Development of SSR Markers by 454 Sequencing in the
Endemic Species Gentianella praecox subsp. bohemica
(Gentianaceae)
Authors: Šurinová, Mária, Brabec, Jiří, and Münzbergová, Zuzana
Source: Applications in Plant Sciences, 5(1)
Published By: Botanical Society of America
URL: https://doi.org/10.3732/apps.1600114
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Applications in Plant Sciences 2017 5(1): 1600114
Applications
in Plant Sciences
PRIMER NOTE
DEVELOPMENT OF SSR MARKERS BY 454 SEQUENCING IN THE
ENDEMIC SPECIES GENTIANELLA PRAECOX SUBSP. BOHEMICA
(GENTIANACEAE)1
MÁRIA ŠURINOVÁ2,3,5, JIřÍ BRABEC4, AND ZUZANA MÜNZBERGOVÁ2,3
2Institute
of Botany of the Czech Academy of Sciences, Zámek 1, 252 43 Průhonice, Czech Republic; 3Department of Botany,
Faculty of Science, Charles University in Prague, Benátská 2, 128 01 Prague, Czech Republic; and 4Muzeum Cheb,
Krále Jiřího z Poděbrad 493/4, 350 11 Cheb, Czech Republic
• Premise of the study: Polymorphic microsatellite loci were developed and used to genotype individuals of Gentianella praecox
subsp. bohemica (Gentianaceae), a highly protected taxon in Europe, to study the genetic structure of the remaining
populations.
• Methods and Results: Thirty-eight primer pairs were successfully amplified; of these, 12 polymorphic microsatellite loci were
developed using a 454 sequencing approach and used to genotype 180 individuals of G. praecox subsp. bohemica from six populations. Allelic richness ranged between one and nine alleles per locus. We detected a high frequency of polyploid individuals
(77.8%). The highest average percentage of heterozygous genotypes was identified for samples from the Hroby population
(75.5%). All loci can also be amplified in the congeneric species G. praecox subsp. praecox, G. amarella subsp. amarella, and
G. obtusifolia subsp. sturmiana.
• Conclusions: These markers will provide knowledge on patterns of gene flow and population genetic structure, which is necessary for current protection actions and for effective conservation of this species in the future.
Key words: genotyping; Gentianaceae; Gentianella praecox subsp. bohemica; microsatellites; polyploidy.
Gentianella praecox (A. Kern. & Jos. Kern.) Dostál
ex E. Mayer subsp. bohemica (Skalický) Holub (IUCN:
e.T161825A5500524) is a strictly biennial herb endemic to the
Bohemian Massif, with most populations occurring in the Czech
Republic but extending to Bavaria (Germany), Upper and Lower
Austria, and Poland. Gentianella Moench (Gentianaceae) is a
highly diverse and taxonomically complicated genus due to seasonal dimorphism, introgression, and hybridization between
closely related species (Winfield et al., 2003; Greimler and Jang,
2007; Plenk et al., 2016). It is expected that G. praecox subsp.
bohemica is tetraploid (Oberdorfer, 1983), but cytotype distribution is unknown. It occurs in seminatural, nutrient-poor grasslands. Strong reduction of population size was recorded during
the 20th century, probably due to land-use intensification or
abandonment of traditional land use, which led to the disintegration of large habitats and fragmentation of original populations.
Gentianella praecox subsp. bohemica is highly protected in Europe (Annexes II and IV of the Habitats Directive; Council of
the European Community, 1992). By using amplified fragment
length polymorphism, Königer et al. (2012) studied the genetic
structure of 11 G. praecox subsp. bohemica populations, but this
taxon is known from 99 localities (Brabec, 2010). For effective
protection of this subspecies, it is necessary to identify populations with high genetic diversity so these populations can be prioritized for protection. Moreover, knowledge about the genetic
structure of all remaining populations will reveal patterns of
gene flow among populations and the potential for inbreeding
depression.
