Molecular and phenotypic characterization of the alternative seasonal growth habit and flowering time in barley (Hordeum vulgare ssp. vulgare L.)

Barley can be classified into three major agronomic types, based on its seasonal growth habit (SGH): spring, winter and alternative. Winter varieties require exposure to vernalization to promote subsequent flowering and are autumn-sown. Spring varieties proceed to flowering in the absence of vernalization and are sown in the spring. The ‘alternative’ (also known as ‘facultative’) SGH is only loosely defined and can be sown in autumn or spring. Here, we investigate the molecular genetic basis of alternative barley. Analysis of the major barley vernalization (VRN-H1, VRN-H2) and photoperiod (PPD-H1, PPD-H2) response genes in a collection of 386 varieties found alternative SGH to be characterized by specific allelic combinations. Spring varieties possessed spring loci at one or both of the vernalization response loci, combined with long-day non-responsive ppd-H1 alleles and wild-type alleles at the short-day photoperiod response locus, PPD-H2. Winter varieties possessed winter alleles at both vernalization loci, in combination with the mutant ppd-H2 allele conferring delayed flowering under short-day photoperiods. In contrast, all alternative varieties investigated possessed a single spring allele (either at VRN-H1 or at VRN-H2) combined with mutant ppd-H2 alleles. This allelic combination is found only in alternative types and is diagnostic for alternative SGH in the collection studied. Analysis of flowering time under controlled environment found alternative varieties flowered later than spring control lines, with the difference most pronounced under short-day photoperiods. This work provides genetic characterization of the alternative SGH phenotype, allowing precise manipulation of SGH and flowering time within breeding programmes, and provides the molecular tools for classification of all three SGH categories within national variety registration processes.

Étude de l'intégrité du génome chloroplastique de l'orge (Hordeum vulgare) en culture de microspores isol��es

Un enjeu actuel en biotechnologie est d’obtenir des plantes haploïdes doubl��es par la technique de la culture de microspores isol��es (CMI). Pourtant, la CMI génère parfois une proportion importante de plantes albinos, laquelle peut atteindre 100 % chez certains cultivars. Des travaux antérieurs ont indiqué que des remaniements du génome chloroplastique seraient à l���origine de cet albinisme. Afin de mieux comprendre ce processus menant à l���albinisme, nous avons entrepris d’étudier l���intégrité du génome chloroplastique au sein de microspores d’orge et de plantes albinos via une approche de séquençage à grande échelle. L���ADN total extrait de microspores à un stade précoce de la CMI, d’une feuille de la plante-mère (témoin), et de feuilles albinos, a été séquencé et les séquences chloroplastiques ont été analysées. Ceci nous a permis de documenter pour la première fois une diminution de l���ADN chloroplastique chez les microspores. De plus une étude de variations structurales a démontré un abaissement généralisé de la quantité de génomes chloroplastiques chez les microspores. Enfin, d’importants remaniements du génome chloroplastique ont été observés chez les plantes albinos, révélant une forte abondance de génomes chloroplastiques altérés de forme linéaire.

Analyse transcriptomique de l'interaction tripartite "Pseudozyma flocculosa-Blumeria graminis f.sp. hordei-Hordeum vulgare"

