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Author(s): Pirjo Tanhuanpää, Sirkka Juhanoja, Linnea Oskarsson, Mari Marstein & Merja 
Hartikainen 
Title: Dear old peonies–for gene banks and gardeners; microsatellite fingerprinting of 
herbaceous peonies in Fennoscandia 
Year: 2021 
Version: Published version 
Copyright:   The Author(s) 2021 
Rights: CC BY 4.0 
Rights url: http://creativecommons.org/licenses/by/4.0/ 
 
Please cite the original version: 
Tanhuanpää, P., Juhanoja, S., Oskarsson, L. et al. Dear old peonies–for gene banks and gardeners; 
microsatellite fingerprinting of herbaceous peonies in Fennoscandia. Genet Resour Crop Evol 
(2021). https://doi.org/10.1007/s10722-021-01201-9 
RESEARCH ARTICLE
Dear old peonies–for gene banks and gardeners;
microsatellite fingerprinting of herbaceous peonies
in Fennoscandia
Pirjo Tanhuanpa¨a¨ . Sirkka Juhanoja . Linnea Oskarsson . Mari Marstein .
Merja Hartikainen
Received: 9 September 2020 / Accepted: 29 April 2021
 The Author(s) 2021
Abstract The genetic diversity of 334 herbaceous
peonies from Fennoscandia was analysed using 18
microsatellites (simple sequence repeats, SSRs). The
samples included peonies mostly from Finnish home
gardens (284) and nurseries (5) as well as from
Norwegian (20) and Swedish (25) peony collections.
The study focused on the following species and
hybrids: Paeonia anomala L., P. 9 hybrida Pall., P.
officinalis ‘Nordic Paradox’ (Marstein 2015), P.
tenuifolia L., and P. 9 festiva Tausch. The 18
microsatellites amplified a total of 249 alleles and
were used to calculate genetic distances between
samples and to build a dendrogram. In the dendro-
gram, samples formed clear groups according to their
species. The outcome from the genetic analysis was
mainly confirmed by preliminary morphological
observations of the Finnish home garden samples
performed within the project and the previous mor-
phological study of peonies in Norwegian clone
archives. The results of the study will be used to
create a Finnish genetic resources collection of the
most diverse and vigorous peonies, and to update the
Norwegian and Swedish collections.
Keywords Gene bank  Genetic diversity  Genetic
resources  Microsatellite  Peony  Simple sequence
repeat (SSR)
Introduction
Peonies (only one genus, Paeonia, in the family
Paeoniacea) are native to Asia, South Europe and the
western parts of North America (Hong 2010). SeveralSupplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/
s10722-021-01201-9.
P. Tanhuanpa¨a¨ (&)  M. Hartikainen
Production Systems, Natural Resources Institute Finland
(Luke), 31600 Jokioinen, Finland
e-mail: pirjo.tanhuanpaa@luke.fi
M. Hartikainen
e-mail: merja.hartikainen@luke.fi
S. Juhanoja
Production Systems, Natural Resources Institute Finland
(Luke), 31600 Jokioinen, Finland
e-mail: ext.sirkka.juhanoja@luke.fi
L. Oskarsson
The Swedish National Gene Bank for Vegetatively
Propagated Horticultural Crops, The Programme for
Diversity of Cultivated Plants, The Swedish University of
Agricultural Sciences, P.O. 57, S-230 53 Alnarp, Sweden
e-mail: linnea.oskarsson@slu.se
M. Marstein 
MiA-Museene I Akershus, Strømsvegen 74,
2010 Strømmen, Norway
e-mail: mmarste@online.no
123
Genet Resour Crop Evol
https://doi.org/10.1007/s10722-021-01201-9(0123456789().,-volV)( 0123456789().,-volV)
thousand years ago, they were used as medicinal plants
by the Chinese, who believed that their roots possessed
medicinal properties (Hsu et al. 1986). They were then
used as ornamental plants from the late 1700s
(Harding 1917), and are currently among the most
popular garden plants in temperate regions. They
come in two types: tree peonies, which are shrubs with
deciduous leaves, and herbaceous peonies. Both types
are mainly multiplied by vegetative propagation, but
some species can be propagated by seed.
The current consensus of the number of known
species in the genus Paeonia is 33 (Christenhusz and
Byng 2016), and they can be divided into three
sections: sect. Moutan, sect. Paeonia, and
sect. Onaepia (Stern 1946). Sect. Moutan contains 9
woody species (e.g. P. suffruticosa Andrews) endemic
to China; sect. Paeonia includes 25 herbaceous
species with the widest distribution, mainly in the
Mediterranean and Eastern Asiatic regions; and
sect. Onaepia two herbaceous species, in western
North America and Mexico (Ji et al. 2012).
The biggest section of peonies, Paeonia, contains
long-lived perennial herbaceous species whose leaves
and stems die during winter but whose roots and
crowns stay underground to resume growth in spring.
Herbaceous peonies are important traditional flowers
in China but are also highly valued as ornamentals in
Europe and USA. They are very diverse in morphol-
ogy and ploidy level (Hao et al. 2016). The basic
chromosome number is five (Dark 1936). Hybridisa-
tion is important in nature and in the development of
new cultivars leading to triploid and tetraploid chro-
mosome numbers.
