Marker Assisted Selection in Wheat Marker Assisted Selection in Wheat - HOME CSREES-USDA

Disease resistance. Insensitivity to the toxins produced by Pyrenophora tritici-repentis and Stagonospora nodorum.

Tsn1

Contributed by Justin Faris (justin.faris@ndsu.edu)

Microsatellites markers for Tsn1

Tsn1 was first mapped to the long arm of chromosome 5B by Faris et al. (1), and high-resolution mapping was recently conducted by Haen et al. (5).

Tsn1 is currently the focus of a map-based cloning effort. During this process, we have developed several SSR markers tightly linked to the Tsn1 gene (Figure 1), which can be used for marker-assisted selection.

The markers Xfcp1 and Xfcp393 / Xfcp394 delimit a 0.4 cM interval including Tsn1. Marker. While Xfcp2 is about 0.4 cM (400 kb) distal to Xfcp394, and it may be useful if Xfcp393 and Xfcp394 fail to reveal polymorphisms.

Figure 1. Genetic map of the SSR markers flanking the Tsn1 locus.

Primers sequences:
Marker: Xfcp1
Forward primer: 5'- ATA ACT CCG TCA CGA CCA CCT CCT CTC AAG -3'
Reverse primer: 5'- CAG TCT GAA AAC GCC ATA CCC G -3'

*Annealing temp: 65°C
*Product size: 402 bp

Marker: Xfcp2
Forward primer: 5'- GTG AGC CCT GGC TGC CTA CTT ATC TCA CTC T -3'
Reverse primer: 5'- GTA GGC ATT TGA AGA TGA GGT AGC AC -3'

Anealing temp: 67°C
*Product size: 498 bp

Marker: Xfcp393
Forward primer:  5'- AGC CAT GAG CTA GCT CGG CTC GAC T -3'
Reverse primer:  5'- GGA AGA TCT GAA CTA GCT CTA CGG AG -3'

Annealing temp: 62°C
*Product size: 333 bp

Marker: Xfcp394
Forward primer: 5'- GTA GCC TGC AGG TAC AAA CTG GA -3'
Reverse primer: 5'- CAG TGT TAA GAA GTG TGT TCT GGT C -3'
Annealing temp: 62°C
*Product size: 383 bp

* Product sizes as determined in Langdon durum.


Final concentrations of the reagents used in the PCR amplification

Reagent Amount

  • DNA (50 ng/ul), 4 µl
  • H2O 13.2 µl
  • 10X PCR buffer 2.5 µl
  • dNTPs (2.5 mM) 2.0 µl
  • MgCl2 (50 mM) 1.0 µl
  • F primer (10 pm/µl) 1.0 µl
  • R primer (10 pm/µl) 1.0 µl
  • Taq (Bioline) 0.3 µl

Total 25 ul

PCR conditions:
  • Denaturing step: 94C, 4 min
  • Amplification step (45 cycles):
    • 94°C, 1 min
    • see primer, 1 min
    • 72°C, 2 min
  • Extension step: 72C, 10 min
Expected products:

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



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