Sue Broughton

Expression level of a gibberellin 20-oxidase gene is associated with multiple agronomic and quality traits in barley

The use of dwarfing genes has resulted in the most significant improvements in yield and adaptation in cereal crops. The allelic dwarfing gene sdw1/denso has been used throughout the world to develop commercial barley varieties. The sdw1 gene has never been used successfully for malting barley, but only for a large number of feed varieties. One of the gibberellin 20-oxidase genes (Hv20ox2) was identified as the candidate gene for sdw1/denso. Semi-quantitative real-time RT-PCR revealed that Hv20ox2 was expressed at different levels in various organs of barley. Transcriptional levels were reduced in leaf blade, sheath, stem and rachis tissue in the barley variety Baudin with the denso gene. Subsequently, the relative expression levels of Hv20ox2 were determined by quantitative real-time RT-PCR in a doubled haploid population and mapped as a quantitative trait. A single expression quantitative trait locus (eQTL) was identified and mapped to its structural gene region on chromosome 3H. The eQTL was co-located with QTLs for yield, height, development score, hectolitre weight and grain plumpness. The expression level of Hv20ox2 was reduced fourfold in the denso mutant, but around 60-fold in the sdw1 mutant, compared to the control variety. The reduced expression level of Hv20ox2 enhanced grain yield by increasing the number of effective tillers, but had negative effects on grain and malting quality. The sdw1 gene can be used only in feed barley due to its severe reduction of Hv20ox2 expression. The gene expression marker for Hv20ox2 can be used to distinguish different alleles of sdw1/denso.

A nonsense mutation in a putative sulphate transporter gene results in low phytic acid in barley

Low phytic acid grains can provide a solution to dietary micronutrient deficiency and environmental pollution. A low phytic acid 1-1 (lpa1-1) barley mutant was identified using forward genetics and the mutant gene was mapped to chromosome 2HL. Comparative genomic analysis revealed that the lpa1-1 gene was located in the syntenic region of the rice Os-lpa-MH86-1 gene on chromosome 4. The gene ortholog of rice Os-lpa-MH86-1 (designated as HvST) was isolated from barley using polymerase chain reaction and mapped to chromosome 2HL in a doubled haploid population of Clipper×Sahara. The results demonstrate the collinearity between the rice Os-lpa-MH86-1 gene and the barley lpa1-1 region. Sequence analysis of HvST revealed a single base pair substitution (C→T transition) in the last exon of the gene in lpa1-1 (M422), which resulted in a nonsense mutation. These results will facilitate our understanding of the molecular mechanisms controlling the low phytic acid phenotype and assist in the development of a diagnostic marker for the selection of the lpa1-1 gene in barley.

Quantitative trait loci controlling kernel discoloration in barley (Hordeum vulgare L.)

Barley kernel discoloration (KD) leads to substantial annual loss in value through downgrading and discounting of malting barley. KD is a difficult trait to introgress into elite varieties as it is controlled by multiple genes and strongly influenced by environment and maturity. As the first step towards marker assisted selection for KD tolerance, we mapped quantitative trait loci (QTLs) controlling KD measured by grain brightness [Minolta L; (Min L)], redness (Min a), and yellowness (Min b) in 7 barley populations. One to 3 QTLs were detected for grain brightness in various populations, and one QTL could account for 5–31% of the phenotypic variation. The QTL located around the centromere region of chromosome 2H was consistently detected in 6 of the 7 populations, explaining up to 28% of the phenotypic variation. In addition, QTLs for grain brightness were most frequently identified on chromosomes 3H and 7H in various populations. Australian varieties Galleon, Chebec, and Sloop contribute an allele to increase grain brightness on chromosome 7H in 3 different populations. A major gene effect was detected for grain redness. One QTL on chromosome 4H explained 54% of the phenotypic variation in the Sloop/Halcyon population, and was associated with the blue aleurone trait. A second QTL was detected on the long arm of chromosome 2H in 3 populations, accounting for 23–47% of the phenotypic variation. The major QTLs for grain yellowness were mapped on chromosomes 2H and 5H. There were strong associations between the QTLs for heading date, grain brightness, and yellowness. The molecular markers linked with the major QTLs should be useful for marker assisted selection for KD.

