Application of microsatellite markers

2.4.1 Application in genotyping, diversity assessment, and breeding

In the last few years microsatellites have become one of the most popular molecular markers used with applications in many different fields. Microsatellites represent single loci molecular markers that combine extensive hypervariability with somatic stability and co-dominant Mendelian inheritance. Besides, SSR regions are abundant and ubiquitously distributed throughout the genome. Since Tautz (1989), and Weber and May (1989) published the first reports on a PCR based strategy for the detection of microsatellite regions and their use as genetic markers, many reports have been published on the application of SSR markers in animal and plant species. In forest tree species specifically, microsatellites are used for a wide range of applications. The first SSR markers developed for a forest tree species were in Pinus radiata (Smith and

Devey, 1994). Some of the main applications of SSR markers are listed below with examples reported in forest tree species:

¾ Genome mapping and characterization of QTLs: STMS analysis has been used for genome mapping of microsatellites and the construction of microsatellite linkage maps for the genomes of different species. Among forest tree species, genome maps including SSR markers have been constructed for some Pinus species (Devey et al., 1996; Devey et al., 1999;

Echt and Nelson, 1997), for Eucalyptus grandis x E. urophylla (Brondani et al., 1998), and for Quercus robur (Barreneche et al., 1998), among others.

In tree species, many linkage maps have been developed to locate quantitative trait loci (QTLs), and to provide a basis for marker-aided selection (MAS). Selecting economically important trees for breeding programs can be a long and costly process when selection is based upon physical traits. MAS programs have the potential to provide a rapid, reliable and effective selection many years earlier in the growth cycle of commercial tree species. The application of SSR markers in MAS programs for the generation of linkage maps, and for the characterization of QTLs, is in an advanced stage especially in Eucalyptus, Pinus and Quercus species (Groover et al., 1994; Barreneche et al., 1998; Scalfi et al., 2004; Isoda et al., 2006; Marcucci Poltri, 2006).

¾ Fingerprinting (Genotyping): The hypervariable nature of microsatellites increases the probability that every individual in a population will have a unique genotype, making microsatellites particularly useful for fingerprinting. Multilocus genotyping systems have been developed for Eucalyptus species (Kirst et al., 1999; Kirst and Grattapaglia, 1999). In Pinus, fingerprinting has been used for clone identification as an important component of breeding programs (Butcher et al., 1999).

¾ Studies of population structure and variation: SSR markers constitute an especially valuable molecular tool for studies on genetic variation within and among populations, for the construction of genetic inventories, for programs of conservation of genetic resources, or for the study of the spatial genetic structure of populations. A wide range of studies have been

reported on population structure and variation based on SSR markers in many different forest tree species. Some examples are Eucalyptus nitens (Byrne et al., 1996), Pseudotsuge menziesii (Viard et al., 2001), Carapa guianensis (Dayanandan et al., 1999); Shorea curtisii (Ujino et al., 1998), Populus tremuloides (Wyman et al., 2003), Pinus strobus (Marquardt and Epperson, 2004), and Fagus sylvatica L. (Vornam et al., 2004).

¾ Phylogenetic studies: Microsatellites are useful for fine-scale phylogenies up to the level of closely related species. They also provide data suitable for phylogeographic studies that seek to explain the concordant biogeographic and genetic histories of the floras. However, there are few phylogenetic studies that use microsatellite markers and most of these studies are based on chloroplast or mitochondrial SSRs. Application of SSR markers in phylogenetic studies have been reported in forest species such as Pinus (Gugerli et al., 2001; Karhu, 2001; Soranzo et al., 1999), Prunus (Xu et al., 2004), and Dipterocarpaceae (Indrioko et al., 2006).

2.4.2. Applications to studies of the reproduction system

Studies on mating systems and gene flow have benefited from the development of biochemical markers, such as isozymes in the 1970s. However, with the development of hypervariable DNA markers, studies of mating systems became more precise.

