Caught red-handed Rc encodes a basic helix-loop-helix protei(8)

the bHLH domain of an1produce null alleles,a parallel to rc in rice(Spelt et al.,2002).

Functional Parallels

In maize,petunia,and Arabidopsis,the bHLH proteins R/B,AN1, and TT8interact with a Myb transcriptional activator(C1,AN2, and TT2,respectively)to promote transcription of the anthocy-anin and proanthocyanidin structural genes.In rice,only Rc has been reported as necessary for the production of colored seeds, although Rd is needed for the seed color to be red.The action of Rd is not consistent with that of the Myb transcription,as a functional copy of Rd is not required for the presence of any color in the pericarp.The Rd locus may be a biosynthetic gene that, when nonfunctional,results in the accumulation of a brown proanthocyanidin precursor.The rice gene OsC1,with similarity to the maize Myb gene C1,has been cloned on rice chromosome 6(Saitoh et al.,2004).This locus is acknowledged to have a role in purple(anthocyanin)rice pigmentation but has not been shown to play a role in red(proanthocyanidin)rice pigmentation.

It is possible that,unlike the anthocyanin regulatory systems in maize,petunia,and Arabidopsis,the bHLH gene encoded by the Rc locus is sufficient to activate the transcription of the rice structural anthocyanin genes alone.The bHLH genes do function as transcription factors in homodimers or heterodimers in animal systems,so there is no inherent reason that proteins containing this domain could not activate transcription on their own(Heim et al.,2003).In maize,the PERICARP COLOR1Myb transcription factor has evolved so that it does not require interaction with a bHLH protein to activate the transcription of genes involved in phlobaphene production(Grotewold et al.,1994).An analogous change may have occurred in rice to allow the bHLH protein to function without an interacting partner.It is more likely,however, that rice also requires an interaction with a Myb transcription factor,but if both the red and white rices used in the genetic studies contain a functional copy of the Myb locus,it would not segregate and hence would not be detected as a contributor to pericarp color.If this is the case,a plant carrying a null allele at the Myb locus and a functional Rc should have white seeds. Despite the differences in function between Rc and In1from maize,the phylogeny of bHLH genes and Ks values support an orthologous relationship.It should be noted that the expected Ks for orthologous sequences(0.65)is also within the range expected of ancient polyploid events in these taxa(Schlueter et al.,2004). Polyploidy could also have produced duplicated regions in the common ancestor of these taxa.If a paralogous region was lost in both the rice and maize lineages after divergence,then Rc rice and In1maize could be ancient paralogs but still have the Ks values we found.Regardless of the possibility of ancient paralogy,In1would still have diverged in function from the other positive regulators of anthocyanin and proanthocyanidin found in clade1. Phenotypic Associations

Red pericarp has long been used as marker for the cluster of domestication traits associated with weedy rice,including dor-mancy and shattering(Gu et al.,2005).Several studies have placed QTLs for dormancy and shattering in the pericentromeric region of chromosome7,encompassing the Rc locus.With the cloning of Rc,it is now possible to ask whether this association is the result of linkage or pleiotrophy.Given the reduced rate of recombination within the rg7.1QTL,it is logical that genes for shattering,dormancy,and pericarp color have simply hitchhiked together in a linkage block.Indeed,fine-mapping of a rice shattering gene in this region has recently shown that this gene is tightly linked to Rc,although it occupies a different position on chromosome7(H.Ji,personal communication).It is also pos-sible that Rc acts pleiotropically,as do many of the other bHLH proteins presented in the phylogeny(Payne et al.,2000;Spelt et al.,2002;Bernhardt et al.,2003;Zhang et al.,2003).Using the recombinant lines generated in this work,we will be able to test these different hypotheses.

Rice and wheat(Triticum aestivum)are similar in that red pericarp in both species can be eliminated by one -parative mapping shows no homology between the position of the Rc gene in rice and the R gene controlling red pericarp in wheat.A reverse genetics approach also failed to locate any ESTs from wheat that map to the Rc locus,although this is not surprising,because no rice or maize ESTs have been found for this locus either.Our work confirms that the Rc transcript only amplifies with a high sensitivity Taq polymerase,and this sug-gests that low transcript abundance may also explain the lack of EST hits in wheat.The R locus in wheat may be orthologous to the Rd gene in rice,given their homologous positions on wheat chromosome3and rice chromosome1.Although the systems look similar phenotypically,molecular genetics analysis sug-gests that the mutations leading to white pericarp occurred at different points in the pathway.

Recombinational Analysis

The rg7.1locus was originally mapped to a7.2-Mb region that included the centromere on rice chromosome7.Given the low frequency of recombination across this region,it was not clear whether positional cloning would be feasible.This study dem-onstrates that even in regions that are recombinationally re-pressed,map-based gene isolation offers a viable approach. The Rc/rg7.1locus is an area rich in gypsy retrotransposons and other repetitive elements().Several pre-vious studies have noted significant repression of recombination associated with the abundance of TEs and other repetitive sequences(Fu et al.,2002;Wu et al.,2003;Shah and Hassan, 2005).Therefore,we expected that the TEs themselves might contribute to the recombinational repression.However,fine-mapping demonstrated that within the401-kb region analyzed in the BC2F6generation,one of the regions with a significantly increased recombination rate was TE-rich.

It has been shown that recombinational break points occur most often in regions with the greatest sequence similarity and that indels(including TEs)decreased the rate of crossovers more than single-nucleotide polymorphisms(Puget et al.,2002).The TE components of varieties may be different,and the lack of similarity would be one explanation for the low recombination in TE-rich regions.A recent study in humans showed that the retrotranspo-sons THE1A and THEA1B are overrepresented in recombinational

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