An efficient and reproducible protocol for production ...

An efficient and reproducible protocol for production of AFLP markers in tree genomes using fluorescent capillary detection Rodrigo Hasbún, Carolina Iturra, Priscila Moraga, Pamela Wachtendorff, Pamela Quiroga & Sofía Valenzuela Tree Genetics & Genomes ISSN 1614-2942 Tree Genetics & Genomes DOI 10.1007/s11295-011-0463-6

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Author's personal copy Tree Genetics & Genomes DOI 10.1007/s11295-011-0463-6

SHORT COMMUNICATION

An efficient and reproducible protocol for production of AFLP markers in tree genomes using fluorescent capillary detection Rodrigo Hasbún & Carolina Iturra & Priscila Moraga & Pamela Wachtendorff & Pamela Quiroga & Sofía Valenzuela

Received: 4 August 2011 / Revised: 2 December 2011 / Accepted: 12 December 2011 # Springer-Verlag 2011

Abstract An optimized protocol for the development and discovery of polymorphic AFLP markers in tree species is described. The protocol was optimized for the production of fluorescently labeled PCR products and analysis using a capillary sequencer. This approach has been demonstrated to be efficient and reproducible for tree species with complex genomes. The most important modification was in the selective amplification step. Instead of using a traditional step down PCR, a fixed and higher annealing temperature was employed, improving the reproducibility and sensitivity of the protocol. The levels of polymorphisms detected with the optimized protocol on three woody species are in agreement with those previously reported in the literature for tree species. Keywords Genetic diversity . Molecular marker . Capillary electrophoresis

Communicated by S. González-Martínez R. Hasbún (*) : C. Iturra : P. Wachtendorff Centro de Biotecnología, Genómica Forestal S.A., Universidad de Concepción, Casilla 160 C, Concepción, Chile e-mail: [email protected] R. Hasbún : P. Moraga : P. Quiroga : S. Valenzuela Facultad de Ciencias Forestales, Universidad de Concepción, Casilla 160 C, Concepción, Chile S. Valenzuela Centro de Biotecnología, Universidad de Concepción, Victoria 631, Casilla 160 C, Concepción, Chile

Introduction The technology of amplified fragment length polymorphism (AFLP) markers allows to simultaneously detect a large number of polymorphisms in different regions of the target genome. Given that AFLP technology has a better reproducibility, resolution, and sensitivity than other techniques for multilocus genomic fingerprinting (e.g., RAPD and ISSR), it has been efficiently used in the identification of genetic variation and applications derived there from Mueller and Wolfenbarger (1999), Meudt and Clarke (2007), Vuylsteke et al. (2007), and Romero et al. (2009). Although AFLP technology has been successfully employed in plants (Bensch and Åkesson 2005), its versatility is limited when it is applied to species with complex genomes such as trees; therefore, it is necessary to optimize its protocols (e.g., Paglia and Morgante 1998; Cervera et al. 2000). The main disadvantage of these protocols is the use of radiolabeled primers, which reduce the work flow flexibility and AFLP analysis (Hartl and Seefelder 1998; Huang and Sun 1999). Numerous variants have been implemented since the original AFLP protocol was published by Vos et al. (1995). The main protocol improvements have included: (1) the use of IRDye® infrared dye (IRD) or other fluorescently labeled oligonucleotide primers instead of radioactive ones, and (2) fragment analysis with an automated DNA sequencer instead of polyacrylamide gel electrophoresis. These modifications increase the efficiency and throughput of the procedure (Hartl and Seefelder 1998; Huang and Sun 1999). AFLP markers generated with the original protocol, but using IRD primers and visualization of fragments by a gel-based sequencer such as a LI-COR DNA Analyzer produced successful results for plant species with genomes of varying complexity (Remington et al. 1999; Klein et al.

