Biallelic Markers Research Articles

Gene-Based Methods to Detect Gene-Gene Interaction in R: The GeneGeneInteR Package

GeneGeneInteR is an R package dedicated to the detection of an association between a case-control phenotype and the interaction between two sets of biallelic markers (single nucleotide polymorphisms or SNPs) in case-control genome-wide associations studies. The development of statistical procedures for searching gene-gene interaction at the SNP-set level has indeed recently grown in popularity as these methods confer advantage in both statistical power and biological interpretation. However, all these methods have been implemented in home made softwares that are for most of them available only on request to the authors and at best have a web interface. Since the implementation of these methods is not straightforward, there is a need for a user-friendly tool to perform gene-based genegene interaction. The purpose of GeneGeneInteR is to propose a collection of tools for all the steps involved in gene-based gene-gene interaction testing in case-control association studies. Illustrated by an example of a dataset related to rheumatoid arthritis, this paper details the implementation of the functions available in GeneGeneInteR to perform an analysis of a collection of SNP sets. Such an analysis aims at addressing the complete statistical pipeline going from data importation to the visualization of the results through data manipulation and statistical analysis.

Population data for 29 biallelic markers on Y chromosome from three groups of Chinese han individuals

Objective29 biallelic markers on Y chromosome were typed in order to compare three groups of Chinese individuals within Han population. Method29 Y chromosome-specific biallelic markers that are polymorphic in Eastern Chinese Han (ECH) population were selected. 343 unrelated ECH male samples were analyzed for four multiplex systems. The detection of genotype was performed on 3130 Genetic Analyzer. Forensic genetic parameters were calculated. Comparison between different populations was performed using Arlequin software. ResultsA total of 64 haplotypes were detected. The haplotype diversity of the 29 loci reached 0.9010. After performing allele frequencies comparison among the Han population in three regions, it was shown that significant differences between: Northwest Chinese Han (NCH) and ECH at two loci, Southern Chinese Han (SCH) and ECH at four markers, SCH and NCH at 17 loci.The data also showed that there were some degrees of genetic differentiation (FST: 0.05˜0.70) at: two loci between ECH and NCH, two loci between ECH and SCH and nine between NCH and SCH. ConclusionsThe presented study shows the usefulness of the Y-chromosomal biallelic markers for forensic purposes. The panel may be suitable for differentiation within Chinese Han populations.

Genetic polymorphism of 190 Y-SNPs in Han population from Jiangsu province, China

Since Y chromosome genetic markers are male-specific and follow strict paternal inheritance, they are powerful tools in forensic genetic. Single nucleotide polymorphism (SNP) is one of the bi-allelic genetic markers, and its allele frequencies are distinct between populations from different regions, which proves that SNPs are associated with ancestry informative markers (AIMs). The Han population accounts for over 90% of the total population in China. However, studies on sub-haplogroup of the population have been insufficient. In this study, a total of 119 unrelated male Han individuals from Liyang, Jiangsu Province were typed by 190 Y-SNPs including 154 sub-haplogroups of O-M175 haplogroup. The haplogroup diversity reached 0.9360 and 41 terminal haplogroups were observed simultaneously, of which O2a2a1a1_CTS201 haplogroup (6.72%) was the most predominant haplogroup. The results demonstrated that 1) most samples (84.87%) from Jiangsu Han population in this study were genotyped into O-M175 haplogroup; and 2) the O2 sub-haplogroup is larger than the O1 sub-haplogroup in Han population from Jiangsu province, China.

Evaluation of 12 Multi-InDel markers for forensic ancestry prediction in Asian populations

Various types of genetic markers have been applied to forensic ancestry inference. Biallelic markers, such as SNPs and InDels, have proven to be optimal choices except for the low information content provided by a single locus. Multi-InDel marker is defined as a specific DNA fragment with several InDel markers located tightly in the physical position. Previous research indicates that multi-InDel markers perform well in population analysis and ancestry inference because of higher degree of polymorphism and remarkable population differences. In this study, a panel consisting of 12 multi-InDel markers was employed to evaluate the general performance in forensic practice and the discrimination power for population analysis. Sample types encountered in routine forensic practice were genotyped to validate the feasibility of regular use. A population study was performed on a total of five Asian populations to verify the discrimination power. Moreover, a double-blind test for ancestry prediction was conducted to assess the predictive capability. In conclusion, these results revealed the significance of multi-InDel markers for population structure stratification. The present panel showed the potential as a valid complementary tool in forensic applications.

