Susceptible Homozygotes Research Articles

Three Novel er1 Alleles and Their Functional Markers for Breeding Resistance to Powdery Mildew (Erysiphe pisi) in Pea.

Powdery mildew caused by Erysiphe pisi DC is a global notorious disease on peas. Deploying resistance pea cultivars is the most efficient and environmentally friendly method for disease control. This study focuses on revealing the resistance genes in three pea germplasms and developing their functional markers for resistance breeding. The identification of resistance genes involved genetic mapping and the sequencing of the pea mildew resistance locus O homolog PsMLO1 gene. To confirm the heredity of three resistant germplasms, they were crossed with susceptible cultivars to generate F1, F2, and F2:3 populations. The F1 generation exhibited susceptibility to E. pisi, whereas the segregation patterns in subsequent generations adhered to the 3:1 (susceptible: resistant) and 1:2:1 (susceptible homozygotes: heterozygotes: resistant homozygotes) ratios, indicating that powdery mildew resistance was governed by a single recessive gene in each germplasm. Analysis of er1-linked markers and genetic mapping suggested that the resistance genes could be er1 alleles in these germplasms. The multiple clone sequencing results of the three homologous PsMLO1 genes showed they were novel er1 alleles, named er1-15, er1-16, and er1-17. The er1-15 and er1-16 were caused by 1-bp deletion at position 335 (A) and 429 (T) in exon 3, respectively, whereas er1-17 was caused by a 1-bp insertion at position 248 in exon 3, causing a frame-shift mutation and premature termination of PsMLO1 protein translation. Their respective functional markers, kompetitive allele-specific PCR (KASP)-er1-15, KASP-er1-16, and KASP-er1-17, were successfully developed and validated in respective mapping populations and pea germplasms. These results provide valuable tools for pea breeding resistance to E. pisi.

Field survey of reproductive modes and sodium channel mutations associated with pyrethroid resistance in Thrips tabaci.

Using PCR-Restriction Fragment Length Polymorphism (RFLP) with mitochondrial cytochrome c oxidase subunit I sequences, we examined the reproductive modes of female adults of Thrips tabaci collected at 54 sites across Japan. Results showed the presence of heteroplasmic insects harboring mitochondria associated with arrhenotoky and thelytoky. Using the insects, we also applied PCR-RFLP to examine the genotypes for the amino acid mutation (T929I) site involved in pyrethroid resistance. Findings showed the presence of thelytokous heterozygotes under the circumstance that most arrhenotokous insects are resistant homozygotes, and many thelytokous insects are susceptible homozygotes. These results suggest that, in the field, genetic exchange occurs between insects through of both reproductive modes. A survey of the genotypes for the other amino acid mutations using nucleotide sequencing showed a decline of insects with an M918T and L1014F pair and an increase of insects with M918L. These results suggest the evolutional progression of amino acid mutations associated with pyrethroid resistance in T. tabaci.

Molecular Characterizations of the er1 Alleles Conferring Resistance to Erysiphe pisi in Three Chinese Pea (Pisum sativum L.) Landraces.

Powdery mildew caused by Erysiphe pisi DC. is a major disease affecting pea worldwide. This study aimed to confirm the resistance genes contained in three powdery mildew-resistant Chinese pea landraces (Suoshadabaiwan, Dabaiwandou, and Guiwan 1) and to develop the functional markers of the novel resistance genes. The resistance genes were identified by genetic mapping and PsMLO1 gene sequence identification. To confirm the inheritance of powdery mildew resistance in the three Landraces, the susceptible cultivars Bawan 6, Longwan 1, and Chengwan 8 were crossed with Suoshadabaiwan, Dabaiwandou, and Guiwan 1 to produce F1, F2, and F2:3 populations, respectively. All F1 plants were susceptible to E. pisi, and phenotypic segregation patterns in all the F2 and F2:3 populations fit the 3:1 (susceptible: resistant) and 1:2:1 (susceptible homozygotes: heterozygotes: resistant homozygotes) ratios, respectively, indicating powdery mildew resistance in the three Landraces were controlled by a single recessive gene, respectively. The analysis of er1-linked markers and genetic mapping in the F2 populations suggested that the recessive resistance genes in three landraces could be er1 alleles. The cDNA sequences of 10 homologous PsMLO1 cDNA clones from the contrasting parents were obtained. A known er1 allele, er1-4, was identified in Suoshadabaiwan. Two novel er1 alleles were identified in Dabaiwandou and Guiwan 1, which were designated as er1-13 and er1-14, respectively. Both novel alleles were characterized with a 1-bp deletion (T) in positions 32 (exon 1) and 277 (exon 3), respectively, which caused a frame-shift mutation to result in premature termination of translation of PsMLO1 protein. The co-dominant functional markers specific for er1-13 and er1-14, KASPar-er1-13, and KASPar-er1-14 were developed and effectively validated in populations and pea germplasms. Here, two novel er1 alleles were characterized and their functional markers were validated. These results provide powerful tools for marker-assisted selection in pea breeding.

