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Peroxidase polymorphism in pubescent oak (Quercus pubescens Willd.) in relation to Q. petraea Matt. Liebl and Q. robur L.
Accepted Manuscript Title: Peroxidase polymorphism in pubescent oak (Quercus pubescens Willd.) in relation to Q. petraea Matt. Liebl and Q. robur L. Author: Maria Krzakowa Kamilla ˙ B˛akowska-Zywicka PII: DOI: Reference:
S1381-1177(17)30003-6 http://dx.doi.org/doi:10.1016/j.molcatb.2017.01.004 MOLCAB 3503
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
12-7-2016 30-12-2016 4-1-2017
˙ Please cite this article as: Maria Krzakowa, Kamilla B˛akowska-Zywicka, Peroxidase polymorphism in pubescent oak (Quercus pubescens Willd.) in relation to Q.petraea Matt.Liebl and Q.robur L., Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2017.01.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Peroxidase polymorphism in pubescent oak (Quercus pubescens Willd.) in relation to Q. petraea Matt. Liebl and Q. robur L. Maria Krzakowa1*, Kamilla Bąkowska-Żywicka2* 1 Department of Genetics, Adam Mickiewicz University, Umultowska 89, 61-614 Poznao, Poland, [email protected] 2 Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznao, Poland, [email protected] *corresponding authors
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Graphical Abstract
2
Highlights
Peroxidase allozymes were analysed in three native species of Quercus from Poland. Our results show an extensive polymorphism of Quercus peroxidases. Q. robur shows low genetic similarity to other Quercus species in question.
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Abstract In spite of the increasing availability of DNA markers with a higher genetic resolution, electrophoretically detectable allozymes still provide the cutting edge in the estimation of genetic variability. Peroxidase was analysed in individual sleeping buds collected in three native species: Quercus pubescens, Q. petraea and Q. robur. The species were characterised with gene and genotype frequencies and showed specific degrees of intra- and inter-population diversity. Genetic variability of marginal population of pubescent oak from Bielinek, the only stand of the species in Poland markedly separated from its compact range, was described by peroxidise polymorphism. Genetic distances among species, statistically examined, showed visible differences between them and a relatively low genetic similarity of Q. robur to the two species in question. Key words: peroxidase, allozymes, genetic distance, Quercus pubescens This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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1. Introduction In Poland there are two important species of oak which are common in Central Europe: pedunculate oak (Quercus robur) and sessile oak (Q. petraea). Both of them are of high economic significance. The pubescent oak (Q. pubescens) of sub-Mediterranean origin has no economic importance, however it is of special interest because of its unusual geographical location. The only stand of Q. pubescens in Poland is situated in postglacial valley of Odra river in Bielinek and protected as forest-steppe preserve. This population is located far away north from the main species range, but within a natural range of two other oak species (Q. robur and Q. petraea). The significant age of some of the Q. pubescens specimens (the oldest tree is over 200 years old), rich fructification and southern character of local biocenosis suggest the natural origin of Bielinek population. Based on the leaf morphology studies Q. pubescens from Bielinek was classified as a hybrid population, however distinct from both Q. petraea and, even more evidently, from Q. robur [1]. Usually, the level of genetic variation of marginal populations differs from those of a continue range [2-4], therefore there is a need for detailed studies of genetic variability of this isolated population. Traditional Mendelian methods of making crosses and scoring the phenotypes of the offspring in one or more generations are however insufficient for a detailed estimate of genetic variability. Moreover, such processes do not always yield precise information on genotype (homozygous dominant vs heterozygote). These limitations can be overcome by using techniques of molecular genetics, allozyme electrophoresis among them. Allozymes are allelic variants of enzymes. Because changes in electric charge and conformation can affect the migration rate of proteins in an electric field, allelic variation can be detected by gel electrophoresis and subsequent enzyme-specific stains. Next, by examining the electrophoretic pattern for a number of enzymes (loci), the genetic variability in the population can be determined and compared with other populations. One of the enzymes frequently used when studying allozyme variations are peroxidases, a haemcontaining enzymes that use hydrogen peroxide as the electron acceptor to catalyse a number of oxidative reactions. Peroxidases are widely distributed in plant species and tissues. Despite the important role of peroxidases in multiple plant physiological processes, such as biosynthesis of and degradation of lignin in cell wall [5-8], induction of auxins metabolism [9-10] and protective reactions, particularly in damaged or pathogen-attacked tissue [11-12], the genetic data are still limited. Peroxidases are usually characterised by monogenic control [13-15], codominant inheritance [16] and monomeric enzyme behaviour. However, in rice (Oryza sativa and O. perennis) leaf tissue [17] as well as in Phragmites australis [18], alleles controlling dimer peroxidases were discovered. It has been shown that electrophoretically detected peroxidase allozymes play an important role as indicators of intra-population variability in forest trees such as Sorbus torminalis [19] and Q. pubescens [20]. Moreover, inter-population differences were described in Fraxinus excelsior [21] and Fagus sylvatica [22]. Peroxidase allozymes have been also utilized in Quercus to distinguish between species and geographic seed sources within a species. Investigations on Q. petraea and Q. robur initiated by U. Olsson already in 1975 indicated that leaf peroxidases can be useful as quantitative biochemical markers in species which show high inter-specific variation [23]. Some years later, peroxidases were included into a large enzymatic investigations in Germany [24]. The genetic structure of Q. pubescens populations with the means of three enzyme systems, including peroxidases, was characterized in our previous studies [20].
