A direct repeat sequence associated with the centromeric retrotransposons in wheat.

A high-density BAC filter of Triticum monococcum was screened for the presence of a centromeric retrotransposon using the integrase region as a probe. Southern hybridization to the BAC digests using total genomic DNA probes of Triticum monococcum, Triticum aestivum, and Hordeum vulgare detected differentially hybridizing restriction fragments between wheat and barley. The fragments that hybridized to genomic DNA of wheat but not to that of barley were subcloned. Fluorescence in situ hybridization (FISH) analysis indicated that the clone pHind258 hybridized strongly to centromeric regions in wheat and rye and weakly to those in barley. The sequence of pHind258 was homologous to integrase and long terminal repeats of centromeric Ty3-gypsy retrotransposons of cereal species. Additionally, pHind258 has a pair of 192-bp direct repeats. FISH analysis indicated that the 192-bp repeat probe hybridized to centromeres of wheat and rye but not to those of barley. We found differential FISH signal intensities among wheat chromosomes using the 192-bp probe. In general, the A-genome chromosomes possess strong FISH signals, the B-genome chromosomes possess moderate signals, and the D-genome chromosomes possess weak signals. This was consistent with the estimated copy numbers of the 192-bp repeats in the ancestral species of hexaploid wheat.

Ito H ，Nasuda S ，Endo TR 《GENOME》

BAC-FISH in wheat identifies chromosome landmarks consisting of different types of transposable elements.

Fluorescence in situ hybridization (FISH) has been widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts making them amenable for FISH mapping. We used BAC-FISH to study genome organization and evolution in hexaploid wheat and its relatives. We selected 56 restriction fragment length polymorphism (RFLP) locus-specific BAC clones from libraries of Aegilops tauschii (the D-genome donor of hexaploid wheat) and A-genome diploid Triticum monococcum. Different types of repetitive sequences were identified using BAC-FISH. Two BAC clones gave FISH patterns similar to the repetitive DNA family pSc119; one BAC clone gave a FISH pattern similar to the repetitive DNA family pAs1. In addition, we identified several novel classes of repetitive sequences: one BAC clone hybridized to the centromeric regions of wheat and other cereal species, except rice; one BAC clone hybridized to all subtelomeric chromosome regions in wheat, rye, barley and oat; one BAC clone contained a localized tandem repeat and hybridized to five D-genome chromosome pairs in wheat; and four BAC clones hybridized only to a proximal region in the long arm of chromosome 4A of hexaploid wheat. These repeats are valuable markers for defined chromosome regions and can also be used for chromosome identification. Sequencing results revealed that all these repeats are transposable elements (TEs), indicating the important role of TEs, especially retrotransposons, in genome evolution of wheat.

Zhang P ，Li W ，Fellers J ，Friebe B ，Gill BS ... -  《CHROMOSOMA》

A Ty3/gypsy retrotransposon-like sequence localizes to the centromeric regions of cereal chromosomes.

A 745 bp sequence (pSau3A9) located at the centromeres of several cereal species was isolated from a sorghum BAC library by Jiang et al. (1996, Proc. Natl Acad. Sci. USA, 93, 14210-14213). We have amplified a partially homologous 809 bp sequence from barely genomic DNA by PCR and localized it to the centromeres of barley, wheat and rye chromosomes by fluorescent in situ hybridization (FISH). Sequence analysis showed this barley homolog of pSau3A9 to have high similarity to the integrase region of the polyprotein gene of Ty3/gypsy group retrotransposons. Using this integrase sequence as a probe, several clones were isolated from a lambda library constructed of genomic barley DNA. One of the lambda clones contained coding regions for all five catalytic sites characteristic of the retrotransposon polyprotein. Two direct repeats flanking the polyprotein gene are homologous to the cereal centromeric sequence described by Aragón-Alcaide et al. (1996, Chromosoma, 105, 261-268) and may represent all or part of the long-terminal repeats (LTRs). Different plasmid subclones containing various regions of the lambda clone were used in FISH to show that the entire polyprotein gene and upstream flanking sequences, including the presumed LTR, are present at barley centromeres. The preferential (or exclusive) localization of an apparently complete retroelement within the centromeric regions of several cereal species raises interesting questions about its role in karyotype evolution and centromere function.

