Association mapping of North American spring wheat breeding germplasm reveals loci conferring resistance to Ug99 and other African stem rust races

Wheat stem rust disease has been primarily controlled by the use of resistant genes
discovered in hexaploid wheat and its related species. However, the Ug99 race group
has defeated many of the widely deployed resistance genes, and thus poses a threat
to wheat production globally. Moreover, several of the previously identified genes
discovered in wild progenitors or landraces are not desirable for their use in resistance
breeding because of linkage drag 3], 44]. Therefore, discovery of loci contributing resistance to Ug99 and other virulent
races in elite breeding germplasm is a clear advantage. The resistance uncovered in
this study, composed of elite germplasm from North American breeding programs, can
provide a great resource for the fight against Ug99 and stem rust in general. As no
SNP markers were significant across all four field environments, differences among
the disease environments with regard to races present, temperature, and other environmental
factors as well as locus by environment interaction are likely involved in this lack
of consistency. Lack of strong correlations among the environments also corroborates
this assumption (Table 1).

Comparison of significant APR Loci with published studies

The map locations of significant SNP markers in our study, obtained from Wang et al. 39], were compared to positions of markers and genes/quantitative trait loci (QTL) reported
in previous mapping studies conducted to uncover loci associated with stem rust resistance.
In this section, we have used from the integrated genetic map consisting of different
marker types generated by Maccaferri et al. 45] to obtain the relative distances between previously reported markers and the significant
markers in our study.

Five significant SNP markers (IWA3120, IWB21176, IWB31027, IWB56771, IWB59663) were detected at position 90 cM on chromosome 1B, of which all except IWA3120 were also detected in Ethiopia 2013. The marker cfd48 reported by Pozniak et al. 46] in a durum wheat (Triticum durum Desf.) GWAS study is located 4 cM from the SNP markers we detected, and could represent
the same locus. Bhavani et al. 47] and Njau et al. 48] both reported the marker wPt-1560 on 1BL to be associated with Ug99 resistance in separate spring wheat RIL populations.
This marker, as well as Sr58, an APR gene for stem rust of wheat 49], 50], are located at a distance of 50 cM from these five SNP markers. QTL on chromosome
2A providing APR to Ug99 have also been mainly reported in durum wheat mapping populations.
Letta et al. 51] detected gwm1045 to be significantly associated with Ug99 resistance in a durum wheat AM panel; and
Haile et al. 52] reported a QTL linked to the marker gwm1198 on 2A that confers resistance to Ug99 in the durum wheat population Kristal/Sebatel.
Neither of these markers was in proximity to the SNP markers detected in our study.
We detected one significant marker, IWB8481, located at 9 cM on chromosome 2D. The only reported QTL on 2D that provides APR
to Ug99 and its derivative races is in the CIMMYT biparental population PBW343/Kiritati
47]. Two Sr genes – Sr32, and Sr46 have been mapped to the short arm of 2D 49], 53], and both provide resistance to Ug99 44]. It should be noted that Sr32 has also been introgressed to 2A and 2B 54], but is not expected to be present in the 250 lines analyzed in this study. We used
the Sr32 markers developed by Mago et al.53] to screen our panel but found the markers to be not predictive of the gene (Additional
file 1). As no reliable marker for Sr46 has been developed, we are unable to distinguish between these two genes and the
marker we found on 2D.

The marker IWA4275 detected on chromosome 2B (position 197 cM) in our study is very close (distance
of 2.7 cM) to the marker wPt-8460, known to be linked to Sr9h in 1956 Rockefeller Foundation cultivar Gabo 56 (CI 14035) 11]. The same marker was also reported by Yu et al. 55] in their association mapping study constituting of CIMMYT spring wheat germplasm.
Sr9h, previously temporarily designated as SrWeb, is derived from the Canadian wheat cultivar ‘Webster’ (RL6201) and confers ASR gene
effective to TTKSK 56]. Markers developed by Rouse et al. 11] showed that Sr9h is present in 13 lines (5 %) in our panel (Additional file 1), implying that IWA4275 could represent the Sr9h locus in our panel. The gene Sr9a is also located on 2BL 55], 57], but is ineffective to Ug99 44].

