Christine A. Hackett

Effects of genotyping errors, missing values and segregation distortion in molecular marker data on the construction of linkage maps

A simulation study was performed to investigate the effects of missing values, typing errors and distorted segregation ratios in molecular marker data on the construction of genetic linkage maps, and to compare the performance of three locus-ordering criteria (weighted least squares, maximum likelihood and minimum sum of adjacent recombination fractions criteria) in the presence of such effects. The study was based upon three linkage groups of 10 loci at 2, 6, and 10 cM spacings simulated from a doubled-haploid population of size 150. Criteria performance were assessed using the number of replicates with correctly estimated orders, the mean rank correlation between the estimated and the true order and the mean total map length. Bootstrap samples from replicates in the maximum likelihood analysis produced a measure of confidence in the estimated locus order. The effects of missing values and/or typing errors in the data are to reduce the proportion of correctly ordered maps, and this problem worsens as the distances between loci decreases. The maximum likelihood criterion is most successful at ordering loci correctly, but gives estimated map lengths, which are substantially inflated when typing errors are present. The presence of missing values in the data produces shorter map lengths for more widely spaced markers, especially under the weighted least-squares criterion. Overall, the presence of segregation distortion has little effect on this population.

Chromatographic classification of commercially available reverse-phase HPLC columns

SummaryThe ever-increasing number of commercially available reversed phases with which the analyst is confronted can cause problems in column selection. These and the non-standard test procedures used by the column manufacturers and packing companies cause further confusion. In order to independently compare and contrast a range of well established C18 stationary phases, we have performed a modified column characterization approach, based on Tanakas methodology, on thirty commonly used phases in our laboratory. These results have been evaluated and presented in various formats as Principal Component Analysis, Cluster Analysis, Tabular Format and Radar Plots in order to assist in observing similarities and differences between phases.The results indicate that, while no two phases are exactly the same (with the exception of “me-to” clones), it is possible to characterize phases into different classes based on their chromatographic performance against selected test probes. The paper illustrates the similarities and differences between these phases.The column characterization described in the paper can form the basis of a rational column selection protocol by either careful matching of the appropriate column characteristics to the analytes physico-chemical properties or by a systematic evaluation of columns from our various categories.

Tri-trophic interactions involving pest aphids, predatory 2-spot ladybirds and transgenic potatoes expressing snowdrop lectin for aphid resistance

Transgenic crops genetically engineered for enhanced insect resistance should be compatible with other components of IPM for the pest resistance to be durable and effective. An experimental potato line was genetically engineered to express an anti-aphid plant protein (snowdrop lectin, GNA), and assessed for possible interactions of the insect resistance gene with a beneficial pest predator. These extended laboratory studies are the first to demonstrate adverse tri-trophic interactions involving a lectin- expressing transgenic crop, a target pest aphid and a beneficial aphidophagous predator. When adult 2-spot ladybirds (Adalia bipunctata[L.]) were fed for 12 days on peach-potato aphids (Myzus persicae Sulzer) colonising transgenic potatoes expressing GNA in leaves, ladybird fecundity, egg viability and longevity significantly decreased over the following 2–3 weeks. No acute toxicity due to the transgenic plants was observed, although female ladybird longevity was reduced by up to 51%. Adverse effects on ladybird reproduction, caused by eating peach-potato aphids from transgenic potatoes, were reversed after switching ladybirds to feeding on pea aphids from non-transgenic bean plants. These results demonstrate that expression of a lectin gene for insect resistance in a transgenic potato line can cause adverse effects to a predatory ladybird via aphids in its food chain. The significance of these potential ecological risks under field conditions need to be further evaluated.

