The Journal of Heredity 2001:92(3)
© 2001 The American Genetic Association 92:260-266
Evidence of Variation in Segregation Patterns Within a Cedrus Population
From the Institut National de la Recherche Agronomique (INRA), Unité de Recherches Forestières Méditerranéennes, av. A. Vivaldi, F-84000 Avignon, France.
Address correspondence to François Lefevre at the address above or e-mail: lefevre{at}avignon.inra.fr
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Abstract |
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We used horizontal starch-gel electrophoresis for a genetic analysis of isozymes within one French Cedrus atlantica stand. Eleven to 29 megagametophytes per tree from 186 trees were assayed. Among the 33 enzyme systems tested, 15 correctly resolved and 8 appeared variable in at least one zone of activity: ACP, GOT, IDH, LAP, MDH, MNR, PGI, and SKDH. They were coded by at least 12 polymorphic loci which were described and tested for Mendelian segregation and linkage. Segregation patterns and linkage relationships were variable in the population. We detected homogeneous segregation distortion for loci Idh, Acp-c, and Got-a over the whole set of segregating progeny. We also found segregation distortions in a significant proportion of progeny for loci Got-b, Mdh-c, Pgi-b, and Skdh. The Acp-c and Got-b loci were linked with an overall map distance of 17 cM, but distance varied drastically among progeny. Both segregation distortions and heterogeneity of recombination frequencies indicate the occurrence of a genetic load in this population.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Globally, woody plant species have the highest level of genetic diversity among all organisms, at least when it is measured with isozymes, most of this diversity being found within populations (Hamrick et al. 1992). Large population size and high rates of gene flow are the main factors that explain this situation. Isozymes and other codominant genetic markers provide valuable information for population biology studies. They are most frequently used to infer "neutral" processes such as drift, gene flow, or mating systems. Segregation analysis is needed to compare the loci and alleles coding for a particular enzyme in different species, or even different populations within a species. Such inference requires knowledge of their mode of inheritance, although generally monogenic, as it can be affected in various ways by selection (Bergmann et al. 1990) or linked deleterious genes.
Conifer tree species also have a much higher mutational load than annual or animal species, although variation among species and populations has been detected (review in Ledig 1986). It is often argued that the same natural mechanisms are responsible for the high genetic diversity and the high genetic load. Deleterious genes may be responsible for inbreeding depression: this principle is used to evaluate the genetic load over the entire genome by measuring inbreeding depression through the concept of "lethal equivalents" (Franklin 1972; Kang et al. 1992; Morton et al. 1956). For breeding or gene conservation purposes, strategies were proposed to monitor diversity and purge the genetic load in the populations by using controlled consanguinity (Eriksson et al. 1993; White et al. 1993). These strategies were discussed by Byers and Waller (1999), arguing that inbreeding depression in small populations can also decrease after fixation rather than purge of deleterious mutations.
Another approach for the study of the genetic load has been proposed by Fu and Ritland (1994): looking at segregation patterns for neutral marker loci in experimental selfed progeny, they make inferences on linked selected loci. They also developed a multiple marker approach to study interactions among selected genes (Fu and Ritland 1996). Studying a controlled progeny with random amplified polymorphic DNA (RAPD) marker, Kuang et al. (1998, 1999a) have recently located a viability gene on the Pinus radiata genome. However, performing controlled crosses in natural populations of a conifer tree species is not always easy; moreover, 2 years are sometimes needed to obtain seeds. We used an alternative approach that consists of studying the variation in segregation patterns among seed trees using megagametophytes.
In this study we present a genetic analysis of enzyme systems within the French Cedrus atlantica stand on Luberon mountain. C. atlantica is one of the four conifer species of the genus Cedrus (Debazac 1964). Its natural range is in the mountain regions of Morocco and Algeria. Seeds were imported to France in the 1860s and it was successfully introduced as a forest tree species into the Mediterranean zone. The oldest artificial cedar forests in France expanded through natural regeneration under more or less intensive silvaculture, leading to significant changes in the ecosystem: from poor forest or overgrazed open field to high forest. These stands have now reached the fourth or fifth generation and they represent an interesting model to study the secondary evolution of an exotic gene pool out of its natural range. Moreover, Cedrus species are drought tolerant (Ducrey 1994) and of particular interest in the perspective of the global climate change expected in this area. Our general objective is the sustainable management of this exotic gene pool.
