# Preliminary Comparative Analysis of Phenological Varieties ofQuercus robur by ISSR-Markers

Authors: Vasiliy Chokheli; Boris Kozlovsky; Mikhail Sereda; Vladimir Lysenko; Igor Fesenko; Tatiana Varduny; Olga Kapralova; Elena Bondarenko
Journal: Journal of Botany
 (2016)

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## Abstract

Quercus robur L. is a valuable wood species having long ontogeny and promising to create long-living artificial plantings of recreational and ameliorative purposes in the steppes zone of Russia and other countries. In this work we have performed the genotyping of varieties ofQuercus robur L. obtained from collection of Botanical Garden of Southern Federal University using intersimple sequence repeat (ISSR) molecular markers. The most polymorphic ISSR-marker (GA) 8YC was found in the collection. The polymorphic DNA markers identified in the present study can be used for the future breeding works to obtain valuable genotypes ofQuercus genus. In addition we have performed DNA fingerprinting of the prospective sample of the varietyQ. robur var.tardiflora Czern.


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## Body

## 1. Introduction

Efficient creation of artificial forests should be based on the use of a genetically diversified planting material of high-quality. Among such a material the English oak (Quercus robur L.) is an important component of artificial forest plantations [1].Q. robur is a widespread species in the natural forest associations [2] of the middle latitudes and mountainous zones of the northern hemisphere and Asia Minor (an area corresponding to the western two-thirds of Turkey), North Africa, the Caucasus, and Europe [3]. The actual area occupied byQ. robur in Russia is located within the subzone of broad-leaved forests and forest-steppe of the European part of the country [4].NowQ. robur cannot be found in the natural associations of West Siberia flora, although oak forests are an essential part of the forest-steppe zone of Western Siberia.Wide distribution ofQ. robur in the area of the continental climate is characterized by its freeze-thaw resistance [5, 6].Q. robur refers to salt-tolerant woody plants growing satisfactorily in saline soils [7]. However, some authors have an opposite view [8, 9]. There are different, conflicting data related toQ. robur. Pogrebnyak showed thatQ. robur is a xeromesophyte [10]. Accordingly to other dataQ. robur is very tolerant to soil conditions and the continental climate but it prefers fertile and well-watered soils [7, 11]. However, it is capable of growing in conditions of high humidity [12]. Drought resistance ofQ. robur is primarily determined by its strong root system, so this species can grow on dry poor, rocky soils.Currently, this type ofQ. robur habitat becomes mobile and reduced in size due to its substitution by other species [3].Significant reduction in the duration of the ontogeny is a serious problem of artificial plantations ofQ. robur [13, 14].In this connection the following varieties ofQ. robur L. (firstly described by Chernyaev [15] in 1858) are of interest:Q. robur var.praecox Czern. andQ. robur var.tardiflora Czern., which differ in time of occurrence of their phenological phases.Two varieties ofQuercus robur were studied:Q. robur var.tardifolia andQ. robur var.praecox, which are cultivated in the Botanical Garden of Southern Federal University.The main diagnostic signs to distinguishQuercus robur var.tardiflora Czern. andQuercus robur var.praecox Czern. are [16] as follows:(1)
Bud, bloom, beginning of shoot growth, and most other phenological phases occur inQuercus robur var.tardiflora Czern. at 2-3 weeks later than inQuercus robur var.praecox Czern..
(2)
In contrast toQuercus robur var.praecox Czern, the senescent leaves ofQuercus robur var.tardiflora Czern. do not fall down, remaining on the shoots until the end of winter.
(3)
Q. robur var.tardiflora is less defeated by pests and diseases.
(4)
