To develop wheat germplasm with resistance to race TTKSK of stem rust, five stem rust resistance genes were used to produce pyramids with up to 4 Sr genes of various combinations by marker-assisted selection (MAS) and doubled haploid (DH) technology. Thirteen desired pyramids with 2 to 4 of the Sr genes were recovered, and wheat cultivar Hoffman was improved by stacking Sr35 and Sr36. TTKSK resistance test showed that pyramided lines with three or four genes plus improved Hoffman with Sr35+Sr36 were all immune to TTKSK. This type of germplasm will be a useful tool for the production of cultivars with long-term sustainability.
Introduction
Stem rust of wheat (Triticum aestivum L.), caused by the fungus Puccinia graminis Pers.: Pers. f. sp. tritici Eriks. & E. Henn (Pgt) has historically been one of the most destructive diseases of wheat worldwide. Over the past several decades, stem rust has been under effective control worldwide by widespread use of resistant cultivars; however, recently most of the commonly used stem rust resistance genes were rendered ineffective against a new Pgt race, TTKSK, and its variants (Singh et al. 2011).
TTKSK (informally known as Ug99) with virulence to Sr31 was first detected in Uganda in 1998 (Pretorius et al. 2000). Since then, the Ug99 race group has been evolving rapidly to overcome other resistance genes. To date, a total 13 races belonging to the Ug99 lineage have been identified and spread to 13 countries of East Africa and the Middle East. Thus, stem rust has returned as a major threat to global wheat production (Bhavani et al. 2019).
At present, most Ug99 resistant genes are originated from wild relatives of wheat (Singh et al. 2011). Sr35 transferred into hexaploid wheat from Triticum monococcum L., is effective against the TTKSK race and its variants (Zhang et al. 2010; Singh et al. 2011). Sr36 was derived from Triticum timopheevi (Zhuk.) Zhuk. and has been deployed in wheat cultivars globally (Tsilo et al. 2008; Jin et al. 2009). It is not only effective against many prevalent Pgt pathotypes but is also effective against all races of the Ug99 race group except TTTSK (Jin et al. 2009; Chemayek et al. 2017). Therefore, it is one of the best available sources of resistance to Ug99 and could be used effectively as a component for Ug99 resistance breeding. Sr33 and Sr43 derived from Aegilops tauschii Coss. and Thinopyrum ponticum (Podp.) Z.-W. Liu & R.R.-C. Wang, respectively, are among the genes effective against Ug99 races (Sambasivam et al. 2008; Niu et al. 2014). The SrCad gene present in the Canadian cultivar AC Cadillac confers moderate resistance to Ug99 and a high level of resistance when combined with Lr34 (Hiebert et al. 2011).
Pyramiding of multiple effective Sr genes in one wheat background is an effective strategy to enhance resistance durability to stem rust. Tools such as molecular markers and doubled haploid (DH) are now readily available to facilitate the pyramiding process (Mago et al. 2011). In the present study, DH technology plus linked markers were used to manipulate five stem rust resistance genes to produce pyramids of up to four Sr genes of various combinations.
Materials and Methods
Plant material and DH plant production
Five wheat cultivars/lines, AC Cadillac (SrCad), Lang (Sr36), RL5405 (Sr33), RL6099 (Sr35), and RWG34 (Sr43) were used as resistant parents to produce two complex crosses: (AC Cadillac/Lang)//(RWG34/RL5405) and RWG34/RL5405//RL6099. Another backcross population, (Hoffman*2/RL6099)//(Hoffman*2/Lang), was produced to improve Hoffman’s stem rust resistance.
The PCR-selected F1 hybrid plants of the above three combinations were used for producing DH populations according to established protocols (Fedak et al. 1997).
DNA extraction and PCR screen
Genomic DNA was extracted from young leaves using the CTAB method. Previously reported PCR markers for these stem rust resistance genes were used for analysis. Markers cfd15, cfa2040 and FSD_RSA were used for Sr33, Sr43, and SrCad, respectively (Sambasivam et al. 2008; Hiebert et al. 2011; Niu et al. 2014). Markers barc51, cfa2076, cfa2193, BF485004, BE405552 were used for Sr35 (Zhang et al. 2010). Markers stm773–2 and gwm319 were used for Sr36 (Tsilo et al. 2008). PCR conditions for amplification were used as described in previous references and PCR products were separated by 2% agarose gel electrophoresis.
Stem rust assay
The DH lines with two/three/four gene combinations were selected and evaluated for seedling resistance to stem rust. Ten plants of each DH line, parents, and susceptible checks were tested with stem rust race TTKSK (Ug99, isolate SA31) inside a biocontainment level 3 lab at the Morden Research and Development Center, Agriculture and Agri-Food Canada. Seedlings were rated for their infection types 14 d post inoculation. Infection types were recorded using a 0–4 scale where plants with an infection type 0–2+ were considered resistant and those with 3–4 were considered susceptible (Stakman 1962).
