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Construction and characterization of a bacterial artificial chromosome (BAC) library of hexaploid wheat (Triticum aestivum L.) and validation of genome coverage using locus-specific primers.
Genome. 2003 Oct; 46(5):870-8.G

Abstract

A BAC library of hexaploid wheat was constructed using the spring wheat cultivar Triticum aestivum L. 'Glenlea'. Fresh shoot tissue from 7- to 10-day-old seedlings was used to obtain HMW DNA. The library was constructed using the HindIII site of pIndigoBAC-5 and the BamHI site of pIndigoBAC-5 and pECBAC1. A total of 12 ligations were used to construct the entire library, which contains over 650 000 clones. Ninety-six percent of the clones had inserts. The insert size ranged from 5 to 189 kb with an average of 79 kb. The entire library was gridded onto 24 high-density filters using a 5 x 5 array. A subset of these membranes was hybridized with two intergenic chloroplast probes and the percentage of clones containing chloroplast DNA (cpDNA) was calculated to be 2.2%. The genome coverage was estimated to be 3.1 x haploid genome equivalents, giving a 95.3% probability of identifying a clone corresponding to any wheat DNA sequence. BAC pools were constructed and screened using markers targeting the Glu-B1 locus (1BL), the hardness loci (5AS, 5BS, 5DS), the leaf rust resistance locus Lr1 (5DL), and the major fusarium head blight QTL locus located on 3BS. These markers were either locus-specific amplicons or microsatellites. A total of 49 BAC clones were identified for 14 markers giving an average of 3.5 clones/marker, thereby corroborating the estimated 3.1x genome coverage. An example using the gene encoding the HMW glutenin Bx7 is illustrated.

Authors+Show Affiliations

Cereal Research Centre, Agriculture and Agri-Food Canada, Winnipeg.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

14608404

Citation

Nilmalgoda, Sasanda D., et al. "Construction and Characterization of a Bacterial Artificial Chromosome (BAC) Library of Hexaploid Wheat (Triticum Aestivum L.) and Validation of Genome Coverage Using Locus-specific Primers." Genome, vol. 46, no. 5, 2003, pp. 870-8.
Nilmalgoda SD, Cloutier S, Walichnowski AZ. Construction and characterization of a bacterial artificial chromosome (BAC) library of hexaploid wheat (Triticum aestivum L.) and validation of genome coverage using locus-specific primers. Genome. 2003;46(5):870-8.
Nilmalgoda, S. D., Cloutier, S., & Walichnowski, A. Z. (2003). Construction and characterization of a bacterial artificial chromosome (BAC) library of hexaploid wheat (Triticum aestivum L.) and validation of genome coverage using locus-specific primers. Genome, 46(5), 870-8.
Nilmalgoda SD, Cloutier S, Walichnowski AZ. Construction and Characterization of a Bacterial Artificial Chromosome (BAC) Library of Hexaploid Wheat (Triticum Aestivum L.) and Validation of Genome Coverage Using Locus-specific Primers. Genome. 2003;46(5):870-8. PubMed PMID: 14608404.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Construction and characterization of a bacterial artificial chromosome (BAC) library of hexaploid wheat (Triticum aestivum L.) and validation of genome coverage using locus-specific primers. AU - Nilmalgoda,Sasanda D, AU - Cloutier,Sylvie, AU - Walichnowski,Andrzej Z, PY - 2003/11/11/pubmed PY - 2004/7/9/medline PY - 2003/11/11/entrez SP - 870 EP - 8 JF - Genome JO - Genome VL - 46 IS - 5 N2 - A BAC library of hexaploid wheat was constructed using the spring wheat cultivar Triticum aestivum L. 'Glenlea'. Fresh shoot tissue from 7- to 10-day-old seedlings was used to obtain HMW DNA. The library was constructed using the HindIII site of pIndigoBAC-5 and the BamHI site of pIndigoBAC-5 and pECBAC1. A total of 12 ligations were used to construct the entire library, which contains over 650 000 clones. Ninety-six percent of the clones had inserts. The insert size ranged from 5 to 189 kb with an average of 79 kb. The entire library was gridded onto 24 high-density filters using a 5 x 5 array. A subset of these membranes was hybridized with two intergenic chloroplast probes and the percentage of clones containing chloroplast DNA (cpDNA) was calculated to be 2.2%. The genome coverage was estimated to be 3.1 x haploid genome equivalents, giving a 95.3% probability of identifying a clone corresponding to any wheat DNA sequence. BAC pools were constructed and screened using markers targeting the Glu-B1 locus (1BL), the hardness loci (5AS, 5BS, 5DS), the leaf rust resistance locus Lr1 (5DL), and the major fusarium head blight QTL locus located on 3BS. These markers were either locus-specific amplicons or microsatellites. A total of 49 BAC clones were identified for 14 markers giving an average of 3.5 clones/marker, thereby corroborating the estimated 3.1x genome coverage. An example using the gene encoding the HMW glutenin Bx7 is illustrated. SN - 0831-2796 UR - https://www.unboundmedicine.com/medline/citation/14608404/Construction_and_characterization_of_a_bacterial_artificial_chromosome__BAC__library_of_hexaploid_wheat__Triticum_aestivum_L___and_validation_of_genome_coverage_using_locus_specific_primers_ DB - PRIME DP - Unbound Medicine ER -