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

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.

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, 4, 5].

For years, the presence of natural P. sylvestris (autochthonous) stands at ‘Serra do Gerês’ (NW of Portugal) was uncertain [6, 7, 8, 9, 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.

2. Methodology

2.1 DNA markers

For the molecular characterisation of different Portuguese pine populations, as dominant DNA markers, we used the: (i) inter-simple sequence repeat (ISSR) [11]; (ii) random amplified polymorphic DNA (RAPD) [12, 13]; and (iii) start codon targeted (SCoT) [14] markers. For the amplification of ISSRs and RAPDs, a concentration of 10 – 25 ng/μL of template DNA was used. The amplification of SCoTs was performed with 60 ng/μL of genomic DNA. The initial concentration of the 10-mer oligonucleotides, SSR and SCoT primers for the amplification of RAPDs, ISSRs and SCoTs was 5 μM. Additional details of reaction mixtures can be found in [15, 16]. For the molecular characterisation of the Portuguese pine populations with ISSRs, RAPDs and SCoTs, we used primers previously developed by others, whose references, along with the primer sequences and amplification conditions used in our previous studies, are presented in Table 1.

Table 1.

Primer sequences used for the amplification of the dominant ISSR, RAPD and SCoT markers in P. sylvestris [15], P. nigra [16] and/or Pinus pinaster [17], and respective amplification conditions.

Table 2 presents the sequences of the SSR primers used to amplify nuclear SSRs (nSSRs), expressed sequence tag-SSRs (EST-SSRs) and/or chloroplastidial-SSRs (cpSSRs) by individual or multiplex PCR in P. sylvestris [18] and/or P. nigra individuals [19].

Table 2.

Microsatellite (SSR) primers used for nSSR, cpSSR or EST-SSR markers amplification in P. sylvestris [18] and/or P. nigra [19], respective sequences and amplification conditions.

Details of the fluorescent dyes used to label the forward primer of each pair, as required for the capillary electrophoresis, along with the composition of the reaction mixtures used for individual or multiplex PCR, and the size of the amplified SSR fragments, in both pine species are available in [18, 19].

2.2 Cytogenetic techniques

Root meristematic cells of P. sylvestris, P. nigra and P. pinaster were cytogenetically analysed by using Classical Cytogenetics, conventional fluorescence in situ hybridisation (FISH) and non-denaturing FISH (ND-FISH). Upon germination, the collection of roots with 1.5-cm length allows the observation of mitotic dividing cells (Figure 1). When the goal of the study is the evaluation of the root mitotic cell cycle, the collected roots are immediately fixed in a solution of absolute ethanol and acetic in the proportion of 3:1 (v/v) and then stained with 2% aceto-carmine for 48 h, to enable the observation of interphase and dividing cells into all mitotic phases for further determination of the cytogenetic parameters, mitotic index (MI) and dividing cells with anomalies (DCA), both expressed in percentage [26, 27] (Figure 1). On the other hand, for the counting of chromosomes or physical mapping of repetitive DNA sequences such as SSRs or rDNA loci, root treatment in ice-cold water (24h – 0°C) or metaphase arresting agents (e.g. 0.05% colchicine or 2 mM 8-hydroxyquinoline) at 4°C or room temperature for a few hours, before root fixation and/or aceto-carmine staining, should be performed [28]. The treatments of roots with ice-cold water and metaphase-arresting agents inhibit the polymerisation of the microtubules of the mitotic spindle, accumulating C-metaphases that enable the evaluation of individual chromosomes [28].

Figure 1.

Schematic representation of the methodology used by [26] to evaluate the effects of induced water stress in the root mitotic cell cycle and nuclear DNA damage in young needles of P. nigra using Classical Cytogenetics and alkaline Comet assay, respectively.

