J Appl Genetics (2011) 52:161–169 and will greatly facilitate the future breeding of anthocyanin- rich hybrid rice varieties. RT-PCR analysis of selected genes RT-PCR analysis was performed on the 17 unknown and hypothetical genes using 27 RNA samples representing each cultivar. Among the 17 genes, nine showed an up- regulated pattern similar to white rice at all seed develop- mental stages tested, while six genes exhibited a down- regulated pattern. The remaining two genes were both up- and down-regulated, and were omitted as a result of small differences with control rice (Fig. 2). Most of the up- and down-regulated genes appear to be related to major biological changes induced in the antho- cyanin biosynthesis pathway. The results of RT-PCR Table 2 RT-PCR primer list of the 15 candidate genes consist- ing of nine up-regulated and six down-regulated genes 167 analysis coincided largely with the expression profiles obtained through the microarray hybridization experiments. Interestingly, four unknown genes (Os07g0184633, Os03g0247300, Os11g0539600, and Os07g0486400) were highly induced during the early heading stage in the two black rice cultivars. This result suggested that these four unknown genes may play a role in anthocyanin production in the early rice heading stage. However, the Os01g0780900 gene is highly induced during late heading stage. Gene Os12g0425800 demonstrates a difference between Heug- jinju and Heugseol. This gene was not expressed in the cv. Dongjin without anthocyanin, was weakly induced in the Heugjinju cultivar with low anthocyanin, and was highly expressed in the Heugseol cultivar with high anthocyanin. Therefore, this gene may play a role in controlling anthocyanin levels. Primer Sequence (5’ → 3’) Product size/bp Locus ID Up-regulation Os11g0532200 Os01g0781600 Os07g0184633 Os03g0247300 Os12g0425800 Os11g0579300 Os11g0539600 Os01g0780900 Os07g0486400 Down-regulation Os06g0170500 Os11g0224900 Os11g0597700 Os11g0226700 Os07g0429700 Os02g0818700 U1 F U1 R U2 F U2 R U3 F U3 R U4 F U4 R U5 F U5 R U6 F U6 R U7 F U7 R U8 F U8 R U9 F U9 R D1 F D1 R D2 F D2 R D3 F D3 R D4 F D4 R D5 F D5 R D6 F D6 R GCCACTGTTCACGTGCCGGA CCCACGGCAACCACGTCTCC GGCTCCAGTGCGGCAGGAGA TCACGCGTCATGTCCACGCC TCCGATCCCTGGGTCGCTCG GGGGCAGCTCAAGGCAAGGT TTCGTCGCCATGGGGTTGCC GCCATCAACCAGCCCGGCAT GACGGGATGGGTCATGCCGGC GAGGACATCAGCCGCCAAGATTC CAGCAGCTGCGGAAGGTTGC ACACGCTCACGCACAGCAGA TGCGTGCGCAAGTGTAGGCT AAGGCGCAGGCTCCATGCAA GACGGGAGGGAGAGGGGTCG TCCCCACGGGGCTCATCAGG GCCGAGTACCCGCAAGGTGG GTTGGAAGCCACGCGCCTCT GGCAAACCCGGTCCACAGCA ACAGGCTCCAAGCGTTCGGC TGCTCACGAGGGTTGCCAGC CAAGCGGCAAGCCCCCACAT TGTCAAGGCCCAACGCCGTG GCTCCGCTGGCGACAAACCT GAGCCATGCAGCGCTGGGAA TGCTGCACCGCACCAAGGTA CATCAGCAGCGGGAGGCAATG GGGACCAACAGATGGATCGAC GCTCCATCGGCTCTGTCACCG CTTGCAGCCATAGCCCCGCA 619 304 397 604 378 622 537 850 607 541 443 788 476 455 520