Biochemical and molecular methods

Original aims of the research

Studies of variation at the biochemical level were started in 1971, soon after the establishment of Northern Research Station, in an attempt to obtain data on genotypic rather than phenotypic diversity. In later years they have been expanded to include molecular techniques. The original aims of the research were to investigate the extent of the biochemical variation which existed between trees, and to discover markers for seed origin and for traits of economic importance. The techniques available at that time were fairly limited and the preliminary work on phenolic compounds was based upon thin-layer chromatography and gel-filtration.

Studies of polyphenol composition

Initial studies of polyphenol composition in Sitka spruce suggested that there were large differences between trees within a provenance, but significant provenance differences were nevertheless found especially for one group of chemicals, the stilbenes. Unfortunately there were strong interactions between phenolic concentrations and planting site.

Use of gas-liquid chromatograph

The installation of a gas-liquid chromatograph (GC) in 1975 enabled biochemical variation to be studied much more rapidly and precisely using the terpene components of conifer resin. It began to emerge that different provenances of lodgepole pine had different biochemical fingerprints. This variation was under strong genetic control and the experimental site and other environmental variables had little effect. Several hundred origins of the species were analysed, obtained both from provenance trials in the UK and Europe and by direct sampling of natural stands in NW America.

The results enabled the natural distribution of Lodgepole pine in NW America to be partitioned into some 15 biochemically distinct regions (Forrest, 1980a). This research effort subsequently generated a practical output over a number of years in the form of a service for the identification of provenances of dubious or unknown origin, by terpene analysis of resin samples taken from a subset of the trees. Similar range-wide studies have been carried out on Sitka spruce, showing clinal biochemical differentiation over its long and very narrow natural distribution. These studies were extended to develop terpene markers for families and for individual clones.

A major study of Scots pine

A major study of the genetic variation existing within the relict native Scots pine woodlands in Scotland was started in 1978, using two distinct marker systems, monoterpene analysis of shoot cortical resin and isozyme analysis of seed endosperm. The aim was to provide a rational basis for the choice of material for the creation and extension of new native woodland areas, since virtually nothing was known of the nature and extent of the genetic diversity of the species within the country.

Remarkably high levels of genetic variation were found, even within the smallest and most depleted sources. Both marker systems identified a small group of woodlands in the northwest as being genetically distinct from all other populations. Terpene analysis was used to divide the country into biochemical regions and to delineate an additional distinctive region in the southwest (Forrest, 1980b). The regional boundaries so defined were used in drawing up the FC's New Native Pinewood Grant Scheme to limit the transfer of planting material, and in this scheme, the two distinct regions were classified as Exclusion Zones (Forrest, 1992).

Further analysis of populations of Eurasian origin led to the formulation of hypotheses of the postglacial migration routes of the species and, in particular, to suggestions of a preglacial origin for the northwest group from a refugial source (Forrest, 1982a; Kinloch et al., 1986).

Identifying biochemical markers for pest and disease  

Attempts have been made to identify biochemical markers for pest and disease resistance in collaborative studies with Pathology and Entomology Branches.

Terpene composition was shown to be associated with the degree of susceptibility of Sitka spruce to the butt-rot fungus Heterobasidion annosum (Forrest, 1982b), but the correlations were complicated by a variety of extraneous sources of variation.

The resistance of Lodgepole pine to Pine Beauty Moth was clearly linked to the composition of terpene volatiles produced by young plants in the laboratory and induced effects of herbivory on resin composition were observed (Leather et al., 1987; Forrest & Leather, 1988). However, in large-scale field experiments on mature crops, these effects were annulled and environmental factors complicated the situation (Watt et al., 1991).

Work was also carried out with Physiology Branch on resistance mechanisms of Sitka spruce to the green spruce aphid. The levels of certain terpenes were important, but also the extent of development of the leaf resin canal system and hence the total resin quantity was at least as important as resin composition.

Isozyme markers

These have been used to study the mating system in a Sitka spruce seed orchard in Perthshire (Cottrell & White, 1995). Estimates of the outcrossing rate were high, even though the orchard was young, and the allozyme systems studied could be used to identify nearly half of the clones unambiguously.

DNA-based techniques 

The work has expanded in the 1990s to encompass a range of DNA-based techniques of molecular analysis. A comprehensive survey of available genetic marker systems was carried out at the beginning of this period of expansion (Forrest, 1994), emphasizing the particular applications of the different types of molecular marker.

Methods incorporating the polymerase chain reaction (PCR), a means of amplifying minute quantities of DNA, using primers of arbitrary sequence (RAPDs) have been applied to generate genetic fingerprints to identify commercial poplar clones (Cottrell et al., 1997a). The same technique has been used to study the genetic diversity of native black poplar in the UK, and has shown that the remaining diversity is low and that a large fraction of the trees are clonal (Cottrell et al., 1997b).

The postglacial colonisation routes of oak in Europe are being studied using DNA markers in the maternally-inherited chloroplast genome and, as a result, we now have a geographical representation of this type of genetic variation for a large number of native oakwoods throughout the UK.

The genetic diversity of a local oakwood has been studied using simple-sequence repeats (SSRs, or microsatellites) in the nuclear genome, as part of the same European project.

Genetic diversity differences in provenance collections of Douglas-fir planted in different European countries are being studied using both terpene and isozyme marker systems, and are being compared with the diversities of the original seed collections from NW America.

Finally, a collaborative project has been initiated to measure the genetic diversity of black poplar throughout Europe using isozymes, chloroplast-DNA, nuclear-DNA SSRs, and AFLPs.

What's of interest

These pages review the work performed by the Forestry Commission and Forest Research on tree improvement following the 50th aniversary of its establishment which passed in 1998.
The genetic background describes the scientific procedures of tree breeding and the technical terms used in the remaining pages.
All species are referred to by their common name in English.