The Journal of Heredity 2001:92(3)
© 2001 The American Genetic Association 92:292-295
Brief Communication |
SSR Markers for Quercus suber Tree Identification and Embryo Analysis
From the INIA-CIFOR, Ctra. de La Coruña Km 7, 28040 Madrid, Spain (Góaomez, Pintos, Aguiriano, and Bueno) and ETSI Montes, UPM, Madrid, Spain (Manzanera).
Address correspondence to M. A. Bueno at the address above or e-mail bueno{at}inia.es.
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Abstract |
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Three Quercus simple sequence repeat (SSR) markers were amplified by polymerase chain reaction (PCR) from nuclear DNA extracts of trees and in vitro-induced haploid embryos from anther cultures of Quercus suber L. These markers were sufficiently polymorphic to identify 10 of 12 trees located in two Spanish natural areas. The same loci have been analyzed in anther-derived haploid embryos showing the parental tree allele segregation. All the alleles were present in the haploid progeny. The presence of diverse alleles in embryos derived from the same anther demonstrated that they were induced on multiple microspores or pollen grains and they were not clonally propagated. Also, diploid cultures and mixtures of haploid-diploid tissues were obtained. The origin of such cultures, either somatic or gametic, was elucidated by SSR markers. All the embryos showed only one allele, corroborating a haploid origin. Allelic composition of the haploid progeny permitted parental identification among all analyzed trees.
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Introduction |
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The embryogenic process originated during anther culture may have different origins, for example, haploid cells such as microspores or pollen grains, or somatic cells from anther tissues. Therefore embryo genetic composition may be of either gametic or sporophytic, depending on which cells are induced. Anthers subjected to stress conditions can become a target for embryo induction (Bueno et al. 1997). When embryogenesis appears as a result of stress, the probability of obtaining haploid embryos from microspores or pollen grains is greater, rendering this method more interesting than embryogenesis induction by plant growth regulators. Data for isolated microspore cultures clearly indicate that hormones are not required for embryogenic induction. In fact, at least benzylaminopurine has been shown to inhibit pollen embryogenesis in tobacco (Kyo and Harada 1990).
Due to the importance of doubled haploid plants for plant breeding, their production through anther culture would be especially useful in species with long generation times and strong inbreeding depression, which make traditional breeding methods impractical. Only a few isolated reports have appeared describing the formation of embryos in anther cultures of forest trees such as Quercus suber (Bueno et al. 1997).
The type of origin, either haploid, dihaploid, or diploid, of anther-derived embryos has been rarely studied in forest tree species or even in other plant species (Hayward et al. 1990; Müller-Starck and Jörgensen 1991). Nevertheless, flow cytometry has permitted the distinction of haploid embryos, diploid embryos, and explants composed of cells of both types, all of them coming from the same anther in cork oak (Q. suber). The proportion of haploid embryos formed in anther cultures has been higher than that of diploids or haploid/diploid mixtures. This has led to the conclusion that the embryos with double DNA content were not heterozygotic diploids with the parental genome but dihaploids. The necessity of testing this assumption led us to use molecular markers, such as simple sequence repeat (SSR).
Codominant molecular markers are more suitable for this study because homozygotic and heterozygotic individuals can be distinguished. For this purpose, isozyme markers were used initially (Müller-Starck and Jörgensen 1991), but presently DNA markers with a higher number of alleles are available, such as microsatellites, that facilitate sample analysis through selective amplification of repeated sequence fragments.
The use of microsatellite markers is generally restricted to species in which they are designed, due to the high degree of homology necessary between primers and sample DNA. Sometimes there are amplifications available for one species derived from closely related species during evolution for which those primers were designed (Fields and Scribner 1997; Primmer et al. 1996; Sun and Kirkpatrick 1996). This is the case for (GA)n microsatellites in the genus Quercus. Most SSRs localized by Steinkellner et al. (1997a, b) in Q. petraea can be polymerase chain reaction (PCR) amplified using the same primers in other oaks and even some SSRs have been found in other species of the Fagaceae.
In the present work, microsatellite markers are used for the first time in cork oak. Those markers can be used for tree identification, genotypic characterization, heterozygosity evaluation, and determination of the ploidy level in anther embryos induced by stress treatments. Furthermore, in the case of highly heterozygotic trees, a test of parental exclusion and genotype identification might be used for identification of the paternal tree of the gametic embryos, based on the analysis of its haploid progeny.
