Proceedings
of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress
Salsomaggiore Terme, Italy - 26/29 September, 2001
ISBN 88-900622-1-5
Poster Abstract
ON THE WAY OF THE
CONSTRUCTION OF DURUM WHEAT (TRITICUM TURGIDUM L. SSP DURUM) BACTERIAL ARTIFICIAL CHROMOSOME (BAC)
LIBRARY: ISOLATION OF HIGH MOLECULAR WEIGHT NUCLEAR DNA
CIFARELLI
R.A., MANGO T., GALLITELLI M. CELLINI F.
Metapontum
Agrobios S.S. Jonica 106, Km 448.2, 75010 Metaponto (MT). Italy
rcifarelli@agrobios.it
durum
wheat, BAC library, HMW DNA
Large-insert BAC libraries are essential
for modern genomics research. These libraries form the foundation for
constructing physical maps, genome sequencing and cloning genes of interest.
To develop durum wheat genome tools aimed
at physical mapping, the identification of molecular markers closely linked to
quality traits loci (QTL), and map-based cloning, we are actively working on
the construction of a BAC library (1). With this purpose we developed a method
to prepare megabased-size DNA, from a tetrapolid wheat Triticum turgidum L.
ssp durum, cv. Ofanto.
As for the library construction, the
quality both of the vector and the genomic inserts is of critical importance.
We prepared megabases-size DNA from wheat
protoplasts (2) and from nuclei (3) using as a source wheat seedlings grown in
greenhouse under dark condition. DNA was partially digested with BamHI
restriction enzyme, then subjected to double size selection by Pulsed Field
Electrophoresis (PFGE), and eventually electroeluted and cloned into
pINDINGO BAC5 ™ vector.
Our purpose is to develop a library
containing at least five genome equivalents and an average insert size of 150
Kb, which will require x00.000 clones.
When compared with the protoplasts agarose
plugs method, high molecular weight (HMW) DNA prepared by wheat nuclei showed:
i) to be enriched by least threefold and ii) a reduction in chloroplast DNA
content. However a higher level of degradation was observed. Preliminary
ligation tests showed the suitability of DNA for BAC cloning.
1 LijavetzkyD., Muzzi G.,
Wicker T., Keller B., Wing R. and J. Dubcovsky Genome (1999), 42:
1176-82
2 Devos K.M. (1989) Electrophoresis 10:267-268
3 Zhang H.B., Zhao X., Ding X., Paterson H. and
Wing R.A. The Plant Journal
(1995) 7(1): 175-184