Stem rust resistance gene Sr28

Information on KASP_IWB1208 contributed by E. Babiker

The assessment of seedling infection types and field stem rust evaluations in Njoro, Kenya to Puccinia graminis sp. tritici race TTKSK showed that a monogenic line for stem rust resistance genes carrying Sr28 had a low infection type at seedling stage and an infection response in the field of type MS-S (moderately susceptible – susceptible) in 2005 and MS-MR (moderately susceptible-moderately resistant) in 2006. Stem rust severity in the Cobb scale ranged from 30 to 40 for the Sr28 line in both years compared to 60-80 for the susceptible checks (1). The Sr28 locus had been previously reported on chromosome arm 2BL (2).

SD 1691 (CI 12499) is a South Dakota breeding line developed during the 1940s. This line displayed adult plant resistance to a bulk of stem rust isolates, including TTKSK, and it was susceptible to several races screened at the seedling stage except races BCCBC, TTKSK, and TTKST (3). SD 1691 has the variety Ceres in its pedigree which is known to possess Sr28 (2).

Seedling screening of progeny derived from SD 1691 crossed to susceptible line LMPG-6 indicated that a single resistance gene to race TTKSK was present. Allelism and race-specificity tests indicated the stem rust resistance gene in SD 1691 was Sr28 (4).

Due to the relative degree of protection Sr28 confers, it is recommend to use this gene as part of gene stacking breeding strategy.

Markers for Sr28

Rouse et al (4) developed a PCR marker derived from a DaRT locus linked to Sr28 and designated it wPt-7004-PCR. This marker and wmc332 flank Sr28 at 5.9 (proximal) and 1.9 cM (distal) respectively in a LMPG-6/SD 1691 mappping population. Both markers were also confirmed to flank Sr28 in two other mapping populations, SD 1691/Gabo 56 and LMPG-6*2/CI 7611.

Babiker et al. in 2016 (5) confirmed the presence of Sr28 in wheat landrace PI 177906, they also developed mapping populutions to fine map this gene using the Illumina based 90K iSelect SNP genotyping platform. Three SNP markers that were tightly linked to Sr28, IWB1208, IWB23232 and IWB57292, were converted to KASP markers for further validation. One of them, KASP_IWB1208, was used to analyze a panel of 96 wheat accessions. The results suggested that this marker is more accurate than the wPt-7004-PCR - wmc332pair.

Primers wPt-7004-PCR and wmc332 

Primer sequences:

wPt-7004-PCR

wPt-7004-PCR-F       5'- CTC CCA CCA AAA CAG CCT AC -3'

wPt-7004-PCR-R       5'- AGA TGC GAA TGG GCA GTT AG -3'

wmc332

wmc332-F       5'- CAT TTA CAA AGC GCA TGA AGC C -3'

wmc332-R       5'- GAA AAC TTT GGG AAC AAG AGC A -3'

PCR conditions:

• Denaturing step: 94°C, 7 min
• Amplification step (35 cycles):
  • 94°C, 60 sec
  • 60°C, 60 sec
  • 72°C, 60 sec
• Extension step: 72°C, 5 min

PCR mix:

• 45 ng genomic DNA
• 1 µmol/L of each primer
• 0.125 mM dNTPs
• 0.05 units/µL Taq DNA polymerase (Qiagen Inc.)
• 1 X PCR buffer

Final volume: 10 µL

Expected products:

Markers wmc332 and wPt-7004-PCR were validated in a panel of 24 hard red spring wheat varieties and seven other wheat lines. 
Amplification products of 214-, 217-, and 220-bp for marker wmc332 were associated with the presence of Sr28. Although the susceptible genetic stock W2691 also yielded an amplicon of 217-bp.  All the other susceptible lines and all the US hard spring wheat cultivars tested yielded products of 208-bp or less.

With wPt-7004-PCR two amplicons of sizes 166- and 194-bp were amplified in all lines. However, susceptible and lines with Sr28can be differentiated by the preferential and repeatable amplification of each product. Preferential amplification of the 194-bp amplicon was associated with the presence of Sr28. In contrast, the susceptible genetic stocks and all the US hard red spring wheat cultivars showed either equal amplification or preferential amplification of the 166-bp amplicon.

KASP marker KASP_IWB1208

Note: primer sequences do not include a tail sequence that correspond with the FAM and HEX dyes

References

1. Characterization of seedling infection types and adult plant infection responses of monogenic Sr gene lines to race TTKS of Puccinia graminis f. sp. tritici. Jin Y, Singh RP, Ward RW, Wanyera R, Kinyua M, Njau P, Fetch T, Pretorius ZA, Yahyaoui A. In: Plant Disease, 2007, 91:1096-1099. DOI:10.1094/PDIS-91-9-1096.

2. Cytogenetic studies in wheat X. Monosomic analysis and linkage studies involving genes for resistance to Puccinia
graminis f. sp. tritici in cultivar Kota. McIntosh RA. In: Heredity, 1978, 41:71-82. DOI:10.1038/hdy.1978.65.

3. Sources of resistance to stem rust race Ug99 in spring wheat germplasm. Rouse MN, Wanyera R, Njau P, Jin Y. In: Plant Disease, 2011, 95:762–766. DOI:10.1094/PDIS-12-10-0940.

4. Identification of markers linked to the race Ug99 effective stem rust resistance gene Sr28 in wheat (Triticum aestivumL.). Rouse MN, Nava IC, Chao S, Anderson JA, Jin Y. In:  Theoretical and Applied Genetics, 2012, 125:877-885. DOI:10.1007/s00122-012-1879-6 

5. Molecular Mapping of Stem Rust Resistance Loci Effective Against the Ug99 Race Group of the Stem Rust Pathogen and Validation of a Single Nucleotide Polymorphism Marker Linked to Stem Rust Resistance Gene Sr28. Babiker EM, Gordon TC, Chao S, Rouse MN, Wanyera R, Acevedo M, Brown-Guedira G, Bonman JM. In:  Phytopathology, 2016, In press. DOI: 10.1094/PHYTO-08-16-0294-R