Haplotype inference from diploid sequence data: evaluating performance using non-neutral MHC sequences

The direct sequencing of PCR products from diploid organisms is problematic because of ambiguities associated with phase inference in multi-site heterozygotes. Several molecular methods such as cloning, SSCP, and DGGE have been developed to empirically reduce diploid sequences to their constitutive haploid components, but in theory these empirical approaches can be supplanted by analytical treatment of diploid sequences. Analytical approaches are more desirable than molecular methods because of the added time and expense required to generate molecular data. A variety of analytical methods have been developed to address this issue, but few have been rigorously evaluated with empirical data. Furthermore, they all assume that the sequences under consideration are evolving in a neutral fashion and assume a moderate number of heterozygous sites. Here, we use non-neutral major histocompatibility complex (MHC) sequences comprised of large numbers of heterozygous sites that are under strong balancing selection to evaluate the performance of the popular Bayesian algorithm implemented by the program PHASE. Our results suggest that PHASE performs admirably with non-neutral sequences of moderate length with numerous heterozygous sites typical of MHC class II sequences. We conclude that analytical approaches to haplotype inference have great potential in large-scale population genetic assays, but recommend groundtruthing analytical results using empirical (molecular) approaches at the outset of population-level analyses.     

Linkage mapping and comparative genomics using next-generation RAD sequencing of a non-model organism

Restriction-site associated DNA (RAD) sequencing is a powerful new method for targeted sequencing across the genomes of many individuals. This approach has broad potential for genetic analysis of non-model organisms including genotype-phenotype association mapping, phylogeography, population genetics and scaffolding genome assemblies through linkage mapping. We constructed a RAD library using genomic DNA from a Plutella xylostella (diamondback moth) backcross that segregated for resistance to the insecticide spinosad. Sequencing of 24 individuals was performed on a single Illumina GAIIx lane (51 base paired-end reads). Taking advantage of the lack of crossing over in homologous chromosomes in female Lepidoptera, 3,177 maternally inherited RAD alleles were assigned to the 31 chromosomes, enabling identification of the spinosad resistance and W/Z sex chromosomes. Paired-end reads for each RAD allele were assembled into contigs and compared to the genome of Bombyx mori (n = 28) using BLAST, revealing 28 homologous matches plus 3 expected fusion/breakage events which account for the difference in chromosome number. A genome-wide linkage map (1292 cM) was inferred with 2,878 segregating RAD alleles inherited from the backcross father, producing chromosome and location specific sequenced RAD markers. Here we have used RAD sequencing to construct a genetic linkage map de novo for an organism that has no previous genome data. Comparative analysis of P. xyloxtella linkage groups with B. mori chromosomes shows for the first time, genetic synteny appears common beyond the Macrolepidoptera. RAD sequencing is a powerful system capable of rapidly generating chromosome specific data for non-model organisms.     

Haplotype phasing: existing methods and new developments

Determination of haplotype phase is becoming increasingly important as we enter the era of large-scale sequencing because many of its applications, such as imputing low-frequency variants and characterizing the relationship between genetic variation and disease susceptibility, are particularly relevant to sequence data. Haplotype phase can be generated through laboratory-based experimental methods, or it can be estimated using computational approaches. We assess the haplotype phasing methods that are available, focusing in particular on statistical methods, and we discuss the practical aspects of their application. We also describe recent developments that may transform this field, particularly the use of identity-by-descent for computational phasing.     

Genetic and physical mapping inBrassica diploid species of a gene cluster defined inArabidopsis thaliana

We report the genetic and physical analysis by pulse field gel electrophoresis (PFGE) in threeBrassica diploid genomes for a cluster of five genes characterized in a selected segment of 15 kb on chromosome 3 ofArabidopsis thaliana, encoding aBradyrhizobium CycJ homologue (At1), a rat p67 translation factor homologue (At2), an Em-like (early methionine) protein (At3), chlorophyll synthase (At4) and a yeast Sac1 homologue (A5). TheArabidopsis gene array was found to be conserved on a single linkage group in each of theBrassica genomes. However, partial complexes were found to be duplicated in other chromosome segments on the same or other linkage groups. Some of the At genes, which could not be genetically mapped because of lack of polymorphism, were assigned to their respective linkage groups by physical mapping. The presence of multiple copies of theA. thaliana gene cluster in the threeBrassica genomes further establishes their complex nature, which results from extensive duplication and chromosomal rearrangement. In general, genetic distances between the At genes agreed with values expected for the physical distances determined inBrassica.     

