Targeting Disease Susceptibility Genes in Wheat Through wide Hybridization with Maize Expressing Cas9 and Guide RNA.

Two genes (TaHRC and Tsn1) conferring susceptibility to Fusarium head blight and tan spot, Septoria nodorum blotch, and spot blotch in wheat were targeted through wide hybridization with maize expressing Cas9 and guide RNA (gRNA). For each gene, two target sites were selected and corresponding gRNA expression cassettes were synthesized and cloned into a binary vector carrying the CRISPR/Cas9-mediated genome editing machinery. The constructed binary vectors were used to transform the hybrid maize Hi-II through an Agrobacterium-mediated approach to generate T0 and T1 plants, which were used to cross with wheat variety Dayn for targeting Tsn1 or the susceptible allele (TaHRC-S) of TaHRC as well as with the near-isogenic line (Day-Fhb1) of Dayn for targeting the resistant allele (TaHRC-R) of TaHRC. Haploid embryos were rescued in vitro from the wide crosses to generate haploid plants. PCR amplification and sequencing indicated that 15 to 33% of the haploid plants contained the target gene with mutations at the target sites. This wheat × maize hybridization combined with genome editing approach provides a useful alternative tool, not only for targeting susceptibility genes to improve disease resistance without regulatory issues, but also for understanding gene function in wheat. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genome-wide association study of fungicide sensitivity in a Fusarium graminearum population collected from North Dakota.

Fusarium head blight (FHB) is a destructive disease of small grains. The disease is predominantly caused by the haploid ascomycete fungus Fusarium graminearum in North America. To understand the genetics of quantitative traits for sensitivity to fungicides in this fungal pathogen, we conducted a genome-wide association study (GWAS) of sensitivity to two demethylation inhibition (DMI) class fungicides, tebuconazole and prothioconazole, using a F. graminearum population of 183 isolates collected between 1981 and 2013 from North Dakota. Baseline sensitivity to tebuconazole and prothioconazole was established using 21 isolates collected between 1981 and 1994. Most fungal isolates were sensitive to both tebuconazole and prothioconazole, however, five isolates showed significantly reduced sensitivity to prothioconazole. GWAS identified one significant marker-trait association (MTA) on chromosome 3 for tebuconazole resistance while six significant MTAs, one on chromosome 1, three on chromosome 2, and two on chromosome 4, were detected for prothioconazole resistance. Functional annotation of the MTA for tebuconazole revealed a candidate gene encoding a basic helix loop helix (bHLH) domain containing protein that reinforces sterol in the fungal membrane. Putative genes for prothioconazole resistance were also identified, which are involved in RNAi, detoxification by ubiquitin-proteasome pathway, and membrane integrity reinforcement. Considering the potential of the pathogen towards overcoming chemical control, continued monitoring of fungal sensitivities to commercially applied fungicides, especially those containing prothioconazole, is warranted to reduce risks of fungicide resistance in the pathogen populations.

Prevalence and Importance of the Necrotrophic Effector Gene ToxA in Bipolaris sorokiniana Populations Collected from Spring Wheat and Barley.

Bipolaris sorokiniana is a necrotrophic fungal pathogen that causes foliar and root diseases on wheat and barley. These diseases are common in all wheat- and barley-growing regions, with more severe outbreaks occurring under warm and humid conditions. B. sorokiniana can also infect a wide range of grass species in the family Poaceae and secrete ToxA, an important necrotrophic effector also identified other wheat leaf spotting pathogens. In this study, the prevalence and virulence role of ToxA were investigated in a collection of 278 B. sorokiniana isolates collected from spring wheat and barley in the Upper Midwest of the United States or other places, including 169 from wheat leaves, 75 from wheat roots, 30 from barley leaves, and 4 from wild quack grass leaves. ToxA was present in the isolates from wheat leaves, wheat roots, and wild grass leaves but was absent from isolates collected from barley leaves. Prevalence of ToxA in wheat leaf isolates (34.3%) was much higher than that in wheat root isolates (16%). Sequencing analysis revealed the presence of two haplotypes, with the majority being BsH2. All ToxA+ isolates produced the functional effector in liquid cultures. Pathogenicity assays revealed that ToxA+ isolates caused significantly more disease on spring wheat lines harboring Tsn1 than their tsn1 mutants, suggesting that the ToxA-Tsn1 interaction plays an important role in spot blotch development. This work confirms the importance of ToxA in B. sorokiniana populations infecting wheat and, thus, the need to eliminate Tsn1 from spring wheat cultivars to reduce susceptibility to spot blotch.

