Undesired fertility restoration in msm1 barley associates with two mTERF genes.

KEY MESSAGE: The novel Rfm3 locus causing undesired fertility restoration in the msm1 cytoplasm of winter barley is located on the short arm of chromosome 6H. Undesired fertility restoration of cytoplasmic male sterile (CMS) mother lines in absence of the functional Rfm1 restorer gene is a significant problem for hybrid breeding in winter barley. Here, we describe that a novel restorer locus on the short arm of chromosome 6H, designated Rfm3, is closely linked to two mitochondrial transcription termination factor family (mTERF) protein coding genes. Genome-wide association studies in a multiparental mapping population revealed that two of the most significantly associated markers are located very close to these genes, with one marker lying directly within one mTERF gene sequence. Sequences of the candidate genes in the parental lines, segregating individuals and an independent set of breeding lines clearly revealed haplotypes discriminating completely sterile, partially fertile and Rfm1-restorer lines. The haplotypes segregate for several single nucleotide polymorphisms, a 6 bp insertion-deletion (InDel) polymorphism and another 2 bp InDel. CMS-unstable genotypes carrying haplotypes associated with undesired fertility restoration showed significantly higher grain setting on bagged spikes when plants were subjected to elevated temperatures during anthesis, indicating a temperature influence on pollen fertility. SNPs associated with desirable Rfm3 haplotypes can be implemented in marker-assisted selection of stable CMS mother lines.

High-resolution mapping of rachis nodes per rachis, a critical determinant of grain yield components in wheat.

KEY MESSAGE: Exploring large genomic data sets based on the latest reference genome assembly identifies the rice ortholog APO1 as a key candidate gene for number of rachis nodes per spike in wheat. Increasing grain yield in wheat is a key breeding objective worldwide. Several component traits contribute to grain yield with spike attributes being among the most important. In this study, we performed a genome-wide association analysis for 12 grain yield and component traits measured in field trials with contrasting agrochemical input levels in a panel of 220 hexaploid winter wheats. A highly significant, environmentally consistent QTL was detected for number of rachis nodes per rachis (NRN) on chromosome 7AL. The five most significant SNPs formed a strong linkage disequilibrium (LD) block and tagged a 2.23 Mb region. Using pairwise LD for exome SNPs located across this interval in a large worldwide hexaploid wheat collection, we reduced the genomic region for NRN to a 258 Kb interval containing four of the original SNP and six high-confidence genes. The ortholog of one (TraesCS7A01G481600) of these genes in rice was ABBERANT PANICLE ORGANIZATION1 (APO1), which is known to have significant effects on panicle attributes. The APO1 ortholog was the best candidate for NRN and was associated with a 115 bp promoter deletion and two amino acid (C47F and D384 N) changes. Using a large worldwide collection of tetraploid and hexaploid wheat, we found 12 haplotypes for the NRN QTL and evidence for positive enrichment of two haplotypes in modern germplasm. Comparison of five QTL haplotypes in Australian yield trials revealed their relative, context-dependent contribution to grain yield. Our study provides diagnostic SNPs and value propositions to support deployment of the NRN trait in wheat breeding.

Wheat Resistances to Fusarium Root Rot and Head Blight Are Both Associated with Deoxynivalenol- and Jasmonate-Related Gene Expression.

Fusarium graminearum is a major pathogen of wheat causing Fusarium head blight (FHB). Its ability to colonize wheat via seedling root infection has been reported recently. Our previous study on Fusarium root rot (FRR) has disclosed histological characteristics of pathogenesis and pathogen defense that mirror processes of spike infection. Therefore, it would be interesting to understand whether genes relevant for FHB resistance are induced in roots. The concept of similar-acting defense mechanisms provides a basis for research at broad Fusarium resistance in crop plants. However, molecular defense responses involved in FRR as well as their relation to spike resistance are unknown. To test the hypothesis of a conserved defense response, a candidate gene expression study was conducted to test the activity of selected prominent FHB defense-related genes in seedling roots, adult plant roots, spikes, and shoots. FRR was examined at seedling and adult plant stages to assess age-related pattern of disease and pathogen resistance. This study offers first evidence for a significant genetic overlap in root and spike defense responses, both in local and distant tissues. The results point to plant development-specific rather than organ-specific determinants of resistance, and suggest roots as an interesting model for studies on wheat-Fusarium interactions.

