Intronic Polymorphism Research Articles

Is KIBRA polymorphism associated with memory performance and cognitive impairment in Alzheimer's disease?

Genetic variations in a common single nucleotide polymorphism in the ninth intron of the KIBRA gene have been linked to memory performance and risk of Alzheimer's disease (AD). We examined the risk of AD related to presence of KIBRA T allele (versus CC homozygote) and to memory performance. The role of established genetic risk factors APOE ε4 and BDNF Met was also considered. Participants were cognitively healthy individuals (n = 19), participants with amnestic mild cognitive impairment (aMCI) due to AD (n = 99) and AD dementia (n = 37) from the Czech Brain Aging Study. Binary and multinomial logistic regressions compared odds of belonging to a certain diagnostic category and multivariate linear regressions assessed associations with memory. KIBRA T allele was associated with increased AD dementia risk (odds ratio [OR] = 5.98, p = 0.012) compared to KIBRA CC genotype. In APOE ε4 negative individuals, KIBRA T allele was associated with a greater risk of both aMCI due to AD (OR = 6.68, p = 0.038) and AD dementia (OR = 15.75, p = 0.009). In BDNF Met positive individuals, the KIBRA T allele was associated with a greater risk of AD dementia (OR = 10.98, p = 0.050). In AD dementia, the association between KIBRA T allele and better memory performance approached significance (β = 0.42; p = 0.062). The link between possessing the KIBRA T allele and better memory reached statistical significance only among BDNF Met carriers (β = 1.21, p = 0.027). Findings suggest that KIBRA T allele may not fully protect against AD dementia but could potentially delay progression of post-diagnosis cognitive deficits.

Chloroplast genome sequencing and divergence analysis of 18 Pyrus species: insights into intron length polymorphisms and evolutionary processes.

Pears constitute an essential temperate crop and are primarily produced through interspecific hybridization owing to self-incompatibility that complicates their breeding history. To address this, we sequenced the complete chloroplast (cp) genomes of 18 Pyrus and one Malus species using the Illumina HiSeq4000 platform. The cp genomes ranged from 159,885 bp to 160,153bp and exhibited a conserved circular DNA structure with an average GC content of 36.5%. Each cp genome contained 127 genes, including 83 protein-coding, 36 tRNA, and 8 rRNA genes. Divergence analysis with mVISTA showed high conservation in the coding regions and notable variations in the non-coding regions. All species shared 17 intron-containing genes, with ycf3 and clpP each having two introns. Five intron-containing genes (ndhB, rpl2, rps12, trnA-UGC, and trnE-UUC) were located in the inverted repeat regions, while trnL-UAA was located in the large single-copy region, with conserved intron lengths across Pomoideae. We identified polymorphic intron sequences in the rpl22, petB, clpP, ndhA, and rps16 genes and designed primers for these regions. Notably, the two Pyrus ussuriensis accessions Doonggeullebae and Cheongdangrori showed intron-length polymorphisms despite being classified as the same species. Phylogenetic analysis of the cp genome sequences revealed two major clusters, indicating distinct maternal lineages and evolutionary origins. This study underscores the importance of cp gene polymorphisms in P. fauriei, P. calleryana, P. ussuriensis, and P. pyrifolia, providing valuable insights into Pyrus evolution as well as aiding in the conservation and breeding of pear germplasm.

Exploring Mitochondrial Heterogeneity and Evolutionary Dynamics in Thelephora ganbajun through Population Genomics.