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Materials and Methods |
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Plant Material
DNA from leaves was obtained from 12 Q. suber trees. Eight trees, located in Cáaaceres, were numbered 1H–8H, and the other four trees, located in Madrid, were labeled J, 1P, 2P, and 3M.
Embryonic tissue for DNA extraction was sampled from Q. suber haploid embryo cultures obtained from tree 3M, following Bueno et al. 1997:
(1)Twelve embryos from two anthers, six from anther 164 and six from anther 169, the haploid origin of which has been assessed by flow cytometry analysis.
(2)Six embryos from anther 104, which have shown a haploid-diploid composition by flow cytometry analysis.
(3)Six embryos from anther 212. In these embryos diploid DNA levels have been observed by flow cytometry analysis.
DNA Isolation, SSR Loci, and Amplification Conditions
DNA from leaves was extracted as described by Ziegenhagen et al. (1993) with a posterior purification with the GENECLEAN® kit (BIO 101). DNA from embryos was extracted as described by Doyle and Doyle (1990).
Three Q. petraea microsatellite loci, (GA)n repeats, were amplified with the primers SsrQpZAG15, SsrQpZAG46, and SsrQpZAG110 designed by Steinkellner et al. (1997a) with the PCR amplification profile as described by Barreneche et al. (1998). These loci were assayed on the basis of their observed heterozygosity and conservation between Quercus species (Steinkellner et al. 1997a,b).
Each 25 µl amplification reaction contained 20 ng of genomic DNA, 0.2 µM of fluorescently labeled forward primer and unlabeled reverse primer (Progenetic), 200 µM each dNTPs, 50 mM KCl, 10 mM Tris-HCl (pH 9), 2.5 mM MgCl2 and 0.5 U of Taq-DNA polymerase (Ecogen). Fluorescent labeled PCR products were separated and analyzed on a semiautomated sequencer (ABI-Prism, Perkin-Elmer). Standards were used for length determination of alleles.
Heterozygosity and Power of Discrimination Indexes
As a measure of the information provided by each locus, heterozygosity was calculated using Nei's (1973) formula: H = n/(n - 1)*1 - 
pi2, where pi is the frequency of allele i in the n analyzed trees.
Power of discrimination for each locus was calculated using the formula PD = 1 - Pi2, where Pi is the frequency of genotype i (Kloosterman et al. 1993).
Mendelian Allele Segregation
Mendelian allele segregation was analyzed by chi-squared test.
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Results |
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DNA amplification in leaf extracts of the trees analyzed was successfully obtained in all three loci. Also, all amplification products had the expected size for all microsatellites (Table 1). It is noteworthy that the extraction system designed for pine needles by Ziegenhagen et al. (1993) has also been applicable to DNA isolation from cork oak leaves, which contain characteristic substances that might interfere with Taq polymerase activity and hamper SSR fragment amplification.
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All loci were polymorphic in cork oak (Table 1) with 3–6 alleles per locus, the average being 4.67 alleles per locus. Table 2 shows the Hardy–Weinberg expected heterozygosity (H), which range between 0.475 and 0.685 per locus. The average value of this index for all three loci studied was 0.576. The power of discrimination between genotypes was 89.6%.
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All three microsatellite loci showed a similar level of polymorphism to that found in other species of the genus Quercus (Steinkellner et al. 1997b). Four alleles were found for locus ssQpZAG15 among the 12 cork oak trees analyzed, in a similar frequency to that found in Q. petraea. Locus ssQpZAG110 had more alleles, while locus ssQpZAG46 had fewer alleles in Q. suber than in Q. petraea. A total of 13 alleles have been found (Table 2), four of which were unique.
Embryo Analysis
Genotype identification of embryos induced in anthers from tree 3M was performed by means of all three microsatellites assayed (Table 3). Six embryos from each anther and four anthers from the same tree were tested for each locus, thus a total of 24 embryos. All alleles of the parent tree were also found in the progeny, but only one allele per locus was amplified in each embryo. The pattern of allelic heredity was analyzed by a chi-squared test, obtaining a 1:1 segregation for locus ssQpZAG46 only (Table 3). A total of five genotypes were differentiated.
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Discussion |
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Three primers designed by Steinkellner et al. (1997a) for microsatellite amplification in Q. petraea were used for DNA analysis of Q. suber.
Tree Analysis
All three loci tested were polymorphic and the average rate of heterozygotic loci found in a set of 12 trees was as high as 55.6%. This is not remarkable if we take into consideration that those markers were chosen for this study on the basis of high polymorphism.