Isozymes have only been recently used to evaluate differentiation among populations of different Cedrus species (Panetsos et al. 1992, 1994; Scaltsoyiannes 1999), but segregation patterns were never tested except for locus Pgi in C. brevifolia (Panetsos et al. 1992). We describe phenotypic banding patterns and their genetic interpretation and we compare our codification of allozymes to that of the existing literature. We pay special attention to the variation of segregation patterns and recombination rates by using a large sample of open-pollinated progeny.
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Materials and Methods |
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We collected open-pollinated seeds from 186 trees sampled in the four age classes which currently produce seeds in this cedar forest (one to four cones per tree). The cones were placed alternatively in water and drought conditions to release seeds which were stored at 4°C. After stratification at 4°C for 4 weeks, we collected megagametophytes from germinating seeds and froze them at -80°C until electrophoresis. We first analyzed 11 to 29 megagametophytes for each seed tree, with a mean number of 16.4 megagametophytes per seed tree analyzed for at least one polymorphic locus. In a second step we increased sample size to 120 and 134 in two progeny for estimation of linkage map distances. We also analyzed some embryos to provide information on enzyme structure. Extraction, migration, and staining protocols derived from Conkle et al. (1982) and Liengsiri et al. (1990), and slightly modified for C. atlantica (Hochu and Fady 1998). We used Pinus resinosa, which is monomorphic for all the enzyme systems studied (Allendorf et al. 1982; Fowler and Morris 1977; Mosseler et al. 1991), as a migration marker. Electrophoresis was stopped when the migration front reached 8 cm from the origin.
We initially stained 33 enzyme systems, but 18 of these had weak or nondetectable activity: ACO (E.C.4.2.1.3); ADH (E.C.1.1.1.1); ALAP (E.C.3.4.11.1); DIA (E.C.1.6.4.3); 
-EST, ß-EST (E.C.3.1.1.1, E.C.3.1.1.1); FUM (E.C.4.2.1.2); GluDH (E.C.1.1.1.47); GlyDH (E.C.1.1.1.29); G3PD (E.C.1.2.1.12); HK (E.C.2.7.1.1); MPI (E.C.5.3.1.8); NADHDH (E.C.1.6.99.3); NADPDH (E.C.1.6.99.1); PEP (E.C.3.4.13.11); PPO (E.C.1.14.18.1); SrDH (E.C.1.1.1.14); UGPP (E.C.2.7.7.9). The remaining 15 enzyme systems were correctly stained (Table 1). For each enzyme system, loci were named with letters and different allozymes were numbered according to their migration distance ranking. To avoid confusion we used quotation marks for the names of loci and alleles found in the literature.
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In conifers, megagametophytes generally consist of haploid tissue containing the same genetic information as the female gamete, although some exceptions have been found by Pichot and El Mâataoui (1997): all isozyme profiles in our sample were consistent with haploid megagametophytes. In the following text we call "progeny" the population of megagametophytes from a single seed tree. For each enzyme we considered seed trees giving monomorphic progeny as homozygous. A tree was considered as heterozygous as soon as segregation was observed, and its genotype was determined by the two allelic forms in segregation: as expected for a diploid species, we never observed more than two variants per locus within a progeny.
We conducted segregation analysis for all heterozygous seed trees with the following steps:
- 1. For each progeny, we computed the individual segregation ratio as
where nai and naj are the observed number of ai and aj gametes; we also computed a 95% confidence interval following a binomial distribution.
- 2. For each locus, we considered sets of homologous progeny descending from heterozygous trees of identical genotypes (e.g., one set for all a1a2 genotypes, another one for a2a3 genotypes); within each set we performed an overall test of Mendelian 1:1 segregation after pooling all megagametophytes and using a chi-squared test. The overall segregation ratio was estimated as 0.50 when the test was not significant, and as
otherwise (where Nai and Naj are the total number of ai and aj gametes within the set).
- 3. Within each set we identified single progeny when the individual segregation ratio significantly differed from the overall segregation ratio of the set.
Because of the large number of progeny and the small sample size per progeny, statistical tests had limited power, and a global homogeneity test among all progeny within a set would not have been relevant. Therefore we used the frequency of single progeny within a set as an indicator of heterogeneity of segregation patterns: this frequency is not expected to exceed 5% if sampling error is the only cause of variation.