Limited exploration in fungal mitochondrial genetics has uncovered diverse inheritance modes. The mitochondrial genomes are inherited uniparentally in the majority of sexual eukaryotes, our discovery of persistent mitochondrial heterogeneity within the natural population of the basidiomycete fungus Thelephora ganbajun represents a significant advance in understanding mitochondrial inheritance and evolution in eukaryotes. Here, we present a comprehensive analysis by sequencing and assembling the complete mitogenomes of 40 samples exhibiting diverse cox1 heterogeneity patterns from various geographical origins. Additionally, we identified heterogeneous variants in the nad5 gene, which, similar to cox1, displayed variability across multiple copies. Notably, our study reveals a distinct prevalence of introns and homing endonucleases in these heterogeneous genes. Furthermore, we detected potential instances of horizontal gene transfer involving homing endonucleases. Population genomic analyses underscore regional variations in mitochondrial genome composition among natural samples exhibiting heterogeneity. Thus, polymorphisms in heterogeneous genes, introns, and homing endonucleases significantly influence mitochondrial structure, structural variation, and evolutionary dynamics in this species. This study contributes valuable insights into mitochondrial genome architecture, population dynamics, and the evolutionary implications of mitochondrial heterogeneity in sexual eukaryotes.

An Integrated Strategy for Analyzing the Complete Complex Integrated Structure of Maize MON810 and Identification of an SNP in External Insertion Sequences.

Genetically modified maize (Zea mays L.) MON810 was approved for importation into China for feed use in 2004; however, the localization data concerning exogenous insertion sequences, which confer insect resistance, have been questionable. MON810 maize plants discovered in northeastern China were used to analyze the molecular characteristics of the exogenous insertion. Using PacBio-HiFi sequencing and PCR assays, we found the insertion was located in chromosome 8, and there was a CaMV35S promoter, hsp70 intron, and insecticide gene cry1Ab, except for genome sequence insertion in the MON810 event. Importantly, the 5' and 3' flanking sequences were located in the region of 55869747-55879326 and 68416240-68419152 on chromosome 5, respectively. The results of this study correct previous results on the genomic localization of the insertion structure for the MON810 event. We also found a single-nucleotide polymorphism (SNP) in the hsp70 intron, which is most likely the first SNP found in a transgenic insertion sequence. PCR amplification in conjunction with Sanger sequencing, allele-specific PCR (AS-PCR), and blocker displacement amplification (BDA) assays were all effective at detecting the base variance. The integrated strategy used in this study can serve as a model for other cases when facing similar challenges involving partially characterized genetic modification events or SNPs.

MIPs: multi-locus intron polymorphisms in species identification and population genomics

The study of species groups in which the presence of interspecific hybridization or introgression phenomena is known or suspected involves analysing shared bi-parentally inherited molecular markers. Current methods are based on different categories of markers among which the classical microsatellites or the more recent genome wide approaches for the analyses of thousands of SNPs or hundreds of microhaplotypes through high throughput sequencing. Our approach utilizes intron-targeted amplicon sequencing to characterise multi-locus intron polymorphisms (MIPs) and assess genetic diversity. These highly variable intron regions, combined with inter-specific transferable loci, serve as powerful multiple-SNP markers potentially suitable for various applications, from species and hybrid identification to population comparisons, without prior species knowledge. We developed the first panel of MIPs highly transferable across fish genomes, effectively distinguishing between species, even those closely related, and populations with different structures. MIPs offer versatile, hypervariable nuclear markers and promise to be especially useful when multiple nuclear loci must be genotyped across different species, such as for the monitoring of interspecific hybridization. Moreover, the relatively long sequences obtained ease the development of single-locus PCR-based diagnostic markers. This method, here demonstrated in teleost fishes, can be readily applied to other taxa, unlocking a new source of genetic variation.

Sulfotransferase 4A1 Coding Sequence and Protein Structure Are Highly Conserved in Vertebrates.

