Methods: genetic analysis of polymorphism of TLR3 (rs3775291) and TLR9 genes (rs352140) was performed in 164 patients with COVID-19, who were divided by degree of severity into three groups: Group 1 with mild, Group 2 with moderate and Group 3 with severe and extremely severe degrees of severity. The control group consisted of 40 healthy donors. Statistical processing of the obtained results was carried out using STATISTICA 12.0 programs (Stat Soft, USA). To assess the difference of groups by qualitative characteristics, the χ2 criterion with Yates correction was used, and in case of violation of its conditions, the χ2 criterion was applied. Yates correction, and if the conditions of its applicability were violated, the two-sided Fisher’s two-sided criterion (RF). The difference between the groups was considered statistically significant at р<0,05. The strength of associations was evaluated in the values of the odds ratio index odds ratio (OR) and 95% confidence interval.> ˂ 0,05. The strength of associations was evaluated in the values of the odds ratio index odds ratio (OR) and 95% confidence interval.Results: the frequency of the ST genotype was significantly higher and the frequency of the TT genotype of the rs3775291 allele of the TLR3 gene was significantly lower in patients with the manifest form of COVID-19 compared with the control group. Frequency analysis of TLR9 (rs352140) in COVID-19 patients showed significantly higher values of the ST genotype and lower values of the CC genotype compared to healthy individuals. Comparative analysis between groups of hospitalized patients depending on severity revealed higher frequency of ST genotype and lower frequency of TT genotype of TLR3 (rs3775291) and TLR9 (rs352140) genes in patients with severe and extremely severe condition.Conclusion: reliable differences in the frequency of occurrence of genotypes of TLR3 (rs3775291) and TLR9 (rs352140) genes in patients with the manifest form of COVID-19 compared to healthy people were revealed. The analysis of nucleotide sequence variants of the studied genes in hospitalized patients depending on the degree of severity also showed significant differences in the frequency of genotypes. Thus, in patients with severe and extremely severe COVID-19, a significant difference in the frequency of occurrence of ST and TT genotypes of TLR3 (rs3775291) and TLR9 (rs352140) genes was revealed compared to patients with mild and moderate COVID-19, which may further have prognostic value in assessing the severity of the disease.

Assessment of the association of the rs12329760 polymorphism of the <i>TMPRSS2</i> gene with acute coronary syndrome in patients with new coronavirus infection

Introduction. Cardiovascular diseases (СVD) rank first in terms of morbidity and mortality not only in the Russian Federation but throughout the world. Since the end of 2019 a pandemic of Severe Acute Respiratory Syndrome (SARS) or new coronavirus infection (NCI) has spread throughout the world, which contributed to the development and course of CVD. Therefore, genetic markers began to be considered as factors potentially influencing CVD, its development and severity. Objective: To evaluate the association of the nucleotide sequence variant rs12329760 of the TMPRSS2 gene with acute coronary syndrome in patients who have had a new coronavirus infection.Material and methods. The study included 100 patients (women – 50, men – 50) with ACS and previous NCI, who were selected on the basis of a positive PCR test for the presence of SARS-CoV-2 in the anamnesis, hospitalized at the regional vascular center No. 7 of the City Clinical Hospital No. 2 of the city of Novosibirsk. Women age was 59.5 ± 7.2 years, men age was 53.5 ± 9.3 years. The diagnosis of ACS was established according to a set of criteria proposed and established by the Russian Society of Cardiology and in accordance with the updated clinical recommendations of the Ministry of Health of the Russian Federation of 2020. Patients underwent clinical and instrumental examination, coronary angiography with possible stenting, as provided for in the standards of medical care and clinical guidelines. The nucleotide sequence variant rs12329760 of the TMPSS2 gene was determined in patients using PCR with further analysis of restriction fragment length polymorphism. The comparison group consisted of 200 patients with ACS without previous NCI (no positive PCR test, no positive antibodies).Results. Carriage of CC, СТ, TT, genotype variants of the nucleotide sequence rs12329760 of the TMPSS2 gene are not associated with an increased risk of developing ACS in patients with previous NCI. When comparing the frequencies of the rs12329760 genotypes of the TMPSS2 gene in groups with ACS with NCI and ACS without NCI, no statistically significant differences were obtained. In the group with ACS with NCI, carriage of the homozygous genotype was more common (p = 0.011).Conclusions. Variants of the nucleotide sequence rs12329760 of the TMPSS2 gene are  not associated with  ACS with previous  NCI.

