BUSCADOR RECOLECTA

Additional file 5: Figure S5. a, Principal component analysis of CD19 + CD21-CD23+ follicular B cells from Rosa26-RRAS2xmb1-Cre mice, CD19 + CD21-CD23+ follicular B cells from WT C57BL/6 J mice and of leukemic CD19 + CD5+, GFPlow and GFPhigh cells from Rosa26-RRAS2xmb1-Cre mice. b, Ingenuity Pathway Analysis (IPA) of differentially expressed genes associated with molecular mechanisms of cancer in leukemic versus normal follicular B cells. Pink-filled symbols: upregulated genes. Green-filled: downregulated genes. Double circle: protein complex; horizontal ellipse: transcription regulator; vertical ellipse: transmembrane receptor, diamond: enzyme; trapezium: transporter; triangle: phosphatase; inverted triangle: kinase; vertical rectangle: G protein-coupled receptor; circle: other. Black arrows: direct interactions; grey/white arrows: indirect interactions. Relationship labels: A: activation; B: binding; C: causation; CO: correlation; E: expression; EC: enzyme catalysis; I: inhibition; L: molecular cleavage; LO: localization; M: biochemical modification; miT: microRNA Targeting; P: phosphorylation/dephosphorylation; PD: protein-DNA binding; PP: protein-protein binding; PR: protein-RNA binding, RB: regulation of binding; RE: reaction; T: transcription; TR: translocation; UB: ubiquitination., Peer reviewed

1 table., Additional file 5: Table S4. Expression data of defense-related genes in leaves of Control and High-Fe plants during M. oryzae infection (48 hpi)., Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

1 table., Additional file 6: Table S5. Expression data (FPKM and Log2FC) of genes involved in the biosynthesis of diterpene phytoalexins and sakuranetin in leaves of Control and High-Fe plants (M. oryzae-infected and mock-inoculated plants)., Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

Additional file 6: Figure S6. a, Mutations found in human cancer involving the RRAS2 gene. Data obtained from cBioPortal (97,250 patients/100669 samples). Refseq: NM_012250. Ensembl: ENST00000256196. CCDS: CCDS7814. Uniprot: RRAS2_HUMAN. Missense mutations (green dots): 36. Truncating mutations (black dots): 6. Splice mutations (orange dots): 5. b, Quantification by RT-qPCR or total mouse (Rras2) and human (RRAS2) mRNA expression in purified splenic CD19+ B cells from Rras2(Q72L)fl/fl xmb1-Cre (Q72L) mice compared to purified B CD19+ B cells from control WT C57BL/6 mice and to CD19 + CD5+ leukemic B cells from Rosa26-RRAS2fl/flxmb1-Cre mice. Results show data obtained in triplicate normalized to the C57BL/6 control for n = 3 mice per group. All mice were 14 month-old. Data show means ± SEM for three mice per group. *p < 0.05; ns. Not significant (one-way ANOVA test). c, Left, quantification by flow cytometry of total B-cell number in spleens of 14 month-old control and Rras2(Q72L)fl/fl xmb1-Cre mice. Right, two-parameter flow cytometry plot showing frequency of IgM + CD5+ cells within CD19+ splenic B cells of control and Rras2(Q72L)fl/fl xmb1-Cre mice. d, Left, concentration of B-cells per microliter in blood of control and Rras2(Q72L)fl/fl xmb1-Cre mice. Right, two-parameter flow cytometry plot showing frequency of CD19 + CD5+ cells within blood B cells of control and Rras2(Q72L)fl/fl xmb1-Cre mice. e, Frequency of marginal zone (MZ) phenotype (CD21high, CD23low), and follicular (CD21low, CD23high) B cells within CD19+ splenic B cells of control and Rras2(Q72L)fl/fl xmb1-Cre mice. f, Phosflow cytometry analysis of different elements from PI3K-Akt-mTOR, Raf-Erk and proximal BCR signaling pathways. Wild-type CD19+ follicular B cells, CD19 + CD5+ leukemic cells from spleens of Rosa26-RRAS2fl/flxmb1-Cre mice and CD19+ non-leukemic B cells from Rras2(Q72L)fl/fl xmb1-Cre are shown. In grey, background fluorescence of the secondary antibodies. All mice were 23 wk-old. Data show means ± SEM from three mice per group. *p < 0.05; **p < 0.01; ****p < 0.0001 (one-way ANOVA test). g, Phosflow cytometry analysis of different elements from PI3K-Akt-mTOR, Raf-Erk and proximal BCR signaling pathways. CD19 + CD5+ leukemic cells from 30 wk-old Rosa26-RRAS2fl/flxmb1-Cre mice are compared with WT Control follicular (CD23highCD21−), marginal zone (MZ, CD23−CD21high), B1a (CD11b + CD5+) and B1b (CD11b + CD5−) spleen B cell populations. Data show means ± SEM from n = 3 mice per group. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant (one-way ANOVA test)., Fundación Científica Asociación Española Contra el Cáncer Ministerio de Ciencia, Innovación y Universidades H2020 European Research Council Instituto de Salud Carlos III Consejería de Educación, Junta de Castilla y León, Peer reviewed