In the Nordic countries, peonies have long been
important as medicinal and ornamental plants. In
Sweden, peonies (P. x festiva Tausch and P. officinalis
L.) are mentioned in medical manuscripts from the
sixteenth century (Larsson 2009). Little is known
about the introduction of peonies in Sweden, but in the
1680s, P. officinalis, P. x festiva, P. peregrina Mill.,
and P. mascula (L.) Mill. were included in the lists of
plants grown in the botanical garden in Uppsala
(Martinsson & Ryman 2007). In the late nineteenth
century, P. 9 festiva ‘Rubra Plena’ was said to be one
of the most common perennials in Swedish gardens
(O.T. 1890). In Norway, peonies were first mentioned
in a Norwegian gardening book, Christian Gartner’s
Horticultura from 1694: «Pæon of all colours»
(Balvoll and Weisaeth 1994). All the peonies covered
in this study grew in the botanic garden in Oslo in
1823. There is even one called hybrida, but we do not
know if it is the P. 9 hybrida Pall. we can find in
Nordic gardens today (Rathke 1823). In Finland,
according to an old written document, peonies have
been grown from the end of the seventeenth century,
as a medicine for epilepsy (Ruoff 2002). In the
nineteenth century, peonies were grown in Finland as
ornamentals and there were seeds from a few different
peony species on the market. Peonies were also
ordered from a nursery in St. Petersburg. Even though
peonies have long been cultivated in Finland, there is
no collection of peony genetic resources as in Norway
and Sweden.
For genetic resources collections, it is very impor-
tant to be able to distinguish species and cultivars. In
addition to tens of peony species, there are a vast
number of different cultivars, 7995 in 2007 (Jaku-
bowski et al. 2007). Identification of different peony
cultivars requires experience in recognising morpho-
logical traits of the plant and the flower, and it takes
from two to 10 years before the plant blooms. In
addition, flower colour may vary depending on
growing site (Zhao et al. 2012). DNA markers can
be used in order to simplify cultivar identification and
to carry it out at an early stage of the plant. Simple
sequence repeat markers (SSRs, microsatellites) were
developed for tree peonies (Gai et al. 2012; Gao et al
2013; Guo et al. 2017; Homolka et al. 2010; Hou et al.
2011a, b; Wang et al. 2009; Wu et al. 2014; Yu et al.
2013; Zhang et al. 2011, 2012) and to a lesser extent
for P. lactiflora Pall. (Cheng et al. 2011; Gilmore et al.
2013; Ji et al. 2014; Li et al. 2011; Sun et al. 2011;
Wan et al. 2020). In addition to identifying cultivars
and species, DNA markers can be used to identify
hybrid origin, to study genetic diversity and relation-
ships, and for linkage mapping.
Finland, Sweden and Norway have always been
strongly connected, both climatically and culturally,
and there has been an active contact across borders. In
all three countries, it is traditional to pass plants along
to friends and relatives, and to take plants with you
when you move from your house. Therefore, it was
justified to carry out a joint study combining plants
from these three countries. In the study, the main
emphasis was on the following species: P. anomala L.,
P. 9 hybrida, P. officinalis ‘Nordic Paradox’ (Mar-
stein 2015), P. tenuifolia L., and P. 9 festiva. The aim
was to collect leaf samples and roots from old peonies
123
Genet Resour Crop Evol
from Finnish home gardens and nurseries (roots for
planting, for subsequent morphological and pheno-
logical observations) and to study their genetic
diversity using SSR markers. The same set of markers
was also used to study genetic diversity of herbaceous
peonies from Norwegian and Swedish peony collec-
tions. The final goal is to create a Finnish collection of
the most diverse and vigorous peonies with a good
ornamental value. In addition, results of the study will
be used to update Norwegian and Swedish collections,
to exclude duplicates and to confirm some identities.
Materials and methods
Plant material
Plant material contained peony samples from Finland,
Norway and Sweden (Figs. 1, 2). In Finland, we first
sought to obtain information about the most rare peony
species grown in private Finnish gardens and nurseries
(Ruoff 2002; Peltola and Koivu 2007). The following
species were selected for the study: P. anomala L.,
P. 9 hybrida, P. officinalis ‘Nordic Paradox’ (Juhan-
nuspioni in Finnish), P. tenuifolia, and P. 9 festiva,
based on their assessed risk of extinction. Every
change in the ownership of a garden will put them at
risk, because new owners seldom have an emotional
connection to the plants in an old garden, and they
often want to simplify and modernise. These species
have been cultivated in Fennoscandia for a long time.
