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Nuclear DNA content and phenotypic traits of the Prunus rootstocks from Poland’s gene resourcesPrunus genties poskiepių iš Lenkijos genetinių išteklių branduolio DNR kiekis ir fenotipiniai požymiai

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The ploidy level can be useful in evaluating reproductive and somatic compatibility, an important parameter in scion and rootstock breeding programs. Evaluation of nuclear DNA content was performed in order to determine ploidy level of 31 rootstocks of genus Prunus for plum, sour cherry and sweet cherry. In the first stage of the research, the conditions of flow cytometry analysis with application of propidium iodide for DNA staining were optimized. Repeatable results and relatively good-quality histograms were obtained using Partec extraction buffer with 1% PVP (polyvinylpyrrolidone) addition, with incubation time from 50 to 90 min. The best internal standard for flow cytometry analysis proved to be tomato (Solanum lycopersicum L.) 2C = 1.96 pg for rootstocks with a nuclear DNA content from 0.55 to 1.64 pg, and soybean (Glycine max (L.) Merr.) 2.50 pg for rootstocks with a larger nuclear DNA contents from 1.8 to 2.3 pg. In five plum rootstocks, 2C values ranged from 0.61 to 0.67 pg indicating their diploid chromosome number. One rootstock was identified as tetraploid (2C = 1.34 pg), and four rootstocks as hexaploids owing to their DNA contents from 2.07 to 2.23 pg. One rootstock was considered a pentaploid due to its 2C value of 1.64 pg which was approximately two and a half times more than in diploid Prunus sp. In nine cherry rootstocks, 2C DNA values ranged from 0.74 to 0.86 pg indicating their diploid chromosome number. Nine cherry rootstocks were identified as triploids with their 2C DNA contents from 1.03 to 1.24 pg. Two cherry rootstocks were considered as tetraploids having 2C DNA of 1.39 pg. The ploidy level of cherry and plum rootstocks was evaluated in relation to morphological and agronomic traits. The tendency to increase the size of stomata and leaves along with an increase in the ploidy level was observed within Prunus rootstock genotypes; however, the correlations between these traits were not so evident. Therefore, stomata and leaf size cannot be considered as a morphological marker indicating ploidy level. The evaluated nuclear DNA contents / ploidy levels as well as stomata and leaf size are the additional descriptors of Prunus rootstocks that can be useful for identifying genotypes. Key words: flow cytometry, hexaploids, pentaploids, ploidy level, plum, cherry rootstocks, tetraploids, triploids.
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ISSN 1392-3196 Zemdirbyste-Agriculture Vol. 107, No. 1 (2020) 71
ISSN 1392-3196 / e-ISSN 2335-8947
Zemdirbyste-Agriculture, vol. 107, No. 1 (2020), p. 71–78
DOI 10.13080/z-a.2020.107.010
Nuclear DNA content and phenotypic traits of the
Prunus rootstocks from Poland’s gene resources
Małgorzata PODWYSZYŃSKA, Mirosław SITAREK,
Agnieszka MARASEK-CIOŁAKOWSKA, Urszula KOWALSKA
Research Institute of Horticulture
Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland
E-mail: malgorzata.podwyszynska@inhort.pl
Abstract
The ploidy level can be useful in evaluating reproductive and somatic compatibility, an important parameter in scion
and rootstock breeding programs. Evaluation of nuclear DNA content was performed in order to determine ploidy
level of 31 rootstocks of genus Prunus for plum, sour cherry and sweet cherry. In the rst stage of the research,
the conditions of ow cytometry analysis with application of propidium iodide for DNA staining were optimized.
Repeatable results and relatively good-quality histograms were obtained using Partec extraction buer with 1%
PVP (polyvinylpyrrolidone) addition, with incubation time from 50 to 90 min. The best internal standard for ow
cytometry analysis proved to be tomato (Solanum lycopersicum L.) 2C = 1.96 pg for rootstocks with a nuclear DNA
content from 0.55 to 1.64 pg, and soybean (Glycine max (L.) Merr.) 2.50 pg for rootstocks with a larger nuclear DNA
contents from 1.8 to 2.3 pg. In ve plum rootstocks, 2C values ranged from 0.61 to 0.67 pg indicating their diploid
chromosome number. One rootstock was identied as tetraploid (2C = 1.34 pg), and four rootstocks as hexaploids
owing to their DNA contents from 2.07 to 2.23 pg. One rootstock was considered a pentaploid due to its 2C value of
1.64 pg which was approximately two and a half times more than in diploid Prunus sp. In nine cherry rootstocks, 2C
DNA values ranged from 0.74 to 0.86 pg indicating their diploid chromosome number. Nine cherry rootstocks were
identied as triploids with their 2C DNA contents from 1.03 to 1.24 pg. Two cherry rootstocks were considered as
tetraploids having 2C DNA of 1.39 pg. The ploidy level of cherry and plum rootstocks was evaluated in relation to
morphological and agronomic traits. The tendency to increase the size of stomata and leaves along with an increase
in the ploidy level was observed within Prunus rootstock genotypes; however, the correlations between these traits
were not so evident. Therefore, stomata and leaf size cannot be considered as a morphological marker indicating
ploidy level. The evaluated nuclear DNA contents / ploidy levels as well as stomata and leaf size are the additional
descriptors of Prunus rootstocks that can be useful for identifying genotypes.
