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Additional file 1: Figure S1. PCA was applied to the Leaf4, Leaf7, and petal data sets to identify trends in shape and size variations among MAGIC lines. The outline of a leaf was represented by the Cartesian coordinates of its 25 points, and similarly, a petal was represented by 25 points in the same way. The GIF showed the data set for each PC expressed in standard deviations from the mean position of the point within the collection of leaves and petals. Figure S2. Range of PC values obtained for the leaf and petal allometric model. The mean of each MAGIC line is represented by a blue diamond, and the bars display the range of all values observed for this line. PC units are standard deviations. Figure S3. Variations along each PC for Leaf4, Leaf7 and Petal allometry within the MAGIC lines. Each histogram represents the distribution of MAGIC lines along one of the PCs from the leaf and petal allometry model. Figure S4. QTL scan of the PCAs for the fourth leaf allometry among MAGIC lines. Figure S5. QTL scan of the PCAs for the seventh leaf allometry among MAGIC lines. Figure S6. QTL scan of the PCAs for petal allometry among MAGIC lines. Figure S7. QTL scan of the PCAs for the fourth leaf and petal allometry covariation among MAGIC lines. Figure S8. QTL scan of the PCAs for the seventh leaf and petal allometry covariation among MAGIC lines. Figure S9. QTL scan of the life-history traits among MAGIC lines. Figure S10. The genetic correlation between life-history traits and the allometry model. The vertical lines represent the QTLs overlapping with the peak SNP within 1 Mb. The different QTLs with the same accession allele conferring maximum effects are shown in red. Figure S11. The promoter sequence alignment and expression analysis of candidate genes. The candidate gene ERECTA (AT2G26330) in the LF7_2.1 locus, obtained the minimum Leaf7.PC2 value in the Bur-0 accession, had specific variation in the Bur-0 promoter sequence and had the lowest expression level in Bur-0. The second candidate gene AGO4 (AT2G27040) in the LF7_2.1, PE_2.5 and LF7PE_3.2 loci had specific variation in the promoter in the Ler-0 accession, with the maximum Leaf7.PC2, Petal.PC2 value and minimum Leaf7-Petal.PC3 value and obtained the highest expression level in Ler-0.

Quantitative analysis of H129-ΔTK-tdT monosynaptic tracing. Helper AAVs and H129-ΔTK-tdT were sequentially injected at VPM (a), nRT (b) or VTA (c) of wild type C57BL/6, PV-Cre or DAT-Cre mice respectively, as described in Fig.4-6. The brains’ corona slices were observed with an interval of 120 μM (every 4 slices), and the amounts of the labeled cell at the indicated brain regions were counted. The starter cells at the injection site were detected at Day32 as tdTomato and GFP co-expressing neurons (opened circle, each circle represents one mouse), and the tdTomato positive neurons at other brain regions were observed at Day 32 (filled triangle, each circle represents one mouse). (PDF 109 kb)

Anterograde transmission of H129-G4 from retina to CNS (a) Schema of the simplified mouse visual pathway. LGN, lateral geniculate nucleus; LP, lateral posterior thalamic nucleus; SC, superior colliculus; V1 and V2, primary and secondary visual cortex. (b-d) H129-G4 tracing from retina. H129-G4 was injected into the right retina of wild-type C57BL/6 mice, and images were obtained at 6 dpi. Representative images at LGN, LP (b) and visual cortex (c) are shown. Selected regions are magnified correspondingly, and representative single neurons are presented (d). (PDF 1100Â kb)

H129-ΔTK-tdT alone in various brain regions. (a) Parameters for H129-ΔTK-tdT injection. (b-g) Representative labeling results. H129-ΔTK-tdT was injected into the indicated brain regions of wild type C57BL/6 as listed in the table (a), and the brains were perfused at 10 dpi. The coronal brain slice throughout the brain were observed after staining for NeuN (blue), GFAP (green) and tdTomato (red). Representative images of upstream and downstream region of the injection sites are shown. The white arrows indicated the injection sites. The neurons in the upstream brain region labeled by virus terminal invasion are indicated by the white arrowheads (e3). OB, olfactory bulb; Pir, piriform cortex; MOE, main olfactory epithelium; M1, primary motor cortex; DG, dentate gyrus; LEC, lateral entorhinal cortex; LGN, lateral geniculate nucleus; V1, primary visual cortex; VTA, ventral tegmental area; LDTg, laterodorsal tegmental nucleus. (PDF 2370 kb)

