Construction and characterization of a plant transformation-competent BIBAC library of the black Sigatoka-resistant banana Musa acuminata cv. Tuu Gia (AA)

Abstract

A plant transformation-competent binary bacterial artificial chromosome (BIBAC) library was constructed from Musa acuminata cv. Tuu Gia (AA), a black Sigatoka-resistant diploid banana. After digestion of high-molecular-weight banana DNA by HindIII, several methods of DNA size selection were tested, followed by ligation, using a vector/insert molar ratio of 4:1. The library consists of 30,700 clones stored in 80 384-well microtiter plates. The mean insert size was estimated to be 100 kb, and the frequency of inserts with internal NotI sites was 61%. The majority of insert sizes fell into the range of 100+/-20 kb, making them suitable for Agrobacterium-mediated transformation. Only 1% and 0.9% of the clones contain chloroplast and mitochondrial DNA, respectively. This is the first BIBAC library for banana, estimated to represent five times its haploid genome (600 Mbp). It was demonstrated by hybridization that the library contains typical members of resistance gene and defense gene families that can be used for transformation of disease susceptible banana cultivars for banana genetic improvement.

Structural and phylogenetic analysis of Pto-type disease resistance gene candidates in banana

“…Whether the banana Pto-RGCs are involved in conferring bacterial resistance as in tomato or are involved in conferring resistance to other types of pathogens will require functional analysis, which could be carried out with genetic complementation or lossof-function experiments. In the case of genetic complementation, the banana Pto-RGCs could be used as probes to screen a banana BIBAC library (Ortiz-Vázquez et al 2005) for the isolation of BIBAC clones containing Pto-RGCs. These Pto-RGC-BIBAC clones could be used to transform a banana disease susceptible cultivar using Agrobacterium tumefaciens (Khanna et al 2004).…”

“…The banana cultivar 'Tuu Gia' was chosen for PCR ampliWcation of Pto-RGC sequences because it shows resistance to a range of banana pathogens, including the most destructive such as M. Wjiensis and F. oxysporum, and also because there is a binary bacterial artiWcial chromosome (BIBAC) genomic library from this cultivar available for banana transformation (Ortiz-Vázquez et al 2005). The plant material was obtained from the international network for the improvement of banana and plantain (INIBAP) Musa Germplasm transit centre in Leuven, Belgium.…”

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.…”

Construction and characterization of a partial binary bacterial artificial chromosome (BIBAC) of Agave tequilana var. azul (2X) and its application for gene identification

“…Concerning the representativeness of the obtained library, Agave presents a medium size genome of 4312 Mpb (Banerjee and Sharma, 1987) compared to other plant species, such as those of Citrus clementina (367 Mpb) (Terol et al, 2008), Musa acuminata (600 Mpb) (Ortiz and Vazquez et al, 2005), Oryza nivara (760 Mpb) (Ammiraju et al, 2006) and Pinus pinaster (23 000 Mpb) (Bautista et al, 2008). High representativeness in the construction of genomic libraries has been reported for several species of plants, for which automatic procedures and equipment has been used.…”

“…The isolated nuclei were later embedded in low melting point (LMP) agarose and arranged in molds that were subjected to analysis according to the study of Ortiz-Vazquez et al (2005). The DNA agarose blocks were subjected to pulsed field electrophoresis (PFGE), using the conditions reported by Tao et al (2002) to remove low molecular weight fragments.…”