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The present study was undertaken to examine the allozyme variability of peroxidases and genetic divergence among populations of native oak species in Poland. We aimed at estimation of the intrapopulation genetic variations and at determination of the degree of inter-specific differences. 2. Material and methods 2.1. Plant material An average of 28.2 threes per population were sampled in five populations of native Quercus species from Poland. The species were represented by the following populations: 1) Q. pubescens – the primary old population (some of the trees are over 200 years old) from Bielinek reserve. Initially grown as one population, however due to the outgrowth of other species, naturally divided into two groups separated geographically. We have therefore treated these groups as two separate populations: located in a proximity to Markocin (29 samples) or Mała Dolinka (28 samples) 2) Q. pubescens – an experimental population initiated in 1994 by dr W. Danielewicz at Poznao University of Life Sciences Arboretum as a progeny of one 200 years old tree, originated from the marginal Bielinek reserve population– 24 samples. The population served for comparison with natural stands. 3) Q. petraea – population from Rogalin - 32 samples 4) Q. robur – population from Rogalin - 28 samples 2.2. Biochemical analyses The isolation and electrophoretic analyses of the peroxidase allozymes (PX, EC 1.11.1.7) were performed as previously described [20], with slight modifications. Briefly, the 1-3 dormant buds were homogenized in the presence of the extraction buffer and the extracts were subjected to 12% starchgel electrophoresis in a lithium-borate buffer system, pH 8.3. The gels were stained as in [25]. 2.3. Statistical analyses Alleles were designated numerically depending on their relative mobility from the origin. Electrophoretically detected phenotypes were used to calculate the genetic parameters: allele frequencies, genotype frequencies, heterozygosity observed (Ho), heterozygosity expected (He) and number of alleles per locus (A). The polymorphism coefficient (Pg) was calculated according to [26]. The genetic distances between populations were calculated on the basis of genotypes frequency [27]. Deviation from Hardy-Weinberg expectations for each locus in each population was estimated with the means of the Wright fixation index (FIS), using the formula FIS=1-Ho/He. The U score, which has been previously shown to be an optimal test for heterozygote excess [28] has been calculated using the R statistical environment. The genetic distances (DN) and genetic similarity (SN) between populations has been calculated according to Nei’s method *29]. Interspecific differences were illustrated with the Nei’s standard gene differentiation coefficient GST (GST =DST/HT, where DST - the inter-population diversity and HT - the total genetic diversity) [30]. A phylogenetic tree has been created based on a distance matrix, using the neighbour-joining method [29] with the bootstrap, using T-Rex (Tree and Reticulogram Reconstruction) web server (www.trex.uqam.ca [31]). Principal coordinate analysis score has been performed using the R statistical environment. 3. Results and discussion
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The establishment of separate loci and their allozymes was performed based on a band patterns on the gels, which confirmed that the peroxidases in Quercus show a monomeric behavior. We have distinguished 5 loci (PX-A to PX-E), each with 2-4 alleles (Fig. 1). PX-D locus was monomorphic, therefore it was excluded from the analyses. 3.1. Genetic diversity and Hardy-Weinberg deviations Locus A of Quercus peroxidases consists of 3 allozymes (A1-A3) distinguishable on gels (Fig. 1). Among all studied populations of Q. pubescens only one allele was present (A2) and it appeared in homozygous form (A2A2) in one individual from Mała Dolinka population and 4 from Markocin (Tables 1-2). This genotype was also the most frequent in Q. robur (frequency of A2A2 genotype is 0.39). Other individuals of Q. pubescens as well as Q. petraea populations were characterized with a null allele (A0), which would mean that this allele is most probably present in the population, however it is not active. Other remaining genotypes, A1A1 and A2A3 and A3A3 have been observed in Q. robur exclusively, with the frequencies of 0.07, 0.29 and 0.25, respectively. Locus B of oak peroxidases was represented by four active allozymes (B1-B4). This locus showed an extensive polymorphism within all tested populations. The most frequent genotype B2B3 was found in all three Q. pubescens populations as well as in Q. petraea and was not present in Q. robur. Homozygous genotype B1B1 was observed only in Q. robur population with frequency of 7%. Other homozygous genotypes were frequent in Q. robur (B2B2 reached up to 0.32 frequency) and Q. petraea (B3B3 - frequency of 0.31). Locus PX-C consists of two allozymes only (C1 and C2). The heterozygous genotype C1C2 was the only one observed in Q. pubescens population from Mała Dolinka and Q. petraea. This genotype also showed a high frequency in Q. pubescens population from Markocin and Q. robur (0.59 and 0.82, respectively). In peroxidase locus E we again observed a null allozyme (as in the A locus) in Q. pubescens population from Mała Dolinka, Q. petraea and Q. robur. Interestingly, all individuals from Q. pubescens Poznao population were characterized as genetically homozygous with E4 allele. Such E4E4 genotype was also very frequent (0.43) in Q. pubescens from Mała Dolinka. Q. robur population was characterized with an absence of E4E4 genotype and low frequency of homozygous individuals in respect to E2E2 genotype. Q. pubescens from Markocin was highly distinguished, with E2E2 and E3E3 mostly present genotypes (with frequencies of 0.45 and 0.28, respectively). Heterozygosity is a major factor in evaluating genetic variation within natural populations. We have therefore analyzed the observed heterozygosity (Ho) and correlated the values with the heterozygosity expected under Hardy-Weinberg equilibrium (He) for all polymorphic peroxidase loci. These values were used for calculation of the fixation index and U score with a corresponding p-value (Table 3). In Q. pubescens populations from Poznao and Mała Dolinka, the fixation index and U score calculated for all polymorphic peroxidase loci was negative, which means that there are more heterozygotes than in population under Hardy-Weinberg equilibrium (HWE). Such heterozygote excess revealed by negative FIS might suggest an adaptive value of the two Q. pubescens populations in Bielinek, as they are isolated from continue range of the species. Therefore, such results strongly suggest that we have an isolate-breaking effect in hand. We observed heterozygote excess revealed by negative FIS also in PX-C locus for Q. pubescens population form Markocin, Q. petraea and Q. robur (Table 3). In other populations, the FIS index was positive, meaning that there were more homozygotes observed. We have also observed clear differences between selected populations when analyzing the polymorphism coefficient (Pg), which is a value of genetic diversity of a population [12] and number 7
of alleles per locus (A) (Table 4). The largest Pg value was observed in locus B for all tested populations and it ranged from 0.56 (Q. robur) to 0.85 (Q. pubescens from Markocin). On the other hand, in locus E we observed significant increase in Pg value for two Q. pubescens populations from Bielinek (0.70 and 0.62 from Markocin and Mała Dolinka population, respectively), when compared to other species (0.28 and 0.36 for Q. petraea and Q. robur, respectively). Moreover, both populations of Q. pubescens from Bielinek reserve were characterized with the highest number of alleles per locus value (A). 3.3. Interspecific genetic relationship Clear differences in allelic frequencies and distribution was noted in the examined populations. Therefore, to further investigate the genetic relationships between particular populations we have calculated the genetic distances (DN) and genetic similarity (SN) according to Nei’s method [29]. Interspecific differences were illustrated by creating a phylogenetic tree from the Nei’s distance matrix, using the neighbour-joining method [31] with the bootstrap. Grouping by the neighbour-joining technique (NJ) pointed into the phylogenetic relationships between the populations. Phylogenetic tree created for all peroxidase loci (Fig. 2A) showed small genetic distance between Q. pubescens populations from Markocin and Poznao. It would mean that these two populations do not differ significantly in means of peroxidase genetics. The most genetically similar to a clade of these two populations is a third Q. pubescens population from Mała Dolinka and Q. petraea. All three Q. pubescens populations are clustering into one clade with a common internal node, which suggests their high genetic similarity. On the other hand, Q. robur was characterized with the longest branch to all remaining populations. This would suggest a large amount of genetic differences in peroxidase loci in Q. robur when compared with other oak species. Bootstrapping of a phylogenetic tree (resampling analysis that involved 1000 iterations) recovered two discussed nodes, however with relatively low support. To gain more insight into the genetic differences between the studied populations within the peroxidases loci, we have performed the principal coordinate analysis of neighbourhood matrix (Fig. 2B). The results observed on a phylogenetic tree (Fig. 2A) were confirmed with the principal coordinate analysis. All Q. pubescens populations and Q. petraea were grouped closely together within the first, most informative coordinate, which suggests their high genetic similarity. Q. robur, in turn was separated from the remaining populations with a long distance. In addition, the indexes of inter-population differentiation were estimated for all peroxidase loci (Table 5). The mean value of the total genetic diversity (HT) for all loci was 0.5674. The calculated value of the Nei’s standard gene differentiation coefficient GST (GST =DST/HT, where DST stands for the inter-population diversity) was in a range from 0.0091 for PX-C locus to 0.5541 for the PX-A locus. A mean value of GST was equal to 0.2798, which means that differentiation within populations is higher than between populations. 4. Conclusions Genetic variability of the three closely related Quercus species clearly shows differences in the composition of their gene pool. Taking into account that peroxidase is, in comparison with other enzyme systems, the easiest to work with, the recognition of the level of genetic variability among Quercus species can be extremely useful for a fast appraisal of the plant material for investigations of natural populations of forest trees.
