Genetic relationships among Arachis species based on AFLP

Amplified Fragment Length Polymorphism (AFLP) was used to establish the genetic relationships among 20 species from seven of the nine sections of genus Arachis. The level of polymorphism among nine accessions of the cultivated peanut, A. hypogaea L., was also evaluated. Three combinations of primers were used to amplify the AFLPs. The fragments were separated in 6% denaturing acrylamide gels. A total of 408 fragments were analyzed. An average of 135.3 fragments per primer combination were scored, and the largest number of fragments was 169 using primer combination Eco RI ACC / Mse I – CTG, while the lowest was 108, with Eco RI ACT / Mse I CTT. In general, the genetic relationships established using AFLPs agreed with the classification established using morphology and crossability data. The results indicated that AFLPs are good markers for establishing the relationships among Arachis species. The polymorphism detected in A. hypogaea by this method was higher than the one found with other markers, like RAPDs and RFLPs. However, our data suggest that the polymorphism detected be using AFLP with only three primer combinations is still too low to be used for any kind of genetic study in this species.


Introduction
The genus Arachis was divided into nine sections by Krapovickas and Gregory (1994).Although those two authors analyzed a large number of accessions of several species, the large number of accessions recently collected broadened the geographic distribution of most sections.New questions about the taxonomic placement and relationships between some species have frequently been raised in this genus, since some accessions show morphological characteristics, which are intermediate between different species.Changes in the taxonomic relationships are likely to happen, when those new accessions are taken into account.If one considers the high probability of new materials being found, to be added to the Arachis germplasm collections, since a relevant effort has been made to broaden this collection, those changes might be quite significant.Thus, a methodology capable of helping to establish the genetic relationships within this genus in a reliable and fast way would be very useful.
AFLP (Amplified Fragment Length Polymorphism), a PCR-based technique (Vos et al., 1995), seems to overcome many of the limitations of the other molecular markers.AFLP has revealed much more polymorphism than RAPD and RFLP, because it allows a larger number of loci to be sampled in a single assay (10 to 50 polymorphic loci), it is more reliable and easier to score than RAPDs, and requires less time to assay a large number of loci.Our objective was to evaluate the usefulness of AFLPs for the establishment of taxonomic relationships among the species of genus Arachis, and to evaluate the polymorphism among cultivars of A. hypogaea.

Plant material
Nine morphologically diverse accessions of A. hypogaea, cultivated in different countries, and 19 wild species of Arachis from seven sections of the genus were analyzed (Table I).

DNA extraction
DNA was extracted from leaves, using the procedure described by Kochert et al. (1991).The DNA quality was checked in 0.8% agarose gels, and its quantity measured by using a 260 nm wavelength.For the AFLP analysis, the DNAs were diluted to 14 ng/µL.

AFLP analysis
The AFLP analysis was performed following the manual of the AFLP kit (Life Technologies), with minor modifications.250 ng (18 µL of the 14 ng/µL DNA dilution) of genomic DNA were digested for 2 hours at 37 °C, using 3 µL of a mix of Eco RI and Mse I.The restriction enzymes were inactivated by heating at 70 °C for 15 min.Eco RI and Mse I adapters were ligated to the restriction fragments, adding to each tube 24 µL of adapter ligation solution, 1 µL of T4 DNA ligase, and incubating the tubes at 20 °C for 2 h.A pre-amplification reaction was performed, mixing 5 µL of a 1:10 dilution of the digested and ligated fragments, 40 µL of pre-amp primer mix, 5 µL of 10 X PCR buffer, and 1 µL of 1 unit/µL Taq DNA polymerase, in a final volume of 51 µL.The following pre-amplification cycle was performed 20 times: 94 °C for 30 s, 56 °C for 60 s, and 72 °C for 60 s.The pre-amplification solution was diluted to 1:50 (v/v), and 3 µL were transferred to a 1.5 mL tube containing 147 µL of TE buffer.A selective amplification was performed, mixing 5 µL of a mix of 5 µL of labeled Eco RI primer (18 µL of Eco RI primer, 10 µL of kinase buffer, 20 µL of alpha 32 P, 2 µL of T4 polynucleotide Kinase) and 45 µL of Mse I primer, 5 µL of a mix of 79 µL of millipore water, 20 µL of 10 X PCR buffer and 1 µL of Taq DNA polymerase, and 5 µL of diluted preamplification solution.The selective amplifications were performed using the three pairs of primers (Eco RI -AGC / Mse I -CTG; Eco RI -ACT / Mse I -CTT; Eco RI -ACC / Mse I -CTG).A touch-down cycle was performed: 1 cycle of 60 s at 94 °C, 60 s at 65 °C and 90 s at 72 °C; followed by 9 cycles of 1 °C lower annealing temperature each cycle, and 23 cycles of 30 s at 94 °C, 30 s at 56 °C and 60 s at 72 °C.The pre and the selective amplifications were performed in a Perkin-Elmer Thermocycler.