The application of n-butanol improves embryo and green plant production in anther culture of Australian wheat (Triticum aestivum L.) genotypes

The objective of this study was to improve the production from anther culture of embryos and green plants in Australian spring wheat genotypes by testing new treatments such as n-butanol, as well as other protocol modifications. To date, the use of n-butanol to enhance embryogenesis has only been tested in two European wheat cultivars; this is the first study which demonstrates its application across a range of breeding crosses. A 5-h treatment using 0.1 or 0.2% (v/v) n-butanol following anther pretreatment on a solid mannitol medium significantly improved the production of embryos, green plants and doubled haploids in a range of Australian wheat crosses and varieties. Green plant production increased between 3- and 6-fold in the crosses Yitpi/2*Bumper, Tammarin Rock/2*Bumper and Tammarin Rock/2*Magenta. The addition of calcium (Ca) and macronutrients to the mannitol pretreatment medium also significantly improved the number of embryos and green plants in varieties and crosses, but only when used in combination with n-butanol treatment. A factorial experiment with four varieties and two treatments (n-butanol and Ca/macronutrients) revealed significant interactions between treatments and genotype. In three of the four varieties, the application of n-butanol resulted in significant increases in embryos and green plants with either pretreatment medium although the best results were obtained with Ca and macronutrients in the pretreatment medium, with 200, 193 and 52 green plants per 100 anthers obtained for Bumper, Gladius and Magenta, respectively. In the variety Fortune however, n-butanol treatment did not improve embryo or green plant production unless it was combined with Ca and macronutrients in the pretreatment medium and then there were dramatic improvements; from 0 to 27 green plants per 100 anthers.

Characterization of the sdw1 semi-dwarf gene in barley

BackgroundThe dwarfing gene sdw1 has been widely used throughout the world to develop commercial barley varieties. There are at least four different alleles at the sdw1 locus.ResultsMutations in the gibberellin 20-oxidase gene (HvGA20ox2) resulted in multiple alleles at the sdw1 locus. The sdw1.d allele from Diamant is due to a 7-bp deletion in exon 1, while the sdw1.c allele from Abed Denso has 1-bp deletion and a 4-bp insertion in the 5’ untranslated region. The sdw1.a allele from Jotun resulted from a total deletion of the HvGA20ox2 gene. The structural changes result in lower gene expression in sdw1.d and lack of expression in sdw1.a. There are three HvGA20ox genes in the barley genome. The partial or total loss of function of the HvGA20ox2 gene could be compensated by enhanced expression of its homolog HvGA20ox1and HvGA20ox3. A diagnostic molecular marker was developed to differentiate between the wild-type, sdw1.d and sdw1.a alleles and another molecular marker for differentiation of sdw1.c and sdw1.a. The markers were further tested in 197 barley varieties, out of which 28 had the sdw1.d allele and two varieties the sdw1.a allele. To date, the sdw1.d and sdw1.a alleles have only been detected in the modern barley varieties and lines.ConclusionsThe results provided further proof that the gibberellin 20-oxidase gene (HvGA20ox2) is the functional gene of the barley sdw1 mutants. Different deletions resulted in different functional alleles for different breeding purposes. Truncated protein could maintain partial function. Partial or total loss of function of the HvGA20ox2 gene could be compensated by enhanced expression of its homolog HvGA20ox1 and HvGA20ox3.

Genome-Wide Association Mapping of Acid Soil Resistance in Barley (Hordeum vulgare L.)

Genome-wide association studies (GWAS) based on linkage disequilibrium (LD) have been used to detect QTLs underlying complex traits in major crops. In this study, we collected 218 barley (Hordeum vulgare L.) lines including wild barley and cultivated barley from China, Canada, Australia, and Europe. A total of 408 polymorphic markers were used for population structure and LD analysis. GWAS for acid soil resistance were performed on the population using a general linkage model (GLM) and a mixed linkage model (MLM), respectively. A total of 22 QTLs (quantitative trait loci) were detected with the GLM and MLM analyses. Two QTLs, close to markers bPb-1959 (133.1 cM) and bPb-8013 (86.7 cM), localized on chromosome 1H and 4H respectively, were consistently detected in two different trials with both the GLM and MLM analyses. Furthermore, bPb-8013, the closest marker to the major Al3+ resistance gene HvAACT1 in barley, was identified to be QTL5. The QTLs could be used in marker-assisted selection to identify and pyramid different loci for improved acid soil resistance in barley.

