Douglas V. Sumerford

Comparative Performance of Single Nucleotide Polymorphism and Microsatellite Markers for Population Genetic Analysis

Microsatellite loci are standard genetic markers for population genetic analysis, whereas single nucleotide polymorphisms (SNPs) are more recent tools that require assessment of neutrality and appropriate use in population genetics. Twelve SNP markers were used to describe the genetic structure of Diabrotica virgifera virgifera (LeConte; Coleoptera: Chrysomelidae) in the United States of America and revealed a high mean observed heterozygosity (0.40 +/- 0.059) and low global F(ST) (0.029). Pairwise F(ST) estimates ranged from 0.007 to 0.045, and all but 2 populations showed significant levels of genetic differentiation (P < or = 0.008). Population parameters and conclusions based on SNP markers were analogous to that obtained by use of microsatellite markers from the identical population samples. SNP-based F(ST) estimates were 3-fold higher than corresponding estimates from microsatellites, wherein lower microsatellite F(ST) estimates likely resulted from an overestimate of migration rates between subpopulations due to convergence of allele size (homoplasy). No significant difference was observed in the proportion of SNP or microsatellite markers loci that were nonneutral within populations. SNP markers provided estimates of population genetic parameters consistent with those from microsatellite data, and their low back mutation rates may result in reduced propensity for error in estimation of population parameters.

Effects of Cry1Ab-Expressing Corn Anthers on Monarch Butterfly Larvae

Abstract Previous studies suggest that exposure to corn, Zea mays L., anthers expressing Bacillus thuringiensis (Bt)-derived protein may have adverse effects on the larvae of monarch butterfly, Danaus plexippus (L.). To examine the potential effects of Bt anthers on monarch butterflies, studies were designed to test toxicity in the laboratory; examine anther distribution in space and time; compare distributions of anthers, pollen, and larval feeding; and measure effects of long-term exposure in the field. In the laboratory, monarch butterfly larvae fed on whole corn anthers, but anther feeding was sporadic. Larvae exposed to 0.3 anther/cm2 fed and weighed less after 4 d compared with larvae exposed to non-Bt anthers. Adverse effects increased with increasing anther density. Monarch butterfly larvae exposed to 0.9 anther/cm2 had reduced feeding, weight, and survival and increased developmental time compared with larvae exposed to non-Bt anthers. Later instars were more tolerant of Bt toxin. For all studies, laboratory testing probably magnified effects because larvae were confined to petri dishes. Field studies showed toxic anther densities are uncommon on milkweed (Asclepias) leaves in and near cornfields during anthesis. Mean anther densities on milkweed leaves in cornfields during peak anthesis were between 0.06 and 0.1 anther/cm2 (≈ 3–5 anthers per leaf). When exposure to a density of five anthers per leaf was tested in field-cage studies, no effects on growth, development, or survival were detected. Based on probability of exposure to toxic densities, Bt anthers alone are not likely to pose a significant risk to monarch butterflies in Iowa.

Mining an Ostrinia nubilalis midgut expressed sequence tag (EST) library for candidate genes and single nucleotide polymorphisms (SNPs)

Genes expressed in lepidopteran midgut tissues are involved in digestion and Bacillus thuringiensis (Bt) toxin resistance traits. Five hundred and thirty five unique transcripts were annotated from 1745 high quality O. nubilalis larval midgut expressed sequence tags (ESTs). Full‐length cDNA sequence of 12 putative serine proteinase genes and 3 partial O. nubilalis aminopeptidase N protein genes, apn1, apn3, and apn4, were obtained, and genes may have roles in plant feeding and Bt toxin resistance traits of Ostrinia larvae. The EST library was not normalized and insert frequencies reflect transcript levels under the initial treatment conditions and redundancy of inserts from highly expressed transcripts allowed prediction of putative single nucleotide polymorphisms (SNPs). Ten di‐, tri‐ or tetranucleotide repeat unit microsatellite loci were identified, and minisatellite repeats were observed within the C‐termini of two encoded serine proteinases. Molecular markers showed polymorphism at 28 SNP loci and one microsatellite locus, and Mendelian inheritance indicated that markers were applicable to genome mapping applications. This O. nubilalis larval midgut EST collection is a resource for gene discovery, expression information, and allelic variation for use in genetic marker development.