Gel electrophoresis
Twenty microliters of loading buffer (98% formamide, 10 mM EDTA, 0.005% of xylene cyanol FF and 0.005% of bromophenol blue) were added to each tube.The PCR products were denatured by heating at 90 °C for 3 min, and then immediately placed on ice.Two microliters of each sample were loaded per track, and the fragments were electrophoresed in 6% acrylamide gels (20:1 acrylamide:bis; 7.5 M urea; 1 X TBE) for 1.30 h at 55 W. The gel was transferred to a 3MM chromatography paper and dried in a gel drier at 80 °C for 2 h.Gels were exposed to XOMAT X-ray film (KODAK) overnight at room temperature.350 Gimenes et al.

Data analysis
The AFLP bands were scored for presence or absence in each accession.Fragments of the same size were considered to have the same sequence.A genetic distance matrix was obtained using the genetic distance described by Link et al. (1995), and used to construct a UPGMA tree.A bootstrap analysis was performed using 1,000 replicates.All analyses were performed using the TREECON computer (program) software (Van de Peer and Watcher, 1994).

Results
A total of 408 fragments were analyzed using the three pairs of primers.One hundred and eight fragments were scored in the essay performed by using the pair of primers Eco RI-ACT / Mse I-CTT, 131 with Eco RI-AGC / Mse I-CTG, and 169 with Eco RI-ACC / Mse I-CTG, with an average of 136 fragments per pair of primers used.Four hundred and six fragments were polymorphic, with an average of 135.3 per reaction.Only two fragments were monomorphic in all samples analyzed.
A total of 94 fragments were detected in the accessions of A. hypogaea, and only six of those fragments were polymorphic (6.4%).Figure 1 shows a polymorphic fragment detected in A. hypogaea with primer combination Eco RI-ACC / Mse I-CTG.
The relationships among the species analyzed are shown in Figure 2. The species were grouped into three major clusters.The first cluster comprised all the species analyzed of section Arachis, the second one comprised the species analyzed of sections Caulorrhizae (A. pintoi and A. repens), Rhizomatosae (A. burkartii and A. glabrata), Heteranthae (A. dardanii), Procumbentes (A. rigonii) and Erectoides (A. paraguariensis, A. major); the third group comprised A macedoi and A. burchellii, both of section Extranervosae.The group formed by the species of section Arachis was subdivided into three subgroups: the first one comprised all the accessions of A. hypogaea, A. monticola, A ipaënsis and A. duranensis, the second one comprised A. kempff-mercadoi, A. diogoi, A. kuhlmannii, A. stenosperma, A. correntina, and the third one comprised A. glandulifera.The bootstrap values ranged from 24% to 100%.
The two analyzed species of section Rhizomatosae did not group together.Arachis burkartii was grouped closer to A. pintoi and A. repens, while A. glabrata was placed closer to A. paraguariensis and A. major, both of section Erectoides.

