Bansal et al (1) found a QTL, designated Qsr.sun-5BL, in an Arina/Forno recombinant inbred lines (RIL) population that was responsible for a 12-15 % reduction in stem rust severity. The donor of this QTL was the Arina parent, a Swiss winter wheat cultivar. This QTL was mapped on the distal region of the long arm of chromosome 5B. On a 1-9 scale of rust response (one, resistant; nine, susceptible) cultivar Arina produced a score of six, equivalent to a moderately susceptible response.
In a following study and to determine the exact location of Qsr.sun-5BL, Bansal et al (2) developed a second mapping population between Arina and Yitpi, an Australian cultivar susceptible to stem rust. These recombinant lines were tested against Australian pathotype Pgt pathotype 34-1,2,7 + Sr38. The rationale for this particular cross was that it had a higher degree of polymorphism than the Arina/Forno cross tested in first place.
In the Arina/Yitpi RIL population the stem rust response locus segregated as a single gene, and it was designated Sr56. By screening and combining results of a set of 30 SSR markers, 16 STS markers derived from DarT markers, 40 STS derived from ESTs and the iSelect 9 K SNP chip, They showed that Sr56 is located on deletion bin 5BL16-0.79-1.00, on an interval flanked by sun209 at 2.6 cm on the proximal end, and sun320 at 1.2 cM, distally. As a result of the incorporation of closer markers the phenotypic variation explained by Sr56 increased from 12-15% to 14-27%.
The two flanking markers were validated on a set of 32 Australian cultivars. Out of these 32, 28 lines did not amplify any Sr56 linked products, three produced false positives for sun209 and a different line produced a false positive for sun320.
Markers for Sr56
sun209
sun209 is a microsatellite marker (SSR) developed by Sidney University based on the analysis of the Brachypodium genome.
Primers sequences:
sun209-F 5'- CTG TAA GGT TCT TTC GGA TTG G -3'
sun209-R 5'- CAT GGT CTT CGA CGA CTT AGT G -3'
Final concentrations of the PCR mix
- 30 ng/µl genomic DNA
- 50 nM forward primer with M13 tail
- 50 nM infared 700 or 800-labelled M13 primer
- 125 nM dNTPs
- 0.04 U/µl Immolase DNA polymerase(Bioline)
- 1X Immolase PCR buffer containing 1.5 mM MgCl2
Reaction volume: 10µl
PCR conditions
- Denaturing step: 95°C, 10 min
- Amplification step (35 cycles):
- 95°C, 15 s
- 60°C, 30 s
- 72°C, 30 s
PCR was performed in a T100™ Thermal Cycler (BIO-RAD USA). Amplification products were separated on a 8% polyacrylamide gel using an Analyzer Gene ReadIR 4200 (Li-COR sequencing system).
sun320
sun320 is an STS marker derived from the wPt-7665 DArT marker.
Primers sequences:
sun320-F 5'- TAG CAA ACG CAA CAA TTT GG -3'
sun320-R 5'- CAT CAG TTT CTA CGG CAG CA G -3'
PCR conditions
- Denaturing step: 95°C, 10 min
- Amplification step (35 cycles):
- 95°C, 30 s
- 60°C, 40 s
- 72°C, 50 s
PCR products were separated on 2% agarose gel.
Expected products
When linked to Sr56, marker sun209 amplifies a 448-bp product, while marker sun320 yields a 448-bp PCR product.
References
1. Genetic mapping of seedling and adult plant stem rust resistance in two European winter wheat cultivars. Bansal UK, Bossolini E, Miah H, Keller B, Park RF, Bariana HS. In: Euphytica, 2008, 164: 821-828. DOI:10.1007/s10681-008-9736-z.
2. Molecular mapping of an adult plant stem rust resistance gene Sr56 in winter wheat cultivar Arina. Bansal U, Bariana H, Wong D, Randhawa M, Wicker T, Hayden M, Keller B. In: Theoretical and Applied Genetics, 2014, 127:1441-1448. DOI:10.1007/s00122-014-2311-1.