The Journal of Heredity 2002:93(1)
© 2002 The American Genetic Association 93:60-63
Brief Communication |
Inheritance and Linkage of Allozymes in a Balkan Endemic, Pinus peuce Griseb.
From the Department of Dendrology, University of Forestry, 10 Kl. Ochridsky Blvd., 1756 Sofia, Bulgaria (Zhelev), and Department of Phytology, Faculty of Forestry, Technical University, Masarykova 24, 96 053 Zvolen, Slovakia (Gömöry and Paule).
Address correspondence to Peter Zhelev at the address above or e-mail: zhelev{at}ltu.acad.bg.
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Abstract |
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This article presents a study of isozyme variation in Pinus peuce Griseb., a Balkan endemic. Among the enzyme systems studied, five were monomorphic and eight were polymorphic in at least one locus. The segregation analysis of the polymorphic loci were consistent with a Mendelian mode of inheritance. No significant deviation from the expected ratio was observed both at the individual and pooled segregation data levels. Segregation patterns were homogeneous across individuals. Two significant linkage groups were found in P. peuce: FEST-2:LAP-2 and 6PG-1:6PG-2, which correspond to the results obtained for other pine species.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Pines (Pinus spp.) are among the most extensively studied forest tree species, due to their wide distribution in temperate and subtropical zones and their economic importance as timber-producing species (Critchfield and Little 1966; Mirov 1967; Vidakovic 1991). Genetic studies on the genus encompass a wide range of approaches and methods, ranging from provenance and progeny testing to the application of biochemical markers, such as terpenes (Hanover 1992), polyphenols (Lebreton 1995), isozymes, DNA, etc. (Müller-Starck et al. 1992). There are some species, however, which have been studied to a lesser extent, mainly due to their relatively limited distribution. Among these species is the Balkan endemic, Pinus peuce Griseb.
P. peuce, referred to as Macedonian pine or Rumelian pine, grows in the mountains of the Balkan peninsula at altitudes of between roughly 800 and 2300 m (Vidakovic 1991). This species possesses numerous remarkable characteristics, such as ecological tolerance and frost resistance, that make it very valuable for forest tree breeding (Mitchell 1996). Because of its limited area of natural distribution, this species requires special attention and implementation of measures for its conservation.
To date, the genetic studies on this species have been limited to monoterpene composition and provenance/progeny performance (Dobrev 1992, 1998). No isozyme study on the species currently exists, with two insignificant exceptions, dealing with single individuals in a botanical garden and bulk seed lot (Bergmann and Gillet 1997; Shurkhal et al. 1992). The objective of the present study is to investigate the genetic control of isozyme variants and possible linkage relationships among loci. This may produce a better understanding of the genetics of this species and could be useful for future studies based on allozyme markers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Material was collected from 42 individuals. Eighteen originated from a natural stand situated in the Vitosha Mountains (42°27'N, 23°12'E) at approximately 1900 m altitude, while the remaining 24 trees were clones originating from a seed orchard in the Yundola Experimental Forest (Rhodopes) at 1600 m altitude (42°04'N, 23°51'E). Cones were harvested in October 1997 in the seed orchard and in September 1998 in the natural stand. Seeds were extracted from the cones of each individual and kept separately at 2–4°C before the analysis.
Seeds were soaked on moist filter paper for several days before the analysis. Since the seeds of this species are characterized by long-term dormancy, no germination was observed. Megagametophytes were homogenized and the enzymes were extracted by Tris-HCl buffer pH 7.3. Also, several milligrams of polyvinyl polypyrrolidone (PVPP 40) were added as a stabilizing agent.
Standard 12% starch-gel electrophoresis was performed using three gel and buffer systems. Electrophoretic procedures and staining recipes followed those described by Cheliak and Pitel (1984) and Conkle et al. (1982), with slight modifications. The buffer systems, the enzyme systems studied, and the loci scored are listed in Table 1.
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Different zones of activity were interpreted as putative gene loci and the isozyme variants in these zones as putative alleles. Zones were numbered according to their mobility, starting from the fastest one. The most frequent allozyme variant in each zone was designated as 100 and the remaining allozymes according to their relative mobility regarding the allele 100.
Genetic control of isozyme variants was tested for consistency with Mendelian inheritance by means of a conventional chi-squared test. Tests were performed on each individual where a putative heterozygous locus was observed, and on the pooled data. Heterogeneity among the segregation patterns of different trees was tested by G test (Sokal and Rohlf 1995).
Linkage relationships among loci were tested using a chi-squared test. The test was performed for each pair of segregating loci, both on individual trees and on pooled data. For the loci having more than two allelic variants, alleles at each locus were bulked into two classes. In the cases when linkage between loci was detected, we estimated the recombination frequency using the binomial estimator y = r/n, where r is the number of observations in the smaller class (coupling or repulsion) and n is the sample size. Calculation of map distances between the linked pairs of loci was performed according to Kosambi (1944).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Inheritance of Allozymes
Among the enzyme systems studied, GOT, IDH, MNR, PGM, and SOD appeared to be monomorphic. The remaining enzymes were polymorphic in at least one individual. The allozyme variants can be presented as follows:
Acid phosphatase (ACP)
One zone of activity with two variants was observed. Since the faster variant was found in one homozygous individual only, no segregation records are available. All the progenies (embryos) of this individual displayed a triple-banded phenotype, which might correspond to a heterozygous genotype for an enzyme with dimeric structure. However, since no test of the Mendelian segregation could be done, our interpretation remains hypothetical.
