RAPD and freezing resistance in Eucalyptus globulus
Marta Fernández R.
Financial support: INNOVA Biobio Grant 03 B1 210 L1.
Keywords: Eucalyptus globulus, freezing resistance, molecular markers, RAPD.
is the second most important forest species in
Labill. is an important species for pulp production. This species
is planted in countries as
practical application of molecular biological techniques in plants
is the genetic identification or fingerprinting through molecular
markers. The technique of molecular markers is based on the detection
of the DNA sequences or combinations that are unique to the individual
plant under study (Henry, 1997). The correct identification
of individuals or clones is important in operational cultivation and
genetic development, since diverse degrees of somaclonal variation
have been detected in different species, under in vitro cultivation
conditions (Keil and Griffin, 1994; Widen
et al. 1994). Consequently, it is essential to have a practical
and economic way to demonstrate clonal identity and fidelity in different
species (Nesbitt et al. 1997). The main advantage
of Random Amplified Polymorphic DNAs (RAPDs) over other techniques
as isozymes is that they yield abundant polymorphism from mature tree
tissue. For Eucalyptus only a relatively small number of polymorphic
allozyme are available (Moran and Bell, 1983), therefore
markers such as RAPDs could be a good alternative. RAPDs are relative
cheap and its development time is minimal compared to other DNA markers
(Skabo et al. 1998). PCR technique based on RAPD
(Whelsh and McClelland, 1990) allows the detection
of multi-locus genetic variations using small primers of arbitrary
sequences. Basically a small quantity of tissue is used, from which
DNA is extracted. Currently RAPDs in
cold resistance in Eucalyptus has been broadly studied in diverse
The objective of the present study is to evaluate the use of RAPD-PCR molecular markers to identify and differentiate freezing sensitive from freezing resistant E. globulus clones.
clones of E. globulus which showed differences in freezing
resistance were studied. These clones were selected from a total of
300 clones of E. globulus which were previously tested under
field conditions. The clones were planted in 2 cold locations in Canteras
were collected from each clone, frozen in liquid nitrogen and stored
reaction conditions used were the following: an initial step of denaturation
From 20 primers tested, 18 generated appropriate amplification patterns. Of these, only primer UBC 210 generated a high background fluorescence. The number of polymorphic bands obtained varied between 6 (OPB 05 primer) to 18 (UBC 237 primer) (Table 2). A total of 213 reproducible bands were scored, out of which, 177 were polymorphic (Table 2).
Of the fragments amplified with primer UBC 218 (Figure 1), two bands were identified that were selective for some of the freezing resistant clones, and was not observed in none of the sensitive clones tested. A band of 768 bp was observed in samples of clones EG 15, EG 09, EG 08 and EG 12 which correspond to four of the eight frost resistant clones studied. Another band of 602 bp was present in three frost resistant clones studied: EG 15, EG 09 and EG 12 (Figure 1).
band of 248 bp was identified when using primer UBC
In contrast with other molecular techniques, such as, SNPs, SSRs, RFLP, DNA sequencing and allozymes, the technique of RAPD is very simple to carry out and it does not require previous knowledge of the genome in study. The use of RAPD has been demonstrated to have several advantages over other techniques of DNA 'fingerprinting' and it has been used for the identification of clones (Keil and Griffin, 1994). It allows to examine a large number of loci and primers are made on aleatory sequences. Despite of its advantages this technique has some practical problems, for example reproducibility (Van Oppen et al. 1996; Jones et al. 1997). However, if it is possible to standardize the DNA extraction procedure and find the optimal PCR conditions, the reproducibility of the patterns should not be a problem. In these study, primers OPF 01 and UBC 210 showed a high fluorescence, background, however it was possibile to obtain good reproducibility of the bands.
It is possible that products of different loci can have similar molecular weight and, for this reason, the identification of the bands can be difficult. Rieseberg (1996) analyzed the homology of 220 co-migranting fragments of RAPD in populations of wild sunflowers, he found that 91% of them turned out to be homologous. Rieseberg (1996) results indicate that fragments of similar size are a good indicator of homology. In our study, a good band separation was obtained. Only when using primer OPF 01, bands of similar molecular weights were obtained. All the primers used in this study, were selected under the approach of having been previously identified for their capability of detecting reproducible polymorphic bands in E. globulus, according to the results found by Nesbitt et al. (1995), after analyzing 140 primers in this specie.
Despite the large number of polymorphic bands found in the 15 clones of E. globulus studied, only primers UBC 218 and UBC 237 generated bands that could be considered as potential markers to identify frost resistant clones. As example, bands of 768, 602 and 248 bp were present in four, three and four of the eight frost resistant clones studied, respectively.
Clones EG 15, EG 09 and EG 12, when amplified with primer UBC 218 showed two polymorphic bands (Figure 1). Comparing these results with those shown in Table 1, it can be observed that clone EG 08 had the largest percentage of damage in the buds (23,0%) and clone EG 12 had the largest percentage of defoliation damage (6.9%).
Primer OPF 01 generated a total of 11 bands, of which 9 were polymorphic and one of these (880 pb), was visualized in all the frost sensitive clones, but also in the frost resistant clone EG 09, therefore it was not considered a selective marker. It is necessary to highlight that this last clone, together with clones EG 08 and EG 10, were classified as freezing resistant (Table 1), although they had the largest percentage of damage in buds and damage for defoliation.
The maximum capacity of a genotype to resist the freezing temperatures is obtained after a hardening process, which is generally reached after the exposure to low temperatures. The hardening capacity to cold will depend on the species in study and of the magnitude of the low temperatures (Alberdi and Corcuera, 1991). The increase of the temperatures during the spring and summer time favors the de-acclimation of the plants, it is for this reason that frosts in the late spring are usually very harmful. Therefore, the dynamics of the acclimation-de-acclimation would be important in the characterization of the genotypes, regarding their frost resistance.
Through the technique of RAPD, which is relatively cheap and requieres small quantities of DNA, it was possible to identify two primers (UBC 218 and UBC 237) that generated polymorphic bands in E. globulus. However due to the small number of clones analysed it can not be concluded that any of the bands could discrimante between freezing tolerant from susceptible clones. Therefore the results obtained must be validated with a larger number of clones and primers.
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