KASP: a high-throughput genotyping system and its applications in major crop plants for biotic and abiotic stress tolerance

This study presents the development of a simple DNA extraction method and a novel PCR-RFLP protocol for genotyping theMALE STERILITY 4(MS4) gene inCryptomeria japonicaas a proof of concept. Traditional CTAB-based DNA extraction methods, while effective, involve hazardous chemicals and require high-speed centrifugation, which are impractical in many field settings. Our approach utilizes a household dish detergent-based buffer, sodium chloride, and polyvinylpyrrolidone K-30 to extract DNA fromC. japonicaneedle leaf tissues—without the need for liquid nitrogen. The simplicity of this method makes it more accessible and environmentally friendly. The extracted DNA was successfully used in PCR-RFLP analysis, targeting a single nucleotide polymorphism in theMS4gene, demonstrating its efficacy for genotyping. The PCR-RFLP markers reliably discriminated between individual genotypes, confirming the practical application of our simple extraction method, even for conifers containing inhibitory substances. This technique is particularly advantageous for use in arboretums and field stations, where the use of hazardous chemicals, specialized equipment, and a draft chamber is limited. Our study contributes to genetic resource management by providing an easy, reliable, and safer method for DNA extraction and genetic analysis.

show abstract

Authenticity Identification of F1 Hybrid Offspring and Analysis of Genetic Diversity in Pineapple

Jia,

Liu,

Lin

et al. 2024

Agronomy

View full text Add to dashboard Cite

Breeding is an effective method for the varietal development of pineapple. However, due to open pollination, it is necessary to conduct authentic identification of the hybrid offspring. In this study, we identified the authenticity of offspring and analyzed the genetic diversity within the offspring F1 hybrids resulting from crosses between ‘Josapine’ and ‘MD2’ by single nucleotide polymorphism (SNP) markers. From the resequencing data, 26 homozygous loci that differentiate between the parents have been identified. Then, genotyping was performed on both the parents and 36 offspring to select SNP markers that are suitable for authentic identification. The genotyping results revealed that 2 sets of SNP primers, namely SNP4010 and SNP22550, successfully identified 395 authentic hybrids out of 451 hybrid offspring. We randomly selected two true hybrids and four pseudohybrids for sequencing validation, and the results have shown that two true hybrids had double peaks with A/G, while pseudohybrids had single peaks with base A or G. Further study showed that the identification based on SNP molecular markers remained consistent with the morphological identification results in the field, with a true hybridization rate of 87.58%. K-means clustering and UPGMA tree analysis revealed that the hybrid offspring could be categorized into two groups. Among them, 68.5% of offspring aggregated with MD2, while 31.95% were grouped with Josapine. The successful application of SNP marker to identify pineapple F1 hybrid populations provides a theoretical foundation and practical reference for the future development of rapid SNP marker-based methods for pineapple hybrid authenticity and purity testing.

show abstract

KASP: a high-throughput genotyping system and its applications in major crop plants for biotic and abiotic stress tolerance

Cited by 5 publications

References 93 publications

Developing simple DNA extraction and PCR-RFLP for MALE STERILITY 4 (MS4) gene in Cryptomeria japonica D. Don: toward an environmentally friendly protocol

Developing simple DNA extraction and PCR-RFLP for MALE STERILITY 4 (MS4) gene in Cryptomeria japonica D. Don: toward an environmentally friendly protocol

Developing simple DNA extraction and PCR-RFLP forMALE STERILITY 4(MS4) gene inCryptomeria japonicaD. Don: Toward an environmentally friendly protocol

Authenticity Identification of F1 Hybrid Offspring and Analysis of Genetic Diversity in Pineapple

Contact Info

Product

Resources

About