Inheritance of isozyme variants in Nothofagus antarctica (G.Forster) Oersted

M.J. Pastorino, P. Marchelli, M. Milleron and L.A. Gallo

Unidad de Genética Forestal - Instituto Nacional de Tecnología Agropecuaria (INTA) EEA Bariloche - CC 277 (8400) S.C. de Bariloche, R.N., Argentina mpastorino@bariloche.inta.gov.ar - fax +54 2944 422731

ABSTRACT

The analysis of the genetic variation and evolutionary processes related to the conservation and use of forest genetic resources requires reliable tools. The aim of the present study is to determine the mode of inheritance of isozyme variants of Nothofagus antarctica, species of economic and ecological importance inhabiting the temperate forests of southern South America. Buds from 56 trees and their offspring were subjected to horizontal starch gel electrophoresis. Good resolution and consistent band patterns were obtained from five out of 15 enzyme systems surveyed. Expected equal number of heterozygous and homozygous seedlings of open pollination from a putative heterozygous mother was tested by the Chi-square test. Superoxide dismutase (SOD) revealed invariant electrophoretic patterns. Glutamate-oxalacetate transaminase (GOT) showed two zones of enzymatic activity, each one encoded by a gene locus with codominant expression and three alleles. Phosphoglucose isomerase (PGI) and phosphoglucomutase (PGM) showed two zones, the most anodal of which were invariant, and six and five alleles respectively in the variable zone. Four confident polymorphic gene markers are thus available for different population genetics studies on Nothofagus antarctica.

Key words: Ñire; Gene markers; Nothofagaceae; Patagonia.

RESUMEN

El análisis de la variación genética y de diversos procesos evolutivos en relación con la conservación y uso de los recursos genéticos forestales requiere la utilización de herramientas confiables. El objetivo del presente estudio es determinar el modo de herencia de variantes isoenzimáticas en Nothofagus antarctica, una especie de los bosques templados de Sudamérica de gran importancia tanto económica como ecológica. Las yemas de 56 árboles adultos y plántulas de su descendencia se analizaron electroforéticamente sobre gel horizontal de almidón. Se obtuvo buena resolución y patrones de bandas consistentes en cinco de los 15 sistemas enzimáticos ensayados. Mediante el test de Chi-cuadrado se puso a prueba la hipótesis esperada de igualdad entre las frecuencias de plántulas homocigotas y heterocigotas en la descendencia de polinización abierta de madres heterocigotas putativas. La enzima superóxido dismutasa (SOD) reveló un patrón electroforético invariable. Glutamato-oxalacetato transaminasa (GOT) mostró dos zonas de actividad enzimática, cada una codificada por un gen-locus codominante y tres alelos. También se observaron dos zonas en fosfoglucosa isomerasa (PGI) y en fosfoglucomutasa (PGM), invariables las más anódicas en ambos casos, y con seis y cinco alelos respectivamente en la zona variable de cada una de estas enzimas. De este modo se dispone de cuatro marcadores génicos polimórficos confiables para llevar a cabo diversos estudios genéticos en Nothofagus antarctica.

Introduction

Nothofagus is an emblematic genus of the temperate forests of southern South America. Among the nine species of this genus found in the Andean Patagonian Forests of Chile and Argentina, Nothofagus antarctica (G.Forster) Oersted, locally called Ñire, is the one with the largest ecological range. It can be found from 36º 30' S in Talca Province (Chile) to 56º S in the islands of Cape Horn (Ramírez et al., 1985), and from the timberline at around 2,000 m a.s.l to near sea level. It can colonize sites with extreme drainage difficulties such as peat bogs, and semiarid sites with less than 500 mm annual precipitation, actually building the ecotone between forest and steppe. According to the National Forest Inventory (Montenegro et al., 2002), the forest type «Ñire Forest» in Argentina covers a surface of 801,937 ha.
Except for Tierra del Fuego Island, where timber phenotypes are common, N. antarctica is hardly taller than 12 m, frequently presents a shrubby shape, and even forms Krummholz in the timberline. Notwithstanding, the ecosystems it dominates are used all over its range, mainly for forestry-pasture production. Since there is a genuine interest for analyzing natural and anthropogenic processes in those ecosystems, reliable genetic tools are increasingly necessary in this regard.
Isozymes have been already used in previous genetic studies on this species, but their mode of inheritance has never been analyzed before (Vidal Russell, 2000; Stecconi et al., 2004; Quiroga et al., 2005). The direct interpretation of zymograms without a previous inheritance analysis can lead to erroneous speculations on population genetics. Bands on a gel are nothing more than phenotypes until an adequate analysis can show that they are encoded by genes; thus the use of the term «gene marker» is better than «genetic marker». The aim of the present study was the determination of isozyme gene markers in N. antarctica.

