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Key Message: Using associative transcriptomics, our study identifies genes conferring resistance to four diverse fungal pathogens in crops, emphasizing key genetic determinants of multi-pathogen resistance. Crops are affected by several pathogens, but these are rarely studied in parallel to identify common and unique genetic factors controlling diseases. Broad-spectrum quantitative disease resistance (QDR) is desirable for crop breeding as it confers resistance to several pathogen species. Here, we use associative transcriptomics (AT) to identify candidate gene loci associated with Brassica napus constitutive QDR to four contrasting fungal pathogens: Alternaria brassicicola, Botrytis cinerea, Pyrenopeziza brassicae, and Verticillium longisporum. We did not identify any shared loci associated with broad-spectrum QDR to fungal pathogens with contrasting lifestyles. Instead, we observed QDR dependent on the lifestyle of the pathogen-hemibiotrophic and necrotrophic pathogens had distinct QDR responses and associated loci, including some loci associated with early immunity. Furthermore, we identify a genomic deletion associated with resistance to V. longisporum and potentially broad-spectrum QDR. This is the first time AT has been used for several pathosystems simultaneously to identify host genetic loci involved in broad-spectrum QDR. We highlight constitutive expressed candidate loci for broad-spectrum QDR with no antagonistic effects on susceptibility to the other pathogens studies as candidates for crop breeding. In conclusion, this study represents an advancement in our understanding of broad-spectrum QDR in B. napus and is a significant resource for the scientific community., (© 2024. The Author(s).)

Key Message: Stripe rust resistance gene YrAet672 from Aegilops tauschii accession CPI110672 encodes a nucleotide-binding and leucine-rich repeat domain containing protein similar to YrAS2388 and both these members were haplotypes of Yr28. New sources of host resistance are required to counter the continued emergence of new pathotypes of the wheat stripe rust pathogen Puccinia striiformis Westend. f. sp. tritici Erikss. (Pst). Here, we show that CPI110672, an Aegilops tauschii accession from Turkmenistan, carries a single Pst resistance gene, YrAet672, that is effective against multiple Pst pathotypes, including the four predominant Pst lineages present in Australia. The YRAet672 locus was fine mapped to the short arm of chromosome 4D, and a nucleotide-binding and leucine-rich repeat gene was identified at the locus. A transgene encoding the YrAet672 genomic sequence, but lacking a copy of a duplicated sequence present in the 3' UTR, was transformed into wheat cultivar Fielder and Avocet S. This transgene conferred a weak resistance response, suggesting that the duplicated 3' UTR region was essential for function. Subsequent analyses demonstrated that YrAet672 is the same as two other Pst resistance genes described in Ae. tauschii, namely YrAS2388 and Yr28. They were identified as haplotypes encoding identical protein sequences but are polymorphic in non-translated regions of the gene. Suppression of resistance conferred by YrAet672 and Yr28 in synthetic hexaploid wheat lines (AABBDD) involving Langdon (AABB) as the tetraploid parent was associated with a reduction in transcript accumulation., (© 2022. Crown.)

Key Message: The first cytological characterization of the 2N v S segment in hexaploid wheat; complete de novo assembly and annotation of 2N v S segment; 2N v S frequency is increasing 2N v S and is associated with higher yield. The Aegilops ventricosa 2N v S translocation segment has been utilized in breeding disease-resistant wheat crops since the early 1990s. This segment is known to possess several important resistance genes against multiple wheat diseases including root knot nematode, stripe rust, leaf rust and stem rust. More recently, this segment has been associated with resistance to wheat blast, an emerging and devastating wheat disease in South America and Asia. To date, full characterization of the segment including its size, gene content and its association with grain yield is lacking. Here, we present a complete cytological and physical characterization of this agronomically important translocation in bread wheat. We de novo assembled the 2N v S segment in two wheat varieties, 'Jagger' and 'CDC Stanley,' and delineated the segment to be approximately 33 Mb. A total of 535 high-confidence genes were annotated within the 2N v S region, with > 10% belonging to the nucleotide-binding leucine-rich repeat (NLR) gene families. Identification of groups of NLR genes that are potentially N genome-specific and expressed in specific tissues can fast-track testing of candidate genes playing roles in various disease resistances. We also show the increasing frequency of 2N v S among spring and winter wheat breeding programs over two and a half decades, and the positive impact of 2N v S on wheat grain yield based on historical datasets. The significance of the 2N v S segment in wheat breeding due to resistance to multiple diseases and a positive impact on yield highlights the importance of understanding and characterizing the wheat pan-genome for better insights into molecular breeding for wheat improvement.

