Mapping of common bunt resistance gene Bt9 in wheat.

KEY MESSAGE: The Bt9 resistance locus was mapped and shown to be distinct from the Bt10 locus. New markers linked to Bt9 have been identified and may be used to breed for resistance towards the seed-borne disease. Increasing organic wheat production in Denmark, and in other wheat-producing areas, in conjunction with legal requirements for organic seed production, may potentially lead to a rise in common bunt occurrence. As systemic pesticides are not used in organic farming, organic wheat production systems may benefit from genetic resistances. However, little is known about the underlying genetic mechanisms and locations of the resistance factors for common bunt resistance in wheat. A double haploid (DH) population segregating for common bunt resistance was used to identify the chromosomal location of common bunt resistance gene Bt9. DH lines were phenotyped in three environments and genotyped with DArTseq and SSR markers. The total length of the resulting linkage map was 2882 cM distributed across all 21 wheat chromosomes. Bt9 was mapped to the distal end of chromosome 6DL. Since wheat common bunt resistance gene Bt10 is also located on chromosome 6D, the possibility of their co-location was investigated. A comparison of marker sequences linked to Bt9 and Bt10 on physical maps of chromosome 6D confirmed that Bt9 and Bt10 are two distinct resistance factors located at the distal (6DL) and proximal (6DS) end, respectively, of chromosome 6D. Five new SSR markers Xgpw4005-1, Xgpw7433, Xwmc773, Xgpw7303 and Xgpw362 and many SNP and PAV markers flanking the Bt9 resistance locus were identified and they may be used in the future for marker-assisted selection.

Molecular breeding of barley for quality traits and resilience to climate change.

Barley grains are a rich source of compounds, such as resistant starch, beta-glucans and anthocyanins, that can be explored in order to develop various products to support human health, while lignocellulose in straw can be optimised for feed in husbandry, bioconversion into bioethanol or as a starting material for new compounds. Existing natural variations of these compounds can be used to breed improved cultivars or integrated with a large number of mutant lines. The technical demands can be in opposition depending on barley's end use as feed or food or as a source of biofuel. For example beta-glucans are beneficial in human diets but can lead to issues in brewing and poultry feed. Barley breeders have taken action to integrate new technologies, such as induced mutations, transgenics, marker-assisted selection, genomic selection, site-directed mutagenesis and lastly machine learning, in order to improve quality traits. Although only a limited number of cultivars with new quality traits have so far reached the market, research has provided valuable knowledge and inspiration for future design and a combination of methodologies to achieve the desired traits. The changes in climate is expected to affect the quality of the harvested grain and it is already a challenge to mitigate the unpredictable seasonal and annual variations in temperature and precipitation under elevated [CO2] by breeding. This paper presents the mutants and encoded proteins, with a particular focus on anthocyanins and lignocellulose, that have been identified and characterised in detail and can provide inspiration for continued breeding to achieve desired grain and straw qualities.

Mutations in genes controlling the biosynthesis and accumulation of inositol phosphates in seeds.

Most of the phosphorus in the resting seed is stored inside protein storage vacuoles as PA (phytic acid; InsP(6)). The biosynthesis and accumulation of PA can be detected beginning from a few days after anthesis and seem to continue during seed development until maturation. The first step in PA biosynthesis is the formation of Ins3P by conversion of glucose 6-phosphate. This is then followed by a sequential and ordered phosphorylation of the remaining five positions of the inositol ring by a number of kinases, resulting in PA. Identification of low-PA mutants in cereals, legumes and Arabidopsis is instrumental for resolving the biosynthetic pathway and identification of genes controlling the accumulation of PA. Mutations in seven genes involved in the metabolism of PA have been identified and characterized among five plant species using induced mutagenesis and insertion elements. Understanding the biosynthetic pathway and genes controlling the accumulation of PA in plant seeds and how PA may balance the free phosphate is of importance for molecular breeding of crop plants, particularly cereals and legumes.

Barley isochorismate synthase mutant is phylloquinone-deficient, but has normal basal salicylic acid level.

Salicylic acid (SA) is an important signaling hormone in plant immunity. It can be synthesized by either the phenylpropanoid pathway or the isochorismate pathway, but mutant studies of this have been scarce in other species than Arabidopsis. Here we identified a mutation that introduced a stop-codon early in the barley gene for isochorismate synthase (ICS). We found that homozygous ics plants wilted if not sprayed with 1,4-dihydroxy-2-naphthoic acid, a precursor of phylloquinone, also synthesized via the isochorismate pathway. Interestingly, ics had unchanged SA, suggesting that the basal level of SA is synthesized via the phenylpropanoid pathway. Previous studies have failed seeing increased SA levels in barley after attack by the powdery mildew fungus, Blumeria graminis f.sp. hordei (Bgh), and indeed, we saw no changes in the interaction of ics with this fungus. Overall, we hope this mutant will be useful for other studies of SA in barley.