Gene-Based Markers in Marker-Assisted Selection to Screen Tomato Genotypes Resistant to Fusarium Wilt, Late Blight, Verticillium Wilt, Leaf Mold, Bacterial Spot and Bacterial Speck

The tomato crop is exposed to serious losses due to infection with several diseases and pests, which threaten tomato production in Egypt and worldwide. Therefore, selecting the tomato germplasm resistant to a specific pathogen by molecular markers closely linked to resistance loci is a desirable goal of this study. In this work, seven co-dominant markers targeting six resistance genes (I-1, Ve, Ph3, Cf-9/Cf-4, Rx4, and Pto) for six main diseases [ fusarium wilt (Fusarium oxysporum f. sp. lycopersici), verticillium wilt (Verticillium dahliae and V. alboatrum), late blight (Phytophthora infestans), leaf mold (Cladosporium fulvum), bacterial spot (Xanthomonas campestris pv. vesicatoria) and bacterial speck (Pseudomonas syringae pv. tomato)], respectively were determined. Theses molecular markers differentiated among 19 tomato genotypes resistant (homozygote/heterozygote) and susceptible (homozygote) to the pathogens. Therefore, this study supplied us with novel tomato lines with resistance to multiple diseases, and their pyramiding inside domesticated tomato cultivars are suggested to apply in the tomato breeding programs of resistance against fungal and bacterial diseases.

Basis and monitoring of methoxyfenozide resistance in the South American tomato pinworm Tuta absoluta

The South American tomato pinworm, Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae), is heavily targeted by insecticide applications. Methoxyfenozide is highly effective against T. absoluta with reduced side effects to natural enemies. This work aimed to (1) genetically and biochemically characterize resistance of the T. absoluta GBN population to methoxyfenozide, (2) establish cross resistance profiles with other insecticide groups and (3) monitor resistance in populations with the goal of improving T. absoluta insecticide resistance management (IRM). Methoxyfenozide resistance was completely recessive, polyfactorial and autosomal. Effective dominance revealed that 10 mg methoxyfenozide/L would be enough to eliminate susceptible homozygotes and heterozygotes, thus used to diagnose resistance in field populations. The synergism of methoxyfenozide toxicity in the resistant population for PBO (SR = 95×), DEF (SR = 51×) and DEM (SR = 45×), suggested monooxygenases, esterases and glutathione S-transferases as resistance mechanisms. However, only monooxygenase activity appeared to be involved in methoxyfenozide resistance. Resistance ratio for methoxyfenozide (2352-fold) after selection and cross-resistance ratios of a lab-selected GBN strain (“GBN-Sel”) were significant relative to a susceptible strain “JDR1-Sus” for tebufenozide (656-fold), cartap hydrochloride (10.68-fold), deltamethrin (4.70-fold), abamectin (2.65-fold), lufenuron (2.22-fold) and indoxacarb (1.92-fold), with negative cross-resistance to spinetoram (0.32-fold). Evidence of control failures was observed in 10 field populations of T. absoluta (mortalities between 13 and 76%), and all populations showed frequencies of resistant phenotypes (percentage survivorship ranging between 4 and 96%). A rational basis for managing resistance to bisacylhydrazines is discussed, along with details of recommended T. absoluta resistance management tactics.

