Esterase polymorphism for genetic diversity analysis of some accessions of a native forage grass , Mesosetum chaseae Luces , from the Brazilian Pantanal

The aim of the present study was to estimate the genetic diversity within the samples of Mesosetum chaseae from the Embrapa Pantanal Germplasm Bank (BAG) and assess how they are genetically structured to guide proposals to: 1) identify native forages for further testing to measure their suitability for sowing in conjunction with or as an alternative to exotic forages, mainly Urochloa humidicola; and 2) improve the species M. chaseae with samples that are maintained in the BAG. Isozyme αand β-esterases were analyzed in 10 accessions collected from different locations in the Nhecolândia sub-region of the Pantanal, and maintained in the BAG. Accessions A11, which showed the highest effective number of alleles, and A32 with the highest average values of expected and observed heterozygosity, were identified as warranting further study as possible options for sowing as pasture forages, as well as for use in recovering poor and degraded areas in the Pantanal region. A high level of population differentiation was detected among the 10 accessions, indicating that they form genetically structured populations and that all accessions are important samples of M. chaseae, which should be maintained in the BAG. Crosses between sample plants with the highest genetic distances are recommended to implement improvement plans with a prospect of broadening the genetic base of the species.


Introduction
The Pantanal region, located in Mato Grosso do Sul State (MS), Central Brazilian Plateau, is the largest wetland in the world and one of the most diverse nature reserves on the planet; it is considered as a World Biosphere Reserve and Natural Heritage by UNESCO.This region is characterized by large pasture fields, with 95% of its area occupied by extensive cattle farms (Godoi Filho 1986).While many exotic species have been introduced to increase productivity, little effort has been made to understand and estimate the potential use of native species such as the grass Mesosetum chaseae Luces (Poaceae family).The most widely sown exotic forage has been the African grass Urochloa humidicola (syn.Brachiaria humidicola) due to its tolerance of poor soils, flooding and trampling, resulting in increases in carrying capacity of the land and farm profitability.However, monocultures in plant communities often result because of the exclusive use of this species, combined with its ability to spread in different landscapes of the Pantanal (Santos et al. 2011).Although scientific research has shown that the effect of U. humidicola on plant diversity is influenced by season and topography in areas experiencing seasonal flooding (Bao et al. 2015), the situation can be different in flood-free areas with soil fertility problems, mainly savannas (Pivello et al. 1999), because few plant species are adapted to such conditions, mainly native grasses (Santos et al. 2011).
Mesosetum chaseae is an important native forage plant in the Pantanal region, and is found mainly in flood-free and temporarily flooded grasslands, showing habitat plasticity and vegetative propagation in these environments (Santos et al. 2002;Alvarez et al. 2004).It is known as "grama-do-cerrado" (grass of the savanna) and is one of the most prominent forage species owing to its productivity (Santos 2001), acceptability by animals (Santos et al. 2002) and drought resistance (Santos et al. 2005).The "grama-do-cerrado" displays good tillering and grows well on poor soils.As it is prevalent under extreme climatic conditions (e.g.severe drought), this species has been considered to have potential for reclaiming degraded areas in the Pantanal.Annual aboveground dry matter production of 2,000-3,800 kg/ha has been recorded under good conditions (Santos et al. 2011), andPinheiro et al. (2005) showed that M. chaseae retained forage quality during ripening and could be used for hay-making.
Studies on quality of seeds, cytogenetics and reproduction (Silva et al. 2011;2012;2013) represent the few investments that have been recently made for the characterization of M. chaseae.In the literature we found no records of molecular level studies.Investigations of genetic divergence at the molecular level of this species, however, are important to guide proposals to: 1) identify native forages for further testing to measure their suitability for sowing with or as an alternative to exotic forages, mainly Urochloa humidicola; and 2) implement plans for improvement of M. chaseae with samples maintained in the Germplasm Bank (Banco Activo de Germoplasma, BAG) of Embrapa Pantanal (Empresa Brasileira de Pesquisa Agropecuária in Corumbá, MS, Brazil).The current study aimed to make a start to document the genetic diversity of the species, and check how the accessions of M. chaseae from the BAG of Embrapa Pantanal are genetically structured to guide further testing in the field and future breeding programs.If suitable accessions can be identified it could provide a platform for promoting the conservation of biodiversity in pastures and the sustainability of native forage resources.Genetic polymorphism in loci for α-and β-esterase isozymes was analyzed to estimate genetic diversity and structure of the M. chaseae accessions.

