MOLECULAR CHARACTERIZATION OF ELEPHANTGRASS ACCESSIONS THROUGH RAPD MARKERS

Elephantgrass pastures are limited by yield variations and reductions in forage quality and availability, thus making the search for genotypes with reduced seasonality a major concern. In order to verify the extent of genetic variability among contrasting cultivars, ten elephantgrass accessions were analyzed through DNA amplification by RAPD technique. A total of 160 DNA bands were generated with the use of 44 random primers and 23% of these bands were monomorphic for all accessions. Gel-obtained binary data (1 for presence and 0 for absence) were used for generating a genetic distance matrix, which was utilized in a UPGMA grouping analysis. Elephantgrass cultivars Cameroon and Vruckwona were the accessions mostly divergent from the others, with an average genetic distance of 0.34. The accessions with the lowest average genetic distances from the others were Pioneiro and CNPGL 27-5, both with a distance of 0.25. Overall, genetic distances ranged from 0.06 to 0.43, indicating little genetic variability for the set of accessions, despite the contrasting morphology of the studied genotypes.


INTRODUCTION
Elephantgrass (Pennisetum purpureum Schum.) is an important forage crop for dairy production systems in the tropics.However, yield fluctuations during the growth season and sharp drops in forage quality and availability during the dry season render the species low in agronomic effectiveness to farmers (PASSOS, 1999).Therefore, the search for genotypes exhibiting reduced seasonality in forage production and improved forage quality becomes of paramount importance for improving elephantgrass-based dairy production systems.
There is substantial agronomic and morphological diversity in elephantgrass germplasm (XAVIER et al., 1995), generally preserved through the occurrence of protogyny, which favors crossfertilization, and through the predominance of vegetative propagation.The germplasm is highly heterozygous and heterogeneous, suggesting the existence of a large amount of genetic variability within the species (HANNA, 1999).However, since the species is an allotetraploid (2n = 4x = 28) with the A and B genomes (PEREIRA et al., 2001), alternative procedures should be sought to better characterize genetic variability of germplasm.
In recent years, fingerprinting systems based on RAPD analysis have been increasingly utilized for detecting genetic polymorphism in several plant genera.The most common uses of the technique for scientific and commercial purposes include the discrimination among species for post-harvest purposes (HOSOKAWA et al., 2000), identification of crop cultivars to settle lawsuits (CONGIU et al., 2000), marker assisted selection establishment of phylogenetic position of natural populations (SGORBATI et al., 2004;TSUJI & OHNISHI, 2001;VICCINI et al., 2004), certification of lack of polymorphism between in vitro and conventionally propagated plant stands (HAO et al., 2004;LAIA et al., 2000;SALVI et al., 2002), and determination of genetic variation in wild and cultivated populations (BIRMETA et al., 2004;GUSTAFSON et al., 1999;SONNANTE et al., 2002).
Few attempts have been carried out to understand genetic variability of elephantgrass based on analyses of genomic DNA.Smith et al. (1989) verified the existence of polymorphism in different populations of elephantgrass through RFLP procedure.Subsequently, RFLP markers were utilized to show genetic uniformity in plants derived from somatic embryos (SHENOY & VASIL, 1992).However, those reports fell short of providing quantification of genetic variability within species, specially regarding genetic distances.Evaluations based on RAPD profiles would be suitable for providing such information due to the high level of polymorphism of this technique.
The purpose of this study was to verify the genetic diversity among morphologically contrasting elephantgrass accessions through DNA amplification patterns obtained by RAPD procedure.

DNA Extraction:
Collected samples, consisting of 300 mg of fresh expanding leaf blade tissue, were immediately placed in labeled plastic bags, immersed in liquid N2, and maintained at -80 o C until use.Subsequently, each sample was reduced to a fine powder through mortar and pestles, with liquid N2 addition, and transferred to a 2.0 mL microcentrifuge tube.DNA extraction was performed using a modified CTAB procedure (FERREIRA & GRATTAPAGLIA, 1995).Sample DNA concentration was estimated by comparing band patterns to lambda DNA standards in agarose gels and also by spectrophotometric absorbance at 260 nm.