Fixed root tips of Pinus spp. were squashed in the presence of 45% or 60% acetic acid for mitotic spread preparation, and after freezing at −80°C, the glass coverslip was removed, and air-drying was allowed. For the detection of the rDNA loci on the P. sylvestris chromosomes, conventional FISH was performed with the cloned 45S rDNA probe, pTa71 [29] that despite being isolated in Triticum aestivum [30], presents homology with the rDNA loci of several plant species. The insert of this clone is an Eco-RI fragment of 9 kb comprising one repeat unit of the rRNA genes 18S–5.8S-25S, the intergenic spacer (IGS) and the internal transcribed spacers 1 and 2 (ITS-1 and ITS-2, respectively) that flank the 5.8S rRNA gene [30, 31]. The cloned rDNA sequence pTa71 is usually labelled by nick translation using commercial kits.

For the physical mapping of oligonucleotide SSR sequences on Scots pine chromosomes, the FISH variant technique, ND-FISH, was used [29, 32]. The SSR sequences can be purchased as synthetic oligonucleotides directly labelled in their 5′-end with fluorochromes (e.g. fluorescein or TAMRA) or indirectly labelled with biotin-16-dUTP or digoxigenin-11-dUTP using commercial Random Primed DNA Labelling kits. ND-FISH allows the hybridisation of single-strand DNA oligonucleotide probes (e.g. telomeric, centromeric or SSR sequences) in a few hours since it does not require chromosomal spread pre-treatments [32, 33]. Despite the simplicity of both hybridisation mixture and ND-FISH protocol (Table 3), the hybridisation signals present high intensity, specificity and resolution [32, 33]. The non-denaturing conditions preserve the chromosomal morphology [33], enabling successive hybridisations with additional DNA probes by ND-FISH or conventional FISH on the same chromosome spread [34]. Although unnecessary for ND-FISH, chromosome spread pre-treatments (Table 3) can be done if successive hybridisations of DNA probes by FISH are envisaged [34].

Main protocol steps	Conventional FISH	ND-FISH
Pre-treatments of the chromosome spreads	Ageing (dry heat for 2 h) RNase (1 h) 2× SSC washes (15 min) Cytoplasm digestion (Pepsin, 10 min) 2× SSC washes (15 min) (Re)fixation of plant material (Paraformaldehyde 10 min) 2× SSC washes (15 min) Dehydration in a series of ethanol solutions (70%, 90%, 100%, 15 min) Air dry	Not required
Hybridisation mixture	Formamide	2× SSC
	20× SSC pH 7.0	2 pmol of each oligonucleotide probe
	50% Dextran sulphate
	Salmon sperm (1 μg/μL)
	10 mM EDTA pH 8.0
	10% SDS
	Probe(s) concentration: 3–7 ng/μL
	Ultra-pure water
Chromosomal denaturing	Temperature: ˃ 65°C; % Formamide: 50%; Final concentration of salt: 2× SSC	Not required
Hybridisation temperature and duration	37°C – overnight	24°C – 2 h
Post-hybridisation washes	At 42°C; Washes (5 min each): 2× SSC, twice in 20% formamide/2× SSC; and twice in 0.01 × SSC; Blocking unspecific sites with Bovine Serum Albumin (BSA) (20 min) where probe molecules bound; Previous step alternates with washes for 3 × 5 min in detection buffer (4× SSC; Tween 20).	At room temperature; Blocking unspecific sites with BSA where probe molecules bound; Previous step alternates with washes for 3 × 5 min in detection buffer (6× SSC) are performed. .
Detection of hybridisation signal	Incubation with specific antibodies (37°C, 1 h), unless the probes were directly labelled with fluorochromes. Previous step alternates with washes for 3 × 5 min in detection buffer (4× SSC; Tween 20).	Incubation with specific antibodies (37°C, 1 h), unless the probes were directly labelled with fluorochromes. Previous step alternates with washes for 3 × 5 min in detection buffer (6× SSC).

Table 3.

Main steps of conventional FISH and ND-FISH protocols.

