Plants were collected in the Denomination of Origin Zone for Agave tequilana ‘Azul’ and in southern Jalisco, México (municipality of Tolimán) for Agave angustifolia ‘Lineño’ and ‘Cimarron’ and in Miraval, Guerrero for Agave cupreata. Three accessions of each species and varieties were used in this work; the accessions were planted in pots containing a mixture of organic soil:sand:vermiculite (3:3:1) and kept under standard greenhouse conditions.

Elongating secondary root tips were treated with 2 mM 8-hydroxyquinoleine for 6 hours at 18 °C, in darkness. Later, root tips were fixed in ethanol:acetic acid (3:1) for 24 hours. Root tips were hydrolyzed with 1 N HCl for 15 minutes at 60 °C, transferred to Schiff’s reagent for 1 hour, and then to 1.8% propionic orcein to stain chromosomes (Moreno-Salazar et al. 2007). Slides were frozen with dry ice (Conger and Fairchild 1953), and mounted in Canada balsam. Twelve of the best cells of each population were photographed by using Technical Pan Film and a Zeiss photomicroscope II (Carl Zeiss AG, Germany).

A negative film was used to draw and measure the chromosome arms and the total genome length. The centromere position was obtained following Levan et al. (1964); arm ratio (r = long arm/short arm) was calculated for each chromosome. Chromosome homology was assigned according to similarities in length and centromere position. In addition, secondary constrictions were useful to distinguish homologous pairs in all populations. Idiograms were constructed according to the arm ratio of the chromosomes, and then grouped in metacentric (m), submetacentric (sm), subtelocentric (st) and telocentric (t) chromosomes. The number of homologous chromosomes was sequentially assigned following chromosome length, for a total number of 30.

Root tips of each three accessions of Agave tequilana ‘Azul’, Agave angustifolia ‘Lineño’ and Agave angustifolia ‘Cimarron’ and Agave cupreata were collected early in the morning, pretreated with satured α-bromonaphthalene solution and kept in ice water overnight, then fixed in ethanol:acetic acid (3:1), for at least 12 hours and stored at -20 °C until use. Root tips were incubated in a pectolytic-enzyme mixture, containing 0.2% (w/v) pectolyase (Sigma, USA), 0.2% (w/v) cellulase Onozuka RS (Yakult, Japan), and 0.2% (w/v) cytohelicase (Sigma) in 10 mM citrate buffer (pH 4.5), at 37 °C for approximately 2 hours. Squash preparations were made in a drop of 45% acetic acid and frozen in liquid nitrogen; the cover slips were removed with a razor blade and slides were dehydrated in absolute ethanol and then air-dried. The best slides were stored at 2–3 °C for up to 1 month.

Total genomic DNA from Agave tequilana ‘Azul’ was extracted from fresh young leaves using the CTAB method (Murray and Thompson 1980). The 5S and 18S rRNA genes were amplified by PCR using the following set of primers as follows: 5SF (5’-CACCAGATCCCATCAGAACT-3’); 5SR (5’-TTAGTCTGGTATGATCGCAC-3’); 18SF (5’-CAAAGATTAAGCCATGCATG-3’) and 18SR (5’-CCCAGAACATCTAAGGGCAT-3’) (Integrated DNA Technologies, USA). Both PCR reactions were performed in 20 µl reactions containing: 5.2 µl mQ water, 2µl Taq buffer 10×, 1 µl 50 mM MgCl₂, 1.6 µl 2.5 mM dNTPs, 2 U Taq polimerase (Life Technologies Corporation, USA), 2.5µl 1 mM of each primer and 50 ng DNA (5µl). Cycling conditions for 5S rDNA were: 94 °C for 4 minutes; 35 cycles of 94 °C for 30 s, 55 °C annealing temperature for 30 s and 72 °C for 30 s, followed by a final extension of 72 °C for 10 minutes. Cycling conditions for 18S rDNA were: 94 °C for 5 minutes; 35 cycles of 94 °C for 30 s, 60 °C annealing temperature for 30 s and 72 °C for 90 s, followed by a final extension of 72 °C for 10 minutes. PCR products were separated by 1% agarose gel electrophoresis in 1× TAE running buffer. Products were visualized by staining with ethidium bromide and the most prominent bands (~1400 bp for 18S and 300-500 bp for 5S) were purified by QIAquick Gel Extraction kit (Qiagen, Germany) according to the manufacturer’s instructions. The purified bands were cloned into pGem^®-T Easy Vector System I (Promega, USA), incubated overnight at 4 °C. Ligation products were transformed into electrocompetent Escherichia coli DH5α cells (Life Technologies Corporation). The recombinant clones were sequenced by LANGEBIO (Cinvestav, Irapuato, Mexico). The sequences were edited with BioEdit version 7.0.9 (Ibis Biosciences, USA) and compared with other sequences available in GenBank (http://www.ncbi.nlm.nih.gov/).

5S and 18S rDNA probes were isolated with the High Pure Plasmid Isolation kit (Roche Diagnostics GmbH, Germany) and labeled with biotin-16-dUTP by nick translation according to the manufacturer’s instructions (Roche Diagnostics GmbH).

