Identification of QTLs conferring resistance to scald (Rhynchosporium commune) in the barley nested association mapping population HEB-25.

BACKGROUND: Barley scald, caused by the fungus Rhynchosporium commune, is distributed worldwide to all barley growing areas especially in cool and humid climates. Scald is an economically important leaf disease resulting in yield losses of up to 40%. To breed resistant cultivars the identification of quantitative trait loci (QTLs) conferring resistance to scald is necessary. Introgressing promising resistance alleles of wild barley is a way to broaden the genetic basis of scald resistance in cultivated barley. Here, we apply nested association mapping (NAM) to map resistance QTLs in the barley NAM population HEB-25, comprising 1420 lines in BC1S3 generation, derived from crosses of 25 wild barley accessions with cv. Barke. RESULTS: In scald infection trials in the greenhouse variability of resistance across and within HEB-25 families was found. NAM based on 33,005 informative SNPs resulted in the identification of eight reliable QTLs for resistance against scald with most wild alleles increasing resistance as compared to cv. Barke. Three of them are located in the region of known resistance genes and two in the regions of QTLs, respectively. The most promising wild allele was found at Rrs17 in one specific wild donor. Also, novel QTLs with beneficial wild allele effects on scald resistance were detected. CONCLUSIONS: To sum up, wild barley represents a rich resource for scald resistance. As the QTLs were linked to the physical map the identified candidate genes will facilitate cloning of the scald resistance genes. The closely linked flanking molecular markers can be used for marker-assisted selection of the respective resistance genes to integrate them in elite cultivars.

Characterisation of barley resistance to rhynchosporium on chromosome 6HS.

KEY MESSAGE: Major resistance gene to rhynchosporium, Rrs18, maps close to the telomere on the short arm of chromosome 6H in barley. Rhynchosporium or barley scald caused by a fungal pathogen Rhynchosporium commune is one of the most destructive and economically important diseases of barley in the world. Testing of Steptoe × Morex and CIho 3515 × Alexis doubled haploid populations has revealed a large effect QTL for resistance to R. commune close to the telomere on the short arm of chromosome 6H, present in both populations. Mapping markers flanking the QTL from both populations onto the 2017 Morex genome assembly revealed a rhynchosporium resistance locus independent of Rrs13 that we named Rrs18. The causal gene was fine mapped to an interval of 660 Kb using Steptoe × Morex backcross 1 S2 and S3 lines with molecular markers developed from Steptoe exome capture variant calling. Sequencing RNA from CIho 3515 and Alexis revealed that only 4 genes within the Rrs18 interval were transcribed in leaf tissue with a serine/threonine protein kinase being the most likely candidate for Rrs18.

Resistance to Rhynchosporium commune in a collection of European spring barley germplasm.

KEY MESSAGE: Association analyses of resistance to Rhynchosporium commune in a collection of European spring barley germplasm detected 17 significant resistance quantitative trait loci. The most significant association was confirmed as Rrs1. Rhynchosporium commune is a fungal pathogen of barley which causes a highly destructive and economically important disease known as rhynchosporium. Genome-wide association mapping was used to investigate the genetic control of host resistance to R. commune in a collection of predominantly European spring barley accessions. Multi-year disease nursery field trials revealed 8 significant resistance quantitative trait loci (QTL), whilst a separate association mapping analysis using historical data from UK national and recommended list trials identified 9 significant associations. The most significant association identified in both current and historical data sources, collocated with the known position of the major resistance gene Rrs1. Seedling assays with R. commune single-spore isolates expressing the corresponding avirulence protein NIP1 confirmed that this locus is Rrs1. These results highlight the significant and continuing contribution of Rrs1 to host resistance in current elite spring barley germplasm. Varietal height was shown to be negatively correlated with disease severity, and a resistance QTL was identified that co-localised with the semi-dwarfing gene sdw1, previously shown to contribute to disease escape. The remaining QTL represent novel resistances that are present within European spring barley accessions. Associated markers to Rrs1 and other resistance loci, identified in this study, represent a set of tools that can be exploited by breeders for the sustainable deployment of varietal resistance in new cultivars.

