Clonal genomic population structure of Beauveria brongniartii and Beauveria pseudobassiana: Pathogens of the common European cockchafer (Melolontha melolontha L.).

Beauveria brongniartii is a fungal pathogen that infects the beetle Melolontha melolontha, a significant agricultural pest in Europe. While research has primarily focused on the use of B. brongniartii for controlling M. melolontha, the genomic structure of the B. brongniartii population remains unknown. This includes whether its structure is influenced by its interaction with M. melolontha, the timing of beetle-swarming flights, geographical factors, or reproductive mode. To address this, we analysed genome-wide SNPs to infer the population genomics of Beauveria spp., which were isolated from infected M. melolontha adults in an Alpine region. Surprisingly, only one-third of the isolates were identified as B. brongniartii, while two-thirds were distributed among cryptic taxa within B. pseudobassiana, a fungal species not previously recognized as a pathogen of M. melolontha. Given the prevalence of B. pseudobassiana, we conducted analyses on both species. We found no spatial or temporal genomic patterns within either species and no correlation with the population structure of M. melolontha, suggesting that the dispersal of the fungi is independent of the beetle. Both species exhibited clonal population structures, with B. brongniartii fixed for one mating type and B. pseudobassiana displaying both mating types. This implies that factors other than mating compatibility limit sexual reproduction. We conclude that the population genomic structure of Beauveria spp. is primarily influenced by predominant asexual reproduction and dispersal.

Spatial and temporal patterns in the population genomics of the European cockchafer Melolontha melolontha in the Alpine region.

The European cockchafer Melolontha melolontha is an agricultural pest in many European countries. Populations have a synchronized 3 or 4 years life cycle, leading to temporally isolated populations. Despite the economic importance and availability of comprehensive historical as well as current records on cockchafer occurrence, population genomic analyses of M. melolontha are missing. For example, the effects of geographic separation caused by the mountainous terrain of the Alps and of temporal isolation on the genomic structure of M. melolontha still remain unknown. To address this gap, we genotyped 475 M. melolontha adults collected during 3 years from 35 sites in a central Alpine region. Subsequent population structure analyses discriminated two main genetic clusters, i.e., the South Tyrol cluster including collections located southeast of the Alpine mountain range, and a northwestern alpine cluster with all the other collections, reflecting distinct evolutionary history and geographic barriers. The "passo di Resia" linking South and North Tyrol represented a regional contact zone of the two genetic clusters, highlighting genomic differentiation between the collections from the northern and southern regions. Although the collections from northwestern Italy were assigned to the northwestern alpine genetic cluster, they displayed evidence of admixture with the South Tyrolean genetic cluster, suggesting shared ancestry. A linear mixed model confirmed that both geographic distance and, to a lower extent, also temporal isolation had a significant effect on the genetic distance among M. melolontha populations. These effects may be attributed to limited dispersal capacity and reproductive isolation resulting from synchronized and non-synchronized swarming flights, respectively. This study contributes to the understanding of the phylogeography of an organism that is recognized as an agricultural problem and provides significant information on the population genomics of insect species with prolonged temporally shifted and locally synchronized life cycles.

Genetic variation and microbiota in bumble bees cross-infected by different strains of C. bombi.

