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Additional file 6. Variation of power as a function of number of allele specific reads per biorep.

Additional file 10: FigureS10. GWAS Manhattan plots for (a) Stearic acid; (b) Nonadecanoic acid; (c) Eicosenoic acid; (d) Docosanoic acid; (e) Tetracosanoic acid; (f) Nervonic acid; (g) Oleic acid; (h) Linoleic acid; (i) Ratio between oleic and linoleic acids. FDR and Bonferroni thresholds are shown by blue and red, respectively.

Additional file 5: FigureS5. (A) Multidimensional scaling plot (two dimensions, 1 - Spearman correlation coefficient between TAGs abundances measured using LC-MS with dilution 1:25 was used as distance). One sample is shown by one point; accessions are shown by different colors; different years are shown by points of different shapes. (B) Minus log10 p-values for the differences between lines (ANOVA) are shown, TAGs are ordered by p-value increase from top left to bottom right. Bonferroni adjusted 0.05 significance level is shown by red line; (C-G) abundances of five selected TAGs are shown across lines and years. Each point represents 1 sample, point shapes, and colors as in (A), lines show per-year averages. This figure complements main Fig. 3.

Additional file 9: FigureS9. Replication experiment on 6 accessions. (Left) Distributions of FAs (top) or TAGs (middle and bottom) by percentages of variance explained by genotype (G), environment (year, E), interaction between genotype and environment (G:E) or percentage of residual variance. Distribution of the same percentages divided by number of degrees of freedom of corresponding factor are shown on the right.

Additional file 6: FigureS6. Replication experiment on 6 accessions: relative abundances of all the fatty acids with significant (FDR

Additional file 1: FigS1. ((A) PCA plots reflecting the relationships between sunflower technical samples based on 15,068 SNPs segregating in the Russian collection. Each dot corresponds to a sunflower technical sample used in the study. Dots are colored by sequencing batch (A) or collection samples were obtained from (B) (C) Nei’s genetic distance matrix between each 2 samples.

Additional file 6: FigureS6. Replication experiment on 6 accessions: relative abundances of all the fatty acids with significant (FDR

Additional file 3: FigureS3. Joint principal component analysis of sunflower accessions genotyped in this study and in Hübner (2019) based on 2345 shared SNPs. The first and the second (A) or the first and the third (B) PCs are shown. Each dot corresponds to a plant accession. Colors indicate the origin (wild/line/landrace). Shapes indicate species.

Additional file 8: FigureS8. Replication experiment on 6 genotypes: relative abundances of all TAGs detected by LC-MS with dilution 1:25 with significant (FDR

Additional file 7: FigureS7. Replication experiment on 6 genotypes: relative abundances of all TAGs detected by LC-MS with dilution 1:3 with significant (FDR

Additional file 2: FigureS2. Linkage disequilibrium (LD) decay plot. (A) Genome-wide LD. Gray dots correspond to a SNP pair, y-axis show r2 between two SNPs calculated using whole dataset. SNP pairs with distance less than 5 Mb are shown. (B) LD per each chromosome. Lines correspond to loess curves; 95% confidence intervals are shown by shades.

Additional file 4 (A) Multidimensional scaling plot (two dimensions, 1 - Spearman correlation coefficient between TAGs abundances measured using LC-MS with dilution 1:3 was used as distance). One sample is shown by one point; accessions are shown by different colors; different years are shown by points of different shapes. (B) Minus log10 p-values for the differences between lines (ANOVA) are shown, TAGs are ordered by p-value increase from top left to bottom right. Bonferroni adjusted 0.05 significance level is shown by red line; (C-G) abundances of five selected TAGs are shown across lines and years. Each point represents 1 sample, point shapes, and colors as in (A), lines show per-year averages. This figure complements main Fig. 3.

Additional file 9: FigureS9. Replication experiment on 6 accessions. (Left) Distributions of FAs (top) or TAGs (middle and bottom) by percentages of variance explained by genotype (G), environment (year, E), interaction between genotype and environment (G:E) or percentage of residual variance. Distribution of the same percentages divided by number of degrees of freedom of corresponding factor are shown on the right.

Additional file 7. Variation of power as a function of the extent of deviation from allelic balance.

