BUSCADOR RECOLECTA

Table S1. List of primer sequences used, related to step 31., Peer reviewed

Supplementary Material 1. Probes used for whole mount in situ hybridisation (ISH). Sequences for synthesized ISH probes., Peer reviewed

10 pages. -- 8 supplementary figures. Figure S1. Phenotypic distributions of leaf accumulation of Cd and Hg in leaves, and relative root growth (RRG) in response to both metals. Corresponding histograms for each phenotype are displayed opposite to the x and y axes. Linear regression and 95% confidence intervals are overlayed on the scatter plot. Heritability (H2) is shown next to each histogram, and Pearson correlation coefficients (r) are shown in each panel. -- Figure S2. Average cross validation errors of 10 Admixture runs for each k between 1 and 10. The yaxis represents the cross-validation error value, the x-axis is k, number of population clusters. The distribution of ancestry components in all samples of the HapMap dataset for the best iteration (lowest cross validation error) was k = 5. -- Figure S3. Population structure analysis identified five admixture components. The population structure was determined using the software Admixture. -- Figure S4. Genome wide association analysis of heavy metal accumulation and tolerance traits (Manhattan plots shown for each trait; output from GEMMA). -- Figure S5. Genomic organization of three tandem ABC transporters (Medtr2g436680.1, Medtr2g436710, Medtr2g436730) on chromosome 2 in the Mt4.0 reference genome (www.medicagohapmap.org). The closest Blast hit to these three genes is the Arabidopsis thaliana ABC transporter ABCC14 (AT3G62700.1). -- Figure S6. The Pleiotropic Drug Resistance 3 gene PDR3 (Medtr5g070320) and Cation Exchanger 3 gene CAX3 (Medtr5g070330) are 5878 bp apart in the Mt4.0 reference genome (www.medicagohapmap.org). -- Figure S7. Density plots showing the genome wide distribution of SNP effect sizes (gray) and the top 1000 SNPs with lowest p-values for each trait (red). The most significant SNPs have the largest effect. -- Figure S8. Effect size and minor allele frequency (MAF) from the top 100 most significant SNPs using the GEMMA package for GWAS (left four panels, one for each trait)., Heavy metals are an increasing problem due to contamination from human sources that and can enter the food chain by being taken up by plants. Understanding the genetic basis of accumulation and tolerance in plants is important for reducing the uptake of toxic metals in crops and crop relatives, as well as for removing heavy metals from soils by means of phytoremediation. Following exposure of Medicago truncatula seedlings to cadmium (Cd) and mercury (Hg), we conducted a genome-wide association study using relative root growth (RRG) and leaf accumulation measurements. Cd and Hg accumulation and RRG had heritability ranging 0.44 – 0.72 indicating high genetic diversity for these traits. The Cd and Hg trait associations were broadly distributed throughout the genome, indicated the traits are polygenic and involve several quantitative loci. For all traits, candidate genes included several membrane associated ATP-binding cassette transporters, P-type ATPase transporters, oxidative stress response genes, and stress related UDP-glycosyltransferases. The P-type ATPase transporters and ATP-binding cassette protein-families have roles in vacuole transport of heavy metals, and our findings support their wide use in physiological plant responses to heavy metals and abiotic stresses. We also found associations between Cd RRG with the genes CAX3 and PDR3, two linked adjacent genes, and leaf accumulation of Hg associated with the genes NRAMP6 and CAX9. When plant genotypes with the most extreme phenotypes were compared, we found significant divergence in genomic regions using population genomics methods that contained metal transport and stress response gene ontologies. Several of these genomic regions show high linkage disequilibrium (LD) among candidate genes suggesting they have evolved together. Minor allele frequency (MAF) and effect size of the most significant SNPs was negatively correlated with large effect alleles being most rare. This is consistent with purifying selection against alleles that increase toxicity and abiotic stress. Conversely, the alleles with large affect that had higher frequencies that were associated with the exclusion of Cd and Hg. Overall, macroevolutionary conservation of heavy metal and stress response genes is important for improvement of forage crops by harnessing wild genetic variants in gene banks such as the Medicago HapMap collection., Peer reviewed