Microsatellite markers present important qualities that make them very desirable and useful molecular markers for determining mating system and gene flow. In forest trees, the first microsatellite markers developed were in Pinus radiata D. Don (Smith and Devey, 1994). They have since been developed from the nuclear genomes of a wide range of temperate and tropical forest trees (Butcher et al., 1999).

SSR markers are now the most important molecular tool for genetic studies for many forest tree species. They serve an important role to estimate genetically effective pollen movement among plants, to assess gene flow within and between populations, to study pollen and/or seed dispersal, to estimate mating system parameters (e.g., outcrossing rate, correlated mating, and biparental inbreeding), and for parentage analysis, among other applications. Next, examples of such applications of SSR markers in forest trees are presented.

There are several studies where microsatellite markers were applied to estimate mating system parameters such as outcrossing and selfing rates, correlated mating, biparental inbreeding, or effective number of fathers in forest tree species. Butcher et al. (1999) reported the used of SSR markers for the estimation of outcrossing rates in natural and breeding populations of Acacia mangium in New Guinea. Collevatti et al. (2001) investigated the mating system of populations of the endangered tropical tree species Caryocar brasiliense using genetic data from ten microsatellite loci to estimate mating system parameters under the mixed mating model. In other tropical tree species from Brazil, Theobroma grandiflorum, Alves et al. (2003) studied the mating system using eight microsatellite loci. Estimation of mating system parameters based on microsatellite markers were also reported in Prunus mahaleb L (García et al., 2005), in Valley oak (Quercus lobata Neé) (Sork et al., 2002), and in Pinus pinaster Ait.

(González-Martínez et al., 2003), among others.

Microsatellite markers can provide insights into the genetic structure of natural populations and gene flow in species with little or no isozyme variation. Echt et al.

(1998) were able to detect variation among populations of Pinus resinosa Ait. using chloroplast microsatellite markers. This forest tree species showed little morphological variation, no isozyme variation, and very limited RAPD variation. Dick et al. (2003) studied the outcrossing rates and pollen dispersal of the tropical tree species Dinizia excelsa using five microsatellite loci and the TwoGener approach, a novel two-generation (parent-offspring) method for the estimation of pollen movement proposed by Smouse et al., (2001). Chase et al. (1996) examined the impacts of forest fragmentation on genetic diversity and gene flow of Pithecellobium elegans using SSR markers.

Parentage analysis allows the detection of the parent or parental couple that could have sired individual seeds or seedlings. It includes the analysis of paternity (i.e.

identification of the father) and maternity (i.e. detection of the mother). Hence, the discriminating power of parental analysis approach provides a direct and realistic estimate of gene movement. The high variability of SSR markers increases the probability that every individual in a population will have a unique genotype, making microsatellites particularly useful for fingerprinting and therefore, for parentage analysis. There is an extensive list of studies where microsatellite markers are used

successfully in parentage analyses in order to assess gene flow, pollen dynamics or seed dispersal in forest tree species. Besides, new studies appear each year. Godoy and Jordano (2001) studied seed dispersal by animals in Prunus mahaleb based on multilocus paternity analysis at SSR markers. Streiff et al. (1999) studied the pollination dynamics in a mixed oak stand comprising Quercus robur L. and Quercus petraea (Matt.) Leibl. using six microsatellite primers and paternity assignment. In Quercus, as well, Dow and Ashley (1996 and 1998) assessed seed dispersal and pollen flow by means of SSRs and parentage analyses. Microsatellites have also been used successfully in paternity analyses in the tropical species Gliricidia to estimate pollen dispersal (Dawson et al., 1997). Tabbener and Cottrell (2003) reported the use of microsatellite markers to assess the incidence of natural hybridization and to provide accurate information regarding the distance traveled by pollen in the genus Populus. In their study, they used seven SSR primers to genotype a group of trees belonging to different species of the genus Populus and their progenies. Then, by means of paternity analysis they estimate the cross between species, degree of introgression, and the distance over which pollen can travel.