FÜLLER, T.N.; TESSELE, C.; BARROS, I.B.I.; BARBOSA NETO, J.F.*
Agronomy Faculty, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 7712, Caixa Postal 776, CEP: 91501-970, Porto Alegre-Brazil * jfbn@ufrgs.br
ABSTRACT: Elionurus muticus naturally occurs in southern Brazil and its economic potential is due to the presence of essential oils. There are few studies about this genus. Thus, the aim of the present study was to characterize native E. muticus populations. The study was performed with five “capim-carona” populations collected in the state of Rio Grande do Sul, totaling 50 plants grown in pots in the Agronomy School. All five E. muticus populations presented variability for phenotypic traits and phenolic compound concentration. The presence of citral was identified in all populations, except in that from the “Morro da Polícia” region. RAPD analysis showed high variability for these populations, allowing the separation of individuals into five groups according to their geographic origin. The highest variability occurred within each population. Based on the results, the populations from São Borja and Agronomy School can be recommended to be used in breeding programs.
Key words: genetic variability, RAPD, essential oils, phenolic compounds
RESUMO: Caracterização fenotípica, fitoquímica e molecular de populações de Elionurus muticus (capim-carona). Elionurus muticus ocorre naturalmente no sul do Brasil e o potencial econômico se deve ao fato da presença de óleos essenciais. Poucos estudos têm sido desenvolvidos para este gênero. Sendo assim, o presente trabalho teve como objetivo caracterizar populações nativas de E. muticus. O trabalho foi realizado com cinco populações de capim-carona coletadas no Rio Grande do Sul, totalizando 50 plantas cultivadas em vasos na Faculdade de Agronomia. As cinco populações de E. muticus apresentaram variabilidade para os caracteres fenotípicos e para concentração de compostos fenólicos. A presença de citral foi identificada em todas as populações, exceto a do Morro da Polícia. A análise de RAPD demonstrou elevada variabilidade para as populações, permitindo a separação dos indivíduos em cinco grupos, sendo possível, de modo geral, agrupá-los de acordo com a origem geográfica. Observou-se também que a maior variabilidade ocorreu dentro de cada população. Os resultados indicaram que as populações São Borja e Faculdade de Agronomia podem ser recomendadas para a utilização em programas de melhoramento.
Palavras-chave: variabilidade genética, RAPD, óleos essenciais, compostos fenólicos
INTRODUCTION
The Elionurus Humb. & Bompl ex Willd genus belongs to the Poaceae family, comprises about 15 species and is common in the tropical and subtropical regions of South America, Africa, Australia (Araújo, 1971; Renvoize, 1978) and temperate Asia (Watson & Dallwitz, 1994). Elionurus muticus occurs naturally in Brazil where it is known as capim-carona (Longhi-Wagner et al., 2001). E. muticus is used as a medicinal and aromatic plant and is known popularly because it has sudorific and fever-reducing properties (Dzingirai et al., 2007). Antioxidant and antibacterial activities of Elionurus sp. had been reported by some authors (Cacciabua et al., 2005; Sabini et al., 2006; Dzigirai et al., 2007; Hess et al., 2007). The essential oil of E. candidus is used as an aromatizing by the cosmetic and domestic cleaning industries (Castro & Ramos, 2003). E. muticus is not suitable for cattle food, because the bitter taste is transmitted to the milk, but young plants can be eaten (Castro & Ramos, 2003; Hess et al.; 2007).
Elionurus muitcus is characterized by high variability in the chemical composition of its essential oil. In Argentina it is classified into five chemical types according to the major compound present in the essential oil, namely neral, geranial, acorenone, Iso-acorenone, and 1.8-cineole. The first two are very important for industrial purposes (Hess et al., 2007; Kolb et al., 2007b). Citral is a mixture of the two geometric isomers geranial and neral. This compound has a strong citric odor (Heydorn et al., 2003) and triggers the greatest interest regarding E. muticus, because it is widely used in the aromatic, food, and cosmetic industries. Citral is used as raw material in the pharmaceutical industry to synthesize a series of ionones. Beta-ionone is specifically used as a starting substance for vitamin A (Koshima et al., 2006). The high demand for essential oil with high citral content, currently supplied by oil grass (Cymbopogon citratus), opens the possibility of using E. muticus as an alternative for oil extraction (Kolb et al., 2007b). The presence of neral and geranial in E. muticus has only been identified in Southern Brazilian populations, in the rest of the country only camphene (11.5%), E-caryophyllene (17.9%) and spathulenol (18.6%) have been reported as the largest components of the oil (Scramim & Saito, 2000).