Exploring genetic polymorphisms and transcript levels of antioxidant and metabolic markers for prediction and monitoring diarrhea in Holstein dairy calves

ABSTRACT The objective of this study was to investigate the genetic polymorphisms and transcript levels of antioxidant and metabolic markers for diarrhea monitoring in Holstein dairy calves. For DNA and RNA extraction, blood samples were taken from 100 female calves (50 with diarrhea and 50 appearing healthy). DNA sequencing and real time PCR for and metabolic genes were performed to identify genetic variants linked to diarrhea susceptibility. Results revealed that there were nucleotide sequence variations in the investigated genes between healthy and diarrheal calves. Keap1, HMOX1, CMPK2, ASPG, FPGT, TNNI3 K, and LPCAT1 were considerably more highly expressed in diarrheic calves than resistant ones. The Nrf2, PRDX2, and PRDX6 genes, however, produced a different pattern. This study provides a practical control strategy to lessen newborn diarrhea in Holstein dairy calves and emphasizes the significance of nucleotide variations along with antioxidant and metabolic gene expression patterns as proxy markers of diarrhea susceptibility/resistance.

Exploring COVID-19 causal genes through disease-specific Cis-eQTLs

Genome-wide association study (GWAS) analysis has exposed that genetic factors play important roles in COVID-19. Whereas a deeper understanding of the underlying mechanism of COVID-19 was hindered by the lack of expression of quantitative trait loci (eQTL) data specific for disease. To this end, we identified COVID-19-specific cis-eQTLs by integrating nucleotide sequence variations and RNA-Seq data from COVID-19 samples. These identified eQTLs have different regulatory effect on genes between patients and controls, indicating that SARS-CoV-2 infection may cause alterations in the human body's internal environment. Individuals with the TT genotype in the rs1128320 region seemed more susceptible to SARS-CoV-2 infection and developed into severe COVID-19 due to the abnormal expression of IFITM1. We subsequently discovered potential causal genes, of the result, a total of 48 genes from six tissues were identified. siRNA-mediated depletion assays in SARS-CoV-2 infection proved that 14 causal genes were directly associated with SARS-CoV-2 infection. These results enriched existing research on COVID-19 causal genes and provided a new sight in the mechanism exploration for COVID-19.

Plant virus diversity in bee and pollen samples from apple (Malus domestica) and sweet cherry (Prunus avium) agroecosystems.

Honey bee (Apis mellifera) pollination is widely used in tree fruit production systems to improve fruit set and yield. Many plant viruses can be associated with pollen or transmitted through pollination, and can be detected through bee pollination activities. Honey bees visit multiple plants and flowers in one foraging trip, essentially sampling small amounts of pollen from a wide area. Here we report metagenomics-based area-wide monitoring of plant viruses in cherry (Prunus avium) and apple (Malus domestica) orchards in Creston Valley, British Columbia, Canada, through bee-mediated pollen sampling. Plant viruses were identified in total RNA extracted from bee and pollen samples, and compared with profiles from double stranded RNA extracted from leaf and flower tissues. CVA, PDV, PNRSV, and PVF coat protein nucleotide sequences were aligned and compared for phylogenetic analysis. A wide array of plant viruses were identified in both systems, with cherry virus A (CVA), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and prunus virus F (PVF) most commonly detected. Citrus concave gum associated virus and apple stem grooving virus were only identified in samples collected during apple bloom, demonstrating changing viral profiles from the same site over time. Different profiles of viruses were identified in bee and pollen samples compared to leaf and flower samples reflective of pollen transmission affinity of individual viruses. Phylogenetic and pairwise analysis of the coat protein regions of the four most commonly detected viruses showed unique patterns of nucleotide sequence diversity, which could have implications in their evolution and management approaches. Coat protein sequences of CVA and PVF were broadly diverse with multiple distinct phylogroups identified, while PNRSV and PDV were more conserved. The pollen virome in fruit production systems is incredibly diverse, with CVA, PDV, PNRSV, and PVF widely prevalent in this region. Bee-mediated monitoring in agricultural systems is a powerful approach to study viral diversity and can be used to guide more targeted management approaches.