DNA electrophoresis:
Amplified DNA fragments were loaded in an 1.5% agarose gel and submitted to electrophoresis for 3 h.The gel was then immersed in a ethidium bromide solution, for 30 min.DNA fragments were visualized under ultra-violet light and gel image was captured by the Eagle-Eye II photo documentation system (Stratagene Inc.), and the images stored as JPEG archives.
Gel scoring and data analysis: Gel images were acquired by RFLPScan software, sample lanes were defined, and DNA bands were marked.Following proper adjustments, bands were exported as "1" for presence and "0" for absence in a text file.Polymorphic Information Content (PIC) for each primer was obtained as: where p ij 2 is the frequency of the i allele in the j locus, 2 is the frequency of the i allele in the j locus of the k primer.
From the binary data, a matrix of genetic distances was generated according to Nei & Li (1979), and a cluster analysis was performed according to UPGMA method.Branch robustness of tree topology was evaluated using bootstrap resampling with 500 replicates.The calculations were performed with BOOD-P software (COELHO, 2003).

RESULTS AND DISCUSSION
The 44 random primers used yielded 160 DNA bands (i.e. an averaged 3.64 bands per primer).A representative primer result is shown in Figure 1, in which 23% of the bands were found to be monomorphic for the 10 elephantgrass accessions.Polymorphic markers including primer PIC and number of polymorphic bands are shown in Table 1.As expected, PIC values showed a very high correlation with the number of polymorphic bands since each marker band is the result of a single amplified locus.
The binary data obtained from the eletrophoretic analysis of DNA amplification products was used to generate a matrix of genetic distances (Table 2).
Based on such analysis, the accessions mostly divergent from the others were Cameroon and Vruckwona, with an average genetic distance of 0.34.The accessions with the lowest average genetic distances from the others were Pioneiro and CNPGL 27-5, both with a distance of 0.25.Overall, genetic distances ranged from 0.06 to 0.43, indicating a small genetic variability despite the fact that these accessions are very contrasting relative to physiological and morphological attributes.It is possible, however, that the small number of accessions might have depressed the overall genetic variability.
The genetic distance coefficients were used to cluster the data using the algorithm of unweighted pair-group method with arithmetic averages (UPGMA) in a dendrogram (Figure 2).Accessions Cameroon and Vruckwona grouped close to each other as expected by their genetic distance (0.06) showed in Table 2.   Ciênc. agrotec.,Lavras,v. 29,n. 3,maio/jun.,  Results are in agreement with RAPD-based work on the genetic diversity of other species reported elsewhere.Martin et al. (1995) examined the relationship among seven wheat cultivars through PCR profiles and concluded that the degree of genetic variability among parents was not an adequate measure for predicting heterosis in the species.Gustafson et al. (1999) characterized genetic diversity and genetic distinctiveness of big bluestem grass with the purpose of assessing genetic erosion.Nietsche et al. (2001) identified thirteen races of the common bean pathogen Phaeoisariopsis griseola and mapped their origin.

CONCLUSIONS
The genetic distance data obtained in this study might be useful as an auxiliary tool in future strategies for crossings and diallel elaboration, so as to evaluate general and specific combination capabilities of desired genotypes.Such molecular data would be also useful for detecting DNA patterns unique for a given accession or set of accessions, which would be utilized as a descriptor with right protection purposes.Finally, our findings demonstrate the feasibility of the RAPD technique for quantifying genetic distances among elephantgrass accessions, although suggesting an overall need for further work with a greater number of genotypes for a better understanding of the genetic variability of the species.

TABLE 1 -
Polymorphic Information Content and number of polymorphic bands for each RAPD primer.

2005 572 TABLE 2 -
Nei's genetic distance matrix of 10 accessions of elephantgrass based on RAPD analysis.