A cell wall invertase modulates resistance to fusarium crown rot and sharp eyespot in common wheat.

Fusarium crown rot (FCR) and sharp eyespot (SE) are serious soil-borne diseases in wheat and its relatives that have been reported to cause wheat yield losses in many areas. In this study, the expression of a cell wall invertase gene, TaCWI-B1, was identified to be associated with FCR resistance through a combination of bulk segregant RNA sequencing and genome resequencing in a recombinant inbred line population. Two bi-parental populations were developed to further verify TaCWI-B1 association with FCR resistance. Overexpression lines and ethyl methanesulfonate (EMS) mutants revealed TaCWI-B1 positively regulating FCR resistance. Determination of cell wall thickness and components showed that the TaCWI-B1-overexpression lines exhibited considerably increased thickness and pectin and cellulose contents. Furthermore, we found that TaCWI-B1 directly interacted with an alpha-galactosidase (TaGAL). EMS mutants showed that TaGAL negatively modulated FCR resistance. The expression of TaGAL is negatively correlated with TaCWI-B1 levels, thus may reduce mannan degradation in the cell wall, consequently leading to thickening of the cell wall. Additionally, TaCWI-B1-overexpression lines and TaGAL mutants showed higher resistance to SE; however, TaCWI-B1 mutants were more susceptible to SE than controls. This study provides insights into a FCR and SE resistance gene to combat soil-borne diseases in common 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 Conserved Hypothetical Gene Is Required but Not Sufficient for Ptr ToxC Production in Pyrenophora tritici-repentis.

The fungus Pyrenophora tritici-repentis causes tan spot, an important foliar disease of wheat worldwide. The fungal pathogen produces three necrotrophic effectors, namely Ptr ToxA, Ptr ToxB, and Ptr ToxC to induce necrosis or chlorosis in wheat. Both Ptr ToxA and Ptr ToxB are proteins, and their encoding genes have been cloned. Ptr ToxC was characterized as a low-molecular weight molecule 20 years ago but the one or more genes controlling its production in P. tritici-repentis are unknown. Here, we report the genetic mapping, molecular cloning, and functional analysis of a fungal gene that is required for Ptr ToxC production. The genetic locus controlling the production of Ptr ToxC, termed ToxC, was mapped to a subtelomeric region using segregating biparental populations, genome sequencing, and association analysis. Additional marker analysis further delimited ToxC to a 173-kb region. The predicted genes in the region were examined for presence/absence polymorphism in different races and isolates leading to the identification of a single candidate gene. Functional validation showed that this gene was required but not sufficient for Ptr ToxC production, thus it is designated as ToxC1. ToxC1 encoded a conserved hypothetical protein likely located on the vacuole membrane. The gene was highly expressed during infection, and only one haplotype was identified among 120 isolates sequenced. Our work suggests that Ptr ToxC is not a protein and is likely produced through a cascade of biosynthetic pathway. The identification of ToxC1 is a major step toward revealing the Ptr ToxC biosynthetic pathway and studying its molecular interactions with host factors.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Cytogenetic and genomic characterization of a novel tall wheatgrass-derived Fhb7 allele integrated into wheat B genome.

KEY MESSAGE: We identified and integrated the novel FHB-resistant Fhb7The2 allele into wheat B genome and made it usable in both common and durum wheat breeding programs without yellow flour linkage drag. A novel tall wheatgrass-derived (Thinopyrum elongatum, genome EE) Fhb7 allele, designated Fhb7The2, was identified and integrated into the wheat B genome through a small 7B-7E translocation (7BS·7BL-7EL) involving the terminal regions of the long arms. Fhb7The2 conditions significant Type II resistance to Fusarium head blight (FHB) in wheat. Integration of Fhb7The2 into the wheat B genome makes this wild species-derived FHB resistance gene usable for breeding in both common and durum wheat. By contrast, other Fhb7 introgression lines involving wheat chromosome 7D can be utilized only in common wheat breeding programs, not in durum wheat. Additionally, we found that Fhb7The2 does not have the linkage drag of the yellow flour pigment gene that is tightly linked to the decaploid Th. ponticum-derived Fhb7 allele Fhb7Thp. This will further improve the utility of Fhb7The2 in wheat breeding. DNA sequence analysis identified 12 single nucleotide polymorphisms (SNPs) in Fhb7The2, Fhb7Thp, and another Th. elongatum-derived Fhb7 allele Fhb7The1, which led to seven amino acid conversions in Fhb7The2, Fhb7Thp, and Fhb7The1, respectively. However, no significant variation was observed in their predicted protein configuration as a glutathione transferase. Diagnostic DNA markers were developed specifically for Fhb7The2. The 7EL segment containing Fhb7The2 in the translocation chromosome 7BS·7BL-7EL exhibited a monogenic inheritance pattern in the wheat genetic background. This will enhance the efficacy of marker-assisted selection for Fhb7The2 introgression, pyramiding, and deployment in wheat germplasm and varieties.