Materials and Methods

Buds and open-pollinated seeds from 10 Nothofagus antarctica trees from each of five natural populations in Nahuel Huapi National Park were collected at the end of summer. Six additional ornamental trees were sampled in San Carlos de Bariloche City. Some additional trees of distant natural populations contributed to the sample only with buds. All buds were kept in plastic bags at- 18º C until laboratory analyses. In order to get seedlings from sampled mother trees, seeds were sown at the end of winter in a greenhouse after two-month stratification in cold humid sand.
Buds from mother trees and their seedlings corresponding to families with more than 20 individuals were subjected to horizontal starch gel electrophoresis.
Laboratory procedures followed those described by Stecconi et al. (2004) with slight modifications. Vegetative extraction buffer I technique from Cheliak and Pitel (1984) was used. Continuous (1) and discontinuous (2) buffer systems were used on routine. (1) electrode: 0.13 M Tris / 0.04 M citric acid till pH 7.4; gel: diluted electrode 1:1.4; starch 11.5% w/v, sucrose 2.7% w/v; for 4.5 h. at 180 mA. (2) electrode:0.3 M boric acid / 0.06 M NaOH till pH 8.2 (Poulik, 1959); gel: 0.07 M Tris / 0.008 M citric acid till pH 8.5; starch 10.5% w/v, sucrose 2% w/v; for 6 h. at 80 mA.
Fifteen enzyme systems were surveyed using the staining solutions reported by Cheliak and Pitel (1984) with slight modifications [Buffer system (1): 6-phosphogluconate dehydrogenase (6- PGDH, E.C.1.1.1.44); malate dehydrogenase (MDH, E.C.1.1.1.37); shikimate dehydrogenase (SKDH or SDH, E.C.1.1.1.25); isocitrate dehydrogenase (IDH, E.C.1.1.1.42); menadione reductase (MR, E.C.1.6.99.2); glucose-6- phosphate dehydrogenase (G6PD, E.C.1.1.1.49); alcohol dehydrogenase (ADH, E.C.1.1.1.1) and acid phosphatase (ACP, E.C.3.1.3.2) - Buffer system (2): glutamate-oxalacetate transaminase (also called aspartate aminotransferase, GOT or AAT, E.C.2.6.1.1); superoxide dismutase (SOD, E.C.1.15.1.1); leucine-amino peptidase (LAP, E.C.3.4.11.1); glutamate dehydrogenase (GDH, E.C.1.4.1.3); diaphorase (DIA, E.C.1.6.4.3); phosphoglucose isomerase (PGI, E.C.5.3.1.9) and phosphoglucomutase (PGM, E.C.2.7.5.1)].
Isozyme nomenclature used was similar to that reported by Harry (1983), e.g. the most anodal zone for GOT is GOT1, and the locus codifying for it (once proved) is Got 1; the most common allele of this locus is Got 1-100, while the other alleles are named by their migration distance relative to it.
We followed the method proposed by Gillet and Hattemer (1989) to prove the mode of inheritance of genetic markers in open pollinated trees, based on the comparison of mothers and their offspring. This method implies first a qualitative approach, namely each seedling must always carry at least one of the maternal alleles. Secondly, a quantitative analysis is performed on the basis that each and every pollen grain arriving to a tree crown has the same probability of fertilizing any of the female gamete (haplo)types produced by that tree. Given that, and assuming regular meiotic segregation during ovule production and absence of differential viability selection in the offspring prior to the genetic analysis, it is expected for a putative heterozygous mother that the number of heterozygous embryos of its offspring (the same heterozygous type as the mother) equals the number of homozygous embryos (the two homozygous types corresponding to the putative mother alleles). In symbols:

Putative maternal genotype: A_iA_j
Expected relation in offspring genotypes:
N_ij = N_ii + N_jj

In presence of a third allele in the effective pollen cloud, it is also expected that in the offspring the number of heterozygotes with that third allele and either of the two maternal alleles is the same. In symbols:

Putative maternal genotype: A_iA_j
Expected relation in offspring genotypes:
N_ik = N_jk (k ≠ i, j)

Frequencies of genotypes observed in seedlings from mother trees sampled were compared to those expected according to the hypothesis by means of a Chi-square goodnessof- fit test. Although the use of seeds as a sample in the offspring is to a certain extent advisable because it minimizes the risk of differential viability selection, the small size of seeds forced us to use seedlings, which makes the preparation of homogenates for enzyme analysis hardly possible. However , this means an advantage since the correspondence of band patterns between adults and offspring can be assured only by using the same tissue, namely buds.