They are typical pass-along plants and not in com-
mercial production. To obtain peony samples from
Finnish home gardens, a call for plants was announced
in 2018–2019. We wanted to collect oral history,
photos and locations of the selected peony species
cultivated in Finland in the 1950s or earlier. Owners of
old peony varieties and landraces were asked to
describe their plants using an online registration form
(www.luke.fi/ilmoitakasvi). This form is still available
and continues to be accessed by owners. Altogether,
690 peony announcements were obtained, and the
samples were given a number with a prefix ‘LUKE’
(referring to the Natural Resources Institute Finland,
Luonnonvarakeskus in Finnish, abbreviation
‘‘Luke’’). A total of 335 plants from the announce-
ments were chosen for the study. Peonies apparently
(based on description and/or photos) not representing
the target species were not chosen. Otherwise,
selection criteria included interesting cultivation his-
tory and, in particular, the age of the plant (more than
60 years old). Leaf samples of the peonies were
requested for DNA analysis, as well as roots for
planting, for later morphological and phenological
observations. Finally, we received leaves from 284
plants (Fig. 1) but the owners did not send roots from
all of them. Roots were planted in pots and kept out-
doors during autumn, and stored indoors at a temper-
ature below ? 5 C during winter. In early spring they
were transferred to a greenhouse and finally planted in
a field at Luke’s experimental station in Piikkio¨
(602530’’N, 02231000’’E) in June 2019. Prelimi-
nary morphological observations were made from 243
plants in the greenhouse, which were classified, when
possible, as different species according to leaf shape,
leaf hairiness, leaf colour, flower shape, and flower
colour. In addition to peonies from home gardens, five
reference samples were included: one P. x festiva
‘Rosea Plena’ (sample number: FIN-2019–75) and
one ‘Alba Plena’ (FIN-2019–74) from a Finnish
nursery, and three P. lactiflora samples (LUKE-5324,
LUKE-5325, LUKE-5326) from Luke’s exhibition
garden Wendla. P. lactiflora samples were included in
order to act as references for this peony group and to
test the functionality of SSRs, which were mainly
derived from this species.
Norwegian samples for the peony collection were
collected through a project financed by the Norwegian
Gene Resources Centre between 2003 and 2008.
Botanists and other professionals visited garden
owners in different parts of Norway, interviewing
them and collecting plants, with a preference for
gardens containing a selection of traditional plants.
The collected plants were planted in separate depart-
ments of the botanical gardens in Kristiansand, Oslo,
Trondheim, and Tromsø, and at some local museums.
Information about the plant’s local growing history
was documented. From the Norwegian collection, 20
samples were selected for the study and leaf samples
were sent to Luke. (Table 1, Fig. 1).
Swedish leaf samples were collected from peonies
preserved in the Swedish National Gene Bank for
Vegetatively Propagated Horticultural Crops. The
gene bank is located at the Swedish University of
Agricultural Sciences and contains 2200 older culti-
vars of fruits, berries, ornamentals, and vegetables.
The gene bank was inaugurated in 2016, and the
cultivars preserved were collected through nationwide
123
Genet Resour Crop Evol
inventories of garden plants grown in Sweden before
1940 or 1950, depending on plant species. The
majority of the cultivars preserved in the gene bank
were collected from private gardens throughout
Sweden, and in addition to documenting the plants,
the histories and traditions associated with them were
also documented. The inventories were initiated and
implemented by the Programme for Diversity of
Fig. 1 Geographical map of the peony samples in the study according to their location to provinces. The class ‘others’ contains peonies
from the following groups: P. officinalis and P. officinalis ‘Mollis
123
Genet Resour Crop Evol
Cultivated Plants, Sweden’s national programme for
plant genetic resources. All in all, 75 accessions of
peonies are preserved in the Swedish National Gene
Bank. Of these, 25 belong to the species selected by
Luke for genetic testing, and leaf samples from these
accessions were sent to Luke in spring 2018 (Table 1,
Fig. 1).
E.Z.N.A SP Plant DNA kit (Omega Bio-tek,
Norcross, GA, USA) was used for DNA extractions
from frozen peony leaves. In some leaf samples, DNA
quality was low (indicated by low A260/A280 and
A260/A230 ratios) and created problems in SSR
amplification. Therefore, DNA from these samples
was further purified using a general protocol of ethanol
precipitation. DNA concentrations were measured
using a NanoDropTM One/OneC Microvolume UV–
Vis Spectrophotometer (Thermo Fisher Scientific Ltd,
Vantaa, Finland).
SSR analyses
For the diversity study we selected 44 SSRs developed
for P. lactiflora and 12 for P. suffruticosa from
different studies (Cheng et al. 2011; Gilmore et al.
2013; Ji et al. 2014; Li et al. 2011; Sun et al. 2011; Wu
et al. 2014). Amplification of SSRs was first tested in
three Paeonia species: P. anomala, P. lactiflora (two
different genotypes), and P. x hybrida. Those ampli-
fying well in this first trial were further analysed for
their polymorphism in five species (16 individuals): P.