Comparing toQuercus robur var.praecox Czern,Q. robur var.tardiflora has a higher and a more regular crown shape, straighter trunk, and a higher quality of wood. It prefers moist habitats.Q. robur var.praecox, which was referred to in an experiment as a typical form, may be found all over the artificial population ofQ. robur in the Botanical Garden.Only two samples have been selected and described from the population in conformity with the phenological cycle ofQ. robur var.tardiflora. The first (N 1) was planted in 1888 and the second (N 2) in 1975.The first sample ofQ. robur var.tardiflora is the most interesting.Q. robur var.tardiflora Czern. (N 1) has a high decorative quality and exceeds other samples in duration of ontogenesis.In contrast to the specimens ofQ. robur var.praecox, planted in similar conditions in the Botanical Garden in 1930, it has no signs of ageing.It differs fromQ. robur var.tardiflora in that it sheds leaves at the time characteristic for deciduous species that significantly enhances its decorative properties.This sample currently has a height of 26 meters and trunk diameter of 108 centimeters.The phenological characteristics ofQ. robur var.tardiflora Czern. andQ. robur var.praecox Czern were showed in Tables 1 and 2.Table 1
Time scale of phenological phases ofQ. roburvarieties.
Phenological phases
Calendar date
Q. r.var. praecox
Q. r.var. tardiflora
Bud
14.IV ± 2.1
21.IV ± 3.4
Beginning of shoot growth
15.IV ± 2.3
24.IV ± 2.7
Leafing
18.IV ± 2.1
1.V ± 2.2
Efflorescence
24.IV ± 2.7
5.V ± 2.7
Full leafy
28.IV ± 2.1
9.V ± 2.1
End of flowering
2.V ± 2.7
7.V ± 2.2
End of shoot growth
20.V ± 4.5
29.V ± 8.2
Secondary shoot growth
15.VI ± 8.2
30.VI ± 14.6
Full maturation of shoots
24.VII ± 6.5
7.VIII ± 8.2
Mass staining
5.X ± 3.4
13.X ± 8.5
Massive maturation of seeds
4.IX ± 5.7
16.IX ± 2.6
Massive fruit abscission
13.IX ± 8.9
21.IX ± 6.4
Massive defoliation
12.X ± 1.3
28.X ± 4.1Table 2
Phenological cycle ofQ. robur varieties in 2014.
Phenological phases
Calendar date
Q. r.var.  praecox N 1
Q. r.var. tardifloraN 1
Q. r.var. tardifloraN 2
Bud
10.IV
24.IV
21.IV
Beginning of shoot growth
14.IV
26.IV
25.IV
Leafing
14.IV
26.IV
25.IV
Efflorescence
20.IV
26.IV
25.IV
Full leafy
7.V
12.V
12.V
End of flowering
30.IV
3.V
2.V
End of shoot growth
15.V
18.V
18.V
Secondary shoot growth
26.V
—
28.V
Full shoot maturation
10.VII
—
10.VII
Mass staining
15.X
15.X
15.X
Massive maturation of seeds
4.IX
7.IX
10.IX
Massive fruit abscission
10.IX
14.IX
14.IX
Massive defoliation
27.X
30.X
Do not fall downThe annual time scale of phenological cycle is shown in Table2.Samples ofQ. robur var.praecox were selected from three different generations of specimens (see Table 2): N 1 and N 2 planted in 1930, N 3 in 1963, and N 4 and N 5 in 1975.Several authors have noted that these varieties have significant differences in the environmental stress resistance, growth, and development [17–23].Q. robur var.tardiflora is a promising and prospective variety, which, however, is already widely used for the artificial formation of green areas in European countries [24]. From such a point of view, this variety has been shown as the most promising that was the result of studies conducted during last few years at the Botanical Garden of the Southern Federal University [13].Obviously, a clear genetic identificationQ. robur var.tardiflora will allow identifying its other, closely allied varieties, as well as identifying the individual, most genetically valuable specimens for the further propagation viain vitro culture and the subsequent bulk producing of planting material.A number of works provide the detailed information on the application of ISSR- and SSR-markers for the study of genetic divergence in the cortical oaks [25, 26] and assessment of phylogenetic relationships [27], as well as on the use of intermicrosatellite DNA markers for genotyping of the familyFagaceae [2, 24, 27–29].The aim of this work is to study the genetic affinity ofQ. robur var.tardiflora Czern. andQ. robur var.praecox Czern. using ISSR-markers, as well as the applicability of different ISSR-markers for genetic analysis of kinship relationships within the genusQuercus.


