Quality traits. Semolina Color

PCR-based marker for Lr19, Sr25 and Y

The Gb primers were developed by Prins et al (7). The germplasm used for that publication was an allosynthetic recombinant produced several years earlier from the original translocation on 7D. An intercalary piece of the translocation was retained that only had Lr19 on it; both Y and Sr25 were lost during recombination, making it a suitable line for breeding in hexaploid lines because the white color of flour would be unaffected. Also the shorter translocation was relocated to 7BL during pairing induction. In spite of these rearrangements, this marker also detects the original Lr19 translocation on 7D (Frans Marais, personal communication).

The PCR marker based on EST BF145935 was originally designed for stem rust resistance gene Sr25, and you can find more details about these gene and marker validation here.

Primer sequences

BF145935

   BF145935-F   5'- CTT CAC CTC CAA GGA GTT CCA C -3’
   BF145935-R   5'- GCG TAC CTG ATC ACC ACC TTG AAG G -3’   

   Gb-F  5'- CAT CCT TGG GGA CCT C -3’
   Gb-R  5'- CCA GCT CGC ATA CAT CCA -3’    

• Denaturing step: 94°C, 4 min
• Amplification step (35 cycles):
• 94°C, 45 sec
• 50°C, 30 sec
• 72°C, 45 sec
• Extension step: 72°C, 7 min

• Denaturing step: 5 minutes at 94°C.
• Amplification step (40 cycles)
• 94°C, 30 sec
• 60°C, 30 sec
• 72°C, 1 min
• Extension step: 5 minutes at 72°C.

Final concentrations of the reagents used in the PCR amplification of Gb.

• 50-100 ng template DNA
• 5 pmol each primer
• 1 unit of Taq DNA polymerase
• 2 µl 15 mM MgCl₂
• 2 µl 10X buffer
• 2 µl 1mM dNTPs

Total volume: 25 µl reaction

Following amplification for Gb, the products are separated in 2% agarose gels at 65 V constant voltage. The diagnostic fragment is 130-bp in length.

Gb

Figure 1. PCR products for Gb primers. 1: Opata (-), 2: 7Ag/7D (+), 3: Pavon (-). 4-12: recombinant lines. Lines 4-6 and 10-11: marker present, lines 7-9 and 12: marker absent.

Marker BF145935 produces fragment sizes of 198 and 180 bp in Sr25 lines and 202 and 180 bp in wheat lines without Sr25 (Fig. 2).

Figure 2. Haplotyping Sr25 in CIMMYT spring lines using marker BF145935. “>” indicates the 198 bp fragment associated with Sr25. CIMMYT entry #11, 12, 42 and 70 showed the 198 fragment, indicating the Sr25-haplotype. CIMMYT #69 shows both 202 and 198 bp fragments, suggesting it is heterozygous.