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Crop Breeding and Applied Biotechnology, v. 3, n. 4, p. 245-254, 2003 245 Morphological and molecular characterization of italian ryegrass populations Caroline M. Castro1*; Antonio C. de Oliveira2; Fernando I. F. de Carvalho2; Manuel de S. Maia2; Luiz Anderson Mattos2 and Fábio Freitas2 1Embrapa Clima Temperado. Rodovia BR 392, Km 78, P.O. Box. 403, CEP 96001-970, Pelotas, RS, Brazil; 2Departamento de Fitotecnia, FAEM/UFPel, P.O. Box 354, CEP 96001-970, Pelotas, RS, Brazil. (* Corresponding Author [email protected]) ABSTRACT Italian ryegrass is the most important temperate grass in Rio Grande do Sul, Brazil. Despite its overall importance, there are no breeding programs for this species in this State. The purpose of this study was to evaluate the existing variability within and between four Italian ryegrass populations, three from Rio Grande do Sul, Brazil, and one from Uruguay. The populations were characterized based on morphological traits such as: number of tillers, canopy diameter, heading date, total leaf area, number of leaves, area/leaf, leaf dry matter, stem dry matter, leaf/stem ratio and dry matter yield. Molecular variation was also characterized using RAPD markers. A large variability was found within the populations (98.41%), which limited the complete separation of populations. However, significant differences were found among populations for traits of great forage interest, such as the number of tillers, canopy and heading date, indicating that the present variability is suitable for the initiation of a breeding program. KEY WORDS: Variability, forage, breeding. INTRODUCTION likely to be highly genetically heterogeneous (Forster et al., 2001). The measurement of genetic variability A large area in the state of Rio Grande do Sul present in germplasm collections is an important (southern Brazil) is covered by natural pastures factor to maintain and use these collections in a better composed mainly of species with spring-summer way (Casler, 1995), as well as to plan strategies to growth, and with a large decrease in production during develop synthetic populations. the cold season (Mota et al., 1981). Among the species Molecular marker technologies have been that were introduced to supply food during winter, successfully applied in the characterization of the Italian ryegrass (Lolium multiflorum Lam.) is the germplasm. The RAPD marker technology, where a most important economically speaking (Maia, 1995), unique primer allows the amplification of multiple cultivated in more than one million hectares. loci in the genome (Tingey and del Tufo, 1993), has The classical breeding of forage species is based on proved to be a good alternative to investigate the the generation of synthetic populations (Vogel and genetic variability in forage species (Huff et al., 1993; Pedersen, 1993). They are expected to produce under Sweenwey, et al., 1996; Huff, 1997; Chai and a wide range of climatic, edaphic and management Sticklen, 1998; Kölliker et al., 1999). conditions (Breese and Hayward, 1972). Therefore, The comparison of molecular and morphological retention of a degree of heterogeneity in selected characterizations is of paramount importance when populations is fundamental for the adaptation of new studying forage crops, since they can be used as cultivars. The success of the British perennial ryegrass complementary tools in the development of breeding cultivar S23 was based on its genetic heterogeneity, programs that are highly dependent on the quality of resulting in a buffering ability and high phenotypic the used germplasm (Loos, 1994a). Traits such as dry performance over a wide range of soil types (Valentine matter yield and forage quality are described as and Charles, 1975). essential in germplasm evaluation programs (Wilkins, The most important temperate forages, such as the 1991). Italian ryegrass, are all cross-pollinated; consequently, This study aimed at evaluating the variability within both natural ecotypes and synthetic populations are and among Italian ryegrass populations grown in Rio 2003, Brazilian Society of Plant Breeding 246 Crop Breeding and Applied Biotechnology, v. 3, n. 4, p. 245-254, 2003 Grande do Sul, Brazil. A molecular characterization population was evaluated with molecular markers. was performed using RAPD markers. A The DNA analysis was optimized by standardizing morphological evaluation was performed based on DNA concentrations through ethidium bromide the following traits: number of tillers/plant, canopy, staining compared to lambda/Hind III markers and heading date, total leaf area, number of leaves, using two independent DNA extractions from each average leaf area, leaf dry weight, stem dry weight, plant. Two grams of plant tissue were extracted using leaf/stem ratio and dry matter yield. the CTAB method (Saghai-Maroof et al., 1984). Further steps in DNA extraction were performed