Within each progeny, we used a Fisher's exact test to detect linkage among pairs of loci. According to Bailey (1961), segregation distortion at both loci may disturb the linkage test: such situations were not considered in this study. We estimated the recombination rate as
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Map distance (d) was estimated following Kosambi (1944):
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Results |
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Isozyme Patterns
We detected no variation in more than 700 megagametophytes for seven enzyme systems (Table 1). These were considered to be monomorphic in our population. The eight other enzyme systems were polymorphic (Table 1, Figure 1).
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ACP. We identified two polymorphic loci, Acp-a and Acp-c. Three other zones of activity were either monomorphic or not clearly resolved.
GOT. We identified two loci, Got-a, including one null allele, and Got-b. The Got-a locus corresponds to "Aat-a" for which Panetsos et al. (1994) detected three alleles in C. atlantica. This locus corresponds to one of the "Got-1" or "Got-2" loci (also called "Aat-1" and "Aat-2") of other conifers (Fady and Conkle 1992); the Got-b locus corresponds to locus "Got-3" of other conifers (Cheliak and Pitel 1985).
IDH. We detected only one zone of activity, interpreted as locus Idh with three alleles. Panetsos et al. (1994) identified two polymorphic loci in C. atlantica, but Scaltsoyiannes (1999) kept only one of these, "Idh-a," which corresponds to our Idh.
LAP. We identified two loci, Lap-a, including one null allele, and Lap-b. Panetsos et al. (1992, 1994) and Scaltsoyiannes (1999) found no polymorphism for Lap-a in their C. atlantica material.
MDH. We could correctly stain only one polymorphic zone of activity interpreted as locus Mdh-c. Two loci were found by Panetsos et al. (1992) in C. atlantica, but correspondence with our zymograms was not clear, and Scaltsoyiannes (1999) could not resolve this enzyme in Cedrus species.
MNR. We considered the three zones of activity as MNR isozymes, although MNR staining may also reveal other enzyme systems in conifer species: DIA, NADPHDH, and NADHDH (Hussendörfer et al. 1995; Yi 1992). We identified two loci, Mnr-a and Mnr-c, but the intermediate zone, Mnr-b, was poorly resolved although polymorphic. We observed one null allele for locus Mnr-a. According to Scaltsoyiannes (1999), Mnr-a corresponds to the locus "Dia-a" revealed after DIA staining.
PGI. We identified one polymorphic locus with five alleles, Pgi-b: alleles b1 and b2 were single-banded, alleles b3, b4, and b5 were triple banded. This corresponds to the patterns observed by Panetsos et al. (1992) and Scaltsoyiannes (1999), although this last author found only four alleles for Pgi-b in C. atlantica. Both authors observed the same single-banded and triple-banded alleles, but no clear explanation could be given.
SKDH. We detected a single Skdh locus with three alleles.
Segregation Analysis
Segregation patterns within each set of progeny are presented in Table 2. We detected significant overall distortion for three loci (Acp-c, Got-a, Idh) (Figure 2). For these loci, single progeny were detected, but their frequency did not exceed 5% as expected at random. The Idh locus showed a clear deviation from the Mendelian ratio, with a constant deficit of allele Idh-1 versus allele Idh-2 in 21 progeny, and a deficit of allele Idh-3 versus allele Idh-2 in 8 of 10 progeny. For Acp-c we observed a deficit of allele Acp-c1 versus allele Acp-c2 in 41 progeny out of 64. We also observed a constant deficit of allele Got-a2, but only three progeny were analyzed for this system.
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, where nai 
is the number of ai (aj) gametes in the progeny of a aiaj seed tree.We found no significant overall deviation from the 1:1 ratio for the other loci. However, we could detect single progeny as previously described: these progeny individually departed from the Mendelian 1:1 ratio (Table 2). The frequency of single progeny within a set was often greater than 5% (up to 29%), supporting the hypothesis of true segregation distortions rather than sampling artifacts.
Linkage Analysis
We tested 25 two-locus associations (Table 3). One significant linkage was found, between Acp-c and Got-b. Recombination rate estimates in two progeny were significantly different: 0.05 versus 0.28. We computed an average estimate of the distance between these loci after pooling the four progeny: d = 17 cM. We also found linkage between Got-b and Pgi-b, but it was only significant in one progeny out of seven, and this should be further checked given the small sample size.
