Dissecting large and complex genomes: flow sorting and BAC cloning of individual chromosomes from bread wheat

The analysis of the complex genome of common wheat (Triticum aestivum, 2n = 6x = 42, genome formula AABBDD) is hampered by its large size (approximately 17 000 Mbp) and allohexaploid nature. In order to simplify its analysis, we developed a generic strategy for dissecting such large and complex geno...

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Bibliographic Details
Published in:	The Plant journal : for cell and molecular biology Vol. 39; no. 6; pp. 960 - 968
Main Authors:	Safar, J, Bartos, J, Janda, J, Bellec, A, Kubalakova, M, Valarik, M, Pateyron, S, Weiserova, J, Tuskova, R, Cihalikova, J
Format:	Journal Article
Language:	English
Published:	Oxford, UK Blackwell Science Ltd 01-09-2004 Blackwell Science Wiley
Subjects:	BAC cloning bacterial artificial chromosomes Biological and medical sciences Chromosome Mapping chromosomes Chromosomes, Artificial, Bacterial Chromosomes, Plant - genetics Cloning, Molecular cytogenetic analysis DNA library flow cytometry Flow Cytometry - methods flow sorting Fundamental and applied biological sciences. Psychology Gene Library Genes, Plant - genetics Genes. Genome genome Genome, Plant grain crops In Situ Hybridization, Fluorescence isolated chromosomes large genomes Life Sciences Molecular and cellular biology Molecular genetics plant genetics Triticum - genetics Triticum aestivum Vegetal Biology wheat Flow cytometry Monocotyledones Genetic marker Genomics Chromosome large genomes Experimental protocol BAC cloning flow sorting isolated chromosomes Gramineae Angiospermae Cytogenetics Spermatophyta wheat Triticum aestivum DNA library chromosome 3B adn génome clonage dna flux isolation de chromosomes cloning flow blé
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Summary:	The analysis of the complex genome of common wheat (Triticum aestivum, 2n = 6x = 42, genome formula AABBDD) is hampered by its large size (approximately 17 000 Mbp) and allohexaploid nature. In order to simplify its analysis, we developed a generic strategy for dissecting such large and complex genomes into individual chromosomes. Chromosome 3B was successfully sorted by flow cytometry and cloned into a bacterial artificial chromosome (BAC), using only 1.8 million chromosomes and an adapted protocol developed for this purpose. The BAC library (designated as TA-3B) consists of 67 968 clones with an average insert size of 103 kb. It represents 6.2 equivalents of chromosome 3B with 100% coverage and 90% specificity as confirmed by genetic markers. This method was validated using other chromosomes and its broad application and usefulness in facilitating wheat genome analysis were demonstrated by target characterization of the chromosome 3B structure through cytogenetic mapping. This report on the successful cloning of flow-sorted chromosomes into BACs marks the integration of flow cytogenetics and genomics and represents a great leap forward in genetics and genomic analysis.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2
ISSN:	0960-7412 1365-313X
DOI:	10.1111/j.1365-313x.2004.02179.x