## 2. Materials and Methods

### 2.1. Plant Material

Samples ofQ. robur var.praecox were selected from three different generations of specimens (see Table 2): N 1 and N 2 planted in 1930; N 3 in 1963; and N 4 and N 5 in 1975. Samples ofQ. robur var.tardiflora were selected from three different generations of specimens. N 1 was planted in 1888 and N 2 in 1975.In addition, in order to predetermine the position of the selected varieties ofQ. robur in the phylogenetic tree of the genus, the following samples were added: one sample ofQ. robur var. “fastigiata” obtained by seeds and two samples ofQ. petraea (Matt.) Liebl. (Russia, the Republic of Adygea) which is a species phylogenetically closest toQ. robur. Furthermore, one sample ofQ. libani Oliv.,Q. macrocarpa Michx, andQ. rubra L., respectively, was taken in the experiment as phylogenetically distant forms (Table 3).Table 3
The total number of samples ofQuercus L.
Species
Variety
Number of samples
Quercus robur
Quercus roburvar. praecox
5
Quercus robur
Quercus roburvar. tardiflora
2
Quercus robur
Q. roburvar. “fastigiata”
1
Quercus libani
—
1
Quercus macrocarpa
—
1
Quercus rubra
—
1
Quercus petraea
—
1


### 2.2. DNA Extraction

Plant samples were obtained in a period from October 2014 to January 2015. Extraction DNA was performed from dormant oak buds freed from bud scales using a Sorb-A-GMO reagent kit (Sintol, Russia).The intersimple sequence repeat- (ISSR-) method was performed for the analysis of the isolated DNA using six oligonucleotide primers (two lines of three primers) with different annealing temperatures (Table4) from “100/9 University British Columbia (UBC)” kit (University of Colombia, USA) [25, 27].Table 4
ISSR primer sequences.
Primer name
Primer sequence
Annealing temperature (°C)
UBC 811
(GA)8C
53
UBC 835
(AG)8YC
52
UBC 841
(GA)8YC
52
UBC 857
(AC)8YG
52
UBC 878
(GGAT)4
53
UBC 880
(GGAG)4
53
Y is any pyrimidine.


### 2.3. ISSR Amplification and Electrophoresis

The PCR mix was prepared calculating the following quantities of reagents per sample: H2O (DD), 15.8 μL; 10xdNTP, 2.5 μL (25 mM); 10x PCR buffer, 2.5 μL; Mg, 2.5 μL (25 mM); mutant Taq-polymerase, 0.2 μL (5 unit/μL); DNA sample, 1 μL and 0.5 μL of primer (10 OU/μL). The total volume of the mixture was 25 μL.Amplification was performed with a Thermal Cycler T100 (BioRad). Amplification protocol is as follows: 1, 94°C, 1.00 min; 2, 94°C, 0.30 s; 3, 52°C/53°C, 0.45 s; 4, 72°C, 2.00 min; 5, 35 cycles from the second point; 6, 72°C, 5.00 min; 7, storage at 12°C.Separation of DNA fragments was performed by electrophoresis on a 2% agarose gel using TBE-buffer (Tris, boric acid, EDTA), at voltage of 100 V and duration 1.5 hours. DNA was stained with SYBR Green I.DNA staining dye produced SYBR Green I of ratio 1μL of dye 5 μL of DNA. Shoot made in GelDoc XR+ system (BioRad) software version 4.1 ImageLab.Length marker of 100–1500 bp DNA fragments was added to 7μL per well.


