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Abstract

The study of the genetic structure of wild plant populations is essential for their management and conservation. Several DNA markers have been used in such studies, as well as isozyme markers. In order to provide a better comprehension of the  results obtained and a comparison between markers which will help choose tools for future studies in natural populations of Oryza ]]> a predominantly autogamous species, this study used both isozymes and microsatellites to assess the genetic diversity and genetic structure of 13 populations, pointing to similarities and divergences of each marker, and evaluating the relative importance of the results for studies of population genetics and conservation. A bulk sample for each population was obtained, by sampling two to three seeds of each plant, up to a set of 50 seeds. Amplified products of eight SSR loci were electrophoresed on non-denaturing polyacrylamide gels, and the fragments were visualized using silver staining procedure. Isozyme analyses were ]]> A=2.83, p=0.71, A_P=3.17, H_o=0.081, H_e=0.351) than isozyme loci (A=1.20, p=0.20, A_P=1.38, H_o=0.006, H_e=0.056). Interpopulation genetic differentiation detected by SSR loci (R_ST=0.631, equivalent to F _ST=0.533) was lower than that obtained with isozymes (F_ST=0.772). However, both markers showed high deviation from Hardy-Weinberg expectations (F_IS=0.744 and 0.899, respectively for SSR and isozymes). The mean apparent outcrossing rate for SSR (=0.14) ]]> =0.043), although both markers detected lower levels of outcrossing in Amazonia compared to the Pantanal. The migrant number estimation was also higher for SSR (Nm=0.219) than isozymes (Nm=0.074), although a small number for both ]]> O. glumaepatula and that microsatellites are powerful for detecting information at the intra-population level, while isozymes are more powerful for inter-population diversity, since clustering of populations agreed with ]]>

Key words: genetic structure, gene flow, isozymes, mating system, SSR.

Resumen

El  estudio de la estructura genética de  poblaciones de plantas silvestres es esencial para su manejo y conservación. Varios  marcadores de ADN e isoenzimas se han utilizado en este tipo de análisis. Con el fin de proporcionar una mejor comprensión de los resultados obtenidos y saber que marcador ]]> Oryza glumaepatula, este trabajo busco evaluar y comparar dos marcadores de ADN, isoenzimas y microsatélites, en la diversidad y estructura genética de 13 poblaciones, destacando las similitudes y divergencias de cada marcador, así como la importancia relativa de los resultados en genética de poblaciones y conservación. Para los SSR, ocho loci SSR fueron evaluados, y los fragmentos se visualizaron ]]> R_ST=0.631, equivalente a F_ST=0.533) fue inferior a la obtenida con las ]]> F_ST=0.772). Ambos marcadores mostraron alta desviación del equilibrio de Hardy-Weinberg (F_IS=0.744 y 0.899, respectivamente, para SSR e  isoenzimas). La tasa media  aparente de cruzamiento para SSR (=0.14) ]]> =0.043), aunque ambos marcadores detectaron niveles más bajos en la tasa de fecundación cruzada para la Amazonia, en comparación con la región del Pantanal.  La  estimación de número de migrantes  también fue mayor para los SSR  (Nm=0.219) que ]]> Nm=0.074).  No se obtuvo  ninguna correlación entre las distancias genéticas y geográficas para los SSR, y para las isoenzimas se obtuvo una correlación positiva entre las distancias  genéticas y geográficas. Llegamos a la conclusión de que estos marcadores son divergentes en la detección de los parámetros de la diversidad genética en O. glumaepatula y ]]>

Palabras clave: estructura genética, flujo de genes, isoenzimas, SSR, sistema de cruzamiento.
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Oryza glumaepatula Steud. (Poaceae) is one of the four wild rice species originated in America, the others being O. latifolia, O. grandiglumis and O. alta ]]> et al. 2003). It is the only diploid wild American species and its importance relies on its use in crosses with O. sativa in plant breeding programs (Brondani et al. 2001, Yoshimura et al. 2010) aiming at introgression of important traits from the wild species and amplifying the genetic ]]>

The Brazilian O. glumaepatula populations are found in the extensive river basins of the Amazon and Pantanal  Matogrossense and the smaller river basins which occur in the states of Goiás and Tocantins (Oliveira 1994, Brondani et al. 2005). This species presents ]]>

