hot spots and that the motif CCTCCCT,found associated with the retrotransposons,has a role in hot spot determination(Myers et al., 2005).In our study,one of the TEs in a region with enhanced recombination near Rc contains a CCTCCCT motif.This offers an alternative explanation for the significant increase in recombina-tion in this particular TE-rich region.

Implications

The cloning of Rc will make possible new methods offighting weedy rice infestations in rice paddies.Red rice is a noxious weed that is currently responsible for losses of as much as $50million per year in the United States(Gealy et al.,2002).It is a perfect mimic of elite varieties,as the red pericarp is not visible until after harvest,when the grains are dehulled.Furthermore,the close association between red pericarp,seed shattering,and dormancy allows it to persist infields for years despite vigorous attempts to remove it.The fact that the pericarp is maternal tissue,so that its color is dependent on the maternal genotype, means that seeds pollinated by red rice can be white(if the maternal plant carries the rc allele),but plants grown from these seeds will produce red seeds.

An immediate application of the work presented here involves the use of perfect markers that specifically target the14-bp functional nucleotide polymorphism within the bHLH gene to screen for red rice contamination within certified seed lots.This will also facilitate the use of genes derived from crosses with wild relatives by allowing breeders to conclusively select against progeny carrying Rc,and to do so before the plants set seed. METHODS

QTL Population Development and QTL Detection

A BC2F2population was constructed for QTL mapping using Oryza sativa subsp tropical japonica cv Jefferson as the recurrent parent and a wild accession of Oryza rufipogon(IRGC-105491from Malaysia)as the donor parent(Thomson et al.,2003).QTL detection was followed as described by Thomson et al.(2003).

Fine-Mapping of rg7.1

Eighteen BC2F2families with red grain but with low levels of dormancy and shattering were used tofine-map the QTL.These families represent a subset of the red-grained phenotypes in the BC2F2population.These segregating families were grown in50-mm-wide3178-mm-deep plastic pots in the Guterman Greenhouse at Cornell University.DNA was extracted using the Matrix Mill method(Paris and Carter,2000).The microsatellite and indel markers were amplified using standard PCR protocols,run on4%polyacrylamide electrophoresis gels,and silver-stained as described(Panaud et al.,1995).Seeds were harvested,and10 to15seeds/plants were dehulled to determine seed color.Color was scored as either present or absent.In each generation,we determined which segments of DNA from the red donor parent,O.rufipogon,were present in all red-seeded progeny and discounted the O.rufipogon segments that appeared in white-seeded progeny as possible gene positions.Only plants heterozygous for the region containing rg7.1were planted for the next generation of screening.A total of5922individuals from BC2F3to BC2F6were genotyped.Molecular Marker Development

The required density of molecular markers in the target region was achieved using previously published SSRs(McCouch et al.,2002)as well as SSR and indel markers developed as part of this study.New markers were designed from the publicly available rice genome sequence(http:// /tigr-scripts/osa1_web/gbrowse/rice;http://143.48.220.116/ genome_browser/index.html)using the SSRIT tool(http://143.48.220. 116/db/searches/ssrtool).Indel markers were designed by sequencing areas from both cv Jefferson and O.rufipogon and aligning the sequences using the SeqMan program of DNAstar(GeneCodes)to identify indels. Primer sequences,map positions,and amplified lengths of the newly developed SSRs and indel markers used in this study are listed in Sup-plemental Table1online.

Recombination Rate Statistics

The recombination rate for each interval was compared with the average using likelihood ratio test statistics and P values for the test that compares the two-parameter model in which the fragment has its own unique recombination rate(while everything else is uniform)with the model in which everything is uniform.The likelihood of each recombination rate¼e l l xi/x i!where x i¼the number of recombinants in interval i,l¼r(length of sequence)(number of plants),and r is the per nucleotide recombination rate and is estimated as(total number of recombinants)/(total length of region)/(number of plants).The P values were corrected for multiple tests using a Bonferroni correction.The RM21177to RM21194interval was significantly lower than the average,even after the recombination rates from the two hot spots with P<0.001were excluded from the average. Sequence Analysis of Red and White Cultivars

Overlapping primers between700and900bp were designed to cover the HLH gene and were used to amplify DNA with Pfu polymerase(Invitro-gen).Amplified products were sequenced at the Cornell Biotechnology Resource Center.

RT-PCR

mRNA was collected from cv Jefferson and O.rufipogon panicles from the following stages and tissues:mature leaves,spikelets before polli-nation,dehulled seeds from the milk/dough stage of seedfilling minus the starchy endosperm,and pericarp and seed coat scraped from the hardened endosperm of mature dried seeds.Total RNA was extracted with the RNAeasy miniplant RNA extraction kit(Qiagen).Total RNA(1m g) was converted into cDNA with reverse transcriptase(Invitrogen),accord-ing to the manufacturer’s instructions.As a control,water was used in place of reverse transcriptase for the reaction.The reaction was diluted threefold,and2m L of cDNA was used for amplification.A total of10m L of PCR products was loaded on1%agarose gels.Primers for RT-PCR were designed to span introns.BLAST searches were used to ensure that primers were specific to the candida …… 此处隐藏：4337字，全部文档内容请下载后查看。喜欢就下载吧 ……