METHODS AND RESULTS
Microsatellite development—Total genomic DNA of 14 individuals (two
individuals per population collected across the whole distribution range) of G.
praecox subsp. bohemica was extracted from dehydrated leaves using the cetyltrimethylammonium bromide (CTAB) method of Lodhi et al. (1994), with all
amounts downscaled 10×. The sequencing facility GenoScreen (Lille, France)
was used to prepare libraries and design primers. Extracted DNA was pooled
for microsatellite library preparation. The fragmented DNA was hybridized
with eight probes (TG, TC, AAC, AAG, AGG, ACG, ACAT, and ACTC) to enrich the DNA library. Sequencing was performed using a GS FLX sequencer
(Roche, 454 Life Sciences, Branford, Connecticut, USA). A total of 19,152
reads were obtained. Raw sequencing data were submitted to the National Center
for Biotechnology Information (NCBI) Sequence Read Archive (accession no.
SRR5113067). QDD software (Meglécz et al., 2009) with default settings was
used to identify microsatellite loci and for design of the microsatellite primers.
A total of 3017 reads contained microsatellite motifs, and 373 candidate
microsatellite loci were identified (Appendix S1), with an average sequence
length of 325 bp. Markers belonged to di-, tri-, tetra-, penta-, and hexanucleotide
1 Manuscript received 13 September 2016; revision accepted 27
November 2016.
This work was supported by grants from Iceland, Liechtenstein, and
Norway (EHP funds 2009–2014) and by the Ministry of the Environment of
the Czech Republic. The authors thank Kristina Plenk and Matthias Kropf
for providing Gentianella praecox subsp. praecox samples and Jana
Kadlecová and Eva Ničová for technical assistance.
5 Author for correspondence: maria.surinova@ibot.cas.cz
doi:10.3732/apps.1600114
Applications in Plant Sciences 2017 5(1): 1600114; http://www.bioone.org/loi/apps © 2017 Šurinová et al. Published by the Botanical Society of America.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC-BY-NC-SA 4.0), which permits
unrestricted noncommercial use and redistribution provided that the original author and source are credited and the new work is distributed
under the same license as the original.
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Applications in Plant Sciences 2017 5(1): 1600114
doi:10.3732/apps.1600114
Šurinová et al.—Gentianella praecox microsatellites
repeats (40.2%, 52.8%, 5.4%, 0.8%, and 0.8%, respectively). Across all candidate loci, 3378 primer pairs (3–15 primer pairs per locus) were designed
using Primer3, as implemented within QDD (Malausa et al., 2011) with amplicon lengths ranging between 90 and 319 bp. For each microsatellite candidate
locus, one primer pair was selected for further analysis. Of these, we selected
50 primer pairs (Appendix S1) recommended by GenoScreen to identify polymorphic markers. Primers were synthesized (Sigma-Aldrich, St. Louis, Missouri,
USA) with M13 tails preceding the 5′ end of the forward primer sequences
(Schuelke, 2000). Six individuals from six populations of G. praecox subsp.
bohemica (Appendix 1) were used to test amplification efficiency and polymorphism. DNA amplification was performed in 10-μL reactions consisting of 5 μL
of QIAGEN Multiplex PCR Master Mix (QIAGEN, Hilden, Germany), 0.25 μL
of each M13-labeled forward, reverse, and fluorolabeled (5′-FAM) M13 primer
(10 μM each in initial volume), 20 ng of DNA dissolved in 1 μL TE buffer, and
3.25 μL of H2O.