A matrix model for density-dependent selection in stage-classified populations, with application to pesticide resistance in Tribolium

The study of eco-evolutionary dynamics is based on the idea that ecological and evolutionary processes may operate on the same, or very similar, time scales, and that interactions of ecological and evolutionary processes may have important consequences. Here we develop a model that combines Mendelian population genetics with nonlinear demography to create a truly eco-evolutionary model. We use the vec-permutation matrix approach, classifying individuals by stage and genotype. The demographic component is female dominant and density-dependent. The genetic component includes random mating by stage and genotype, and arbitrary effects of genotype on the demographic phenotype. Mutation is neglected. The result is a nonlinear matrix population model that projects the stage × genotype distribution. We show that the results can include bifurcations of population dynamics driven by the response to selection. We present analytical criteria that determine whether one allele excludes the other or if they persist in a protected polymorphism. The results are based on local stability analysis of the homozygous boundary equilibria.As an example, we use a density-dependent stage-classified model of the flour beetle Tribolium castaneum. Our model permits arbitrary life-cycle complexity and nonlinearity. Tribolium has developed resistance to the pesticide malathion due to a dominant allele at a single autosomal locus. Using parameters reported from laboratory experiments, we show that the model successfully describes the dynamics of both resistant and susceptible homozygotes, and the outcome of a selection experiment containing both alleles. Stability analysis of the boundary equilibria confirms that the resistant allele excludes the susceptible allele, even in the absence of malathion, agreeing with previously reported results.

The role of windows of selection and windows of dominance in the evolution of insecticide resistance in human disease vectors.

Persistent insecticides sprayed onto house walls, and incorporated into insecticide‐treated bednets, provide long‐acting, cost‐effective control of vector‐borne diseases such as malaria and leishmaniasis. The high concentrations that occur immediately postdeployment may kill both resistant and susceptible insects. However, insecticide concentration, and therefore killing ability, declines in the months after deployment. As concentrations decline, resistant insects start to survive, while susceptible insects are still killed. The period of time after deployment, within which the mortality of resistant individuals is lower than that of susceptible ones, has been termed the “window of selection” in other contexts. It is recognized as driving resistance in bacteria and malaria parasites, both of which are predominantly haploid. We argue that paying more attention to these mortality differences can help understand the evolution of insecticide resistance. Because insects are diploid, resistance encoded by single genes generates heterozygotes. This gives the potential for a narrower “window of dominance,” within the window of selection, where heterozygote mortality is lower than that of susceptible homozygotes. We explore the general properties of windows of selection and dominance in driving resistance. We quantify their likely effect using data from new laboratory experiments and published data from the laboratory and field. These windows can persist months or years after insecticide deployments. Differential mortalities of resistant, susceptible and heterozygous genotypes, after public health deployments, constitute a major challenge to controlling resistance. Greater attention to mortality differences by genotype would inform strategies to reduce the evolution of resistance to existing and new insecticides.

Molecular identification and chromosomal localization of new powdery mildew resistance gene Pm11 in oat

The appropriate selection of various traits in valuable plants is very important for modern plant breeding. Effective resistance to fungal diseases, such as powdery mildew, is an example of such a trait in oats. Marker-assisted selection is an important tool that reduces the time and cost of selection. The aims of the present study were the identification of dominant DArTseq markers associated with a new resistance gene, annotated as Pm11 and derived from Avena sterilis genotype CN113536, and the subsequent conversion of these markers into a PCR-based assay. Among the obtained 30,620 silicoDArT markers, 202 markers were highly associated with resistance in the analysed population. Of these, 71 were selected for potential conversion: 42 specific to resistant and 29 to susceptible individuals. Finally, 40 silicoDArT markers were suitable for primer design. From this pool, five markers, 3 for resistant and 2 for susceptible plants, were selected for product amplification in the expected groups. The developed method, based on 2 selection markers, provides certain identification of resistant and susceptible homozygotes. Also, the use of these markers allowed the determination of heterozygotes in the analysed population. Selected silicoDArT markers were also used for chromosomal localization of new resistance genes. Five out of 71 segregating silicoDArT markers for the Pm11 gene were found on the available consensus genetic map of oat. Five markers were placed on linkage groups corresponding to Mrg12 on the Avena sativa consensus map.

