Genetics of pinus pinaster Aiton with cytoplasmic and nuclear markers

Tese de Doutoramento apresentada no Department of Forest Genetics and Plant Phisiology da Swedish University of Agricultural Sciences, em Umea

This thesis summarizes and discusses results of three studies in which biochemical and molecular techniques were used to study the genetic variation in Pinus pinaster. In particular, the investigation focused on: (i) the within- and among-population genetic diversity in the region hypothesised as a putative refugium for the species during the last glaciation; (ii) the comparison of nuclear and cytoplasmic estimates of diversity within and between two regions of the species; and (iiii) the design of a test for provenance identification using knowledge about the levels of genetic variation between the two regions. The distribution of the genetic variation of P. pinaster in Portugal, as revealed by chloroplast microsatellites (cpSSR), indicated that there are low levels of differentiation among populations and that the diversity is found mainly within populations. No discernable geographic pattern was found. Evidences of strong anthropogenic influence associated with extensive gene flow could explain these findings. Fossil, charcoal and palynological records supported the presence of the species in Portugal before and during the last glaciation; therefore, the hypothesis of a putative refugium in this country cannot be excluded. The genetic variation of 24 populations from France and Portugal was investigated with amplified fragments length polymorphisms (AFLPs) and cpSSRs. Both types of markers could discriminate between the two provenances and the diversity of the French provenance was higher compared with that from Portugal. Similar differentiation estimates were found with nuclear and cytoplasmic markers. Extensive gene flow could account for this result, but higher mutation rates and homoplasy at cpSSR loci are not to be excluded. Despite the different modes of inheritance, a high correlation was found between the genetic distances matrices with both types of markers, which suggests that migration surpassed genetic drift in moulding the genetic structure of this species in the regions studied. A provenance diagnostic test was designed, based on cpSSRs, to screen the putative origin of stands of P. pinaster in southwestern France and compared with the currently used terpene-based test. Five stands of unknown origin were diagnosed with both tests. The cpSSR-based test proved to be faster and more accurate to determine if stands were of French or northwest Iberian (Portugal and Galicia) origin. The result obtained was probably due to the higher capability of the DNA-based markers to discriminate between both provenances, compared to that of the terpene markers.