Evolution, diversity, and function of the disease susceptibility gene Snn1 in wheat.

Septoria nodorum blotch (SNB), caused by Parastagonospora nodorum, is a disease of durum and common wheat initiated by the recognition of pathogen-produced necrotrophic effectors (NEs) by specific wheat genes. The wheat gene Snn1 was previously cloned, and it encodes a wall-associated kinase that directly interacts with the NE SnTox1 leading to programmed cell death and ultimately the development of SNB. Here, sequence analysis of Snn1 from 114 accessions including diploid, tetraploid, and hexaploid wheat species revealed that some wheat lines possess two copies of Snn1 (designated Snn1-B1 and Snn1-B2) approximately 120 kb apart. Snn1-B2 evolved relatively recently as a paralog of Snn1-B1, and both genes have undergone diversifying selection. Three point mutations associated with the formation of the first SnTox1-sensitive Snn1-B1 allele from a primitive wild wheat were identified. Four subsequent and independent SNPs, three in Snn1-B1 and one in Snn1-B2, converted the sensitive alleles to insensitive forms. Protein modeling indicated these four mutations could abolish Snn1-SnTox1 compatibility either through destabilization of the Snn1 protein or direct disruption of the protein-protein interaction. A high-throughput marker was developed for the absent allele of Snn1, and it was 100% accurate at predicting SnTox1-insensitive lines in both durum and spring wheat. Results of this study increase our understanding of the evolution, diversity, and function of Snn1-B1 and Snn1-B2 genes and will be useful for marker-assisted elimination of these genes for better host resistance.

Identification of Sr67, a new gene for stem rust resistance in KU168-2 located close to the Sr13 locus in wheat.

KEY MESSAGE: Sr67 is a new stem rust resistance gene that represents a new resource for breeding stem rust resistant wheat cultivars Re-appearance of stem rust disease, caused by the fungal pathogen Puccinia graminis f. sp. tritici (Pgt), in different parts of Europe emphasized the need to develop wheat varieties with effective resistance to local Pgt populations and exotic threats. A Kyoto University wheat (Triticum aestivum L.) accession KU168-2 was reported to carry good resistance to leaf and stem rust. To identify the genomic region associated with the KU168-2 stem rust resistance, a genetic study was conducted using a doubled haploid (DH) population from the cross RL6071 × KU168-2. The DH population was phenotyped with three Pgt races (TTKSK, TPMKC, and QTHSF) and genotyped using the Illumina 90 K wheat SNP array. Linkage mapping showed the resistance to all three Pgt races was conferred by a single stem rust resistance (Sr) gene on chromosome arm 6AL, associated with Sr13. Presently, four Sr13 resistance alleles have been reported. Sr13 allele-specific KASP and STARP markers, and sequencing markers all showed null alleles in KU168-2. KU168-2 showed a unique combination of seedling infection types for five Pgt races (TTKSK, QTHSF, RCRSF, TMRTF, and TPMKC) compared to Sr13 alleles. The phenotypic uniqueness of the stem rust resistance gene in KU168-2 and null alleles for Sr13 allele-specific markers showed the resistance was conferred by a new gene, designated Sr67. Since Sr13 is less effective in hexaploid background, Sr67 will be a good source of stem rust resistance in bread wheat breeding programs.

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.

Genome-wide association analyses of leaf rust resistance in cultivated emmer wheat.

KEY MESSAGE: Fifteen and eleven loci, with most loci being novel, were identified to associate with seedling and adult resistances, respectively, to the durum-specific races of leaf rust pathogen in cultivated emmer. Leaf rust, caused by Puccinia triticina (Pt), constantly threatens durum (Triticum turgidum ssp. durum) and bread wheat (Triticum aestivum) production worldwide. A Pt race BBBQD detected in California in 2009 poses a potential threat to durum production in North America because resistance source to this race is rare in durum germplasm. To find new resistance sources, we assessed a panel of 180 cultivated emmer wheat (Triticum turgidum ssp. dicoccum) accessions for seedling resistance to BBBQD and for adult resistance to a mixture of durum-specific races BBBQJ, CCMSS, and MCDSS in the field, and genotyped the panel using genotype-by-sequencing (GBS) and the 9 K SNP (Single Nucleotide Polymorphism) Infinium array. The results showed 24 and nine accessions consistently exhibited seedling and adult resistance, respectively, with two accessions providing resistance at both stages. We performed genome-wide association studies using 46,383 GBS and 4,331 9 K SNP markers and identified 15 quantitative trait loci (QTL) for seedling resistance located mostly on chromosomes 2B and 6B, and 11 QTL for adult resistance on 2B, 3B and 6A. Of these QTL, one might be associated with leaf rust resistance (Lr) gene Lr53, and two with the QTL previously reported in durum or hexaploid wheat. The remaining QTL are potentially associated with new Lr genes. Further linkage analysis and gene cloning are necessary to identify the causal genes underlying these QTL. The emmer accessions with high levels of resistance will be useful for developing mapping populations and adapted durum germplasm and varieties with resistance to the durum-specific races.