1 figure., Cultivated strawberry, Fragaria  ×  ananassa, has a complex aroma due to the presence of more than 350 volatile organic compounds (VOCs). However, a mixture of only 19 compounds, called Key Volatile Compounds (KVC), can impart the main strawberry aroma. The octoploid nature of the cultivated strawberry species (2n = 8x = 56) adds complexity to the heritance of the accumulation of the volatiles responsible for aroma. An F1 population cross between two breeding parental lines, FC50 and FD54, was phenotyped for aroma by SPME GCMS during six harvests. A total of 58 compounds were identified: 33 esters, nine terpenes, seven aldehydes, four lactones, two furans, one acid, one alkane and one alcohol, of which 16 were KVCs. A total of 179 QTLs were found, and 85 of these were detected in at least three harvests, of which 50 QTLs were considered major (LOD > 4.0) and detected in five or six analyzed harvests. Several clusters of ester QTLs associated with fruity aroma were discovered, such as QTLs for esters that share hexanoate group that were mapped in LG4A (Hexanoate_4A), those that share acetate and octyl groups in LG6A (Acetate_6A and Octyl_6A) or those with the same methyl group in LG7B (Methyl_7B). Different terpene QTLs associated with floral aroma appear grouped in a cluster in LG3C (Terpene_3C). Some of these clusters of QTLs were validated in a second F2 population, a cross of “Camarosa” and “Dover,” that was also phenotyped for three years. Selected SNPs from floral and fruity aroma QTLs were tested in a third population, which will most likely be useful for marker-assisted breeding (MAB)., Peer reviewed

The supporting information: Figure S1: Intra-group outcomes changes at different locations. Bar plots representing the mean difference and SD difference between pre and post intervention assessment of pressure pain threshold (A), conditioned pain modulation (B), and temporal summation (C)., Peer reviewed

1 figure., Cultivated strawberry, Fragaria  ×  ananassa, has a complex aroma due to the presence of more than 350 volatile organic compounds (VOCs). However, a mixture of only 19 compounds, called Key Volatile Compounds (KVC), can impart the main strawberry aroma. The octoploid nature of the cultivated strawberry species (2n = 8x = 56) adds complexity to the heritance of the accumulation of the volatiles responsible for aroma. An F1 population cross between two breeding parental lines, FC50 and FD54, was phenotyped for aroma by SPME GCMS during six harvests. A total of 58 compounds were identified: 33 esters, nine terpenes, seven aldehydes, four lactones, two furans, one acid, one alkane and one alcohol, of which 16 were KVCs. A total of 179 QTLs were found, and 85 of these were detected in at least three harvests, of which 50 QTLs were considered major (LOD > 4.0) and detected in five or six analyzed harvests. Several clusters of ester QTLs associated with fruity aroma were discovered, such as QTLs for esters that share hexanoate group that were mapped in LG4A (Hexanoate_4A), those that share acetate and octyl groups in LG6A (Acetate_6A and Octyl_6A) or those with the same methyl group in LG7B (Methyl_7B). Different terpene QTLs associated with floral aroma appear grouped in a cluster in LG3C (Terpene_3C). Some of these clusters of QTLs were validated in a second F2 population, a cross of “Camarosa” and “Dover,” that was also phenotyped for three years. Selected SNPs from floral and fruity aroma QTLs were tested in a third population, which will most likely be useful for marker-assisted breeding (MAB)., Peer reviewed