Results and Discussion

Seedlings were essential in order to perform the quantitative test for the inheritance analysis of isozyme bands. Although we sown a total of 150 g, that is more than 120,000 seeds (Premoli, 1991), we got only 543 seedlings. By cutting small samples of seeds from different trees and populations, we observed that the percentage of empty seeds was quite high (86 %) and variable, even without any single full seed in some samples. The low number of seedlings obtained was detrimental to the quantitative test. In several enzymes, the interpretation of the zymograms was not evident, and the lack of samples with family structure did not allow to explain the patterns observed.
All phenotypes observed in enzyme zymograms with good resolution are shown in Figures 1 and 2.

Figure 1: Electrophoretic patterns of SOD and GOT isozyme systems found in Nothofagus antarctica buds by means of Poulik (1959) buffer system and their corresponding genetic interpretation

Figure 2: Electrophoretic patterns of PGI and PGM isozyme systems found in Nothofagus antarctica buds by means of Poulik (1959) buffer system and their corresponding genetic interpretation

GOT

Two variable zones of activity are recognized in GOT enzyme system. The most anodal one is proposed to be encoded by a gene with three different alleles: Got1-100, Got1-113 and Got1- 86, being the last two extremely infrequent. Heterozygotes presented the typical three-banded phenotype of a dimeric enzyme. Since no seedling could be obtained from heterozygous mothers, the quantitative test could not be performed. However, the offspring of four homozygous mothers Got1-100/100 showed all the seedlings with at least one Got1-100 allele (Table 1).

Table 1: Frequencies of Got1, Got2, Pgi, and Pgm2 genotypes in the offspring of four Nothofagus antarctica mothers, and Chi-square value of the goodness-of-fit test corresponding to the hypothesis N₁₁ + N₂₂ = N₁₂

The other zone is not easy to interpret and its mode of inheritance could only be proved by means of the quantitative test. Three alleles are proposed: Got2-100, Got2-130, and Got2-76, being the latter rarely observed. The unexpected phenotype of the heterozygote 100/130 hindered its interpretation, since it showed two faint bands of the same intensity, instead of the three ones expected. Nevertheless, a putative heterozygous mother segregated in three phenotypes in its offspring, being one of them equal to that of the mother. This observation led to the hypothesis proposed, which was then proved through the quantitative test (Table 1).This enzyme seems to be inactive in Nothofagus nervosa (Phil.) Dim. et Mil. bud tissue. However, Marchelli and Gallo (2000) succeeded in proving the genetic control of three active zones observed in embryos, each encoded by a single locus. In Nothofagus obliqua (Mirb.) Oerst., three zones were also observed, two of which proved to be genetically controlled (Azpilicueta and Gallo, submitted).

PGI

Two zones of enzymatic activity can be distinguished in PGI, the most anodal of which is invariant, and the other one showing six alleles: Pgi2-100, Pgi2-108, Pgi2-120, Pgi2-82, Pgi2- 78, and Pgi2-60. All putative heterozygotes presented the typical three-banded phenotypes of dimeric enzymes. The quantitative test could not be performed since no seedling was obtained from heterozygous mothers. Data from the offspring of four homozygous mothers are presented in Table 1, supporting the qualitative analysis. The same patterns were described by Vidal Russell (2000) and Stecconi et al. (2004), but with only two electrophoretic variants in the first case, and four in the second study.
Two invariant zones were described for N. nervosa and N. obliqua. However, the single band observed in the less anodal zone presented different migration distance for each species and a heterozygous pattern was recognized in natural hybrids between them (Gallo et al., 1997). The inheritance analysis could therefore be performed in the offspring of hybrid individuals.
Two zones of enzymatic activity were also described in other four South American Nothofagus species: N. dombeyi (Mirb.) Oerst., N. betuloides (Mirb.) Oerst., N. nitida (Phil.) Krasser (Premoli, 1997) and N. pumilio (Poepp. et Endl.) Krasser (Premoli, 2003), the most anodal invariant in the four species. The same pattern was observed in the New Zealand Nothofagus species N. truncata (Col.) Ckn., N. solandri var solandri (Hook. f.) Oerst., N. solandri var cliffortioides (Hook. f.) Poole and N. menziesii (Hook. f.) Oerst., while the two zones resulted monomorphic in N. fusca (Hook. f.) Oerst. (Haase, 1993).

