American Journal of Botany: e277–e279. 2011.
American Journal of Botany: e277–e279, 2011; http://www.amjbot.org/ © 2011 Botanical Society of America
The genus Petunia is commonly known worldwide as the
garden petunia , an artifi cial hybrid obtained in the beginning of
the 19th century from a crossing between P. integrifolia (Hook.)
Schinz & Thell. and P. axillaris (Lam.) Britton, Sterns &
Poggenb. ( Stehmann et al., 2009 ). Garden petunias are one of the
world ’ s most important ornamental plants, with seed trading gen-
erating millions of dollars annually. There is great potential for
enhancing this cultivated species utilizing native Petunia species
as a source of genetic variability and agronomic features ( Gerats
and Vandenbussche, 2005 ). Conservation programs for native
species of the genus are urgently needed to ensure appropriate
management of this genetic resource. Petunia shows very low
variability in nuclear sequence markers ( Chen et al., 2007 ), both
among and within species ( Kulcheski et al., 2006 ). Mitochon-
drial regions, which are usually capable of discriminating among
species of the same genus, do not show any variability in Petunia
( Kulcheski et al., 2006 ). Therefore, there is an urgent need for
new, more variable genetic markers for this genus.
Petunia integrifolia is composed of a complex of morphologi-
cally similar species that differ in habitat use, geographical distri-
bution, and minor details in fl oral and vegetative structures. All
species present 2 n = 14, and chromosome counts assign the basic
number of x = 7. This similarity has led to many changes in tax-
onomy and species delimitation in the past. In this work, we
adopt the most recent classifi cation, based also on molecular
data ( Stehmann et al., 2009 ). Therefore, the P. integrifolia com-
plex comprises fi ve taxonomic entities: P. bajeensis T. Ando &
Hashim., P. infl ata R. E. Fr., P. integrifolia subsp. depauperata
(R. E. Fr.) Stehmann, P. integrifolia subsp. integrifolia ,
and P. interior T. Ando & Hashim. These taxa have one of the
largest distributions of Petunia species, found in southern Brazil,
Uruguay, Paraguay, and Argentina ( Stehmann et al., 2009 ). In
this study, we describe the isolation and characterization of 11
loci for P. integrifolia subsp. depauperata and test their transfer-
ability to the other members of the complex.
METHODS AND RESULTS
Genomic DNA was extracted from an individual of P. integrifolia subsp.
depauperata according to Roy et al. (1992), and repeat motifs were isolated using
an enrichment technique ( Beheregaray et al., 2004 ). Briefl y, genomic DNA was
digested with Rsa I and Hae III, and fragments linked to two oligo adaptors. Bioti-
nylated oligo probes (dGT) 10 , (dGA) 10 , (dAGAT) 10 , (dAACT) 10 , and (dACAT) 10
were hybridized to the digested DNA and selectively retained using streptavidin
magnetic particles (Promega, Madison, Wisconsin, USA). PCRs were performed
on the microsatellite-enriched eluate using one of the oligo adaptors as a primer.
The enriched library was purifi ed using an UltraClean 15 DNA Purifi cation
Kit (MO BIO Laboratories, Carlsbad, California, USA), linked into pCR
2.1-TOPO vector (Invitrogen, Carlsbad, California, USA) and transformed into
One Shot TOP10 Chemically Competent Cells (Invitrogen). The plasmid DNA
was PCR-amplifi ed using M13( − 20) forward and M13( − 40) reverse primers,
purifi ed, and 279 positive clones were sequenced with MegaBACE 1000 auto-
mated sequencer (GE Healthcare Biosciences, Pittsburgh, Pennsylvania, USA).
A total of 28 clones presented perfect unique microsatellites, but only 13 were
suitable for primer design using Primer3 (Rozen and Skaletsky, 2000 ; http://
1 Manuscript received 13 April 2011; revision accepted 16 May 2011.
The authors thank Conselho Nacional de Desenvolvimento Cient í fi co e
Tecnol ó gico (CNPq), Funda ç ao de Amparo à Pesquisa do Estado do Rio
Grande do Sul (FAPERGS), and the Australian Research Council (ARC)
for fi nancial support and grants.
5 Author for correspondence: firstname.lastname@example.org
6 These authors have contributed equally to this work, the order of
authorship being arbitrary.