Key words: ow cytometry, hexaploids, pentaploids, ploidy level, plum, cherry rootstocks, tetraploids, triploids.
Introduction
It is well known that within one genus, the
species and even cultivars of the same species dier
in the content of nuclear DNA which translates into a
ploidy level. It is believed that nuclear DNA content is
a specic feature and its assessment by ow cytometry
can be helpful in dierentiating taxa. For many Prunus
genotypes used as rootstocks for plum and cherry trees
collected in gene resources of the Research Institute of
Horticulture in Skierniewice, Poland, the nuclear DNA
content is unknown. There are over 400 to 430 Prunus
species, of which approximately one hundred is important
for horticulture and breeding (Das et al., 2011). The
species are grouped into four subgenera: Prunus, Cerasus,
Amygdalus and Emplectocladus (Angiosperm Phylogeny
Group, 2009). The main Prunus crops are peach, plum,
apricot, cherry, almond and their various hybrids, e.g.,
plumcot. The Prunus taxa are mainly cultivated as fruit
crops and several species are grown as ornamental plants.
Prunus crops like many other fruit crops are propagated
by grafting and grown on rootstocks. Grafting onto
rootstocks enables the grower to determine the tree’s
eventual size and improve environmental adaptability,
such as tolerance to wet / dry soil conditions, acidity /
alkalinity of soil or even hot/cold air temperature and
resistance to various pests and diseases (Das et al., 2011).
The basic chromosome number in Prunus is x
= 8 (Das et al., 2011). Somatic chromosome number of
various Prunus species varies from diploid to hexaploid
(Table 1). Most of the species are diploid (2n = 2x= 16),
e.g., P. dulcis, P. armeniaca, P. avium, P. canescens,
P. cerasifera, P. lannesiana, P. mahaleb, P. munsoniana,
P. persica, P. pumila and P. tomentosa. Some of these
species (P. avium, P. cerasifera and P. spinosa) occur
in dierent polyploid forms. There are also several
tetraploids (2n = 4x = 32) such as P. cerasus, P. fruticosa
and P. maackii, and hexaploids (2n = 6x = 48) such as
Please use the following format when citing the article:
Podwyszyńska M., Sitarek M., Marasek-Ciołakowska A., Kowalska U. 2020. Nuclear DNA content and phenotypic traits of the
Prunus rootstocks from Poland’s gene resources. Zemdirbyste-Agriculture, 107 (1): 71–78. DOI 10.13080/z-a.2020.107.0010
72 Nuclear DNA content and phenotypic traits of the Prunus rootstocks from Poland’s gene resources
P. domestica and P. domestica var. insititia. Some of these
Prunus species have been used for selecting improved
rootstock genotypes or served as parental genotypes for
creation of hybrid rootstock cultivars combining desired
traits (Das et al., 2011).
At present, many rootstocks with dierent
vigour and behaviour are available for plum, sour cherry
and sweet cherry trees. The wide genetic diversity and
adaptability of rootstocks allow dierent vegetative-
productive behaviours in an orchard. Eects of rootstock
on the tree growth, tree productivity and fruit quality
of dierent stone fruit crops was reported in numerous
publications (Murri et al., 2013; Sitarek, 2017; Usenik
et al., 2017).
The natural polyploid formation within the
genus Prunus results from both somatic chromosome
doubling and union of unreduced gametes with the latter
considered most important polyploidisation mechanism
(Ramsey, Schemske, 1998; Wang et al., 2018).
Since the nuclear DNA content and ploidy
level of many Prunus rootstocks are unknown, the
evaluation of these traits was performed in order to
provide information for germplasm conservation and
breeding. In this study, 2C DNA value (somatic nuclear
DNA content) / ploidy level of Prunus rootstocks was
evaluated in relation to stomata and leaf size as well
as agronomic trait such as growth vigour. If the size of
stomata and leaves and vigour were positively correlated
with the level of ploidy, these features could be used as
easy-to-use ploidy markers. Such knowledge would also
be helpful in selecting parents for crosses in order to
obtain hybrids with specic traits, e.g., dwarng or semi
dwarng eect on tree vigour.
Materials and methods
Plant material. Studies were carried out in
2017–2018 in the Research Institute of Horticulture,
Skierniewice, Poland. Thirty one Prunus genotypes, 11
rootstocks for plum trees and 20 rootstocks for cherry and
sweet cherry, gathered in genetic resources of the Research
Institute of Horticulture were used for the research.
Evaluation of nuclear DNA content. In the rst
stage of the research, the conditions of ow cytometry
(FCM) analysis with application of propidium iodide (PI)
for DNA staining FCM were optimized: type of buer
for nuclei extraction and incubation time (from 15 to
120 min) as well as selection of internal standards (plant
genotypes with known nuclear DNA content). Analysis
of genome size was done using FCM. Samples were
taken in mid-July from six leaves collected randomly
from one plant of each genotype. Leaf tissue (0.5−1 cm2)
was chopped together with a piece (0.5–1 cm2) of plant
internal standard in a Petri dish in 1.5 mL nuclei isolation
Galbraith’s buer (Galbraith et al., 1983) or Partec
buer (Sysmex Partec GmbH, Germany) with slight
modication according to Śliwińska (2008) to which
propidium iodide (50 μg mL-1), RNase (50 μg mL-1)
and 1% PVP (polyvinylpyrrolidone) were added. As the
internal standards, the young leaves of tomato (Solanum
lycopersicum L.) ‘Stupicke’ were used (2C DNA = 1.96
pg (Doležel et al., 1992), soybean (Glycine max L. Merr.)
