Sharon Westcott

GA-20 oxidase as a candidate for the semidwarf gene sdw1/denso in barley.

The barley sdw1/denso gene not only controls plant height but also yield and quality. The sdw1/denso gene was mapped to the long arm of chromosome 3H. Comparative genomic analysis revealed that the sdw1/denso gene was located in the syntenic region of the rice semidwarf gene sd1 on chromosome 1. The sd1 gene encodes a gibberellic acid (GA)-20 oxidase enzyme. The gene ortholog of rice sd1 was isolated from barley using polymerase chain reaction. The barley and rice genes showed a similar gene structure consisting of three exons and two introns. Both genes share 88.3% genomic sequence similarity and 89% amino acid sequence identity. A single nucleotide polymorphism was identified in intron 2 between barley varieties Baudin and AC Metcalfe with Baudin known to contain the denso semidwarf gene. The single nucleotide polymorphism (SNP) marker was mapped to chromosome 3H in a doubled haploid population of Baudin × AC Metcalfe with 178 DH lines. Quantitative trait locus analysis revealed that plant height cosegregated with the SNP. The sdw1/denso gene in barley is the most likely ortholog of the sd1 in rice. The result will facilitate understanding of the molecular mechanism controlling semidwarf phenotype and provide a diagnostic marker for selection of semidwarf gene in barley.

Identification of QTLs associated with salinity tolerance at late growth stage in barley

Salinity is a major abiotic stress to barley (Hordum vulgare L.) growth and yield. In the current study, quantitative trait loci (QTL) for yield and physiological components at the late growth stage under salt stress and non-stress environments were determined in barley using a double haploid population derived from a cross between CM72 (salt-tolerant) and Gairdner (salt-sensitive). A total of 30 QTLs for 10 traits, including tiller numbers (TN), plant height, spikes per line (SPL), spikes per plant (SPP), dry weight per plant, grains per plant, grain yield, shoot Na+ (NA) and K+ concentraitions (K) in shoot, and Na+/K+ ratio (NAK), were detected, with 17 and 13 QTLs under non-stress and salt stress, respectively. The phenotypic variation explained by individual QTL ranged from 3.25 to 29.81%. QTL flanked by markers bPb-1278 and bPb-8437 on chromosomes 4H was associated with TN, SPL, and SPP under salt stress. This locus may be useful in the breeding program of marker-assisted selection for improving salt tolerance of barley. However, QTLs associated with NA, K, and NAK differed greatly between non-stress and salt stress environments. It may be suggested that only the QTLs detected under salt stress are really associated with salt tolerance in barley.

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.

Discovery of Novel Bmy1 Alleles Increasing β-Amylase Activity in Chinese Landraces and Tibetan Wild Barley for Improvement of Malting Quality via MAS

China has a large barley germplasm collection which has not been well characterized and is therefore underutilized. The Bmy1 locus encoding the β-amylase enzyme on chromosome 4H has been well characterized in the worldwide barley germplasm collections due to its importance in the malting and brewing industry. The Bmy1 locus was chosen as an indicator to understand genetic potential for improvement of malting quality in Chinese landraces and Tibetan wild barley. The genetic diversity of 91 barley accessions was assessed using allele specific Multiplex-ready molecular markers. Eight accessions were further sequenced, based on the Multiplex-ready marker diversity for Bmy1 in the germplasm. Six of the eight accessions clustered together in a unique group, and showed similarities to ‘Haruna Nijo’, wild barley accession PI296896 and ‘Ashqelon’. Sequence comparisons with the known Bmy1 alleles identified not only the existing 13 amino acid substitutions, but also a new substitution positioned at A387T from a Chinese landrace W127, which has the highest β-amylase activity. Two new alleles/haplotypes namely Bmy1-Sd1c and Bmy1-Sd5 were designated based on different amino acid combinations. We identified new amino acid combination of C115, D165, V233, S347 and V430 in the germplasm. The broad variation in both β-amylase activity and amino acid composition provides novel alleles for the improvement of malting quality for different brewing styles, which indicates the high potential value of the Chinese landraces and Tibetan wild barley.