Pedigree relationships to control inbreeding in optimum-contribution selection realise more genetic gain than genomic relationships

BackgroundWe tested the premise that optimum-contribution selection with pedigree relationships to control inbreeding (POCS) realises at least as much true genetic gain as optimum-contribution selection with genomic relationships (GOCS) at the same rate of true inbreeding.MethodsWe used stochastic simulation to estimate rates of true genetic gain realised by POCS and GOCS at a 0.01 rate of true inbreeding in three breeding schemes with best linear unbiased predictions of breeding values based on pedigree (PBLUP) and genomic (GBLUP) information. The three breeding schemes differed in number of matings and litter size. Selection was for a single trait with a heritability of 0.2. The trait was controlled by 7702 biallelic quantitative-trait loci (QTL) that were distributed across a 30-M genome. The genome contained 54,218 biallelic markers that were used in GOCS and GBLUP. A total of 6012 identity-by-descent loci were placed across the genome in base populations. Unique alleles at these loci were used to calculate rates of true inbreeding. Breeding schemes were run for 10 discrete generations. Selection candidates were genotyped and phenotyped before selection.ResultsPOCS realised more true genetic gain than GOCS at a 0.01 rate of true inbreeding in all combinations of breeding scheme and prediction method. POCS realised 14 to 33% more true genetic gain than GOCS with PBLUP in the three breeding schemes. It realised 1.5 to 5.7% more true genetic gain than GOCS with GBLUP.ConclusionsPOCS realised more true genetic gain than GOCS because it managed expected genetic drift without restricting selection at QTL. By contrast, GOCS penalised changes in allele frequencies at markers that were generated by genetic drift and selection. Because these marker alleles were in linkage disequilibrium with QTL alleles, GOCS restricted changes in allele frequencies at QTL. This provides little incentive to use GOCS and highlights that we have more to learn before we can control inbreeding using genomic relationships in selective-breeding schemes. Until we can do so, POCS remains a worthy method of optimum-contribution selection because it realises more true genetic gain than GOCS at the same rate of true inbreeding.

Y-Chromosome Marker Characterization of Epipaleolithic and Neolithic Groups of Southern India

The aim of the study is to investigate the Y-chromosome unique event polymorphisms (UEPs), to know the origin and past demographic history of paternal lineages, genetic relatedness, and admixtures in two tribal populations of Southern India. A total of 106 male samples from two Dravidian speaking tribal populations of Southern India: Urali Kuruman (n = 50) and Melakudiya (n = 56) were analyzed. A set of 30 bi-allelic UEP markers of the non-recombining region of the Y-chromosome was sequenced by the Sanger sequencing method. The phylogenetic analysis of the two populations revealed six Y-chromosome haplogroups: C, F*, H, K*, L*, and R2. The Urali Kuruman Y-chromosome lineage was predominantly of native origin clustering with other Dravidian tribes of the region, whereas the Melakudiya Y-chromosome lineage clustered with the people of Near East, and other Indo-European speakers of India.

Estimating Relatedness Between Malaria Parasites.