The bumblebee Bombus terrestris is commonly infected by a trypanosomatid gut parasite Crithidia bombi. This system shows a striking degree of genetic specificity where host genotypes are susceptible to different genotypes of parasite. To a degree, variation in host gene expression underlies these differences, however, the effects of standing genetic variation has not yet been explored. Here we report on an extensive experiment where workers of twenty colonies of B. terrestris were each infected by one of twenty strains of C. bombi. To elucidate the host's genetic bases of susceptibility to infection (measured as infection intensity), we used a low-coverage (~2 x) genome-wide association study (GWAS), based on angsd, and a standard high-coverage (~15x) GWAS (with a reduced set from a 8 x 8 interaction matrix, selected from the full set of twenty). The results from the low-coverage approach remained ambiguous. The high-coverage approach suggested potentially relevant genetic variation in cell surface and adhesion processes. In particular, mucin, a surface mucoglycoprotein, potentially affecting parasite binding to the host gut epithelia, emerged as a candidate. Sequencing the gut microbial community of the same bees showed that the abundance of bacterial taxa, such as Gilliamella, Snodgrassella, or Lactobacillus, differed between 'susceptible' and 'resistant' microbiota, in line with earlier studies. Our study suggests that the constitutive microbiota and binding processes at the cell surface are candidates to affect infection intensity after the first response (captured by gene expression) has run its course. We also note that a low-coverage approach may not be powerful enough to analyse such complex traits. Furthermore, testing large interactions matrices (as with the full 20 x 20 combinations) for the effect of interaction terms on infection intensity seems to blur the specific host x parasite interaction effects, likely because the outcome of an infection is a highly non-linear process dominated by variation in individually different pathways of host defence (immune) responses.

Transcriptome-wide SNPs for Botrychium lunaria ferns enable fine-grained analysis of ploidy and population structure.

Ferns are the second most diverse group of land plants after angiosperms. Extant species occupy a wide range of habitats and contribute significantly to ecosystem functioning. Despite the importance of ferns, most taxa are poorly covered by genomic resources and within-species studies based on high-resolution markers are entirely lacking. The genus Botrychium belongs to the family Ophioglossaceae, which includes species with very large genomes and chromosome numbers (e.g., Ophioglossum reticulatum 2n = 1520). The genus has a cosmopolitan distribution with 35 species, half of which are polyploids. Here, we establish a transcriptome for Botrychium lunaria (L.) Sw., a diploid species with an extremely large genome of about ~19.0-23.7 Gb. We assembled 25,677 high-quality transcripts with an average length of 1,333 bp based on deep RNA-sequencing of a single individual. We sequenced 11 additional transcriptomes of individuals from two populations in Switzerland, including the population of the reference individual. Based on read mapping to reference transcript sequences, we identified 374,463 single nucleotide polymorphisms (SNPs) segregating among individuals for an average density of 14 SNPs per kilobase. We found that all 12 transcriptomes were most likely from diploid individuals. The transcriptome-wide markers provided unprecedented resolution of the population genetic structure, revealing substantial variation in heterozygosity among individuals. We also constructed a phylogenomic tree of 92 taxa representing all fern orders to ascertain the placement of the genus Botrychium. High-quality transcriptomic resources and SNP sets constitute powerful population genomic resources to investigate the ecology, and evolution of fern populations.

Species ecology explains the spatial components of genetic diversity in tropical reef fishes.

Generating genomic data for 19 tropical reef fish species of the Western Indian Ocean, we investigate how species ecology influences genetic diversity patterns from local to regional scales. We distinguish between the α, ß and γ components of genetic diversity, which we subsequently link to six ecological traits. We find that the α and γ components of genetic diversity are strongly correlated so that species with a high total regional genetic diversity display systematically high local diversity. The α and γ diversity components are negatively associated with species abundance recorded using underwater visual surveys and positively associated with body size. Pelagic larval duration is found to be negatively related to genetic ß diversity supporting its role as a dispersal trait in marine fishes. Deviation from the neutral theory of molecular evolution motivates further effort to understand the processes shaping genetic diversity and ultimately the diversification of the exceptional diversity of tropical reef fishes.

Quantitative trait locus analysis of parasitoid counteradaptation to symbiont-conferred resistance.