The following PCR protocol was performed using an Eppendorf Mastercycler pro S Thermal Cycler (Eppendorf, Hamburg, Germany): an initial denaturation step at 95°C for 15 min; followed by 25 cycles of denaturation (95°C for
20 s), annealing (59°C for 30 s), and extension (72°C for 20 s); followed by 10
cycles of denaturation (95°C for 30 s), annealing (53°C for 45 s), and extension
(72°C for 45 s); and a final extension at 72°C for 10 min. Thirty-eight primer
pairs (76%) were successfully amplified. Due to allele dosage uncertainty in
polyploid individuals, preliminary statistics included determination of polymorphic information content (PIC) for each locus by PICcalc (Nagy et al.,
2012). Based on PIC, 20 (53%) of the 38 primer pairs were selected for detailed
variability screening on 36 individuals of G. praecox subsp. bohemica (two individuals from each population). Based on the multiplex PCR performance and
variability screening, 12 polymorphic primer pairs were identified.
To confirm primer specificity for these 12 loci, we ran PCRs for each primer
pair separately under the same conditions described in the next paragraph. PCR
products were purified using the QIAquick PCR Purification Kit (QIAGEN) and
cloned using pGEM-T Vector Systems II (Promega Corporation, Madison, Wisconsin, USA) in accordance with the manufacturer’s instructions, but downscaled to half reactions. Approximately 10 colonies per sample were transferred
into 20 μL of ddH20 and denatured at 95°C for 10 min. These served as templates for subsequent PCR amplifications for sequencing. Sequencing was performed by the commercial company SEQme (Dobříš, Czech Republic), and the
resulting sequences were aligned using MAFFT 7.017 (Katoh et al., 2002) as
implemented in Geneious 8.1.6 (Kearse et al., 2012). Repeat motifs with variation in number of repeats were confirmed in the obtained sequences. GenBank
TABLE 1.
GbM11
GbM34
GbM3
GbM12
GbM19
GbM38
GbM5
GbM2
GbM48
GbM39
GbM43
Genotyping—Total DNA was extracted from 180 G. praecox subsp. bohemica
individuals from six populations and from 114 individuals from eight populations of three closely related taxa (Appendix 1) for initial primer screening. DNA
amplification was carried out in three multiplex reactions consisting of 2.5 μL of
QIAGEN Multiplex PCR Master Mix and 10 ng of DNA dissolved in 0.5 μL of
TE buffer. For multiplex mix I (MM I), the PCR contained 1.1 μL of primer mix
(10 μM each in initial volume) and 0.9 μL of H2O, for MM II the PCR consisted
of 1.1 μL of primer mix (10 μM each in initial volume) and 0.9 μL of H2O, and
for MM III the PCR contained 0.7 μL of primer mix (10 μM each in initial volume) and 1.3 μL of H2O. The sequence, labeling, motif information, final volumes, and PCR product size range are given in Table 1. The following PCR
protocol was performed using an Eppendorf Mastercycler pro S Thermal Cycler:
an initial denaturation step at 95°C for 15 min; followed by 35 cycles of denaturation (95°C for 20 s), annealing (59°C for 30 s), and extension (72°C for 20 s);
and a final extension at 72°C for 10 min. PCR products were diluted with ddH2O
in these ratios: 1 : 2 (PCR product of MM I and MM II PCRs : ddH2O), 1 : 9 (PCR
product of MM III PCR : ddH2O). Each PCR product (1 μL) was mixed with
11 μL of a 120 : 1 solution of formamide : size standard (GeneScan 500 LIZ;
Thermo Fisher Scientific, Waltham, Massachusetts, USA). Fragment lengths
were determined by capillary gel electrophoresis with an ABI 3130 Genetic Analyzer using GeneMapper 4.0 (Thermo Fisher Scientific). Using SPAGeDi (Hardy
and Vekemans, 2002), we calculated the number of alleles per locus, which ranged
between one and nine (Table 2). All markers were polymorphic in all G. praecox
subsp. bohemica populations, except marker GbM48, which was monomorphic
in the Zidkovi population. The highest average percentage of heterozygous genotypes was identified for individuals from the Hroby population (75.5%) and the
lowest percentage for individuals from the Zidkovi population (50.5%). We detected a high frequency of polyploid individuals (77.8%). The observed heterozygote excess is likely caused by the fact that the species is tetraploid.