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2000; Myburg et al. 2001; Komulainen et al. 2003; Ukrainetz et al. 2008). In the case of fluorescent AFLP markers, several commercial kits have been developed to visualize amplified fragments using a capillary sequencer, e.g., Genetic Analyzer (Applied Biosystems) or GenomeLab (Beckman Coulter). In these cases, successful results have been obtained in plants that have genomes with a low level of repetitive sequences or small genomes (Vendrame et al. 1999; Abedinia et al. 2000; Schnell et al. 2001; Gonçalves et al. 2005; Karudapuram and Larson 2005; Grundt et al. 2006; Parisod and Bonvin 2008; Zhang et al. 2010). Use of commercial kits or alternative protocols for the generation of fluorescent AFLP markers in sequencer capillary systems has yielded unsatisfactory results for species with complex genomes (Kim et al. 2003, 2005, 2011; Colbeck et al. 2008). Until now, no efficient protocol for fluorescent AFLP marker genotyping using capillary gel electrophoresis for species with complex genomes is available. In addition, the existing commercial kits for AFLP genotyping are inapplicable to certain plant species, especially those with large and complex genomes such as conifers. The optimized protocol described here was based on the original procedure implemented by Vos et al. (1995) and the adapted AFLP protocol described for the ABI PRISM™ fluorescent dye labeling (Wolf et al. 2004). The final protocol was the result of an optimization process in order to achieve high reproducibility and sensitivity together with a wide range of fragment resolution using a capillary sequencer (3130xl Genetic Analyzer).

Plant samples and reagents Plant samples corresponded to needle or young leaf tissues of three tree species: Pinus radiata (n030), Eucalyptus globulus (n030), and Nothofagus alpina (n08). Genomic DNA was extracted using the DNeasy Plant Mini kit (QIAGEN Inc., Valencia, CA, USA) according to the instructions of the manufacturer. Total genomic DNA was quantified using a Nanodrop ND-1000 UV/Vis (Nanodrop Technologies, Wilmington, DE, USA) spectrophotometer and qualitatively analyzed by agarose gel electrophoresis. Restriction endonucleases (EcoRI, MseI) and T4 DNA Ligase were obtained from New England Biolabs (NEB, Ipswich, MA, USA). Taq DNA Polymerase and Platinum Taq DNA Polymerase were purchased from Invitrogen (Invitrogen Brazil Ltda., São Paulo, Brazil). dNTPs were obtained from Fermentas (Fermentas International Inc., Burlington, Canada). EcoRI and MseI adaptors were equimolar mixtures of two oligonucleotides, which were custom synthesized at Integrated DNA Technologies (IDT Inc., Coralville, IA, USA) according to Vos et al. (1995). The pairs of complementary oligonucleotides were mixed, heated at 95°C for 5 min to denature, and then cooled gradually at 3°C/min to renature completely. Oligonucleotides used for the preselective amplification were Eco+0 (5′-GACTGCGTACCAATTC) and Mse+0 (5′-GATGAGTCCTGAGTAA), and those used for the selective amplification contained three or four additional selective nucleotides (Table 1). Selective oligonucleotides were synthesized and labeled at the 5′-end with a fluorescent dye at Applied Biosystems Custom Oligonucleotide

Table 1 Total number of AFLP markers and number of polymorphisms (within parentheses) generated in each species per selective oligonucleotide combination Oligonucleotide combination

P. radiata (n030)

Scoring error rate (%)a

Oligonucleotide combination

E. globulus (n030)

N. alpina (n08)

ACA/AGCT ACA/CCCG ACA/CCGA ACC/AGGC ACC/CCGA ACC/CCTA ACG/AGAC ACG/AGCT ACG/CCAA ACG/CCCA ACG/CCTC ACG/CCTG All combined Polymorphic loci

96 (43) 69 (48) 118 (76) 90 (33) 63 (40) 124 (68) 84 (57) 75 (25) 73 (25) 79 (43) 87 (51) 68 (43) 1,026 (552) 53.1%

1.7 0.5 1.8 1.7 0.6 2.8 0.6 0.7 1.1 1.0 1.8 0.7 1.2

ACA/CAT ACA/CCG ACA/CTG ACC/CAT ACC/CCG ACC/CTG ACG/CAT ACG/CCG ACG/CTG ACT/CAT ACT/CCG ACT/CTG All combined Polymorphic loci

