164 software (Zeeberg et al. 2003). The statistical significance of these matches was assessed by calculating p-values using the one-sided Fisher exact test for the number of categorized GO terms in the total analysis. False discovery rate (FDR) values were collected from less than 0.05. The selected genes were analyzed by average linkage clustering to group genes with similar function using SAS/STAT (SAS Institute Inc., Cary, NC, USA, http://www.sas.com). Transcription factors were evaluated and clustered using the hyper-geometric analysis method. Candidate genes in the hyper-geometric analyses were defined as those with p-values less than 0.05 according to ANOVA assessment using GeneSpring GX 11 software (Agilent Technologies Inc., Santa Clara, CA, USA, http:// www.chem.agilent.com/). Results and Discussion To evaluate anthocyanin biosynthesis in rice cultivars, we performed a four-stage screen. First, we tested the hypothesis that comparison between cv. Dongjin and high anthocyanin cultivars, such as Dongjin/Heugjinju, Dongjin/Heugseol, and Heugjinju/Heugseol, express the same genes at three time points following the heading stage. Second, COG and GO analysis were performed to classify the genes functionally and identify orthologous genes involved in anthocyanin biosynthesis. Third, transcription factors involved in anthocyanin pigmenta- tion were evaluated and clustered by the hyper-geometric distribution analysis method. Finally, selected unknown and predicted genes related to anthocyanin biosynthesis were verified by reverse transcription-polymerase chain reaction (RT-PCR). Data collection and p-value analysis Microarray analysis was performed to assess three factors (i.e., a white cultivar and two black rice cultivars) and three treatments (i.e., seed developmental stages) in triplicate. From this experiment, a total of 852,876 intensity items was yielded. We screened 12,673 genes to identify those with greater than a 2-fold up- or down-regulation in each combination of the three rice cultivars and three seed developmental stages. The number of genes significantly upregulated in each cultivar during the three seed develop- mental stages ranged from 528 to 3,695. The number of down-regulated genes ranged between 295 and 1,782. The number of significant up-regulated genes for each develop- mental stage was higher than that of down-regulated genes. In the Dongjin/Heugjinju combination, 1,089 upregulated genes were significantly expressed in the heading+ 7 days stage, and this number increased to 3,695 genes at the heading +21 days stage. In the Dongjin/Heugseol combina- J Appl Genetics (2011) 52:161–169 tion, the expression of 928 upregulated genes increased to 2,305 genes at the heading + 21 days stage. In the Heugjinju/Heugseol combination, the expression of 528 upregulated genes increased to 1,682 genes at the heading+ 21 days stage. Finally, 365 genes were upregulated in the two black rice cultivars at all developmental stages and showed a significant correlation (r= 0.6503, p= 0.0325), suggesting that these genes may be associated with pigmentation metabolism. We also identified 57 genes that demonstrated downregulation and showed a significant correlation (r= 0.4391, p = 0.0453). We assumed these genes were related to inhibition of pigmentation production or anthocyanin metabolism. Clusters of orthologous groups (COGs) analysis COGs analysis was performed on the 12,673 candidate genes using the NCBI/COGs database (http://www.ncbi. nlm.nih.gov/COG/). The upregulated genes were classified as general function (21.6%), signal transduction (12.3%), and posttranslational modification, protein, and chaperones (8.2%). Of the regulated genes, 3,271 genes (25.8%) were poorly characterized, and 617 of those genes (4.8%) had an unknown function. The COGs related to anthocyanin function were assumed due to the presence of highly conserved genes between two specific categories: post- translational modification, protein turnover, and chaperones (8.2%) and carbohydrate transport and metabolism (4.7%). GO analysis associated with anthocyanin production The processes that were most affected during anthocyanin production were determined by GO analysis. First, GoMiner categorizes each gene according to its GO terms and mode of gene expression. Modes of expression are denoted as under, over, and change to represent genes that were downregulated, upregulated, or both, respectively. With this analysis, 1,558 and 295 genes were identified as orthologous to Arabidopsis genes and up- or downregu- lated, respectively. GoMiner analysis demonstrated that these genes were involved in 25, 19, and 12 biological processes, cellular components, and molecular functions, respectively. Figure 1 shows the composition ratio of genes in the each category. Candidate genes associated with anthocyanin pigmentation Genes identified through COGs and GO analysis were compared to the rice genome database at http://rgp.dna. affrc.go.jp/E/IRGSP/ and the rice genome system supported by NAAS website http://nabic.naas.go.kr/. Comparison with these databases enabled elimination of genes related