Stripe rust resistance gene Yr78

In recent years new races of Puccinia striiformis f. sp. tritici, the causative agent of stripe rust, appeared in different world locations with a more aggressive virulence profile, and they rendered many resistance genes ineffective.

 A paper published by Maccaferri et al. (1) reported the results of a genome-wide association study (GWAS) of resistance to stripe rust in a large collection of hexaploid spring wheat cultivars in six different environments in the western USA. This GWAS identified ten loci that were highly significant in most of the environments. One of these loci, designated QYr.ucw-6B, mapped on chromosome 6B in a different location from other known stripe rust resistance genes (2). This locus was designated Yr78, an adult plant resistance and non-race specific gene. The authors determined that SNP locus IWA7257 maps 0.6-0.66 cM distal to Yr78 and it could be useful for marker assisted selection (see next section).

Later, Dang et al (3) produced a higher resolution map of Yr78 and mapped it within a 0.05-cM interval that corresponds to an 11.16 Mb region between loci TraesCS6B02G116200 and TraesCS6B02G118000 in the “Chinese Spring” genome (RefSeq v1.1), which contains 15 genes. Low recombination rates were observed in this segment, possibly due to the presence of the un-assembled nucleolus organizer NOR-B2 region within the  Yr78 candidate gene region. The unassembled NOR-B2 region has been estimated to be between 37.3 Mb (4) and 61.2 Mb (5), which indicates that the Yr78 candidate gene region is much larger than in the current CS RefSeq.v1.1. The comparative analysis of polymorphisms between susceptible and resistant genotypes revealed five major haplotypes. Out of these, the H1 haplotype, which likely originated in the European spelta wheats, was found associated with Yr78. This haplotype was found in approximately 30% of the hexaploid cultivars scanned, but not in any of the tetraploid wheat lines analyzed (3).  The authors developed two molecular markers for the H1 haplotype that can be used for breeding programs (see next section).

Markers for Yr78

IWA7257

QYr.ucw-6B  (Yr78) was determined to be linked to SNP locus IWA7257 (2), which is located in position 92,462,100 of assembly RefSeq v1.1, and maps 0.6-0.66 cM distal to Yr78.

Allele-specific and common primers for IWA7257 (QYr.ucw-6B) KASP assay:

The capital letters indicate the VIC and FAM tails, IWA7257_Rev_A_VIC is the primer specific for QYr.ucw-6B and the allele-specific nucleotides are shown in bold.

Primer assay mix (100 μl):

• VIC primer (100 mM), 12 μl
• FAM primer (100 mM), 12 μl
• common primer (100 mM), 30 μl
• distilled water, 46 μl.

KASP assays were performed in a 5.07 μl reaction volume containing:

• 2x KASP Master Mix, 2.5 μl
• KASP primer assay mix, 0.07 μl
• genomic DNA at 5–50 ng/μl,  2.5 μl

A two-step touchdown PCR was carried out using the following conditions (KASP master mix):

• 94 °C for 15 min,
• 10 cycles of touchdown starting each with 94 °C for 20 s
• followed by annealing from 61 to 55 °C for 1 min (dropping 0.6 °C per cycle)
• 26 cycles of 94 °C for 20 s, annealing at 55 °C for 1 min.

If a PACE master mix is used, the touchdown at the annealing steps goes from 65 to 57 °C, dropping 0.8 °C per cycle; and instead of the following 26 cycles at fixed temperature, between 30 to 40 cycles at 57 °C are needed.

The length of the amplicon is 77 bp. KASP results were analyzed with a FLUOstar Omega F plate reader (BMG LABTECH, Ortenberg, Germany) using the software KlusterCaller (LGC Genomics, Teddington, UK).

Diagnostic SNPs for resistant haplotype H1

Dang et al. (3) selected two SNPs (SNP-106,540,703; SNP-108,227,904) that discriminated the H1 haplotype from the H2, H3, H4, and H5 haplotypes and developed two codominant KASP markers,  CDM158 and CDM160-2 respectively. These markers were successfully tested in a diverse panel of lines. These two markers represent an improvement over IWA7257 because historic recombination events were detected between IWA7257 and Yr78

Primers:

Capital letters indicate the VIC and FAM tails. The primers labelled with VIC are Yr78 specific, and the allele-specific nucleotides are shown in bold.

References

1. A genome-wide association study of resistance to stripe rust (Puccinia striiformis f. sp. tritici) in a worldwide collection of hexaploid spring wheat (Triticum aestivum L.).  Maccaferri M, Zhang J, Bulli P, Abate Z, Chao S, Cantu D, Bossolini E, Chen X, Pumphrey M, Dubcovsky J. In: G3: Genes, Genomes, Genetics, 2015, 5:449-65. DOI:10.1534/g3.114.014563.

2. Validation and characterization of a QTL for adult plant resistance to stripe rust on wheat chromosome arm 6BS (Yr78). Dong Z, Hegarty JM, Zhang J, Zhang W, Chao S, Chen X, Zhou Y, Dubcovsky, J, 2017. In: Theoretical and Applied Genetics, 130:2127-2137. DOI:10.1007/s00122-017-2946-9.

3. High‐resolution mapping of Yr78, an adult plant resistance gene to wheat stripe rust. Dang C, Zhang J, Dubcovsky J. In: The plant genome, 2022, Jun,15(2):e20212. DOI:10.1002/tpg2.20212.

4. Fine structure and transcription dynamics of bread wheat ribosomal DNA loci deciphered by a multi‐omics approach. Tulpová Z, Kovařík A, Toegelová H, Navrátilová P, Kapustová V, Hřibová E, Vrána J, Macas J, Doležel J, Šimková H. In: The Plant Genome, 2022,15(1), p.e20191. DOI:10.1002/tpg2.20191.

5. Structural features of two major nucleolar organizer regions (NORs), Nor-B1 and Nor-B2, and chromosome-specific rRNA gene expression in wheat. Handa H, Kanamori H, Tanaka T, Murata K, Kobayashi F, Robinson SJ, Koh CS, Pozniak CJ, Sharpe AG, Paux E, Consortium IWGS, Wu J, Nasuda S. In: The Plant Journal, 2018,  96:1148-1159. DOI:10.1111/tpj.14094.