]]> The Oryza genus presents different reproductive systems such as outcrossing, inbreeding, intermediate crossing rates and vegetative reproduction. Several studies have indicated that O. glumaepatula is a self-fertilizing species (Akimoto et al. 1998, Buso et al. 1998, Ge et al. 1999), but recently studies have reported that this species has different outcrossing rates in different populations, with values ranging from 9.3% to 30% (Brondani et al. 2005, Karasawa et al. ]]>

The knowledge of genetic diversity and genetic structure of populations is essential for understanding species evolution, the genetics of natural populations, as well as the adoption of collecting strategies ]]> in situ or ex situ management, restoration and conservation practices (Slatkin 1987, Vigouroux et al. 2008). Several molecular techniques are available for the detection of genetic variability in natural populations (Agarwal et al. 2008). Among the different molecular markers, the first to be established were the isozymes in the 60s (Lewontin & Hubby 1966). Isozyme markers have been widely used in different genetic studies in the genus Oryza, including studies of ]]>

With the advent of PCR in the late 1980s, which made the analysis and genotyping straightforward, microsatellites, characterized by high heterozygosity and the presence of multiple alleles, became the marker of choice in genome mapping and also in population genetics studies and related areas (Ellegren 2004). Microsatellites or simple sequence ]]> Oryza glumaepatula natural populations, including the genetic structure and diversity (Brondani et al. 2005, ]]>

Studies comparing genetic structure of natural populations with these two markers (isozymes and microsatellites) have been conducted for quite a few species, such as Sorghum ]]> (Djé et al. 1999), Elymus caninus (Sun et al. 2001), oaks (Quercus spp.) (Curtu et al.2007), and Euterpe edulis (Conte et al. 2008). But the only report to-date comparing isozymes and microsatellite markers in the genus Oryza was conducted for O. rufipogon, a predominantly cross-pollinated ]]> O. glumaepatula, this study used both isozyme and microsatellite markers to assess (1) the level and distribution of genetic diversity; (2) the distribution of this diversity within and among populations, pointing ]]> O. glumaepatula.

Materials and Methods

]]> Populations   studied   and   sampling: Thirteen O.  glumaepatula populations  were assessed in this study, belonging to the wild rice collection in the Genetics Department of the Luiz de Queiroz College of Agriculture, University of São Paulo. The populations were selected from three regions in Brazil: (I) Amazon region, in Amazonas and Roraima States, with eight populations originating from the Purus, Solimões, Japurá, ]]> Table 1, Fig. 1). These populations were sampled during two ]]>

Each population ]]>

DNA extraction and amplification of SSR loci: Total genomic DNA was extracted from adult lyophilized leaves of individual plants using the CTAB method according to Hoisington et al. (1994), modified by Karasawa et al. (2007a). DNA was quantified on 4% (w/v) polyacrylamide gels and bands were revealed using the silver nitrate staining procedure (Bassam ]]> Table 2).

For each PCR reaction, 30ng of genomic DNA from individual plants were used in a 12μL volume containing 0.3μM of each primer, 0.25mM of each dNTP, 1.5mM of MgCl₂, 10mM Tris-HCl, and 0.6 unit of Taq DNA ]]>
]]> Isozyme analyses procedures: Isozyme analyses were conducted in polyacrylamide gels, under a discontinuous system. The gel and electrode buffers used were a basic buffer (Hames 1996), with pH 8.8 for the 5.5% resolving gel and pH 6.8 for the 3.5% stacking gel.

The newest expanded ]]>

From a total of 10 promising enzymatic systems (Veasey et al. 2008), four were selected in this study due to the presence of higher band ]]>

For the statistical analysis, the GDA program (Lewis & Zaykin  2000) was used to estimate allelic and genotypic frequencies, number of alleles per locus ( A), observed heterozygosities (H_o), gene diversity (H_e), and fixation indices (f). Genotypic frequencies obtained from both markers were submitted to Fisher’s exact test considering the Hardy-Weinberg equilibrium, as defined by Weir (1996), using the TFPGA software (Miller 1997).
]]> Wright’s F statistics were estimated considering a random model, defined according to Weir (1996), where the sampled populations were considered representative of the species and with a common evolutionary history. These estimates (F_IS, F_ST e F_IT) ]]> STAT (Goudet 1995). Confidence intervals (95%) were also obtained for each of these estimates. Private alleles were also identified with the GDA program (Lewis & Zaykin 2000).

