Microsatellite primers for Ficus racemosa and Ficus rubiginosa

We developed 11 polymorphic microsatellite loci each for the figs Ficus (Sycomorus) racemosa and Ficus (Urostigma) rubiginosa from AG- and TG-enriched genomic libraries. These 22 loci were investigated for cross-species amplification and polymorphism in 17–21 F. racemosa and 16–24 F. rubiginosa individuals from Townsville, Australia. Observed heterozygosities range from 0.12 to 0.90 in F. racemosa and from 0.25 to 1.0 in F. rubiginosa.

Inference on microsatellite mutation processes in the invasive mite, Varroa destructor, using reversible jump Markov chain Monte Carlo

Varroa destructor is a parasitic mite of the Eastern honeybee Apis cerana. Fifty years ago, two distinct evolutionary lineages (Korean and Japanese) invaded the Western honeybee Apis mellifera. This haplo-diploid parasite species reproduces mainly through brother sister matings, a system which largely favors the fixation of new mutations. In a worldwide sample of 225 individuals from 21 locations collected on Western honeybees and analyzed at 19 microsatellite loci, a series of de novo mutations was observed. Using historical data concerning the invasion, this original biological system has been exploited to compare three mutation models with allele size constraints for microsatellite markers: stepwise (SMM) and generalized (GSM) mutation models, and a model with mutation rate increasing exponentially with microsatellite length (ESM). Posterior probabilities of the three models have been estimated for each locus individually using reversible jump Markov Chain Monte Carlo. The relative support of each model varies widely among loci, but the GSM is the only model that always receives at least 9% support, whatever the locus. The analysis also provides robust estimates of mutation parameters for each locus and of the divergence time of the two invasive lineages (67,000 generations with a 90% credibility interval of 35,000-174,000). With an average of 10 generations per year, this divergence time fits with the last post-glacial Korea Japan land separation. (c) 2005 Elsevier Inc. All rights reserved.

Characterization and PCR multiplexing of polymorphic microsatellite loci in cashew (Anacardium occidentale L.) and their cross-species utilization

Cashew (Anacardium occidentale L.) is the most economically important tropical nut crop in the world, and yet there are no sequence tagged site (STS) markers available for its study. Here we use an automated, high-throughput system to isolate cashew microsatellites from a non-enriched genomic library blotted onto membranes at high density for screening. Sixty-five sequences contained a microsatellite array, of which 21 proved polymorphic among a closely related seed garden population of 49 genotypes. Twelve markers were suitable for multiplex analysis. Of these, 10 amplified in all three related tropical tree species tested: Anacardium microcarpum, Anacardium pumilum and Anacardium nanum.

Allelic size standards and reference genotypes to unify international cocoa (Theobroma cacao L.) microsatellite data

Standardisation of microsatellite allele profiles between laboratories is of fundamental importance to the transferability of genetic fingerprint data and the identification of clonal individuals held at multiple sites. Here we describe two methods of standardisation applied to the microsatellite fingerprinting of 429 Theobroma cacao L. trees representing 345 accessions held in the worlds largest Cocoa Intermediate Quarantine facility: the use of a partial allelic ladder through the production of 46 cloned and sequenced allelic standards (AJ748464 to AJ48509), and the use of standard genotypes selected to display a diverse allelic range. Until now a lack of accurate and transferable identification information has impeded efforts to genetically improve the cocoa crop. To address this need, a global initiative to fingerprint all international cocoa germplasm collections using a common set of 15 microsatellite markers is in progress. Data reported here have been deposited with the International Cocoa Germplasm Database and form the basis of a searchable resource for clonal identification. To our knowledge, this is the first quarantine facility to be completely genotyped using microsatellite markers for the purpose of quality control and clonal identification. Implications of the results for retrospective tracking of labelling errors are briefly explored.

An enhanced microsatellite map of diploid Fragaria

A total of 45 microsatellites (SSRs) were developed for mapping in Fragaria. They included 31 newly isolated codominant genomic SSRs from F. nubicola and a further 14 SSRs, derived from an expressed sequence tagged library (EST-SSRs) of the cultivated strawberry, F. × ananassa. These, and an additional 64 previously characterised but unmapped SSRs and EST-SSRs, were scored in the diploid Fragaria interspecific F2 mapping population (FV×FN) derived from a cross between F. vesca 815 and F. nubicola 601. The cosegregation data of these 109 SSRs, and of 73 previously mapped molecular markers, were used to elaborate an enhanced linkage map. The map is composed of 182 molecular markers (175 microsatellites, six gene specific markers and one sequence-characterised amplified region) and spans 424 cM over seven linkage groups. The average marker spacing is 2.3 cM/marker and the map now contains just eight gaps longer than 10 cM. The transferability of the new SSR markers to the cultivated strawberry was demonstrated using eight cultivars. Because of the transferable nature of these markers, the map produced will provide a useful reference framework for the development of linkage maps of the cultivated strawberry and for the development of other key resources for Fragaria such as a physical map. In addition, the map now provides a framework upon which to place transferable markers, such as genes of known function, for comparative mapping purposes within Rosaceae.