Insect hosts and parasitoids are engaged in an intense struggle of antagonistic coevolution. Infection with heritable bacterial endosymbionts can substantially increase the resistance of aphids to parasitoid wasps, which exerts selection on parasitoids to overcome this symbiont-conferred protection (counteradaptation). Experimental evolution in the laboratory has produced counteradapted populations of the parasitoid wasp Lysiphlebus fabarum. These populations can parasitize black bean aphids (Aphis fabae) protected by the bacterial endosymbiont Hamiltonella defensa, which confers high resistance against L. fabarum. We used two experimentally evolved parasitoid populations to study the genetic architecture of the counteradaptation to symbiont-conferred resistance by QTL analysis. With simple crossing experiments, we showed that the counteradaptation is a recessive trait depending on the maternal genotype. Based on these results, we designed a customized crossing scheme to genotype a mapping population phenotyped for the ability to parasitize Hamiltonella-protected aphids. Using 1835 SNP markers obtained by ddRAD sequencing, we constructed a high-density linkage map consisting of six linkage groups (LGs) with an overall length of 828.3 cM and an average marker spacing of 0.45 cM. We identified a single QTL associated with the counteradaptation to Hamiltonella in L. fabarum on linkage group 2. Out of 120 genes located in this QTL, several genes encoding putative venoms may represent candidates for counteradaptation, as parasitoid wasps inject venoms into their hosts during oviposition.

Dioecy Is Associated with High Genetic Diversity and Adaptation Rates in the Plant Genus Silene.

About 15,000 angiosperm species (∼6%) have separate sexes, a phenomenon known as dioecy. Why dioecious taxa are so rare is still an open question. Early work reported lower species richness in dioecious compared with nondioecious sister clades, raising the hypothesis that dioecy may be an evolutionary dead-end. This hypothesis has been recently challenged by macroevolutionary analyses that detected no or even positive effect of dioecy on diversification. However, the possible genetic consequences of dioecy at the population level, which could drive the long-term fate of dioecious lineages, have not been tested so far. Here, we used a population genomics approach in the Silene genus to look for possible effects of dioecy, especially for potential evidence of evolutionary handicaps of dioecy underlying the dead-end hypothesis. We collected individual-based RNA-seq data from several populations in 13 closely related species with different sexual systems: seven dioecious, three hermaphroditic, and three gynodioecious species. We show that dioecy is associated with increased genetic diversity, as well as higher selection efficacy both against deleterious mutations and for beneficial mutations. The results hold after controlling for phylogenetic inertia, differences in species census population sizes and geographic ranges. We conclude that dioecious Silene species neither show signs of increased mutational load nor genetic evidence for extinction risk. We discuss these observations in the light of the possible demographic differences between dioecious and self-compatible hermaphroditic species and how this could be related to alternatives to the dead-end hypothesis to explain the rarity of dioecy.

Isolation and Comparative Genomic Analysis of Reuterin-Producing Lactobacillus reuteri From the Chicken Gastrointestinal Tract.

Lactobacillus reuteri is a natural inhabitant of selected animal and human gastrointestinal tract (GIT). Certain strains have the capacity to transform glycerol to 3-hydroxypropionaldehyde (3-HPA), further excreted to form reuterin, a potent antimicrobial system. Reuterin-producing strains may be applied as a natural antimicrobial in feed to prevent pathogen colonization of animals, such as in chicken, and replace added antimicrobials. To date, only seven L. reuteri strains isolated from chicken have been characterized which limits phylogenetic studies and host-microbes interactions characterization. This study aimed to isolate L. reuteri strains from chicken GIT and to characterize their reuterin production and antimicrobial resistance (AMR) profiles using phenotypic and genetic methods. Seventy strains were isolated from faces, crops and ceca of six chicken from poultry farms and samples from slaughterhouse. Twenty-five strains were selected for further characterization. Draft genomes were generated for the new 25 isolates and integrated into a phylogenetic tree of 40 strains from different hosts. Phylogenetic analysis based on gene content as well as on core genomes showed grouping of the selected 25 L. reuteri chicken isolates within the poultry/human lineage VI. Strains harboring pdu-cob-cbi-hem genes (23/25) produced between 156 mM ± 11 and 330 mM ± 14 3-HPA, from 600 mM of glycerol, in the conditions of the test. All 25 chicken strains were sensitive to cefotaxime (MIC between 0.016 and 1 µg/mL) and penicillin (MIC between 0.02 and 4 µg/mL). Akin to the reference strains DSM20016 and SD2112, the novel isolates were resistant to penicillin, possibly associated with identified point mutations in ponA, pbpX, pbpF and pbpB. All strains resistant to erythromycin (4/27) carried the ermB gene, and it was only present in chicken strains. All strains resistant to tetracycline (5/27) harbored tetW gene. This study confirms the evolutionary history of poultry/human lineage VI and identifies pdu-cob-cbi-hem as a frequent trait but not always present in this lineage. L. reuteri chicken strains producing high 3-HPA yield may have potential to prevent enteropathogen colonization of chicken.