Key Message: Novel rust resistance genes LrP and YrP from Ae. peregrina identified on chromosome 5D and the linked markers will aid deployment of these genes in combination with other major/minor genes. Aegilops peregrina, a wild tetraploid relative of wheat with genome constitution UUSS, displays genetic variation for resistance to leaf and stripe (yellow) rust. The wheat Ae. peregrina introgression line, IL pau16058, harbouring leaf and stripe rust resistance, was crossed with wheat cv. WL711 to generate an F 2:3 mapping population. Inheritance studies on this population indicated the transfer of dominant co-segregating resistance to leaf and stripe rust. Ethyl methane sulphonate mutagenesis of IL pau16058 identified independent loss-of-function mutants for leaf and stripe rust resistance, indicating that the leaf and stripe rust resistance is controlled by independent genes, herein designated LrP and YrP, respectively. A high-resolution genetic map of LrP and YrP was constructed using the Illumina Infinium iSelect 90K wheat array and resistance gene enrichment sequencing (RenSeq) markers. The map spans 4.19 cM on the distal-most region of the short arm of chromosome 5D, consisting of eight SNP markers and one microsatellite marker. LrP and YrP co-segregated with markers BS00163889 and 5DS44573_snp and was flanked distally by the SNP marker BS00129707 and proximally by 5DS149010, defining a 15.71 Mb region in the RefSeq v1.0 genome assembly.

Key Message: We identified two novel wheat stem rust resistance genes, Sr-1644-1Sh and Sr-1644-5Sh in Aegilops sharonensis that are effective against widely virulent African races of the wheat stem rust pathogen. Stem rust is one of the most important diseases of wheat in the world. When single stem rust resistance (Sr) genes are deployed in wheat, they are often rapidly overcome by the pathogen. To this end, we initiated a search for novel sources of resistance in diverse wheat relatives and identified the wild goatgrass species Aegilops sharonesis (Sharon goatgrass) as a rich reservoir of resistance to wheat stem rust. The objectives of this study were to discover and map novel Sr genes in Ae. sharonensis and to explore the possibility of identifying new Sr genes by genome-wide association study (GWAS). We developed two biparental populations between resistant and susceptible accessions of Ae. sharonensis and performed QTL and linkage analysis. In an F 6 recombinant inbred line and an F 2 population, two genes were identified that mapped to the short arm of chromosome 1S sh , designated as Sr-1644-1Sh, and the long arm of chromosome 5S sh , designated as Sr-1644-5Sh. The gene Sr-1644-1Sh confers a high level of resistance to race TTKSK (a member of the Ug99 race group), while the gene Sr-1644-5Sh conditions strong resistance to TRTTF, another widely virulent race found in Yemen. Additionally, GWAS was conducted on 125 diverse Ae. sharonensis accessions for stem rust resistance. The gene Sr-1644-1Sh was detected by GWAS, while Sr-1644-5Sh was not detected, indicating that the effectiveness of GWAS might be affected by marker density, population structure, low allele frequency and other factors.

Frost resistance-H2 (Fr-H2) is a major QTL affecting freezing tolerance in barley, yet its molecular basis is still not clearly understood. To gain a better insight into the structural characterization of the locus, a high-resolution linkage map developed from the Nure × Tremois cross was initially implemented to map 13 loci which divided the 0.602 cM total genetic distance into ten recombination segments. A PCR-based screening was then applied to identify positive bacterial artificial chromosome (BAC) clones from two genomic libraries of the reference genotype Morex. Twenty-six overlapping BACs from the integrated physical-genetic map were 454 sequenced. Reads assembled in contigs were subsequently ordered, aligned and manually curated in 42 scaffolds. In a total of 1.47 Mbp, 58 protein-coding sequences were identified, 33 of which classified according to similarity with sequences in public databases. As three complete barley C-repeat Binding Factors (HvCBF) genes were newly identified, the locus contained13 full-length HvCBFs, four Related to AP2 Triticeae (RAPT) genes, and at least five CBF pseudogenes. The final overall assembly of Fr-H2 includes more than 90 % of target region: all genes were identified along the locus, and a general survey of Repetitive Elements obtained. We believe that this gold-standard sequence for the Morex Fr-H2 will be a useful genomic tool for structural and evolutionary comparisons with Fr-H2 in winter-hardy cultivars along with Fr-2 of other Triticeae crops.