Cytosolic sulfotransferases (SULTs) are Phase 2 drug-metabolizing enzymes that catalyze the conjugation of sulfonate to endogenous and xenobiotic compounds, increasing their hydrophilicity and excretion from cells. To date, 13 human SULTs have been identified and classified into five families. SULT4A1 mRNA encodes two variants: (1) the wild type, encoding a 284 amino acid, ~33 kDa protein, and (2) an alternative spliced variant resulting from a 126 bp insert between exon 6 and 7, which introduces a premature stop codon that enhances nonsense-mediated decay. SULT4A1 is classified as an SULT based on sequence and structural similarities, including PAPS-domains, active-site His, and the dimerization domain; however, the catalytic pocket lid 'Loop 3' size is not conserved. SULT4A1 is uniquely expressed in the brain and localized in the cytosol and mitochondria. SULT4A1 is highly conserved, with rare intronic polymorphisms that have no outward manifestations. However, the SULT4A1 haplotype is correlated with Phelan-McDermid syndrome and schizophrenia. SULT4A1 knockdown revealed potential SULT4A1 functions in photoreceptor signaling and knockout mice display hampered neuronal development and behavior. Mouse and yeast models revealed that SULT4A1 protects the mitochondria from endogenously and exogenously induced oxidative stress and stimulates cell division, promoting dendritic spines' formation and synaptic transmission. To date, no physiological enzymatic activity has been associated with SULT4A1.

Erectile dysfunction in cardiovascular patients: A prospective study of the eNOS gene T‐786C, G894T, and INTRON variable number of the tandem repeat functional interaction

AbstractBackgroundCardiovascular disease induces erectile dysfunction modulated by endothelial nitric oxide synthase enzyme and an impaired ejection fraction that restricts penis vascular congestion. However, the mechanisms regulating endothelial dysfunction are not understood.ObjectivesExploring the functional impact of endothelial nitric oxide synthase genetic polymorphisms on erectile dysfunction and drug therapy optimization in high‐risk cardiovascular disease patients.Materials and methodsPatients with erectile dysfunction symptoms and candidates for andrology therapy were included (n = 112). Clinical data and endothelial nitric oxide synthase rs1799983 (G894T) and rs2070744 (T‐786C), genotyped by fluorescence polarization assays, were registered. The 27‐bp variable number of the tandem repeat polymorphism in intron 4 (intron4b/a) was analyzed by polymerase chain reaction‐restriction fragment length polymorphism. Association analyses were run with the R‐3.2.0 software.ResultsA significant association between endothelial nitric oxide synthase 786‐TT (p = 0.005) and the aa/ac of intron 4 variable number of the tandem repeat (p = 0.02) with higher erectile dysfunction susceptibility was observed in cardiovascular disease patients (60 ± 9 years, 66% severe erectile dysfunction, 56% ejection fraction). After 3‐months of phosphodiesterase type 5 inhibitors, erectile dysfunction (International Index of Erectile Function, 50 ± 16 scores, the International Index of Erectile Function‐Erectile Function 21 ± 10 scores, p < 0.001) and sexual quality of life (modified Sexual Life Quality Questionnaire 55 ± 23 scores, p < 0.001) had significantly improved. The cardiovascular ejection fraction was influenced positively with better sexual quality of life (0.1941), and also in the endothelial nitric oxide synthase G894‐T allele (p = 0.076) carriers, which could merit future analyses. Erectile dysfunction was present as the primary clinical manifestation in 62% of cases, with cardiovascular disease occurring concurrently. Only former smokers and obese subjects debuted prior to cardiovascular disease than to erectile dysfunction.ConclusionsOur study provides comprehensive insights into the functional interaction linking endothelial nitric oxide synthase gene polymorphisms, erectile function, and ejection fraction in high‐risk cardiovascular disease patients. Future therapeutic strategies could target endothelial nitric oxide synthase activity by including lifestyle changes and epigenetic modulations.

Genetic association of FTO gene polymorphisms with obesity and its related phenotypes: A case-control study.

