The two genes, C (chromogen for anthocyanin) and A (anthocyanin activator) are known to be basic genes for forming anthocyanin pigment in different organs of rice, and P (colored apiculus) is also essential for the pigmentation in the apiculus (Takahashi 1957, 1982). Series of multiple alleles at these loci, especially at the C locus, contribute varying degrees of apiculus coloration and some alleles at the C locus are responsible for so called tawny color without the A (Takahashi 1982). In addition, the genetic diversity for anthocyanin pigmentation seems to be higher in cultivated rice than the wild progenitors (Oka 1989). We intend to investigate the allelic differentiation at the C locus in order to understand the genetic diversity preserved in cultivated rice.

The C locus is located on the short arm of chromosome 6 and is linked to the wx which also shows allelic differentiation among rice cultivars. Based on synteny of maps between rice and maize, the region including these genes is present on chromosome 9 of maize and the maize C1 anthocyanin regulatory gene is located in the region. The C1 encodes a MYB like protein factor which activates the transcription of a number of structural genes involved in anthocyanin pigment biosynthetic pathway (Paz-Ares et al. 1987). The rice homologue (Os-C1) of the maize C1 was cloned from cDNA library of Purpleputtu seedlings (Reddy et al. 1998). The present study was carried out to compare the genomic sequences of the Os-C1 of tawny-colored (T65wx, Japonica type) and colorless (IR36 and Patpaku, Indica type) lines. DNA fragments were amplified with PCR and were sequenced, showing the presence of three exons as expected. The results revealed that the two colorless lines had a 10-bp deletion and T65wx had a non-synonymous substitution in the 3rd exon, when compared with the OsC1 from Purpleputtu. This suggested that the deletion might be responsible for colorless apiculus since the two colorless lines had the same deletion. Regarding the location of the Os-C1, 5 NILs which carried different segments of chromosome 6 from Patpaku were examined. Comparisons by sequencing revealed that 2 colorless NILs (A and D) had a 10-bp deletion and 3 colored

NILs (F, G and I) had the same sequence as that of T65wx (Fig. 1). This indicates that the difference in the Os-C1 is related to the phenotype for apiculus coloration and the Os-C1 is located on chromosome 6.

To locate the Os-C1 precisely, 67 F₂ plants of T65wx x IR36 were analyzed using RFLP markers on the short arm of chromosome 6. The 10-bp deletion in the 3rd exon was analyzed with PCR products using primers of #3 and #4 (Fig. 1) which were separated on 5% acrylamide gel. The difference in fragment size was easily detected and the heterozygotes showed two-bands. The Os-C1 was linked to RZ588 marker with map distance of 6.2 cM and was located between RZ588 and G200. It was also linked to the wx with 27.1 cM which was consistent with the recombination value reported in Indica-Japonica crosses (Oka 1989), suggesting that the Os-C1 is a candidate for the C although further studies are needed. Comparisons of the Os-C1 among lines with different alleles at the C locus are now in progress.

References

Oka, H.I., 1989. Distribution of gene diversity in Indica and Japonica rice varieties and their wild progenitors. RGN 6: 70-71.

Paz-Aryes, J., D. Ghosal, U. Wienand, P.A. Peterson and H. Saedler, 1987. The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. EMBO J. 6: 3553-3558.

Reddy, V.S., B.E. Scheffler, U. Wienand, S.R. Wessler and A.R. Reddy, 1998. Cloning and characterization of the rice homologue of the maize C1 anthocyanin regulatory gene. Plant Mol. Biol. 36: 497-498.

Takahashi, M.-E., 1957. Analysis on apiculus color genes essential to anthocyanin coloration in rice. J. Fac. Agr. Hokkaido Univ., 50: 266-362.

Takahashi, M.-E., 1982. Gene analysis and its related problems. J. Fac. Agr. Hokkaido Univ., 61: 91-142.