Design and In Silico Evaluation of a PCR Marker for Fall Armyworm Resistance
DOI:
https://doi.org/10.65339/ijsair.V2.I2.288Keywords:
Maize (Zea mays L.), Fall Armyworm (Spodoptera Frugiperda), Candidate Gene, Molecular Marker, PCR Amplification, Sequence AlignmentAbstract
Fall armyworm (Spodoptera frugiperda) has become a significant constraint in corn (Zea mays L.) production, necessitating the development of molecular tools to support the identification of potential resistance-associated loci. This study presents the preliminary development and in silico validation of a candidate gene-derived molecular marker targeting a putative chromosome 8 locus associated with previously reported resistance-linked regions. Primer design was based on the NC_050103.1 genomic reference sequence, supported by existing genome-wide association study (GWAS) evidence. The designed primer pair was evaluated through in silico PCR, which produced a predicted amplicon corresponding to the targeted genomic region. Sequence analysis showed 88.24% identity with the reference sequence and homology to a Zea mays clone sequence (EU969166.1), supported by BLAST results indicating 95% similarity, an E-value of 3e-44, and 75% query coverage. Multiple sequence alignment using ClustalW showed varying levels of nucleotide identity among the reference sequence, homologous clone, in silico product, and experimentally derived amplicon. Identity values ranged from 82.58% to 100%, indicating partial conservation of the targeted locus across sequences. Conserved regions were observed, although moderate divergence suggests possible genetic variation or closely related genomic loci within chromosome 8. Overall, the findings provide preliminary evidence that the designed marker may target a partially conserved chromosome 8 genomic region potentially associated with resistance-linked loci. Further validation across diverse corn germplasm is required to assess its specificity, stability, and suitability for marker-assisted selection in fall armyworm resistance studies.
References
Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403–410. https://doi.org/10.1016/S0022-2836(05)80360-2.
Day, R., Abrahams, P., Bateman, M., et al. (2017). Fall armyworm: Impacts and implications for Africa. Outlooks on Pest Management, 28(5), 196–201. https://doi.org/10.1564/v28_oct_02.
Doyle, J. J., & Doyle, J. L. (1990). Isolation of plant DNA from fresh tissue. Focus, 12, 13–15.
ExPASy. (n.d.). Translate tool (Expasy Translate). Swiss Institute of Bioinformatics. https://web.expasy.org/translate/
Goergen, G., Kumar, P. L., Sankung, S. B., Togola, A., & Tamò, M. (2016). First report of outbreaks of fall armyworm in Africa. PLOS ONE, 11(10), e0165632. https://doi.org/10.1371/journal.pone.0165632.
Kelly, L. A., Mezulis, S., Yates, C. M., Wass, M. N., & Sternberg, M. J. E. (2015).
The Phyre2 web portal for protein modeling, prediction and analysis.
Nature Protocols, 10(6), 845–858. https://doi.org/10.1038/nprot.2015.053.
Sievers, F., & Higgins, D. G. (2018). Clustal Omega for accurate multiple sequence alignment. Protein Science, 27(1), 135–145. https://doi.org/10.1002/pro.3290.
Tamura, K., Stecher, G., & Kumar, S. (2021). MEGA11: Molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, 38(7), 3022–3027. https://doi.org/10.1093/molbev/msab120.
Warburton, M. L., Woolfolk, S. W., Smith, J. S., Hawkins, L. K., Castaño-Duque, L., LeBar, M. D., & Williams, W. P. (2022). Genes and genetic mechanisms contributing to fall armyworm resistance in corn. The Plant Genome. https://doi.org/10.1002/tpg2.20311.
Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., & Madden, T. L. (2012). Primer-BLAST: A tool to design target-specific primers for PCR. BMC Bioinformatics, 13, 134. https://doi.org/10.1186/1471-2105-13-134.
