Genetic diversity in section Rhizomatosae of the genus Arachis ( Fabaceae ) based on microsatellite markers

The genus Arachis (Fabaceae) native to South America, contains 80 species divided into nine sections, three of which contain species of special economic importance such as the cultivated peanut (Arachis hypogaea), belonging to the section Arachis, and some perennial forage species from sections Caulorrhizae and Rhizomatosae. We used microsatellite markers to assay genetic variability among 77 accessions of four species from section Rhizomatosae, the diploid Arachis burkartii (2n = 2x = 20) and the tetraploid Arachis glabrata, Arachis pseudovillosa and Arachis nitida (2n = 4x = 40). A total of 249 alleles were found in the fifteen loci analyzed and a high degree of intra and interspecific polymorphism was detected. The lowest intraspecific variation occurred in Arachis burkartii, while the smallest estimated interspecific value was between A. nitida and A. pseudovillosa and the largest was between A. burkartii and A. nitida. High observed heterozygosity was detected in A. glabrata. The diploid accessions grouped in one cluster and the tetraploid accessions in another. It was possible to distinguish all 77 accessions and the genetic distance between accessions could not be correlated with geographic origin.

The herbaceous perennial A. glabrata, commonly called the rhizoma peanut or perennial forage peanut, is a wild species from which several commercial tropical for-age cultivars have been developed (Prine et al., 1981(Prine et al., , 1986)), including the Florigraze and Arbrook cultivars used in the USA as an alternative to alfalfa because they contain high levels of proteins and have high disease and pest resistance (French et al., 1994).In Australia, A. glabrata is valued as high quality forage with the ability to spread through swards of aggressive summer-growing grass species (Bowman et al., 1998).Furthermore, A. glabrata also shows multiple disease resistance and has been considered as a potential source of genes to genetic improvement of A. hypogaea (Mallikarjuna and Sastri, 2002).However, A. glabrata cultivars have a narrow genetic basis because they have been selected from a restricted number of available accessions (Prine, 1972;Cook and Crosthwaite, 1994).There are many A. glabrata accessions available in germplasm collections and the evaluation of this material could identify accessions with novel characteristics, such as growth at lower temperatures and in more humid soils, which may result in new cultivars (Prine and French, 1993).netic variability has been evaluated using isozymes (Maass and Ocampo, 1995) and random amplification of polymorphic DNA (RAPD) analysis (Nóbile et al., 2004).Although these studies have shown high variability in rhizomatous species and accessions these molecular markers have produced no information on the mating system of the species because of the low polymorphism of isozymes and dominance effects in RAPD analysis.Furthermore, comparison between studies is difficult because isoenzyme and RAPD markers have medium to low reproducibility (Jones et al., 1997).Moreover, RAPD analysis is also sensitive to the reaction conditions and the possible co-migration of RAPD fragments with similar sizes but different sequences cannot be excluded (Tang et al., 2005).
Microsatellites or simple sequence repeats (SSR) are stretches of a short nucleotide sequence that can be repeated many times in tandem (Casacuberta et al., 2000).These molecular markers have been used for the characterization of genetic variability and analysis of the genetic relationships among plant species (Mörchen et al., 1996;Khlestkina et al., 2004).Microsatellite markers have several advantages: they are very abundant and thoroughly distributed in eukaryotic genomes (Tóth et al., 2000); microsatellite primer pairs can be used in related species (Katzir et al., 1996); and microsatellite markers provide a higher incidence of detectable polymorphisms than other molecular markers such as restriction fragment length polymorphism (RFLP) and RAPD markers (Powell et al., 1996).
The objective of the study described in this paper was to use microsatellite markers to evaluate the variability and genetic relationships between the four species of the section Rhizomatosae, paying particular attention to the forage species A. glabrata.