AJB Primer Notes & Protocols in the Plant Sciences
ISOLATION, CHARACTERIZATION, AND CROSS-AMPLIFICATION OF
MICROSATELLITE MARKERS FOR THE PETUNIA INTEGRIFOLIA
(SOLANACEAE) COMPLEX 1
Raquel A. Kriedt 2,6 , Aline M. C. Ramos-Fregonezi 2,6 , Luciano B. Beheregaray 3 ,
Sandro L. Bonatto 4 , and Loreta B. Freitas 2,5
2 Molecular Evolution Laboratory, Department of Genetics, Universidade Federal do Rio Grande do Sul, CP 15053, 91501-970
Porto Alegre, Rio Grande do Sul, Brazil; 3 Molecular Ecology Laboratory, School of Biological Sciences, Flinders University,
GPO Box 2100, Adelaide 5001, South Australia, Australia; and 4 Genomic and Molecular Biology Laboratory, Pontif í cia
Universidade Cat ó lica do Rio Grande do Sul, Ipiranga 6681, 90610-001 Porto Alegre, Rio Grande do Sul, Brazil
• Premise of the study : Microsatellite markers were developed for Petunia integrifolia subsp. depauperata with an intent to clarify taxo-
nomic questions on the P. integrifolia complex, and to identify a purple-fl owered parent of P. hybrida .
• Methods and Results : We characterized 11 microsatellite loci by screening primers developed using an SSR-enriched library.
Genotyping of two populations resulted in eight polymorphic loci. Cross-species transferability was tested for other members
of the P. integrifolia complex.
• Conclusions : The development of these markers may contribute to population genetics studies in Petunia , and cross-amplifi cation
among related species could be a useful tool for research on hybridization and introgression.
Key words: congeneric transferability; microsatellite; Petunia integrifolia complex.
American Journal of Botany
using GENETIC PROFILER 2.0 (GE Healthcare). Analysis of allele numbers,
expected and observed heterozygosity, Hardy – Weinberg equilibrium (HWE),
and genotypic disequilibrium were performed in GENEPOP 4.0 ( Raymond and
Rousset, 1995 ). Tests for null alleles were performed in MICRO-CHECKER
2.2.3 ( Van Oosterhout et al., 2004 ).
All PCR products met the expected sizes based on sequence information,
and the number of bands per individual was consistent with the diploidy condi-
tion of these species (one or two bands per locus per individual). Two loci
(PID2F2 and PID3G3) signifi cantly deviated from HWE in both populations,
and another two loci (PID1D6 and PID1F1) showed signifi cant deviations for
the Garopaba population ( Table 1 ). These results are likely a consequence of
the nonrandom mating system. Although this species is an outcrosser, it presents
Primers were tested for amplifi cation in two populations of P. integrifolia
subsp. depauperata from the Brazilian Coastal Plain (Taim, n = 20; Garopaba,
n = 23). Ten of the 13 primer pairs were successfully amplifi ed, and eight of
them showed polymorphism and were therefore further characterized. Amplifi -
cations were performed in a 15 µ L reaction containing ~10 – 100 ng of template
DNA and 200 µ M of each dNTP (Invitrogen). Reactions included 2 pmol fl uo-
rescent-labeled M13( − 21) primer and reverse primer, 0.4 pmol forward primer
with 5 ′ -M13( − 21) tail, 2.0 mM MgCl 2 (Invitrogen), and 0.5 U Taq Platinum
DNA polymerase and its reaction buffer (Invitrogen). Table 1 lists the se-
quences and the annealing temperature of each primer pair. Fragment analysis
was performed on the MEGABACE 1000, with ET-ROX 550 size ladder (GE
Healthcare). Fragment length and microsatellite genotyping were determined
Table 1. Characterization of microsatellite loci indicating GenBank accession number, repeat motif, primer sequence, annealing temperature ( T a ), allele
size range (bp), and number of alleles per locus ( A ) for Petunia integrifolia subsp. depauperata , as well as expected heterozygosity ( H e ) and observed
heterozygosity ( H o ) for the two analyzed populations.
