Linkage drag analysis in three Aegilops speltoides introgressions carrying Sr47 in modern durum and hard red spring wheat germplasm.

KEY MESSAGE: Yield and quality tests of wheat lines derived from RWG35 show they carry little, or no linkage drag and are the preferred source of Sr47 for stem rust resistance. Three durum wheat (Triticum turgidum L. subsp. durum) lines, RWG35, RWG36, and RWG37 carrying slightly different Aegilops speltoides introgressions, but each carrying the Sr47 stem rust resistance gene, were backcrossed to three durum and three hard red spring (HRS) wheat (Triticum aestivum L.) cultivars to produce 18 backcross populations. Each population was backcrossed to the recurrent parent six times and prepared for yield trials to test for linkage drag. Lines carrying the introgression (S-lines) were compared to euploid sibling lines (W-lines) and their parent. Yield trials were conducted from 2018 to 2021 at three locations. Three agronomic and several quality traits were studied. In durum, lines derived from RWG35 had little or no linkage drag. Lines derived from RWG36 and RWG37 still retained linkage drag, most notably involving yield and thousand kernel weight, but also test weight, falling number, kernel hardness index, semolina extract, semolina protein content, semolina brightness, and peak height. In HRS wheat, the results were more complex, though the general result of RWG35 lines having little or no linkage drag and RWG36 and RWG37 lines retaining linkage drag still applied. But there was heterogeneity in the Glenn35S lines, and Linkert lines had problems combining with the Ae. speltoides introgressions. We concluded that introgressions derived from RWG35 either had eliminated linkage drag or any negative effects were minor in nature. We recommend that breeders who wish to incorporate Sr47 into their cultivars should work exclusively with germplasm derived from RWG35.

Haplotype variants of Sr46 in Aegilops tauschii, the diploid D genome progenitor of wheat.

KEY MESSAGE: Stem rust resistance genes, SrRL5271 and Sr672.1 as well as SrCPI110651, from Aegilops tauschii, the diploid D genome progenitor of wheat, are sequence variants of Sr46 differing by 1-2 nucleotides leading to non-synonymous amino acid substitutions. The Aegilops tauschii (wheat D-genome progenitor) accessions RL 5271 and CPI110672 were identified as resistant to multiple races (including the Ug99) of the wheat stem rust pathogen Puccinia graminis f. sp. tritici (Pgt). This study was conducted to identify the stem rust resistance (Sr) gene(s) in both accessions. Genetic analysis of the resistance in RL 5271 identified a single dominant allele (SrRL5271) controlling resistance, whereas resistance segregated at two loci (SR672.1 and SR672.2) for a cross of CPI110672. Bulked segregant analysis placed SrRL5271 and Sr672.1 in a region on chromosome arm 2DS that encodes Sr46. Molecular marker screening, mapping and genomic sequence analysis demonstrated SrRL5271 and Sr672.1 are alleles of Sr46. The amino acid sequence of SrRL5271 and Sr672.1 is identical but differs from Sr46 (hereafter referred to as Sr46_h1 by following the gene nomenclature in wheat) by a single amino acid (N763K) and is thus designated Sr46_h2. Screening of a panel of Ae. tauschii accessions identified an additional allelic variant that differed from Sr46_h2 by a different amino acid (A648V) and was designated Sr46_h3. By contrast, the protein encoded by the susceptible allele of Ae. tauschii accession AL8/78 differed from these resistance proteins by 54 amino acid substitutions (94% nucleotide sequence gene identity). Cloning and complementation tests of the three resistance haplotypes confirmed their resistance to Pgt race 98-1,2,3,5,6 and partial resistance to Pgt race TTRTF in bread wheat. The three Sr46 haplotypes, with no virulent races detected yet, represent a valuable source for improving stem resistance in wheat.

Characterization of synthetic wheat line Largo for resistance to stem rust.

