Identification and Preliminary Analysis of Several Centromere‐associated Bacterial Artificial Chromosome Clones from a Diploid Wheat Library

Abstract

Although the centromeres of some plants have been investigated previously, our knowledge of the wheat centromere is still very limited. To understand the structure and function of the wheat centromere, we used two centromeric repeats (RCS1 and CCS1-5ab) to obtain some centromere-associated bacterial artificial chromosome (BAC) clones in 32 RCS1-related BAC clones that had been screened out from a diploid wheat (Triticum boeoticum Boiss.; 2n=2x=14) BAC library. Southern hybridization results indicated that, of the 32 candidates, there were 28 RCS1-positive clones. Based on gel blot patterns, the frequency of RCS1 was approximately one copy every 69.4 kb in these 28 RCS1-positive BAC clones. More bands were detected when the same filter was probed with CCS1-5ab. Furthermore, the CCS1 bands covered all the bands detected by RCS1, which suggests that some CCS1 repeats were distributed together with RCS1. The frequency of CCS1 families was once every 35.8 kb, nearly twice that of RCS1. Fluorescence in situ hybridization (FISH) analysis indicated that the five BAC clones containing RCS1 and CCS1 sequences all detected signals at the centromeric regions in hexaploid wheat, but the signal intensities on the A-genome chromosomes were stronger than those on the B-and/or Dgenome chromosomes. The FISH analysis among nine Triticeae cereals indicated that there were A-genomespecific (or rich) sequences dispersing on chromosome arms in the BAC clone TbBAC5. In addition, at the interphase cells, the centromeres of diploid species usually clustered at one pole and formed a ring-like allocation in the period before metaphase. Centromeres play critical roles in the faithful segregation of sister chromatids during the period of mitosis and meiosis (Presting et al. 1998;Jin et al. 2004). Although they have the same function, the DNA sequences of centromeric regions are not conserved among species (Clarke 1990;Houben and Schubert 2003;Ito et al. 2004;Jin et al. 2004). Owing to highly heterochromatic structure and complex repeats, it is more difficult to clone, map, sequence, and assemble the centromeres than the low-copy number DNA. Therefore, the centromere has become the last barrier of entire genome sequencing at present (Zhang et al. 2004b).In plants, considerable achievements have been made with regard to centromeres of model plants over the past few years. Highly repetitive "satellite" arrays and dispersed transponsable elements have been detected in most centromeres studied, where the former is organized into long stretches, interrupted by the latter and located at the core region . Studies have shown that the centromeres of Arabidopsis thaliana consist of a 178 ± 1 bp pAL1 satellite and Athila retroelement (Copenhaver et al. 1999;Houben and Schubert 2003). In rice (Oryza sativa L.), the DNA sequences in centromeres are composed of 155/156 bp CentO satellite and Centromeric Retrotransposon of BAC Clones Associated with Wheat Centromeres 349 Rice (CRR; Dong et al. 1998;Cheng et al. 2002;Jiang et al. 2003;Zhang et al. 20...

Structure and dynamics of retrotransposons at wheat centromeres and pericentromeres

“…We have also isolated two clones associated with wheat centromeres from the BAC library of Triticum boeoticum. One contains both centromeric and dispersed multi-copy elements, whereas a second has been found to hybridize in situ specifically to the centromeric regions of wheat (Chen et al 2002;Liu et al 2006). We report here the complete sequence of these two BAC clones and reveal that centromeric/pericentromeric sequences have evolved dynamically during speciation and allopolyploidization in wheat and its relatives.…”

“…Using the cereal centromeric universal repetitive sequence RCS1 ) and the cereal centromeric sequence CCS1 (Aragón-Alcaide et al 1996) as probes, 28 centromere-associated BAC clones were identified out of 3,072 clones screened (Chen et al 2002;Liu et al 2006), two of which (TbBAC5 and TbBAC30) were selected for detailed analysis. Based on their NotI, PmeI, SwaI, and PacI restriction fingerprints, their insert sizes were estimated to be 90 kb and 83.5 kb, respectively.…”

“…FISH and fiber-FISH FISH analysis was perfomed as described by Liu et al (2006). The probes were labeled with digoxigenin-11-dUTP or biotin-16-dUTP via nick translation.…”

Molecular and Cytogenetic Characterization of Wheat Introgression Lines Carrying the Stem Rust Resistance Gene Sr39

“…We used the genomic in situ hybridization (GISH) procedures as described by Cai et al (1998) and Liu et al (2006) Seeds were germinated in Petri dishes at 22°C for about 24 h. Roots of 1 to 2 cm in length were collected and pretreated in ice water for 20 to 24 h. After pretreatment, the roots were fixed in a 3:1 mixture of ethanol and acetic acid for 48 h at room temperature. To prepare the slide, root tips were transferred into 1% acetic carmine for 2 to 3 min and then root tips were squashed in 45% acetic acid under a cover glass.…”

Uniqueness of the Gossypium mustelinum Genome Revealed by GISH and 45S rDNA FISH

“…The in situ hybridization (ISH) technique, developed more than 30 years ago (Gall and Pardue ; John et al ), has proved to be a powerful technique in cytological biology studies. Jiang and Gill () stressed florescent in situ hybridization (FISH) as one of the most important techniques in plant molecular cytogenetic research, because it allows DNA sequences to be mapped directly on chromosomes (Cheng et al , ; Dai et al ; Liu et al ; Jiang and Bikram ; Tang et al ). Genomic in situ hybridization (GISH) is one of the most effective methods to obtain integrated information of DNA biology on genomic chromosomes, and is therefore powerful in its ability to provide data on genomic evolution and specific relationships (Tan et al ; Liu et al ; Zhou et al ).…”