Association Mapping of Resistance to Tan Spot in the Global Durum Panel.

Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr), is an important disease of durum and common wheat worldwide. Compared with common wheat, less is known about the genetics and molecular basis of tan spot resistance in durum wheat. We evaluated 510 durum lines from the Global Durum Wheat Panel (GDP) for sensitivity to the necrotrophic effectors (NEs) Ptr ToxA and Ptr ToxB and for reaction to Ptr isolates representing races 1 to 5. Overall, susceptible durum lines were most prevalent in South Asia, the Middle East, and North Africa. Genome-wide association analysis showed that the resistance locus Tsr7 was significantly associated with tan spot caused by races 2 and 3, but not races 1, 4, or 5. The NE sensitivity genes Tsc1 and Tsc2 were associated with susceptibility to Ptr ToxC- and Ptr ToxB-producing isolates, respectively, but Tsn1 was not associated with tan spot caused by Ptr ToxA-producing isolates, which further validates that the Tsn1-Ptr ToxA interaction does not play a significant role in tan spot development in durum. A unique locus on chromosome arm 2AS was associated with tan spot caused by race 4, a race once considered avirulent. A novel trait characterized by expanding chlorosis leading to increased disease severity caused by the Ptr ToxB-producing race 5 isolate DW5 was identified, and this trait was governed by a locus on chromosome 5B. We recommend that durum breeders select resistance alleles at the Tsr7, Tsc1, Tsc2, and the chromosome 2AS loci to obtain broad resistance to tan spot.

A protein kinase-major sperm protein gene hijacked by a necrotrophic fungal pathogen triggers disease susceptibility in wheat.

Septoria nodorum blotch (SNB), a disease caused by the necrotrophic fungal pathogen Parastagonospora nodorum, is a threat to wheat (Triticum aestivum) production worldwide. Multiple inverse gene-for-gene interactions involving the recognition of necrotrophic effectors (NEs) by wheat sensitivity genes play major roles in causing SNB. One interaction involves the wheat gene Snn3 and the P. nodorum NE SnTox3. Here, we used a map-based strategy to clone the Snn3-D1 gene from Aegilops tauschii, the D-genome progenitor of common wheat. Snn3-D1 contained protein kinase and major sperm protein domains, both of which were essential for function as confirmed by mutagenesis. As opposed to other characterized interactions in this pathosystem, a compatible Snn3-D1-SnTox3 interaction was light-independent, and Snn3-D1 transcriptional expression was downregulated by light and upregulated by darkness. Snn3-D1 likely emerged in Ae. tauschii due to an approximately 218-kb insertion that occurred along the west bank of the Caspian Sea. The identification of this new class of NE sensitivity genes combined with the previously cloned sensitivity genes demonstrates that P. nodorum can take advantage of diverse host targets to trigger SNB susceptibility in wheat.

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.

A triple threat: the Parastagonospora nodorum SnTox267 effector exploits three distinct host genetic factors to cause disease in wheat.

Parastagonospora nodorum is a fungal pathogen of wheat. As a necrotrophic specialist, it deploys effector proteins that target dominant host susceptibility genes to elicit programmed cell death (PCD). Here we identify and functionally validate the effector targeting the host susceptibility genes Snn2, Snn6 and Snn7. We utilized whole-genome sequencing, association mapping, gene-disrupted mutants, gain-of-function transformants, virulence assays, bioinformatics and quantitative PCR to characterize these interactions. A single proteinaceous effector, SnTox267, targeted Snn2, Snn6 and Snn7 to trigger PCD. Snn2 and Snn6 functioned cooperatively to trigger PCD in a light-dependent pathway, whereas Snn7-mediated PCD functioned in a light-independent pathway. Isolates harboring 20 SnTox267 protein isoforms quantitatively varied in virulence. The diversity and distribution of isoforms varied between populations, indicating adaptation to local selection pressures. SnTox267 deletion resulted in the upregulation of effector genes SnToxA, SnTox1 and SnTox3. We validated a novel effector operating in an inverse-gene-for-gene manner to target three genetically distinct host susceptibility genes and elicit PCD. The discovery of the complementary gene action of Snn2 and Snn6 indicates their potential function in a guard or decoy model. Additionally, differences in light dependency in the elicited pathways and upregulation of unlinked effectors sheds new light onto a complex fungal necrotroph-host interaction.

