ijms International Journal of Molecular Sciences Int. J. Mol. Sci. 1422-0067 Molecular Diversity Preservation International (MDPI) 10.3390/ijms130911343 ijms-13-11343 Short Note Isolation and Characterization of Microsatellite Markers for Passiflora contracta CazéAna Luíza R.1† KriedtRaquel A.1† BeheregarayLuciano B.2 BonattoSandro L.3 FreitasLoreta B.1* Laboratory of Molecular Evolution, Department of Genetics, Federal University of Rio Grande do Sul, PO Box 15053, 91501-970 Porto Alegre, RS, Brazil; E-Mails: analuizacaze@gmail.com (A.L.R.C.); raquelkriedt@yahoo.com (R.A.K.) Molecular Ecology Laboratory, School of Biological Sciences Flinders University, GPO Box 2100, Adelaide 5001, Australia; E-Mail: luciano.beheregaray@flinders.edu.au Laboratory of Genomic and Molecular Biology, Pontifical Catholic University of Rio Grande do Sul, Ipiranga 6681, 90610-001 Porto Alegre, RS, Brazil; E-Mail: slbonatto@pucrs.br Author to whom correspondence should be addressed; E-Mail: loreta.freitas@ufrgs.br; Tel.: +55-51-3308-6731; Fax: +55-51-3308-9823.

These authors contributed equally to this work.

 2012 12 09 2012 13 9 11343 11348 27 08 2012 07 09 2012 08 09 2012 © 2012 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. 2012

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Passiflora contracta Vitta (Passifloraceae) is an endemic species of the Atlantic Rainforest, one of the most species-rich ecoregions in the world, although extremely endangered. We have developed an enriched microsatellite library in order to fine-scale studies of the genetic structure of P. contracta. Twelve pairs of microsatellite primers were designed, and seven loci were successfully amplified and characterized by genotyping two wild populations of P. contracta. All seven loci were polymorphic, with an average number of alleles found being 4.8 and 5 per population. The cross-species transferability was tested using sister species Passiflora ovalis Vell. Ex Roemer. The development of these markers will contribute to the studies of population genetics in P. contracta as well as future studies concerning diversity patterns in the Atlantic Rainforest, and may also help to establish strategies for the conservation of this species.

 nuclear microsatellites Passiflora contracta conservation genetics 1. Introduction

Passiflora contracta Vitta (Passifloraceae) is an endemic species of the Atlantic Rainforest, one of the most species-rich ecoregions in the world. Originally covering 15% of Brazil, this endangered ecosystem has been reduced to less than 8% of its coverage. Despite the anthropic interference, the Atlantic Rainforest still harbors impressively high levels of endemism and diversity [1], characteristic of areas of long-term climate stability, also known as refugia [2]. Although refugia have been proposed in the Atlantic Rainforest [3], these have not been tested by studies of the intraspecific plant genetic diversity due to the lack of information on the genetic diversity of the Atlantic Forest’s vegetation. P. contracta is an excellent species for such a study. This woody vine is distributed along the coast of Brazil, ranging from the Pernambuco to Espirito Santo states (~07–21°S latitude) [4]. The species is characterized by its chiropterophily syndrome, which is not a common feature in the Passifloraceae family [5].

The identification of high-resolution genetic markers within P. contracta is an important step to develop fine-scale investigations of endemism-rich ecosystems and is also an interesting tool for testing the refugia hypothesis. Therefore, the aim of this study was to develop and characterize microsatellite markers in P. contracta and to test their transferability to its sister species, Passiflora ovalis Vell. Ex Roemer (Passifloraceae).

 2. Results and Discussion

A total of seven of the 12 primer pairs were successfully amplified; all of them were polymorphic and presented alleles in the expected size range for the two wild populations: Linhares-ES (19°24′S, 40°28′W) and Maraú-BA (14°07′50″S, 38°59′55″W) (Table 1). The characteristics of the microsatellite loci and variability measures across the two wild populations are described in Table 2. The number of alleles per population ranged from two to nine (Linhares) and from three to eight (Maraú). The average alleles found for each population was 5 and 4.8, respectively. The observed and expected heterozygosity ranged from 0.31 to 0.84 (Linhares), and 0.38 to 0.67 (Maraú). Two loci, PC6F7 (Linhares) and PC7H11 (Maraú), showed significant deviations from the Hardy-Weinberg equilibrium (p < 0.007). These findings may be a consequence of the high inbreeding in both populations, which could result from habitat fragmentation since species populations are small and, in general, restricted to preserved areas. Alternatively, the HWE deviation could be due to null alleles, which were indicated by MICRO-CHECKER 2.2.3 for the deviating loci (PC6F7) of the Linhares population, and also for the deviating loci (PC7H11) of the Maraú population. Null alleles were not detected for the remaining loci. One pair of loci (PC5E11 and PC6G11) showed significant linkage disequilibrium for the Linhares population after Bonferroni correction (p < 0.002). However, with no additional information, the physical linkage of the loci cannot be distinguished from disequilibrium due to population processes as nonrandom mating [6].

