SFP genotyping from affymetrix arrays is robust but largely detects cis-acting expression regulators.

The recent development of Affymetrix chips designed from assembled EST sequences has spawned considerable interest in identifying single-feature polymorphisms (SFPs) from transcriptome data. SFPs are valuable genetic markers that potentially offer a physical link to the structural genes themselves. However, most current SFP prediction methodologies were developed for sequenced species although SFPs are particularly valuable for species with complex and unsequenced genomes. To establish the sensitivity and specificity of prediction, we explored four methods for identifying SFPs from experiments involving two tissues in two commercial barleys and their doubled-haploid progeny. The methods were compared in terms of numbers of SFPs predicted and their ability to identify known sequence polymorphisms in the features, to confirm existing SNP genotypes and to match existing maps and individual haplotypes. We identified >4000 separate SFPs that accurately predicted the SNP genotype of >98% of the doubled-haploid (DH) lines. They were highly enriched for features containing sequence polymorphisms but all methods uniformly identified a majority of SFPs ( approximately 64%) in features for which there was no sequence polymorphism while 5% mapped to different locations, indicating that "SFPs" mainly represent polymorphism in cis-acting regulators. All methods are efficient and robust at predicting markers for gene mapping.

Constructing genetic linkage maps under a tetrasomic model.

An international consortium has launched the whole-genome sequencing of potato, the fourth most important food crop in the world. Construction of genetic linkage maps is an inevitable step for taking advantage of the genome projects for the development of novel cultivars in the autotetraploid crop species. However, linkage analysis in autopolyploids, the kernel of linkage map construction, is theoretically challenging and methodologically unavailable in the current literature. We present here a theoretical analysis and a statistical method for tetrasomic linkage analysis with dominant and/or codominant molecular markers. The analysis reveals some essential properties of the tetrasomic model. The method accounts properly for double reduction and incomplete information of marker phenotype in regard to the corresponding phenotype in estimating the coefficients of double reduction and recombination frequency and in testing their significance by using the marker phenotype data. Computer simulation was developed to validate the analysis and the method and a case study with 201 AFLP and SSR markers scored on 228 full-sib individuals of autotetraploid potato is used to illustrate the utility of the method in map construction in autotetraploid species.

Precision and high-resolution mapping of quantitative trait loci by use of recurrent selection, backcross or intercross schemes.

Dissecting quantitative genetic variation into genes at the molecular level has been recognized as the greatest challenge facing geneticists in the twenty-first century. Tremendous efforts in the last two decades were invested to map a wide spectrum of quantitative genetic variation in nearly all important organisms onto their genome regions that may contain genes underlying the variation, but the candidate regions predicted so far are too coarse for accurate gene targeting. In this article, the recurrent selection and backcross (RSB) schemes were investigated theoretically and by simulation for their potential in mapping quantitative trait loci (QTL). In the RSB schemes, selection plays the role of maintaining the recipient genome in the vicinity of the QTL, which, at the same time, are rapidly narrowed down over multiple generations of backcrossing. With a high-density linkage map of DNA polymorphisms, the RSB approach has the potential of dissecting the complex genetic architecture of quantitative traits and enabling the underlying QTL to be mapped with the precision and resolution needed for their map-based cloning to be attempted. The factors affecting efficiency of the mapping method were investigated, suggesting guidelines under which experimental designs of the RSB schemes can be optimized. Comparison was made between the RSB schemes and the two popular QTL mapping methods, interval mapping and composite interval mapping, and showed that the scenario of genomic distribution of QTL that was unlocked by the RSB-based mapping method is qualitatively distinguished from those unlocked by the interval mapping-based methods.

A demonstration of a 1:1 correspondence between chiasma frequency and recombination using a Lolium perenne/Festuca pratensis substitution.