Taim a (n = 20) Garopaba b (n = 23)
motifPrimer sequence (5 ′ – 3 ′ )
Accession No. T a ( ° C)
(bp) A H e H o A H e H o
PID1D6 (TGG) 6 F: TGGCTATAGAGGAACATACCAATAG
JF720334 62 263 – 27550.7600.50030.626 0.350 d
PID1F1 c (CT) 7 JF720335 62171 – 1753 0.664 0.4703 0.5520.173 d
PID1G6(TG) 7 JF72033663 2161 0.000 0.0001 0.000 0.000
PID2F2(TC) 12 JF720337 58 235 – 2415 0.841 0.375 d 2 0.3690.000 d
PID3C4 (CT) 13 JF72033863247 – 2595 0.7840.5005 0.7250.904
PID3G3(TAGA) 7 JF72033958 180 – 2569 0.8460.055 d 4 0.500 0.100 d
PID3G5(TTC) 8 JF720340 62 1701 0.0000.0001 0.0000.000
PID3G7 c (CA) 10 JF72034159 170 – 1763 0.342 0.26630.416 0.523
PID3H7(GAA) 6 JF72034258132 – 13520.2120.07630.4480.473
PID4C6 (GAA) 13 JF7203436217610.0000.00010.0000.000
PID4G8(CA) 8 JF720344 58228 – 23020.5060.63610.0000.000
a Taim, Rio Grande do Sul, Brazil (30 ° 32 ′ 32 ″ S, 52 ° 34 ′ 34 ″ W).
b Garopaba, Santa Catarina, Brazil (28 ° 01 ′ 26 ″ S, 48 ° 36 ′ 50 ″ W).
c Linked loci in one population after correction for multiple tests ( P < 0.0009).
d Deviation from Hardy – Weinberg equilibrium after correction for multiple tests ( P < 0.004).
Table 2. Transspecies amplifi cation of microsatellite markers developed for Petunia integrifolia subsp. depauperata in four congeneric taxa of the native
range. Allele size range (bp) and number of alleles ( A ) are given.
P. bajeensis (n = 18) P. infl ata (n = 42)
P. integrifolia subsp.
integrifolia (n = 30) P. interior (n = 42)
PrimerSize range (bp) A Size range (bp) A Size range (bp) A Size range (bp) A
263 – 272
161 – 175
247 – 263
170 – 173
129 – 132
173 – 176
257 – 278
171 – 181
212 – 218
247 – 267
129 – 135
176 – 179
228 – 232
260 – 278
169 – 175
216 – 218
251 – 263
170 – 176
129 – 135
228 – 230
257 – 278
169 – 189
212 – 218
249 – 265
234 – 238
126 – 132
230 – 268
Note : NA = no amplifi cation.
e279 Download full-text
AJB Primer Notes & Protocols — PETUNIA INTEGRIFOLIA microsatellites
short-distance seed dispersal, which may cause populations to be constituted
of genetically close individuals ( Stehmann et al., 2009 ). The results could
alternatively be related to null alleles, with MICRO-CHECKER suggesting the
presence of null alleles in deviating loci ( P < 0.004). One pair of loci showed
signifi cant linkage disequilibrium for one population after Bonferroni correc-
tion ( P < 0.0009). However, with no additional information, physical linkage of
loci cannot be distinguished from disequilibrium due to population processes
such as nonrandom mating ( Hedrick, 2005 ).
Amplifi cation and variability were also tested in P. infl ata , P. interior , P.
integrifolia subsp. integrifolia , and P. bajeensis . Of the 11 primer pairs tested,
seven successfully amplifi ed PCR products in these species, 10 amplifi ed in at
least one of the species, and one did not result in any PCR product ( Table 2 ) .
For P. infl ata , 10 loci were amplifi ed in at least 10 individuals, while for the
other species the transferability success was lower. Of these 10 loci, eight ex-
hibited equal or higher levels of polymorphism in the transferred species than
in the source species (except for P. bajeensis ). The latter was not expected
based on studies that compare the behavior for homologous SSRs in plant spe-
cies ( Jarne and Lagoda, 1996 ).
This is the fi rst study to report SSR markers for the P. integ-
rifolia complex. Our results showed better transferability of
the tested markers to P. infl ata and P. interior than to other
species. Most of the loci developed here might prove to be use-
ful to address a range of questions on genetic diversity and
structure, speciation, and migration, especially within P. in-
fl ata , P. interior , and P. integrifolia subsp. integrifolia . The
development of these markers may contribute to different ar-
eas of study in Petunia. Also, information on population dy-
namics of the species may help establish strategies for a
conservation priority of population groups that best represent
the history of these species.
