APPLICATIONS FOR MICROSATELLITE MARKERS IN THE DOMESTICATION AND CONSERVATION OF FOREST TREES¹

by

P. A. Butcher², J. C. Glaubitz and G. F. Moran
CSIRO Forestry and Forest Products
PO Box E4008, Kingston ACT 2604
Australia

INTRODUCTION

Reliable information on the distribution of genetic variation is a prerequisite for sound selection, breeding and conservation programs for forest trees. Genetic variation of a species is assessed either by measuring morphological and metric characters in the field or by studying molecular markers in the laboratory. Laboratory techniques have, until recently, relied on estimates of genetic diversity and mating system parameters from population surveys using isozymes. Isozymes continue to provide a relatively simple and inexpensive method of obtaining genetic information, however their application is limited by the number of enzyme loci, their low levels of variability in some species, and the fact that they only reveal variation in protein-coding genes. The development of DNA markers, including RAPDs (Random Amplified Polymorphic DNA), RFLPs (Restriction Fragment Length Polymorphism), AFLPs (Amplified Fragment Length Polymorphism) and microsatellites, has overcome limitations on the number of variable loci and provided the tools to study variation in coding, non-coding, and highly variable regions of both nuclear or organelle genomes.

Microsatellites are a type of DNA marker which are coming into prominence for individual genotyping and studies of gene flow in forest trees. Microsatellites, or simple sequence repeats (SSRs), consist of segments of DNA containing numerous tandem repeats of a short "motif" sequence, usually of one to six bases (e.g. CACACACA...). They are assayed by polymerase chain reaction (PCR) amplification (Mullis and Faloona 1987), using primers designed to match unique sequences flanking the tandem repeat. The PCR process makes a large number of copies of the target DNA segment containing the microsatellite sequence.

The utility of microsatellites is due to their high variability, together with the ability to semi-automate their analysis and scoring.. Microsatellites are codominant markers (heterozygotes can be distinguished from homozygotes) and are therefore far more informative for genotyping individuals and for linkage mapping than dominant markers such as RAPDs and AFLPs. However the number of microsatellite sequences in the genome is limited, restricting their use for mapping when compared with the potentially unlimited number of RFLP, RAPD and AFLP loci. Other limitations to their use are firstly, the effort and expense required for their development and secondly, evidence that their flanking sequences may not be highly conserved across species in some genera so that markers are not transferable across more distantly related species (Echt et al. 1996; Decroocq et al. 1997; Karhu et al., 1999). In population studies, the high variability detected within populations using microsatellites may in fact reduce the power to detect differences among populations. Caution has therefore been advised on the application of statistical procedures developed for less variable markers (i.e. isozymes) to microsatellite data (Hedrick 1999).

APPLICATION OF MICROSATELLITES TO FOREST TREES

The first microsatellites developed in forest trees were in Pinus radiata (Smith and Devey 1994). They have since been developed from the nuclear genomes of a range of temperate and tropical forest trees (Table 1). Microsatellites from the chloroplast genome have also been isolated in several Pinus species (Powell et al. 1995; Cato and Richardson 1996; Vendramin et al. 1996) and Abies alba (Vendramin and Ziegenhagen 1997). The chloroplast genome is paternally inherited in most gymnosperms, offering opportunities for comparative studies of pollen- vs. seed-mediated gene flow and paternity testing (Kent and Richardson, 1997).

Table 1: Microsatellites loci characterised in forest trees

The main areas where microsatellite markers are being applied in forest trees include studies of genetic diversity in natural and breeding populations, particularly in species with low levels of isozyme variation, gene flow, pollen and/or seed dispersal and mating systems. As these parameters are relevant to the conservation of forest genetic resources, microsatellites are being used to monitor genetic impacts of forest management practices and of fragmentation. In domestication programs microsatellites can be used for germplasm identification and to assist with the construction of genetic linkage maps, with the eventual goal of performing marker-assisted-selection.

GENOTYPING USING MICROSATELLITES (DNA FINGERPRINTING)

The higher variability of microsatellites compared with isozymes increases the probability that every individual in a population will have a unique genotype, making microsatellites particularly useful for fingerprinting and monitoring pollen flow or seed dispersal. It should also make these markers extremely sensitive to changes in population breeding size or structure and to changes in dispersal rates (Slatkin 1995). In the rainforest tree Pithecellobium elegans, Chase et al. (1996a) were able to distinguish 80% of individuals in a population using only three microsatellite loci compared with 37% using six isozyme loci. There were also a higher number of microsatellite alleles that were apparently restricted to single populations, increasing the power to detect gene flow among populations. Similarly, in a survey of 20 unrelated individuals from five natural populations of Acacia mangium, the expected heterozygosity calculated from five microsatellite loci was three times that of RFLPs tested on the same individuals and 30 times that of isozymes in a previous population survey (Table 2) (Decroocq et al. 1997). Seventy-five percent of individuals could be distinguished with the five microsatellite loci and unique alleles were detected in three of the five populations.

