Construction and characterisation of a hexaploid wheat (Triticum aestivum L.) BAC library from the reference germplasm 'Chinese Spring'

Hexaploid wheat (Triticum aestivum L.) is one of the most important crops in the world with 582,691,612 million tonnes produced in 2001 on one third of the world's cultivated area. It has a very large genome (16700 Mb) that is 35 times that of rice. To develop genomic resources for positional cloning and structural genomics in wheat, a joint collaboration was set up between BBSRC (UK) and INRA (France) to construct a bacterial artificial chromosome (BAC) library from the wheat cultivar Chinese Spring with Hind III as the cloning site. The BAC library consists of 1,200,000 clones (3125 384-well microtitre plates). A random sampling of 1000 clones representing different ligations indicated an average insert size of 130 kilobases (kb) (ranging from 45 to 350 kb) and 3% empty clones (either empty vectors or no growth of colony). Screening the colony filters for organellar DNA content indicated a low level of contamination with chloroplast and mitochondria DNA (0.49% and 0.15% respectively). The library gives 9.3 haploid genome equivalents allowing a 99.99% probability of recovering any specific sequence of interest. Clones were gridded onto high-density filters using the Q-PIX and Q-Bot robots (Genetix limited, Hamphire, UK) in a 4 x 4, 5 x 5 and 6 x 6 double-spotted array on 22.5 cm 2 filters. Filters and clones are publicly available, being unencumbered in terms of IP, and will be provided on request.

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