High throughput, high resolution selection of polymorphic microsatellite loci for multiplex analysis

Background Large-scale genetic profiling, mapping and genetic association studies require access to a series of well-characterised and polymorphic microsatellite markers with distinct and broad allele ranges. Selection of complementary microsatellite markers with non-overlapping allele ranges has historically proved to be a bottleneck in the development of multiplex microsatellite assays. The characterisation process for each microsatellite locus can be laborious and costly given the need for numerous, locus-specific fluorescent primers. Results Here, we describe a simple and inexpensive approach to select useful microsatellite markers. The system is based on the pooling of multiple unlabelled PCR amplicons and their subsequent ligation into a standard cloning vector. A second round of amplification utilising generic labelled primers targeting the vector and unlabelled locus-specific primers targeting the microsatellite flanking region yield allelic profiles that are representative of all individuals contained within the pool. Suitability of various DNA pool sizes was then tested for this purpose. DNA template pools containing between 8 and 96 individuals were assessed for the determination of allele ranges of individual microsatellite markers across a broad population. This helped resolve the balance between using pools that are large enough to allow the detection of many alleles against the risk of including too many individuals in a pool such that rare alleles are over-diluted and so do not appear in the pooled microsatellite profile. Pools of DNA from 12 individuals allowed the reliable detection of all alleles present in the pool. Conclusion The use of generic vector-specific fluorescent primers and unlabelled locus-specific primers provides a high resolution, rapid and inexpensive approach for the selection of highly polymorphic microsatellite loci that possess non-overlapping allele ranges for use in large-scale multiplex assays.

Nomenclature in the dock. Overclassification leads to instability: a case study in the horticulturally important genus Cyclamen (Myrsinaceae)

Nine different classifications have been produced in the last 70 years for the horticulturally valuable genus Cyclamen, a small genus with fewer than 30 species. These classifications, generated by intuitive methods and cladistic analyses, incorporated a total of four infrageneric ranks above that of species and were based on data from morphology, cytology and DNA sequencing. Our results, based on cladistic analyses of three independent data sources − nrDNA ITS, cpDNA trnL intron and morphological data − reveal good resolution only in nrDNA sequence data. However, when these three data sources are combined they provide stronger resolution and support for three major clades, only one of which, subgenus Psilanthum, has been consistently supported in previous classifications. The differing infrageneric classifications produced in Cyclamen result from varying taxon sampling, differing interpretation of morphological data, changes in the sources and analysis of data, and inconsistent application of names. Extensive subdivision of small genera in the absence of adequate data that could provide evidence for consistent patterns of relationship is premature and leads to a proliferation of names.© 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 339-349.

A genetic linkage map of microsatellite, gene-specific and morphological markers in diploid Fragaria

Diploid Fragaria provide a potential model for genomic studies in the Rosaceae. To develop a genetic linkage map of diploid Fragaria, we scored 78 markers (68 microsatellites, one sequence-characterised amplified region, six gene-specific markers and three morphological traits) in an interspecific F2 population of 94 plants generated from a cross of F.vesca f. semperflorens × F. nubicola. Co-segregation analysis arranged 76 markers into seven discrete linkage groups covering 448 cM, with linkage group sizes ranging from 100.3 cM to 22.9 cM. Marker coverage was generally good; however some clustering of markers was observed on six of the seven linkage groups. Segregation distortion was observed at a high proportion of loci (54%), which could reflect the interspecific nature of the progeny and, in some cases, the self-incompatibility of F. nubicola. Such distortion may also account for some of the marker clustering observed in the map. One of the morphological markers, pale-green leaf (pg) has not previously been mapped in Fragaria and was located to the mid-point of linkage group VI. The transferable nature of the markers used in this study means that the map will be ideal for use as a framework for additional marker incorporation aimed at enhancing and resolving map coverage of the diploid Fragaria genome. The map also provides a sound basis for linkage map transfer to the cultivated octoploid strawberry.

Cross-species amplification and phylogenetic reconstruction using fragaria microsatellite primers

Twenty-eight microsatellite primer pairs developed from Fragaria vesca ‘Rügen’ were applied to sixteen accessions representing eight diploid Fragaria species. The number of alleles generated, the power of discrimination and the percentage of accessions where no PCR product could be amplified were calculated for each locus for the thirteen non-F. vesca accessions. A phylogeny was then generated for the species accessions sampled, using the presence or absence of alleles at the polymorphic loci as character states. Despite the problems inherent in phylogeny reconstruction from microsatellite data, the phylogeny showed some congruence with a previously published phylogeny of Fragaria, based on nucleotide sequence data. However, relationships inferred from microsatellite allele data were relatively unresolved and poorly supported. The genetic basis of allelic polymorphisms at specific loci was investigated through direct sequencing of the PCR products amplified by three primer pairs. The potential utility of sequence data generated from microsatellite loci in evolutionary studies of closely related species groups is briefly explored.