FTO gene belongs to the non-heme Fe (II) and 2 oxoglutarate-dependent dioxygenase superfamily. Polymorphisms within the first intron of the FTO gene have been examined across various populations, yielding disparate findings.The present study aimed to determine the impact of two intronic polymorphisms FTO 30685T/G (rs17817449) and -23525T/A (rs9939609) on the risk of obesity in Punjab, India. Genotypic and biochemical analysis were done for 671 unrelated participants (obese=333 and non-obese=338) (age≥18 years). Genotyping of the polymorphisms was done by PCR-RFLP method. However, 50% of the samples were sequenced by Sanger sequencing. Both the FTO variants 30685 (TT vs GG: odds ratio (OR), 2.30; 95% confidence interval (CI), 1.39-3.79) and -23525 (TT vs AA: odds ratio (OR), 2.78; 95% confidence interval (CI), 1.37-5.64) showed substantial risk towards obesity by conferring it 2 times and 3 times, respectively. The analysis by logistic regression showed a significant association for both the variants 30685T/G (rs17817449) and -23525T/A (rs9939609) (OR=2.29; 95%CI: 1.47-3.57) and (OR=5.25; 95% CI: 2.68-10.28) under the recessive genetic model, respectively. The haplotype combination TA (30685; -23525) develops a 4 times risk for obesity (P=0.0001). Among obese, the G allele of 30685T/G and A- allele of -23525T/A showed variance in Body mass index (BMI), waist circumference (WC), waist-to-height ratio(WHtR), systolic blood pressure (SBP), diastolic blood pressure (DBP) and triglyceride(TG). The present investigation indicated that both the FTO 30685T/G (rs17817449) and -23525T/A (rs9939609) polymorphisms have a key impact on an individual's vulnerability to obesity in this population.

Development of chromosome-specific potential intron polymorphism markers in watermelon

Development of chromosome-specific potential intron polymorphism markers in watermelon

Genetic Variability and Genetic Differentiation of Populations in the Grooved Carpet Shell Clam (Ruditapes decussatus) Based on Intron Polymorphisms

Genetic Variability and Genetic Differentiation of Populations in the Grooved Carpet Shell Clam (Ruditapes decussatus) Based on Intron Polymorphisms

Mutations and intron polymorphisms in voltage-gated sodium channel genes of different geographic populations of Culex pipiens pallens/Culex pipiens quinquefasciatus in China

BackgroundCulex pipiens pallens and Culex pipiens quinquefasciatus are the dominant species of Culex mosquitoes in China and important disease vectors. Long-term use of insecticides can cause mutations in the voltage-gated sodium channel (vgsc) gene of mosquitoes, but little is known about the current status and evolutionary origins of vgsc gene in different geographic populations. Therefore, this study aimed to determine the current status of vgsc genes in Cx. p. pallens and Cx. p. quinquefasciatus in China and to investigate the evolutionary inheritance of neighboring downstream introns of the vgsc gene to determine the impact of insecticides on long-term evolution.MethodsSampling was conducted from July to September 2021 in representative habitats of 22 provincial-level administrative divisions in China. Genomic DNA was extracted from 1308 mosquitoes, the IIS6 fragment of the vgsc gene on the nerve cell membrane was amplified using polymerase chain reaction, and the sequence was used to evaluate allele frequency and knockdown resistance (kdr) frequency. MEGA 11 was used to construct neighbor-joining (NJ) tree. PopART was used to build a TCS network.ResultsThere were 6 alleles and 6 genotypes at the L1014 locus, which included the wild-type alleles TTA/L and CTA/L and the mutant alleles TTT/F, TTC/F, TCT/S and TCA/S. The geographic populations with a kdr frequency less than 20.00% were mainly concentrated in the regions north of 38° N, and the geographic populations with a kdr frequency greater than 80.00% were concentrated in the regions south of 30° N. kdr frequency increased with decreasing latitude. And within the same latitude, the frequency of kdr in large cities is relatively high. Mutations were correlated with the number of introns. The mutant allele TCA/S has only one intron, the mutant allele TTT/F has three introns, and the wild-type allele TTA/L has 17 introns.ConclusionsCx. p. pallens and Cx. p. quinquefasciatus have developed resistance to insecticides in most regions of China. The neighboring downstream introns of the vgsc gene gradually decreased to one intron with the mutation of the vgsc gene. Mutations may originate from multiple mutation events rather than from a single origin, and populations lacking mutations may be genetically isolated.Graphical

