Stem rust resistance gene Sr24

Contributed by I.Z. Lowe and M.A. Soria

Markers for Sr24

Sr24 offers resistance to most races of stem rust, including the virulent race Ug99 (TTKSK) now established in East Africa and Ethiopia. Incidents of virulence to this major resistance gene has been reported in South Africa (1) and India (2). Also, Sr24 is not effective against a more recent variant of UG99, designated TTKST.

From their examination of the rust-resistant hexaploid wheat Agent, Smith et al. (3) first determined Sr24 to reside on the 3DL chromosome, within a spontaneous translocation from the 3Ag chromosome of Agropyron elongatum.  In 1973, Sears developed more recombinant lines, successfully introducing a much smaller A. elongatum translocation segment containing Sr24 into the 3DL chromosome.  This truncated segment broke the linkage between Sr24 and red grain color observed in Agent, allowing the subsequent introgression of Sr24 into white-grained wheats. Sr24 was also discovered in the wheat variety Amigo, located in an A. elongatumsegment within a translocation derived from rye.  In Amigo-type resistance varieties, Sr24 is found on 1BS rather than 3DL (1BL.1BS-3Ae#1 translocation).

The leaf rust resistance gene Lr24, also found within the A. elongatum translocation mentioned above, is linked to Sr24 in all of the recombinant types discussed.

There are several different molecular markers available for Sr24, including an SSR (BARC71) and two AFLPs (Sr24#12 and Sr24#50) (1).  The Sr24#12 AFLP marker has been found completely linked to Sr24. The Sr24#50 marker failed to predict the presence of Sr24 in some germplasm. Standard PCR primers have been developed for both fragments.

Primers sequences:

Xbarc71

BARC71-F       5'- GCG CTT GTT CCT CAC CTG CTC ATA -3'

BARC71-R       5'- GCG TAT ATT CTC TCG TCT TCT TGT TGGT T -3'

Sr24#12

Sr24#12-F       5'- CAC CCG TGA CAT GCT CGT A -3'

Sr24#12-R       5'- AAC AGG AAA TGA GCA ACG ATG T -3'

Sr24#50

Sr24#50-F       5'- CCC AGC ATC GGT GAA AGA A -3'

Sr24#50-R       5'- ATG CGG AGC CTT CAC ATT TT -3'

PCR conditions for BARC71:

  • Denaturing step: 94°C, 5 min
  • Amplification step (37 cycles):
    • 94°C, 30 sec
    • 56°C, 30 sec
    • 72°C, 30 sec
  • Extension step: 72°C, 10 min

PCR conditions for Sr24#12:

  • Denaturing step: 94°C, 5 min
  • Touchdown step 1 (7 cycles, 1ºC down each cycle):
    • 92°C, 30 sec
    • 62°C, 30 sec
    • 72°C, 30 sec
  • Amplification step (30 cycles):
    • 92°C, 30 sec
    • 59°C, 30 sec
    • 72°C, 30 sec
  • Extension step: 72°C, 10 min

PCR conditions for Sr24#50:

  • Preamplification step: 20°C, 1 min
  • Denaturing step: 94°C, 3 min
  • Amplification step (30 cycles):
    • 94°C, 30 sec
    • 63°C, 30 sec
    • 72°C, 40 sec
  • Extension step: 72°C, 10 min

PCR reaction mix

For a 20-µl PCR reaction:

  • 12 µl dH20
  • 2 µl DNA (50-75 ng/µl)
  • 4 µl 5X premix (see below)
  • 0.5 µl forward primer (10 µM)
  • 0.5 µl reverse primer (10 µM)
  • 1 µl Taq

5X premix (makes 1000 ul):

  • 160 µl dH2sub>0
  • 10 µl each dNTP (100 mM)
  • 500 µl Taq buffer (10X, see below)
  • 300 µl MgCl2 (25 mM)

10X Taq buffer (makes 1000 µl):

  • 100 µl Tris-HCl (1 M, pH 8.8)
  • 250 µl KCl (2 M)
  • 10 µl Tween 20 (10%)
  • 640 µl dH20


Expected products:

Xbarc71

Xbarc71 is the most distal marker mapped on the long arm of chromosome 3D of wheat, and all of the varieties carrying Sr24amplified a pair of diagnostic bands (103 bp and 85 bp), as shown below.  While most of the susceptible lines lacking Sr24 amplified a 107-bp fragment, some non-Sr24 wheat varieties carried different alleles (e.g. see lanes 5, 6, and 10 in the gel image).

Sr24 - Barc71
PCR amplification for Xbarc71 linked to Sr24 locus. Products were separated in a 6% polyacrylamide gel. Lane 1: Molecular ladder (L), indicating a 100-bp fragment.  Lanes 2, 3, 13, and 15: The pair of 103-bp and 85-bp bands that is characteristic of the presence of Sr24.  All other lanes: No indication of the presence of Sr24.

 

Sr24#12

STS Sr24#12 is a dominant marker that amplifies a single 500-bp band linked to Sr24.

Sr24 - #12
PCR amplification with Sr24#12. Products were separated on a 1% agarose gel. Lane 9: Molecular ladder (L).  Lanes 1, 2, and 5: The 500-bp band that is characteristic of the presence of Sr24.  All other lanes: No indication of the presence of Sr24.
Sr24#50

STS Sr24#50 is a dominant marker that amplifies a single band of 200-bp linked to Sr24.

 

Conditions presented here should be considered only as a starting point of the PCR optimization for individual laboratories.

References

1. Development of PCR markers for the selection of wheat stem rust resistance genes Sr24 and Sr26 in diverse wheat germplasm. Mago R, Bariana HS, Dundas IS, Spielmeyer W, Lawrence GJ, Pryor AJ, Ellis JG. In: Theoretical and Applied Genetics, 2005, 111:496-504. DOI: 10.1007/s00122-005-2039-z

2. A pathotype of Puccinia graminis f. sp. tritici on Sr24 in India. Bhardwaj SC, Nyar SK, Prashar M, Kumar J, Menon MK, Singh SB. In: Cereal Rusts and Powdery Mildews Bulletin, 1990, 18:35-38.

3. Registration of agent wheat. Smith EL, Schlehuber AM, Young HC Jr, Edwards LH. In: Crop Science, 1968, 8:511-512.

Tags

stem rust resistance