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5. Acknowledgements We would like to thank Mrs. Barbara Malchrowicz for her skilful technical assistance. 6. References [1] W. Danielewicz, K. Bąkowska, M. Krzakowa, Variability of downy oak (Quercus pubescens Willd.) marginal population in Bielinek (north-western Poland) in al traits of leaves, Rocznik Dendrol. 50 (2002) 43-48. [2] B. Wójkiewicz, M. Litkowiec, W. Wachowiak, Contrasting patterns of genetic variation in core and peripheral populations of highly outcrossing and wind pollinated forest tree species, AoB Plants. 8 (2016) e-pub ahead of print [3] V.V. Potenko, O.G. Koren', V.P. Verkholat, Genetic variation and differentiation in population of Japanese emperor oak (Quercus dentata Thunb.) and Mongolian oak (Quercus mongolica fisch. ex ledeb.) in the south of the Russian far east, Russian J. Gen. 43 (2007) 387–395. [4] P. Jimenez, D. Agundez, R. Alia, L. Gil, Genetic variation in central and marginal populations of Quercus suber L, Silvae Genetica 48 (1999) 278-284. [5] H. Grisebach, Lignins, in: E.E. Conn (Ed), The biochemistry of plants, Academic Press, New York, 1981, pp. 457–478. [6] M. Maeder, R. Fuessl, Role of peroxidase in lignification of tobacco cells, Plant Physiol. 70 (1982) 1132–1134. [7] L.M. Lagrimini, Wound-induced deposition of polyphenols in transgenic plants overexpressing peroxidase, Plant Physiol. 96 (1991) 577–583. *8+ J. Łobarzewski, Peroxidase role in lignin biotransformation, in: H. Greppin, C. Penel, Th. Gaspar (Eds), Molecular and Physiological Aspects of Plant Peroxidases, University of Geneva, Switzerland 1986, pp. 231-246. [9] R.L. Hinnman , J. Lang, Peroxides catalyzed oxidation of indole-3-acetic acid, Biochemistry 4 (1965) 144–158. [10] I. Gazaryan, L.M. Lagrimini, Tobacco anionic peroxidase overexpressed in transgenic plants. II. Aerobic oxidation of indole-3-acetic acid, Phytochemistry 42 (1996) 1271–1280. [11] X. Ye, S. Pan, J. Kuc, Activity, isozyme pattern, and cellular localization of peroxidase as related to systemic resistance of tobacco to blue mold (Peronospora tabacina) and tobacco mosaic virus, Phytopathology 80 (1990) 1295–1298. [12] P.F. Dowd, L.M. Lagrimini, The role of peroxidase in host insect defenses, in: N. Carozzi, M. Koziel (Eds), Transgenic plants for control of insect pests, Taylor and Francis, New York, 1997, pp 195–223. [13] C. Benito, M. Perez de la Vega, J. Salinas, The inheritance of wheat kernel peroxidases, J. Hered. 71 (1980) 416-418. [14] P. Garcia, M. Perez de la Vega, C. Benito, The inheritance of rye seed peroxidases, Theor. Appl. Genet. 61 (1982) 341-351. [15] D. Talukdar, Allozyme variations in leaf esterase and root peroxidase isozymes and linkage with dwarfing genes in induced dwarf mutants of grass pea (Lathyrus sativus L.), Int. J. Genet. Mol. Biol. 2 (2010) 112-120. [16] J.F. Wendel, N.F. Weeden, Visualization and interpretation of plant isozymes, in: D.E. Soltis, P.S. Soltis (Eds), Isozymes in Plant Biology, Chapman and Hall, London, 1990, pp. 5-45. [17] B.B. Shai, Y.E.Chu, H.J. Oka, Analysis of gene controlling peroxidise isozymes in Oryza sativa and O. perennis, Japan J. Genetics 44 (1969) 421-328. 9
[18] M. Krzakowa, Genetic diversity of Phragmites australis (cav.) Trin. ex Steud. revealed by electrophoretically detected differences in peroxidises, in: C. Obinger, U. Eberman, C. Penel, H. Greppin (Eds), Plant Peroxidases: Biochemistry and Physiology, University of Geneva, Geneva, 1996, pp. 184-189. [19] M. Krzakowa, L. Bednorz, Electrophoretic evidence of monomorphism in the Wild Service Tree, Sorbus torminalis (L.) Crantz. population from “Bytyoskie Brzęki” preserve (western Poland), Rocznik Dendrol. 47 (1999) 45-54. [20] M. Krzakowa, K. Bąkowska, W. Danielewicz, Genetic variation patterns in marginal population of pubescence oak (Quercus pubescence Willd.) in Bielinek, on the Odra riverside, Ecol. Questions 4 (2004) 77-82. [21] M. Krzakowa, K. Przybył, Peroxidase polymorphism in Ash tree (Fraxinus excelsior L.), in: M. Acosta, J.N. Rodrigez-Lopez, M.A. Pedreno (Eds), Plant peroxidases: Biochemistry and Physiology, University of Murcia, Espana, 2003, pp. 147-151. [22] M. Krzakowa, J. Matras, Genetic variability among beech (Fagus sylvatica L.) populations from the Sudety Mountains, in respect of peroxidase and malate dehydrogenase loci, J. Appl. Genet. 46 (2005) 271-277. [23] U. Ollson, Peroxidise isozymes in Quercus petraea and Quercus robur, Bot. Not. 128 (1975) 408411. [24] G. Mueller-Starck, S. Herzog, H.H. Httamer, Intra- and inter-populational genetic variation in juvenile populations of Quercus robur L and Quercus petraea Liebl, Ann. Sci. For. 50 (1993) 233-244. [25] C.R. Show, R. Prasad, Starch gel electrophoresis of enzymes, a compilation of recipes, Bioch. Gen. 4 (1970) 297-320. [26] A.L. Kahler, R. Allard, M. Krzakowa, C.F. Wehrharn, E. Nevo, Associations between isozyme phenotypes and environment in the slender wild oat (Avena barbatta) in Israel, Theor. Appl. Genet. 56 (1980) 31-47. [27] M. Nei, Analysis of gene diversity in subdivided populations, Proc. Natl. Acad. Sci. U. S. A. 70 (1973) 3321–3323. [28] F. Rousset, M. Raymond, Testing heterozygote excess and deficiency, Genetics 140 (1995) 14131419. [29] M. Nei, Genetic distance between populations, Am. Nat. 106 (1972) 283–292. [30] R. Sokal, C. Michener, A statistical method for evaluating systematic relationships, University of Kansas Science Bulletin 38 (1958) 1409–1438. [31] V. Makarenkov, T-Rex: reconstructing and visualizing phylogenetic trees and reticulation networks, Bioinformatics 17 (2001) 664-668.
