AFLP analysis of genetic variability in three reproductive forms of Agave tequilana
Análisis AFLP de la variabilidad genética en tres formas de reproducción de Agave tequilana
María Jazmín Abraham-Juárez1, Rafael Ramírez-Malagón2, Katia del C. Gil-Vega2 y June Simpson1*
1 Department of Plant Genetic Engineering, CINVESTAV-IPN, Campus Gto. Apdo. Postal 629, 36821, Irapuato, Guanajuato, México. Tel.: +52 462 623 9667; Fax: +52 462 623 9650 *Corresponding author (jsimpson@ira.cinvestav.mx)
]]> 2 Instituto de Ciencias Agrícolas, Universidad de Guanajuato, Apdo. Postal 311. 36820, Irapuato, Guanajuato. México.
Recibido: 11 de Noviembre de 2008.
Aceptado: 27 de Agosto de 2009.
Abstract
The Agave genus belongs to the Agavaceae family and contains around 200 species with diverse uses. Agave tequilana is one of the most important species at the industrial level in México since sugars accumulated in the stems of these plants are used to produce the spirit called tequila. Agave production is supported by intensive clonal propagation and the suppression of the sexual reproduction, leading to reduction in genetic variability and a greater susceptibility to plagues and diseases in commercial plantations. Nevertheless, this study with plants from a field in Guanajuato, México, carried out using AFLP markers shows that differences at the molecular level exist between offsets and bulbils produced asexually from the same mother plant (75.08 and 86.06 % polymorphic loci, respectively). Although a significant level of polymorphism is observed between rhizome offsets, levels are even higher between bulbils, reaching levels comparable to those found between plantlets produced from seeds (90.1 %). We propose a more detailed study of the factors causing genetic variability between asexually propagated individuals of Agave tequilana.
Key words: Agave tequilana, AFLP, genetic variability, asexual reproduction.
Resumen
]]> El género Agave pertenece a la familia Agavaceae y cuenta con alrededor de 200 especies, con diversos usos. Agave tequilana es una de las especies más importantes a nivel industrial en México, ya que los azúcares que se acumulan en sus tallos son usados para producir la bebida alcohólica llamada tequila. La producción de agave es soportada por una intensiva propagación clonal y supresión de la reproducción sexual, lo cual ha conducido a reducción en la variabilidad genética y mayor susceptibilidad a plagas y enfermedades en plantaciones comerciales. No obstante, este estudio con plantas de un campo de cultivo del Estado de Guanajuato, México, llevado a cabo con marcadores AFLP, muestra que existen diferencias a nivel molecular entre hijuelos y bulbillos producidos asexualmente provenientes de la misma planta madre (75.08 % y 86.06 % de loci polimórficos, respectivamente). Aunque se observa un nivel significativo de polimorfismo entre hijuelos de rizoma, los niveles son aún más altos entre bulbillos con valores comparables a los encontrados entre plantas producidas por semilla (90.1 %). Por ello se propone un estudio más detallado de los factores que están causando la variabilidad genética entre individuos de Agave tequilana propagados asexualmente.Palabras clave: Agave tequilana, AFLP, variabilidad genética, reproducción asexual.
INTRODUCTION
The Agavaceae family has nine genera and around 300 species, is endemic to America and its center of origin and diversity is located in México where 76 % of all known species are found (García-Mendoza, 2007). Currently the economic importance of these species is based on the extraction of fibers and production of alcoholic beverages such as tequila and mezcal (Valenzuela, 1997), but they are also of ecological importance (Good-Avila et al., 2006). The requirements of the tequila industry and restrictions of origin denomination have caused an intensive clonal selection and severe reduction of sexual reproduction of A. tequilana Weber, var. Azul (Palomino et al., 2003). This has resulted in a reduction in genetic variability, leading to concerns of susceptibility to plagues and diseases in commercial plantations (Gil-Vega et al., 2001).
