435 For. Snow Landsc. Res. 76, 3: 435–438 (2001)
Evaluation of Portuguese chestnut stands by RAPDs
Rita Costa Seabra1, Ana Margarida Simões1, José Baeta1, M. Salomé Pais2
1 Estação Florestal Nacional Nacional. INIA, R. Borja, 2 1399-051 Lisboa, Portugal
2 Laboratório de Biotecnologia Vegetal. ICAT, Campo Grande 1749-016 Lisboa, Portugal rseabra@efn.net.novis.pt
Abstract
The aim of this study is to evaluate the genetic pool of Portuguese chestnut populations, managed for different purposes. Young leaves were collected in fruit orchards, coppice and high forest stands. The statistical analysis performed with RAPD data showed that the individuals collected in fruit orchards are less differentiated, while the high forest and coppice stands showed greater genetic differentiation.
Keywords: chestnut, RAPDs, orchards, high forest, coppice, Castanea sativa, Portugal
1 Introduction
Castanea sativa is an economically important species currently occupying more than 1 700 000 ha in southern Europe. It covers important areas in Italy, Turkey, Spain, Portugal, France, Greece and in the United Kingdom.
During the past 100 years both European and American chestnut species have suffered severely from root rot, known as ink disease, caused by Phytophthora cinnamomiRands and P. cambivora(Petri) Buiss. In Portugal, the disease has been responsible for the decline of the chestnut-producing area by 50 000 ha since 1950.
In recent decades there has been an increased interest in chestnut cultivation in Portugal.
According to the last inventory in the continental country, the total area of chestnut for fruit production was 14 359 ha in 1986 and 19 881 ha in 1998. The average production of chestnut fruit was near one ton per hectare (Estatísticas Agrícolas 1999). 96% of Portuguese chest- nut-growing areas (orchards and forest) are concentrated in the northern central region of Portugal.
Chestnut cultivation is very important in Portugal for both nut and wood production.
Nevertheless, until now there has been no information on the levels of genetic variability in orchard used for nut production, high forest (wood production) and coppice populations (wood production).
The aim of this study is to evaluate the gene pool of the species Castanea sativaMill. in Portugal, in order to make an inventory of Portuguese chestnut genetic resources. To this end, sixteen populations of chestnut, collected in the Trás-os-Montes Region (northern Portugal), were analysed by RAPD. The inventory will lead to the establishment of conser- vation priorities, and will allow more precise planning of future chestnut forestation pro- grammes.
436 Rita Costa Seabra et al.
2 Material and methods
Eighty-one trees in total, collected in 16 different types of populations with different manage- ment, were sampled. In the fruit orchards, the samples were collected in 40 different trees, from eight different populations in eight different sites (A, B, C, D, E, F, G, H), corresponding to five trees in each site. In the high forest stands, the samples were collected in sixteen different trees in three different populations in sites I, J and K. In the coppice stands, the samples were collected from twenty-five trees from different populations in five different sites (L, M, N, O and P). The DNA was isolated from young leaves using the Plant DNeasy kit (Qiagen). Eight arbitrary primers (Operon Technologies) were selected and used in the PCR reactions. The amplification conditions were previously optimised for chestnut plant material (SEABRA2000). Each amplifi - cation re action was repeated three times in order to guarantee the reproducibility of the RAPD profiles.
For the statistical analysis the data were organised in a computer file as binary matrices, where
“absence of a band” was coded as 0 and “presence of band” coded as 1. Canonical Discriminant Analysis (CDA) was performed for the different populations studied (fruit orchards, high forest and coppice stands), according to the geographic origins of the groups, comparing these groups with the variables associated with the bands obtained from the RAPD analysis.
3 Results
The CDA performed for fruit orchards shows that the populations from sites A, F and H were very well defined, and were significantly different from the population at the centre (χ2for site A = 59.73; for site F = 91.68 and for site H = 63.69, being χ2 = 9.21, where p< 0.01). The remaining populations from sites B, C, D, E and G, did not show differentiation from the centre population (χ2for site B = 0.83 for site C = 3.36 for site D = 2.41 for site E = 2.81 and for site G = 9.52). When considering sites A, F and H, the individuals in group H appear to be more homogeneous, and were clearly distinct from those in the centre. In group F a genetic proximity between four individuals was observed. Sample number 29 was very far from both the four individuals of the group, and the central group. Finally, in group A, three individuals (2, 3 and 4) were found to be more closely related than individuals 1 and 5 (Fig. 1).
2 4
Site H 29
0.8 0.6 0.4 0.2 0 –0.2 –0.4 –0.6 –0.8
–1.5 –1 –0.5 0 0.5 1
1 5
3 Site A Site F
Component 1
Component 2
Fig. 1. Canonical Discriminant Analysis performed for fruit orchard stands showing that populations from sites A, F and H are significantly different from those at the centre. Components 1 and 2 explain 50% of the total variation.
437 For. Snow Landsc. Res. 76, 3 (2001)
For the high forest all three populations were well differentiated. The population from site K showed greater genetic homogeneity between individuals compared to the popu - lations from sites I and J. The individuals from population I were the most genetically heterogeneous (Fig. 2).
Site I
Site J 2
1
0
–1
–2 –1.5
Site K
Component 1
Component 2
Fig. 2. Canonical Discriminant Analysis performed for the high forest stands shows that the populations from sites I, J and K were well differentiated. Components 1 and 2 explain the total variation.
2
Site P 1.5
0.5
–1 0.4 1.1
Site L
Component 1
Component 2
–0.5
1.5
–1.7 –0.3 1.8
Fig. 3. Canonical Discriminant Analysis performed for coppice stands shows that populations from sites L and P were the most differentiated. Components 1 and 2 explain 70% of the total variation.
In the coppice stands, the values of χ2obtained for all five populations were significant (χ2for Group L = 142.08; for Group M = 10.47; for Group for N = 11.13; for Group O = 56.68 and for Group P = 148.24, where χ2= 9.21, and p< 0.01). The populations from sites L and P were the most differentiated (Fig. 3).
The individuals collected in fruit orchards were less genetically differentiated than those in the high forest and coppice stands.
438 Rita Costa Seabra et al.
4 Discussion
The genetic characterisation of different chestnut populations performed in this study has provided interesting information about the variation in different chestnut populations managed for either nut production in fruit orchards or wood production in high forest or coppice stands. The fruit orchard stands showed less genetic differentiation than the high forest and coppice stands, even though three groups were very different from the rest of the individuals in the other five groups. These five groups may have been less differentiated because chestnut trees grown for fruit production are normally multiclonal plantations in terms of their grafted parts and the majority of individuals belongs to the same variety (26/40). Similar results have been obtained for Greek populations of Castanea sativa (Drouzas, pers. comm.).
The populations managed as high forest are very differentiated. They constitute the only populations managed as high forest in Portugal, with trees that are 50 to 60 years old. This makes them very interesting candidate populations for conservation. All the coppice stand groups showed differentiation, especially groups L and P. All the coppice stands have rotation periods of 15 years. Group L (coppice stands) is in the same location as group K (high forest stands) and both groups showed a high genetic homogeneity (Fig. 2 and 3).
In this study the genetic differentiation within orchards, high forest and coppice stands was evaluated. The next step will be to evaluate the genetic differentiation among these populations.
5 References
Estatísticas Agrícolas Instituto Nacional de Estatística (1987–1999).
SEABRA, R.C., 2000: Caracterização Molecular do Género Castanea.Silva lusitana 8, 2: 149–164.
Accepted 4.3.02
