FISSR-PCR: a simple and sensitive assay for highthroughput genotyping and genetic mapping

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Inter-Simple Sequence Repeat PCR (ISSR-PCR) is a common technique for detecting polymorphic DNA markers.
• Traditional ISSR-PCR methods involve radioactive labeling or standard gel electrophoresis, which can be time-consuming and less sensitive.
• There is a need for more efficient and informative molecular marker techniques.

Purpose of the Study:

• To enhance the informativity, sensitivity, and speed of ISSR-PCR.
• To introduce a novel method, Fluorescent ISSR-PCR (FISSR-PCR), for improved genetic analysis.
• To demonstrate the utility of FISSR-PCR for generating species- and varietal-specific markers.

Main Methods:

• Incorporation of fluorescent nucleotides into the PCR reaction.
• Resolution of PCR products using an ABI 377 automated sequencer.
• Analysis of marker inheritance and segregation in an F2 population.

Main Results:

• FISSR-PCR demonstrated a two-fold increase in detectable amplified fragments (informativeness).
• FISSR-PCR required half the amount of template DNA compared to regular ISSR-PCR.
• FISSR markers were shown to be inherited and segregated in a Mendelian fashion.
• The method proved effective in generating numerous species- and varietal-specific markers.

Conclusions:

• FISSR-PCR significantly improves upon traditional ISSR-PCR by increasing informativity and sensitivity while reducing DNA input.
• This technique is highly effective for generating specific genetic markers in plants, insects, parasites, and infectious organisms.
• FISSR-PCR is a valuable tool for large-scale variety screening and high-throughput genotyping, especially in genomes with limited microsatellite data.