Variabilidad genética dentro de la raza mexicana de aguacate (Persea americana Mill.) determinada por ISSRs
H. Cuiris–Pérez1, H. Guillén–Andrade1*, M. E. Pedraza–Santos1, J. López–Medina1 e I. Vidales–Fernández2.
1 Unidad de Investigaciones Avanzadas en Agrobiotecnología, Facultad de Agrobiología "Presidente Juárez", Universidad Michoacana de San Nicolás de Hidalgo. Paseo de la Revolución esquina Berlín, Col. Emiliano Zapata, Uruapan, Michoacán, C. P. 60180, México. Correo–e: hguillenandrade@prodigy.net.mx. (*Autor responsable).
2 Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Av. Latinoamericana Núm. 1101, Col. Revolución, Uruapan, Michoacán, C. P. 60080, México.
Recibido: 11 de marzo, 2009 ]]> Aceptado: 21 de julio, 2009
Resumen
El presente estudio se efectuó para determinar la existencia de diversidad genética dentro de una colección de germoplasma de aguacate (Persea americana Mill.) perteneciente al Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Campo Experimental Uruapan (CEFAP–Uruapan). Se investigó la relación parental entre 77 accesiones (231 plantas) de la raza Mexicana con el uso de siete ISSRs (Inter Simple Sequence Repeat microsatellites, en inglés). En total se detectaron 154 loci. El porcentaje de polimorfismo varió de 82.3 a 95.4, con un número de bandas entre 17 y 25 dentro de las accesiones. El análisis de similitud genética reveló la formación de dos grupos principales, uno con once subgrupos y el otro con tres subgrupos. La similitud genética fue más alta entre las accesiones 237 (Atlixco, Puebla) y XTC01 (Uruapan, Michoacán), mientras que las accesiones 532 (Atlixco, Puebla) y 369 (Chilchota, Michoacán) fueron las más disímiles. No se encontraron duplicados en los genotipos analizados. En general, el presente estudio demostró la utilidad del análisis mediante ISSRs para la determinación de diversidad genética en aguacate.
Palabras clave: microsatélites, germoplasma, similitud genética.
Abstract
The present study was undertaken in order to asses the existing genetic diversity within a germplasm collection of avocado (Persea americana Mill.) kept at the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP) Campo Experimental Uruapan (CEFAP–Uruapan). The parental relationship among 77 accessions (231 plants) of the Mexican race was investigated by using seven Inter Simple Sequence Repeat microsatellites (ISSRs). A total of 154 loci were detected. Percentage polymorphism ranged from 82.3 to 95.4, with number of bands ranging from 17 to 25 within accessions. Genetic similarity analysis revealed formation of two major groups, one with eleven subgroups and the other one with three subgroups. Genetic similarity was highest between accession 237 (Atlixco, Puebla) and accession XTC01 (Uruapan, Michoacan), while accessions 532 (Atlixco, Puebla) and 369 (Chilchota, Michoacan) were the most dissimilar ones. None of the accessions were found to be duplicates. In general, the present study demonstrated the usefulness of ISSRs analysis for determination of genetic diversity in avocado.
Key words: microsatellites, germplasm, genetic similarity.
]]> INTRODUCTION
Avocado (Persea americana Mill.) is grown under diverse environmental conditions all over the world. Mexico is the world's leading producer of this crop, where 105,477 ha and total production of 1.134 MMT were reported in 2006 with 84% of avocado acreage occurring in the state of Michoacán. The several activities involved in the production chain of avocado generates over 40,000 permanent and over 60,000 seasonal jobs each year in Michoacán (Guillén–Andrade et al., 2007).
Since avocado is an outcrossing species, great genetic diversity has been generated throughout the evolution process of this crop. Natural selection first and man–driven selection later has lead to the development of genotypes adapted to diverse environments (Ben Ya'acov et al., 1992a, b and c). Therefore, the opportunities to exploit the existing genetic resources of avocado are almost endless (Bergh, 1992; Schieber and Zentmyer, 1992). Of the three major horticultural groups existing in avocado (Scora and Bergh, 1992), the Mexican race outstands for its high (15 to 20%) oil content (Knight, 2002), making individuals within this race attractive for breeding purposes. Although the genetic diversity within the Mexican race is thought to be wide, the lack of information with regards to characterization and correct identification of germplasm collections somehow limits the efforts on genetic improvement of avocado in México.
