Targeted Sequencing of the Short Arm of Chromosome 6V of a Wheat Relative Haynaldia villosa for Marker Development and Gene Mining

The short arm of chromosome 6V (6VS) of Haynaldia villosa has been used in wheat breeding programs to introduce Pm21 resistance gene against powdery mildew (Pm) and some other genes. In this this study, 6VS was flow-sorted from wheat-H. villosa ditelosomic addition line Dt6VS and sequenced by Illumina technology. An assembly of 230.39 Mb was built with contig N50 of 9.788 bp. In total, 3.276 high-confidence genes were annotated and supported by RNA sequencing data. Repetitive elements represented 74.91% of the 6VS assembly. The 6VS homologous genes were identified on homologous group 6 in six Triticeae species confirming their synteny relationships. Out of 45 NB-ARC domain proteins identified on 6VS, 15 were upregulated and might also be involved in the innate immunity of H. villosa to Pm. High thousand grain weight (TGW) for 6VS/6AL translocation line was not attributable to GW2-6V gene. Based on the intron size differences, 119 intron-target (IT) markers were developed to trace the 6VS chromatins introduced into wheat background. The assembled 6VS genome sequence and the developed 6VS specific IT markers in this work will facilitate the gene mining and utilization of agronomic important genes on 6VS.

The Exocyst Complex Subunit EXO70E1-V From Haynaldia villosa Interacts With Wheat Powdery Mildew Resistance Gene CMPG1-V

EXO70 belongs to the exocyst complex subunit that plays a critical role in regulating plant cell polarity establishment and defense response. A previous study proved that the E3 ligase CMPG1-V from Haynaldia villosa, a diploid wheat relative, positively regulates the resistance to wheat powdery mildew (Pm), caused by fungus Blumeria graminis f.sp tritici (Bgt). In this study, a member of EXO70 superfamily named EXO70E1-V was isolated from H. villosa, and EXO70E1-V interacted with CMPG1-V were shown by yeast two-hybrid (Y2H), pull-down assay, bimolecular fluorescence complementation (BiFC) assay, and luciferase complementation imaging (LCI) assay. It is localized in various subcellular organs, i.e., plasma membrane (PM) and endoplasmic reticulum. Co-expression of EXO70E1-V and CMPG1-V showed dot-like structure fluorescence signals that were mainly in PM and nucleus. Expression of EXO70E1-V was relatively higher in leaf and was significantly induced by Bgt infection and exogenous application of hormones such as salicylic acid. Transient or stable overexpression of EXO70E1-V could not enhance/decrease the Pm resistance level, suggesting overexpression of EXO70E1-V alone has no impact on Pm resistance in wheat.

The Regulatory Network of CMPG1-V in Wheat–Blumeria graminis f. sp. tritici Interaction Revealed by Temporal Profiling Using RNA-Seq

Wheat powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is a prevalent fungal disease. The diploid wheat relative Haynaldia villosa (H. villosa) showed broad-spectrum resistance (BSR) to Pm. A previous study reported an E3 ligase gene, CMPG1-V from H. villosa, showing BSR to Pm. To elucidate the regulatory network mediated by CMPG1-V, in this study, gene expression profiling of CMPG1-V transgenic plant (CMPG1-VOE) and its receptor Yangmai 158 was analyzed and compared after Bgt inoculation at four infection stages. GO and KEGG analysis revealed obvious reprogramming of SA and ABA signaling, starch/sucrose metabolism, and photosynthesis in CMPG1-VOE, compared with those in Yangmai 158. Transcripts of SA synthesis genes SARD1 and UGT, signaling factors TGA and PRs, and SnRKs in ABA signaling were specifically upregulated in CMPG1-VOE rather than Yangmai 158. Transcripts of LHCII in photosynthesis, GLUC and TPP in starch/sucrose metabolism were also induced distinctly in CMPG1-VOE. WGCNA analysis showed crucial regulatory candidates of CMPG1-V, involving serine/threonine-protein kinase in phosphorylation, glucosyltransferase in flavonoid biosynthesis, defense factor WRKYs, and peroxidase in oxidative stress. Our results facilitate the deciphering of the resistant regulatory network of CMPG1-V and the identification of key candidates which might be employed in breeding programs.

Targeted sequencing of the short arm of chromosome 6V of a wheat relative Haynaldia villosa for marker development and gene mining

Background: Short arm of chromosome 6V (6VS) of Haynaldia villosa has been used in wheat breeding programs to introduce Pm21 resistance gene against powdery mildew and some other genes. Results: In this work, 6VS was isolated from a wheat ( Triticum aestivum ) - 6VS telosome addition line by flow cytometric sorting and sequenced by illumina technology. The assembly length was 230.39 Mb with contig N50 of 9,788 bp. The sequence annotation identified 3,276 high confidence genes supported by RNA sequencing data, representing about 2.3% of the chromosome arm sequence; repetitive elements accounted for 74.91% of the arm sequence. Sequences homologous to 6VS genes were identified on short arms of chromosomes 6A of T. urartu , 6D of Aegilops tauschii , 6A and 6B of T. dicoccoides , 6A, 6B and 6D of T. aestivum and 6H of Hordeum vulgare , revealing synteny relationships among these chromosome arms. Based on differences in intron size between the homologous genes on 6VS and 6AS/6BS/6DS of T. aestivum , 222 primer pairs were designed. Out of them, 120 amplified 6VS-specific products and are suitable as intron-target (IT) markers to trace the 6VS chromatin introduced into wheat. Conclusions: The results obtained and markers developed in this work will facilitate introduction of important genes to common wheat from its wild relative, while reducing the presence of unfavorable genes due to linkage drag.

Targeted sequencing of the short arm of chromosome 6V of a wheat relative Haynaldia villosa for marker development and gene mining

Background: Short arm of chromosome 6V (6VS) of Haynaldia villosa has been used in wheat breeding programs to introduce Pm21 resistance gene against powdery mildew and some other genes. Results: In this work, 6VS was isolated from a wheat ( Triticum aestivum ) - 6VS telosome addition line by flow cytometric sorting and sequenced by illumina technology. The assembly length was 230.39 Mb with contig N50 of 9,788 bp. The sequence annotation identified 3,276 high confidence genes supported by RNA sequencing data, representing about 2.3% of the chromosome arm sequence; repetitive elements accounted for 74.91% of the arm sequence. Sequences homologous to 6VS genes were identified on short arms of chromosomes 6A of T. urartu , 6D of Aegilops tauschii , 6A and 6B of T. dicoccoides , 6A, 6B and 6D of T. aestivum and 6H of Hordeum vulgare , revealing synteny relationships among these chromosome arms. Based on differences in intron size between the homologous genes on 6VS and 6AS/6BS/6DS of T. aestivum , 222 primer pairs were designed. Out of them, 120 amplified 6VS-specific products and are suitable as intron-target (IT) markers to trace the 6VS chromatin introduced into wheat. Conclusions: The results obtained and markers developed in this work will facilitate introduction of important genes to common wheat from its wild relative, while reducing the presence of unfavorable genes due to linkage drag.