Cultivar Axe was crossed with a susceptible genotype Nybing-3, a selection from cultivar Nyabing. F1 seeds were grown and harvested separately for producing the F2 population. Individual F2 seeds were planted 10 cm apart in the field and each plant was harvested separately. A single spike was harvested from each greenhouse grown F3 family to generate an F4 population. Similarly, single head harvest from F4 generation led to the production of an F5 population. Single seed from each F5 line was grown and the whole plant was harvested to raise an F6 generation. The final set of 151 lines is referred to as the Axe/Nyb recombinant inbred line (RIL) population.
The Axe/Nyb RIL population was screened in the greenhouse against the most prevalent Pst pathotype 134E16A+Yr17+Yr27+ (Plant Breeding Institute culture no. 617). Eight to 10 seeds from each RIL (four RILs per pot) were sown in 9-cm diameter pots filled with a potting mixture comprising of composted pine bark and sand in a 2:1 ratio, followed by fertilizer treatment (25g Aquasol®; Hortico Pty. Ltd., Revesby, NSW, Australia /10L of water for 100 pots). Seedlings were fertilised weekly with Urea at the same rate as Aquasol. Inoculations were carried out at the 4th leaf stage in a specialized inoculation chamber by atomising urediniospores of Pst pathotype 134E16 A+Yr17+Yr27+ suspended in light mineral oil (Isopar L, 5 mg spores 10 ml-1 of oil as solvent) using a hydrocarbon propellant pressure pack. After inoculation, plants were moved to luke-warm water-filled steel trays covered with polythene hoods in the incubation room set at 9-12 °C. Following 24 hours of incubation, plants were transferred to microclimate rooms (automated temperature and irrigation control) set at 17 °C. Rust response assessments were performed 16 -18 days after inoculation using a 0 to 4 scale described in McIntosh et al. (1995.) The Axe/Nyb RILs were classified as homozygous resistant (HR), homozygous susceptible (HS) and segregating (this class can have very low frequency and sometime could represent a mixture).
DNA from 10-12 days old seedings of each Axe/Nyb RIL and parents was extracted and quantified following a modified CTAB method outlined in Bansal et al. (2014).
Chromosomal location of stripe rust resistance
For identification of chromosomal location of the resistance locus, selective genotyping was carried out on eight resistant and eight susceptible RILs using an Illumina iSelect 90K Infinium SNP genotyping array (Wang et al. 2014). The stripe rust response-linked SNPs were converted into kompetitive allele-specific polymerase chain reaction (KASP) assays and were tested on parental lines (Axe and Nyb) using the protocol described by LGC (Laboratory of the Government Chemist) genomics, UK (www.biosearchtech.com/ngs). The KASP markers which gave clear clusters were genotyped on the entire RIL population for construction of a linkage map.
All KASP assays in this study were genotyped following the procedure described in Pakeerathan et al. (2019).
Detailed mapping using a targeted genotyping-by-sequencing (tGBS) assay
Once the genomic region that controls stripe rust resistance was identified through selective genotyping, we used targeted genotyping-by-sequencing (tGBS) assay for saturation of the map. This service was provided by Agriculture Victoria, Agribio, Bundoora, Victoria. The purpose of the tGBS assay was to capture additional polymorphisms between parents by sequencing the genomic region of interest. The genetic map was constructed using the R package ASMap (Taylor and Butler 2017) and imputations for missing SNP data were performed using Impute v2.2 (Sargolzaei et al. 2014).
An automated bioinformatic pipeline PolyMarker (Ramirez-Gonzalez et al. 2015) was used for designing KASP markers from the SNPs associated with stripe rust resistance identified through tGBS mapping. Allele specific primers A1 and A2 were tagged with diagnostic sequences for fluorescent dyes FAM (gaaggtgaccaagttcatgct) and HEX (gaaggtcggagtcaacggatt) at their 5ʹ, respectively.
A set of seven KASP markers from the tGBS assay were genotyped on the entire Axe/Nyb RIL population (154 lines) and these markers were incorporated into the initial map. Following this, a large mapping population of 1032 F2 plants (2064 gametes) from an Axe/Nyb cross was developed. DNA from this population was extracted using a modified SDS (sodium dodecyl sulfate) DNA extraction protocol (M. Pourkheirandish personal communication). Leaf tissue from individual F2 plants was collected in 96 wells plate and two ball bearings and 450µl of extraction buffer (100mM Tris-HCl pH 8.0, 50mM EDTA pH 8.0, 500mM NaCl and 10mM 2-mercaptoethanol) was added in each well and samples were crushed in a tissue lyser (1600 MiniGTM) for 3 minutes. After crushing, 60µl of 10% SDS buffer was added and samples were inverted vigorously for homogenous mixing. Samples were then incubated at 65 °C for 60 min and the plate was centrifuged for 1 min at 3600 rpm. After centrifugation, 200µl of 7.5 M ammonium acetate was added and the plate was shaken vigorously, followed by incubation for 60 min at 4 °C. After centrifugation at 1000 rpm for 1 min, 300µl of chloroform was added and the plate was again shaken vigorously, followed by centrifugation for 10 min at 4800 rpm. One hundred µl of supernatant was added to100µl of chilled isopropanol in new plate and mixed gently. After centrifugation for 10 min at 4800 rpm, the isopropanol was removed and the pellet was resuspended in 100µl of 1M Tris HCl (pH 8) containing 10mM of RNase, with incubation at 37 °C for 1-2 hours.
Yr75 Flanking markers were tested on a large F2 population. Recombinants were phenotyped in the F3 generation against Pst pathotype 134E16A+17+27 to refine the location of the stripe rust resistance locus.
Statistical analysis and genetic mapping
Chi-squared (χ2) analysis was used to identify segregation distortion among markers located near the resistance locus. The KASP and SSR marker data were converted to ‘A’ for Axe allele and ‘B’ for Nyb allele and ‘H’ for heterozygotes for mapping. Genetic linkage maps were constructed using MapManager QTXb20 (Manly et al. 2001) with the Kosambi map function (Kosambi 1943) and then presented graphically using MapChart version 2.3 (Voorrips 2002). The physical position of markers that flanked the resistance locus and structural variation in the genomic region was investigated using the tool Pretzel (Keeble-Gagnère et al. 2019) which compares genetic and physical maps.