Molecular markers have proven to be useful in clarifying genetic relationship among individuals in avocado germplasm. Techniques include both minisatellites (Davis et al., 1998; Fiedler et al., 1998; Lavi et al., 1991; Librada–Alcaraz and Hormaza, 2007; Mhameed et al., 1996) and microsatellites (Rodriguez et al., 2007; Shnell et al., 2003; Shnell et al., 2007). In addition to germplasm characterization, genetic maps have been constructed using molecular markers (Sharon et al., 1997; Viruel et al., 2007). In relation with other crops, however, the use of molecular marker technology for use in avocado is still scarce. Therefore, the objective of this study was to generate information at the DNA level regarding the genetic diversity of 77 Mexican race avocado accessions kept at the Field Experiment Station at Uruapan (CEFAP–Uruapan) germplasm collection belonging to the National Institute for Forestry, Livestock and Agricultural Research (INIFAP).
MATERIALS AND METHODS
Genetic material. Leaf material of a total of 231 plants representing 77 accessions of avocado (Persea americana Mill.) belonging in the Mexican race were used in the present study. Description of the plant material is presented in Table 1.
DNA isolation. DNA extraction was performed on 400 mg samples of leaf tissue according to the procedure described by Clarke et al. (1989). Isolated DNA was quantified on a DR/4000 U spectrophotometer (HACHTM) by using 40 μL DNA and 1960 μL TE solution of each sample. Readings were made at 260 and 280 nanometers length waves. DNA was standardized at 200 μM and then stored at 4 °C until use.
]]> Microsatellite markers and DNA amplification. Seven inter simple sequence repeat (ISSR) markers (Zietkiewickz et al., 1994) were used in the present study. Microsatellite number and primer sequence are listed in Table 2. PCR amplification reactions were carried out in a total volume of 20 μL which consisted of 4.0 μL genomic DNA (40 ng), 1.5 mL dNTP mixture (187 mM each dNTP), 2.0 μL ISSR primer (1.0 mM), 2.2 μL 1X Taq buffer, 0.2 μL DNA Taq polymerase (1 U·mL–1), 0.8 μL MgCl2 (2.0 mM), and 9.3 μL ddH2O. Thermal cycling profile consisted of the following: initial heating at 94 °C for 4 min, 35 cycles of denaturing at 94 °C for 1 min, annealing at 52 °C for 1 min, extension at 72 °C for 2 min, and a final extension for 5 min at 72 °C. PCR was carried out on a MJ Research (DNA Engine Tetrad®) thermoclycer. Primer pairs were run individually.Electrophoresis. Polyacrylamide gels (37.5:1) at 6% were use on a 38 x 50 cm vertical Sequio–Gen GT (BIO–RAD®) electrophoresis system. Buffer solution consisted of 1X TBE (0.09 M Tris–boric acid and 2 mM EDTA). As polymerizing agents, 25% ammonium persulfate and 0.11% TEMED® were added to achieve a final concentration of 0.028%. For every 20 μL amplification product 5 μL 5X sample gel buffer–xilene cyanole staining solution (1M Tris pH 8.0, 0.5 M EDTA pH 8.0, 25% sucrose, bromophenol blue, 2 mg·mL–1, and xilene cyanole 2 mg·ml–1) were added; approximately 7 μL of each sample were loaded and then run at 120 W for 3 h. Detection of amplified fragments was achieved by staining with silver nitrate.
Data analysis. Band number (alleles) and degree of polymorphism, revealed by each microsatellite marker (locus) on the polyacrylamide gels, for each avocado accession were scored on the basis of the presence (1) or absence (0) of band at each locus of the sample. A 0 and 1 matrix was created for computing Nei and Li distances (Nei and Li, 1979). Molecular data were recorded by the HyperMap Data software (Hoisington, 1993). Genetic similarity coefficients were calculated by using the Numerical Taxonomy and Multivariate Analysis System (NTSYS–PC) software, version 2.02 (Rohlf, 1993). Genetic distances were calculated with the help of the Similarity Genetic Data (SIMGEND) procedure. Dendrograms were constructed with the Sequential, Agglomerative, Hierarchical, and Nested (SAHN) clustering method (Sneath and Sokal, 1973).
RESULTS AND DISCUSSION
Due to the high level of polymorphism observed in preliminary studies, only seven ISSR markers were used in the analysis of seven avocado accessions collected in seven states of the Mexican Republic. There were 154 polymorphic fragments (alleles) in total; the number of bands and the polymorphism information content (PIC) produced by each primer ranged from 17 to 25 and 82 to 95%, respectively (Table 2). A polymorphic primer was one which presented at least one different band among the 77 genotypes; that is, polymorphic bands were those that were missing in at least one of the genotypes analyzed. An example of the allelic diversity is given in Figure 1 for the (AG)8C primer. Highest band number (25) and percentage polymorphism (95.4%) were obtained by both (AG)8C and (GA)8C primers. On the other hand, primer (CT)8RC produced the lowest band number (17) while primer (AG)8T had the lowest PIC value (82.3%). In general, the high PIC value (92.64%) indicates, on one hand, the existence of great genetic variability within the analyzed avocado accessions; in the other hand, these findings also indicate the usefulness of ISSRs as a tool in distinguishing genotypes with diverse origins. However, the loci detected in this study were not associated with any morphological trait, an approach that would be worth taking into consideration for future studies.