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Discussion |
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All monomorphic enzymes in this study were analyzed for the first time in Cedrus, except 6-Pgd, which was also found to be monomorphic in C. atlantica by Panetsos et al. (1994), although Scaltsoyiannes (1999) found some polymorphism in a population from Morocco. Twelve loci were identified for the eight polymorphic enzymes. Acp-a, Acp-c, Mdh, Mnr-c, and Skdh were described for the first time. Got-a, Got-b, Lap-a, Lap-b, and Pgi-b correspond to previously described loci (Panetsos et al. 1992, 1994; Scaltsoyiannes 1999), although some new alleles were identified. Idh and Mnr-a might be related respectively to the "Idh-a" and "Dia-a" loci described by Scaltsoyiannes (1999) but this has to be further checked. Altogether, these 12 polymorphic loci revealed 33 alleles.
In our population we detected null alleles at three loci (Got-b, Lap-a, and Mnr-a) for the first time in C. atlantica. Such alleles are more easily detected using haploid megagametophytes; however, since we collected megagametophytes after seed germination, null alleles with strong deleterious effect might remain undetected. Null alleles are commonly reported for GOT, LAP, and DIA loci in conifers (Adams et al. 1990; Allendorf et al. 1982; Harry 1986; Morgante et al. 1993; Muona et al. 1987). Scaltsoyiannes (1999) found a null allele for his "Dia-a" locus, which corresponds to our Mnr-a, but not in his C. atlantica populations.
We observed significant overall segregation distortion for 3 of the 12 polymorphic loci. In these cases, the distribution of the segregation ratio was unimodal; the alleles in deficit were also the rarest alleles in the population (Acp-c1, Idh-1 and Idh-3, Got-a2). Segregation distortion in favor of the most common alleles has often been reported in conifers for Got and Acp loci (review in Strauss and Conkle 1986). Segregation distortion in Idh was also mentioned (Geburek and Wang 1990; Harry 1986). For five other loci, although there was no overall distortion, we could identify single progeny deviating from the Mendelian ratio, sometimes at high frequency. In that case, a single allele could be found either in deficit or in excess in different progeny. These deviations might have different causes.
As pointed out by Strauss and Conkle (1986), the overrepresentation of the most common allele may result from a scoring error of poorly resolved enzyme systems: this was not the case with Idh and Got-b, which always gave clear patterns; for Acp-c, staining was sometimes weak, but particular attention was paid to scoring the gels taking into account even the faint bands. Another misinterpretation might be related to the "genetic mosaicism" of trees: somatic mutations can lead to a chimeric structure along the crown of a tree (Gill 1987; Klekowski and Kasarinova-Fukshansky 1984; White 1979); such mutations affecting gametophytic lineage could result in distorted segregation in a mixture of seeds collected from different cones. However, only somatic mutations have been reported (Whitham and Slobodchikoff 1981) and, to our knowledge, no evidence of somatic mutations included in reproductive tissues has been reported. Furthermore, strong homoplasy should be invoked to account for the groups of identical genotypes we deal with. Therefore we reject this hypothesis.
Strauss and Conkle (1986) also reported increased segregation distortions for interpopulation hybrids due to hybrid dysgenesis (deleterious effects of interpopulation crossing). Indeed, this cedar forest has probably been founded using a mixture of several Algerian seed sources, therefore interpopulation hybrids may have been formed in the second and subsequent generations. Hybrid dysgenesis could thus explain part of the distorted segregations. However, this does not explain segregation distortion observed in the founder generation: as an example, segregation distortion between Idh-1 and Idh-2 alleles was homogeneous among the four age classes. Furthermore, hybrid dysgenesis would be expected when strong differentiation exists among provenances, which was not found by Scaltsoyiannes (1999) in C. atlantica.