Additional file 3. Variation of type I error as a function of number of simulations, number of allele specific reads per bioreps and extent of deviation from allelic balance.

Additional file 22: Supporting experimental procedures. Method S1. Reagents, Method S2. GBS library preparation, Method S3. Lipid extraction and UPLC-MS.

Additional file 12: FigureS12. Data clean up using blank samples. Top panels show dependence of average log2 seed sample intensity of FAs and TAGs (two dilutions) on average log2 intensity of same lipids in blank samples (see Methods). Straight and dashed lines correspond to equal intensities in both types of sample and to two-fold higher concentration in seed samples compared to blanks, respectively. Bottom panels show the same lipids as top panels in coordinates of total FA (TAG) chain length (x-axis), and number of double bounds (y-axis), point size is proportional to log2 average intensity in seed samples. Only lipids with log2(sample/blank) > 1 were used in analysis, remaining FAs and TAGs (shown in red) were filtered out.

Additional file 11: FigureS11. LD blocks containing significant SNPs. Each panel represents one significant association, traits and chromosome names where significant loci is located are shown in panel titles. Each panel schematically shows associated loci with all detected SNPs. LD-blocks detected by Haploview software are shown by heatmaps, r2 (%) for each SNP pair is shown by color and numbers.

Additional file 5. Variation of power as a function of number of simulations.

Additional file 1. Additional Methods. This file contains the section additional methods, in which we summarize the definition of the Bayesian model used in this work. The model has been previously published and described, and we provide in additional methods a brief summary just to facilitate readers.

Additional file 23: AppendixS1. Description of the lines used in the study.

Additional file 7: FigureS7. Replication experiment on 6 genotypes: relative abundances of all TAGs detected by LC-MS with dilution 1:3 with significant (FDR

Additional file 4. Variation of power as a function of number of bioreps.

Additional file 11:Bland Altman plots for each biological replicate. (PDF 4 MB)

Authors’ original file for figure 5

Additional file 1:Overlapping exons combined into single genomic region. D. melanogaster ovo gene (Flybase ID BFgn0003028) used as an example of combining overlapping exons into a single genome region for mapping purposes. Format PDF. View with Adobe. (PDF 93 KB)

Authors’ original file for figure 1

Additional File 4. Bayesian inference and Gibbs sampling.

Authors’ original file for figure 4

Authors’ original file for figure 6

Additional file of RNA-seq: technical variability and sampling

Additional file 10:Length of the Exon does not explain disagreement in technical replicates. Coverage plots of the c167 cell lines data. The Y axis is the average coverage across all technical replicates. A bar is drawn if the exon is present (at any coverage level) in that technical replicate. The 10th percentile represents that bottom 10% of the exons in length while the 90th percentile represents the top 10% of exons by length. Format PDF. View with Adobe (PDF 745 KB)

Additional File 1. Absolute values of correlations between phenotypic traits.

Additional File 3. Maximum Likelihood estimates.

Authors’ original file for figure 2

Authors’ original file for figure 3

Additional File 5. Root mean square error.

Supplementary material describing Papatsenko-Levine formalism with description of regulatory framework, full derivation of the solver for the general form of the model, runtime comparison between solvers, model fit to simulated data, convergence properties and output for models D7 & D8. (PDF 728 kb)

The genomics of non-model invertebrates remains challenging. Every step from sampling to final genome annotation has unexpected complications. We sequenced two marine invertebrate genomes: the Manila clam (Ruditapes philippinarum) genome, and the moon jellyfish (Aurelia aurita) genome, using both Illumina and PacBio platforms. We failed to assemble those genomes using all available conventional assemblers or more sophisticated assembly pipelines. Initially we explained this by very high heterozygosity rate and highly repeated genome composition. However during the stage of coverage-wise polishing of PacBio-only assembly by Illumina reads we observed many cases of complex structural variants that cannot be explained by diploid heterozygosity or repeats. After manual check of these regions we found the presence of two types of similar sequences but different in read coverage. We developed a software that analyzes all forks in de Bruijn graphs constructed from Illumina reads and we found that both the clam and the jellyfish genomes additionally to the main 20x coverage have many fragments with a 4x coverage. We manually checked the difference between 20x and 4x fragments finding that they are very similar in sequence but do not have classical heterozygosity difference. We found multiple indels that cannot be explained by Illumina errors or observed natural heterozygosity or by presence of DNA from gametes. We hypothesize that these two genomes are subject to chromatin diminution—a chromosomal fragmentation, followed by the elimination of part of the chromosome during mitosis. We have developed a specific assembly pipeline to trying to solve this issue.

