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Euphytica 97: 143–149, 1997. c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Identification of polymorphic DNA markers in cultivated peanut (Arachis hypogaea L.) Guohao He & Channapatna S. Prakash Center for Plant Biotechnology Research, Tuskegee University, School of Agriculture and Home Economics, Milbank Hall, Tuskegee, AL 36088, U.S.A.; ( author for correspondence) Received 19 June 1996; accepted 13 January 1997

Key words: amplified fragment length polymorphism (AFLP) markers, DNA amplification fingerprinting (DAF), groundnut, polymerase chain reaction (PCR), random amplified polymorphic DNA (RAPD) markers

Summary The detection of DNA polymorphism in cultivated peanut (Arachis hypogaea L.) is reported here for the first time. The DNA amplification fingerprinting (DAF) and amplified fragment length polymorphism (AFLP) approaches were tested for their potential to detect genetic variation in peanut. The AFLP approach was more efficient as 43% of the primer combinations detected polymorphic DNA markers in contrast to 3% with the DAF approach. However, the number of polymorphic bands identified using primers selected in both approaches was comparable. In the DAF study, when 559 primers of varying types were screened, 17 (mostly 10-mer types) detected polymorphism producing an average of 3.7 polymorphic bands per primer with a total of 63 polymorphic markers. In the AFLP study, when 64 primer combinations (three selective nucleotides) corresponding to restriction enzymes Eco RI and Mse I were screened, 28 detected polymorphism. On an average, 6.7% of bands obtained from these 28 primer pairs were polymorphic resulting in a total of 111 AFLP markers. Our results demonstrate that both AFLP and DAF approaches can be employed to generate DNA markers in peanut and thus have potential in the marker-assisted genetic improvement and germplasm evaluation of this economically important crop. Introduction The cultivated peanut or groundnut (Arachis hypogaea L.) is unique among crop species because earlier studies using random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) approaches have found no DNA variation among its genotypes (Kochert et al., 1991; Halward et al., 1992; Paik-Ro et al., 1992). Similarly, isozyme and seedprotein studies have observed limited variation among peanut cultivars (e.g., Lu & Pickersgill, 1993; Stalker et al., 1994). However, substantial diversity exists among cultivated peanut genotypes for various morphological, physiological and agronomic traits (Stalker, 1992). Nearly 15,000 germplasm accessions of cultivated peanut and related species are maintained at ICRISAT (India), while USDA (Griffin, GA) has 8,500 accessions. It is thus intriguing that a genus with substantial

variation for phenotypic traits such as plant habit, seed color, and resistance to biotic and abiotic factors possess no detectable variation at the DNA level. The DNA amplification fingerprinting (DAF) approach is a variation of the RAPD technique (Williams et al., 1990), but is relatively more informative because of the use of altered reaction conditions, shorter primers, and silver staining (CaetanoAnoll´es et al., 1991). The new AFLP (amplified fragment length polymorphism) procedure detects a large number of polymorphic DNA markers in a relatively short time and is thus an useful technique when highthroughput is desired (Vos et al., 1995). AFLP markers are more reliable and reproducible compared to RAPD markers and less cumbersome than the RFLP technique. The genomic DNA is first digested by restriction endonucleases, and the DNA fragment ends are ligated to synthetic oligonucleotide adapters, and amplified by PCR using primers that contain the common oli-

144 Table 1. Description of peanut genotypes employed for screening DAF and AFLP primers No. #

A. hypogaea subspecies

Botanical variety

USDA plant intr. no.

Country of origin

1 2 3 4 5 6

hypogaea hypogaea fastigiata fastigiata fastigiata fastigiata

hypogaea hypogaea fastigiata aequatoriana aequatoriana fastigiata

497302 475871 262023 497633 497630 502033

Bolivia Brazil Paraguay Ecuador Ecuador Peru

Table 2. Summary of primers tested in DAF and AFLP studies for the identification of DNA polymorphism in cultivated peanut Type of primer

DAF Primers 8-mer 7-mer+hairpin 10-mer Simple sequence repeats Total (DAF) Total (AFLP)

gos homologous to the adapters and with one to three arbitrary selective nucleotides at the 30 end. This study aimed to identify polymorphic DNA markers in cultivated peanut through DAF and AFLP approaches, and to increase the possibility of DAF marker identification by optimizing PCR reaction and DNA detection conditions.