On 2B, we also detected five SNP markers: IWA8534, IWB23660, IWB25868, IWB69631, and IWB25869 located at 126 cM. Based on the consensus map published by Maccaferri et al. 45], these markers are located at a distance of 4 cM from wmc332, which is linked to Sr2858]. We used two markers: wmc33258] and a newly developed SNP marker (Michael Pumphrey, personal communication) to investigate
if Sr28 was present in our panel. While both markers have only been tested on a limited panel
of lines and are not confirmed as diagnostic, we detected that up to 20 lines (8 %)
could possess Sr28 (Additional file 1). Thus, our marker-trait association may be detecting Sr28 on this 2B region.

Nine SNP markers were detected on the short arm of chromosome 3B with 8 SNPs located
at positional range of 11.5 – 14.1 cM and one additional SNP at 32.2 cM. These 8 SNPs
in the range 11.5 – 14.1 cM may be proximal to Sr2, a highly important APR gene for stem rust of wheat 3], 59]. Upon marker screening, it was found that 22 lines (9 %) in the panel contain Sr2 (Additional file 1). This gene is used extensively in the CIMMYT spring wheat breeding program, and
is shared by some US breeding programs that also incorporated this gene in their germplasm
for broad-spectrum resistance. It is possible that the SNP at 32.2 cM is associated
with Ug99 resistance that has been observed near the Sr12 locus 60]. Another stem rust APR gene, Sr5761], is located on chromosome 7D. Screening of the panel using the sequence-tagged site
marker developed by Lagudah et al. 62] showed that 97 lines (39 %) could contain Sr57. For other two stem rust APR genes: Sr55, located on 4D 63] and Sr56, located on 5B 64], no diagnostic markers are available. As no SNP markers were detected on chromosomes
4D and 5B during the analysis, we believe these genes are not present in our mapping
panel.

Several QTL located on chromosome 4A that provide resistance to Ug99 have been reported
in association mapping studies 55], 65], 66], and in biparental studies 47] in CIMMYT germplasm. These sources of stem rust resistance are not located in the
vicinity of the SNPs IWB46973, IWB56556, and IWB67877 detected also on 4A in our study. Similarly, QTL on chromosome 5A providing resistance
against Ug99 have been reported in biparental and association mapping studies 46], 47]. However, chromosome positions of the QTL and significant loci reported in these
studies differ from those detected in our study.

We detected only one significant SNP (IWA233) on 6AS. Mapped at 66 cM, this SNP is located away (100 cM) from the marker gwm617 reported by Pozniak et al. 46], and from the marker Sr26#43 linked to Sr26, which provides resistance to the Ug99 and its derivative races 55], 67]. Marker screening confirmed that Sr26 is absent in the panel under study (Additional file 1). Several QTL effective to Ug99 and its derivative races have also been discovered
on chromosome 6B 4], 49]. Of the reported QTL, the DArT marker wPt-6116 in the AM study conducted by Yu et al. 65] is located very close to the significant markers detected in this study: 1.1 cM from
IWB24757 and 2.2 cM from IWB45581. The gene Sr11 is located on 6BL, but is ineffective to Ug99 and its derivative races 3], 5]. Likewise, several QTL have been reported on 7A that provide field resistance to
Ug99 46], 47], 51], 52], 68]. However, none of the reported QTL or positions of significant marker effects coincide
with the significant markers detected in this study. Two 7B SNP markers – IWB47548 and IWA4175 – were significantly associated with resistance to Ug99. Letta et al. 51] have reported loci associated with resistance to Ug99 in durum wheat germplasm, however
they are located at a large distance (50 cM) from both markers in our study.

The significant SNP markers associated with APR to Ug99 reported in this study provide
several resistance loci to fight the disease, of which some are likely novel. Validation
of the significant markers in all chromosomes is essential to confirm the identity
of the associated resistance loci as well as to test their usefulness in marker assisted
resistance breeding in breeding programs.

Comparison of significant seedling-resistance Loci with existing resistance genes

The results of the GWAS for seedling-resistance in this study were compared with previous
findings for ASR to stem rust of wheat. As the discovered SNPs are suspected to be
linked primarily with existing or putatively novel resistance genes, a search for
similarities in chromosomal location with known resistance genes was emphasized.