Understanding the relatedness of individuals within or between populations is a common goal in biology. Increasingly, relatedness features in genetic epidemiology studies of pathogens. These studies are relatively new compared to those in humans and other organisms, but are important for designing interventions and understanding pathogen transmission. Only recently have researchers begun to routinely apply relatedness to apicomplexan eukaryotic malaria parasites, and to date have used a range of different approaches on an ad hoc basis. Therefore, it remains unclear how to compare different studies and which measures to use. Here, we systematically compare measures based on identity-by-state (IBS) and identity-by-descent (IBD) using a globally diverse data set of malaria parasites, Plasmodium falciparum and P. vivax, and provide marker requirements for estimates based on IBD. We formally show that the informativeness of polyallelic markers for relatedness inference is maximized when alleles are equifrequent. Estimates based on IBS are sensitive to allele frequencies, which vary across populations and by experimental design. For portability across studies, we thus recommend estimates based on IBD. To generate estimates with errors below an arbitrary threshold of 0.1, we recommend ∼100 polyallelic or 200 biallelic markers. Marker requirements are immediately applicable to haploid malaria parasites and other haploid eukaryotes. C.I.s facilitate comparison when different marker sets are used. This is the first attempt to provide rigorous analysis of the reliability of, and requirements for, relatedness inference in malaria genetic epidemiology. We hope it will provide a basis for statistically informed prospective study design and surveillance strategies.

Formula omitted] and the triangle inequality for biallelic markers

The population differentiation statistic FST, introduced by Sewall Wright, is often treated as a pairwise distance measure between populations. As was known to Wright, however, FST is not a true metric because allele frequencies exist for which it does not satisfy the triangle inequality. We prove that a stronger result holds: for biallelic markers whose allele frequencies differ across three populations, FSTnever satisfies the triangle inequality. We study the deviation from the triangle inequality as a function of the allele frequencies of three populations, identifying the frequency vector at which the deviation is maximal. We also examine the implications of the failure of the triangle inequality for four-point conditions for placement of groups of four populations on evolutionary trees. Next, we study the extent to which FST fails to satisfy the triangle inequality in human genomic data, finding that some loci produce deviations near the maximum. We provide results describing the consequences of the theory for various types of data analysis, including multidimensional scaling and inference of neighbor-joining trees from pairwise FST matrices.

The horse Y chromosome as an informative marker for tracing sire lines

Analysis of the Y chromosome is the best-established way to reconstruct paternal family history in humans. Here, we applied fine-scaled Y-chromosomal haplotyping in horses with biallelic markers and demonstrate the potential of our approach to address the ancestry of sire lines. We de novo assembled a draft reference of the male-specific region of the Y chromosome from Illumina short reads and then screened 5.8 million basepairs for variants in 130 specimens from intensively selected and rural breeds and nine Przewalski’s horses. Among domestic horses we confirmed the predominance of a young’crown haplogroup’ in Central European and North American breeds. Within the crown, we distinguished 58 haplotypes based on 211 variants, forming three major haplogroups. In addition to two previously characterised haplogroups, one observed in Arabian/Coldblooded and the other in Turkoman/Thoroughbred horses, we uncovered a third haplogroup containing Iberian lines and a North African Barb Horse. In a genealogical showcase, we distinguished the patrilines of the three English Thoroughbred founder stallions and resolved a historic controversy over the parentage of the horse ‘Galopin’, born in 1872. We observed two nearly instantaneous radiations in the history of Central and Northern European Y-chromosomal lineages that both occurred after domestication 5,500 years ago.

Genetic association with high-resolution climate data reveals selection footprints in the genomes of barley landraces across the Iberian Peninsula.

Landraces are local populations of crop plants adapted to a particular environment. Extant landraces are surviving genetic archives, keeping signatures of the selection processes experienced by them until settling in their current niches. This study intends to establish relationships between genetic diversity of barley (Hordeum vulgare L.) landraces collected in Spain and the climate of their collection sites. A high‐resolution climatic data set (5 × 5 km spatial, 1‐day temporal grid) was computed from over 2,000 temperature and 7,000 precipitation stations across peninsular Spain. This data set, spanning the period 1981–2010, was used to derive agroclimatic variables meaningful for cereal production at the collection sites of 135 barley landraces. Variables summarize temperature, precipitation, evapotranspiration, potential vernalization and frost probability at different times of the year and time scales (season and month). SNP genotyping of the landraces was carried out combining Illumina Infinium assays and genotyping‐by‐sequencing, yielding 9,920 biallelic markers (7,479 with position on the barley reference genome). The association of these SNPs with agroclimatic variables was analysed at two levels of genetic diversity, with and without taking into account population structure. The whole data sets and analysis pipelines are documented and available at https://eead-csic-compbio.github.io/barley-agroclimatic-association. We found differential adaptation of the germplasm groups identified to be dominated by reactions to cold temperature and late‐season frost occurrence, as well as to water availability. Several significant associations pointing at specific adaptations to agroclimatic features related to temperature and water availability were observed, and candidate genes underlying some of the main regions are proposed.