Chromosomes counterstaining with DAPI. Store at 4°C. Observation on the epifluorescence microscope.

Unfortunately, the Comet assay is rarely performed in plants when compared to animal species, but the gathered results can complement and enrich Plant Cytogenetics studies as demonstrated before [26] (Figure 1). Figure 1 presents a scheme with the main steps of the alkaline Comet assay that was performed, as far as we know, for the first time, in P. nigra [26].

Additional details about the alkaline Comet assay procedure and analysis performed in P. nigra can be found in [26].

3. (Cyto)molecular studies in Pinus spp.

3.1 Pinus sylvestris

P. sylvestris (Scots pine) is the conifer with the broadest area of natural distribution, which extends from the British Isles to the Siberian taiga and occupies Southern Europe [35, 36, 37, 38, 39, 40, 41, 42]. Portugal constitutes the westernmost limit of the Scots pine natural distribution area.

The various P. sylvestris refugees that resulted from the Holocene Epoch greatly influenced the genetic diversity and adaptation of the species found in multiple ecological niches [5, 40]. In the last decades, the increased temperature has enhanced growth and reproduction at the northern limit of its distribution area. In contrast, in its southern boundary, water stress has decreased growth and, in some cases, massive mortality.

In the scope of a multidisciplinary R&D project entitled “P. sylvestris in Portugal: the Southwest end or just the end?” (PTDC/AGR-CFL/110988/2009) supported by the FCT – Portuguese Foundation for the Science and Technology and executed at UTAD, a total of nine Scots pine populations were characterised. In the first approach, we performed the molecular characterisation of seven Scots pine populations located in the North and Centre of Portugal, recognised as planted stands and representative of the species distribution in our country. To assess the intra- and inter-population genetic variability, genetic structure and relationships, we used RAPD and ISSR markers [15]. Furthermore, the RAPD and ISSR patterns achieved in the Portuguese samples were compared with others produced in Scots pine individuals from native populations of different countries [15]. The global RAPD and ISSR data analysis revealed higher genetic variability within rather than among populations and a highly differentiated genetic structure [15]. The genetic relationships among the Scots pine populations from different provenances indicated higher similarity between the Portuguese and German individuals, suggesting that the plant material used in the establishment of the P. sylvestris stands at the North and Centre of Portugal probably had origin in Central Europe [15]. Scots pine untraceable seeds have been traded and planted across Europe for a long time [43, 44]. The molecular characterisation of Scots populations in different countries can provide valuable information for traceability, certification of forest reproductive material, and definition of germplasm conservation and management strategies [5].

The two putative native Scots pine populations from ‘Serra do Gerês’ (NW Portugal) [6, 8, 9] were characterised by the codominant, multi-allelic and highly reproducible nSSR and cpSSR markers [18]. The nSSRs are highly efficient for plant genotyping and have been widely used in population genetics studies [5]. The cpSSRs exploit the DNA polymorphism within the chloroplast genome that is haploid and inherited by the male parent in Pinus spp.. Since it shows a lower mutation rate than the nuclear genome, it can provide insights about founder effects, genetic drift, historical bottlenecks and other evolutionary events [5, 42, 45]. DNA markers targeting the mitochondrial genome that is maternally inherited and spread by the seeds are also helpful in population genetic studies allowing the exploitation of the history and postglacial recolonisation routes [5 and references therein]. Using nSSR markers, [18] reported higher intra-population polymorphism than among populations. Several studies based on these neutral DNA markers have reported similar results for Pinus spp. [5, 19]. The cpSSRs identified distinct haplotypes in the Portuguese populations, resulting in their clustering apart from different European native populations used for comparison [18]. Besides, the two Portuguese populations also showed genetic differentiation between them [18]. Such a study supported the hypothesis of the existence of native Scots pine populations in Portugal and our country as the westernmost limit of the species’ natural distribution [18]. The high genetic diversity found in the Portuguese Scots pine populations [15, 18] can explain its adaptive potential to the Mediterranean temperate climate corroborating its broad adaptive potential and phenotypic plasticity, reported mainly for this species with the highest natural distribution area among the conifers [39]. However, as noted earlier for other marginal P. sylvestris populations located in the limits of the natural distribution area of the species, such individuals may show phenotypic variations due to the adaptation pressure to a unique environment, along with cytogenetic instability [[46] and references therein]. Therefore, we analysed mitotic chromosome spreads prepared with fixed root-tips upon germination of seeds collected in the native populations of ‘Serra do Gerês’ [29]. As expected for most of the conifer genera and particularly a species from genus Pinus [47], the karyotype of the Scots pine individuals from ‘Serra do Gerês’ presented 2n = 2x = 24 chromosomes with similar morphology and size (Figure 2a).