Fine mapping of the Rrs1 resistance locus against scald in two large populations derived from Spanish barley landraces.

KEY MESSAGE: In two Spanish barley landraces with outstanding resistance to scald, the Rrs1 Rh4 locus was fine mapped including all known markers used in previous studies and closely linked markers were developed. Scald, caused by Rhynchosporium commune, is one of the most prevalent barley diseases worldwide. A search for new resistance sources revealed that Spanish landrace-derived lines SBCC145 and SBCC154 showed outstanding resistance to scald. They were crossed to susceptible cultivar Beatrix to create large DH-mapping populations of 522 and 416 DH lines that were scored for disease resistance in the greenhouse using two R. commune isolates. To ascertain the pattern of resistance, parents and reference barley lines with known scald resistance were phenotyped with a panel of differential R. commune isolates. Subpopulations were genotyped with the Illumina GoldenGate 1,536 SNP Assay and a large QTL in the centromeric region of chromosome 3H, known to harbour several scald resistance genes and/or alleles, was found in both populations. Five SNP markers closest to the QTL were converted into CAPS markers. These CAPS markers, together with informative SSR markers used in other scald studies, confirmed the presence of the Rrs1 locus. The panel of differential scald isolates indicated that the allele carried by both donors was Rrs1 Rh4 . The genetic distance between Rrs1 and its flanking markers was 1.2 cM (11_0010) proximally and 0.9 cM (11_0823) distally, which corresponds to a distance of just below 9 Mbp. The number and nature of scald resistance genes on chromosome 3H are discussed. The effective Rrs1 allele found and the closely linked markers developed are already useful tools for molecular breeding programs and provide a good step towards the identification of candidate genes.

A major QTL for resistance against Fusarium head blight in European winter wheat.

We report on the verification of a resistance quantitative trait locus (QTL) on chromosome 1BL (now designated Qfhs.lfl-1BL) which had been previously identified in the winter wheat cultivar Cansas. For a more precise estimation of the QTL effect and its influence on plant height and heading date lines with a more homogeneous genetic background were created and evaluated in four environments after spray inoculation with Fusarium culmorum. Qfhs.lfl-1BL reduced FHB severity by 42% relative to lines without the resistance allele. This QTL did not influence plant height, but significantly delayed heading date by one day. All of the most resistant genotypes of the verification population carried this major QTL displaying its importance for disease resistance. This resistance QTL has not only been found in the cultivar Cansas, but also in the three European winter wheat cultivars Biscay, History and Pirat. A subsequent meta-analysis confirmed the presence of a single QTL on the long arm of chromosome 1B originating from the four mentioned cultivars. Altogether, the results of the present study indicate that Qfhs.lfl-1BL is an important component of FHB resistance in European winter wheat and support the view that this QTL would be effective and valuable in backcross breeding programmes.

Fine mapping, physical mapping and development of diagnostic markers for the Rrs2 scald resistance gene in barley.

The Rrs2 gene confers resistance to the fungal pathogen Rhynchosporium secalis which causes leaf scald, a major barley disease. The Rrs2 gene was fine mapped to an interval of 0.08 cM between markers 693M6_6 and P1D23R on the distal end of barley chromosome 7HS using an Atlas (resistant) x Steffi (susceptible) mapping population of 9,179 F(2)-plants. The establishment of a physical map of the Rrs2 locus led to the discovery that Rrs2 is located in an area of suppressed recombination within this mapping population. The analysis of 58 barley genotypes revealed a large linkage block at the Rrs2 locus extending over several hundred kb which is present only in Rrs2 carrying cultivars. Due to the lack of recombination in the mapping population and the presence of a Rrs2-specific linkage block, we assume a local chromosomal rearrangement (alien introgression or inversion) in Rrs2 carrying varieties. The variety analysis led to the discovery of eight SNPs which were diagnostic for the Rrs2 phenotype. Based on these SNPs diagnostic molecular markers (CAPS and pyrosequencing markers) were developed which are highly useful for marker-assisted selection in resistance gene pyramiding programmes for Rhynchosporium secalis resistance in barley.

Deciphering the genetic basis for vitamin E accumulation in leaves and grains of different barley accessions.