, Jost's D, and FST are similarly constrained by allele frequencies: A mathematical, simulation, and empirical study.

Statistics and Jost's D have been proposed for replacing FST as measures of genetic differentiation. A principal argument in favour of these statistics is the independence of their maximal values with respect to the subpopulation heterozygosity HS , a property not shared by FST . Nevertheless, it has been unclear if these alternative differentiation measures are constrained by other aspects of the allele frequencies. Here, for biallelic markers, we study the mathematical properties of the maximal values of and D, comparing them to those of FST . We show that and D exhibit the same peculiar frequency-dependence phenomena as FST , including a maximal value as a function of the frequency of the most frequent allele that lies well below one. Although the functions describing , D, and FST in terms of the frequency of the most frequent allele are different, the allele frequencies that maximize them are identical. Moreover, we show using coalescent simulations that when taking into account the specific maximal values of the three statistics, their behaviours become similar across a large range of migration rates. We use our results to explain two empirical patterns: the similar values of the three statistics among North American wolves, and the low D values compared to and FST in Atlantic salmon. The results suggest that the three statistics are often predictably similar, so that they can make quite similar contributions to data analysis. When they are not similar, the difference can be understood in relation to features of genetic diversity.

Quantitative Trait Loci for Freezing Tolerance in a Lowland x Upland Switchgrass Population.

Low-temperature related abiotic stress is an important factor affecting winter survival in lowland switchgrass when grown in northern latitudes in the United States. A better understanding of the genetic architecture of freezing tolerance in switchgrass will aid the development of lowland switchgrass cultivars with improved winter survival. The objectives of this study were to conduct a freezing tolerance assessment, generate a genetic map using single nucleotide polymorphism (SNP) markers, and identify QTL (quantitative trait loci) associated with freezing tolerance in a lowland × upland switchgrass population. A pseudo-F2 mapping population was generated from an initial cross between the lowland population Ellsworth and the upland cultivar Summer. The segregating progenies were screened for freezing tolerance in a controlled-environment facility. Two clonal replicates of each genotype were tested at six different treatment temperatures ranging from −15 to −5°C at an interval of 2°C for two time periods. Tiller emergence (days) and tiller number were recorded following the recovery of each genotype with the hypothesis that upland genotype is the source for higher tiller number and early tiller emergence. Survivorship of the pseudo-F2 population ranged from 89% at −5°C to 5% at −15°C with an average LT50 of −9.7°C. Genotype had a significant effect on all traits except tiller number at −15°C. A linkage map was constructed from bi-allelic single nucleotide polymorphism markers generated using exome capture sequencing. The final map consisted of 1618 markers and 2626 cM, with an average inter-marker distance of 1.8 cM. Six significant QTL were identified, one each on chromosomes 1K, 5K, 5N, 6K, 6N, and 9K, for the following traits: tiller number, tiller emergence days and LT50. A comparative genomics study revealed important freezing tolerance genes/proteins, such as COR47, DREB2B, zinc finger-CCCH, WRKY, GIGANTEA, HSP70, and NRT2, among others that reside within the 1.5 LOD confidence interval of the identified QTL.

Comparison of non-parametric methods in genomic evaluation of discrete traits

The prediction performance of SVM and Bayesian methods in genomic evaluation of discrete traits was compared in different scenarios of heritability level (0.1, 0.3, and 0.5), number of quantitative trait loci, QTL (60, 300 and 600) and reference population size (1000, 2000 and 5000 individuals). To do this, a genome comprised of 3 chromosomes was simulated on which 6000 bi-allelic SNP markers were distributed. The QTL effects were modeled with gamma distribution was used to model. Pearson's correlation between the true and predicted genomic breeding values was used as the measure of the predictive accuracy. Results showed the significant effect of heritability on prediction accuracy in a way that by increasing heritability, the accuracy of prediction increased significantly. However, the accuracy of prediction was not affected by the number of QTLs. In addition, by increasing the size of the reference population from 1000 to 5000 individuals, the accuracy of prediction increased. Among different kernel functions used to construct SVM, the radial function had higher predictive performance and therefore was used to analysis data. In all the scenarios studied, BayesA outperformed SVM, especially where the heritability level was low. Therefore, the BayesA was recommended for genomic selection of discrete traits. In addition, it was recommended that in different scenarios of genomic selection, different methods should be compared and the best method should be applied for genomic selection.