Genome Sequence Resources for the Maize Pathogen Fusarium temperatum Isolated in Poland.

Fusarium temperatum (Scaufl. & Munaut) is one of the most important fungal pathogens that cause ear and stalk rots in maize. In this study, we sequenced genomes of two F. temperatum isolates (KFI615 and KFI660) isolated from corn ears in Poland. A total of 110.3 and 116.3 million 100-nucleotide paired-end clean reads were obtained for KFI615 and KFI660, which were assembled into 20 and 18 scaffolds with an estimated genome size of 45.21 and 45.00 Mb, respectively. These genome sequences provide important resources for understanding pathogenicity and biology of the pathogens within the Fusarium fujikuroi complex.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A genome-wide genetic linkage map and reference quality genome sequence for a new race in the wheat pathogen Pyrenophora tritici-repentis.

Pyrenophora tritici-repentis is an ascomycete fungus that causes tan spot of wheat. The disease has a worldwide distribution and can cause significant yield and quality losses in wheat production. The fungal pathogen is homothallic in nature, which means it can undergo sexual reproduction by selfing to produce pseudothecia on wheat stubble for seasonal survival. Since homothallism precludes the development of bi-parental fungal populations, no genetic linkage map has been developed for P. tritici-repentis for mapping and map-based cloning of fungal virulence genes. In this work, we created two heterothallic strains by deleting one of the mating type genes in each of two parental isolates 86-124 (race 2) and AR CrossB10 (a new race) and developed a bi-parental fungal population between them. The draft genome sequences of the two parental isolates were aligned to the Pt-1C-BFP reference sequence to mine single nucleotide polymorphisms (SNPs). A total of 225 SNP markers were developed for genotyping the entire population. Additionally, 75 simple sequence repeat, and two gene markers were also developed and used in the genotyping. The resulting linkage map consisted of 13 linkage groups spanning 5,075.83 cM in genetic distance. Because the parental isolate AR CrossB10 is a new race and produces Ptr ToxC, it was sequenced using long-read sequencing platforms and de novo assembled into contigs. The majority of the contigs were further anchored into chromosomes with the aid of the linkage maps. The whole genome comparison of AR CrossB10 to the reference genome of M4 revealed a few chromosomal rearrangements. The genetic linkage map and the new AR CrossB10 genome sequence are valuable tools for gene cloning in P. tritici-repentis.

Thyroid nodule recognition using a joint convolutional neural network with information fusion of ultrasound images and radiofrequency data.

OBJECTIVE: To develop a deep learning-based method with information fusion of US images and RF signals for better classification of thyroid nodules (TNs). METHODS: One hundred sixty-three pairs of US images and RF signals of TNs from a cohort of adult patients were used for analysis. We developed an information fusion-based joint convolutional neural network (IF-JCNN) for the differential diagnosis of malignant and benign TNs. The IF-JCNN contains two branched CNNs for deep feature extraction: one for US images and the other one for RF signals. The extracted features are fused at the backend of IF-JCNN for TN classification. RESULTS: Across 5-fold cross-validation, the accuracy, sensitivity, specificity, and area under the receiver operating characteristic curve (AUROC) obtained by using the IF-JCNN with both US images and RF signals as inputs for TN classification were respectively 0.896 (95% CI 0.838-0.938), 0.885 (95% CI 0.804-0.941), 0.910 (95% CI 0.815-0.966), and 0.956 (95% CI 0.926-0.987), which were better than those obtained by using only US images: 0.822 (0.755-0.878; p = 0.0044), 0.792 (0.679-0.868, p = 0.0091), 0.866 (0.760-0.937, p = 0.197), and 0.901 (0.855-0.948, p = .0398), or RF signals: 0.767 (0.694-0.829, p < 0.001), 0.781 (0.685-0.859, p = 0.0037), 0.746 (0.625-0.845, p < 0.001), 0.845 (0.786-0.903, p < 0.001). CONCLUSIONS: The proposed IF-JCNN model filled the gap of just using US images in CNNs to characterize TNs, and it may serve as a promising tool for assisting the diagnosis of thyroid cancer. KEY POINTS: • Raw radiofrequency signals before ultrasound imaging of thyroid nodules provide useful information that is not carried by ultrasound images. • The information carried by raw radiofrequency signals and ultrasound images for thyroid nodules is complementary. • The performance of deep convolutional neural network for diagnosing thyroid nodules can be significantly improved by fusing US images and RF signals in the model as compared with just using US images.