Validation of genetic risk scores for hypertension in the Central Russian population

Aim. To validate and evaluate the accuracy of 4 genetic risk scores (GRSs) for hypertension (HTN), previously created on European samples, on a population sample of the Ivanovo Oblast.Material and methods. For genetic analysis, targeted next-generation sequencing was used on a sample of the Central Russia (n=1682) based on the biobank collection. Four GRSs associated with HTN, previously developed for the European population, were selected for validation. The coefficient of determination and the area under the ROC curve were used as quality metrics for regression models. Additional validation was carried out to include all nucleotide sequence variants, regardless of linkage disequilibrium level. A combined GRS was compiled based on coefficients from individual GRSs using the clumping + thresholding (C+T) method.Results. The study demonstrated that the predictive value of previously developed GRSs when used for Central Russian population is lower than in the original studies. The proportion of explained variance was 0,5-0,8%. The best predictive ability (proportion of explained variance — 2,5%) was demonstrated using previously developed GRSs (Evangelou E, et al., 2018), which includes the largest number of nucleotide sequence variants (n=852).Conclusion. GRSs for HTN, developed on European samples, is not recommended for Russian population without preliminary validation. To create original GRSs, combining statistical parameters (β-coefficients and p-value) from different GRS is not recommended.

Differential amplification of the subtelomeric satellite DNA JcSAT1 in the genus Jatropha L. (Euphorbiaceae).

Satellite DNAs (satDNAs) are highly repetitive sequences that occur in virtually all eukaryotic genomes and can undergo rapid copy number and nucleotide sequence variation among relatives. After chromosomal mapping of the satDNA JcSAT1, it was found a large accumulation at subtelomeres of Jatropha curcas (subgenus Curcas), but an absence of these monomers in J. integerrima (subgenus Jatropha). This fact suggests a dynamic scenario for this satellite repeat in Jatropha genomes. Here, we used a multitasking approach (sequence analysis, DNA blotting and chromosomal mapping) to investigate the molecular organization and chromosomal abundance and distribution of JcSAT1 in a broader group of species from the subgenus Jatropha (J. gossypiifolia, J. mollissima, J. podagrica, and J. multifida) in addition to J. curcas, with the aiming of understanding the evolution of this satDNA. Based on the analysis of BAC clone sequences of J. curcas, a large array (~ 30kb) of 80 homogeneous monomers of JcSAT1 was identified in BAC 23J11. The monomer size was conserved (~ 358bp) and contained a telomeric motif at the 5' end. PCR amplification coupled with a Southern blot revealed the presence of JcSAT1-like sequences in all species examined. However, a large set of genome copies was identified only in J. curcas, where a ladder-like pattern with multimers of different sizes was observed. In situ hybridization of BAC 23J11 confirmed the subtelomeric pattern for J. curcas, but showed no signals on chromosomes of species from the subgenus Jatropha. Our data indicate that JcSAT1 is a highly homogeneous satDNA that originated from a region near the telomeres and spread throughout the chromosomal subtermini, possibly due to frequent ectopic recombination between these regions. The abundance of JcSAT1 in the genome of J. curcas suggests that an amplification event occurred either at the base of the subgenus Curcas or at least in this species, although the repeat is shared by all species of the genus studied so far.