Resistance breeding is an effective approach against wheat stem rust caused by Puccinia graminis f. sp. tritici (Pgt). The synthetic hexaploid wheat line Largo (pedigree: durum wheat "Langdon" × Aegilops tauschii PI 268210) was found to have resistance to a broad spectrum of Pgt races including the Ug99 race group. To identify the stem rust resistance (Sr) genes, we genotyped a population of 188 recombinant inbred lines developed from a cross between the susceptible wheat line ND495 and Largo using the wheat Infinium 90 K SNP iSelect array and evaluated the population for seedling resistance to the Pgt races TTKSK, TRTTF, and TTTTF in the greenhouse conditions. Based on genetic linkage analysis using the marker and rust data, we identified six quantitative trait loci (QTL) with effectiveness against different races. Three QTL on chromosome arms 6AL, 2BL, and 2BS corresponded to Sr genes Sr13c, Sr9e, and a likely new gene from Langdon, respectively. Two other QTL from PI 268210 on 2DS and 1DS were associated with a potentially new allele of Sr46 and a likely new Sr gene, respectively. In addition, Sr7a was identified as the underlying gene for the 4AL QTL from ND495. Knowledge of the Sr genes in Largo will help to design breeding experiments aimed to develop new stem rust-resistant wheat varieties. Largo and its derived lines are particularly useful for introducing two Ug99-effective genes Sr13c and Sr46 into modern bread wheat varieties. The 90 K SNP-based high-density map will be useful for identifying the other important genes in Largo.

Function and evolution of allelic variations of Sr13 conferring resistance to stem rust in tetraploid wheat (Triticum turgidum L.).

The resistance gene Sr13 is one of the most important genes in durum wheat for controlling stem rust caused by Puccinia graminis f. sp. tritici (Pgt). The Sr13 functional gene CNL13 has haplotypes R1, R2 and R3. The R1/R3 and R2 haplotypes were originally designated as alleles Sr13a and Sr13b, respectively. To detect additional Sr13 alleles, we developed Kompetitive allele specific PCR (KASP™) marker KASPSr13 and four semi-thermal asymmetric reverse PCR markers, rwgsnp37-rwgsnp40, based on the CNL13 sequence. These markers were shown to detect R1, R2 and R3 haplotypes in a panel of diverse tetraploid wheat accessions. We also observed the presence of Sr13 in durum line CAT-A1, although it lacked any of the known haplotypes. Sequence analysis revealed that CNL13 of CAT-A1 differed from the susceptible haplotype S1 by a single nucleotide (C2200T) in the leucine-rich repeat region and differed from the other three R haplotypes by one or two additional nucleotides, confirming that CAT-A1 carries a new (R4) haplotype. Stem rust tests on the monogenic, transgenic and mutant lines showed that R1 differed from R3 in its susceptibility to races TCMJC and THTSC, whereas R4 differed from all other haplotypes for susceptibility to TTKSK, TPPKC and TCCJC. Based on these differences, we designate the R1, R3 and R4 haplotypes as alleles Sr13a, Sr13c and Sr13d, respectively. This study indicates that Sr13d may be the primitive functional allele originating from the S1 haplotype via a point mutation, with the other three R alleles probably being derived from Sr13d through one or two additional point mutations.

Mapping and characterization of two stem rust resistance genes derived from cultivated emmer wheat accession PI 193883.

KEY MESSAGE: Two stem rust resistance genes identified on chromosome arms 2BL and 6AL of the cultivated emmer wheat accession PI 193883 can be used for protecting modern varieties against Ug99 strains. The wheat research community consistently strives to identify new genes that confer resistance to stem rust caused by the fungal pathogen Puccinia graminis f. sp. tritici Eriks & E. Henn (Pgt). In the current study, our objective was to identify and genetically characterize the stem rust resistance derived from the cultivated emmer accession PI 193883. A recombinant inbred line population developed from a cross between the susceptible durum wheat line Rusty and PI 193883 was genotyped and evaluated for reaction to Pgt races TTKSK, TRTTF, and TMLKC. Two QTLs conferring resistance were identified on chromosome arms 2BL (QSr.fcu-2B) and 6AL (QSr.fcu-6A). The stem rust resistance gene (Sr883-2B) underlying QSr.fcu-2B was recessive, and based on its physical location it is located proximal to the Sr9 region. QSr.fcu-6A was located in the Sr13 region, but PI 193883 is known to carry the susceptible haplotype S4 for Sr13, indicating that the gene underlying QSr.fcu-6A (Sr883-6A) is likely a new allele of Sr13 or a gene residing close to Sr13. Three IWGSC scaffold-based simple sequence repeat (SSR) and two SNP-based semi-thermal asymmetric reverse PCR (STARP) markers were developed for the Sr883-2B region, and one STARP marker was developed for Sr883-6A. Sr883-2B was epistatic to Sr883-6A for reaction to TTKSK and TRTTF, and the two genes had additive effects for TMLKC. These two genes and the markers developed in this research provide additional resources and tools for the improvement in stem rust resistance in durum and common wheat breeding programs.

