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Genetic diversity
Caroline Agufa
Introduction – why conduct molecular analysis?
Morphological, ethnobotanical and physiological characteristics are often very
informative in characterising levels and patterns of genetic variation within tree species.
However, these traits represent only a small portion of the genome of an organism and
are often influenced by environmental factors, which limit their utility in describing the
potential complex genetic structures that may exist within tree taxa. Although
environmental variation can be reduced in controlled field trials, the number of
morphological and physiological characters available for describing genetic variation
remain few. In addition, their patterns of inheritance remain poorly understood.
In order to compliment these techniques, therefore, molecular genetic markers are now
often used to provide more mechanistic information on genetic variation within tree
species. Techniques include isozyme analysis and, more recently, methods based on the
polymerase chain reaction (PCR), which can provide large numbers of ‘neutral’ genetic
markers that are inherited in a Mendelian and (normally) codominant manner (Avise
1994) (see below)
These techniques provide information on the underlying diversity and genetic constitution of
tree populations and the factors, such as mating systems, selection and the extent of pollen
and seed flow, which determine this diversity (Anon 1991). An understanding of these
factors allows more optimal genetic management strategies to be employed for the tree
species. For example, unique and genetically diverse populations suitable for field trial
evaluation, conservation and distribution to users can be identified. The utility of molecular
analysis is best understood to be a consideration of a few examples (see Box 1)
Techniques for molecular genetic analysis
Although a wide range of techniques is available for molecular analysis of genetic
variation (Avise 1994), the most commonly applied are isozyme and PCR-based
approaches:
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Isozyme analysis
Isozymes are different forms of an enzyme with the same biological activity. Isozyme
analysis relies on the fact that non-denatured proteins with different net charges migrate
at different rates through starch or acrylamide gels across which an electric current is
passed. Extracts from fresh leaf or other plant samples are applied to a gel, followed by
an electric current. The gel is then stained with specific histochemicals that detect the
activity of particular enzymes. Different allelic forms of the same enzyme migrate at
different rates through the gel and can be detected by their different positions after
staining. Normally, isozyme markers are inherited in a Mendelian and codominant
manner and hence are useful as markers for molecular population genetic studies.
PCR analysis
PCR is a recently developed technique that allows the selective in vitro amplification of
DNA, such that particular sequences of interest can be greatly enriched over
background levels. Amplification of target sequences occurs to such an extent that
products can be directly visualized when on agarose or acrylamide gel matrices that, by
the application of an electric current, separate DNA products based on their size.
Differences between individuals at target sequences are assessed through determining
the presence or absence of a product of particular size on the gel. PCR has a number of
advantages over isozyme analysis. It does not require enzyme activity in a sample and
relies on very small quantities of DNA, which can be extracted from fragments of dried
leaf, facilitating field collection and the analysis of herbarium specimens. In addition,
because PCR directly assesses the DNA of an organism, a much wider portion of the
genome can be assessed than with isozyme analysis, which relies on gene expression.
Furthermore, individual PCR-based loci often show much greater allelic variation than
isozyme loci, allowing a wider range of population genetic questions to be addressed. A
great variety of different approaches for the detection of genetic variation are based on
PCR. These techniques include amplified fragment length polymorphism (AFLP) (Vos
et al. 1995), random amplified polymorphic DNA (RAPD) (Williams et al. 1990),
restriction fragment length polymorphism-PCR (RFLP-PCR) (Karl & Avise 1992) and
simple sequence repeat (SSR) (White &Powell 1997) analyses. Each of these
approaches has it’s own particular advantages and disadvantages. In particular, RAPDs
and AFLPs, unlike SSRs and RFLP-PCR, may be applied to previously unstudied taxa
because they do not require DNA sequence information. However, unlike SSR and
RFLP-PCR techniques, which reveal codominant markers, RAPDs and AFLPs are
normally dominant markers. Dominance is a disadvantage because it precludes direct
analysis of heterozygosity at single loci within individual trees.