anomala, P. lactiflora, P. x hybrida (four genotypes),
P. officinalis (two genotypes), and P. x festiva (three
genotypes), and in five samples with undefined species
from Finnish home gardens. Eighteen SSRs (Table 2),
which amplified well and were polymorphic, were
selected for final analyses. The SSRs were amplified in
three PCR reactions according to results from the
Multiplex Manager v1.2 program (http://
P. anomala 
P. officinalis P. lacflora P. x hybrida 
P. x fesva ’Rubra plena’ P. x fesva ’Rosea plena’ 
P. officinalis ’Mollis’ P. officinalis ’Nordic Paradox’ P. tenuifolia 
Fig. 2 Photos of different peony species taken by Mari Marstein, except P. tenuifolia by Mikko Uusi-Honko
123
Genet Resour Crop Evol
Table 1 Twenty peony samples from the Norwegian collection (NOR) and 25 from the Swedish collection (SWE)
Species/hybrid Cultivar Municipality Province Accession number
P. x festiva ’Rubra Plena’ 4213 Tvedestrand Agder NOR-UiA-2003–0248
’Rubra Plena’ 4206 Farsund Agder NOR-UiA-2006–0135
’Rubra Plena’ 3034 Nes Viken (Akershus) NOR-GH-2008–09
’Rubra Plena’ 1813 Bro¨nno¨y Nordland NOR-UiT-2002–56
’Rubra Plena’ 1837 Melo¨y Nordland NOR-UiT-2002–298
’Rubra Plena’ 1806 Svolvær Nordland NOR-UiT-2015–399
’Rubra Plena’ Tranemo Va¨stra Go¨taland SWE-2018–1
cf ’Rubra Plena’ Falun Dalarna SWE-2018–2
’Rubra Plena’ Floda Dalarna SWE-2018–3
’Rosea Plena’ 4215 Lillesand Agder NOR-UiA-2001–1028
’Rosea Plena’ 5053 Indero¨y Tro¨ndelag NOR-NTNU-2004–501
’Rosea Plena’ 3033 Ullensaker Viken (Akershus) NOR-GH-2007–17
’Rosea Plena’ 5402 Harstad Troms NOR-UiT-2010–70
cf ’Rosea Plena’ Hasslo¨ Blekinge SWE-2018–4
cf ’Mutabilis Plena’ Klintehamn Gotland SWE-2018–5
P. officinalis ’Nordic Paradox’ 3034 Nes Viken (Akershus) NOR-GH-1980–01
’Nordic Paradox’ 5037 Levanger Tro¨ndelag NOR-NTNU-2005–254
’Nordic Paradox’ Tro¨no¨dal Ga¨vleborg SWE-2018–23
’Nordic Paradox’ Sidensjo¨ Va¨sternorrland SWE-2018–24
3026 Aurskog-Ho¨land Viken (Akershus) NOR-GH-2006–23
’Mollis’ 5401 Tromso¨ Troms NOR-UiT-2004–207
’Mollis’ 5401 Tromso¨ Troms NOR-UiT-2004–181
’Mollis’ 5401 Tromso¨ Troms NOR-UiT-2010–153
P. officinalis? Filipstad Va¨rmland SWE-2018–21
P. officinalis? Gustavs Dalarna SWE-2018–22
P. x hybrida 3030 Lillestro¨m Viken (Akershus) NOR-GH-2009–09
1849 Hamaro¨y Nordland NOR-UiT-2004–120
0729 Færder Vestfold NOR-UiT-1993–982
Kristinehamn Va¨rmland SWE-2018–6
Falun Dalarna SWE-2018–7
Hagfors Va¨rmland SWE-2018–8
Smedjebacken Dalarna SWE-2018–9
Ka¨larne Ja¨mtland SWE-2018–10
Gagnef Dalarna SWE-2018–11
Borensberg O¨stergo¨tland SWE-2018–12
Ta¨by Stockholm SWE-2018–13
Odensbacken O¨rebro SWE-2018–14
Gyttorp O¨rebro SWE-2018–15
Dyltabruk O¨rebro SWE-2018–16
Brevens Bruk O¨rebro SWE-2018–17
O¨jebyn Norrbotten SWE-2018–18
Delsbo Ga¨vleborg SWE-2018–19
Grunnebacka Va¨rmland SWE-2018–20
P. anomala 3007 Ringerike Viken (Buskerud) NOR-GH-2009–10
O¨stersund Ja¨mtland SWE-2018–25
123
Genet Resour Crop Evol
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IC
1
2
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–
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0
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6
–
0
.3
5
5
A
T
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T
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y
4
P
.
la
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(2
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5
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IC
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0
0
.3
0
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0
.4
9
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0
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C
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A
C
C
T
123
Genet Resour Crop Evol
multiplexmanager.com). To separate and visualise
amplified products, an ABI PRISM 310 Genetic
Analyzer (Thermo Fisher Scientific Ltd, Vantaa, Fin-
land) was used. The forward primer of each primer
pair was labelled with a fluorescent dye, FAMTM (5-
carboxyfluorescein), NEDTM, VIC or PET. The
PCR amplification conditions were as follows: 32
cycles of 30 s at 95 C, 90 s at 57 C, and 30 s at 72 C
in a BioRad C1000 Thermal Cycler (Bio-Rad, Her-
cules, California, USA). The program started with an
initial denaturation step of 5 min at 95 C and was
followed by a final extension step of 30 min at 60 C.
The PCR amplification was performed in a total vol-
ume of 10 ll, containing 5 ll Master Mix from Qiagen
Type-it Microsatellite PCR Kit (Qiagen, Helsinki,
Finland), 5 ng of DNA, and 67–400 nM in each primer.
PCR products were diluted 1/50 for the ABI runs.
GeneMapper software 5 was used for allele size
estimation.
Data analyses
The study contained plants with different and often
unknown ploidy levels, and it was impossible to know
the dosages of the SSR alleles. In addition, some SSRs
might also represent multiple loci (P05 and Pae100,
Gilmore et al. 2013). Therefore, each SSR allele was
treated as a separate marker locus and a binary code (1/
0) was used for the presence or absence of allele peaks.