Molecular and Cytogenetic Characterization of Six Wheat-Aegilops markgrafii Disomic Addition Lines and Their Resistance to Rusts and Powdery Mildew.

Aegilops markgrafii (Greuter) Hammer is an important source of genes for resistance to abiotic stresses and diseases in wheat (Triticum aestivum L.). A series of six wheat 'Alcedo'-Ae. markgrafii chromosome disomic addition lines, designated as AI(B), AII(C), AIII(D), AV(E), AIV(F), and AVIII(G) carrying the Ae. markgrafii chromosomes B, C, D, E, F, and G, respectively, were tested with SSR markers to establish homoeologous relationships to wheat and identify markers useful in chromosome engineering. The addition lines were evaluated for resistance to rust and powdery mildew diseases. The parents Alcedo and Ae. markgrafii accession 'S740-69' were tested with 1500 SSR primer pairs and 935 polymorphic markers were identified. After selecting for robust markers and confirming the polymorphisms on the addition lines, 132 markers were considered useful for engineering and establishing homoeologous relationships. Based on the marker analysis, we concluded that the chromosomes B, C, D, E, F, and G belong to wheat homoeologous groups 2, 5, 6, 7, 3, and 4, respectively. Also, we observed chromosomal rearrangements in several addition lines. When tested with 20 isolates of powdery mildew pathogen (Blumeria graminis f. sp. tritici) from five geographic regions of the United States, four addition lines [AIII(D), AV(E), AIV(F), and AVIII(G)] showed resistance to some isolates, with addition line AV(E) being resistant to 19 of 20 isolates. The addition lines were tested with two races (TDBJ and TNBJ) of the leaf rust pathogen (Puccinia triticina), and only addition line AI(B) exhibited resistance at a level comparable to the Ae. markgrafii parent. Addition lines AII(C) and AIII(D) had been previously identified as resistant to the Ug99 race group of the stem rust pathogen (Puccinia graminis f. sp. tritici). The addition lines were also tested for resistance to six United States races (PSTv-4, PSTv-14, PSTv-37, PSTv-40, PSTv-51, and PSTv-198) of the stripe rust pathogen (Puccinia striiformis f. sp. tritici); we found no resistance either in Alcedo or any of the addition lines. The homoeologous relationships of the chromosomes in the addition lines, molecular markers located on each chromosome, and disease resistance associated with each chromosome will allow for chromosome engineering of the resistance genes.

Physical mapping of DNA markers linked to stem rust resistance gene Sr47 in durum wheat.

