Caught red-handed Rc encodes a basic helix-loop-helix protei(3)

terminus.Myb domains exhibit DNA binding ability and function in protein–protein interactions.These proteins are encoded by the PURPLE PLANT1/COLORED ALEURONE1(Pl1/C1)genes in maize(Zea mays),by the ANTHOCYANIN2(AN2)and AN4genes in petunia(Petunia hybrida)(Spelt et al.,2000),and by the TRANSPARENT TESTA2(TT2)gene in Arabidopsis thaliana(Nesi et al.,2001).

The Myb proteins have been shown to interact with a basic helix-loop-helix(bHLH)protein in each of the three model sys-tems.In Arabidopsis,TT8encodes a bHLH protein(Baudry et al., 2004),whereas petunia has two bHLH proteins involved in anthocyanin regulation,AN1and JAF13(Spelt et al.,2000).In maize,several genes belonging to the RED/BOOSTER(R1/B1) families encode these proteins(Goff et al.,1992).These genes have different tissue specificities and exhibit no activation do-mains or DNA binding activity alone(Goff et al.,1992).Recent experiments in maize suggest that R functions in part to free C1 from interaction with a repressor protein as well as to recognize R-specific anthocyanin promoter elements(Hernandez et al., 2004).Maize also contains a bHLH protein,INTENSIFIER1(In1), whose dominant allele acts as a negative regulator of pigmen-tation(Burr et al.,1996).

In the vegetative tissue of maize,only one member from each of the Myb and bHLH families has been shown to be required for pigmentation.However,in petunia,Arabidopsis,and maize seeds,genes encoding a WD40protein are also required for the expression of anthocyanin biosynthetic genes.These proteins are encoded by the TRANSPARENT TESTA GLABRA1gene in Arabidopsis,by AN11in petunia,and by PALE ALEURONE COLOR1in maize(de Vetten et al.,1997;Baudry et al.,2004; Carey et al.,2004).They have been shown to physically interact with the bHLH protein in petunia and Arabidopsis(Walker et al., 1997;Sompornpailin et al.,2002).In petunia and Arabidopsis, other regulatory factors,such as TT1,a zincfinger protein (Sagasser et al.,2002),and ANTHOCYANINLESS2,a homeo-domain protein(Kubo et al.,1999),have also been described where loss of function results in a complete lack of pigmentation. In addition to being constitutively expressed,anthocyanin and proanthocyanidins can be induced by stresses,including cold, drought,and UV light.The regulatory elements that control these processes are only beginning to be understood.Recent studies have found that the basic domain/leucine zipper family of tran-scription factors,together with the Myb genes,play a role in induced expression(Ithal and Reddy,2004;Hartmann et al.,2005). We report here the cloning of a bHLH gene underlying a quantitative trait locus(QTL)for rice pericarp color.The QTL colocalizes with the mutant Rc.A frame shift deletion before the bHLH domain results in a knockout of proanthocyanidin pro-duction,leading to white rice.

RESULTS

Rough Mapping of QTLs and the Rc Mutant

Previous QTL mapping in this laboratory identified a single, significant QTL associated with red grain(rg7.1)on chromosome 7(Figure1B,i).This QTL was identified in two independent BC2populations derived from crosses between an accession of O.rufipogon(IRGC-105491)from Malaysia and,in one case,a U.S.tropical japonica cultivar,Jefferson,and in the other case,a widely planted tropical indica cultivar,IR64.The log of the odds scores associated with the rg7.1QTL peaks in these two populations were99and33,respectively,and the QTL was detected in multiple environments(Septiningsih et al.,2003).The peak of both QTLs corresponded to the previously mapped position of the mutant locus,brown pericarp,Rc(Kinoshita, 1998).All of the BC2F1plants had red seeds,indicating that the rg7.1locus is dominant for red color,with the dominant allele donated by the O.rufipogon ing the cv Jefferson/ O.rufipogon population,rg7.1encompassed a5.1-centimorgan (cM)region that represented;7.2Mb straddling the border of the centromere on chromosome7(Figure1B,i).The genetic/ physical distance in this region averages1.4Mb/cM,much above the genome average of200to250kb/cM,as expected for a pericentromeric region(Zhao et al.,2002;Wu et al.,2003).An investigation of the genotype–phenotype relationship in285 BC2F2families demonstrated that all18families with red grain contained the O.rufipogon allele at either RM125or the adjacent marker,RG30,suggesting that the gene underlying rg7.1lay between these two markers.

Fine-Mapping of rg7.1

Tofine-map the gene,1410BC2F3plants were genotyped using markersflanking the QTL.The72recombinant plants were genotyped using six markers within the QTL region to locate the recombination break points more precisely,and the seed color of each recombinant line was recorded.Nineteen new simple-sequence repeat(SSR)and insertion/deletion(indel)markers were developed to help define break points across the region (see Supplemental Table1online).To narrow the region respon-sible for rg7.1in each successive generation,we determined which segments of DNA from the red donor parent,O.rufipogon, were shared in all red-seeded progeny and eliminated from further consideration the O.rufipogon segments that appeared in white-seeded progeny.Because the trait is dominant,both the heterozygous and the homozygous O.rufipogon classes had identical phenotypes and therefore were grouped together dur-ingfine-mapping.

In thefinal BC2F6generation,4000plants were genotyped and three classes of informative recombinants were identified, which narrowed the rg7.1QTL to an18.5-kb region(Figure1B, iii).Class1consisted of a single plant with a break point defined by markers RID13and RM21197,located between thefirst and second exons of the gene LOC_Os07g11020.1,as illustrated in panel iv of Figure1B.Upstream of RID13,these plants inherited O.rufipogon DNA,and downstream of RM21197,they were homozygous for cv Jefferson alleles,with the break point delimited to the region between the two markers.Because these plants had white seeds,we concluded that rg7.1could not be located in the region upstream of RID13,which was heterozy-gous for O.rufipogon DNA.Recombinant class2consisted of22 individuals having recombination break points between RID14 and RID15.This14-kb region was highly repetitive,precluding the development of additional markers to help resolve the

Rc Encodes bHLH Protein Red Rice285