Multi-donor × elite-based populations reveal QTL for low-lodging wheat.

KEY MESSAGE: Low-lodging high-yielding wheat germplasm and SNP-tagged novel alleles for lodging were identified in a process that involved selecting donors through functional phenotyping for underlying traits with a designed phenotypic screen, and a crossing strategy involving multiple-donor × elite populations. Lodging is a barrier to achieving high yield in wheat. As part of a study investigating the potential to breed low-lodging high-yielding wheat, populations were developed crossing four low-lodging high-yielding donors selected based on lodging related traits, with three cultivars. Lodging was evaluated in single rows in an early generation and subsequently in plots in 2 years with contrasting lodging environment. A large number of lines lodged less than their recurrent parents, and some were also higher yielding. Heritability for lodging was high, but the genetic correlation between contrasting environments was intermediate-low. Lodging genotypic rankings in single rows did not correlate well with plots. Populations from the highest lodging background were genotyped (90 K iSelect BeadChip array). Fourteen markers on nine chromosomes were associated with lodging, differing under high- versus low-lodging conditions. Of the fourteen markers, ten were found to co-locate with previously identified QTL for lodging-related traits or at homoeologous locations for previously identified lodging-related QTL, while the remaining four markers (in chromosomes 2D, 4D, 7B and 7D) appear to map to novel QTL for lodging. Lines with more favourable markers lodged less, suggesting value in these markers as a selection tool. This study demonstrates that the combination of donor functional phenotyping, screen design and crossing strategy can help identify novel alleles in germplasm without requiring extensive bi-parental populations.

Organophosphate and pyrethroid hydrolase activities of mutant Esterases from the cotton bollworm Helicoverpa armigera.

Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes' active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4-6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively.

Heterologous expression and biochemical characterisation of fourteen esterases from Helicoverpa armigera.

Esterases have recurrently been implicated in insecticide resistance in Helicoverpa armigera but little is known about the underlying molecular mechanisms. We used a baculovirus system to express 14 of 30 full-length esterase genes so far identified from midgut cDNA libraries of this species. All 14 produced esterase isozymes after native PAGE and the isozymes for seven of them migrated to two regions of the gel previously associated with both organophosphate and pyrethroid resistance in various strains. Thirteen of the enzymes obtained in sufficient yield for further analysis all showed tight binding to organophosphates and low but measurable organophosphate hydrolase activity. However there was no clear difference in activity between the isozymes from regions associated with resistance and those from elsewhere in the zymogram, or between eight of the isozymes from a phylogenetic clade previously associated with resistance in proteomic and quantitative rtPCR experiments and five others not so associated. By contrast, the enzymes differed markedly in their activities against nine pyrethroid isomers and the enzymes with highest activity for the most insecticidal isomers were from regions of the gel and, in some cases, the phylogeny that had previously been associated with pyrethroid resistance. Phospholipase treatment confirmed predictions from sequence analysis that three of the isozymes were GPI anchored. This unusual feature among carboxylesterases has previously been suggested to underpin an association that some authors have noted between esterases and resistance to the Cry1Ac toxin from Bacillus thuringiensis. However these three isozymes did not migrate to the zymogram region previously associated with Cry1Ac resistance.

How many genetic options for evolving insecticide resistance in heliothine and spodopteran pests?

The widely accepted paradigm for the development of insecticide resistance in field populations of insects is of selection for one or a very few genes of major effect. Limited genetic mapping data for organophosphate and pyrethroid resistance in heliothine and spodopteran pests generally agrees with this paradigm. However, other biochemical and transcriptomic data suggest a more complex set of changes in multiple P450 and esterase gene/enzyme systems in resistant strains of these species. We discuss possible explanations for this paradox, including the likely embedding of these genes in regulatory cascades and emerging evidence for their arrangement in large clusters of closely related genes. We conclude that there could indeed be an unusually large number of genetic options for evolving resistance in these species.