S1 Fig. Fas2 requirement in imaginal discs. (A) Gynandromorph (y fas2eB112/R(1)2) displaying a Fas2-deficient right side (y fas2eB112, labeled with the yellow marker, green outline). (B) Coupled-MARCM Fas2-deficient clones (fas2eB112, fas2– labeled with GFP) and their control Fas2-normal twins (fas2+ labeled with mtdTomato) in a wing imaginal disc at puparium formation. The Fas2-deficient clones are missing or consist of single cells, but their fas2+ twins can be larger than average WT clones, reminiscent of the Minute effect. (C) fas2– coupled-MARCM clones (labeled with GFP, green) show a grow deficit in imaginal disc derivatives compared to their control twins (mtdTomato, red) but do much better in abdominal histoblasts. Compare the number and size of fas2– clones (GFP) with that of their twin fas2+ controls (mtdTomato) in notum vs. abdomen. The picture corresponds to an adult just after eclosion. (D) Coupled-MARCM fas2eB112 clones (GFP) and twin control clones (mtdTomato) in a wing disc stained with anti-pH3 antibody to reveal mitosis. The red outline marks the rim of 2–3 cells around the Fas2-deficient clone (used to quantify the mitotic index). (E) Notum of an adult individual with the whole left heminotum covered by control clone territory (sn, fas2+ outlined in red), but without any detectable Fas2-deficient twin (which would have been labeled with y f36a, fas2—). (F) At least some Fas2-deficient cell clones survived in the adult. The picture shows a y fas2eB112 f36a clone (green arrow) and its sn3 control twin (red arrows point to sn3 bristles). The clone was induced in 2nd instar larva. (G) y fas2eB112 f36a M+ clones in a Minute heterozygous background are able to grow normally and differentiate epidermis in the adult (green arrows point to y fas2eB112 f36a chaetes). (H) Eye clone deficient for Fas2 induced in a Minute genetic background. fas2eB112 M+ null clones (without GFP) could grow more normally in the M–/M+ heterozygous background (labeled with Ubi-GFP). Genotype: y fas2eB112 FRT18A/Ubi-GFP M(1)OspFRT18A; hs-FLP/+. The Minute technique consists in creating normal Minute+ clones in a heterozygous Minute—/Minute+ mutant individual. The normal Minute+ cells grow faster than the Minute—/Minute+ heterozygous background cells. Thus, simply reducing the proliferation rate of the cells outside of the fas2—clones allows them to reach a more normal size. Bar is 50μm. (I) Expression of cleaved Caspase 3 in fas2– null clones is limited to a fraction of cells (arrow). Bar is 50 μm. (J) Quantification of fas2—clone size (grey bar) and their control twins (fas2+, white bar) in the adult notum, and rescue of fas2– MARCM clones by expression of UAS-fas2GPI and UAS-fasTRM compared to fas2—M+ clones and genetic conditions reducing apoptosis (grey bars). Clones were induced in 1st instar larva. The adult size of fas2—Minute+ clones (fas2—M+) in a Minute—/Minute+ heterozygous genetic background approached the size of the normal fas2+ control clones. Fas2-deficient MARCM clones (fas2eB112, fas2—) growing in a Df(3L)H99/+ genetic background displayed a little significant (depending on the statistical test used, t or Mann-Whitney) normalization of clone size in the adult notum. Expression of the Drosophila-inhibitor of apoptosis (UAS-diap) in fas2—MARCM clones (fas2—diapGOF) had a similar effect, while expression of UAS-P35 had no effect. Clone size is represented as number of chaetes. N is number of clones. (K) Inhibition of the JNK signaling pathway in MS1096-GAL4/+; UAS-fas2RNAi#34084/UAS-bskRNAi#32977 did not cancel the reduction in wing size compared to MS1096-GAL4/+; UAS-bskRNAi#32977/+ controls. Wing size in μm2/103. N is number of individuals., S2 Fig. Quantification of the expression of JNK activity reporters. (A) Left, quantification of TRE-DsRed signal in en-GAL4 UAS-GFP/TRE-DsRed (fas2+) and en-GAL4 UAS-GFP/TRE-DsRed; UAS-fas2RNAi#34084/+ (fas2RNAi) wing imaginal discs. The TRE-DsRed signal was amplified using an anti-RFP antibody, and the signal in the anterior compartment was subtracted to the signal in the Posterior compartment in each disc to normalize for differences in staining. Right, quantification of puc-LacZ signal in en-GAL4 UAS-GFP/+; puc-LacZ/+ (fas2+) and en-GAL4 UAS-GFP/+; UAS-fas2RNAi#34084/puc-LacZ (fas2RNAi) wing imaginal discs. The signal in the anterior compartment was subtracted to the signal in the Posterior compartment in each disc to normalize for differences in staining. N is number of wing imaginal discs. (B) Left, quantification of TRE-DsRed signal in fas2eB112FRT19A; TRE-DsRed/; Tub-GAL4 UAS-GFP/+ MARCM null cell clones (fas2–) and fas2eB112 FRT18A; TRE-DsRed/+ Minute+ null cell clones (fas2– M+) induced in fas2eB112 FRT18A/Ubi-GFP M(1)Osp FRT18A; TRE-DsRed/+ wing imaginal discs. The signal in the background was subtracted to the signal in the clone in each disc. Right, quantification of puc-LacZ signal in fas2eB112FRT19A; Tub-GAL4 UAS-GFP/puc-LacZ MARCM null cell clones (fas2–) and fas2eB112 FRT18A; puc-LacZ/hs-FLP Minute+ null cell clones (fas2– M+) induced in fas2eB112 FRT18A/Ubi-GFP M(1)Osp FRT18A; puc-LacZ/hs-FLP wing imaginal discs. The signal in the background was subtracted to the signal in the clone in each disc. N is number of clones., S3 Fig. Fas2 functions via the EGFR pathway. (A) Left, WT adult head. Right, the hypomorphic fas2eB112/fas2e76 combination displayed the absence or size reduction of ocelli, as well as loss of bristles in the dorsal head, an alteration reminiscent of Egfr torpedo alleles (Egfrtop). (B) Quantification of fluorescent ppERK antibody signal (ratio of pixels with a signal level higher than 100, out of 255 levels) in the eye disc of ey-driven fas2RNAi (#34084) FLP-OUTs (green bar) and their control CyO siblings (white bar). N is number of eye imaginal discs. (C) Left, ey-driven fas2RNAi (#34084) FLP-OUT. Right, ey-driven fas2RNAi (#34084) FLP-OUT in an Egfrt1/+ heterozygous background. (D) Suppressors of the fas2-null MARCM-clone phenotype in the adult. MARCM fas2eB112 clones labeled with yellow and forked were induced in combinations expressing UAS-insertions for components of different growth signaling pathways. Expression of activated components of the EGFR signaling pathway (RasV12, RafGOF and PI3K - Dp110-) and over-expression of Yki produced a significant correction in the size of fas2—clones in the adult notum. In contrast, expression of activated-FGFR (λHtl), which shares most downstream effectors with EGFR, activated-InR (InRR418P), activated-Notch (NINTRA) and myristoylated-Src did not cause a significant suppression. Clone size is number of marked microchaetes. N is number of clones., S4 Fig. JNK is required for the Hippo pathway increased signaling in the fas2 LOF condition. Quantification of ex-LacZ reporter expression in en-GAL4/+; fas2RNAi#34084 wing imaginal discs. The intensity of expression of the ex-LacZ reporter (measured as grey average in the red channel) is similar in the anterior and posterior compartments of each control wing imaginal disc (giving a posterior/anterior signal ratio close to 1.0). Expression of UAS-fas2RNAi (#34084) in the posterior compartment causes a strong increase of ex-LacZ expression compared with the anterior compartment in the same disc (ex-LacZ P/A signal ratio). While inhibition of the JNK pathway (UAS-bskRNAi, #32977) in the posterior compartment slightly increases ex-LacZ P/A signal ratio, it strongly suppresses the increase caused by the inhibition of Fas2 expression. N is number of wing imaginal discs., S5 Fig. Fas2 dependence on EGFR function. (A) Fas2 and EGFR are expressed by all cells in imaginal discs. A Fas2::GFP protein trap [13] shows colocalization with EGFR (ImageJ Colocalization plug-in, Pearson´s correlation: 0,3146). (B) Homozygous Egfrtop1 imaginal discs showed a lower expression of Fas2 (red channel, labeled with the anti-Fas2 1D4 antibody) than the imaginal discs from their heterozygous siblings (Egfrtop1/CyO, GFP)., S6 Fig. Fas2 expression is directly regulated by EGFR. (A) A Fas2::GFP protein trap line shows expression of Fas2 in all cells of imaginal discs, with a maximum in differentiating retinal cells. (B) FLPOUT UAS-LacZ clones (red signal) expressing activated-EGFR (λEgfr) display a dramatic increase in Fas2::GFP expression. Note that the saturation of the Fas2::GFP expression prevents the visualization of the normal Fas2::GFP expression in the other cells of the wing disc (compare to Fig 7A which is stained with the 1D4 antibody that only recognizes the TRM isoforms of Fas2). (C) Wing imaginal disc FLPOUT UAS-LacZ clones (red signal) expressing activated-EGFR (λEgfr) plus bskRNAi314767 display a Fas2::GFP signal similar to activated-EGFR clones. (D) Eye imaginal disc FLPOUT UAS-LacZ clones (red signal) expressing activated-EGFR (λEgfr) plus bskRNAi#31476 display a similarly strong Fas2::GFP signal. Note that the saturation of the Fas2::GFP signal only permits a very faint visualization of the normal Fas2 peak of expression in the differentiating retina., S1 Text. List of Strains used for experiments., Peer reviewed

