Caught red-handed Rc encodes a basic helix-loop-helix protei(5)

synthesis.As illustrated in Figure1B,panel iv,a recombination break point between RM21197and RID12in recombinant class3 had eliminated the promoter region and thefirst two exons of the bHLH gene as the source of the functional nucleotide poly-morphism,leaving only exons3to7within the18.5-kb target region.In addition to the bHLH protein,two putative transposon proteins,LOC_Os07g11040.1and LOC_Os07g11030.1,both of the CACTA type,En/Spm subclass,were also present within the 18.5-kb target region.These were eliminated from further consid-eration based on three lines of evidence.First,the sequences of both of these proteins have>100BLAST hits in the rice genome, with>98%identity over the entire length of the sequences. Mutations in either of these highly repeated genes would have no phenotypic consequences,because many other copies would remain functional in the genome.Second,domain analysis showed that LOC_Os07g11040.1contains a transposase domain and LOC_Os07g11030.1contains a proteinase domain,neither of which has been found in any of the regulatory or biosynthetic proteins responsible for anthocyanin/proanthocyanidin pigmen-tation in plants.Third,although TEs can be responsible for phenotypic changes if they are inserted within functional genes, neither of the two TEs within the rg7.1QTL region shows any evidence of having disrupted any other genes.Therefore,based on our positional analysis and in silico functional interpretation,we postulated that the bHLH gene was responsible for red pericarp at the rg7.1locus.We proceeded to test this hypothesis using three additional lines of evidence:sequence comparison of parental lines,sequence comparison of an allelic series,and expression analysis of the bHLH gene.

Sequence Comparison of the bHLH Gene

We sequenced the bHLH locus in both mapping parents, O.sativa(cv Jefferson)and O.rufipogon,to search for sequence changes that could explain the observed change in pericarp color.Having eliminated the promoter of the bHLH gene through recombination,we focused on changes that could affect the protein sequence.Six indels and22single-nucleotide polymor-phisms were detected across the genomic sequence.We also compared the sequences of the mapping parents with the publicly available cv Nipponbare sequence.The cv Jefferson allele was identical to the Nipponbare sequence,both of which are japonica cultivars having white seeds.

Keeping in mind the possibility that annotation from the Nipponbare sequence might provide a gene model that differed from the dominant allele,we annotated the allele obtained from O.rufipogon and compared it with the gene model available at TIGR(/tdb/e2k1/osa1).In the Nipponbare annotation,the39end of exon5and the59end of exon6were truncated relative to the O.rufipogon gene model,predicting an mRNA in Nipponbare that was513bp shorter than the mRNA predicted from the O.rufipogon annotation(Figure3C).To confirm the accuracy of the different gene models,we amplified a segment of cDNA from both O.rufipogon and cv Jefferson within the only region in which the gene models differed.The cDNA amplicons were both400bp,the size expected from the O.rufipogon annotation(Figure3A).Sequencing of this amplicon from O.rufipogon confirmed the splice sites predicted from the O.rufipogon annotation.When the polymorphisms between cv Jefferson and O.rufipogon were aligned with the new gene model,10of the sequence polymorphisms fell within the coding sequence,and5of those are expected to affect the protein sequence(Figure4).

To help identify which of the sequence polymorphisms be-tween the parents was responsible for the altered function of the gene,we also sequenced the bHLH locus in H75,an Rc mutant stock belonging to the japonica subspecies(Figure1A).H75,like O.rufipogon,carries a functional allele,but it is much more closely related to cv Jefferson than to O.rufipogon.Thus,a sequence comparison between H75and cv Jefferson was ex-pected to help eliminate some of the nonfunctional polymor-phisms detected between the parents in the bHLH gene.We found that the coding sequence of the bHLH allele in H75was identical to the cv Jefferson sequence except for a14-bp indel in exon6(Figure4).This14-bp sequence was present in the H75 stock as well as in O.rufipogon,but it was deleted in cv Jefferson and cv Nipponbare.The deletion induces a frame shift in the sequence,resulting in two premature stop codons before the end of exon6.The stop codons truncate the protein before the bHLH domain.Given that this deletion was the only differ-ence between the alleles of LOC_Os07g11020.1in the H75 mutant stock(pigmented seeds)and the japonica cultivars cv Jefferson and cv Nipponbare(white seeds),that its location in exon6is consistent with the recombinational data,and that it would have a clear and important impact on gene function,we conclude that the14-bp deletion is the only apparent reason

for Figure3.Expression Analysis of Rc.

(A)Transcripts of Rc and actin detected by RT-PCR in leaves,panicles before fertilization,pericarp from grains in the milk or dough stage of filling,and pericarp from mature seeds from both cv Jefferson(J;white seeds)and O.rufipogon(R;red seeds).

(B)Short transcripts of Rc detected by RT-PCR from the milk and dough stages offilling from cv Jefferson(J)and O.rufipogon(R)run out on polyacrylamide.

(C)Gene models from TIGR version3using the cv Nipponbare sequence and FgenesH prediction using the O.rufipogon sequence.The regions of mRNA amplified are indicated by horizontal lines,the longer one used in (A)and the shorter one used in(B).The arrow indicates the location of the 14-bp deletion.

Rc Encodes bHLH Protein Red Rice287