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Preparation of sub-genomic fractions enriched for particular chromosomes in polyploid wheat

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Biologia Plantarum

Abstract

Flow-sorted chromosomes have been used to simplify analyses of complex plant genomes. In bread wheat, majority of studies involve cultivar Chinese Spring, a genotype chosen for sequencing. Telosomic lines developed from this cultivar enable isolation by flow sorting chromosome arms, which represent less than 3.4 % of the genome. However, access to other wheat cultivars is needed to allow mapping and cloning useful genes. In these cultivars, cytogenetic stocks are not readily available and only one chromosome (3B) can be sorted. Remaining chromosomes form composite peaks on flow karyotypes and cannot be sorted. In order to overcome this difficulty, we tested a pragmatic approach in which composite chromosome peaks are dissected to smaller sections. The analysis of chromosome composition in sorted fractions confirmed feasibility of obtaining sub-genomic fractions comprising only a few chromosomes. Usually one of the chromosomes was more abundant and the frequencies of dominant chromosomes in sorted fractions ranged from 16 % (chromosome 7B) to 80 % (chromosome 2B). The enrichment factor, calculated as the relative proportion of chromosomal DNA in the wheat genome to the proportion of chromosomal DNA in a sorted fraction, ranged from 3.2-fold (7B) to 16.4-fold (5D). At least a 5-fold enrichment can be obtained for 17 out of 21 wheat chromosomes. Moreover, we show that 15 out of the 21 chromosomes can be sorted without being contaminated by their homoeologs. These observations provide opportunities for constructing sub-genomic large-insert DNA libraries, optical mapping, and targeted sequencing selected genome regions in various cultivars of wheat. The availability of fractions enriched for chromosomes of interest and free of contaminating homoeologs will increase the efficiency of research projects and reduce their costs as compared to whole genome approaches. The same methodology should be feasible in other plants where single chromosome types cannot be sorted.

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Abbreviations

DAPI:

4′,6-diamidino-2-phenylindole

FISH:

fluorescence in situ hybridization

FISHIS:

FISH in suspension

PRINSES:

primed in situ labelling en suspension

RIL:

recombinant inbred line

STS:

sequence-tagged site

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  1. Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Šlechtitelů 31, CZ-78371, Olomouc, Czech Republic

    J. Vrána, M. Kubaláková, J. Číhalíková, M. Valárik & J. Doležel

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  1. J. Vrána
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  2. M. Kubaláková
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  3. J. Číhalíková
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  4. M. Valárik
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  5. J. Doležel
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Correspondence to J. Doležel.

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Acknowledgements: We warmly acknowledge the help provided by R. Šperková and Z. Dubská in chromosome sorting. We are grateful to P. Sourdille (INRA, Clermont-Ferrand, France) for providing seeds of bread wheat cv. Chinese Spring and Renan, K. Järve (the Tallinn Technical University, Tallinn, Estonia) for seeds of cv. Tähti, and B.S. Gill (the Kansas State University, Manhattan, USA) for seeds of double ditelosomic line 4A of cv. Chinese Spring. This work was supported by a grant award LO1204 from the National Program of Sustainability I.

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Vrána, J., Kubaláková, M., Číhalíková, J. et al. Preparation of sub-genomic fractions enriched for particular chromosomes in polyploid wheat. Biol Plant 59, 445–455 (2015). https://doi.org/10.1007/s10535-015-0522-1

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