Abiotic Stress and Agronomic Traits

Root Biomass and Drought Tolerance

We thank Javier Zuñiga and Gina Brown-Guedira for their contributions to this section.

PCR markers for rye translocations.

These primers were designed to detect 1BL.1RS and 1AL.1RS translocations. No amplification occurs in wheat lines without any of those translocations. For more information on the NOR marker, see Ref. 9.

RIS F  5'- TAA TTT CTG CTT GCT CCA TGC -3'
RIS R  5'- ACT GGG GTG CAC TGG ATT AG -3'

RYE-NOR-F  5'- GCA TGT AGC GAC TAA CTC ATC -3'
RYE-NOR-R  5'- CCC AGT TTT CCA TGT CGC -3'

Final concentrations of the reagents used in the PCR amplification

• 25 ng template DNA. IMPORTANT: DNA may require previous RNAse treatment to see PCR product on gels
• 0.040 µM each primer
• 0.4 unit of Taq DNA polymerase
• 2 mM MgCl₂
• 80 µM each dNTP

Total volume: 25 µl reaction

• Denaturing step: 94°C, 4 min
• Amplification step (30 cycles)
• 94°C, 15 sec
• 65°C, 45 sec
• 72°C, 45 sec
• Extension step: 7 minutes at 72°C.

Following amplification, PCR products are separated on 1.5-2% agarose gels, and stained with ethidium bromide.

Expected products for RIS primers: 110-bp (all 1BL.1RS and 1AL.1RS translocation lines tested) .

Expected products for NOR primers: 400, 600, 700 and 800 -bp, although most of the lines amplify only three bands.

PCR products for RIS primers: wheat lines carrying 1BL.1RS or 1AL.1RS translocations yield a 110-bp band (green arrowhead). Lines without translocation do not amplify. M, size markers; K, Kavkaz, control line carrying a rye translocation; CS, Chinese Spring, control line without the translocation

PCR products for NOR primers: wheat lines carrying 1BL.1RS or 1AL.1RS translocations yield three or four different bands (yellow arrowheads). Lines without translocation do not amplify. M, size markers; K, Kavkaz, control line carrying a rye translocations; CS, Chinese Spring, control line without the translocation

Microsatellite marker Xscm9

The PCR markers SCM were developed to reveal microsatellite loci in rye (10). Some of them have also been found in wheat. SCM9 was located on chromosome arm 1RS, and polymorphic loci in wheat were found on chromosome arms 1AL and 1BL.

SCM9-F  5'- TGA CAA CCC CCT TTC CCT CGT -3'
SCM9-R  5'- TCA TCG ACG CTA AGG AGG ACC C -3'

Final concentrations of the reagents used in the PCR amplification

• 50 ng template DNA.
• 10 µM each primer
• 2.5 unit of Taq DNA polymerase
• 1.75 mM MgCl₂
• 200 µM each dNTP
• 1X PCR buffer

Total volume: 15 µl reaction

• Denaturing step: 94°C, 2 min
• Amplification step (40 cycles)
• 95°C, 60 sec
• 60°C, 60 sec
• 72°C, 60 sec
• Extension step: 5 minutes at 72°C.

Rye DNA amplification with SCM9 primers yields a 220-bp band. Amplification in wheat renders different products for 1AL.1RS and 1BL.1RS translocations.