Genetics of mycorrhizal symbiosis in winter wheat (Triticum aestivum).

Bread wheat (Triticum aestivum) is a major staple food and therefore of prime importance for feeding the Earth's growing population. Mycorrhiza is known to improve plant growth, but although extensive knowledge concerning the interaction between mycorrhizal fungi and plants is available, genotypic differences concerning the ability of wheat to form mycorrhizal symbiosis and quantitative trait loci (QTLs) involved in mycorrhization are largely unknown. Therefore, a diverse set of 94 bread wheat genotypes was evaluated with regard to root colonization by arbuscular mycorrhizal fungi. In order to identify genomic regions involved in mycorrhization, these genotypes were analyzed using the wheat 90k iSelect chip, resulting in 17 823 polymorphic mapped markers, which were used in a genome-wide association study. Significant genotypic differences (P < 0.0001) were detected in the ability to form symbiosis and 30 significant markers associated with root colonization, representing six QTL regions, were detected on chromosomes 3A, 4A and 7A, and candidate genes located in these QTL regions were proposed. The results reported here provide key insights into the genetics of root colonization by mycorrhizal fungi in wheat.

A saturated SNP linkage map for the orange wheat blossom midge resistance gene Sm1.

KEY MESSAGE: SNP markers were developed for the OWBM resistance gene Sm1 that will be useful for MAS. The wheat Sm1 region is collinear with an inverted syntenic interval in B. distachyon. Orange wheat blossom midge (OWBM, Sitodiplosis mosellana Géhin) is an important insect pest of wheat (Triticum aestivum) in many growing regions. Sm1 is the only described OWBM resistance gene and is the foundation of managing OWBM through host genetics. Sm1 was previously mapped to wheat chromosome arm 2BS relative to simple sequence repeat (SSR) markers and the dominant, sequence characterized amplified region (SCAR) marker WM1. The objectives of this research were to saturate the Sm1 region with markers, develop improved markers for marker-assisted selection (MAS), and examine the synteny between wheat, Brachypodium distachyon, and rice (Oryza sativa) in the Sm1 region. The present study mapped Sm1 in four populations relative to single nucleotide polymorphisms (SNPs), SSRs, Diversity Array Technology (DArT) markers, single strand conformation polymorphisms (SSCPs), and the SCAR WM1. Numerous high quality SNP assays were designed that mapped near Sm1. BLAST delineated the syntenic intervals in B. distachyon and rice using gene-based SNPs as query sequences. The Sm1 region in wheat was inverted relative to B. distachyon and rice, which suggests a chromosomal rearrangement within the Triticeae lineage. Seven SNPs were tested on a collection of wheat lines known to carry Sm1 and not to carry Sm1. Sm1-flanking SNPs were identified that were useful for predicting the presence or absence of Sm1 based upon haplotype. These SNPs will be a major improvement for MAS of Sm1 in wheat breeding programs.

Insights Into Triticum aestivum Seedling Root Rot Caused by Fusarium graminearum.

Fusarium graminearum is one of the most common and potent fungal pathogens of wheat (Triticum aestivum), known for causing devastating spike infections and grain yield damage. F. graminearum is a typical soil-borne pathogen that builds up during consecutive cereal cropping. Speculation on systemic colonization of cereals by F. graminearum root infection have long existed but have not been proven. We have assessed the Fusarium root rot disease macroscopically in a diverse set of 12 wheat genotypes and microscopically in a comparative study of two genotypes with diverging responses. Here, we show a 'new' aspect of the F. graminearum life cycle, i.e., the head blight fungus uses a unique root-infection strategy with an initial stage typical for root pathogens and a later stage typical for spike infection. Root colonization negatively affects seedling development and leads to systemic plant invasion by tissue-adapted fungal strategies. Another major outcome is the identification of partial resistance to root rot. Disease severity assessments and histological examinations both demonstrated three distinct disease phases that, however, proceeded differently in resistant and susceptible genotypes. Soil-borne inoculum and root infection are considered significant components of the F. graminearum life cycle with important implications for the development of new strategies of resistance breeding and disease control.