The Parastagonospora nodorum necrotrophic effector SnTox5 targets the wheat gene Snn5 and facilitates entry into the leaf mesophyll.

Parastagonospora nodorum is an economically important necrotrophic fungal pathogen of wheat. Parastagonospora nodorum secretes necrotrophic effectors that target wheat susceptibility genes to induce programmed cell death (PCD). In this study, we cloned and functionally validated SnTox5 and characterized its role in pathogenesis. We used whole genome sequencing, genome-wide association study (GWAS) mapping, CRISPR-Cas9-based gene disruption, gain-of-function transformation, quantitative trait locus (QTL) analysis, haplotype and isoform analysis, protein modeling, quantitative PCR, and laser confocal microscopy to validate SnTox5 and functionally characterize SnTox5. SnTox5 is a mature 16.26 kDa protein with high structural similarity to SnTox3. Wild-type and mutant P. nodorum strains and wheat genotypes of SnTox5 and Snn5, respectively, were used to show that SnTox5 not only targets Snn5 to induce PCD but also facilitates the colonization of the mesophyll layer even in the absence of Snn5. Here we show that SnTox5 facilitates the efficient colonization of the mesophyll tissue and elicits PCD specific to host lines carrying Snn5. The homology to SnTox3 and the ability of SnTox5 to facilitate the colonizing of the mesophyll also suggest a role in the suppression of host defense before PCD induction.

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.

Identification of a major dominant gene for race-nonspecific tan spot resistance in wild emmer wheat.

KEY MESSAGE: A single dominant gene found in tetraploid and hexaploid wheat controls broad-spectrum race-nonspecific resistance to the foliar disease tan spot caused by Pyrenophora tritici-repentis. Tan spot is an important foliar disease of durum and common wheat caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis. Genetic studies in common wheat have shown that pathogen-produced necrotrophic effectors interact with host genes in an inverse gene-for-gene manner to cause disease, but quantitative trait loci (QTLs) with broad race-nonspecific resistance also exist. Less work has been done to understand the genetics of tan spot interactions in durum wheat. Here, we evaluated a set of Langdon durum-wild emmer (Triticum turgidum ssp. dicoccoides) disomic chromosome substitution lines for reaction to four P. tritici-repentis isolates representing races 1, 2, 3, and 5 to identify wild emmer chromosomes potentially containing tan spot resistance genes. Chromosome 3B from the wild emmer accession IsraelA rendered the tan spot-susceptible durum cultivar Langdon resistant to all four fungal isolates. Genetic analysis indicated that a single dominant gene, designated Tsr7, governed resistance. Detailed mapping experiments showed that the Tsr7 locus is likely the same as the race-nonspecific QTL previously identified in the hexaploid wheat cultivars BR34 and Penawawa. Four user-friendly SNP-based semi-thermal asymmetric reverse PCR (STARP) markers cosegregated with Tsr7 and should be useful for marker-assisted selection of resistance. In addition to 3B, other wild emmer chromosomes contributed moderate levels of tan spot resistance, and, as has been shown previously for tetraploid wheat, the Tsn1-Ptr ToxA interaction was not associated with susceptibility. This is the first report of a major dominant gene governing resistance to tan spot in tetraploid wheat.

Dissection and physical mapping of wheat chromosome 7B by inducing meiotic recombination with its homoeologues in Aegilops speltoides and Thinopyrum elongatum.