Insights into the genetic architecture of sexual dimorphism from an interspecific cross between two diverging Silene (Caryophyllaceae) species.

The evolution of sexual dimorphism in species with separate sexes is influenced by the resolution of sexual conflicts creating sex differences through genetic linkage or sex-biased expression. Plants with different degrees of sexual dimorphism are thus ideal to study the genetic basis of sexual dimorphism. In this study we explore the genetic architecture of sexual dimorphism between Silene latifolia and Silene dioica. These species have chromosomal sex determination and differ in the extent of sexual dimorphism. To test whether QTL for sexually dimorphic traits have accumulated on the sex chromosomes and to quantify their contribution to species differences, we create a linkage map and performed QTL analysis of life history, flower and vegetative traits using an unidirectional interspecific F2 hybrid cross. We found support for an accumulation of QTL on the sex chromosomes and that sex differences explained a large proportion of the variance between species, suggesting that both natural and sexual selection contributed to species divergence. Sexually dimorphic traits that also differed between species displayed transgressive segregation. We observed a reversal in sexual dimorphism in the F2 population, where males tended to be larger than females, indicating that sexual dimorphism is constrained within populations but not in recombinant hybrids. This study contributes to the understanding of the genetic basis of sexual dimorphism and its evolution in Silene.

Genomic Variation among Strains of Crithidia bombi and C. expoeki.

In this study, we sequenced and analyzed the genomes of 40 strains, in addition to the already-reported two type strains, of two Crithidia species infecting bumblebees in Alaska and Central Europe and demonstrated that different strains of Crithidia bombi and C. expoeki vary considerably in terms of single nucleotide polymorphisms and gene copy number. Based on the genomic structure, phylogenetic analyses, and the pattern of copy number variation, we confirmed the status of C. expoeki as a separate species. The Alaskan populations appear to be clearly separated from those of Central Europe. This pattern fits a scenario of rapid host-parasite coevolution, where the selective advantage of a given parasite strain is only temporary. This study provides helpful insights into possible scenarios of selection and diversification of trypanosomatid parasites.IMPORTANCE A group of trypanosomatid flagellates includes several well-studied medically and economically important parasites of vertebrates and plants. Nevertheless, the vast majority of trypanosomatids infect only insects (mostly flies and true bugs) and, because of that, has attracted little research attention in the past. Of several hundred trypanosomatid species, only four can infect bees (honeybees and bumblebees). Because of such scarcity, these parasites are severely understudied. We analyzed whole-genome information for a total of 42 representatives of bee-infecting trypanosomatids collected in Central Europe and Alaska from a population genetics point of view. Our data shed light on the evolution, selection, and diversification in this important group of trypanosomatid parasites.

Population genomic evidence for plant glacial survival in Scandinavia.