Association of M470V polymorphism of CFTR gene with variability of clinical expression of asthma: Case-report study

Introduction and ObjectivesAsthma is a complex genetic disorder. Several genes have been found associated with asthma. The cystic fibrosis transmembrane conductance regulator (CFTR) gene is one of them. The aim of this study was to perform a comparative analysis of the genotype and allele frequency distributions of the biallelic marker M470V within the CFTR gene on mutant and wide chromosomes. Patients and methodsThe molecular approach consists in the genotyping of the M470V marker by the PCR-RFLP technique in 105 asthmatic patients, aged between four months and 17 years, and 105 healthy subjects. ResultsWe found a significant difference in the genotype frequencies between the two studied groups (χ2=9.855, P=0.007). The V/V genotype was over represented in the asthmatic group as compared to the controls (32.38% vs. 16.19%). Whereas, the M/V genotype is more frequent in healthy subjects (40.95% vs. 28.71%). We also noted a significant difference in allelic distribution of M470V with associated diseases (χ2=9.610, P=0.022). ConclusionsThe present study is the first report on the distribution of the M470V polymorphism in asthmatic Tunisian patients. We noticed that the M470V variant could modulate the clinical phenotype of asthmatic patients. This preliminary study will establish the molecular basis of this disease in Tunisia.

High cryptic species diversity is revealed by genome-wide polymorphisms in a wild relative of banana, Musa itinerans, and implications for its conservation in subtropical China

BackgroundSpecies delimitation is a challenging but essential task in conservation biology. Morphologically similar species are sometimes difficult to recognize even after examination by experienced taxonomists. With the advent of molecular approaches in species delimitation, this hidden diversity has received much recent attention. In addition to DNA barcoding approaches, analytical tools based on the multi-species coalescence model (MSC) have been developed for species delimitation. Musa itinerans is widely distributed in subtropical Asia, and at least six varieties have been documented. However, the number of evolutionarily distinct lineages remains unknown.ResultsUsing genome resequencing data of five populations (making up four varieties), we examined genome-wide variation and found four varieties that were evolutionary significant units. A Bayesian Phylogenetics and Phylogeography (BP&P) analysis using 123 single copy nuclear genes support three speciation events of M. itinerans varieties with robust posterior speciation probabilities; However, a Bayes factor delimitation of species with genomic data (BFD*) analysis using 1201 unlinked single nucleotide polymorphisms gave decisive support for a five-lineage model. When reconciling divergence time estimates with a speciation time scale, a modified three-lineage model was consistent with that of BP&P, in which the speciation time of two varieties (M. itinerans var. itinerans and M. itinerans var. lechangensis) were dated to 26.2 kya and 10.7 kya, respectively. In contrast, other two varieties (M. itinerans var. chinensis and M. itinerans var. guangdongensis) diverged only 3.8 kya in the Anthropocene; this may be a consequence of genetic drift rather than a speciation event.ConclusionOur results showed that the M. itinerans species complex harbours high cryptic species diversity. We recommend that M. itinerans var. itinerans and M. itinerans var. lechangensis be elevated to subspecies status, and the extremely rare latter subspecies be given priority for conservation. We also recommend that the very recently diverged M. itinerans var. chinensis and M. itinerans var. guangdongensis should be merged under the subspecies M. itinerans var. chinensis. Finally, we speculate that species delimitation of recently diverged lineages may be more effective using genome-wide bi-allelic SNP markers with BFD* than by using unlinked loci and BP&P.