Tocopherols and tocotrienols, commonly referred to as vitamin E, are essential compounds in food and feed. Due to their lipophilic nature they protect biomembranes by preventing the propagation of lipid-peroxidation especially during oxidative stress. Since their synthesis is restricted to photosynthetic organisms, plant-derived products are the major source of natural vitamin E. In the present study the genetic basis for high vitamin E accumulation in leaves and grains of different barley (Hordeum vulgare L.) accessions was uncovered. A genome wide association study (GWAS) allowed the identification of two genes located on chromosome 7H, homogentisate phytyltransferase (HPT-7H) and homogentisate geranylgeranyltransferase (HGGT) that code for key enzymes controlling the accumulation of tocopherols in leaves and tocotrienols in grains, respectively. Transcript profiling showed a correlation between HPT-7H expression and vitamin E content in leaves. Allele sequencing allowed to decipher the allelic variation of HPT-7H and HGGT genes corresponding to high and low vitamin E contents in the respective tissues. Using the obtained sequence information molecular markers have been developed which can be used to assist smart breeding of high vitamin E barley varieties. This will facilitate the selection of genotypes more tolerant to oxidative stress and producing high-quality grains.

Towards systems genetic analyses in barley: Integration of phenotypic, expression and genotype data into GeneNetwork.

BACKGROUND: A typical genetical genomics experiment results in four separate data sets; genotype, gene expression, higher-order phenotypic data and metadata that describe the protocols, processing and the array platform. Used in concert, these data sets provide the opportunity to perform genetic analysis at a systems level. Their predictive power is largely determined by the gene expression dataset where tens of millions of data points can be generated using currently available mRNA profiling technologies. Such large, multidimensional data sets often have value beyond that extracted during their initial analysis and interpretation, particularly if conducted on widely distributed reference genetic materials. Besides quality and scale, access to the data is of primary importance as accessibility potentially allows the extraction of considerable added value from the same primary dataset by the wider research community. Although the number of genetical genomics experiments in different plant species is rapidly increasing, none to date has been presented in a form that allows quick and efficient on-line testing for possible associations between genes, loci and traits of interest by an entire research community. DESCRIPTION: Using a reference population of 150 recombinant doubled haploid barley lines we generated novel phenotypic, mRNA abundance and SNP-based genotyping data sets, added them to a considerable volume of legacy trait data and entered them into the GeneNetwork http://www.genenetwork.org. GeneNetwork is a unified on-line analytical environment that enables the user to test genetic hypotheses about how component traits, such as mRNA abundance, may interact to condition more complex biological phenotypes (higher-order traits). Here we describe these barley data sets and demonstrate some of the functionalities GeneNetwork provides as an easily accessible and integrated analytical environment for exploring them. CONCLUSION: By integrating barley genotypic, phenotypic and mRNA abundance data sets directly within GeneNetwork's analytical environment we provide simple web access to the data for the research community. In this environment, a combination of correlation analysis and linkage mapping provides the potential to identify and substantiate gene targets for saturation mapping and positional cloning. By integrating datasets from an unsequenced crop plant (barley) in a database that has been designed for an animal model species (mouse) with a well established genome sequence, we prove the importance of the concept and practice of modular development and interoperability of software engineering for biological data sets.

Considering causal genes in the genetic dissection of kernel traits in common wheat.

