Genomic Organization of Wheat Centromere

摘要

A large-insert DNA clone, R11H, which showed strong hybridization signals on the wheat centromeres, was screened from the binary cosmid library of Aegilops squarrosa by FISH (fluorescence in situ hybridization). The R11H clone contains Tγ3/gypsy retrotransposon-related sequences; both integrase and CCS1 family sequences were identified. These characteristics are also common in other cereals, In the case of NotI digestion of the R11H clone followed by PFGE (Pulsed Field Gel Electrophoresis) analysis, one single main band consisting of three 31-kb fragments was observed in addition to other bands of insert. We subcloned the three 31-kb NotI fragments and several BamHI, Eco RI, and ApaI fragments, and partially mapped the 92-kb region cloned in R11H. Sequence analysis of the subclones resulted in finding of the other dispersed repeats, that made it difficult to assemble the subclones. We also isolated additional 23 large-insert DNA clones which contained the integrase sequences. The total number of the integrase sequences per haploid genome of Ae. squarrosa (1C = 4700 Mb) were estimated to be 500, about 70 copies per chromosome. Most of them contained the CCS1 sequences, and showed hybridization signals on the centromeres in FISH analysis as in the case of R11H. Based on the number of the integrase repeats in the clones determined by DNA gel blot analysis, we concluded that the retrotransposon-like sequences are tandemly repeated in wheat centromeres in ca. 55-kb interval on an average. This conclusion is consistent with the results of FISH analysis on the extended DNA fibers.