Gerry C. Douglas

Characterization and primer development for amplification of chloroplast microsatellite regions of Fraxinus excelsior

This study reports the characterization of chloroplast DNA (cpDNA) variation of Fraxinus excelsior at five loci and the successful development of primer pairs for the amplification of three of these containing mononucleotide microsatellites. We detected high levels of haplotype variation among provenances of Fraxinus around Europe and within Ireland.

Genetic characterisation of oak seedlings, epicormic, crown and micropropagated shoots from mature trees by RAPD and microsatellite PCR

Abstract DNA was isolated from seedlings of Quercus robur, collected from a single provenance, and from epicormic, crown shoots and in vitro shoots from a single tree of Q. petraea using a CTAB method of extraction. DNA was obtained in sufficient quantity and purity, from 13 out of 30 seedlings, and from all isolations from epicormic and in vitro shoots (2.5–10.0 μg/g fresh/ weight). Smearing was minimised at a primer concentration of 0.12 μM with Taq polymerase at 0.5 unit/reaction. Nine primers produced 142 bands, 28 of which were polymorphic. A similarity index showed that 11 seedlings were closely related with high coefficients (0.85–0.90), but each could be identified from another using only 9 primers (OPA-02 and -05, OPG-04 and -05, OPE-01, -02, -03, -08, -09). DNA was isolated from crown, epicormic and in vitro leaves originating from a single 150-yr old tree of Q. petraea and analysed by randomly amplified polymorphic DNA (RAPD) and microsatellites. With each primer, a characteristic RAPD pattern was obtained, and it was common to all six epicormic shoots derived from different parts of a single branch of this tree; also to the shoots from the crown of the same tree with OPE1 OPA-05, OPA-08, OPA-01, OPA-02, OPA-04, OPA-05, OPG-02, OPG-10, OPE-12. Similarly, the RAPD pattern obtained from shoot cultures in vitro, derived from individual nodes of epicormic shoots produced by six different branch segments, were uniform for each of 15 primers. This work was repeated using microsatellite PCR. Three microsatellite loci AG16, AG 1/2 and AG 1/5 were amplified by PCR. It showed a uniformity of these microsatellite loci in shoots from the crown of the tree, and from epicormic shoots cultures derived from six different sections of branch.

Thank you for not flowering: conservation genetics and gene flow analysis of native and non-native populations of Fraxinus (Oleaceae) in Ireland

The risks of gene flow between interfertile native and introduced plant populations are greatest when there is no spatial isolation of pollen clouds and phenological patterns overlap completely. Moreover, invasion probabilities are further increased if introduced populations are capable of producing seeds by selfing. Here we investigated the mating system and patterns of pollen-mediated gene flow among populations of native ash (Fraxinus excelsior) and mixed plantations of non-native ash (F. angustifolia and F. excelsior) as well as hybrid ash (F. excelsior × F. angustifolia) in Ireland. We analysed the flowering phenology of the mother trees and genotyped with six microsatellite loci in progeny arrays from 132 native and plantation trees (1493 seeds) and 444 potential parents. Paternity analyses suggested that plantation and native trees were pollinated by both native and introduced trees. No signs of significant selfing in the introduced trees were observed and no evidence of higher male reproductive success was found for introduced trees compared with native ones either. A small but significant genetic structure was found (φft=0.05) and did not correspond to an isolation-by-distance pattern. However, we observed a significant temporal genetic structure related to the different phenological groups, especially with early and late flowering native trees; each phenological group was pollinated with distinctive pollen sources. Implications of these results are discussed in relation to the conservation and invasiveness of ash and the spread of resistance genes against pathogens such as the fungus Chalara fraxinea that is destroying common ash forests in Europe.

Molecular and Morphological Characterization of Reciprocal F1 Hybrid Ash (Fraxinus excelsior × Fraxinus angustifolia, Oleaceae) and Parental Species Reveals Asymmetric Character Inheritance

Hybridization between Fraxinus excelsior and Fraxinus angustifolia is common. However, identifying hybrids in natural populations is difficult because the closely related parental species share many morphological characters and the inheritance pattern of these characters in hybrids is unknown. We evaluated how morphological characters are inherited and whether morphological and molecular markers can efficiently discriminate artificial first-generation hybrids. Reciprocal F1 hybrids of F. excelsior with F. angustifolia were examined using six microsatellite DNA marker loci and 14 morphological characters. Plants were divided into four groups (F. angustifolia, F. excelsior, the F1 hybrid with F. angustifolia as the maternal parent, and the F1 hybrid with F. excelsior as the maternal parent). The F1 hybrids showed intermediate morphology in most characters, and the range of variation overlapped with the parental species. Canonical discriminant analysis using only the morphological traits separated the four groups without any overlap between the two parental species. F1 hybrids from different maternal parent species could therefore be distinguished. A further analysis that combined molecular and morphological traits allowed clear separation of the four groups and strongly confirmed the a priori defined groups. Our results suggest that intermediate characters can be expected in F1 hybrids of ash but differences may be observed due to maternal/paternal effects.

Alley coppice—a new system with ancient roots

ContextCurrent production from natural forests will not satisfy future world demand for timber and fuel wood, and new land management options are required.AimsWe explore an innovative production system that combines the production of short rotation coppice in wide alleys with the production of high-value trees on narrow strips of land; it is an alternative form of alley cropping which we propose to call ‘alley coppice’. The aim is to describe this alley coppice system and to illustrate its potential for producing two diverse products, namely high-value timber and energy wood on the same land unit.MethodsBased on a comprehensive literature review, we compare the advantages and disadvantages of the alley coppice system and contrast the features with well-known existing or past systems of biomass and wood production.ResultsWe describe and discuss the basic aspects of alley coppice, its design and dynamics, the processes of competition and facilitation, issues of ecology, and areas that are open for future research.ConclusionBased on existing knowledge, a solid foundation for the implementation of alley coppice on suitable land is presented, and the high potential of this system could be shown.

High levels of variation in Salix lignocellulose genes revealed using poplar genomic resources

BackgroundLittle is known about the levels of variation in lignin or other wood related genes in Salix, a genus that is being increasingly used for biomass and biofuel production. The lignin biosynthesis pathway is well characterized in a number of species, including the model tree Populus. We aimed to transfer the genomic resources already available in Populus to its sister genus Salix to assess levels of variation within genes involved in wood formation.ResultsAmplification trials for 27 gene regions were undertaken in 40 Salix taxa. Twelve of these regions were sequenced. Alignment searches of the resulting sequences against reference databases, combined with phylogenetic analyses, showed the close similarity of these Salix sequences to Populus, confirming homology of the primer regions and indicating a high level of conservation within the wood formation genes. However, all sequences were found to vary considerably among Salix species, mainly as SNPs with a smaller number of insertions-deletions. Between 25 and 176 SNPs per kbp per gene region (in predicted exons) were discovered within Salix.ConclusionsThe variation found is sizeable but not unexpected as it is based on interspecific and not intraspecific comparison; it is comparable to interspecific variation in Populus. The characterisation of genetic variation is a key process in pre-breeding and for the conservation and exploitation of genetic resources in Salix. This study characterises the variation in several lignocellulose gene markers for such purposes.