The transferability of the markers to the sister species P. ovalis was tested for all 12 primer pairs, and showed a low efficiency, with just two loci (PC6G11 and PC7H11) being amplified. This result reinforces the differentiation between these species that were previously considered as a single one.

 3. Experimental Section 3.1. Microsatellite-Enriched Library Construction and Isolation of Microsatellite Markers

Genomic DNA was extracted from an individual of P. contracta using the Nucleo Spin Plant II kit (Macherey-Nagel, Düren, Germany), and the repetitive motifs were isolated using an enrichment technique [7] in which the genomic DNA was digested with RsaI and HaeIII and the resulting fragments were linked to two oligonucleotide adaptors. Biotinylated oligonucleotide probes (dGT)10, (dGA)10, (dAGAT)10, (dAACT)10, and (dACAT)10 were hybridized with the digested DNA and selectively restrained by streptavidin magnetic particles (Promega, Madison, Wisconsin, USA). The selected DNA fragments were eluted in 25 μL ultra pure water and amplified by PCR in a total volume of 50 μL. Reactions were conducted with 50 ng of eluted DNA, 1× Colourless GoTaq Reaction Buffer (Promega), 200 mM dNTPs (Promega), 40 pmol of “oligo adapter A” as primer (Sigma-Aldrich, St. Louis, MO, USA), 1.5 mM MgCl2, 5 U of GoTaq Flexi DNA Polymerase (Promega). The PCR conditions were as follows: An initial denaturation at 94 °C for 5 min followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min and extension at 72 °C for 2 min, with a final extension at 72 °C for 5 min. The enriched library was purified using an UltraClean 15 DNA Purification Kit (MO BIO Laboratories, Carlsbad, CA, USA), linked to the pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA) and transformed into One Shot TOP10 Chemically Competent Cells (Invitrogen). The plasmid DNA was PCR-amplified using 16 pmol M13(-20) forward and M13(-40) (Sigma-Aldrich), reverse primers, 2.5 U GoTaq Flexi DNA polymerase (Promega), 200 μM of each dNTP (Promega), 2.5 mM MgCl2 (Promega), 1× GoTaq Colourless Reaction Buffer (Promega), and 1 μL of transformed cells grown in 100 μL liquid broth LB. The PCR conditions were as follows: An initial denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 3 min, with a final extension at 72 °C for 5 min. A total of 163 positive PCR fragments were purified and sequenced using a MegaBACE™ 1000 automated sequencer (GE Healthcare Biosciences, Pittsburgh, PA, USA), following the DYEnamic™ ET terminator sequencing premix kit with terminal fluorescent labeled protocol according to the conditions were as follows: 4 μL of DYEnamic™ ET terminator sequencing premix, 5 μM of forward/reverse primer, 40 ng of purified PCR products, and ultra pure water to complete a 10 μL volume. This reaction was submitted to 95 °C for 20 s, 50 °C for 15 s and 60 °C for 1 min. A total of 23 clones presented perfect unique microsatellites, but only 12 were suitable for primer design using Primer 3 software [8].

 3.2. Genotyping and Data Analysis

The primers were tested for amplification in two wild populations of P. contracta species, Linhares-ES (19°24′S, 40°28′W), n = 20 and Maraú-BA (14°07′50″S, 38°59′55″W), n = 20, and 10 individuals of P. ovalis were tested for cross-amplification. The amplifications were performed in a 15 μL reaction containing ~10 ng template DNA, 1× Taq Platinum reaction buffer (Invitrogen), 200 μM each dNTP (Invitrogen), 2 pmol FAM fluorescently labeled M13(-21) primer [9] and reverse primer, 0.4 pmol forward primer with a 5′-M13(-21) tail, 2.0 mM MgCl2 (Invitrogen), and 0.5 U Taq Platinum DNA polymerase (Invitrogen). The PCR conditions for SSR were as follows: An initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 20 s, annealing at primer-specific temperatures (50–55 °C) see (Table 2) for 45 s, and extension at 72 °C for 1 min, with a final extension at 72 °C for 15 min. The repeat motif, primer sequences, labeling dye, annealing temperature (Ta °C), and allele size range in base pair, with the M13 tail included, of each primer pair are listed in Table 1.

The fragments were analyzed using MegaBACE™ 1000, based on the ET-ROX 550 size ladder (GE Healthcare). The fragment length and microsatellite genotyping were determined using GENETIC PROFILER 2.0 (GE Healthcare). The allele numbers, expected and observed heterozygosity, Hardy-Weinberg Equilibrium (HWE), and genotypic disequilibrium analyses were performed using ARLEQUIN version 3.5 [10] and FSTAT [11]. MICRO-CHECKER 2.2.3 [12] was used to test for null alleles.

 4. Conclusions

The development of polymorphic microsatellite markers will contribute to the population genetic studies of P. contracta, particularly with regard to comparative studies of diversity patterns in the Atlantic Rainforest. These markers may also help to establish strategies for the conservation of priority population groups of this species that inhabits an extremely endangered ecosystem.