Materials and methods Plant material Six divergent genotypes of cultivated peanut from three botanical varieties were employed (Table 1). Seeds were obtained from the USDA Plant Genetic Resources Conservation Unit, Griffin, GA. DNA isolation Total genomic DNA was extracted from the peanut seedlings (10–15 d) using the urea-based protocol of Chen and Dellaporta (Chen & Dellaporta, 1994). The A260/280 readings for the DNA samples were 1.6 to 1.8. PCR amplification DAF: The PCR reaction components for DAF were optimized to obtain complex but clear DNA profiles in a reproducible manner from peanut. The PCR reaction mix for DAF (10 L) contained template DNA (75 ng), primer (0.3 M), Taq DNA polymerase Stoffel Fragment (Perkin Elmer; 5 U), mg++ (2.5 mM), buffer (1X) and overlaid with a drop of mineral oil. Amplifications were performed in a MJ Research thermal cycler (96well type) for 35 cycles after an initial denaturation at 94  C for 5 min and a final extension at 72  C for 5

No. of pimers tested

No. of primers detecting polymorphism

68 7 379 105 559

1 1 15 – 17

64

28

min (Caetano-Anoll´es et al., 1991). For arbitrary and mini-hairpin primers, each cycle consisted of 5 sec at 94  C, 20 sec at either 35  C or 45  C (depending on the primer; see Table 3) and 30 sec at 72  C. For SSR primers, each cycle was 1 min at 94  C, 1 min at 55  C and 2 min at 72  C. AFLP: 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. Restriction enzymes EcoRI and MseI were used to digest 50 ng l 1 of peanut genomic DNA and the reaction mix was subjected to the ligation of adapters followed by preamplification. The preamplified DNA was not diluted prior to selective amplification. The selective amplification was conducted using two primers each with three selective nucleotides (64 primer combinations) corresponding to the EcoRI and MseI linkers obtained from the Life Technologies Inc. Gel electrophoresis DAF: After amplification, DNA fragments were separated in a vertical electrophoresis system using a polyacrylamide-based vinyl polymer (GeneAmp; Perkin Elmer, Norwalk, CT) as described earlier (Gowda et al., 1996) . AFLP: The gel electrophoresis for AFLP products was as described by Vos et al (1995) but employing a 6% denaturing polyacrylamide gel (19:1 acrylamide: bis; 7.5 M urea; 1X TBE buffer). Electrophoresis was performed at constant power, 55W for about 2 h.

145 Silver staining for DNA visualization Both DAF and AFLP gels were silver stained using a modified procedure of Bassam et al. (Bassam et al., 1991) but with an initial oxidizing step to reduce the background and to obtain clearer bands (Gowda et al., 1996). Primer screening In the DAF study, 559 primers were screened including those with arbitrary sequence (8-, 10-mer, and 7-mer + 3- or 4-mer mini-hairpin) (Williams et al., 1990; Caetano-Anolles & Gresshoff, 1994) and simple sequence repeats known to detect polymorphism in other plant species (Gupta et al., 1994; Wang et al., 1994). Among the 10-mer primers tested, 300 were from University of British Columbia (UBC, Vancouver, Canada) and 79 from Operon Technologies (Alameda, CA). The SSR primers tested were a set of 100 UBC primers (series# 9), consisting of anchored 2-base repeats and unanchored 3-, 4-, and 5-base repeats (Dr. John Hobbs, Pers. Comm.). In addition, we also screened following simple sequence repeat primers: (1) single (GC)2 (CA)8 ; (2) duplex (ACAG)4 with (GATA)4 ; and (3) telomere-specific duplex (TTTAGGG)4 with (CCCTAAA)4 (Kolchinsky & Gresshoff, 1994). For the AFLP Study, 64 primer combinations (8 X 8) were screened; the +3 selective nucleotide sequence of these primers are shown in Table 4.

