Modified Allele-Specific qPCR (ASQ) Genotyping

Zinc finger knuckle genes are associated with tolerance to drought and dehydration in chickpea (Cicer arietinum L.)

“…The allele discrimination and genotyping results are clear using this ASQ method with medium throughput working effectively in 96-or 384-well microplates and qPCR instruments. Successful results for the ASQ method were recorded earlier in the genotyping of barley (Kalendar et al, 2022), sugar beet, and tomato (Amangeldiyeva et al, 2023).…”

“…The ASQ method was used for plant genotyping following the protocol described recently (Amangeldiyeva et al, 2023). In brief, option A with a short 4-bp tag was used, and two allele-specific forward primers and one reverse primer were developed targeting two SNPs, Ca04468-SNP1 and Ca07571-SNP4.…”

“…Primers and universal probes were ordered from Sigma-Merck (Australia). Master-mix preparation was the same as described by Amangeldiyeva et al (2023). Each reaction had a 10-µl cocktail in total and was loaded in a 96-well BioRad microplate sealed with clear tape prior to amplification in a CFX96 Real-Time PCR Detection System (Bio-Rad, USA) with automatically recorded fluorescence using the described protocol (Amangeldiyeva et al, 2023).…”

“…Master-mix preparation was the same as described by Amangeldiyeva et al (2023). Each reaction had a 10-µl cocktail in total and was loaded in a 96-well BioRad microplate sealed with clear tape prior to amplification in a CFX96 Real-Time PCR Detection System (Bio-Rad, USA) with automatically recorded fluorescence using the described protocol (Amangeldiyeva et al, 2023). Amplification of FAM and HEX was checked and controlled, whereas genotyping results were determined in a post-run step automatically using the software CFX Manager accompanying the qPCR instrument.…”

“…In general, it is much faster to work with medium throughput methods, for example, based on fluorescence (Förster) resonance energy transfer (FRET). Recently, allele-specific qPCR (ASQ) methods for plant genotyping have been developed that are also suitable for chickpea (Kalendar et al, 2022;Amangeldiyeva et al, 2023). Microarray technology employing thousands of simultaneous reactions on a microchip represents the next generation of plant genotyping (Varshney et al, 2012), and diversity array technology (DArT) is also very powerful and popular for the identification of SNP and haplotypes (a group of closely located SNPs) in genetic regions of entire genomes (James et al, 2008;Deres and Feyissa, 2022), including chickpea (Roorkiwal et al, 2014;Thudi et al, 2014).…”