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Figure Captions
Figure 1. Schematic diagram of electrophoretically detected peroxidase allozymes in Quercus. Migration distance is shown in cm.
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Figure 2. Visualization of genetic relations within Quercus species. (A) Phylogenetic tree for all polymorphic peroxidase loci and (B) principal coordinate analysis.
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Locality and sample size (N) Q. pubescens Allele Poznań (N=24)
Markocin (N=29)
Q. petraea Mała Dolinka (N=28)
Rogalin (N=32)
Q. robur Rogalin (N=28)
A0
1.00
0.86
0.96
1.00
0.00
A1
0.00
0.00
0.00
0.00
0.07
A2
0.00
0.14
0.04
0.00
0.53
A3
0.00
0.00
0.00
0.00
0.40
B1
0.08
0.10
0.07
0.09
0.07
B2
0.46
0.29
0.38
0.16
0.73
B3
0.35
0.24
0.43
0.39
0.16
B4
0.10
0.36
0.13
0.36
0.02
C1
0.50
0.60
0.50
0.50
0.59
C2
0.50
0.40
0.50
0.50
0.41
E0
0.00
0.00
0.43
0.84
0.78
E1
0.00
0.03
0.04
0.00
0.00
E2
0.00
0.53
0.10
0.06
0.13
E3
0.00
0.38
0.00
0.00
0.07
E4
1.00
0.05
0.09
0.09
0.02
Table 1. Allele frequencies for all polymorphic peroxidase loci among Quercus populations examined.
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Locality and sample size (N) Q. pubescens Genotype Poznań (N=24)
Markocin (N=29)
Q. petraea Mała Dolinka (N=28)
Rogalin (N=32)
Q. robur Rogalin (N=28)
A0A0
1.00
0.86
0.96
1.00
0.00
A1A1
0.00
0.00
0.00
0.00
0.07
A2A2
0.00
0.14
0.04
0.00
0.39
A2A3
0.00
0.00
0.00
0.00
0.29
A3A3
0.00
0.00
0.00
0.00
0.25
B1B1
0.00
0.00
0.00
0.00
0.07
B1B2
0.12
0.00
0.07
0.00
0.00
B1B3
0.04
0.03
0.07
0.03
0.00
B1B4
0.00
0.19
0.00
0.16
0.57
B2B2
0.12
0.10
0.10
0.06
0.32
B2B3
0.38
0.17
0.46
0.03
0.00
B2B4
0.17
0.20
0.00
0.16
0.00
B3B3
0.13
0.10
0.04
0.31
0.00
B3B4
0.04
0.07
0.26
0.09
0.04
B4B4
0.00
0.10
0.00
0.10
0.00
C1C1
1.00
0.31
0.00
0.00
0.18
C1C2
0.00
0.59
1.00
1.00
0.82
C2C2
0.00
0.10
0.00
0.00
0.00
E0E0
0.00
0.00
0.43
0.84
0.79
E1E1
0.00
0.00
0.04
0.00
0.00
E1E3
0.00
0.07
0.00
0.06
0.11
E2E2
0.00
0.45
0.10
0.00
0.00
14
E2E3
0.00
0.14
0.00
0.00
0.00
E2E4
0.00
0.03
0.00
0.00
0.04
E3E3
0.00
0.28
0.00
0.00
0.07
E4E4
1.00
0.03
0.43
0.09
0.00
Table 2. Genotype frequencies for all polymorphic peroxidase loci among Quercus populations examined.
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A PX Locus A B C E
Ho 0.00 0.75 1.00 0.00
Poznań (N=24) He F U 0.00 0.00 ND 0.65 -0.15 -8.98 0.50 -1.00 ND 0.00 0.00 ND
p ND 0.194 ND ND
Ho 0.00 0.65 0.59 0.24
Locality and sample size (N) Q. pubescens Markocin (N=29) He F U p 0.24 0.00 29 0.000 0.71 0.08 4.35 0.262 0.48 -0.23 -6.52 0.232 0.57 0.58 35.75 0.002
Ho 0.00 0.86 1.00 0.00
Mała Dolinka (N=28) He F U 0.69 0.00 28 0.65 -0.32 -17.67 0.50 -1.00 ND 0.62 0.00 84
p 0.000 0.014 ND 0.000
B Locality and sample size (N) PX Locus A B C E
Ho 0.00 0.47 1.00 0.00
He 0.00 0.69 0.50 0.28
Q. petraea Rogalin (N=32) F 0.00 0.32 -1.00 0.00
U
p
ND 20.31 ND 64
ND 0.020 ND 0.000
Ho 0.29 0.36 0.82 0.04
He 0.55 0.43 0.48 0.36
Q. robur Rogalin (N=28) F U 0.47 38.35 0.16 21.85 -0.71 -19.52 0.89 52
p 0.000 0.003 0.000 0.000
Table 3. The heterozygosity expected (He) and observed (Ho) as well as fixation index (F) values and U score (U) with corresponding p-value (p) for all polymorphic peroxidase loci among Quercus populations examined. The negative F and U report an excess of homozygotes whereas negative F and U report an excess of heterozygotes.
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Locality and sample size (N) PX Locus
Q. pubescens Poznań (N=24)
Markocin (N=29)
Mała Dolinka (N=28)
Q. petraea
Q. robur
Rogalin (N=32)
Rogalin (N=28)
Pg
A
Pg
A
Pg
A
Pg
A
Pg
A
A
0.00
1
0.24
2
0.69
2
0.00
1
0.69
3
B
0.78
4
0.85
4
0.70
4
0.81
4
0.56
4
C
0.00
2
0.55
2
0.00
2
0.00
2
0.29
2
E
0.00
1
0.70
4
0.62
4
0.28
3
0.36
4
Table 4. The polymorphism coefficient (Pg) values and number of alleles per locus (A) for all polymorphic peroxidase loci among Quercus populations examined.
17
Peroxidase
HT
DST
GST
locus A
0.3869 0.2144 0.5541
B
0.6951 0.0679 0.0977
C
0.4970 0.0045 0.0091
E
0.6906 0.3167 0.4585
Mean value 0.5674 0.1509 0.2798
Table 5. Indexes of inter-populational differentiation between Quercus populations based on 4 polymorphic peroxidases loci. HT - total genetic diversity, DST - interpopulational diversity , GST Nei’s standard gene differentiation coefficient.