Transfer of sclerotinia resistance from wild relative of Brassica oleracea into Brassica napus using a hexaploidy step.

KEY MESSAGE: Sclerotinia resistance was transferred into rapeseed from a wild relative of Brassica oleracea (B. incana) using hexaploids derived from crosses between B. incana and rapeseed as a bridge. A high level of resistance against Sclerotinia sclerotiorum has been documented in wild Brassica oleracea, but not in cultivated rapeseed (Brassica napus). To transfer sclerotinia resistance from a wild relative into rapeseed, a strategy was proposed using hexaploids (AACCCC) derived from crosses between the wild B. oleracea-related B. incana genotype 'C01' and the Chinese rapeseed variety 'Zhongshuang 9' as a bridge. Progenies (BC1F1) generated by backcrossing the hexaploid to 'Zhongshuang 9' could be generated with a high crossability (average 18.3 seeds per pod). Seventy-three individuals in BC1F1 were firstly screened for resistance with five molecular markers linked to the major resistance QTL on chromosome C09 in 'C01', and 11 individuals harboring resistance loci were selected to develop vegetative clones. Of these, five exhibited significantly higher resistance than 'Zhongshuang 9' and the most resistant individual was chosen to develop the BC1F2 progeny. Finally, five individual genotypes with nearly twofold higher resistance than 'Zhongshuang 9' were found among 100 BC1F2 individuals by using marker-assisted selection and resistance evaluation. Hereof, one rapeseed-type individual with 38 chromosomes and good self-fertility (15.0 ± 3.56 seeds/pod) was identified. Our results indicate that the proposed strategy is effective for transferring sclerotinia resistance from a wild relative of B. oleracea into rapeseed.

High resolution mass spectrometry imaging of plant tissues: towards a plant metabolite atlas.

Mass spectrometry (MS) imaging provides spatial and molecular information for a wide range of compounds. This tool can be used to investigate metabolic changes in plant physiology and environmental interactions. A major challenge in our study was to prepare tissue sections that were compatible with high spatial resolution analysis and therefore dedicated sample preparation protocols were established and optimized for the physicochemical properties of all major plant organs. We combined high spatial resolution (5 µm), in order to detect cellular features, and high mass accuracy (<2 ppm root mean square error), for molecular specificity. Mass spectrometry imaging experiments were performed in positive and negative ion mode. Changes in metabolite patterns during plant development were investigated for germination of oilseed rape. The detailed localization of more than 90 compounds allowed assignment to metabolic processes and indicated possible functions in plant tissues. The 'untargeted' nature of MS imaging allows the detection of marker compounds for the physiological status, as demonstrated for plant-pathogen interactions. Our images show excellent correlation with optical/histological examination. In contrast to previous MS imaging studies of plants, we present a complete workflow that covers multiple species, such as oilseed rape, wheat seed and rice. In addition, different major plant organs and a wide variety of compound classes were analyzed. Thus, our method could be used to develop a plant metabolite atlas as a reference to investigate systemic and local effects of pathogen infection or environmental stress.

Unravelling the genetic complexity of sorghum seedling development under low-temperature conditions.

Sorghum is a promising alternative to maize for bioenergy production in Europe; however, its use is currently limited by poor adaptation to low temperatures during and after germination. We collected multi-trait phenotype data under optimal and suboptimal temperatures in a genetically diverse recombinant inbred line (RIL) mapping population showing contrasting segregation patterns for pre- and post-emergence chilling tolerance. Germination, emergence, seedling development, root architecture and seedling survival were assessed in two different seedlots. Emergence and root establishment were found to be the key determinants of development and survival under chilling stress. Highly interactive epistatic quantitative trait loci (QTL) hotspots, including a previously unknown QTL on Sb06 with a significant effect on prolonged chilling survival, were found to regulate different physiological mechanisms contributing to maintenance of growth and development despite the chilling temperatures. The major QTL regions harbour promising candidate genes with known roles in abiotic stress tolerance. Identification of loci in the QTL hotspot regions conferring maintenance of cell division and growth under early chilling stress represents a promising step towards breeding for successful establishment of sorghum in temperate climates.