Genetic factors controlling thousand-kernel weight (TKW) were characterized for their association with other seed traits, including kernel width, kernel length, ratio of kernel width to kernel length (KW/KL), kernel area, and spike number per m2 (SN). For this purpose, a genetic map was established utilizing a doubled haploid population derived from a cross between German winter wheat cultivars Pamier and Format. Association studies in a diversity panel of elite cultivars supplemented genetic analysis of kernel traits. In both populations, genomic signatures of 13 candidate genes for TKW and kernel size were analyzed. Major quantitative trait loci (QTL) for TKW were identified on chromosomes 1B, 2A, 2D, and 4D, and their locations coincided with major QTL for kernel size traits, supporting the common belief that TKW is a function of other kernel traits. The QTL on chromosome 2A was associated with TKW candidate gene TaCwi-A1 and the QTL on chromosome 4D was associated with dwarfing gene Rht-D1. A minor QTL for TKW on chromosome 6B coincided with TaGW2-6B. The QTL for kernel dimensions that did not affect TKW were detected on eight chromosomes. A major QTL for KW/KL located at the distal tip of chromosome arm 5AS is being reported for the first time. TaSus1-7A and TaSAP-A1, closely linked to each other on chromosome 7A, could be related to a minor QTL for KW/KL. Genetic analysis of SN confirmed its negative correlation with TKW in this cross. In the diversity panel, TaSus1-7A was associated with TKW. Compared to the Pamier/Format bi-parental population where TaCwi-A1a was associated with higher TKW, the same allele reduced grain yield in the diversity panel, suggesting opposite effects of TaCwi-A1 on these two traits.

Are PECTIN ESTERASE INHIBITOR Genes Involved in Mediating Resistance to Rhynchosporium commune in Barley?

A family of putative PECTIN ESTERASE INHIBITOR (PEI) genes, which were detected in the genomic region co-segregating with the resistance gene Rrs2 against scald caused by Rhynchosporium commune in barley, were characterized and tested for their possible involvement in mediating resistance to the pathogen by complementation and overexpression analysis. The sequences of the respective genes were derived from two BAC contigs originating from the susceptible cultivar 'Morex'. For the genes HvPEI2, HvPEI3, HvPEI4 and HvPEI6, specific haplotypes for 18 resistant and 23 susceptible cultivars were detected after PCR-amplification and haplotype-specific CAPS-markers were developed. None of the tested candidate genes HvPEI2, HvPEI3 and HvPEI4 alone conferred a high resistance level in transgenic over-expression plants, though an improvement of the resistance level was observed especially with OE-lines for gene HvPEI4. These results do not confirm but also do not exclude an involvement of the PEI gene family in the response to the pathogen. A candidate for the resistance gene Rrs2 could not be identified yet. It is possible that Rrs2 is a PEI gene or another type of gene which has not been detected in the susceptible cultivar 'Morex' or the full resistance reaction requires the presence of several PEI genes.

Pm50: a new powdery mildew resistance gene in common wheat derived from cultivated emmer.

Fungal diseases of wheat, including powdery mildew, cause significant crop, yield and quality losses throughout the world. Knowledge of the genetic basis of powdery mildew resistance will greatly support future efforts to develop and cultivate resistant cultivars. Studies were conducted on cultivated emmer-derived wheat line K2 to identify genes involved in powdery mildew resistance at the seedling and adult plant growth stages using a BC(1) doubled haploid population derived from a cross between K2 and susceptible cultivar Audace. A single gene was located distal to microsatellite marker Xgwm294 on the long arm of chromosome 2A. Quantitative trait loci (QTL) analysis indicated that the gene was also effective at the adult plant stage, explaining up to 79.0 % of the variation in the progeny. Comparison of genetic maps indicated that the resistance gene in K2 was different from Pm4, the only other formally named resistance gene located on chromosome 2AL, and PmHNK54, a gene derived from Chinese germplasm. The new gene was designated Pm50.

Is DNA methylation an epigenetic contribution to transcriptional regulation of the bovine endometrium during the estrous cycle and early pregnancy?

Epigenetic events controlling the transcriptional regulation of genes involved in endometrial function during the estrous cycle and early pregnancy have only sparsely been investigated. We analyzed the gene expression of DNA methyltransferases and the most prominent endocrine transcriptional mediator estrogen receptor alpha (ESR1) in the bovine endometrium of heifers at 0, 12 and 18 days following estrous and at day 18 after insemination. The luminometric methylation assay for the investigation of global DNA methylation and an elegant combination of methylation-sensitive high resolution melting and pyrosequencing for local methylation levels of ESR1 were deployed. In spite of differential gene expression of ESR1 among groups, no differences in endometrial ESR1 DNA methylation during neither estrous cycle nor early pregnancy were determined. Global DNA methylation prevailed at similar low levels in endometrium, likely controlled by the observed moderate DNMT3b expression. Thus, the epigenetic contribution of DNA methylation influencing endometrial function seems rather limited. However, because a control tissue expressing only minute amounts of ESR1 transcripts was locally significantly higher methylated, DNA methylation might contribute to an appropriate tissue-specific expression status underlying further specific control mechanisms of gene transcription.