A single chromosome of the grass species Festuca pratensis has been introgressed into Lolium perenne to produce a diploid monosomic substitution line 2n = 2x = 14. The chromatin of F. pratensis and L. perenne can be distinguished by genomic in situ hybridization (GISH), and it is therefore possible to visualize the substituted F. pratensis chromosome in the L. perenne background and to study chiasma formation in a single marked bivalent. Recombination occurs freely in the F. pratensis/L. perenne bivalent, and chiasma frequency counts give a predicted map length for this bivalent of 76 cM. The substituted F. pratensis chromosome was also mapped with 104 EcoRI/Tru91 and HindIII/Tru91 amplified fragment length polymorphisms (AFLPs), generating a marker map of 81 cM. This map length is almost identical to the map length of 76 cM predicted from the chiasma frequency data. The work demonstrates a 1:1 correspondence between chiasma frequency and recombination and, in addition, the absence of chromatid interference across the Festuca and Lolium centromeres.

The association of flowering time quantitative trait loci with duplicated regions and candidate loci in Brassica oleracea.

A population of 150 doubled haploid lines of rapid cycling Brassica oleracea, derived from an F1 from a var. alboglabra x var. italica cross, was scored for flowering time in two trials. Using information on 82 mapped molecular markers, spread evenly across the nine linkage groups, QTL were identified at six locations; one each on linkage groups O2 and O3 and two each on linkage groups O5 and O9. In total, these QTL explained 58 and 93% of the genetical variation in the two trials. Three of these QTL, on linkage groups O2, O3, and O9, were situated in regions showing considerable homology both with each other and with chromosome regions of B. nigra that have been shown to affect flowering time. These same regions are all homologous to a single tract of Arabidopsis chromosome 5, which contains a number of the flowering-related genes, one or more of which may be candidates for the QTL found in Brassica.

Physical and genetic mapping in the grasses Lolium perenne and Festuca pratensis.

A single chromosome of the grass species Festuca pratensis has been introgressed into Lolium perenne to produce a diploid monosomic substitution line 2n = 2x = 14. In this line recombination occurs throughout the length of the F. pratensis/L. perenne bivalent. The F. pratensis chromosome and recombinants between it and its L. perenne homeologue can be visualized using genomic in situ hybridization (GISH). GISH junctions represent the physical locations of sites of recombination, enabling a range of recombinant chromosomes to be used for physical mapping of the introgressed F. pratensis chromosome. The physical map, in conjunction with a genetic map composed of 104 F. pratensis-specific amplified fragment length polymorphisms (AFLPs), demonstrated: (1) the first large-scale analysis of the physical distribution of AFLPs; (2) variation in the relationship between genetic and physical distance from one part of the F. pratensis chromosome to another (e.g., variation was observed between and within chromosome arms); (3) that nucleolar organizer regions (NORs) and centromeres greatly reduce recombination; (4) that coding sequences are present close to the centromere and NORs in areas of low recombination in plant species with large genomes; and (5) apparent complete synteny between the F. pratensis chromosome and rice chromosome 1.

Identification of quantitative trait loci controlling developmental characteristics of Brassica oleracea L.

A segregating population of F(1)-derived doubled haploid (DH) lines of Brassica oleracea was used to detect and locate QTLs controlling 27 morphological and developmental traits, including leaf, flowering, axillary bud and stem characters. The population resulted from a cross between two very different B. oleracea crop types, an annual cauliflower and a biennial Brussels sprout. A principal component analysis (PCA), based on line means, allowed all the traits to be grouped into distinct categories according to the first five Principal Components. These were: leaf traits (PC1), flowering traits (PC2), axillary bud traits (PC3 and 5) and stem traits (PC4). Between zero and four putative QTL were located per trait, which individually explained between 6% and 43% of the additive genetic variation, using the multiple-marker regression approach to QTL mapping. For lamina width, bare petiole length and stem length two QTL with opposite effects were detected on the same linkage groups. Intra- and inter-specific comparative mapping using RFLP markers identified a QTL on linkage group O8 accounting for variation in vernalisation, which is probably synonymous with a QTL detected on linkage group N19 of Brassica napus. In addition, a QTL for petiole length detected on O3 of this study appeared to be homologous to a QTL detected on another B. oleracea genetic map (Camargo et al. 1995).

Why do essential proteins tend to be clustered in the yeast interactome network?