Considering that the mutation process in microsatellite loci is not in ]]> F_ST parameter (Slatkin 1995) developed specifically for microsatellite data (R_ST) was also estimated. Parameters R_ST and gene flow (Nm) were estimated using the R_STCal program (Goodman 1997). Dendrograms were ]]>

Patterns of spatial variation were analyzed using Pearson’s coefficient of correlation (r) between Nei’s genetic distances matrix (Nei 1978) and the matrix of geographic distances (shortest distance between two ]]> = (1-F_IS)/(1+F_IS). The parameter  was also estimated for each population [ = (1-f)/(1+f)] (Weir 1996)

Results

]]> Genetic diversity levels: All SSR loci showed polymorphism (Table 2) while isozymes were monomorphic for two of the six loci, Aat3 and Gdh (Table 3). A total of 81 alleles were found for the SSR markers, varying from five to 21 alleles per locus ( Table 2), while 11 alleles were found for the isozyme markers, varying from one to three alleles per locus (Table 3). For both markers, the mean expected heterozygosity or gene diversity was higher than the mean observed heterozygosity (H_o=0.085 and H_e=0.761 for SSR; H _o=0.007 and H_e=0.247 for isozymes) (Tables 2 and 3).

Considering ]]> Table 4). The average number of alleles per locus per population ( ) was 1.20 and 2.83, with means over loci of 1.83 and 10.12, respectively, and the average number of alleles per polymorphic locus () of 1.83 and 3.17, respectively, for isozymes and microsatellites (Table 4).
]]> No private alleles were detected for the isozyme markers, probably due to the small number of alleles and small number of enzyme loci, whereas the SSR markers were effective in detecting private alleles (Fig. 2). There was only one private allele detected in the Xingu region, 19 in the Amazon and eight in the Pantanal region (Fig. 2B). At the ]]> Fig. 2A).
 
The average observed heterozygosity (H_o) and gene diversity (H_e) ]]> Table 4). On the other hand, when considering H_o and H_e as the mean over loci, we detected H_o=0.007 and 0.085, and H_e=0.247 ]]> H_o and H_e levels, within each of the three regions, was maintained (Table 5). The apparent  outcrossing  rate () estimated  by these markers showed ]]> Table 4) and regional (Table 5) levels, but both markers showed lower levels in the Amazon region (1.9% and 2.8%), while the  highest  levels occurred in the Pantanal region,  with  5.2%  and  10.5%,  respectively, for isozymes and microsatellites (Table  5). However, the markers were not in agreement in the case of the Xingu ]]>

Genetic population structure: Mean inbreeding registered within each population (f) was higher, in general, for the isozymes (0.910) when compared to microsatellites (0.771) (Table 4). This tendency was maintained for both markers also at the regional level (Table 5). The estimates of F statistics revealed that total inbreeding coefficient ]]> IT) in this species is very high, 0.974 for isozymes and 0.895 for microsatellites (Table 6), and that the main factor promoting the deviation from Hardy-Weinberg equilibrium is the mating system (F_IS=0.899 and 0.774, respectively, for isozymes and microsatellites). However, although of a lower magnitude, but also expressive, the genetic differentiation among populations ( F_ST) contributed for the total inbreeding levels observed, with the isozyme markers showing a value of 0.772 for this parameter, while the level registered for the SSR markers was 0.533 (or 0.631 for the corrected value of R_ST). On the other hand, the number of migrants per generation (Nm = ¼ [(1/F _ST or R_ST)-1]) observed for both markers was small, only 0.074 for the isozymes and 0.146 for the SSR marker (Table 6).

