genes were identified as being unique and 23 other genes had a total of 87 additional gene family members. Identification of 72 Genes for Hierarchical Clustering Analysis RiceCyc (http://pathway.gramene.org/RICE/class-instances? object = Pathways) is a web-based tool curated by Gramene (http://www.gramene.org/) and provides biochemical/metabolic pathways. Seven pathways in RiceCyc are associated with 7 mutants identified in this study. U11 is a part of the ammonia assimilation pathway and two genes in this pathway are displayed. U1 is a part of the MEP pathway and eight genes in this pathway are displayed. U10 is a part of the carotenoid biosynthesis pathway and eleven genes in this pathway are displayed. P9-1 is a part of the nitrate assimilation pathway and one gene in this pathway is displayed. P5-1 is a part of the photorespiration pathway and eight genes in this pathway are displayed. U3 is a part of the photosynthetic light reaction pathway by PSI and PSII and fifteen genes in this pathway are displayed. P13-1 is a part of the Vitamin C biosynthesis pathway and eight genes in this pathway are displayed (Figure 4; Table S4). The abscisic acid (ABA; there are 3 genes), chlorophyll (9 genes), and vitamin E biosynthetic (5 genes) pathways mediated by precursor or final product of carotenoid biosynthesis are added for co-expression analysis [31,82] (Table S4). Unique sequences or predominantly expressed gene family members were selected for this analysis. Single-step reactions unlinked to these or other pathways were established for the other genes for which mutant phenotypes were identified during this study, i.e. U4 (Os02g57030), U5 (Os03g04470), and Ca1 (P2-1, Os1g45274) (see, for example, Table 2). Pathway Analyses Incorporating Gene Expression Profiling Data The pathways used in this study were developed using Gramene RiceCyc (http://pathway.gramene.org/RICE/class-instances? object = Pathways). Candidate genes in the pathways having evidence of expression, such as expressed sequence tags (ESTs) or full length cDNAs, were available at the above website. Additional candidate genes for which no evidence of gene expression was available were selected based on Arabidopsis best-hit genes homologous to TIGR version 5 gene models. Probable gene family members of all candidate genes were checked against Table S7 which lists all rice gene family members. For the co-expression analysis, we selected 72 genes in 13 biochemical/metabolic pathways or reactions associated with the 10 genes for which we had identified mutant phenotypes in this study (Figure 4). As a result, 10 gene clusters were identified. By using Cytoscape software, we deduced relationships among the different biochemical pathways or reactions (Figure S7). To do this, we considered the 10 gene clusters classified by co-expression analysis in Figure 4 as different functional groups. (The twelve boxes with different colors indicate the different biochemical pathways or reactions in Figure S7. Of them, the gray-colored box indicates reactions of unknown genes U4 and U5). In addition, co- expressed gene-clusters in Figure 4 were marked with 10 differently colored lines in Figure S7. Each line indicates an enzyme reaction carried out by the product of one of 72 genes in Figure 4. These gene products are numbered in the 10 biochemical pathways with different colored rectangular boxes (Figure S7). Each rectangular box (node) indicates chemical compounds in the pathway. Functional Analysis of Singleton and Gene Families Supporting Information Figure S1 Previously Published Defective Phenotypes Associat- ed with Unique Genes or Gene Family Members Predominantly Expressed in the Light involved in the Rice Chlorophyll Biosynthesis Pathway. Mutations in genes ChlH, ChlI, ChlD, ChlM and Cao1 in the rice chloroplast biosynthesis pathway showed leaf- color defective phenotypes. White box indicates normalized spot intensity in the light and black box indicates normalized spot intensity in the dark. Numeric numbers in X-axis indicate normalized spot intensity from NSF45K array. Arrows indicate predominantly light-induced gene family members (i.e. ChlM1 and Cao1). ChlH, magnesium-chelatase subunit H family protein gene at step 1; ChlI, magnesium-chelatase subunit I family protein gene at step 1; ChlD, magnesium chelatase ATPase subunit D protein gene at step 1; ChlM1 and ChlM2, magnesium-protoporphyrin O- methyltransferase genes at step 2; and Cao1 and Cao2, chlorophyll a oxygenase genes at step 7. Found at: doi:10.1371/journal.pgen.1000164.s001 (1.38 MB EPS) Figure S2 Strategy for Functional Validation of Candidate Genes. Found at: doi:10.1371/journal.pgen.1000164.s002 (0.97 MB JPG) Figure S3 Expression Patterns of 37 Rice Candidate Genes Selected For Functional Validation Based on the NSF45K Light vs. Dark Dataset. (A) Differential gene expression patterns for 12 unique genes. (B) Differential gene expression patterns for 13 gene families which include one member predominantly expressed in the light. (C) Differential gene expression patterns for 12 gene families without one predominantly expressed gene family member in the light. Arrows indicate genes for which the homozygous mutant resulted in a defective phenotype. Triangles indicate genes for which somaclonal variations mask the phenotypes in progenies homozygous for an insertional mutation in the gene. Genes which have only asterisks in (B) and sharps in (C), when homozygous for an insertional mutation, also did not segregate with a defective phenotype. Found at: doi:10.1371/journal.pgen.1000164.s003 (0.48 MB JPG) Figure S4 Examples of Mutant Lines for which Phenotypes were Not Determined Due to Apparent Somaclonal Variations. Red arrows indicate homozygous progenies. WT indicates segregants showing normal growth phenotypes. U indicates unique genes; GF, existence of gene family; P, predominantly light-induced gene family members; and NP, not predominantly light-induced gene family members. These phenotypes were repeatedly observed at least two times. Found at: doi:10.1371/journal.pgen.1000164.s004 (0.42 MB JPG) Figure S5 Phenotype Associated with the Mutation in A Not Predominantly Light-induced Gene (Os07g05000, NP8-2). Line 3A-03008 has a T-DNA insertion in rice oxidoreductase gene and the T-DNA insertional homozygous lines showed pale green pheno- type. A black arrow indicates a homozygous progeny. Found at: doi:10.1371/journal.pgen.1000164.s005 (0.09 MB JPG) Figure S6 Arabidopsis Microarray Data of Wild Type and pif3 Mutants Treated with Dark and Red Light for 32 Genes Which were Screened for Identifying Mutants in Photomorphogenic Signaling Pathway and their Gene Family Members. Arabidopsis microarray data of wild type and pif3 mutants treated with dark and 1 hour red light are used for this analysis [88]. Left panel shows average log2 fold changes of red light over dark in wild type, red light over dark in pif3 mutant, wild type over pif3 mutant in dark, and wild type over pif3 mutant in dark. Middle in this figure displays average spot intensity of dark in wild type and pif3 mutant, PLoS Genetics | www.plosgenetics.org 15 August 2008 | Volume 4 | Issue 8 | e1000164