Creation of a BAC resource to study the structure and evolution of the banana (Musa balbisiana) genome

Abstract

The first bacterial artificial chromosome (BAC) library of the banana species Musa balbisiana 'Pisang Klutuk Wulung' (PKW BAC library) was constructed and characterized. One improved and one novel protocol for nuclei isolation were employed to overcome problems caused by high levels of polyphenols and polysaccharides present in leaf tissues. The use of flow cytometry to purify cell nuclei eliminated contamination with secondary metabolites and plastid DNA. Furthermore, the usefulness of the inducible pCC1BAC vector to obtain a higher amount of BAC DNA was demonstrated. The PKW BAC library represents nine haploid genome equivalents of M. balbisiana and its mean insert size is 135 kb. It consists of two sublibraries, of which the first one (SN sublibrary with 24,960 clones) was prepared according to an improved standard nuclei isolation protocol, whereas the second (FN sublibrary with 11,904 clones) was obtained from flow-sorted nuclei. Screening with 12 RFLP probes, which were genetically anchored to 8 genetic linkage groups of the banana species Musa acuminata, revealed an average of 11 BAC clones per probe, thus confirming the genome coverage estimated based on the insert size, as well as a high level of conservation between the two species of Musa. Localization of selected BAC clones to mitotic chromosomes using FISH indicated that the BAC library represented a useful resource for cytogenetic mapping. As the first step in map-based cloning of a genetic factor that is involved in the activation of integrated pararetroviral sequences of Banana streak virus (BSV), the BSV expressed locus (BEL) was physically delimited. The PKW BAC library represents a publicly available tool, and is currently used to reveal the integration and activation mechanisms of BSV sequences and to study banana genome structure and evolution.

The bacterial artificial chromosome (BAC) library of the narrow-leafed lupin (Lupinus angustifolius L.)

“…That resulted not only in an almost complete absence of organellar DNA but also in a very high molecular weight of isolated DNA. The improved protocol was used to create a genomic BAC library of banana with low cytoplasmic contamination [40]. In this study, the same protocol resulted in contamination lower than 0.1%.…”

“…The procedure of the lupin BAC library construction was based on the protocol described by Šáfář et al [40]. Agarose plugs were washed twice for 1 h in TE buffer, cut into small pieces and equilibrated in digestion buffer (1×HindIII buffer supplemented with 4 mM spermidine) on ice for 60 min.…”

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

“…cloned in bacterial artificial chromosomes (BAC) (Hanson et al 1995, Lapitan et al 1997, Kim et al 2002, Mokroš et al 2006. Most of BAC libraries comprise clones carrying inserts 70 -200 kb long (Vilarinhos et al 2003, Šafář et al 2004, Ortiz-Vazquez et al 2005. If the detection limit of FISH on plant mitotic chromosomes is 10 kb (Lehfer et al 1993, Valárik et al 2004) the inserts of the BAC clones should be easy to detect.…”

Proteomic Analysis of Barley Cell Nuclei Purified by Flow Sorting

“…A protocol aimed at yielding suspensions of intact chromosomes [Doležel et al, 1992] is also suitable for the isolation of nuclei; the mild fixation with formaldehyde does not compromise the quality of the DNA in the context of preparing DNA libraries [Šafář et al, 2004;Kasprzak et al, 2006]. The method offers the opportunity to purify nuclei suitable for proteomic analysis in a single step, thereby avoiding the time-consuming fractionation steps included in more conventional protocols; it also reduces the risk of contamination by non-nuclear proteins.…”