Inheritance and mapping of 2n-egg production in diploid alfalfa.

The production of eggs with the sporophytic chromosome number (2n eggs) in diploid alfalfa (Medicago spp.) is mainly associated with the absence of cytokinesis after restitutional meiosis. The formation of 2n eggs through diplosporic apomeiosis has also been documented in a diploid mutant of M. sativa subsp. falcata (L.) Arcang. (2n = 2x = 16), named PG-F9. Molecular tagging of 2n-egg formation appears to be an essential step towards marker-assisted breeding and map-based cloning strategies aimed at investigating and manipulating reproductive mutants of the M. sativa complex. We made controlled crosses between PG-F9 and three wild type plants of M. sativa subsp. coerulea (Less.) Schm. (2n = 2x = 16) and then hand-pollinated the F1 progenies with tetraploid plants of M. sativa subsp. sativa L. (2n = 4x = 32). As a triploid embryo block prevents the formation of 3x progenies in alfalfa because of endosperm imbalance, and owing to the negligible selfing rate, seed set in 2x-4x crosses was used to discriminate the genetic capacity for 2n-egg production. F1 plants that exhibited null or very low seed sets were classified as normal egg producers and plants with high seed sets as 2n-egg producers. A bulked segregant analysis (BSA) with RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat), and AFLP (amplified fragment length polymorphism) markers was employed to identify a genetic linkage group related to the 2n-egg trait using one of the three F1 progenies. This approach enabled us to detect a paternal ISSR marker of 610 bp, generated by primer (CA)8-GC, located 9.8 cM from a putative gene (termed Tne1, two-n-eggs) that in its recessive form determines 2n eggs and a 30% recombination genomic window surrounding the target locus. Eight additional RAPD and AFLP markers, seven of maternal, and one of paternal origin, significantly co-segregated with the trait under investigation. The minimum number of quantitative trait loci (QTLs) controlling seed set in 2x-4x crosses was estimated by ANOVA and regression analysis. Four maternal and three paternal independent molecular markers significantly affected the trait. A paternal RAPD marker allele, mapped in the same linkage group of Tne1, explained 43% of the variation for seed set in 2x-4x crosses indicating the presence of a major QTL. A map of the PG-F9 chromosome regions carrying the minor genes that determine the expression level of 2n eggs was constructed using selected RAPD and AFLP markers. Two of these genes were linked to previously mapped RFLP loci belonging to groups 1 and 8. Molecular and genetic evidence support the involvement of at least five genes.     

Microsatellite-centromere mapping in Cynoglossus semilaevis using gynogenetic diploid families produced by the use of homologous and non-homologous sperm

Twenty-one microsatellite markers were studied in three meiogynogenetic families of Cynoglossus semilaevis gunther for centromere mapping using half-tetrad analysis. Among the 13 mapped loci, 10 were estimated to be located in the telomeric region, one in the centromeric region, and two in the intermediate region of the chromosome. This study provides a basis for constructing a linkage map of C. semilaevis .     

The development of a bin mapping population and the selective mapping of 103 markers in the diploid Fragaria reference map.

We have identified a set of plants (the bin set) to permit "selective" or "bin" mapping using the diploid strawberry mapping population FV x FN, derived from the F2 cross F. vesca 815 x F. nubicola 601, which has been used to develop the Fragaria reference map. The bin set consists of 8 plants: the F. vesca 815 parent, the F1 hybrid individual, and 6 seedlings of the F2 population. This bin set divides the 578 cM of the diploid Fragaria genome into 46 bins, the largest mapping bin being 26 cM in length and the average bin size being 12.6 cM. To validate the FV x FN bin set, we used it to locate 103 loci into bins on the FV x FN map. These loci comprised 61 previously described SSRs, 38 new SSRs developed in this investigation from Fragaria x ananassa genomic DNA, EST and gene sequences, and 4 ripening-related genes developed for Prunus. The 103 markers were located to bins on all 7 linkage groups of the Fragaria map and a new mapping bin was identified with the novel markers, demonstrating that the map covers the majority of the diploid Fragaria genome and that the 6 bin-set seedlings selected were appropriate for bin mapping using this progeny.     

