Are rye centromeric repetitive sequences confined in the centromere of rye chromosomes?

Are rye centromeric repetitive sequences confined in the centromere of rye chromosomes?

Masashi Tsuchida, Yadav P. Gyawali and Takashi R. Endo

Laboratory of Plant Genetics, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Corresponding author: Takashi R. Endo

E-mail: trendo@kais.kyoto-u.ac.jp

Tsuchida et al. (2008) cytologically dissected rye chromosome 1R in common wheat by the gametocidal system and established 55 common wheat lines carrying structurally rearranged 1R chromosomes, such as terminal deletions and translocations between 1R and wheat chromosomes. They also reported that seven of the 55 lines carry Robertsonian translocations between 1R and wheat. However, it is difficult to draw a distinction between the breakages in the centromere and those in the pericentromeric regions by microscopy. Therefore, it was not known which centromere, that of rye or that of wheat or both, those translocations chromosomes had.

Francki (2001) isolated and characterized a rye centromeric repetitive element and showed by FISH that the sequence (3.4 kbp repetitive sequence, pAWRC. 1), representing a diverged family of retrotransposon-like elements, called the Bilby family, localized within the centoromeres of all rye chromosomes and no signal was detected on wheat chromosomes. By using the rye centromeric repeat, we attempted to elucidate the centromere identity of the rearranged 1R chromosomes whose breakpoints were thought to be located in the centromere. We used 20 1Rⁱ dissection lines developed by Tsuchida et al. (2008). Eight of them had telocentric chromosomes and 12 lines carried translocations between 1Rⁱ and wheat chromosomes including: The translocations in seven lines were of Robertsonian type and those in the remaining five lines carried lRⁱL segments translocated onto wheat chromosomes, which were used as negative controls (Table 1). We also employed euploid rye ‘Imperial’, and 1Rⁱ, 1Rⁱ L and 1Rⁱ S addition lines of common wheat ‘Chinese Spring’ as positive controls, and euploid barley ‘Betzes’. These lines were obtained from National BioResource Project-Wheat, Japan (http://www.shigen.nig.ac.jp/wheat/komugi/top/top.jsp). In addition, we used eight dissection lines of another 1R chromosome derived from a wheat cultivar Burgas 2 with a 1R (1B) substitution (designated 1R^Br in this paper) together with a positive control of 1R^Br addition line of common wheat (Table 1, unpublished).

We designed a primer pair from the sequence of pAWRC. 1 and conducted PCR analysis. The PCR amplification of pAWRC. 1 was seen in all 15 1Rⁱ dissection lines carrying rearranged 1Rⁱ chromosomes with breakpoints in the centromere, four out of the eight 1R^Br dissection lines and all positive controls (Table 1, Fig. 1). This result suggested that those 15 rearranged 1Rⁱ and four rearranged 1R^Br chromosomes had their breakpoints in the middle of the centromeric region and that the other four rearranged 1R^Br chromosomes had their breakpoints in the vicinity of, but not in, the centromere. As expected, pAWRC. 1 was not amplified in wheat and barley, and three 1Rⁱ dissection lines 1Rⁱ-73 and 1Rⁱ -71, and 1Rⁱ -60, but unexpectedly, it was amplified in 1Rⁱ-11 and 1Rⁱ-69 (Table 1, Fig. 2). Judging from the FISH/GISH images (Fig. 2) and from the PCR analysis by Tsuchida et al. (2008), it is obvious that these two lines did not have the rye centromere: the 1Rⁱ-11 line lacked all PCR markers on the 1Rⁱ short arm and two proximal PCR markers on the 1Rⁱ long arm; the 1Rⁱ-69 line lacked all PCR markers on the 1Rⁱ short arm and one proximal PCR markers on the 1Rⁱ long arm. In these lines there may have been rye centromere segments that were too small to be detected by cytological observation. If this is the case, we may be able to exploit such minichromosomes to create artificial chromosomes by a top-down approach (see a review, Houben and Schubert 2007). The other possibility is that the 1Rⁱ long arm contained the same centromeric sequences in some region other than the centromere. If this is the case, it might be said the long arm of 1Rⁱ was originated from a complicated structural rearrangements, like a pericentromeric inversion involving the centromeric sequences. We are planning to elucidate which is the case by examining the segregating progeny of the 1Rⁱ-69 and 1Rⁱ-11 lines that were hemizygous for the critical rearranged 1Rⁱ chromosomes.

Generally, it is difficult to prove that so-called centromere-specific sequences are really confined to the centromere or the primary constriction. Molecular analysis is highly sensitive to detect the presence of certain centromeric sequences but it is helpless to tell the chromosomal location of such sequences. At present in situ hybridization is the only way to know the chromosomal location of centromeric sequences. It is a powerful tool when such centromeric sequences are concentrated in a chromosomal region, e.g. the primary constriction but otherwise it is also helpless to detect dispersed centromeric sequences. Combined with PCR analysis, rye-wheat translocation chromosomes, like 1Rⁱ-69 and 1Rⁱ-11, that have the wheat centromeres and rye chromosomal segments are useful in proving whether or not candidate sequences for the rye centromere are really confined to the centromere. Whatever the case may be, the result of the study we are planning to start would bring a fascinating development in the study of the cereal centromere structure.

References

Houben A, Schubert I (2007) Engineered plant minichromosomes: A resurrection of B chromosomes? Plant Cell 19: 2323-2327.

Francki M (2001) Identification of Bilby, a diverged centromeric Ty1-copia retrotransposon family from cereal rye (Secale cereale L.). Genome 44: 266-274.

Tsuchida M, Fukushima T, Nasuda S, Masoudi-Nejad A, Ishikawa G, Nakamura T, Endo TR (2008) Dissection of rye chromosome 1R in common wheat. Genes & Genetic Systems 83: (in press).