Genetic Resources and Crop Evolution 48: 347–352, 2001.  2001 Kluwer Academic Publishers. Printed in the Netherlands.

347

Evaluation of genetic relationships among botanical varieties of cultivated peanut (Arachis hypogaea L.) using AFLP markers Guohao He* and Channapatna Prakash Center for Plant Biotechnology Research College of Agricultural, Environmental and Natural Sciences Tuskegee University, Tuskegee, AL 36088, USA; * Author for correspondence (e-mail: hguohao@ tusk.edu; phone: 334 727 8459; fax: 334 727 8552) Received 15 November 1999; accepted in revised form 22 March 2000

Key words: AFLP markers, Botanical varieties, Cultivated peanut, Genetic diversity, Polymorphism

Abstract Forty-four accessions of cultivated peanut (Arachis hypogaea L.) representing six botanical varieties of two subspecies along with three accessions of the wild relative A. monticola Krapov et Rigoni were evaluated for their genetic relationships using the AFLP marker technology. Fifteen AFLP primer pairs (EcoRI /MseI) generated 28 distinct polymorphic markers that were employed to develop unique profiles of all accessions and to construct a phenogram. The results showed that the botanical varieties aequatoriana and peruviana were closer to subspecies hypogaea than subspecies fastigiata Waldr. to which they belong, and the wild A. monticola was not distinct from the cultivated A. hypogaea. Although the extent of genetic diversity in peanut is low compared to many other crops, our studies show that by employing the AFLP approach, sufficient DNA variation can be detected in the cultivated peanut germplasm to conduct evolutionary studies.

Introduction The cultivated peanut or groundnut (Arachis hypogaea L.) is an important legume used for its oil and protein content. It consists of two subspecies, hypogaea and fastigiata Waldr., which are further classified into six botanical varieties based on their morphology and growth habits (Krapovickas and Gregory 1994). Botanical varieties ‘hypogaea’ and ‘hirsuta’ belong to ssp. hypogaea while varieties ‘fastigiata’, ‘peruviana’, ‘aequatoriana’ and ‘vulgaris’ belong to ssp. fastigiata (Table 1). DNA-based markers provide a reliable means of estimating the genetic relationships between genotypes and taxonomic groups as compared to morphological markers (Gepts 1993). However, molecular markers have not aided studies on genetic relationships in cultivated peanut because of the limited detectable polymorphism using protein and DNA markers in this crop. Singh et al. (1991, 1994) found very limited or no variation among the accessions of the cultivated peanut using seed protein profiles.

Similarly, isozyme markers could detect no differences within either subspecies of A. hypogaea, and the two subspecies varied only by a very few such markers (Grieshammer and Wynne 1990; Lu and Pickersgill 1993; Lacks and Stalker 1993). The RFLP and RAPD techniques have detected very little polymorphism among accessions of cultivated peanut (Kochert et al. 1991; Lanham et al. 1994; Halward et al. 1992; Paik-Ro et al. 1992; Garcia et al. 1995), and thus could not be employed in genetic studies of peanut varieties. We have, however, recently shown that AFLP technique (Vos et al. 1995) detects DNA polymorphism in the cultivated peanut (He and Prakash 1997). For continued improvement of the cultivated peanut through breeding, an understanding of genetic diversity helps in determining the extent of variation in the germplasm. Precise understanding of the degree of genetic relationships between genotypes and botanical varieties of peanut may provide insights into the domestication and evolution of this crop. Further, it would have immediate tangible impact on peanut

348 Table 1. Country of origin and USDA principle introduction number of 44 accessions of A. hypogaea and three lines of A. monticola. Species

Subspecies

Botanical variety

Country of origin

PI or Ac No.