Identification of Fungal Species Associated with Crown and Root Rots of Wheat and Evaluation of Plant Reactions to the Pathogens in North Dakota.

Common root rot (CRR) and crown rot (CR), caused by Bipolaris sorokiniana and Fusarium species, respectively, can cause significant yield losses in cereal crops. To assess the prevalence, incidence, and severity of these diseases in North Dakota, wheat samples were collected from spring wheat fields across the state in 2012, 2013, and 2014. Based on subcrown internode symptoms, a greater incidence and severity of CRR was observed in 2012 (warm and dry year) than in 2013 and 2014. Also, the Northwestern Glaciated Plains and Northwestern Great Plains ecoregions showed greater CRR incidence and severity compared with the Northern Glaciated Plains and Lake Agassiz Plains ecoregions in the state. B. sorokiniana and Fusarium species, including Fusarium acuminatum, Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, Fusarium equiseti, Fusarium pseudograminearum, Fusarium oxysporum, Fusarium redolens, Fusarium sporotrichioides, and Fusarium solani were isolated and identified from the root and crown tissues of the wheat samples. B. sorokiniana was isolated more frequently than other fungal species in all sampled years and ecoregions of North Dakota. F. acuminatum, F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. pseudograminearum, and F. redolens were pathogenic causing infections on seedlings of the two wheat genotypes (ND652 and Alsen), whereas isolates of F. oxysporum and F. solani were nonpathogenic and considered as secondary invaders associated with the root and CR diseases. Evaluation of some spring wheat genotypes for reactions to one B. sorokiniana isolate at seedling and adult plant stages, and one F. culmorum isolate at the seedling stage, indicated that susceptibility to these pathogens varied among different wheat genotypes tested. This study provides useful information on fungal species associated with CRR and CR of wheat in North Dakota and on resistant/susceptible reactions of some spring wheat lines to the different fungal isolates evaluated.

First Report of Fusarium equiseti Causing Seedling Death on Sugar Beet in Minnesota, USA.