Inheritance of resistance to Fusarium head blight in three European winter wheat populations.

Fusarium head blight (FHB) resistance is of particular importance in wheat breeding programmes due to the detrimental effects of this fungal disease on human and animal health, yield and grain quality. Segregation for FHB resistance in three European winter wheat populations enabled the identification of resistance loci in well-adapted germplasm. Populations obtained from crosses of resistant cultivars Apache, History and Romanus with susceptible semi-dwarfs Biscay, Rubens and Pirat, respectively, were mapped and analysed to identify quantitative trait loci (QTL) for FHB severity, ear emergence time and plant height. The results of the present study together with previous studies in UK winter wheat indicated that the semi-dwarfing allele Rht-D1b seems to be the major source for FHB susceptibility in European winter wheat. The high resistance level of the cultivars Romanus and History was conditioned by several minor resistance QTL interacting with the environment and the absence of Rht-D1b. In contrast, the semi-dwarf parents contributed resistance alleles of major effects apparently compensating the negative effects of Rht-D1b on FHB reaction. The moderately resistant cultivar Apache contributed a major QTL on chromosome 6A in a genome region previously shown to carry resistance loci to FHB. A total of 18 genomic regions were repeatedly associated with FHB resistance. The results indicate that common resistance-associated genes or genomic regions are present in European winter wheats.

Mapping of extreme resistance to PVY (Ry (sto)) on chromosome XII using anther-culture-derived primary dihaploid potato lines.

The inheritance of extreme resistance to PVY (Ry (sto)) by a single dominant locus was confirmed by obtaining a 1:1 segregation ratio in a virus inoculation test with 28 resistant (Ryry) to 29 susceptible (ryry) anther culture-derived dihaploid lines (2n=2x=24) from cv. "Assia" (2n=4x=48) having extreme resistance derived from Solanum stoloniferum in simplex constitution (Ryryryry). Twelve Ry (sto) markers selected in AFLP assays using bulked segregant analysis were applied to 106 tested potato cultivars from Germany, The Netherlands and Poland and 19 potato cultivars were identified by these markers as extremely resistant to PVY in alignment with phenotypic data. The locus for extreme resistance (Ry (sto)) to PVY was mapped on chromosome XII co-segregating with the SSR marker STM 0003. The utility of anther-culture derived dihaploid potatoes for genetic marker development was demonstrated. Marker transferability from diploids to tetraploids provides an optimistic potential for marker-assisted selection in potato breeding programs.

The gene ENHANCER OF PINOID controls cotyledon development in the Arabidopsis embryo.

During Arabidopsis embryo development, cotyledon primordia are generated at transition stage from precursor cells that are not derived from the embryonic shoot apical meristem (SAM). To date, it is not known which genes specifically instruct these precursor cells to elaborate cotyledons, nor is the role of auxin in cotyledon development clear. In laterne mutants, the cotyledons are precisely deleted, yet the hypocotyl and root are unaffected. The laterne phenotype is caused by a combination of two mutations: one in the PINOID (PID) gene and another mutation in a novel locus designated ENHANCER OF PINOID (ENP). The expression domains of shoot apex organising genes such as SHOOT MERISTEMLESS (STM) extend along the entire apical region of laterne embryos. However, analysis of pid enp stm triple mutants shows that ectopic activity of STM does not appear to cause cotyledon obliteration. This is exclusively caused by enp in concert with pid. In pinoid embryos, reversal of polarity of the PIN1 auxin transport facilitator in the apex is only occasional, explaining irregular auxin maxima in the cotyledon tips. By contrast, polarity of PIN1:GFP is completely reversed to basal position in the epidermal layer of the laterne embryo. Consequently auxin, which is believed to be essential for organ formation, fails to accumulate in the apex. This strongly suggests that ENP specifically regulates cotyledon development through control of PIN1 polarity in concert with PID.