Based on the Dice coefficient, a dissimilarity index
between samples was counted with DARwin software
version 6.0.014 (Dissimilarity Analysis and Repre-
sentation for Windows, Perrier and Jacquemoud-
Collet 2006) using a bootstrap value of 1000 replica-
tions. The dissimilarity matrix was used for building
an unweighted neighbour-joining (NJ, Saitou and Nei
T
a
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(b
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)
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)
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th
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cFig. 3 The dendrogram of 334 peony samples, of which 25 are
from Sweden (prefix SWE), 20 from Norway (prefix NOR), and
the rest from Finland: 284 from home gardens (prefix LUKE)
and 5 references (LUKE-5324, LUKE-5325, and LUKE-
5326 = P. lactiflora, FIN-2019–74 = P. x festiva ‘Alba Plena’,
FIN-2019–75 = P. x festiva ‘Rosea Plena’). Confidence levels
greater or equal to 50% from bootstrap analysis of 1000
replicates are indicated. Eight or more identical genotypes have
been united under a single name (duplicate groups 1–7, number
of samples in parenthesis) to facilitate interpretation of the
dendrogram. The individual sample names in the duplicate
groups are presented in Table S1
123
Genet Resour Crop Evol
SWE-2018-10
SWE-2018-11
SWE-2018-12
SWE-2018-13
SWE-2018-14
NOR-UiT-2002-56
NOR-UiT-2004-120
NOR-UiT-2004-207
NOR-UiT-2004-181
NOR-UiT-2010-70
NOR-UiT-2010-153
NOR-UiT-2015-399
SWE-2018-15
NOR-UiT-1993-982
SWE-2018-16
SWE-2018-17
SWE-2018-18
SWE-2018-19
SWE-2018-20
SWE-2018-21
SWE-2018-22
SWE-2018-25
SWE-2018-3
NOR-UiA-2001-1028
SWE-2018-4
SWE-2018-5
NOR-NTNU-2004-501
NOR-GH-2006-23
NOR-GH-2007-17
NOR-GH-2008-09
NOR-GH-2009-09
NOR-GH-2009-10
LUKE-5326
LUKE-5325
LUKE-5324
SWE-2018-6
SWE-2018-7
SWE-2018-8
SWE-2018-9
FIN-2019-74
FIN-2019-75
LUKE-1228
LUKE-1229
LUKE-126
LUKE-13
LUKE-135
LUKE-1903
LUKE-21
duplicate group 6 (8)
LUKE-222
LUKE-2247
LUKE-277
LUKE-2815
LUKE-2816
LUKE-288
LUKE-3236
LUKE-3425
LUKE-3447
LUKE-3454
LUKE-3463
LUKE-3467
LUKE-3513
LUKE-3604
LUKE-3872
LUKE-4047
LUKE-42
LUKE-4224
LUKE-4338
LUKE-4380
LUKE-4384
LUKE-4387
LUKE-4400
LUKE-4405
LUKE-4409
LUKE-4414
LUKE-4415
LUKE-4417
LUKE-4433
LUKE-4434
LUKE-4437
LUKE-4438
LUKE-4443
LUKE-4447
LUKE-4450
LUKE-4451
LUKE-4454
duplicate group 1 (12)
LUKE-4468
LUKE-4471
LUKE-4472
LUKE-4478
LUKE-4479
LUKE-4481
LUKE-4483
LUKE-4484
LUKE-4488
LUKE-4497
LUKE-4500
LUKE-4501
LUKE-4502
LUKE-4504
LUKE-4510
LUKE-4511
LUKE-4515
LUKE-4518
LUKE-4519
LUKE-4528
LUKE-4539
LUKE-4543
LUKE-4551
LUKE-4562
LUKE-4565
LUKE-457
LUKE-4583
LUKE-4588
LUKE-4592
LUKE-4607
LUKE-4615
LUKE-4618
LUKE-4619
LUKE-4620
LUKE-4621
LUKE-4627
LUKE-4639
LUKE-4649
LUKE-4666
LUKE-4670
LUKE-4672
LUKE-4675
LUKE-4680
LUKE-4682
LUKE-4683
LUKE-4685
LUKE-4702
LUKE-4704
LUKE-4710
LUKE-4712
LUKE-4716
LUKE-4723
LUKE-4724
LUKE-4729
LUKE-4733
LUKE-4734
LUKE-4735
LUKE-4736
LUKE-4738
LUKE-4739
LUKE-4742A
LUKE-4742B
LUKE-4744
LUKE-4745
LUKE-4752
LUKE-4762
LUKE-4763
LUKE-4764
LUKE-4766
LUKE-4767
LUKE-4768
LUKE-4773
LUKE-4774
LUKE-4781
LUKE-4790
LUKE-4791
LUKE-4792
LUKE-4793
LUKE-4797
LUKE-4798
LUKE-4800
duplicate group 3 (15)
LUKE-4804
LUKE-4806
LUKE-4807
LUKE-4817
LUKE-4819
LUKE-4828
LUKE-483
LUKE-4831
LUKE-4841
duplicate group 5 (27)
LUKE-4893
LUKE-4898
LUKE-4901
LUKE-4903
LUKE-4904
LUKE-4917
LUKE-4921
LUKE-4923
duplicate group 4 (12)
LUKE-4926
LUKE-4927
LUKE-4928
LUKE-4938
LUKE-4939
LUKE-4940
LUKE-4946
LUKE-4971
LUKE-5001
duplicate group 7 (23)
LUKE-5009
LUKE-5021
LUKE-5022
LUKE-5023
LUKE-5025
LUKE-5039
duplicate group 2 (26)
LUKE-5050
LUKE-5051
LUKE-5052
LUKE-5068
LUKE-5134
LUKE-5135
LUKE-5160
LUKE-5161
LUKE-5164
LUKE-5165
LUKE-5166
LUKE-5167
LUKE-70
97
100
96
62
82
85
82
62
99
99
100
63
77
66
50
100
58
100
52
64
52
58
72
98
100
100
81
80
63
50
66
100
100
100
84
67
51
59
100
86
100
99
87
100
96
100
50
62
56
65
96
96
73
60
99
63
66
79
97
76
66
53
100
72
76
57
100
62
61
69
99
72
50
65
53
57
52
72
P. x fesva
P. anomala
P. x hybrida
P. tenuifolia
P. officinalis
’Nordic Paradox’/
P. officinalis
P. officinalis ’Mollis’
P. lacflora
123
Genet Resour Crop Evol
1987) tree. Polymorphism information content (PIC)
of the SSRs was calculated in a free online computer
program (Abuzayed et al. 2017) using the formula
from Roldan-Ruiz et al. (2000).