The level of polymorphism observed in this work was in agreement with results found in other studies (Fang and Roose, 1997; Guillén–Andrade et al., 2000; Salimath et al., 1995; Wolff et al., 1995; Yamamoto et al., 2007) in which the same type of primers have been used. Wang et al. (1994) point out that in plants the most common microsatellite sequences are the dinucleotides (AT/AT) , followed by the sequences (GT/TG)n. In grapes, the most common sequences were the dinucleotides (CA/GT)n (Thomas and Scott, 1993). In the present work, based on the number of primers and the number of bands generated by them, the most common sequences found in the avocado accessions belonging in the Mexican race were the dinucleotides (AG/ AC)n and (CT/RC)n; the number of bands found in this work, however was lower in relation with the number of bands found in other studies (Fang and Roose, 1997; Prevost and Wilkinson, 1999).
Genetic differentiation among and relatedness of accessions
]]> ISSR data were used to generate a genetic distance matrix. Genetic distance values among accessions ranged from cero (closely–related material) to one (none–related material). From the genetic distance (Nei and Li, 1979) matrix a dendrogram was constructed (Figure 2). Two major groups were clearly distinguished. Group I was composed of 11 subgroups: subgroup Ia encompassed accessions collected in Puebla, Guanajuato, Veracruz, Michoacán, Oaxaca, and the State of Mexico; subgroup I was formed by accessions of Veracruz and Puebla; subgroup Ic shared a Chiapas collection with subgroup Ib and was formed by Michoacán and Veracruz collections; subgroup Id grouped two accessions, one collected in Morelos and the other one in Michoacán and it also was related to Ic by a Chiapas collection; in subgroup I two collections of Puebla, three of the State of México and one of Guanajuato were associated; subgroup If was made up by one collection each of Puebla and Veracruz; subgroup Ig grouped two collections of Nuevo Leon and one of Michoacán; in subgroup Ih two collections of Michoacán and one of the State of México were involved; subgroup Ii was made up with three collections of Michoacán, one of Oaxaca, one of Nuevo Leon, one of Puebla, and one of Morelos, and also shared a collection with Puebla; subgroup Ij was formed by Puebla and Veracruz collections; and subgroup Ik grouped one collection each of Puebla and Chiapas. We must point out that in subgroup Ia the greatest genetic similarity between genotypes was for accessions 237 (Atlixco, Puebla) and XTC01 (Uruapan, Michoacán).In Group II a more clearly distinguishable association among accessions was observed; three subgroups were formed. Subgroup IIa grouped accessions collected in Chiapas, Puebla, State of México, Veracruz, Oaxaca, Aguascalientes, and Michoacán. In this subgroup highest genetic similarity was for accessions 332 (Jalancingo, Veracruz), 334 (Altotongo, Veracruz), 356 (Aguascalientes, Aguascalientes), 541 (Xalostepec, Puebla), 535 (Atlixco, Puebla), and 649 (Teposcolula, Oaxaca). Subgroup IIb was made up with accessions of Puebla, Aguascalientes, State of México, Michoacán, and Oaxaca, with all seven accessions collected in the State of Mexico being the most predominant ones. In the end, accessions 532 (Atlixco, Puebla) and 369 (Chilchota, Michoacán) had the greatest genetic dissimilarity.
The usefulness of ISSR microsatellites to detect genetic variation, even among closely related individuals, was confirmed in this research and was in agreement with other works in avocado (Clegg et al., 1999; Librada–Alcaraz and Hormaza, 2007; Rodríguez et al., 2007; Schnell et al., 2007) and other crops (Nybom and Hall, 1991; Yamamoto et al., 2007). It is confirmed that ISSRs overcome technical limitations of RFLP and RAPD markers (Prevost and Wilkinson, 1999; Ratnaparkhe et al., 1998), with results similar to those obtained with AFLPs (Lanham and Brennam, 1999; Viruel et al., 2007).
The polymorphism detected in this work with the ISSR markers is an indication of the high genetic variation existing among avocado accessions within the Mexican race. This can be explained by the high interest in exploiting avocado germplasm for commercial use, mainly in central and southern México, generating a constant movement of this species throughout the Country in seek of new production areas. The diversifying soil and climatic conditions of this Country plus the fact that avocado is an outcrossing species have all favored a certain degree of genetic variation within his species. These results clearly indicate the wide genetic variation existing in the avocado germplasm collection at the CEFAP–Uruapan experiment station. In addition, none of the accessions were found to be duplicates, therefore there is no need to discard any of the individuals; these should be kept and used for breeding purposes.
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