KEY MESSAGE: Markers linked to stem rust resistance gene Sr47 were physically mapped in three small Aegilops speltoides chromosomal bins. Five markers, including two PCR-based SNP markers, were validated for marker-assisted selection. In durum wheat (Triticum turgidum subsp. durum), the gene Sr47 derived from Aegilops speltoides conditions resistance to race TTKSK (Ug99) of the stem rust pathogen (Puccinia graminis f. sp. tritici). Sr47 is carried on small interstitial translocation chromosomes (Ti2BL-2SL-2BL·2BS) in which the Ae. speltoides chromosome 2S segments are divided into four bins in genetic stocks RWG35, RWG36, and RWG37. Our objective was to physically map molecular markers to bins and to determine if any of the molecular markers would be useful in marker-assisted selection (MAS). Durum cultivar Joppa was used as the recurrent parent to produce three BC2F2 populations. Each BC2F2 plant was genotyped with markers to detect the segment carrying Sr47, and stem rust testing of BC2F3 progeny with race TTKSK confirmed the genotyping. Forty-nine markers from published sources, four new SSR markers, and five new STARP (semi-thermal asymmetric reverse PCR) markers, were evaluated in BC2F2 populations for assignment of markers to bins. Sr47 was mapped to bin 3 along with 13 markers. No markers were assigned to bin 1; however, 7 and 13 markers were assigned to bins 2 and 4, respectively. Markers Xrwgs38a, Xmag1729, Xwmc41, Xtnac3119, Xrwgsnp1, and Xrwgsnp4 were found to be useful for MAS of Sr47. However, STARP markers Xrwgsnp1 and Xrwgsnp4 can be used in gel-free systems, and are the preferred markers for high-throughput MAS. The physical mapping data from this study will also be useful for pyramiding Sr47 with other Sr genes on chromosome 2B.

Development of a diagnostic co-dominant marker for stem rust resistance gene Sr47 introgressed from Aegilops speltoides into durum wheat.

KEY MESSAGE: A robust and diagnostic STS marker for stem rust resistance gene Sr47 was developed and validated for marker-assisted selection. Stem rust (caused by Puccinia graminis f. sp. tritici, Pgt) resistance gene Sr47, originally transferred from Aegilops speltoides to durum wheat (Triticum turgidum subsp. durum) line DAS15, confers a high level of resistance to Pgt race TTKSK (Ug99). Recently, the durum Rusty 5D(5B) substitution line was used to reduce the Ae. speltoides segment, and the resulting lines had Sr47 on small Ae. speltoides segments on wheat chromosome arm 2BL. The objective of this study was to develop a robust marker for marker-assisted selection of Sr47. A 200-kb segment of the Brachypodium distachyon genome syntenic with the Sr47 region was used to identify wheat expressed sequence tags (ESTs) homologous to the B. distachyon genes. The wheat EST sequences were then used to develop sequence-tagged site (STS) markers. By analyzing the markers for polymorphism between Rusty and DAS15, we identified a co-dominant STS marker, designated as Xrwgs38, which amplified 175 and 187 bp fragments from wheat chromosome 2B and Ae. speltoides chromosome 2S segments, respectively. The marker co-segregated with the Ae. speltoides segments carrying Sr47 in the families from four BC2F1 plants, including the parent plants for durum lines RWG35 and RWG36 with the pedigree of Rusty/3/Rusty 5D(5B)/DAS15//47-1 5D(5B). Analysis of 62 durum and common wheat cultivars/lines lacking the Sr47 segment indicated that they all possessed the 175-bp allele of Xrwgs38, indicating that it was diagnostic for the small Ae. speltoides segment carrying Sr47. This study demonstrated that Xrwgs38 will facilitate the selection of Sr47 in durum and common wheat breeding.

Homoeology of Thinopyrum junceum and Elymus rectisetus chromosomes to wheat and disease resistance conferred by the Thinopyrum and Elymus chromosomes in wheat.

Thirteen common wheat "Chinese Spring" (CS)-Thinopyrum junceum addition lines and three common wheat "Fukuhokomuji"(Fuku)-Elymus rectisetus addition lines were characterized and verified as disomic additions of a Th. junceum or E. rectisetus chromosome in the wheat backgrounds by fluorescent genomic in situ hybridization. Another Fuku-E. rectisetus addition line, A1048, was found to contain multiple segregating E. rectisetus chromosomes. Seven partial CS-Th. junceum amphiploids were identified to combine 12-16 Th. junceum chromosomes with CS wheat chromosomes. The disomic addition lines AJDAj5, 7, 8, 9, and HD3508 were identified to contain a Th. junceum chromosome in homoeologous group 1. Two of them, AJDAj7 and AJDAj9, had the same Th. junceum chromosome. AJDAj2, 3, and 4 contained a Th. junceum chromosome in group 2, HD3505 in group 4, AJDAj6 and AJDAj11 in group 5, and AJDAj1 probably in group 6. The disomic addition lines A1026 and A1057 were identified to carry an E. rectisetus chromosome in group 1 and A1034 in group 5. E. rectisetus chromosomes in groups 1-6 were detected in A1048. The homoeologous group of the Th. junceum chromosome in HD3515 could not be determined in this study. Several Th. junceum and E. rectisetus chromosomes in the addition lines were found to contain genes for resistance to Fusarium head blight, tan spot, Stagonospora nodorum blotch, and stem rust (Ug99 races). Understanding of the homoeology of the Th. junceum and E. rectisetus chromosomes with wheat will facilitate utilization of the favorable genes on these alien chromosomes in wheat improvement.

