This study combines neutrality tests and environmental correlations to identify nonneutral patterns of evolution in candidate genes related to drought stress in two closely related Mediterranean conifers, Pinus pinaster Ait. and P. halepensis Mill. Based on previous studies, we selected twelve amplicons covering six candidate genes that were sequenced in a large sample spanning the full range of these two species. Neutrality tests relatively robust to demography (DHEW compound test and maximum likelihood multilocus Hudson-Kreitman-Aguadé test) were used to detect selection events at different temporal scales. Environmental associations between variation at candidate genes and climatic variables were also examined. These combined approaches detected distinct genes that may be targeted by selection, most of them specific to only one of the two conifers, despite their recent divergence (<10 Ma). An exception was 4-coumarate: CoA ligase, a gene involved in the production of various important secondary products that appeared to play a role in local adaptation processes of both pines. Another remarkable result was that all significant environmental correlations involved temperature indices, highlighting the importance of this climatic factor as a selective driver on Mediterranean pines. The ability to detect natural selection at the DNA sequence level depends on the nature and the strength of the selection events, on the timescale at which they occurred, and on the sensitivity of the methods to other evolutionary forces that can mimic selection (e.g., demography and population structure). Using complementary approaches can help to capture different aspects of the evolutionary processes that govern molecular variation at both intra-and interspecific levels.

Innes Centre, Colney, Norwich NR4 7UH, United Kingdom (E.B., C.M.)Virus-induced gene silencing (VIGS) is a powerful tool for reverse genetics in tomato (Solanum lycopersicum). However, the irregular distribution of the effects of VIGS hampers the identification and quantification of nonvisual phenotypes. To overcome this limitation, a visually traceable VIGS system was developed for fruit, comprising two elements: (1) a transgenic tomato line (Del/Ros1) expressing Antirrhinum majus Delila and Rosea1 transcription factors under the control of the fruitspecific E8 promoter, showing a purple-fruited, anthocyanin-rich phenotype; and (2) a modified tobacco rattle virus VIGS vector incorporating partial Rosea1 and Delila sequences, which was shown to restore the red-fruited phenotype upon agroinjection in Del/Ros1 plants. Dissection of silenced areas for subsequent chemometric analysis successfully identified the relevant metabolites underlying gene function for three tomato genes, phytoene desaturase, TomloxC, and SlODO1, used for proof of concept. The C-6 aldehydes derived from lipid 13-hydroperoxidation were found to be the volatile compounds most severely affected by TomloxC silencing, whereas geranial and 6-methyl-5-hepten-2-one were identified as the volatiles most severely reduced by phytoene desaturase silencing in ripening fruit. In a third example, silencing of SlODO1, a tomato homolog of the ODORANT1 gene encoding a myb transcription factor, which regulates benzenoid metabolism in petunia (Petunia hybrida) flowers, resulted in a sharp accumulation of benzaldehyde in tomato fruit. Together, these results indicate that fruit VIGS, enhanced by anthocyanin monitoring, can be a powerful tool for reverse genetics in the study of the metabolic networks operating during fruit ripening.