We also tested cross-amplification of these loci in three other Gentianella taxa:
G. praecox subsp. praecox, G. amarella (L.) Börner subsp. amarella, and G.
obtusifolia (F. W. Schmidt) Holub subsp. sturmiana (A. Kern. & Jos. Kern.) Holub.
We tested 114 individuals from eight populations (Appendix 1). DNA amplification
was carried out in three multiplex reactions as described above. Tests for crossamplification in the three congeneric taxa resulted in successful amplification of up
to seven of the 12 polymorphic loci (Table 2). These results (Table 3) demonstrate
that these primer pairs may be of broad utility throughout Gentianella.
Characteristics of 12 polymorphic loci designed for genotyping of Gentianella praecox subsp. bohemica.
Locusa
GbM46
accession numbers of identified sequences for 12 loci of G. praecox subsp.
bohemica are provided in Table 1.
Primer sequences (5′–3′)
F:
R:
F:
R:
F:
R:
F:
R:
F:
R:
F:
R:
F:
R:
F:
R:
F:
R:
F:
R:
F:
R:
F:
R:
a Optimal
CAACCACAAGAAGCTTCCAA
GCATTGCCAACAGATGCAG
TGGTTTGATTTCAGACCCTTG
CAGGTTGCCCTACCAAGATG
GAAGCGTCCGTTTCAGTTTC
GCTTAGAGCCCAAGATACCTAGA
AGTTGAGAATTGGCCTGGAG
GATGCATTGGAAGCAGGATT
ATCAGGCATTGCCATTAAGC
GAGATTCATAGGTGGCGAGG
GGAATTCCTTGTGAAGCCAG
TTGCTGCTTCTTTTCCATGA
TTTCAAGGTTGCTTTTGGCT
GCCTTGTGTTAAATTAGTTGCAG
CTCCTTCCCTTTTCCCAAAC
GCTTATGTCGCAGTGCAGAA
GGGAGAAGCGAGTTCAAAAG
AAGCTGCTAAACTTCAATACTTGC
ACCGAAGGCAGTTTCAACAC
CCAACAAACTTAGCTACCTTAGCA
AACAGAGCAAAAACAAAAACAGG
CAAGAAAGCAATGAATCCCC
AATCATGTCCAGCTCAGCCT
GCCGACGTAGAATGTTTGGT
Repeat motif
PCR product size
range (bp)
Fluorescent
label
(CTT)4
81–129
PET
(TTG)16
138–180
(TGT)5
Volume of forward
primer (μL)
Multiplex
GenBank
accession no.
0.1
I
KX420610
PET
0.25
I
KX420608
119–152
NED
0.075
I
KX420611
(GTAT)5
134–174
VIC
0.125
I
KX420606
(AC)5
96–108
VIC
0.15
II
KX420604
(GAG)8
136–202
VIC
0.225
II
KX420609
(AGA)6
129–162
NED
0.075
II
KX420612
(AG)8
158–180
PET
0.1
II
KX420615
(GGA)13
147–180
VIC
0.075
III
KX420607
(GGA)3
84–93
NED
0.175
III
KX420613
(AGA)8
79–94
VIC
0.05
III
KX420614
(CCT)4
158–185
NED
0.05
III
KX420605
annealing temperature was 59°C for all loci.
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Results of initial primer screening of 12 microsatellite loci developed in Gentianella praecox subsp. bohemica and congeners.
Species
G. praecox subsp.
bohemica
Population codea
HROBY
PODVORI
POLNA
VYSNY
ZIDKOVI
VANIC
G. amarella subsp.
amarella
VANIC
ČER.S.
