Analysis of Genetic Variability among Phalaenopsis Species and Hybrids Using Amplified Fragment Length Polymorphism

Phalaenopsis is the second most valuable potted plant in the United States. Information on the genetic diversity and relationships among species and hybrids is important for breeding purposes and species conservation. In this study, genetic variability of 16 Phalaenopsis species and hybrids was analyzed using amplified fragment length polymorphism (AFLP) markers. Ten AFLP primer combinations amplified 1353 DNA fragments ranging in size from 100 to 350 bp and 1285 (95%) of them were polymorphic. The genetic similarity among Phalaenopsis species and hybrids ranged from 0.298 to 0.774 based on Dice coefficient. The dendrogram derived by the unweighted pair group method with arithmetic mean analysis clustered the germplasm into two main groups. Bootstrap values for the groups supported 70% of the clustering. A significant linear relationship (r = 0.724, P < 0.0001) was observed between known pedigrees and AFLP-derived genetic similarity for 136 pairwise comparisons of Phalaenopsis species and hybrids. The results of this study demonstrate the usefulness of AFLP analysis in Phalaenopsis and its potential application in breeding and species conservation. Orchids are not only beautiful, but also commercially important and occupy the second place in the potted flowering plant market in the United States (Griesbach, 2002). According to the U.S. Department of Agriculture (USDA) floriculture crops 2007 survey, the potted orchid industry was valued at $126 million (USDA, 2008). Among potted orchids, Phalaenopsis comprises the majority of commercial orchids (50% to 90%) in the United States as a result of their long-lasting flowers, easy care, and low cost (Griesbach, 2002; Laws, 2004). The orchid family is unique because hybridization is possible not only between species, but also between members of related genera. Moreover, hybrids can interbreed with other species to create novel combinations (Tan and Hew, 1995). As a result of these features, genetic relationships are sometimes difficult to evaluate. However, accurate information on genetic relationships among species and hybrids is needed for the conservation and use of breeding materials. Through intensive breeding activity, 3500 Odontoglossum hybrids and intergenerics, 4300 Cattleyas hybrids and intergenerics, and over 5000 Paphiopedilum crosses had been made by 1945 (American Orchid Society, 2002). However, only 140 Phalaenopsis hybrids were registered through 1945 (Challis, 1996). An average of 100 new Phalaenopsis hybrids was registered in the Royal Horticulture Society every 2 months from 1976 through 2008, underscoring the importance of novel Phalaenopsis hybrids for market demands. Despite the economic importance of Phalaenopsis, the genetic potential of Phalaenopsis has not been fully exploited. An assessment of genetic variability is important for the use of genetic resources and for determining the uniqueness of genotypes to protect breeders’ intellectual property rights and to exploit heterosis (Franco et al., 2001). Genetic diversity can be determined using phenotypic variation and/or molecular markers. Morphological characteristics have limitations because environmental factors and the developmental stage of the plant may influence their expression. In contrast to morphological markers, molecular markers based on DNA polymorphism are more informative, independent of environmental conditions and unlimited in number (Agarwal et al., 2008). Several types of molecular markers are available; however, amplified fragment length polymorphism (AFLP) markers have notable advantages such as reproducibility, high levels of polymorphism that can be detected in a single reaction, genomewide distribution of markers, and no need for prior DNA sequence information compared with other DNA-based markers (Vos et al., 1995). AFLP analysis uses selective amplification of a subset of restriction enzyme-digested DNA fragments to generate a unique fingerprint of a particular genome (Mueller and Wolfenbarger, 1999). AFLP markers have been used in the assessment of genetic relationships of a wide range of species, including ornamentals such as Caladium (Loh et al., 1999), Calathea (Chao et al., 2005), Hemerocallus (Tomkins et al., 2001), Cornus (Smith et al., 2007), and Received for publication 2 Sept. 2008. Accepted for publication 14 Oct. 2008. This project was supported in part by a grant from the U.S. Department of Agriculture (USDA 2003-38891-02112), USDA HATCH funds (135816) as well as through operating funds provided by the Commonwealth of Virginia. We thank the Institute for Advanced Learning and Research (IALR) for supplying materials. We also thank Rubina Ahsan for helpful technical advice. Y.K. Chang was a graduate student at the Department of Horticulture at Virginia Tech. The research was conducted at ISRR in MJI laboratory and coadvised by REV. Corresponding author. E-mail: [email protected]. 58 J. AMER. SOC. HORT. SCI. 134(1):58–66. 