Meta-Analysis of Association of SNP 19 In-del Polymorphism at the CAPN 10 Gene with Type2 Diabetes Mellitus

The CAPN 10 gene encoding the larger subunit of the ubiquitously expressing calpain protein has been found to play a role in the incidence of Type2 diabetes mellitus (T2DM). Several intronic polymorphisms of the CAPN10 gene and the linked haplotypic combinations have been implicated in T2DM. The SNP 19 locus of the gene lying within intron 6 exhibits an in-del polymorphism and has been found to be associated with T2DM in several studies. However, the results have not been constant. Keeping this in view, the present work was carried out to understand the association of the intronic SNP 19 in-del polymorphism at the CAPN 10 gene with T2DM through meta-analysis of the case-control studies. All statistical tests based on PRISMA and PROSPERO were performed on R studio (4.2.3). However, the pooled odds ratio (OR) estimates did not reveal any significant association between polymorphism at the SNP 19 locus of CAPN 10 gene and T2DM. Therefore, it is concluded that polymorphism at the SNP19 locus of CAPN 10 gene alone has no role to play in the etiology of T2DM.

Определение соматических мутаций в гене EZH2 и оценка их прогностической значимости при фолликулярной лимфоме 1–3А цитологического типа

Aim. To determine the incidence and prognostic value of mutations in exon 16 of EZH2 as well as those of polymorphism с.1582-21А>G (rs2072407) in EZH2 in patients with follicular lymphoma (FL) grades 1–3А in relation to morphologic and cytogenetic tumor characteristics.
 Materials & Methods. The prospective cohort study conducted by the National Research Center for Hematology from January 2017 to April 2021 enrolled 80 patients with newly diagnosed FL grades 1/2 and 3А. The median follow-up was 53 months. Molecular and cytogenetic analyses were based on biopsy samples of lymph nodes obtained before chemotherapy. The mutation status of exon 16 in EZH2 and the presence of intronic polymorphism rs2072407 in EZH2 were examined by Sanger sequencing method. Translocation t(14;18)(q32;q21) was detected by karyotyping or FISH.
 Results. Mutations in exon 16 of EZH2 (mutEZH2) were identified in 10/80 (13 %) patients. All patients showed missense mutation in codon 646 of EZH2. Translocation t(14;18) was detected in 45/80 (56 %) cases. Poor outcome in the cohort with no t(14;18) was observed 3 times more often than in the group of patients with t(14;18) (p = 0.0001). The presence of t(14;18) was associated with favorable prognosis irrespective of either the mutation status of exon 16 in EZH2 or the FL grade. The analysis of the polymorphism rs2072407 status yielded the following genotypes: AA in 24 % (n = 19), AG in 42 % (n = 34), and GG in 34 % (n = 27) of cases. The variants АА and AG were associated with higher risk of death (hazard ratio 2.9; 95% confidence interval 1.2–10.6; p = 0.01), whereas the genotype GG was associated with wtEZH2 (10 % vs. 37 %) and favorable prognosis (p = 0.065).
 Conclusion. Significant biological markers for favorable prognosis in FL appeared to be the presence of t(14;18)(q32;q21) and GG genotype of polymorphism rs2072407 in EZH2. The previously identified prognostic factors (grade 3А, bulky tumor lesions > 6 cm, Ki-67 > 35 %, and a short interval between symptom onset and chemotherapy start) were incorporated into a new unified personalized predictive (index PPI) FL model by supplementing it with two additional biological markers: the presence of t(14;18)(q32;q21) and GG genotype of polymorphism rs2072407. This approach may increase the prognostic value of the new personalized design which will provide the basis for risk-adapted algorithms for FL treatment.