Identification of RAPD markers linked to a Rhynchosporium secalis resistance locus in barley using near-isogenic lines and bulked segregant analysis

Three hundred random sequence 10-mer primers were used to screen a pair of near-isogenic lines of barley and their donor parent for markers linked to genes conferring resistance to Rhynchosporium secalis. One primer was identified which reproducibly generated a product, SC10-65-H400, from the donor parent and the Rhynchosporium-resistant near-isogenic line but not from the recurrent parent. Segregation analysis on a barley doubled haploid population and examination of a further three near-isogenic lines, their donor and recurrent parents confirmed that this marker was linked to the Rhynchosporium resistance locus (Rh) on chromosome 3L. The presence or absence of SC10-65-H400 was subsequently used along with the resistance phenotype to identify two groups of individuals in the doubled haploid population which possessed alternative alleles at both loci and defined a genetic interval between these two markers. Based on that information two bulked DNA samples were constructed by combining equal amounts of DNA from five individuals from each group. The two bulks and doubled haploid parental lines were screened with 700 10-mer primers. Seven products were identified which were present in the ‘resistant’ bulk and parent and were absent in the susceptible samples. Segregation analysis established their association with Rh. In addition co-segregation of the linked markers with a set of chromosome arm specific RFLPs confirmed the location of the Rh locus on the long arm of barley chromosome 3.

Construction of Reference Chromosome-Scale Pseudomolecules for Potato: Integrating the Potato Genome with Genetic and Physical Maps

The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker−based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new ~936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (~93%) of the 723 Mb genome assembly and 37,482 (~96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal “pseudomolecules”.

Effect of substrate composition on production of volatile organic compounds from Trichoderma spp. Inhibitory to wood decay fungi

Abstract The production of volatile organic compounds (VOCs) by two Trichoderma isolates grown on sand infused with either malt extract broth or a minimal medium with the carbon:nitrogen levels similar to that found in Scots pine wood was examined. The VOCs were trapped in tubes packed with chromatographic adsorbant, then analysed by Integrated Automated Thermal Desorption-GC-MS. A total of 45 individual VOCs were recorded. Principle Component Analysis was used to establish that the production of individual VOCs was dependent on both the Trichoderma isolated and the growth media type. The inhibitory effect of the VOCs from the two Trichoderma isolates was also tested on four common wood decay fungi. Although VOCs from both Trichoderma species grown on a malt agar medium consistently inhibited the growth of all four target fungi, effects were negligible when the isolates were grown on a minimal agar medium. The importance of the production of individual VOCs on the inhibition of the wood decay fungi and the implications for the biological control of such fungi are discussed.

Analysis of simple sequence repeats (SSRs) in wild barley from the Fertile Crescent: associations with ecology, geography and flowering time

Wild barley, Hordeum spontaneum C. Koch, is the progenitor of cultivated barley, Hordeum vulgare. The centre of diversity is in the Fertile Crescent of the Near East, where wild barley grows in a wide range of conditions (temperature, water availability, day length, etc.). The genetic diversity of 39 wild barley genotypes collected from Israel, Turkey and Iran was studied with 33 SSRs of known map location. Analysis of molecular variance (AMOVA) was performed to partition the genetic variation present within from the variation between the three countries of origin. Using classification tree analysis, two (or three) specific SSRs were identified which could correctly classify most of the wild barley genotypes according to country of origin. Associations of SSR variation with flowering time and adaptation to site-of-origin ecology and geography were investigated by two contrasting statistical approaches, linear regression based on SSR length variation and linear regression based on SSR allele class differences. A number of SSRs were significantly associated with flowering time under four different growing regimes (short days, long days, unvernalised and vernalised). Most of the associations observed could be accounted for by close linkage of the SSR loci to earliness per se genes. No associations were found with photoperiodic and vernalisation response genes known to control flowering in cultivated barley suggesting that different genetic factors may be active in wild barley. Novel genomic regions controlling flowering time in wild barley were detected on chromosomes 1HS, 2HL, 3HS and 4HS. Associations of SSRs with site-of-origin ecological and geographic data were found primarily in genomic regions determining plant development. This study shows that the analyses of SSR variation by allele class and repeat length are complementary, and that some SSRs are not necessarily selectively neutral.