Gene order is largely collinear in the small-grained cereals, a feature which has proved helpful in both marker development and positional cloning. The accuracy of a virtual gene order map ("genome zipper") for barley (Hordeum vulgare), developed by combining a genetic map of this species with a large number of gene locations obtained from the maps constructed in other grass species, was evaluated here both at the genome-wide level and at the fine scale in a representative segment of the genome. Comparing the whole genome "genome zipper" maps with a genetic map developed by using transcript-derived markers, yielded an accuracy of >94 %. The fine-scale comparison involved a 14 cM segment of chromosome arm 2HL. One hundred twenty-eight genes of the "genome zipper" interval were analysed. Over 95 % (45/47) of the polymorphic markers were genetically mapped and allocated to the expected region of 2HL, following the predicted order. A further 80 of the 128 genes were assigned to the correct chromosome arm 2HL by analysis of wheat-barley addition lines. All 128 gene-based markers developed were used to probe a barley bacterial artificial chromosome (BAC) library, delivering 26 BAC contigs from which all except two were anchored to the targeted zipper interval. The results demonstrate that the gene order predicted by the "genome zipper" is remarkably accurate and that the "genome zipper" represents a highly efficient informational resource for the systematic identification of gene-based markers and subsequent physical map anchoring of the barley genome.

The barley Rdg2a locus confers resistance to the leaf stripe pathogen Pyrenophora graminea and, in the barley genotype Thibaut, it is composed of a gene family with three highly similar paralogs. Only one member of the gene family (called as Rdg2a) encoding for a CC-NB-LRR protein is able to confer resistance to the leaf stripe isolate Dg2. To study the genome evolution and diversity at the Rdg2a locus, sequences spanning the Rdg2a gene were compared in two barley cultivars, Thibaut and Morex, respectively, resistant and susceptible to leaf stripe. An overall high level of sequence conservation interrupted by several rearrangements that included three main deletions was observed in the Morex contig. The main deletion of 13,692 bp was most likely derived from unequal crossing over between Rdg2a paralogs leading to the generation of a chimeric Morex rdg2a gene which was not associated to detectable level of resistance toward leaf stripe. PCR-based analyses of genic and intergenic regions at the Rdg2a locus in 29 H. vulgare lines and one H. vulgare ssp. spontaneum accession indicated large haplotype variability in the cultivated barley gene pool suggesting rapid and recent divergence at this locus. Barley genotypes showing the same haplotype as Thibaut at the Rdg2a locus were selected for a Rdg2a allele mining through allele re-sequencing and two lines with polymorphic nucleotides leading to amino acid changes in the CC-NB and LRR encoding domains, respectively, were identified. Analysis of nucleotide diversity of the Rdg2a alleles revealed that the polymorphic sites were subjected to positive selection. Moreover, strong positively selected sites were located in the LRR encoding domain suggesting that both positive selection and divergence at homologous loci are possibly representing the molecular mechanism for the generation of high diversity at the Rdg2a locus in the barley gene pool.

Soil-borne barley yellow mosaic virus disease, caused by different strains of Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV), is one of the most important diseases of winter barley (Hordeum vulgare L.) in Europe and East Asia. The recessive resistance gene rym11 located in the centromeric region of chromosome 4HL is effective against all so far known strains of BaMMV and BaYMV in Germany. In order to isolate this gene, a high-resolution mapping population (10,204 meiotic events) has been constructed. F2 plants were screened with co-dominant flanking markers and segmental recombinant inbred lines (RILs) were tested for resistance to BaMMV under growth chamber and field conditions. Tightly linked markers were developed by exploiting (1) publicly available barley EST sequences, (2) employing barley synteny to rice, Brachypodium distachyon and sorghum and (3) using next-generation sequencing data of barley. Using this approach, the genetic interval was efficiently narrowed down from the initial 10.72 % recombination to 0.074 % recombination. A marker co-segregating with rym11 was developed providing the basis for gene isolation and efficient marker-assisted selection.

The increasing amount of available expressed gene sequence data makes whole-transcriptome analysis of certain crop species possible. Potato currently has the second largest number of publicly available expressed sequence tag (EST) sequences among the Solanaceae. Most of these ESTs, plus other proprietary sequences, were combined and used to generate a unigene assembly. The set of 246,182 sequences produced 46,345 unigenes, which were used to design a 44K 60-mer oligo array (Potato Oligo Chip Initiative: POCI). In this study, we attempt to identify genes controlling and driving the process of tuber initiation and growth by implementing large-scale transcriptional changes using the newly developed POCI array. Major gene expression profiles could be identified exhibiting differential expression at key developmental stages. These profiles were associated with functional roles in cell division and growth. A subset of genes involved in the regulation of the cell cycle, based on their Gene Ontology classification, exhibit a clear transient upregulation at tuber onset indicating increased cell division during these stages. The POCI array allows the study of potato gene expression on a much broader level than previously possible and will greatly enhance analysis of transcriptional control mechanisms in a wide range of potato research areas. POCI sequence and annotation data are publicly available through the POCI database ( http://pgrc.ipk-gatersleben.de/poci ).