Quaternary glaciations have played a major role in shaping the genetic diversity and distribution of plant species. Strong palaeoecological and genetic evidence supports a postglacial recolonization of most plant species to northern Europe from southern, eastern and even western glacial refugia. Although highly controversial, the existence of small in situ glacial refugia in northern Europe has recently gained molecular support. We used genomic analyses to examine the phylogeography of a species that is critical in this debate. Carex scirpoidea Michx subsp. scirpoidea is a dioecious, amphi-Atlantic arctic-alpine sedge that is widely distributed in North America, but absent from most of Eurasia, apart from three extremely disjunct populations in Norway, all well within the limits of the Weichselian ice sheet. Range-wide population sampling and variation at 5,307 single nucleotide polymorphisms show that the three Norwegian populations comprise unique evolutionary lineages divergent from Greenland with high between-population divergence. The Norwegian populations have low within-population genetic diversity consistent with having experienced genetic bottlenecks in glacial refugia, and host private alleles that probably accumulated in long-term isolated populations. Demographic analyses support a single, pre-Weichselian colonization into Norway from East Greenland, and subsequent divergence of the three populations in separate refugia. Other refugial areas are identified in North-east Greenland, Minnesota/Michigan, Colorado and Alaska. Admixed populations in British Columbia and West Greenland indicate postglacial contact. Taken together, evidence from this study strongly indicates in situ glacial survival in Scandinavia.

Genomic imprinting mediates dosage compensation in a young plant XY system.

Sex chromosomes have repeatedly evolved from a pair of autosomes. Consequently, X and Y chromosomes initially have similar gene content, but ongoing Y degeneration leads to reduced expression and eventual loss of Y genes1. The resulting imbalance in gene expression between Y genes and the rest of the genome is expected to reduce male fitness, especially when protein networks have components from both autosomes and sex chromosomes. A diverse set of dosage compensating mechanisms that alleviates these negative effects has been described in animals2-4. However, the early steps in the evolution of dosage compensation remain unknown, and dosage compensation is poorly understood in plants5. Here, we describe a dosage compensation mechanism in the evolutionarily young XY sex determination system of the plant Silene latifolia. Genomic imprinting results in higher expression from the maternal X chromosome in both males and females. This compensates for reduced Y expression in males, but results in X overexpression in females and may be detrimental. It could represent a transient early stage in the evolution of dosage compensation. Our finding has striking resemblance to the first stage proposed by Ohno6 for the evolution of X inactivation in mammals.

Has adaptation occurred in males and females since separate sexes evolved in the plant Silene latifolia?

The evolution of separate sexes may involve changed expression of many genes, as each sex adapts to its new state. Evidence is accumulating for sex differences in expression even in organisms that have recently evolved separate sexes from hermaphrodite or monoecious (cosexual) ancestors, such as some dioecious flowering plants. We describe evidence that a dioecious plant species with recently evolved dioecy, Silene latifolia, has undergone adaptive changes that improve functioning in females, in addition to changes that are probably pleiotropic effects of male sterility. The results suggest pervasive adaptations as soon as males and females evolve from their cosexual ancestor.

Teosinte in Europe - Searching for the Origin of a Novel Weed.

A novel weed has recently emerged, causing serious agronomic damage in one of the most important maize-growing regions of Western Europe, the Northern Provinces of Spain. The weed has morphological similarities to a wild relative of maize and has generally been referred to as teosinte. However, the identity, origin or genetic composition of 'Spanish teosinte' was unknown. Here, we present a genome-wide analysis of single-nucleotide polymorphism (SNP) data for Spanish teosinte, sympatric populations of cultivated maize and samples of reference teosinte taxa. Our data are complemented with previously published SNP datasets of cultivated maize and two Mexican teosinte subspecies. Our analyses reveal that Spanish teosinte does not group with any of the currently recognized teosinte taxa. Based on Bayesian clustering analysis and hybridization simulations, we infer that Spanish teosinte is of admixed origin, most likely involving Zea mays ssp. mexicana as one parental taxon, and an unidentified cultivated maize variety as the other. Analyses of plants grown from seeds collected in Spanish maize fields and experimental crosses under controlled conditions reveal that hybridization does occur between Spanish teosinte and cultivated maize in Spain, and that current hybridization is asymmetric, favouring the introgression of Spanish teosinte into cultivated maize, rather than vice versa.