Absence of labeled cells upstream of nRT-PV neurons. H129-Î TK-tdT and AAV-DIO-TK-GFP were injected into the nRT of PV-Cre mice as shown in Fig.5. The animals were perfused Day 32, and the coronal brain slices throughout the entire brains were observed. The representative images of S1, the upstream region of nRT, are shown. (PDF 929Â kb)

Application of H129-G4 in tree shrew (a) Comparison of mouse and tree shrew brains. The brains of adult mouse and tree shrew are imaged with top (left) or side view (middle) after perfusion and fixation. The average size and weight of the brains are presented (right) as mean ± SD (standard deviation) from 5 animals in each group. (b-g) Tracing results H129-G4 in tree threw M1 circuit. H129-G4 and CTB were injected into the M1of adult tree shrews, and the brains were perfused at 6 dpi. Representative images of the coronal brain sections are presented, and the boxed regions are displayed with a higher magnification. M1, primary motor cortex; IRd, infraradiata dorsalis; Pir, piriform cortex; Pu, putamen; Cl, claustrum (Cl); PC, paracentral thalamic nucleus; VL, ventrolateral thalamic nucleus; V1, primary visual cortex. (h) A representative H129-G4 labeled single neuron in tree threw. A representative GFP-labeled neuron around the injection site is shown, and the magnified images of the apical (h1-h3) and basal dendrites (h4) are presented in the right panels. (PDF 3120 kb)

In vitro replication of the H129-ΔTK-tdT. To determine the growth property of H129-ΔTK-tdT which lacks TK, fetal mouse hippocampal and cortical neurons (Neuron) or Vero cells were infected with H129-ΔTK-tdT or the TK competent strain H129-tdT at an MOI of 0.02. At the indicated time point, virus titers in the cell culture were determined by standard plaque forming assay. Shown is the representative data from 3 independent experiments, and presented as the Mean ± SD from triplicates. (PDF 183 kb)

The microfluidic plate (a) The schematic structure diagram of the microfluidic system. (b) Axons through the microchannels. Freshly isolated fetal mouse hippocampal and cortical neurons were seeded into one chamber of the microfluidic plate, and cultured for 7 days with positive hydrostatic pressure in the soma chamber. Then the plate was disassembled and stained with antibodies against Tau and Map2. Shown is the representative image from 3 plates. (c) Pre- and post-synaptic markers in the afferent chamber. Neurons were sequentially plated into both chambers at Day 1 and Day 5 respectively, and cultured for additional 7 days with positive hydrostatic pressure in the efferent chamber. The plate was disassembled on Day 12 and stained for pre-synaptic marker synaptophysin (SYP) and post-synaptic marker PSD-95. The nuclei were counterstain with Hoechst dye. Shown is the representative image from 3 plates. (d) No inter-compartment leakage between the chambers. Vero cells were cultured in one chamber with positive hydrostatic pressure, and H129-G4 was added into the opposite chamber to a final concentration of 2.5 × 109 pfu/ml. The GFP signal in the Vero cell culture chamber was monitored, and show is the representative image at 72 hpi. (PDF 676 kb)

Invasion and transmission of H129-G4 in the VPM-S1 circuit. (a) Simplified schema of the VPM-S1 circuit. VPM, ventral posteromedial thalamic nucleus; nRT, nucleus of reticular thalamus; S1, primary somatosensory cortex; IV, V and VI, layer 4, 5 and 6 of the cortex. (b) Representative tracing results of H129-G4 in VPM-S1 circuit. H129-G4 (1 × 106 pfu in 200 nl) was injected into the VPM of wild-type C57BL/6 mice together with Alexa Fluor 594-conjugated CTB (CTB, red). The animals were perfused at the indicated time points, and representative images of the coronal brain slice at the VPM-S1 regions are shown. The boxed areas are magnified and presented in the right panel. The layers of the S1 cortex were determined according to NeuN staining and indicated by the dotted lines. (PDF 503 kb)