Table 2: Comparison of levels of genetic diversity detected in 20 unrelated individuals of Acacia mangium with different markers

MONITORING BREEDING PROGRAMS

Estimating outcrossing rates in natural and breeding populations - a case study of Acacia mangium

Acacia mangium is a pioneer tree which occurs naturally in Australia, Papua New Guinea, Irian Jaya and on islands of the Moluccas. Over the last 20 years it has become the most widely planted species in South East Asia for pulp and paper production. Its introduction to South East Asia as a plantation species is often cited as an example of the decline in performance that can occur when such introductions are founded from a narrow genetic base (Simons 1992; Butcher et al. 1996). Seed production stands were established at Subanjeriji, Sumatra which supplied the majority of planting stock for Indonesian plantations during the 1980s. The seed stands were established from populations in the Daintree region which are characterised by relatively low levels of genetic diversity (Butcher et al. 1998) and low growth rates (Harwood and Williams 1992) compared with populations from far north Queensland and Papua New Guinea. In provenance trials 30 month old progeny from Subanjeriji produced stem volumes that were 70-80% less than progeny from a New Guinea provenance (Turvey 1995).

To help explain the poor performance of Subanjeriji and its source populations, microsatellite markers have been used to examine the breeding system in the natural populations and the seed stand. The increased polymorphism at microsatellite loci gives greater power to detect outcrossing events. Outcrossing rates were determined for populations from Daintree in Australia, Bimadebun in New Guinea, and Aru in the Moluccas and compared with the Subanjeriji seed stand using six microsatellite markers. The population from Daintree had very high levels of inbreeding (70%) in contrast with populations from New Guinea and Aru where there was no evidence of inbreeding. Outcrossing rates in Subanjeriji were the same as those in their putative source populations in the Daintree region, suggesting that inbreeding may be a contributing factor to their poor performance. The differences among populations of A. mangium have major implications for breeding programs. Predictions of genetic gain from recurrent selection in seed orchards are based on the assumption of random mating; if however a high proportion of the seed is produced from selfing the gains from second generation seed will be far less than expected. In addition, use of a constant correlation coefficient to estimate heritability and genetic gain from provenance-progeny trials will lead to biased estimates. This example demonstrates the problems that can arise when breeding programs are based on a limited sample of a species' geographic range and without knowledge of the breeding system.

Seed orchard management

Microsatellites can be used in seed orchard management for estimating pollen contamination from outside sources as well as for studying mating patterns and male fertility variation. Such studies involve determining male parentage of seed produced in the orchard (female parentage is known). The power to discriminate among males is a function of the number of markers, the amount of allelic variability at individual loci, the frequencies of alleles in the population, and the number of males in the population.

In oaks (Quercus robur) microsatellites were used to determine the genetic relationships among selected trees (Lefort et al. 1998). Using nine loci it was established that five selected trees were not closely related and would therefore form a suitable seed source for an advanced breeding program. In a second study, nine microsatellite loci were used to detect seed contaminants and confirm half-sib relationships in seedlots from single Q. robur trees (Lexer et al. 1999).

Management of advanced breeding programs

Germplasm identification is an important component of advanced breeding programs, which rely on controlled crosses or on the correct identification of clones for mass propagation programs. Significant error levels have been detected in controlled-crosses pedigrees using microsatellite markers.

In Pinus radiata three microsatellites have been used to identify incorrectly identified individuals in two controlled-cross pedigrees that were planted at three trial sites in south-eastern Australia. Two percent of the progeny were incorrectly identified in one pedigree, while 20% of progeny were incorrectly identified in the second pedigree (J.C. Bell and G.F. Moran, CSIRO unpub. data). The majority of the rogues were found in a single trial suggesting that errors occurred at the stage of trial establishment rather than during pollination. In Eucalyptus nitens pedigrees, the time that flowers were bagged following pollination had a significant effect on contamination from foreign pollen. The error rate increased from zero in a pedigree where bags were left in place for two weeks following pollination to 20% in a second pedigree where bags were removed one week after pollination (M. Byrne and G.F. Moran, CSIRO unpub. data). These studies illustrate that microsatellite markers can, not only be used to detect errors in breeding pedigrees, but can also help identify the source of errors. They could also be used to reduce the error rates in trials by screening plants prior to trial establishment.

