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RFLP analysis allows for the identification of alfalfa ecotypes

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Three seed lots, obtained from different ‘foundation farms’, for each of the alfalfa, Medicago sativa L., ecotypes ‘Vogherese’ and ‘Maremmana’, together with the variety ‘Iside’ as a control, were studied to test the possibility of distinguishing them through restriction fragment length polymorphism analysis. Twenty plants per seed lot were analysed with 25 DNA probes that yielded 155 informative polymorphic fragments. Variance partitioning showed that within-population variability accounted for nearly 98% of the total variance, while a small but significant contribution to the total variance was due to among-lot variability. ‘Vogherese’ was more homogeneous than ‘Maremmana’, as a consequence of the greater environmental homogeneity of the adaptation area of the former ecotype compared with the latter. Bulk analysis yielded eight variety-specific bands, 12 bands present in both ecotypes and absent in the variety and one band specific of ‘Maremmana’. Results are discussed in relation to the practical use of molecular markers for alfalfa ecotype identification.

Cultivar identification by molecular markers has been attempted since the early 1980s, when the use of restriction fragment length polymorphisms (RFLPs) was proposed for the first time ( Soller and Beckmann 1983). Since then, new classes of markers such as random amplified polymorphic DNAs (RAPDs) ( Williams et al. 1990), microsatellites ( Morgante and Olivieri 1993) and amplified fragment length polymorphisms (AFLPs) ( Vos et al. 1995) have been reported and almost all the major crop species have been investigated. However, irrespective of the types of markers used, cultivar identification has been carried out mainly in autogamous species ( Keim et al. 1989, Hongtrakul et al. 1997, Paull et al. 1998, Zhu et al. 1998) or clonally propagated species ( Görg et al. 1992, Guilford et al. 1997). Few papers report results on allogamous, seed-propagated species ( Huff et al. 1993, Huff 1997, Warnke et al. 1997). Outcrossing seed-propagated species are heterogeneous populations at equilibrium. Their populations or cultivars can only be differentiated based on differences in marker frequencies, a necessity which imposes the use of a high number of markers and plants. Alfalfa (lucerne), Medicago sativa L., a major forage crop throughout the world, is an outcrossing seed-propagated species whose populations have a genetic structure complicated by the tetrasomic inheritance, and by a rate of selfing which varies according to the environmental conditions. In general, alfalfa cultivars are synthetic varieties generated by intercrossing selected plants and by advancing their offspring through limited generations of random matings.

A significant percentage of alfalfa cultivated in Italy is represented by local landraces (ecotypes). An ecotype is defined as a population growing in a specific geographical environment, whose individuals share a common gene pool. The adaptation zone of an ecotype is the region where it has evolved under local climatic, pedological and biotic conditions and, in the case of cultivated ecotypes, also under anthropic pressure. The production of the core seed, assuring the stability of ecotype characteristics over time, is entrusted to ‘foundation farms’. At present, Italian alfalfa ecotypes may cover large geographical regions and the problem arises as to whether they still conform to the definition of ecotype. This situation requires the development of a method for the unambiguous and reproducible identification of alfalfa ecotypes. RFLP markers were first used by Brummer et al. (1991) to differentiate three synthetic North American alfalfa varieties; more recent papers report the effective distinctiveness of varieties and landraces either by considering the fraction of shared bands in pairwise comparisons of individual plants ( Crochemore et al. 1996, Pupilli et al. 1996) or based on differences on band frequencies ( Pupilli et al. 1996, Gherardi et al. 1998). In all the cases to date, accession-specific markers were detected at very low frequency so that a large number of individuals had to be analysed.

Bulking DNA of fresh tissue from several plants allows one to identify markers linked to any specific trait in a heterogeneous population segregating for the trait of interest ( Michelmore et al. 1991). Such a method has been proposed to differentiate heterogeneous populations in alfalfa, but this approach only allows for the discrimination of populations with very diverse origins ( Kidwell et al. 1994) or of varieties and breeding populations with different genetic bases ( Yu and Pauls 1993). Pupilli et al. (1996) emphasized the necessity of increasing the number of bulked individuals to overcome a strong sampling effect resulting from rarely-occurring RFLP fragments.

Despite the published results, an in-depth study on the practical use of molecular markers for the discrimination of alfalfa commercial ecotypes is still not available. In this paper, two ecotypes were studied to assess whether RFLP molecular markers are effective in distinguishing them, using both individual and bulked plant samples.
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Keywords: Medicago sativa; bulk analysis; ecotype identification; molecular markers

Document Type: Research Article

Affiliations: 1: Instituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere del CNR, Via della Madonna alta 130, 1-06128 Perugia, Italy; 2: Istituto Sperimentale per le Colture Foraggere, Viale Piacenza 29, 1-20075 Lodi, Italy

Publication date: 01 January 2000

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