Figure 2.

Normal (a) and irregular (b, c) C-metaphase cells of P. sylvestris (2n = 2× = 24) observed in DAPI-counterstained chromosome spreads, prepared with ice-cold water treated roots, showing: (a) 24 chromosomes with similar size and shape; (b) anomalies like one ring chromosome (arrow) and polycentric chromosomes (arrowheads indicating one of those chromosomes as an example); and (c) centric (arrow) and acentric (arrowhead) chromosomal fragments.

Previous cytogenetic studies done in Scots pine from marginal populations were based on Classical Cytogenetics techniques and allowed analyses at the morphometric, mitotic, meiotic and nucleolar activity levels [46, 47]. The karyotype of Scots pine was referred to as composed of 11 pairs of metacentric (I – X, XII) and one pair (XI) of submetacentric chromosomes [46, 47]. Mitotic and meiotic instabilities and chromosomal irregularities in individuals from marginal Scots pine populations were reported [46, 47]. To analyse the chromosomes of the Scots pine individuals from ‘Serra do Gerês’, we used the Molecular Cytogenetics techniques, conventional FISH and ND-FISH, performed with the 45S rDNA pTa71 and 14 SSR sequences, respectively, as probes [29]. The root chromosome spreads showed anomalies such as ring chromosomes, polycentric chromosomes (with additional constrictions beyond the centromere), centric and acentric chromosomal fragments [29] (Figure 2b and c). These anomalies were similar to those reported earlier for marginal populations of Scots pine and other conifers [[46] and references therein]. The marginal populations are subjected to different selection pressures than those within the central distribution area [29]. The stressful environmental conditions might be responsible for the molecular data achieved in the two native Scots pine populations from ‘Serra do Gerês’, namely, the high intra-population genetic variability and higher differentiation relative to the populations from Central Europe used for comparison [18]. The high molecular instability can be the origin of the observed cytogenetic irregularities due to the occurrence of DNA strand breakage in response to stress factors.

The pTa71 probe detected 14 rDNA loci on the short and long arms of seven Scots pine chromosome pairs [29]. Among the 14 tested SSR probes, eight of them, based on di-, tri- and tetranucleotide repeat motifs, revealed hybridisation signals on centromeric, pericentromeric, telomeric, subtelomeric and/or interstitial regions of the Scots pine chromosomes [29]. The SSR (AG)₁₀ showed a more discriminative hybridisation pattern through the 24 Scots pine chromosomes and allowed their karyotyping [29].

The Pinus spp. genomes are physically large, as expressed by their DNA contents [48], and highly complex. The high amount of repetitive DNA sequences of the pine genomes provides a vast adaptive potential, and its analysis can be valuable for evolutionary studies. Hizume et al. [49] demonstrated that using telomeric, centromeric and rDNA sequences as FISH probes constituted reliable cytogenetic markers and allowed the identification of individual chromosomes in four pine species. The widely dispersed and abundant SSRs and retrotransposon-based sequences could also comprise excellent choices for FISH probes enabling additional karyotypes and/or new evolutionary clues among the genomes of pines and other conifers.

