Disease resistance. Leaf and stripe rust resistance

Lr37-Yr17-Sr38

There are two markers available for detecting the presence of the fragment containing the genes Lr37-Yr17-Sr38. The first is a PCR marker, dominant for the 2NS segment, while the other is a CAPS (cleavage amplified polymorphic sequence) marker. The CAPS marker requires an extra step of digestion with a restriction enzyme after the PCR amplification, but has two advantages, it is codominant and can be used with DNA of lower quality than the PCR marker.

PCR marker specific for the N genome:

The pair of primers VENTRIUP-LN2 was designed to amplify preferentially the N-allele of Xcmwg682. The 3' end from the left primer VENTRIUP matches a point mutation at position 46 that differentiates the N-allele from the other three wheat alleles, while the 3' end of the right primer LN2 matches a TT indel at positions 261-262 that is specific for the N and A-alleles (see sequences).

VENTRIUP 5'- AGG GGC TAC TGA CCA AGG CT -3'
LN2      5'- TGC AGC TAC AGC AGT ATG TAC ACA AAA -3'

• 1X Taq buffer (Mg²⁺ free)
• dNTP: 200 µM each
• MgCl₂: 1.5 mM
• primers: 0.2 µM each
• Taq polymerase: 1.0 U
• DNA input: 120 ng/reaction

Final volume: 25 µL

• Denaturing step: 94°C, 45 sec
• Amplification step (30 cycles)
  • 94°C 45 sec
  • 65°C 30 sec
  • 72°C 60 sec
• Extension step: 72°C 7 min

These two primers are expected to amplify a 259-bp fragment in plants carrying one or two doses of the 2NS translocation.

PCR amplification with 2NS specific primers VENTRIUP and LN2. Genomic DNAs were extracted from Madsen, Yecora Rojo (YR) and six backcross individual plants. AA: absence of 2NS translocations, AN: heterozygosity, NN: homozygous for the translocation. The arrow indicates the 2NS specific 262-bp PCR amplification product. M: molecular size markers.

The authors also developed a TaqMan® assay for this allele which can be used in high throuput breeding program.

CAPS marker specific for the A genome:

The 259-bp PCR product from primers VENTRIUP-LN2 is a dominant marker and therefore cannot differentiate heterozygous from homozygous 2NS individuals. A marker to differentiate these two classes is a desirable tool for selection of 2NS homozygous plants in F₂ segregating populations or after self-pollination of the heterozygous BC plants from the last cycle of a backcrossing program. One way to differentiate the homozygous 2NS plants from the heterozygous is by the absence of the 2A-allele in the homozygous 2NS plants. A CAPS marker for the 2A-allele was developed to complement the 2N-allele specific primers: primers URIC and LN2 amplify preferentially the N and A-alleles of Xcmwg682. These two alleles can then be differentiated by the presence of a Dpn II restriction site at position 110 that is present in the A and B genomes and is absent in the D and N genomes (see sequences).

URIC 5'- GGT CGC CCT GGC TTG CAC CT -3'
LN2  5'- TGC AGC TAC AGC AGT ATG TAC ACA AAA -3'

The reagents and concentrations are the same as for the previous set of primers.

• Denaturing step: 94°C, 45 sec
• Amplification step (38 cycles)
  • 94°C 45 sec
  • 64°C 30 sec
  • 72°C 60 min
• Extension step: 72°C 7 min

Following amplification with CAPS primers, 5µL of the PCR reaction were digested with restriction enzyme Dpn II by adding 5 U of enzyme to the PCR product. Samples were separated by electrophoresis in 2% agarose gel and visualized using ethidium bromide and UV light.

Amplification of wheat genomic DNA from heterozygous 2AS/2NS plants with CAPS primers URIC and LN2 amplify two fragments of 285-bp (N-allele) and 275-bp (A-allele). Although the size difference between these two fragments is small, they can be clearly separated in polyacrylamide gels as well as in 4% Metaphor agarose gels. However, both alleles can be better differentiated after digestion with Dpn II: an undigested 285-bp band corresponds to the N genome PCR product and the 166 and 109-bp fragments corresponded to the digested PCR product of the A genome.

CAPS marker for the A and N genomes. PCR fragments were amplified with primers URIC – LN2 followed by Dpn II digestion. Del-2AS5 is a deletion line lacking the distal 22% of the 2AS arm and the targeted Xcmwg682 locus; N2BT2D and N2DT2A are Chinese Spring nullisomic-tetrasomic lines. The remaining six lanes are individuals from an F2 backcross population. “A” and “N” : A and N genomes respectively. The arrows indicate the undigested PCR amplification product of 2N genome (285-bp) and digested fragments (166 and 109 bp) from the A genome. M: molecular size markers.

The presence of the Dpn II restriction site was tested in a diverse set of 18 cultivars. After digestion with Dpn II, the 285-bp undigested fragment was not observed in any of these cultivars indicating that the Dpn II restriction site was conserved in the A genome from all the genotypes

Cultivars tested: