162 been characterized in several plant species (Holton and Cornish 1995). The main pigments comprising anthocyanin of dark purple rice are cyanidin (cyanidin-3-O-β-glucoside) and peonicin (peonidin-3-O-β-glucoside) (Ryu et al. 1998; Abdel-Aal et al. 2006). Recently, anthocyanins have been recognized as health- promoting food ingredients due to their antioxidant activity (Nam et al. 2006; Philpott et al. 2006), and anticancer (Hyun and Chung 2004), hypoglycemic (Tsuda et al. 2003), and anti-inflammatory effects (Tsuda et al. 2002). For example, pigment-supplemented diets of black rice reduced oxidative stress in mice (Xia et al. 2003) and its pigment fraction may have antiatherogenic activity (Xia et al. 2006). A recent report showed that black rice is a good source of fiber, minerals, and several important amino acids (Zhang et al. 2005), and there is increased interest in alternative sources of anthocyanins due to a rising demand for economical sources of natural and stable pigments (Hu et al. 2003; Zhang et al. 2004). The examined cv. Heugjinju has the richest anthocyanin content among dark purple rice cultivars, with the highest cyanidin-3-glucoside (C3G) pigment content of these culti- vars (Ryu et al. 2000; Kim et al. 2007). C3G is known to have high antioxidative function and is the most powerful among the 14 main anthocyanins (Ding et al. 2006). Orthology assignment in pigmentation biosynthesis is a critical prerequisite of numerous comparative genomics procedures (Dessimoz et al. 2006). The clusters of orthologous groups (COGs) analysis is delineated by comparing protein sequences encoded in complete genomes, representing major phylogenetic lineages. It is important that identification of orthologs is derived from the COGs methodology (Remm et al. 2001). The Gene Ontology (GO) is a major bioinformatics initiative with the aim of standardizing the representation of genes involved in the identification of orthologs (Ashburner et al. 2000). Recently, a number of studies have demonstrated that microarray analyses can be used to examine changes in genome-wide gene expression. Microarray experiments have been used to analyze gene expression changes in a number of crop species, including rice (Kim et al. 2009; Kim et al. 2010; Troester et al. 2009). Here, we investigated anthocyanin gene expression used by black rice to gain insight into the causes of pigment production. Our results will greatly facilitate future breed- ing of hybrid varieties of rice rich in anthocyanin. Materials and methods Rice The white rice used was the cv. Dongjin. Black rice was a Heugjinju and Heugseol cultivar. Three rice cultivars were J Appl Genetics (2011) 52:161–169 cultivated under controlled conditions in an experimental field of the National Academy of Agricultural Science (NAAS). Heugjinju represents the first breeding cultivar of black rice in Korea (Moon et al. 1998). Heugseol is a new black pericarp cultivar for high anthocyanin, which was developed by the National Institute of Crop Science in 2008 (Choi et al. 2008). It was derived as a crossbreed between Seolgaeng/Heugjinju. Experimental design This experiment was designed to assess three factors (i.e., one white and two black rice cultivars) and three treatments (i.e., three seed developmental stages, heading +7 days, + 14 days, and +21 days), in triplicate. The samples were harvested from research plots in the experimental fields in 2009. They were manually hulled and ground to obtain a fine powder using a cyclone mixer mill (HMF-590; Hanil, Seoul, Korea) and a mortar and pestle. The milled rice powders were kept at –80°C before RNA extraction. The results of anthocyanin analysis showed total anthocyanin content of Heugjinju cultivar to be 295 mg/ 100 g and Heugseol to be 417 mg/100 g. The major components for both consisted of cyanidin-3-O-glucoside (C3G) and peonidin-3-O-glucoside. The distribution ratio of individual anthocyanins showed Heugjinju to have 95.4% C3G and Heugseol, 94.7% C3G. In contrast, the Dongjin cultivar (white seed) showed no detectable anthocyanins. Therefore, the experiments were performed with Dong- jin (white color seed of no anthocyanin), Heugjinju (black color seed of lower anthocyanin) and Heugseol (black color seed of higher anthocyanin). We assumed that two black cultivars would be useful for comparing the gene expres- sion of anthocyanins because they have a similar genetic background but very different anthocyanin content. RNA extraction Total RNA was extracted from seeds of two black rice cultivars, Dongjin and Heugjinju, and the wild type Dongjin cultivar. We performed at least three replicates for each treatment. Frozen samples were homogenized with a mortar and pestle in liquid N2. The ground powder was transferred to empty falcon tubes on liquid N2 until the homogenization procedure was ready to be performed, and 0.5 mL of RLC buffer (Qiagen, Hilden, Germany) was added. Because of the high starch content of rice seed, the homogenates were shaken with a vortex for only 10 s, and then plant debris was pelleted by centrifugation. Super- natants were extracted using an RNeasy Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instruc- tions. The RNA samples were further purified using