Pyrethroid resistance in the pest mite, Halotydeus destructor: Dominance patterns and a new method for resistance screening

An L1024F substitution in the para gene, which encodes a subunit of the voltage-gated sodium channel, has been implicated in pyrethroid resistance in a mite pest, Halotydeus destructor, which attacks rape and other grain crops. A high-resolution melt (HRM) genotyping assay was developed for testing the relative pyrethroid susceptibility of different para genotypes and for high-throughput field screening of resistant alleles. The L1024F mutation was found to be incompletely recessive in phenotypic laboratory bioassays with the pyrethroid pesticide, bifenthrin. While the resistance ratio of heterozygotes (RS) to susceptible homozygotes (SS) was <6 in 24 h bioassays, the resistant homozygotes (RR) (with a resistance ratio > 200,000) survived the recommended field rate of bifenthrin (100 mgL−1). HRM genotyping of mites from field populations across Australia indicated the presence of resistant alleles in Western Australia and South Australia, but not in Victoria and New South Wales. The assay developed will be useful for routine screening of pyrethroid resistance, and the dominance relationships established here point to useful resistance management strategies involving the maintenance of reservoirs of susceptible mites to dilute resistant homozygotes in a population.

Target-site mutations (AChE-G119S and kdr) in Guangxi Anopheles sinensis populations along the China-Vietnam border

BackgroundIn South Asia, the epidemiology of malaria is complex, and transmission mainly occurs in remote areas near international borders. Vector control has been implemented as a key strategy in malaria prevention for decades. A rising threat to the efficacy of vector control efforts is the development of insecticide resistance, thus it is important to monitor the type and frequency of insecticide resistant alleles in the disease vectors such as An. sinensis along the China-Vietnam border. Such information is needed to synthesize effective malaria vector control strategies.MethodsA total of 208 adults of An. sinensis, collected from seven sites in southwest Guangxi along the China-Vietnam border, were inspected for the resistance-conferring G119S mutation in acetylcholinesterase (AChE) by PCR-RFLP (polymerase chain reaction restriction fragment length polymorphism) and kdr mutations in the voltage-gated sodium channel (VGSC) by sequencing. In addition, the evolutionary origin of An. sinensis vgsc gene haplotypes was analyzed using Network 5.0.ResultsThe frequencies of mutant 119S of AChE were between 0.61–0.85 in the seven An. sinensis populations. No susceptible homozygote (119GG) was detected in three of the seven sites (DXEC, LZSK and FCGDX). Very low frequencies of kdr (0.00–0.01) were detected in the seven populations, with most individuals being susceptible homozygote (1014LL). The 1014F mutation was detected only in the southeast part (FCGDX) at a low frequency of 0.03. The 1014S mutation was distributed in six of the seven populations with frequencies ranging from 0.04 to 0.08, but absent in JXXW. Diverse haplotypes of 1014L and 1014S were found in An. sinensis along the China-Vietnam border, while only one 1014F haplotype was detected in this study. Consistent with a previous report, resistant 1014S haplotypes did not have a single origin.ConclusionsThe G119S mutation of AChE was present at high frequencies (0.61–0.85) in the An. sinensis populations along the China-Vietnam border, suggesting that the vector control authorities should be cautious when considering carbamates and organophosphates as chemicals for vector control. The low frequencies (0.00–0.11) of kdr in these populations suggest that pyrethroids remain suitable for use against An. sinensis in these regions.

Frequency of the L1014F Mutation in the Sodium Channel Gene, in Culex quinquefasciatus (Diptera: Culicidae) Populations From Rural and Urban Areas of Yucatan State, Mexico.

Culex quinquefasciatus Say (Diptera: Culicidae) is a mosquito species that has attracted a lot of attention from a medical and veterinary point of view; however, little is known about the frequency of L1014F mutations that have been found in the sodium channel gene, with this being a target for DDT and pyrethroid insecticides. The distribution and frequency of the L1014F mutation in Cx. quinquefasciatus populations was determined in rural and urban areas of Yucatan, Mexico from January 2015 to March 2016. Nine hundred fifty adult females out of 17,727 immature states were collected and analyzed in all sites sampled (n = 10). Susceptible homozygotes were identified (L1014/L1014) in 12% (114/950), heterozygous individuals (F1014/L1014) in 34% (323/950), and mutated homozygotes (F1014/F1014) in 54% (513/950) during the dry and rainy seasons. In this work, study areas with a high frequency of L1014F mutation were identified. These findings may help guarantee a more effective and efficient use of the resources available for the control of this vector.

