Stem rust resistance gene Sr44 was derived from a partial wheat-Thinopyrum intermedium amphiploid and six derived disomic chromosome addition lines in the French wheat cultivar ‘Vilmorin 27’ background produced by Cauderon et al. in 1973 (1). Later Friebe et al characterized the chromosomal organization of these lines (2). The original name of this gene was SrAgi but was renamed as Sr44in 1996 (3). Sr44 confers resistance the Ug99 race complex including races TTKSK, TTSKT, and TTTSK (4).
Sr44 maps on the short arm of the Th. intermedium 7J#1S short chromosome arm, and when transferred to wheat it was present in non-compensating translocations involving chromosome 7D. However, these non-compensating translocations cannot be used for breeding because of their instability. More recently Liu et al. (4) produced a line with a homozygous compensating wheat-Th. intermedium T7DL•7J#1S Robertsonian translocation which carries Sr44 on the 7J#1S fragment. This line was designated TA5657 and also harbors two wheat-wheat Robertsonian translocations (T5BL.7BL and T5BS.7BS). Although this line is stable it still contains large fragments of Th. intermedium chromatin, so it might not be appropriate for breeding programs. The same authors who produced TA5657 are currently developing a chromosome engineering program to shorten the Th. intermedium segment using ph1b induced homoeologous recombination.
Markers for Sr44
To track the transfer of T7DL•7J#1S Liu et al. (4) developed two CAPS markers, Xbe404728 and Xbe473884, derived from ESTs BE404728 and BE473884. The former maps on the centromeric bin C-7BS1-0.27, and the latter on the distal bin 7AS1-0.89-1.00.
Primers sequences:
Xbe404728
be404728-F 5'- GGT GGT GCC TGT CAA GAT T -3'
be404728-R 5'- TTG ATG GAT CCT GGC TTA GG -3'
Xbe473884
be473884-F 5'- GGC TAT CTC TGG CGC TAA AA -3'
be473884-R 5'- TCC ACA AAC AAG TAG CGC C -3'
PCR conditions:
- Denaturing step: 94°C, 10 min
- Touchdown cycles (decrease 0.5°C/cycle for 10 cycles):
- 94°C, 20 sec
- 63-58.5°C, 45 sec
- 72°C, 120 sec
- Amplification cycles (35 cycles)
- 94°C, 20 sec
- 58°C, 20 sec
- 72°C, 120 sec
- Extension step: 72°C, 10 min
PCR and restriction digestion mixes:
For a 15 µl PCR reaction:
- 90 ng genomic DNA
- 1x PCR buffer (Bioline USA Inc)
- 2 mM MgCl2sub>
- 0.25 mM dNTPs
- 5 pmol forward and reverse primer
- 0.02 unit/µl Taq DNA polymerase (Bioline USA Inc)
The PCR products were digested with restriction enzyme MspI. A total of 5 µl of digestion mixture (3.25 µl of ddH2O, 1.5 µl of 10X NEB buffer 4, 0.15 µl of 100x BSA, 0.1 µl of enzyme stock solution) was added to 10 µl PCR products and incubated for 2 h at 37°C.
Expected products:
CAPS products were resolved on 1.5 % agarose gels and visualized by ethidium bromide staining under UV light.
References
1. The resistance to wheat rusts of Agropyron intermedium and its use in wheat improvement. Cauderon Y, Saigne B, Dauge M. In: Sears ER, Sears LMS (eds) Proceedings of the 4th international wheat genetics symposium, Columbia, Missouri, 1973, pp 401–407.
2. C-banding and in situ hybridization analyses of Agropyron intermedium, a partial wheat x Ag. intermedium amphiploid, and six derived chromosome addition lines. Friebe B, Mukai Y, Gill BS, Cauderon Y. In: Theoretical and Applied Genetics, 1992, 84:899–905
3. Characterization of wheat alien translocations conferring resistance to diseases and pests: current status. Friebe B, Jiang J, Raupp WJ, McIntosh RA, Gill BS. In: Euphytica, 1996, 91:59–87
4. Development and characterization of a compensating wheat-Thinopyrum intermedium Robertsonian translocation with Sr44 resistance to stem rust (Ug99). Liu W, Danilova TV, Rouse MN, Bowden RL, Friebe B, Gill BS, Pumphrey MO. Theoretical and Applied Genetics, 2013, 126:1167–1177. DOI 10.1007/s00122-013-2044-6