Short communication: Development of a new polymorphic genetic marker in Araucaria araucana (Mol) K. Koch

Seed storage proteins have been used as genetic marker in forest species to evaluate genetic variability, demon-strating its effectiveness both in conifers and broad-leaved. In conifers, megagametophyte storage proteins are particularly useful because of their haploid nature. The aim of this study was to determine whether these proteins could be used as a new marker of genetic diversity in Araucaria araucana, one of the oldest conifers of South America and a representative symbol of Chilean forest biodiversity. For this, megagametophytes from two A. araucana populations were assessed to identify polymorphic bands and to obtain a preliminary estimation of the genetic diversity. The results revealed that globulin is the best fraction for measuring the variability in the species, due to their high level of variation (20 identified bands, 11 of them polymorphic). Both populations showed high genetic diversity, with more than 92% of the variation within populations. The study highlighted that these proteins can be used to measure the genetic diversity in A. araucana , providing good information to ensure the preservation of the species genetic resources.

*Corresponding author: ge2macum@uco.es Received: 10-05-11. Accepted: 11-01-12 Abbreviations used: AA (homozygotic tree for the absence of a band); CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora); Gst (genetic differentiation among populations); Hs (genetic diversity within populations); Ht (genetic diversity over all populations); PA (heterozygotic tree for the allele responsible of a band); PP (homozygotic tree for the allele responsible of a band). A new polymorphic genetic marker in Araucaria araucana Seed storage proteins have been used as genetic marker in plants for evaluating the genetic variability of many species. Their main advantages as markers are a high polymorphism level, simple genetic control, environmental independency and their economic, easy and fast analysis (Gepts, 1990). These proteins are present in seeds of all plant species, because they are sources of amino acids for synthesis processes that occur during germination. Moreover, their variations have no repercussion on any physiological function, thus allowing these random changes to be maintained.
The seed storage proteins are classified in four fractions according to their solubility: albumins, globulins, prolamins and glutelins (Osborne, 1924). These proteins have been widely studied in cereals due to its relations to bread-making quality (Payne et al., 1982). These studies have shown high levels of genetic variability, highlighting their possibility in the study of genetic resources (Metakovsky et al., 2000) and varietal identification (Zillman & Bushuk, 1979).
The role of these proteins as genetic marker in forest species has been scarcely explored despite their demonstrated effectiveness in evaluating genetic diversity both in broad-leaved and conifers. In this respect, the protein fraction used as a marker is different depending of the species: albumins are used in Abies pinsapo Boiss. (Martín et al., 2010a), globulins in Pinus pinea L. (Alvarez et al., 2004) and Castanea sativa Mill., (Alvarez et al., 2003) and glutelins in Quercus ilex L.  and Nothofagus spp. (Martín et al., 2010b). Furthermore, in conifers this technique is particularly favourable given that seed storage proteins constitute an important component of the megagametophyte, a haploid tissue derived from the female gametophyte that surrounds the embryo in a mature seed. Thus, analysis of the segregation pattern in parental trees may enable the determination of the genetic control and linkage relationships without making crosses (Neale & Adams, 1981;El-Kassaby et al., 1987).
Araucaria araucana (Mol) K. Koch, or monkeypuzzle tree, is one of the oldest conifers of South America and is part of the impressive landscape in native forest ecosystems because of its umbrella-shaped adult crown. Moreover, it is considered a representative symbol of Chilean forest biodiversity given its endemicity and longevity (Hoffmann et al., 2001). The current distribution of the species is fragmented and occurs mainly in the Andean region at the frontier of Chile and Argentina and in two disjunct populations in the Coastal Cordillera (Nahuelbuta Cordillera) of Chile.
Thus, the current populations are remnants of a more extensive former distribution that was severely diminished by the intense exploitation of the species in the past, the impact of domestic herbivores, seed harvest and natural perturbations (Veblen, 1982;Burn, 1993). For these reasons, the species is included in Appendix I of the Convention on International Trade of Endangered Species of Wild Flora and Fauna (CITES) and is listed as a vulnerable species by the International Union for Conservation of Nature (IUCN, 1996).
Until now, there is no a complete diagnosis about the situation and distribution of the genetic resources of the species and the available information is contradictory, in some cases. On the one hand, studies using markers such as isozymes (Gallo, 2003;Ruiz et al., 2007) and RAPDs (Bekessy et al., 2002(Bekessy et al., , 2003 have indicated that genetic diversity is high and unaffected by the ecological heterogeneity of the species. Conversely, a recent study based on chloroplast and mitochondrial markers reported the low variation in the species (Marchelli et al., 2010). On the other hand, an expedition carried out in the distribution range of the species revealed high fragmentation in some Araucaria forests, indicating low or nonexistent regeneration (Drake et al., 2009) and corroborating the hypothesis that these Araucaria stands are in decline phase and will be lost in a short period of time (Drake, 2004).
