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The Chloroplast Psbk-Psbi Intergenic Region, a Potential Genetic Marker

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The Chloroplast Psbk-Psbi Intergenic Region, a Potential Genetic Marker HORTSCIENCE 49(10):1244–1252. 2014. chloroplast trnG intron, trnH-psbA, and trnC-ycf6 intergenic spacers in Anthurium has recently resulted in the reclassification The Chloroplast psbK-psbI Intergenic and improved understanding of the various subgeneric sections and species relationships Region, a Potential Genetic Marker in this genus (Carlsen, 2011; Carlsen and Croat, 2013; Croat and Carlsen, 2013). This for Broad Sectional Relationships is the most comprehensive molecular phy- logeny of Anthurium to date, providing a con- temporary framework for identifying species, in Anthurium their origins, interrelationships, evolution, Jon Y. Suzuki1, Tracie K. Matsumoto, Lisa M. Keith, and diversification. Lemnaceae-specific chlo- and Roxana Y. Myers roplast DNA markers for the rpoB, rpoC1, matK,andrbcL gene regions, and the USDA, ARS, Daniel K. Inouye U.S. Pacific Basin Agricultural Research trnH-psbA, atpF-atpH,andpsbK-psbI inter- Center, Tropical Plant Genetic Resources and Disease Research Unit, 64 genic regions were recently developed (Wang Nowelo Street, Hilo, HI 96720 et al., 2010) based on a recommendation by the Consortium for the Barcode of Life (CBOL) Additional index words. Araceae, barcode, CBOL, genotyping, germplasm, ornamentals, (CBOL Plant Working Group, 2009), which phylogeny, sectional groupings allowed evaluation of DNA barcoding markers Abstract. Nuclear and chloroplast genetic markers have been extensively used for plant for species identification in this family. identification and molecular taxonomy studies. The efficacy of genetic markers to be used as In this study, we further explored the DNA barcodes is under constant evaluation and improvement with identification of new marker genes used in Lemna to supplement barcodes that provide greater resolution and efficiency of amplification for specific species those presently used for molecular systematic groups as well as distantly related plants. In this study, chloroplast DNA genetic markers for studies in Anthurium as a result of their Anthurium, the largest genus in the Araceae family, were adapted from chloroplast markers relatively close phylogenetic relationship previously designed for Lemna minor, a member of the same plant family. Primers for (Rothwell et al., 2004). Although we did chloroplast region trnH-psbA, previously used for molecular systematic studies in Anthurium, not have access to a large collection of live as well as primers for the rpoB, rpoC1, psbK-psbI, matK, rbcL,andatpF-atpH regions, all accessioned species of Anthurium, dried leaf located within the large single copy sequence in the chloroplast genome, were evaluated and material of species from previously identified found to efficiently amplify target sequences when using DNA of varied quality and collections was obtained from various sour- concentration extracted from silica-dried leaves of selected accessioned species of Anthurium. ces and stored on silica. The goal of this study The trnH-psbA, psbK-psbI,andatpF-atpH intergenic region primers were further evaluated was to determine the appropriateness of using using Anthurium species spanning different subgeneric groups. Of the intergenic region dried leaf material as the source of amplifi- primers tested, psbK-psbI primers were the most robust, yielding well-defined amplicons able DNA and for further development of across Anthurium species that were consistent, with exceptions, within sectional groupings. diagnostic gene markers. Approaches for Application of the psbK-psbI region amplicon as a visual marker for surveying sectional applying known phylogenetic information relationships in Anthurium is novel and serves as a model for the development of a diagnostic for practical diagnostic genotyping of An- method for genotyping plants and testing for sample integrity from among species or thurium species or germplasm in collections germplasm collections. This work further demonstrates the use of dried plant tissue banks as were also investigated. a genetic reference and information resource to support basic research as well as ornamental plant characterization and improvement. Materials and Methods DNA extraction from Anthurium plant leaves. Anthurium leaves from various sour- Research at the USDA ARS Pacific Basin A. amnicola, A. antioquiense, A. antrophyoides, ces that had been stored in silica at ambient Agricultural Research Center (PBARC) Trop- A. armeniense, A. formosum, A. hoffmannii, A. temperature were used as initial material for ical Plant Genetic Resources and Disease kamemotoanum,andA. nymphaeifolium,as DNA extractions. Approximately 20 mg of Research unit in Hilo, HI, includes support well as hybrids between species