Results
Fifty-six previously reported SSRs were evaluated in
order to study genetic diversity in herbaceous peonies.
Based on their amplification and polymorphism, the
18 best SSRs (Table 2) were used for final analyses of
334 peony samples. Two of the selected SSRs
contained a trinucleotide repeat, and the rest dinu-
cleotide repeats. The PIC values of the SSRs varied
from 0.08 (Pmg180) to 0.26 (Sy4) with a mean of 0.16.
Six of the selected SSRs (33%) were developed from
P. suffruticosa and 12 (67%) from P. lactiflora; those
from P. suffruticosa produced more alleles (mean 18
vs. 12/SSR) but their PIC value was lower (mean 0.13
vs. 0.18) than in P. lactiflora SSRs. The 18 SSRs
amplified 249 alleles (markers) in total, and the
number of alleles per SSR varied from 4 (Pae115) to
33 (PS004).
Genetic distances between samples were visualised
with an NJ tree (Fig. 3). The samples formed clear
groups, which were named according to previously
identified species samples (‘references’) from Nor-
way, Sweden, and Finland (Table 3): 1) P. x lactiflora,
2) P. officinalis ‘Nordic Paradox’/P. officinalis, 3) P.
officinalis ‘Mollis’, 4) P. x festiva (based only on
morphological observations, no references), 5) P. x
hybrida, 6) P. anomala, and 7) alleged P. tenuifolia. In
addition, one yellow-flowered peony (LUKE-4338)
did not clearly cluster into any group. There were
duplicates in all groups, the amount varying from 0 to
75% among the samples from Finnish home gardens
(Table 3, Table S1). All the reference samples fell into
their corresponding groups. The two uncertain P.
officinalis samples (SWE-2018–21 and SWE-
2018–22, Table 1) from Sweden proved to be P.
officinalis. Some subgroupings could also be observed
within each group, e.g. in P. officinalis ‘Nordic
Paradox’/P. officinalis group, there were clearly
separate subgroups for P. officinalis ‘Nordic Paradox’
and for P. officinalis; in addition, three separate
samples (LUKE-5021, LUKE-4607, and LUKE-4793)
did not cluster into either subgroup.
Only ten of the 18 SSRs selected for final analyses
were amplified and polymorphic in all species
(Table 4). Therefore, even though the total number
of polymorphic markers was 249, the number in each
Table 3 The number of samples in each peony group in the NJ tree. One sample (LUKE-4338) did not clearly cluster into any group
Group no Group name Total
no. of
samples
Reference samples from Samples from Finnish home gardens
Morphology described
Finland Norway Sweden Total Different
genotypes
Total Inconsistencya
1 P. lactiflora 79 3 76 49 (65%) 57 0
2 P. officinalis ’Nordic
Paradox’ / P. officinalis
77
- P. officinalis ’Nordic Paradox’ 68 2 2 64 16 (25%) 54 0
- P. officinalis 6 1 2 3 2 (67%) 1 0
- separate samples 3 3 3 (100%) 0 0
3 P. officinalis ’Mollis’ 8 3 5 5 (100%) 5 1
4 P. x festiva 71 2 10 5 54 26 (48%) 42 0
5 P. x hybrida 56 3 15 38 10 (26%) 35 0
6 P. anomala 35 1 1 33 32 (97%) 28 1
7 P. tenuifolia 7 7 3 (43%) 4 0
Total 333 5 20 25 283 146 226 2
ainconsistency between genetic analysis and morphological evaluation
123
Genet Resour Crop Evol
group varied greatly, and discrimination between
samples within a group was based on 38 (P. x
festiva)–116 (P. anomala) markers. In P. anomala
and P. lactiflora the number of polymorphic SSRs and
the number of alleles were the highest among all
groups. All species contained private alleles (Table 4),
i.e. alleles that were not found in other species,
however, they were very seldom (only three markers)
amplified from all samples within a species.
