Analysis Of A Centromeric BAC Clone TbBAC30 From Triticum Boeoticum Boiss.

Centromeres are responsible for sister chromatid cohesion and are the sites forkinetochore assembly and spindle fiber attachment, thereby enabling faithfulsegregation of chromosomes during cell division. Although these functions areconserved among all eukaryotes, there is no conservation of centromeric DNAsequences: different organisms have strikingly different centromeric DNAs. Triticumgenus consists of diploid, tetraploid and hexaploid, which is one of the best modelsfor studying genome evolution in allopolyploidization. In the tribe Triticeae,knowledge on centromeric DNA sequence is limited. No research on large-scalesequencing and analysis of centromeric regions in Triticum has been reported to date.We had constructed a bacterial artificial chromosome (BAC) library of T. boeoticumBoiss. and screened out 28 centromere-associated BAC clones that associated withcentromere by dot-blot and Southern hybridization methods with RCS1 and CCS1 asprobes. In this dissertation, one BAC clone, TbBAC30, was certified to be located atthe centromeric region by fluorescence in situ hybridization (FISH) analysis andselected to be further studied. One shotgun library of TbBAC30 was constructed,sequenced and assembled. As a result, one continuous contig was obtained andannotated by sequence blast. Repetitive sequences were the major components of thisclone. A few repetitive elements were used to do FISH and Southern hybridizationanalyses in several cereals. Such analyses will help to study the DNA compositions ofcentromeric regions in genus Triticum. It will also be helpful to discuss the dynamicsof centromeric sequences in species speciation and evolution of polyploids. And atlast, it will provide some academic proofs to reveal the chromosome action in meiosisof polyploid species. The main conclusions were as follows:1. FISH and Southern hybridization analyses proved that TbBAC30 was acentromere-associated BAC clone. The signals were distributed almost evenly oneach chromosome of hexaploid wheat.2. One shotgun subclone library of TbBAC30 was constructed. The full length ofTbBAC30 was 84,332 bp, obtained by sequence assembling and manipulation.The whole sequence was certified by analysis of 9 individual restriction enzyme'sdigestions and 6 pairwise enzyme combinations' digestions. It was feasible to sequence some centromeric regions of wheat in large scale by shotgun method.3. It was the first discovery of complete centromeric retrotransposons of wheat(CRW) in genus Triticum. They were high homology to the centromericretrotransposons of barley (cereba). In TbBAC30, there were 6 complete and 5truncated CRW elements, with several segments of non-CRW retrotransposos.The DNA sequences in centromeric region were complex.4. According to the similarity of the two LTRs in each intact retrotransposon, theestimated invading time of these elements were from 0.16 to 0.86 MYA.Considering the differentiation time of diploid ancestors of wheat (2.5-4.5 MYA),the centromeric DNA was still in dynamics during and even after the formation of T.boeoticum.5. Southern hybridization analysis suggested CRW had amplified during the formationof the tetraploid and hexaploid wheats.6. It was analyzed on the similarities and differences of autonomous andnon-autonomous CRW. The LTR of autonomous CRW consisted of homologoussequences of 365 bp, CCS1 and 192 bp repeats.7. CRW was shared by all the three subgenomes of hexaploid wheat, which wouldhelp to guarantee the unification of the whole genome.