Material and Methods
Samples of M. chaseae were collected from different locations in Nhecolândia, a sub-region of the Brazilian Pantanal, and are maintained in the germplasm collection at Embrapa Pantanal (Figure 1).Table 1 indicates the original collection sites according to the Universal Transverse Mercator (UTM) system.
Seeds from each M. chaseae accession were collected and distributed for germination in 500-mL pots containing sterile soil.The resulting plants were kept at room temperature, watered daily and used for the study.Leaves were collected from 10-30 plants of each accession; the plants were individually evaluated through electrophoresis.Leaf pieces (200 mg) from each plant were homogenized with a glass rod in an Eppendorf micro-centrifuge tube with the use of 60 μL extraction solution prepared with 1.0 M phosphate buffer pH 7.0, containing 5% PVP-40, 1.0 mM EDTA, 0.5% βmercaptoethanol and 10% glycerol solution and maintained in a cold chamber.After homogenization, the samples were centrifuged at 25,000 rpm (48,200 x g) for 30 min, at 4 °C, in a Juan 23 MRi (Thermo Scientific, USA) centrifuge, and the supernatant was used for each sample.Polyacrylamide gels (12%) were used to analyze the esterase isozymes (EST; EC 3.1.1)according to the method of Frigo et al. (2009).The polyacrylamide gel was prepared with 0.375 M Tris-HCl, pH 8.8 as buffer.A 6.2-mL volume of acrylamide/bis-acrylamide solution (30 g acrylamide and 0.8 g bis-acrylamide dissolved in 100 mL of double-distilled water), 4.0 mL 1.5 M Tris-HCl, pH 8.0, 6.2 mL double-distilled water, 320 μL ammonium persulfate 2% and 16 μL TEMED [Tetramethylethylenediamine] were used to separate the gel.The stacking gel was prepared with 3.0 mL acrylamide/bis-acrylamide (5 g acrylamide and 0.25 g bisacrylamide dissolved in 50 mL double-distilled water), 3.0 mL 0.24 M Tris-HCl, pH 6.8, 30 mL double-distilled water, 250 μL ammonium persulfate (2%) and 3 μL TEMED.
The electrophoresis (PAGE, polyacrylamide gel electrophoresis) was performed for 5 h at 4 °C, and a constant voltage of 200 V.The running buffer was 0.125 M Tris/0.0959M glycine, pH 8.3.
The esterases were identified through procedures previously described by Sala et al. (2011).The gels were soaked for 30 min in 50 mL 0.1 M sodium phosphate, pH 6.2, at room temperature.Esterase activity was visu-alized by placing the gels for 1 h in a staining solution prepared with 50 mL of sodium phosphate solution, 30 mg of β-naphthyl acetate, 40 mg of α-naphthyl acetate, 60 mg of Fast Blue RR salt and 5 mL of N-propanol.The polyacrylamide gels were dried as described by Sala et al. (2011), and kept at room temperature for 60 min in a mixture of 7.5% acetic acid and 10% glycerol embedded in 5% gelatin.They were then placed between 2 sheets of wet cellophane paper stretched on an embroidering hoop and left to dry for 24-48 h.
The genetic variability in the M. chaseae samples was analyzed with POPGENE 1.32 Computer Software (Yeh et al. 1999) for the analysis of allele frequencies, mean observed heterozygosity (Ho) and expected heterozygosity (He) and mean number of alleles per locus (Na), effective number of alleles per polymorphic locus (Ne), percentage of polymorphic loci (%P), χ 2 test for deviation from Hardy-Weinberg equilibrium.The F statistic of Sewall Wright (Wright 1965), the deficit of heterozygotes (FIS and FIT), and the genetic diversity between the 10 samples (FST) were also estimated using POPGENE 1.32.To explore the hierarchical partitioning of genetic variation within and among the samples, we performed an Analysis of Molecular Variance www.tropicalgrasslands.info (AMOVA, GenAlEx 6.2; Peakall and Smouse 2006).
The genetic identity (Nei 1973) and distances among the M. chaseae samples were calculated by UPGMA grouping.Polymorphisms from α/β-esterase loci were also analyzed using STRUCTURE software 2.0 (Pritchard and Wen 2003), which evaluates the level of genetic admixture between the 160 M. chaseae plants.
The genotypes were clustered, with the number of clusters (K) ranging from 2 to 10, and were tested using the admixture model with a burn-in period of 5,000 repeats followed by 50,000 Markov Chain Monte Carlo (MCMC) repeats, considering the presence and absence of alleles across the sample.The true number of populations (K) is often identified using the maximal value of ∆ (K) returned by the software.The most probable number (K) of sub-populations was identified as described by Evanno et al. (2005).The display of the graphical output of the results STRUCTURE was taken as input data using the STRUCTURE HARVESTER, a website and software that are used to visualize STRUCTURE outputs and to implement the Evanno method (Earl and von Holdt 2012) to display a graphical representation.
A comparison between the diversity parameters in the 10 accessions (Table 2) showed that the number of effective alleles is higher in A11 (Ne = 2.7383) than in the other M. chaseae accessions.The observed and expected mean heterozygosities were highest in accession A32 (Ho = 0.75;He = 0.5972).Departure from Hardy-Weinberg equilibrium was observed in 28 of 40 tests (70%) in 10 accessions, resulting from an excess of heterozygous samples at the Est-1, Est-3 and Est-4 loci (Table 3); the fixation index (FIS) was negative at the Est-1, Est-3 and Est-4 loci.An excess of heterozygous plants (Ho˃He) occurred in accessions A11, A17, A31 and A32 (Table 2).The deficit of heterozygous plants due to inbreeding within sub-populations was only 4.3% (FIS = 0.0432) (Table 3).
The correlation between the genetic distance matrix and the cophenetic matrix calculated by the Mantel test (Rohlf 1989) was low (r 2 = 0.004 for 1,000 permutations), indicating that the level of genetic differentiation between frequencies of alleles in the Est-1, Est-2, Est-3 and Est-4 loci of the plants from the 10 M. chaseae accessions was not related to the geographical distance separating the plants.
The clustering of the 160 plants from 10 accessions according to a model-based Bayesian algorithm is shown in Figure 4.Each bar in the graph represents a plant and its inferred proportion of allele admixture.The colors represent 3 different clusters corresponding to differential allele proportions in each plant.The optimal K value determined by Bayesian analysis indicated that the plants were grouped into 3 clusters (∆K2 = 0.00; ∆K3 = 33.7349;∆K4 = 0.72333; ∆K5 = 1.7489; ∆K6 = 1.1321; ∆K7 = 1.5338; ∆K8 = 10.4170;∆K9 = 0.1642; ∆K10 = 0.00).The bar plot obtained for the K value (K = 3; ΔK = 33.7349),and the results were consistent with the evidence of plants with a mixture of ancestral genome of either 2 or 3 groups, and plants with genome predominantly of 1 of the groups.At least 78% of the plants from accession A32 were in the green cluster and at least 72% of the plants of accessions A8 and A11 were in the red cluster.Plants from accessions A1, A9 and A31 contain predominantly a mixture of genome of the ancestral groups blue and green, those in A17 a mixture of ancestral groups blue and red and those in A4, A5 and A24 a mixture of genome of the groups blue, green and red (Table 5).
Bayesian analysis indicated that the most probable number of sub-populations is 3 in the M. chaseae accessions from the Nhecolândia sub-region kept in the germplasm collection at Embrapa Pantanal.In accession A32 there are predominantly plants ascending from one sub-population (green cluster), in accessions A8 and A11 there are predominantly plants ascending from another sub-population (red cluster), while in accessions A4, A5 and A24 there are plants ascending from the 3 sub-populations.