The cluster analysis, based on Nei´s genetic distances (Nei 1978) ]]> Aat-1 and Aat-2 loci, different alleles predominated in each of the two regions, Pantanal and the Amazon (Fig.  3A). The Xingu population (XI-1) and the Pantanal  ]]> Skd-1 locus and from all the others for presenting a high frequency of the a3 allele at Pgm-1 locus.
]]> However, data from microsatellites tended to exhibit a random clustering, not in agreement with the geographic origins (Fig. 3B). These results were confirmed in the correlation tests between genetic and geographic distances obtained for each marker. In this sense, correlations between genetic and geographic distances showed a positive and significant result (r=0.6594, p<0.05) when estimated with ]]>

Discussion

]]> Genetic diversity levels: This study compared the levels of diversity assessed with isozymes and microsatellite markers in 13 O. glumaepatula populations in order to establish the potential of each marker for a predominantly inbreeding wild rice species. The results of the analysis of microsatellite and isozyme loci are consistent with the principles of each marker, considering the fact that microsatellites showed a wide range of alleles at each locus whereas isozymes showed only one to ]]>

In this study, isozymes showed only 42.4% to 43.5% of the ]]> ) and average number of alleles per polymorphic locus (), respectively. Similarly, we found that microsatellites detected private alleles, whereas no private alleles were found with isozyme markers, which may be due to a lower number of apparent alleles per locus and a lower number of loci used in ]]>

The levels of observed heterozygosity (H_o) and gene diversity (H_e) assessed with isozymes represented, on average, 7.4% and 15.9% of those observed with microsatellites, respectively.  However, when gene diversity was considered over loci we could verify that the amount is ]]> H_e) ]]> O. rufipogon populations from China. Similarly, higher levels of polymorphism revealed by microsatellites when compared to isozymes were found in natural populations of Euterpe edulis Mart. in Brazil (Conte et al. 2008), oaks (Quercus spp.) in West-Central Romania (Curtu et al. 2007), in a natural Elymus caninus ]]> Sorghum bicolor L.) landraces in North-Western Morocco (Djè et al. 1999).

Population genetic structure: Isozyme markers showed similar inbreeding levels (f) as microsatellites for populations, regions and for total ]]> F_IT), except for the Xingu population. The existence of inbreeding within populations showed, with both markers, that this species has an inbred maternal family structure within populations and that this structure was established primarily by the reproductive system (F_IS), and that most of the total diversity is located among families within populations. In fact, the reproductive ]]> F_ST). Gao et al. (2002b) also found an agreement in this type of result with both markers, but in O. rufipogon ]]> F_ST). Measures of genetic structure, such as Wright´s F statistics, or Nei’s coefficient of gene differentiation (G_ST) (Nei 1973), were similar for the two sets of markers (isozymes and SSR) assessed in natural populations of Sorghum ]]> (Djé et al. 1999), Elymus caninus (Sun et al. 2001) and Euterpe edulis (Conte et al. 2008).

Previous studies on genetic structure conducted in the genus Oryza have ]]> F_ST (G_ST) values, but largely agree that the mode of reproduction of the species has generated prominent effect on the differentiation observed. In O. glumaepatula, studies using allozymes showed F_ST   values of 0.346 (Akimoto et al. 1998), 0.310 ]]>

O. rufipogon studies with isozymes showed genetic structure levels ]]> F_ST) dependent on the developmental stages (seed, young and adults plants) and the life cycle (annual or perennial). Annual populations showed F_ST values varying from 0.085 to 0.208 for seeds, from 0.087 to 0.145 for young plants and 0.193 for adults, while among the perennial populations higher values were found, ranging from 0.327 to 0.366 at the seed level, from 0.129 to 0.356 for young plants and from 0.360 to ]]> F_ST values of 0.289 for intermediate populations, 0.396 for perennials and 0.600 for annuals. Gao et al. (2000) and Gao et al. (2002a) reported values such as 0.310 and 0.254 in perennial populations with isozymes. With microsatellites, Gao (2004) obtained an F_ST value of 0.246, whereas when comparing isozymes and ]]> O. officinalis perennial populations, an F_ST value of 0.442 was reported (Gao 2005).

