Dissecting the Complex Genome of Crested Wheatgrass by Chromosome Flow Sorting

Abstract

Core Ideas Flow sorting enabled to dissect the genome of crested wheatgrass into chromosomes. This is a powerful approach to access the complex genome of a wheat wild relative. Chromosome genomics will support alien introgression breeding of the crop. Wheatgrass (Agropyron sp.) is a potential source of beneficial traits for wheat improvement. Among them, crested wheatgrass [A. cristatum (L.) Gaertn.] comprises a complex of diploid, tetraploid, and hexaploid forms with the basic genome P, with some accessions carrying supernumerary B chromosomes (Bs). In this work, we applied flow cytometry to dissect the complex genome of crested wheatgrass into individual chromosomes to facilitate its analysis. Flow karyotypes obtained after the analysis of 4ʹ,6‐diamidino‐2‐phenylindole (DAPI)‐stained mitotic chromosomes of diploid and tetraploid accessions consisted of three peaks, each corresponding to a group of two or three chromosomes. To improve the resolution, bivariate flow karyotyping after fluorescent labeling of chromosomes with fluorescein isothiocyanate (FITC)‐conjugated probe (GAA)7 microsatellite was applied and allowed discrimination and sorting of P genome chromosomes from wheat–crested wheatgrass addition lines. Chromosomes 1P–6P and seven telomeric chromosomes could be sorted at purities ranging from 81.7 to 98.2% in disomics and from 44.8 to 87.3% in telosomics. Chromosome 7P was sorted at purities reaching 50.0 and 39.5% in diploid and tetraploid crested wheatgrass, respectively. In addition to the whole complement chromosomes (A), Bs could be easily discriminated and sorted from a diploid accession at 95.4% purity. The sorted chromosomes will streamline genome analysis of crested wheatgrass, facilitating gene cloning and development of molecular tools to support alien introgression into wheat.

Uncovering homeologous relationships between tetraploid Agropyron cristatum and bread wheat genomes using COS markers

“…Our results based on the chromosomal location of COS markers may indicate that chromosomal inversions are more abundant in the P genome of tetraploid A. cristatum than those of the diploid form, which could be the consequence of the polyploidization-induced genome reorganization (Ma et al 2004; Han et al 2005, 2017; Zhang et al 2013). In line with these studies, Said et al (2019) observed differences in the FISH signal pattern between chromosomes flow sorted from the diploid and tetraploid accessions, and the differences were more pronounced on the chromosomes flow sorted from tetraploid A. cristatum . A recent development in the flow-cytometric sorting of P genome chromosomes from the wheat– A. cristatum addition and ditelosomic lines opens the way for shot gun sequencing and high-resolution structure analysis of A. cristatum chromosomes (Said et al 2019).…”

“…In line with these studies, Said et al (2019) observed differences in the FISH signal pattern between chromosomes flow sorted from the diploid and tetraploid accessions, and the differences were more pronounced on the chromosomes flow sorted from tetraploid A. cristatum . A recent development in the flow-cytometric sorting of P genome chromosomes from the wheat– A. cristatum addition and ditelosomic lines opens the way for shot gun sequencing and high-resolution structure analysis of A. cristatum chromosomes (Said et al 2019).…”

Development of DNA Markers From Physically Mapped Loci in Aegilops comosa and Aegilops umbellulata Using Single-Gene FISH and Chromosome Sequences

“…Prior to the flow cytometric analysis, the chromosomes were labeled by fluorescence in situ hybridization in suspension (FISHIS) using 5′-FITC-GAA 7 -FITC-3′ oligonucleotides (Sigma, Saint Louis, MO, United States) according to Giorgi et al ( 2013 ) and stained by DAPI (4′,6-diamidino 2-phenylindole) at 2 μg/ml. Chromosome analysis and sorting were carried out using a FACSAria II SORP flow cytometer and sorter (Becton Dickinson Immunocytometry Systems, San José, CA, United States) as described by Molnár et al ( 2016 ) and Said et al ( 2019 ). Bivariate flow karyotypes FITC vs. DAPI fluorescence were acquired for each sample and two batches of 25,000–76,000 copies of each chromosome were sorted into PCR tubes containing 40 μl sterile deionized water.…”

Chromosome analysis and sorting

“…Agropyron cristatum (GAA) 7 [42] Dasypirum villosum (H. villosa) (GAA) 7 [61] Dasypyrum villosum (H. villosa) pTa71 (rDNA) [74] Triticum aestivum (GAA) 7 (AG) 12 [61] Triticum aestivum (GAA) 7 [75] Triticum aestivum (transgenic lines) (GAA) 7 [76] Triticum. dicoccoides (GAA) 7 [52] Triticum durum (AG) 12 (AAT) 7 (AAC) 5 [61] Triticum durum (AG) 12 [74] Triticum militinae (GAA) 7 [51] Triticum monococcum (ACC) 5 + (GAA) 7 [50] Triticum timopheevii (GAA) 7 Unpublished F I G U R E 5 Setting sort windows (red rectangles) to isolate particular chromosomes from bread wheat (Triticum aestivum, 2n = 6x = 42) cv.…”