We detected 23 SNPs in our germplasm panel that were significantly associated with
race TTKSK resistance at the seedling stage. The SNP marker IWA642 mapped at 67.7 cM on 1D is relatively close to Sr50, a gene that provides resistance to the Ug99 group of races 28]. The seedling resistance genes SrCad and SrTmp are considered to be present in the panel used in this study. SrCad is a stem rust resistance gene derived from the Canadian wheat lines ‘Peace’ and
‘AC Cadillac’, and is effective to Ug99 and its derivative races 69]. Located on chromosome 6D, this gene confers a highly resistant reaction (IT of 1
to 12) to TTKSK in seedling stages, and is moderately resistant to Ug99 in field nurseries.
SrCad has not been shown to be different than Sr42 in either map position or resistance specificity 70]. SrTmp is another gene resistant to TTKSK yet no SNPs were detected on 4B where the SrTmp gene is thought to be located 3]. Additional data suggest that SrTmp may be located on 6DS at a similar location to Sr42/SrCad71]. We used two markers: SSR marker cfd4972] and a SNP marker (Gao et al., unpublished) to screen the panel for presence/absence of Sr42. Results indicated that at least 71 or more lines in the panel could carry this gene,
yet the markers did not support each other (Additional file 1). Neither marker results also corroborate our TTKSK seedling screening results. We
are not aware of any study carried out on broad germplasm to determine if these two
markers are diagnostic or even predictive. From our results, it appears that they
are neither diagnostic nor predictive of Sr42/SrCad. Similarly, Sr9h has a resistant reaction to race TTKSK at the seedling stages (1 to 2 infection type)
56]. The presence of Sr9h was confirmed by marker screening, as discussed above. Except for the likely presence
of Sr9h, the position of the loci conferring Ug99 resistance in this study suggest that different
genes than the ones discussed above could be present in our panel. We suspect that
association mapping is limited by the low frequency of resistance loci (only 15 (6 %)
of 250 lines resistant to TTKSK), leading to lack of detection of SNPs significantly
associated with SrCad or SrTmp. Since no ASR genes effective to Ug99 are known to exist on chromosomes 3B and 5B,
our findings indicate that the North American breeding germplasm might contain previously
undiscovered important sources of resistance to the disease.

The genes Sr24 and Sr36 are resistant to the race TTKSK, yet no SNPs associated with resistance to this race
were detected in the chromosomes containing these genes. Sr24, located on 3DL, is widely used in Mexico and the USA; Sr36, located on 2BS, is known to be present in wheat lines in the USA 3]. Lack of detection of these genes can be attributed to either 1) representative germplasm
with these genes are not present in our GWAS panel; or 2) if present, the allele frequency
is very low which does not pass our stringent analysis filters. Upon marker screening
(Additional file 1), we discovered that Sr36 is not present in our panel; and only 7 lines (3 %) contain Sr24, confirming our assumptions.

Of the 77 mapped SNPs significantly associated with resistance to TRTTF, 57 SNPs were
located on the short arm of chromosome 6A (position range 2 cM – 26 cM). These markers
are most likely linked to the gene Sr8a which is located on 6AS and is effective to the race TRTTF 34], 73]. Similarly, the SNP marker IWB48466 located on the long arm of 7A (217 cM) is in the same region as the stem rust resistance
gene Sr22. This gene was introgressed into 7AL of hexaploid wheat from its diploid relative
Triticum boeoticum74], and is effective against TRTTF 75]. Marker screening of the GWAS panel with a robust sequence tagged site (STS) marker
developed by Periyannan et al. 76] confirmed that Sr22 is not present in the panel. Sr31, while ineffective against TTKSK, is effective against TRTTF, and is located on 1BL
75], 77]. Our GWAS results detected 13 significant SNPs, all on the short arm of chromosome
1B (position range 44 cM – 65 cM). Given the presence of CIMMYT lines in our panel
and the widespread use of Sr31 in breeding programs, screening of lines with Sr31 with these markers is needed to determine if the markers are linked to Sr31, or if a novel source of resistance to TRTTF is located on 1BS.

