Microsatellites from Fosterella christophii (Bromeliaceae) by de Novo Transcriptome Sequencing on the Pacific Biosciences RS Platform Author(s): Tina Wöhrmann, Bruno Huettel, Natascha Wagner and Kurt Weising Source: Applications in Plant Sciences, 4(1) Published By: Botanical Society of America DOI: http://dx.doi.org/10.3732/apps.1500084 URL: http://www.bioone.org/doi/full/10.3732/apps.1500084 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. 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Applications in Plant Sciences 2016 4(1): 1500084 Applications in Plant Sciences PRIMER NOTE MICROSATELLITES FROM FOSTERELLA CHRISTOPHII (BROMELIACEAE) BY DE NOVO TRANSCRIPTOME SEQUENCING ON 1 THE PACIFIC BIOSCIENCES RS PLATFORM TINA WÖHRMANN2,4, BRUNO HUETTEL3, NATASCHA WAGNER2, AND KURT WEISING2 2Systematics and Morphology of Plants, Institute of Biology, University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany; and 3Max Planck-Genome-centre Cologne, Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Cologne, Germany • Premise of the study: Microsatellite markers were developed in Fosterella christophii (Bromeliaceae) to investigate the genetic diversity and population structure within the F. micrantha group, comprising F. christophii, F. micrantha, and F. villosula. • Methods and Results: Full-length cDNAs were isolated from F. christophii and sequenced on a Pacific Biosciences RS plat- form. A total of 1590 high-quality consensus isoforms were assembled into 971 unigenes containing 421 perfect microsatel- lites. Thirty primer sets were designed, of which 13 revealed a high level of polymorphism in three populations of F. christophii, with four to nine alleles per locus. Each of these 13 loci cross-amplified in the closely related species F. micrantha and F. villosula, with one to six and one to 11 alleles per locus, respectively. • Conclusions: The new markers are promising tools to study the population genetics of F. christophii and to discover species boundaries within the F. micrantha group. Key words: Bromeliaceae; Fosterella christophii; microsatellites; Pacific Biosciences; single-molecule real-time (SMRT) sequencing; transcriptome. Fosterella christophii Ibisch, R. Vásquez & J. Peters, F. METHODS AND RESULTS micrantha (Lindl.) L. B. Sm., and F. villosula (Harms) L. B. Sm. form a well-circumscribed species group within the ge- Total RNA was isolated from fresh leaves of one F. christophii plant nus Fosterella L. B. Sm., known as the F. micrantha group (NW09.030-11) using the RNeasy Plus Micro Kit (QIAGEN, Venlo, The Neth- (Pitcairnioideae; Bromeliaceae) (Wagner et al., 2013). The erlands). RNA quality and quantity were assessed by capillary electrophoresis three species are morphologically very similar terrestrial on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA). Polyadenylated RNA was isolated with the NEBNext Poly(A) mRNA Mag- rosette plants with small, whitish, insect-pollinated flowers netic Isolation Module (New England Biolabs, Ipswich, Massachusetts, USA), (Peters, 2009). Such high levels of similarity are surprising, followed by an integrity check via capillary electrophoresis. An aliquot of 1 ng given that F. micrantha is endemic to Central America, whereas of poly(A) RNA was selected as an input for cDNA synthesis with a SMARTer the other two species reside in the Bolivian Andes. Controlled PCR cDNA Synthesis Kit (Clontech Laboratories, Mountain View, California, pollination experiments indicated that all three species are USA). A SMRTbell library was prepared as recommended by Pacific Biosci- self-compatible but also form viable hybrids (Wagner et al., ences (PacBio, Menlo Park, California, USA). The amplified cDNA was size- fractionated on agarose gels, and fragments with insert sizes >1.5 kb were 2015). To investigate the genetic diversity and differentiation recovered. SMRTbell templates were bound to polymerases using the PacBio in this closely related species complex, we used Pacific Bio- DNA/polymerase binding kit P4 and v2 primers. Polymerase-template com- sciences’ single-molecule real-time (SMRT) technology (Eid plexes were bound to magnetic beads using a MagBead Kit (PacBio, part #100- et al., 2009) to develop a set of genic microsatellite markers in 133-600). Sequencing was carried out on the PacBio RS II sequencer using C2 F. christophii. sequencing reagents with a movie length of 180 min. Full-length cDNAs were identified with the PacBio SMRT analysis software (version 2.2.0). High-quality sequences were achieved by running the protocol with a filter for a minimum of three full passes of a cDNA and discarding all non–full length cDNAs and chimeric products. The read output was further trimmed and assembled into unigenes using CAP3 (Huang and Madan, 1999). 