GENETIC LINKAGE MAPPING AND CHARACTERISATION OF QTLS

Genetic linkage maps can be used to locate genes affecting traits of economic importance, or for studies comparing chromosome organization between species. Molecular markers in chromosomal regions that are strongly associated with a quantitative trait can be used for early selection. The potential benefits of marker-aided selection (MAS) are greatest for traits that are difficult or expensive to measure (for example wood quality traits and pulp yield) and for traits which only appear under certain conditions such as resistance to a particular pest, pathogen or abiotic factor such as salinity. Mapping and MAS tends to be used only in species of high commercial value and has the most potential in clonal breeding programs where any additional gains can be multiplied.

The high variability of microsatellite markers, together with the option to partially automate the analysis and scoring of segregating loci makes them ideally suited to genetic linkage mapping. However, their application in mapping forest trees has been limited by the availability of markers - a consequence of the time and effort required for their development, together with limitations on the number of microsatellite sequences in the genome and on the transferability of markers across species.

Microsatellites have been placed on genetic linkage maps in Eucalyptus grandis x E. urophylla (19 loci), (Brondini et al. 1998), Pinus radiata (16 loci) and P. taeda (11 loci) (Devey et al. 1999), P. strobus (5 loci) (Echt and Nelson 1997), Quercus robur (18 loci) (Barrenche et al. 1998) and Acacia mangium (30 loci) (Butcher, unpub. data), in combination with other markers, for example RFLPs and RAPDs. The data from mapping indicates that, in addition to generally being more informative than other marker types, microsatellite loci are evenly dispersed throughout the genome.

CONSERVATION OF THE GENETIC RESOURCES OF FOREST TREES

While the use of microsatellites for paternity analysis in mating system studies and for genetic linkage mapping requires few assumptions beyond Mendelian inheritance, their interpretation in population and evolutionary studies requires assumptions about the nature and rate of the mutational process that generated the various alleles (see reviews by Jarne and Lagoda, 1996; Goldstein and Pollack 1997; Nielsen and Palsboll 1999). The non-amplification of alleles has also been reported from microsatellite data, resulting in apparent heterozygote deficiencies and upwardly biased inbreeding coefficients in population studies (for example White and Powell 1997a; Fisher et al. 1998; Thomas et al. 1999).

In a comparative study of isozymes and microsatellites in oaks (Quercus robur and Q. petraea), Streiff et al. (1998) found that both markers uncovered similar large-scale spatial distributions of diversity. Despite the higher variability of microsatellite loci (average A = 21.7 for microsatelites cf A = 4.3 for isozymes), little evidence of spatial genetic structuring in either species was detected - consistent with evidence of extensive pollen flow, over long distances and from multiple origins as shown by paternity analysis (Streiff et al. 1999). Similarly, in Melaleuca alternifolia a survey of 500 individuals from natural populations using 5 microsatellite loci (Rossetto et al. 1999b) failed to identify spatial genetic structure beyond that previously identified in 100 individuals using 17 isozyme loci (Butcher et al. 1992).

Microsatellites can, however, provide insights into the genetic structure of natural populations and gene flow in species with little or no allozyme variation. Using microsatellites from the chloroplast genome Echt et al. (1998) were able to detect variation among populations of Pinus resinosa, a forest tree species with little morphological variation, no allozyme variation and very limited RAPD variation. The pattern of variation indicated little gene flow between populations of P. resinosa which has a disjunct distribution. Genetically distinctive populations with high levels of chloroplast variation could be targeted for conservation of the species' genetic resources and for breeding programs. With proper sampling, chloroplast microsatellite markers could be used to trace paths of post-Pleistocene migration, and to identify populations that are the most evolutionarily divergent (Echt et al. 1998). It would be interesting to see if such populations are also most divergent for nuclear genomic and quantitative traits.

MONITORING THE EFFECTS OF FOREST MANAGEMENT PRACTICES

There have been several comparative studies of microsatellites and other nuclear markers which indicate that microsatellites are more sensitive indicators of fine scale genetic structure. In a study of the effects of different harvesting practices on genetic diversity in Eucalyptus sieberi forest in south-eastern Australia, Glaubitz et al. (1999) reported genetic distances between nearby stands estimated from microsatellites were 9 times higher than from RFLP data. The microsatellites displayed twice as much variablity as the RFLPs. The topology of a dendrogram based on microsatellites should therefore be less sensitive to sampling error due to the much larger underlying genetic distances. In a similar study in Pinus contorta genetic distances between unharvested, planted and naturally regenerated stands were only slightly higher for microsatellite loci than RAPD loci (Thomas et al. 1999). Both studies reported no significant difference in the levels of genetic diversity in unharvested stands versus regeneration, and no difference among regeneration techniques.

