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

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 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), microsatellite-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.

Genotyping and identification of six date palm (Phoenix dactylifera L.) cultivars<br>of the Gaza Strip by random amplification of polymorphic DNA

“…This agrees with many previous works which discussed the RAPD reproducibility and sensitivity when changing the PCR reaction components (Sharma et al, 1995;Skorić et al, 2012). Therefore, the optimal RAPD conditions (concentration of the primer, MgCL2, Taq DNA polymerase and dNTPs; the amount of DNA and amplification conditions) were maintained for fingerprinting and identification of date palm in the study.…”

New applications of low-C0t DNA as a DNA fingerprint probe

“…Both PCR fingerprinting methods require no prior DNA sequence knowledge of the genome and often generate discriminatory fingerprinting profiles. Another technique based on microsatellite DNA sequences is called microsatellite-primed PCR (MP-PCR [13,14]). With the increasing amount of sequencing data, another variant to this approach is the use of 5¢-and 3¢-anchored microsatellite primers in MP-PCR [15].…”

Chickpea (Cicer arietinum L.)

“…Over the past 15 years, electrophoretic data based on seed storage protein (Ladizinsky and Adler, 1975;Vairinhos and Murray, 1983; and isozymes (Kazan and Muehlbauer, 1991;Labdi et al, 1996;Tayyar and Waines, 1996;Gargav andGaur, 2001, Sudupak and have also been applied to systematic studies in Cicer. More recently, DNA-based techniques (Patil et al, 1995;Sharma et al, 1995;Ahmad, 1999;Choumane et al, 2000;Iruela et al, 2002;Sudupak et al, 2002;Rajesh et al, 2003;Javedi and Yamaguchi, 2004a, b;Nguyen et al, 2004;Sudupak, 2004; have provided many new approaches to compare aspects of genome relationships in ways not previously possible. Among the annual Cicer species, there is a consensus that C. reticulatum and C. echinospermum are the wild species most closely related to the domesticated C. arietinum.…”