Finally, segregation distortion can be due to selection. Bush and Smouse (1991, 1992) reviewed evidence of reduced fitness associated with rare alleles in Pinus species. In our case, the selection may operate during gametogenesis or sporogenesis: megagametophytes have the genotype of the ovules that yield viable embryos, and we observed a high proportion of empty seeds in cedar cones. Direct selection would be on the allozyme allele itself, whereas indirect selection would be due to a hitchhiking effect. Since linkage disequilibrium is generally low in natural forest stands, indirect selection should lead to heterogeneity of segregation patterns among unrelated seed trees, some being in coupling phase, others in repulsion phase (Cheliak and Pittel 1985): this might explain why some progeny deviate from the expected 1:1 ratio, whereas overall deviation among several progeny is not significant. The particular case of the Pgi-b locus is interesting because such single progeny were found with three different genotypes (b2b4, b2b5, b4b5), and the same isozyme allele was found either in deficit or in excess depending on the progeny. Therefore Pgi-b, as well as Got-b, Lap-a, Lap-b, Mdh-c, Mnr-a, Mnr-c, and Skdh might be linked to deleterious genes in our population. But assuming low gametic disequilibrium, indirect selection would not account for the systematic deviation we observed on Idh, Acp-c, and Got-a. For these loci, the most likely hypothesis would be direct selection on some allozymes.
Linkage between Acp-c and Got-b has rarely been documented in forest tree studies. However, it was previously reported for Pinus taeda by Conkle (1979), with a map distance of 17cM, and by Adams and Joly (1980) with a map distance of 63 cM. Comparable loci (Acp-c and Got-c) were also linked in Quercus petrea (Müller-Starck et al. 1996). In our Cedrus population, recombination estimates in two progeny were very different, although the distance averaged across progeny was exactly that of Conkle (1979). Recombination rate should be considered as a variable trait among individuals, even within a population. Hadad et al. (1996) estimated the heritability of recombination rate in maize: it ranged from h2 = 0.2 to 0.69 depending on the linkage group, and selection was efficient to increase recombination. Chromosome rearrangements could be responsible for important variation of recombination rates among individuals (Barrett et al. 1987; Lewandowski et al. 1992; Strauss and Conkle 1986). In our case, although both progeny segregated following a Mendelian 1:1 ratio for both loci, we previously concluded from the segregation analysis that deleterious genes might occur near the Acp-c and Got-b loci: although it has been shown that the recombination fraction would not be affected by a single deleterious gene involved in zygotic selection (Kuang et al. 1999b), this could happen when two viability genes are involved (Lorieux et al. 1995).
Null alleles, deviations from Mendelian expectations, and variations of recombination ratios were detected in this population. Among other hypotheses, natural selection against deleterious alleles should be invoked to explain at least part of the segregation patterns. The evolution of genetic diversity and mutational load among generations will be further investigated.
Linkage groups can be used to identify synteny and study evolution in taxonomic groups (Weeden and Wendel 1989). Linkage has often been reported between "Pgi-2" and "Aat-1" (also called "Got-1") in Pinus sp. (review in Conkle 1979) and Picea sp. (King and Dancik 1983; Muona et al. 1987), whereas it has been observed between "Pgi-2" and "Aat-2" ("Got-2") in Pseudotsuga sp. (Adams et al. 1990), Calocedrus sp. (Harry 1986), Abies sp. (Neale and Adams 1981), and Larix sp. (Cheliak and Pittel 1985). This could be genetic evidence of taxonomic divergence between the more primitive Pinus–Picea group and other conifers (Cheliak and Pittel 1985). Unfortunately the linkage between Got-a and Pgi-b could not be tested in this study due to the lack of double heterozygous trees. Moreover, Got-a could not be clearly related to the "Aat-1" or "Aat-2" locus of other conifer species. The linkage observed between Got-b and Pgi-b in a single progeny on a very small sample size should be checked. It has never been reported before in conifers, and only once in angiosperms (Müuller-Starck et al. 1996).
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Acknowledgments |
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We thank two anonymous reviewers for their helpful comments on a previous version of the manuscript. A. Aliberti, G. Bettachini, A. Giai-Checa, B. Jouaud, F. Rei, and J. Thévenet were involved in seed collection and processing; I. Hochu and T. Nieuwjaer participated as laboratory assistants. This work was part of the project "Adaptation and selection of Mediterranean Pinus and Cedrus for sustainable afforestation of marginal lands" FAIR CT95-0097 financed by the Commission of the European Communities DGVI.
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Footnotes |
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Corresponding Editor: James L. Hamrick
Received February 25, 2000
Accepted January 15, 2001
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References |
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