Chromosome 1DS is known to harbor multiple Sr genes 49], and could be represented by the two SNPs that were detected on 1DS in our analysis.
We also discovered markers on 5DL and 6BS associated with resistance to TRTTF. As
no ASR genes effective against TRTTF are known to exist on 5DL and 6BS, the North
American elite breeding germplasm likely possesses novel genes for resistance to the
Yemeni stem rust race TRTTF.

One-hundred and nine SNPs associated with seedling resistance to the newly detected
Ethiopian stem rust race TKTTF were detected on five chromosomes: 1AS, 4AL, 5AL, 6BL,
and 7AS. The 52 6BL SNPs distributed in the positional range of 109 cM – 123 cM likely
represent the gene Sr11 which is effective to this race. Fifty-one significant SNPs were located on 4AL (142 cM
– 164 cM) possibly indicative of resistance gene Sr7a (TKTTF is virulent to Sr7b). No ASR genes are known to be located on chromosome 5AL, and therefore the germplasm
under study may possess a new source of resistance to the race TKTTF in this region.
APR QTL providing resistance to the Ug99 and its derivative races have been detected
in the 1AS region 49], 55]. Additionally, the gene Sr1RS ^Amigo
is located on the 1RS.1AL rye chromosome arm translocation. Chromosome 7AS does not
possess any known ASR genes, yet APR QTL effective to Ug99 and its derivative races
have been detected in the region 49], 68].

None of the SNPs associated with seedling resistance for the three races were common,
suggesting that none of the genes in this material are broadly effective. Further
studies involving development of populations for fine mapping and allelism tests are
required to elaborate and confirm the nature of the genetic mechanisms controlling
the resistance to these three rust races.

Using breeding lines in GWAS

One of the main advantages of conducting association mapping on a panel consisting
of breeding germplasm is to explore the genetic composition of the lines, and estimate
the effects of significantly associated loci with the trait(s) of interest. The discovery
of significant SNPs can allow for tagging of lines that are enriched for alleles associated
with the trait, and their use in gene introgression for resistance breeding. More
importantly, as the lines used in this AM study are elite, they possess the desired
agronomic traits, and are adapted to the desired regions. This helps in avoiding the
problems that could otherwise arise from linkage drag, when more diverse germplasm
is used to introgress alleles of interest. Singh et al. 5] have reported that up to 95 % of germplasm from global seed collections and breeding
programs are susceptible to Ug99. As Ug99 and its derivative races have not yet been
observed in North America, it is prudent to prepare for their possible arrival by
developing resistant varieties. Discovery of resistant sources in existing breeding
programs can speed up the process of gene introgression into elite lines, gene pyramiding
for elevated resistance to the disease, and possible identification of diagnostic
markers that can be used in marker assisted resistance breeding. Germplasm sharing
among the breeding programs for this purpose, at least within the US, is plausible
given the genetic similarity among the lines, as observed in Fig. 2. The availability of SNP alleles associated with reduced disease severity (as well
as increased severity) in both adult plant and seedling stages (Additional file 1) should be useful for breeders to make decisions about selection of lines to be used
as parents in their breeding programs. Breeders may also use the significant SNP markers
we have provided to design assays for possible marker assisted selection or screening
of resistant materials in their own breeding programs. Additionally, Table 6 has been populated with a list of lines that exhibited high levels of APR to Ug99
(Table 6), and seedling resistance to TTKSK (Table 7). The complete genotypic and phenotypic data presented in this study have also been
made available on The Triticeae Toolbox (T3 webportal) with the goal of facilitating
line selection based on Sr marker associations. We are confident that the North American wheat breeding programs
can fortify the stem rust resistance in their germplasm by capitalizing on the information
provided in this study.

Table 6. Elite spring wheat lines from North American breeding programs that exhibit high level
of adult plant resistance (APR) to Ug99 in four field environments

Table 7. Elite spring wheat lines from North American breeding programs that exhibit high level
of seedling resistance to race TTKSK. For each line, the observed seedling infection
type (IT) for each race and the corresponding value on the linear scale are presented
under the column ‘IT’ and ‘Linear Score’, respectively