BackgroundUnlike Drosophila but similar to other arthropod and vertebrate embryos, the flour beetle Tribolium castaneum develops everted limb buds during embryogenesis. However, the molecular processes directing the evagination of epithelia are only poorly understood.ResultsHere we show that the newly discovered genes Tc-flipflop1 and Tc-flipflop2 are involved in regulating the directional budding of appendages. RNAi-knockdown of Tc-flipflop results in a variety of phenotypic traits. Most prominently, embryonic limb buds frequently grow inwards rather than out, leading to the development of inverted appendages inside the larval body. Moreover, affected embryos display dorsal closure defects. The Tc-flipflop genes are evolutionarily non-conserved, and their molecular function is not evident. We further found that Tc-RhoGEF2, a highly-conserved gene known to be involved in actomyosin-dependent cell movement and cell shape changes, shows a Tc-flipflop-like RNAi-phenotype.ConclusionsThe similarity of the inverted appendage phenotype in both the flipflop- and the RhoGEF2 RNAi gene knockdown led us to conclude that the Tc-flipflop orphan genes act in a Rho-dependent pathway that is essential for the early morphogenesis of polarised epithelial movements. Our work describes one of the few examples of an orphan gene playing a crucial role in an important developmental process.Electronic supplementary materialThe online version of this article (10.1186/s12983-017-0234-9) contains supplementary material, which is available to authorized users.

Drought represents a major abiotic stress factor negatively affecting growth, yield and tuber quality of potatoes. Quantitative trait locus (QTL) analyses were performed in cultivated potatoes for drought tolerance index DRYM (deviation of relative starch yield from the experimental median), tuber starch content, tuber starch yield, tuber fresh weight, selected transcripts and metabolites under control and drought stress conditions. Eight genomic regions of major interest for drought tolerance were identified, three representing standalone DRYM QTL. Candidate genes, e.g., from signaling pathways for ethylene, abscisic acid and brassinosteroids, and genes encoding cell wall remodeling enzymes were identified within DRYM QTL. Co-localizations of DRYM QTL and QTL for tuber starch content, tuber starch yield and tuber fresh weight with underlying genes of the carbohydrate metabolism were observed. Overlaps of DRYM QTL with metabolite QTL for ribitol or galactinol may indicate trade-offs between starch and compatible solute biosynthesis. Expression QTL confirmed the drought stress relevance of selected transcripts by overlaps with DRYM QTL. Bulked segregant analyses combined with next-generation sequencing (BSAseq) were used to identify mutations in genes under the DRYM QTL on linkage group 3. Future analyses of identified genes for drought tolerance will give a better insight into drought tolerance in potatoes.

Background: The evolutionarily conserved odd-skipped related genes oddskipped (odd), drumstick (drm), sister of odd and bowel (sob), and brother-ofodd-with-entrails-limited (bwl) act downstream of the Notch pathway in various insect tissues including the appendages and the gut. While the function of some of these genes have been analyzed in the adult Tribolium beetle, the expression during and their requirement for embryonic development is not known. Results: We describe here the embryonic expression patterns of drm, sob, and bwl and analyze the RNAi knockdown phenotypes with emphasize on the appendages and the hindgut. We show that in Tribolium, drm acts independently of other odd-family members in the formation of legs, hindgut, and the dorsal epidermis. Moreover, we establish drm and sob as further markers for segment borders in the appendages that include the gnathobasic mandibles. Conclusions:We conclude that the regulatory interrelationship among the odd genes differs between Tribolium and Drosophila, where odd and drm seem to act redundantly. In Tribolium, the genes drm and sob uncover the relict of a precoxal joint incorporated in the lateral body wall.

RNA interference (RNAi)-mediated knockdown serves as an effective technique for the functional analysis of developmental genes that is well established in many organisms. In the beetle Tribolium castaneum, double-stranded RNA is applied by simple injection and distributes systemically within the tissue. Thus, systematic testing for RNAi specificity and efficiency is easily possible in this organism. Generally, the use of non-overlapping dsRNA fragments yielding qualitatively identical phenotypes is the method of choice to verify target-specific knockdown effects. Here, we show that UTR-specific RNAi results in different effects regarding quality, severity and penetrance when compared to RNAi fragments directed at the coding region. Furthermore, when using 3'UTR-specific dsRNA, we first describe the Distal-less antenna-to-leg transformation phenotype in the Tribolium larva, which has only been observed in the adult beetle and Drosophila so far. In addition, we unexpectedly observed sterility effects caused by 3'UTR-specific knockdown of the Tribolium-Sp8 orthologue that is not seen when dsRNA targeted a sequence within the coding-region or the 5'UTR that itself led to early embryonic lethality. We conclude that targeting UTR sequences by region-specific RNAi can reveal unexpected new aspects of gene function applicable in basic research and crop protection.