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.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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3567-z) contains supplementary material, which is available to authorized users.

Characterization of Chromosomal Rearrangement in New Wheat—Thinopyrum intermedium Addition Lines Carrying Thinopyrum—Specific Grain Hardness Genes

“…intermedium, and Zhong 5, using a sodium dodecyl sulfate (SDS) protocol [14]. PCR-based Landmark Unique Gene (PLUG) primers [33] and CINAU (Cytogenetics Institute, Nanjing Agricultural University, Nanjing, China) markers [34] were designed from rice genomic DNA sequences specific for the wheat A, B, and D syntenic regions. Polymerase chain reaction (PCR) was performed in an Icycler Thermal Cycler (Bio-Rad Laboratories, Emeryville, CA, USA) in a 25 µ: reaction, containing 10 mmol Tris-HCl (pH 8.3), 2.5 mmol MgCl 2 , 200 µmol of each dNTP, 100 ng template DNA, 0.2 U Taq polymerase (Takara, Japan), and 400 nmol of each primer.…”

“…We analyzed the amplification patterns of the DNAs from Zhong5, Zhong 2, Hy36, Hy37, and CS, respectively, by using 350 PLUG markers [33] which were located on seven wheat homoeologous groups and 890 CINAU markers [34] derived from all D. villosum chromosomes (Figure 4). The 51 markers from the short arm of group 5, and the 44 markers from the short arm of group 3 generated Th.…”

Sequencing flow-sorted short arm of Haynaldia villosa chromosome 4V provides insights into its molecular structure and virtual gene order

“…However, due to the lower density of markers used in determination of translocated alien fragments, they only generated a physical maps with limited resolution, which consisting of 13 bins. Facilitated by the availability of 4VS scaffolds, a total of 235 PCR-based STS markers were developed [ 29 ] which will dramatically increase the physical map density. Once the resistant gene was fine-mapped, the synteny-based 4VS genome-zipper will be especially helpful for resistance candidate genes prediction.…”

“…The draft sequence obtained will be used to characterize the genomic composition of 4VS including repetitive sequences and gene content, identify microRNA (miRNA) precursors and perform genome-zipper analysis to find syntenic regions among genomes of Triticeae homoeologous group 4 and other sequenced grasses. The sequences can also be used to develop cytogenetic and PCR-based 4VS specific markers [ 29 ], which have the potential use to trace and define alien chromosome in 4VS small fragment translocation lines. The 4VS survey sequence will provide an outline of genome features for H. villosa and facilitate candidate genes discovery on 4VS.…”

Scale development and utilization of universal PCR-based and high-throughput KASP markers specific for chromosome arms of rye (Secale cereale L.)

“…The wheat relatives have significant genetic diversity and abundant valuable genes, therefore can play an important role in wheat improvement [3]. To date, many elite alien genes and desirable traits have been transferred into common wheat through hybridization and chromosome engineering, such as disease resistance, superior yield-related traits, salt and drought tolerance [4][5][6][7].…”