Bivalve molluscs are a highly successful and ancient Class, including over 20,000 known species, and they are an interesting group for evolutionary and biodiversity studies. Bivalves represent a good model for studying adaptation to anoxia/hypoxia, salinity, and temperature, and they are useful bioindicators for monitoring the concentration of pollutants and heavy metals in the water. They also make up an important source of food all over the world, with a production corresponding to ~20% of the global aquaculture yield; clams are first in production, followed by oysters, mussels, and scallops. A striking feature of bivalves—and the main reason behind this project—is the presence of an unusual mitochondrial inheritance system: the Doubly Uniparental Inheritance (DUI), so far detected in ~50 bivalve species, belonging to seven families. In DUI species, two mitochondrial genomes (mtDNAs) are present: one is transmitted through eggs (F-type, for female-inherited), the other through sperm (M-type, for male-inherited), and the amino acid p-distance between conspecific M and F genomes ranges from 10% to over 50%. DUI provides a unique and privileged point of view for studying several fundamental aspects of eukaryote biology. In DUI systems: i) males are naturally heteroplasmic, with two very divergent mtDNAs; ii) it is possible to follow germ line mitochondria during development (to study mitochondrial inheritance and bottleneck); iii) mitochondria are under selection for male functions (e.g.: spermatogenesis, sperm swimming); iv) there are two coexisting mitochondrial genomes in the same nuclear background (coevolution, conflicts). All these interesting biological features are in sharp contrast with the lack of genomic resources about bivalve molluscs.Here we present the draft genome of the DUI species Ruditapes philippinarum (the Manila clam). DNA from a male individual was sequenced with x40 Illumina HiSeq and with x30 PacBio RSII. We have tried to assembly this dataset with all available hybrid or PacBio only assembly pipelines. The best results were obtained with PacBio reads assembled by Canu assembler with contig N50 76 kb, and 39.92% completed and 74.60% partial genes according to CEGMA. We annotated families of tandem and dispersed repeats, we found a new highly repeated dispersed element, and characterised the major families of satellite DNA. We report the results of the first analyses as well as the technical challenges we faced, especially during the phase of de novo assembly.