KEY MESSAGE: We constructed a homoeologous recombination-based bin map of wheat chromosome 7B, providing a unique physical framework for further study of chromosome 7B and its homoeologues in wheat and its relatives. Homoeologous recombination leads to the dissection and diversification of the wheat genome. Advances in genome sequencing and genotyping have dramatically improved the efficacy and throughput of homoeologous recombination-based genome studies and alien introgression in wheat and its relatives. In this study, we aimed to physically dissect and map wheat chromosome 7B by inducing meiotic recombination of chromosome 7B with its homoeologues 7E in Thinopyrum elongatum and 7S in Aegilops speltoides. The special genotypes, which were double monosomic for chromosomes 7B' + 7E' or 7B' + 7S' and homozygous for the ph1b mutant, were produced to enhance 7B - 7E and 7B - 7S recombination. Chromosome-specific DNA markers were developed and used to pre-screen the large recombination populations for 7B - 7E and 7B - 7S recombinants. The DNA marker-mediated preselections were verified by fluorescent genomic in situ hybridization (GISH). In total, 29 7B - 7E and 61 7B - 7S recombinants and multiple chromosome aberrations were recovered and delineated by GISH and the wheat 90 K SNP assay. Integrated GISH and SNP analysis of the recombinants physically mapped the recombination breakpoints and partitioned wheat chromosome 7B into 44 bins with 523 SNPs assigned within. A composite bin map was constructed for chromosome 7B, showing the bin size and physical distribution of SNPs. This provides a unique physical framework for further study of chromosome 7B and its homoeologues. In addition, the 7B - 7E and 7B - 7S recombinants extend the genetic variability of wheat chromosome 7B and represent useful germplasm for wheat breeding. Thereby, this genomics-enabled chromosome engineering approach facilitates wheat genome study and enriches the gene pool of wheat improvement.

Genome-wide association mapping of tan spot resistance in a worldwide collection of durum wheat.

KEY MESSAGE: Resistance to tan spot in durum wheat involves race-nonspecific QTL and necrotrophic insensitivity gene. Tan spot, caused by the necrotrophic fungus Pyrenophoratritici-repentis, is a major foliar disease on all cultivated wheat crops worldwide. Compared to common wheat, much less work has been done to investigate the genetic basis of tan spot resistance in durum. Here, we conducted disease evaluations, necrotrophic effector (NE) sensitivity assays and a genome-wide association study using a collection of durum accessions. The durum panel segregated for the reaction to disease inoculations and NE infiltrations with eighteen accessions being highly resistant to all races and most of them insensitive to both PtrToxA and PtrToxB. Over 65,000SNP markers were developed from genotyping-by-sequencing for the association mapping. As expected, sensitivity to PtrToxA and PtrToxB was mapped to the chromosome arms 5BL and 2BS, respectively. For the fungal inoculations, a quantitative trait locus (QTL) on chromosome 3B was associated with resistance to all races and likely corresponds to the race-nonspecific resistance QTL previously identified in common wheat. The Tsn1locus was not significantly associated with tan spot caused by the PtrToxA-producing isolates Pti2 and 86-124, but the Tsc2 locus was significantly associated with tan spot caused by the PtrToxB-producing isolate DW5. Another QTL on chromosome arm 1AS was associated with tan spot caused by the PtrToxC-producing isolate Pti2 and likely corresponds to the Tsc1 locus. Additional QTL for specific races was identified on chromosome 1B and 3B. Our work highlights the complexity of genetic resistance to tan spot and further confirms that the Ptr ToxA-Tsn1 interaction plays no significant role in disease development in tetraploid wheat.

Partitioning and physical mapping of wheat chromosome 3B and its homoeologue 3E in Thinopyrum elongatum by inducing homoeologous recombination.

KEY MESSAGE: We performed homoeologous recombination-based partitioning and physical mapping of wheat chromosome 3B and Th. elongatum chromosome 3E, providing a unique physical framework of this homoeologous pair for genome studies. The wheat (Triticum aestivum, 2n = 6x = 42, AABBDD) and Thinopyrum elongatum (2n = 2x = 14, EE) genomes can be differentiated from each other by fluorescent genomic in situ hybridization (FGISH) as well as molecular markers. This has facilitated homoeologous recombination-based partitioning and engineering of their genomes for physical mapping and alien introgression. Here, we constructed a special wheat genotype, which was double monosomic for wheat chromosome 3B and Th. elongatum chromosome 3E and homozygous for the ph1b mutant, to induce 3B-3E homoeologous recombination. Totally, 81 3B-3E recombinants were recovered and detected in the primary, secondary, and tertiary homoeologous recombination cycles by FGISH. Comparing to the primary recombination, the secondary and tertiary recombination shifted toward the proximal regions due to the increase in homology between the pairing partners. The 3B-3E recombinants were genotyped by high-throughput wheat 90-K single nucleotide polymorphism (SNP) arrays and their recombination breakpoints physically mapped based on the FGISH patterns and SNP results. The 3B-3E recombination physically partitioned chromosome 3B into 38 bins, and 429 SNPs were assigned to the distinct bins. Integrative analysis of FGISH and SNP results led to the construction of a composite bin map for chromosome 3B. Additionally, we developed 22 SNP-derived semi-thermal asymmetric reverse PCR markers specific for chromosome 3E and constructed a comparative map of homoeologous chromosomes 3E, 3B, 3A, and 3D. In summary, this work provides a unique physical framework for further studies of the 3B-3E homoeologous pair and diversifies the wheat genome for wheat improvement.