Afin d’améliorer nos pratiques agricoles dans le contexte d’une agriculture durable, plusieurs agents de lutte biologique (ALB) ont été développés, testés et sont maintenant utilisés dans le monde pour combattre les pertes de rendements causées par les maladies. Blumeria graminis f. sp. hordei ( Bgh) est l���agent pathogène responsable du blanc de l���orge et peut réduire les rendements de cette culture jusqu’à 40%. Un champignon épiphyte, Pseudozyma flocculosa, a été découvert et identifié en 1987 en association étroite avec le blanc du trèfle. Les chercheurs ont alors remarqué que ce champignon exhibait une forte activité antagoniste contre le blanc en détruisant les structures de l���agent pathogène. Suite à d’autres travaux, il est apparu que ce comportement antagoniste était dirigé contre tous les membres des Erysiphales et semblait lié à la synthèse d’un glycolipide antifongique soit la flocculosine. Toutefois, on n’est toujours pas parvenus à associer l���efficacité de l���ALB avec la production de ce glycolipide. Ces observations suggèrent que d’autres facteurs seraient impliqués lorsque les deux protagonistes, l���ALB et le blanc, sont en contact. L���objectif principal de ce projet était donc de chercher d’autres mécanismes mol��culaires pouvant expliquer l���interaction P. flocculosa-blanc et orge, en faisant une analyse transcriptomique compl��te des trois protagonistes en même temps. L���interaction tripartite a été échantillonnée à différents temps suivant l���inoculation de P. flocculosa sur des feuilles d’orge présentant déjà une intensité de blanc d’environ 50%. Les échantillons de feuilles prélevés ont ensuite été utilisés pour l���extraction de l���ARN qui ont été ensuite transformés en ADNc pour la préparation des librairies. Cinq répliquats ont été effectués pour chaque temps et le tout a été séquencé à l���aide de séquençage par synthèse Illumina HiSeq. Les séquences obtenues (reads) ont ensuite été analysées à l���aide du logiciel CLC Genomics Workbench. Brièvement, les séquences obtenues ont été cartographiées sur les trois génomes de référence. Suite à la cartographie, les analyses d’expression ont été conduites et les gènes exprimés de façon différentielle ont été recherchés. Cette étape a été conduite en portant une attention particulière aux gènes codant pour un groupe de protéines appel��es CSEP pour “candidate secreted effector proteins” qui seraient possiblement impliquées dans l���interaction tripartite. Parmi les protéines exprimées de façon différentielle en présence du blanc ou en absence de ce dernier, nous avons pu constater que certaines CSEP étaient fortement exprimées en présence du blanc. Ces résultats sont prometteurs et nous offrent une piste certaine pour l���élucidation des mécanismes impliqués dans cette interaction tripartite.

Relationship Between Hardness Genes and Quality in Barley (Hordeum vulgare)

Barley (Hordeum vulgare) genotypes were sequenced for polymorphism in the hardness genes, these being the three hordoindoline (hin a, hin b1 and hin b2) genes. The variation in haplotype was determined by sequencing for single nucleotide polymorphisms (SNPs). Polymorphism between each gene was then compared to grain hardness (three methods), malt quality characteristics (hot water extract and friability) and cattle feed quality. Two haplotypes were found in a set of forty barley genotypes. For hin a, two alleles were present, namely hin a1 and hin a2. However, there was no specific hin a allele that was associated with grain hardness, malt and feed quality. Barley has two hin b genes, namely hin b1 and hin b2, and the genotypes tested here had one of two alleles for each gene. However, there were no obvious effects on hardness or quality from either of these hin b alleles. Unlike wheat, where a clear relationship has been demonstrated between a number of SNPs in the wheat hardness genes and quality (soft or hard wheat), there was no such relationship for barley. Despite the wide range in hardness, malt and feed quality, there were only two haplotypes for each of the hin a, hin b1 and hin b2 genes and there was no clear relationship between grain hardness, malt or feed quality. The genotypes used in this study demonstrated that there was a low level of polymorphism in hardness genes in current commercial varieties as well as breeding lines and these polymorphisms had no impact on quality.

Occurrence of LINE, gypsy-like, and copia-like retrotransposons in the clonally propagated sweet potato (Ipomoea batatas L.)