Recently, the debate on the centrality-lethality rule is resolved by the "second-generation" high-throughput Y2H data from the yeast interactome network, which suggests no significant correlation between the degree of connectedness and essentiality of proteins. However, it is still not clear why essential proteins strongly tend to interact with each other. Previously, the concept of essential protein-protein interactions was proposed to explain the mechanism underlying the clustering of essential proteins. In this article we show that 67 to 75% of the excessive interactions between essential proteins (IBEPs) in the yeast interactome network can be attributed to interactions within protein complexes characterised by the same deletion phenotype for subunits within the complex. Furthermore, 20 to 78% of the excess in IBEPs are caused by the strongly modular structure of the network and by variation in protein essentiality among modules. Not only do proteins function as an interactive network in cellular processes, but furthermore, many proteins do not take part in the network alone, but integrate into protein complexes and many functionally related complexes integrate into modules. So, the local structure of protein complexes as both functional and structural modules are the main contributory factors in the clustering of essential proteins.

Does sequence polymorphism of FLC paralogues underlie flowering time QTL in Brassica oleracea?

Previous locations of flowering time (FT) QTL in several Brassica species, coupled with Arabidopsis synteny, suggest that orthologues of the genes FLC, FY or CONSTANS might be the candidates. We focused on FLC, and cloned paralogous copies in Brassica oleracea, obtained their genomic DNA sequences, and confirmed their locations relative to those of known FT-QTL by genetical mapping. They varied in total length mainly due to the variable size of the first and last introns. A high level of identity was observed among Brassica FLC genes at the amino acid level but non-synonymous differences were present. Comparative analysis of the promoter and intragenic regions of BoFLC paralogues with Arabidopsis FLC revealed extensive differences in overall structure and organisation but showed high conservation within those segments known to be essential in regulating FLC expression. Four B. oleracea FLC copies (BoFLC1, BoFLC3, BoFLC4 and BoFLC5) were located to their respective linkage groups based on allelic sequence variation in lines from a doubled haploid population. All except BoFLC4 were within the confidence intervals of known FT-QTL. Sequence data indicated that relevant non-synonymous polymorphisms were present between parents A12DHd and GDDH33 for BoFLC genes. However, BoFLC alleles segregated independently of FT in backcrosses while the study provided evidence that BoFLC4 and BoFLC5 contain premature stop codons and so could not contribute to flowering time variation. Therefore, there is strong evidence against any of the 4 BoFLC being FT-QTL candidates in this population.

Physical organization of the major duplication on Brassica oleracea chromosome O6 revealed through fluorescence in situ hybridization with Arabidopsis and Brassica BAC probes.

The close relationship between Brassica oleracea and Arabidopsis thaliana has been used to explore the genetic and physical collinearity of the two species, focusing on an inverted segmental chromosome duplication within linkage group O6 of B. oleracea. Genetic evidence suggests that these segments share a common origin with a region of Arabidopsis chromosome 1. Brassica oleracea and Arabidopsis bacterial artificial chromosome probes have been used for fluorescence in situ hybridization analysis of B. oleracea pachytene chromosomes to further characterize the inverted duplication. This has been highly effective in increasing the local resolution of the cytogenetic map. We have shown that the physical order of corresponding genetic markers is highly conserved between the duplicated regions in B. oleracea and the physical lengths of the regions at pachytene are similar, while the genetic distances are considerably different. The physical marker order is also well conserved between Arabidopsis and B. oleracea, with only one short inversion identified. Furthermore, the relative physical distances between the markers in one segment of B. oleracea and Arabidopsis have stayed approximately the same. The efficacy of using fluorescence in situ hybridization, together with other forms of physical and genetic mapping, for elucidating such issues relating to synteny is discussed.

QTL analysis: further uses of 'marker regression'.

