Genome Size in the Arenaria ciliata Species Complex (Caryophyllaceae), with Special Focus on Northern Europe and the Arctic.

The main aim of the present study has been the completion of genome size data for the diverse arctic-alpine A. ciliata species complex, with special focus on the unexplored arctic taxon A. pseudofrigida, the north-European A. norvegica, and A. gothica from Gotland (Sweden). Altogether, 46 individuals of these three Nordic taxa have been sampled from seven different regions and their genome size estimated using flow cytometry. Three other alpine taxa in the A. ciliata complex (A. multicaulis, A. ciliata subsp. ciliata, and A. ciliata subsp. bernensis) were also collected and analyzed for standardization purposes, comprising 20 individuals from six regions. A mean 2c value of 1.65 pg of DNA was recorded for A. pseudofrigida, 2.80 pg for A. norvegica, and 4.14 pg for A. gothica, as against the reconfirmed 2c value of 1.63 pg DNA for the type taxon A. ciliata subsp. ciliata. Our results presenting the first estimations of genome sizes for the newly sampled taxa, corroborate ploidy levels described in the available literature, with A. pseudofrigida being tetraploid (2n = 4x = 40), A. norvegica possessing predominantly 2n = 8x = 80, and A. gothica with 2n = 10x = 100. The present study also reconfirms genome size and ploidy level estimations published previously for the alpine members of this species complex. Reflecting a likely complex recent biogeographic history, the A. ciliata species group comprises a polyploid arctic-alpine species complex characterized by reticulate evolution, polyploidizations and hybridizations, probably associated with rapid latitudinal and altitudinal migrations in the Pleistocene-Holocene period.

Genome Size in the Arenaria ciliata Species Complex (Caryophyllaceae), with Special Focus on A. ciliata subsp. bernensis, a Narrow Endemic of the Swiss Northern Alps.

The genus Arenaria (Caryophyllaceae) comprises approximately 300 species worldwide; however, to date, just six of these taxa have been investigated in terms of their genome size. The main subject of the present study is the A. ciliata species complex, with special focus on A. ciliata subsp. bernensis, an endemic plant occurring in the Swiss Northern Alps. Altogether, 16 populations and 77 individuals of the A. ciliata complex have been sampled and their genome sizes were estimated using flow cytometry, including A. ciliata subsp. bernensis, A. ciliata s.str., A. multicaulis, and A. gothica. The Arenaria ciliata subsp. bernensis shows the highest 2c-value of 6.91 pg of DNA, while A. gothica showed 2c = 3.69 pg, A. ciliata s.str. 2c = 1.71 pg, and A. multicaulis 2c = 1.57 pg. These results confirm the very high ploidy level of A. ciliata subsp. bernensis (2n = 20x = 200) compared to other taxa in the complex, as detected by our chromosome counting and previously documented by earlier work. The genome size and, thus, also the ploidy level, is stable across the whole distribution area of this taxon. The present study delivers additional support for the taxonomic distinctiveness of the high alpine endemic A. ciliata subsp. bernensis, which strongly aligns with other differences in morphology, phylogeny, phenology, ecology, and plant communities, described previously. In affirming these differences, further support now exists to re-consider the species status of this taxon. Upgrading to full species rank would significantly improve the conservation prospects for this taxon, as, because of its precise ecological adaptation to alpine summit habitats, the A. ciliata subsp. bernensis faces acute threats from accelerated climate warming.

Potato in the age of biotechnology.

Biotechnology-based tools are now widely used to enhance and expand the traditional remit of potato in food production. By modifying its functionality, the capacity of the potato to produce, for example, therapeutic or industrial compounds is now a reality, and its ability to resist disease can also be radically improved. Two developments have been crucial to expanding the role of potato: the recent advances in the fields of structural and functional potato genomics and the ability to integrate genes of interest into the potato genome. In this review we discuss how both developments have diversified the remit of this crop.

Rapid identification of chloroplast haplotypes using High Resolution Melting analysis.

We have evaluated High Resolution Melting (HRM) analysis as a method for one-step haplotype identification in phylogeographic analysis. Using two adjoined internal amplicons (c. 360 and 390 bp) at the chloroplast rps16 intron (c. 750 bp) we applied HRM to identify haplotypes in 21 populations of two European arctic-alpine herb species Arenaria ciliata and Arenaria norvegica (Caryophyllaceae). From 446 accessions studied, 20 composite rps16 haplotypes were identified by the melting-curve protocol, 18 of which could be identified uniquely. In a comparative sensitivity analysis with in silico PCR-RFLP, only seven of these 20 haplotypes could be identified uniquely. Observed in vitro experimental HRM profiles were corroborated by in silico HRM analysis generated on uMelt(SM) . In silico mutation analysis carried out on a 360 bp wild-type rps16I amplicon determined that the expected rate of missed single-nucleotide polymorphisms (SNP) detection in vitro was similar to existing evaluations of HRM sensitivity, with transversion SNPs being more likely to go undetected compared to transition SNPs. In vitro HRM successfully discriminated between all amplicon templates differing by two or more base changes (352 cases) and between 11 pairs of amplicons where the only difference was a single transition or transversion SNP. Only one pairwise comparison yielded no discernable HRM curve difference between haplotypes, and these samples differed by one transversion (C/G) SNP. HRM analysis represents an untapped resource in phylogeographic analysis, and with appropriate primer design any polymorphic locus is potentially amenable to this single-reaction method for haplotype identification.

