Competitive allele specifi c PCR (KASP): features, the interpretation of the results

The use of competitive allele-specific PCR is becoming a popular method for mass crop genotyping. The development of markers for SNPs in polyploid organisms requires additional validation and testing when developing primers for further use. In this work, we propose an algorithm for testing and interpreting the performance of KASP markers on wheat using existing common wheat genomic assemblies. Using the described algorithm, six KASP-markers on VIVIPAROUS-1, MOTHER OF FT AND TFL1, TGW6-A1, PSY-A1, Dreb-B1 and leaf rust resistance locus Lr14a were analyzed. The most promising KASP markers by bioinformatic analysis and PCR were the markers for VIVIPAROUS-1, MOTHER OF FT AND TFL1 genes.

1. Ayalew H., Tsang P.W., Chu C., Wang J., Liu S., Chen C., Ma X.F. Comparison of TaqMan, KASP and rhAmp SNP genotyping platforms in hexaploid wheat. PLOS ONE. 2019; 14 (5): e0217222.

2. Brinton J., Ramirez-Gonzalez R.H., Simmonds J., Wingen L., Orford S., S. Griffiths, 10 Wheat Genome Project, Haberer G., Spannagl M., Walkowiak S., Pozniak C., Uauy C. A haplotype-led approach to increase the precision of wheat breeding. Communications Biology. 2020; 3 (1): 712.

3. Darrier B., Colas I., Rimbert H., Choulet F., Bazile J., Sortais A., Jenczewski E., Sourdille P. Location and Identification on Chromosome 3B of Bread Wheat of Genes Affecting Chiasma Number. Plants. 2022; 11 (17): 2281.

4. Duan X., Yu H., W. Sun Ma J., Zhao Y., Yang R., Ning T., Li Q., Liu Q., Guo T., Yan M., Tian J., Chen J. A major and stable QTL controlling wheat thousand grain weight: identification, characterization, and CAPS marker development. Molecular Breeding. 2020; 40 (7): 68.

5. Food and Agriculture Organization of the United Nations. Selected indicators in Russion Federation, 2020. [Electronic source]. URL: https://www.fao.org/faostat/en/#country/185

6. Hanig M., Gao F., Liu J., Wen W., Zhang Y., A Rasheed., Xia X., He Z., Cao S. TaTGW6-A1, an ortholog of rice TGW6, is associated with grain weight and yield in bread wheat. Molecular Breeding. 2016; 36 (1): 1.

7. He X.Y., He Z.H., Zhang L.P., Sun D.J., Morris C.F., Fuerst E.P., Xia X.C. Allelic variation of polyphenol oxidase (PPO) genes located on chromosomes 2A and 2D and development of functional markers for the PPO genes in common wheat. Theoretical and Applied Genetics. 2007; 115 (1): 47–58.

8. He. X.Y., Zhang Y.L., He Z.H., Wu Y.P., Xiao Y.G., Ma C.X., Xia X.C. Characterization of phytoene synthase 1 gene (Psy1) located on common wheat chromosome 7A and development of a functional marker. Theoretical and Applied Genetics. 2008; 116 (2): 213–221.

9. Kaur B., Mavi G.S., Gill M.S., Saini D.K. Utilization of KASP technology for wheat improvement. Cereal Research Communications. 2020; 48 (4): 409–421.

10. Kumar S., Kumar M., Mir R.R., Kumar R. Advances in Molecular Markers and Their Use in Genetic Improvement of Wheat. Physiological, Molecular, and Genetic Perspectives of Wheat Improvement. Cham: Springer International Publishing, 2021: 139–174.

11. Lei L., Zhu X., Wang S., Zhu M., Carver B.F., Yan L. TaMFT-A1 Is Associated with Seed Germination Sensitive to Temperature in Winter Wheat. PLoS ONE. 2013; 8 (9): e73330.

12. Poland J.A., Brown P.J., Sorrells M.E., Jannink J. – L. Development of High-Density Genetic Maps for Barley and Wheat Using a Novel Two-Enzyme Genotyping-by-Sequencing Approach. PLoS ONE. 2012; 7 (2): e32253

13. Ramirez-Gonzalez R.H., Uauy C., Caccamo M. PolyMarker: A fast polyploid primer design pipeline: Fig. 1. Bioinformatics. 2015; 31 (12): 2038–2039.

14. Rasheed A., Wen W., Gao F., Zhai S., Jin H., Liu J., Guo Q., Zhang Y., Dreisigacker S., Xia X., He Z. Development and validation of KASP assays for genes underpinning key economic traits in bread wheat. Theoretical and Applied Genetics. 2016; 129 (10): 1843–1860.

15. Rogers S.O., Bendich A.J. Extraction of total cellular DNA from plants, algae and fungi. Plant Molecular Biology Manual. Dordrecht: Springer Netherlands, 1994: 183–190.

16. Shavrukov Y. Comparison of SNP and CAPS markers application in genetic research in wheat and barley. BMC Plant Biology. 2016; 16 (S1): 11.

17. Tan C., Assanga S., Zhang G., Rudd J.C., Haley S.D., Xue Q., Ibrahim A., Bai G., Zhang X., Byrne P., Fuentealba M.P., Liu S. Development and Validation of KASP Markers for Wheat Streak Mosaic Virus Resistance Gene Wsm2. Crop Science. 2017; 57 (1): 340–349.

18. Tariq H. A novel and high throughput wheat (Triticum aestivum L.) genotyping using Kompetitive Allele Specific PCR assay for genes underpinning major economic attributes. Pakistan Journal of Agricultural Sciences. 2021; 58 (06): 1799–1807.

19. Terracciano I., Maccaferri M., Bassi F., Mantovani P., Sanguineti M.C., Salvi S., Šimková H., Doležel J., Massi A., Ammar K., Kolmer J., Tuberosa R. Development of COS-SNP and HRM markers for high-throughput and reliable haplotype-based detection of Lr14a in durum wheat (Triticum durum Desf.). Theoretical and Applied Genetics. 2013; 126 (4): 1077–1101.

20. Walkowiak S., Gao L., Monat C. et al. Multiple wheat genomes reveal global variation in modern breeding. Nature. 2020; 588 (7837): 277–283.

21. Wei B., Jing R., Wang C., Chen J., Mao X., Chang X., Jia J. Dreb1 genes in wheat (Triticum aestivum L.): development of functional markers and gene mapping based on SNPs. Molecular Breeding. 2009; 23 (1): 13–22.

22. Winfield M.O., Allen A.M., Burridge A.J. et al. High-density SNP genotyping array for hexaploid wheat and its secondary and tertiary gene pool. Plant Biotechnology Journal. 2016; 14 (5): 1195–1206.

23. Yang Y., Zhao X.L., Xia L.Q., Chen X.M., Xia X.C., Yu Z., He Z.H., Röder M. Development and validation of a Viviparous-1 STS marker for pre-harvest sprouting tolerance in Chinese wheats. Theoretical and Applied Genetics. 2007; 115 (7): 971–980.