In May 2019, sugar beet (Beta vulgaris L.) seedlings with symptoms of wilting and root tip discoloration and necrosis were found in Moorhead (46.5507° N, 96.4208° W), Minnesota, USA. Roots of infected seedlings were surface sterilized with 10% bleach for 15 seconds, rinsed with sterile distilled water and cultured on water agar (MA Mooragar®, Inc, CA) for 3 days at 23 ± 2°C. Isolates were transferred to carnation leaf agar (CLA) and incubated at room temperature (22°C) under fluorescent light for 14 days. Abundant macroconidia were produced in sporodochia. Macroconidia were 5- to 7-septate, slightly curved at the apex, and ranged from 35 to 110 ×1.2 to 3.8 µm. No microconidia were produced. Chlamydospores with thick, roughened walls were observed in chains or in clumps, and were ellipsoidal or subglobose. Single spore was transferred from CLA to potato dextrose agar (HIMEDIA Laboratories, India) produced abundant white mycelium and was pale brown where the colony was in contact with the media. The morphological features of the isolates were consistent with Fusarium equiseti (Corda) Sacc. (Leslie and Summerell 2006, Li et al. 2015). Genomic DNAs (NORGEN BIOTEK CORP, Fungi DNA Isolation Kit #26200) of two representative isolates were used for polymerase chain reaction (PCR). The second largest subunit of RNA polymerase (RPB2) was amplified by PCR with primers 5f2/7cr (O'Donnell et al. 2010). The amplified PCR product was sequenced and deposited in GenBank (accession number MW048778). A BLAST search in Genbank and the Fusarium MLST database showed 100% sequence alignment to F. equiseti with accession MK077037.1 and NRRL 25795, respectively. Pathogenicity testing was done using three sugar beet seedlings (Hilleshög proprietary material, Hilleshög Seed, LLC, Halsey, OR 97348) at cotyledonary stage grown in a pot (4˝×4˝×6˝) with six replicates. Seedlings were inoculated with F. equiseti conidial suspension (104 conidia ml-1 for 8 minutes) by the root dip method (Hanson, 2006). Mock inoculated plants were dipped in sterile water. Inoculated and control plants were placed in the greenhouse at 25 ± 2°C, and 75 to 85% relative humidity. One week later, inoculated seedlings showed root tip tissue discoloration similar to those observed in the field and non-inoculated seedlings were symptomless. This study was repeated. The fungus was re-isolated from diseased roots and confirmed to be F. equiseti based on morphological characters. Fusarium equiseti was reported on freshly harvested and stored beet in Europe but was not found to be pathogenic (Christ et al. 2011). Strausbaugh and Gillen (2009) reported the association of F. equiseti and root rot of sugar beet but did not report pathogenicity. This pathogen is reported in several crops including edible beans that is grown in rotation with sugar beet in several production areas (Jacobs et al. 2018). The most important Fusarium species reported to cause significant economic damage to sugar beet include F. oxysporum and F. secorum (Secor et al. 2014, Webb et. al. 2012). The presence of another pathogenic Fusarium species in sugar beet will require monitoring to determine how widespread it is and whether current commercial cultivars are resistant. To our knowledge, this is the first report of F. equiseti causing disease on sugar beet seedlings in Minnesota, USA.

Molecular Mapping of Loci Conferring Susceptibility to Spot Blotch and Resistance to Powdery Mildew in Barley Using the Sequencing-Based Genotyping Approach.

Spot blotch (SB) caused by Bipolaris sorokiniana and powdery mildew (PM) caused by Blumeria graminis f. sp. hordei are two important diseases of barley. To map genetic loci controlling susceptibility and resistance to these diseases, a mapping population consisting of 138 recombinant inbred lines (RILs) was developed from the cross between Bowman and ND5883. A genetic map was constructed for the population with 852 unique single nucleotide polymorphism markers generated by sequencing-based genotyping. Bowman and ND5883 showed distinct infection responses at the seedling stage to two isolates (ND90Pr and ND85F) of Bipolaris sorokiniana and one isolate (Race I) of Blumeria graminis f. sp. hordei. Genetic analysis of the RILs revealed that one major gene (Scs6) controls susceptibility to Bipolaris sorokiniana isolate ND90Pr, and another major gene (Mla8) confers resistance to Blumeria graminis f. sp. hordei isolate Race I, respectively. Scs6 was mapped on chromosome 1H of Bowman, as previously reported. Mla8 was also mapped to the short arm of 1H, which was tightly linked but not allelic to the Rcs6/Scs6 locus. Quantitative trait locus (QTL) analysis identified two QTLs, QSbs-1H-P1 and QSbs-7H-P1, responsible for susceptibility to spot blotch caused by Bipolaris sorokiniana isolate ND85F in ND5883, which are located on chromosome 1H and 7H, respectively. QSbs-7H-P1 was mapped to the same region as Rcs5, whereas QSbs-1H-P1 may represent a novel allele conferring seedling stage susceptibility to isolate ND85F. Identification and molecular mapping of the loci for SB susceptibility and PM resistance will facilitate development of barley cultivars with resistance to the diseases.

Fine mapping of a dominant gene conferring resistance to spot blotch caused by a new pathotype of Bipolaris sorokiniana in barley.