Results Detecting peanut DNA polymorphism with the DAF approach Many parameters were studied to ensure that peanut DAF profiles are detected in an informative and reproducible manner. The DNA isolation procedure (the urea-based approach) and the PCR-reaction components concentration of template DNA (7.5 ng L 1 ), primer (0.3 M for 10-mer) and mg++ (2.5 mM); and the type of Taq polymerase (truncated AmpliTaq, Stoffel fragment) were critical in obtaining a large number of clear, reproducible DAF bands with all types of primers tested (data not shown). The use of vinylpolymer of polyacrylamide along with silver staining further enabled an improved separation and detection

Figure 1. DNA amplification fingerprinting (DAF) patterns of cultivated peanut genotypes representing six botanical varieties obtained using primers UBC 306 (GTC CTC GTAG). Amplified products were separated through electrophoresis on vinyl-polymer of polyacrylamide (200 v) and visualized by modified silver-staining. The identity of peanut botanical varieties in lanes 1 to 6 correspond to the numbers shown in Table 1. Left border lane (m) shows molecular weight markers in base pairs. Arrows show polymorphic bands.

of DNA fragments-especially those with similar size and with variable intensities. Among 559 DAF primers screened, 17 detected polymorphism in the peanut genotypes tested (Table 2). None of the primers with simple sequence repeats tested including those with anchored ends and telomere-specific sequences detected any polymorphism. Among the mini-hairpin primers tested, one primer was informative but produced only one polymorphic band. Similarly 8-mer primers were less efficient in detecting polymorphism, as only 1 among 68 tested was informative. Operon primers when used individually detected no polymorphism, but a combination of Operon L2+L3 primers produced one polymorphic band (Table 3). The most successful detection of polymorphism in peanut was clearly achieved by the use of UBC 10-mer primers especially those in the 300 series. Among 17 DAF primers that detected polymorphism in peanut, 15 were 10-mer primers from UBC. Overall, the use of 17 selected primers produced an average of nearly 18 bands/primer/genotype out of which 3.7 bands were polymorphic (Table 3). Six of these primers produced bands that were at least 25% polymorphic. It was, however, critical to choose the appropriate annealing temperature in DAF reac-

146 Table 3. Primers detecting polymorphic DNA markers from the DAF study in cultivated peanut1

1 2

Primer Code

Primer sequence (50 –30 )

Anneal temp ( C)

No. of bands2

No. of Bands polymorphic

% Bands polymorphic

OPL-2+ OPL-3 OR 57 Hairpin 29 UBC 3 UBC 23 UBC 25 UBC 302 UBC 306 UBC 332 UBC 336 UBC 346 UBC 348 UBC 355 UBC 391 UBC 400 UBC 776 UBC 788

TGG GCG TCAA + CCA GCA GCTT GTC CAT CG GCG AAG C-CTC CCT GGG CTTA CCC GCC TTCC ACA GGG CTCA CGG CCC ACGT GTC CTC GTAG AAC GCG TAGA GCC ACG GAGA TAG GCG AACG CAC GGC TGCG GTA TGG GGCT GCG AAC CTCG GCC CTG ATAT CTT CCC TCCT CCT TCC CTCT

45

22

1

4.5

35 45 35 35 35 35 35 35 35 45 45 45 35 35 35 35

16 19 17 20 16 14 16 16 15 20 22 17 14 21 16 17

4 1 6 5 2 2 6 5 3 2 5 6 4 6 2 3

25.0 5.2 35.3 25.0 12.5 14.3 37.5 31.3 20.0 10.0 22.7 35.2 28.6 28.6 12.5 17.6

Mean

-

-

17.5

3.7

21.5

Identified by screening 559 primers on six genotypes of cultivated peanut (see Table 1) Maximum number of bands observed in a peanut genotype.