To assess the complex situation of this species, it is necessary to first implement the information on its genetic diversity using all available means. Thus, the aim of this study was to determine whether seed storage proteins could be used as new marker of genetic diversity in the species.
Twenty-one trees from two populations of A. araucana from both the Chilean and Argentinean sides of the Andean Cordillera were collected for the study. The Chilean population was situated in the Cunco District (38° 57' S; 71° 40' W) and the Argentinean in Lanin National Park (39° 47' S; 71° 40' W). This material comes from a collecting mission carried out in 2007 in the distribution range of the species (Drake et al., 2009). Eight megagametophytes per tree were used for the analysis, because this is an optimum number of megagametophytes for estimating the homozygotic or heterozygotic nature of each locus in conifers (Morris & Spieth, 1978).
Megagametophytes were isolated by removing both the seed coat and the embryo. The lipids were removed with diethyl ether (50:1 v/w, 1 h, 4°C), followed by a second extraction with acetone (50:1 v/w, 1 h, 4°C).
Proteins were fractionated in vertical SDS-PAGE slabs in a discontinuous Tris-HCl-SDS buffer system (pH 6.8/8.8) at a 12% polyacrylamide concentration (w/v, C=1.28%). The Tris-HCl/glycine buffer system of Laemmli (1970) was used. Electrophoresis was performed at a constant current of 30 mA/gel at 18°C for 10 min after the tracking dye migrated off the gel. Gels were stained overnight with 12% (w/v) trichloroacetic acid solution containing 5% (v/v) ethanol and 0.05% (w/v) Coomassie Brilliant Blue R-250. Destaining was carried out with tap water.
The Mendelian inheritance of each polymorphic band in the megagametophytes from all polymorphic trees for each band was tested for goodness of fit to the expected 1:1 (presence:absence) segregation using the chi-square test. The eight megagametophytes from each tree were used to determine if the mother tree was homozygotic for the allele responsible for the band (PP), homozygotic for the absence of the band (AA) or heterogygotic (PA). Genetic variability parameters as the percentage of polymorphic bands (P), total genetic diversity over all populations (Ht), the average genetic diversity within populations (Hs) and genetic differentiation between populations (Gst) were calculated according to Nei (1973) using Popgene software, version 1.32 (Yeh et al., 1997).
Among the four protein fractions, globulins showed the highest and the clearest resolution between bands with no detected ghost bands and other contaminations.
Moreover, the method was highly reliable and repetitive, and these bands were consistently present. These results agree with those reported in the study of other conifers that indicated globulins as the main stored fraction in P. pinea (Allona et al., 1994) and as the best fraction for the analysis of the genetic diversity in the species (Alvarez et al., 2004).
Five zones were identified in the gel, classified according to a molecular weight marker used as reference: A (upper 66 kDa), B (66-45 kDa), C (45-30 kDa), D (30-14 kDa) and E (below 14 kDa) (Fig. 1). The identified bands were named with the letter corresponding to the zone where they were located and a number in function of their mobility.
The analysis of the evaluated accessions for globulins showed up to 20 consistently scored bands, 11 of which were polymorphic (55%). The differences between bands were mainly due to mobility although these differences were also due to intensity in some cases. Four, two and five polymorphic bands were identified in zones C, D and E, respectively, whereas bands were tenuous in zones A and B and no polymorphism was detected (Fig. 1). The number of detected bands in the species was low compared with other conifers such as P. pinea and A. pinsapo; however, the percentage of polymorphic bands was significantly higher (Alvarez et al., 2004;Martín et al., 2010a).
The presence or absence of each polymorphic band in the megagametophytes of all heterozygotic trees fit a 1:1 ratio, with no significant deviation from the expected segregation in any case (results not shown). For each band, the genotype of the studied trees was obtained along with the corresponding frequency (Table 1). All polymorphic bands were present in both populations; the exception was band 5E, which was absent in the Lanin population. In general, all bands displayed a wide distribution among the populations, except for band 3C that showed the lowest frequency (19.0%); however, the frequency of this band was more than double in the Cunco District than in the Lanin National Park (27.3% vs 10.0%) ( Table 1).
A preliminary evaluation of the genetic diversity was carried out in two populations of the species. The total genetic diversity was considerably high (Ht = 0.429), being this value slightly higher in the Cunco District than in the Lanin National Park (0.431 and 0.374, respectively). The differentiation among populations (Gst) was 7.1%, indicating that 92.9% of the variation was within populations. When comparing the results obtained using this marker with those achieved with  isozymes, the genetic diversity was similar to that described by Gallo (2003) for the species in Argentina but significantly higher than that detected by Ruiz et al. (2007) for the species in Chile.
In conclusion, the current study highlighted that the globulin fraction is a good marker to measure the genetic diversity in A. araucana. Its use may provide an additional tool to complement the information provided by other genetic markers and to set up lines of action to ensure the preservation of the species genetic resources.