### 2.4. Statistical Analysis

Сomputer processing of the derived electropherograms was performed using the program PyElph 1.4, followed by its presentation in the form of a matrix of binary data. Being dominant markers, the ISSRs were scored as presence (1) or absence (0) in order to construct binary matrices for statistical analysis. Only reproducible fragments (in repeated experiments) were considered, whereas the variation in intensity was ignored. Computer analysis of matrices was conducted using MS Excel. Hierarchical cluster analysis of the data was performed by Neighbor-Joining (NJ) and Unweighted Pair Group Average (UPGMA) methods [30], using programs TREECON (version 1.3b) and PyElph 1.4 [31].


## 2.1. Plant Material

Samples ofQ. robur var.praecox were selected from three different generations of specimens (see Table 2): N 1 and N 2 planted in 1930; N 3 in 1963; and N 4 and N 5 in 1975. Samples ofQ. robur var.tardiflora were selected from three different generations of specimens. N 1 was planted in 1888 and N 2 in 1975.In addition, in order to predetermine the position of the selected varieties ofQ. robur in the phylogenetic tree of the genus, the following samples were added: one sample ofQ. robur var. “fastigiata” obtained by seeds and two samples ofQ. petraea (Matt.) Liebl. (Russia, the Republic of Adygea) which is a species phylogenetically closest toQ. robur. Furthermore, one sample ofQ. libani Oliv.,Q. macrocarpa Michx, andQ. rubra L., respectively, was taken in the experiment as phylogenetically distant forms (Table 3).Table 3
The total number of samples ofQuercus L.
Species
Variety
Number of samples
Quercus robur
Quercus roburvar. praecox
5
Quercus robur
Quercus roburvar. tardiflora
2
Quercus robur
Q. roburvar. “fastigiata”
1
Quercus libani
—
1
Quercus macrocarpa
—
1
Quercus rubra
—
1
Quercus petraea
—
1


## 2.2. DNA Extraction

Plant samples were obtained in a period from October 2014 to January 2015. Extraction DNA was performed from dormant oak buds freed from bud scales using a Sorb-A-GMO reagent kit (Sintol, Russia).The intersimple sequence repeat- (ISSR-) method was performed for the analysis of the isolated DNA using six oligonucleotide primers (two lines of three primers) with different annealing temperatures (Table4) from “100/9 University British Columbia (UBC)” kit (University of Colombia, USA) [25, 27].Table 4
ISSR primer sequences.
Primer name
Primer sequence
Annealing temperature (°C)
UBC 811
(GA)8C
53
UBC 835
(AG)8YC
52
UBC 841
(GA)8YC
52
UBC 857
(AC)8YG
52
UBC 878
(GGAT)4
53
UBC 880
(GGAG)4
53
Y is any pyrimidine.


## 2.3. ISSR Amplification and Electrophoresis

The PCR mix was prepared calculating the following quantities of reagents per sample: H2O (DD), 15.8 μL; 10xdNTP, 2.5 μL (25 mM); 10x PCR buffer, 2.5 μL; Mg, 2.5 μL (25 mM); mutant Taq-polymerase, 0.2 μL (5 unit/μL); DNA sample, 1 μL and 0.5 μL of primer (10 OU/μL). The total volume of the mixture was 25 μL.Amplification was performed with a Thermal Cycler T100 (BioRad). Amplification protocol is as follows: 1, 94°C, 1.00 min; 2, 94°C, 0.30 s; 3, 52°C/53°C, 0.45 s; 4, 72°C, 2.00 min; 5, 35 cycles from the second point; 6, 72°C, 5.00 min; 7, storage at 12°C.Separation of DNA fragments was performed by electrophoresis on a 2% agarose gel using TBE-buffer (Tris, boric acid, EDTA), at voltage of 100 V and duration 1.5 hours. DNA was stained with SYBR Green I.DNA staining dye produced SYBR Green I of ratio 1μL of dye 5 μL of DNA. Shoot made in GelDoc XR+ system (BioRad) software version 4.1 ImageLab.Length marker of 100–1500 bp DNA fragments was added to 7μL per well.