Genetic Diversity in Laboratory Colonies of Western Corn Rootworm (Coleoptera: Chrysomelidae), Including a Nondiapause Colony

Abstract Laboratory-reared western corn rootworms, Diabrotica virgifera virgifera, from colonies maintained at the North Central Agricultural Research Laboratory (NCARL) in Brookings, SD, are used extensively by many researchers in studies of the biology, ecology, behavior, and genetics of this major insect pest. A nondiapause colony developed through artificial selection in the early 1970s is particularly attractive for many studies because its generation time is much shorter than that of typical diapause colonies. However, the nondiapause colony has been in culture for ≈190 generations without out-crossing. We compared variation at six microsatellite loci among individuals from the NCARL nondiapause colony (≈190 generations), main diapause colony (≈22 generations), four regional diapause colonies (3–8 generations), and four wild populations. Genetic diversity was very similar among the diapause laboratory colonies and wild populations. However, the nondiapause colony showed ≈15–39% loss of diversity depending on the measure. Pairwise estimates of FST were very low, revealing little genetic differentiation among laboratory colonies and natural populations. The nondiapause colony showed the greatest genetic differentiation with an average pairwise FST of 0.153. There was little evidence that the laboratory colonies had undergone genetic bottlenecks except for the nondiapause colony. The nondiapause colony has suffered a moderate loss in genetic diversity and is somewhat differentiated from wild populations. This was not unexpected given its history of artificial selection for the nondiapause trait, and the large number of generations in culture. In contrast, the results indicate that the diapause colonies maintained at NCARL are genetically similar to wild populations.

A Core Set of Microsatellite Markers for Western Corn Rootworm (Coleoptera: Chrysomelidae) Population Genetics Studies

Abstract Interest in the ecological and population genetics of the western corn rootworm, Diabrotica virgifera virgifera LeConte, has grown rapidly in the last few years in North America and Europe. This interest is a result of a number of converging issues related to the increasing difficulty in managing this pest and the need to characterize and understand gene flow in the context of insect resistance management. One of the key components needed for successful population genetics studies is the availability of suitable molecular markers. Using a standard group of microsatellite markers enables researchers from different laboratories to directly compare and share their data, reducing duplication of effort and facilitating collaborative work among laboratories. We screened 22 candidate microsatellite loci against five criteria to create a core set of microsatellite markers for D. v. virgifera population genetics studies. The criteria for inclusion were moderate to high polymorphism, unambiguous readability and repeatability, no evidence of null alleles, apparent selective neutrality, and no linkage between loci. Based on our results, we recommend six microsatellite markers to be included as a core set in future population genetics studies of D. v. virgifera along with any other microsatellite or genetic markers. As more microsatellites are developed, those meeting the criteria can be added to the core set. We encourage other groups of researchers with common interests in a particular insect species to develop their own core sets of markers for population genetics applications.

Estimating the frequency of Cry1F resistance in field populations of the European corn borer (Lepidoptera: Crambidae)

BACKGROUND Transgenic corn hybrids that express toxins from Bacillus thuringiensis (Bt) have suppressed European corn borer populations and reduced the pest status of this insect throughout much of the US corn belt. A major assumption of the high-dose/refuge strategy proposed for insect resistance management and Bt corn is that the frequency of resistance alleles is low so that resistant pests surviving exposure to Bt corn will be rare. RESULTS The frequency of resistance to the Cry1F Bt toxin was estimated using two different screening tools and compared with annual susceptibility monitoring based on diagnostic bioassays and LC50 and EC50 determinations. An F1 screening approach where field-collected individuals were mated to a resistant laboratory strain and progeny were assayed to determine genotype revealed that resistance alleles could be recovered even during the first year of commercially available Cry1F corn (2003). Estimates of frequency from 2003-2005 and 2006-2008 indicated that, although allele frequency was higher than theoretical assumptions (0.0286 and 0.0253 respectively), there was no indication that the frequency was increasing. Similar estimates in 2008 and 2009 using an F2 screening approach confirmed the presence of non-rare resistance alleles (frequency ≈ 0.0093 and 0.0142 for 2008 and 2009, respectively). The results of both screening methods were in general agreement with the observed mortality in diagnostic bioassays and LC50 and EC50 determinations. CONCLUSIONS These results are consistent with previous modeling results, suggesting that the high-dose/refuge strategy that is in place for Bt corn may be effective in delaying resistance evolution even when a relatively high frequency of resistance alleles exists.

A novel class of miniature inverted repeat transposable elements (MITEs) that contain hitchhiking (GTCY)(n) microsatellites.