New perspectives to the enterotoxigenic E. coli F4 porcine infection model: Susceptibility genotypes in relation to performance, diarrhoea and bacterial shedding

Enterotoxigenic E. coli (ETEC), causing post-weaning diarrhoea, is a major problem in weaned piglets. Individual animal responses to ETEC infection show high variability in animal experiments. Two studies were designed to optimize the ETEC F4ac infection model in piglets by combining the genotype susceptibility with performance, diarrhoea incidence and bacterial shedding. The studies were performed with respectively 120 and 80 male piglets that were tested for susceptibility or resistance towards ETEC O149:F4ac by a DNA marker based test. Three different genotypes were observed; resistant (RR), susceptible heterozygote (RS) and susceptible homozygote (SS). Piglets, were orally infected with an inoculum suspension (containing 1.5E8 CFU/ml ETEC F4ac) at day 0, 1 and 2 of the study. Performance, diarrhoea incidence and bacterial shedding were followed for 21days.In the first week after challenge a difference in average daily gain was observed between resistant and susceptible piglets in both studies. For the complete study period no significant differences were observed. Diarrhoea incidence was significantly higher in susceptible pigs compared to the resistant pigs in the first week after challenge. Bacterial shedding was much higher in the susceptible pigs and ETEC excretion lasted longer. ETEC was hardly detected in the faecal material of the resistant pigs. In conclusion, susceptible pigs showed higher diarrhoea incidence and higher numbers of faecal ETEC shedding in the first week after challenge compared to resistant pigs. The DNA marker based test can be used to select pigs that are susceptible for ETEC for inclusion in ETEC infection model, resulting in less animals needed to perform infection studies.

Composition and genetics of malaria vector populations in the Central African Republic.

BackgroundIn many African countries malaria has declined sharply due to a synergy of actions marked by the introduction of vector control strategies, but the disease remains a leading cause of morbidity and mortality in Central African Republic (CAR). An entomological study was initiated with the aim to characterize the malaria vectors in Bangui, the capital of CAR, and determine their vector competence.MethodsA cross-sectional entomological study was conducted in 15 sites of the district of Bangui, the capital of CAR, in September–October 2013 and a second collection was done in four of those sites between November and December 2013. Mosquitoes were collected by human landing catch (HLC) indoors and outdoors and by pyrethrum spray catch of indoor-resting mosquitoes. Mosquitoes were analysed for species and multiple other attributes, including the presence of Plasmodium falciparum circumsporozoite protein or DNA, blood meal source, 2La inversion karyotype, and the L1014F kdr insecticide resistance mutation.ResultsOverall, 1292 anophelines were analysed, revealing a predominance of Anopheles gambiae and Anopheles funestus, with a small fraction of Anopheles coluzzii. Molecular typing of the An. gambiae complex species showed that An. gambiae was predominant (95.7 %) as compared to An. coluzzii (2.1 %), and Anopheles arabiensis was not present. In some areas the involvement of secondary vectors, such as Anopheles coustani, expands the risk of infection. By HLC sampling, An. funestus displayed a stronger endophilic preference than mosquitoes from the An. gambiae sister taxa, with a mean indoor-capture rate of 54.3 % and 67.58 % for An. gambiae sister taxa and An. funestus, respectively. Human biting rates were measured overall for each of the species with 28 or 29 bites/person/night, respectively. Both vectors displayed a strong human feeding preference as determined by blood meal source, which was not different between the different sampling sites. An. coustani appears to be highly exophilic, with 92 % of HLC samples captured outdoors. The mean CSP rate in head-thorax sections of all Anopheles was 5.09 %, and was higher in An. gambiaes.l. (7.4 %) than in An. funestus (3.3 %). CSP-positive An. coustani were also detected in outdoor HLC samples. In the mosquitoes of the An. gambiae sister taxa the kdr-w mutant allele was nearly fixed, with 92.3 % resistant homozygotes, and no susceptible homozygotes detected.ConclusionsThis study collected data on anopheline populations in CAR, behaviour of vectors and transmission levels. Further studies should investigate the biting behaviour and susceptibility status of the anophelines to different insecticides to allow the establishment of appropriate vector control based on practical entomological knowledge.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1431-2) contains supplementary material, which is available to authorized users.

