Development of intron targeting (IT) markers specific for chromosome arm 4VS of Haynaldia villosa by chromosome sorting and next-generation sequencing

Abstract

Background: Haynaldia 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.

Results: This 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.

Conclusion: The 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.

Keywords: Haynaldia villosa; Intron polymorphism; Molecular marker; Triticum aestivum.

Fig. 1

Schematic representation of the development system of PCR-based IT markers specific for the chromosome 4VS of Haynaldia villosa and 8% non-denaturing poly-acrylamide gels electrophoresis of PCR product of IT marker CINAU687. a Schematic representation of the development system of PCR-based IT markers specific for the chromosome 4VS of Haynaldia villosa. b 8% non-denaturing poly-acrylamide gels electrophoresis of PCR product of IT marker CINAU687. Grey boxes represented the exons and grey lines represented the introns. Red line, light blue line, deep blue line and light green line showed the intron 7 region of gene fragment AEGTA27279 corresponding to the intron regions of the scaffold of IWGSC_CSS_4AL_scaff_7149580, IWGSC_CSS_4BS_scaff_4952162, IWGSC_CSS_4DS_scaff_2323695 and Hecate_SCF226, respectively). The base number of intron 7 in the gene fragment of subgenome AA, BB, DD and VV was 658 bp, 367 bp, 415 bp and 468 bp, respectively

Fig. 2

8% non-denaturing poly-acrylamide gels electrophoresis of PCR products. Arrows show 4VS-specific fragments