18
S1381-1177(17)30003-6 http://dx.doi.org/doi:10.1016/j.molcatb.2017.01.004 MOLCAB 3503
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
12-7-2016 30-12-2016 4-1-2017
˙ Please cite this article as: Maria Krzakowa, Kamilla B˛akowska-Zywicka, Peroxidase polymorphism in pubescent oak (Quercus pubescens Willd.) in relation to Q.petraea Matt.Liebl and Q.robur L., Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2017.01.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Peroxidase polymorphism in pubescent oak (Quercus pubescens Willd.) in relation to Q. petraea Matt. Liebl and Q. robur L. Maria Krzakowa1*, Kamilla Bąkowska-Żywicka2* 1 Department of Genetics, Adam Mickiewicz University, Umultowska 89, 61-614 Poznao, Poland, [email protected] 2 Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznao, Poland, [email protected] *corresponding authors
1
Graphical Abstract
2
Highlights
Peroxidase allozymes were analysed in three native species of Quercus from Poland. Our results show an extensive polymorphism of Quercus peroxidases. Q. robur shows low genetic similarity to other Quercus species in question.
3
Abstract In spite of the increasing availability of DNA markers with a higher genetic resolution, electrophoretically detectable allozymes still provide the cutting edge in the estimation of genetic variability. Peroxidase was analysed in individual sleeping buds collected in three native species: Quercus pubescens, Q. petraea and Q. robur. The species were characterised with gene and genotype frequencies and showed specific degrees of intra- and inter-population diversity. Genetic variability of marginal population of pubescent oak from Bielinek, the only stand of the species in Poland markedly separated from its compact range, was described by peroxidise polymorphism. Genetic distances among species, statistically examined, showed visible differences between them and a relatively low genetic similarity of Q. robur to the two species in question. Key words: peroxidase, allozymes, genetic distance, Quercus pubescens This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
4
1. Introduction In Poland there are two important species of oak which are common in Central Europe: pedunculate oak (Quercus robur) and sessile oak (Q. petraea). Both of them are of high economic significance. The pubescent oak (Q. pubescens) of sub-Mediterranean origin has no economic importance, however it is of special interest because of its unusual geographical location. The only stand of Q. pubescens in Poland is situated in postglacial valley of Odra river in Bielinek and protected as forest-steppe preserve. This population is located far away north from the main species range, but within a natural range of two other oak species (Q. robur and Q. petraea). The significant age of some of the Q. pubescens specimens (the oldest tree is over 200 years old), rich fructification and southern character of local biocenosis suggest the natural origin of Bielinek population. Based on the leaf morphology studies Q. pubescens from Bielinek was classified as a hybrid population, however distinct from both Q. petraea and, even more evidently, from Q. robur [1]. Usually, the level of genetic variation of marginal populations differs from those of a continue range [2-4], therefore there is a need for detailed studies of genetic variability of this isolated population. Traditional Mendelian methods of making crosses and scoring the phenotypes of the offspring in one or more generations are however insufficient for a detailed estimate of genetic variability. Moreover, such processes do not always yield precise information on genotype (homozygous dominant vs heterozygote). These limitations can be overcome by using techniques of molecular genetics, allozyme electrophoresis among them. Allozymes are allelic variants of enzymes. Because changes in electric charge and conformation can affect the migration rate of proteins in an electric field, allelic variation can be detected by gel electrophoresis and subsequent enzyme-specific stains. Next, by examining the electrophoretic pattern for a number of enzymes (loci), the genetic variability in the population can be determined and compared with other populations. One of the enzymes frequently used when studying allozyme variations are peroxidases, a haemcontaining enzymes that use hydrogen peroxide as the electron acceptor to catalyse a number of oxidative reactions. Peroxidases are widely distributed in plant species and tissues. Despite the important role of peroxidases in multiple plant physiological processes, such as biosynthesis of and degradation of lignin in cell wall [5-8], induction of auxins metabolism [9-10] and protective reactions, particularly in damaged or pathogen-attacked tissue [11-12], the genetic data are still limited. Peroxidases are usually characterised by monogenic control [13-15], codominant inheritance [16] and monomeric enzyme behaviour. However, in rice (Oryza sativa and O. perennis) leaf tissue [17] as well as in Phragmites australis [18], alleles controlling dimer peroxidases were discovered. It has been shown that electrophoretically detected peroxidase allozymes play an important role as indicators of intra-population variability in forest trees such as Sorbus torminalis [19] and Q. pubescens [20]. Moreover, inter-population differences were described in Fraxinus excelsior [21] and Fagus sylvatica [22]. Peroxidase allozymes have been also utilized in Quercus to distinguish between species and geographic seed sources within a species. Investigations on Q. petraea and Q. robur initiated by U. Olsson already in 1975 indicated that leaf peroxidases can be useful as quantitative biochemical markers in species which show high inter-specific variation [23]. Some years later, peroxidases were included into a large enzymatic investigations in Germany [24]. The genetic structure of Q. pubescens populations with the means of three enzyme systems, including peroxidases, was characterized in our previous studies [20].