1 table., Additional file 7: Table S6. Expression data (FPKM and Log2FC) of genes involved in Fe homeostasis in leaves of Control and High-Fe plants (M. oryzae-infected and mock-inoculated plants)., Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

1 table., Additional file 8: Table S7. Statistics for RNAseq analysis of leaves from Control and High-Fe plants, mock-inoculated and M. oryzae-inoculated (48 hpi)., Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

1 table., Additional file 9: Table S8. List of oligonucleotides used in this study., Consejo Superior de Investigaciones Cientificas (CSIC), Peer reviewed

Additional file 7: Figure S7. Ingenuity Pathway Analysis (IPA) of differentially expressed genes associated with mTOR signaling, immunological development, and G1-S checkpoint regulation in leukemic versus normal follicular B cells. Pink-filled symbols: upregulated genes. Green-filled: downregulated genes. Double circle: protein complex; horizontal ellipse: transcription regulator; vertical ellipse: transmembrane receptor, diamond: enzyme; trapezium: transporter; triangle: phosphatase; inverted triangle: kinase; vertical rectangle: G protein-coupled receptor; circle: other. Black arrows: direct interactions; grey/white arrows: indirect interactions. Relationship labels: A: activation; B: binding; C: causation; CO: correlation; E: expression; EC: enzyme catalysis; I: inhibition; L: molecular cleavage; LO: localization; M: biochemical modification; miT: microRNA Targeting; P: phosphorylation/dephosphorylation; PD: protein-DNA binding; PP: protein-protein binding; PR: protein-RNA binding, RB: regulation of binding; RE: reaction; T: transcription; TR: translocation; UB: ubiquitination., Fundación Científica Asociación Española Contra el Cáncer Ministerio de Ciencia, Innovación y Universidades H2020 European Research Council Instituto de Salud Carlos III Consejería de Educación, Junta de Castilla y León, Peer reviewed

Fundación Científica Asociación Española Contra el Cáncer Ministerio de Ciencia, Innovación y Universidades H2020 European Research Council Instituto de Salud Carlos III Consejería de Educación, Junta de Castilla y León, Peer reviewed