87 (40) – 60 (22) 124 (57) 34 (21) 52 (28) 97 (44) 38 (24) – 99 (42) – – 591 (278) 49.4%

59 (31) 22 (10) 35 (12) 44 (21) – 38 (23) 35 (12) 14 (8) 25 (16) 62 (30) – 30 (17) 364 (180) 50.4%

a

Calculated from the eight individuals that were reassayed as 100×(number of discordant scores on three independent analyses)/(number of scored markers×number of individuals)

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Synthesis Service (Applied Biosystems Inc, Foster City, CA, USA). 3130xl Genetic Analyzer Sequencer, 3130xl/ 3100 Genetic Analyzer Capillary Array 36 cm (part number 4315931), POP-4™ Polymer for 3130/3130xl Genetic Analyzers (part number 4352755), Hi-Di formamide, the internal size standard GeneScan™ 500 LIZ®, and the software GeneMapper® v. 4.0 were purchased from Applied Biosystems Inc. Identification of commercial products in this paper was done in order to specify the experimental procedure, but does not imply endorsement nor recommendation.

Production of fluorescence-labeled AFLP markers The genomic DNA digestion and ligation of adapters were performed in a single step. Each reaction was conducted in a 11-μl volume containing 50 ng of sample DNA, 1× T4 DNA ligase buffer (50 mM Tris–HCl, 10 mM MgCl2, 10 mM DTT, 1 mM ATP), 55 ng BSA, 50 μM NaCl, 5 U EcoRI, 1 U MseI, 10 cohesive end units T4 DNA ligase, 5 pmol EcoRI adaptor, and 50 pmol MseI adaptor pairs. The mixture was incubated for 2 h at 37°C, and then diluted to 22 μl with low TE buffer (1 mM Tris–Cl; 0.1 mM EDTA; pH 8.0) and stored at −20°C until use. Preselective amplification was performed using 3 μl of the diluted digestion–ligation with a total volume of 25 μl. The reaction mixture volume contained 1× PCR buffer, 1.5 mM MgCl 2 , 0.12 mM of each dNTP, 0.2 μM Eco+0 oligonucleotide, 0.2 μM Mse+0 oligonucleotide, and 0.5 U Taq DNA polymerase. PCR amplification was carried out with a preliminary denaturation of 2 min at 94°C, then 25 cycles of 20 s at 94°C, 30 s at 60°C, and 2 min at 72°C. This preselective amplification was compared with amplification using Eco+2/Mse+2 oligonucleotides. Preselective amplifications were visualized on agarose gel to check amplification. Finally, amplification products were diluted 1:10 with low TE buffer. A set of selective oligonucleotide combinations with Eco+3/Mse+4 were used for P. radiata and Eco+3/Mse+3 for E. globulus and N. alpina (Table 1) in order to validate of optimized protocol and test the transferability to other tree species. Selective amplification was done using 2.5 μl from the diluted preselective amplification as template in reactions containing 1× PCR buffer, 1.5 mM MgCl 2 , 0.1 mM of each dNTP, 0.1 μM Eco+ACN oligonucleotide, 0.5 μM Mse+3 or Mse+4 oligonucleotide, and 0.5 U Platinum Taq DNA polymerase in a total volume of 12.5 μl. Amplification was performed with an initial denaturation of 2 min at 94°C, then 30 cycles with the following cycle profile: 20 s at 94°C, 60 s at 66°C, and 2 min at 72°C, followed by 30 min at 60°C to avoid split peaks.