‘Polanka’ 2C DNA = 2.50 pg (Doležel et al., 1994) or
maize (Zea mays L.) CE-777 2C DNA = 5.43 pg (Lysak,
Doležel, 1998) were used. The seeds of reference plants
were kindly provided by Institute of Experimental
Botany, Czech Republic. Optimization of FCM analysis
was performed with reference plants of Prunus genotypes
(external standards) with the known chromosome
numbers: diploid P. cerasifera var. divaricata Led.
‘Anna’ (myrobalan) (2n = 2x = 16), triploid sweet cherry
rootstock GiSelA 3 (P. cerasus ‘Schattenmorelle’ ×
P. canescens) (2n = 3x = 24) and haxaploid P. domestica
‘Eruni’ (2n = 6x = 48).
After adding 1.5 mL of the isolation buer, the
samples were ltered through a 30 μm lter and incubated
for 50–60 min in room temperature. The uorescence of
the nuclei was measured using analyser CyFlow Ploidy
(Sysmex Partec) with an Nd-YAG green laser at 532 nm.
Data were analysed by means of software CyView
(Sysmex Partec). The 2C DNA content of a sample was
calculated as the sample peak mean divided by the mean
of the standard plant peak and multiplied by the amount
of DNA of the standard plant. Samples with at least 5000
nuclei were measured for six leaves of each plant with two
runs from each nuclei isolation extract. The ploidy level
of rootstocks was evaluated in relation to stomata and leaf
size and vigour. The latter feature was classied as weak,
intermediate vigour, vigorous or very vigorous, depending
on the impact of the rootstock on the tree growth in the
orchard, described according to the literature (Murri et al.,
2013; Sitarek, 2017; Usenik et al., 2017).
In Table 1, the nuclear 2C DNA contents and/or
ploidy levels according to dierent authors are presented
for the Prunus species which are present in pedigree of
the rootstocks for plums and cherries evaluated in our
study. This information was helpful in indicating ploidy
levels of Prunus genotypes based on FCM analysis and
was also used in discussion.
Phenotype evaluation. Leaves were collected in
late May and June. Leaf area was measured for randomly
collected 30 leaves, with an optical planimeter Area
Meter AM 350 (ADC Bioscientic, UK). The leaves
for measurement of stomata length were collected in
mid-June. The abaxial epidermis was isolated from the
middle part of the leaves with a transparent adhesive tape
and stained with toluidine blue and mounted on slides
for microscopic observations according to the procedure
of Dyki and Habdas (1996). The stomata measurements
were determined for ve leaves (×20 stomata) of each
genotype using a Nikon Eclipse 80i microscope with
the program NIS-Elements BR 2.30 (Nikon Instruments
Europe BV, The Netherlands) at 400× magnication.
Statistical analysis. Data of all parameters were
analysed with analysis of variance (ANOVA)-nested
design. All calculations were done with the package
Statistica, version 10 (StatSoft Inc., USA). The means
were compared by Tukey’s test at p = 0.05.
Results
Repeatable results and relatively good-quality
histograms were obtained using Partec extraction buer
with 1% PVP addition, with incubation time from 50 to
130 min. The best internal standard for FCM analysis of
the rootstocks for plums and cherries with a nuclear DNA
content from about 0.7 to 1.4 pg proved to be Solanum
lycopersicum (2C = 1.96 pg), of which peaks 2C and 4C
did not coincide with the peaks of the genotypes tested
(Fig. 1). However, peaks of S. lycopersicum overlapped
partially with the peaks of Prunus genotypes with larger
genomes tested. For rootstock genotypes with a nuclear
DNA contents from 1.8 to 2.3 pg, Glycine max (2.50 pg)
was selected as the internal standard. In the second stage,
the optimized method and well-chosen internal standards
allowed to assess with high precision the nuclear DNA
content / ploidy level of 31 Prunus genotypes of the
rootstocks for plums and cherries.