Considering the actual climate change scenario and projections, among other extreme environmental episodes, an increase in the frequency and severity of heat waves and drought episodes is expected [50]. A temperature window, a typical pattern of the Mediterranean Pinus spp., greatly influences pine seed germination and seedling emergence [51]. On the other hand, water stress, particularly the summer drought, is a limiting factor for seedling survival, reducing the forestry area, changing distribution patterns, compromising the success of afforestation programmes, and, ultimately, the persistence of pine stands [26, 52, 53, 54]. When we induced three water availability regimes (watering, moderate water stress and drought) to Scots pine populations from five European provenances (‘Gerês’, ‘Puebla de Lillo’, ‘Montes Universales’, Germany and Sweden), we verified that the Portuguese one, represented by ‘Ribeira das Negras’ (‘Gerês’), had higher tolerance to moderate water stress and drought than the remaining ones [53]. The Portuguese individuals presented the highest stability in photosynthetic reactions and better photochemical and metabolic behaviours under drought [53]. Besides, the relative expression ratio of three water stress-responsive genes (abaH, lp3, PAL1) in individuals from ‘Gerês’ were lower but increased gradually with the scarcity of water, followed by those of German [53]. These molecular results were exciting regarding our earlier study that assigned Germany as a potential origin of the plant material used to establish the allochthonous Scots pine populations in the North and Centre of Portugal [15]. The Portuguese germplasm of Scots pine, including the native populations of ‘Gerês’ and the planted ones, have the ability and high adaptive potential to respond to water deficit conditions [53]. On the other hand, the authors verified that the Swedish Scots pine individuals responded faster to moderate and severe water stress revealing the lowest tolerance among the studied provenances [53]. Also, the young needles of the Portuguese Scots pine individuals have physiological and morpho-anatomic parameters more suited to environments with low water availability than those of the remaining provenances [53]. Overall, the genetic, physiological and morpho-anatomic traits evidenced by the ‘Gerês’ population allowed us to suggest that the Portuguese germplasm is well adapted to the temperate climate and responds better and gradually to water stress relative to the other tested European provenances [53]. This information is precious concerning the conservation and use of germplasm in abroad regions where the afforestation, adaptation and survival of Scots pine have been or will be compromised by climate change. Likewise, in Portugal, Scots pine can be used for the afforestation of high-altitude areas to which Pinus pinaster is not adapted and where the risk of pinewood nematode attack is minor.

Abiotic and biotic stresses and other natural and artificial factors have been declining the forest area throughout Europe, bringing negative consequences to the forestry industry. Concerning the excellent adaptation of Scots pine to the SW end of Europe and the need for alternative resinous species for the wood and resin industry, we characterised the physical, chemical and mechanical properties of wood sampled in individuals from the allochthonous Portuguese P. sylvestris populations using X-ray microdensitometry, near-infrared (NIR) spectrometry and bending tests, to infer about its quality [55, 56]. The wood density and radial growth expressed by the ring width of the Portuguese-planted Scots pine individuals were higher than those reported for populations from different northern European regions [55, 56]. Nevertheless, these wood traits did not supplant those exhibited by P. pinaster, constituting the main resinous species exploited for industrial purposes in Portugal [55]. The mechanical and physical traits were positively correlated, evidencing that trees with high radial growth can also have excellent mechanical performance [56]. Overall, the Portuguese Scots pine wood evidenced high quality, almost similar to the one of P. pinaster [55, 56], reinforcing the need for conservation and use of this germplasm with benefits at the ecological and economic level in Portugal and abroad.

3.2 Pinus nigra

The distribution of P. nigra (Black pine) in Portugal is restricted to six sites in the North and Centre of the country where adult, regular, mature and planted (allochthonous) stands can be found [16, 19, 57, 58].