5
The present study was undertaken to examine the allozyme variability of peroxidases and genetic divergence among populations of native oak species in Poland. We aimed at estimation of the intrapopulation genetic variations and at determination of the degree of inter-specific differences. 2. Material and methods 2.1. Plant material An average of 28.2 threes per population were sampled in five populations of native Quercus species from Poland. The species were represented by the following populations: 1) Q. pubescens – the primary old population (some of the trees are over 200 years old) from Bielinek reserve. Initially grown as one population, however due to the outgrowth of other species, naturally divided into two groups separated geographically. We have therefore treated these groups as two separate populations: located in a proximity to Markocin (29 samples) or Mała Dolinka (28 samples) 2) Q. pubescens – an experimental population initiated in 1994 by dr W. Danielewicz at Poznao University of Life Sciences Arboretum as a progeny of one 200 years old tree, originated from the marginal Bielinek reserve population– 24 samples. The population served for comparison with natural stands. 3) Q. petraea – population from Rogalin - 32 samples 4) Q. robur – population from Rogalin - 28 samples 2.2. Biochemical analyses The isolation and electrophoretic analyses of the peroxidase allozymes (PX, EC 1.11.1.7) were performed as previously described [20], with slight modifications. Briefly, the 1-3 dormant buds were homogenized in the presence of the extraction buffer and the extracts were subjected to 12% starchgel electrophoresis in a lithium-borate buffer system, pH 8.3. The gels were stained as in [25]. 2.3. Statistical analyses Alleles were designated numerically depending on their relative mobility from the origin. Electrophoretically detected phenotypes were used to calculate the genetic parameters: allele frequencies, genotype frequencies, heterozygosity observed (Ho), heterozygosity expected (He) and number of alleles per locus (A). The polymorphism coefficient (Pg) was calculated according to [26]. The genetic distances between populations were calculated on the basis of genotypes frequency [27]. Deviation from Hardy-Weinberg expectations for each locus in each population was estimated with the means of the Wright fixation index (FIS), using the formula FIS=1-Ho/He. The U score, which has been previously shown to be an optimal test for heterozygote excess [28] has been calculated using the R statistical environment. The genetic distances (DN) and genetic similarity (SN) between populations has been calculated according to Nei’s method *29]. Interspecific differences were illustrated with the Nei’s standard gene differentiation coefficient GST (GST =DST/HT, where DST - the inter-population diversity and HT - the total genetic diversity) [30]. A phylogenetic tree has been created based on a distance matrix, using the neighbour-joining method [29] with the bootstrap, using T-Rex (Tree and Reticulogram Reconstruction) web server (www.trex.uqam.ca [31]). Principal coordinate analysis score has been performed using the R statistical environment. 3. Results and discussion
6
The establishment of separate loci and their allozymes was performed based on a band patterns on the gels, which confirmed that the peroxidases in Quercus show a monomeric behavior. We have distinguished 5 loci (PX-A to PX-E), each with 2-4 alleles (Fig. 1). PX-D locus was monomorphic, therefore it was excluded from the analyses. 3.1. Genetic diversity and Hardy-Weinberg deviations Locus A of Quercus peroxidases consists of 3 allozymes (A1-A3) distinguishable on gels (Fig. 1). Among all studied populations of Q. pubescens only one allele was present (A2) and it appeared in homozygous form (A2A2) in one individual from Mała Dolinka population and 4 from Markocin (Tables 1-2). This genotype was also the most frequent in Q. robur (frequency of A2A2 genotype is 0.39). Other individuals of Q. pubescens as well as Q. petraea populations were characterized with a null allele (A0), which would mean that this allele is most probably present in the population, however it is not active. Other remaining genotypes, A1A1 and A2A3 and A3A3 have been observed in Q. robur exclusively, with the frequencies of 0.07, 0.29 and 0.25, respectively. Locus B of oak peroxidases was represented by four active allozymes (B1-B4). This locus showed an extensive polymorphism within all tested populations. The most frequent genotype B2B3 was found in all three Q. pubescens populations as well as in Q. petraea and was not present in Q. robur. Homozygous genotype B1B1 was observed only in Q. robur population with frequency of 7%. Other homozygous genotypes were frequent in Q. robur (B2B2 reached up to 0.32 frequency) and Q. petraea (B3B3 - frequency of 0.31). Locus PX-C consists of two allozymes only (C1 and C2). The heterozygous genotype C1C2 was the only one observed in Q. pubescens population from Mała Dolinka and Q. petraea. This genotype also showed a high frequency in Q. pubescens population from Markocin and Q. robur (0.59 and 0.82, respectively). In peroxidase locus E we again observed a null allozyme (as in the A locus) in Q. pubescens population from Mała Dolinka, Q. petraea and Q. robur. Interestingly, all individuals from Q. pubescens Poznao population were characterized as genetically homozygous with E4 allele. Such E4E4 genotype was also very frequent (0.43) in Q. pubescens from Mała Dolinka. Q. robur population was characterized with an absence of E4E4 genotype and low frequency of homozygous individuals in respect to E2E2 genotype. Q. pubescens from Markocin was highly distinguished, with E2E2 and E3E3 mostly present genotypes (with frequencies of 0.45 and 0.28, respectively). Heterozygosity is a major factor in evaluating genetic variation within natural populations. We have therefore analyzed the observed heterozygosity (Ho) and correlated the values with the heterozygosity expected under Hardy-Weinberg equilibrium (He) for all polymorphic peroxidase loci. These values were used for calculation of the fixation index and U score with a corresponding p-value (Table 3). In Q. pubescens populations from Poznao and Mała Dolinka, the fixation index and U score calculated for all polymorphic peroxidase loci was negative, which means that there are more heterozygotes than in population under Hardy-Weinberg equilibrium (HWE). Such heterozygote excess revealed by negative FIS might suggest an adaptive value of the two Q. pubescens populations in Bielinek, as they are isolated from continue range of the species. Therefore, such results strongly suggest that we have an isolate-breaking effect in hand. We observed heterozygote excess revealed by negative FIS also in PX-C locus for Q. pubescens population form Markocin, Q. petraea and Q. robur (Table 3). In other populations, the FIS index was positive, meaning that there were more homozygotes observed. We have also observed clear differences between selected populations when analyzing the polymorphism coefficient (Pg), which is a value of genetic diversity of a population [12] and number 7
of alleles per locus (A) (Table 4). The largest Pg value was observed in locus B for all tested populations and it ranged from 0.56 (Q. robur) to 0.85 (Q. pubescens from Markocin). On the other hand, in locus E we observed significant increase in Pg value for two Q. pubescens populations from Bielinek (0.70 and 0.62 from Markocin and Mała Dolinka population, respectively), when compared to other species (0.28 and 0.36 for Q. petraea and Q. robur, respectively). Moreover, both populations of Q. pubescens from Bielinek reserve were characterized with the highest number of alleles per locus value (A). 3.3. Interspecific genetic relationship Clear differences in allelic frequencies and distribution was noted in the examined populations. Therefore, to further investigate the genetic relationships between particular populations we have calculated the genetic distances (DN) and genetic similarity (SN) according to Nei’s method [29]. Interspecific differences were illustrated by creating a phylogenetic tree from the Nei’s distance matrix, using the neighbour-joining method [31] with the bootstrap. Grouping by the neighbour-joining technique (NJ) pointed into the phylogenetic relationships between the populations. Phylogenetic tree created for all peroxidase loci (Fig. 2A) showed small genetic distance between Q. pubescens populations from Markocin and Poznao. It would mean that these two populations do not differ significantly in means of peroxidase genetics. The most genetically similar to a clade of these two populations is a third Q. pubescens population from Mała Dolinka and Q. petraea. All three Q. pubescens populations are clustering into one clade with a common internal node, which suggests their high genetic similarity. On the other hand, Q. robur was characterized with the longest branch to all remaining populations. This would suggest a large amount of genetic differences in peroxidase loci in Q. robur when compared with other oak species. Bootstrapping of a phylogenetic tree (resampling analysis that involved 1000 iterations) recovered two discussed nodes, however with relatively low support. To gain more insight into the genetic differences between the studied populations within the peroxidases loci, we have performed the principal coordinate analysis of neighbourhood matrix (Fig. 2B). The results observed on a phylogenetic tree (Fig. 2A) were confirmed with the principal coordinate analysis. All Q. pubescens populations and Q. petraea were grouped closely together within the first, most informative coordinate, which suggests their high genetic similarity. Q. robur, in turn was separated from the remaining populations with a long distance. In addition, the indexes of inter-population differentiation were estimated for all peroxidase loci (Table 5). The mean value of the total genetic diversity (HT) for all loci was 0.5674. The calculated value of the Nei’s standard gene differentiation coefficient GST (GST =DST/HT, where DST stands for the inter-population diversity) was in a range from 0.0091 for PX-C locus to 0.5541 for the PX-A locus. A mean value of GST was equal to 0.2798, which means that differentiation within populations is higher than between populations. 4. Conclusions Genetic variability of the three closely related Quercus species clearly shows differences in the composition of their gene pool. Taking into account that peroxidase is, in comparison with other enzyme systems, the easiest to work with, the recognition of the level of genetic variability among Quercus species can be extremely useful for a fast appraisal of the plant material for investigations of natural populations of forest trees.