1 figure., Cultivated strawberry, Fragaria  ×  ananassa, has a complex aroma due to the presence of more than 350 volatile organic compounds (VOCs). However, a mixture of only 19 compounds, called Key Volatile Compounds (KVC), can impart the main strawberry aroma. The octoploid nature of the cultivated strawberry species (2n = 8x = 56) adds complexity to the heritance of the accumulation of the volatiles responsible for aroma. An F1 population cross between two breeding parental lines, FC50 and FD54, was phenotyped for aroma by SPME GCMS during six harvests. A total of 58 compounds were identified: 33 esters, nine terpenes, seven aldehydes, four lactones, two furans, one acid, one alkane and one alcohol, of which 16 were KVCs. A total of 179 QTLs were found, and 85 of these were detected in at least three harvests, of which 50 QTLs were considered major (LOD > 4.0) and detected in five or six analyzed harvests. Several clusters of ester QTLs associated with fruity aroma were discovered, such as QTLs for esters that share hexanoate group that were mapped in LG4A (Hexanoate_4A), those that share acetate and octyl groups in LG6A (Acetate_6A and Octyl_6A) or those with the same methyl group in LG7B (Methyl_7B). Different terpene QTLs associated with floral aroma appear grouped in a cluster in LG3C (Terpene_3C). Some of these clusters of QTLs were validated in a second F2 population, a cross of “Camarosa” and “Dover,” that was also phenotyped for three years. Selected SNPs from floral and fruity aroma QTLs were tested in a third population, which will most likely be useful for marker-assisted breeding (MAB)., Peer reviewed

