Prediction of mitochondrial genome-wide variation through sequencing of mitochondrion-enriched extracts

Authors: FISHER, KELSEY - Iowa State University; BRADBURY, STEVEN - Iowa State University; COATES, BRAD - US Department Of Agriculture (USDA)

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/19/2020
Publication Date: 11/5/2020
Citation: Fisher, K.E., Bradbury, S.P., Coates, B.S. 2020. Prediction of mitochondrial genome-wide variation through sequencing of mitochondrion-enriched extracts. Scientific Reports. 10.Article 19123. https://doi.org/10.1038/s41598-020-76088-0.
DOI: https://doi.org/10.1038/s41598-020-76088-0

Interpretive Summary: Damage to corn inflicted by pest insect feeding results in reduced yields and grain quality worldwide. For the last decade, growers have used transgenic corn hybrids expressing crystalline (Cry) protein toxins to lessen the amount of insect feeding damage. However, larvae of the European corn borer (ECB) have recently developed resistance to the Cry1F toxin expressed by transgenic corn, renewing their threat to corn production in the United States. To better predict how resistance moves within ECB populations, a greater understanding of ECB population structure is needed. A team of scientists (ARS and University) developed a method to enrich cell extracts for the mitochondrion, an organelle inherited separately from the nucleus, that has its own DNA. High throughput methods were used to effectively and efficiently sequence the mitochondrial genome. Sequence differences found between insect from different locations or strains can be used to establish population structures. Comparison of entire mitochondrial genomes predicted a greater number of mutations not described in prior studies that used partial genome sequence data, thus providing comparatively more accurate estimates for population genetic analyses. The methods from this study can be applied to virtually any organism; insect, animal, or plant. Results of this research will be of interest to university and industry stakeholders concerned about the impact of pest insect movement and migration on the sustainability of control tactics and spread of Bt resistance across the ECB population.

Technical Abstract: Variation among maternally-inherited mitochondrial DNA (MtD) haplotypes can be used to estimate inter-population diversity, female dispersal, and phylogenetic relationships. Despite utility, relatively economical and high-throughput methods for assessing haplotype variation based on entire MtD genome sequences are lacking. To partially address this shortfall, methods were developed to enrich cell extracts for mitochondria by differential centrifugation prior to DNA extraction and applied with eight families from Ostrinia nubilalis. Real-time quantitative PCR of DNA extracts from mitochondrion-enriched fractions using mitochondrial cytochrome c oxidase subunit I primers resulted in significantly lower cycle threshold (CT) estimates compared to paired nuclear fractions (p < 0.001). Subsequent normalization with respect to a single copy nuclear gene indicated an 86.1-fold mitochondrial DNA enrichment. Read counts estimated from alignment of mitochondrial fraction-specific short-read data for each family to the reference O. nubilalis mitochondrial genome (accession AF442957.1) were 48.1-fold greater compared to reads from unenriched controls. A total of 79 substitutions, insertions, or deletions were predicted among short read alignments, and corresponding de novo assembled mitochondrial genome sequences for each maternal lineage, where 33 of 54 (61.1%) within protein-coding genes were nonsynonymous and an insertion/duplication of a single Ile codon in the ATP synthase subunit 8 gene from a single family. Compared to the Sanger sequencing-derived reference (AF442957.1), all eight de novo MtD genome assemblies contained the AT-rich control region and corrected the 28 C-terminal residues of the NADH gene to that congruent with phylogenetically related species. Our pipeline provides accessibility of whole mitochondrial genome sequence data for applications within sustainable agricultural and natural resource management frameworks.