Abstract
BackgroundHaynaldia villosa (L.) Schur (syn. Dasypyrum villosum L. Candargy, 2n = 14, genome VV) is the tertiary gene pool of wheat, and thus a potential resource of genes for wheat improvement. Among other, wheat yellow mosaic (WYM) resistance gene Wss1 and a take-all resistance gene were identified on the short arm of chromosome 4 V (4VS) of H. villosa. We had obtained introgressions on 4VS chromosome arm, with the objective of utilizing the target genes. However, monitoring these introgressions has been a daunting task because of inadequate knowledge as to H.villosa genome, as reflected by the lack of specific markers.ResultsThis study aims to develop 4VS-specific markers by combination of chromosome sorting and next-generation sequencing. The short arm of chromosome 4VS of H.villosa was flow-sorted using a FACSVantage SE flow cytometer and sorter, and then sequenced by Illumina sequencing. The sequence of H. villosa 4VS was assembled by the software Hecate, and then was compared with the sequence assemblies of wheat chromosome arms 4AL, 4BS and 4DS and Ae. tauschii 4DS, with the objectives of identifying exon-exon junctions and localizing introns on chromosome 4VS of H. villosa. The intron length polymorphisms suitable for designing H. villosa primers were evaluated with criteria. Consequently, we designed a total of 359 intron targeting (IT) markers, among which 232 (64.62%) markers were specific for tracing the 4VS chromatin in the wheat background.ConclusionThe combination of chromosome sorting and next-generation sequencing to develop specific IT markers for 4VS of H. villosa has high success rate and specificity, thus being applicable for the development of chromosome-specific markers for alien chromatin in wheat breeding.
Highlights
Introduction2n = 14, genome VV) is the tertiary gene pool of wheat, and a potential resource of genes for wheat improvement
Shotguns sequencing of H. villosa chromosome 4VS and its assembly The analysis of DAPI-stained, chromosome suspensions prepared from a wheat-H. villosa 4VS addition line resulted in histograms with five peaks of fluorescence intensity
In this study, we flow-sorted a ditelosomic addition wheat-H. villosa line to isolate the short arm of the H.villosa 4VS chromosome with ~89% purity identified by fluorescence in situ hybridization (FISH) using a FACSVantage SE flow cytometer and sorter
Summary
2n = 14, genome VV) is the tertiary gene pool of wheat, and a potential resource of genes for wheat improvement. Wheat yellow mosaic (WYM) resistance gene Wss and a take-all resistance gene were identified on the short arm of chromosome 4 V (4VS) of H. villosa. We had obtained introgressions on 4VS chromosome arm, with the objective of utilizing the target genes. Monitoring these introgressions has been a daunting task because of inadequate knowledge as to H.villosa genome, as reflected by the lack of specific markers. There is urgent need to exploit a system for high-throughput method for developing molecular markers to identify alien introgression and translocation lines. The PLUG markers can identify homology between wheat and alien chromosomes, being useful in marker assisted selection (MAS), comparative genomics, alien chromosome tracing, taxonomic studies and genotyping [9,10,11,12]
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