In general, Agave species are monocarpic, semelparous, and can be propagated by seeds, rhizome offsets and inflorescence bulbils. Agave tequilana Weber var. 'Azul' may be propagated by all three forms, although reproduction is mainly achieved through rhizome offsets (Granados, 1993). Crossing and recombination between genotypes of Agave with desirable agronomic traits has been limited by asexual reproduction and the long life cycle of the plants. It is important to determine the degree of genetic variability between populations. If it is enough high it could be possible to select plants with economically important characteristics.
For molecular marker analysis, the AFLP (Amplified Fragment Length Polymorphism) technique is useful and has the advantages that no previous knowledge of the genome under study is necessary, little DNA is required and differences at level of species and subspecies may be detected (Vos et al., 1995). Normally, for plant genome analysis between 50 and 100 markers are obtained for each combination of oligonucleotide primers (Simpson, 1997).
Until now, reports for Agave tequilana have only described studies in which the genetic variability between rhizome offsets was determined, but not of bulbils or plants produced from seeds. Recently the level of polymorphism between nine A. tequilana varieties and rhizome offsets from different plants was reported (Gil-Vega et al., 2006). This report also showed that the 'Manso', 'Azul listado', 'Moraleno' and 'Siguin' cultivars are closely related genetically to A. tequilana Weber var. Azul. These results agree with another report based on the levels of ploidy and DNA nuclear content (Palomino et al., 2003) in which eight varieties of A. tequilana were analyzed, and with a more recent analysis using the transposon display technique (Bousios et al., 2007) where the same grouping of varieties reported in Gil et al. (2006), was obtained.
Genetic variability in other asexually propagated Agave species such as A. fourcroydes (Infante et al., 2003) and Agave angustifolia (Barraza-Morales et al., 2006; Sanchez-Teyer et al., 2009), has also been reported in addition to other asexually propagated plants including banana (Loh et al., 2000) and also between plants propagated by somatic embryogenesis such as Coffea arabica (Sanchez-Teyer et al., 2003).
In this work the levels of genetic variability within offsets, bulbils and seed-propagated Agave tequilana Weber var. 'Azul' plants were compared using AFLP analysis.
]]>MATERIALS AND METHODS
Plant material
Samples were taken from leaf tissue of offsets, bulbils and germinated seedlings (15 individuals of each type) obtained from the same mother plant located in a commercial plantation at Ex-Hacienda de Silva, Romita, Guanajuato, México. Samples were frozen in liquid nitrogen and stored at -70 °C until processed.
DNA extraction
The CTAB DNA miniprep method was used (2.5 % CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris HCl pH 8, 0.02 % of β-mercaptoethanol) (Edwards, 2001).
AFLP analysis
The Li-cor Biosciences IRDyeTM Fluorescent AFLPR Kit for Large Plant Genome Analysis was used based on Vos et al. (1995). In the selective amplification four primer combinations were used, where primers EcoRI were fluorescently labeled: Msel + CAG/EcoRI + ACC, Msel + CAG/EcoRI + AGG, Msel + CAA/EcoRI + ACC and MseI + CAA/EcoRI + AGC. Bands were detected using a Li-cor model 4200 DNA analyzerTM, (Lincoln, Nebraska).
Data analysis
Amplified fragment patterns were analyzed with SAGAMX software to produce binary matrix with (+) for presence and (-) for absence of bands, and then were analyzed in the software Free Tree (Hampl et al. 2001) using the Simple Matching coefficient (Skroch et al., 1992). A dendrogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA), and the bootstrap values were calculated based on 1000 resampled trees (Felsenstein, 1985) using Free Tree. Levels of polymorphism were determined as described Infante et al. (2003) and Gil et al. (2006) (Method 1), where the number of polymorphic fragments is divided by total number of fragments analyzed and expressed as a percentage. The second was reported by Newbury et al. (2000) (Method 2), in which the number of polymorphic fragments is divided by the number of amplified fragments and multiplied by the number of individuals and expressed as percentage.
]]>RESULTS AND DISCUSSION
This is the first study in Agave tequilana that describes the levels of genetic variability found between offspring produced by two different methods of asexual reproduction (offsets and bulbils) from the same mother plant and comparing them with plants sexually produced.