Genetic Status of Exotic Chinese Carp Hypophthalmichthys molitrix and Hypophthalmichthys nobilis through Mitochondrial Gene Cytochrome C Oxidase Subunit I (COI) in Bangladesh

The use of mitochondrial gene Cytochrome C Oxidase subunit I (mtCOI) has emerged as a promising technique in study of genetic variation and phylogenetic relationship of different group of fishes. This work reports the first example of the study of genetic status of two exotic Chinese carp, silver carp, Hypophthalmichthys molitrix and bighead carp, Hypophthalmichthys nobilis in Bangladesh through the analyses of mtCOI gene. The highest nucleotide sequence divergence occurred between sequenced H. nobilis N3 and reference H. molitrix from India which was 1.208 and the lowest nucleotide divergence occurred in sequenced H. nobilis N1 and sequenced H. molitrix M3 with their reference counterparts from Bangladesh, India and China (0.002). The phylogenetic tree analysis showed that one of the H. nobilis (H. nobilis N3) formed a sister group with H. molitrix M2 with 100% sequence similarities. Otherwise, all sequenced and reference H. molitrix and H. nobilis showed separate clade in the tree. Therefore, we can conclude that the exotic H. molitrix and H. nobilis maintain distinct population status with lower genetic variation. However, we should take essential measures to reduce inbreeding in future.

Dual RNA-sequencing of Fusarium head blight resistance in winter wheat.

Fusarium head blight (FHB) is a devastating fungal disease responsible for significant yield losses in wheat and other cereal crops across the globe. FHB infection of wheat spikes results in grain contamination with mycotoxins, reducing both grain quality and yield. Breeding strategies have resulted in the production of FHB-resistant cultivars, however, the underlying molecular mechanisms of resistance in the majority of these cultivars are still poorly understood. To improve our understanding of FHB-resistance, we performed a transcriptomic analysis of FHB-resistant AC Emerson, FHB-moderately resistant AC Morley, and FHB-susceptible CDC Falcon in response to Fusarium graminearum. Wheat spikelets located directly below the point of inoculation were collected at 7-days post inoculation (dpi), where dual RNA-sequencing was performed to explore differential expression patterns between wheat cultivars in addition to the challenging pathogen. Differential expression analysis revealed distinct defense responses within FHB-resistant cultivars including the enrichment of physical defense through the lignin biosynthesis pathway, and DON detoxification through the activity of UDP-glycosyltransferases. Nucleotide sequence variants were also identified broadly between these cultivars with several variants being identified within differentially expressed putative defense genes. Further, F. graminearum demonstrated differential expression of mycotoxin biosynthesis pathways during infection, leading to the identification of putative pathogenicity factors.

<i>DEPDC5</i>-related familial focal epilepsy

Focal epilepsy is the most common type of epilepsy accounting for 60–70% of all cases of this pathology. We present two familial cases of focal epilepsy associated with a nucleotide sequence variant in DEPDC5 gene. Clinical and ancestry examination was performed by using instrumental (magnetic resonance imaging, video-electroencephalography) and genetic testing methods. The nucleotide sequence variants in DEPDC5 gene were found in two probands and paired fathers with epilepsy. Focal cortical dysplasia was detected only in the father of Proband 1 as well as Proband 2 with resistant epilepsy and severe cognitive deficit. Hence, such clinical cases confirm that pathogenic variants in DEPDC5 gene are related with familial focal epilepsy, which clinical manifestation may depend on the type of identified mutation. The study of genotype-phenotype correlations is necessary to apply proper therapy. Before surgical treatment of epilepsy, the genetic testing by whole exome or whole genome sequencing should be performed.

Genomic Analysis of Sindbis Virus Reveals Uncharacterized Diversity within the Australasian Region, and Support for Revised SINV Taxonomy.

Sindbis virus (SINV) is a widely dispersed mosquito-borne alphavirus. Reports of Sindbis disease are largely restricted to northern Europe and South Africa. SINV is frequently sampled in Australian mosquito-based arbovirus surveillance programs, but human disease has rarely been reported. Molecular epidemiological studies have characterized six SINV genotypes (G1-G6) based on E2 gene phylogenies, mostly comprising viruses derived from the African-European zoogeographical region and with limited representation of Australasian SINV. In this study, we conducted whole genome sequencing of 66 SINV isolates sampled between 1960 and 2014 from countries of the Australasian region: Australia, Malaysia, and Papua New Guinea. G2 viruses were the most frequently and widely sampled, with three distinct sub-lineages defined. No new G6 SINV were identified, confirming geographic restriction of these viruses to south-western Australia. Comparison with global SINV characterized large-scale nucleotide and amino acid sequence divergence between African-European G1 viruses and viruses that circulate in Australasia (G2 and G3) of up to 26.83% and 14.55%, respectively, divergence that is sufficient for G2/G3 species demarcation. We propose G2 and G3 are collectively a single distinct alphavirus species that we name Argyle virus, supported by the inapparent or mild disease phenotype and the higher evolutionary rate compared with G1. Similarly, we propose G6, with 24.7% and 12.61% nucleotide and amino acid sequence divergence, is a distinct alphavirus species that we name Thomson's Lake virus.