ISSN 1392-3196 Zemdirbyste-Agriculture Vol. 107, No. 1 (2020) 73
Table 1. Ploidy levels and nuclear DNA contents of reference Prunus genotypes, which are the parental genotypes for
rootstock for plum, cherry and sweet cherry trees
Genotype Ploidy
level
References for
ploidy level
Nuclear 2C DNA
content (pg),
references
References for
nuclear DNA
content*
P. amygdalus Batsch, syn. Prunus dulcis,
almond B, D, Pr 0.66 B
P. armeniaca L., apricot Dic, D, Pr 0.61, 0.60 Ar, Dic, Di
P. avium (L.) L., sweet cherry 2×, 3×, 4× Dic, D, Pr, W 0.70, 0.67 Ar, Dic, Di
P. canescens Bois, hoary cherry D, Pr
P. cerasifera, cherry plum 2×, 2× + 1, 3×, 4×, 6× D
P. cerasus L., sour cherry Dic, D, Pr 1.24, 1.36–1.42 Ar, Dic, Di
P. domestica L., plum Dic, D, Z 1.83, 1.83, 1.86, 2.10 Ar, Dic, Di, Z
P. domestica ssp. insititia L., damson 3×, 6× D, BT, Pr, Z 2.14, 1.99–2.13 Z, BT
P. fruticosa Pall., European dwarf cherry D, Pr
P. incisa Thunb., Fuji cherry Pr
P. maackii, manchurian cherry P, Pr 0.96–0.97 P
P. lannesiana (Carriere) E.H. Wilson,
syn. P. serrulata var. lannesiana,
Japanese cherry
2×, 3× Pr, W
P. mahaleb L., mahaleb cherry D, Pr
P. munsoniana, W.Wight & Hedrick,
wild-goose plum E, Pr
P. persica (L.) Batsch,
peach
3× (polyploidistion)
4× (polyploidisation)
Dic, D, Pr
0.54–0.55
0.58–0.64
0.98
1.23
Ar, Dic, Di
B
B
B
P. psudocerasus Lindl., Cambridge cherry 4×, 5×, 6× G, W
P. pumila L., sand cherry Pr
P. salicina Lindl., Japanese plum 2×, 4× D, BT, Pr, W 0.44–0.97 BT
P. spinosa L., blackthorn 2×, 3×, 4×, 5×, 6× D, O, Pr, Z 1.40 Z
P. tomentosa Thunb., nanking cherry D, W 0.57 B
GiSelA V
* – the letters in columns indicate references for ploidy levels or nuclear DNA contents: Ar – Arumuganathan, Earle (1991), B –
Baird et al. (1994), BT – Ben Tamarzizt et al. (2015), D – Das et al. (2011), Dic – Dickson et al. (1992), Di – Dirlewanger et al.
(2009), G – Gu et al. (2014), O – Okie, Weinberger (1996), P – Pooler et al. (2012), Pr Prunus Vulnerability Statement (2017),
V – Vujović et al. (2012), W – Wang et al. (2018), Z – Žabka et al. (2018).
2× – diploid plum rootstock VVA-1, 3× – triploid cherry rootstock GiSelA 6, 4× – tetraploid cherry rootstock LC-52, 5× – pentaploid
plum rootstock ‘Mariana P 8.13’, 6× – hexaploid plum rootstock ‘Eruni’
Figure 1. Histograms of nuclear DNA / ploidy level estimation using ow cytometry (FCM) with internal standards:
Solanum lycopersicum (2C DNA = 1.96 pg) for the taxa of nuclear DNA content below 1.70 pg and Glycine max
(2C DNA = 2.50 pg) for hexaploid genomes (2.07–2.24 pg)
74
For the reference Prunus genotypes of the known
chromosome number, 2C DNA values were 0.68 pg for
diploid P. cerasifera var. divaricata Led. ‘Anna’, 1.18
pg for triploid GiSelA 3 (P. cerasus ‘Schattenmorelle’
× P. canescens) and 2.16 pg for hexaploid P. domestica
‘Eruni’ (Table 2). In four plum rootstock genotypes
(‘Ferciana Ishtara’, VVA-1, ‘Druzba’ and GF 667),
2C values ranged from 0.61 to 0.67 pg indicating their
diploid chromosome number. One rootstock ‘Fereley
Jaspi’ was identied as tetraploid (2C = 1.34 pg) and
three rootstocks as hexaploids (‘St. Julien A’, ‘Pixy’ and
GF 655/2) owing to their DNA contents from 2.07 to
2.23 pg. The rootstock ‘Mariana P 8.13’ was considered
pentaploid due to its 2C value of 1.64 pg, which was
approximately two and a half times more than in diploid
Prunus sp. (2C DNA = 0.66 pg).
In nine rootstocks for cherry trees: F 12/1,
PiKu 1, Piku 3, INRA SL 64, ‘Maxma Delbard 14
Brokforest’, ‘Ferci SL 405’, GM 79, LC-13 and L-2, 2C
DNA values ranged from 0.55 to 0.86 pg indicating their
diploid chromosome number (Table 2). Nine rootstocks
were identied as triploids: ‘Colt’, GiSelA 3, GiSelA 5,
GiSelA 6, P-HL A, P-HL C, PiKu 4, VSL-1 and VSL-2,
with their 2C DNA contents from 1.03 to 1.24 pg. Two
rootstocks were considered tetraploid: WC-13 and LC-
52, having 2C DNA of 1.39 pg.