Rhynchospio cf. foliosa Imajima, 1991 (Fig. 1) Rhynchospio foliosa Imajima, 1991: 14 ���17, figs 6, 7. Material. USA, Oregon, Newport, sea water pump of the Hatfield Marine Science Center, 44.6223 ��N, 124.0433 ��W, coll. J.W. Chapman, 20 Oct 2010, MIMB 28 104 (2). Synopsis. Up to 22 mm long, 1 mm wide for 90 chaetigers. Prostomium with two conical pointed fronto-lateral horns. Caruncle low and indistinct, decreasing in height and ending at posterior margin of chaetiger 1. Occipital antenna absent. Two pairs of dark red eyes arranged trapezoidally, comprising one pair of small median eyes and one pair of slightly larger lateral eyes situated anteriorly and set wider apart. Nuchal organs metameric, one pair of metamers on posterior half of each chaetiger ending on about chaetiger 25. Chaetiger 1 with capillaries and postchaetal lamellae in both rami; notopodial capillaries directed upwards, about 1.5 times as long as those on succeeding chaetigers. Notopodia with only capillary chaetae throughout body. Sabre chaetae in neuropodia as gradual transformation of inferior capillaries on chaetigers 25���30; chaetae with narrow limbation and granulation on distal part of shaft. Hooks in neuropodia from chaetigers 17���18, with only outer hood, tridentate, with two small upper teeth arranged in line above main fang; shaft slightly curved, with weak constriction in upper part. Branchiae from chaetiger 2 onwards, large, broad and foliaceous on anterior chaetigers, gradually becoming narrower and smaller on posterior chaetigers, flattened, with surfaces oriented parallel to body axis, free from notopodial lamellae. Pygidium with one pair of ventral cirri and up to 15 pairs of thinner and longer dorsal cirri; ventral cirri with dark pigment diffused in basal part; dorsal cirri transparent. Simultaneous hermaphrodites with sperm in chaetigers 13���34 and oocytes from chaetiger 36 onwards. Oogenesis intraovarian; vitellogenesis occurring when oocytes growing attached to segmental blood vessels. Vitellogenic oocytes subspherical, up to about 50 ��m in diameter, with honey-combed envelope without vesicles. Developed coelomic oocytes oval, up to 75 x 125 ��m in diameter, with germinal vesicle about 40 ��m and single nucleolus 12 ��m in diameter; envelope about 6 ��m thick, with external honey-combed surface and 20���30 vesicles (cortical alveoli) each 6���7 ��m in diameter associated with inner surface (Fig. 1 A, B). Spermatids joined in tetrads; spermatozoa ect-aquasperm with conical acrosome about 1.5 ��m long and 1 ��m in diameter, ovoid nucleus 2 ��m long and 1.5 ��m in diameter, a few spherical mitochondria, and a long flagellum; acrosome with transparent central part and a small dark spherical structure in anterior part (Fig. 1 C, D). Remarks. Rhynchospio foliosa was originally described by Imajima (1991) from a single complete individual (18 mm long, about 1 mm wide for 79 chaetigers, with sabre chaetae in neuropodia from chaetiger 15 and hooks from chaetiger 17) from Usujiri Bay, Hokkaido Is., Japan. The species has never been redescribed or reported from outside of the type locality. Individuals from Oregon match the description of R. foliosa in most diagnostic features but differ in having up to 32 pygidial cirri instead of 18. This difference may be due to ontogenetic or individual variability which remain unknown in this species, but such variability has been documented in other similar spionids (Radashevsky 2012). No Rhynchospio with numerous pygidial cirri and broad and foliaceous branchiae similar to those from the Hatfield Marine Science Center, Newport, Oregon has been reported from the Pacific coast of North America. Whether the worms from Newport belong to a rare new species native in Oregon or result from an introduction of R. foliosa from Japan or elsewhere, remains uncertain. Herein, we therefore tentatively refer to them as R. cf. foliosa., Published as part of Radashevsky, Vasily I., Pankova, Victoria V. & Nuzhdin, Sergey V., 2016, Molecular analysis of six Rhynchospio Hartman, 1936 species (Annelida: Spionidae) with comments on the evolution of brooding within the group, pp. 579-590 in Zootaxa 4127 (3) on pages 583-584, DOI: 10.11646/zootaxa.4127.3.10, http://zenodo.org/record/255112, {"references":["Imajima, M. (1991) Spionidae (Annelida, Polychaeta) from Japan. VI. The genera Malacoceros and Rhynchospio. Bulletin of the National Science Museum, Tokyo, Series A (Zoology), 17 (1), 5 - 17."]}

Radashevsky, Vasily I., Pankova, Victoria V., Nuzhdin, Sergey V. (2016): Molecular analysis of six Rhynchospio Hartman, 1936 species (Annelida: Spionidae) with comments on the evolution of brooding within the group. Zootaxa 4127 (3): 579-590, DOI: http://doi.org/10.11646/zootaxa.4127.3.10