The results in our study with O.glumaepatula populations, ]]> F_ST=0.533) with isozymes (F_ST=0.772), are in accordance with the results obtained by Gao et al. (2002b) for perennial outcrossing O. rufipogon populations, although these values were lower than those found in O. glumaepatula. It is difficult to ]]> F_ST values found with isozyme loci compared with microsatellites was caused by stochastic processes or as an indirect effect of selection. According to a survey by Frankel et al. (1995), the genetic diversity among populations (G_ST) with isozymes should be lower in cross-pollinating and perennial plants, and higher in autogamous and annual plants, which may explain the results ]]>

Due to the mode of reproduction of this species, the number of migrants (Nm) estimated promoting gene flow among populations was small, only 0.074 for isozymes and 0.146 for microsatellites. Gene flow is important in the evolution of plant populations, because the geographical variation observed in morphology and gene frequencies of a ]]>

Cluster analysis grouped the populations according to their geographical origin with isozymes, whereas a pattern of random clustering of populations not consistent with the geographical origin ]]> O. rufipogon, found that information obtained by both markers were not completely correlated. The grouping of maternal plants of Elymus caninus based on isozymes and microsatellites were also not ]]>
 
In our study, we verified a positive correlation between genetic and geographical distances for isozymes and a negligible negative correlation when estimated with microsatellites. A positive correlation between genetic and geographical distances, when the genetic markers ]]>

Our results showed that microsatellites generate a higher volume of intra-population genetic diversity information compared to those obtained with isozymes in this species. Microsatellites showed 130% more alleles and 3.5 times higher rate of polymorphism, being more ]]>

In summary, it can be said that SSR markers showed more power for investigating neutral intra-population diversity, as expected. At the inter-population level, however, despite the relatively small number of loci used, isozymes led to better results, since clustering of populations agreed with the expectations based on the geographic distribution of the populations.

Acknowledgments

This research was supported by grants and scholarships provided by Fundação de Amparo à Pesquisa do Estado de ]]>

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*Correspondencia: Marines M. G. Karasawa: Departamento de Ciências Biológicas e da Terra, Universidade Federal de Alfenas (UNIFAL), 37300-000 Alfenas, Minas Gerais, Brazil; mgniechk@yahoo.com.br
Roland Vencovsky: Departamento de Genética, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, ESALQ/ USP, Caixa Postal 83, 13400-970 Piracicaba, São Paulo, Brazil; rvencovs@esalq.usp.br
Cynthia M. Silva: Departamento de Genética, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, ESALQ/ USP, Caixa Postal 83, 13400-970 Piracicaba, São Paulo, Brazil; cynthia.esalq@gmail.com
Daruska C. Cardim: SEMASA-Serviço Municipal de Saneamento Ambiental de Santo André, R. José Caballero, 143, 09040-210 Santo André, São Paulo, Brazil; daruskacardim@yahoo.com.br
Eduardo de A. Bressan: Centro de Energia Nuclear na Agricultura, CENA/USP, Piracicaba, SP, Brazil; ebressan@cena.usp.br
Giancarlo C.X. Oliveira: Departamento de Genética, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, ESALQ/ USP, Caixa Postal 83, 13400-970 Piracicaba, São Paulo, Brazil; gcxolive@gmail.com 
Elizabeth A. Veasey: Departamento de Genética, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, ESALQ/ USP, Caixa Postal 83, 13400-970 Piracicaba, São Paulo, Brazil; eaveasey@esalq.usp.br ]]>
1.    Departamento de Ciências Biológicas e da Terra, Universidade Federal de Alfenas (UNIFAL), 37300-000 Alfenas, Minas Gerais, Brazil; mgniechk@yahoo.com.br
2.    Departamento de Genética, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, ESALQ/ USP, Caixa Postal 83, 13400-970 Piracicaba, São Paulo, Brazil; rvencovs@esalq.usp.br, cynthia.esalq@gmail.com, eaveasey@esalq.usp.br, gcxolive@gmail.com
3.    SEMASA-Serviço Municipal de Saneamento Ambiental de Santo André, R. José Caballero, 143, 09040-210 Santo André, São Paulo, Brazil; daruskacardim@yahoo.com.br
4.    Centro de Energia Nuclear na Agricultura, CENA/USP, Piracicaba, SP, Brazil; ebressan@cena.usp.br

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