Towards map-based cloning of a Hessian fly resistance gene H34 in wheat

Abstract

Wheat is a staple food crop worldwide and insect damage is a major constraint for its production. Among the insects, Hessian fly (HF, Mayetiola destructor) is a destructive pest that significantly reduces wheat grain yield. To date, 37 HF resistant genes have been named, but diagnostic markers for these genes are lacking, which hampers their deployment in wheat breeding. HF resistance gene H34 on the short arm of chromosome 6B was one of the genes from a U.S. winter wheat Clark. To finely map H34, a cross was made between two F₁₂ recombinant inbred lines (RIL115-S and RIL118-R) derived from Ning7840 x Clark. RIL118-R carries the resistance allele and RIL118-S carries the susceptibility allele at H34. Screening 286 (RIL115-S x RIL118-R) F₃ lines using flanking Kompetitive Allele Specific PCR (KASP) markers identified five heterogenous inbred families (HIFs) segregating at H34. The first round of screening of 159 homozygous recombinant plants from five different HIFs using the KASP markers delimited H34 to a 5.0 Mb interval. Genotype-by-sequencing (GBS) analysis of the four pairs of near-isogenic lines (NILs) from the selected HIFs identified additional SNPs in the H34 region that further narrowed the H34 region to 1.3 Mb after screening 75 additional homozygous recombinant NILs. RNA-sequencing (RNA-seq) of the four pairs of NILs identified three differentially expressed genes (DEGs) in the H34 interval and they were considered as the putative H34 candidate genes for further study. Using the sequences of the DEGs and GBS-SNPs identified in the H34 interval, seven KASP markers were designed and validated to be diagnostic in a US winter wheat panel of 203 lines. These markers can be used in gene pyramiding of H34 with other HF resistance genes using marker-assisted selection (MAS) in the U.S. wheat-breeding programs. Furthermore, studying mechanism of HF resistance in wheat using RNA-seq data revealed that genes encoding defense proteins, stress-regulating transcription factors, and secondary metabolites were strongly up regulated within the first 48 hours of larval feeding, revealing an early defense in resistant wheat plants in response to larval attack. Also, HF feeding on resistant plants triggered the secretions of R-gene receptors by HF to initiate a hypersensitive response (HR) in the plants. This HR response resulted in production of reactive oxygen species (ROS) to up regulate the downstream genes involved in cell wall fortification and activation of different transcription factors (TFs), which prevents HF to access the nutrients in the resistant plants and eventually results in the death of HF larvae. The new knowledge generated in this study will aid in better understanding of HF-resistant mechanisms and developing new crop improvement strategies to increase HF resistance in wheat.