High-throughput genomics in sorghum: from whole-genome resequencing to a SNP screening array.

With its small, diploid and completely sequenced genome, sorghum (Sorghum bicolor L. Moench) is highly amenable to genomics-based breeding approaches. Here, we describe the development and testing of a robust single-nucleotide polymorphism (SNP) array platform that enables polymorphism screening for genome-wide and trait-linked polymorphisms in genetically diverse S. bicolor populations. Whole-genome sequences with 6× to 12× coverage from five genetically diverse S. bicolor genotypes, including three sweet sorghums and two grain sorghums, were aligned to the sorghum reference genome. From over 1 million high-quality SNPs, we selected 2124 Infinium Type II SNPs that were informative in all six source genomes, gave an optimal Assay Design Tool (ADT) score, had allele frequencies of 50% in the six genotypes and were evenly spaced throughout the S. bicolor genome. Furthermore, by phenotype-based pool sequencing, we selected an additional 876 SNPs with a phenotypic association to early-stage chilling tolerance, a key trait for European sorghum breeding. The 3000 attempted bead types were used to populate half of a dual-species Illumina iSelect SNP array. The array was tested using 564 Sorghum spp. genotypes, including offspring from four unrelated recombinant inbred line (RIL) and F2 populations and a genetic diversity collection. A high call rate of over 80% enabled validation of 2620 robust and polymorphic sorghum SNPs, underlining the efficiency of the array development scheme for whole-genome SNP selection and screening, with diverse applications including genetic mapping, genome-wide association studies and genomic selection.

Genomics-based high-resolution mapping of the BaMMV/BaYMV resistance gene rym11 in barley (Hordeum vulgare L.).

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.

Stage-specific genotype-by-environment interactions determine yield components in wheat.

In cereal crops, environmental fluctuations affect different physiological processes during various developmental phases associated with the formation of yield components. Because these effects are coupled with cultivar-specific phenology, studies investigating environmental responses in different cultivars can give contradictory results regarding key phases impacting yield performance. To dissect how genotype-by-environment interactions affect grain yield in winter wheat, we estimated the sensitivities of yield components to variation in global radiation, temperature and precipitation in 220 cultivars across 81 time-windows ranging from double ridge to seed desiccation. Environmental sensitivity responses were prominent in the short-term physiological subphases of spike and kernel development, causing phenologically dependent, stage-specific genotype-by-environment interactions. Here we reconcile contradicting findings from previous studies and show previously undetected effects; for example, the positive impact of global radiation on kernel weight during canopy senescence. This deep insight into the three-way interactions between phenology, yield formation and environmental fluctuations provides comprehensive new information for breeding and modelling cereal crops.

Jasmonate and ethylene dependent defence gene expression and suppression of fungal virulence factors: two essential mechanisms of Fusarium head blight resistance in wheat?

BACKGROUND: Fusarium head blight (FHB) caused by Fusarium species like F. graminearum is a devastating disease of wheat (Triticum aestivum) worldwide. Mycotoxins such as deoxynivalenol produced by the fungus affect plant and animal health, and cause significant reductions of grain yield and quality. Resistant varieties are the only effective way to control this disease, but the molecular events leading to FHB resistance are still poorly understood. Transcriptional profiling was conducted for the winter wheat cultivars Dream (moderately resistant) and Lynx (susceptible). The gene expressions at 32 and 72 h after inoculation with Fusarium were used to trace possible defence mechanisms and associated genes. A comparative qPCR was carried out for selected genes to analyse the respective expression patterns in the resistant cultivars Dream and Sumai 3 (Chinese spring wheat). RESULTS: Among 2,169 differentially expressed genes, two putative main defence mechanisms were found in the FHB-resistant Dream cultivar. Both are defined base on their specific mode of resistance. A non-specific mechanism was based on several defence genes probably induced by jasmonate and ethylene signalling, including lipid-transfer protein, thionin, defensin and GDSL-like lipase genes. Additionally, defence-related genes encoding jasmonate-regulated proteins were up-regulated in response to FHB. Another mechanism based on the targeted suppression of essential Fusarium virulence factors comprising proteases and mycotoxins was found to be an essential, induced defence of general relevance in wheat. Moreover, similar inductions upon fungal infection were frequently observed among FHB-responsive genes of both mechanisms in the cultivars Dream and Sumai 3. CONCLUSIONS: Especially ABC transporter, UDP-glucosyltransferase, protease and protease inhibitor genes associated with the defence mechanism against fungal virulence factors are apparently active in different resistant genetic backgrounds, according to reports on other wheat cultivars and barley. This was further supported in our qPCR experiments on seven genes originating from this mechanism which revealed similar activities in the resistant cultivars Dream and Sumai 3. Finally, the combination of early-stage and steady-state induction was associated with resistance, while transcript induction generally occurred later and temporarily in the susceptible cultivars. The respective mechanisms are attractive for advanced studies aiming at new resistance and toxin management strategies.

