Identification of quantitative trait loci associated with leaf rust resistance in rye by precision mapping.

BACKGROUND: Leaf rust (LR) is among the most destructive fungal diseases of rye (Secale cereale L.). Despite intensive research using various analytical and methodological approaches, such as quantitative trait locus (QTL) mapping, candidate gene expression analysis, and transcriptome sequencing, the genetic basis of the rye immune response to LR remains unclear. RESULTS: A genome-wide association study was employed to detect QTLs controlling the immune response to LR of rye. A mapping population, G38A, was constructed by crossing two inbred lines: 723 (susceptible to LR) and JKI-NIL-Pr3 (a donor of the LR resistance gene Pr3). For genotyping, SNP-DArT and silico-DArT markers were used. Resistance phenotyping was conducted by visual assessment of the infection severity in detached leaf segments inoculated with two isolates of Puccinia recondita f. sp. secalis, namely, 60/17/2.1 (isolate S) in the main experiment and 86/n/2.1_5x (isolate N) in the validation experiment, at 10 and 17 days post-infection (dpi), respectively. In total, 42,773 SNP-DArT and 105,866 silico-DArT markers were included in the main analysis including isolate S, of which 129 and 140 SNP-DArTs and 767 and 776 silico-DArTs were significantly associated (p ≤ 0.001; - log10(p) ≥ 3.0) with the immune response to LR at 10 and 17 dpi, respectively. Most significant markers were mapped to chromosome 1R. The number of common markers from both systems and at both time points occupying common chromosomal positions was 37, of which 21 were positioned in genes, comprising 18 markers located in exons and three in introns. This gene pool included genes encoding proteins with a known function in response to LR (e.g., a NBS-LRR disease resistance protein-like protein and carboxyl-terminal peptidase). CONCLUSION: This study has expanded and supplemented existing knowledge of the genetic basis of rye resistance to LR by (1) detecting two QTLs associated with the LR immune response of rye, of which one located on the long arm of chromosome 1R is newly detected, (2) assigning hundreds of markers significantly associated with the immune response to LR to genes in the 'Lo7' genome, and (3) predicting the potential translational effects of polymorphisms of SNP-DArT markers located within protein-coding genes.

Identification and characteristics of wheat Lr orthologs in three rye inbred lines.

The genetic background of the immune response of rye to leaf rust (LR), although extensively studied, is still not well understood. The recent publication of the genome of rye line Lo7 and the development of efficient transcriptomic methods has aided the search for genes that confer resistance to this disease. In this study, we investigated the potential role of rye orthologs of wheat Lr genes (Lr1, Lr10, Lr21, Lr22a, and RGA2/T10rga2-1A) in the LR seedling-stage resistance of inbred rye lines D33, D39, and L318. Bioinformatics analysis uncovered numerous Lr orthologs in the Lo7 genome, namely, 14 ScLr1, 15 ScRga2, and 2 ScLr21 paralogs, and 1 each of ScLr10 and ScLr22a genes. The paralogs of ScLr1, ScRga2, and ScLr21 were structurally different from one another and their wheat counterparts. According to an RNA sequencing analysis, only four wheat Lr gene orthologs identified in the Lo7 genome (ScLr1_3, ScLr1_4, ScLr1_8, and ScRga2_6) were differentially expressed; all four were downregulated after infection with compatible or incompatible isolates of Puccinia recondita f. sp. secalis (Prs). Using a more precise tool, RT-qPCR, we found that two genes were upregulated at 20 h post-infection, namely, ScLr1_4 and ScLr1_8 in lines D33 and D39, respectively, both of which have been found to be resistant to LR under field conditions and after treatment with a semi-compatible Prs strain. We were unable to discern any universal pattern of gene expression after Prs infection; on the contrary, all detected relationships were plant genotype-, Prs isolate-, or time-specific. Nevertheless, at least some Lr orthologs in rye (namely, ScLr1_3 ScLr1_4, ScLr1_8, and ScRga2_6), even though mainly downregulated, may play an important role in the response of rye to LR.

How Machine Learning Methods Helped Find Putative Rye Wax Genes Among GBS Data.

