Development of novel microsatellite DNA markers for first insight into the population genetic structure of bean leaf beetle ootheca mutabilis (Coleoptera: Chrysomelidae) in Uganda

Abstract

Ootheca mutabilis leaf beetles have emerged economically important pests on common bean due to their voracious feeding. The adults defoliate leaves and larvae feed on roots leading to high yield losses and complete crop loss during high infestations. Despite the economic losses caused, population genetics of these beetles have remained unexplored. This study explored the population genetic structure of O. mutabilis beetles using microsatellite DNA markers. To design unique microsatellite markers for use, next generation sequences of O. mutabilis genomic DNA were assembled de novo and the microsatellites were identified, designed, and commercially synthesized. A total of 19,356 microsatellite markers were identified and respective primers designed out of which 81 primers of repeat motif; di, tri, and tetra-nucleotides were selected for synthesis. The primers were optimized and finally, five polymorphic loci were found most appropriate for use in this study. These primers were used to analyze samples from five O. mutabilis populations. Overall, the average expected heterozygosity was higher than the average observed heterozygosity indicating higher heterozygosity than it is seen in O. mutabilis samples. The highest genetic variation of 61 % was found within the individuals. The second highest genetic variation of 37 % was found among the individuals and the least genetic variation of 2% was found among the populations. The Fst 0.024, p = 0.001 among all the populations indicated low but significant genetic differentiation among beetles showing that there is allele sharing. Isolation by distance (r = -0.071, p 0.046) showed an insignificant correlation between the geographical and genetic distances. Also, cluster analysis did not separate samples according to their populations. Interestingly, the samples which were exhibiting different colours clustered together in the cluster analysis. In conclusion, this study has shed light on the genetic structure status of O. mutabilis. There will be a need to increase the sampling area to include other locations in Africa and as well to increase the number of markers.