8
5. Acknowledgements We would like to thank Mrs. Barbara Malchrowicz for her skilful technical assistance. 6. References [1] W. Danielewicz, K. Bąkowska, M. Krzakowa, Variability of downy oak (Quercus pubescens Willd.) marginal population in Bielinek (north-western Poland) in al traits of leaves, Rocznik Dendrol. 50 (2002) 43-48. [2] B. Wójkiewicz, M. Litkowiec, W. Wachowiak, Contrasting patterns of genetic variation in core and peripheral populations of highly outcrossing and wind pollinated forest tree species, AoB Plants. 8 (2016) e-pub ahead of print [3] V.V. Potenko, O.G. Koren', V.P. Verkholat, Genetic variation and differentiation in population of Japanese emperor oak (Quercus dentata Thunb.) and Mongolian oak (Quercus mongolica fisch. ex ledeb.) in the south of the Russian far east, Russian J. Gen. 43 (2007) 387–395. [4] P. Jimenez, D. Agundez, R. Alia, L. Gil, Genetic variation in central and marginal populations of Quercus suber L, Silvae Genetica 48 (1999) 278-284. [5] H. Grisebach, Lignins, in: E.E. Conn (Ed), The biochemistry of plants, Academic Press, New York, 1981, pp. 457–478. [6] M. Maeder, R. Fuessl, Role of peroxidase in lignification of tobacco cells, Plant Physiol. 70 (1982) 1132–1134. [7] L.M. Lagrimini, Wound-induced deposition of polyphenols in transgenic plants overexpressing peroxidase, Plant Physiol. 96 (1991) 577–583. *8+ J. Łobarzewski, Peroxidase role in lignin biotransformation, in: H. Greppin, C. Penel, Th. Gaspar (Eds), Molecular and Physiological Aspects of Plant Peroxidases, University of Geneva, Switzerland 1986, pp. 231-246. [9] R.L. Hinnman , J. Lang, Peroxides catalyzed oxidation of indole-3-acetic acid, Biochemistry 4 (1965) 144–158. [10] I. Gazaryan, L.M. Lagrimini, Tobacco anionic peroxidase overexpressed in transgenic plants. II. Aerobic oxidation of indole-3-acetic acid, Phytochemistry 42 (1996) 1271–1280. [11] X. Ye, S. Pan, J. Kuc, Activity, isozyme pattern, and cellular localization of peroxidase as related to systemic resistance of tobacco to blue mold (Peronospora tabacina) and tobacco mosaic virus, Phytopathology 80 (1990) 1295–1298. [12] P.F. Dowd, L.M. Lagrimini, The role of peroxidase in host insect defenses, in: N. Carozzi, M. Koziel (Eds), Transgenic plants for control of insect pests, Taylor and Francis, New York, 1997, pp 195–223. [13] C. Benito, M. Perez de la Vega, J. Salinas, The inheritance of wheat kernel peroxidases, J. Hered. 71 (1980) 416-418. [14] P. Garcia, M. Perez de la Vega, C. Benito, The inheritance of rye seed peroxidases, Theor. Appl. Genet. 61 (1982) 341-351. [15] D. Talukdar, Allozyme variations in leaf esterase and root peroxidase isozymes and linkage with dwarfing genes in induced dwarf mutants of grass pea (Lathyrus sativus L.), Int. J. Genet. Mol. Biol. 2 (2010) 112-120. [16] J.F. Wendel, N.F. Weeden, Visualization and interpretation of plant isozymes, in: D.E. Soltis, P.S. Soltis (Eds), Isozymes in Plant Biology, Chapman and Hall, London, 1990, pp. 5-45. [17] B.B. Shai, Y.E.Chu, H.J. Oka, Analysis of gene controlling peroxidise isozymes in Oryza sativa and O. perennis, Japan J. Genetics 44 (1969) 421-328. 9
[18] M. Krzakowa, Genetic diversity of Phragmites australis (cav.) Trin. ex Steud. revealed by electrophoretically detected differences in peroxidises, in: C. Obinger, U. Eberman, C. Penel, H. Greppin (Eds), Plant Peroxidases: Biochemistry and Physiology, University of Geneva, Geneva, 1996, pp. 184-189. [19] M. Krzakowa, L. Bednorz, Electrophoretic evidence of monomorphism in the Wild Service Tree, Sorbus torminalis (L.) Crantz. population from “Bytyoskie Brzęki” preserve (western Poland), Rocznik Dendrol. 47 (1999) 45-54. [20] M. Krzakowa, K. Bąkowska, W. Danielewicz, Genetic variation patterns in marginal population of pubescence oak (Quercus pubescence Willd.) in Bielinek, on the Odra riverside, Ecol. Questions 4 (2004) 77-82. [21] M. Krzakowa, K. Przybył, Peroxidase polymorphism in Ash tree (Fraxinus excelsior L.), in: M. Acosta, J.N. Rodrigez-Lopez, M.A. Pedreno (Eds), Plant peroxidases: Biochemistry and Physiology, University of Murcia, Espana, 2003, pp. 147-151. [22] M. Krzakowa, J. Matras, Genetic variability among beech (Fagus sylvatica L.) populations from the Sudety Mountains, in respect of peroxidase and malate dehydrogenase loci, J. Appl. Genet. 46 (2005) 271-277. [23] U. Ollson, Peroxidise isozymes in Quercus petraea and Quercus robur, Bot. Not. 128 (1975) 408411. [24] G. Mueller-Starck, S. Herzog, H.H. Httamer, Intra- and inter-populational genetic variation in juvenile populations of Quercus robur L and Quercus petraea Liebl, Ann. Sci. For. 50 (1993) 233-244. [25] C.R. Show, R. Prasad, Starch gel electrophoresis of enzymes, a compilation of recipes, Bioch. Gen. 4 (1970) 297-320. [26] A.L. Kahler, R. Allard, M. Krzakowa, C.F. Wehrharn, E. Nevo, Associations between isozyme phenotypes and environment in the slender wild oat (Avena barbatta) in Israel, Theor. Appl. Genet. 56 (1980) 31-47. [27] M. Nei, Analysis of gene diversity in subdivided populations, Proc. Natl. Acad. Sci. U. S. A. 70 (1973) 3321–3323. [28] F. Rousset, M. Raymond, Testing heterozygote excess and deficiency, Genetics 140 (1995) 14131419. [29] M. Nei, Genetic distance between populations, Am. Nat. 106 (1972) 283–292. [30] R. Sokal, C. Michener, A statistical method for evaluating systematic relationships, University of Kansas Science Bulletin 38 (1958) 1409–1438. [31] V. Makarenkov, T-Rex: reconstructing and visualizing phylogenetic trees and reticulation networks, Bioinformatics 17 (2001) 664-668.
