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Structural Features of the Endogenous CHS Silencing and Target Loci in the Soybean Genome

Dep. of Crop Sciences, 384 ERML, 1201 W. Gregory Dr., Univ. of Illinois, Urbana, IL 61801. Sequence data from this article have been deposited in GenBank under the accession numbers: EF623853, EF623854, EF623855, EF623856, EF623857, EF623858, EF623859, EF623860

^* Corresponding author (l-vodkin{at}uiuc.edu).

The I locus in soybean [Glycine max (L.) Merr.] has been shown to be a naturally occurring duplication of chalcone synthase (CHS) genes inhibiting seed coat pigmentation in a trans-dominant manner via sequence specific degradation of CHS7/CHS8 transcripts. To better understand the dominant negative effect on seed-coat pigmentation, the relative organization of the eight member CHS multigene family was investigated. The bacterial artificial chromosome (BAC) library from the soybean cultivar Williams 82, harboring the silencing iⁱ allele, was screened exhaustively for different members of the multigene CHS family. Out of the 11 identified clones, 6 were completely sequenced and annotated. A total of 91 genes were predicted in the 540 kilobase (kb) of the gene-rich fraction of the soybean genome represented by these six BACs. While some members of the multigene family preferentially existed in clusters (CHS1, CHS3, CHS4, CHS5, and CHS9), the others existed as single copies. More important, CHS7 and CHS8, the target transcripts for siRNA mediated gene silencing in the seed coats, were present as single copies on different chromosomes, while the region surrounding the silencing iⁱ allele was found to encompass at least 230 kb containing tandem inverted repeats of CHS1, CHS3, CHS4, CHS5, and CHS9.

Abbreviations: BAC, bacterial artificial chromosome • CHS, chalcone synthase • EST, expressed sequence tag • kb, kilobase • LRR, leucine rich repeat • LTR, long terminal repeat • MLG, molecular linkage group • NCBI, National Center for Biotechnology Information • NR, nonredundant • ORF, open reading frame • PCR, polymerase chain reaction • RFLP, restriction fragment length polymorphism • RT-PCR, reverse transcriptase-polymerase chain reaction • TAG, tandemly arrayed duplicated gene • UTR, untranslated region

Received for publication October 2, 2007.