A novel dwarfing mutation in a green revolution gene from Brassica rapa

Mutations in the biosynthesis or signaling pathways of gibberellin (GA) can cause dwarfing phenotypes in plants, and the use of such mutations in plant breeding was a major factor in the success of the Green Revolution. DELLA proteins are GA signaling repressors whose functions are conserved in different plant species. Recent studies show that GA promotes stem growth by causing degradation of DELLA proteins via the ubiquitin-proteasome pathway. The most widely utilized dwarfing alleles in wheat (Triticum aestivum; e.g. Rht-B1b and Rht-D1b) encode GA-resistant forms of a DELLA protein that function as dominant and constitutively active repressors of stem growth. All of the previously identified dominant DELLA repressors from several plant species contain N-terminal mutations. Here we report on a novel dwarf mutant from Brassica rapa (Brrga1-d) that is caused by substitution of a conserved amino acid in the C-terminal domain of a DELLA protein. Brrga1-d, like N-terminal DELLA mutants, retains its repressor function and accumulates to high levels, even in the presence of GA. However, unlike wild-type and N-terminal DELLA mutants, Brrga1-d does not interact with a protein component required for degradation, suggesting that the mutated amino acid causes dwarfism by preventing an interaction needed for its degradation. This novel mutation confers nondeleterious dwarf phenotypes when transferred to Arabidopsis (Arabidopsis thaliana) and oilseed rape (Brassica napus), indicating its potential usefulness in other crop species.     

Dominant and pleiotropic effects of a GAI gene in wheat results from a lack of interaction between DELLA and GID1

Dominance, semidominance, and recessiveness are important modes of Mendelian inheritance. The phytohormone gibberellin (GA) regulates many plant growth and developmental processes. The previously cloned semidominant GA-insensitive (GAI) genes Reduced height1 (Rht1) and Rht2 in wheat (Triticum aestivum) were the basis of the Green Revolution. However, no completely dominant GAI gene has been cloned. Here, we report the molecular characterization of Rht-B1c, a dominant GAI allele in wheat that confers more extreme characteristics than its incompletely dominant alleles. Rht-B1c is caused by a terminal repeat retrotransposons in miniature insertion in the DELLA domain. Yeast two-hybrid assays showed that Rht-B1c protein fails to interact with GA-INSENSITIVE DWARF1 (GID1), thereby blocking GA responses and resulting in extreme dwarfism and pleiotropic effects. By contrast, Rht-B1b protein only reduces interaction with GID1. Furthermore, we analyzed its functions using near-isogenic lines and examined its molecular mechanisms in transgenic rice. These results indicated that the affinity between GID1 and DELLA proteins is key to regulation of the stability of DELLA proteins, and differential interactions determine dominant and semidominant gene responses to GA.     

Novel Genetic Variants of GA-Insensitive Rht-1 Genes in Hexaploid Wheat and Their Potential Agronomic Value

This study has found numerous novel genetic variants of GA-insensitive dwarfing genes with potential agricultural value for crop improvement. The cultivar, Spica is a tall genotype and possesses the wild-type genes of Rht-A1a, Rht-B1a and Rht-D1a. The cultivar Quarrion possesses a null mutant in the DELLA motif in each of the 3 genomes. This is a first report of a null mutant of Rht-A1. In addition, novel null mutants which differ from reported null alleles of Rht-B1b, Rht-B1e and Rht-D1b have been found in Quarrion, Carnamah and Whistler. The accession, Aus1408 has an allele of Rht-B1 with a mutation in the conserved ‘TVHYNP’ N-terminal signal binding domain with possible implications on its sensitivity to GA. Mutations in the conserved C-terminal GRAS domain of Rht-A1 alleles with possible effects on expression have been found in WW1842, Quarrion and Drysdale. Genetic variants with putative spliceosomal introns in the GRAS domain have been found in all accessions except Spica. Genome-specific cis-sequences about 124 bp upstream of the start codon of the Rht-1 gene have been identified for each of the three genomes.     

Wild-type alleles of Rht-B1 and Rht-D1 as independent determinants of thousand-grain weight and kernel number per spike in wheat

The utilization of dwarfing genes Rht-B1b and Rht-D1b in wheat significantly increased grain yield and contributed to the “green revolution”. However, the benefit of Rht-B1b and Rht-D1b in drought environments has been debated. Although quantitative trait loci (QTL) for kernel number per spike (KN) and thousand-grain weight (TGW) have been found to be associated with Rht-B1 and Rht-D1, the confounding effect of environmental variation has made a direct association difficult to find. In this study, we used a doubled haploid population (225 lines) of Westonia × Kauz, in which both Rht-B1b (Kauz) and Rht-D1b (Westonia) segregated. The purpose of the study was to determine the interaction of Rht-B1 and Rht-D1 with grain yield components, namely KN and TGW, and to investigate genotype-by-environment interactions in glasshouse and field trials conducted in 2010 and 2011 in Western Australia. A genetic map of 1,156 loci was constructed using 195 microsatellite markers, two gene-based markers for Rht-B1 and Rht-D1, and 959 single nucleotide polymorphisms. The major QTL for TGW and KN were strongly linked to Rht-B1 and Rht-D1 loci and the positive effects were associated with the wild-type alleles, Rht-B1a and Rht-D1a. The major QTL of TGW were on chromosome 2D and 4B. The significant genetic effects (14.6–22.9 %) of TGW indicated that marker-assisted selection for TGW is possible, and markers gwm192a (206 bp) or gwm192b (236 bp) can be used as indicators of high TGW. For KN, one major QTL was detected on chromosome 4D in the analysis across three environments. The association of the wild-type alleles Rht-B1a and Rht-D1a in drought environments is discussed.     