Evidence of genotype dependency within Agrobacterium tumefaciens in relation to the integration of vector backbone sequence in transgenic Phytophthora infestans-tolerant potato.

In this study the effect of Agrobacterium tumefaciens genotype of two strains AGL1 and LBA4404 was investigated in regard to the propensity for backbone integration during the transformation of potato for blight tolerance conferred by the resistant to blight (RB) gene carried by the vector pCLDO4541. A PCR based walking approach was employed to identify left and right backbone sequences as well as for selected genes carried on the plasmid backbone. It was found that adjacent to the left border insertion site, the integration of backbone sequence was greater for AGL1 than for LBA4404; however, the opposite was observed with regards to the right border T-DNA junction. Considering both T-DNA borders LBA4404 was found to have a two fold greater integration potential for backbone than the AGL1. The possibility of only backbone integration in T-DNA negative plants was also investigated with the average rate of integration between the two strains calculated at 4.2% with LBA4404 recording a three fold greater occurrence of backbone integration than AGL1. In summary, evidence of Agrobacterium genotype dependency showed that LBA4404 has greater potential to integrate non-T-DNA vector sequence than AGL1 and this should be taken into account when utilising the listed A. tumefaciens genotypes in generating transgenic potato. Additionally, the application of a PCR and primer walking system proved to be reliable and allows for fine detailed studies of backbone sequence integration of transgenic plant.

Facilitating co-existence by tracking gene dispersal in conventional potato systems with microsatellite markers.

Based on international findings, Irish co-existence guidelines for the cultivation of GM potato stipulate that an isolation distance of 20 m is required to minimize the spread of transgenic pollen in accordance with required labeling thresholds. As potato tolerant to Phytophthora infestans is the most applicable GM crop from an Irish context, we tested the efficacy of this isolation distance under Irish environmental conditions using the conventional variety Désirée as a pollen donor and the male-sterile variety British Queen as a pollen receptor. Gene flow was determined by scoring for berry presence on receptor plants and confirmed using a microsatellite marker system designed to assess paternity in F(1) seedlings. 99.1% of seedlings recovered were identified as having Désirée paternity. Whereas 19.9% (140/708) of total berries formed on receptor plants occurred at a distance of 21 m from the pollen source, only 4 of these berries bore viable true potato seed (TPS), from which 23 TPS germinated. TPS-bearing berry formation was negatively correlated with distance from the pollen source, and although overall distribution of berries and seeds was non-random across the plot, no significant correlation was evident with respect to wind direction. Microsatellite markers were also used to confirm that the foraging beetle Meligethes aeneus is a vector for the transmission of potato pollen, but a more detailed statistical analysis of this dataset was limited by inclement weather during the trial. To conclude, we recommend that a two-tiered system be established in regard to establishing isolation distances for the experimental trial and commercial cultivation of GM potato in Ireland, and that responsible crop management be adopted to minimize the establishment of TPS-derived volunteers, which we have noted will emerge through a rotation as a result of pollen-mediated gene flow.

Tracing field hybridization in Ryegrass species using microsatellite and morphological markers.

We have assessed the utility of morphological and microsatellite markers for tracing field hybridization between Lolium multiflorum and Lolium perenne in cereal-enclosed gene flow plots. The presence of awns on the inflorescence of F(1) hybrids was found to be a reliable, but underscoring, indicator of L. multiflorum paternity in L. perenne derived seed as determined by inheritance of species-specific alleles at the microsatellite locus 'H01 H06' in these progeny. A positive correlation was evident in the experimental treatment between the number of pollen donor plants in a given plot and the frequency of hybrid F(1) seed harvested from pollen receptor plants in that plot. These experiments have established the utility of naturally occurring heritable markers for the measurement of gene flow rates in field Ryegrass populations, with particular significance for risk assessment modeling of potential gene flow from transgenic grass cultivars.

Employing a composite gene-flow index to numerically quantify a crop's potential for gene flow: an Irish perspective.

Guidelines to ensure the efficient coexistence of genetically modified (GM) and conventional crops are currently being considered across the European Union. The purpose of this strategy is to describe the measures a farmer must adopt to minimize the admixture of GM and non-GM crops. Minimizing pollen/seed-mediated gene flow between GM and non-GM crops is central to successful coexistence. However no system is currently available to permit the numeric quantification of a crop's propensity for pollen/seed-mediated gene flow. The provision of such a system could permit a background level of gene flow, specific for a particular conventional crop, to be calculated. Here we present a gene flow index model implemented using the principal arable crops in Ireland as a model dataset. The objective of this research was to establish a baseline gene flow data set for Ireland's primary conventional crops through the provision of a simple numerical index. This Gene Flow Index (GFI) incorporates four strands of crop-mediated gene flow (crop pollen-to-crop, crop pollen-to-wild, crop seed-to-volunteer and crop seed-to-feral) into a format that permits the calculation of a crop's gene flow potential. Responsive to regional parameters, we have applied the model to sugar beet, oilseed rape, potato, ryegrass, maize, wheat and barley. We propose that the attained indices will highlight those crops that require additional measures in order to minimize gene flow in accordance with anticipated coexistence guidelines.