MONITORING THE EFFECTS OF FOREST FRAGMENTATION

The impacts of forest fragmentation on genetic diversity and gene flow have been examined in several species using microsatellites. In Pithecellobium elegans (Chase et al. 1996b) and Gliricidia sepium (Dawson et al. 1997) evidence from microsatellites of pollen flow between isolated trees in pasture and forest fragments indicates such trees make an important contribution to the genetic diversity of the contiguous undisturbed forest. In P. elegans most mating events were not with nearest neighbours and pollen flow was not diminished by physical isolation. In Swietenia humilis, a tropical hardwood, comparison of trees from cleared pasture with those in adjacent undisturbed forest using microsatellites revealed similar levels of genetic diversity despite fewer alleles in the pasture trees (White and Powell 1997a). The difference in the number of alleles could reflect sampling as more than twice as many trees were sampled from the undisturbed forest.

Studies of the mating system and of pollen dispersal are sensitive to short-term perturbations in the pollination system while diversity in the adult trees may give a clearer indication of the reproductive potential within forest fragments. In Symphonia globulifera comparisons were made among the adult, sapling and seedling stages in rainforest remnants and continuous forest (Aldrich et al. 1998). Genetic diversity was higher in seedlings in the remnant forest patches than in adjacent continuous forest; this was attributed to the concentration of seeds by foraging bats. However, higher levels of inbreeding were evident in the fragmented forest and seedlings from the forest remnants were differentiated from those in the continuous forest. It was concluded that reductions in the number of adults can lead to rapid changes in genetic composition following fragmentation, particularly when these changes occur in conjunction with pre-existing genetic structure.

In addition to providing data on the fine scale genetic structure in populations of forest trees, the higher allelic diversity of microsatellites has created opportunities for the use of analytical methods which allow more detailed inferences to be made about both evolutionary parameters and historical events (reviewed by Luikart and England 1999). Powerful likelihood methods are being developed that can simultaneously estimate the approximate date and rate of a recent reduction or increase in effective population size (Ne). Assignment tests estimate dispersal among populations using a direct method which can be compared with indirect methods - the difference between the two indicates the extent to which rare and unpredictable events have been important in the recent history of the species (Slatkin 1987). Estimates of interpopulation dispersal rates are particularly useful for detecting introgression of genes from other species. The power of these approaches will increase with the number and variablity of loci - making possible estimates of rates of dispersal among populations, identification of individual dispersers and reconstruction of genetic lineages in natural populations (Waser and Strobeck 1998).

CONCLUSIONS

Microsatellite markers provide a powerful tool for addressing genetic questions related to breeding where genetic discrimination is paramount. In species such as A. mangium and P. resinosa with low levels of allozyme diversity, these markers can be used to gain more information on the level and distribution of variation and the breeding system. This is particularly important for conservation programs, which aim to conserve as much diversity as possible in order to ensure the long term viability of species.

In advanced breeding programs, the increased variability of microsatellite markers compared with protein and other DNA markers improves our ability to discriminate among individuals. Parentage of seed orchard progeny can be determined and controlled crosses can be verified. Microsatellite markers have proved particularly useful for identifying errors and their probable source in controlled crossing programs. They are being used to develop more powerful genetic linkage maps. Such maps will lead to a greater understanding of genome organisation within and between species and hopefully the genetic basis of quantitative variation.

The higher variability of microsatellites has led to the development of more powerful analytical procedures for assigning genotypes to source populations and identifying recent immigrants into populations. This area of research is of interest with the expansion of plantings using exotic germplasm and ensuing concerns regarding the contamination of local gene pools. The applications of microsatellites in evolutionary studies remain limited, however, by questions regarding the mutational model by which they evolve. The accuracy of inferences made from microsatellite data in population genetic and evolutionary studies relies upon a realistic model of the molecular evolution of these loci.

ACKNOWLEDGEMENTS

Charlie Bell and Chris Harwood provided useful comments on the manuscript. The support of the Australian Seed Tree Centre, CSIRO is gratefully acknowledged.

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1. Received August 1999. Original in English.
2. Corresponding author P.A. Butcher. CSIRO Forestry and Forest Products, PO Box E4008, Kingston, ACT 2604. Tel.. +616 281 8289 Fax: +616 281 8233 Email: [email protected]