In spite of the demand, little is known about the chemical composition, biological activity, and cultivation of E. muticus. The establishment of breeding programs could provide better knowledge and could enable exploitation of existing resources, developing new populations with essential oil production ability and favorable agronomic traits for cultivation. High genetic and morphological variability was detected in E. muticus (Hess et al., 2007; Kolb et al., 2007b). The success of any breeding program depends basically on the genetic variability of the parents involved (Allard, 1960). Therefore, before starting breeding itself, it is essential to characterize the genetic variability of the plant populations for the target trait (Nodari & Guerra, 2000).
The aim of this study was to analyze the variability of morphological traits, phenolic compounds, and the identification of citral in the essential oil in five Elionurus muticus populations collected in southern Brazil.
MATERIAL AND METHOD Plant material
Natural Elionurus muticus (Spreng.) Kuntze populations were collected in several regions in Rio Grande do Sul State (Brazil). Voucher specimen for all collections were deposited at the Natural Science Institut Herbarium of the Botanical Department of Universidade Federal do Rio Grande do Sul (UFRGS). The number of individuals collected and the number of herbarium catalogue were shown at Table 1.The set of individuals collected in a determined locality was considered a population and the individuals consisted of each clump. Two populations were collected in Porto Alegre, forming the populations from Morro da Polícia and Morro Santana. Another two came from the locations of São Borja and São Francisco de Paula. In addition, an E. muticus population available for teaching purposes at the Agronomy Faculty campus of UFRGS was also used in the analysis. The plants collected were transplanted to bags with soil and kept in a greenhouse at an average temperature of 20ºC for further analysis.
Agronomic characterization
The individuals were at the same development state when the agronomic traits were analyzed. The traits were analyzed in the individual plants were plant height (cm), which was measured from the base of the plant to the top of the leaves; leaf width (mm), measured with a digital pachymeter, consisting on the largest part of the leaf; regrowth (cm), defined as plant height 30 days after cutting; shoot fresh weight (g); leaf curling; growth habit; the clumb habit was observed being attributed values: 0=erect, 1=semi-prostate e 2=prostate; and inflorescence presence. The results were submitted to variance analysis (ANOVA) and the means were separated by Tukey test (α=0,05).
Phenolic compounds
The phenolic compounds were analyzed using 3g of dried leaves macerated with 20 mL of methanol 80%. The concentration of phenolic compounds was assessed by the Folin-Ciocaulteau method according to Arnaldos et al. (2001). After 30 min incubation at 25°C in the dark, the absorbancies of the samples were obtained from a spectrophotometer at 765 nm (λ). Gallic acid was used as standard to establish the calibration curve. Three replications per individual were used to quantify the phenolic compounds. The data was submitted to the variance analysis (ANOVA) and the means were separated by Tukey test (α=0.05).
Essential oil
The oil was extracted from shoots by hydrodestillation in a Clevenger apparatus for three hours. The aqueous phase was extracted with ethylic ether and the oil was dried with magnesium sulfate. The oil contents were analyzed using gas chromatography in a Hewlett-Packard 5890 chromatographer with flame ionization detector, using an HP17A capillary column (30m x 0.25mm x 0.25μm), with hydrogen as the carrier gas (1.0 mL min-1) and programmed temperatures of 50°C for five minutes to 250°C for 30 minutes, with a rate of 15°C per minute. The oil was also analyzed and identified by mass spectrometry in a Shimadzu GC-17A chromatographer coupled to a Shimadzu QP5050 selective mass detector, using a HP17A (30m x 0.25mm x 0.25μm) capillary column, with hydrogen as the carrier gas (1,0ml/min) under the conditions reported above. The chemical constituents were identified by the retention indices, which were obtained by co-injecting the oil samples with a C11 – C24 linear hydrocarbon mixture and used to identify the compounds present, together with spectrum mass data and comparison with the literature (Adams, 1995) and complemented by computerized comparison of the apparatus library and literature. Quantitative analysis of each oil component (expressed in percent) was carried out by peak area normalization measurements.
Molecular analysis
The DNA was extracted from the leaves following the method reported by Harberer (1998). DNA was quantified in 1.6% agarose gel and the standard solutions were prepared at a concentration of 2,0 ng/ul. The RAPD reactions were amplified using a method adapted from Ferreira & Gratapagllia (1998). The DNA was amplified using a program that consisted of 40 cycles of 1’30’’ at 94ºC, 50’’ at 94ºC, 1’ at 35.5ºC, 2’ at 72ºC, 10’ at 72ºC, and 24h at 4ºC. The DNA Ladder marker 100 pb (Invitrogen) was used as a molecular weight standard. The amplified DNA fragments were separated in 1.6% agarose gel, with migration of 3 h in a horizontal cube. The fragments were stained with ethidium bromide (0,5µm/ml), visualized under ultraviolet light and photographed. The individuals studied were genotyped based on band presence. The genetic variability for each population was estimated according to the proportion of polymorphic loci (P: 0.95 criterion), the observed (Ho) and expected (He) mean heterozygosity per locus, and the f value (inbreeding coefficient or Fis according to Weir & Cockerham (1984). F statistics (Wright, 1978) were estimated in order to quantify levels of genetic diversity within and among populations and to infer the degree of population subdivision. The analyses were performed using the TFPGA software package (Miller, 1997). The similarity among genotypes was estimated using Jaccard Coefficient and the UPGMA method was used for clustering using the NTSYS software (Rohlf, 1997). The partition of the variability among and within populations was estimated by analysis of molecular variance (AMOVA) (Excoffier et al., 1992). The analyses were carried out following a model to correct allele frequencies estimated from dominant data (Lynch & Milligan, 1994).