A morphological study of peonies in the Norwegian
clone archives was conducted in 2018 and 2019 using
12 specific characters. The results are published in a
48-page report on the MiA website–Museums in
Akershus (https://dms-cf-05.dimu.org/file/
03349w5fjobV). Fifteen of the Norwegian samples
in the present DNA study were included in the mor-
phological study and there was complete agreement
between morphology and DNA markers; all the
specimens fell into the expected groups in the den-
drogram. Preliminary morphological evaluation from
the Finnish home garden samples was carried out in
the greenhouse in Piikkio¨ from 243 samples. The
species could not be defined from 17 of the plants due
to poor growth or because the plant did not bloom at
all. From the remaining 226 samples, only two
(LUKE-4940 and LUKE-4387) gave controversial
results compared to genetic analysis (Table 3). LUKE-
4940 clustered in the dendrogram to P. anomala group
but was (clearly) separate from the other samples. The
SSRs worked in this sample partly as in P. anomala
and partly as in P. x hybrida: P05 amplified normally
as in P. anomala (does not work in P. x hybrida) but on
the other hand, Sy2 did not amplify and Sy4 was
monomorphic as in P. hybrida (Table 4). Morpho-
logically LUKE-4940 seemed to be P. x hybrida,
however, also containing characters from P. anomala.
In fact, LUKE-4940 can be P. intermedia C.A. Mey.,
which has long been thought to be a subspecies of P.
anomala, even though Hong (2010) thinks that it is a
species of its own. LUKE-4387 clustered genetically
into the P. officinalis ‘Mollis’ group but morphologi-
cally into P. officinalis ‘Nordic Paradox’, however,
this plant did not bloom in the greenhouse. According
to the photo sent by the owner, LUKE-4387 seems to
be ‘Mollis’, so the genetic result is correct. The mor-
phological identification of samples in the ‘Mollis’
group was not straightforward but the five samples
from home gardens were classified as undefined. Only
one of these plants flowered in the greenhouse, and it
seemed to be ‘Mollis’. The final identification of most
of the samples according to morphological and phe-
nological observations from two years of field trials
will be reported later in another article. However,
some of the samples did not survive the first winter,
which diminishes the number of morphological
results.
Discussion
Genetic diversity in peony samples from Swedish and
Norwegian peony collections, and from Finnish home
gardens and nurseries was assessed with 18 SSRs. The
objective of the call for old peonies from Finnish home
Table 4 Amplification of 18 SSRs in different peony species groups. Groups with less than 10 samples have been omitted (P.
tenuifolia, 7 samples and P.officinalis ’Mollis’ group, 8 samples)
Group
no
Group name No. of polymorphic
SSRs
No. of polymorphic
alleles
No. of private
alleles
SSRs not
amplified
Monomorphic
SSRs
1 P. lactiflora 17 90 36 Sy1
2 P. officinalis ’Nordic
Paradox’ /
15 77 (43)a 20 (15)b Pae115, Sy5 Sy1
P. officinalis
4 P. x festiva 14 38 8 Sy2, Sy5 Pae03, Sy1
5 P. x hybrida 15 45 3 P06, Sy2 Sy4
6 P. anomala 17 116 35 P06
aFourty-three if the three separate samples (see text) are not included
bFifteen if the three separate samples (see text) are not included
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Genet Resour Crop Evol
gardens was to obtain the following species: P.
anomala, P. 9 hybrida, P. officinalis ‘Nordic Para-
dox’, P. tenuifolia, and P. 9 festiva. In addition to
these, samples representing P. lactiflora and P.
officinalis were also obtained. In the dendrogram,
different species were clearly separated into their own
groups and the identity of a group could be ascertained
using Finnish reference samples and previously iden-
tified samples from Norwegian and Swedish collec-
tions. The separation into different species groups was
facilitated due to some SSRs being group-specific, e.g.
they did not amplify at all or were monomorphic in
certain groups. But on the other hand, due to a lower
number of polymorphic markers in some groups, it
was perhaps not possible to differentiate between
samples, which led to a high number of duplicates
(about half of the samples from Finnish home gardens
were duplicates). This might of course also represent a
real situation: well-growing peonies have spread out
across Finland for decades because people have given
peony roots to each other. On the other hand, in the P.
anomala group, nearly all samples from home gardens
and nurseries were of a different genotype, and only
two samples were genetically identical. This can be
explained by the highest number of polymorphic
markers in the P. anomala group and the fact that this
species is mainly propagated by seeds.
The informativeness level of markers can be
assessed using PIC values, which reflect the diversity
and distribution of alleles. In the present study, the PIC
values were mostly in the category of low (\ 0.25,
Botstein et al. 1980), the mean being 0.16. One reason
for this is that the SSRs were developed in a different
species than the ones in which they were used, and
therefore, did not amplify or were monomorphic in
some species groups. In addition, SSRs had to be
scored as dominant markers due to unknown ploidy
levels, and this also diminishes PIC values. In a
comparable study of rhubarb, PIC values were similar,
varying from 0.05 to 0.16 with a mean of 0.12
(Tanhuanpa¨a¨ et al. 2019).
There has been controversy over the species’
identity within the P. anomala complex, which
contains herbaceous peonies in Central Asia, Siberia,
and adjacent North Eastern European regions (Hong
and Pan 2004). P. x hybrida of Pallas in this complex
was, according to A. P. de Candolle (1818), a garden
hybrid between P.anomala and P.tenuifolia, also
occurring in the wild (Stern 1946). On the other hand,
Anderson (1818) regarded P. x hybrida as synony-
mous with P. tenuifolia for the first time and, after
taxonomic revision, Hong and Pan (2004) were of the
same opinion. In our study, P. x hybrida, P. anomala,
and P. tenuifolia belonged to a bigger cluster, within
which they each formed their own subgroups, sug-
gesting that P. x hybrida and P. tenuifolia are different
species. However, because there were only 7 P.
tenuifolia samples, and they only represented three
different genotypes, more samples are needed to verify
this observation.