A. hypogaea L.

hypogaea

hypogaea

Bolivia Bolivia Bolivia Argentina Argentina Brazil Mexico Peru Peru Mexico Mexico Peru Peru Peru Brazil Brazil Brazil Bolivia Paraguay Paraguay Paraguay Argentina Mexico Peru Peru Bolivia Bolivia Ecuador Ecuador Ecuador Ecuador Ecuador Ecuador Ecuador Ecuador Ecuador Paraguay Paraguay Brazil Uruguay Uruguay Uruguay Argentina Argentina Argentina Argentina Argentina

468222 468248 497302 468191 497253 475871 576631 501297 501296 576613 576617 502020 476206 501985 476052 536263 536269 475914 493678 262023 493309 493813 576609 502045 502053 540833 540835 497633 497624 497626 497659 497625 497632 497615 497630 497634 494005 494024 494052 536232 536290 536234 493985 493984 468196 21769 30063

¨ hirsuta Kohler

fastigiata Waldron

fastigiata

peruviana Krapov. et W.C. Gregory

aequatoriana Krapov. et W.C. Gregory

vulgaris Harz

A. monticola Krapov. et Rigoni

improvement through identification of appropriate parents to ensure a broad genetic base in the cultivar development. This is especially critical in the U.S. considering that many commercial peanut cultivars have a very narrow genetic base and thus are predisposed to disease and pest epidemics although their genetic base has widened considerably during past 20

years (Knauft and Gorbet 1989). The DNA profiling could also aid in the better management of genetic resources through precise identification of the germplasm and in eliminating or pooling together duplicate collections. This study employed AFLP markers to: (1) evaluate the phylogenetic relationships among six botanical

349 varieties of the cultivated peanut; and (2) determine the nature of the genetic relationships between the wild A. monticola and the cultivated A. hypogaea. We used peanut landraces collected from diverse geographic locations in its centers of origin and diversity (in South America) as they represent true botanical varieties uncorrupted by modern crop breeding.

Materials and methods Forty-four accessions of the cultivated peanut representing six botanical varieties from two subspecies of A. hypogaea were chosen for evaluation (Table 1). This sample consists of diverse genotypes based on their phenotypic traits and geographic origin. Three accessions from A. monticola, a wild species and the only other tetraploid species of section Arachis in addition to A. hypogaea, were also included in the evaluation (Table 1). Seeds were provided by Dr. Roy Pittman, USDA Plant Genetic Resources Conservation Unit, Griffin, GA. Genomic DNA was extracted from seedlings (1015d) using the urea-based protocol of Chen et al. (1994). The AFLP procedure was performed following the protocol described by Vos et al. (1995) and the product manual supplied by Life Technologies Inc. (Gaithersburg, MD, USA) with minor modifications. Briefly, genomic DNA (0.5 mg) was digested by the restriction enzyme combination EcoR I / Mse I at 37 8C for 2 h. Next, a solution containing 5 pMol EcoR I adapters, 50 pMol Mse I adapters, 1 u T4 DNA ligase, 1 3 buffer (10 mM Tris-HCl pH 7.5, 10 mM Mg-acetate, 50 mM K-acetate) was added, and Table 2. Primer pairs tested in the AFLP analysis of peanut germplasm. E-aac / M-caa * E-aac / M-cag * E-aag / M-cac * E-aag / M-cag * E-aag / M-ctt E-aca / M-caa * E-aca / M-ctg E-acc / M-cac * E-acc / M-cag * E-acc / M-cta * E-acg / M-caa E-acg / M-cac E-acg / M-cat * E-acg / M-ctc *

E-acg / M-ctg * E-acg / M-ctt * E-acg / M-cag * E-act / M-cag E-act / M-cta * E-act / M-ctt E-act / M-caa * E-agg / M-caa E-agg / M-cac E-agg / M-cag E-agg / M-cat E-agg / M-cta E-agg / M-ctt E-agc / M-ctt *

The primer pairs which produced clear polymorphic bands in this study.