KEY MESSAGE: We fine-mapped and physically anchored a dominant gene (Rbs7) conferring resistance to spot blotch caused by a new pathotype of Bipolaris sorokiniana in a genomic interval of 304 kb on barley chromosome 6H. Spot blotch, caused by Bipolaris sorokiniana, is an economically important disease on barley in the Upper Midwest region of the USA and Prairie Provinces of Canada. A new pathotype (pathotype 7, represented by isolate ND4008) of B. sorokiniana has been identified, which is highly virulent on barley cultivars with resistance to other pathotypes of the fungus. In this study, we fine-mapped a dominant gene conferring resistance to pathotype 7 in the barley line PI 235186. Genetic analysis of the F1 and F2 plants from a cross between PI 356741 (highly susceptible to ND4008) and PI 235186 (highly resistant to ND4008) indicated that a single dominant gene (Rbs7) controls the resistance in PI 235186. This result was confirmed by genetic analysis of the F2:3 families and a recombinant inbred line (RIL) population derived from the same cross. Bulked segregant analysis using simple sequence repeat markers localized Rbs7 on the short arm of chromosome 6H. Additional DNA markers were developed from the 6H pseudomolecule sequence of barley cv. Morex and mapped to the genomic region carrying Rbs7 using the RIL population and F2 recombinants derived from the PI 356741 × PI 235186 cross. Rbs7 was fine-mapped between two markers (M13.06 and M13.37), which spans a physical distance of 304 kb on Morex chromosome 6H. These results provide a foundation for future cloning of the resistance gene and development of user-friendly molecular markers that can be used for development of spot-blotch-resistant cultivars in barley breeding programs.

Molecular mapping of QTL for Fusarium head blight resistance introgressed into durum wheat.

KEY MESSAGE: The major QTL for FHB resistance from hexaploid wheat line PI 277012 was successfully introgressed into durum wheat and minor FHB resistance QTL were detected in local durum wheat cultivars. A combination of these QTL will enhance FHB resistance of durum wheat. Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of durum wheat. To combat the disease, great efforts have been devoted to introgress FHB resistance from its related tetraploid and hexaploid wheat species into adapted durum cultivars. However, most of the quantitative trait loci (QTL) for FHB resistance existing in the introgression lines are not well characterized or validated. In this study, we aimed to identify and map FHB resistance QTL in a population consisting of 205 recombinant inbred lines from the cross between Joppa (a durum wheat cultivar) and 10Ae564 (a durum wheat introgression line with FHB resistance derived from the hexaploid wheat line PI 277012). One QTL (Qfhb.ndwp-2A) from Joppa and two QTL (Qfhb.ndwp-5A and Qfhb.ndwp-7A) from 10Ae564 were identified through phenotyping of the mapping population for FHB severity and DON content in greenhouse and field and genotyping with 90K wheat Infinium iSelect SNP arrays. Qfhb.ndwp-2A explained 14, 15, and 9% of the phenotypic variation, respectively, for FHB severity in two greenhouse experiments and for mean DON content across the two greenhouse environments. Qfhb.ndwp-5A explained 19, 10, and 7% of phenotypic variation, respectively, for FHB severity in one greenhouse experiment, mean FHB severity across two field experiments, and mean DON content across the two greenhouse experiments. Qfhb.ndwp-7A was only detected for FHB severity in the two greenhouse experiments, explaining 9 and 11% of the phenotypic variation, respectively. This study confirms the existence of minor QTL in North Dakota durum cultivars and the successful transfer of the major QTL from PI 277012 into durum wheat.

The gene conferring susceptibility to spot blotch caused by Cochliobolus sativus is located at the Mla locus in barley cultivar Bowman.

KEY MESSAGE: We identified, fine mapped, and physically anchored a dominant spot blotch susceptibility gene Scs6 to a 125 kb genomic region containing the Mla locus on barley chromosome 1H. Spot blotch caused by Cochliobolus sativus is an important disease of barley, but the molecular mechanisms underlying resistance and susceptibility to the disease are not well understood. In this study, we identified and mapped a gene conferring susceptibility to spot blotch caused by the pathotype 2 isolate (ND90Pr) of C. sativus in barley cultivar Bowman. Genetic analysis of F1 and F2 progeny as well as F3 families from a cross between Bowman and ND 5883 indicated that a single dominant gene (designated as Scs6) conferred spot blotch susceptibility in Bowman. Using a doubled haploid (DH) population derived from a cross between Calicuchima-sib (resistant) and Bowman-BC (susceptible), we confirmed that Scs6, contributed by Bowman-BC, was localized at the same locus as the previously identified spot blotch resistance allele Rcs6, which was contributed by Calicuchima-sib and mapped on the short arm of chromosome 1H. Using a genome-wide putative linear gene index of barley (Genome Zipper), 13 cleaved amplified polymorphism markers were developed from 11 flcDNA and two EST sequences and mapped to the Scs6/Rcs6 region on a linkage map constructed with the DH population. Further fine mapping with markers developed from barley genome sequences and F2 recombinants derived from Bowman × ND 5883 and Bowman × ND B112 crosses delimited Scs6 in a 125 kb genomic interval harboring the Mla locus on the reference genome of barley cv. Morex. This study provides a foundational step for further cloning of Scs6 using a map-based approach.

