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Construction and characterization of a bacterial artificial chromsome library of hexaploid wheat (Triticum aestivum L) genotype Chinese Spring

Bo Shen1, Lynne McIntyre1, Evans Lagudah2 and Chunji Liu1

1 CSIRO Plant Indusries, Queensland Bioscences Precent, 306 Carmody Road, St Lucis, Brisbane 4067, Australia. www.csiro.au
2
CSIRO Plant Indusries, GPO Box 1600, Canberra, ACT 2601, Australia. www.csiro.au
Corresponding author: chunji.liu@csiro.au

Abstract

We have constructed a hexaploid wheat bacterial artificial chromosome (BAC) library using the genotype ‘Chinese Spring’. The library consists of 395136 clones sorted in 1029 384-well plates. About 92% of these clones contain inserts with an estimated average size of 157 kb. Clones containing chloroplast DNA were estimated to be 0.5%. Thus these BAC clones provide an estimated 3.4x genome equivalents. Of these, 221,184 clones have been robotically arrayed onto 24 high-density nylon filters. Screening of the filters with 21 single-copy RFLP probes identified 106 positive BAC clones. The average number of BAC clones identified from the 221,184 grided clones by these probes agreed with that of the expected from the estimated genome coverage of the clones tested. Given the vast range of precise genetic stocks, the numerous DNA libraries and the sequence data derived from this genotype, the CS BAC library could be highly valuable in wheat research.

Media summary

A BAC library of wheat genotype ‘Chinese Spring’ was developed by CSIRO plant Industries, providing a highly valuable resource for wheat research.

Keywords

bacterial artificial chromsome (BAC) library – Triticum aestivum - wheat

Introduction

BAC (bacterial artificial chromosome) libraries have been extensively used in physical mapping (Mozo et al., 1999), positional cloning (Giraudat et al., 1992), comparative mapping and genome evolution study (Dubcovsky et al., 2001, Klein et al., 2003, Sorrells et al., 2003), and developing polymorphic markers for targeted genome regions (Cregan et al., 1999). Although with only a very short history, BAC libraries have now been constructed for all species of agronomic importance. In wheats and their close relatives, BAC libraries have been reported for the diploid T. tauschii (with D genome) (Moullet et al. 1999), for the diploid T. monococcum (Am genome) (Lijavetzky et al. 1999), and for the tetraploid T turgidum (AB genome) (Cenci et al. 2003). In hexaploid wheat T. aestivum, a bulked BAC library was reported for genotype ‘Hartog’ (Ma et al, 2000) and gridded BAC libraries were reported for genotypes ‘Renan’ (Chalhoub et al. 2002) and ‘Glenlea’ (Nilmalgoda et al. 2003).

A vast range of aneuploids was constructed by ER Sears (1954, 1966) using the hexaploid wheat genotype ‘CS’, an achievement regarded as unequalled in its versatility, practicality, and creativity in any other species (Kimber 2001). Many other genetic stocks, including the numerous intervarietal single chromosome substitutions (Law et al. 1988), wheat-alien substitutions and additions (Gale and Miller 1988), and the deletions stocks (Endo and Gill 1996), were produced based on this genotype. These genetic stocks have been extensively exploited for mapping genes/markers in wheat. As a result, the genotype ‘CS’ has become the reference base for all wheat cytogenetics (Riley 1991) and is the most commonly used genotype for the generations of different DNA libraries, such as the EST libraries (Bartels and Thompson, 1983; Ainsworth et al., 1995; Kate et al. 2002). Thus, a CS BAC library would be highly valuable in wheat genome research. We report such a library in this paper.

Materials and Methods

Construction of CS BAC clones

Vector used in this study was pIndigoBAC-5 (Epicentre). Methods used for preparation of the CS BAC library were basically the same as described by Ma et al. (2000), with the only exception that a pseudo-double size fractionation by PFGE as described by Osoegawa et al (1998) was used for the separation of partially digested DNA.

BAC library screening

For filter screening, all probes were gel purified using QIAEXII kit and labelled with a-32P dCTP by the method of random priming (Feinberg and Vogelstein 1983). Filters were prehybridized at 65oC in hybridization buffer (1x Denhardt’s solution and 1x HSB solution) for 4-16 hours and hybridization carried out at 65oC overnight. Membrane were first washed in 2x SSC, 1% SDS for 45 minutes, followed by washing with 0.1x SSC, 0.1%SDS 2-3 times depending on signal strength. Filters were exposed to Kodark O-mat K Film for 24-96 hours depending on signal levels. Positive clones were addressed against an array template generated by a Qpix robot.

Results and discussion

Construction of CS BAC library

All clones picked for the CS BAC library were derived from ligations obtained using a single preparation of HMW DNA. The only difference between the ligations is the molar ratios of DNA vs vector used, which vary between 1:5 to1:10.

A total number of 395136 clones were picked, of which 221184 were grided on high density filters. Insert sizes of clones were analysed by randomly checking 247 clones. These clones contain inserts ranging from 40kb to 350kbp, with an average of 157kb. About 8% of clones contain no inserts. The distribution of the insert size was shown in fig 1.

(a)

(b)

Figure 1. Analysis of randomly selected wheat BAC clones. a.) BAC DNA was digested with NotI and separated by pulsed-.eld gel electrophoresis. The vector band are indicated by arrow. b.) Insert size distribution of randomly selected BAC clones of wheat BAC library. The insert sizes of 247 randomly selected clones were determined following NotI digestion of clone DNA.

To estimate the percentage of chloroplast clones in the library, two rice chloroplast-specific probes were screened against 2 high-density filters that contain a total of 18432 wheat BAC clones. These probes detected 87 positive BAC clones. Thus, the percentage of chloroplast clones contained in the wheat BAC library was estimated to be approximately 0.5%. Based on the estimated genome size of wheat (16,700 Mb/1C; Bennett and Leitch 1995), the BAC library provides an estimated 3.4x genome equivalents of the hexaploid wheat genome.

Screening BAC library using wheat probes

To further evaluate the quality of the BAC library, 21 single-copy RFLP probes were tested against the 24 high-density filters containing 221,184 BAC clones. These clones detected a total of 106 positive clones, with an average of 5.1 clones for each probe. This result agrees with the expected one from the estimated genome coverage of the library.

Conclusion

We reported here a fully characterized wheat BAC library that was constructed using the hexaploid wheat genotype ‘CS’. This library contains 395136 clones with an average insert size of 157kb. It provides some 3.4x genome equivalents that translate to a 96.7% of probability to identify any unique wheat sequences. Coupling with the wealth of precise genetic stocks, the numerous numbers/types of libraries, and the large quantity of mapping and sequence data accumulated in this genotype, the CS BAC library could be highly valuable in facilitating wheat genome research.

Acknowledgements

We are grateful to Tom Magner, Miki Miyagi for their help in the preparation of the library, and to Steve Brumbley (BSES) for the use of robotic equipments.

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