Identification of genetic variants of bGH, GHR and IGF1 gene and their association with lactation persistency in Tharparkar cattle

Persistency can be explained as number of days during which the high level of milk production is maintained after the peak milk yield. The aim of this study was to identify genetic variants of bovine-Growth Hormone (bGH), Growth Hormone Receptor (GHR) and Insulin like Growth Factor1 (IGF1) gene and their association with lactation persistency in Tharparkar cattle. Study was carried out using data of daily milk yield, monthly test days, total milk yield and peak yield of 372 lactation records of 190 Tharparkar cattle maintained at Livestock Research Centre of ICAR-National Dairy Research Institute from 1996-2019. Lactation persistency was estimated using Woods and Wilminks method and its least squares means were 2.37±0.08 and -5.4±0.56, respectively. Investigations on polymorphism were performed using seven reported primers for amplifying targeted regions of bGH, GHR and IGF1 gene. In PCR-RFLP, bGH-MspI RFLP had shown polymorphism in intron 3 and its genotypic frequencies were TT 0.49, CT 0.39 and CC 0.12. The allelic frequency of C and T were 0.31 and 0.69, respectively. The bGH-AluI RFLP had shown monomorphism in exon 5. GHR-AluI had shown polymorphism in exon 10 with only two genotypes AA and AG with frequency of 0.92 and 0.08, respectively. The frequency of A and G allele was 0.96 and 0.04, respectively. In GHR-StuI and IGF1-TasI in promoter region, RsaI in exon 3 and SnaB I in exon 1 had shown monomorphism. No significant association was found between genetic variants of bGH and GHR with lactation persistency in Tharparkar cattle.

Molecular basis of DEL phenotype in the Indian population: Insights from next-generation sequencing analysis of two cases

The DEL phenotype represents an intriguing and challenging aspect of blood group serology. This condition is characterized by an extremely weak expression of the D antigen on red blood cells, to the extent that it often eludes detection through routine serological methods. Identifying the DEL phenotype necessitates more specialized techniques, such as adsorption and elution tests, to reveal the presence of the D antigen. This distinctive phenotype underscores the complexity and subtlety of blood group genetics and highlights the importance of using advanced methods to accurately classify individuals with this condition, as their ability to form anti-D antibodies can have clinical implications during transfusion and pregnancy scenarios. There is a paucity of data for the DEL phenotype in the Indian population, and the molecular basis has not been elucidated yet. Our investigation delves into the genetic underpinnings of two distinct DEL phenotype cases that pose challenges for resolution through conventional serological techniques. We employ next-generation amplicon sequencing to unravel the intricate genetic landscape underlying these cases. In the D-negative donor, the DEL phenotype was first identified serologically, which was subsequently confirmed by molecular analysis. In the second case, it was associated with an anti-D antibody in a D-positive patient. Initial data analysis unveiled a substantial reduction in coverage across the exonic segments of the RHD gene in both samples, suggesting the potential presence of RHD exon deletions. On both occasions, we identified a homozygous intronic RHD polymorphism that is well established to be linked to the RHD* 01EL.32/RHD*DEL32 variant.

Association of fat mass and obesity associate (FTO) single nucleotide polymorphisms in the first intron and obesity risk among Indonesians

Obesity is one of the global pandemics characterized by an excessive fat buildup due to disruption of energy homeostasis in the body. As obesity is a risk factor for many other non-communicable diseases such as diabetes and coronary heart disease, it is crucial to understand the risk factors that contribute to the pathogenesis of obesity. Although obesity is mainly caused due to unhealthy lifestyles, genetic predisposition also plays a part in the pathogenesis of obesity. Individuals who carry risk alleles for genes that control energy balance in the body have a greater risk of developing obesity. Fat mass and obesity associate (FTO) is a gene strongly correlated with obesity and is widely expressed in the hypothalamus. This gene is predicted to have 89 common variations that affect obesity-related phenotypes. Among Indonesians, the three most studied single nucleotide polymorphisms (SNPs) in the first intron of the FTO gene are rs1421085, rs17817449, and rs9939609. They are strongly associated with obesity’s related traits such as weight gain, fat mass, body mass index (BMI), waist, and hip sizes. rs993609 is the most studied among diverse ethnicities in Indonesia, with AA genotype and allele A as a risk allele.