Data S1. Supporting Information., Peer reviewed

1 figure., Cultivated strawberry, Fragaria  ×  ananassa, has a complex aroma due to the presence of more than 350 volatile organic compounds (VOCs). However, a mixture of only 19 compounds, called Key Volatile Compounds (KVC), can impart the main strawberry aroma. The octoploid nature of the cultivated strawberry species (2n = 8x = 56) adds complexity to the heritance of the accumulation of the volatiles responsible for aroma. An F1 population cross between two breeding parental lines, FC50 and FD54, was phenotyped for aroma by SPME GCMS during six harvests. A total of 58 compounds were identified: 33 esters, nine terpenes, seven aldehydes, four lactones, two furans, one acid, one alkane and one alcohol, of which 16 were KVCs. A total of 179 QTLs were found, and 85 of these were detected in at least three harvests, of which 50 QTLs were considered major (LOD > 4.0) and detected in five or six analyzed harvests. Several clusters of ester QTLs associated with fruity aroma were discovered, such as QTLs for esters that share hexanoate group that were mapped in LG4A (Hexanoate_4A), those that share acetate and octyl groups in LG6A (Acetate_6A and Octyl_6A) or those with the same methyl group in LG7B (Methyl_7B). Different terpene QTLs associated with floral aroma appear grouped in a cluster in LG3C (Terpene_3C). Some of these clusters of QTLs were validated in a second F2 population, a cross of “Camarosa” and “Dover,” that was also phenotyped for three years. Selected SNPs from floral and fruity aroma QTLs were tested in a third population, which will most likely be useful for marker-assisted breeding (MAB)., Peer reviewed