The movement of miniature inverted repeat transposable elements (MITEs) modifies genome structure and function. We describe the microsatellite‐associated interspersed nuclear element 2 (MINE‐2), that integrates at consensus WTTTT target sites, creates dinucleotide TT target site duplications (TSDs), and forms predicted MITE‐like secondary structures; a 5′ subterminal inverted repeat (SIR; AGGGTTCCGTAG) that is partially complementary to a 5′ inverted repeat (IR; ACGAAGCCCT) and 3′‐SIRs (TTACGGAACCCT). A (GTCY)n microsatellite is hitchhiking downstream of conserved 5′MINE‐2 secondary structures, causing flanking sequence similarity amongst mobile microsatellite loci. Transfection of insect cell lines indicates that MITE‐like secondary structures are sufficient to mediate genome integration, and provides insight into the transposition mechanism used by MINE‐2s.

Bacillus thuringiensis Cry1Ac resistance frequency in tobacco budworm (Lepidoptera: Noctuidae).

ABSTRACT The tobacco budworm, Heliothis virescens (F.) (Lepidoptera Noctuidae), is one of the most important pests of cotton, Gossypium hirsutum L., that has become resistant to a wide range of synthetic insecticides. Cry1Ac-expressing cotton has proven its effectiveness against this insect since its introduction in North America in 1996. However, the constant exposure of tobacco budworm to this protein toxin may result in the development of resistance to it. To estimate the frequency of alleles that confer resistance to a 1.0 µg of Bacillus thuringiensis Cry1Ac diagnostic concentration in field-collected insects, the second generation (F2) of 1,001 single-pair families from seven geographical regions representing 2,202 alleles from natural populations was screened in 2006 and 2007 without finding major resistant alleles. Neonates of 56 single-pair families were able to develop to second instar on the diagnostic concentration in the initial screen, but only seven of these lines did so again in a second confirmatory screen. Minor resistance alleles to Cry1Ac may be quite common in natural populations of H. virescens. Our estimated resistance allele frequencies (0.0036 – 0.0263) were not significantly different from a previously published estimate from 1993. There is no evidence that H. virescens populations have become more resistant to Cry1Ac.

Growth, Development, and Survival of Nosema pyrausta-Infected European Corn Borers (Lepidoptera: Crambidae) Reared on Meridic Diet and Cry1Ab

Abstract Transgenic corn, Zea mays L., hybrids expressing crystal protein endotoxin genes from Bacillus thuringiensis Berliner are an increasingly popular tactic for managing the European corn borer, Ostrinia nubilalis (Hübner), in North America. O. nubilalis populations also are often vulnerable to the ubiquitous entomopathogenic microsporidium Nosema pyrausta (Paillot). We examined the effect of feeding meridic diet incorporated with purified Cry1Ab on growth, development, and survival of Nosema-infected and uninfected neonate O. nubilalis. Infected larvae developed more slowly than uninfected larvae. Increasing the concentration of Cry1Ab in diet reduced larval development, and this effect was amplified by microsporidiosis. Infected larvae weighed significantly less than uninfected larvae. The relationship among Nosema infection, Cry1Ab concentration, and larval weight was fitted to an exponential function. The LC50 of infected larvae was one-third that of uninfected larvae, indicating that infected larvae are more vulnerable to toxin. This work has implications for resistance management of O. nubilalis and demonstrates that it is important to determine whether N. pyrausta is present when testing susceptibility of larvae to transgenic corn hybrids.

A single major QTL controls expression of larval Cry1F resistance trait in Ostrinia nubilalis (Lepidoptera: Crambidae) and is independent of midgut receptor genes

The European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae), is an introduced crop pest in North America that causes major damage to corn and reduces yield of food, feed, and biofuel materials. The Cry1F toxin from Bacillus thuringiensis (Bt) expressed in transgenic hybrid corn is highly toxic to O. nubilalis larvae and effective in minimizing feeding damage. A laboratory colony of O. nubilalis was selected for high levels of Cry1F resistance (>12,000-fold compared to susceptible larvae) and is capable of survival on transgenic hybrid corn. Genetic linkage maps with segregating AFLP markers show that the Cry1F resistance trait is controlled by a single quantitative trait locus (QTL) on linkage group 12. The map position of single nucleotide polymorphism (SNP) markers indicated that midgut Bt toxin-receptor genes, alkaline phosphatase, aminopeptidase N, and cadherin, are not linked with the Cry1F QTL. Evidence suggests that genes within this genome interval may give rise to a novel Bt toxin resistance trait for Lepidoptera that appears independent of known receptor-based mechanisms of resistance.