FISSR-PCR: a simple and sensitive assay for highthroughput genotyping and genetic mapping.
The recently developed Inter-Simple Sequence Repeat PCR (ISSR-PCR) or microsatellite primed PCR or Simple Sequence Repeat (SSR)-Anchored PCR technique detects polymorphic markers in a wide variety of genomes. Usually the ISSR primers are either 5' end-labeled with gamma[32P]ATP or one of the alpha[32P] labeled dNTPs is added to the PCR reaction and the PCR products are resolved on PAGE and autoradiographed. Alternatively, cold PCR products are resolved on agarose gel electrophoresis. In the present study, we show that informativity, sensitivity and speed of the ISSR-PCR can be substantially enhanced by adding fluorescent nucleotide in the PCR reaction followed by resolution of PCR products on an ABI 377 automated sequencer. The informativeness, measured as a number of detectable amplified fragments, was two-fold higher and the quantity of required template DNA is two-fold lower than the regular ISSR-PCR. We have termed this method as FISSR-PCR and show its usefulness in generating large number of species and varietal specific markers in plants, insects, parasites of insects and human and various infectious organisms. Further, we show that the FISSR markers are inherited and segregated in Mendelian fashion as demonstrated on a panel of 99 F2 offspring derived from a cross of two divergent silkworm strains. The FISSR-PCR marker assay could be a method of choice for large scale screening of varieties/cultivars and highthroughput genotyping in mapping of genomes where microsatellite information is scanty or absent.
Nagaraju
J
J
Laboratory of Molecular Genetics, Centre for DNA Fingerprinting and Diagnostics, Nacharam, Hyderabad, India. jnagaraju@www.cdfd.org.in
Kathirvel
M
M
Subbaiah
E V
EV
Muthulakshmi
M
M
Kumar
L D
LD
D000818:Animals; D001331:Automation; D012831:Bombyx / Q000235:genetics*; D002874:Chromosome Mapping / Q000379:methods*; D003433:Crosses, Genetic; D002523:Edible Grain / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry; D005828:Genetics, Population; D023281:Genomics / Q000379:methods; D018895:Microsatellite Repeats; D009711:Nucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011110:Polymorphism, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods
D005456:Fluorescent Dyes; D009711:Nucleotides