Retrotransposons are a class of transposable elements that represent a major fraction of the repetitive DNA of most eukaryotes. Their abundance stems from their expansive replication strategies. We screened and isolated sequence fragments of long terminal repeat (LTR), gypsy-like reverse transcriptase (rt) and gypsy-like envelope (env) domains, and two partial sequences of non-LTR retrotransposons, long interspersed element (LINE), in the clonally propagated allohexaploid sweet potato (Ipomoea batatas (L.) Lam.) genome. Using dot-blot hybridization, these elements were found to be present in the ~1597 Mb haploid sweet potato genome with copy numbers ranging from ~50 to ~4100 as observed in the partial LTR (IbLtr-1) and LINE (IbLi-1) sequences, respectively. The continuous clonal propagation of sweet potato may have contributed to such a multitude of copies of some of these genomic elements. Interestingly, the isolated gypsy-like env and gypsy-like rt sequence fragments, IbGy-1 (~2100 copies) and IbGy-2 (~540 copies), respectively, were found to be homologous to the Bagy-2 cDNA sequences of barley (Hordeum vulgare L.). Although the isolated partial sequences were found to be homologous to other transcriptionally active elements, future studies are required to determine whether they represent elements that are transcriptionally active under normal and (or) stressful conditions.

Assessing for genetic and environmental effects on ruminant feed quality in barley ( Hordeum vulgare )

Grain samples from a combined intermediate and advanced stage barley breeding trial series, grown at two sites in two consecutive years were assessed for detailed grain quality and ruminant feed quality. The results indicated that there were significant genetic and environmental effects for “feed” traits as measured using grain hardness, acid detergent fibre (ADF), starch and in-sacco dry matter digestibility (ISDMD) assays. In addition, there was strong genotypic discrimination for the regressed feed performance traits, namely Net Energy (NE) and Average Daily Gain (ADG). There was considerable variation in genetic correlations for all traits based on variance from the cultivars used, sites or laboratory processing effects. There was a high level of heritability ranging from 89% to 88% for retention, 60% to 80% for protein and 56% to 68% for ADF. However, there were only low to moderate levels of heritability for the feed traits, with starch 30–39%, ISDMD 55–63%, ADF 56–68%, particle size 47–73%, 31–48% NE and ADG 44–51%. These results suggest that there were real differences in the feed performance of barleys and that selection for cattle feed quality is potentially a viable option for breeding programs.

Measurement of genetic and environmental variation in barley (Hordeum vulgare) grain hardness

In this study, we assessed a broad range of barley breeding lines and commercial varieties by three hardness methods (two particle size methods and one crush resistance method (SKCS—Single-Kernel Characterization System), grown at multiple sites to see if there was variation in barley hardness and if that variation was genetic or environmentally controlled. We also developed near-infrared reflectance (NIR) calibrations for these three hardness methods to ascertain if NIR technology was suitable for rapid screening of breeding lines or specific populations. In addition, we used this data to identify genetic regions that may be associated with hardness. There were significant (p<0.05) genetic effects for the three hardness methods. There were also environmental effects, possibly linked to the effect of protein on hardness, i.e. increasing protein resulted in harder grain. Heritability values were calculated at >85% for all methods. The NIR calibrations, with R2 values of >90%, had Standard Error of Prediction values of 0.90, 72 and 4.0, respectively, for the three hardness methods. These equations were used to predict hardness values of a mapping population which resulted in genetic markers being identified on all chromosomes but chromosomes 2H, 3H, 5H, 6H and 7H had markers with significant LOD scores. The two regions on 5H were on the distal end of both the long and short arms. The region that showed significant LOD score was on the long arm. However, the region on the short arm associated with the hardness (hordoindoline) genes did not have significant LOD scores. The results indicate that barley hardness is influenced by both genotype and environment and that the trait is heritable, which would allow breeders to develop very hard or soft varieties if required. In addition, NIR was shown to be a reliable tool for screening for hardness. While the data set used in this study has a relatively low variation in hardness, the tools developed could be applied to breeding populations that have large variation in barley grain hardness.