Discussion
The expected heterozygosity in Est-1, Est-2, Est-3 and Est-4 loci of M. chaseae ranged between 0.3937 and 0.6013 (plants from accessions A9 and A32).The mean He value (0.6153) of the plants from 10 accessions distributed across the Nhecolândia sub-region was high.Accession A11, showing the highest effective number of alleles (Ne = 2.7383), and A32 the highest value of mean expected and observed heterozygosity (He = 0.5972; Ho = 0.7500) present the highest genetic diversity.Accessions A11 and A32 may be, therefore, recommended for crossings to enlarge the genetic basis of M. chaseae in future breeding programs.In addition, these accessions should be planted in a range of environments to determine their suitability for increasing pasture areas planted to native species, as well as for recovering poor and degraded areas in the Pantanal region.Allendorf and Luikart (2007) suggested that the level of heterozygosity is an important indicator of the amount of adaptive genetic variation in a plant population.High heterozygosity implies a high chance for divergent selection.Populations with high heterozygosity may have been derived through different selective pressures throughout their geographical distribution and may persist under climatic or seasonal changes that may eliminate other populations.Studies of phenotypic characteristics in some samples of M. chaseae showed that accession A11 stands out for the large number of green leaves and a larger number of emerging leaves (Santos et al. 2007).High heterozygosity and production of green leaves are important features supporting the potential use of A11 for expansion of pastures and restoration of degraded areas, provided that it also has other desirable attributes such as high nutritional value, and adaptation to drought, mismanagement, disease and insect pests, which are yet to be evaluated.
High genetic diversity (He˃0.50)was also detected in accessions A1, A4, A5, A8, A17, A24 and A31, indicating that these samples may have potential for use in breeding programs and warrant further study.High heterozygosity would indicate that the plant is likely to have a substantial amount of genetic variation to withstand different selective pressures.An excess of heterowww.tropicalgrasslands.info zygous plants (Ho˃He) was observed in accessions A11, A17, A31 and A32.The deficit of heterozygotes (Ho<He) was higher in A24 than in A1, A4, A5, A8 and A9.Higher density and productivity, excellent coverage with a higher number of tillers/m 2 , and fewer dry leaves have been described as phenotypic characteristics for accessions A5, A9 and A24 (Santos et al. 2007).
Analyses of the genetic divergence among all 10 accessions showed a global value (FST) of 0.1693.According to Wright (1978), values of FST ranging from 0.15 to 0.25 indicate high genetic differentiation between populations.On the other hand, the estimated mean level of genetic divergence among populations for Poaceae is higher than 0.20 (Hamrick and Godt 1996).Thus, in the case of M. chaseae, an outcrossing grass (Silva et al. 2013), the genetic differentiation among accessions may be considered as moderate The genetic structure observed in the accessions of M. chaseae is also evident in the bar plot clustering of the 160 plants from the 10 accessions showing 3 subpopulations.The 3 probable sub-populations indicated by Bayesian analysis seem not associated with the original habitat of each accession.Plants of the green group (accession A32) originated from areas of non-flooded grassland and open grassland areas, while plants of the blue group originated from savanna (accessions A17 and A31) and the edge of the cordillera (accession A9).The genetically structured populations showing genetic diversity are all important samples of M. chaseae, which should be maintained in the Germplasm Bank of Embrapa Pantanal.
Despite the genetic structure observed in the accessions of M. chaseae, the analysis of polymorphic α/βesterase loci has indicated a significant gene flow (Nm = 1.2265) between the plants of the 10 accessions, suggesting the occurrence of outcrossing between the samples (Govindaraju 1989).The Bayesian analysis showed a mixture of plants of 3 (green, blue and red) clusters in the 10 accessions.The reproductive system in M. chaseae may be the main vector for the suggestive exchange of alleles.In M. chaseae, reproduction (tested in protected flowers) indicated allogamy (Silva et al. 2013).In addition, the high gene flow may be explained by a mixture of samples among accessions due to the dispersion of seeds that may occur in certain areas exposed to occasional flooding periods.
The genetically structured accessions of M. chaseae showing high genetic diversity are according to their wide distribution in the Pantanal region, their habitat plasticity and vegetative development in these environments as shown by Santos et al. (2002) and Alvarez et al. (2004).Mesosetum chaseae specimens had a remarkably high number of alleles per locus and greater genetic diversity than those reported within populations of other grasses (Godt and Hamrick 1998).The combinations of characteristics such as: tropical region, regional range, perennial life form, sexual mode of reproduction, outcrossing breeding system, and seed dispersal mechanism, may explain the great genetic diversity in samples of M. chaseae.The low number of loci analyzed and the high genetic diversity generally ascribed to the esterase isozymes may also explain the high genetic diversity observed in our study compared with other grasses (Godt and Hamrick 1998).