In vitro replication of the H129-ΔTK-tdT. To determine the growth property of H129-ΔTK-tdT which lacks TK, fetal mouse hippocampal and cortical neurons (Neuron) or Vero cells were infected with H129-ΔTK-tdT or the TK competent strain H129-tdT at an MOI of 0.02. At the indicated time point, virus titers in the cell culture were determined by standard plaque forming assay. Shown is the representative data from 3 independent experiments, and presented as the Mean ± SD from triplicates. (PDF 183 kb)

Quantitative analysis of H129-ΔTK-tdT monosynaptic tracing. Helper AAVs and H129-ΔTK-tdT were sequentially injected at VPM (a), nRT (b) or VTA (c) of wild type C57BL/6, PV-Cre or DAT-Cre mice respectively, as described in Fig.4-6. The brains’ corona slices were observed with an interval of 120 μM (every 4 slices), and the amounts of the labeled cell at the indicated brain regions were counted. The starter cells at the injection site were detected at Day32 as tdTomato and GFP co-expressing neurons (opened circle, each circle represents one mouse), and the tdTomato positive neurons at other brain regions were observed at Day 32 (filled triangle, each circle represents one mouse). (PDF 109 kb)

Invasion and transmission of H129-G4 in the VPM-S1 circuit. (a) Simplified schema of the VPM-S1 circuit. VPM, ventral posteromedial thalamic nucleus; nRT, nucleus of reticular thalamus; S1, primary somatosensory cortex; IV, V and VI, layer 4, 5 and 6 of the cortex. (b) Representative tracing results of H129-G4 in VPM-S1 circuit. H129-G4 (1 × 106 pfu in 200 nl) was injected into the VPM of wild-type C57BL/6 mice together with Alexa Fluor 594-conjugated CTB (CTB, red). The animals were perfused at the indicated time points, and representative images of the coronal brain slice at the VPM-S1 regions are shown. The boxed areas are magnified and presented in the right panel. The layers of the S1 cortex were determined according to NeuN staining and indicated by the dotted lines. (PDF 503 kb)

Anterograde transmission of H129-G4 from retina to CNS (a) Schema of the simplified mouse visual pathway. LGN, lateral geniculate nucleus; LP, lateral posterior thalamic nucleus; SC, superior colliculus; V1 and V2, primary and secondary visual cortex. (b-d) H129-G4 tracing from retina. H129-G4 was injected into the right retina of wild-type C57BL/6 mice, and images were obtained at 6 dpi. Representative images at LGN, LP (b) and visual cortex (c) are shown. Selected regions are magnified correspondingly, and representative single neurons are presented (d). (PDF 1100Â kb)

Terminal invasion of H129-G4 in vitro (a) Dose related incidence of H129-G4 terminal invasion. Fetal mouse hippocampal and cortical neurons were cultured as described above, H129-G4 was added to the terminal chamber at the indicated final concentrations, and GFP signal was monitored daily. Representative images at 48 hpi from 3 plates at each concentration group are shown. The labeled neurons are indicated with the dotted boxes and magnified in the lower panels. Scale bar = 100 μm. (b) Census of H129-G4 terminal invasion. Hippocampal and cortical neurons, trigeminal ganglion (TG) and dorsal root ganglion neurons (DRG) were cultured in microfluidic plates at the indicated cell amount. H129-G4 was added to the axonal terminal chamber at different final concentrations, and the GFP positive neurons in each plate were counted. Data were from 3 plates under each condition, and results are presented as mean ± SD. (c) Terminal invasion of VSV. Similarly, VSV-GFP was added into the terminal chamber of hippocampal and cortical neurons to a final concentration of 1 × 107 pfu/ml. The representative image from 3 plates at 48 hpi is shown. Scale bar = 100 μm. (PDF 429 kb)