RESULT AND DISCUSSION
E. muticus has been poorly characterized, especially in southern Brazil, where the studies were mostly related to taxonomic descriptions (Araújo, 1971; Renvoize, 1978; Longhi-Wagner, 2001; Watson & Dallwitz, 1994). In the present study, the five populations assessed showed phenotypic variability and populations collected in different locations could be differentiated based on the phenotype. From the traits evaluated, only plant height did not differ significantly among the populations (Table 2). The other traits were significantly different among the populations, showing the morphological variability that exists in the populations collected in different regions. The standard deviation observed in most of the traits was large when compared to the population means, indicating variability among the individuals within each population.
The Agronomy Faculty (AF), Morro da Polícia (MP) and São Borja (SB) populations had narrower leaves compared to the others (Table 2). Variability for the leaf curl trait has been reported for other species (Soster et al., 2004; Rosa et al., 2006). Regarding regrowth, the São Borja population presented the fastest regrowth, while Morro da Polícia population presented the slowest regrowth. The standard deviation was larger in the populations with slower regrowth (Table 2). Significant difference for the regrowth has also been reported for other species (Simioni et al., 1999; Scheffer-Basso et al., 2001). The plants of the São Francisco de Paula population presented the highest mean for the shoot fresh weight, but did not differ statistically from the São Borja population (Table 2). However, the phenotypic standard deviation was very strong for this character, demonstrating the presence of large variability among individuals. The total weight of the shoot is a character related to plant architecture, it is important to note that the populations studied showed no variation for height. Therefore, this difference in weight can be justified by the large leaf curl observed in the São Francisco de Paula population. The direct relationship expected between plant height and shoot fresh weight was also not detected by other authors, who associated the difference in shoot fresh weight to other traits related to the lateral expansion of the plants (Blank et al., 2004; Bortolini et al., 2006). Regarding leaf curling, there was a predominance of curled leaves in the Agronomy Faculty and São Borja populations. The other populations presented about 20% individuals with curled leaves. Regarding the vegetative habit of the populations studied, all the individuals in the Agronomy Faculty population and 93.3% of the Morro Santana population presented prostrate habit. In contrast, the São Francisco de Paula population did not present any individuals with prostrate habit and the other populations presented about 50% of the individuals with prostrate habit. The variability for this trait has been reported by other authors (Bortolini et al., 2006).
No population presented 100% flowering individuals. No plants from São Francisco de Paula populations flowered during the study period and the higher number of flowering plants was observed in the São Borja population with 82% flowering individuals. San Francisco de Paula is the location that has the highest altitude and lower average temperature compared to the other sites studied in this work. Some plants require exposure to environmental conditions suitable for flowering to occur. The main environmental factors affecting flowering are day length and temperature (Taiz & Zeiger, 2006). E. muticus is a grass that blooms in spring, compliant behavior and species that require winter chilling hours for the induction of flowering. Therefore, because the analysis of populations have been held at a warmer environment, the population of San Francisco de Paula may not have received the number of chilling hours necessary for the induction of flowering, the plants remaining in stage season. The phenotype is the result of interaction between genotype and environment (Allard, 1960). Therefore, the influence of the environment is reduced and the differences observed for these phenotypic characters can be attributed to the effect of genotype.
The concentration of phenolic compounds varied depending on the population assessed. The population with least value for phenolic compounds concentration was from São Francisco de Paula (Table 2). The other populations did not differ statistically for the trait and presented means around 9.4mg/g. The concentrations obtained differed from those reported by other authors. Dzingirai et al. (2007) reported a concentration of 46.8mg/g of total phenolic compounds analyzing the whole plant of E. muticus (Spreng.) Kuntze. On the other hand, Dzingirai et al. (2007) reported 0.68mg/g from the whole plant and 0.41mg/g from the root of E. muticus (Spreng.) Kuntze. These data show the large variability for phenolic compounds concentration which is dependent on the environment (Kutchan, 2001) and plant genotype (Asami et al., 2003).