Prevalence of bovine dermatophilosis and disease-associated alleles in zebu Goudali cattle and their Italian Simmental crosses ranching in the western highland plateau savannah of Cameroon.

Abundance of native pastures makes Cameroon's western highland savannah (WHS) a hotspot for low-input beef-type cattle. Dumbo Ranch is central to cattle seed stock multiplication in WHS and holds that Dermatophilus congolensis infection undermines production. The bovine BoLA-DRB3 has been variously demonstrated as the principal gene of the major histocompatibility locus associated with immunity and resistance to dermatophilosis in cattle. We studied the profile of dermatophilosis prevalence in zebu Goudali (G) and its Simmental composite, SimGoud (SG), at Dumbo Ranch and determined the distribution of a dermatophilosis-associated susceptibility allele of the BoLA-DRB3 gene by allele-specific polymerase chain reaction (PCR). We recorded a 42% prevalence of dermatophilosis in the studied cohort (337 animals). Dermatophilosis was more common in older cattle than in cattle ≤36months (p ≤ 0.05). G was more affected compared to SG, because of the prevalence of the disease in the oldest animals and the age distribution of the experimental subjects. No susceptible homozygote was observed. About 85 and 15% of the cohort carried the homozygous resistant and heterozygous condition, respectively. This genotype distribution was not affected by cattle type. The study confirms the presence of dermatophilosis among G and SG cattle in WHS. However, there was no correlation between the presence of the disease-associated susceptible allele considered and clinical manifestation. Screening for this dermatophilosis resistance-associated allele of BoLA-DRB3 gene appeared not useful for selection of G and SG in WHS.

Fitness costs and stability of Cry1Fa resistance in Brazilian populations of Spodoptera frugiperda.

The presence of fitness costs of resistance to Bacillus thuringiensis (Bt) insecticidal proteins in insect populations may delay or even reverse the local selection of insect resistance to Bt transgenic crops, and deserves rigorous investigation. Here we assessed the fitness costs associated with Cry1Fa resistance in two strains of fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), derived from field collections in different Brazilian regions and further selected in the laboratory for high levels of resistance to Cry1Fa using leaves of TC1507 corn. Fitness components were compared using paired resistant and susceptible strains with similar genetic backgrounds and F1 generations from reciprocal crosses, all of them reared on non-transgenic corn leaves. No apparent life history costs in the larval stage were observed in the Bt-resistant strains. Moreover, the resistance remained stable for seven generations in the absence of selection, with no decrease in the proportion of resistant individuals. Larval respiration rates were also similar between resistant and susceptible homozygotes, and heterozygotes displayed respiration rates and demographic performance equal or superior to those of susceptible homozygotes. In combination, these results indicate the lack of strong fitness costs associated with resistance to Cry1Fa in the fall armyworm strains studied. These findings suggest that Cry1Fa resistance in S. frugiperda populations is unlikely to be counterselected in Cry1Fa-free environments. © 2016 Society of Chemical Industry.

Type of fitness cost influences the rate of evolution of resistance to transgenic Bt crops