The standard approach to genetic mapping was supplemented by machine learning (ML) to establish the location of the rye gene associated with epicuticular wax formation (glaucous phenotype). Over 180 plants of the biparental F2 population were genotyped with the DArTseq (sequencing-based diversity array technology). A maximum likelihood (MLH) algorithm (JoinMap 5.0) and three ML algorithms: logistic regression (LR), random forest and extreme gradient boosted trees (XGBoost), were used to select markers closely linked to the gene encoding wax layer. The allele conditioning the nonglaucous appearance of plants, derived from the cultivar Karlikovaja Zelenostebelnaja, was mapped at the chromosome 2R, which is the first report on this localization. The DNA sequence of DArT-Silico 3585843, closely linked to wax segregation detected by using ML methods, was indicated as one of the candidates controlling the studied trait. The putative gene encodes the ABCG11 transporter.

Changes in benzoxazinoid contents and the expression of the associated genes in rye (Secale cereale L.) due to brown rust and the inoculation procedure.

Benzoxazinoids (BXs) are secondary metabolites with diverse functions, but are primarily involved in protecting plants, mainly from the family Poaceae, against insects and fungal pathogens. Rye is a cereal crop that is highly resistant to biotic stresses. However, its susceptibility to brown rust caused by Puccinia recondita f. sp. secalis (Prs) is still a major problem affecting its commercial production. Additionally, the genetic and metabolic factors related to this disease remain poorly characterized. In this study, we investigated whether and to what extent the brown rust infection and the inoculation procedure affect the contents of specific BXs (HBOA, GDIBOA, DIBOA, GDIMBOA, DIMBOA, and MBOA) and the expression of genes related to BX (ScBx1-5, ScIgl, and Scglu). We revealed that treatments with water and a urediniospore suspension usually downregulate gene expression levels. Moreover, HBOA and DIBOA contents decreased, whereas the contents of the remaining metabolites increased. Specifically, the MBOA content increased more after the mock treatment than after the Prs treatment, whereas the increase in GDIBOA and GDIMBOA levels was usually due to the Prs infection, especially at two of the most critical time-points, 17 and 24 h post-treatment. Therefore, GDIBOA and GDIMBOA are glucosides that are important components of rye defence responses to brown rust. Furthermore, along with MBOA, they protect rye against the stress associated with the inoculation procedure used in this study.

Identification and mapping of a new recessive dwarfing gene dw9 on the 6RL rye chromosome and its phenotypic effects.

The introduction of high-yielding semi-dwarf varieties of wheat into cultivation has led to a "green revolution." This has required intensive research into various sources of dwarfism in wheat. However, there has been very little advancement in research on dwarfing genes in rye in comparison to wheat or barley. So far, three dominant dwarfing genes (Ddw1, Ddw3, and Ddw4) and three recessive genes (ct1, ct2, and np) have been characterized and precisely mapped in rye. There is no complete catalog of dwarfing genes available in rye. This paper presents an identification of the source of dwarfism and preliminary characterization of the new recessive gene dw9 from the BK-1 line. The gene was mapped on the long arm of the 6R chromosome and belongs to the GA-insensitive group. The initial characterization of the influence of this gene on morphological traits shows that it significantly affects the decrease of yielding trait parameters. A full evaluation can be performed after detailed breeding studies.

Effective BAC clone anchoring with genotyping-by-sequencing and Diversity Arrays Technology in a large genome cereal rye.

Identification of bacterial artificial chromosome (BAC) clones containing specific sequences is a prerequisite for many applications, such as physical map anchoring or gene cloning. Existing BAC library screening strategies are either low-throughput or require a considerable initial input of resources for platform establishment. We describe a high-throughput, reliable, and cost-effective BAC library screening approach deploying genotyping platforms which are independent from the availability of sequence information: a genotyping-by-sequencing (GBS) method DArTSeq and the microarray-based Diversity Arrays Technology (DArT). The performance of these methods was tested in a very large and complex rye genome. The DArTseq approach delivered superior results: a several fold higher efficiency of addressing genetic markers to BAC clones and anchoring of BAC clones to genetic map and also a higher reliability. Considering the sequence independence of the platform, the DArTseq-based library screening can be proposed as an attractive method to speed up genomics research in resource poor species.