Rhynchospio darwini Radashevsky, 2015 Rhynchospio darwini Radashevsky, 2015: 684 ���686, figs 33, 34. Material. Australia, Northern Territory, Timor Sea, Beagle Gulf, Fannie Bay, Darwin, Bullocky Point, 12.4356 ��S, 130.8323 ��E, intertidal, muddy sand, coll. V.I. Radashevsky, 3 Sep 2013, NTM W025648 (holotype), MIMB 28105 (paratype). Synopsis. Up to 5 mm long, 0.3 mm wide for 60 chaetigers. Prostomium with two conical fronto-lateral horns, extending posteriorly to end of chaetiger 1 as a low, indistinct caruncle. Occipital antenna absent. Two pairs of small red eyes arranged trapezoidally. Nuchal organs metameric; first pair of metamers on chaetiger 1 as curved ciliary bands on either side of low caruncle; successive metamers as one pair of ciliary bands on posterior half of each chaetiger ending on at least chaetiger 15. Chaetiger 1 with long capillaries and small postchaetal lamellae in both rami. Notopodia with only capillary chaetae throughout body. Sabre chaetae in neuropodia from chaetigers 11���12. Hooks in neuropodia from chaetigers 11���12, with only outer hood, tridentate, with two small upper teeth arranged in line above main fang; shaft slightly curved, without constriction in upper part. Branchiae from chaetiger 2 through most of body, flattened, with surfaces oriented parallel to body axis, free from notopodial postchaetal lamellae. Pygidium with one pair of ventral cirri and two pairs of thinner and slightly longer dorsal cirri. Simultaneous hermaphrodites with sperm in chaetigers 11���14 and oocytes from chaetiger 15 to chaetigers 25���30. Oocytes subspherical, with thin and smooth envelope less than 1 ��m thick, without vesicles. Spermatozoa with long and thin nucleus. Remarks. Two Rhynchospio species were originally described from Australia by Blake and Kudenov (1978), R. australiana from Western Australia, and R. glycera from New South Wales. Both species were described based on single individuals, never re-described and not reported from other localities. Gamete morphology and reproductive biology are not known for both. Adults of R. darwini from Northern Territory, Australia, appear very similar to those of R. nhatrangi from Nhatrang Bay, Vietnam (see Radashevsky 2007 a). Worms from these two localities have prostomia, pygidia and gametes of similar shape, and sabre chaetae, nuchal organs and branchiae of similar morphology and arrangement. They differ, however, in that mature individuals of R. nhatrangi have falcate unidentate hooks in neuropodia of chaetigers 11���14, and tridentate hooks from chaetiger 15 onwards, whereas mature individuals of R. darwini have only tridentate hooks in neuropodia from chaetigers 11���12., Published as part of Radashevsky, Vasily I., Pankova, Victoria V. & Nuzhdin, Sergey V., 2016, Molecular analysis of six Rhynchospio Hartman, 1936 species (Annelida: Spionidae) with comments on the evolution of brooding within the group, pp. 579-590 in Zootaxa 4127 (3) on page 582, DOI: 10.11646/zootaxa.4127.3.10, http://zenodo.org/record/255112, {"references":["Radashevsky, V. I. (2015) Spionidae (Annelida) from Lizard Island, Great Barrier Reef, Australia: the genera Aonides, Dipolydora, Polydorella, Prionospio, Pseudopolydora, Rhynchospio, and Tripolydora. Zootaxa, 4019 (1), 635 - 694. http: // dx. doi. org / 10.11646 / zootaxa. 4019.1.22","Blake, J. A. & Kudenov, J. D. (1978) The Spionidae (Polychaeta) from southeastern Australia and adjacent areas with a revision of the genera. Memoirs of the National Museum of Victoria, 39, 171 - 280.","Radashevsky, V. I. (2007 a) Morphology and biology of a new Rhynchospio species (Polychaeta: Spionidae) from the South China Sea, Vietnam, with the review of Rhynchospio taxa. Journal of Natural History, London, 41 (17), 985 - 997. http: // dx. doi. org / 10.1080 / 00222930701376717"]}

Background Many genes produce multiple transcripts due to alternative splicing or utilization of alternative transcription initiation/termination sites. This 'transcriptome expansion' is thought to increase phenotypic complexity by allowing a single locus to produce several functionally distinct proteins. However, sex, genetic and developmental variation in the representation of alternative transcripts has never been examined systematically. Here, we describe a genome-wide analysis of sex-specific expression of alternative transcripts in Drosophila melanogaster. Results We compared transcript profiles in males and females from eight Drosophila lines (OregonR and2b, and 6 RIL) using a newly designed 60-mer oligonucleotide microarray that allows us to distinguish a large proportion of alternative transcripts. The new microarray incorporates 7,207 oligonucleotides, satisfying stringent binding and specificity criteria that target both the common and the unique regions of 2,768 multi-transcript genes, as well as 12,912 oligonucleotides that target genes with a single known transcript. We estimate that up to 22% of genes that produce multiple transcripts show a sex-specific bias in the representation of alternative transcripts. Sexual dimorphism in overall transcript abundance was evident for 53% of genes. The X chromosome contains a significantly higher proportion of genes with female-biased transcription than the autosomes. However, genes on the X chromosome are no more likely to have a sexual bias in alternative transcript representation than autosomal genes. Conclusion Widespread sex-specific expression of alternative transcripts in Drosophila suggests that a new level of sexual dimorphism at the molecular level exists.