Although widely dispersed throughout Europe, the wood quality and growth of P. nigra were not very studied. Still, it has the potential to be explored, as revealed earlier by its physical, chemical and mechanical traits, with the advantage that it could be used for the reforestation of mountainous areas where other resinous species with higher economic importance, such as P. pinaster are not adapted [57, 58]. In fact, during the 19th century, P. nigra was used to reforesting degraded European landscapes due to its adaption to shallow rocky soils and xerophytic sites [59].

The scattered distribution of this pine species through Southern Europe, Asia Minor, Corsica and Sicily islands, and NW Africa led to high inter-population variation at the genetic and biochemical levels and high adaption to different ecological niches [60, 61, 62, 63]. Consequently, its infraspecific taxonomic classification has been revised, and the most accepted comprises the existence of six subspecies and some of them with regional varieties. Five P. nigra subspecies were assigned to populations distributed through Eurasia: (i) dalmatica (Balkans); (ii) laricio (Corsica, Sicily and Calabria); (iii) nigra (Alps); (iv) pallasiana (Turkey and Crimea) and (v) salzmannii (France and Spain) [10, 62, 64, 65, 66, 67, 68], whereas the (vi) subspecies mauretanica (Maire and Peyerimh) Heywood was ascribed to the NW Africa [69].

The origin and infraspecific taxonomy of the plant material used in establishing the Portuguese-planted stand 50–90 years ago are unknown [16]. In the 1980s, a phenological characterisation of the Portuguese P. nigra stands classified them as belonging to the subspecies laricio, salzmannii and nigra [70]. Therefore, while the molecular characterisation of the six Portuguese P. nigra stands, currently representative of the species distribution in our country, we also analysed samples from different European provenances and with certified infraspecific taxonomy taken into consideration the three subspecies assigned earlier [16, 70]. Firstly, we used the dominant ISSR and Start Codon Targeted (SCoT) markers to evaluate the genetic diversity, relationships and structure and extrapolate the Portuguese populations’ infraspecific taxonomy [16]. Both ISSR and SCoT markers revealed higher genetic similarity among the Portuguese populations and those from abroad with provenances belonging to the subspecies laricio [16], one of the subspecies assigned to Portugal by [70]. Nevertheless, when we individually considered the ISSR and SCoT data, the former marker system revealed higher genetic similarity with the variety corsicana, and the second, with the variety calabrica, both from subspecies laricio [16]. The genetic relationships and differentiation of the six Portuguese P. nigra populations revealed two main clusters, supporting the possible use of plant material from two different origins in establishing the stands performed in the past century [16]. These results were later reinforced by the use of the codominant nSSRs and cpSSRs, which structured the six Portuguese P. nigra populations into two main genetic clusters but with low differentiation (F_ST = 0.04) [19] matching our previous hypothesis of plant material from two varieties belonging to the same subspecies. Additionally, and similarly to the results achieved with the dominant DNA markers, the SSRs also evidenced a higher genetic diversity within (95%) rather than among (5%) populations [19]. The molecular data achieved in the Portuguese P. nigra populations suggested a broad adaptive potential that could be explored if we regard its ability to be used for the reforestation of mountainous areas, reinforcing its ecological value in Portugal and abroad. Also, this species has potential for the forestry industry and can constitute an alternative resinous species considering the results achieved by characterising their wood’s physical, chemical and mechanical traits [57, 58]. Some authors pointed out that the natural regeneration in P. nigra is complex [59, 71], and it can be even more difficult in dry soils, which considering the climate projections, could be problematic for the species’ survival. Hence, we evaluated the water stress tolerance of different infraspecific taxa of P. nigra based on the analysis of the root mitotic cell cycle and alkaline Comet assay [26] (Figure 1). In this latter study, we imbibed P. nigra seeds of different provenances with certified infraspecific taxonomy to aqueous solutions of 10% and 20% polyethylene glycol (PEG) (seed osmopriming) during four days to mimic water stress and then allowed their germination on distilled water (Figure 1). Roots were collected and immediately fixed upon germination to analyse the effect of induced water stress in the mitotic cell division [26] (Figure 1). We verified that this abiotic stress delayed the seed germination, induced mitotic anomalies in the meristematic root cells, and caused nuclear DNA damage as detected in needles of young seedlings weeks after the interruption of the osmotic stress induction [26]. This result suggested a stress memory transmission between plant tissues and can also explain the high rate of seedling mortality widely reported to different Pinus spp. [59]. These adverse effects were more pronounced in the subspecies laricio variety corsicana, to which the 20% PEG treatment was lethal [26], which showed high genetic similarity with the Portuguese populations [16]. The evidence of differential water stress tolerance among the studied P. nigra infraspecific subspecies and varieties is fascinating. It should be exploited in-depth in additional taxa, particularly under climate change scenarios.