Genetic dissection of the temperature dependent emergence processes in sorghum using a cumulative emergence model and stability parameters.

Among the major limitations for cultivating biomass sorghum in temperate regions is low temperature in spring that results in low and non-uniform emergence. The adaptation of sorghum to tropical and subtropical highlands gives hint of genetic variation in cold tolerance during emergence. The objective of the present study was to detect marker-trait associations for parameters describing the emergence process under different temperature regimes. A diversity set comprising 194 genotypes was tested in nine controlled environments with temperatures ranging from 9.4 to 19.9 °C. The genotypes were fingerprinted with 171 DArT markers. A piecewise linear regression model carried out on cumulative emergence was used to estimate genotype mean performance across environments and to carry out stability analysis on the parameters of the regression model. Base temperature (T (b)) and thermal time required for emergence (E (TS)) were determined based on median time to emergence data. Identified QTL positions were compared to marker-trait associations for final emergence percentages under low (FEP(cold)) and normal (FEP(normal)) temperatures. QTL for mean final emergence percentage (FEP), FEP(cold) and FEP(normal,) T (b) and E (TS) were detected on SBI-01. Other QTL-rich regions were located on SBI-03, SBI-04, SBI-06, SBI-08, and SBI-09. Marker-trait associations for T (b) and E (TS) co-localized to QTL for the across environment stability of FEP and the median time to emergence or emergence rate, respectively. We conclude that genome regions on six chromosomes highly influencing cold tolerance during emergence are promising for regional association studies and for the development of stable markers for marker-assisted selection.

Seedling development in a Brassica napus diversity set and its relationship to agronomic performance.

Brassica napus L. is the leading European oilseed crop and has therefore a high economical importance. The objectives of our study were to examine (1) the patterns of phenotypic diversity in a species-wide B. napus germplasm set of 518 inbreds with respect to various seedling development, agronomic, and seed quality traits as well as (2) the interrelationship of the examined traits and their use in selection on correlated traits. The B. napus germplasm set was evaluated in greenhouse and field trials for several seedling development, agronomic, and seed quality traits. The traits were highly correlated within the individual trait categories and moderately correlated between the different trait categories. We observed differences in phenotypic diversity among the examined eight germplasm types. The reduction of phenotypic diversity was on average more pronounced for the seedling development traits than for the agronomic and seed quality traits, suggesting that plant breeders need to introgress new genetic variation with respect to the former.

A knockout mutation in the lignin biosynthesis gene CCR1 explains a major QTL for acid detergent lignin content in Brassica napus seeds.