The selective amplification using four primer combinations produced 293 amplified fragments in each group of analyzed plants ranging in size from 50 to 500 bp. Levels of polymorphism determined by two different methods appear in Table 1. Although the levels of polymorphism determined by the different methods are numerically very different, the relationship between the three different types of samples is the same. Plants produced from seeds showed the highest levels of polymorphism, bulbils intermediate and offsets the lowest levels.
Figure 1 shows the dendrogram obtained using the simple matching coefficient and UPGMA. Two main groups supported by boostrap values are observed. The nodes defining each group showed high bootstrap values indicating that the topology of the dendrogram shown in the figure is robust. Group A contained all samples asexually reproduced and group S all samples from germinated seedlings. Group A showed two sub-groups, R and B, which included samples from offsets and bulbils, respectively. Levels of similarity within groups in the dendrogram confirm the results obtained by calculating the levels of polymorphism that individuals reproduced by seeds are genetically more diverse than those reproduced asexually.
Detailed analysis of the AFLP images revealed markers that are unique to one of the three groups of plants and therefore specific to a single reproductive group. An example is shown in Figure 2, where a 102 pb band in the M-CAA, E-ACC primer combination is present in the bulbil samples but absent in offsets and seed derived plants. This suggests that the mechanisms producing the genetic variability in bulbils and offsets act independently in different parts of the mother plant.
Several previous reports have described the occurrence of genetic variation in different asexually propagated plant species (Crouch et al., 2000; Loh et al., 2000; Pornon et al., 2000; Infante et al., 2003, Rottenberg and Parker, 2004; Wang et al., 2004; Gil-Vega et al., 2006; Barraza-Morales et al., 2006 and Sanchez-Teyer et al., 2009) as shown in Table 2. Comparison of the levels of polymorphism observed in different studies is hampered by the different population types and different methods used to calculate variation. But by comparing polymorphism level in Agave species we can observe that in A. fourcroydes and A. tequilana these are lower than in A. angustifolia, and these differences could be because populations analyzed in A. angustifolia are wild, so being heterogeneous populations. Although the two methods used in this study to calculate the level of polymorphism produced by each reproductive strategy lead to numerically large different results, the relative levels of polymorphism between each group remained constant. This suggests that both methods are informative, although care should be taken to apply the same method when comparing different reports. In general, levels of polymorphism were higher than other reports in Agave.
Of the two methods of asexual reproduction, offsets showed lowest levels of variability. These results were reflected in the similarity values of the dendrogram where the samples also grouped according to reproductive mechanism.
Although it was expected that the sexually propagated individuals would be genetically distinct to those produced asexually, it is interesting that the bulbil and offset samples also form discrete groups. These groups are determined by markers which are specific to or highly represented within each. This may be related to the developmental processes of the plant. Offsets are produced throughout the life cycle of the plant on rhizomes or directly from the stem, whereas bulbils are only formed on the mature floral scape when sexual reproduction has failed. Different mutations may accumulate in certain cell lineages throughout the long life cycle of the plant leading to differences in genotype when bulbils and offsets develop on different plant tissues.
Preliminary data (Abraham-Juarez et al., unpublished observation) suggest that new meristems form to produce bulbils on the floral scape and many hundreds of these may form. This implies a massive developmental reprogramming and initiation of cell division which may lead to errors during replication or mitosis, resulting in an increase in detectable changes at the DNA level. These changes may be single base substitutions, deletions or insertions or involve rearrangement of larger fragments or whole chromosomes.
]]> This study confirms previous reports of variation in asexually propagated A. tequilana plants and shows that significant variation may also be found between individuals obtained from the same mother plant. These results suggest that genotype variability could be maintained and even increased in A. tequilana by inducing and propagating bulbils on selected plants, but at the same time avoid the much higher levels of variability produced by sexual recombination which could produce undesirable phenotypic traits. Since in vitro propagation is being exploited as an alternative to produce plants for commercial plantations, it will be essential to determine the levels of variability produced by this process. It will also be of interest to determine the molecular nature of the underlying mutations and the mechanisms which cause them.
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