Fluorescent esterase (FEST)
Two zones of activity were observed, but only the slower one was interpretable. The faster zone was not represented well enough to allow a correct interpretation. The results showed that the enzyme variants in the second zone (FEST-2) could be interpreted as expressing the gene action in a locus represented by three alleles.
Glutamate dehydrogenase (GDH)
One zone of activity was observed, represented by two alleles. The faster allele was found in only one individual from the seed orchard=mYundola=mand also in the pollen pool in the orchard. Among the other white pines, the enzyme was polymorphic only in P. longaeva (Hiebert and Hamrick 1983).
Leucine aminopeptidase (LAP)
This enzyme system showed two zones of activity coding for two putative allozyme loci. Both loci were polymorphic and each was represented by two alleles. The occurrence of null alleles, which is quite a common phenomenon in LAP isozymes, was not found in P. peuce.
Malate dehydrogenase (MDH)
Four putative allozyme loci were resolved (MDH-1, MDH-2, MDH-3, and MDH-4). The fastest, most intensely stained one (MDH-1) was monomorphic. The second and third zones were partially overlapping, which made interpretation difficult. The most plausible explanation for the patterns observed is that the two loci form an interlocus heterodimer. Locus MDH-2 was monomorphic, while the next locus (MDH-3) possessed two alleles, the faster one partially overlapping with MDH-2. This interpretation was subsequently proved by the segregation analysis. Gene duplication with subsequent forming of heterodimeric enzymes in MDH is common in several members of Pinaceae (El-Kassaby 1981).
Four double-banded allelic variants were found in the fourth zone (MDH-4), where the lower variant was always more intensely stained, allowing a reliable interpretation. The fastest and the slowest alleles were represented at a low frequency.
6-phosphogluconate dehydrogenase (6PG)
This enzyme system was represented by two loci, the faster one with two alleles and the slower one with three alleles, one of which was a null allele (in fact, there was a very faintly stained band with a relative mobility of approximately 92). Bergmann and Gillet (1997) reported three loci in P. peuce, but no segregation analysis was performed in their study because they used a bulk seedlot.
Phosphoglucose isomerase (PGI)
Two zones of activity were found in PGI; however, only the lower one was interpretable. Unfortunately it did not occur in all the heterozygous trees due to technical reasons. Two zones were also observed in other five-needle pines [see Beaulieu and Simon (1994) for references].
Shikimate dehydrogenase (SkDH)
This enzyme system expressed its activity in two zones, both with two alleles. The first zone was, in most cases, not clear enough to allow reliable interpretation and therefore we present a single-tree segregation record.
Segregation Analysis
No significant deviation from the expected Mendelian segregation ratio was observed in all the polymorphic loci concerning both the pooled data (Table 2) and the single-tree segregation records (results not shown). A single exception is acid phosphatase, where the segregation analysis was not possible due to the homozygosity of the tree bearing the rare allele (see above). The heterogeneity G test also did not reveal significant differences among the segregation patterns of the heterozygous trees studied (Table 2). Finally, we could suggest that even though some allozymes were not analyzed in enough individuals, their genetic control was proved and they can be used in further studies.
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Linkage Analysis
Of 55 possible two-locus combinations, we tested 32. Significant linkage was found among two pairs of loci only: FEST-2:LAP-2 and 6PG-1:6PG-2 (Table 3). The level of significance is higher in the first case (P = .0014), while in the second case (6PG-1:6PG-2) it is at the threshold level of significance (P = .049). Applying Bonferroni correction in the first case did not change the significance level, while in the second case this correction was not applicable, as only one tree was analyzed.
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In spite of the different significance level, the recombination fractions calculated were quite similar: 0.4022 ± 0.03 for FEST-2:LAP-1 and 0.3978 ± 0.05 for 6PG-1:6PG-2. These recombination values indicate weak linkage between the two pairs of loci. In the second case, however, only one double-heterozygous tree was available, and further testing is required to confirm the possible linkage. The map distances calculated are 55.56 cM for the pair FEST-2:LAP-1 and 54.34 cM for the pair 6PG-1:6PG-2. Based on our results, it is not possible to conclude whether these linkage groups are situated on the same chromosome or not.
In three combinations (LAP-1:MDH-4, LAP-2:MDH-4, and LAP-2:SkDH-2), slightly significant deviations from the expected ratios were found in individual trees, but these deviations were not confirmed when testing the pooled data (results not shown). The two linkage locus pairs in P. peuce were also found in other conifers. The first linkage locus pair (FEST-2:LAP-1) was also reported for other white pines (e.g., Eckert et al. 1981; Politov et al. 1989), while the second pair (6PG-1:6PG-2) was found in one tree only and therefore a comparison with other studies is not informative.
Knowledge of the linkage relationships among allozyme loci is a necessary prerequisite before using the allozymes in studies of population genetics and mating systems. Without information about the linkage among loci, inferences from such studies could be significantly biased.
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Acknowledgments |
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We thank Prof. B. Bogdanov from the University of Forestry, Sofia, Bulgaria, for allowing us to collect seeds in the seed orchard and to G. Slavov, who collected the cones from the orchard. Thanks are also due to Zuzana Slancikova for her skillful technical assistance. The study was performed during the visit of P. Zhelev to the Technical University, Zvolen, Slovakia. Financial support was provided by the Slovak Ministry of Education within the bilateral government contract with Bulgaria (to P.Z.). The study was partially supported by research grant 1/7056/20 from the Slovak Grant Agency for Science.
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Footnotes |
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Corresponding Editor: James L. Hamrick
Received September 17, 2001
Accepted November 26, 2001
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References |
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