2009. Impatiens (Carr et al., 2003). The objectives of this study were to determine the level of genetic variability in Phalaenopsis species and hybrids using AFLP fingerprinting and to estimate the correlation of genetic relatedness with the known pedigree in various hybrids. Materials and Methods PLANT MATERIAL. Two commercially available Phalaenopsis species and 14 hybrids were used in this study (Table 1). The germplasm represents a range of flower size and colors available in the U.S. market. All the plants were maintained at 20 to 30 C with 70% humidity in the greenhouse. DNA EXTRACTION. Genomic DNA was extracted from fresh Phalaenopsis leaves according to Doyle and Doyle (1990) with some modifications. Plant tissue (0.5 g) was pulverized in liquid nitrogen and immediately transferred into a 50-mL tube containing 10 mL extraction buffer [2% CTAB, 100 mM Tris (pH .8.0), 1.4 M NaCl, 20 mM EDTA, 0.2% 2-mercaptoethanol, and 4% polyvinyl pyrrolidine]. The samples were incubated at 60 C for 1 h and 10 mL chloroform:isoamyl alcohol (24:1) was added to each sample and mixed. The samples were centrifuged at 12,000 g for 10 min. The aqueous phase was transferred to a clean tube. DNA was precipitated with 5 mL of ice-cold isopropanol and stored at –20 C overnight. The samples were centrifuged at 5000 g for 10 min and the supernatant was discarded. The DNA pellet was resuspended in 20 mL wash buffer (10 mM NH4OAc, 75% ethanol) for 20 min and centrifuged at 12,000 g for 5 min. The DNA was dissolved in TE buffer (100 mL) containing 100 mg of RNase and incubated at 37 C for 1 h. DNA concentration was measured using a NanoDrop (Thermo Fisher Scientific, Waltham, MA) and quality was checked by electrophoresis on a 0.8% agarose gel in TBE buffer. AMPLIFIED FRAGMENT LENGTH POLYMORPHISM ANALYSIS. AFLP analysis was carried out according to Vos et al. (1995) with some modifications. Genomic DNA (500 ng) was restriction-digested with EcoRI and MseI and EcoRI and MseI adapters were ligated in a final volume of 11 mL. The reaction contained 1· T4 ligase buffer [50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 10 mM dithiothreitol, 1 mM ATP] [New England Biolabs (NEB), Ipswich, MA], 0.05 M NaCl, 0.045 mg mL BSA, 1 mM EcoRI adapter, 5 mM MseI adapter, 5 U EcoRI (NEB), 5 U MseI (NEB), and 1 U T4 DNA ligase (NEB). The reactions were gently mixed and incubated at 37 C for 3 h. After restriction and ligation, the reaction mixture was diluted 10-fold with 0.1· TE buffer [10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA]. Preselective amplification was carried out in a final volume of 13 mL consisting of 1· polymerase chain reaction (PCR) buffer [100 mM Tris-HCl (pH 8.3), 500 mM KCl], 2.0 mM MgCl2, 0.2 mM dNTP, 10 mM EcoRI+A primer, 10 mM MseI+C primer, and 3 mL of diluted restriction-ligation product. PCR was carried out in a MyCycler thermal cycler (Bio-Rad Laboratories, Hercules, CA) programmed for 72 C for 2 min followed by 20 cycles of 94 C for 20 s, 56 C for 30 s, and 72 C for 2 min and a final incubation of 72 C for 2 min and 60 C for 30 min. The preselective amplification PCR products were diluted 10-fold in 0.1· TE buffer and used as a template for selective amplification. Selective amplification was carried out in 8 mL reaction volume containing 1· PCR buffer [100 mM Tris-HCl (pH 8.3), 500 mM KCl], 2.0 mM MgCl2, 0.2 mM dNTPs, 0.625 mM of D4 WellRED dye-labeled EcoRI primer (E+3), 0.625 mM MseI primer (M+3), 0.2 U of JumpStart Taq DNA polymerase (Sigma-Aldrich, St. Louis, MO), and 2 mL of diluted preselective amplification product. The PCR amplification consisted of an initial denaturation step of 94 C for 2 min followed by the first cycle of 94 C for 20 s, 66 C for 30 s, 72 C for 2 min, and 1 C decrease in annealing temperature in each of the next nine cycles. This was followed by 25 cycles of 94 C for 30 s, 56 C for 30 s, and 72 C for 3 min. The reactions were incubated at 60 C for 30 min before electrophoresis. PCR products were diluted two-fold with sample loading solution (Beckman-Coulter, Fullerton, CA) and 1.5 mL of diluted reaction products were added to 40 mL of sample loading solution (Beckman-Coulter). DNA size standard 600 (Beckman-Coulter) was also added to each sample. The samples were electrophoresed and detected using a Beckman-Coulter CEQ 8800 Genetic Analysis System. The Frag-4 module of CEQ was used to size all the fragments using internal DNA size standard. DATA ANALYSIS. All AFLP fragments were scored as binary data (1, peak present; 0, peak absent) along with their sizes. The binary scores were manually compared with the electropherograms to reconfirm presence or absence of peaks. NTSYS-pc software version 2.20 (Rohlf, 2005) was used to calculate the genetic distance or similarity between the samples. A cluster analysis was performed using unweighted pair group method with arithmetic mean (UPGMA) based on the Dice index (Nei and Li, 1979). This analysis was also compared using the FreeTree software package (Hampl et al., 2001). Bootstrap values (based on 1000 resamplings) were used to estimate the reliability of the clustering pattern. Coefficients of coancestry for all possible pairs of Phalaenopsis species and hybrids were calculated according to Weir et al. (2006) based on pedigree information in the Royal Horticulture Society (RHS) orchid hybrid registration database information system (RHS, 2002). Simple linear correlation analysis was conducted to determine the association between coancestry coefficient and genetic similarity values using JMP software (version 5.1; SAS Institute, Cary, NC).