The essential oil yield was similar among the populations studied, except for the São Francisco de Paula population that produced a lower yield (Table 2). The other populations presented average values around 0.70%. Similar values were reported by Silou et al. (2006) for shoot fresh weight of E. hensii K. Schum. The authors also assessed the oil yield of the flowers and roots that presented an average yield of 1.0% and 0.4%, respectively. Other authors have reported lower values for oil yield in Elionurus species. Mevy et al. (2002) obtained an average yield of 0.45% for shoot fresh weight of E. elegans Kunth. Hess et al. (2007) assessed differences in the oil yield of E. muticus (Spreng.) Kuntze according to the season of the year and observed that the highest mean oil yield was during the spring (0.37%) followed by the winter (0.29%), summer (0.25%) and fall (0.23%). These values were similar to those reported for the São Francisco de Paula population. Variability in the essential oil yield values has also been reported for other aromatic species such as Cymbopogon spp. Spreng. (Khanuja et al., 2005) and Ocimum basilicum L. (Blank et al., 2004). A preliminary chemical analysis of the oil identified citral in all the populations, except for the Morro da Polícia population (Table 3).
Citral has been reported in Elionurus by other authors, but these compounds were more frequent in Argentina and Uruguay (Mevy et al., 2002; Cacciabua et al., 2005; Kolb et al., 2007a; Sabini et al., 2006). Studies in Brazil have been concentrated in the Central region and to date there has been no report of the presence of citral in the plants studied in these regions (Scramim et al., 2000; Hess et al., 2007). In the present study, the plants used were collected in Rio Grande do Sul, where climatic conditions are similar to those of Argentina and Uruguay because of the geographic location. These conditions (colder regions) probably favor the production of these compounds by the plants.
The variations in RAPD band profiles of the 50 genotype studied resulted in 188 consistent molecular markers for analysis. This number of bands was obtained by the amplification of 16 primers which generated an average number of 11.7 markers per primer. Morro Santana population presented higher percentage of polymorphic molecular markers whereas São Francisco de Paula and Agronomy Faculty populations presented the lower percentage (Table 4). The genetic variation observed in E. muticus was high. The observed heterozigosity (Nei’s diversity index) was high for all populations (from 0.12 to 0.37) (Table 4). The results for f (from 0.027 to 0.105) indicated that the populations are panmmitic.
The dendrogram based on the Jaccard coefficient showed that generally there was a relationship depending on the geographic origin (Figure 1). The average similarity among all the genotypes (0.33) was the cutting point used in the analysis of the dendrogram and indicated the formation of five groups. There was a clear subdivision in the second and largest group that reflected geographic origin. The cophenetic coefficient was 0.85. The most similar individuals, with a value of 0.85, were MP32 and MP33 from Morro da Polícia. The relationship between genetic variability and geographic distribution has been observed in several aromatic plants as Hesperozygis ringens Benth (Fracaro & Echeverrigaray, 2006), Phalaris minor Retz grass (McRoberts et al., 2005), and Camellia sinensis (L.) Kuntze (Yao et al., 2008).
The analysis of molecular variance showed that the higher variability fraction observed was within each population, among its constituent individuals, representing 94% of the total variance (Table 5). The high intrapopulational variability suggested the occurrence of allogamy in the species studied. Several authors have reported high frequency of intrapopulational variability with cross-pollination (Yao et al., 2008; Silva et al., 2006). Populations were significantly differentiated from each other (FST = 0.30), meaning that there was a reduction in the gene flow among the populations that induced an increase in the genetic differentiation (Table 4). High values of genetic differentiation for natural populations have been reported for other species (Ferreyra et al., 2004; Ross-Ibarra et al., 2008). Populations inbreeding coefficient (f) were low, suggesting cross pollination.
TABLE 5. Analysis of molecular variance (AMOVA) in five E. muticus populations collected at Agronomy Faculty (AF), Morro da Polícia (MP), Morro Santana (MS), São Borja (SB), and São Francisco de Paula (SFP).
The E. muticus populations studied presented variability for agronomic and chemical traits. The population from São Francisco de Paula presented the greatest difference compared to the others. This can be observed in the phenotypic analysis where San Francisco de Paula presented, in most cases, different values from other populations. Thus this population would not be indicated for use in a breeding program. In spite of this, this population was the most different and may represent a source of variability for conservation in germplasm banks. In the case of establishing a breeding program, the breeder should concentrate efforts on the São Borja and Agronomy Faculty populations. The São Borja population presented high citral concentrations, accentuated variability and good performance for the traits assessed. The Agronomy Faculty population can also be highlighted based on the citral concentration but it presented less variability due to more reduced sampling.
ACKNOWLEDGMENT
The authors acknowledge Coordination of Improvement of Superior Education (Capes) and National Council on Cientific and Technological Development (CNPq) for financial support, Ana Carolina Nunes for the germplasm, and Dra Fernanda Bered for valuable suggestions.
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