PGM

We also found two zones of enzymatic activity in PGM, from which only the less anodal is variable. This electrophoretic pattern matches the hypothesis of two genes, the variable one with five alleles: Pgm2-100, Pgm2-133, Pgm2-96, Pgm2-90, and Pgm2-75. The heterozygous individuals showed a two-banded phenotype, what agrees with the expectation for a monomeric enzyme. This qualitative interpretation of the zymograms was proved by means of the quantitative test proposed by Gillet and Hattemer (1989) (Table 1).
Azpilicueta and Gallo (submitted) found two zones in zymograms of N. nervosa, the most anodal of which was variable, then tested and proved to be genetically controlled. The same zone pattern was described by Haase (1993) for N. truncata, N. fusca, N. s. var solandri, N. s. var cliffortioides, and N. menziesii; the less anodal zone in this last species showed to be polymorphic. However, only one zone was observed in N. dombeyi, N. betuloides, and N. nitida (Premoli, 1997), which resulted monomorphic in the three species.

SOD

Zymograms for this enzyme presented a monomorphic single-banded pattern in a hundred of trees corresponding to 13 different natural populations (Figure 1). Two monomorphic zones have been reported for N. truncata, N. fusca, N. s. var solandri, N. s. var cliffortioides, and N. menziesii (Haase, 1993).

Not-resolved and monomorphic enzymes

MDH enzyme showed a very complex pattern, with four zones of activity, presumably variable the two in the middle. Intergenic hybrid bands are probably formed between those putative genes. However we could not resolve the system since, besides its complexity, the bands were not reliable enough to be confidently scored, not even to propose a qualitative hypothesis. We assayed the laboratory protocols proposed by Ranker et al. (1989), and the use of fresh leaves for the homogenates, but we did not succeed in getting better results. Two zones of activity were reported in a previous study (Vidal Russell, 2000), one of them variable, with three electrophoretic variants.
Enzymes MR, G6PD, ACP, LAP, GDH, and DIA presented bad resolution in the initial electrophoreses, and were therefore discarded. Vidal Russell (2000) did not obtain confident results either for MR or GDH.
Enzymes IDH, 6-PGDH, ADH, and SKDH showed a weak and inconsistent band pattern in our zymograms, which could not be interpreted although variation was observed. Two zones of enzymatic activity were found for IDH in a previous study (Vidal Russell, 2000), only one of them variable, with two electrophoretic variants. Two variable zones were reported for 6-PGDH and ADH in the same study, with two electrophoretic variants in each of them. One variable zone was observed for SKDH, with three variants (Vidal Russell, 2000). In all of these reported polymorphisms, the frequency of the observed electrophoretic variants was quite unbalanced.

Final considerations

In spite of its great economic and ecological importance, Nothofagus antarctica is not yet commercially planted. The genetic study of forest tree species which are not yet domesticated implies an extra effort since several details referred to their autoecology and manipulation under laboratory conditions remain still unknown. The extremely small size of seeds did not allow us to analyze embryos, and their reduced germination capacity limited the availability of vegetative material from the offspring. Some extra difficulties were related to laboratory procedures. Contrary to our experience with other Nothofagus species, the activity of most of the enzymes in N. antarctica buds decreases with time, although refrigerated at -18ºC. Doubtful electrophoreses could not be repeated, and several enzymes with potential good resolution and presumably polymorphic such as MDH, 6-PGDH, and ADH had to be discarded (on the other side, enzymes like PGI resulted quite robust in this regard).
Buds collected at the end of spring showed more enzymatic activity of enzymes assayed than those collected at the end of summer, that is, flushing buds resulted better than recently formed buds.
Three monomorphic bands were discarded as genetic markers due to the impossibility of testing its genetic control (PGI1, PGM1, and SOD). Two variable zones with patterns similar to those of other related species were discarded because some infrequent phenotypes did not match the simplest hypotheses (MDH2 and MDH3). Rigorousness in markers determination is crucial and proper of this early stage, since the delay in such a decision could lead to great effort waste or even erroneous speculations on population genetics.
In conclusion, four confident polymorphic genic markers are now available to contribute to answer several genetic questions relevant for guiding the conservation and domestication of Nothofagus antarctica.

Acknowledgements

The authors wish to thank Abel Martínez for his assistance in bud and seed collection and management of nursery activities. This research study was funded by Instituto Nacional de Tecnología Agropecuaria (INTA), Project PATNO13 «Productividad y efectos ambientales en ñirantales: plantaciones con Pino Oregón y sistemas silvopastoriles».
M. Pastorino and P. Marchelli are Researchers to the Consejo Nacional de Investigaciones Científicas y Técnicas-CONICET, Argentina.

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