A variety of approaches are available for identifying the location and effect of QTL in segregating populations using molecular markers. However, these have problems in distinguishing two linked QTL, particularly in relation to the size of the test statistic when many independent tests are performed. An empirical method for obtaining the distribution of the test statistic for specific datasets is described, and its power for demonstrating the inadequacy of a single-QTL model is explored through computer simulation. The method is an extension of the previously described technique of 'marker regression', and it is applied here to demonstrate two situations in which it may be useful. Firstly, we examine the power of the technique to distinguish two, linked QTL from one and compare this ability with that of two contemporary methods, 'Mapmaker/QTL' and 'regression mapping'. Secondly, we show how to combine information from two, or more, populations that may be segregating for different marker loci in a given linkage group. This is illustrated for two populations having in common just two linked marker loci although the sharing of loci is not a pre-requisite. Empirical tests are used to determine whether the same or different QTL are segregating and, if they are the same QTL, whether they are the same alleles. Evidence is discussed which suggests that the upper limit to the number of QTL that can be located for any single quantitative trait in a segregating populations is 12.

Maximum likelihood estimation of linkage between a marker gene and a quantitative trait locus. II. Application to backcross and doubled haploid populations.

The algorithm for estimating both the recombination fraction between a marker gene and a locus affecting a quantitative trait, and also the means and variances of the QTL genotypes, is extended to backcross and doubled haploid populations. The simulation experiments show that estimates of these parameters can be obtained with acceptable accuracy and results are compared with those obtained using F2 populations studied previously (Luo & Kearsey, 1989).

QTL analysis: a simple 'marker-regression' approach.

A method to locate quantitative trait loci (QTL) on a chromosome and to estimate their additive and dominance effects is described. It applies to generations derived from an F1 by selfing or backcrossing and to doubled haploid lines, given that marker genotype information (RFLP, RAPD, etc.) and quantitative trait data are available. The method involves regressing the additive difference between marker genotype means at a locus against a function of the recombination frequency between that locus and a putative QTL. A QTL is located, as by other regression methods, at that point where the residual mean square is minimised. The estimates of location and gene effects are consistent and as reliable as conventional flanking-marker methods. Further applications include the ability to test for the presence of two, or more, linked QTL and to compare different crosses for the presence of common QTL. Furthermore, the technique is straightforward and may be programmed using standard pc-based statistical software.

Maximum likelihood estimation of linkage between a marker gene and a quantitative locus.

A maximum likelihood approach is developed for estimating the recombination fraction in a segregating population (F2), between a marker gene and a locus affecting a quantitative trait as well as estimating the means and variances of the three genotypes of the quantitative trait. The experimental results from computer simulations show that even with experimental sizes of 500, estimates of the parameters can be obtained by aid of the codominant marker gene as long as the heritability of the quantitative trait in question is not less than 0.10. However at low heritabilities the variances of estimates are very large.

Hybrid dysgenesis in Drosophila: correlation between dysgenic traits.

Crosses between laboratory stocks and extractions from wild populations have recently been shown to produce non-reciprocal genetic aberrations commonly termed hybrid dysgenesis, which appear to arise from a nuclear cytoplasmic interaction. Female sterility is one aspect which has been investigated and both poor egg production (GD sterility) and low hatchability (SF sterility) have been shown to contribute. It has previously been suggested that these characters may have an independent action and causation. The results presented in this paper however, show a high degree of correlation in the response of SF and GD sterility to various developmental temperature regimes, with both forms of sterility showing an increase as the developmental temperature rises. For each character, the whole of the life cycle appears to be affected by changes in the developmental temperature although two stages were identified as being particularly sensitive. The results therefore suggest that these two characters have a common cause and the relationship between these and other dysgenic traits is discussed.

QTL analysis in plants; where are we now?

We have briefly reviewed the methods currently available for QTL analysis in segregating populations and summarized some of the conclusions arising from such analyses in plant populations. We show that the analytical methods locate QTL with poor precision (10-30 cM), unless the heritability of an individual QTL is high. Also the estimates of the QTL effects, particularly the dominance effects tend to be inflated because only large estimates are significant. Estimates of numbers of QTL per trait are generally low (< 8) for individual trials. This may suggest that there are few QTL but probably reflects the power of the methods. There is no large correlation between the numbers of QTL found and the amount of the variation explained. Of those cases where dominance is measurable, dominance ratios are often > 1, but seldom significantly greater. These latter cases need further analysis. Many QTL map close to candidate genes, and there is growing evidence from synteny studies of corresponding chromosome regions carrying similar QTL in different species. However, unreliability of QTL location may suggest false candidates.