G. obtusifolia subsp.
sturmiana
PP PILA
KOCEL
G. praecox subsp.
praecox
BUBE
GIEE
LEOE
GbM46
GbM11
GbM34
GbM3
GbM12
GbM19
GbM38
GbM5
GbM2
GbM48
GbM39
GBm43
A
Aind
% Het
A
Aind
% Het
A
Aind
% Het
A
Aind
% Het
A
Aind
% Het
A
Aind
% Het
A
Aind
% Het
A
Aind
% Het
A
Aind
% Het
A
Aind
% Het
A
Aind
% Het
A
Aind
% Het
A
Aind
% Het
A
Aind
% Het
3
2.3
96.7
6
2.3
86.7
4
1.43
40
7
1.73
73.3
5
2.7
100
9
2.8
96.7
4
1.63
60
4
2.53
96.7
4
1.43
43.3
4
1.77
63.3
5
1.5
50
6
2.6
100
1
1.8
75
2
1.79
79.2
6
2.3
86.7
4
1.9
86.7
5
2.07
80
3
1.73
73.3
2
1.77
76.7
4
2.27
100
3
1.67
50
1
1
0
5
2.3
90
4
2.5
100
3
1.1
10
2
1.71
62.5
1
1.25
25
2
1
0
4
1.43
40
4
1.43
36.7
4
1.83
73.3
2
1.5
50
4
1.1
10
3
1.03
6.7
2
1.17
16.7
1
1
0
4
1.6
60
3
1.7
70
1
1
0
3
1.21
20.8
2
1
0
2
1
0
7
1.73
73.3
3
1.47
46.7
4
1.5
50
3
1.13
13.3
6
1.37
36.7
3
1.53
53.3
3
1.33
33.3
1
1
0
6
1.5
50
3
1.6
60
2
1.3
30
4
1.29
29.2
2
1
0
1
1.13
12.5
5
2.7
100
5
1.7
56.7
4
2.17
83.3
5
2.13
83.3
2
1.53
53.3
4
1.63
60
4
2.17
100
2
2
100
2
1.7
70
4
2.3
100
3
2.3
100
4
1.54
45.8
2
1.05
5
2
1.83
83.3
9
2.8
96.7
5
1.9
70
5
2
80
6
2.63
100
4
2.3
100
5
2.77
96.7
4
2.17
100
2
2
100
7
2.6
90
7
2.6
100
2
2
100
3
1.71
66.7
1
1
0
1
1
0
4
1.63
60
4
1.27
26.7
4
1.43
43.3
3
1.53
53.3
2
1.03
3.3
4
1.67
66.7
4
1.17
16.7
1
1
0
4
1.6
60
4
1.8
80
1
1
0
2
1.21
20.8
1
1.4
40
1
1.04
4.2
4
2.53
96.7
4
2.57
100
4
2.47
100
5
2.6
100
3
2.03
100
4
2.47
100
3
2.17
100
2
2
100
2
1.2
20
3
1.8
70
2
2
100
2
2.29
100
2
2
20
2
2
100
4
1.43
43.3
5
1.33
33.3
6
1.5
50
3
1.5
50
3
1.13
13.3
3
1.43
43.3
4
2
83.3
2
2
100
2
1.1
10
1
1
0
1
1
0
4
1.25
25
3
1
0
3
1.13
12.5
4
1.77
63.3
2
1.2
20
3
1.3
30
3
1.27
26.7
1
1
0
3
1.53
46.7
2
1.83
83.3
2
2
100
3
1.4
40
1
1
0
1
1
0
5
1.21
20.8
3
1.25
25
4
1.04
4.2
5
1.5
50
3
1.3
30
2
1.13
13.3
3
1.5
50
2
1.17
16.7
3
1.57
56.7
3
1.17
16.7
1
1
0
3
1.4
40
2
1.1
10
1
1
0
3
1.17
16.7
3
1.15
15
2
1.08
8.3
6
2.6
100
5
2.57
100
5
2.43
100
4
2.23
100
3
2.1
96.7
4
2.53
100
7
2.83
100
3
3
100
4
2.6
100
4
2.8
100
2
2
100
2
2.46
100
2
2.3
20
2
2.13
100
Note: A = number of alleles; Aind = mean number of alleles per individual; % Het = percentage of heterozygous genotypes.
code refers to collecting locality. Detailed information is provided in Appendix 1.
b Due to allele ambiguity in polyploids, the H : H ratio is replaced by the percentage of heterozygous genotypes.
o
e
a Population
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Šurinová et al.—Gentianella praecox microsatellites
RANK
Primer/Indexesb
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TABLE 2.