as previously described (Yang et al., 1996). MATERIAL AND METHODS Thirty primers from University of British Columbia, Plant Materials UBC 1 to 30, were screened to find those most effective for producing polymorphic bands in these Three Italian ryegrass populations from Rio Grande populations. The six primers showing the best do Sul, named “Dom Pedrito” (DP), “Pantano amplification products (UBC 2: CCTGGGCTTG; Grande” (PG) and “Pedro Osório” (PO), and one from 3: CCTGGGCTTA; 4: CCTGGGCTGGG; 9: Uruguay, cultivar “La Estanzuela-284” (LE-284) CCTGCGCTTA; 12: CCTGGGTCCA; 13: were used in this study. Selection criteria for the CCTGGGTGGA) were chosen. RAPD reactions Brazilian populations were their good seed quality were performed in 25 µl, with 2.5 ng genomic DNA; and performance, based in the information provided 0.2 mM dNTPs, 0.2 mM cresol red; 0.5 mM primer; by Fonsêca (1997). The cultivar “La Estanzuela-284” 2.5 mM MgCl ; 50 mM KCl; 10 mM Tris-HCl pH was chosen because it is largely cultivated in Rio 2 8.0; 1 mL/mL tritonX-100 and 1 U Taq polymerase Grande do Sul. (Pharmacia Biotech Inc.). The amplifications were performed on a thermocycler Morphological Characterization (MJ Research, Inc.) consisting of denaturation at 94 o o Each population consisted of 300 plants, planted at C (180s), followed by 44 cycles at 94 C (60s), 38 o o a distance of 0.30 m from each other, laid out in the C (60s) and 72 C (90s). In the end, samples were o field in a randomized complete block design with submitted to 5 min at 72 C for a final extension. three replications containing plots of 100 plants Amplified fragments were separated in 1.4 % (w/v) each. A random sample of 30 individuals per agarose gels, stained with ethidium bromide and replication was used for measuring most visualized on a UV light. morphological traits. The number of tillers/plant and the canopy diameter were measured in all plants 98 Statistical Analysis days after sowing, at the beginning of the elongation stage (Moore and Moser, 1995). The canopy The morphological data were subjected to a Levene’s diameter was determined as the average between two test (Snedecor and Cochran, 1980) to verify the diameter/plant measurements. The heading date was homogeneity of phenotypic variance, and then measured by the number of days after sowing until analysed using the GLM procedure of the SAS the first tiller anthesis. The leaf area and the number statistical package (SAS Institute, 1990). When the of leaves per plant were measured in the laboratory differences between populations were significant, the in an area meter (Model LI-3100, LI-COR, Lincoln, average values of each population for each trait were NE) one week after anthesis. Leaves and stems were tested with the Least Significant Difference test (LSD, dried separately at 65oC with forced air until a P≤0.05). constant weight. Dry matter yield was obtained by The populations were also compared pair wise in adding leaf and stem weights. The leaf/steam ratio relation to the proportion of genotypes found in was obtained by dividing the leaf weight by the stem the interval above the general mean of the trait weight. within a population. For this procedure, a chi- square analysis was performed according to Snedecor and Cochran (1980), with a 2x2 Molecular Characterization contingency table as: A minimum of 95 plants chosen by chance from each 2003, Brazilian Society of Plant Breeding Crop Breeding and Applied Biotechnology, v. 3, n. 4, p. 245-254, 2003 247 Population A Population B Total Plants above the general mean ƒi=1 ƒ i=2 X Plants below the general mean ƒ i=3 ƒ i=4 Y Total W K Z 2 4 1 χ 2 = ()f − F ∑ i = 1 Fi where ƒ was the observed frequence and F was the expected frequence obtained by the formula: (W.X ) (K.X ) (W.Y ) (K.Y ) F1 = F2 = F3 = F4 = Z Z Z Z For the molecular analysis, only bands present in both data transformation. Significant differences were extractions were scored as present (1) or absent (0) found through the analysis of variance in the number on a given genotype and recorded in a binomial of tillers, canopy diameter and heading date traits. matrix. Based on the molecular data, the genetic The “DP” population had more tillers per plant distance between individuals was estimated as (59.52) than “LE-284” at 48.29 (Table 1). These Euclidean distance (E) according to Excoffier et al. populations were also different from the other two (1992) and Huff et al. (1993), populations in the study. “PG” and “PO” populations did not differ, with 53.76 and 52.45 tillers/plant, (1− 2n ) E = n xy respectively. The “PG” population had a significantly 2n larger canopy (61.75 cm in diameter) than the other three populations. The four populations showed where n is the total number of polymorph bands, and significant differences for the heading date. The nxy is the number of bands shared by the individuals x earliest was “LE-284”, with an average flowering time and y.