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Research Project: Enhanced Resistance of Maize to Aspergillus flavus Infection, Aflatoxin Accumulation, and Insect Damage

Location: Corn Host Plant Resistance Research

Title: Development and validation of diagnostic Kompetitive allele-specific PCR (KASP) markers for quality control (QC) genotyping in a collection of four rice species

Author
item GOUDA, ARNAUD - Africa Rice Center (AFRICARICE)
item Warburton, Marilyn
item DJEDATIN, GUSTAVE - The National University Of Science, Technology, Engineering And Mathematics (UNSTIM) Of Abomey
item KPEKI, SÈDJRO - Africa Rice Center (AFRICARICE)
item WAMBUGU, PETERSON - Kenya Agricultural And Livestock Research Organization
item GNIKOUA, KARLIN - Africa Rice Center (AFRICARICE)
item NDJIONDJOP, MARIE - Africa Rice Center (AFRICARICE)

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/30/2021
Publication Date: 9/20/2021
Citation: Gouda, A.C., Warburton, M.L., Djedatin, G.L., Kpeki, S.B., Wambugu, P.W., Gnikoua, K., Ndjiondjop, M.N. 2021. Development and validation of diagnostic Kompetitive allele-specific PCR (KASP) markers for quality control (QC) genotyping in a collection of four rice species. Scientific Reports. 11:18617. https://doi.org/10.1038/s41598-021-97689-3.
DOI: https://doi.org/10.1038/s41598-021-97689-3

Interpretive Summary: There are four economically important rice species conserved in the genebank of the Africa Rice Center. Some are superficially very similar, and some are hybrids of each other, so proper classification can be difficult. However, it is important to properly classify all entries in the gene bank, to ensure the collection is as complete and diverse as possible, and to help breeders choose the correct gene bank entries when they wish to seek new and useful traits from the many entries. A previous study found many DNA markers that could be used to distinguish different species of rice and even more closely related sub-species, but the way these DNA markers must be run in the lab are time consuming and costly. Thus, this article shows how some of those markers were converted to Kompetitive allele-specific PCR (KASP) DNA markers, which are much simpler, faster, and cheaper to run. This will help in genebank curation and rice breeding.

Technical Abstract: Morphological identification of closely related rice species particularly those in the Oryza AA genome group presents major challenges and has usually resulted in cases of misidentification. Recent work by this group identified diagnostic single nucleotide polymorphic (SNPs) markers specific for several rice species and subspecies based on DArTseq-based next-generation sequencing technology (“DArTseq”). These SNPs can be used for quality control (QC) analysis in rice breeding and germplasm maintenance programs. Here, we present the DArTseq-based diagnostic SNPs converted into Kompetitive allele-specific PCR (KASPar or KASP) assays and validated a subset of them for low-cost routine genotyping quality control (QC) analysis. Out of the 224 species/subspecies’ diagnostic SNPs tested, 158 of them produced working KASP assays, a conversion success rate of 71%. Two validation experiments were run with 87 of the 158 SNP markers to ensure the assays amplified, were polymorphic, and distinguished the five species/subspecies tested. Based on these validation test results, we recommend a panel of 36 SNP markers that clearly delineate O. barthii, O. glaberrima, O. longistaminata, O. sativa spp. indica and japonica. The KASP assays provide a flexible, rapid turnaround and cost-effective tool that facilitates germplasm curation and management of these four Oryza AA genome species across multiple genebanks.