Despite the large number of animal complete mitochondrial genomes currently available in public databases, knowledge about mitochondrial genomics in invertebrates is uneven. This paper reports, for the first time, the complete mitochondrial genome of the grooved carpet shell,Ruditapes decussatus, also known as the European clam.Ruditapes decussatusis morphologically and ecologically similar to the Manila clamRuditapes philippinarum, which has been recently introduced for aquaculture in the very same habitats ofRuditapes decussatus, and that is replacing the native species. Currently the production of the European clam is almost insignificant, nonetheless it is considered a high value product, and therefore it is an economically important species, especially in Portugal, Spain and Italy. In this work we: (i) assembledRuditapes decussatusmitochondrial genome from RNA-Seq data, and validated it by Sanger sequencing; (ii) analyzed and characterized theRuditapes decussatusmitochondrial genome, comparing its features with those of other venerid bivalves; (iii) assessed mitochondrial sequence polymorphism (SP) and copy number variation (CNV) of tandem repeats across 26 samples. Despite using high-throughput approaches we did not find evidence for the presence of two sex-linked mitochondrial genomes, typical of the doubly uniparental inheritance of mitochondria, a phenomenon known in ∼100 bivalve species. According to our analyses,Ruditapes decussatusis more genetically similar to species of the Genus Paphia than to the congenericRuditapes philippinarum, a finding that bolsters the already-proposed need of a taxonomic revision. We also found a quite low genetic variability across the examined samples, with few SPs and little variability of the sequences flanking the control region (Largest Unassigned Regions (LURs). Strikingly, although we found low nucleotide variability along the entire mitochondrial genome, we observed high levels of length polymorphism in the LUR due to CNV of tandem repeats, and even a LUR length heteroplasmy in two samples. It is not clear if the lack of genetic variability in the mitochondrial genome ofRuditapes decussatusis a cause or an effect of the ongoing replacement ofRuditapes decussatuswith the invasiveRuditapes philippinarum, and more analyses, especially on nuclear sequences, are required to assess this point.

Gene expression variation within species is relatively common, however, the role of natural selection in the maintenance of this variation is poorly understood. Here we investigate low and high latitude populations of Drosophila melanogaster and its sister species, D. simulans, to determine whether the two species show similar patterns of population differentiation, consistent with a role for spatially varying selection in maintaining gene expression variation. We compared at two temperatures the whole male transcriptome of D. melanogaster and D. simulans sampled from Panama City (Panama) and Maine (USA). We observed a significant excess of genes exhibiting differential expression in both species, consistent with parallel adaptation to heterogeneous environments. Moreover, the majority of genes showing parallel expression differentiation showed the same direction of differential expression in the two species and the magnitudes of expression differences between high and low latitude populations were correlated across species, further bolstering the conclusion that parallelism for expression phenotypes results from spatially varying selection. However, the species also exhibited important differences in expression phenotypes. For example, the genomic extent of genotype × environment interaction was much more common in D. melanogaster. Highly differentiated SNPs between low and high latitudes were enriched in the 3’ UTRs and CDS of the geographically differently expressed genes in both species, consistent with an important role for cis-acting variants in driving local adaptation for expression-related phenotypes., Author Summary While gene expression variation in natural populations is common, the population genetic processes responsible for the maintenance of this variation remain obscure. Here we study geographic differences in gene expression in recently established low and high latitude populations of two closely related species of Drosophila. We observe substantial parallelism in expression differences and expression plasticity between populations, which supports the idea that spatially varying selection correlated with latitude contributes to the maintenance of gene expression variation in these species. Comparison of inter-population sequence differentiation and expression differentiation suggests that cis-acting variants play a role in geographic expression differentiation.