Conor Meade

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.

GM Crop Cultivation in Ireland: Ecological and Economic Considerations

Like many states in the European Union, Ireland has yet to fully commit itself to genetically modified (GM) crop technology. The general position of the Irish Government is ‘positive but precautionary’. However, with the European-wide de-facto moratorium on commercial production of GM crops now ended, many strategically important decisions regarding the commercial deployment of such crops and their co-existence with conventional/organic crops need to be considered. To date, little research on the environmental impact of GM crops has been carried out in Ireland, and the provision of relevant local information lags far behind that available in other countries in the European Union. In this paper, we discuss much of the new ecological and economic data that have emerged since the moratorium on GM crops was introduced in 1998, assess the likely impacts of pest-oriented GM crops should they be introduced to Ireland and examine criteria for post-release monitoring. We also describe the likely commercial demand for these crops and the consequent priorities for ecological research. We argue that the impact of GM technology needs to be assessed in relation to the environmental impact of modern agriculture as a whole. Public unease in relation to this technology may be addressed if adequate resources are made available for independent Irish research on the issue.

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 uMeltSM. 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.

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.

Intraspecific variation among isolates of the entomopathogenic nematode Steinernema feltiae from Bull Island, Ireland

The application of large numbers of entomopathogenic nematodes (EPN) to control insect pests of agriculture is likely to have an impact on the local EPN fauna, yet little is known about the intraspecific relationships between EPN populations, particularly with regard to phylogeny and outbreeding. Here we assess the fitness, with regards to fecundity, host insect mortality and time taken to produce progeny, of isolates of Steinernema feltiae from Bull Island, Ireland. Exon-primed, intron-crossing (EPIC) PCR was used to examine intraspecific phylogenies between S. feltiae isolates, and identified up to three possible colonisation events of Bull Island. EPIC-PCR grouped two isolates, 33.D.(2) and 59.F.(2), separately from the remaining ten S. feltiae isolates These same two isolates consistently performed poorly in all fitness assessments. Following the crossbreeding of all isolates in Galleria mellonella , the number of host cadavers exhibiting emerging infective juveniles was significantly fewer than expected and there were significant differences between isolates in the number of days until progeny were observed. Host insect mortality varied between 40 and 87%. Such intraspecific variation may be a result of adaptation to different microhabitats of Bull Island, which in turn may be accentuated by laboratory culture practices.

Characterization of diverse ploidy in the arctic-alpine Arenaria ciliata species complex (Caryophyllaceae) using shoot meristem staining and flow cytometry analysis of archived frozen tissue: Ploidy analysis in Arenaria ciliata

Ploidy levels were analyzed in 21 European populations of the Arenaria ciliata complex using baseline chromosome counts derived from Feulgen staining of HCl-treated shoot meristems and calibrated flow-cytometry analysis of fresh and archival frozen tissue. Calibration with two to three control samples of different ploidy facilitated rapid identification of ploidy states in unknown samples. Observed ploidy levels varied from 2N = 40–200, with the majority of populations showing 2N = 40–80. High-altitude populations collectively showed the full range of ploidy states, but at low elevations only lower ploidy levels were observed. Populations with the highest observed ploidy contained the greatest observed phylogenetic diversity in the western and eastern Alps. Multiple polyploidization events are inferred in the continental European metapopulation, with lower, more stable ploidy characteristic of the west and north. The method deployed provides an effective approach to ploidy analysis for archival desiccated/frozen tissue samples from biogeographic collections.