Unraveling biotypes of the northern house mosquito, Culex pipiens s.l. (Diptera: Culicidae): molecular differentiation and morphometric analysis.

Geometric morphometrics was used to determine whether geographic isolation could explain differences in wing size and shape between and within continental (27°S to 41°S) and insular (Rapa Nui) populations of Culex pipiens s.s. Linnaeus and their biotypes (f. pipiens and f. molestus). Molecular protocols based on polymorphisms in the second intron of nuclear locus ace-2 (acetylcholinesterase-2) were used to differentiate Cx. pipiens s.s. from Cx. quinquefasciatus Say, and an assay based on polymorphisms in the flanking region of a microsatellite locus (CQ11) was used to identify biotypes. Culex pipiens f. molestus and hybrids shared larval habitats in all continental sites, while Cx. pipiens f. pipiens was found in 5 of the 10 sites. Only biotype molestus was found in Rapa Nui (Easter Island) Pipiens and molestus biotypes occur sympatrically in aboveground locations, and only molestus was found in the underground site (ME). Biotype molestus was dominant in rural locations and preferably anthropophilic. These results agree with the ecological descriptions previously reported for the biotypes of Cx. pipiens s.s. Procrustes ANOVA only showed differences in centroid size between biotypes in females and males and did not show significant differences in wing shape. However, we found significant differences among the geographic areas in the centroid size and wing shape of both females and males. Particularly, the population of Rapa Nui Island had shorter wings than the continental populations. The results highlight the effects of geographic and environmental processes on morphotypes in vector mosquitoes.

The Development of Multiplex PCR-RFLP of FTO Genetics Variants

Background: Major health concern throughout the whole world is the increasing rate of obesity, either in elder group or younger group of multiracial ethnics that are believed to be associated with genetic factor that plays important role in the pathogenesis of obesity. The very first gene to be associated with obesity is fat mass and obesity-associated (FTO) gene with the involvement from many single nucleotide polymorphisms (SNPs) in the first intron of FTO. Early detection of obesity-related SNPs is very crucial in the effort of reducing obesity rate which is expected to increase by 2025.This has led to the urge of developing a method to detect individual with predisposing factors towards obesity, with the hope that this method will eventually helpful in assisting healthcare authorities to rapidly detect individual who are at high risk of being obese. Multiplex PCR-RFLP is a method for rapid detection of obesity-related SNPs using gene sequence amplifying technique and optimization done using gradient PCR technique. Thus, this study was conducted to evaluate the potential of multiplex PCR-RFLP to amplify FTO genetic variants. Materials and methods: A total of 14 UniSZA students were recruited to obtain 2 ml of peripheral whole blood for DNA extraction. Single gradient PCR dan multiplex PCR were employed to determine the optimum annealing temperature for all assigned FTO SNPs as rs9939609, rs1421085 and rs17817288. The PCR products were run on 1% of agarose gel electrophoresis and visualized under UV Gel doc Image Analyzer. These PCR products were introduced to restriction enzymes for the cutting site on genetic variation namely Bfal, HpyCH4V, HpyCH4IV for rs9939609, rs1421085 and rs17817288, respectively. Next, the PCR fragments were identified on 2% agarose gel electrophoresis under UV Gel Doc Image Analyzer. Results: The optimum annealing temperature to amplify all FTO SNPs obtained through gradient PCR was 60°C. The amplification of FTO rs9939609, rs17817288 and rs1421085 regions, showed specific size product of 151 bp, 140 bp, and 131 bp respectively. In RFLP, the fragments after restriction enzyme digestion produced homozygous wild type, heterozygous and homozygous mutant type for genotype characterization based on allele cut site by specific restriction enzyme namely BfaI, HpyCG4IV and HpyCH4V, respectively. Conclusion: This study may become preliminary data on the potential of multiplex PCR-RFLP in provide rapid diagnosis to amplify multiple FTO genetic variants.