Despite the apparent advantages of PCR, isozyme analysis continues to be widely used
because it is relatively cheap in comparison to PCR-based methods, which require
expensive reagents such as the thermostable Taq polymerase required for DNA
amplification. Thus, in any particular study, the molecular approach used to detect
genetic variation depends on the questions being addressed and the resources available
for analysis. It should also be noted that although molecular markers can provide a
wealth of genetic information, data must always be interpreted in the context of
morphological, physiological, distribution or other data available from field studies,
since a synergy is likely to exist between data sets.
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Molecular analysis and genetic variation
Molecular studies indicate that, for most tree species, a significant degree of molecular
genetic variation partitions among tree populations. However, compared to other
plants, molecular genetic differentiation among populations as a function of
geographical distance is relatively low (Hamrick et al.1992).
Overall levels of molecular genetic differentiation among tree populations may be
relatively low because of the predominantly out-breeding nature of most tree species
(Hamrick et al. 1992). Outcrossing prevents the build up of local genetic structure by
reducing related matings and thereby promoting longer distance gene flow. In many
cases, trees are self-incompatible, and rely on cross-pollination for seed production.
Differentiation among tree populations may also be low because of long distance gene
movement during pollination or seed dispersal, which further limits the build up of local
genetic structure that can result from local matings.
Most molecular genetic studies have been undertaken on temperate trees. However, in
recent years, emphasis on studying tropical tree populations has increased. Many of
these studies have employed PCR-based techniques to assess variation. Despite an
increase in the number of tropical studies, the general focus of research is still on
temperate trees. Considering their importance as reservoirs of global biodiversity, tree
species of South America, Africa and Asia all remain very poorly studied.
Although tropical trees generally show relatively low differentiation among populations
within their native ranges, there are clear exceptions to this rule. For example, Calliandra
calothyrsus (Meso-America), Faidherbia albida (African savannah), Prunus africana
(Afromontane forest) and Sesbania sesban (African savannah, particularly flood plain
and water-logged sites) all show extensive among population differentiation within their
native ranges. These results may indicate a general taxonomic under-differentiation of
tropical trees when compared to temperate trees.
Molecular genetic variation and the impact of tree
cultivation
To date, very few studies have evaluated the impact of cultivation on the genetic
variation of tree species. Partly, this reflects the complexity of devising suitable
strategies that allow direct comparison between wild and cultivated material to be made.
Clearly, cultivation must have a significant impact on the distribution and level of
genetic variation in tree species. Those studies that do provide information in this area
have normally studied landraces as part of a wider survey of natural and introduced
populations (Chamberlain et al. 1996). Levels of genetic variation in cultivated material
are generally lower than in wild populations, possibly reflecting the initial sampling of a
small number of trees during introduction. Since most tree species are out-breeding, a
narrow genetic base in cultivated material can have serious negative implications for
sustainable utilization. Severe inbreeding depression may result. In addition, less
adaptive capacity is available to service changes either in user requirements or
environmental conditions (Simons et al. 1994). In the future, as natural tree
populations recede and tree cultivation increases, greater attention will need to be paid
to the genetic diversity of trees in farmers fields, in order that appropriate strategies for
sustainable utilization within this environment are developed.
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Limitations to molecular genetic analysis
Molecular analysis of genetic variation does not normally provide information on the
field performance of populations. This is because molecular markers are by nature
‘neutral’ indicators of underlying genetic variation, rather than linked to any one
character trait. Molecular markers can be linked to phenotypic characters by a process
of ‘mapping’, but this is a difficult and time consuming process that is not normally
undertaken with trees.
As a result, the molecular analysis of tree populations should not normally be envisaged
as a substitute for the field evaluation of intraspecific phenotypic and physiological
variation in important character traits of tree taxa. Often, molecular and field evaluation
techniques are most effective when they are combined.
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References
Anon (1991) Structure of genetic variation. In: Managing global genetic resources: Forest trees.
Committee on Managing Global Genetic Resources: Agricultural Imperatives, Board on
Agriculture National Research Council, National Academy Press, Washington, USA. Pp
51-72.
Avise JC (1994) Molecular markers, natural history and evolution. Chapman and Hall, New
York, USA.
