Genetic diversity analysis in the section Caulorrhizae (genus Arachis) using microsatellite markers

Diversity in 26 microsatellite loci from section Caulorrhizae germplasm was evaluated by using 33 accessions of A. pintoi Krapov. & W.C. Gregory and ten accessions of Arachis repens Handro. Twenty loci proved to be polymorphic and a total of 196 alleles were detected with an average of 9.8 alleles per locus. The variability found in those loci was greater than the variability found using morphological characters, seed storage proteins and RAPD markers previously used in this germplasm. The high potential of these markers to detect species-specific alleles and discriminate among accessions was demonstrated. The set of microsatellite primer pairs developed by our group for A. pintoi are useful molecular tools for evaluating Section Caulorrhizae germplasm, as well as that of species belonging to other Arachis sections.


Introduction
The genus Arachis comprises nine taxonomic sections, viz., Arachis, Caulorrhizae, Erectoides, Extranervosae, Heteranthae, Procumbentes, Rhizomatosae, Trierectoides and Triseminatae, (Krapovickas and Gregory (1994), and includes both annual and perennial species. In this genus, most secies are acceptable as versatile forage plants. Nevertheless, more recent studies have provided abundant information on the potential and effective commercial use of accessions from the sections Caulorrhizae and Rhizomatosae (Loch and Ferguson, 1999;Teguia, 2000). Section Caulorrhizae is represented by only two stoloniferous species, Arachis pintoi Krapov. & Gregory and Arachis repens Handro. Both are native of valleys of the rivers Jequitinhonha, Araçuai, São Francisco and Paranã, the latter a tributary of the Tocantins, in Central Brazil.
Lately, the number of accessions available in both species has increased, with the current maintenance of over 150 in the Arachis Germplasm Bank (EMBRAPA Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil). Furthermore, a program for agronomic appraisal and production of intra-and inter-specific hybrids from section Caulorrhizae, as well as progenies from accessions with high forage potential, has been developed (Carvalho S, PhD Thesis, UNESP, São Paulo, 2000). The significant genetic variability in available germplasm, both in accessions and hybrids, requires conservation, investigation and economical exploitation (Gimenes et al., 2000).
Several genetic markers have been used to estimate the genetic variability in species of section Caulorrhizae, including morphological characters (Monçato L, MSc Dissertation, UNESP, São Paulo, 1995), seed storage proteins (Bertozo and Valls, 2001), isozymes (Maass et al., 1993) and RAPDs (Gimenes et al., 2000) These markers were useful for the characterization of genetic variation in both species, but they offered limited informative content since some detected low levels of polymorphism (morphological characters, isozymes and seed proteins). RAPDs, on the other hand, yielded more complex band patterns (RAPDs). Due to their limitations, these markers were incapable of providing relevant information regarding important points for the conservation and use of the species, such as an estimate of the cross-pollination rate, identification of hybrids among species, and accurate estimation of genetic variability.
From recent studies, 18 microsatellite markers from A. pintoi have been described. The utility of these markers in evaluating genetic variability in section Caulorrhizae (20 accessions of A. pintoi and five of A. repens) has been demonstrated (Palmieri et al., 2002(Palmieri et al., , 2005. In the present study, we used 19 previously described microsatellite markers and seven new primer pairs to estimate genetic variation in accessions of A. pintoi and A. repens.

Plant material
Thirty-three accessions of A. pintoi and ten of A. repens were analyzed (Table 1). The samples were obtained from Dr. José F.M. Valls, curator of Wild Arachis Germplasm Bank, EMBRAPA Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil, and from Dr. Sandremir de Carvalho, the Fundação Faculdade de Agronomia "Luiz Meneghel", Bandeirantes, PR, Brazil. In the ArLag (Arachis sp.) accession, collected at Botucatu, SP, Brazil, the morphological type appeared to be closer to A. repens accessions, although definitive botanical identification was not possible.