Summary The evolution of resistance to pesticides by insect pests is a significant challenge for sustainable agriculture. For transgenic crops expressing Bacillus thuringiensis (Bt), crystalline (Cry) toxins resistance evolution may be delayed by the high‐dose/refuge strategy in which a non‐toxic refuge is planted to promote the survival of susceptible insects. The high‐dose/refuge strategy may interact with fitness costs associated with resistance alleles to further delay resistance. However, while a diverse range of fitness costs are reported in the field, they are typically represented as a fixed reduction in survival or viability which is insensitive to ecological conditions such as competition. Furthermore, the potential dynamic consequences of restricting susceptible insects to a refuge which represents only a fraction of the available space have rarely been considered.We present a generalized discrete time model which utilizes dynamic programming methods to derive the optimal management decisions for the control of a theoretical insect pest population exposed to Bt crops. We consider three genotypes (susceptible homozygotes, resistant homozygotes and heterozygotes) and implement fitness costs of resistance to Bt toxins as either a decrease in the relative competitive ability of resistant insects or as a penalty on fecundity. Model analysis is repeated and contrasted for two types of density dependence: uniform density dependence which operates equally across the landscape and heterogeneous density dependence where the intensity of competition scales inversely with patch size and is determined separately for the refuge and Bt crop.When the planting of Bt is decided optimally, fitness costs to fecundity allow for the planting of larger areas of Bt crops than equivalent fitness costs that reduce the competitive ability of resistant insects.Heterogeneous competition only influenced model predictions when the proportional area of Bt planted in each season was decided optimally and resistance was not recessive. Synthesis and applications. The high‐dose/refuge strategy alone is insufficient to preserve susceptibility to transgenic Bacillus thuringiensis (Bt) crops in the long term when constraints upon the evolution of resistance are not insurmountable. Fitness costs may enhance the delaying effect of the refuge, but the extent to which they do so depends upon how the cost is realized biologically. Fitness costs which apply independently of other variables may be more beneficial to resistance management than costs which are only visible to selection under a limited range of ecological conditions.

A novel er1 allele and the development and validation of its functional marker for breeding pea (Pisum sativum L.) resistance to powdery mildew.

A novel er1 allele, er1 -7, conferring pea powdery mildew resistance was characterized by a 10-bp deletion in PsMLO1 cDNA, and its functional marker was developed and validated in pea germplasms. Pea powdery mildew caused by Erysiphe pisi DC is a major disease worldwide. Pea cultivar 'DDR-11' is an elite germplasm resistant to E. pisi. To identify the gene conferring resistance in DDR-11, the susceptible Bawan 6 and resistant DDR-11 cultivars were crossed to produce F1, F2, and F(2:3) populations. The phenotypic segregation patterns in the F2 and F(2:3) populations fit the 3:1 (susceptible:resistant) and 1:2:1 (susceptible homozygotes:heterozygotes:resistant homozygotes) ratios, respectively, indicating that resistance was controlled by a single recessive gene. Analysis of er1-linked markers in the F2 population suggested that the recessive resistance gene in DDR-11 was an er1 allele, which was mapped between markers ScOPE16-1600 and c5DNAmet. To further characterize er1 allele, the cDNA sequences of PsMLO1 from the parents were obtained and a novel er1 allele in DDR-11 was identified and designated as er1-7, which has a 10-bp deletion in position 111-120. The er1-7 allele caused a frame-shift mutation, resulting in a premature termination of translation of PsMLO1 protein. A co-dominant functional marker specific for er1-7 was developed, InDel111-120, which co-segregated with E. pisi resistance in the mapping population. The marker was able to distinguish between pea germplasms with and without the er1-7. Of 161 pea germplasms tested by InDel111-120, seven were detected containing resistance allele er1-7, which was verified by sequencing their PsMLO1 cDNA. Here, a novel er1 allele was characterized and its an ideal functional marker was validated, providing valuable genetic information and a powerful tool for breeding pea resistance to powdery mildew.

Estimation of allele-specific Ace-1 duplication in insecticide-resistant Anopheles mosquitoes from West Africa.