Genotypes dier signicantly in the leaf size and
stomata length (Tables 2–3, Figs 2–3). Within rootstocks
Table 2. Nuclear DNA contents and ploidy levels of Prunus genotype rootstocks for plum, sour cherry and sweet
cherry trees in relation to the leaf area, stomata length and vigour level
Genotype Parentage Nuclear
2C DNA
content pg
Ploidy
level
Leaf area
cm2
Stomata length
µm
Vigour
level
Rootstocks for plum trees
VVA-1 (Krymsk 1) P. tomentosa ×
P. cerasifera 0.61 ± 0.003 16.3 d-c 24.1 ± 2.38 d intermediate vigour
Druzba P. pumila × P. armeniaca 0.65 ± 0.006 15.2 d-g 22.1 ± 2.39 e very vigorous
Ferciana Ishtara P. salicina ‘Belsiana’ ×
(P. cerasifera × P. persica)0.67 ± 0.013 20.9 cd 21.4 ± 2.98 e vigorous
GF 677 (P. persica × P. amygdalus) 0.67 ± 0.000 26.3 ab 30.2 ± 3.08 a vigorous
Myrobalan P. cerasifera var. divaricata 0.68 ± 0.005 11.4 g 15.9 ± 3.32 g very vigorous
Fereley Jaspi P. salicina ‘Methley’ ×
P. spinosa 1.34 ± 0.017 14.8 fg 21.3 ± 2.43 e vigorous
Mariana P 8.13 P. cerasifera × P. munsoniana 1.64 ± 0.039 20.1 d 19.6 ± 1.99 f very vigorous
Pixy P. insititia 2.07 ± 0.250 22.4 bc 26.8 ± 2.64 c weak
Eruni P. domestica 2.16 ± 0.057 30.8 a 21.7 ± 2.81 e intermediate vigour
GF 655/2 P. insititia 2.21 ± 0.029 19.9 c-e 28.3 ± 2.54 b intermediate vigour
St. Julien A P. insititia 2.23 ± 0.017 19.3 c-f 24.8 ± 2.50 d intermediate vigour
Rootstocks for sour cherry and sweet cherry trees
LC-13 P. avium × P. cerasus 0.55 ± 0.008 13.3 k 18.9 ± 2.98 kl intermediate vigour
Ferci SL 405 P. mahaleb 0.75 ± 0.013 18.9 h-k 25.7 ± 3.36 c-e vigorous
INRA SL 64 P. mahaleb 0.75 ± 0.006 19.7 h-k 25.2 ± 2.39 d-f vigorous
L-2 (Krymsk 7) P. lannesiana 0.77 ± 0.010 15.9 jk 23.7 ± 2.02 gh intermediate vigour
PiKu 3 (4.83) P. pseudocerasus ×
(P. canescens × P. incisa)0.77 ± 0.010 21.1 h-j 24.9 ± 2.45 d-g vigorous
Maxma Delbard 14
Brokforest P. avium × P. mahaleb 0.79 ± 0.006 32.2 d-f 24.1 ± 2.59 f-h vigorous
GM 79 (Camil) P. canescens 0.80 ± 0.021 33.6 de 20.1 ± 2.34 k intermediate vigour
PiKu 1 (4.20) P. avium ×
(P. canescens × P. tomentosa)0.81 ± 0.008 20.2 h-k 23.4 ± 2.31 hi weak
F 12/1 P. avium 0.86 ± 0.013 45.3 b 22.3 ± 2.07 ij very vigorous
Colt P. avium (F299/2) ×
P. pseudocerasus 1.03 ± 0.039 57.8 a 24.5 ± 2.88 e-h very vigorous
PiKu 4 (4.22) (P. canescens × P. tomentosa)
× P. avium 1.12 ± 0.022 31.6 d-f 22.2 ± 2.39 ij intermediate vigour
VSL-1 P. fruticosa × P. lannesiana 1.12 ± 0.029 22.3 g-i 25.0 ± 2.79 d-f intermediate vigour
VSL-2 (Krymsk 5) P. fruticosa × P. lannesiana 1.12 ± 0.029 15.1 jk 26.1 ± 3.16 cd vigorous
GiSelA 5 (146/2) P. cerasus ‘Schattenmorelle’
× P. canescens 1.13 ± 0.017 29.4 d-f 21.8 ± 2.45 j intermediate vigour
GiSelA 6 (148/1) P. cerasus ‘Schattenmorelle’
× P. canescens 1.14 ± 0.005 29.0 e-g 27.0 ± 3.58 bc intermediate vigour
GiSelA 3 (209/1) P. cerasus ‘Schattenmorelle’
× P. canescens 1.18 ± 0.005 21.0 h-j 26.2 ± 2.54 cd weak
P-HL A (84) P. avium × P. cerasus 1.20 ± 0.015 41.9 bc 25.7 ± 2.91 c-e intermediate vigour
P-HL C (6) (P. avium × P. cerasus) 1.24 ± 0.037 48.3 b 25.2 ± 2.52 d-f weak
LC-52 (Krymsk 6) P. cerasus ‘Lyubskaya’ ×
(P. cerasus ‘Michurin’ ×
P. maackii)
1.39 ± 0.010 25.6 fg 30.8 ± 3.02 b intermediate vigour
WC-13 P. cerasus ‘Vladimirskaya’ ×
(P. cerasus ‘Michurin’ ×
P. maackii)
1.39 ± 0.012 36.7 cd 27.8 ± 3.02 b intermediate vigour
Note. The mean separation within columns by Tukey’s test at p = 0.05; the means followed by the same letter do not dier at p = 0.05.