Source of microsatellites primer pairs
Nineteen primer pairs had already been described by Palmieri et al. (2002Palmieri et al. ( , 2005 and Hoshino et al. (2006), and seven new ones are described herein (Table 2). All the microsatellites used were isolated by applying libraryenrichment protocol adapted from Kijas et al. (1994). The Primer 3 (Rozen and Skaletsky, 2000) program was employed for designing all the primer pairs, according to the following criteria: Tm of 50 to 60°C (Tm difference between each primer within a pair was maintained below 3°C), length of PCR products ranging from 100 to 350 bp and GC-content maintained around 50%. All primer pairs were synthesized by Invitrogen, SP, Brazil. BLAST searches were performed for all microsatellite sequences using blastx program to determine whether the microsatellites were associated with conserved gene regions (Altschul et al., 1997). These searches were based on the full-length sequence from which the primer pairs were designed.

DNA extraction
Genomic DNA was extracted using the protocol described by Grattapaglia and Sederoff (1994) with minor modifications as to DNA precipitation. DNA quality was checked with electrophoresis in 1% agarose gels, and concentration estimated by spectrophotometry (Spectronic, Inc., Rochester, NY, USA).

DNA amplification and electrophoresis
PCR reactions contained 15 ng of genomic DNA, 1U of Taq DNA polymerase (Amersham Biosciences), 1x PCR buffer (200 mM Tris pH 8.4, 500 mM KCl), 1.5-2.0 mM MgCl 2 , 200 mM of each dNTP, and 0.4 mM of each primer, in a final reaction volume of 10 mL. All PCR amplifications were carried out in a PTC100 thermocycler (MJ Research, Inc., Watertown, MA, USA). PCR conditions were 96°C for 5 min, followed by 32 cycles of 96°C for 30 s, X ºC for   45 s, 72°C for 1 min, with a final extension of 10 min at 72°C. The X value for each primer pair is shown in Table 2. PCR reactions were mixed with equal volumes of loading buffer (95% formamide, 0.01% bromophenol blue, 0.01% xylene cyanol, 0.5% NaOH 0.2 M), denatured at 95°C for 5 min, cooled on ice and loaded onto the gel. PCR products were separated in denaturing polyacrylamide gels (6% acrylamide/bisacrylamide, 29:1, 5 M urea in TBE, pH 8.3) at 60 W for 4 h in 1x TBE buffer. DNA fragments were visualized by silver staining. The silver staining procedure consisted of 10 min in 10% ethanol/1% acetic acid solution, staining for 15 min in 0.2% (w/v) silver nitrate solution, and rinsing for 30 s in deionized water, and developing in 30 g/L of NaOH/10 mL/L of 37% formaldehyde solution for about 10 min or until bands became visible.