Background Identification of variation in Ace-1 copy number and G119S mutation genotype from samples of Anopheles gambiae and Anopheles coluzzii across West Africa are important diagnostics of carbamate and organophosphate resistance at population and individual levels. The most widespread and economical method, PCR–RFLP, suffers from an inability to discriminate true heterozygotes from heterozygotes with duplication. MethodsIn addition to PCR–RFLP, in this study three different molecular techniques were applied on the same mosquito specimens: TaqMan qPCR, qRTPCR and ddPCR. To group heterozygous individuals recorded from the PCR–RFLP analysis into different assumptive genotypes K-means clustering was applied on the Z-scores of data obtained from both the TaqMan and ddPCR methods. The qRTPCR analysis was used for absolute quantification of copy number variation.ResultsThe results indicate that most heterozygotes are duplicated and that G119S mutation must now be regarded as a complex genotype ranging from primarily single-copy susceptible Glycine homozygotes to balanced and imbalanced heterozygotes, and multiply-amplified resistant Serine allele homozygotes. Whilst qRTPCR-based gene copy analysis suffers from some imprecision, it clearly illustrates differences in copy number among genotype groups identified by TaqMan or ddPCR. Based on TaqMan method properties, and by coupling TaqMan and ddPCR methods simultaneously on the same type of mosquito specimens, it demonstrated that the TaqMan genotype assays associated with the K-means clustering algorithm could provide a useful semi-quantitative estimate method to investigate the level of allele-specific duplication in mosquito populations.ConclusionsAce-1 gene duplication is evidently far more complex in An. gambiae and An. coluzzii than the better-studied mosquito Culex quinquefasciatus, which consequently can no longer be considered an appropriate model for prediction of phenotypic consequences. These require urgent further evaluation in Anopheles. To maintain the sustained effectiveness carbamates and organophosphates as alternative products to pyrethroids for malaria vector control, monitoring of duplicated resistant alleles in natural populations is essential to guide the rational use of these insecticides.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-1026-3) contains supplementary material, which is available to authorized users.

Distribution and frequency of G119S mutation in ace-1 gene within Anopheles sinensis populations from Guangxi, China.

BackgroundMalaria is one of the most serious vector-borne diseases in the world. Vector control is an important measure for malaria prevention and elimination. However, this strategy is under threat as disease vectors are developing resistance to insecticides. Therefore, it is important to monitor mechanisms responsible for insecticide resistance. In this study, the presence of G119S mutation in the acetyl cholinesterase-encoding gene (ace-1) was investigated in nine Anopheles sinensis populations sampled across Guangxi Zhuang Autonomous Region China.MethodsPCR–RFLP (polymerase chain reaction-restriction fragment length polymorphism) method was used to genotype each individual adult of An. sinensis. Direct sequencing of PCR products was performed to verify the accuracy of PCR–RFLP genotyping result. Population genetics analysis was conducted using Genepop programme.ResultsThe frequencies of susceptible homozygotes, heterozygotes and resistant homozygotes in the nine populations ranged between 0–0.296, 0.143–0.500 and 0.333–0.857, respectively. Overall, a high frequency (0.519–0.929) of mutant 119S allele was observed and the genotype frequency of the ace-1 gene of An. sinensis was at Hardy–Weinberg equilibrium in each of the nine examined populations.ConclusionThe G119S mutation has become fixed and is widespread in An. sinensis field populations in Guangxi, China. These findings are useful in helping design strategies for An. sinensis control.

Resistance to Phytophthora Infestans in Three Solanum Nigrum F3 Families

AbstractSolanum nigrumis a self-pollinating, hexaploid weed and one of a fewSolanaceaespecies native to Europe. It used to be described as a non-host forPhytophthora infestans. However, now it is known that, like its distant relatives: potato (Solanum tuberosumL.) and tomato (Solanum lycopersicumL.),S. nigrumcan suffer from potato late blight caused by this pathogen. Both susceptible and resistantS. nigrumgenotypes have been previously identified and inheritance of resistance originating from one accession has been described based on population of F2plants and 15 F3lines. The goal of this study was to evaluate resistance of three families of F3lines, originating from crosses between a susceptible and three different resistantS. nigrumaccessions followed by two self-pollinations. Parental accessions were tested for the spectrum of late blight resistance against 48P. infestansisolates. The three families consisted of 106, 96 and 115 F3lines, respectively, and from each line 20 plants were tested for resistance toP. infestans. Laboratory detached leaf assays were performed in two dates and two replications of three leaves each. Segregation of the trait within the line allowed us to distinguish hetero- and homozygous lines. In one F3family, the ratio of resistant homozygotes: heterozygotes: susceptible homozygotes was 1:2:1, indicating that a single gene is most likely underlying the late blight resistance in this case. In the other two, observed segregations of the trait significantly deviated from this model suggesting more complex inheritance patterns.