Molecular Mapping of Fusarium Head Blight Resistance in the Spring Wheat Line ND2710.

ND2710 is a hard red spring wheat line with a very high level of resistance to Fusarium head blight (FHB). It was selected from the progeny of a cross between ND2603 (an advanced breeding line derived from the Sumai 3/Wheaton cross) and Grandin (a spring wheat cultivar). The FHB resistance of ND2710 is presumably derived from Sumai 3 because the other parents (Grandin and Wheaton) are very susceptible to FHB. To identify and map the quantitative trait loci (QTL) for FHB resistance in ND2710, we developed a mapping population consisting of 233 recombinant inbred lines (RILs) from the cross between ND2710 and the spring wheat cultivar Bobwhite. These RILs along with their parents and checks were evaluated for reactions to FHB in three greenhouse experiments and one field experiment during 2013 to 2014. The population was also genotyped with the wheat 90K iSelect single-nucleotide polymorphism (SNP) assay, and a genetic linkage map was developed with 1,373 non-cosegregating SNP markers, which were distributed on all 21 wheat chromosomes spanning 914.98 centimorgans of genetic distance. Genetic analyses using both phenotypic and genotypic data identified one major QTL (Qfhb.ndwp-3B) on the short arm of chromosome 3B, and three minor QTL (Qfhb.ndwp-6B, Qfhb.ndwp-2A, and Qfhb.ndwp-6A) on 6B, 2A, and 6A, respectively. The major QTL on 3B was detected in all experiments and explained 5 to 20% of the phenotypic variation, while the three minor QTL on 6B, 2A, and 6A explained 5 to 12% phenotypic variation in at least two experiments, except for Qfhb.ndwp-2A, which was only detected in the field experiment. Qfhb.ndwp-3B and Qfhb.ndwp-6B were mapped to the genomic regions containing Fhb1 and Fhb2, respectively, confirming that they originated from Sumai 3. The additive effect of the major and minor QTL may contribute to the high level of FHB resistance in ND2710. The SNP markers closely linked to the FHB resistance QTL will be useful for marker-assisted selection of FHB resistance in wheat breeding programs.

Toward a better understanding of the genomic region harboring Fusarium head blight resistance QTL Qfhs.ndsu-3AS in durum wheat.

KEY MESSAGE: New molecular markers were developed and mapped to the FHB resistance QTL region in high resolution. Micro-collinearity of the QTL region with rice and Brachypodium was revealed for a better understanding of the genomic region. The wild emmer wheat (Triticum dicoccoides)-derived Fusarium head blight (FHB) resistance quantitative trait locus (QTL) Qfhs.ndsu-3AS previously mapped to the short arm of chromosome 3A (3AS) in a population of recombinant inbred chromosome lines (RICLs). This study aimed to attain a better understanding of the genomic region harboring Qfhs.ndsu-3AS and to improve the utility of the QTL in wheat breeding. Micro-collinearity of the QTL region with rice chromosome 1 and Brachypodium chromosome 2 was identified and used for marker development in saturation mapping. A total of 42 new EST-derived sequence tagged site (STS) and simple sequence repeat (SSR) markers were developed and mapped to the QTL and nearby regions on 3AS. Further comparative analysis revealed a complex collinearity of the 3AS genomic region with their collinear counterparts of rice and Brachypodium. Fine mapping of the QTL region resolved five co-segregating markers (Xwgc1186/Xwgc716/Xwgc1143/Xwgc501/Xwgc1204) into three distinct loci proximal to Xgwm2, a marker previously reported to be closely linked to the QTL. Four other markers (Xwgc1226, Xwgc510, Xwgc1296, and Xwgc1301) mapped farther proximal to the above markers in the QTL region with a higher resolution. Five homozygous recombinants with shortened T. dicoccoides chromosomal segments in the QTL region were recovered by molecular marker analysis and evaluated for FHB resistance. Qfhs.ndsu-3AS was positioned to a 5.2 cM interval flanked by the marker Xwgc501 and Xwgc510. The recombinants containing Qfhs.ndsu-3AS and new markers defining the QTL will facilitate utilization of this resistance source in wheat breeding.

Comparative genome structure, secondary metabolite, and effector coding capacity across Cochliobolus pathogens.

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP-encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.