Slide pretreatment. Slides were incubated in RNase A (100 μg ml^-1 in 2× SSC) for 1 hour at 37 °C, and washed with 2× SSC for 15 minutes. Then, the slides were incubated in 0.01 M HCl for two minutes and followed by treatment in pepsin (5 μg ml^-1) in 0.01M HCl for 10 minutes at 37 °C. Afterwards, the slides were washed in 2× SSC for 10 minutes and incubated in 4% paraformaldehyde for 10 minutes at room temperature. Finally, the slides were dehydrated in ethanol series (70%, 90%, and absolute ethanol for 3 minutes each), and air-dried.

Probe hybridization. Hybridization was carried by using a mixture consisting of 20× SSC, formamide, 50% sodium dextran sulphate, 10% sodium dodecyl sulphate, and 25-50 ng/slide of each probe. DNA probes were denatured by heating the hybridization mixture at 70 °C for 10 minutes and then placing it on ice for at least 10 minutes. For each slide, 40 μl of the hybridization mixture were used. Slides were denatured at 80 °C for 5 minutes. The slides were then placed in a pre-warmed humid chamber and incubated overnight at 37 °C. Slides were washed at 37 °C in 2× SSC for 15 minutes, 0.1× SSC at 42 °C for 30 minutes, and 2× SSC at room temperature for 10 minutes.

Signal detection. Biotin-labeled probes were detected with streptavidin-Alexa Fluor⁵⁴⁶ conjugate (Life Technologies Corporation) and amplified with biotinylated goat-antistreptavidin (Vector Laboratories, USA). Chromosomes were counterstained with DAPI solution (1 μg ml^-1), and one drop of Vectashield antifade (Vector Laboratories) was added before examination under a Leica DMRA2 microscope (Leica Microsystems, Germany) equipped with epifluorescent illumination and coupled to an Evolution QEi Camera (Media-Cybernetics, USA), and the images were analyzed with the Image-Pro software (Media-Cybernetics) and enhanced with Photoshop (Adobe Systems Incorporated, USA).

The partial amplification of 18S rDNA generated one band, which was cloned into electrocompetent Escherichia coli DH5α cells and a single clone was isolated, which after sequencing showed a fragment of 1424 bp (GenBank: KF159807) and a maximal identity of 100 % with Agave tequilana cultivar Azul (GenBank: GU980213.1) and Agave ghiesbreghtii K.Koch, 1862 voucher Chase 3467(K) (GenBank: HM640709.1) according to BLASTn analysis (nucleotide blast) at the NCBI database. The partial amplification of 5S rDNA generated one band, which was cloned into electrocompetent Escherichia coli DH5α cells and one clone was isolated, which after sequencing showed a fragment of 436 bp (GenBank: KF159808) and a maximal identity of 97% with Arabidopsis thaliana (Linnaeus, 1753) clone CIC YAC 9A12 and 9A5 5S ribosomal RNA gene (GenBank: AF198223.1), according to BLASTn analysis (nucleotide blast) at the NCBI database.

The physical mapping of 5S and 18S rDNA from Agave tequilana ‘Azul’ were investigated by fluorescent in situ hybridization (FISH) (Fig. 1). FISH experiments with both probes labeled with biotin and detected as a red signals, showed that the number of sites of rDNA were constant among all the species under study. 5S rDNA loci were located in both arms of small chromosome pair in each species (Fig. 1). The hybridization sites of cloned 18S rDNA were associated with the secondary constriction of a large chromosome pair in each species, being a subtelocentric chromosome pair in Agave tequilana ‘Azul’ and a telocentric chromosome pair in Agave cupreata and Agave angustifolia ‘Lineño’ and ‘Cimarron’ (Fig. 1).

All the studied species were diploids with 2n = 2x = 60, confirmed by chromosome counting, considering the basic chromosome number x = 30 for the genus, and showed a bimodal karyotype with five pairs of large chromosomes and 25 pairs of small chromosomes. Karyotype analysis of Agave species is summarized in Table 1, and where it can be seen that all species showed different karyotypic formulae as well as a secondary constriction in one large chromosome pair; in Agave tequilana ‘Azul’ it was observed in pair 1, in Agave cupreata in pair 3, in Agave angustifolia ‘Lineño’ in pair 5 and in Agave angustifolia ‘Cimarron’ in pair 2.

FISH data were integrated in idiograms, indicating the number and position of rDNA loci (Fig. 2). 5S rDNA loci always were located in a proximal region on both arms of a small chromosome in each species, whereas 18S rDNA loci always were located in the interstitial region of a large chromosome. Fig. 2a shows a hybridization signal of 18S rDNA in Agave tequilana ‘Azul’ on pair 1, while the 5S rDNA signals are on both arms of pair 10; in Agave cupreata (Fig. 2b), the hybridization signal of 18S rDNA is on pair 3, while the 5S rDNA signals are on both arms of pair 8; in Agave angustifolia ‘Lineño’ and ‘Cimarron’ (Fig. 2c-d), the hybridization signal of 18S rDNA is on pair 5 and 2, respectively, while the 5S rDNA signals are on both arms of pair 11 in both varieties.