Population Genomics of Parallel Adaptation in Threespine Stickleback using Sequenced RAD Tags

Next-generation sequencing technology provides novel opportunities for gathering genome-scale sequence data in natural populations, laying the empirical foundation for the evolving field of population genomics. Here we conducted a genome scan of nucleotide diversity and differentiation in natural populations of threespine stickleback (Gasterosteus aculeatus). We used Illumina-sequenced RAD tags to identify and type over 45,000 single nucleotide polymorphisms (SNPs) in each of 100 individuals from two oceanic and three freshwater populations. Overall estimates of genetic diversity and differentiation among populations confirm the biogeographic hypothesis that large panmictic oceanic populations have repeatedly given rise to phenotypically divergent freshwater populations. Genomic regions exhibiting signatures of both balancing and divergent selection were remarkably consistent across multiple, independently derived populations, indicating that replicate parallel phenotypic evolution in stickleback may be occurring through extensive, parallel genetic evolution at a genome-wide scale. Some of these genomic regions co-localize with previously identified QTL for stickleback phenotypic variation identified using laboratory mapping crosses. In addition, we have identified several novel regions showing parallel differentiation across independent populations. Annotation of these regions revealed numerous genes that are candidates for stickleback phenotypic evolution and will form the basis of future genetic analyses in this and other organisms. This study represents the first high-density SNP–based genome scan of genetic diversity and differentiation for populations of threespine stickleback in the wild. These data illustrate the complementary nature of laboratory crosses and population genomic scans by confirming the adaptive significance of previously identified genomic regions, elucidating the particular evolutionary and demographic history of such regions in natural populations, and identifying new genomic regions and candidate genes of evolutionary significance

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

High bicarbonate assimilation in the dark by Arctic bacteria

10 páginas, 4 figuras, 1 tabla.Although both autotrophic and heterotrophic microorganisms incorporate CO2 in the dark through different metabolic pathways, this process has usually been disregarded in oxic marine environments. We studied the significance and mediators of dark bicarbonate assimilation in dilution cultures inoculated with winter Arctic seawater. At stationary phase, bicarbonate incorporation rates were high (0.5–2.5 μg C L−1 d−1) and correlated with rates of bacterial heterotrophic production, suggesting that most of the incorporation was due to heterotrophs. Accordingly, very few typically chemoautotrophic bacteria were detected by 16S rRNA gene cloning. The genetic analysis of the biotin carboxylase gene accC putatively involved in archaeal CO2 fixation did not yield any archaeal sequence, but amplified a variety of bacterial carboxylases involved in fatty acids biosynthesis, anaplerotic pathways and leucine catabolism. Gammaproteobacteria dominated the seawater cultures (40–70% of cell counts), followed by Betaproteobacteria and Flavobacteria as shown by catalyzed reporter deposition fluorescence in situ hybridization (CARDFISH). Both Beta- and Gammaproteobacteria were active in leucine and bicarbonate uptake, while Flavobacteria did not take up bicarbonate, as measured by microautoradiography combined with CARDFISH. Within Gammaproteobacteria, Pseudoalteromonas-Colwellia and Oleispira were very active in bicarbonate uptake (ca. 30 and 70% of active cells, respectively), while the group Arctic96B-16 did not take up bicarbonate. Our results suggest that, potentially, the incorporation of CO2 can be relevant for the metabolism of specific Arctic heterotrophic phylotypes, promoting the maintenance of their cell activity and/or longer survival under resource depleted conditions.This work is a contribution to the International Polar Year – Circumpolar Flaw Lead system study (IPY-CFL 2007/2008) lead by D. Barber (University of Manitoba) supported through grants from the Canadian IPY Federal Program Office, the National Sciences and Engineering Research Council, grant BOREAL (CLG2007-28872-E/ANT) from the Spanish Ministry of Science and Innovation to C.P.-A., and grants from the Swedish Research Council to S.B and L.A.S. L.A.S. was supported by a Marie Curie Intraeuropean Fellowship (CHEMOARC PIEF-GA-2008- 221121), E.O.C by the Spanish grant CGL2009-13318- BOS, and P. E. G by a Marie Curie grant (CRENARC MEIF-CT-2007-040247).Peer reviewe

Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis

Plant vacuoles are multi-functional, developmentally varied and can occupy up to 90% of plant cells. The N-terminal propeptide (NTPP) of sweet potato sporamin and the C-terminal propeptide (CTPP) of tobacco chitinase have been developed as models to target some heterologous proteins to vacuoles but so far tested on only a few plant species, vacuole types and payload proteins. Most studies have focused on lytic and protein-storage vacuoles, which may differ substantially from the sugar-storage vacuoles in crops like sugarcane. Our results extend the evidence that NTPP of sporamin can direct heterologous proteins to vacuoles in diverse plant species and indicate that sugarcane sucrose-storage vacuoles (like the lytic vacuoles in other plant species) are hostile to heterologous proteins. A low level of cytosolic NTPP-GFP (green fluorescent protein) was detectable in most cell types in sugarcane and Arabidopsis, but only Arabidopsis mature leaf mesophyll cells accumulated NTPP-GFP to detectable levels in vacuoles. Unexpectedly, efficient developmental mis-trafficking of NTPP-GFP to chloroplasts was found in young leaf mesophyll cells of both species. Vacuolar targeting by tobacco chitinase CTPP was inefficient in sugarcane, leaving substantial cytoplasmic activity of rat lysosomal beta-glucuronidase (GUS) [ER (endoplasmic reticulum)-RGUS-CTPP]. Sporamin NTPP is a promising targeting signal for studies of vacuolar function and for metabolic engineering. Such applications must take account of the efficient developmental mis-targeting by the signal and the instability of most introduced proteins, even in storage vacuoles