Disease resistance. Leaf and stripe rust resistance

Lr37-Yr17-Sr38

TaqMan® assay for Lr37 (7):

A PCR assay was developed using TaqMan® technology as a high-throughput alternative for selection of the 2NS/2AS translocation in large segregating populations in breeding programs that have access to real time PCR equipment. This technology uses real time fluorogenic detection of PCR products and therefore does not require gels to visualize the PCR products, facilitating automation of the MAS process.
Reactions were performed using a system composed by a pair of primers (VENTRIUP - LN2) with the probe W2NS-55T. The probe included a single point mutation at bp 17 that differentiated the 2NS allele from the 2A, 2B and 2D alleles (see sequences). The probe was labeled with the fluorescent reporter dye 6-carboxy-fluorescein (FAM) at the 5' end and the quencher 6-carboxy-N,N,N',N'-tetramethyl-rhodamine (TAMRA) at the 3' end. When the target sequence is being amplified, the 5' nuclease activity of the Taq DNA polymerase cleaves the fluorogenic probe included into the PCR reaction. The physical separation of the fluorescent reporter dye from the quencher causes an increase in its fluorescence intensity that is detected in "real time" by the quantitative PCR equipment.

Primers:

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

Probe:

W2NS-55T 5'- FAM-TGT TTG GTT CCT ATC TCC TTC CTG GTC CTG-TAMRA -3'

Final concentrations of the products used in the PCR reaction:

primers: 0.4 µM each
probe: 0.08 µM
1X TaqMan® Universal PCR Mastermix (Applied Biosystems) :

10 mM Tris-HCl (pH 8.3)
50 mM KCl
5 mM MgCl₂
2.5 mM deoxynucleotide triphosphates
0.625 U AmpliTaq Gold DNA polymerase per reaction
0.25 U AmpErase UNG per reaction

DNA input: 200 ng/reaction

Final volume: 25 µL

PCR conditions

The samples were placed in 96 well plates and amplified in an ABI Prism 7700 Sequence Detection System (Applied Biosystems).

2 min at 50°C
10 min at 95°C
Amplification step (40 cycles)

95°C 15 sec
60°C 60 sec

Expected products:

The figure below shows the amplification signal obtained from 24 individuals with known AA, AN, and NN genotypes using TaqMan® system. The analysis of variance for the amplification signal showed significant differences among genotypes (P= 0.002). The response was linear as evidenced by a significant linear response (P=0.006) and a non-significant quadratic response (P=0.48).

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