Capillary gel electrophoresis and data scoring After selective amplification, 1.7 μl of PCR product was mixed with loading buffer (10 μl Hi-Di formamide and 0.1 μl GenScan 500 LIZ molecular weight marker). Samples were loaded on the automated DNA capillary sequencer AB 3130xl, using a 36-cm column and POP-4™ Polymer. The run module FragmentAnalysis36_POP-4 with some modifications in the electrophoresis parameters was used: injection time 10 s, injection voltage 10 kV, and running voltage 13 kV. The dye set used was DS-33 (filter set G5). Analysis of raw fluorescent AFLP data was performed and visualized using Genemapper v.4.0 software. A minimum fluorescence threshold value of 200 was chosen. Each unique peak in the chromatogram was interpreted as an independent genetic locus without considering those loci represented by overlapping peaks. Polymorphic peaks were those that differed within species, and at least two samples had the corresponding amplification. The peaks between 50 and 500 bp were included in the analysis and differences in intensity were taken into account.

Development of an optimized protocol using P. radiata In order to detect AFLP markers for tree species with complex genomes, a fluorescent labeling and capillary gel electrophoresis protocol was established using samples of P. radiata as a model species. The most important modification with respect to the original AFLP protocol was in the selective amplification step. Instead of using a step down PCR, a fixed and higher annealing temperature was employed. Different fixed annealing temperatures covering the range of the original step down PCR (58°C, 62°C, and 66°C) had evident quantitative and qualitative effects on the band profile (Fig. 1). The number of well separated peaks and high intensity peaks increased with annealing temperature. This result suggests that the binding efficiency of the selective oligonucleotide is affected negatively by lower annealing temperatures, influencing the reliability of the AFLP profile through preferential amplification of a subset of fragments. In theory, by using a step down PCR, initial higher temperatures give a greater specificity for oligonucleotide binding and final lower temperatures allow a more efficient amplification of the specific products formed during initial cycles. However, results obtained using fixed annealing temperatures showed that the AFLP profile is consistent with higher annealing temperatures. As described by Vos et al. (1995), no more than two selective nucleotides should be added to the EcoRI primers between two consecutive PCR amplifications to reduce background in the fingerprint and to ensure a high selectivity. However the optimized protocol produced identical

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Fig. 1 AFLP electropherograms of two different P. radiata samples. The optimized protocol using three different annealing temperatures (58°C, 62°C, and 66°C) for the selective amplification is shown. A

magnified view of some peaks is shown in the right panel. The filled green peaks show representative polymorphisms detected at the higher annealing temperature

fingerprints either using Eco+0/Mse+0 or Eco+2/Mse+2. According to the selective combination used, 0 to 2 fragments showed differences, i.e., presence using Eco+0/Mse+0 and absence using Eco+2/Mse+2 (Fig. 2) or vice versa. The protocol developed by Remington et al. (1999) for IRD-labeled AFLP and detection on gel-based sequencers (i.e., LI-COR IR2 automated sequencer) was used to compare with the optimized protocol. The entire production process of AFLP markers was performed according to Remington et al. (1999) and analyzed in the AB 3130xl sequencer. Comparison of the electropherograms demonstrated that the optimized protocol is more efficient for capillary gel electrophoresis than the Remington protocol (Fig. 3). The latter produced detectable peaks in the range of 50–300 bp, with single overamplified peaks around 130 bp, while peaks observed above 300 bp had low signal. These results are similar to those reported in other studies using the protocol of Remington with capillary detection system which only considered peaks ranging from 70 to 280 bp (Kim et al. 2003, 2011). Therefore, AFLP analysis system based on capillary gel electrophoresis requires specific protocols. Separation of DNA fragments by capillary gel electrophoresis has advantages over the classical slab gel-based separations in terms of speed, resolution, and performance. This mode of detection offers high sensitivity and improved

selectivity for samples containing fluorescent tags (Liu and Chen 2000). Most capillary gel electrophoresis systems utilize electrokinetic injection, which is highly sensitive to the sample/matrix mixture. The AFLP sample may vary in terms of some physicochemical properties (e.g., conductivity, salt content, pH, presence of contaminants), which can modify the migration velocity of analytes (Terabe et al. 2006). Optimization of AFLP samples for fluorescence detection in a capillary electrophoresis system can be complicated in a general context, and the amplification products of AFLP reactions should be specifically generated for analysis using this system, as demonstrated in this work using the optimized protocol.