Genetic mapping of QTLs controlling horticultural traits in diploid roses

A segregating progeny set of 96 F₁ diploid hybrids (2n=2x=14) between Blush Noisette (D10), one of the first seedlings from the original Champneys Pink Cluster, and Rosa wichurana (E15), was used to construct a genetic linkage map of the rose genome following a pseudo-testcross mapping strategy. A total of 133 markers (130 RAPD, one morphological and two microsatellites) were located on the 14 linkage groups (LGs) of the D10 and E15 maps, covering total map lengths of 388 and 260 cM, respectively. Due to the presence of common biparental markers the homology of four LGs between parental maps (D10-1/E15-1 to D10-4/E15-4) could be inferred. Four horticulturally interesting quantitative traits, flower size (FS), days to flowering (DF), leaf size (LS), and resistance to powdery mildew (PM) were analysed in the progeny in order to map quantitative trait loci (QTLs) controlling these traits. A total of 13 putative QTLs (LOD>3.0) were identified, four for FS, two for flowering time, five for LS, and two for resistance to PM. Possible homologies between QTLs detected in the D10 and E15 maps could be established between Fs1 and Fs3, Fs2 and Fs4, and Ls1 and Ls3. Screening for pairwise epistatic interactions between loci revealed additional, epistatic QTLs (EQTLs) for DF and LS that were not detected in the original QTL analysis. The genetic maps developed in this study will be useful to add new markers and locate genes for important traits in the genus providing a practical resource for marker-assisted selection programs in roses.     

QTL mapping in outbred tetraploid (and diploid) diallel populations

Over the last decade, multiparental populations have become a mainstay of genetics research in diploid species. Our goal was to extend this paradigm to autotetraploids by developing software for quantitative trait locus (QTL) mapping in connected F1 populations derived from a set of shared parents. For QTL discovery, phenotypes are regressed on the dosage of parental haplotypes to estimate additive effects. Statistical properties of the model were explored by simulating half-diallel diploid and tetraploid populations with different population sizes and numbers of parents. Across scenarios, the number of progeny per parental haplotype (pph) largely determined the statistical power for QTL detection and accuracy of the estimated haplotype effects. Multiallelic QTL with heritability 0.2 were detected with 90% probability at 25 pph and genome-wide significance level 0.05, and the additive haplotype effects were estimated with over 90% accuracy. Following QTL discovery, the software enables a comparison of models with multiple QTL and nonadditive effects. To illustrate, we analyzed potato tuber shape in a half-diallel population with three tetraploid parents. A well-known QTL on chromosome 10 was detected, for which the inclusion of digenic dominance lowered the Deviance Information Criterion (DIC) by 17 points compared to the additive model. The final model also contained a minor QTL on chromosome 1, but higher-order dominance and epistatic effects were excluded based on the DIC. In terms of practical impacts, the software is already being used to select offspring based on the effect and dosage of particular haplotypes in breeding programs.     

Isolation of molecular markers from specific chromosomal intervals using DNA pools from existing mapping populations.

We present a general method for isolating molecular markers specific to any region of a chromosome using existing mapping populations. Two pools of DNA from individuals homozygous for opposing alleles for a targeted chromosomal interval, defined by two or more linked RFLP markers, are constructed from members of an existing mapping population. The DNA pools are then screened for polymorphism using random oligonucleotide primers and PCR (1). Polymorphic DNA bands should represent DNA sequences within or adjacent to the selected interval. We tested this method in tomato using two genomic intervals containing genes responsible for regulating pedicle abscission (jointless) and fruit ripening (non-ripening). DNA pools containing 7 to 14 F2 individuals for each interval were screened with 200 random primers. Three polymorphic markers were thus identified, two of which were subsequently shown to be tightly linked to the selected intervals. The third marker mapped to the same chromosome (11) but 45 cM away from the selected interval. A particularly attractive attribute of this method is that a single mapping population can be used to target any interval in the genome. Although this method has been demonstrated in tomato, it should be applicable to any sexually reproducing organism for which segregating populations are being used to construct genetic linkage maps.     

