There are two STS markers available for detecting the presence of Yr5: STS-7/STS-8 and STS-9/STS-10. The PCR protocol is the same for both sets of primersPrimers:
STS-7 / STS-8
STS-7: 5'- GTA CAA TTC ACC TAG AGT -3' STS-8: 5'- GCA AGT TTT CTC CCT ATT -3'
STS-9 / STS-10
STS-9: 5'- AAA GAA TAC TTT AAT GAA -3' STS-10: 5'- CAA ACT TAT CAG GAT TAC -3'PCR conditions:
A 2.5 min ramp time is used between the 94°C denaturation and the 45°C annealing steps; for all other temperature transitions, the fastest possible ramp is used. The number of amplification cycles can be reduced to 40 if a more sensitive detection system is used.
Final concentrations of the products used in the PCR reaction:
Final volume: 15 µlExpected products:
PCR products are separated in 5% denaturing polyacrylamide gel. When using the set of primers STS-7 and STS-8, Avocet produces a band of 472 bases and Yr5-Avocet a band of 478 bases. The other marker STS-9 / STS-10 yields a product of 433 for Avocet and 439 for Yr5-Avocet.
Some cultivars are not polymorphic for the STS markers described above. Besides, polyacrylamide gels are more difficult to prepare and develop than agarose gels. Chen et al. (In press) analyzed the sequence of the fragments amplified by the STS markers and found a single base pair polymorphism that generates a Dpn II restriction site in the DNA of susceptible parents. This restriction site was used to develop a CAPS marker that was tested in a large set of cultivarsProcedure:
Some laboratories reported difficulties amplifying with primer STS-9. As an alternative, the PCR amplification can be done using primers pairs STS-7/STS-8 or STS-7/STS-10. In both cases the digestion procedure is the same as indicated before, and the products are co-dominant.Expected products:
After digestion the resistant line Yr5-Avocet yields five restriction fragments (289 bp, 63 bp, 56 bp, 20 bp
and 12 bp), while Avocet and other non-Yr5 genotypes produce four restriction fragments (182 bp, 102 bp,
20 bp and 12 bp). The presence/absence pattern of the 289-bp fragment from the Yr5 line and the 182-bp fragment
from the susceptible lines can be scored in agarose gels.