Comparative Analysis of Subtelomeric Heterochromatin in Allium Fistulosum L., Allium Cepa L. And Allium Wakegi using Bac-Fish
Kiseleva, A.V.; Kirov, I.V.; Pavlenko, O.S.; Romanov, D.V.; Khrustaleva, L.I.
Izvestiya Timiryazevskoi sel'skokhozyaistvennoi akademii 2013(4): 23-31
Genomes of onions are poorly studied because of their large sizes, high frequency of duplications and increased heterozygosity. The creation of BAC (bacterial artificial chromosome) libraries with large inserts of genomic DNA and physical mapping of BAC clones on the chromosomes will significantly accelerate the studying of onion genomes. We constructed a BAC library of Allium fistulosum. Special attention was paid to the search in the BAC library of clones carrying telomeric and/or subtelomeric DNA sequences as for Allium the sequence of telomeric end and therefore mechanism for maintaining of chromosome stability and preservation of telomere lengths endings after each round of replication remains unknown. We carried out BAC library screening with DNA-probing in order to detect a common subtelomeric repeat for Allium species. Four BAC clones were identified. Cross dot blot analysis of BAC library with Cot-1 fractions of A. cepa and A. fistulosum identified one BAC clone 5.12.7 with weak homology to the Cot-1 fraction of A. cepa. The selected five BAC clones were physically mapped by fluorescence in situ hybridization (FISH) on chromosomes of A. fistulosum, A. cepa and A. wakegi. Four clones that carry the common subtelomeric repeat, were localized in the distal end of chromosomes in all three species. End-sequencing of the BAC clones and bioinformatic analysis of the sequence data revealed their high homology to the common onion subtelomeric repeat and to the genome survey sequences (GSS) of A. cepa. One BAC clone revealed homology to the common subtelomeric repeat at both ends. Three BAC clones had homology from the one end to the common subtelomeric repeat and from the other to the GSS of A. cepa. Alignment of DNA sequences with the homology to the GSS to each other did not reveal common sequences, which gives ground for a suggestion that these BAC clones originated from different arms or chromosomes. FISH analysis of BAC clone 5.12.7 revealed strong signals in the subtelomeric region only on chromosomes of A. fistulosum. FISH analysis of BAC clone 5.12.7 on A. wakegi, which is a natural hybrid between A. cepa and A. fistulosum, revealed strong signals on the chromosomes of A. fistulosum and weak signals on the chromosomes of A. cepa. Using the PCR product with primers on the common subtelomeric repeat and genomic DNA of A. cepa and A. fistulosum as a block-DNA in FISH probing with BAC clone 5.12.7 on A. wakegi, the signals were not observed on chromosomes of A. cepa. ВАС-FISH on A. cepa with block-DNA on the common subtelomeric repeat also did not show any hybridization signals. This may point to the presence in BAC clone 5.12.7 species-specific subtelomeric repeat. End-sequencing of this clone revealed homology only to the GSS of A. cepa. Further whole sequencing of the BAC clone 5.12.7 will allow us to identify the species-specific subtelomeric repeat in A. fistulosum.