The genus Cryptanthus (Bromeliaceae) is endemic to Brazil. Many of its currentlyrecognized 66 species are narrow endemics that are threatened by habitat destruction.Molecular markers are needed to evaluate the extent and distribution of genetic diversityin rare Cryptanthus species, which would be a prerequisite for taking appropriateconservation measures. Here we describe the development of plastid microsatellitemarkers (cpSSRs) for Cryptanthus. PCR primers specific for 34 cpSSR loci in Dyckiamarnier-lapostollei were initially tested for their functionality in Cryptanthus schwackeanus. PCR was successful for 29 loci, and 13 loci were shown to harbour extended stretches of mononucleotide repeats. Seven loci were further characterized bygenotyping Cryptanthus samples at the level of populations and species, and six lociproved to be polymorphic among 30 individuals of each of the two endangered species C. schwackeanus and C. warren-loosei, respectively. All primers cross-amplified in other genera from three subfamilies of Bromeliaceae

Benko-Iseppon, Ana Maria

dos Santos Cruz, Geyner Alves

Krapp, Florian

Weising, Kurt

WÃƒÂ¶hrmann, Tina

English

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 Research in Plant Biology, 3(2): 18-21, 2013                                                         ISSN : 2231-5101  www.resplantbiol.com   Received: 19.1.2013; Revised: 24.3.2013; Accepted: 26.3.2013 Short Communication A set of variable plastid SSR markers for the genus Cryptanthus (Bromeliaceae)  Florian Krapp1*, Geyner Alves dos Santos Cruz2, Tina Wöhrmann1, Ana Maria Benko-Iseppon2 & Kurt Weising1  1Plant Molecular Systematics, Department of Sciences, University of Kassel, D-34132 Kassel, Germany 2Genetics Department, CCB, Universidade Federal de Pernambuco (UFPE), Av. Prof. Moraes Rego, 1235, 50.670-420, Recife, PE, Brazil *Corresponding author e-mail: floriankrapp@gmx.de  The genus Cryptanthus (Bromeliaceae) is endemic to Brazil. Many of its currently recognized 66 species are narrow endemics that are threatened by habitat destruction. Molecular markers are needed to evaluate the extent and distribution of genetic diversity in rare Cryptanthus species, which would be a prerequisite for taking appropriate conservation measures. Here we describe the development of plastid microsatellite markers (cpSSRs) for Cryptanthus. PCR primers specific for 34 cpSSR loci in Dyckia marnier-lapostollei were initially tested for their functionality in Cryptanthus schwackeanus. PCR was successful for 29 loci, and 13 loci were shown to harbour extended stretches of mononucleotide repeats. Seven loci were further characterized by genotyping Cryptanthus samples at the level of populations and species, and six loci proved to be polymorphic among 30 individuals of each of the two endangered species C. schwackeanus and C. warren-loosei, respectively. All primers cross-amplified in other genera from three subfamilies of Bromeliaceae.  Keywords : Bromeliaceae; Bromelioideae; Cryptanthus; cpSSR; microsatellites; population genetics  The genus Cryptanthus Otto & A. Dietr. (Bromeliaceae) comprises 66 species that are endemic to Brazil. Cryptanthus species can be found in a variety of habitats, ranging from semi-humid locations in Atlantic Forests to the semi-arid Campos Rupestres, which form exposed “rocky fields” in the Cerrado and Caatinga biomes of eastern Brazil. The genus is distributed from Rio Grande do Norte in the north to Rio de Janeiro in the south, forming a centre of biodiversity in the states of Minas Gerais and Espírito Santo (Ramirez-Morillo, 1996; Ramirez-Morillo & Brown, 2001; Luther, 2008; Forzza et al., 2011). In general, Cryptanthus species have narrow distribution areas, and many of them are micro-endemics. Due to ongoing deforestation, 26 Cryptanthus species have become quite rare and are now included in the list of endangered species, such as the particularly vulnerable C. schwackeanus Mez (Martinelli et al., 2008). Cryptanthus species from the Campos Rupestres of Minas Gerais and Bahia are additionally threatened by mining, anthropogenic fires, and grazing. Here, the ongoing habitat destruction is compromising the survival of e.g. C. warren-loosei Leme, a micro-endemic species of that habitat in Bahia (Versieux et al., 2008). The successful implementation of conservation measures for Cryptanthus Krapp et al. / Research in Plant Biology, 3(2): 18-21, 2013   19  requires some basic knowledge about the extent and distribution of genetic diversity within and among species. Such knowledge can be collected via appropriate population genetic surveys. Suitable molecular genetic marker systems are however needed for this purpose. Here we describe the development of a set of highly polymorphic chloroplast microsatellite markers (cpSSRs) for Cryptanthus, based on previously analyzed plastome sequences from another bromeliad, Dyckia marnier-lapostollei L.B.Sm. (Krapp et al., 2012).  