Caught red-handed Rc encodes a basic helix-loop-helix protei(10)

1999)to identify similar regions,with extensive manual adjustment. Alignment was undertaken to initially preserve the reading frame of each sequence.Some gaps were later introduced that disrupted small sections of the reading frame to minimize the total number of events (substitutions,insertions,or deletions)required to transform one se-quence to another.Only some regions of the sequences could be con?dently aligned across all taxa,including the bHLH domain(;1kb). Further alignment was accomplished among sequences within the three main clades found in our analyses,with gaps for the remaining sequences (;500bp).Gaps were treated as missing in all analyses.Indels were not coded as additional characters.

Analyses

The aligned matrix was examined for the presence of secondary signal(s) using reverse successive weighting,implemented in RSW1.1a(Trueman, 1998).Using a parsimony bootstrap cutoff of70%and1000fast bootstrap replicates,no evidence of secondary signal was found.Further analyses were conducted with equal character weighting using parsimony imple-mented in PAUP*(Swofford,1998)as well as Bayesian analysis using HKY and GTRþG models implemented in Mr Bayes(Huelsenbeck and Ronquist,2001)version3.1.1to examine the sensitivity of the resulting phylogenetic inference to some model and method variation.Of1449 aligned positions,810(55%)were parsimony-informative.A single most parsimonious tree of3629steps was found after a heuristic search using 100random addition sequence replicates holding a maximum of100,000 trees(maximum not reached);Bayesian analyses for both models used10 chains and were run for5million generations,saving a tree every1000 generations.Two replicate runs were compared with check convergence using the likelihood versus generation plot.Full mixing was evident from almost the?rst few generations,so an arbitrary100burnin trees were discarded before producing the consensus topologies and clade posterior probabilities.The parsimony tree differed slightly from the Bayesian majority rule tree(Figure5).The parsimony-preferred positions of taxa are shown by arrows where they connect to other branches. Synonymous changes per synonymous site(Ks)were calculated for Rc rice and In1maize(Zea mays)sequences along with sequences from two ?anking genes from both species,No Apical Meristem putative protein and putative cellulose synthase sequences.We used the method of Yang and Nielsen(2000)implemented in PAML(Yang,2000;Yang and Nielsen, 2000)to test whether these values were consistent with that expected for orthologous sequences,around Ks¼0.65,assuming a synonymous substitution rate of0.65310ÿ9(Gaut et al.,1996)and the age of divergence of rice and maize at50million years ago(Gaut,2002).Two approaches were considered when deciding how different Ks values could be from the expected values and not be signi?cantly different.In the ?rst approach,we compared two standard errors above and below the estimated Ks for each gene with the expected value of0.65.This represents an approximation of the95%con?dence interval around each estimate.The other approach we used was to treat the expected value as the mean of a distribution of Ks values,and therefore an estimate for any one pair of genes.Extending a similar approach that has been used in comparisons of genes duplicated by polyploidy(Zhang et al., 2003),we might expect the variation for90%of genes duplicated by the same event(here speciation)to show2.6-fold variation in Ks.Therefore,a range of0.361to0.939around0.65can be expected for most genes. Accession Numbers

Sequence data for the genomic sequence of Rc can be found in the GenBank/EMBL data libraries under the following accession numbers: H75,DQ204735;cv Jefferson,DQ204736;O.ru?pogon,DQ204737; Surjamkuhi,DQ204738.The cDNA fragment ampli?ed from O.ru?pogon has accession number DQ315482.Supplemental Data

The following materials are available in the online version of this article.

Supplemental Table1.Primers Developed for Fine-Mapping.

Supplemental Figure1.Sequence Alignments Used in Phylogenetic Analysis.

Supplemental Figure2.Ks Ranges for Rc–In1Surrounding Gene Pairs.

ACKNOWLEDGMENTS

We thank W.De Jong and J.J.Doyle for critical reading of the manuscript and L.Swales for administrative assistance.The Rc mutant stocks were provided by H.J.Koh.We are grateful to Scott Williamson for statistical consultation about hot spot analysis and to Dave Gealey for helpful discussions and for providing information about?eld problems associ-ated with weedy red rice.This material is based upon work supported by National Science Foundation Grant0110004.B.E.P.acknowledges support from National Science Foundation Grant0321664.

Received October6,2005;revised December13,2005;accepted December19,2005;published January6,2006.

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292The Plant Cell

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