plant material and cultivation
We used 12 cultivars and four wild-types of lima bean (Fabaceae: Phaseolus lunatus L.). Seeds were provided by the Institute of Plant Genetics and Crop Plant Research (IPK) in Gatersleben, Germany; the Centro Internacional de Agricultura Tropical (CIAT), Recta Cali-Palmira, Colombia; the MPI for Chemical Ecology, Jena, Germany; Betty Benrey from the University of Neuchâtel, Switzerland; and Marco Gross from the University of Hamburg, Germany. Additional seeds were directly collected from native populations of lima bean growing in different parts of the state of Oaxaca, Mexico (see Table S1 in Supplementary material for detailed information on the accessions used).
Plants used for quantification of their defences were cultivated under greenhouse conditions (16 : 8 L : D with a photon flux density of 350–450 µmol s−1 m−2 at the plant container and 800–950 µmol s−1 m−2 on the top of the plants, depending on natural radiation). Additional light was provided by 400 watt high-pressure sodium lamps (Son-Targo 400, Philips®). To avoid effects by hot spots under the lamps, plants were moved every 3 days. Day/night temperature and ambient relative air humidity were set to 25 °C : 20 °C and 60–70%, respectively. Plants were fertilized with nitrogen–phosphate fertilizer (Blaukorn®-Nitrophoska®-Perfekt, Compo GmbH) twice a week (3 mg pot−1) and cultivated in plant-containers of 18 cm in diameter in a 1 : 1 mixture of standard substrate (TKS®-1-Instant, Floragard®) and sand (grain size 0.5–2.0 mm). Thirty plants per accession were cultured (full sibs). Plants used for the experiments were 5 weeks old and had developed five to seven trifoliate secondary leaves.
volatile organic compounds (vocs)
Plants were induced for volatile production by spraying 10 mL of a 1-mmol L−1 aqueous solution of jasmonic acid (JA) per plant at 09:00 AM. Control plants were sprayed with 10 mL of water. Plants were sprayed until runoff, subsequently allowed to dry (c. 30 min), before the procedure was repeated. After drying, the plant part bearing the secondary leaves was placed in a PET bag (‘Bratschlauch’, Toppits®, Minden, Germany; this material does not emit detectable amounts of volatiles even after exposure to temperatures up to 150 °C; Fig. 1). Both ends of the bag were tied avoiding shoot damage. Primary leaves of individual plants were placed in separate PET bags (Fig. 1). The six highest and five lowest cyanogenic accessions were analysed for volatile emission from primary leaves. Bagged plants remained in their respective pot and were placed in the greenhouse under the same ambient conditions as for plant cultivation.
Volatiles were collected continuously over 24 h on charcoal filters (1.5 mg charcoal, CLSA-Filters, Le Ruissaeu de Montbrun, France) using air circulation in closed loop stripping as described previously (Donath & Boland 1995). After 24 h, volatiles were eluted from the carbon filter with dichloromethane (40 µL) containing 1-bromodecane (200 ng µL−1) as internal standard (IS). Samples were analysed on a GC-Trace mass spectrometer (Trace GC Ultra DSQ; Thermo Electron, Austin, TX). The program for separation [Rtx5-Ms column (Restek, Philadelphia, PA), 15 m × 0.25 mm; 0.25 µm coating] was 40 °C initial temperature (2 min), 10 °C min−1 to 200 °C, then 30 °C min−1 to 280 °C with He (constant flow 1.5 mL min−1) as carrier gas. Compounds were identified by comparison to standard substances (Fluka Seelze, Germany) and with the Nist 05 library (Xcalibur 1.4 software; Thermo Electron Corp., Austin, TX). Individual compounds (peak areas) were quantified with respect to the peak area of the IS (1-bromodecane), and quantities are presented as a percentage of the IS area. Only compounds for which reference substances were available were included in quantitative analysis (cis-3-hexenyl acetate, 2-ethylhexan-1-ol, cis-β-ocimene, linalool, cis-3-hexanol butyrate, methyl salicylate, cis-3-hexenyl isovalerate, indole, cis-jasmone, β-caryophyllene, methyl jasmonate). The quantitative data on volatile production were divided by the leaf fresh weight of the respective plant in order to calculate total volatile mass released per gram fresh mass.
All accessions investigated for their cyanogenic features and their VOC emission were included in an AFLP analysis and combined with a larger set of wild-types of Phaseolus lunatus L., Phaseolus coccineus L., Phaseolus microcarpus Martius and Phaseolus vulgaris L.. Two individuals per accession were analysed, resulting in a total of 72 specimens (see Table S1). Total genomic DNA was extracted from finely ground (Qiagen Tissue Lyser) plant material using the Qiagen Plant Mini Kit (Qiagen, Santa Clarita, CA) and following the manual's instructions. The restriction and ligation mix with a total volume of 20 µL contained 1× T4 buffer, 0.1 mm NaCl, 1 mm BSA, 1 U Mse1, 5 U EcoRI, 1 U T4 DNA ligase, 1 µL MseI adapter, 1 µL EcoR1 adapter and 15 µL of genomic DNA. Incubation was 3 h at 37 °C followed by 10 min at 72 °C to stop the reaction. Consecutively, samples were diluted 1 : 9 with 180 µL TE buffer (0.1 mm EDTA).