The biological activity of a recombinantly expressed (His)(6)-tagged peanut allergen (rAra h 1) is unaffected by endotoxin removal.

The application of recombinant (His)(6)-tagged proteins in cell culture assays is associated with problems due to lipopolysaccharide (LPS) contamination. LPS stimulates cells of the immune system, thereby masking antigen-specific activation of T cells. Due to the affinity of LPS for histidine it is associated with difficulties to remove LPS from recombinant (His)(6)-tagged proteins. Here we describe that the Triton X-114 phase separation method can be used to remove LPS from (His)(6)-tagged proteins and that the recombinant proteins retain their biological activity.

TILLING in Brachypodium distachyon.

TILLING is a low-cost screening method that allows for identification of mutations in a gene-of-interest within a range of few base pairs. TILLING can be applied to mutant populations or to plant collections of cultivars, landraces or crop wild relatives (Eco-TILLING). The method is based on the Cel1 enzyme cleavage of mismatches in PCR products amplified with labeled primers. The cleavage can be detected due to the labeled primers by different methods including capillary electrophoresis. Here, we introduce the development of the mutant population BRACHYLIFE and present a Brachypodium TILLING protocol based on fluorescing primers for PCR, enzymatic cleavage, and detection with Applied Biosystems 3130xl Genetic Analyzer.

QTLs and Potential Candidate Genes for Heat Stress Tolerance Identified from the Mapping Populations Specifically Segregating for Fv/Fm in Wheat.

Despite the fact that Fv/Fm (maximum quantum efficiency of photosystem II) is the most widely used parameter for a rapid non-destructive measure of stress detection in plants, there are barely any studies on the genetic understanding of this trait under heat stress. Our aim was to identify quantitative trait locus (QTL) and the potential candidate genes linked to Fv/Fm for improved photosynthesis under heat stress in wheat (Triticum aestivum L.). Three bi-parental F2 mapping populations were generated by crossing three heat tolerant male parents (origin: Afghanistan and Pakistan) selected for high Fv/Fm with a common heat susceptible female parent (origin: Germany) selected for lowest Fv/Fm out of a pool of 1274 wheat cultivars of diverse geographic origin. Parents together with 140 F2 individuals in each population were phenotyped by Fv/Fm under heat stress (40°C for 3 days) around anthesis. The Fv/Fm decreased by 6.3% in the susceptible parent, 1-2.5% in the tolerant parents and intermediately 4-6% in the mapping populations indicating a clear segregation for the trait. The three populations were genotyped with 34,955 DArTseq and 27 simple sequence repeat markers, out of which ca. 1800 polymorphic markers mapped to 27 linkage groups covering all the 21 chromosomes with a total genome length of about 5000 cM. Inclusive composite interval mapping resulted in the identification of one significant and heat-stress driven QTL in each population on day 3 of the heat treatment, two of which were located on chromosome 3B and one on chromosome 1D. These QTLs explained about 13-35% of the phenotypic variation for Fv/Fm with an additive effect of 0.002-0.003 with the positive allele for Fv/Fm originating from the heat tolerant parents. Approximate physical localization of these three QTLs revealed the presence of 12 potential candidate genes having a direct role in photosynthesis and/or heat tolerance. Besides providing an insight into the genetic control of Fv/Fm in the present study, the identified QTLs would be useful in breeding for heat tolerance in wheat.

Genome-wide association mapping in winter barley for grain yield and culm cell wall polymer content using the high-throughput CoMPP technique.

A collection of 112 winter barley varieties (Hordeum vulgare L.) was grown in the field for two years (2008/09 and 2009/10) in northern Italy and grain and straw yields recorded. In the first year of the trial, a severe attack of barley yellow mosaic virus (BaYMV) strongly influenced final performances with an average reduction of ~ 50% for grain and straw harvested in comparison to the second year. The genetic determination (GD) for grain yield was 0.49 and 0.70, for the two years respectively, and for straw yield GD was low in 2009 (0.09) and higher in 2010 (0.29). Cell wall polymers in culms were quantified by means of the monoclonal antibodies LM6, LM11, JIM13 and BS-400-3 and the carbohydrate-binding module CBM3a using the high-throughput CoMPP technique. Of these, LM6, which detects arabinan components, showed a relatively high GD in both years and a significantly negative correlation with grain yield (GYLD). Overall, heritability (H2) was calculated for GYLD, LM6 and JIM and resulted to be 0.42, 0.32 and 0.20, respectively. A total of 4,976 SNPs from the 9K iSelect array were used in the study for the analysis of population structure, linkage disequilibrium (LD) and genome-wide association study (GWAS). Marker-trait associations (MTA) were analyzed for grain yield and cell wall determination by LM6 and JIM13 as these were the traits showing significant correlations between the years. A single QTL for GYLD containing three MTAs was found on chromosome 3H located close to the Hv-eIF4E gene, which is known to regulate resistance to BaYMV. Subsequently the QTL was shown to be tightly linked to rym4, a locus for resistance to the virus. GWAs on arabinans quantified by LM6 resulted in the identification of major QTLs closely located on 3H and hypotheses regarding putative candidate genes were formulated through the study of gene expression levels based on bioinformatics tools.