Materials and methods DNA was isolated from leaves of individual plants following Tel-Zur et al. (1999). Thirty-four primer pairs that flank cpSSR loci in Dyckia marnier-lapostollei were initially tested on DNA from a single accession of C. schwackeanus. PCRs were carried out in 10 µL volumes in a Biometra T-Gradient cycler, using the indirect fluorescence labelling procedure described by Schuelke (2000). Each assay contained approximately 1 ng of template DNA, 1x Mango-Taq reaction buffer (Bioline), 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.04 µM forward primer carrying a 5'-M13 tail, 0.16 µM of M13 forward primer with fluorescent 5'-IRD700 modification, 0.16 µM unlabeled reverse primer, 0.5 µg/µl BSA and 0.05 U Mango-Taq DNA polymerase (Bioline). All loci were amplified using a standard PCR program with an initial denaturation at 80°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, primer annealing at 52°C for 1 min and elongation at 65°C for 2 min. Final extension was performed at 65°C for 10 min. Samples were electrophoresed on denaturing 6% polyacrylamide gels in 1xTBE buffer, using an automated sequencer (Li-Cor 4300, Li-Cor Biosciences). Fragment sizes were determined by eye with the help of an external size standard, as outlined by Wöhrmann et al. (2012). Allele sizes were validated by repeated PCR amplification of selected samples. PCR products for one accession of C. schwackeanus were sequenced directly, using a Thermo Sequenase Primer Cycle Sequencing Kit (GE Healthcare) and an automated sequencer (Li-Cor 4300, Li-Cor Biosciences), following the protocols supplied by the manufacturers.  Results and discussion Twenty-nine of the 34 loci showed distinct bands on agarose when tested with a single accession of C. schwackeanus. The presence and copy number of A/T mononucleotide repeats in the PCR products were validated by single nucleotide sequence analyses (Guicking et al., 2008). A/T repeats with N≥10 were found at 13 loci, which were further evaluated by amplifying an extended set of Cryptanthus individuals from three species, C. schwackeanus, C. warren-loosei and C. sergipensis I. Ramírez. Seven loci proved to be suitable for further study, based on the extent of variation within species and populations, the rate of successful PCR amplification, and the ease of sizing. Primer sequences, locus characteristics and GenBank accession numbers are shown in Table 1. These seven loci were further characterized by genotyping 30 individuals (three populations) of each of the two endangered species C. schwackeanus and C. warren-loosei as well as several samples from related species and genera. The results are summarized in Table 2. Six loci were polymorphic within C. schwackeanus with two or three alleles each, resulting in a total of eight different haplotypes. Three loci also showed polymorphisms within populations. Six loci were polymorphic in C. warren-loosei, with two or three alleles per locus combining into five different haplotypes. All six loci were polymorphic in at least one population. The seven cpSSR loci revealed between four and eight alleles within a total of five species of Cryptanthus, and all primers amplified well in ten other genera of Bromelioideae, five of Pitcairnioideae, and one of Puyoideae (Table 2).   Krapp et al. / Research in Plant Biology, 3(2): 18-21, 2013   20   Table 1 Characteristics of seven chloroplast microsatellite markers (cpSSRs) developed for Cryptanthus shown with GenBank accession numbers of the sequences of the locus in C. schwackeanus (sequenced for individual 01 of the population from Serra da Piedade). Primer sequences are derived from Dyckia marnier-lapostollei (Krapp et al., 2012)  Locus Primer sequence (5'-3′) Position Motif Size (bp) GenBank Accession No. Crypt_cpSSR_01 fwd: 5'-CCTATTACAGAGATGGTGCG-3' rev: 5'-TTTCTCGTAAGACTGAGGGC-3' ycf3 intron 2/exon 3 intronic/exonic (T)9 69 KC111433 Crypt_cpSSR_02 fwd: 5'-GTTCCCAGTAAGAACCAACC-3' rev: 5'-CTCAATAATTTCACATTTCC-3' rpoC1 Intron intronic (T)13 104 KC111434 Crypt_cpSSR_03 fwd: 5'-TTGTTGTGGTATCTTTCGCC-3' rev: 5'-CAAGATTTCTCTGATACCCG-3' psbB-psbT