Analysis and mapping of randomly chosen bacterial artificial chromosome clones from hexaploid bread wheat

Abstract

The current view of wheat genome composition is that genes are compartmentalized into gene-rich and gene-poor regions. This model can be tested by analyzing randomly selected bacterial artificial chromosome (BAC) clones for gene content, followed by placement of these BACs onto physical and genetic maps. Map localization could be difficult for BACs that consist entirely of repeated elements. We therefore developed a technique where repeat junctions are used to generate unique markers. Four BAC clones from hexaploid wheat variety Chinese Spring were randomly selected and sequenced at 4- to 6-fold redundancy. About 50% of the BAC sequences corresponded to previously identified repeats, mainly LTR-retrotransposons, whereas most of the remaining DNA consisted of sequences with unknown origin or function. The average gene content was <1%, although each BAC contained one or two identified genes. Repeat boundaries were amplified and used to map each clone to a chromosome arm. Extrapolation from wheat-rice comparative knowledge suggests that three of the four BAC clones originate from "gene-rich" regions of the wheat genome. Nevertheless, because these BACs carry only a single gene (two BACs) or two genes (one BAC), the predicted gene density is approximately 1 gene per 75 kb, which is considerably lower than previously estimated gene densities (one gene per 5-20 kb) for gene-rich regions in wheat. This analysis of randomly selected wheat BAC clones suggests that genes are more evenly distributed in wheat than previously believed and substantiates the need for large-scale random BAC sequencing to determine wheat genome organization.

Fig. 1.

Primer design across a repeat boundary on BAC TA1353M1 and an agarose gel showing the mapping of the corresponding amplification product on a set of 21 NT lines. The absence of the fragment in the line nullisomic for 3B (N3B) allocates BAC TA1353M1 to chromosome 3B.

Fig. 2.

Separation of the amplification products generated by primer set TA1426L2_16F2/R2 in the group 5 NT lines by SSCP. A comparison of the banding patterns in the N5A, N5B, and N5D lines shows that specific amplification products are absent in the N5B line, which indicates that chromosome 5B is the origin of BAC clone TA1426L2.