biologia plantarum

Biologia plantarum 59:445-455, 2015 | DOI: 10.1007/s10535-015-0522-1

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

J. Vrána¹, M. Kubaláková¹, J. Číhalíková¹, M. Valárik¹, J. Doležel^1,*

¹ Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Olomouc, Czech Republic

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.

Keywords: chromosome sorting; complexity reduction; genome analysis

Subjects: sub-genomic fractions; chromosome sorting; genome analysis; FISH; flow cytometric analysis; polyploid wheat

Received: December 7, 2014; Revised: February 28, 2015; Accepted: March 4, 2015; Published: September 1, 2015  Show citation