Data collection and analysis
Fragment sizes were estimated by comparison with a 10-bp DNA ladder (Life Technologies) using Gene Profiler 4.03 for Windows software, evaluation edition (Scanalytics, Inc., Fairfax, VA, USA). Bands with the same mobility were considered identical. Assuming the absence of null alleles, the presence of only one fragment of a given microsatellite indicated homozygosis. The Ap172 primer pair amplified a putative duplicate locus, and for this reason the amplification of two independent loci for this marker was considered. PopGene software (version 1.31; Yeh et al., 1999) was used to estimate genetic diversity based on the following indexes: polymorphic information content, allele number (observed and effective) per locus, allelic frequencies, observed (H O ) and expected (H E ) heterozygosities. Allelic polymorphic information content (PIC) was calculated for each microsatellite locus using the formula: , where p i and p j are the frequencies of the i th and j th alleles in the population (Weber, 1990). PIC values provided an estimate of the discriminatory power of a marker by taking into account, not only the number of alleles at a locus, but also their relative frequencies in the population under study. Markers with a large number of alleles occurring at equal frequencies will always have the highest PIC values (Senior et al., 1998). Effective alleles per locus (n e ) were calculated according to Weir (1989) with the formula 1/ (1 -H E ). H E , the expected heterozygosity per locus, is equal to 1 2 -å p i i , where p i is the frequency of the i th allele at the locus. The Unweighted Pair-Group Method was applied for cluster analysis, using Arithmetic Averages (UPGMA) based on unbiased genetic distance measures (Nei, 1978).
The number of monomorphic loci was high by accounting that each primer pair that did not allow detection of polymorphism was adjacent to regions containing a high number of repeats, these ranging from 19 (Ap32) to 58 (Ap35) repeats. Among the ones that did not detect any polymorphism four are described in this paper and two (Ap32 and Ap38) were previously used in three studies on genetic variability in Arachis Hoshino et al., 2006;Angelici et al., 2008), all with similar results. We tested the latter two primer pairs because Hoshino et al. (2006) studied only one accession of each species of section Caulorrhizae, whereas Bravo et al. (2006) and Angelici et al. (2008) used these two primers in other sections of genus Arachis. Thus, we expected additional information from these primers by using samples of the species from which they had been isolated. It may be that the areas targeted by the two primer pairs are within conserved regions of the genome. There was no similarity between the sequences used to design primers for these six microsatellites and any nucleotide or protein sequence in GenBank.
The Ap172 primer pair amplified a putative duplicated locus. At first, the double-band pattern was interpreted as a technical artifact, but after several attempts to optimize the amplification reaction, the band pattern still remained, thereby implying locus duplication. Amplification of duplicated loci has been observed in several species, such as Glycine max (L.) Merr. (Powell et al., 1996;Peakall et al., 1998), Zea mays L. (Senior et al., 1998), Vigna radiata (Kumar et al. 2002) and Cicer arietinum L. (Sethy et al., 2003). In rice and sunflowers, the amplification of double-band patterns has also been attributed to the occurrence of a duplication process within the genome itself, as well as to the evolution of families of repetitive sequences (Akagi et al., 1998;Paniego et al., 2002). In the amphidiploid A. hypogaea, amplification of duplicated loci was reported by Hopkins et al. (1999), and duplication at several genomic regions by Burow et al. (2001). Despite A. pintoi and A. repens being diploid species, gene duplication is not rare in the genus Arachis, and it could have happened to Ap172.
In this study, only Ap172 and Ap176 sequences showed similarity at the amino acid level to seryl-tRNA synthetase (57% identity, 76% similarity) and lipoxygenase (41% identity, 47% similarity) of plants, respec-tively. These stretches of similarity are localized adjacent to microsatellite sequences (data not shown). A like occurrence was reported by Peakall et al. (1998) in soybean. These authors found a similarity of 96% at the amino acid level between a microsatellite sequence and a seryl-tRNA synthetase of Arabidopsis thaliana. These data seem to be in agreement with observations from several authors (Tóth et al., 2000;Li et al., 2002;Morgante et al., 2002), in the sense that microsatellite sequences are present both in coding and non-coding regions of nuclear and organellar genomes.