the solution was incubated at 20 8C for 2 h. After ligation, the reaction mixture was diluted by 1:10 with TE (10 mM Tris-HCl, 0.1 mM EDTA pH 8.0). The first step of the PCR amplification was performed with pre-amp primer obtained from Life Technologies Inc. The first amplified DNA was not diluted prior to the second PCR amplification. The second amplification was conducted using two primers each with three selective nucleotides. Our previous study showed that a polymorphism among cultivated peanut could be detected by 28 out of a total 64 primer combinations tested (He and Prakash 1997). These 28 primer combinations were used in this study (Table 2). After amplification, DNA fragments were separated in a 6% denaturing polyacrylamide gel. Electrophoresis was performed at a temperature range from 48 8C to 52 8C for about 2.5 h. Gels were silver stained using a modified procedure of Bassam et al. (1991) but with an oxidizing step prior to staining to reduce the background and to obtain clearer bands (He et al. 1994). Gels were scored for presence or absence of polymorphic bands. Cluster analysis was performed by using NTSYS 1.7 software with Jaccard’s coefficient as similarity index and the unweighted paired group method (UPGMA) to construct a phenogram (Rohlf 1992).

Results Among the 28 AFLP primer combinations tested on the cultivated peanut germplasm, 15 pairs produced clear polymorphic bands. We identified 28 distinct AFLP markers using these primer pairs that differentiated peanut genotypes both between and within subspecies (Table 2). Based on these 28 DNA markers, unique DNA fingerprint profiles of each accession of cultivated peanut were developed and a phenogram depicting relationships among the cultivated peanuts was constructed. The 44 peanut lines and three lines of A. monticola used in this study fell into two major groups separating at about 52% similarity coefficient (Figure 1). The first group (A), included four botanical varieties, of which two varieties ‘hypogaea’ and ‘hirsuta’ belong to ssp. hypogaea and other two varieties ‘aequatoriana’ and ‘peruviana’ belong to ssp. fastigiata. Ten accessions within group (A) formed the first subgroup comprised mostly of botanical varieties of peruviana and aequatoriana with one hypogaea line and one fastigiata line. The second subgroup

350 within group (A) consisted of 15 accessions from varieties of hypogaea and hirsuta, 3 lines of A. monticola and 2 accessions of aequatoriana. The accessions within this subgroup seems to be progressive aggregations to the first subgroup. The third

subgroup was the smallest and included an accession of aequatoriana and two of peruviana. The second group (B) was clearly more distinct as it included all the accessions from the two botanical varieties fastigiata and vulgaris, belonging to ssp.

Figure 1. Phenogram of 44 accessions of A. hypogaea and 3 accessions of A. monticola based on AFLP markers

351 fastigiata. No accessions of ssp. hypogaea were in the group (B).

Discussion The cultivated peanut is an allotetraploid and because of little detectable genetic variation in its germplasm, Halward et al. (1991) suggested that this crop may have evolved recently from its diploid ancestors through rapid speciation (Ayala 1982). Under selection pressure imposed preferentially by crop breeding and agroecological adaptation, considerable morphological variation in agronomic traits may be produced but are accompanied by minor alterations at the DNA or protein level (Halward et al. 1991; ShattuckEidens et al. 1990). By using the AFLP technology, we have successfully detected DNA polymorphism in peanut and employed it here for the first time to gain insights into genetic relatedness among botanical varieties of peanut. In this study, the AFLP technique detected not only the variation between the subspecies of A. hypogaea but also within the subspecies underscoring its utility as a reliable and efficient method of identification of DNA polymorphism in cultivated peanut. Recently we have constructed a framework genetic map of cultivated peanut using AFLP markers (data not shown). Based on the morphological and physiological traits, rarer peanut botanical varieties aequatoriana and peruviana have been classified as belonging to ssp. fastigiata which also includes the more common botanical varieties fastigiata and vulgaris. Our study using DNA markers shows that accessions of varieties aequatoriana and peruviana are much closer to ssp. hypogaea and A. monticola rather than ssp. fastigiata to which they belong under the present classification. Our results based on DNA polymorphism are thus not consistent with the dichotomy of A. hypogaea subspecies. All the accessions of vulgaris and most of fastigiata (with one exception; PI 502020 in group A which may have been misclassified) clustered together in group B. Our results thus raise questions on the recently expanded classification of peanut botanical varieties. The relationship between the wild species A. monticola and the cultivated A. hypogaea has not been well determined. Both of them are tetraploids and Krapovickas et al. (1974) suggested A. monticola could have originated through crossing between A. hypogaea and a wild diploid. Pickersgill (1986) and