Presting GG ，Malysheva L ，Fuchs J ，Schubert I ... -  《PLANT JOURNAL》

Centromeric distribution of 350-family in Dasypyrum villosum and its application to identifying Dasypyrum chromatin in the wheat genome.

Variants of the 350-family sequences were isolated from Dasypyrum villosum genomic DNA. Although the consensus sequence shared 86% homology to p380 and 84% homology to pHvNAU62 of D. villosum, a 63 bp long region including variable microsatellite repeat (TG)(7-9) was found to be absent in pHvNAU62 or much different from p380. This sequence also shared 67% homology to the 350-family of rye. The representative clone pDvTU383 was localized on the D. villosum chromosomes by using the sequential C-banding and fluorescence in situ hybridization (FISH). Combining with the probes of pTa71 and pTa794 containing 18S-5.8S-28S rDNA (NOR) and 5S rDNA multigene families, respectively, the pDvTU383 probe allowed identification of all seven pairs of D. villosum chromosomes. The pair of the 1V chromosomes fluoresced strong in situ hybridization signals of 18S-5.8S-28S rRNA genes in the NORs of 1VS, and the pair of the 5V chromosomes had weak signals of 5S rRNA genes at the end of 5VS. The pair of the 4V chromosomes showed sub-telomeric signals of pDvTU383 probe on the both arms, however, the signal of the short arm is weaker than that of the long arm. The pair of the 7V chromosomes showed no signals of pDvTU383 on the both sub-telomeric regions. The pair of the 6V chromosomes showed a characteristic interstitial signal of pDvTU383 on the short arm. The pairs of the 2V and 3V chromosomes were distinguished by the difference of interstitial signals of pDvTU383 on their long arms. Furthermore, hybridization signals of pDvTU383 were also visualized on centromeres of all chromosomes. Based on the above D. villosum chromosomal patterns made by FISH, one pair of the 4V chromosomes originated from D. villosum were found in a trigeneric hybrid line involving wheat, Dasypyrum and Thinopyrum. The sequence pDvTU383 provides an effective probe for further analysis of the D. villosum genome.

Yuan WY ，Tomita M 《HEREDITAS》

A tandem repetitive sequence located in the centromeric region of common wheat (Triticum aestivum) chromosomes.

Although Tail-family sequences are present in the subtelomeric region of Leymus racemosus, it became apparent in the present study that such sequences are also present in the centromeric region of common wheat (Triticum aestivum). These sequences hybridized to all chromosomes with various degrees of signal strength. FISH using Tail and Ty3/gypsy, a conservative sequence in cereal centromeres, revealed a complicated arrangement of both sequences in all wheat chromosomes at once. Unlike the Arabidopsis centromeres characterized by massive tandem arrays of 180-bp family with flanking paracentromeric retrotransposons in all chromosomes, wheat chromosomes showed various arrangement patterns of Tail and Ty3/gypsy sequences depending on the chromosome; Tail-family sequences were scattered in many wheat centromeres as isolated colonies instead of forming uninterrupted solid tandem arrays. This pattern may have resulted from retrotransposon insertion within pre-existing Tail-tandem arrays or a two-step amplification mechanism of the Tail family where each Tail colony was amplified to form arrays independently after the insertion of Tail-family sequences along the entire centromere. Although sequence analysis of centromeric Tail repeats in wheat and subtelomeric Tail repeats in L. racemosus showed variable and conservative regions between the two repeats, they did not show a distinctive difference phylogenically. The widespread presence of tandem repetitive sequences in the eucaryotic centromere suggests a significant role for them in centromeric formation.

Kishii M ，Nagaki K ，Tsujimoto H 《CHROMOSOME RESEARCH》