In Eragrostis curvula, a valuable native forage grass in Africa, the esterase pattern proved useful as an additional criterion for identifying the individual taxa making up the so-called Eragrostis curvula complex (E.curvula, E. conferta, E. robusta, E. chloromelas and E. lehmanniana) and for evaluating their reciprocal relationships (Poverene and Curvetto 1989).Isozymes (including esterases) were used for genetic characterization of different samples of elephant grass (Pennisetum purpureum), a species of forage widely distributed in Brazil after its introduction to the country (Daher et al. 1997), although the values for heterozygosity were not presented.When the isozymes αand β-esterase were used to analyze the forages Brachiaria brizantha (now: Urochloa brizantha), B. plantaginea, B. humidicola and B decumbens (now: U. plantaginea, U. humidicola and U. decumbens, respectively), a clear distinction between the 4 species was possible (Martins et al. 1999).Using the esterase system also made it possible to identify quickly and accurately the patterns of 7 cultivars of elephant grass (Pennisetum purpureum) and their hybrids with Pearl millet (P.americanum) from the germplasm bank of elephant grass (Freitas et al. 2000).
Higher genetic variability within than between samples also has been described for other forage species of the Poaceae family such as Bromus auleticus, using isozymes (esterases and peroxidases) and random amplified polymorphism of DNA (Yanaka et al. 2005).Since the year 2000, besides isozymes, DNA markers have also been used as tools to investigate the genetic diversity in forages.Stylosanthes macrocephala, a tropical forage legume native to Brazil, showed high polymorphism of random amplified DNA segments (RAPD), but the levels of heterozygosity and genetic divergence among accessions (FST) were not reported (Barros et al. 2005).Recently, the characterization of microsatellite markers in the grass, Paspalum atratum, has been considered a potential and promising tool for genetic diversity studies of Paspalum species (Cidade et al. 2010).Association of Single-Nucleotide Polymorphism markers with candidate genes in mapping of forage quality traits in perennial ryegrass was reported in Gill et al. (2012).
Estimates of heterozygosity are important indicators of the adaptive potential of populations for increasing grazing areas, and also for investigations for improvement of the species.Plants showing characteristics of interest (e.g.resistance to drought, greater leaf weight, early development) and high levels of heterozygosity are promising sources of inbred lines for breeding programs.As the levels of heterozygosity of the 10 accessions of M. chaseae have been estimated in our study, identification of features of interest for each sample would be necessary before mounting a breeding program for the species.The values of genetic identity (Nei 1973) and distances between the accessions estimated in our study (Table 4) are important information to assist in formulating a breeding program for the species.Crosses between accessions with the highest genetic distances (A9 and A11: 0.7260; A8 and A9: 0.6324; and A8 and A32: 0.5760) would broaden the genetic base of the species.Only 10 combinations among the accessions (A1 and A4, A1 and A5, A1 and A31, A4 and A5, A4 and A9, A4 and A17, A4 and A31, A5 and A31, A9 and A24 and A17 and A31) showed a very high level of genetic identity (I˃0.90).Lower levels of genetic identity have been reported for other forage species: 0.0 to 0.857 and 0.2916 to 0.7142 in samples of Pennisetum purpureum (Daher et al. 1997;Freitas et al. 2000), and 0.0 to 0.50 in 16 samples of Bromus auleticus (Yanaka et al. 2005).
Analysis of the genetic polymorphism in loci for αand β-esterase isozymes of M. chaseae accessions kept in the Germplasm Bank of Embrapa Pantanal was important for selecting accessions for further testing to determine their potential for use in recovering degraded areas, and for sowing as grazing pasture areas in the Pantanal region.The study has shown that, although the accessions presented a very high level of genetic identity (I˃0.90), the samples form genetically structured populations (0.15<Fst<0.25).Field testing of accessions in a range of environments is warranted to determine if they possess other desirable attributes, e.g.persistence, drought-tolerance, high nutritive value, and disease-and pest-resistance, before they could be recommended for widespread sowing.Since there were no identical samples, all should be maintained in the Germplasm Bank of Embrapa Pantanal.Crossing plants from accessions with the highest genetic distances is recommended for the improvement of this species, with the prospect of broad-ening the genetic base and opening possibilities for the emergence of new characteristics of interest.