Applications in Plant Sciences 2017 5(1): 1600114
doi:10.3732/apps.1600114
Šurinová et al.—Gentianella praecox microsatellites
TABLE 3. Allele size ranges obtained during cross-amplification trials of
microsatellite loci isolated from Gentianella praecox subsp. bohemica
and tested in three additional taxa.
Locus
G. amarella subsp.
amarella
G. praecox subsp.
praecox
G. obtusifolia subsp.
sturmiana
111–126
159–168
131–134
142–170
100–102
142–154
137–143
168–176
150–177
84–87
85–91
167–179
114–117
150–165
128–131
134–158
100–106
142–157
137–146
168–176
150–156
87–90
82–91
164–176
114–126
138–168
119–131
134–170
100–108
142–178
134–153
168–178
147–153
84–90
79–85
167–176
GbM46
GbM11
GbM34
GbM3
GbM12
GbM19
GbM38
GbM5
GbM2
GbM48
GbM39
GbM43
CONCLUSIONS
We developed and successfully multiplexed 12 polymorphic
markers in several taxa of Gentianella. These polymorphic loci
will be valuable for the future management of the extremely
rare G. praecox subsp. bohemica.
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APPENDIX 1. Accession information for Gentianella species used in this study.a
Species name
G. praecox (A. Kern. & Jos. Kern.) Dostál
ex E. Mayer subsp. bohemica (Skalický) Holub
G. praecox subsp. bohemica
G. praecox subsp. bohemica
G. praecox subsp. bohemica
G. praecox subsp. bohemica
G. praecox subsp. bohemica
G. amarella (L.) Börner subsp. amarella
G. amarella subsp. amarella
G. obtusifolia (F. W. Schmidt) Holub subsp.
sturmiana (A. Kern. & Jos. Kern.) Holub
G. obtusifolia subsp. sturmiana
G. obtusifolia subsp. sturmiana
G. praecox subsp. praecox
G. praecox subsp. praecox
G. praecox subsp. praecox
Population code
HROBY
PODVORI
POLNA
VYSNY
ZIDKOVI
VANIC
VANIC
ČER.S.
PP PILA
KOCEL
RANK
BUBE
GIEE
LEOE
Collection locality
Country
n
Latitude
Longitude
Hroby
CZ
30
49.3932222
14.85622
Podvoří
Polná in the Šumava Mountains
Vyšný
Olešnice in the Orlické Mountains
Nature Reserve Opolenec
Nature Reserve Opolenec
Kouty nad Desnou
Pila u Karlových Varů
CZ
CZ
CZ
CZ
CZ
CZ
CZ
CZ
30
30
30
30
30
6
10
10
48.8356111
48.7928056
48.8266944
50.3619444
49.0866667
49.0866667
50.1225
50.1747222
14.20819
14.14997
14.30211
16.28389
13.79706
13.79706
17.16111
12.92694
Kocelovice
Rankovice
Buchberg, Lower Austria
Gießhübl, Lower Austria
Leopolds, Lower Austria
CZ
CZ
AU
AU
AU
10
10
24
20
24
49.475
50.0067778
48.376944
48.320833
48.427778
13.82444
12.84208
15.39722
15.36306
15.28611
Note: AU = Austria; CZ = Czech Republic; n = number of individuals.
a Because all investigated species are rare and highly protected, it was not possible to sample whole plants for herbarium vouchers. Leaf samples were
collected in the field for up to five individuals per population and were dried in silica gel before performing DNA extraction. The leaf samples and DNA
extracts were deposited at the Institute of Botany of the Czech Academy of Sciences, Průhonice, Czech Republic.
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