DNA extraction and amplification
For each accession, we extracted total DNA from the leaves of a single plant using the procedure described by Doyle and Doyle (1987) and the minor modifications given by Ferreira and Grattapaglia (1996).
The 15 microsatellite primer pairs used in this study (Table 2) were isolated from three different species from three Arachis sections: six from A. hypogaea (Ah) in section Arachis, five from Arachis pintoi (Ap) in section Caulorrhizae and four from A. glabrata (Ag) in section Rhizomatosae.The isolation and characterization of microsatellites Ap40 and Ap176 have been described by Palmieri et al. (2002) and Ap33 by Palmieri et al. (2005).Microsatellites Ah3, Ah7, Ah11, Ah21, Ah126 and Ah282 were isolated by Gimenes et al. (2007) while microsatellites Ap32, Ap38 Ag39, Ag151, Ag167 and Ag171 were described by Moretzsohn et al. (2005).For each plant, individual amplification was carried out in a reaction mixture containing 15 ng of total DNA, 0.3 mM of each primer, 0.375 μM of each dNTP, 0.5 units of Taq DNA polymerase (GE Healthcare, USA), 1 X amplification buffer and appropriate concentration of MgCl 2 (1.5 mM for Ah3, Ah7, Ah21, Ah126, Ap176; 2.0 mM for Ap32, Ap33, Ap38, Ap40; and 2.5 mM for Ah11, Ag39 Ag151, Ag167, Ag171, Ah282) and made up to 10 μL with deionized water.All amplifications were performed in a PTC100 thermal cycler (MJ Research, United Kingdom).The amplification parameters used consisted of 5 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 45 s at 50 °C, and 1 min at 72 °C.The PCR products were denatured by adding 7 μL of a loading buffer (95% formamide, 5% NaOH 0.2 M, 0.25% bromophenol blue) and heating for 5 min at 90 °C, separated on 4% (w/v) 82 Angelici et al.  polyacrylamide denaturing gels at 30 W and 1100-1500 V for about 2.5 h and stained with silver according to a commercial protocol (Promega, Madison, USA).

Data analysis
For each sample the amplified fragments were scored as present or absent.A genetic distance matrix was generated using the Nei and Li Genetic Distance Coefficient (Nei and Li, 1979).Genetic relationships between the 77 accessions were viewed in a neighbor-joining tree (Saitou and Nei, 1987).All analyses were computed with the TREECON 1.3 software (Van der Peer and Watcher, 1994).The mean genetic distance coefficient for each species was estimated by calculating the mean genetic distance for each pairwise comparison in each species.

Results
Band patterns formed by one, two, three and four fragments were observed in the samples.Patterns formed by one or two fragments were observed for A. burkartii and from one to four fragments in A. glabrata, A. nitida and A. pseudovillosa (data not shown).The number of fragments observed in the patterns was in line with the expected pattern for a locus in species with the corresponding ploidy levels.The data indicated that A. burkartii is diploid, with a maximum of two alleles, while the others are tetraploid with up to four putative alleles.Therefore, accessions presenting a single amplified fragment were considered homozygote while those presenting more than one fragment were considered heterozygote for the locus in question.
We detected 249 observed alleles (A o ) in the 15 loci amplified, of which three loci (20%) were monomorphic (Ap32, Ap33 and Ap38) for all the species while locus Ag167 was monomorphic only for A. burkartii and Ap176 for A. pseudovillosa only (Table 2).In the 12 polymorphic loci (Ag151, Ag167, Ag171, Ag39, Ah11, Ah126, Ah21, Ah282, Ah3, Ah7, Ap176 and Ap40) the observed number of alleles ranged from 10 for Ap176 to 26 for Ah21, with the mean number of observed alleles per locus being 20.5 while the overall mean observed heterozygosity per locus (H o *) ranged from 0.2365 for Ap176 to 0.9420 for Ah126 (Table 2).The six primer pairs developed for A. hypogaea (section Arachis) detected polymorphism for all four spe-cies of section Rhizomatosae.Three of the five primer pairs developed for A. pintoi (section Caulorrhizae) detected only monomorphic loci in the species from section Rhizomatosae.We also found that the Ap40 locus was polymorphic in the four species studied, while locus Ap176 was polymorphic for A. glabrata, A. nitida and A. burkartii and monomorphic only for A. pseudovillosa.Three primer pairs (Ag39, Ag151 and Ag171) of the four pairs developed for A. glabrata were polymorphic for the other three species of section Rhizomatosae, and the Ag167 locus was monomorphic only for A. burkartii and polymorphic for the three tetraploid species (A.glabrata, A. nitida and A. pseudovillosa).
The general mean observed heterozygosity per species (H o ') for the polymorphic loci analyzed was highest (H o ' = 0.6947) for A. pseudovillosa and lowest (H o ' = 0.4444) for A. burkartii (Table 3).In addition, the mean number of putative alleles (A p ) per locus (A p /A D ) ranged from 4.81 for A. pseudovillosa to 15.92 for A. glabrata, while the percentage of different band patterns per total of accession analyzed (NBP/N A ) ranged from 65.95% for A. glabrata to 89.00% for A. pseudovillosa (Table 3).
The mean genetic distances within accessions of each species and between the species were high (Table 4).Diploid A. burkartii showed the lowest intraspecific variation of all the species analyzed.The smallest estimated interspecific value (0.7106) was between A. nitida and A. pseudovillosa and the largest (0.8279) was between A. burkartii and A. nitida.
The band presence and absence data matrix was used to produce a dendrogram, which showed that no pair of accessions presented 100% similarity and that the accessions were distributed in two large clusters (Figure 1).One cluster was formed by the accessions of the diploid species 84 Angelici et al. A. burkartii while the accessions of the tetraploid species A. glabrata, A. nitida and A. pseudovillosa formed another cluster made up of two sub-clusters, the first, subdivided into several small clusters, consisted of 55 of the 58 A. glabrata accessions and the second contained the tetraploid species (A.nitida and A. pseudovillosa) and the three remaining A. glabrata accessions (Figure 1).The relationship between the number of randomly sampled accessions from the A. glabrata and the number of alleles detected is presented in Figure 2. The number of alleles increased to 178 when 27 accessions were sampled and from then onwards the number of alleles tended to stabilize and about two alleles were added with each new accession.