Individual and population diversity of 20 representative olfactory receptor genes in pigs

Understanding the influence of genetic variations in olfactory receptor (OR) genes on the olfaction-influenced phenotypes such as behaviors, reproduction, and feeding is important in animal biology. However, our understanding of the complexity of the OR subgenome is limited. In this study, we analyzed 1120 typing results of 20 representative OR genes belonging to 13 OR families on 14 pig chromosomes from 56 individuals belonging to seven different breeds using a sequence-based OR typing method. We showed that the presence of copy number variations, conservation of locus-specific diversity, abundance of breed-specific alleles, presence of a loss-of-function allele, and low-level purifying selection in pig OR genes could be common characteristics of OR genes in mammals. The observed nucleotide sequence diversity of pig ORs was higher than that of dogs. To the best of our knowledge, this is the first report on the individual- or population-level characterization of a large number of OR family genes in livestock species.

Transmission Dynamics of Imported Vaccine-Origin PRRSV-2 within and between Commercial Swine Integrations in Hungary.

This study reports on the molecular epidemiology of Ingelvac-PRRS-MLV-associated cases in Hungary for the period 2020-2021. Field epidemiology investigations led the experts to conclude that imported pigs, which were shipped through transit stations in Denmark, introduced the vaccine virus. The movement of fatteners and the neglect of disease control measures contributed to the spread of the virus to PRRS-free pig holdings in the vicinity. Deep sequencing was performed to genetically characterize the genes coding for the virion antigens (i.e., ORF2 through ORF7). The study isolates exhibited a range of 0.1 to 1.8% nucleotide sequence divergence from the Ingelvac PRRS MLV and identified numerous polymorphic sites (up to 57 sites) along the amplified 3.2 kilo base pair genomic region. Our findings confirm that some PRRSV-2 vaccine strains can accumulate very high number of point mutations within a short period in immunologically naive pig herds.

Molecular Characterization of Lyssaviruses Originating from Domestic and Wild Cats Provides an Insight on the Diversity of Lyssaviruses and a Risk of Rabies Transmission to Other Susceptible Mammals and Humans in South Africa.

Rabies is one of the most significant public and veterinary health problems, causing approximately 59,000 human deaths annually in the developing countries of Asia and Africa. The aetiologic agent, a viral species of the Lyssavirus genus, is highly neurotropic and has a wide host range, including terrestrial mammals and several Chiropteran species. The Lyssavirus mokola (MOKV) was first isolated in the late 1960s from organ pools of shrews (Crocidura flavescens manni) in the Mokola forest (Nigeria). To date, at least 30 MOKV isolations have been confirmed, all exclusively from Africa, with 73% from southern Africa. There is limited knowledge about the epidemiology of MOKV, and the reservoir host species is unknown. Here, we report on the molecular characterization of rabies viruses originating from both domestic and African wild cats. A partial region of the lyssavirus genome, encoding the nucleoprotein, was amplified and sequenced. Nucleotide sequence analysis demonstrated that 98% of cats were infected with both the canid and mongoose rabies virus variants, as well as a rare lyssavirus, Lyssavirus mokola, from a domestic cat from Eswatini. Furthermore, the nucleotide sequence divergence between the recently identified MOKV isolate and the historical Lyssavirus mokola isolates ranged from 6.8% to 8.3%. This study further highlights the association between the potential host species of Lyssavirus mokola and the domestic cat as an incidental host, and the important role cats may play in rabies transmission dynamics in the country. Therefore, continuous vaccination of domestic cats against rabies is crucial, even after the elimination of dog-mediated rabies, as spillover related to sylvatic rabies cycles is likely to occur.

