SNP-based markers have been widely used to identify tolerant varieties harboring major genes related to abiotic stress tolerance. Here, we developed Fluidigm markers for the core set of SNPs underlying tolerance to abiotic stresses such as salinity, drought, anaerobic germination and submergence. The core set of SNPs was selected from the major genes and/or QTLs for the abiotic stresses previously reported in rice; Saltol for salinity, qDTY2.2 and qDTY4.1 for drought, OsTPP7 for anaerobic germination, and Sub1A for submergence tolerance. First, a total of 17 KASP markers were developed and converted to Fluidigm markers. The developed Fluidigm markers were applied to genotypic screening of 172 domestic and abroad varieties. The phylogenetic analysis has revealed that the majority of varieties can be largely grouped into two clusters, which correspond to domestic and foreign categories. This observation could be attributed to the fact that most tolerance genes for abiotic stresses have been inherited from indica varieties. The developed Fluidigm marker set would be used for screening genotypes tolerant to major abiotic stresses in the rice plant breeding process.

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The anaerobic germination 1 (AG1) locus, which confers tolerance to the anaerobic germination of rice seed, has been previously identified and a gel-based InDel DNA marker developed for marker-assisted selection (MAS). However, there is a need for marker development for high-throughput genotyping in order to increase the breeding efficiency. Single nucleotide polymorphisms (SNPs)-based Kompetitive Allele Specific PCR (KASP) markers enable high-accuracy and high-throughput analyses for the genotyping of AG1 locus. In the present study, we developed one AG1 InDel-specific and four flanking KASP markers, which can be used as the foreground and recombination markers, respectively, for the AG1 locus. Of those, three KASP markers developed were validated with Dasan-AG1 (BC₃F₂) segregating lines; the AG1 InDel-specific KASP marker was 100% consistent with the existing AG1 gel-based marker, while the two flanking markers showed 70.3 and 66.7 percent consistency to the AG1 gel-based marker, respectively. In addition, the five KASP markers developed were further used to genotype 78 Korean and 95 foreign rice varieties. The genotyping results with 172 varieties revealed that most of the Korean varieties possessed the AG1 locus; however, a wide variation was observed in the foreign varieties for AG1 locus. Hence, the KASP markers developed for AG1 locus show promise as useful tools for expediting the breeding of varieties tolerant to anaerobic germination stress via high-throughput genotyping.

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• KASP: a high-throughput genotyping system and its applications in major crop plants for biotic and abiotic stress tolerance
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• Development of SNP Marker Set to Select Varieties Tolerant to Multiple Abiotic Stresses in Rice
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The SUBMERGENCE 1 (SUB1) locus, conferring tolerance to complete inundation, was identified, and gel-based DNA markers, AEX1 and GnS2, were previously developed for marker-assisted breeding (MAB). However, a high throughput and high specific method, at low cost, is still required to detect Sub1 alleles. Kompetitive Allele Specific PCR (KASP) markers enable high throughput analysis for a large number of seeds, as well as detection of both alleles, in a single reaction. In this study, we developed KASP markers that can distinguish specific alleles at Sub1A loci based on single nucleotide polymorphisms (SNPs). Marker validations were carried out by genotyping of a segregating population with the developed KASP markers. The results from KASP assay and gel-based marker analysis were consistent for Sub1A alleles. KASP markers developed for Sub1A would be helpful due to high accuracy, low cost, and a high throughput genotyping feature in MAB.

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  Molecular Biology Reports.2024;[Epub]     CrossRef
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• Development of Kompetitive Allele Specific PCR Markers for Anaerobic Germination 1 Locus in Rice
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