Submitted to: Cellular and Molecular Biology of Soybean Biennial Conference
Publication Type: Proceedings
Publication Acceptance Date: 6/30/2002
Publication Date: 8/12/2002
Citation: Clough, S.J., Gregoire, R., Chan, W., Marek, L., Shoemaker, R.C., Vodkin, L. Sequence analysis of the i locus in williams 82. Cellular and Molecular Biology of Soybean Biennial Conference. p. 603.
Technical Abstract: A key enzyme of the flavonoid biosynthetic pathway is chalcone synthase (CHS). CHS is the last enzyme of a 4-step catabolic pathway that converts the amino acid phenylalanine into chalcone, a complex aromatic molecule that is the starting material for various plant secondary compounds including lignon, anthocyanin pigments, phytoalexins, and flavones. In soybean, CHS is present as a gene family consisting of at least 7 members. We used a fragment of CHS4 to probe soybean BAC filters of Williams 82 genomic DNA. Several different BACs hybridized to the CHS probe and one clone, 104J7, was chosen to be sequenced. BAC 104J7 was restriction digested with either MunI, EcoRI, or HindIII and fragments were subcloned into the vector pGEM3Zf+. A diagnostic sequence was obtained by sequencing all subclones with universal primers off the vector. This sequence data was used to identify unique clones and to verify that soybean DNA was present. Unique clones were fully sequenced by primer walking and contigged using the software program Sequencher. Gaps were filled by either sequencing directly off the BAC, or by identification of new subclones using different restriction enzymes (either SstI, SstII, Sau3A, PstI, or AccI). BAC 104J7 is approximately 105,000 bases and gene rich. The I-locus, consisting of CHS4, CHS3, and CHS1 is present on 104J7 in duplication, which has complicated sequencing efforts. Further annotation of this locus, as well as that of several neighboring genes on this BAC clone, will be presented.