Useful Molecular and Cytogenetic Approaches in Population Genetics Studies of Pine Species

*Ana Carvalho, Maria João Gaspar, Alexandra Dias, José Luís Lousada, Maria Emília Silva and José Lima-Brito*

### **Abstract**

In the last decade, we characterised Portuguese populations of *Pinus sylvestris* L., *Pinus nigra* Arnold and *Pinus pinaster* Aiton by using different molecular and cytogenetic approaches. DNA markers helped assess intra- and inter-population genetic variability, extrapolation of phylogenies, provenances and/or infraspecific taxonomy. Quantitative real-time polymerase chain reaction (qRT-PCR), Classical Cytogenetics techniques and/or Comet assay were valuable to assign origins, infraspecific *taxa* or populations more tolerant to water stress. Seed germination tests coupled with the cytogenetic analysis of root cell division provided clues about the impact of fire recurrence on the natural regeneration ability and root growth, respectively. Molecular Cytogenetics contributed to detecting chromosomal anomalies commonly detected in individuals from peripheral populations relative to the species' natural distribution area. The studies briefly described in this chapter integrated multidisciplinary R&D projects whose assembled results allowed inferences about the adaptive potential of the analysed *Pinus* spp. and relevant information for the definition of strategies concerning germplasm conservation, management, use, and, ultimately, genetic improvement.

**Keywords:** adaptation, cytogenetics, DNA markers, genetic resources, *Pinus* spp.

### **1. Introduction**

Pine species are an essential component of the European forests. The distribution of pine forests depends on climate change, human action, refuges location, geographical barriers and largely, from their ability of natural regeneration and adaptive potential, with the latter being highly influenced by the tree's genetic diversity [1, 2].

In Portugal (Southwest of Europe), the lowland Mediterranean *Pinus pinaster* Aiton (Maritime pine) is the dominant pine species, highly economically important for resin and timber production. The highland Mediterranean *Pinus sylvestris* L. (Scots pine) and *Pinus nigra* Arnold (Black pine) can be found in scattered small-sized populations distributed in the North and Centre of Portugal. Scots pine and Black pine also have economic and ecological importance in Portugal and other European forest ecosystems [3–5].

For years, the presence of natural *P. sylvestris* (autochthonous) stands at 'Serra do Gerês' (NW of Portugal) was uncertain [6–10]. It was believed that only artificial *P. sylvestris* stands resulting from planting towards timber production and prevention of soil erosion existed [8]. Such suspicions arose from the lack of historical background for *P. sylvestris*. The exact origin of the plant material used in the planting of the artificial (allochthonous) stands of *P. sylvestris* and *P. nigra* was also unclear. Nonetheless, the studies our team developed so far clarified these issues partially. Through this chapter, we will present a brief description of the molecular and cytogenetic studies that we developed during the last decade, in the Portuguese populations of *P. sylvestris*, *P. nigra* and *P. pinaster*, with the aid and collaboration of various research colleagues, students and technicians from UTAD and other national and international institutions, in the scope of different multidisciplinary R&D projects.