Genetics of quantitative traits in Arabidopsis thaliana.

The genetic control of 22 quantitative traits, including developmental rates and sizes, was examined in generations of Arabidopsis thaliana derived from the cross between the ecotypes, Columbia (Col) and Landsberg erecta (Ler). The data were obtained from three sets of families raised in the same trial: the 16 basic generations, that is, parents, F(1)'s, F(2)'s, backcrosses, recombinant inbred lines (RILs) and a triple test cross (TTC), the latter produced by crossing the RILs to Col, Ler and their F(1). The data were analysed by two approaches. The first (approach A) involved traditional generation mean and variance component analysis and the second (B), based around the RILs and TTC families, involved marker-based QTL analysis. From (A), genetic differences between Col and Ler were detected for all traits with moderate heritabilities. Height at flowering was the only trait to show heterosis. Dominance was partial to complete for all height traits, and there was no overdominance but there was strong evidence for directional dominance. For most other traits, dominance was ambidirectional and incomplete, with average dominance ratios of around 80%. Epistasis, particularly of the duplicate type that opposes dominance, was a common feature of all traits. The presence of epistasis must imply multiple QTL for all traits. The QTL analysis located 38 significant effects in four regions of chromosomes I, II, IV and V, but not III. QTL affecting rosette size and leaf number were identified in all four regions, with days to maturity on chromosomes IV and V. The only QTL for height was located at the expected position of the erecta gene (chromosome II; 50 cM), but the additive and dominance effects of this single QTL did not adequately explain the generation means. The possible involvement of other interacting height QTL is discussed.

Theoretical basis for genetic linkage analysis in autotetraploid species.

Linkage analysis in autotetraploid species has been an historical challenge in quantitative genetics theory and is a stumbling block that urgently needs to be removed in the rapidly emerging genome research on this species, such as cultivated potato. This article presents theory of a full model of tetrasomic linkage and develops a statistical framework for the linkage analysis. The model considers both double reduction and recombination, the most essential features of tetrasomic inheritance with linked loci, whereas the statistical method takes appropriate account of the major complexities in analyzing both dominant and codominant molecular marker data during map reconstruction in tetraploid species. These complexities include the problems arising from multiple dosage of allelic inheritance, the null allele, allelic segregation distortion, mixed bivalent and quadrivalent pairing in meiosis, and incomplete information of marker phenotype data. The theoretical analysis established the relationship between the coefficients of double reduction at linked loci, which is essential in the present tetrasomic linkage analysis and in assessing the impact of double reduction on the evolution of tetraploid populations. The statistical method, based on the combination of theoretical analysis and a computer-based algorithm, provided analytical tools for predicting the maximum-likelihood estimates of the model parameters. A simulation study showed the feasibility of a practical implementation of the method, detailed the procedure of the analysis, validated the power and reliability in the parameter estimation, and compared the present method with those proposed in the current literature.

Prediction of the performance of inbred lines derived from a population cross in autumn-sown onions (Allium cepa L.).

A design and model are presented to allow the prediction, in early generations, of the mean and distribution of recombinant inbred lines derived from a cross between two parental populations or partially inbred lines. The procedure has been tested in autumn-sown onions (in the UK) using a wide cross between the openpollinated Japanese cultivar, Senshyu, and a partially inbred line derived from the European cultivar, Rawska. The early generations used for prediction included the first self-pollinated generation of the two parental populations and the F3 generation produced from the hybrid population. The predictions were tested by reference to the field performance of a random array of inbred lines, which were produced by single-seed descent (SSD) and had been selfed for three generations. The early generations, used for prediction, and a sample of SSD lines were raised alongside each other in each of two seasons. Within each season, good agreement was found between the predicted and observed performance of the recombinant inbred lines for three characters - yield, quality and maturity. This is used as evidence of the validity of the genetical model and the assumptions made. The effects of genotype x environment interactions prevented predictions made in one season being reliably applied to those made in the other and, therefore, reduce the attraction of this type of prediction study to the plant breeder.