The current European P. nigra forests are ageing, present decline or constitute fire-prone ecosystems. In the absence of seed trees, the persistence of Black pine stands depends on direct replacement by planting, which is usually chosen but costly, or natural regeneration that, despite preferable, is hard due to various interacting factors [59]. Natural forestry regeneration is a slow and difficult-to-predict process [72], but it is determinant in the stand persistence [73]. Despite the development of predicting models based on different climate scenarios to simulate regeneration and to help in decision-making concerning forest management [72], the characterisation of marginal pine populations based on the approaches reviewed here is still essential and opportune. The conservation and use of P. nigra germplasm with high water stress tolerance, high genetic diversity, known provenance and wide adaptive potential constitute valuable resources to be used in the planting or sowing of Black pine stands in Portugal and abroad.

3.3 P. pinaster

Different authors have been reporting the solid genetic control of the resin chemical components and yield in various pine species, including P. pinaster, which constitutes the main conifer species exploited for resining in Europe [74, 75]. Efforts have been made to correlate the genotypic and phenotypic data concerning resin yield and quality in different pine species [[75, 76] and references therein]. However, in a first approach, the genetic characterisation of maritime pine stands installed for resining by using molecular markers can be helpful in the selection of elite trees for further establishment of high-resin yielding seed orchards to implement tree breeding programmes towards the improvement of resin yield and quality [17, 74]. A few years ago, our team sampled vascular cambium in 200 adult P. pinaster individuals from two populations in the North of Portugal (‘Paredes de Coura’ and ‘Vila Pouca de Aguiar’) used for resin production and analysed their genomic DNA with the neutral molecular markers ISSR and SCoT [17]. Both marker systems revealed high intra-population genetic diversity and high inter-population genetic differentiation. The intra-population ISSR and SCoT polymorphism was more elevated in ‘Vila Pouca de Aguiar’ [17]. Upon comparison of the molecular data with the resin yield values achieved for two consecutive years, we verified that the population of ‘Paredes de Coura’ that showed lower intra-population ISSR and SCoT polymorphism presented higher resin productivity [17]. Besides, the trees with the highest resin yields gave similar ISSR and SCoT molecular patterns, showing the closest genetic relationships and enabling their selection as elite trees based on these approaches [17]. While low intra-population genetic variation for resin production seems advantageous, high genetic diversity is required to ensure adaptation to stressful factors and species survival. Maritime pine stands are mainly found in the North of Portugal and constitute fire-prone habitats with complex management [77]. This pine species developed evolutionary strategies to cope with fire, such as serotonin protecting the cones’ seeds from high temperatures, ensuring their natural regeneration and maintenance of high intra-population genetic diversity [78, 79].To test this latter hypothesis, [60] characterised juvenile P. pinaster individuals (5–6 years) naturally regenerated in stands that burned once and twice and compared with adult pine trees from an unburned stand. The authors verified that, in the short term, the Nei’s gene diversity (expected heterozygosis) was higher in the individuals from the twice-burned stand and that fire recurrence did not cause genetic erosion. Considering the future increase in wildfires frequency and water deficit, particularly in the Mediterranean region [50], and the influence of these factors on the natural regeneration of P. pinaster [80], recently, we analysed seeds from postfire naturally regenerated stands that burned once, twice and three times between the 1990s and 2005 [27]. In the long term, fire recurrence did not influence the seed germination index but delayed the mean germination time significantly, even during recovery [27]. To the same seeds, 10% and 20% polyethylene glycol (PEG) solutions were added before and during germination to analyse how fire recurrence, induced water stress and their interaction impacted seed germination and root cell division [27]. The water stress induction did not significantly influence the germination index of the P. pinaster seeds [27]. The fire recurrence negatively impacted the regularity of root mitosis and increased the frequency of irregular cells, mainly in seeds collected in stands that burned twice and thrice [27]. The water stress induced with 10% PEG (osmotic potential: −0.4 MPa) significantly increased the frequency of mitotic anomalies in the root-tip cells [27]. Regardless of the fire regime, seeds exposed to 20% PEG (−0.8 MPa) did not germinate or presented root growth inhibition a few days after the beginning of germination [27]. Anomalies in root cell division might hinder the growth of the radicular system, constituting a significant problem in dry soils.

