The potential of microsatellites for hybridization- and polymerase chain reaction-based DNA fingerprinting of chickpea (Cicer arietinum L.) and related species

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Abstract
The genetic variability in agronomically important chickpea accessions (Cicer arietinum L.) as detected by single‐locus restriction fragment length polymorphism (RFLP) probes, random amplified polymorphic DNA (RAPD) and isoenzyme markers, is rather low. Recently, highly polymorphic microsatellites became the markers of choice for linkage mapping and population studies. We are currently following two main strategies to exploit the variability of microsatellites and adjacent sequences for genetic studies in chickpea. (i) In an approach referred to as oligonucleotide fingerprinting, microsatellite‐complementary oligonucleotides were employed as multilocus probes for in‐gel hybridization. A total of 38 different probes representing di‐, tri‐ and tetranucleotide repeats were used to analyze variability between and within four accessions of C. arietinum. Hybridization signals were obtained with 35 probes. While the abundance and level of polymorphism of the different target sequences varied considerably, distinct, intraspecifically informative banding patterns were obtained with the majority of probes and all restriction enzymes tested. No obvious correlation existed between abundance, fingerprint quality, and sequence characteristics of a particular motif. (ii) In a recently developed strategy called microsatellite primed polymerase chain reaction (MP‐PCR), micro‐satellite‐complementary oligonucleotides serve as single PCR primers for genomic DNA templates. We tested the general applicability of MP‐PCR by amplifying DNA samples from tomato, chickpea and two related annual Cicer species with a variety of di‐, tri‐ and tetranucleotide repeat primers. Most but not all primers generated distinct fingerprint‐like banding patterns after agarose gel electrophoresis and ethidium bromide staining of the amplification products. Since the method proved to be sensitive to reaction conditions in a way similar to RAPD analysis, we increased the PCR specificity by the introduction of a modified “touch‐down” protocol. In chickpea, touch‐down MP‐PCR generated highly reproducible banding patterns which predominantly revealed interspecific polymorphisms. The potential of different microsatellite‐based strategies for genome analysis in chickpea is discussed.