Nuclear DNA content and phenotypic traits of the Prunus rootstocks from Poland’s gene resources
ISSN 1392-3196 Zemdirbyste-Agriculture Vol. 107, No. 1 (2020) 75
for plums, the smallest stomata with size of 15.9 and 19.6
µm, were recorded for diploid myrobalan and pentaploid
‘Mariana P 8.13’ and the largest stomata, 28.3 and
30.2 µm, for hexaploid GF 655/2 and diploid GF 677,
respectively. The intermediate stomata lengths ranging
from 21.3 to 24.1 µm were observed for three diploid, one
tetraploid and one hexaploid plum rootstocks. Within the
plum rootstocks of the smallest leaves (11.4–16.3 cm2),
there were three diploids, including myrobalan, and one
tetraploid genotype. Among the plum rootstocks having
intermediate and larger leaves (19.3–30.8 cm2), there
were two diploids, one pentaploid and four hexaploids
with the largest leaves recorded for hexaploid ‘Eruni’.
Within cherry rootstocks, the correlation of
stomata size and ploidy level was more evident (Tables
2–3). Among 10 genotypes of the smaller stomata (18.9–
24.9 µm), there were eight diploids and two triploids
while within cherry rootstocks of larger stomata (25.0–
30.8 µm), there were two diploids, six triploids and two
tetraploids. However, the tendency in the increase of
leaf size with the increase in the ploidy level was not so
clear. The smallest leaves were observed for diploid LC-
13 (13.3 cm2) and triploid VSL-2 (15.1 cm2) while the
largest leaves 48.3 and 57.8 cm2 were found for triploids
P-HL C and ‘Colt’, respectively. In turn, tetraploids WC-
13 and LC-52 had leaves of intermediate size.
Figure 2. Stomata of plum rootstocks, from the left: diploid myrobalan (with very small and the incidental larger
stomata), hexaploid ‘Eruni’ and hexaploid GF 655/2 with large stomata (bar = 25 µm)
Figure 3. Leaves of cherry rootstocks: diploids LC-13
and ‘Maxma Delbard’ (2×), triploids VSL-1 and ‘Colt’
(3×) and tetraploids LC-53 and WC-13 (4×)
Table 3. Eect of ploidy level on leaf area and stomata length of Prunus rootstocks
Rootstocks for plum trees Rootstocks for sour cherry and sweet cherry trees
ploidy level leaf area
cm2
stomata length
µm ploidy level leaf area
cm2
stomata length
µm
18.1 bc 22.5 b 24.3 b 23.6 c
14.8 c 21.3 b 32.9 a 24.8 b
20.1 ab 19.6 c 31.2 a 29.3 a
23.1 a 25.4 a
p0.00 0.00 0.00 0.00
Note. The mean separation within columns by Tukey’s test at p = 0.05; the means followed by the same letter do not dier at p = 0.05.
In general, comparison of averages for individual
ploidy levels showed a tendency to increase the size of
stomata along with an increase in the level of ploidy
(Table 3). Such tendency was observed in rootstocks
for sweet and sour cherries. In plum rootstocks, only
hexaploids had on average signicantly larger stomata
and leaves compared to other ploidy levels. In rootstocks
for cherries, triploid and tetraploid levels had on average
signicantly larger stomata and leaves compared to
diploid level. It should be noted that pentaploids and
hexaploids were not detected among cherry rootstocks.
Discussion
The ploidy level as well as DNA content of
Prunus sp. was rst estimated using FCM analysis by
Arumuganathan and Earle (1991). These authors assessed
the nuclear DNA amount for six species of Prunus,
reporting 2C DNA values 0.54–0.70 pg for diploids,
1.24–1.42 pg for tetraploids and 1.83 pg for hexaploids.
Later, Dickson et al. (1992) evaluated 2C DNA of nine
Prunus species and cultivars, and evaluations of these
authors for particular ploidy levels were very similar.
The authors mentioned above found that the genome
size showed little variation within the diploid species of
genus Prunus with the smallest value for P. persica and
the largest for P. avium.
In our study, similar variation in 2C DNA values
of diploid taxa especially within cherry rootstocks was
observed. These values ranged from 0.55 pg for LC-13
to 0.86 pg for F 12/1. Besides, the latter was selected
from P. avium. And this species was reported to have
larger genome compared to other diploid species
(Arumuganathan, Earle, 1991; Dickson et al., 1992). Such
dierences in nuclear DNA contents between cultivars
or species possessing the same chromosome number
76
and belonging to the same genus are widely known in
other genera, e.g., Lilium (Van Tuyl, Boon, 1997), Malus
(Korban et al., 2009; Podwyszyńska et al., 2016); Tulipa
(Zonneveld, 2009) and Narcissus (Zonneveld, 2010).
The nuclear 2C DNA content is therefore considered as
one of the taxa descriptors.
Our analysis showed that F 12/1 selected
from P. avium had 2C DNA of 0.86 pg while the values
reported by other authors (Arumuganathan, Earle,
1991; Dickson et al., 1992) were lower (0.70 pg). Such
dierences between 2C values obtained for the same
species and reported by various authors resulted probably
from various FCM methods, in which dierent buers
and incubation times as well as various DNA staining
methods and internal standards were used. It was reported
that in Prunus, nuclear DNA content evaluation was
performed using the following internal standards: chicken
erythrocytes of 2C = 2.33 pg (Arumuganathan, Earle,
1991; Dickson et al., 1992; Baird et al., 1994), Glycine
max 2C = 2.50 pg (Pooler et al., 2012; Ben Tamarzizt et al.
2015; Žabka et al., 2018) and Zea mays with 2C = 5.43
pg (Jedrzejczyk, Sliwinska, 2010). In our study, Solanum
lycopersicum of 2C = 1.96 pg was considered the most
suitable internal standard for evaluation of ploidy levels
from diploid to pentaploid levels of Prunus genotypes.