intergenic (T)10 64 KC111435 Crypt_cpSSR_04 fwd: 5'-TNAATCAATATGGCGAAGGC-3' rev: 5'-ATTCCCTCACGCTTGGCGCC-3' clpP Intron 2 intronic (T)10 79 KC111436 Crypt_cpSSR_05 fwd: 5'-TTTTTGTTATGGGTATTCCC-3' rev: 5'-ACAAACAGAAAAGAGAGGGC-3' atpF intron intronic (T)12 71 KC111437 Crypt_cpSSR_06 fwd: 5'-CTTCCATTTATCCATATCCC-3' rev: 5'-AAAATAAATCTGATTATGGG-3' rpl16 Intron intronic (T)14 67 KC111438 Crypt_cpSSR_07 fwd: 5'-GTGGATTTATTTTTTGTCCC-3' rev: 5'-AGACCCCGGGCTCGAGGACG-3' rps3-rpl22 intergenic (TA)4(T)13 170 KC111439  Table 2. Observed allele sizes (bp) at seven chloroplast microsatellite loci in C. schwackeanus and C. warren-loosei, each represented by three populations with N=10. Allele numbers and allele size range among five different Cryptanthus species (C. acaulis Beer, C. fosterianus L.B. Sm., C. microglazioui I. Ramírez, C. schwackeanus and C. warren-loosei (between one and 30 individuals each) and cross-amplification in the subfamilies Pitcairnioideae, Bromelioideae (excluding Cryptanthus) and Puyoideae.  Locus C. schwackeanus C. warren-loosei Cryptanthus subfamily Pitcairni-oideae subfamily Bromeli-oideae Puya Serra da Piedade* Serra da Calçada* Serra da Brígida* Jacobina* Buraco do Possidônio(MCH)* Guariba (MCH)* allele number (size range in bp) Crypt_cpSSR_01 69, 71 69,71 69,71 80 75, 80 79, 80 8 (69-80) 2 (69-70) 5 (68-74) 1 (69) Crypt_cpSSR_02 104 105 104 101 101, 104 101 6 (100-105) 3 (101-104) 5 (96-109) 1 (101) Crypt_cpSSR_03 64 64 64 65 64, 65 63, 64 4 (61-65) 2 (63-64) 5 (62-69) 1 (64) Crypt_cpSSR_04 79 80, 81 80 79 78, 79 78 5 (78-86) 3 (78-80) 5 (78-84) 1 (81) Crypt_cpSSR_05 71, 72 72 73 68 68 68 4 (68-73) 4 (67-71) 4 (67-70) 1 (67) Crypt_cpSSR_06 67 70 62 63 62, 63 63, 64 6 (61-70) 4 (60-65) 5 (60-65) 1 (63) Crypt_cpSSR_07 170 172 176 170 169, 170 170 7 (161-176) 5 (156-163) 8 (161-179) 1 (159) * Population (sampling location)  Krapp et al. / Research in Plant Biology, 3(2): 18-21, 2013   21   Acknowledgements The authors thank R Louzada, DSB Pinangé and MGL Wanderley for help in the field work and J Peters, N Schütz and the Botanical Gardens of Heidelberg and Vienna for providing plant material. F Krapp and G Cruz are supported by fellowship grants of the Otto-Braun-Fonds (Melsungen, Germany) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Brazil), respectively. This work was supported by DAAD/CAPES in the frame of a PROBRAL project, and by PNADB/CAPES.  References Forzza RC, Costa A, Siqueira Filho JA, Martinelli G. 2011. Bromeliaceae in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. Available from http://floradobrasil.jbrj.gov.br /2011/FB016586.  Guicking D, Kröger-Kilian T, Weising K, Blattner FR. 2008. Single nucleotide sequence analysis: a cost- and time-effective protocol for the analysis of microsatellite- and indel-rich chloroplast DNA regions. Mol. Ecol. Resour. 8:62-65. Krapp F, Wöhrmann T, Pinangé DSB, Benko-Iseppon AM, Huettel B, Weising K. 2012. A set of plastid microsatellite loci for the genus Dyckia (Bromeliaceae) derived from 454 pyrosequencing. Am. J. Bot. 99(12):e470-e473. Luther, HE. 2008. An alphabetical list of bromeliad binominals, ed. 11. The Bromeliad Society International. Martinelli G, Vieira CM, Gonzalez M, Leitman P, Piratininga A, Costa AF, Forzza RC. 2008. Bromeliaceae da Mata Atlântica Brasileira: Lista de Espécies, Distribuição e Conservação. Rodriguesia 59:209-258. Ramírez-Morillo IM. 1996. Systematics, phylogeny, chromosome number and evolution of Cryptanthus (Bromeliaceae). PhD thesis, University of Missouri, Saint Louis, Missouri, USA. Ramírez-Morillo IM, Brown GK. 2001. The origin of the low chromosome number in Cryptanthus (Bromeliaceae). Syst. Bot. 26:722-726. Schuelke M. 2000. An economic method for the fluorescent labeling of PCR fragments. Nat. Biotechnol. 18:233-234. Tel-Zur N, Abbo S, Myslabodski D, Mizrahi Y. 1999. Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae). Plant Mol. Biol. Rep. 17:249-254. Versieux LM, Wendt T, Louzada RB, Wanderley MGL. 2008. Bromeliaceae da Cadeia do Espinhaço. Megadiversidade 4:99-110. Wöhrmann T, Wagner N, Krapp F, Huettel B, Weising K. 2012. Development of microsatellite markers in Fosterella rusbyi (Mez) L.B. Sm. (Bromeliaceae) using 454 pyrosequencing. Am. J. Bot. 99(4):e160–e163.  

A set of variable plastid SSR markers for the genus Cryptanthus (Bromeliaceae)

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A set of variable plastid SSR markers for the genus Cryptanthus (Bromeliaceae)

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