Qiaojun Jia

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.

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 New QTL for Plant Height in Barley (Hordeum vulgare L.) Showing No Negative Effects on Grain Yield

Introduction Reducing plant height has played an important role in improving crop yields. The success of a breeding program relies on the source of dwarfing genes. For a dwarfing or semi-dwarfing gene to be successfully used in a breeding program, the gene should have minimal negative effects on yield and perform consistently in different environments. Methods In this study, 182 doubled haploid lines, generated from a cross between TX9425 and Naso Nijo, were grown in six different environments to identify quantitative trait loci (QTL) controlling plant height and investigate QTL × environments interaction. Results A QTL for plant was identified on 7H. This QTL showed no significant effects on other agronomic traits and yield components and consistently expressed in the six environments. A sufficient allelic effect makes it possible for this QTL to be successfully used in breeding programs.

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.

Molecular characterization and functional analysis of barley semi-dwarf mutant Riso no. 9265.

Backgroundsdw1/denso is one of the most important and useful semi-dwarf genes in barley breeding. At least four sdw1/denso alleles have been reported and HvGA20ox2 is considered as the candidate gene. Up to date, results of studies have not univocally proven the genetic relationship between sdw1/denso and HvGA20ox2.ResultsIn the present study, a complete deletion of Morex_contig_40861 including both HvGA20ox2 and Mloc_56463 genes was identified at the sdw1 locus from a semi-dwarf mutant Riso no. 9265. Expression of the genes encoding gibberellin biosynthesis (HvGA20ox1 and HvGA3ox2) were increased in the mutant compared to the wild type Bomi, while the expression of GA catabolic gene HvGA2ox3 was decreased. Over-expression of HvGA20ox2 could rescue the semi-dwarf phenotype and increase GAs concentration.ConclusionsWe confirmed that a GA biosynthetic enzyme HvGA20ox2, acted as GA 20-oxidase, is the functional gene for the sdw1/denso semi-dwarfism. Lose of HvGA20ox2 is partially compensated by HvGA20ox1 and further feedback is regulated by gibberellin. We also deduced that the sdw1/denso allele itself affects later heading owing to its reduced endogenous GAs concentration.

Toward Identification of Black Lemma and Pericarp Gene Blp1 in Barley Combining Bulked Segregant Analysis and Specific-Locus Amplified Fragment Sequencing

Black barley is caused by phytomelanin synthesized in lemma and/or pericarp and the trait is controlled by one dominant gene Blp1. The gene is mapped on chromosome 1H by molecular markers, but it is yet to be isolated. Specific-locus amplified fragment sequencing (SLAF-seq) is an effective method for large-scale de novo single nucleotide polymorphism (SNP) discovery and genotyping. In the present study, SLAF-seq with bulked segregant analysis (BSA) was employed to obtain sufficient markers to fine mapping Blp1 gene in an F2 population derived from Hatiexi No.1 × Zhe5819. Based on SNP screening criteria, a total of 77,542 polymorphic SNPs met the requirements for association analysis. Combining two association analysis methods, the overlapped region with a size of 32.41 Mb on chromosome 1H was obtained as the candidate region of Blp1 gene. According to SLAF-seq data, markers were developed in the target region and were used for mapping the Blp1 gene. Linkage analysis showed that Blp1 co-segregated with HZSNP34 and HZSNP36, and was delimited by two markers (HZSNP35 and HZSNP39) spanning 8.1 cM in 172 homozygous yellow grain F2 plants of Hatiexi No.1 × Zhe5819. More polymorphic markers were screened in the reduced target region and were used to genotype the population. As a result, Blp1 was delimited within a 1.66 Mb on chromosome 1H by the upstream marker HZSNP63 and the downstream marker HZSNP59. Our results demonstrated the utility of SLAF-seq-BSA approach to identify the candidate region and discover polymorphic markers at the specific targeted genomic region.

Gene Deletion in Barley Mediated by LTR-retrotransposon BARE

A poly-row branched spike (prbs) barley mutant was obtained from soaking a two-rowed barley inflorescence in a solution of maize genomic DNA. Positional cloning and sequencing demonstrated that the prbs mutant resulted from a 28 kb deletion including the inflorescence architecture gene HvRA2. Sequence annotation revealed that the HvRA2 gene is flanked by two LTR (long terminal repeat) retrotransposons (BARE) sharing 89% sequence identity. A recombination between the integrase (IN) gene regions of the two BARE copies resulted in the formation of an intact BARE and loss of HvRA2. No maize DNA was detected in the recombination region although the flanking sequences of HvRA2 gene showed over 73% of sequence identity with repetitive sequences on 10 maize chromosomes. It is still unknown whether the interaction of retrotransposons between barley and maize has resulted in the recombination observed in the present study.

Identification and Fine Mapping of a White Husk Gene in Barley (Hordeum vulgare L.)

Barley is the only crop in the Poaceae family with adhering husks at maturity. The color of husk at barely development stage could influence the agronomic traits and malting qualities of grains. A barley mutant with a white husk was discovered from the malting barley cultivar Supi 3 and designated wh (white husk). Morphological changes and the genetics of white husk barley were investigated. Husks of the mutant were white at the heading and flowering stages but yellowed at maturity. The diastatic power and α-amino nitrogen contents also significantly increased in wh mutant. Transmission electron microscopy examination showed abnormal chloroplast development in the mutant. Genetic analysis of F2 and BC1F1 populations developed from a cross of wh and Yangnongpi 5 (green husk) showed that the white husk was controlled by a single recessive gene (wh). The wh gene was initially mapped between 49.64 and 51.77 cM on chromosome 3H, which is syntenic with rice chromosome 1 where a white husk gene wlp1 has been isolated. The barley orthologous gene of wlp1 was sequenced from both parents and a 688 bp deletion identified in the wh mutant. We further fine-mapped the wh gene between SSR markers Bmac0067 and Bmag0508a with distances of 0.36 cM and 0.27 cM in an F2 population with 1115 individuals of white husk. However, the wlp1 orthologous gene was mapped outside the interval. New candidate genes were identified based on the barley genome sequence.