Preselective PCR was conducted with primers complementary to the adapter sequences and having one additional nucleotide on their 3′ end. The preselective primer sequence complementary to the EcoRI end was: 5′-GACTGCGTACCAATTCA-3′ (EcoRI-A). The MseI preselective primer sequence was: 5′-GATGAGTCCTGAGTAAC (MseI-C). Underlined letters correspond to the first selective nucleotide. The preselective PCR was performed in a 25-µL reaction containing 1× PCR buffer (Roche, Basel, Switzerland), 1.5 mm MgCl2, 1× BSA, 0.16 mm of each dNTP, 0.3 µm of each primer, 1 U of Taq DNA Polymerase (Roche) and 5 µL of the restricted and ligated DNA (after dilution). Samples were amplified on a MJ Research DYAD with the following PCR profile: initial denaturation for 2 min at 94 °C followed by 26 cycles of 94 °C for 1 min, 56 °C for 1 min (primer annealing), 72 °C for 1 min and a final elongation step of 10 min at 72 °C; holding temperature was set at 4 °C. Amplification products were diluted with 175 µL TE buffer (0.1 mm EDTA).
Eight primer combinations were used for the selective PCR of the DNA restriction fragments (Table 1). EcoRI primers (Applied Biosystems, Forster City, CA) were labelled with fluorescing dye. The PCR reaction mixture consisted of 1× PCR buffer (Roche), 1.5 mm MgCl2, 1× BSA, 0.16 mm of each dNTP, 0.3 µm of the MseI-C++ primer, 0.1 µm of the labelled EcoRI-A++ primer, 0.6 U of Taq DNA Polymerase (Roche) and 2.5 µL of the PCR product derived from the preselective PCR in a total reaction volume of 10 µL. Thermal cycling parameters were: initial denaturation of 2 min at 94 °C followed by 12 cycles of 30 s at 94 °C, 30 s at 65 °C (–0.7 °C per cycle), and 2 min at 72 °C, 25 cycles of 30 s at 94 °C, 30 s at 56 °C and 2 min at 72 °C, and a final elongation step of 30 min at 72 °C; holding temperature was set at 4 °C.
Table 1. Level of polymorphism found in a group of wild Phaseolus as well as wild and cultivated Phaseolus lunatus accessions and genotypes by means of AFLPs as indicated by primer combination
|Primer combination||Dye||Number of bands|
|Analysed||Polymorph||Per accession||Polymorph for P. lunatus||Per P. lunatus acc.|
|EcoRI-CAA/MseIAAG||NED|| 93|| 93|| 5–58|| 72|| 36–58|
|EcoRI-CTC/MseI-ACC||6-FAM|| 73|| 73|| 1–26|| 46|| 15–38|
|EcoRI-CTC/MseIAAG||NED|| 95|| 93|| 6–50|| 75|| 20–50|
|EcoRI-CTT/MseI-AGC||VIC|| 46|| 46|| 3–22|| 34|| 7–22|
|EcoRI-CCG/MseI-ACG||VIC|| 60|| 60|| 2–26|| 54|| 9–26|
|EcoRI-CCG/MseI-ACC||6-FAM|| 37|| 37|| 1–16|| 28|| 2–14|
|EcoRI-CCG/MseI-AAG||NED|| 56|| 56|| 8–26|| 36|| 10–26|
|EcoRI-CCG/MseI-AGA||PET|| 49|| 49|| 12–26|| 39|| 17–26|
Samples were scored on an ABI 3730 with 9.0 µL HiDi formamide and 0.3 µL LIZ 500 ladder (Applied Biosystems), and 0.7 µL of the product. Fragments were analysed using the Peakscanner 1.0 software (Applied Biosystems).
A binary matrix reflecting the presence (1) or absence (0) of each AFLP band was generated for each specimen. Only bands between 100–500 bp were included in the analysis. Genetic similarities among all accessions were calculated using the program paup* (Swofford 2003) after the Nei–Li model (Nei & Li 1979), which is S = 2a/(2a + b + c), where a, bands shared by both individuals; b, bands present in individual (1) but not in (2), and c, bands present in individual (2) but not (1). Dendrograms were visualized using TreeView (Page 1996). For each primer combination one dendrogram was calculated (data not shown) and one was calculated for all primer combinations together. The correlation coefficient between similarity matrices for each primer was 0.99, indicating that each primer combination provided similar information about this group of accessions.