White rust: Plant-microbe interaction and plant breedingperspective on Indian mustard

White rust is an important disease of Indian mustard which results in appreciable yield losses. A monogenic dominant gene has been responsible for resistance against this pathogen in B. juncea. In the pathogen, avirulence has been observed to be dominant, governed by a single gene. R gene (resistance gene) present in resistant lines of B. juncea, encodes CC-NB-LRR (coiled coil nucleotide binding leucine rich repeat) protein whereas, susceptible lines have truncated LRR domain of the same protein. Resistant and moderately resistant lines have higher amount of chlorophyll, sugars and total phenols than susceptible cultivars, throughout crop growth. Increased accumulation of phytoalexins such as spirobrassinin, cyclobrassinin and rutalexin have been observed in resistant lines compared to, susceptible ones upon infection by fungi. Molecular markers tagged to R gene are being used in resistance breeding programmes. Scientists have developed intron polymorphism (IP) markers namely;At5g41560, At5g41940 and At2g36360 for positive selection of white rust resistance and, simple sequence repeat (SSR) marker, At2g34700, for negative selection of resistance in different rust resistant sources. The major issue in use of these markers is their transferability as they do not show association with R gene in different set of crosses.

Development of Allium cepa potential intron polymorphism markers for molecular breeding of Alliums

Allium cepa is a widely grown crop for its spice and culinary properties. For molecular breeding of Allium cepa, mining and utilization of various sequence-based markers have been widely reported. Unfortunately, none has reported its molecular breeding using potential intron polymorphism (PIP) markers. Due to advantage of intron positioning prediction and practical utility, the PIP markers used for screening the possible polymorphism and cross-transferability in the Alliums. Screening results showed that among 500 Allium cepa potential intron polymorphism (AcPIP) markers, 275 are cross-transferable and polymorphic. Among the 275 AcPIP, 111 polymorphic markers were physically mapped on to 1st to 8th chromosomes of the Allium cepa. Out of the 275 AcPIP containing functionally significant markers, 118 were mapped in onion with Arabidopsis proteome. A set of 10 Alliums were utilized to interpret the polymorphic possibilities of the 500 AcPIP markers. In totality, 55 % AcPIP markers were polymorphic and cross-transferable among the Alliums. The polymorphic information content (PIC) of AcPIP markers ranged from 0.03 to 0.47 and heterozygosity index (H) varied between 0.16 and 0.80. The constructed phylogenetic tree based on the AcPIP markers of 10 Alliums revealed different clusters due to differences in their taxonomic positions. Out of 275 AcPIP markers, 10 AcPIP markers genotyping based Jaccard dissimilarity-based NJ tree of 96 individuals of Allium cepa showed two distinct groups (high total soluble solid; HTSS and low total soluble solid; LTSS). NJ tree and dissimilarity matrix reveal that group I genotypes are distinct, and dissimilar from group II and III genotypes hence it seems that group II and III genotype evolve from group I genotypes. Further we identified nine diverse Allium cepa subsample among which, genotype number 74 has HTSS and could be used for identification and introgression of HTSS coding genes in elite cultivars. Further DNA fingerprint of Alliums with 20 AcPIP markers suggested geographical reach of the released onion varieties. Hence results suggest that 275 AcPIP markers may be useful for accelerating the breeding programme of the Alliums and other species.