4. Conclusions

This chapter reviewed molecular and cytogenetic approaches that, individually or combined, integrated multidisciplinary projects and/or postgraduate studies developed at UTAD and focused on Portuguese populations of P. sylvestris, P. pinaster and P. nigra. We are confident that the information we acquired through the last decade concerning genetic diversity, structure and relationships, extrapolation of provenances and infraspecific taxonomy, along with the estimation of their ability to respond to water stress or fire recurrence, can give new insights about the adaptive potential of the Portuguese pine germplasm that can be explored at the national and international levels. The gathered information is helpful for decision-making regarding the definition of strategies for silvicultural practices, conservation and use of genetic resources, and implementation of breeding programmes, particularly for marginal pine populations that inhabit ecosystems in the limits of their natural distribution, experiencing unfavourable climatic conditions that might increase the risk of seed recruitment and competition for resources, threaten the stands and species persistence.

Acknowledgments

The authors acknowledge the contribution of all researchers’ colleagues, field technicians and students from UTAD and other national and international institutions involved in the execution of the cited works. Additionally, the authors acknowledge the funding sources that enabled the execution of the cited works, namely: (i) the Portuguese Foundation for the Science and Technology (FCT – “Fundação para a Ciência e a Tecnologia”) which supported the R&D project PTDC/AGR-CFL/110988/2009 co-financed by the European Fund of Regional Development (FEDER) – COMPETE-QREN program (leaded by J. Lima-Brito); projects UID/AGR/04033/2019, UIDB/04033/2020 and POCI-01-0145-FEDER-006958-FEDER/COMPETE/POCI - Operational Competitiveness and Internationalisation Program attributed to CITAB/UTAD; the postdoctoral grant SFRH/BPD/68932/2010 (attributed to A. Carvalho) and the doctoral grants SFRH/BD/91781/2012 (attributed to A. Dias) and SFRH/BD/98777/2013 (supervised by J. Lima-Brito, J. Lousada and M.J. Gaspar) co-financed by the Social European Fund (FSE) under the POPH-QREN program; (ii) the COST Action FP1202 “Strengthening conservation: a key issue for adaptation of marginal/peripheral populations of forest trees to climate change in Europe (MaP-FGR)” which financed a Short Term Scientific Mission to A. Dias; and (iii) Project RESINPROVE supported by EU funds under the Program of Rural Development in the Continent (PRODER – “Programa de Desenvolvimento Rural do Continente”). Ana Carvalho acknowledges the FCT and UTAD for her contract as a researcher under the scope of the D.L. no. 57/2016 and Law no. 57/2017.

The article processing charges of this chapter were supported by national funds provided by the Portuguese Foundation for the Science and Technology (FCT – “Fundação para a Ciência e a Tecnologia”) to the research unit CITAB under the project UIDB/04033/2020.