13 pages. -- Supplementary Figure 1. Breeding scheme used in the marked-assisted backcross introgression of the Saltol QTL from FL478 (indica) into the background of the rice variety OLESA (temperate japonica rice). -- Supplementary Figure 2. Polymorphism obtained with the SKC10 SSR marker visualized by agarose gel analysis. (A) Saltol QTL region showing the SKC10 SSR marker and relevant salt-related genes positions. (B) PCR products obtained from the Saltol donor (FL478), the recurrent (OLESA) parent and 4 representative introgression lines derived from FL478 x OLESA crosses (BC2F1). C-, negative control, He, heterozygous, Ho, homozygous. Primers are indicated in Supplementary Table 2. -- Supplementary Figure 3. Graphical representation of the genotypes of the Saltol-introgressed rice lines (BC3F3). Genotyping was carried out by KASPar analysis. SNPs are indicated in columns according to their chromosomal location (in mega base-pairs, Mb). Introgression lines (IL1 to IL31) are clustered in four groups (I to IV) depending on the BC3F1 parent from which they derive. The Saltol QTL location (and length) is indicated in the upper part. Homozygous donor (FL478) and recurrent (OLESA) alleles are depicted in blue and white, respectively. The KASPar markers used in this study are listed in Supplementary Tables 1 and 2. -- Supplementary Figure 4. SES score of parental lines (FL478, OLESA) hydroponically grown in modified Yoshida solution containing different NaCl concentrations (60 mM, 80 mM and 100 mM) for 14 days. Box plots show the distribution of SES scores in each line and condition (15 plants/genotype each experiment; T-test, * P < 0.05). Values above each box indicate the mean SES score . -- Supplementary Figure 5. Characterization of salt tolerant introgression lines. (A) Standard evaluation system (SES) scores of visual salt injury of the 30 ILs. Evaluation was performed after 14 days of salt treatment (80 mM NaCl). SES scores are shown as the percentage of plants at each score value. 1, highly tolerant; 3, tolerant; 5, moderately tolerant; 7, sensitive; 9, highly sensitive. ILs are clustered in four groups (I to IV) depending on the BC3F1 parent used. ILs were evaluated in successive rounds, with 5 plants and 10 plants in control and salt conditions respectively in each experiment, and most salt-sensitive ILs were discarded in the following assays. A total of six independent experiments were carried out with the most salt-tolerant ILs. (B) Representative images of IL22 and IL13 plants and parental lines in control and salt conditions after 14 days of treatment. -- Supplementary Figure 6. Plant growth of parental lines (FL478, OLESA) and IL22 plants hydroponically grown in modified Yoshida solution containing 80 mM NaCl. Control plants were not supplemented with NaCl. The leaf number of each genotype at different times of salt treatment is indicated. At least 6 plants per genotype and condition were assayed. -- Supplementary Figure 7. Samples analysed by RNASeq, and comparisons of data sets from each genotype (IL22, OLESA) and condition (control, salt-treated). -- Supplementary Figure 8. Singular enrichment analysis of introgressed indica genes (chromosome 1, blocks 1 and 2, and chromosome 3) using AgriGO (Tian et al., 2017). For a full list of gene IDs, see Supplementary Table 5. -- Supplementary Figure 9. Singular enrichment analysis of japonica genes up-regulated in IL22 plants at 24 h of salt treatment (80 mM NaCl) using AgriGO (Tian et al., 2017). For a full list of gene IDs, see Supplementary Table 8. -- Supplementary Figure 10. Singular enrichment analysis of japonica genes up-regulated in OLESA plants at 24 h of salt treatment (80 mM NaCl) using AgriGO (Tian et al., 2017). For a full list of gene IDs, see Supplementary Table 8. -- Supplementary Figure 11. Singular enrichment analysis of japonica genes down-regulated in IL22 plants at 24 h of salt treatment (80 mM NaCl) using AgriGO (Tian et al., 2017). For a full list of gene IDs, see Supplementary Table 8. -- Supplementary Figure 12. Singular enrichment analysis of japonica genes down-regulated in OLESA plants at 24 h of salt treatment (80 mM NaCl) using AgriGO (Tian et al., 2017). For a full list of gene IDs, see Supplementary Table 8. -- Supplementary Figure 13. Mapman analysis of japonica genes up- and down-regulated in IL22 and OLESA plants at 24 h of salt treatment (80 mM NaCl). Regulation overview, stress and transport schemes are shown. Color scale (yellow to blue) represents the log2 fold change of salt vs. control conditions., Rice is the most salt sensitive cereal crop and its cultivation is particularly threatened by salt stress, which is currently worsened due to climate change. This study reports the development of salt tolerant introgression lines (ILs) derived from crosses between the salt tolerant indica rice variety FL478, which harbors the Saltol quantitative trait loci (QTL), and the salt-sensitive japonica elite cultivar OLESA. Genotyping-by-sequencing (GBS) and Kompetitive allele specific PCR (KASPar) genotyping, in combination with step-wise phenotypic selection in hydroponic culture, were used for the identification of salt-tolerant ILs. Transcriptome-based genotyping allowed the fine mapping of indica genetic introgressions in the best performing IL (IL22). A total of 1,595 genes were identified in indica regions of IL22, which mainly located in large introgressions at Chromosomes 1 and 3. In addition to OsHKT1;5, an important number of genes were identified in the introgressed indica segments of IL22 whose expression was confirmed [e.g., genes involved in ion transport, callose synthesis, transcriptional regulation of gene expression, hormone signaling and reactive oxygen species (ROS) accumulation]. These genes might well contribute to salt stress tolerance in IL22 plants. Furthermore, comparative transcript profiling revealed that indica introgressions caused important alterations in the background gene expression of IL22 plants (japonica cultivar) compared with its salt-sensitive parent, both under non-stress and salt-stress conditions. In response to salt treatment, only 8.6% of the salt-responsive genes were found to be commonly up- or down-regulated in IL22 and OLESA plants, supporting massive transcriptional reprogramming of gene expression caused by indica introgressions into the recipient genome. Interactions among indica and japonica genes might provide novel regulatory networks contributing to salt stress tolerance in introgression rice lines. Collectively, this study illustrates the usefulness of transcriptomics in the characterization of new rice lines obtained in breeding programs in rice., Peer reviewed