Combining Nucleotide Sequence Variants and Transcript Levels of Immune and Antioxidant Markers for Selection and Improvement of Mastitis Resistance in Dromedary Camels

The immune and antioxidant genetic factors that could converse with mastitis susceptibility in dromedary camels were looked at in this research. Of 120 female dromedary camels (60 healthy, and 60 with mastitis) were utilised. Each camel’s jugular vein was pierced to obtain five millilitres of blood. The blood was placed within tubes containing sodium fluoride or EDTA anticoagulants to obtain whole blood and extract DNA and RNA. The immunological (OTUD3, TLR2, TLR4, STAB2, MBL2, TRAPPC9, and C4A) and antioxidant (CAT, SOD3, PRDX6, OXSR1, NDUFS6, SERP2, and ST1P1) genes’ nucleotide sequence polymorphisms between healthy and mastitis affected she-camels were discovered using PCR-DNA sequencing. Fisher’s exact test revealed that camel groups with and without mastitis had noticeably different odds of all major nucleotide alterations propagating (p < 0.01). Mastitic camels were significantly more likely to express the OTUD3, TLR2, TLR4, STAB2, MBL2, TRAPPC9, C4A, OXSR1, SERP2, and ST1P1 genes (p < 0.05). However, CAT, SOD3, PRDX6, and NDUFS6 genes elicited a different pattern. The results may be used to develop management strategies and support the significance of nucleotide differences and gene expression patterns in these markers as indicators of the incidence of mastitis.

Evaluation of Various Analytical Methods for Quantification of Rabies Antigen

Background: Rabies is a zoonotic viral disease caused by rabies virus. Rabies virus has special inclination towards central nervous system (CNS) where it causes life threatening acute encephalitis. Perhaps being extremely fatal, rabies can be 100% prevented by early, effective and complete post exposure prophylaxis (PEP). PEP for rabies include immediate toileting of bite wound, administration of rabies immunoglobulins (severe exposures), and a full course of anti-rabies vaccination (ARV). As ensuring the absence of any variations in the primary nucleotide sequence of the viral backbone after successive propagation in human cell lines is crucial to ensure that the expression vector does not produce variants of the antigen during production of vaccine, the present study was conducted with an aim to evaluate various analytical methods for quantification of rabies antigen. Method: Various analytical methods the Western Blot technique, the Sodium Dodecyl Sulphate Agarose Gel Electrophoresis(SDS-PAGE), ELLA method, the Enzyme-linked immunosorbent assay (ELISA) and extinction coefficient were used for quantification of rabies antigen. Results: When results of these tests were analyzed, it was observed that these analytical methods are easy, cost effective, accurate and most importantly rapid for quantification of rabies antigen. Conclusion: On the basis of observations noted in this study, Western Blot technique, the Sodium Dodecyl Sulphate Agarose Gel Electrophoresis(SDS-PAGE), ELLA method, the Enzyme-linked immunosorbent assay (ELISA) and extinction coefficient can be reliably recommended as analytical methods for quantification of rabies antigen..

Polymorphism in FAE1gene paralogs of Brassica juncea associated with erucic acid content

