Localization of BAC clones on mitotic chromosomes of Musa acuminata using fluorescence in situ hybridization

Abstract

A bacterial artificial chromosome (BAC) library of banana (Musa acuminata) was used to select BAC clones that carry low amounts of repetitive DNA sequences and could be suitable as probes for fluorescence in situ hybridization (FISH) on mitotic metaphase chromosomes. Out of eighty randomly selected BAC clones, only one clone gave a single-locus signal on chromosomes of M. acuminata cv. Calcutta 4. The clone localized on a chromosome pair that carries a cluster of 5S rRNA genes. The remaining BAC clones gave dispersed FISH signals throughout the genome and/or failed to produce any signal. In order to avoid the excessive hybridization of repetitive DNA sequences, we subcloned nineteen BAC clones and selected their 'low-copy' subclones. Out of them, one subclone gave specific signal in secondary constriction on one chromosome pair; three subclones were localized into centromeric and peri-centromeric regions of all chromosomes. Other subclones were either localized throughout the banana genome or their use did not result in visible FISH signals. The nucleotide sequence analysis revealed that subclones, which localized on different regions of all chromosomes, contained short fragments of various repetitive DNA sequences. The chromosome-specific BAC clone identified in this work increases the number of useful cytogenetic markers for Musa.

Molecular Analysis and Genomic Organization of Major DNA Satellites in Banana (Musa spp.)

“…Our findings extend significantly previous results of cytogenetic mapping of 45S and 5S rRNA genes and a single copy BAC clone 2G17 in Musa [25], [28], [29], [30], [31]. The 45S rDNA cluster localized to NORs in all accessions and the number of loci corresponded to their ploidy.…”

“…A BAC clone 2G17, which was originally identified as a single copy clone on mitotic chromosomes of M. acuminata ‘Calcutta 4’ [30] was localized on one chromosome pair in all diploid accessions with three exceptions. Four signals were observed on mitotic chromosomes of M. balbisiana ‘Honduras’.…”

“…BAC clone 2G17 [30] was labeled by Dig-11-dUTP or Bio-16-dUTP Nick Translation (Roche Applied Science, Penzberg, Germany) according to manufacturer’s instructions. FISH probes for 45S rDNA and 5S rDNA were obtained by labeling Radka 1 DNA clone (45S rDNA) and Radka 2 DNA clone (5S rDNA) [31] with digoxigenin-11-dUTP or biotin-16-dUTP (Roche Applied Science).…”

“…Apart from identifying parental chromosomes, the paucity of chromosome-specific landmarks and markers hampers studies on karyotype evolution and chromosome behavior in hybrids. To date, only a few DNA sequences and probes such as rDNA, some DNA repeats and BAC clones were found useful to study the organization of plant nuclear genomes at cytogenetic level [29], [30], [31].…”

Cytogenetic analysis of hybrids derived from wheat and Tritipyrum using conventional staining and genomic in situ hybridization

“…Although generating a segregated population from wheat and synthetic allohexaploids usually involves backcrossing of F 1 plants to one of the parents, few attempts were reported for the production of segregating population without backcrossing (Hassani et al 2003) because the F 1 plants are highly sterile. Cytological markers such as C-banding, fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and molecular markers can be used in chromosome identification and screening addition, substitution and recombinant lines (Mullan et al 2005, Silkova et al 2006, Qi et al 2007, Anugrahwati et al 2008, Hřibová 2008, Landjeva et al 2008. GISH is among the most powerful techniques to identify alien chromatin in the wheat background.…”