Results
Improvement of Canadian wheat cultivar Hoffman
To produce gene combinations (Sr35+Sr36) in the Hoffman background, PCR-selected BC1F1 plants from the cross Hoffman*2/ RL6099 (Sr35) and Hoffman*2/ Lang (Sr36) were crossed. Then F1 plants from the cross combination (Hoffman*2/ RL6099)//(Hoffman*2/ Lang) were screened by PCR for the Sr35+Sr36 genotype. Fifteen F1 plants with Sr35+Sr36 genotype were used to produce the DH lines that could generate four genotypes, Sr35+Sr36, Sr35, Sr36, and null. Finally, 16 of 62 DH lines with genotypes Sr35+Sr36 were obtained (Table 1).
Table 1.
Frequencies of gene combinations in douple haploid (DH) lines derived from three hybrid combinations in this study.
Production of plants with different gene combinations
The F1 plants from the crosses (AC Cadillac/Lang)//(RWG34/RL5405) and RWG34/RL5405//RL6099 were screened by PCR to confirm the presence of the respective gene combinations. Seven F1 plants carrying Sr33+Sr36+SrCad+Sr43 and six F1 plants carrying Sr33+Sr35+Sr43 were used to make DH lines by the wheat × maize pollination method. At the end, 46 DH lines with 14 genotypes and 81 DH lines with 8 genotypes were developed, respectively, from the two crosses (Table 1). In total, 13 desired gene combinations were obtained: SrCad + Sr36 + Sr43 + Sr33; SrCad + Sr36 + Sr43; SrCad + Sr36 + Sr33; SrCad + Sr43 + Sr33; Sr36 + Sr43 + Sr33; SrCad + Sr36; SrCad + Sr43; Sr36 + Sr43; Sr36 + Sr33; Sr43 + Sr33; Sr35 + Sr33 + Sr43; Sr35 + Sr33; Sr35 + Sr43.
TTKSK resistance
DH lines with the above 14 desired gene combinations were evaluated for TTKSK resistance and the results were summarised as Table 2. The pyramided lines with two Sr genes confer a high-level of resistance to TTKSK. All the DH lines with pyramids of three or four Sr genes (SrCad, Sr35, Sr36, Sr43, and Sr33) conferred immunity to TTKSK. In addition, the improved Hoffman with pyramids of Sr35 and Sr36 also conferred immunity to TTKSK.
Table 2.
TTKSK infection type of different gene combinations.
Discussion
Stem rust race TTKSK (Ug99) has unique virulence which renders most current wheat varieties susceptible worldwide. It has virulence to most Sr genes of common wheat origin and some Sr genes from alien relatives. Development of new germplasm with effective Sr genes is one effective way to control Ug99 spread, especially breeding cultivars with multiple gene combinations through marker-assisted selection (MAS) to enhance the resistance durability (Bhavani et al. 2019). Stem rust resistance genes Sr33, Sr35, Sr36, and Sr43 are all alien Sr genes that provide resistance to TTKSK (Singh et al. 2011). SrCad present in Canadian cultivar AC Cadillac, confers the seedling resistance to Ug99 in Canadian wheat cultivars (Hiebert et al. 2011). In the present study, the above five stem rust resistance genes were used to produce pyramids of up to four genes of various combinations by using MAS and DH technology. And DH lines with pyramids of SrCad + Sr36 + Sr43 + Sr33, SrCad + Sr36 + Sr43, SrCad + Sr36 + Sr33, SrCad + Sr43 + Sr33, Sr36 + Sr43 + Sr33, and Sr35 +Sr33 + Sr43 were extremely effective against TTKSK (Table 2). These results suggest that pyramiding multiple resistance genes into a single line is an effective way to produce wheat germplasm with high resistance.
Hoffman is a Canadian commercial wheat cultivar with many good traits, especially high yield; however, it is susceptible to TTKSK and related races. To enhance its stem rust resistance, we have used a MAS-based backcross breeding strategy to stack two Sr genes (Sr35 and Sr36) in Hoffman. Although Sr36 is ineffective against one race of Ug99 lineage TTTSK, it is still effective against Ug99 and its other variants (Jin et al. 2009; Bhavani et al. 2019). Moreover, Sr36 is effective against other prevailing Pgt pathotypes (Chemayek et al. 2017). Finally, three improved wheat lines Hoffman (Sr35+Sr36), Hoffman (Sr35) and Hoffman (Sr36) were obtained (Table 1). The TTKSK resistance test result showed that pyramided lines with genotype Sr35+Sr36 had immunity to TTKSK, which indicated enhanced resistance compared with the parent ‘Hoffman’ (Table 2). Therefore, disease resistance of wheat cultivars can be improved by combining multiple resistance genes through a MAS-based backcross breeding strategy.
It is virtually impossible to produce a large stack of disease resistance genes by phenotyping alone. This study has shown, as have others, that it is feasible to produce pyramids with a combination of up to four Sr genes by selecting out of reasonable-sized DH populations (Mago et al. 2011). The pyramids produced in this study all proved to be highly resistant following inoculation with TTKSK and can be used as the donors of multiple Sr genes (Liu et al. 2020). As a result, the pyramided lines with three or four Sr genes and improved Hoffman with Sr35 and Sr36 can be used for future wheat breeding. New molecular tools have been developed for the more efficient detection, identification and mapping of unique alien disease resistance genes. This increasing supply of disease resistance genes will increase the scope of gene pyramiding by increasing the potential for producing gene pyramids of increasing complexity.
Acknowledgements
The authors acknowledge the timely advice and excellent facilities supplied by the staff at Ottawa Research and Development Centre.