Likelihood-based estimation of microsatellite mutation rates

Microsatellites are widely used in genetic analyses, many of which require reliable estimates of microsatellite mutation rates, yet the factors determining mutation rates are uncertain. The most straightforward and conclusive method by which to study mutation is direct observation of allele transmissions in parent-child pairs, and studies of this type suggest a positive, possibly exponential, relationship between mutation rate and allele size, together with a bias toward length increase. Except for microsatellites on the Y chromosome, however, previous analyses have not made full use of available data and may have introduced bias: mutations have been identified only where child genotypes could not be generated by transmission from parents' genotypes, so that the probability that a mutation is detected depends on the distribution of allele lengths and varies with allele length. We introduce a likelihood-based approach that has two key advantages over existing methods. First, we can make formal comparisons between competing models of microsatellite evolution; second, we obtain asymptotically unbiased and efficient parameter estimates. Application to data composed of 118,866 parent-offspring transmissions of AC microsatellites supports the hypothesis that mutation rate increases exponentially with microsatellite length, with a suggestion that contractions become more likely than expansions as length increases. This would lead to a stationary distribution for allele length maintained by mutational balance. There is no evidence that contractions and expansions differ in their step size distributions.

Classification of instability modes in a model of aluminium reduction cells with a uniform magnetic field

A unified view on the interfacial instability in a model of aluminium reduction cells in the presence of a uniform, vertical, background magnetic field is presented. The classification of instability modes is based on the asymptotic theory for high values of parameter β, which characterises the ratio of the Lorentz force based on the disturbance current, and gravity. It is shown that the spectrum of the travelling waves consists of two parts independent of the horizontal cross-section of the cell: highly unstable wall modes and stable or weakly unstable centre, or Sele’s modes. The wall modes with the disturbance of the interface being localised at the sidewalls of the cell dominate the dynamics of instability. Sele’s modes are characterised by a distributed disturbance over the whole horizontal extent of the cell. As β increases these modes are stabilized by the field.

Experimental model of the interfacial instability in aluminium reduction cells

A solution has been found to the long-standing problem of experimental modelling of the interfacial instability in aluminium reduction cells. The idea is to replace the electrolyte overlaying molten aluminium with a mesh of thin rods supplying current down directly into the liquid metal layer. This eliminates electrolysis altogether and all the problems associated with it, such as high temperature, chemical aggressiveness of media, products of electrolysis, the necessity for electrolyte renewal, high power demands, etc. The result is a room temperature, versatile laboratory model which simulates Sele-type, rolling pad interfacial instability. Our new, safe laboratory model enables detailed experimental investigations to test the existing theoretical models for the first time.

Initiation of deep convection at marginal instability in an ensemble of mesoscale models: a case-study from COPS

The present study investigates the initiation of precipitating deep convection in an ensemble of convection-resolving mesoscale models. Results of eight different model runs from five non-hydrostatic models are compared for a case of the Convective and Orographically-induced Precipitation Study (COPS). An isolated convective cell initiated east of the Black Forest crest in southwest Germany, although convective available potential energy was only moderate and convective inhibition was high. Measurements revealed that, due to the absence of synoptic forcing, convection was initiated by local processes related to the orography. In particular, the lifting by low-level convergence in the planetary boundary layer is assumed to be the dominant process on that day. The models used different configurations as well as different initial and boundary conditions. By comparing the different model performance with each other and with measurements, the processes which need to be well represented to initiate convection at the right place and time are discussed. Besides an accurate specification of the thermodynamic and kinematic fields, the results highlight the role of boundary-layer convergence features for quantitative precipitation forecasts in mountainous terrain.

Generation of inertia-gravity waves in the rotating thermal annulus by a localised boundary layer instability

Waves with periods shorter than the inertial period exist in the atmosphere (as inertia-gravity waves) and in the oceans (as Poincaré and internal gravity waves). Such waves owe their origin to various mechanisms, but of particular interest are those arising either from local secondary instabilities or spontaneous emission due to loss of balance. These phenomena have been studied in the laboratory, both in the mechanically-forced and the thermally-forced rotating annulus. Their generation mechanisms, especially in the latter system, have not yet been fully understood, however. Here we examine short period waves in a numerical model of the rotating thermal annulus, and show how the results are consistent with those from earlier laboratory experiments. We then show how these waves are consistent with being inertia-gravity waves generated by a localised instability within the thermal boundary layer, the location of which is determined by regions of strong shear and downwelling at certain points within a large-scale baroclinic wave flow. The resulting instability launches small-scale inertia-gravity waves into the geostrophic interior of the flow. Their behaviour is captured in fully nonlinear numerical simulations in a finite-difference, 3D Boussinesq Navier-Stokes model. Such a mechanism has many similarities with those responsible for launching small- and meso-scale inertia-gravity waves in the atmosphere from fronts and local convection.