Introgression and characterization of a goatgrass gene for a high level of resistance to ug99 stem rust in tetraploid wheat.

The transfer of alien genes to crop plants using chromosome engineering has been attempted infrequently in tetraploid durum wheat (Triticum turgidum L. subsp. durum). Here, we report a highly efficient approach for the transfer of two genes conferring resistance to stem rust race Pgt-TTKSK (Ug99) from goatgrass (Aegilops speltoides) to tetraploid wheat. The durum line DAS15, carrying the stem rust resistance gene Sr47 derived from Ae. speltoides, was crossed, and backcrossed, to durum 5D(5B) aneuploids to induce homeologous pairing. After a final cross to 'Rusty' durum, allosyndetic recombinants were recovered. The Ae. speltoides chromosomal segment carrying Sr47 was found to have two stem rust resistance genes. One gene conditioning an infection type (IT) 2 was located in the same chromosomal region of 2BS as Sr39 and was assigned the temporary gene symbol SrAes7t. Based on ITs observed on a diverse set of rust races, SrAes7t may be the same as Sr39. The second gene conditioned an IT 0; and was located on chromosome arm 2BL. This gene retained the symbol Sr47 because it had a different IT and map location from other stem rust resistance genes derived from Ae. speltoides. Allosyndetic recombinant lines carrying each gene on minimal alien chromosomal segments were identified as were molecular markers distinguishing each alien segment. This study demonstrated that chromosome engineering of Ae. speltoides segments is feasible in tetraploid wheat. The Sr47 gene confers high-level and broad spectrum resistance to stem rust and should be very useful in efforts to control TTKSK.

Targeted introgression of a wheat stem rust resistance gene by DNA marker-assisted chromosome engineering.

Chromosome engineering is a useful strategy for transfer of alien genes from wild relatives into modern crops. However, this strategy has not been extensively used for alien gene introgression in most crops due to low efficiency of conventional cytogenetic techniques. Here, we report an improved scheme of chromosome engineering for efficient elimination of a large amount of goatgrass (Aegilops speltoides) chromatin surrounding Sr39, a gene that provides resistance to multiple stem rust races, including Ug99 (TTKSK) in wheat. The wheat ph1b mutation, which promotes meiotic pairing between homoeologous chromosomes, was employed to induce recombination between wheat chromosome 2B and goatgrass 2S chromatin using a backcross scheme favorable for inducing and detecting the homoeologous recombinants with small goatgrass chromosome segments. Forty recombinants with Sr39 with reduced surrounding goatgrass chromatin were quickly identified from 1048 backcross progenies through disease screening and molecular marker analysis. Four of the recombinants carrying Sr39 with a minimal amount of goatgrass chromatin (2.87-9.15% of the translocated chromosomes) were verified using genomic in situ hybridization. Approximately 97% of the goatgrass chromatin was eliminated in one of the recombinants, in which a tiny goatgrass chromosome segment containing Sr39 was retained in the wheat genome. Localization of the goatgrass chromatin in the recombinants led to rapid development of three molecular markers tightly linked to Sr39. The new wheat lines and markers provide useful resources for the ongoing global effort to combat Ug99. This study has demonstrated great potential of chromosome engineering in genome manipulation for plant improvement.