Only hexaploid taxa required another internal standard
plant, namely Glycine max. Thus the peaks for 2C
Prunus hexaploids overlapped with S. lycopersicum 2C
pick, both having similar nuclear DNA contents.
In our study, ve rootstocks for plum and nine
rootstocks for cherries were identied as diploids, which
correspond to their origin from diploid parents. Among
diploid rootstocks, myrobalan was selected from diploid
P. cerasifera var. divaricata and the cherry rootstocks
such as ‘Ferci SL 405’ and INRA SL 64, L-2, GM 79
and F 12/1 were the selections of the following diploid
species: P. mahaleb, P. lannesiana, P. canescens and
P. avium, respectively. In turn, other diploid cherry
rootstocks are generally the hybrids of diploid parental
species mentioned above and some other diploids such as
P. incisa, P. pseudocerasus and P. tomentosa. Only diploid
PiKu 3 has more complicated pedigree, which comprises
two diploid and one tetraploid genotypes. In the case of
triploid cherry rootstocks, all of them are the hybrids
of diploid and tetraploid species, with one exception of
PiKu 4, which has in its pedigree three diploid species.
In the latter, one of the parent genotypes is P. avium for
which, except of diploid, also triploid and tetraploid
forms are known (Das et al., 2011). On the other hand,
PiKu 4 can be a hybrid of only diploid parents where one
of them could be a donor of unreduced gamete 2n. Such
phenomenon is widespread within Prunus genus (Wang
et al., 2018).
In the case of plum rootstocks evaluated in our
study, their ploidy levels correspond well with their origin.
All of the diploid rootstock genotypes were selected
from diploid species or are the interspecic hybrids of
diploid species. Tetraploid ‘Fereley Jaspi’ has probably
tetraploid parental forms of P. salicina and P. spinose. In
pentaploid plum rootstock ‘Mariana P 8.13’, probably
one of the parents is tetraploid or hexaploid form of
P. cerasifera since such forms are often found (Das et al.,
2011). It has been documented that autopolyploids of
P. pseudocerasus can produce during meiosis multiple
fertile gametes with complete chromosome sets, e.g., 4n,
leading to formation of pentaploids (Ramsey, Schemske,
1998). We suppose that similar phenomenon could occur
also in autopolyploid P. cerasifera, which, apart from
diploid P. munsoniana, was the second parent of the
plum rootstock P 8.13. In turn, all accessions identied
as hexaploids (four plum rootstocks) were selections of
hexaploid P. domestica or P. domestica var. insititia.
For genotypes within a species and genus, the
higher ploidy level is generally associated with increased
sizes of stomata and leaves as, e.g., in tea (Wachira,
1994), banana and plantain (Vandenhout et al., 1995) or
mulberry (Laltanmawii, Roychowdhuri, 2010). Although,
in Prunus rootstocks, comparison of averages for ploidy
levels showed a certain tendency to increase the stomata
and leaf size along with an increase in the ploidy level;
however, the correlations between these traits were not
so evident within the genus Prunus (plum and cherry
rootstocks) as compared to the high positive correlation
between nuclear DNA amount and stomatal length found
for the cultivars of the species Malus × domestica (Tatum
et al., 2005; Korban et al., 2009; Podwyszyńska et al.,
2016). This probably results from the fact that the genus
Prunus includes many various species and hybrids, for
which phenotypic and genetic variability is much higher
compared to variability of the cultivars within one species
such, e.g., Malus × domectica. Thus, in plum rootstocks,
the smallest (19.6 µm) stomata were found in pentaploid
(P 8.13) and in contrast, the largest (30.2 µm) stomata
were recorded in one of the diploid genotype (GF 677).
So stomata and leaf size cannot be considered as a
morphological marker indicating ploidy level for Prunus
rootstocks. Moreover, there is no correlation between
ploidy level and vigour of individual rootstocks.
The evaluated nuclear DNA contents / ploidy
levels as well as stomata and leaf size are the additional
descriptors of Prunus rootstock genotypes that can
be useful for identifying genotypes. In addition, it is
well known that polyploids with an odd number of
chromosome sets, e.g., triploids or pentaploids, are
low-fertile or infertile (Bharadwaj, 2015). Therefore,
the knowledge about the ploidy level of a given Prunus
genotype can be useful for planning breeding programs.
It can be helpful in the selection of parental forms for
crosses and allows predicting the reproductive abilities
of hybrids.
Conclusions
1. Genome sizes of 31 Prunus of rootstocks
for plum and cherry trees were evaluated using ow
cytometry (FCM) analysis. In plum rootstocks, ve
diploid, one tetraploid, one pentaploid and four hexaploid
genotypes were detected while in rootstocks for cherry
trees, nine diploid, nine triploid and two tetraploid
genotypes were identied.
2. The tendency to increase the size of stomata
and leaves along with an increase in the ploidy level
is observed within Prunus rootstocks for cherry trees.
In plum rootstocks, only hexaploids have on average
signicantly larger stomata and leaves compared to other
ploidy levels. However, the correlations between these
traits are not so evident. Therefore, stomata and leaf
size cannot be considered as a morphological marker
indicating ploidy level.