 Acknowledgments

We thank CNPq, FAPERGS and the ARC for financial support and grants.

 References MyersN.MittermeierR.A.MittermeierC.G.FonsecaG.A.B.KentJ.Biodiversity hotspots for conservation prioritiesNature200040385385810.1038/3500250110706275 PranceG.T.Origin and evolution of Amazon floraInterciencia19783207222 CarnavalA.C.MoritzC.Historical climate modelling predicts patterns of current biodiversity in the Brazilian Atlantic forestJ. Biogeogr2008351187120110.1111/j.1365-2699.2007.01870.x VittaF.A.BernacciL.C.A new species of Passiflora in section Tetrastylis (Passifloraceae) and two overlooked species of Passiflora from BrazilBrittonia200456899510.1663/0007-196X(2004)056[0089:ANSOPI]2.0.CO;2 BuzatoS.FrancoA.L.Tetrastylis ovalis: A second case of bat-pollinated passionflower (Passifloraceae)Plant Syst. Evol199218126126710.1007/BF00937450 HedrickP.W.Genetics of Populations3rd edJones Bartlett PublishersBoston, MA, USA2005737 BeheregarayL.B.MöllerL.M.SchwartzT.S.ChaoN.L.CacconeG.Microsatellite markers for the cardinal tetra Paracheirodon axelrodi, a commercially important fish from central AmazoniaMol. Ecol. Notes2004433033210.1111/j.1471-8286.2004.00686.x RozenS.SkaletskyHPrimer3 on the WWW for General Users and for Biologist ProgrammersBioinformatics Methods and ProtocolsKrawetzS.MisenerS.Humana PressTotowa, NJ, USA2000365386 SchuelkeM.An economic method for the fluorescent labeling of PCR fragment. A poor man’s approach to genotyping for research and high-throughput diagnosticsNat. Biotechnol20001823323410.1038/7270810657137 ExcoffierL.G.L.LischerH.E.L.Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and WindowsMol. Ecol. Res20101056456710.1111/j.1755-0998.2010.02847.x GoudetJ.FSTAT ver 1.2: A computer program to calculate F-StatisticsJ. Hered199586485486 Van OosterhoutC.HutchinsonW.F.WillsD.P.M.ShipleyP.Micro-checker ver2.2.3: Software for identifying and correcting genotyping errors in microsatellite dataMol. Ecol. Notes2004453553810.1111/j.1471-8286.2004.00684.x Tables

Characteristics of seven microsatellite markers for Passiflora contracta. For each locus, the name, repeat motif, primer sequence, labeling dye, annealing temperature (Ta), allele size range (bp) and GenBank accession number are shown.

Locus Repeat motif Primer sequence (5′–3′) Dye Ta (°C) Size (bp) Genbank
PC5E11 (AC)7(AG)6 F:CTGGTCTTGGATTGTCCTTTGR:CAAAGTAACTGGTGAGCTTAGGG FAM 54 158–170 JX575753
PC6E8 (GT)8 F:TTGCAAATGATAACAAAACACGR:TATCTCGGATTCCCAAAACC FAM 53 165–181 JX575754
PC6G11 (GA)10 F:ACTGGAAGTCAAACGGTGAGR:GGTGGCTCGAAATTCAAATC FAM 52 207–229 JX575755
PC7H11 (CTT)13 F:TGAAATCCCTGTTGTGTGACTCR:TCCTGAGGGGAGCTGTAGTG FAM 53 169–175 JX575759
PC6D6 (CT)9 F:TTTTTGTGAAGGTAATTTGTCAR:CATGTTGCCTCCATGTTTGA FAM 50 162–168 JX575757
PC7C12 (AC)7 F:TGAAATCCCTGTTGTGTGACTCR:TCCTGAGGGGAGCTGTAGTG FAM 55 179–195 JX575758
PC6F7 (CT)8 F:AACGCATTTTTCAGTTTCTGCR:TGAGACTCCCATTCACCAAG FAM 53 230–248 JX575756

Characterization of microsatellite loci indicating number of alleles per locus (A); expected (HE) and observed (HO) heterozygosity for the two analyzed populations, Linhares-ES (19°24′S, 40°28′W) and Maraú-BA (14°07′50″S, 38°59′55″W), of Passiflora contracta.

Passiflora contracta
Locus Linhares-ES Maraú-BA
A HE HO A HE HO
PC5E11# 6 0.82011 0.64286 5 0.63678 0.60000
PC6E8 2 0.31452 0.37500 5 0.46508 0.55556
PC6G11# 4 0.37619 0.44444 8 0.58571 0.44444
PC7H11 3 0.60484 0.75000 3 0.65238 0.38889*
PC6D6 4 0.64368 0.53333 4 0.64308 0.53846
PC7C12 7 0.79637 0.68750 5 0.67137 0.56250
PC6F7 9 0.84135 0.41176* 4 0.57619 0.50000
mean 5 4.8

disequilibrium linkage after Bonferroni correction to Linhares-ES population (p < 0.002);

deviation from Hardy-Weinberg equilibrium (p < 0.007).