Figure 3 .
Figure 3. Similarity among the 10 accessions of Mesosetum chaseae from the Nhecolândia sub-region maintained in the germplasm collection of Embrapa Pantanal, based on UPGMA cluster analysis (Nei 1978) of the allele polymorphism at Est-1, Est-2, Est-3 and Est-4 loci.

Figure 4 .
Figure 4. Bar plot-like population structure, based on polymorphism at the Est-1, Est-2, Est-3 and Est-4 loci for 10 accessions (A1, A4, A5, A8, A11, A17, A24, A31 and A32) of Mesosetum chaseae from the Nhecolândia sub-region maintained in the germplasm collection of Embrapa Pantanal within the K clusters.Each plant is represented by a single vertical bar broken in K colored segments (K = 3), with lengths proportional to each of the K inferred clusters.Each color represents the proportion of fragments for each individual, represented by a vertical bar.

Table 1 .
Accessions of Mesosetum chaseae maintained in the germplasm collection of Embrapa Pantanal, location of the collection, habitat and geographic coordinates.

Table 2 .
Number of alleles (Na) and number of effective alleles (Ne) per polymorphic locus, mean observed heterozygosity (Ho) and expected heterozygosity (He), and % of polymorphic α/β-esterase loci in the plants of 10 accessions of Mesosetum chaseae maintained in the germplasm collection of Embrapa Pantanal.

Table 5 .
Proportion of the plants from 10 accessions of Mesosetum chaseae from the Nhecolândia sub-region maintained in the germplasm collection of Embrapa Pantanal in each group (K = 3), and number of plants per accession.