Performance of optimized protocol and application to different tree species The optimized protocol was tested using additional genotypes of P. radiata and as well as other two tree species (E. globulus and N. alpina) which have smaller genome size (Bennett and Leitch 2010). Specific selective oligonucleotide primer combinations were used for each species (Table 1). Only descriptive statistics (average, standard deviation, and range) on polymorphisms observed in the different species are reported in this paper (Table 1). The AFLP data

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Fig. 2 Electropherograms of three P. radiata AFLP samples for the primer combination ACG/AGAC produced using Eco+0/Mse+0 or Eco+AC/ Mse+AG preselective reactions. The filled green peaks show fingerprint differences between reactions

obtained showed that the average percentage of polymorphic loci per oligonucleotide pair was 53±14% (range 33–70%) in

P. radiata, 49±9% (range 37–63%) in E. globulus, and 50± 12% (range 34–64%) in N. alpina. Percentage of polymorphic

Fig. 3 Representative electropherograms of two different P. radiata AFLP samples generated by a protocol for gel-based sequencer (Remington et al. 1999), and the optimized protocol for capillary gel electrophoresis

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AFLP loci reported in Pinus monticola was 64.7% (Kim et al. 2011), 61.9% in Pinus sylvestris (Lerceteau and Szmidt 1999), 78% in Araucaria angustifolia (Stefenon et al. 2007) and 48% in a hybrid of Eucalyptus grandis × E. globulus (Myburg et al. 2001). Although comparisons of AFLP polymorphism in different species depend on different variables, polymorphism of AFLP data previously reported in the literature for woody species is in agreement with our results. Quantifying genotyping error rates is an essential component of an AFLP study (Meudt and Clarke 2007). The error rate for AFLP loci, based on repeated tests on new DNA extracts from the same individuals, has been estimated to be between 2% and 5% (Mueller and Wolfenbarger 1999; Busch et al. 2000), but specific protocols for limiting the extent of such errors are not a standard practice (Bonin et al. 2004). Using the optimized protocol, genotyping error rates were determined for each primer combination in P. radiata according to Herrera and Bazaga (2009). The AFLP profiles obtained for eight unrelated genotypes of P. radiata with three different DNA extractions each showed extremely high reproducibility with error rates close to 1% (Table 1). Colbeck et al. (2008) using a similar protocol concluded that AFLP profiles seem highly sensitive to the quality of DNA and hence the sample storage and preparation methods.

Conclusion The development and discovery of efficient polymorphic molecular markers and their use in genetic studies have led to the development of several cost-effective protocols that can yield reliable information for different taxa, of differing genome size or complexity. Among PCR-based fingerprinting techniques, AFLP is known for its universal applicability (does not require specific oligonucleotides for each species), with a high reproducibility and discriminative power between and within species. New and more precise methodologies for detection and analysis of fragments have been developed such as capillary electrophoresis that require adjustment and optimization of existing protocols for use without dependency on genome complexity. This paper reports an optimized protocol that provides a highly reproducible profile of well-resolved AFLP markers with low genotyping error rates. Furthermore, the transferability of the optimized protocol to other tree species was demonstrated, showing results on three different woody species including a species with a recognized complex genome (P. radiata) and two species with smaller genome size and complexity (E. globulus and N. alpina). These species are important for forestry in different parts of the world and techniques for genomic DNA fingerprinting can yield significant economical and ecological benefits. This protocol

will provide a start point for researchers interested in using AFLP markers applied to tree species using fluorescently labeled PCR products and detection by a capillary sequencer. Acknowledgments The authors gratefully acknowledge the support from INNOVA-CHILE (grant number 05CTE04-03) and Genómica Forestal. Ross Whetten is gratefully acknowledged for the critical reading of this manuscript and discussions throughout this work.

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The AFLP process is covered by patents and patent applications held by Keygene NV (Wageningen, Netherlands), and users of the technique should either purchase a commercial kit that confers a license to use the technique or contact Keygene directly regarding licensing.