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Figure Captions
Figure 1. Schematic diagram of electrophoretically detected peroxidase allozymes in Quercus. Migration distance is shown in cm.
11
Figure 2. Visualization of genetic relations within Quercus species. (A) Phylogenetic tree for all polymorphic peroxidase loci and (B) principal coordinate analysis.
12
Locality and sample size (N) Q. pubescens Allele Poznań (N=24)
Markocin (N=29)
Q. petraea Mała Dolinka (N=28)
Rogalin (N=32)
Q. robur Rogalin (N=28)
A0
1.00
0.86
0.96
1.00
0.00
A1
0.00
0.00
0.00
0.00
0.07
A2
0.00
0.14
0.04
0.00
0.53
A3
0.00
0.00
0.00
0.00
0.40
B1
0.08
0.10
0.07
0.09
0.07
B2
0.46
0.29
0.38
0.16
0.73
B3
0.35
0.24
0.43
0.39
0.16
B4
0.10
0.36
0.13
0.36
0.02
C1
0.50
0.60
0.50
0.50
0.59
C2
0.50
0.40
0.50
0.50
0.41
E0
0.00
0.00
0.43
0.84
0.78
E1
0.00
0.03
0.04
0.00
0.00
E2
0.00
0.53
0.10
0.06
0.13
E3
0.00
0.38
0.00
0.00
0.07
E4
1.00
0.05
0.09
0.09
0.02
Table 1. Allele frequencies for all polymorphic peroxidase loci among Quercus populations examined.
13
Locality and sample size (N) Q. pubescens Genotype Poznań (N=24)
Markocin (N=29)
Q. petraea Mała Dolinka (N=28)
Rogalin (N=32)
Q. robur Rogalin (N=28)
A0A0
1.00
0.86
0.96
1.00
0.00
A1A1
0.00
0.00
0.00
0.00
0.07
A2A2
0.00
0.14
0.04
0.00
0.39
A2A3
0.00
0.00
0.00
0.00
0.29
A3A3
0.00
0.00
0.00
0.00
0.25
B1B1
0.00
0.00
0.00
0.00
0.07
B1B2
0.12
0.00
0.07
0.00
0.00
B1B3
0.04
0.03
0.07
0.03
0.00
B1B4
0.00
0.19
0.00
0.16
0.57
B2B2
0.12
0.10
0.10
0.06
0.32
B2B3
0.38
0.17
0.46
0.03
0.00
B2B4
0.17
0.20
0.00
0.16
0.00
B3B3
0.13
0.10
0.04
0.31
0.00
B3B4
0.04
0.07
0.26
0.09
0.04
B4B4
0.00
0.10
0.00
0.10
0.00
C1C1
1.00
0.31
0.00
0.00
0.18
C1C2
0.00
0.59
1.00
1.00
0.82
C2C2
0.00
0.10
0.00
0.00
0.00
E0E0
0.00
0.00
0.43
0.84
0.79
E1E1
0.00
0.00
0.04
0.00
0.00
E1E3
0.00
0.07
0.00
0.06
0.11
E2E2
0.00
0.45
0.10
0.00
0.00
14
E2E3
0.00
0.14
0.00
0.00
0.00
E2E4
0.00
0.03
0.00
0.00
0.04
E3E3
0.00
0.28
0.00
0.00
0.07
E4E4
1.00
0.03
0.43
0.09
0.00
Table 2. Genotype frequencies for all polymorphic peroxidase loci among Quercus populations examined.
15
A PX Locus A B C E
Ho 0.00 0.75 1.00 0.00
Poznań (N=24) He F U 0.00 0.00 ND 0.65 -0.15 -8.98 0.50 -1.00 ND 0.00 0.00 ND
p ND 0.194 ND ND
Ho 0.00 0.65 0.59 0.24
Locality and sample size (N) Q. pubescens Markocin (N=29) He F U p 0.24 0.00 29 0.000 0.71 0.08 4.35 0.262 0.48 -0.23 -6.52 0.232 0.57 0.58 35.75 0.002
Ho 0.00 0.86 1.00 0.00
Mała Dolinka (N=28) He F U 0.69 0.00 28 0.65 -0.32 -17.67 0.50 -1.00 ND 0.62 0.00 84
p 0.000 0.014 ND 0.000
B Locality and sample size (N) PX Locus A B C E
Ho 0.00 0.47 1.00 0.00
He 0.00 0.69 0.50 0.28
Q. petraea Rogalin (N=32) F 0.00 0.32 -1.00 0.00
U
p
ND 20.31 ND 64
ND 0.020 ND 0.000
Ho 0.29 0.36 0.82 0.04
He 0.55 0.43 0.48 0.36
Q. robur Rogalin (N=28) F U 0.47 38.35 0.16 21.85 -0.71 -19.52 0.89 52
p 0.000 0.003 0.000 0.000
Table 3. The heterozygosity expected (He) and observed (Ho) as well as fixation index (F) values and U score (U) with corresponding p-value (p) for all polymorphic peroxidase loci among Quercus populations examined. The negative F and U report an excess of homozygotes whereas negative F and U report an excess of heterozygotes.
16
Locality and sample size (N) PX Locus
Q. pubescens Poznań (N=24)
Markocin (N=29)
Mała Dolinka (N=28)
Q. petraea
Q. robur
Rogalin (N=32)
Rogalin (N=28)
Pg
A
Pg
A
Pg
A
Pg
A
Pg
A
A
0.00
1
0.24
2
0.69
2
0.00
1
0.69
3
B
0.78
4
0.85
4
0.70
4
0.81
4
0.56
4
C
0.00
2
0.55
2
0.00
2
0.00
2
0.29
2
E
0.00
1
0.70
4
0.62
4
0.28
3
0.36
4
Table 4. The polymorphism coefficient (Pg) values and number of alleles per locus (A) for all polymorphic peroxidase loci among Quercus populations examined.
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Peroxidase
HT
DST
GST
locus A
0.3869 0.2144 0.5541
B
0.6951 0.0679 0.0977
C
0.4970 0.0045 0.0091
E
0.6906 0.3167 0.4585
Mean value 0.5674 0.1509 0.2798
Table 5. Indexes of inter-populational differentiation between Quercus populations based on 4 polymorphic peroxidases loci. HT - total genetic diversity, DST - interpopulational diversity , GST Nei’s standard gene differentiation coefficient.
18