Discussion
Four hundred and eight fragments were detected using three pairs of primers, 406 (99%) being polymorphic, taking all the species into account.The RAPD analysis of five species of section Arachis using six primers showed an average of 19.3 polymorphic loci (Lanham et al., 1992).Lanham et al. (1994) analyzed seed storage protein variation in seventy-two accessions of 22 species of sections Arachis, Erectoides, Extranervosae and Triseminatae and found a total of 24 polymorphic bands.
The results suggest that AFLP is very useful in establishing the genetic relationships among Arachis species.As a rule, the grouping of the species based on AFLP data agreed with the classification established by using morphology, geographic distribution and cross-compatibility (Krapovickas and Gregory, 1994), despite the small number of accessions analyzed of each species.AFLP data were also consistent with the existing knowledge of the systematics of other plant species (Hill et al., 1996;Paul et al., 1997).
The species of section Arachis were grouped into three clusters.The first one comprised A. hypogaea (AABB), A. monticola (AABB), A. duranensis (AA) and A. ipaënsis (BB).The close relationship of the diploid species A. ipaënsis and A. duranensis with A. hypogaea is consistent with previous studies (Kochert et al., 1991;Kochert et  al ., 1996), which also appointed these diploid species as being the most probable donors of the genomes of A. hypogaea.The second group comprised the A genome species, and the third one comprised A. glandulifera, which has a D genome (Stalker, 1991).The data agreed with the cytogenetic classification, that is a good evidence of crossability among A genomes species.
The other sections with more than one species analyzed were also well characterized.Arachis pintoi and A. repens (section Caulorrhizae) were grouped very close to each other.Hybrids between A. pintoi and A. repens show a pollen fertility level of 86.9% (Gregory and Gregory, 1979).RAPD analysis of several accessions of A. pintoi and A. repens indicated that these two species are closely related on molecular grounds (Gimenes et al., 2000).The species of section Erectoides (A. paraguariensis, A. major) were grouped with A. glabrata, that belongs to section Rhizomatosae.Section Extranervosae was also characterized, having A. macedoi and A. burchellii grouped together.The exception was section Rhizomatosae, which had A. glabrata and A. burkartii put apart in the tree.Arachis glabrata is tetraploid, while A. burkartii is diploid, and it is known that the latter one does not cross with any other species of genus Arachis (Gregory and Gregory, 1979).Thus, the evidence suggests that these two species are not closely related and, along the evolution and diversification of this genus, rhizomes may have appeared independently, at least twice.
Six fragments were polymorphic in A. hypogaea cultivars.The polymorphism detected using this molecular marker is still very low, when compared to other crops.For instance, in an analysis of 14 rice accessions, 27.8% (147/529) of loci detected were polymorphic (Mackill et al., 1996).The percentage of polymorphic markers found in A. hypogaea (6.4%) was similar to the one found in another AFLP analysis of variation within 6 divergent genotypes from three botanical varieties of A. hypogaea (He and Prakash, 1997).Because A. hypogaea may have arisen recently from a single polyploidization event (Halward et al., 1991), it is likely that a simple nucleotide substitution, rather than gross differences in few loci, may account for the variation in the cultivated peanut (Kochert et al., 1996;He and Prakash, 1997).The microsatellite loci analysis of A. hypogaea, where alleles differed from each other in a  few bases, showed higher polymorphism than RFLP and RAPDs (Hopkins et al., 1999).Thus, the data presented in this and in other studies indicate that techniques which allow the detection of few to single nucleotide modifications, such as SNPs (Single Nucleotide Polymorphisms), will have to be used in A. hypogaea to identify informative loci.

Genetic relationships among Arachis species 351 Figure 1 -
Figure 1 -Polymorphism among the accessions of A. hypogaea using the primers Eco RI -ACC/ Mse I -CTG.

Figure 2 -
Figure 2 -UPGMA tree showing the relationships among the 20 species of seven sections of the genus Arachis.

Table I -
Accessions of A. hypogaea and other Arachis species analyzed.