Breeding for low erucic acid is a major objective to improve the edible oil quality in Brassica juncea. FAE1, theintron-less, 1521-bp gene encoding fatty acid elongase, genetically control the biosynthesis of erucic acid in oilseed Brassicas. The Brassica juncea being an amphidiploid, contains two paralogs of this gene (FAE1.1 and FAE1.2). Allelic variations in the gene such as SNPs and InDels are predominantly responsible for variability in erucic acid content in the seed, among the Brassica genotypes. The coding sequence of FAE1 gene paralogs were sequenced from low (LEA) and high (HEA) erucic acid genotypes which had a few SNPs that differentiated LAE and HEA genotypes. Two SNPs in FAE1.1 at position 591 and 1265 and one in FAE1.2 at 237, which led to a change in the recognition site of Hpy99I, BglII and MnlI restriction enzymes, respectively, converted into CAPS markers to differentiate LEA and HEA genotypes. The efficacy of these CAPS markers was found 100 per cent when validated in Brassica juncea, B. rapa and B. nigra genotypes and used in back-cross breeding. However, owing to functional complexity of CAPS in routine analysis; nucleotide sequence variability in the promoter region of FAE1 gene paralogs were expedited to develop simple PCR based markers. The upstream regions of FAE were sequenced from LEA cultivar Pusa Mustard30 and HEA cultivar Pusa Bold. A28-bp deletion in the promoter of FAE1.1 and a 340- bp insertion of a transposonlike element in the FAE1.2 gene promoter was discovered in LEA genotype in comparison to HEA. Markers based on these sequence variabilities in the promoter regions of FAE1.1 and FAE1.2 were found to completely co-segregate with the seed erucic acid content. These markers would be effectively used in marker assisted selection for development of low erucic acid cultivars in B. juncea.

Distribution of large lungworms (Nematoda: Dictyocaulidae) in free-roaming populations of red deer Cervus elaphus (L.) with the description of Dictyocaulus skrjabini n. sp.

Lungworms of the genus Dictyocaulus are causative agents of parasitic bronchitis in domestic and wild ungulates. This study investigates the distribution, morphology and genetic diversity of D. cervi and a new lungworm species, Dictyocaulus skrjabini n. sp. infecting red deer Cervus elaphus, fallow deer Dama dama and moose Alces alces in Poland and Sweden. The study was conducted on 167 red deer from Poland and on the DNA of lungworms derived from 7 fallow deer, 4 red deer and 2 moose collected in Sweden. The prevalence of D. cervi and D. skrjabini n. sp. in dissected red deer in Poland was 31.1% and 7.2%, respectively. Moreover, D. skrjabini n. sp. was confirmed molecularly in 7 isolates of fallow deer lungworms and 1 isolate of red deer lungworms from Sweden. Dictyocaulus skrjabini n. sp. was established based on combination of their distinct molecular and morphological features; these included the length of cephalic vesicle, buccal capsule (BC), buccal capsule wall (BCW), distance from anterior extremity to the nerve ring, the width of head, oesophagus, cephalic vesicle, BC and BCW, as well as the dimensions of reproductive organs of male and female. Additionally, molecular analyses revealed 0.9% nucleotide sequence divergence for 1,605 bp SSU rDNA, and 16.5–17.3% nucleotide sequence divergence for 642 bp mitochondrial cytB between D. skrjabini n. sp. and D. cervi, respectively, and 18.7–19% between D. skrjabini n. sp. and D. eckerti, which translates into 18.2–18.7% amino acid sequence divergence between D. skrjabini n. sp. and both lungworms.

GATA4 rs61277615, rs73203482, and rs35813172 in Newborns with Transposition of the Great Arteries.

Congenital heart disease is the most common malformative pathology in newborns, with a worldwide incidence at 0.4-5%. We investigated the possible relationship between variations in nucleotide sequences and specific cardiac malformations in the GATA-binding factor 4 (GATA4) exon 1 region by using Sanger sequencing. Forty-four newborns from a third-level neonatal intensive care unit who were diagnosed with nonsyndromic, ductal-dependent congenital heart disease (i.e., transposition of the great arteries or ductal-dependent coarctation of the aorta) were enrolled. Their DNA was extracted using commercial methods and tested using the multiplex ligation-dependent probe amplification (MLPA) technique. The Sanger sequencing for GATA4 exon 1 in the newborns' DNA identified rs61277615, rs73203482, and rs35813172 variants not reported in the ClinVar archive of human variations in newborns previously diagnosed with transposition of the great arteries (n=5) and coarctation of the aorta (n=1). The identification of these novel variants in newborns with transposition of the great arteries or ductal-dependent coarctation of the aorta may be the first step in determining the variants' contribution to the occurrence of congenital heart disease. However, these results may be inconclusive, since the observed variants within GATA4 gene were not previously reported.