A total of 196 putative alleles were detected at 20 polymorphic loci. It was assumed that fragments of different lengths were different alleles. The number of alleles ranged from two at Ap45 to 23 at Ap18 (a mean of 9.8 alleles/locus) ( Table 3). The effective number of alleles ranged from 1.07 at Ap45 to 16.7 at Ap18 (Table 4). In A. pintoi, 174 alleles were detected distributed among the 19 polymorphic loci (mean of 9.2 alleles/locus), their fragment sizes ranging from 140 bp (Ap161) to 306 bp (Ap152). In A. repens accessions, 99 alleles, with fragment sizes ranging from 140 bp (Ap161) to 304 bp (Ap33), were detected among 19 polymorphic loci (mean 5,2 alleles/locus) (Table 3). Ninety-nine alleles (49%) were exclusively present in A. pintoi and twenty-one alleles (10.7%) were found in A. repens accessions only. Seventy-ninealleles (40.3%) were shared between the two species (data not shown). On using RAPDs, Gimenes et al. (2000) obtained lower values for exclusive fragments for these two species (22% in A. pintoi and 5% in A. repens) and a higher value for shared fragments (73%). Based on these results, they discussed the difficulty in justifying the separation into two 114 Palmieri et al. species. Our data could reinforce a separation of these species into two taxa, as the higher values observed were due to the codominance and informativeness of microsatellite markers, thereby allowing us to distinguish and better estimate the genetic diversity within the analyzed germplasm.
Data on allelic polymorphic information content (PIC), and observed (H O ) and expected (H E ) heterozygosities per locus are presented in Table 4. PIC values ranged from 0.0651 at Ap45 to 0.9369 at Ap18, with an average value of 0.6423 when considering 20 polymorphic loci (Table 4). Average observed heterozygosities at 20 loci for the whole A. pintoi and A. repens sample were 0.5788, 0.5820 and 0.5861, respectively (Table 4), and average expected heterozigosities for the whole sample, A. pintoi and A. repens accessions were 0.6753, 0.6553 and 0.6202, respectively (Table 4). Mean values of observed heterozygosity (H O ) were lower than the H E values estimated from allele frequencies. At some loci, H O values were higher than H E (Ap22, Ap23, Ap154, Ap172a, Ap172b, Ap187, and Ap190). The variability observed in A. pintoi could be the consequence of crosses between different ac-cessions that had been vegetatively maintained at experimental plots. Thus, the high observed heterozygosity at some loci could be attributed to the presence of parentals carrying different alleles, thereafter being sustained through the vegetative propagation methods used in conserving accessions.
The dendrogram showing the relationships among A. pintoi and A. repens accessions is presented in Figure 1. Cluster analysis allowed the discrimination of all individuals from the two species. Such differentiation was also obtained using RAPD markers (Gimenes et al., 2000). However, microsatellites should be the marker of choice because they are much more effective and have higher reproducibility since longer primer pairs are used instead of unique short primers that allows multiple loci amplification, which makes the analysis difficult.
Three major groups (I, II and III) were formed in the tree. In general, A. pintoi accessions were positioned in all the three major groups, with a mean genetic distance among them of 0.295, ranging from 0.064 (between NP s/nº and WPn 128) to 0.566 (between W 34 and CIAT 17434 -Maní Genetic diversity in Arachis 115 The longest genetic distance (0.582) was obtained between the accessions CIAT 17434 -Maní Mejorador (A. pintoi) and WPn 215 (A. repens), whereas the shortest (0.064) was between two A. pintoi accessions (NP s/nº and WPn 128). The VSa 7394 (A. pintoi) accession, the most diverse, was positioned outside the three major groups (Figure 1). Tree analysis showed that the species could not be characterized based on polymorphism detected by using 20 microsatellite loci, since accessions of each species were not entirely grouped together. Likewise, Bravo et al. (2006) and Hoshino et al. (2006) did not resort to microsatellites when characterizing Arachis species. They pointed out that this was probably due to: 1 -high microsatellite-detected polymorphism, requiring larger samples for adequate representation of species variability; and 2 -the existence of homoplasies (fragments of the same size but from different loci that have no common origin). These same factors could possibly have affected the results obtained in this study. However, we believe the main reason is that crossability in A. pintoi and A. repens is high (86.7%, Krapovickas and Gregory, 1994), these being considered by some authors as a single species (Gimenes et al., 2000). As mentioned above, differentiation between A. repens and A. pintoi, as observed in the present study, was greater than that observed by Gimenes et al. (2000). We consider this to be a relevant result, because it shows that the primary gene pool of these species probably has a wider base than was detected by the RAPD data.
It has been demonstrated that the set of microsatellite markers previously described and used here provides a powerful tool for germplasm characterization analysis of A. pintoi and A. repens species. Among the primer pairs presented in this study, 21 are readily available. These primers could be useful in all the steps from conservation to the use of germplasm. The existence of duplicates, mislabeling and loss of integrity due to physical contamination, crosspollination or genetic drift are realities, so these markers could be used as an aid in evaluating these events in the germplasm collection. Furthermore, they could also be used in identifying accessions and cultivars and for selecting parents for hybridization.