Disease resistance. Leaf Rust Resistance.

Lr51

CAPS Marker for Lr51

Helguera et al. (2) developed a CAPS (Cleaveged Amplified Polymorphic Sequence) marker for the AGA7 gene located within the T. speltoides translocation containing Lr51. Primers S30-13L and AGA7-759R were designed to amplify preferentially AGA7 alleles from the S and B genomes, and to discriminate against the A and D genomes. Since the T. speltoides segment does not recombine with the wheat chromosomes in a Ph background, only one marker is enough to follow the segment in a breeding program.

   S30-13L     5'- GCA TCA ACA AGA TAT TCG TTA TGA CC -3' 
   AGA7-759R   5'- TGG CTG CTC AGA AAA CTG GAC C -3'    

• 100 ng genomic DNA
• 0.2 µM each primer
• 200 µM each dNTP
• 1U Taq DNA polymerase (Promega)
• 1x Taq polymerase buffer (Promega)
• 1.5 mM MgCl₂

Total volume: 25 µl

• Denaturing step: 94°C, 4 min
• Amplification step (40 cycles):
• 94°C, 45 s
• 52°C, 45 s
• 72°C, 60 s
• Extension step: 72°C, 10 min

10 µl of the PCR amplification products were digested with restriction enzymes Pst I or BamH I (Promega) by adding 5 units of enzyme to the PCR product. The mix was incubated for 2 h at 37°C.

After digestion samples were separated by electrophoresis in 2% agarose gel and visualized using ethidium bromide and UV light. Pst I cut only the S genome amplification product (397-bp and 422-bp), whereas BamH I cut only the B genome amplification product (672-bp and 111-bp).

The amplification products from primers S30-13L /AGA7-759R from the B (783-bp) and S (819-bp) alleles can be also separated using polyacrylamide gels (14 %) without digestion.

Undigested and Pst I digested PCR amplification products. 1-3) Chinese Spring nullisomic-tetrasomic lines. 1) N1AT1B, 2) N1BT1D, 3) N1DT1A, 4) Neepawa, 5) Neepawa*6/Triticum speltoides F-7-3. The red arrowhead indicates the B-genome product . The cyan arrowhead indicates the digestion productsfrom the S genome.

BamH I digestion of PCR products. Nee: Neepawa. NeeF7: Neepawa*6/Triticum speltoides F-7-3. Labels "BS" and "SS" indicate the genomes present at the Lr51 locus in different BC6F2 plants. Parental line Neepawa (Nee, ane 7) has a BB genotype. The orange arrowhead indicates the 819-bp PCR amplification product from the S genome allele and the green arrowhead shows the larger of the two BamH I digested fragments from the B genome allele of XAga7.

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