Statistical analysis of the time-course of Biolog substrate utilization

Abstract Growth of a microbial community on a particular Biolog substrate can be assessed crudely as presence or absence of colour development. More refined is an assessment based on the maximum colour reached after a particular incubation period. The rate of colour development leading up to that maximum can also give useful information about the utilization of the substrate. The statistical analysis and comparison of such time-course profiles is complicated by the multivariate nature of the data. We describe two methods for examining such profiles: 1. a comparison of similarities between profiles from different substrates, using cluster analysis and principal coordinate analysis; 2. an analysis of summary statistics of the profiles, using principal component analysis to identify substrates whose profiles distinguish different treatments. We illustrate these methods with data from a complex microbial community. Cluster analysis and principal coordinate analysis are found to be useful for identifying groups of substrates with similar patterns of reactions. In particular we can identify substrates where no colour development is produced by the microbial communities. The area under the colour development profile summarises the profile in a single statistic, and this may be used in a principal component analysis to identify differences between sample treatments, and the substrates responsible for these.

GENETIC MAPPING OF QUANTITATIVE TRAIT LOCI FOR TRAITS WITH ORDINAL DISTRIBUTIONS

Statistical methods for mapping quantitative trait loci relative to genetic markers are now well established for continuous traits with normal distributions. However, many traits of economic importance are recorded on a discrete, ordinal scale. Here we describe a model developed for the analysis of ordinal traits, such as degree of difficulty in calving or categories of plant disease resistance. The model estimates the distance from the quantitative trait locus to neighbouring genetic markers, and also genetic parameters, either as gene effects on an underlying continuous scale or as probabilities of the observed categories. The model is tested on simulated data and is compared with an analysis based on mixtures of normal distributions. The ordinal model is found to estimate the parameters more accurately, especially when the number of categories is small or when only one linked marker is available.

Identification of AFLP and SSR markers associated with quantitative resistance to Globodera pallida (Stone) in tetraploid potato (Solanum tuberosum subsp. tuberosum) with a view to marker-assisted selection

Abstract Seventy eight clones from the cross between SCRI clone 12601ab1 and cv Stirling were used to explore the possibility of genetical linkage analysis in tetraploid potato (Solanum tuberosum subsp. tuberosum). Clone 12601ab1 had quantitative resistance to Globodera pallida Pa2/3 derived from S. tuberosum subsp. andigena. The strategy adopted involved identifying single- (simplex) and double- (duplex) dose AFLP markers in the parents from segregation ratios that could be unambiguously identified in their offspring, detecting linkage between a marker and a putative quantitative trait locus (QTL) for resistance, and placing the QTL on the linkage map of markers. The numbers of scorable segregating markers were 162 simplex ones present only in 12601ab1, 87 present in Stirling, and 32 present in both; and 72 duplex markers present only in 12601ab1 and 45 present in Stirling. The total map length was 990.9 cM in 12601ab1 and 484.6 cM in Stirling. A QTL with a resistance allele present in double dose (QQqq) in 12601ab1 was inferred from the associations between resistance scores (square root of female counts) and two duplex markers linked in coupling, which, in turn, were linked in coupling to four simplex markers also associated with resistance, but to a lesser degree. The largest marker class difference was the one for the duplex marker P61M34=15. It accounted for 27.8% of the phenotypic variance in resistance scores, or approximately 30% of the genotypic variance. Subsequently, this duplex marker was found to be linked in coupling with a duplex SSR allele Stm3016=a, whose locus was shown to be on chromosome IV in a diploid reference mapping population. The other QTLs for resistance segregating in the progeny were not identified for one or more of the following reasons: the markers did not cover the whole of the genome, there were unfavourable repulsion linkages between the QTLs and markers, or the gene effects were not large enough to be detected in an experiment of the size conducted. It is concluded that prospects appear good for detecting QTLs and using marker-assisted selection in a tetraploid potato breeding programme, provided that, in future, the population size is increased to over 250 and more SSR markers are used to complement the AFLPs; the same is likely to be true for other autotetraploid crops.