Contributed by Long-Xi Yu and Mark Sorrells
Sr22 was previously mapped on the long arm of chromosome 7A (Khan et al., 2005). Three linked markers, cfa2019, cfa2123 and BARC121 (Miranda et al., 2007), were used for haplotyping this locus in our study (Yu et al. 2010).

Primers sequences and PCR conditions:

CFA2123

5'-cggtctttgtttgctctaaacc-3’

5'-accggccatctatgatgaag-3’

PCR conditions

• Denaturing step: 94°C, 3 min
• Touchdown cycles (decrease 1°C/cycle for 15 cycles):
  • 94°C, 30 sec
  • 65-51°C, 30 sec
  • 72°C, 45 sec
• Amplification cycles (25 cycles)
  • 94°C, 30 sec
  • 50°C, 30 sec
  • 72°C, 45 sec
• Extension step: 72°C, 7 min

CFA2019

5'-gacgagctaactgcagaccc-3’

5'-ctcaatcctgatgcggagat-3’

PCR conditions

• Denaturing step: 94°C, 3 min
• Amplification step (35 cycles):
  • 94°C, 30 sec
  • 60°C, 30 sec
  • 72°C, 45 sec

Extension step: 72°C, 7 min

BARC121

5'-actgatcagcaatgtcaactgaa-3’

5'-ccggtgtctttcctaacgctatg-3’

PCR conditions

• Denaturing step: 94°C, 3 min
• Amplification step (35 cycles):
  • 94°C, 45 sec
  • 50°C, 30 sec
  • 72°C, 45 sec
• Extension step: 72°C, 7 min

Expected products:

PCR amplification by cfa2019 and cfa2123 showed 235 and 245 bp fragments, respectively in the Sr22 source germplasm, ‘Sr22TB’. BARC121 amplified two polymorphic bands of 215 and 230 bp in most germplasm. However, only the 215 bp fragment was amplified in ‘Sr22TB’, indicating that only the 215 fragment was specific to Sr22, while the 230 bp fragment was not associated with Sr22. Therefore, the haplotype for Sr22 was “235-245-215” for markers cfa2019, cfa2123 and BARC121, respectively (Yu et al., 2010). Since these markers are not completely diagnostic and may produce false positive result. Care must be taken when interpreting the result.