Single nucleotide polymorphism analysis using KASP assay reveals genetic variability in Rotylenchulus reniformis

Authors: KHANAL, CHURAMANI - Louisiana State University; KULARATHNA, MANJULA - Lincoln University - New Zealand; Ray, Jeffery - Jeff; Stetina, Salliana - Sally; MCGAWLEY, EDWARD - Louisiana State University; OVERSTREET, CHARLES - Louisiana State University

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/4/2018
Publication Date: 6/13/2019
Citation: Khanal, C., Kularathna, M.T., Ray, J.D., Stetina, S.R., McGawley, E.C., Overstreet, C. 2019. Single nucleotide polymorphism analysis using KASP assay reveals genetic variability in Rotylenchulus reniformis. Plant Disease. 103:1835-1842. https://doi.org/10.1094/PDIS-11-18-1975-RE.
DOI: https://doi.org/10.1094/PDIS-11-18-1975-RE

Interpretive Summary: The reniform nematode is a microscopic worm that lives in the southern United States and feeds on roots of two economically important crops, cotton and soybean. Scientists are working to develop cotton and soybean varieties with resistance to this pest, but progress is slow because we do not yet have a clear understanding of how much variation there is in nematode populations, and how that variability affects the utility and longevity of resistant varieties. To gain a better understanding of genetic diversity in reniform nematode populations, we extracted DNA from 26 different reniform nematode isolates representing Louisiana, Mississippi, Arkansas, South Carolina, Georgia, Hawaii, and Alabama. We used a technique called single nucleotide polymorphisms (SNPs) that looks for one change in a very long sequence of nucleotides that make up the genetic code; an example would be a very long sentence that is identical except in one version of the sentence “is” replaces “it” – one letter has been substituted. We identified 162 likely places where these single changes in the genetic code exist among reniform nematode isolates. We used the surrounding nucleotide sequence information to develop 31 SNP assays and tested them to determine if they could distinguish within isolates from the same state as well as between isolates from different states. We successfully documented both within-state and between-state variability in the first study of its kind to report the use of SNP assays to test for genetic variability in reniform nematode. Scientists may be able to build on the information from this study to find SNPs associated with traits of interest in the nematode, such as those associated with the ability of nematode to reduce plant yield, that would be extremely useful in resistance breeding programs.

Technical Abstract: This study employed single nucleotide polymorphisms (SNPs) to determine genetic variability present in 26 isolates of Rotylenchulus reniformis from Louisiana, Mississippi, Arkansas, South Carolina, Georgia, Hawaii, and Alabama. Genomic DNA from reniform nematode was extracted and increased quantitatively using the process of whole genome amplification. Over 162 putative SNPs were identified of which 31 were tested using a competitive allele-specific PCR (KASP) genotyping assay. Of the SNPs tested, 13, 17, and 19 SNPs revealed genetic variability within reniform nematode isolates from Louisiana, Mississippi, and Arkansas, respectively. Seven SNPs elucidated genetic differences among isolates of reniform nematode from Louisiana, Mississippi, and Arkansas. Eight SNPs determined genetic variability among individual isolates from South Carolina, Georgia, Hawaii, and Alabama. This study is the first to report genetic variability in geographic isolates of reniform nematode employing a SNP assay. This study also demonstrated that SNP markers can be used to evaluate R. reniformis isolates and could be useful to assess their genetic diversity, origin, and distribution. Such information would be extremely useful in resistance breeding programs.