## 2.4. Statistical Analysis

Сomputer processing of the derived electropherograms was performed using the program PyElph 1.4, followed by its presentation in the form of a matrix of binary data. Being dominant markers, the ISSRs were scored as presence (1) or absence (0) in order to construct binary matrices for statistical analysis. Only reproducible fragments (in repeated experiments) were considered, whereas the variation in intensity was ignored. Computer analysis of matrices was conducted using MS Excel. Hierarchical cluster analysis of the data was performed by Neighbor-Joining (NJ) and Unweighted Pair Group Average (UPGMA) methods [30], using programs TREECON (version 1.3b) and PyElph 1.4 [31].


## 3. Results and Discussion

In our work, we used six ISSR primers to test genetic affinity of phenological formsQ. robur. The obtained data showed that marker UBC 878 has a lower specificity as compared with all varieties ofQ. robur.There was a slight amplification of PCR product in this case that, however, did not affect the analysis of the obtained amplicon spectra (Figure1).Figure 1
PCR detection of oak trees using the primer UBC 878. 1,Q. r. var.praecox1; 2,Q. r. var.praecox 2; 3,Q. r. var.praecox3; 4,Q. r. var.praecox 4; 5,Q. r. var.praecox 5; 6,Q. r. var.tardiflora 1; 7,Q. r. var.tardiflora 2; 8,Q. r. f.fastigiata; 9,Q. macrocarpa; 10,Q. libani; 11,Q. rubra; 12,Q. petraea 1; 13,Q. petraea 2; 14,Q. petraea 3; M, the number of the base pair.Markers UBC 841 and UBC 880 showed the highest sampling specificity (Figures2 and 3).Figure 2
PCR detection of oak trees using the primer UBC 841. 1,Q. r. var.praecox 1; 2,Q. r. var.praecox 2; 3,Q. r. var.praecox 3; 4,Q. r. var.praecox 4; 5,Q. r. var.praecox 5; 6,Q. r. var.tardiflora 1; 7,Q. r. var.tardiflora 2; 8,Q. r. f.fastigiata; 9,Q. macrocarpa; 10,Q. libani; 11,Q. rubra; 12,Q. petraea 1; 13,Q. petraea 2; 14,Q. petraea 3; M, the number of the base pair.Figure 3
PCR detection of oak trees using the primer UBC 880. 1,Q. r. var.praecox 1; 2,Q. r. var.praecox 2; 3,Q. r. var.praecox 3; 4,Q. r. var.praecox 4; 5,Q. r. var.praecox 5; 6,Q. r. var.tardiflora 1; 7,Q. r. var.tardiflora 2; 8,Q. r. f.fastigiata; 9,Q. macrocarpa; 10,Q. libani; 11,Q. rubra; 12,Q. petraea 1; 13,Q. petraea 2; 14,Q. petraea 3; M, the number of the base pair.A similar result was obtained previously by López-Aljorna and coauthors in the study ofQuercus suber L. [25].When analyzing the obtained matrix it was determined that the total number of the amplicons did not exceed 12 (UBC 835) and was not less than 2 (UBC 857). The average number of amplified fragments was 7 of five primers. The quantity of monomorphic (unique) fragments ranged from 5 (UBC 857) to 10 (UBC 880). The average percentage of polymorphism for primer UBC 811 was 93.6%; for the UBC 835 88.8%; for UBC 841 85.9%; for UBC 857 54.0%; for UBC 880 91.6% (Table5).Table 5
Analysis of information content of the primers.
Name of primers
UBC 811
UBC 835
UBC 841
UBC 857
UBC 880
Maximum number of amplicons
10
12
11
8
11
Minimum number of amplicons
6
3
3
2
5
Monomorphic fragments (unique), total number
7
8
9
5
10
Average percentage of polymorphism of ISSR
93.6
88.8
85.9
54.0
91.6The obtained results allowed constructing the dendrogram of a genetic similarity of the material (Figures4 and 5).Figure 4
Dendrogram representing the genetic affinity ofQuercus built by UPGMA on the basis of the electropherogram of primer 841.Figure 5
Dendrogram representing the genetic affinity ofQuercus built by NJ on the basis of the electropherogram of primer 841.The obtained dendrogram was compared to the taxonomic position of the studied species within the genus. Intrageneric taxonomy of these species is shown in Figure6 [32, 33].Figure 6
Dendrogram of intrageneric taxonomy of the genus oak (Quercus).Dendrograms obtained by different methods (Figures4 and 5) were matched with an intrageneric taxonomy of the studied species (Figure 6). In result, the preference was given to the Neighbor-Joining (NJ) method.It should be noted here that both methods, NJ and UPGMA, allow clearly identifying clade of«
Q. r. var. praecox
» and «
Q. petraea
». Evidently, both these methods will be effective in analyzing the polymorphism in collection of samples fromQ. robur andQ. petraea, two closely related species.The dendrogram of Figure5 showed three principal groups:(i)
Group 1 withQ. libani Oliv. andQ. rubra L. samples. Both species are most taxonomically distant fromQ. robur.
(ii)
Group 2«
Q. r. var. praecox
», which includes all samples of the variety, as well as sample N 2 (Q. r. var.tardiflora) that is logical as far as this sample belongs to the same seed generation as that ofQ. r. var.praecox N 4 and N 5 (all samples of these oaks have a common origin).
(iii)
Group 3«
Q. petraea
», which also includes samples ofQ. robur “fastigiata” andQ. macrocarpa that does not contradict to the genera phylogeny.
(iv)
Q. r. var.tardiflora N 1 out of clades.It is significant that in result of dendrogram construction by UPGMA method (Figure4) this sample appeared to be in a more kinship with North American species,Q. rubra andQ. macrocarpa (one clade), than withQ. r. var.praecox.