Chamberlain JR, Galwey NW, Simons AJ (1996) Population structure in Glicidia sepium
(Leguminosae) as revealed by isozyme variation. Silvae Genetica, 45, 112-118
Dawson IK, Powell W (1999) Genetic variation in the Afromontane tree Prunus africana,
and endangered medicinal species. Molecular Ecology, 8, 151-156.
Hamrick JL, Godt MJ, Sherman-Broyles SL (1992) Factors influencing levels of genetic
diversity in woody plant species. New Forests, 6, 95-124.
Karl SA, Avise JC (1992) Balancing selection at allozyme loci in oysters: implications
from nuclear RFLPs. Science, 256, 100-102
Russell JR, Weber JC, Booth A, Powell W, Sotelo-Montes C, Dawson IK (1999)
Genetic variation of Calycophyllum Spruceanum in the Peruvian Amazon Basin, revealed by
amplified fragment length polymorphism (AFLP) analysis. Molecular Ecology, 8, 199-204.
Simons AJ, MacQueen DJ, Stewart JL (994) Strategic concepts in the domestication of
non-industrial trees. In: Leakey RRB, Newton AC (eds) Tropical trees: The Potential
for Domestication and the Rebuilding of Forest Resources. HMSO, London, UK. pp
99-102.
Vos P, Hogers R, Bleeker M, Reijans M, van de Lee T, Hornes M, Frijters A, Pot J,
Peleman J, Kuiper M, Zabeau M (1995) AFLP: a new technique for DNA
fingerprinting. Nucleic Acids Research, 23, 4407-4414.
White G, Powell W (1997) Isolation and characterization of microsatellite loci in
Swietenia humilis (Meliaceae): an endangered tropical hardwood species. Molecular
Ecology, 6, 851-860.
Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA
polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic
Acids Research, 18, 6531-6535.
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Calycophyllum spruceanum
In a study undertaken by ICRAF and partners of the timber tree Calycophyllum spruceanum
from the Peruvian Amazion Basin (Russell et al. 1999), AFLP analysis allowed the
determination of diverse populations suitable for cultivation and on-farm conservation. In
addition, stands particularly worthy of in situ conservation (as being unique and diverse) were
identified. The role of water in mediating seed dispersal in the species was also investigated.
If important, populations at river confluences were expected to be more diverse and therefore
particularly worthy of collection and conservation. However, no significant evidence for watermediated seed dispersal was obtained.
Prunus africana
The afromontane medicinal tree Prunus africana is heavily over-exploited in Cameroon and
Madagascar, with populations in danger of extinction. Employing RAPD analysis, Dawson and
Powell (1999) assessed populations from a number of African countries. Unique populations
particularly worthy of in situ conservation in Madagascar and Cameroon were determined. The
overall large genetic distances among populations from different countries indicated the importance
of rangewide collection for proper evaluation.
Irvingia gabonensis and I. Wombulu
The tropical rainforest trees Irvingia gabonensis and I. Wombulu were identified as priorities
for research in West Africa and have been the subject of extensive germplasm collections.
RAPD and RFLP-PCR analysis were used to assess genetic variation in collections from
Nigeria, Cameroon and Gabon. Results allowed targeting of new collections to particular
geographic areas and identified unique and diverse populations as priorities for ex- and in- situ
conservation. Individuals misclassified as the alternate species during germplasm collection
were reassigned. No evidence of hybridisation between the species was obtained, suggestion
that either species may safely be distributed in the natural range of the alternate species
during domestication.
Inga edulis
Recently, the Royal Botanic Garden Edinburgh and ICRAF began a project to measure
genetic variation in natural- and farmer-stands of Inga edulis, an important fruit and firewood
tree in the Peruvian Amazon Basin, which is also for soil fertility improvement. This project
will determine the effects of domestication on genetic structure and what the likely long-term
effects of cultivation of the species are expected to be on the sustainability of production. In
addition, it will indicate what interventions may be made farmer-stands more representative of
diversity in the species for conservation purposes. Similar work is planned in the same region
on Bactris gasipaes, an important palm tree, which will study variation among colonist and
Amerindian communities in cultivated material. Through these studies, models for genetic
management that maintain on-farm diversity and ensure the productivity of agroforestry
systems will be developed.
Box 1. Applications of molecular genetic analysis of trees for selected species.