1 Manuscript received 18 July 2015; revision accepted 24 September A total of 1590 high-quality consensus isoforms with an average size of 2015. 1322 bp were assembled into 971 unigenes. BatchPrimer3 (You et al., 2008) The authors thank the Botanical Gardens of Hannover and Heidelberg, was applied to detect perfect microsatellites, accepting minimum thresholds of Dr. Jule Peters (University of Kassel, Kassel, Germany), Dr. Nicole Schütz seven repeat units for di-, six for tri-, five for tetra-, and four for penta- and (Stuttgart State Museum of Natural History, Stuttgart, Germany), and Dr. hexanucleotide repeats, respectively. A total of 421 microsatellites were pres- Ivón Ramírez-Morillo (Centro de Investigación Científica de Yucatán, ent in 275 unigenes. Motif types are compiled in Appendix S1. Flanking se- Mérida, Yucatán, Mexico) for kindly providing plant material and Sascha quences of appropriate quality and length were present at 335 microsatellite Herwig for assistance in the laboratory. loci. 4 Author for correspondence: [email protected] Microsatellite-flanking primers were designed using the BatchPrimer3 interface (You et al., 2008), applying the following criteria: length ranging doi:10.3732/apps.1500084 from 18 to 23 nucleotides, product size ranging from 100 to 300 bp, annealing Applications in Plant Sciences 2016 4(1): 1500084; http://www.bioone.org/loi/apps © 2016 Wöhrmann et al. Published by the Botanical Society of America. This work is licensed under a Creative Commons Attribution License (CC-BY-NC-SA). 1 of 4 Applications in Plant Sciences 2016 4(1): 1500084 Wöhrmann et al.—Fosterella christophii microsatellites doi:10.3732/apps.1500084 TABLE 1. Characteristics of 22 microsatellite loci and flanking primer pairs developed for Fosterella christophii. Allele GenBank SSR a,b c Locus Primer sequences (5′–3′) Repeat motif size (bp) Ta (°C) accession no. BLASTX description % location Foc_01 F: CCTCACTATCGCTCACTACAT (AG)15 146 55.2 KT036677 PREDICTED: cinnamoyl-CoA reductase 1-like isoform 80 5′UTR R: GTCACGCACACCAACTTC X1 [Phoenix dactylifera] Foc_02 F: GAGGCATTGGGTTTTTCT (TC)17 147 55.3 KT036678 No match found — — R: AGATCTGCGGCTACATCTC Foc_03 F: CCTTATTCCCCAAATCATAAA (AG)17 148 55.8 KT036679 PREDICTED: tetraspanin-3-like [Musa acuminata 80 5′UTR R: CTACCTCCTCTTCCTCTTCCT subsp. malaccensis] Foc_04 F: GCCATTGAGTTCACCAAGT (AG)20 145 55.5 KT036680 PREDICTED: tricetin 3′,4′,5′-O-trimethyltransferase 77 3′UTR R: ACAACCCAAGCAATAATAACA [Phoenix dactylifera] Foc_05 F: CTTCTCCTTCTCCTCCATCT (TCG)7 136 55.4 KT036681 PREDICTED: ribonuclease 2 [Musa acuminata 78 5′UTR R: AATAGGTCAGAGGATTTGAGC subsp. malaccensis] Foc_06 F: CGTCAATCTCAATCCCTTC (CCT)7 138 55.3 KT036682 PREDICTED: secretory carrier-associated membrane 88 5′UTR R: ACCTGCACTACTCAGAGGAA protein 2-like isoform X1 [Elaeis guineensis] Foc_07 F: TTCATCGCCTCTCGTTTAT (CTC)7 130 57.9 KT036683 PREDICTED: uncharacterized protein LOC103707719 86 5′UTR R: CTTCGCCGTACCTCCAGTAG isoform X2 [Phoenix dactylifera] Foc_09* F: TAAAGGGAGAGAGAGGAAGAA (AGA)8 168 55.3 KT581625 PREDICTED: phosphoribulokinase, chloroplastic 93 5′UTR R: GATGAGCTGCTGCTTCTG [Pyrus ×bretschneideri] Foc_10* F: CTCCTTTTTCCTTTTCCTTTA (AGG)8 140 54.6 KT581626 Ubiquitin-conjugating enzyme 32 [Theobroma cacao] 87 5′UTR R: CCGTGTTCTTCTTGTTGTACT Foc_11* F: GAGGGTAAATTTCTCTGCTTC (GAA)9 204 55.2 KT581627 Best match <75% sequence similarity — — R: TACGATGTACAGCTAGGGATG Foc_12 F: CACAAATGTGCTCTTCTGG (TCT)14 153 55.0 KT036684 Best match <75% sequence similarity — — R: CGTGGGATCTCTATCGTG Foc_15* F: GAGGACTTCGGTGTAATTTGT (ATTTT)4 185 55.2 KT581628 Best match <75% sequence similarity — — R: CCAACGGAAGAGTTCATAATA Foc_16** F: CTCAGCTGAACAATTCTGAG (GAAGA)5 194 54.3 KT581629 PREDICTED: sec-independent protein translocase 90 5′UTR R: ACTTGGAGATGGAAGATAAGG protein TATC, chloroplastic-like [Oryza brachyantha] Foc_17* F: GCCATTGTCCAGAAGTCC (TCCTC)4 153