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Wheat Information Service
Number 85: 35-42 (1997)
Research article

DNA amplification fingerprinting (DAF) as a new source of molecular markers in bread wheat

Arnab Sen, H.S. Balyan, P.C. Sharma, B. Ramesh, Alok Kumar, Joy K. Roy, Rajeev K. Varshney and P.K. Gupta*

Molecular Biology Laboratory, Department of Agricultural Botany, Ch. Charan Singh University, Meerut 250 004, India


Summary

Polymorphism in seven bread wheat genotypes (differing for four quality traits) was studied using DAF. Ten arbitrary, 8- mer, GC rich, linear primers and ten 1 1-mer minihairpin primers, each having a 3-mer core sequence at 3' end, were utilized for DNA amplification. Nine linear primers and four mini-hairpin primers produced characteristic fingerprinting patterns involving polymorphism. The remaining primers gave poor DAF profiles with high background smear, which was associated with relatively higher GC content of these primers. It could partly also be due to large genome size of bread wheat. Both linear as well as mini-hairpin primers producing good DAF profiles, also revealed polymorphic DAF products, some of which were unique to a genotype. Contrary to earlier claims, neither the average number, nor the proportion of polymorphic products obtained with mini-hairpin primers suggested their superiority over the linear primers. In view of our earlier unpublished results with RAPDs and MP-PCR on the same set of genotypes, giving no reproducible polymorphism, we conclude that DAF technology may be a useful tool for detecting polymorphism in a difficult crop such as bread wheat.

Key words: arbitrary primers, DAF, fingerprinting, PAGE, bread wheat


Introduction

A variety of molecular markers are available for detecting DNA polymorphism among animal and plant materials. In crop plants, their use in tagging genes for marker aided selection has been demonstrated (for a review see Mohan et al. 1997). These markers may be hybridization based markers or PCR based markers and include (i) RFLPs using various types of probes, (ii) microsatellites or simple sequence repeats (SSRs) (Jefferys et al. 1985; Weber and May 1989; Litt and Luty 1989; Tautz, 1989; Akkaya et al. 1992; Morganty and Oliviari 1993), (iii) minisatellites (Jefferys et al. 1988; Vassart et al- 1987; Broun et al. 1992; Stockton et al. 1992), and (iv) RAPDs (Williams et al. 1990), AP-PCR (Welsh and McClelland 1990) and DAF (Caetano-Anolles et al. 1991). The last category of markers are also collectively called as MAAP (Multiple Arbitrary Amplicon Profiling) (Caetano-Anolles et al. 1991 ). The lack of prior knowledge of DNA sequences and lack of characterization of the probes restricts the use of RFLPs for detecting polymorphism. Similarly, the requirement of prior knowledge of flanking sequences for designing primers limits the use of minisatellites and microsatellites. Markers of MAAP family, however, make use of arbitrary primers and therefore, can be used without any prior knowledge of DNA sequences. However, among these markers of MAAP family also, markers like RAPDs proved to be of limited value in a crop like bread wheat due to detection of low polymorphism (Penner et al. 1995). DNA Amplification Fingerprinting (DAF) has recently been found to be promising in many plant materials. DAF makes use of very short (may be 5-mer but typically 7-8-mer) GC rich primers. The utility of short oligonucleotides for DNA amplification, producing genotype specific, characteristic DNA profiles was first demonstrated by Caetano-Anolles et al. (1991). It was shown that these complex DNA profiles can detect genetic differences in a wide variety of organisms including bacteria, fungi, plants (soybean, bermudagrass, centipedegrass, etc.) and humans (Caetano-Anolles et al. 1991, 1995; Gresshoff and MacKenzie 1994; Prabhu and Gresshoff 1994; Weaver et al. 1995; Gresshoff et al. 1997). Following PCR amplification, DAF products can be separated on polyacrylamide gel. Silver staining of the gels reveals DNA amplification profiles with high multiplex ratio (Caetano-Anolles et al. 1991, 1995; Prabhu and Gresshoff 1994; CaetanoAnolles and Gresshoff, 1996). A new class of DAF primers referred to as minihairpin primers have also been used and a few recent reports suggested their superiority over linear DAF primers (Caetano-Anolles and Gresshoff 1994; 1996). In view of the above, and due to failure of RFLPs and RAPDs to detect polymorphism among wheat varieties (Chao et al. 1989; Penner et al- 1995), we utilized DAF technology in bread wheat for the first time to cheek its suitability for detection of polymorphism and for their subsequent use in tagging of some quality traits for molecular marker aided selection in plant breeding.
* Corresponding author

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