Discussion
All the accessions studied could be differentiated with the primer pairs used, indicating that microsatellite markers are highly polymorphic and discriminative in Arachis species of the section Rhizomatosae.These same characteristics of microsatellite primers have previously been detected in many plant species, including the apricot Prunus armeniaca L. (Hormaza, 2002), the tetraploid potato Solanum tuberosum L. (McGregor et al., 2000) and a wild and cultivated wheat collection (Medini et al., 2005).Furthermore, studies comparing molecular markers such as Amplified Fragment Length Polymorphisms (AFLP), RAPD, RFLP and microsatellites have already indicated that AFLP and microsatellite markers are highly reproducible within and between laboratories (Rafalski and Tingey, 1993;Cordeiro et al., 2000).Apart from reproducibility, we also found that a further advantage of the microsatellites used in our study was the cross-species transferability of these markers between Arachis species belonging to different sections (Arachis, Caulorrhizae and Rhizomatosae).
The high frequency of A. glabrata accessions with several heterozygous loci indicated that this is a characteristic commonly present in this species and is not due to any other factor, such as a long period of cultivation in greenhouses, outside the area of natural distribution.High heterozygosity is not an expected characteristic among Arachis species, since they are generally considered autogamous, although this is not necessarily the case for species in the least investigated taxonomic sections of this genus.For example, the rhizomatous species A. glabrata has some pecu-