P101 Chimerism monitoring by next-generation sequencing: A multiplatform comparison

Accurate monitoring of the chimeric status after stem cell transplantation is essential for early detection of relapse and at the moment mainly performed by STR or qPCR. The major disadvantages of STR are the laborious data analysis and poor sensitivity. The qPCR technique enables a much quicker workflow and better sensitivity. However a disadvantage is the need of a pre-transplant sample for each monitoring event. Performing chimerism monitoring by NGS will eliminate these limitations while allowing for a multiplexed setup, reducing the amount of needed lab work and DNA. The workflow can be combined with routinely applied NGS HLA typing in clinical laboratories. PCR amplification primers have been designed for a set of biallelic markers. These markers were tested on a number of artificial chimeric samples in a range between 0.01–100% of positive DNA mixed with negative DNA. Amplicons were applied in the respective NGSgo library preparation workflows and run on Illumina MiSeq, Ion Torrent S5 and Oxford Nanopore MinION systems. NGS data was analysed with customized analysis tools designed to quantitate the two variants of each marker. Each marker was tested on a small panel of non-chimeric samples (Coriell Institute) and resulted in clear homo- or heterozygous patterns for all markers. Results of the different platforms on the artificial chimeric samples in a range of 0.01%–100% were compared. Sensitivity was determined for each sequencing platform. A read depth threshold was established for each of the platforms to achieve a minimum sensitivity of 0.05%. Results including difference between sequencing platforms will be discussed further. The results demonstrate that NGS-based chimerism monitoring is feasible for a wide range of NGS platforms, with a high sensitivity and wide dynamic range. E.E. Bouwmans: 5. Employee; Company/Organization; GenDx. L.A. van de Pasch: 5. Employee; Company/Organization; GenDx. M.T. Penning: 5. Employee; Company/Organization; GenDx. E. Rozemuller: 5. Employee; Company/Organization; GenDx. 6. Stock Shareholder; Company/Organization; GenDx. 7. Other (Identify); Company/Organization; Founder of GenDx.

Inference of species phylogenies from bi-allelic markers using pseudo-likelihood.

MotivationPhylogenetic networks represent reticulate evolutionary histories. Statistical methods for their inference under the multispecies coalescent have recently been developed. A particularly powerful approach uses data that consist of bi-allelic markers (e.g. single nucleotide polymorphism data) and allows for exact likelihood computations of phylogenetic networks while numerically integrating over all possible gene trees per marker. While the approach has good accuracy in terms of estimating the network and its parameters, likelihood computations remain a major computational bottleneck and limit the method’s applicability.ResultsIn this article, we first demonstrate why likelihood computations of networks take orders of magnitude more time when compared to trees. We then propose an approach for inference of phylogenetic networks based on pseudo-likelihood using bi-allelic markers. We demonstrate the scalability and accuracy of phylogenetic network inference via pseudo-likelihood computations on simulated data. Furthermore, we demonstrate aspects of robustness of the method to violations in the underlying assumptions of the employed statistical model. Finally, we demonstrate the application of the method to biological data. The proposed method allows for analyzing larger datasets in terms of the numbers of taxa and reticulation events. While pseudo-likelihood had been proposed before for data consisting of gene trees, the work here uses sequence data directly, offering several advantages as we discuss.Availability and implementationThe methods have been implemented in PhyloNet (http://bioinfocs.rice.edu/phylonet).

Molecular Characterization of Mung Bean Germplasm from the USDA Core Collection Using Newly Developed KASP‐based SNP Markers

