Abstract
BackgroundThe cultivated strawberry Fragaria ×ananassa is one of the most economically-important soft-fruit species. Few structural genomic resources have been reported for Fragaria and there exists an urgent need for the development of physical mapping resources for the genus. The first stage in the development of a physical map for Fragaria is the construction and characterisation of a high molecular weight bacterial artificial chromosome (BAC) library.MethodsA BAC library, consisting of 18,432 clones was constructed from Fragaria vesca f. semperflorens accession 'Ali Baba'. BAC DNA from individual library clones was pooled to create a PCR-based screening assay for the library, whereby individual clones could be identified with just 34 PCR reactions. These pools were used to screen the BAC library and anchor individual clones to the diploid Fragaria reference map (FV×FN).FindingsClones from the BAC library developed contained an average insert size of 85 kb, representing over seven genome equivalents. The pools and superpools developed were used to identify a set of BAC clones containing 70 molecular markers previously mapped to the diploid Fragaria FV×FN reference map. The number of positive colonies identified for each marker suggests the library represents between 4× and 10× coverage of the diploid Fragaria genome, which is in accordance with the estimate of library coverage based on average insert size.ConclusionThis BAC library will be used for the construction of a physical map for F. vesca and the superpools will permit physical anchoring of molecular markers using PCR.
Highlights
The cultivated strawberry Fragaria ×ananassa is one of the most economicallyimportant soft-fruit species
This bacterial artificial chromosome (BAC) library will be used for the construction of a physical map for F. vesca and the superpools will permit physical anchoring of molecular markers using PCR
PCR analysis of BAC library pools and superpools All markers screened on the superpools of the F. vesca 'Ali Baba' BAC library amplified a PCR product of the expected size in relation to amplicons generated from F. vesca genomic DNA, and when single positive colonies were identified, all contained the expected PCR marker, indicating no cross-contamination in the library
Summary
High molecular weight DNA preparation A total of 20 g of young leaf tissue from the Fragaria vesca f. semperflorens cultivar 'Ali Baba' that had been given 72 h dark treatment at 16°C, was snap-frozen in liquid nitrogen and ground to a fine powder. The supernatant was removed and 2 ml of NIB (without BME) was added along with an equal volume of 1.5% low-melting temperature agarose. The plugs were rinsed twice with 0.1% BME followed by a double wash for 60 min each time with 10 mM Tris, 1 mM EDTA. Size selection of Fragaria DNA was performed with two consecutive rounds of pulse field gel electrophoresis (PFGE), conducted at 12°C, with 1 - 40 sec switch time, 6 V/cm, 120 deg angle for 18 hrs on a CHEF-DR-II system (Bio-Rad, Hercules, CA, USA). A second size selection was performed on the three sections but the DNA was allowed to electrophorese into low melting point agarose with 3 - 5 sec switch time. DNA from each of six plates (2,304 BAC clones each) was pooled to create eight superpools for prescreening of the library. The four clones identified by each positive PCR were grown overnight and used as template for PCR following the procedures described above, and single positive BAC clones were identified following electrophoresis and visualization over UV light
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