Virus resistance. Wheat Streak Mosaic Virus (WSMV)

Wsm1

Detection of the Agropyron intermedium segment carrying Wsm1

This protocol is based on the information presented by Jamie Sherman during the coordination meeting at PAGX, you can also see Ref. 3

Sequences of the J15 primers (Wsm1) :

This sequence-tagged-site (STS) PCR primers are specific for the A. intermedium segment containing the Wsm1 gene

J15L:   5'- GTA GCA GGG GAA GCT GAA GA -3'

J15R:   5'- CCG AGC TCA CAC GCT AAT TT -3'

Sequences of the G43 primers :

These primers are used as positive control of the PCR reaction (J15 will not amplify when the A. intermedium segment is absent)

G43L:   5'- GGC GCA TGC ACC AAA ATG TT -3'

G43R:   5'- ACC TTG TCG TGC ATA GGA AC -3'  

PCR amplification conditions:

1. Denaturing step: 94°C, 4 min
2. 30 cycles of: [94°C 1 min, 45°C 1 min, 72°C 1.2 min]
3. Extension step: 72°C 7 min

Final volume: 50 ul

Final concentrations of the products used in the PCR reaction:

• KCl: 50mM
• Tris-HCl: 10mM
• Triton X-100: 0.1%
• dNTP: 50 uM each
• MgCl₂: 1.5 mM
• primers: 0.4 uM each
• Taq polymerase: 0.6 units
• DNA input: 100 ng/reaction

• Fragment amplified by J15 (Wsm1): 400 bp
• Fragment amplified by G43: 700 bp