Evolution of sex-biased gene expression in a dioecious plant.

Separate sexes and sex-biased gene expression have repeatedly evolved in animals and plants, but the underlying changes in gene expression remain unknown. Here, we studied a pair of plant species, one in which separate sexes and sex chromosomes evolved recently and one which maintained hermaphrodite flowers resembling the ancestral state, to reconstruct expression changes associated with the evolution of dioecy. We found that sex-biased gene expression has evolved in autosomal and sex-linked genes in the dioecious species. Most expression changes relative to hermaphrodite flowers occurred in females rather than males, with higher and lower expression in females leading to female-biased and male-biased expression, respectively. Expression changes were more common in genes located on the sex chromosomes than the autosomes and led to feminization of the X chromosome and masculinization of the Y chromosome. Our results support a scenario in which sex-biased gene expression evolved during the evolution of dioecy to resolve intralocus sexual conflicts over the allocation of resources.

SEX-DETector: A Probabilistic Approach to Study Sex Chromosomes in Non-Model Organisms.

We propose a probabilistic framework to infer autosomal and sex-linked genes from RNA-seq data of a cross for any sex chromosome type (XY, ZW, and UV). Sex chromosomes (especially the non-recombining and repeat-dense Y, W, U, and V) are notoriously difficult to sequence. Strategies have been developed to obtain partially assembled sex chromosome sequences. Most of them remain difficult to apply to numerous non-model organisms, either because they require a reference genome, or because they are designed for evolutionarily old systems. Sequencing a cross (parents and progeny) by RNA-seq to study the segregation of alleles and infer sex-linked genes is a cost-efficient strategy, which also provides expression level estimates. However, the lack of a proper statistical framework has limited a broader application of this approach. Tests on empirical Silene data show that our method identifies 20-35% more sex-linked genes than existing pipelines, while making reliable inferences for downstream analyses. Approximately 12 individuals are needed for optimal results based on simulations. For species with an unknown sex-determination system, the method can assess the presence and type (XY vs. ZW) of sex chromosomes through a model comparison strategy. The method is particularly well optimized for sex chromosomes of young or intermediate age, which are expected in thousands of yet unstudied lineages. Any organisms, including non-model ones for which nothing is known a priori, that can be bred in the lab, are suitable for our method. SEX-DETector and its implementation in a Galaxy workflow are made freely available.

Fungal Infection Induces Sex-Specific Transcriptional Changes and Alters Sexual Dimorphism in the Dioecious Plant Silene latifolia.

Sexual dimorphism, including differences in morphology, behavior and physiology between females and males, is widespread in animals and plants and is shaped by gene expression differences between the sexes. Such expression differences may also underlie sex-specific responses of hosts to pathogen infections, most notably when pathogens induce partial sex reversal in infected hosts. The genetic changes associated with sex-specific responses to pathogen infections on the one hand, and sexual dimorphism on the other hand, remain poorly understood. The dioecious White Campion (Silene latifolia) displays sexual dimorphism in floral traits and infection with the smut fungus Micobrotryum lychnidis-dioicae induces a partial sex reversal in females. We find strong sex-specific responses to pathogen infection and reduced sexual dimorphism in infected S. latifolia. This provides a direct link between pathogen-mediated changes in sex-biased gene expression and altered sexual dimorphism in the host. Expression changes following infection affected mainly genes with male-biased expression in healthy plants. In females, these genes were up-regulated, leading to a masculinization of the transcriptome. In contrast, infection in males was associated with down-regulation of these genes, leading to a demasculinization of the transcriptome. To a lesser extent, genes with female-biased expression in healthy plants were also affected in opposite directions in the two sexes. These genes were overall down-regulated in females and up-regulated in males, causing, respectively, a defeminization in infected females and a feminization of the transcriptome in infected males. Our results reveal strong sex-specific responses to pathogen infection in a dioecious plant and provide a link between pathogen-induced changes in sex-biased gene expression and sexual dimorphism.