within the leaf dry weight was placed into Lysing for the improvement of tropical plant orna- sections Porphyrochitonium and Cardio- Matrix D tubes (MP Biomedicals) and ho- mental and fruit crops, including cut and lonchium, and intersectional hybrids between mogenized for 20 s in MP Fast Prep-24 potted Anthurium for the Hawaiian floral Calomystrium and Cardiolonchium, among Buffer PL1. RNaseA obtained from Nucleo- industry. Anthurium are the top cut flower in others (Kamemoto and Kuehnle, 1996). One SpinÒ Plant II kit (Macherey Nagel USA, Hawaii with sales in 2012 of 348,000 dozen of the ongoing research goals at PBARC is the Bethlehem, PA) was then added to each tube flower stems valued at $3.34 million and development of molecular resources to identify following recommended volumes and the 35,000 potted Anthurium were sold for a value origins of ornamental and horticultural diver- mixture was homogenized for an additional of $267,000 (USDA, National Agriculture sity at the genome and molecular biological 20 s. The contents of each tube were then Statistics Service and Hawaii Department of levels (Bliss and Suzuki, 2012) to support centrifuged and transferred to a NucleoSpinÒ Agriculture Agricultural Development Divi- improvement of cultivars through breeding filter placed in a collection tube and purified sion, 2013). The genus Anthurium, the largest and biotechnological applications (Matsumoto using NucleoSpinÒ Plant II following the of the Araceae family, is comprised of 905 et al., 2013). manufacturer’s recommendations. DNA described species that range from southern In tropical ornamentals, including Anthurium, yields were 5.3 to 347 ng·mL–1 in a total Mexico to northern Argentina (Carlsen, 2011; very few tools, simple or complex, for molecular volume of 50 mL. Govaerts et al., 2014), but based on the evaluation of germplasm are available. Chlo- Primer design. Anthurium chloroplast number of estimated new species to be de- roplast gene markers have been used exten- gene marker primers were adapted from scribed, this figure may increase to 1500 taxa sively for molecular identification in plants, the Lemna minor chloroplast DNA barcode (T.B. Croat, unpublished data; Boyce and although their use for species-level resolution primer sequence (Wang et al., 2010) and Croat, 2012). Many modern commercial is dependent on the genes used and the target Anthurium polymerase chain reaction Anthurium cultivars are derived from interfer- particular family of plants being studied (PCR) product lengths estimated using se- tile hybrids, mainly from species within sec- (Shaw et al., 2005, 2007). Sequence analysis quences obtained from high-throughput se- tion Calomystrium,includingA. andraeanum, of the nuclear CHS gene intron and the quencing of chloroplast-enriched DNA from 1244 HORTSCIENCE VOL. 49(10) OCTOBER 2014 | BREEDING,CULTIVARS,ROOTSTOCKS, AND GERMPLASM RESOURCES Table 1. Anthurium species chloroplast gene marker primers adapted from Lemna minor chloroplast DNA barcode primer sequences (Wang et al., 2010) based on A. andraeanum Hort. ‘New Pahoa Red’ chloroplast DNA sequences.z Anthurium Target Lemna minor andraeanum Primer Primer gene regions amplicon (bp) amplicon (bp) Primers Primer sequence length Tm trnH-psbA 300 335 *Aa.trnH-psbA.F 5#-GTGATGCATGAACGTAATGCTC-3# 22 60.8 Lm.trnH-psbA.F 5#-GTTATGCACGAACGTAATGCTC-3# LmAa.trnH-psbA.R 5#-CGCGCGTGGTGGATTCACAATCC-3# 23 68.1 rpoB 406 426 LmAa.rpoB.F 5#-ATGCAGCGTCAAGCAGTTCCGTTCC-3# 25 67.9 LmAa.rpoB.R 5#-TCGGATGTGAAAAGAAGTATA-3# 21 54.8 rpoC1 509 529 *Aa.rpoC1.F 5#-GGCAAAGAGGGAAGATTCCG-3# 20 62.4 Lm.rpoC1.F 5#-GGAAAAGAGGGAAGATTCCG-3#; LmAa.rpoC1.R 5#-CAATTAGCATATCTTGAGTTGG-3# 22 57.1 psbK-psbI 544 541 *Aa.psbK-psbI.F 5#-TTAGCCTTTGTTTGGCAAG-3# 19 55.8 Lm.psbK-psbI.F 5#-TTAGCATTTGTTTGGCAAG-3# LmAa.psbK-psbI.R 5#-AAAGTTTGAGAGTAAGCAT-3# 19 51.5 rbcL 580 599 *Aa.rbcL.F 5#-GTAAAATCAAGTCCACCGCG-3# 20 60.4 Lm.rbcL.F 5#-GTAAAATCAAGTCCACCACG-3#; *Aa.rbcL.R 5#-ATGTCACCACAAACAGAAACTAAAGC-3# 26 61.4 Lm.rbcL.R 5#-ATGTCACCACAAACAGAGACTAAAGC-3# atpF-atpH 675 750 LmAa.atpF-atpH.F 5#-ACTCGCACACACTCCCTTTCC-3# 21 64.5 LmAa.atpF-atpH.R 5#-GCTTTTATGGAAGCTTTAACAAT-3# 23 55.6 matK 862 889 *Aa.matK.F 5#-CGTACCGTACTTTTATGTTTACGAG-3# 25 61.3 Lm.matK.F 5#-CGTACTGTACTTTTATGTTTACGAG-3#; *Aa.matK.R 5#-ATCCCATCCATCTGGAAATCCTGGTTC-3# 27 66.1 Lm.matK.R 5#-ATCCGGTCCATCTAGAAATATTGGTTC-3# zDNA bases shown in bold denote base pairs that differ between Anthurium (*Aa.xx) and L. minor (Lm.xx) primers; LmAa.xx denotes primer sequences identical at the corresponding target gene regions of L. minor and A. andraeanum. Primer Tm was based on a general formula provided by the manufacturer (see ‘‘Materials and Methods’’). A. andraeanum Hort. ‘New Pahoa Red’ matK are coding genes, encoding the beta sizes (Table 1). PCR primer sequences for (Suzuki, unpublished data). Primers were and N-terminus of the beta’s subunit, re- rpoB and
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