QTL mapping of resistance to tan spot induced by race 2 of Pyrenophora tritici-repentis in tetraploid wheat.

KEY MESSAGE: A total of 12 QTL conferring resistance to tan spot induced by a race 2 isolate, 86-124, were identified in three tetraploid wheat mapping populations. Durum is a tetraploid species of wheat and an important food crop. Tan spot, caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr), is a major foliar disease of both tetraploid durum wheat and hexaploid bread wheat. Understanding the Ptr-wheat interaction and identifying major QTL can facilitate the development of resistant cultivars and effectively mitigate the negative effect of this disease. Over 100 QTL have already been discovered in hexaploid bread wheat, whereas few mapping studies have been conducted in durum wheat. Utilizing resistant resources and identifying novel resistant loci in tetraploid wheat will be beneficial for the development of tan spot-resistant durum varieties. In this study, we evaluated four interconnected tetraploid wheat populations for their reactions to the race 2 isolate 86-124, which produces Ptr ToxA. Tsn1, the wheat gene that confers sensitivity to Ptr ToxA, was not associated with tan spot severity in any of the four populations. We found a total of 12 tan spot-resistant QTL among the three mapping populations. The QTL located on chromosomes 3A and 5A were detected in multiple populations and co-localized with race-nonspecific QTL identified in other mapping studies. Together, these QTL can confer high levels of resistance and can be used for the improvement in tan spot resistance in both hexaploid bread and durum wheat breeding. Two QTL on chromosomes 1B and 7A, respectively, were found in one population when inoculated with a ToxA knockout strain 86-124ΔToxA only, indicating that their association with tan spot was induced by other unidentified necrotrophic effectors, but under the absence of Ptr ToxA. In addition to removal of the known dominant susceptibility genes, integrating major race-nonspecific resistance loci like the QTL identified on chromosome 3A and 5A in this study could confer high and stable tan spot resistance in durum wheat.

Pyrenophora tritici-repentis Race 4 Isolates Cause Disease on Tetraploid Wheat.

The ascomycete fungus Pyrenophora tritici-repentis is the causal agent of tan spot of wheat. The disease can occur on both common wheat (Triticum aestivum) and durum wheat (T. turgidum ssp. durum) and has potential to cause significant yield and quality losses. The fungal pathogen is known to produce necrotrophic effectors (NEs) that act as important virulence factors. Based on the NE production and virulence on a set of four differentials, P. tritici-repentis isolates have been classified into eight races. Race 4 produces no known NEs and is avirulent on the differentials. From a fungal collection in North Dakota, we identified several isolates that were classified as race 4. These isolates caused no or little disease on all common wheat lines including the differentials; however, they were virulent on some durum cultivars and tetraploid wheat accessions. Using two segregating tetraploid wheat populations and quantitative trait locus mapping, we identified several genomic regions significantly associated with disease caused by two of these isolates, some of which have not been previously reported. This is the first report that race 4 is virulent on tetraploid wheat, likely utilizing unidentified NEs. Our findings further highlight the insufficiency of the current race classification system for P. tritici-repentis.

Genetic analysis of threshability and other spike traits in the evolution of cultivated emmer to fully domesticated durum wheat.

Genetic mutations in genes governing wheat threshability were critical for domestication. Knowing when these genes mutated during wheat evolution will provide more insight into the domestication process and lead to further exploitation of primitive alleles for wheat improvement. We evaluated a population of recombinant inbred lines derived from a cross between the durum variety Rusty and the cultivated emmer accession PI 193883 for threshability, rachis fragility, and other spike-related traits. Quantitative trait loci (QTL) associated with spike length, spikelets per spike, and spike compactness were primarily associated with known genes such as the pleiotropic domestication gene Q. Interestingly, rachis fragility was not associated with the Q locus, suggesting that this trait, usually a pleiotropic effect of the q allele, can be influenced by the genetic background. Threshability QTL were identified on chromosome arms 2AS, 2BS, and 5AL corresponding to the tenacious glume genes Tg2A and Tg2B as well as the Q gene, respectively, further demonstrating that cultivated emmer harbors the primitive non-free-threshing alleles at all three loci. Genetic analysis indicated that the effects of the three genes are mostly additive, with Q having the most profound effects on threshability, and that free-threshing alleles are necessary at all three loci to attain a completely free-threshing phenotype. These findings provide further insight into the timeline and possible pathways of wheat domestication and evolution that led to the formation of modern day domesticated wheats.

