Disease resistance. Leaf Rust Resistance

Lr34 - Yr18

Contributed by Marcelo A. Soria, Wenjun Zhang and Jorge Dubcovsky

STS marker csLV34

Primers sequences:

   csLV34F   5'- GTT GGT TAA GAC TGG TGA TGG -3' 
   csLV34R   5'- TGC TTG CTA TTG CTG AAT AGT -3' 

• Denaturing step: 94°C, 5 min
• Amplification step (40 cycles):
• 94°C, 45 s
• 55°C, 30 s
• 72°C, 60 s
• Extension step: 72°C, 7 min

The Lr34 allele yields a 150-bp product, and a 229-bp band is amplified in non-Lr34 germplasm.

Other markers

Previously to the development of STS marker csLV34 and the positional cloning of Lr34, there were several markers available that mapped farther away and were validated in some populations. Suenaga et al. (7) had determined that Xgwm295 and Xgwm130 were closely linked to each other at the center of the slow rusting QTL. Later, Schnurbusch et al. (10) analyzing an Arina x Forno population found that the region containing Lr34 and flanked by Xbarc352 (distal) and Xgwm295 (proximal) spanned 17.7 cM. Below we provide protocols for using this markers.

Microsatellite marker Xgwm295

The microsatellite locus Xgwm295 maps close to the Lr34 locus but its detection is sometimes difficult to optimize. Details of the PCR protocol can be obtained from the WMS295 probe record in GrainGenes.

Manilal William, personal communication: the Xgwm295 allele putatively linked to Lr34/Yr18 has a size of approximately 250-bp.

Microsatellite markers Xgwm130 and Xbarc352

The microsatellite loci Xgwm130 and Xbarc352 flank the Lr34 locus. In some crosses these set of markers provides clearer results than Xgwm295. This pair of markers was tested in several lines carrying Lr34: Sunvale, Janz, Annuello, Mulgara, Lang, Babbler, Cook, Sunland, Oxley, Sunco, Cunnincham and Anza,

Xgwm130:

   WMS130-L   5'- AGC TCT GCT TCA CGA GGA AG  -3' 
   WMS130-R   5'- CTC CTC TTT ATA TCG CGT CCC -3' 
   

Xbarc352:

   barc352-L   5'- CCC TTT CTC GCT CGC CTA TCC C  -3' 
   barc352-R   5'- CTG TTT CGC CCA ATC TCG GTG TG -3' 
   

• Genomic DNA: 50 ng
• Primers: 5 pM each
• dNTPs: 100 µM each
• Taq DNA polymerase: 1U
• 10x PCR buffer
• 1.5 mM MgCl₂

Total volume: 20 µl

Xgwm130:

• Denaturing step: 94°C, 5 min
• Amplification step (45 cycles):
• 94°C, 20 s
• 58°C, 20 s
• 72°C, 30 s
• Extension step: 72°C, 7 min

Xbarc352:

• Denaturing step: 94°C, 4 min
• Amplification step (38 cycles):
• 94°C, 30 s
• 60°C, 30 s
• 72°C, 60 s
• Extension step: 72°C, 5 min

All the gels shown below are 6% polyacryalmide gels stained with ethidium bromide and visualized under UV light (method).

Xgwm130:

Xgwm130 is in a proximal position relative to Lr34 (ref. 10 and also see maps in the main section). One potential problem with this marker is that it detects more than one locus. The red arrowhead indicates a putative identification of the band cosegregating with Lr34. The information provided should be considered preliminary.

Please read carefully the communications below from people also working with this marker. Input from users is welcome.

R.Rahaman, H.Bariana, M.Shriflou and P. Sharp personal communication: gwm130 showed a band of 133-bp in 21 genotypes carrying Lr34, whereas a band of 135-bp is amplified in 20 genotypes lacking Lr34. The Lr34 carrying cultivar Suneca was an exception, where a 120 bp-band was amplified.

Manilal William, personal communication: In some populations (Fukokumughi x Oligoculm and Avocet x Parula) the Xgwm130 allele is scored as a codominant band, while in other populations (Frontana x Inia66) it has been scored as a dominant band, which is present in Inia66 and absent in Frontana carrying Lr34/Yr18). The apparent size of the +/- allele is about 110. In multiple mapping populations it was not possible to map any allele of Xgwm130 on 7A.

Microsatellite marker Xgwm130. F2 lines derived from a cross Anza-Lr34 x Yecora Rojo. AA: F2 line homozygous for the Xgwm130 allele in the Anza-Lr34 line; YY: F2 line homozygous for the Xgwm130 allele from Yecora rojo. AY: heterozygous line. The red arrowhead shows the position of the Xgwm130 allele linked to Lr34.	Microsatellite marker Xgwm130. Lines containing Lr34 and the putative linked allele of Xgwm130 (red arrowhead).

Xgwm130 alleles in Chinese spring (CS), and nullisomic lines N7A, N7B and N7D. The red arrowhead shows the position of the Xgwm130 allele linked to Lr34, which is absent in line N7D.

Xbarc352:

Microsatellite marker Xbarc352. F2 lines derived from a cross Anza-Lr34 x Yecora Rojo. AA: F2 line homozygous for the Xbarc352 allele in the Anza-Lr34 line; YY: F2 line homozygous for the Xbarc352 allele in Yecora rojo. AY: heterozygous line. The red arrowhead shows the position of the Xbarc352 allele linked to Lr34.	Microsatellite marker Xbarc352. Lines containing Lr34 and the linked allele of Xbarc352 (red arrowhead).

Xbarc352 in Chinese spring (CS), Anza and nullisomic lines N7A and N7B. The red arrowhead shows the position of the Xbarc352 allele linked to Lr34, which is absent in line N7D.

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