Beheregaray , L. B. , L. M. M ö ller , T. S. Schwartz , N. L. Chao , and
G. Caccone . 2004 . Microsatellite markers for the cardinal tetra
Paracheirodon axelrodi , a commercially important fi sh from central
Amazonia. Molecular Ecology Notes 4 : 330 – 332 .
Chen , S. , K. Matsubara , T. Omori , H. Kokubun , H. Kodama , H.
Watanabe , G. Hashimoto , et al . 2007 . Phylogenetic analysis of the
genus Petunia (Solanaceae) based on the sequence of the HF1 gene.
Journal of Plant Research 120 : 385 – 397 .
Gerats , T. , and M. Vandenbussche . 2005 . A model system for compara-
tive research: Petunia. Trends in Plant Science 10 : 251 – 256 .
Hedrick , P. W. 2005 . Genetics of populations. Jones and Bartlett
Publishers, Boston, Massachusetts, USA.
Jarne , P. , and P. J. L. Lagoda . 1996 . Microsatellites, from molecules to
populations and back. Trends in Ecology & Evolution 11 : 424 – 429 .
Kulcheski , F. R. , V. C. Muschner , A. P. Lorenz-Lemke , J. R.
Stehmann , S. L. Bonatto , F. M. Salzano , and L. B. Freitas .
2006 . Molecular phylogenetic analysis of Petunia Juss. (Solanaceae).
Genetica 126 : 3 – 14 .
Raymond , M. , and F. Rousset . 1995 . GENEPOP (version 1.2): Population
genetics software for exact tests and ecumenicism. The Journal of
Heredity 86 : 248 – 249 .
Rozen , S. , and H. J. Skaletsky . 2000 . Primer3 on the WWW for
general users and for biologist programmers. In S. Krawetz and S.
Misener [eds.], Bioinformatics methods and protocols: Methods in
mole cular biology, 365 – 386. Humana Press, Totowa, New Jersey, USA.
Stehmann , J. R. , A. P. Lorenz-Lemke , L. B. Freitas , and J. Semir .
2009 . The genus Petunia. In T. Gerats and J. Strommer [eds.], Petunia:
Evolutionary, developmental and physiological genetics, 1 – 28.
Springer, New York, New York, USA.
Van Oosterhout , C. , W. F. Hutchinson , D. P. M. Wills , and P.
Shipley . 2004 . Micro-checker: Software for identifying and cor-
recting genotyping errors in microsatellite data. Molecular Ecology
Notes 4 : 535 – 538 .
Appendix 1. Information on voucher specimens deposited in the herbarium of Universidade Federal de Minas Gerais, Belo Horizonte, Brazil (BHCB).
Taxon Voucher specimensLocality in Brazil
Petunia bajeensis T. Ando & Hashim.
Petunia infl ata R. E. Fr.
Bag é , Rio Grande do Sul, 31 ° 24 ′ 35 ″ S, 54 ° 38 ′ 03 ″ W
S ã o Luiz Gonzaga, Rio Grande do Sul, 28 ° 27 ′ 24 ″ S, 55 ° 07 ′ 24 ″ W
Santo Cristo, Rio Grande do Sul, 27 ° 50 ′ 16 ″ S, 54 ° 38 ′ 03 ″ W
Taim, Rio Grande do Sul, 30 ° 32 ′ 32 ″ S, 52 ° 34 ′ 34 ″ W
Garopaba, Santa Catarina, 28 ° 01 ′ 26 ″ S, 48 ° 36 ′ 50 ″ W
Cachoeira do Sul, Rio Grande do Sul, 30 ° 27 ′ 11 ″ S, 52 ° 56 ′ 08 ″ W
Quara í , Rio Grande do Sul, 30 ° 26 ′ 14 ″ S, 56 ° 20 ′ 06 ″ W
Dois Irm ã os das Miss õ es, Rio Grande do Sul, 27 ° 37 ′ 40 ″ S, 53 ° 33 ′ 53 ″ W
Panambi, Rio Grande do Sul, 28 ° 20 ′ 14 ″ S, 53 ° 34 ′ 02 ″ W
S ã o Luiz Gonzaga, Rio Grande do Sul, 28 ° 24 ′ 22 ″ S, 54 ° 41 ′ 28 ″ W
Petunia integrifolia (Hook.) Schinz & Thell. subsp.
depauperata (R. E. Fr.) Stehmann
Petunia integrifolia (Hook.) Schinz & Thell. subsp.
Petunia interior T. Ando & Hashim.