Table 4. Number of detectable and polymorphic DNA fragments among AFLP primer combinations detecting DNA polymorphism in cultivated peanut1 EcoRI primers

MseI primers CAA2 CAC

AAC AAG ACA ACC ACG ACT AGC AGG % Pol. Loci

91 (4)4 61 (4)

CAG

CAT

CTA

CTC

72 (7) 39 (2)

90 (4) 4.5

CTT

91 (3)

85 (3) 64 (3) 92 (5)

CTG

42 (3) 56 (5) 60 (4)

52 (3) 7.0

34 (2) 45 (5) 34 (3)

54 (3) 44 (7)

35 (2) 8.1

35 (2) 11.4

41 (2)

49 (6)

4.9

9.9

75 (5) 51 (2) 5.6

72 (8) 65 (4) 78 (5) 53 (5) 7.0

% Pol. Loci3 6.7 4.7 4.7 6.9 9.3 6.4 6.4 5.7 6.7

1

Data shown only for those primer combinations detecting polymorphism. Sequences shown refer to the selective nucleotides at the 30 end of primers 3 Mean percentage of polymorphic fragments per primer pair among six peanut lines tested. 4 Figures refer to number of fragments detectable per lane while those in parenthesis refer to the number of polymorphic fragments per lane. 2

tions (Table 3), because certain primers required the use of higher 45  C than the usual 35  C to detect polymorphism. The typical DAF profiles of six peanut genotypes are shown in Figure 1. A mixture of intensely staining primary bands and less-intense secondary bands were seen in the detectable range of 150 to 3000

bp. Eight bands were polymorphic (arrows; Figure 1), and among these one band (220 bp; lanes 4 and 5; Figure 1) was specific to the botanical variety aequatoriana while another band was found only in the fastigiata (210 bp; lanes 3 and 6; Figure 1). The DAF study

147 was repeated with the selected 17 primers, and results obtained were highly reproducible. Detecting peanut DNA polymorphism with the AFLP approach Eight primers corresponding to the EcoRI adapter and eight corresponding to MseI adapter (all with 3 selective nucleotides) were tested in 64 possible combinations on peanut genotypes. The DNA polymorphism was detected in the peanut with 28 of the 64 AFLP primer pairs tested (Tables 2 and 4; Figure 2). These 28 AFLP primer pairs cumulatively detected 111 polymorphic loci (Table 4) and this rate is comparable to the 63 markers detected by DAF approach from 19 primers. An average of 57.8 bands per primer pair were detectable and 3.96 bands per primer pair were polymorphic (6.8%). Among the AFLP primers corresponding to the EcoRI adapter, ACG and AGG were the most informative while among those corresponding to the MseI adapter, CAA, CAG and CTT were superior in identifying polymorphism in peanut (Table 4). Two AFLP gels are shown in Figure 2. The bands detectable on the polyacrylamide gel using silver staining were between 50 bp to 1250 bp. Four polymorphic bands were observed with the primers EcoRI+AGG and MseI +CAA (arrows in panel A; Figure 2) while three such bands were seen with the primers EcoRI+ACA and MseI+CAA (arrows in panel B; Figure 2). Certain bands were specific to the botanical varieties. For instance, a 800 bp band obtained using the primers EcoRI+AGG and MseI +CAA was present in the hypogaea (lanes 1 and 2) but not in the other two botanical varieties from the subspecies fastigiata (panel B; Figure 2).

Discussion The AFLP approach was more efficient in detecting DNA polymorphism in peanut as 43% of the AFLP primer pairs identified polymorphism compared to 3% of the DAF primers. The superiority of the AFLP approach versus DAF is that the large number of bands are detectable in a single lane of the AFLP gel, which increases the possibility of finding polymorphic markers per lane although intrinsically the AFLP procedure does not necessarily detect more polymorphism than the DAF approach. However, once an informative primer or primer pair has been identified, both AFLP and DAF approaches yielded fairly similar lev-

Figure 2. AFLP profiles of cultivated peanut genotypes. Panel A was obtained using the primer pair EcoRI+AGG and MseI +CAA and the Panel B was with primer pairs EcoRI+ACA and MseI+CAA. The identity of peanut botanical varieties in lanes 1 to 6 in both panels correspond to the numbers shown in Table 1. The AFLP reaction protocol followed was similar to that described by Vos et al. (1995). Bands were detected directly on the polyacrylamide gel using silver staining. Molecular weight markers in base pairs are shown in the left. Arrows show polymorphic bands.