Inheritance and genetic mapping of tuber eye depth in cultivated diploid potatoes

Tuber eye depth of the potato (Solanum tuberosum L.) is an important trait for the processing quality and appearance of potatoes. In the present study, we used a cultivated diploid potato family (12601) of 107 plants to dissect the mode of inheritance and to map the gene(s) controlling the trait. The family segregated for both eye depth (deep vs shallow) and tuber shape (round vs long) traits. The deep eye (Eyd) phenotype was found to be associated with round tubers (Ro) in most progeny clones. Further evaluation of this population with molecular markers including simple sequence repeats, amplified fragment length polymorphism, and sequence-characterized amplified regions revealed that the primary locus for eye depth is located on chromosome 10. This map location was confirmed by evaluating a second diploid family (12586). The results of this study led to the following conclusions: (1) there is a major locus controlling the eye depth trait; (2) deep eye (Eyd) is dominant to shallow (eyd); (3) the Eyd/eyd locus is located on chromosome 10; and (4) the Eyd/eyd locus is closely linked with the major locus for tuber shape (Ro/ro), at a distance of about 4 cM.     

Efficient control of population structure in model organism association mapping

Genomewide association mapping in model organisms such as inbred mouse strains is a promising approach for the identification of risk factors related to human diseases. However, genetic association studies in inbred model organisms are confronted by the problem of complex population structure among strains. This induces inflated false positive rates, which cannot be corrected using standard approaches applied in human association studies such as genomic control or structured association. Recent studies demonstrated that mixed models successfully correct for the genetic relatedness in association mapping in maize and Arabidopsis panel data sets. However, the currently available mixed-model methods suffer from computational inefficiency. In this article, we propose a new method, efficient mixed-model association (EMMA), which corrects for population structure and genetic relatedness in model organism association mapping. Our method takes advantage of the specific nature of the optimization problem in applying mixed models for association mapping, which allows us to substantially increase the computational speed and reliability of the results. We applied EMMA to in silico whole-genome association mapping of inbred mouse strains involving hundreds of thousands of SNPs, in addition to Arabidopsis and maize data sets. We also performed extensive simulation studies to estimate the statistical power of EMMA under various SNP effects, varying degrees of population structure, and differing numbers of multiple measurements per strain. Despite the limited power of inbred mouse association mapping due to the limited number of available inbred strains, we are able to identify significantly associated SNPs, which fall into known QTL or genes identified through previous studies while avoiding an inflation of false positives. An R package implementation and webserver of our EMMA method are publicly available.     

Frequency of spontaneous and induced recessive mutations in a diploid strain of Aspergillus nidulans

The spontaneous and UV-induced frequencies of recessive mutations have been studied in a diploid strain of Aspergillus nidulans, by the p-fluoro-phenylalanine (FPA) and 8-azaguanine (8-AZA) resistance tests, on their resting or germinating conidia.

Observed frequencies are in the order of magnitude of those expected, which have been calculated considering the observed mutation frequencies in the diploid strain as well as mitotic recombination frequencies.

We also review some papers which claim to have found higher rates of recessive mutations in mammalian cell lines; in some cases no really higher rates are evident and the authors' conclusions often rest on misinterpretation of their own data.     

Quantitative trait loci and candidate gene mapping of aluminum tolerance in diploid alfalfa

Aluminum (Al) toxicity in acid soils is a major limitation to the production of alfalfa (Medicago sativa subsp. sativa L.) in the USA. Developing Al-tolerant alfalfa cultivars is one approach to overcome this constraint. Accessions of wild diploid alfalfa (M. sativa subsp. coerulea) have been found to be a source of useful genes for Al tolerance. Previously, two genomic regions associated with Al tolerance were identified in this diploid species using restriction fragment length polymorphism (RFLP) markers and single marker analysis. This study was conducted to identify additional Al-tolerance quantitative trait loci (QTLs); to identify simple sequence repeat (SSR) markers that flank the previously identified QTLs; to map candidate genes associated with Al tolerance from other plant species; and to test for co-localization with mapped QTLs. A genetic linkage map was constructed using EST-SSR markers in a population of 130 BC₁F₁ plants derived from the cross between Al-sensitive and Al-tolerant genotypes. Three putative QTLs on linkage groups LG I, LG II and LG III, explaining 38, 16 and 27% of the phenotypic variation, respectively, were identified. Six candidate gene markers designed from Medicago truncatula ESTs that showed homology to known Al-tolerance genes identified in other plant species were placed on the QTL map. A marker designed from a candidate gene involved in malic acid release mapped near a marginally significant QTL (LOD 2.83) on LG I. The SSR markers flanking these QTLs will be useful for transferring them to cultivated alfalfa via marker-assisted selection and for pyramiding Al tolerance QTLs.     