Mung bean [Vigna radiata (L.) Wilczek] is an important, nutritious legume food crop for tropical and subtropical countries with high value in Asia and worldwide potential. However, the genetic improvement of mung bean is slowed by low polymorphism of previous molecular markers, even microsatellites. The goals of this study were to develop Kompetitive Allele Specific Polymerase Chain Reaction (KASP)‐based single‐nucleotide polymorphism (SNP) markers and use them for characterization of 94 cultivated mung bean genotypes from the USDA originating in 27 countries across 10 regions of the world, all being cultivars rather than wild accessions. We targeted 42 SNPs from previous sequencing and converted them into 20 robust KASP assays. Of these, 18 were polymorphic among the mung bean cultivars, with 38 alleles total and 1.9 alleles per locus average. The polymorphism information content of the newly developed markers ranged from zero for monomorphic markers to 0.375 for the most diverse biallelic polymorphic marker (MBkSNP_39) and averaged 0.250 across all loci. Population structure analysis grouped 90% of the accessions into two subpopulations, with 10% belonging to an admixture group, but did not follow geographic origins of the germplasm, suggesting no clear center of origin and blending of the subpopulations. An analysis of molecular variance revealed 22% of genetic variation among subpopulations and 78% within subpopulations. The first two axes of region‐wide principle coordinate analysis explained 81.26% variation of total variation, indicating the existence of genetic diversity among groups. The SNP markers of this study can be used in molecular breeding of mung bean and are the first to work with KASP detection.

Maximization of Markers Linked in Coupling for Tetraploid Potatoes via Monoparental Haploids

Haploid potato populations derived from a single tetraploid donor constitute an efficient strategy to analyze markers segregating from a single donor genotype. Analysis of marker segregation in populations derived from crosses between polysomic tetraploids is complicated by a maximum of eight segregating alleles, multiple dosages of the markers and problems related to linkage analysis of marker segregation in repulsion. Here, we present data on two monoparental haploid populations generated by prickle pollination of two tetraploid cultivars with Solanum phureja and genotyped with the 12.8 k SolCAP single nucleotide polymorphism (SNP) array. We show that in a population of monoparental haploids, the number of biallelic SNP markers segregating in linkage to loci from the tetraploid donor genotype is much larger than in putative crosses of this genotype to a diverse selection of 125 tetraploid cultivars. Although this strategy is more laborious than conventional breeding, the generation of haploid progeny for efficient marker analysis is straightforward if morphological markers and flow cytometry are utilized to select true haploid progeny. The level of introgressed fragments from S. phureja, the haploid inducer, is very low, supporting its suitability for genetic analysis. Mapping with single-dose markers allowed the analysis of quantitative trait loci (QTL) for four phenotypic traits.

Influence of polymorphisms in toll-like receptors (TLRs) on malaria susceptibility in low-endemic area of the Atlantic Forest, São Paulo, Brazil

In low-endemic areas for malaria transmission, asymptomatic individuals play an important role as reservoirs for malarial infection. Understanding the dynamics of asymptomatic malaria is crucial for its efficient control in these regions. Genetic host factors such as Toll-like receptor (TLR) polymorphisms may play a role in the maintenance or elimination of infection. In this study, the effect of TLR polymorphisms on the susceptibility to malaria was investigated among individuals living in the Atlantic Forest of São Paulo, Southern Brazil. A hundred and ninety-five Brazilian individuals were enrolled and actively followed up for malaria for three years. Twenty-four polymorphisms in five toll-like receptor (TLR) genes were genotyped by RFLP, direct sequencing or fragment analysis. The genotypes were analyzed for the risk of malaria. Ongoing Plasmodium vivax or P. malariae infection, was identified by the positive results in PCR tests and previous P. vivax malaria, was assumed when antiplasmodial antibodies against PvMSP119 were detected by ELISA. An evaluation of genomic ancestry was conducted using biallelic ancestry informative markers and the results were used as correction in the statistical analysis. Nine SNPs and one microsatellite were found polymorphic and three variant alleles in TLR genes were associated to malaria susceptibility. The regression coefficient estimated for SNP TLR9.-1237.T/C indicated that the presence of at least one allele C increased, on average, 2.3 times the malaria odds, compared to individuals with no allele C in this SNP. However, for individuals with the same sex, age and household, the presence of at least one allele C in SNP TLR9.-1486.T/C reduced, on average, 1.9 times the malaria odds, compared to individuals with no allele C. Moreover, this allele C plus an S allele in TLR6.P249S in individuals with same sex, age and ancestry, reduced, on average, 4.4 times the malaria odds. Our findings indicate a significant association of TLR9.-1237.T/C gene polymorphism with malarial infection and contribute to a better knowledge of the role of TLRs in malaria susceptibility in an epidemiological setting different from other settings.