Semiautomated sequencer output revealed only a single peak for loci of homozygous trees and two peaks of different size in the case of heterozygotes. It was concluded that this system is adequate for size determination of microsatellites (Bredemeijer et al. 1998).
Microsatellite polymorphism has provided a new approach to the genetic analysis of cork oak and an efficient tree identification system, due to the high discrimination power obtained for genotypic differentiation. In our case, only three loci were necessary to identify 10 of 12 trees tested. Two fully homozygotic trees for all three loci, with the same allelic composition, were found (namely 2H and 1P).
On the other hand, three individuals were heterozygotic for all three loci. A high degree of heterozygosity has already been detected for oaks by means of isozyme analysis. In Q. petraea and Q. robur, for instance, a population of 1606 juvenile plants showed a 21.9 and 21.3% heterozygosity, respectively (Müller-Starck 1990).
Embryo Analysis
We obtained a haploid progeny of pollen embryos through anther culture from tree 3M, which has a high degree of heterozygosity. Previous studies on the ploidy level of cork oak anther embryos were performed by flow cytometry to determine their origin, either gametic or somatic. A high percentage of embryos were in fact haploid, confirming their origin from microspores or pollen grains. Nevertheless, some exceptions were found, revealing a diploid genome, such as embryos from anther 212 or a mixture of haploid and diploid genome, such as in anther 104. One of the aims of our analysis by microsatellite markers was to prove the hypothesis of a spontaneous duplication of the haploid genome. The results obtained in this work verify this hypothesis. Four anther cultures previously analyzed by flow cytometry for ploidy level determination were chosen. From these anthers, six embryos per anther were analyzed by microsatellite amplification, corroborating the allelic pattern of the parent tree (Table 3). Both alleles from each locus were found in the progeny and different haplotype combinations have been obtained.
As is shown in Table 3, diploid anther embryos had only one allele per locus, revealing a homozygotic genome for all loci tested. These results confirm the applicability of microsatellite markers as indicators of the ploidy level in embryo regeneration from anther cultures, as it was previously performed by isozymes in Q. petraea (Müller-Starck and Jörgensen 1991). Again, in the case of haploid-diploid embryos induced on anther 104, a single allelic combination (124, 192, and 222 bp) was observed.
Both alleles of locus ssQpZAG46 (190 and 192 bp) segregated 1:1 in the haploid embryo progeny induced on anther 164. Also for dihaploid embryos from anther 212, both alleles of ssQpZAG15 (120 and 124 bp) were present.
The use of microsatellite markers has permitted the verification of the hypothesis that anther embryos are induced from different microspores or pollen grains, as it has been proved by the diverse genetic composition of embryos from the same anther. This confirms previous results obtained by isozymes and RAPDs (Bueno et al. 2000).
Parental Tree Analysis by Descendant Embryo Analysis
Allele segregation in the haploid descendants can be used for the heterozygosity analysis of an individual so that direct DNA analysis is not necessary for the parental tree. The high rate of polymorphism observed also permitted the identification of the parent tree by parental exclusion.
In this case locus ssQpZAG15 has two alleles, of 120 and 124 bp each, which are present in trees 1H, 3H, 4H, 7H, J, and 3M, excluding other trees as possible fathers. The locus ssQpZAG46 has two alleles pres[chent in the embryos, of 190 and 192 bp, which are present in trees 8H and 3M only. The combination of both exclusion criteria reveals 3M as the parental tree. The same result can be obtained with locus ssQpZAG110, with alleles of 222 and 238 bp, only present in trees 3H and 3M. The principle of parental exclusion could be applied in our embryo cultures and only two loci were sufficient for parental identification.
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Conclusions |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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Nuclear DNA microsatellites are an adequate system for the tree identification in cork oak thanks to a high discrimination power among genotypes. Both alleles of each SSR locus were inherited by anther embryos, and Mendelian segregation (1:1) could be statistically proved by chi-squared test in one case. The homozygosity of both haploid and diploid anther embryos has been proved by microsatellite markers, revealing a certain rate of spontaneous DNA duplication. Embryo origin from multiple microspores or pollen grains inside a cork oak anther has been found by the different genetic composition of those embryos. The parent tree genome can be deduced from the haploid embryo progeny. SSR markers were used for the first time in cork oak, corroborating their applicability for many genetic analyses.
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Acknowledgments |
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This study was supported by INIA project SC 98-081.
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Footnotes |
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Corresponding Editor: James L. Hamrick
Received October 2, 1999
Accepted November 11, 2000
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References |
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