Transgene expression and Bt protein content in transgenic Bt maize (MON810) under optimal and stressful environmental conditions.

Bt protein content in transgenic insect resistant (Bt) maize may vary between tissues within plants and between plants growing under different environmental conditions. However, it is unknown whether and how Bt protein content correlates with transgene expression, and whether this relationship is influenced by stressful environmental conditions. Two Bt maize varieties containing the same transgene cassette (MON 810) were grown under optimal and stressful conditions. Before and during stress exposure, the upper leaves were analysed for transgene expression using quantitative RT-PCR and for Bt content using ELISA. Under optimal conditions there was no significant difference in the transgene expression between the two investigated Bt maize varieties whereas Bt protein content differed significantly. Transgene expression was correlated with Bt protein content in only one of the varieties. Under stressful environmental conditions we found similar transgene expressions as under optimal conditions but Bt content responded differently. These results suggest that Bt content is not only controlled by the transgene expression but is also dependent on the genetic background of the maize variety. Under stressful conditions the concentration of Bt protein is even more difficult to predict.

Gene regulatory variation mediates flowering responses to vernalization along an altitudinal gradient in Arabidopsis.

Steep environmental gradients provide ideal settings for studies of potentially adaptive phenotypic and genetic variation in plants. The accurate timing of flowering is crucial for reproductive success and is regulated by several pathways, including the vernalization pathway. Among the numerous genes known to enable flowering in response to vernalization, the most prominent is FLOWERING LOCUS C (FLC). FLC and other genes of the vernalization pathway vary extensively among natural populations and are thus candidates for the adaptation of flowering time to environmental gradients such as altitude. We used 15 natural Arabidopsis (Arabidopsis thaliana) genotypes originating from an altitudinal gradient (800-2,700 m above sea level) in the Swiss Alps to test whether flowering time correlated with altitude under different vernalization scenarios. Additionally, we measured the expression of 12 genes of the vernalization pathway and its downstream targets. Flowering time correlated with altitude in a nonlinear manner for vernalized plants. Flowering time could be explained by the expression and regulation of the vernalization pathway, most notably by AGAMOUS LIKE19 (AGL19), FLOWERING LOCUS T (FT), and FLC. The expression of AGL19, FT, and VERNALIZATION INSENSITIVE3 was associated with altitude, and the regulation of MADS AFFECTING FLOWERING2 (MAF2) and MAF3 differed between low- and high-altitude genotypes. In conclusion, we found clinal variation across an altitudinal gradient both in flowering time and the expression and regulation of genes in the flowering time control network, often independent of FLC, suggesting that the timing of flowering may contribute to altitudinal adaptation.

Identification of internal reference genes for gene expression normalization between the two sexes in dioecious white Campion.

Quantitative real time (qRT)-PCR is a precise and efficient method for studying gene expression changes between two states of interest, and is frequently used for validating interesting gene expression patterns in candidate genes initially identified in genome-wide expression analyses, such as RNA-seq experiments. For an adequate normalisation of qRT-PCR data, it is essential to have reference genes available whose expression intensities are constant among the different states of interest. In this study we present and validate a catalogue of traditional and newly identified reference genes that were selected from RNA-seq data from multiple individuals from the dioecious plant Silene latifolia with the aim of studying gene expression differences between the two sexes in both reproductive and vegetative tissues. The catalogue contains more than 15 reference genes with both stable expression intensities and a range of expression intensities in flower buds and leaf tissues. These reference genes were used to normalize expression differences between reproductive and vegetative tissues in eight candidate genes with sex-biased expression. Our results suggest a trend towards a reduced sex-bias in sex-linked gene expression in vegetative tissues. In this study, we report on the systematic identification and validation of internal reference genes for adequate normalization of qRT-PCR-based analyses of gene expression differences between the two sexes in S. latifolia. We also show how RNA-seq data can be used efficiently to identify suitable reference genes in a wide diversity of species.