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ARS Home » Plains Area » Fargo, North Dakota » Edward T. Schafer Agricultural Research Center » Cereal Crops Research » Research » Research Project #438336

Research Project: Localization and Identification of Barley Resistance Genes to Net Form Net Blotch

Location: Cereal Crops Research

Project Number: 3060-21000-038-032-T
Project Type: Trust Fund Cooperative Agreement

Start Date: Jun 15, 2020
End Date: Jun 14, 2022

Objective:
1) Fine mapping of the major QTL on 7H, QRptm-7H-119-137 (QRptm-7H hereafter). 2) Identification of candidate genes for QRptm-7H. 3) Development of user-friendly markers to assist barley breeding for resistance to spot form net blotch (SFNB). 4) Field and greenhouse test to confirm if the resistance induced by disruption of QRptm-7H is effective at both seedling and adult stages.

Approach:
1) Generation segregating population using Pinnacle (S) and PI67381 (R) Pinnacle is widely used by breeding program of the cooperator, but it is susceptible many Ptt isolates, including 4 geographically different isolates collected from ND (FGO), Montana (CA17 and PA17), and Australia (SG1) while PI 67381 is resistant to these four isolates. Genetic analysis revealed that spot form net blotch (SFNB) susceptibility is controlled by the major QTL QRptm-7H and two minor QTL on 2H and 3H in Pinnacle (Tamang et al., 2019). Cooperator provided F2s and recombinant inbred lines (RILs) derived from Pinnacle x PI67381. A total of 200 F2s will be genotyped with flanking markers for the three loci, and the individuals segregating only at the 7H locus will be selected to develop near-isogenic lines (NILs). Meanwhile, 180 RILs will be used to identify heterozygous residuals at the 7H locus for construction of NIL. 2) Fine mapping of QRptm-7H At least 1000 RILs will be used for genetic mapping. Inoculum isolate preparation and inoculation will follow the methods established by Nepane et al. (2015). Briefly, two-week-old seedlings growing in a greenhouse will be spray-inoculated, followed by incubation for 24 hours in a mist chamber. The inoculated plants will be then transferred to a growth chamber. Ninety-four RILs will be used first for SNP genotyping with the barley 50k iSelect SNP Array. To assisted breeding selection, the linked SNPs will be converted to PCR-based markers, such as semi-thermal asymmetric reverse PCR (STARP) markers. 3) Data Analysis and interpretation Disease reactions will be evaluated seven days after inoculation using a 1 to 5 scale as described by Neupane et al. (2015), where 1 is highly resistant and 5 is highly susceptible. The average disease severities from three independent replicates of the phenotyping assays will be used for mapping of SFNB resistance/susceptibility. A permutation test with 1000 iterations will be performed to find a LOD threshold for QTL analysis at a significance level of a = 0.05 and 0.01. Linkage analysis and QTL mapping will be conducted using the software program Joinmap. 4) Identification of candidate genes for QRptm-7H. Gene annotation will be conducted with the genome sequence at the QRptm-7H region, and potential candidate genes will be identified for functional validation. User-friendly markers will be developed for the candidate genes. 5) Field and greenhouse test of QRptm-7H at both seedling and adult stages Near-isogenic lines will be used for field and greenhouse test to confirm if the 7H locus is effective at both seedling and adult stages. Three replicates with a completely randomized design will be employed.