Genetic study in Aedes (Stegomyia) aegypti (Linnaeus, 1762) from Londrina (Paraná State, Brazil): an approach to population structure and pyrethroid resistance

Abstract Londrina is the fourth most populous city in southern Brazil. Its subtropical weather with rain in all seasons, as well as its high population density, make the city perfect for the Aedes aegypti (Linnaeus, 1762) life cycle. Over the last few years, Londrina presented high infestation indexes and was one of the cities with the most reported cases of dengue. Uncontrolled use of synthetic insecticides may influence the mosquito's genetic composition. In this paper, we studied mitochondrial DNA and kdr mutations in Aedes aegypti. The analysis of the ND4 gene in 330 specimens showed the presence of 27 haplotypes. The pyrethroid resistance alleles (kdr) evaluated are present in the collected populations, with a 50% frequency of the Val1016Ile and 48% of the Phe1534Cys mutations. Such analysis of the mutations in the populations collected at the State University of Londrina's campus - a microenvironment that differs from the rest of the city - showed frequencies of 57% and 62%, respectively. The low gene flow observed, Nm = 0.11 and Nm = 0.10, along with the elevated differentiation, Fst = 0.19 and Fst = 0.18, among populations suggest an influence of genetic drift. The strong presence of resistance alleles kdr in the city is evident, which demonstrates that even with the interruption of the use of pyrethroids by the National Dengue Control Program, resistance may be maintained due to domestic use. Thus, the results have shown the need for genetic monitoring, alongside other entomological surveillance monitoring tools, to create strategies of mosquito control.

Detecting acetyl-coenzyme a carboxylase resistance gene in rice (Oryza sativa L.).

Herbicides inhibiting acetyl-coenzyme A carboxylase (ACCase) are very effective in controlling grass weeds including weedy-rice in paddy rice production systems. The ACCase inhibitor affects the enzyme by blocking fatty acid biosynthesis resulting in plant death. The herbicide resistance in rice is conferred by a single point mutation with an amino acid substitution of the carboxyl transferase domain of the ACCase gene. An assay based on the tetra-primer ARMS-PCR method was developed to detect the SNP G2027T that causes a tryptophan-cysteine substitution in the gene encoding chloroplastic ACCase in rice. The protocol was tested in 453 rice samples from a segregant population for validation of the assay. This technique can be exploited to monitor resistant lines in rice breeding programs to detect homozygous or heterozygous resistant genotypes and homozygous susceptible genotypes. The presence of resistant ACCase allele(s) can be detected with rapidity, simplicity, at low cost and can be used in any molecular biology laboratory with minimal equipment.

First report of the Phe1534Cys kdr mutation in natural populations of Aedes albopictus from Brazil.

BACKGROUND: Knockdown resistance (kdr), caused by alterations in the voltage-gated sodium channel (NaV), is one of the mechanisms responsible for pyrethroid (PY) resistance. In the Asian tiger mosquito, Aedes albopictus, at least four different mutations were described in the IIIS6 NaV segment in populations from Asia, North America and Europe. In contrast, in Aedes aegypti at least 12 non-synonymous mutations have been reported at nine different codons, mostly in the IIS6 and IIIS6 NaV segments. The Phe1534Cys kdr mutation in the IIIS6 NaV segment is the most prevalent in populations of Ae. aegypti worldwide, also found in Ae. albopictus from Singapore. Herein, we investigated the DNA diversity corresponding to the IIS6 and IIIS6 NaV segments in natural populations of Ae. albopictus from Brazil. METHODS: DNA from eight Brazilian Ae. albopictus natural populations were individually extracted and pooled by states of origin, amplified, cloned and sequenced for the corresponding IIS6 and IIIS6 NaV segments. Additionally, samples from each location were individually genotyped by an allelic specific PCR (AS-PCR) approach to obtain the genotypic and allelic frequencies for the 1534 NaV site. RESULTS: No non-synonymous substitutions were observed in the IIS6 sequences. However, the Phe1534Cys kdr mutation was evidenced in the Ae. albopictus NaV IIIS6 segment sequences from Paraná (PR) and Rondônia (RO) states, but not from Mato Grosso (MT) state. The 1534Cys kdr allele varied from 3% (Marilena/PR and Porto Velho/RO) to 10% (Foz do Iguaçu/PR). To our knowledge, this paper reports the first occurrence and provides distribution data of a possible kdr mutation in Ae. albopictus in South America. CONCLUSION: The emergence of a likely kdr mutation in Ae. albopitus natural populations is a signal of alert for vector control measures since PY are the most popular insecticides adopted by residents. Additionally, once the kdr allele is present, its frequency tends to increase faster under exposition to those compounds. Although the Asian tiger mosquito is not incriminated as an important vector of dengue, chikungunya and Zika viruses in South America, its importance in this regard has been extensively discussed since Ae. albopictus is rapidly spreading and can also migrate between sylvatic and urban environments. Therefore, insecticide resistance monitoring initiatives should also be extended to Ae. albopictus in Brazil in order to maintain chemical compounds as an efficient vector control tool when needed.