## 4. Conclusions

Analyzing the obtained matrix it was determined that the maximum number of amplicons is 12 (UBC 835) and minimum number of amplicons is 2 (UBC 857). The average percentage of polymorphism for primer UBC 811 was 93.6%; for UBC 835 88.8%; for UBC 841 85.9%; for UBC 857 54.0%; for UBC 880 91.6%.It was showed by analyzing the obtained dendrogram (Figure5) thatQ. libani andQ. rubra samples form Group 1. It is not contrary to the intrageneric taxonomy of the genus oak. Group 2 includes all samples of the varietyQ. robur var.praecox and sample of the varietyQ. robur var.tardiflora N 2. All samples of these oaks have a common origin. The sampleQ. robur var.tardiflora N 2 belongs to the same seed generation as that ofQ. robur var.praecox N 4 andQ. robur var.praecox N 5. Group 3 includes all samples ofQ. petraea and also includes the samples ofQ. robur “fastigiata” andQ. macrocarpa that does not contradict to the genera phylogeny (Figure 6). Sample ofQ. robur “fastigiata” belongs to the seed generation ofQ. robur var.praecox. SampleQ. r. var.tardiflora N 1 is out of clades.Analysis of polymorphism of intersimple sequence repeats can be used successfully for DNA fingerprinting of varieties ofQ. robur. The revealed polymorphism of two samples ofQ. robur. var.tardiflora indicated that similar phenological forms can be based on genetically far-distinct populations, lines, generations, and forms within theQ. robur species. Therefore the phenological features ofQ. robur should not always be considered to be related with its valuable biological and economical characteristics, as it has been done in a number of studies [17–23]. It was shown also that the sample N 1 ofQ. r. var.tardiflora from the artificial population of the Botanical Garden of SFU has unique characteristics of its phenology, living longevity, and characteristics obtained by ISSR-analysis.



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