Stem rust resistance gene Sr35

Contributed by W. Zhang, M. Pumphrey and J. Dubcovsky

The stem rust resistance gene Sr35 was originally transferred from Triticum monococcum to hexaploid wheat (1) and is effective against the TTKSK (Ug99) race of of Puccinia graminis sp. tritici (2) and its variants, TTKST and TTTSK. During the field tests in Kenya in 2005 and 2006 this gene showed resistant to moderately resistant infections responses with relatively low disease severity (2). Using two T. monococcum mapping populations it was shown that Sr35 maps on the long arm of chromosome 3Am between markers XBF483299 and XCJ656351in a region of 2.2 to 3.1 cM, depending on the population (3). Additionally, crossing the hexaploid line Marquis*5/G2919 (a T. monococcum accession carrying Sr35) to two susceptible hexaploid cultivars, it was confirmed that the T. monococcum fragment containing Sr35 is located on chromosome arm 3AL in T. aestivum. Because of its alien origin this fragment has none or very low recombination with the T. aestivum chomosomes, reducing the genetic distances. For example, the distance between marker Xbarc69, outside the fragment on the proximal side, and Xgwm480, the most distal marker within the fragment is 42 cM in the T. aestivum consensus map while the distance decreases to 2.2 cM - 4.1 cM in maps generated from crosses between Marquis*5/G2919 and T. aestivum (3).

Saintenac et al. (4) cloned the Sr35 gene from T. monococcum and showed it codes for a coiled coil-nucleotide binding-leucine rich repeat product (CNL). The Sr35 was found in a region containing five intact genes coding for the same type of proteins (CNL1, CNL2, CNL4, CNL6 and CNL9). Analyzing mutants induced by ethyl-methanesulfonate, the natural variation in a number of accessions and transgenic plants the authors could demonstrate that CNL9 is Sr35. CNL proteins are involved in the detection of pathogen attacks of different types.

Dominant perfect marker for Sr35

A marker located in the genomic region spanning the Sr35 locus was recently developed (Dubcovsky and Zhang, personal communication). This is a dominant perfect marker. The primer sequences are:

NL9F5:       5'- CTC ATC AAC TGC TTG AGC GAA C -3'

NL9R6:       5'- GTA TCT AGC GAA CCT CAA TCG -3'

The recommended annealing temperature for this pair of primers is 56°C. The germplasm carrying Sr35 amplifies a 719-bp band (dominant marker).

Other markers for Sr35

A number of polymorphic SSR and EST-derived markers were found on the T. monococcum fragment containing Sr35 and transferred to hexaploid wheat. They are shown in the blue box in the map on the right. Since this fragment has reduced recombination in hexaploid wheat, all the markers in the Table are useful for marker assisted selection. Still, it is better to use the closest polymorphic marker to Sr35 to reduce the risk of occasional recombination, and to check the final material with a second marker on the other side of Sr35.

Marker

Type

Primer sequences

Notes

Band size 
(Marquis Sr35 / Marquis)

Xcfa2193

SSR

CFA2193-F  ACATGTGATGTGCGGTCATT
CFA2193-R TCCTCAGAACCCCATTCTTG

Anneal. temp.: 60°C

243 / 230

XBE423242

Indel

BE424242-F TCTGACCAATGCAAAATGGA

BE424242-R GCTGATTGGCTTGGAAGGTA

Touch down from 63°C to 59°C (9 cycles of 20 sec, -0.5°C each)
Amplification at 58°C (34 cycles of 20 sec each)

430 / 426

Xwmc559

SSR

WMC559-F ACACCACGAATGATGTGCCA 
WMC559-R ACGACGCCATGTATGCAGAA
Anneal. temp.: 61°C

~225 / 235

XBF485004

Indel

BE485004-F TGCAGAATGCGTTCCTTCTA

BE485004-R GGCCAGAGAATTTCTTGAGG

Touch down from 63°C to 59°C (9 cycles of 20 sec, -0.5°C each)
Amplification at 58°C (34 cycles of 20 sec each)

Null / 597

Xcfa2170

SSR

CFA2170-F TGGCAAGTAACATGAACGGA
CFA2170-R ATGTCATTCATGTTGCCCCT
Anneal. temp.: 60°C

~180 / 190

XAK335187

InDel

AK335187-F GGTTCAACATCGTCGGACAG
AK335187-R CCAGCACGACGTACTTGGAG
Anneal. temp.: 58°C

~205 / 205, 290

Xcfa2076

SSR

CFA2076-F CGAAAAACCATGATCGACAG 
CFA2076-R ACCTGTCCAGCTAGCCTCCA
Anneal. temp.: 60°C

~180 / 182

XBE405552

Indel

BE405552-F CACCATCTTCGTCACCATCA

BE405552-R CACAGTGCAGCGAACAGAT

Touch down from 63°C to 59°C (9 cycles of 20 sec, -0.5°C each)
Amplification at 58°C (34 cycles of 20 sec each)

377 / 371

Xwmc169

SSR

WMC169-F TACCCGAATCTGGAAAATCAAT
WMC169-R TGGAAGCTTGCTAACTTTGGAG
Anneal. temp.: 61°C

143 / 153

Xgwm480

SSR

GWM480-F TGCTGCTACTTGTACAGAGGAC
GWM480-R CCGAATTGTCCGCCATAG
Anneal. temp.: 60°C

null / 188

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

References

1. Cytogenetical studies in wheat .XIII. Sr35 - a 3rd Gene from Triticum monococcum for resistance to Puccinia graminis triticiMcIntosh RA, Dyck PL, The TT, Cusick J, Milne DL. In: Zeitschrift für Pflazenzüchtung, 1984, 92:1-14.

2. 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, Pretorius ZA. Plant Disease, 2007, 91:1096-1099. DOI:10.1094/PDIS-91-9-1096.

3. Genetic maps of stem rust resistance gene Sr35 in diploid and hexaploid wheat. Zhang W, Olson E, Saintenac C, Rouse M, Abate Z, Jin Y, Akhunov E, Pumphrey M, Dubcovsky J. In: Crop Science, 2010, 50: 2464-2474. DOI:10.2135/cropsci2010.04.0202

4. Identification of wheat gene Sr35 that confers resistance to Ug99 stem rust race group. Saintenac C, Zhang W, Salcedo A, Rouse MN, Trick HN, Akhunov E, Dubcovsky J.In: Science, published online 27 June 2013. DOI:10.1126/science.1239022.