Characterization of a Thermo-Inducible Chlorophyll-deficient mutant in barley

Leaf color is an important trait for not only controlling crop yield but also monitoring plant status under temperature stress. In this study, a thermo-inducible chlorophyll-deficient mutant, named V-V-Y, was identified from a gamma-radiated population of the barley variety Vlamingh. The leaves of the mutant were green under normal growing temperature but turned yellowish under high temperature in the glasshouse experiment. The ratio of chlorophyll a and chlorophyll b in the mutant declined much faster in the first 7–9 days under heat treatment. The leaves of V-V-Y turned yellowish but took longer to senesce under heat stress in the field experiment. Genetic analysis indicated that a single nuclear gene controlled the mutant trait. The mutant gene (vvy) was mapped to the long arm of chromosome 4H between SNP markers 1_0269 and 1_1531 with a genetic distance of 2.2 cM and a physical interval of 9.85 Mb. A QTL for grain yield was mapped to the same interval and explained 10.4% of the yield variation with a LOD score of 4. This QTL is coincident with the vvy gene interval that is responsible for the thermo-inducible chlorophyll-deficient trait. Fine mapping, based on the barley reference genome sequence, further narrowed the vvy gene to a physical interval of 0.428 Mb with 11 annotated genes. This is the first report of fine mapping a thermo-inducible chlorophyll-deficient gene in barley.

Towards the identification of a gene for prostrate tillers in barley (Hordeum vulgare L.)

Tiller angle, an important agronomic trait, contributes to crop production and plays a vital role in breeding for plant architecture. A barley line V-V-HD, which has prostrate tillers during vegetative growth and erect tillers after booting, is considered the ideal type for repressing weed growth and increasing leaf area during early growth. Genetic analysis identified that the prostrate trait in V-V-HD is controlled by a single gene. A double haploid population with 208 lines from V-V-HD × Buloke was used to map the prostrate growth gene. Ninety-six SNP markers were used for primary mapping, and subsequently, SSR and InDel markers were used for fine mapping. The gene was fine-mapped to a 3.53 Mb region on chromosome 3HL between the markers InDelz3028 and InDelz3032 with 52 candidate genes located in this region. Gene annotation analysis of the 52 genes within the target region indicated that a gene involved in zinc-ion binding (gene ID HORVU3Hr1G090910) is likely to be the candidate gene for prostrate growth in V-V-HD, and is linked to the denso/sdw gene. Association analysis showed that prostrate plants were shorter, flowered later.

Early growth stages salinity stress tolerance in CM72 x Gairdner doubled haploid barley population

A doubled haploid (DH) population of barley (Hordeum vulgare L.) generated from salinity tolerant genotype CM72 and salinity sensitive variety Gairdner was studied for salinity stress tolerance at germination, seedling emergence and first leaf full expansion growth stages. Germination study was conducted with deionized water, 150 mM and 300 mM NaCl treatments. Seedling stage salinity tolerance was conducted with three treatments: control, 150 mM NaCl added at seedling emergence and first leaf full expansion growth stages. Results from this study revealed transgressive phenotypic segregations for germination percentage and biomass at seedling stage. Twelve QTL were identified on chromosomes 2H–6H each explaining 10–25% of the phenotypic variations. A QTL located at 176.5 cM on chromosome 3H was linked with fresh weight per plant and dry weight per plant in salinity stress induced at first leaf full expansion growth stage, and dry weight per plant in salinity stress induced at seedling emergence. A stable QTL for germination at both 150 and 300 mM salinity stress was mapped on chromosome 2H but distantly located from a QTL linked with seedling stage salinity stress tolerance. QTL, associated markers and genotypes identified in this study play important roles in developing salinity stress tolerant barley varieties.

Barley and Wheat Share the Same Gene Controlling the Short Basic Vegetative Period

Basic vegetative period (BVP) is an important trait for determining flowering time and adaptation to variable environments. A short BVP barley mutant is about 30 d shorter than its wild type. Genetic analysis using 557 F2 individuals revealed that the short BVP is governed by a single recessive gene (BVP-1) and was further validated in 2 090 F3 individuals. The BVP-1 gene was first mapped to barley chromosome 1H using SSR markers. Comparative genomic analysis demonstrated that the chromosome region of BVP-1 is syntenic to rice chromosome 5 and Brachypodium chromosome 2. Barley ESTs/genes were identified after comparison with candidate genes in rice and Brachypodium; seven new gene-specific markers were developed and mapped in the mapping populations. The BVP-1 gene co-segregated with the Mot1 and Ftsh4 genes and was flanked by the gene-specific markers AK252360 (0.2 cM) and CA608558 (0.5 cM). Further analysis demonstrated that barley and wheat share the same short BVP gene controlling early flowering.