Lu and Pickersgill (1993) suggested that A. monticola and A. hypogaea are probably not distinct species. Lanham et al. (1994) pointed out A. monticola may have arisen by hybridization of two subspecies of A. hypogaea. Singh et al. (1991) detected little difference in protein profiles between A. monticola and A. hypogaea. Our study using DNA markers clearly shows that wild A. monticola is not genetically distinct from the cultivated A. hypogaea. Further, A. monticola is closer to the subspecies hypogaea than to subspecies fastigiata. This conclusion is consistent with the supposed phylogenetic position of the ssp. hypogaea and its relationship with the wild A. monticola. Paik-Ro et al. (1992) also observed that A. monticola was more closely related to subspecies hypogaea than to subspecies fastigiata using RFLP markers. Singh et al. (1994) also found that A. monticola hybridized more readily with subspecies hypogaea than with subspecies fastigiata. Many peanut genotypes originating from a geographic region grouped together suggesting the influence of geographic isolation on genetic polymorphism. For instance, most genotypes from Ecuador grouped together, although most genotypes from Paraguay and Uruguay were scattered into different subgroups. Similarly, genotypes from Brazil, Bolivia and Argentina were spread over both groups A and B. Many previous studies could detect a very low DNA variation in peanut presumably because they tested fewer primers, employed RFLP or RAPD methods which are less efficient in detecting polymorphism compared to the AFLP and used modern, highly-related cultivars for testing polymorphism. Our study reported here and an earlier one (He and Prakash 1997), shows that cultivated peanut does contain considerable genetic variation and DNA markers can be gainfully employed to assess genetic diversity and to measure the extent of genetic relationship among genotypes.

Acknowledgements Contribution 314 from the George Washington Carver Agricultural Experiment Station. We thank Dr David Williams (IPGRI, Colombia) for suggestions and critical evaluation of the manuscript. We also thank Dr Jose Valls (EMBRAPA, Brazil) and Dr A. K. Singh (NBPGR, India) for helpful comments on the manuscript. Assistance of Dr Channabyre Gowda and Mr Martis Watts is acknowledged. Research supported by

352 a competitive grant from USDA / CSREES under the 1890 Institutional Capacity Building Program and NASA.