Microsatellites in Rhizomatosae 85
Table 4 -The mean genetic distance estimates (Nei and Li, 1979)  liarities, scattered wild populations occasionally produce seeds while only a small quantity of seed, if any, is sporadically found when this plant is cultivated in greenhouses and nurseries (Valls, 1996), making vegetative propagation by cuttings the normal method of artificial multiplication of this species.The absence of seeds in plants maintained in germplasm banks may be related to incompatibility systems (Cook and Crosthwaite, 1994;French et al., 1994), since A. glabrata pollen viability appears to be high and is not in itself seen to be an obstacle to seed production (Niles and Quesenberry, 1992).It thus appears that there is a high frequency of cross-pollination in the original area of A. glabrata distribution, resulting in high heterozygosity among A. glabrata accessions which is retained during vegetative propagation.We found that the tetraploid species of section Rhizomatosae presented higher heterozygosity levels than the diploid species A. burkartii.However, this was not observed when the same species were assessed using RAPD markers (Nóbile et al., 2004), this difference between studies probably being due to the greater ability of microsatellite markers to detect polymorphism and amplified polymorphic loci in tetraploid species which appeared null when assessed with RAPD markers.
The genetic distance estimates calculated by us were high (Table 4), indicating a large amount of genetic variability in section Rhizomatosae species, as expected in wild Arachis species.High variability has also been detected in the germplasm of wild Arachis species in other studies using isozyme (Maass and Ocampo, 1995), RAPD (Nóbile et al., 2004), RFLP (Gimenes et al., 2002) and microsatellite (Moretzsohn et al., 2004;Hoshino et al., 2006;Gimenes et al., 2007) markers.The high genetic variability which we found in A. glabrata supports the work by Maass and Ocampo (1995), who analyzed four isoenzyme systems (alpha-esterase, acid phosphatase, glutamate oxaloacetate transaminase and diaphorase) in 12 A. glabrata accessions from the International Tropical Agriculture Center germplasm bank (CIAT, Colombia).In addition, Nóbile et al. (2004) used RAPD markers to assess 57 A. glabrata accessions, 11 A. burkartii accessions, 11 A. nitida accessions and 1 A. pseudovillosa accession from the CENARGEN Arachis Germplasm Bank and also detected high variability in the species.
In our study, it is interesting to note that the number of general and specific alleles detected were similar to the number of accessions analyzed of each of the four rhizomatous species, reflecting not only the different extent of the geographic distribution of the different species but also the fact that not only is A. glabrata germplasm preferentially collected because it is a potential forage but it has a much greater ability to survive for many years under greenhouse or nursery conditions than the other three section Rhizomatosae species.
As in the study of the genetic integrity of the Gatersleben Triticum aestivum L. germplasm bank by Börner et al. (2000), we found that the accessions of the species already represented in the CENARGEN Arachis germplasm bank and the new accessions collected may be efficiently monitored by microsatellite markers for the maintenance and preservation of the genetic variability of this material.
Our study showed that the species investigated shared 124 (50.2%) alleles, a relatively large number.Differentiation between the species is, therefore, due not only to the specific alleles but also to the differences in the frequencies of the shared alleles in each species (Table 3).This was the case for A. pseudovillosa and A. nitida, which presented a very small number of specific alleles (Table 3).In the dendrogram (Figure 1) the first cluster contains tetraploid species which were separated from the diploid species in the second cluster, confirming the results of RAPD analysis (Nóbile et al., 2004).The larger subcluster formed in the first cluster included 55 accessions that belong exclusively to A. glabrata while the second smaller subcluster did not distinguish the species perfectly.Although A. nitida was clearly separated from the other accessions, the A. pseudovillosa and A. glabrata accessions in this subcluster did not separate perfectly.These results support those of Krapovickas and Gregory (1994), who reported natural hybrids of these two species, and the results of the RAPD analysis carried out by Nóbile et al. (2004).Valls (1996) pointed out that no hybrid exists between the diploid and tetraploid species of section Rhizomatosae, which seems to indicate that different genomes are present in the tetraploid and diploid species (Stalker and Moss, 1987).However, many of the accessions used in these crossing attempts were not good seed producers, and unsuccessful hybridization may have other causes that do not involve genetic or genomic incompatibility (Valls, 1996).
As Figure 2 shows, A. glabrata harbors a large number of alleles and the allele content in a random sample can be monitored using microsatellites loci that would help the formation of a core collection of this species.However, it is necessary to point out that, although extensive, the present sampling did not include representatives of wild popula-  tions from Argentina (A. burkartii and A. glabrata), Uruguay (A. burkartii), and southern Paraguay (A. glabrata, except for the `Arbrook' cultivar).Southern Paraguay and the adjacent Province of Corrientes in Argentina harbor the morphologically distinct A. glabrata var.hagenbeckii, with narrow leaflets, in sympatry with local types of A. glabrata var.glabrata (Krapovickas and Gregory, 1994).Much more scarce, A. pseudovillosa occurs also in the Department of Amambay, Paraguay, near the Brazilian border, but only a few Brazilian accessions are available and this species is poorly represented in gene banks.Further efforts to cover such gaps in our knowledge of the genetic diversity of section Rhizomatosae using the microsatellite primers described in the present paper are obviously needed.
In conclusion, the microsatellite markers used allowed us to distinguish the species and accessions of section Rhizomatosae, detected high variation accessions and can be very useful for monitoring the genetic variability in germplasm banks when establishing core collections.

Figure 2 -
Figure 2 -The relationship between the number of randomly sampled Arachis glabrata accessions and the number of alleles detected.

Table 1 -
Accessions (n = 77) of the four Arachis species in section Rhizomatosae analyzed using microsatellite markers.

Table 2 -
Results obtained for analysis of 77 accessions from the four Arachis species in section Rhizomatosae.The table shows the locus, primer sequence, number of observed alleles (A o ), number of different band patterns observed (NBP), mean observed heterozygosity for each species and locus (H o ) and the overall mean observed heterozygosity per locus (H o *).The source species categories and the loci are ordered by decreasing overall H o value, with zero indicating no heterozygosity.o Arachis species in section Rhizomatosae

Table 3 -
Statistical analysis of the data for 77 accessions of the four Arachis species in section Rhizomatosae, showing the number of accessions (N A ), total alleles detected (A D ), number of specific alleles (A s ), ratio of putative alleles (A p ) to total alleles detected (A p /A D ), general mean observed heterozygosity per species (H o '), mean number of different band patterns (NBP') and percentage of different band patterns per number of accessions (NBP' /N A , %).
for the four Arachis species in section Rhizomatosae.
A. glabrata A. nitida A. burkartii A. pseudovillosaFigure1-Neighbor joining tree showing the relationships between accessions of the four Arachis species in section Rhizomatosae.The accession numbers are those shown in Table1.