148 els of polymorphism: an average of 3.96 polymorphic markers per primer pair in AFLP versus 3.31 polymorphic markers per primer in DAF (Tables 3 and 4). Thus, despite the substantial initial effort, the DAF approach may still be effective in DNA marker studies of peanut because the procedure involves fewer steps and thus requires less time, expense and labor compared to the AFLP approach. Furthermore, the DAF procedure is less demanding in its template DNA quality than the AFLP procedure where relatively pure DNA samples are needed. However, AFLP approach is generally more tolerant to changes in PCR reaction conditions and thus has higher reproducible rate compared to DAF or RAPD procedures (Vos et al., 1995). Although the published AFLP protocol calls for the use of radioactive labeling to detect the DNA fragments (Vos et al., 1995), we have adopted the non-radioactive silver staining of gels to detect such fragments. This procedure while avoiding the hazardous radioactivity involves fewer steps, less expensive but comparable to radiolabeling in sensitivity. Lack of detectable DNA markers in peanut has hindered marker-assisted genetic studies of this crop. Our study demonstrates for the first time that DNA variation exists among peanut genotypes and can be detected using appropriate techniques including the use of informative primers. The success of our study in identifying polymorphic DNA markers relative to earlier studies in peanut may be due to many factors: the use of AFLP approach; and in the DAF study the use of optimized PCR reaction conditions, screening of a large number of primers, choice of primer sequences, use of a novel gel matrix that provides a superior resolution of DNA fragments, sensitive silver-staining procedure for DNA detection, and the use of genetically diverse peanut genotypes for screening. Earlier studies could not detect any polymorphism among cultivated peanut presumably because of the fewer primers they tested. For instance, Halward et al. (1991 and 1992) employed 16 primers and found no polymorphism among cultivated peanut although the wild Arachis species displayed considerable diversity. Although this study has identified many primers detecting polymorphism in peanut (totally 174 polymorphic loci), the percentage of polymorphic markers relative to the total number of primers screened was small (0.11% in DAF and 1.7% in AFLP). Thus the level of genetic variation in cultivated peanut when compared to other crops is relatively low; for instance, more than 50% of the 8-mer DAF primers tested on sweetpotato detected polymorphism (He et al., 1995).

This supports the notion that Arachis hypogaea may have arisen recently as a single polyploidization event (Halward et al., 1991). The low DNA polymorphism in peanut in contrast to the high diversity for agronomic traits may be due to the selective neutrality of molecular markers while morphological traits have been subjected to intense selection (Gepts, 1993). In a evolutionarily nascent species like cultivated peanut it is likely that simple nucleotide substitutions rather than gross differences account for variation among genotypes. The AFLP technique is relatively more efficient in detecting single nucleotide changes (at sites for restriction and selective amplification) compared to RFLP or RAPD procedures, and may explain the successful detection of polymorphism by this approach. Because earlier studies failed to detect DNA polymorphism within the cultivated peanut, a cross between two diploid wild Arachis species has been employed to construct a genetic map of Arachis (Halward et al., 1993). Such a map is useful due to the colinearity among related genomes and can also be employed to monitor introgression of traits from alien to cultivated species. Nevertheless, it is desirable to develop a genetic map of the cultivated peanut, especially because alien species have not been used much in the breeding of modern peanut cultivars (Isleib & Wynne, 1992) . Development of a genetic map of the cultivated peanut may also enrich the existing map of Arachis and thus facilitate an accelerated improvement of this crop. Availability of molecular markers will also enable detailed investigation of the peanut genome with immediate practical applications in cultivar identification through DNA fingerprinting, genetic diversity analysis and in understanding the domestication history of this crop.

Acknowledgments We thank Mr. Matand Kanyand and Mr. Martis Watts for technical assistance, Dr. A.K. Singh for advice, Dr. David Williams for selecting peanut genotypes, Dr. Roy Pittman for supplying seeds, Dr. Caetano Anoll´es for providing mini-hairpin primers and Dr. Julie Vogel for assistance with AFLP studies. Contribution # 257 from the George Washington Carver Agricultural Experiment Station. Research supported by grants from the USDA under the OICD (58-319R1-028) and the CSREES programs (94-38814-0475), and NASA (NAGW-2940). Mention of trademark or proprietary names is intended only for exact descrip-

149 tion and should not be taken to imply approval to the exclusion of other products that may be suitable.

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