Development and characterization of wheat-sea wheatgrass (Thinopyrum junceiforme) amphiploids for biotic stress resistance and abiotic stress tolerance.

KEY MESSAGE: Development of a complete wheat-Thinopyrum junceiforme amphiploid facilitated identification of resistance to multiple pests and abiotic stress derived from the wild species and shed new light on its genome composition. Wheat production is facing numerous challenges from biotic and abiotic stresses. Alien gene transfer has been an effective approach for wheat germplasm enhancement. Thinopyrum junceiforme, also known as sea wheatgrass (SWG), is a distant relative of wheat and a relatively untapped source for wheat improvement. In the present study, we developed a complete amphiploid, 13G819, between emmer wheat and SWG for the first time. Analysis of the chromosome constitution of the wheat-SWG amphiploid by multiple-color genomic in situ hybridization indicated that SWG is an allotetraploid with its J1 genome closely related to Th. bessarabicum and Th. elongatum, and its J2 genome was derived from an unknown source. Two SWG-derived perennial wheat lines, 14F3516 and 14F3536, are partial amphiploids and carry 13 SWG chromosomes of mixed J1 and J2 genome composition, suggesting cytological instability. We challenged the amphiploid 13G819 with various abiotic and biotic stress treatments together with its emmer wheat parent. Compared to its emmer wheat parent, the amphiploid showed high tolerance to waterlogging, manganese toxicity and salinity, low nitrogen and possibly to heat as well. The amphiploid 13G819 is also highly resistant to the wheat streak mosaic virus (temperature insensitive) and Fusarium head blight. All three amphiploids had solid stems, which confer resistance to wheat stem sawflies. All these traits make SWG an excellent source for improving wheat resistance to diseases and insects and tolerance to abiotic stress.

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.

Cloning and characterization of the homoeologous genes for the Rec8-like meiotic cohesin in polyploid wheat.

BACKGROUND: Meiosis is a specialized cell division critical for gamete production in the sexual reproduction of eukaryotes. It ensures genome integrity and generates genetic variability as well. The Rec8-like cohesin is a cohesion protein essential for orderly chromosome segregation in meiotic cell division. The Rec8-like genes and cohesins have been cloned and characterized in diploid models, but not in polyploids. The present study aimed to clone the homoeologous genes (homoeoalleles) for Rec8-like cohesin in polyploid wheat, an important food crop for humans, and to characterize their structure and function under a polyploid condition. RESULTS: We cloned two Rec8-like homoeoalleles from tetraploid wheat (TtRec8-A1 and TtRec8-B1) and one from hexaploid wheat (TaRec8-D1), and performed expression and functional analyses of the homoeoalleles. Also, we identified other two Rec8 homoeoalleles in hexaploid wheat (TaRec8-A1 and TaRec8-B1) and the one in Aegilops tauschii (AetRec8-D1) by referencing the DNA sequences of the Rec8 homoeoalleles cloned in this study. The coding DNA sequences (CDS) of these six Rec8 homoeoalleles are all 1,827 bp in length, encoding 608 amino acids. They differed from each other primarily in introns although single nucleotide polymorphisms were detected in CDS. Substantial difference was observed between the homoeoalleles from the subgenome B (TtRec8-B1 and TaRec8-B1) and those from the subgenomes A and D (TtRec8-A1, TaRec8-A1, and TaRec8-D1). TtRec8-A1 expressed dominantly over TtRec8-B1, but comparably to TaRec8-D1, in polyploid wheat. In addition, we developed the antibody against wheat Rec8 and used the antibody to detect Rec8 cohesin in the Western blotting and subcellular localization analyses. CONCLUSIONS: The Rec8 homoeoalleles from the subgenomes A and D are transcriptionally more active than the one from the subgenome B in polyploid wheat. The structural variation and differential expression of the Rec8 homoeoalleles indicate a unique cross-genome coordination of the homoeologous genes in polyploid wheat, and imply the distinction of the wheat subgenome B from the subgenomes A and D in the origin and evolution.