Development and application of KASP marker for high throughput detection of AhFAD2 mutation in peanut

Background: Cultivated peanut (Arachis hypogaea L.) is a major oilseed crop worldwide. Fatty acid composition of peanut oil may affect the flavor and shelf life of the resulting food products. Oleic acid and linoleic acid are the major fatty acids of peanut oil. The conversion from oleic acid to linoleic acid is controlled by theΔ12 fatty acid desaturase (FAD) encoded byAhFAD2AandAhFAD2B, two homoeologous genes from A and B subgenomes, respectively. One nucleotide substitution (G:C→A:T) ofAhFAD2Aand an "A" insertion ofAhFAD2Bresulted in high-oleic acid phenotype. Detection ofAhFAD2mutation had been achieved by cleaved amplified polymorphic sequence (CAPS), real-time polymerase chain reaction (qRT-PCR) and allele-specific PCR (AS-PCR). However, a low cost, high throughput and high specific method is still required to detectAhFAD2genotype of large number of seeds. Kompetitive allele specific PCR (KASP) can detect both alleles in a single reaction. The aim of this work is to develop KASP for detectionAhFAD2genotype of large number of breeding materials. Results: Here, we developed a KASP method to detect the genotypes of progenies between high oleic acid peanut and common peanut. Validation was carried out by CAPS analysis. The results from KASP assay and CAPS analysis were consistent. The genotype of 18 out of 179 BC4F2seeds was aabb. Conclusions: Due to high accuracy, time saving, high throughput feature and low cost, KASP is more suitable fordeterminingAhFAD2genotype than other methods.

Modified allele-specific PCR improves HER2 Ile655Val detection by reducing genotyping errors

Background: A reliable method to detect gene polymorphisms must be established to eliminate genotyping errors due to false PCR amplification. In the previous study, we have developed AS-PCR (Allele Specific-Polymerase Chain Reaction) to detect HER2 Ile655Val gene polymorphism with good specificity and sensitivity, yet it produces some errors. This study is aimed to eliminate the source of genotyping errors mainly by betaine treatment and PCR program modification. Methods: Genotyping errors produced by AS-PCR was qualitatively and quantitatively evaluated using two genomic DNA that each contained AA genotype and GG genotype of HER2 Gene. Betaine treatment or PCR program modification was tested to eliminate the occurrence of genotyping errors during AS-PCR amplification. Results: The types of genotyping errors exhibited by HER2 Ile655Val AS-PCR are diverse, ranging from LDO (Locus Drop Out), preferential amplification to ADO (Allele Drop Out). The rate of genotyping errors was from 10% to 50% depending on the amount and ratio of DNA template and the annealing temperature of PCR. In the mixed DNA template model, the betaine treatment has shown to reduce ADO only in preferentially amplified GG genotype amplicon. Alternatively, reducing the template of the heterozygous DNA by half ( -0.5 ng of DNA template) for such case has effectively recovered the AS-PCR from ADO. Furthermore, increasing the denaturation temperature to 96 °C with an annealing time of 40 s at the first 10 cycles of AS-PCR has succeeded in eliminating severe preferential amplification of AA genotype amplicon by preventing the DNA template with GG genotype from forming into a G-quadruplex structure. The guideline offered in this study has been successfully applied for clinical samples of breast cancer that show preferential amplification. Conclusion: Betaine and the modifying AS-PCR program can reduce significantly genotyping errors making AS-PCR for HER2 Ile655Val detection more reliable to be used as a molecular tool for genotyping purpose (AU)

Design of an allele-specific PCR assay to genotype the rs12255372 SNP in a pilot study of association between common TCF7L2 polymorphisms and type 2 diabetes in Venezuelans

Objective The global burden of diabetes mellitus will impact strongly American countries in the coming decades. Type 2 diabetes mellitus (T2DM) is a multifactorial disease and the basis for its genetic susceptibility remains not fully understood. Different population studies have demonstrated that variants of the TCF7L2 gene are strongly associated with an increased risk of T2DM. Moreover, institutions or countries with limited budget to conduct genetic research need cost effective methods for detecting DNA variants. Subjects and methods We standardized a rapid and simple allele-specific PCR method for genotyping the rs12255372 single nucleotide polymorphism (SNP) in a pilot study exploring the association of three TCF7L2 polymorphisms (rs7903146, rs12255372 and DG10S478) with T2DM in 70 patients and 73 controls from Venezuela. Results The performance of the designed allele-specific PCR reaction for rs12255372 genotyping was reliable and accurate. Patients carrying the TCF7L2 rs7903146 T allele (CT + TT genotypes) and heterozygous CT genotype had a significantly higher risk for T2DM (OR = 2.9 and 2.3, respectively). Although rs12255372 and DG10S478 risk alleles predominated in T2DM group no statistical significance was found. Conclusions We developed a novel allele-specific PCR method for easier and rapid detection of rs12255372 polymorphism without the use of expensive instrumentation and reagents. Our study in a relatively small sample of the Venezuelan population replicated the association of the rs7903146 SNP with T2DM. Further studies with larger sample size and more biochemical data should be conducted to explore the genetic basis of T2DM susceptibility in Venezuela.