Anisotropy induced by macroscopic boundaries: surface-normal mapping using diffusion-weighted imaging

In MRI, macroscopic boundaries lead to a diffusion-related increase in signal intensity near them—an effect commonly referred to as edge-enhancement. In diffusion-weighted imaging protocols where the signal attenuation due to diffusion results predominantly from the application of magnetic field gradients, edge-enhancement will depend on the orientation of these diffusion gradients. The resulting diffusion anisotropy can be exploited to map the direction normal to the macroscopic boundary. Simulations suggest that the hypothesized anisotropy may be within observable limits even when the voxel contains no boundary itself—hence, the name remote-anisotropy. Moreover, for certain experimental parameters there may be significant phase cancellations within the voxel that may lead to an edge detraction effect. When this is avoided, the eigenvector corresponding to the smallest eigenvalue of the diffusion tensor obtained from diffusion-tensor imaging can be used to create surface-normal maps conveniently. Experiments performed on simple geometric constructs as well as real tissue demonstrate the feasibility of using the edge-enhancement mechanism to map orientations orthogonal to macroscopic surfaces, which may be used to assess the integrity of tissue and organ boundaries noninvasively.     

Haplotype mapping of a major quantitative-trait locus for fetal hemoglobin production, on chromosome 6q23.

Fetal hemoglobin (Hb F) and fetal cell (FC) levels in adults show considerable variation and are influenced by several genetic variants; the major determinants appear to be unlinked to the beta-globin gene cluster. Recently, a trans-acting locus controlling Hb F and FC production has been mapped to chromosome 6q23 in an Asian Indian kindred that includes individuals with heterocellular hereditary persistence of Hb F (HPFH) associated with beta thalassemia. We have extended the kindred by 57 members, bringing the total studied to 210, and have saturated the region with 26 additional markers. Linkage analysis showed tight linkage of the quantitative-trait locus (QTL) to the anonymous markers D6S976 (LOD score 11.3; recombination fraction .00) and D6S270 (LOD score 7.4; recombination fraction .00). Key recombination events now place this QTL within a 1-2-cM interval spanning approximately 1.5 Mb between D6S270 and D6S1626. Furthermore, haplotype analysis has led to a reevaluation of the genealogy and to the identification of additional relationships in the kindred.     

Genetic dissection of drought tolerance and recovery potential by quantitative trait locus mapping of a diploid potato population

Potato is the third most important staple food crop in terms of consumption, yet it is relatively susceptible to yield loss because of drought. As a first step towards improving drought tolerance in this crop, we set out to identify the genetic basis for drought tolerance in a diploid potato mapping population. Experiments were carried out under greenhouse conditions in two successive years by recording four physiological, seven growth and three yield parameters under stress and recovery treatments. Genotypes showed significant variation for drought and recovery responses. The traits measured had low to moderately high heritabilities (ranging from 22 to 74?%). A total of 47 quantitative trait loci (QTL) were identified, of which 28 were drought-specific, 17 under recovery treatment and two under well-watered conditions. The majority of these growth and yield QTL co-localized with a QTL for maturity on chromosome 5. Four QTL for ??¹³C, three for chlorophyll content and one for chlorophyll fluorescence (F _v/F _m) were found to co-localize with yield and other growth trait QTL identified on other chromosomes. Several multi-year and multi-treatment QTL were detected and QTL?×?environment interaction was found for ??¹³C. To our knowledge, this is the first comprehensive QTL study on water deficit and recovery potential in potato.     

Genetic and physical mapping inBrassica diploid species of a gene cluster defined inArabidopsis thaliana

We report the genetic and physical analysis by pulse field gel electrophoresis (PFGE) in threeBrassica diploid genomes for a cluster of five genes characterized in a selected segment of 15 kb on chromosome 3 ofArabidopsis thaliana, encoding aBradyrhizobium CycJ homologue (At1), a rat p67 translation factor homologue (At2), an Em-like (early methionine) protein (At3), chlorophyll synthase (At4) and a yeast Sac1 homologue (A5). TheArabidopsis gene array was found to be conserved on a single linkage group in each of theBrassica genomes. However, partial complexes were found to be duplicated in other chromosome segments on the same or other linkage groups. Some of the At genes, which could not be genetically mapped because of lack of polymorphism, were assigned to their respective linkage groups by physical mapping. The presence of multiple copies of theA. thaliana gene cluster in the threeBrassica genomes further establishes their complex nature, which results from extensive duplication and chromosomal rearrangement. In general, genetic distances between the At genes agreed with values expected for the physical distances determined inBrassica.