H129-ΔTK-tdT alone in various brain regions. (a) Parameters for H129-ΔTK-tdT injection. (b-g) Representative labeling results. H129-ΔTK-tdT was injected into the indicated brain regions of wild type C57BL/6 as listed in the table (a), and the brains were perfused at 10 dpi. The coronal brain slice throughout the brain were observed after staining for NeuN (blue), GFAP (green) and tdTomato (red). Representative images of upstream and downstream region of the injection sites are shown. The white arrows indicated the injection sites. The neurons in the upstream brain region labeled by virus terminal invasion are indicated by the white arrowheads (e3). OB, olfactory bulb; Pir, piriform cortex; MOE, main olfactory epithelium; M1, primary motor cortex; DG, dentate gyrus; LEC, lateral entorhinal cortex; LGN, lateral geniculate nucleus; V1, primary visual cortex; VTA, ventral tegmental area; LDTg, laterodorsal tegmental nucleus. (PDF 2370 kb)

Invasion and transmission of H129-G4 in the visual pathway. Different amount of H129-G4 was injected into the left LGN (a) or V1 (b) of wild-type C57BL/6 mice together with CTB, respectively. The animals were perfused at the indicated time points, and the coronal brain slices throughout the entire brains were observed. Representative images at V1, LGN and retinas are presented. (PDF 1400Â kb)

The microfluidic plate (a) The schematic structure diagram of the microfluidic system. (b) Axons through the microchannels. Freshly isolated fetal mouse hippocampal and cortical neurons were seeded into one chamber of the microfluidic plate, and cultured for 7 days with positive hydrostatic pressure in the soma chamber. Then the plate was disassembled and stained with antibodies against Tau and Map2. Shown is the representative image from 3 plates. (c) Pre- and post-synaptic markers in the afferent chamber. Neurons were sequentially plated into both chambers at Day 1 and Day 5 respectively, and cultured for additional 7 days with positive hydrostatic pressure in the efferent chamber. The plate was disassembled on Day 12 and stained for pre-synaptic marker synaptophysin (SYP) and post-synaptic marker PSD-95. The nuclei were counterstain with Hoechst dye. Shown is the representative image from 3 plates. (d) No inter-compartment leakage between the chambers. Vero cells were cultured in one chamber with positive hydrostatic pressure, and H129-G4 was added into the opposite chamber to a final concentration of 2.5 × 109 pfu/ml. The GFP signal in the Vero cell culture chamber was monitored, and show is the representative image at 72 hpi. (PDF 676 kb)

Terminal invasion of H129-G4 in vitro (a) Dose related incidence of H129-G4 terminal invasion. Fetal mouse hippocampal and cortical neurons were cultured as described above, H129-G4 was added to the terminal chamber at the indicated final concentrations, and GFP signal was monitored daily. Representative images at 48 hpi from 3 plates at each concentration group are shown. The labeled neurons are indicated with the dotted boxes and magnified in the lower panels. Scale bar = 100 μm. (b) Census of H129-G4 terminal invasion. Hippocampal and cortical neurons, trigeminal ganglion (TG) and dorsal root ganglion neurons (DRG) were cultured in microfluidic plates at the indicated cell amount. H129-G4 was added to the axonal terminal chamber at different final concentrations, and the GFP positive neurons in each plate were counted. Data were from 3 plates under each condition, and results are presented as mean ± SD. (c) Terminal invasion of VSV. Similarly, VSV-GFP was added into the terminal chamber of hippocampal and cortical neurons to a final concentration of 1 × 107 pfu/ml. The representative image from 3 plates at 48 hpi is shown. Scale bar = 100 μm. (PDF 429 kb)

Invasion and transmission of H129-G4 in the visual pathway. Different amount of H129-G4 was injected into the left LGN (a) or V1 (b) of wild-type C57BL/6 mice together with CTB, respectively. The animals were perfused at the indicated time points, and the coronal brain slices throughout the entire brains were observed. Representative images at V1, LGN and retinas are presented. (PDF 1400Â kb)