Nuclear DNA content and phenotypic traits of the Prunus rootstocks from Poland’s gene resources
ISSN 1392-3196 Zemdirbyste-Agriculture Vol. 107, No. 1 (2020) 77
3. There is no correlation between ploidy level
and vigour of individual rootstocks.
4. The evaluated nuclear DNA contents / ploidy
levels as well as stomata and leaf size are the additional,
useful descriptors for identifying Prunus rootstock
genotypes.
Acknowledgements
This work was performed in the frame of
multiannual programme on preservation of gene bank
resources nanced by the Polish Ministry of Agriculture
and Rural Development: task 1.3 “Collecting,
preservation in ex situ collections, cryoconservation,
evaluation, documentation and using of gene bank
resources of horticultural crops”.
Received 02 06 2018
Accepted 14 06 2019
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ISSN 1392-3196 / e-ISSN 2335-8947
Zemdirbyste-Agriculture, vol. 107, No. 1 (2020), p. 71–78
DOI 10.13080/z-a.2020.107.010
Prunus genties poskiepių iš Lenkijos genetinių išteklių
branduolio DNR kiekis ir fenotipiniai požymiai
M. Podwyszyńska, M. Sitarek, A. Marasek-Ciołakowska, U. Kowalska
Skernevicių sodininkystės ir daržininkystės tyrimų institutas, Lenkija
Santrauka
Ploidiškumo lygis gali būti naudingas vertinant reprodukcinį ir somatinį suderinamumą, kuris yra svarbus parametras
įskiepių ir poskiepių selekcinėse programose. Branduolio DNR kiekio įvertinimas buvo atliktas siekiant nustatyti
31 Prunus genties (slyvos, vyšnios ir trešnės) vaismedžių ploidiškumą. Pirmajame tyrimo etape buvo optimizuotos
srovinės citometrijos analizės sąlygos naudojant propidžio jodidą (PJ) DNR dažymui. Pakartojami rezultatai ir
palyginti geros kokybės histogramos buvo gauta naudojant ekstrahavimo buferį, papildytą 1 % polivinilpirolidono
(PVP), inkubuojant nuo 50 iki 90 minučių.
Geriausias srovinės citometrijos analizės vidinis standartas poskiepiams, kurių branduolio DNR kiekis yra nuo
0,55 iki 1,64 pg, buvo valgomasis pomidoras (Solanum lycopersicum L.) 2C = 1,96 pg, o poskiepiams su didesniu
branduolinės DNR kiekiu (1,8–2,3 pg) gauruotoji soja (Glycine max (L.) Merr.) 2,50 pg. Penkiuose slyvos
poskiepiuose 2C vertės svyravo nuo 0,61 iki 0,67 pg; tai rodė jų diploidinį chromosomų skaičių. Vienas poskiepis
buvo identikuotas kaip tetraploidas (2C = 1,34 pg), keturi poskiepiai – kaip heksaploidai, kurių DNR kiekis
buvo nuo 2,07 iki 2,23 pg. Vienas poskiepis identikuotas kaip pentaploidas dėl jo 1, 64 pg 2C vertės; tai buvo
apie 2,5 karto daugiau nei diploidinėje Prunus sp. Devyniuose vyšnios poskiepiuose 2C DNA vertės svyravo nuo
0,74 iki 0,86 pg; tai rodė diploidinį chromosomų skaičių. Devyni vyšnios poskiepiai buvo identikuoti kaip
triploidai, kurių 2C DNR kiekis svyravo nuo 1,03 iki 1,24 pg. Du vyšnios poskiepiai identikuoti kaip tetraploidai,
kurių 2C DNR kiekis buvo 1,39 pg. Vyšnios ir slyvos poskiepių ploidiškumo lygis buvo įvertintas atsižvelgiant
į morfologinius ir agronominius požymius. Prunus poskiepio genotipuose didėjant ploidiškumo lygiui nustatyta
žiotelių ir lapų didėjimo tendencija, tačiau koreliacijos tarp šių požymių nebuvo tokia akivaizdi. Taigi, žiotelių ir
lapų dydis negali būti laikomas morfologiniu žymekliu, rodančiu ploidiškumo lygį. Įvertintas branduolinės DNR
kiekis / ploidiškumo lygis, taip pat ir žiotelių ir lapų dydis yra papildomi Prunus poskiepių deskriptoriai, kurie gali
būti naudingi identikuojant genotipus.
Reikšminiai žodžiai: heksaploidai, pentaploidai, ploidiškumo lygis, slyva, srovinė citometrija, tetraploidai,
triploidai, vyšnios poskiepiai.
Nuclear DNA content and phenotypic traits of the Prunus rootstocks from Poland’s gene resources
... The basic chromosome number in Prunus is x = 8 [4]. The somatic chromosome number of various Prunus species varies from diploid to hexaploid [4][5][6][7]. Most of the species are diploid (2n = 2x = 16), e.g., P. armeniaca, P. avium, P. canescens, P. cerasifera, P. mahaleb, P. persica, P. spinosa, and P. tomentosa. ...
... Samples with at least 2000 nuclei were measured for two leaves of each plant. As external standard of known ploidy levels, diploid cultivar 'Santa Rosa' [5] and hexaploid P. domestica 'Eruni' were used [7]. ...
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