Association Mapping in Scandinavian Winter Wheat for Yield, Plant Height, and Traits Important for Second-Generation Bioethanol Production.

A collection of 100 wheat varieties representing more than 100 years of wheat-breeding history in Scandinavia was established in order to identify marker-trait associations for plant height (PH), grain yield (GY), and biomass potential for bioethanol production. The field-grown material showed variations in PH from 54 to 122 cm and in GY from 2 to 6.61 t ha(-1). The release of monomeric sugars was determined by high-throughput enzymatic treatment of ligno-cellulosic material and varied between 0.169 and 0.312 g/g dm for glucose (GLU) and 0.146 and 0.283 g/g dm for xylose (XYL). As expected, PH and GY showed to be highly influenced by genetic factors with repeatability (R) equal to 0.75 and 0.53, respectively, while this was reduced for GLU and XYL (R = 0.09 for both). The study of trait correlations showed how old, low-yielding, tall varieties released higher amounts of monomeric sugars after straw enzymatic hydrolysis, showing reduced recalcitrance to bioconversion compared to modern varieties. Ninety-three lines from the collection were genotyped with the DArTseq(®) genotypic platform and 5525 markers were used for genome-wide association mapping. Six quantitative trait loci (QTLs) for GY, PH, and GLU released from straw were mapped. One QTL for PH was previously reported, while the remaining QTLs constituted new genomic regions linked to trait variation. This paper is one of the first studies in wheat to identify QTLs that are important for bioethanol production based on a genome-wide association approach.

A comparative study of the allergenic potency of wild-type and glyphosate-tolerant gene-modified soybean cultivars.

A large proportion of soybean cultivars grown in the USA are now genetically modified varieties and concern has been raised about the safety of these products for consumers. A study of the impact on allergenic potency in soybeans, comparable except for the newly introduced gene (CP4 EPSPS), was performed using soybean-sensitized patients. The allergenicity of 18 different (10 GM and 8 WT) soybean extracts was examined blindly by the following three methods: A) Sera from patients with specific IgE against soybean were used to determine concentrations inducing 50% RAST inhibition; B) Histamine release induced by the extracts was examined using blood from sensitized patients; C) SPT was performed on sensitized patients with all 18 extracts. All three methods showed variations in the allergenic potency between the individual extracts but allergenic potential was not affected by presence of the transgene. By using standard in vitro methods and SPT for determination of allergenicity we were not able to detect any significant difference in the allergenic potency between GM and WT soybeans.

Barbarea vulgaris linkage map and quantitative trait loci for saponins, glucosinolates, hairiness and resistance to the herbivore Phyllotreta nemorum.

Combined genomics and metabolomics approaches were used to unravel molecular mechanisms behind interactions between winter cress (Barbarea vulgaris) and flea beetle (Phyllotreta nemorum). B. vulgaris comprises two morphologically, biochemically and cytologically deviating types, which differ in flea beetle resistance, saponin and glucosinolate profiles, as well as leaf pubescence. An F2 population generated from a cross between the two B. vulgaris types was used to construct a B. vulgaris genetic map based on 100 AFLP and 31 microsatellite markers. The map was divided into eight linkage groups. QTL (quantitative trait loci) analysis revealed a total of 15 QTL affecting eight traits, including nine QTL for four saponins, two QTL for two glucosinolates, two QTL for hairiness, and two QTL for flea beetle resistance. The two QTL for resistance towards flea beetles in B. vulgaris co-localized with QTL for the four saponins associated with resistance. Furthermore, global QTL analysis of B. vulgaris metabolites identified QTL for a number of flavonoid glycosides and additional saponins from both resistant and susceptible types. The transcriptome of the resistant B. vulgaris type was sequenced by pyrosequencing, and sequences containing microsatellites were identified. Microsatellite types in B. vulgaris were similar to Arabidopsis thaliana but different from Oryza sativa. Comparative analysis between B. vulgaris and A. thaliana revealed a remarkable degree of synteny between a large part of linkage groups 1 and 4 of B. vulgaris harboring the two QTL for flea beetle resistance and Arabidopsis chromosomes 3 and 1. Gene candidates that may underlie QTL for resistance and saponin biosynthesis are discussed.