The cultivar name of some reference samples was
known (Table 1). Samples under the same cultivar
name are expected to have the same genotype due to
vegetative propagation. However, this was not always
the case. P. x festiva cultivars ‘Rosea Plena’ and
‘Rubra Plena’ seemed not to be uniform and they did
not even cluster into their own groups. However,
differences between samples were small because the
number of polymorphic SSRs in the ‘Rosea Plenas’
and ‘Rubra Plenas’ was not large, 3 and 8, respec-
tively. In addition, there was uncertainty in the
interpretation of some SSRs. Therefore, more markers
would be needed to confirm the genetic result. On the
other hand, this might also reflect a real situation
because seed propagation of peonies was rather
common earlier. Further, the definition of the Swedish
‘Rosea Plena’ and ‘Mutabilis Plena’ was not defini-
tive. The three Norwegian P. officinalis ‘Mollis’
samples were not identical, either, but according to
the importer’s diaries, both seeds and living plants
have been imported and the plants have been propa-
gated from seeds for sale in Norway, which might be a
reason for the variation. P. officinalis ‘Mollis’ samples
in Norway are twice as tall (about 80 cm) as in mid-
Sweden. Of the four P. officinalis ‘Nordic Paradox’
samples, one from Norway fell into duplicate Group 2
and the other from Norway and the two from Sweden
into duplicate Group 3. However, the difference was
only due to one somewhat uncertain allele and
therefore, these four samples can be regarded as the
same genotype.
There are several studies on genetic diversity in tree
peonies (Gao et al. 2013; Guo et al. 2018; He et al.
2020; Ji et al. 2012; Wang et al. 2014; Wu et al. 2014)
but very few in herbaceous peony species and
cultivars, and especially in European cultivars. Gil-
more et al. (2013) used 21 SSRs to distinguish 93
cultivars in tree, intersectional and herbaceous
123
Genet Resour Crop Evol
peonies, of which the last one was further separated
into three species groups.
Conclusions
Most of the peony accessions that were morpholog-
ically evaluated grouped as expected in the dendro-
gram. This confirms that the genetic method used is
reliable and will be a good base for updating
Norwegian and Swedish collections and choosing
specimens for the Finnish gene resources collection
and for the market. There is some genetic variation
within the different species. Further morphological
and phenological studies will assist in choosing which
specimens should be included in the collection and
which specimens would be best suited for the market.
Acknowledgements The authors wish to thank Marja-Riitta
Araja¨rvi, Minna Kavander, Pirkko Nyka¨nen, Hannu Ojanen, and
Anneli Virta for their excellent technical assistance. Finnish
citizens and nurseries are thanked for providing us with the plant
material. Vesa Koivu and Ahti Valli have provided valuable
information on the traditional peony species. The nursery,
Pionien koti, has kindly put photos at the project’s disposal. The
Association of Finnish Nursery Growers is thanked for
providing the network of peony growers. The Finnish National
Plant Genetic Resources Programme is acknowledged for
supporting the call for plants and data management. Thanks to
the Norwegian UiA Natural History Museum and Botanical
Garden, NTNU Ringve Botanical Garden, UiT Tromsø Arctic-
Alpine Botanical Garden and MiA – Museums in Akershus for
providing plant material. Botanists Brynhild Mørkved and
Martin Hajman in Tromsø have kindly helped with information
concerning P. officinalis ‘Mollis’. Thanks to the owners of
Swedish private gardens who donated plants of their older
peonies and contributed so generously to the gene bank
collection.
Author contributions Pirjo Tanhuanpa¨a¨ Conceptualisation,
Methodology, Investigation, Resources, Writing–Original
Draft, Writing–Review & Editing, and Visualisation. Sirkka
Juhanoja: Conceptualisation, (Methodology), Investigation,
Writing–Original Draft, and Writing–Review & Editing.
Linnea Oskarsson: Investigation, Resources, Writing–Original
Draft, and Writing–Review & Editing. Mari Marstein:
Investigation, Resources, Writing–Original Draft, and
Writing–Review & Editing, Funding acquisition. Merja
Hartikainen: Conceptualisation, (Methodology), Investigation,
Resources, Writing–Original Draft, Writing–Review & Editing,
Project Administration, and Funding Acquisition. All authors
read and approved the final manuscript.
Funding Open access funding provided by Natural Resources
Institute Finland (LUKE). Maiju ja Yrjo¨ Rikalan Puutarhasa¨a¨tio¨
and Nikolai ja Ljudmila Borisoffin Puutarhasa¨a¨tio¨ have financed
the study. The Norwegian Agriculture Agency provided
financial support for collecting the Norwegian material.
Data availability The datasets generated during the study are
available from the corresponding author on reasonable request.
Declarations
Conflicts of interest The authors declare that they have no
known competing financial interests or personal relationships
that could have appeared to influence the work reported in this
paper.
Open Access This article is licensed under a Creative Com-
mons Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any med-
ium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative
Commons licence, and indicate if changes were made. The
images or other third party material in this article are included in
the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons licence and your
intended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/.
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