References Ayala F.J. 1982. Population and Evolutionary Genetics. Benjamin / Cummings Publishing Company, Inc, Menlo Park, CA. Bassam B.J., Gaetano-Anolles G. and Gresshoff P.M. 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Analytical Biochemistry 196: 80–83. Chen J. and Dellaporta S. 1994. Urea-based plant DNA miniprep. In: Freeling M (ed.)Walbot V., The Maize Handbook. SpringerVerlag, New York, pp. 526–528. Garcia G.M., Stalker H.T. and Kochert G. 1995. Introgression analysis of an interspecific hybrid population in peanuts (Arachis hypogaea L.) using RFLP and RAPD markers. Genome 38: 166–176. Gepts P. 1993. The use of molecular and biochemical markers in crop evolution studies. Evolutionary Biology 27: 51–94. Grieshammer U. and Wynne J.C. 1990. Isozyme variability in mature seeds of U. S. peanut cultivars and collections. Peanut Science 18: 72–75. Halward T.M., Stalker H.T., LaRue E. and Kochert G. 1991. Genetic variation detectable with molecular markers among unadapted germplasm resources of cultivated peanut and related wild species. Genome 34: 1013–1020. Halward T.M., Stalker H.T., LaRue E. and Kochert G. 1992. Use of single-primer DNA amplification in genetic studies of peanut (Arachis hypogaea L.). Plant Molecular Biology 18: 315–325. He G.H., Prakash C.S., Jarret R.L., Tuzun S. and Qiu J. 1994. Comparison of gel matrices for resolving PCR-amplified DNA fingerprint profiles. PCR Meth. Appl. 4: 50–51. He G.H. and Prakash C.S. 1997. Identification of polymorphic DNA markers in cultivated peanut (Arachis hypogaea L.). Euphytica 97: 143–149. Knauft D.A. and Gorbet D.W. 1989. Genetic diversity among peanut cultivars. Crop Sci. 29: 1417–1422. Kochert G., Halward T., Branch W.D. and Simpson C.E. 1991. RFLP variability in peanut (Arachis hypogaea L.) cultivars and wild species. Theor. Appl. Genet. 81: 565–570.

Krapovickas A., Fernandez A. and Seeligman P. 1974. Recuperacion de la fertilidad en un hibrido interspeifico esteril de Arachis (Leguminosae). Bonplandia 3: 129–142. Krapovickas A. and Gregory W.C. 1994. Taxonomia del genero Arachis (Leguminosae). Bonplandia 8: 1–186. Lacks G.D. and Stalker H.T. 1993. Isozyme analyses of Arachis species and interspecific hybrids. Peanut Science 20: 76–81. Lanham P.G., Fennell S., Moss J.P. and Powell W. 1992. Detection of polymorphic loci in Arachis germplasm using random amplified polymorphic DNAs. Genome 35: 885–889. Lanham P.G., Forster B.P., McNicol P., Mossand J.P. and Powell W. 1994. Seed storage protein variation in Arachis species. Genome 37: 487–496. Lu J. and Pickersgill B. 1993. Isozyme variation and species relationships in peanut and its wild relatives (Arachis L.Leguminosae). Theor. Appl. Genet. 85: 550–560. Paik-Ro O.G., Smith R.L. and Knauft D.A. 1992. Restriction fragment length polymorphism evaluation of six peanut species within the Arachis section. Theor. Appl. Genet. 84: 201–208. Pickersgill B. 1986. Evolution of hierarchical variation patterns under domestication and their taxonomic treatment. In: Style S.T (ed.), Infraspecific Classification of Wild and Cultivated Plants. Clarendon Press, Oxford, pp. 191–209. Rohlf F.J. 1992. Numerical Taxonomy and Multivariate Analysis System. Applied Biostatistics Inc., New York. Shattuck-Eidens D.M., Bell R.N., Neuhausen S.L. and Helentjaris T. 1990. DNA sequence variation within maize and melon: Observation from polymerase chain reaction amplification and direct sequency. Genetics 126: 207–217. Singh A.K. and Moss J.P. 1984. Utilization of wild relatives in genetic improvement of Arachis hypogaea L. 5: Genome analysis in section Arachis and its implications in gene transfer. Theor. Appl. Genet. 68: 355–365. Singh A.K., Sivaramakrishnan S., Mengesha M.H. and Ramaiah C.D. 1991. Phylogenetic relations in section Arachis based on seed protein profile. Theor. Appl. Genet. 82: 593–597. Singh A.K., Santosh Gurt and Jambunathan R. 1994. Phylogenetic relationships in the genus Arachis based on seed protein profiles. Euphytica 74: 219–225. Vos P., Hogers R., Bleeker M., Reijans M., Van de Lee T., Hornes M. et al. 1995. AFLP: A new technique for DNA fingerprinting. Nucleic Acids Research 21: 4407–4414.