QTLs and their interaction determining different heading dates of barley in Australia and China

Heading date is a major determinant of the regional and seasonal adaptation of barley varieties. The dogma is that introduced germplasm is more likely to be adapted if it is derived from a similar latitude. However, barley germplasm introduced from similar latitudes of South-East Asia is extremely early heading in the Australian environments and vice versa. A doubled-haploid population from a cross of an Australian barley Galleon and a Japanese barley Haruna Nijo was evaluated for heading date in Australia (Perth, 31°56′S) and China (Wuhan, 30°33′N) under normal autumn sowing, late sowing in the field, and extended-light glasshouse conditions. One major QTL was identified on chromosome 5H under the three conditions in China. The single QTL accounted for up to 50% of phenotypic variation for heading date. The Australian variety contributed to late heading date. Two QTLs on chromosomes 4H and 5H were detected for controlling heading date in Australia. The QTL/QTL interaction contributed up to 35.8% of phenotypic variation for heading date in Australia, which is the major reason for the extremely early heading date of the Japanese variety in the Australian environment. The chromosome 5H QTL was detected at the same chromosomal location when the population was grown in either China or Australia. In both environments the Australian variety contributed to the late heading date. Selection against the Japanese alleles of chromosomes 4H and 5H QTLs could eliminate the extremely early genotype in Australia and selection against the Australian allele of chromosome 5H QTL could eliminate the extremely late genotype in China when Australian and Japanese germplasms are used in the breeding programs.

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.

A major QTL controlling adult plant resistance for barley leaf rust

Race-specific resistance genes (Rph) for leaf rust (Puccinia hordei) are often overcome by new pathotypes with matching virulence. Adult plant resistance (APR) is considered potentially more durable for controlling barley leaf rust. Previous studies established that the cultivar Pompadour carried APR to leaf rust. A doubled haploid population (DH) of 200 lines developed from a cross Pompadour/Stirling, and the parents were phenotyped for leaf rust resistance at five field experimental sites in three agricultural zones in Australia. Using a linkage map of SSR and DArT molecular markers, a major QTL associated with the leaf rust resistance was identified on the short arm of chromosome 5H. This QTL explained between 31% and 86% of the phenotypic variation for the APR at different sites. A PCR-based molecular marker was developed and mapped at 1.6 cM to the APR gene. The present study provides new genetic material and a molecular tool for breeding new varieties with adult plant leaf rust resistance using marker-assisted selection.

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.

Molecular marker-assisted backcrossing breeding: an example to transfer a thermostable β-amylase gene from wild barley

Molecular marker-assisted backcrossing (MABC) is widely recommended for transferring favorable alleles from a donor to an elite variety. The question remains whether MABC is an effective approach to developing a competitive commercial variety. Here, we illustrate the transfer of a thermostable β-amylase allele Sd3 from wild barley into a commercial barley variety Gairdner. The elite lines were chosen for the Regional Crop Variety Test that followed a standard conventional breeding process. The results demonstrated that the Sd3 allele not only increased enzyme thermostability but dramatically enhanced diastatic power, an important malting quality trait. The BC1F1 individuals had a fundamental impact on the comprehensive agronomic and quality traits of the final progenies, demonstrating the importance of screening at the early stage of backcrossing in MABC. There was sufficient genetic variation in the BC3F3 families to select other malting quality and agronomic traits. Ten individual breeding lines with improved β-amylase thermostability also had improved yields and grain plumpness. Three elite lines with improved malting quality and agronomic traits were selected to provide a parental line to incorporate the wild barley allele for breeding a commercial variety. A new strategy should be considered that uses marker-assisted selection and backcrossing to transfer a favorable allele from a wild parent.