Seed coat phenolic compounds represent important antinutritive fibre components that cause a considerable reduction in value of seed meals from oilseed rape (Brassica napus). The nutritionally most important fibre compound is acid detergent lignin (ADL), to which a significant contribution is made by phenylpropanoid-derived lignin precursors. In this study, we used bulked-segregant analysis in a population of recombinant inbred lines (RILs) from a cross of the Chinese oilseed rape lines GH06 (yellow seed, low ADL) and P174 (black seed, high ADL) to identify markers with tight linkage to a major quantitative trait locus (QTL) for seed ADL content. Fine mapping of the QTL was performed in a backcross population comprising 872 BC(1)F(2) plants from a cross of an F(7) RIL from the above-mentioned population, which was heterozygous for this major QTL and P174. A 3:1 phenotypic segregation for seed ADL content indicated that a single, dominant, major locus causes a substantial reduction in ADL. This locus was successively narrowed to 0.75 cM using in silico markers derived from a homologous Brassica rapa sequence contig spanning the QTL. Subsequently, we located a B. rapa orthologue of the key lignin biosynthesis gene CINNAMOYL CO-A REDUCTASE 1 (CCR1) only 600 kbp (0.75 cM) upstream of the nearest linked marker. Sequencing of PCR amplicons, covering the full-length coding sequences of Bna.CCR1 homologues, revealed a locus in P174 whose sequence corresponds to the Brassica oleracea wild-type allele from chromosome C8. In GH06, however, this allele is replaced by a homologue derived from chromosome A9 that contains a loss-of-function frameshift mutation in exon 1. Genetic and physical map data infer that this loss-of-function allele has replaced a functional Bna.CCR1 locus on chromosome C8 in GH06 by homoeologous non-reciprocal translocation.

Correlations between genetic, morphological, and chemical diversities in a germplasm collection of the medicinal plant Origanum vulgare L.

In total, 42 accessions of Origanum vulgare L., mostly originating from Europe, were evaluated, to detect molecular, quantitative morphological, and chemotype polymorphisms and to discover possible correlations between them. Twelve traits related to morphological characteristics were measured. The components in the essential oils were identified by GC/MS analysis, and the oil contents of 18 major compounds were determined. A total of 477 molecular polymorphisms including 214 AFLP (amplified fragment length polymorphism) and 263 SAMPL (selectively amplified microsatellite polymorphic loci) were used for genotyping. Euclidean distances of morphological and chemotypic data and genetic distances (1 - Dice's similarity) of molecular markers were compared by applying Mantel tests to ascertain the congruencies between them. A relatively high correlation between chemotypic patterns and genetic markers was identified, while a lower correlation was found between the morphological and genetic matrices. Pairwise analyses of correlation among all traits showed that the stem diameter was correlated to the essential-oil yield and the carvacrol content. Cluster analysis, population inference, and principal component analysis revealed a broad genetic and chemical variation among the accessions. The knowledge of these diversities, found in this study, will allow a plant improvement of Origanum vulgare related to pharmaceutical and spice uses.

QTL for fibre-related traits in grain × sweet sorghum as a tool for the enhancement of sorghum as a biomass crop.

Compared to maize and temperate grasses, sorghum has received less attention in terms of improving cell wall components. The objectives of this study were to identify quantitative trait loci (QTL) with main effects, epistatic and pleiotropic effects along with QTL × environment (QE) interactions controlling fibre-related traits in sorghum. Neutral detergent fibre (NDF), acid detergent fibre (ADF), acid detergent lignin (ADL), cellulose, hemicellulose, fresh leaf mass, stripped stalk mass, dry stalk mass, fresh biomass and dry biomass were analysed from a population of 188 grain × sweet sorghum recombinant inbred lines. A genetic map consisting of 157 DNA markers was constructed, and QTL were detected using composite interval mapping (CIM). CIM detected more than 5 additive QTL per trait explaining 7.1-24.7% of the phenotypic variation. Abundant co-localization of these QTL was observed across all chromosomes, and the highest cluster was identified on chromosome 6. Searching for candidate genes using the confidence interval of our QTL clusters reveals that these clusters might comprise a set of genes that are tightly linked. Some QTL showed multiple effects; however, the allele for each trait was favouring the parent with the increasing effect. QE interactions were observed for QTL showing multiple effects. Additive × additive interaction was observed for 7 out of 10 traits, indicating the importance of epistatic analysis. However, the phenotypic variation explained by digenic interactions was lower compared to the individual QTL. Our results indicate that various genetic components contribute to fibre-related traits and should be considered during the enhancement of sorghum for lignocellulosic biomass.