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

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.

Multiple displacement amplification of the DNA from single flow–sorted plant chromosome

“…Based on these results and those of the present work, we suggest coupling of chromosome flow sorting with MDA in microfluidic chips to develop a high‐throughput pipeline for preparation of chromosome‐derived DNA samples. Flow sorting is an efficient approach to prepare chromosome fractions enriched for particular chromosomes or chromosome groups (Vrána et al ., ). The samples with a reduced proportion or absence of chromosomes that are not targeted in the particular study, and are free of nuclear and cellular remnants, would be ideal samples for MDA in microfluidic chips, where the chromosomes are distributed one by one into separate chambers and amplified with significantly reduced bias.…”

Dissecting the Complex Genome of Crested Wheatgrass by Chromosome Flow Sorting

“…The order of chromosomes from the shortest to the largest (4P, 3P, 1P, 7P, 5P, 6P, and 2P) agrees with the assignment of chromosome groups to Peaks I (1P, 3P, 4P), II (5P, 6P) and III (2P, 7P) on monoparametric (DAPI‐) flow karyotypes. Although this result indicates that it is not possible to obtain a single type chromosome fractions, it shows that it is possible to dissect the complex genome into groups of some specific chromosomes and thus to simplify downstream analyses, as demonstrated in bread wheat by Vrána et al (2015).…”

Dissecting the U, M, S and C genomes of wild relatives of bread wheat (Aegilops spp.) into chromosomes and exploring their synteny with wheat

“…To overcome the difficulty to sort particular chromosomes, Vrána et al . () suggested dissecting composite chromosome peaks representing several chromosomes into smaller sections enriched for the chromosomes of interest, while Cápal et al . () developed a protocol for sequencing single flow‐sorted chromosomes.…”