Correlation between NIRS generated and chemically measured feed quality data in barley (Hordeum vulgare), and potential use in QTL analysis identification

A study was performed to investigate the value of near infrared reflectance spectroscopy (NIRS) as an alternate method to analytical techniques for identifying QTL associated with feed quality traits. Milled samples from an F6-derived recombinant inbred Tallon/Scarlett population were incubated in the rumen of fistulated cattle, recovered, washed and dried to determine the in-situ dry matter digestibility (DMD). Both pre- and post-digestion samples were analysed using NIRS to quantify key quality components relating to acid detergent fibre, starch and protein. This phenotypic data was used to identify trait associated QTL and compare them to previously identified QTL. Though a number of genetic correlations were identified between the phenotypic data sets, the only correlation of most interest was between DMD and starch digested (r = -0.382). The significance of this genetic correlation was that the NIRS data set identified a putative QTL on chromosomes 7H (LOD = 3.3) associated with starch digested. A QTL for DMD occurred in the same region of chromosome 7H, with flanking markers fAG/CAT63 and bPb-0758. The significant correlation and identification of this putative QTL, highlights the potential of technologies like NIRS in QTL analysis.

QTL associated with barley (Hordeum vulgare) feed quality traits measured through in situ digestion

Barley (Hordeum vulgare) is a major feed source for the intensive livestock industry. Competitiveness against other cereal grains depends largely on the price per unit of expressed feed quality. The traits which contribute to feed quality in barley are largely quantitative in nature but little is known about their genetic control. A study to identify the quantitative trait loci (QTLs) associated with feed quality was performed using a F6-derived recombinant inbred barley population. Samples from each line were incubated in the rumen of fistulated cattle, recovered, washed and dried for determination of in situ dry matter digestibility. Additionally, both pre- and post-digestion samples were analysed to quantify the content of key quality components relating to acid detergent fibre, total starch and protein. The data was used to identify trait-associated QTLs. Genetic analysis identified significant QTLs on chromosomes 2H, 5H and 7H. Genetic markers linked to these QTL should provide an effective tool for the selection and improvement of feed barley in the future.

Antioxidant principles of Tanacetum vulgare L. aerial parts

The methanolic extract of aerial parts of Tanacetum vulgare L. (Asteraceae) and its fractions were investigated for antioxidant activity. The crude extract displayed DPPH radical scavenging effects with an EC50 value of 37 +/- 1.2 microg/mL (n=3). Activity-guided fractionations of the crude extract resulted in the isolation of three antioxidant compounds; 3,5-O-dicaffeoylquinic acid (3,5-DCQA), axillarin and luteolin. 3,5-DCQA was the major constituent with antioxidant activity (IC50 = 9.7 microM) comparable with that of the standard quercetin (IC50 = 8.8 microM). Though the isolated compounds were previously known for their antioxidant effects, this is the first report on the identification of 3,5-DCQA from Tanacetum vulgare. The displayed potent antioxidant activity of the crude extract and isolated active principles is in support of the traditional medicinal uses of the plant for disease conditions such as wound healing, rheumatic arthritis and other inflammatory conditions.

Measuring root traits in barley (Hordeum vulgare ssp vulgare and ssp spontaneum) seedlings using gel chambers, soil sacs and X-ray microtomography

Root characteristics of seedlings of five different barley genotypes were analysed in 2D using gel chambers, and in 3D using soil sacs that were destructively harvested and pots of soil that were assessed non-invasively using X-ray microtomography. After 5 days, Chime produced the greatest number of root axes (similar to 6) and Mehola significantly less (similar to 4) in all growing methods. Total root length was longest in GSH01915 and shortest in Mehola for all methods, but both total length and average root diameter were significantly larger for plants grown in gel chambers than those grown in soil. The ranking of particular growth traits (root number, root angular spread) of plants grown in gel plates, soil sacs and X-ray pots was similar, but plants grown in the gel chambers had a different order of ranking for root length to the soil-grown plants. Analysis of angles in soil-grown plants showed that Tadmore had the most even spread of individual roots and Chime had a propensity for non-uniform distribution and root clumping. The roots of Mehola were less well spread than the barley cultivars supporting the suggestion that wild and landrace barleys tend to have a narrower angular spread than modern cultivars. The three dimensional analysis of root systems carried out in this study provides insights into the limitations of screening methods for root traits and useful data for modelling root architecture.