Contrasting effects of dwarfing alleles and nitrogen availability on mineral concentrations in wheat grain

Background and aim

Concentrations of essential minerals in plant foods may have declined in modern high-yielding cultivars grown with large applications of nitrogen fertilizer (N). We investigated the effect of dwarfing alleles and N rate on mineral concentrations in wheat.

Methods

Gibberellin (GA)-insensitive reduced height (Rht) alleles were compared in near isogenic wheat lines. Two field experiments comprised factorial combinations of wheat variety backgrounds, alleles at the Rht-B1 locus (rht-B1a, Rht-B1b, Rht-B1c), and different N rates. A glasshouse experiment also included Rht-D1b and Rht-B1b+D1b in one background.

Results

In the field, depending on season, Rht-B1b increased crop biomass, dry matter (DM) harvest index, grain yield, and the economically-optimal N rate (N _opt ). Rht-B1b did not increase uptake of Cu, Fe, Mg or Zn so these minerals were diluted in grain. Nitrogen increased DM yield and mineral uptake so grain concentrations were increased (Fe in both seasons; Cu, Mg and Zn in one season). Rht-B1b reduced mineral concentrations at N _opt in the most N responsive season. In the glasshouse experiment, grain yield was reduced, and mineral concentrations increased, with Rht allele addition.

Conclusion

Effects of Rht alleles on Fe, Zn, Cu and Mg concentrations in wheat grain are mostly due to their effects on DM, rather than of GA-insensitivity on N _opt or mineral uptake. Increased N requirement in semi-dwarf varieties partly offsets this dilution effect.     

Identity of the Rht-11 and Rht-B1e reduced plant height genes

The Rht (reduced plant height) genes provided a basis for the ??green revolution??, which led to a substantial increase in wheat productivity. More than 20 Rht genes are now known in wheat, Rht-B1 and Rht-11 being among the most important ones. Analysis of a segregating F2 population and molecular analysis of the common wheat cultivars carrying the Rht-11 gene showed that the Rht-11 and Rht-B1e genes are identical.     

Rht-1 and Ppd-D1 associations with height, GA sensitivity, and days to heading in a worldwide bread wheat collection

Reduced height (Rht)-1 and Photoperiod (Ppd) have major effects on the adaptability of bread wheat (Triticum aestivum) to specific environments. Ppd–D1a is a photoperiod insensitive allele that reduces time to flowering. The gibberellin (GA) insensitive alleles Rht–B1b and Rht–D1b shorten plant stature and were important components of the ‘green revolution’. Two additional Rht–B1 alleles were recently identified that contain a 160 or 197 bp insertion upstream of the coding region and may affect plant height or GA sensitivity Wilhelm et al. (Theor Appl Gen doi:10.1007/s00122-013-2088-7, 2013b). We determined the frequency of the five alleles in a worldwide core collection of 372 wheat accessions (372CC) and estimated their effects on height, days to heading, and GA sensitivity when the collection was grown in pots outdoors or in the glasshouse. This revealed that each allele was widespread geographically with frequencies ranging from 0.12 to 0.25. Ppd-D1a was associated with significant (p ≤ 0.05) reductions in days to heading and height relative to photoperiod sensitive Ppd-D1b. Relative to wild type, Rht-B1b and Rht-D1b each resulted in significant reductions in height (approximately 30 %) and GA sensitivity. The 160 and 197 bp alleles were associated with significant height reductions of 18 and 12 %, respectively, and with non-significant reductions in GA sensitivity relative to wild type. Two statistical methods were developed and used to estimate GA sensitivity of the 372CC accessions, but novel GA insensitive alleles were not identified. Further characterization of the Rht-B1 insertion alleles is required, but our results suggest these may enable fine adjustments in plant height.     

Whole Genome Association Mapping of Plant Height in Winter Wheat (Triticum aestivum L.)

The genetic architecture of plant height was investigated in a set of 358 recent European winter wheat varieties plus 14 spring wheat varieties based on field data in eight environments. Genotyping of diagnostic markers revealed the Rht-D1b mutant allele in 58% of the investigated varieties, while the Rht-B1b mutant was only present in 7% of the varieties. Rht-D1 was significantly associated with plant height by using a mixed linear model and employing a kinship matrix to correct for population stratification. Further genotyping data included 732 microsatellite markers, resulting in 770 loci, of which 635 markers were placed on the ITMI map plus a set of 7769 mapped SNP markers genotyped with the 90 k iSELECT chip. When Bonferroni correction was applied, a total of 153 significant marker-trait associations (MTAs) were observed for plant height and the SSR markers (−log₁₀ (P-value) ≥4.82) and 280 (−log₁₀ (P-value) ≥5.89) for the SNPs. Linear regression between the most effective markers and the BLUEs for plant height indicated additive effects for the MTAs of different chromosomal regions. Analysis of syntenic regions in the rice genome revealed closely linked rice genes related to gibberellin acid (GA) metabolism and perception, i.e. GA20 and GA2 oxidases orthologous to wheat chromosomes 1A, 2A, 3A, 3B, 5B, 5D and 7B, ent-kaurenoic acid oxidase orthologous to wheat chromosome 7A, ent-kaurene synthase on wheat chromosome 2B, as well as GA-receptors like DELLA genes orthologous to wheat chromosomes 4B, 4D and 7A and genes of the GID family orthologous to chromosomes 2B and 5B. The data indicated that besides the widely used GA-insensitive dwarfing genes Rht-B1 and Rht-D1 there is a wide spectrum of loci available that could be used for modulating plant height in variety development.     

Identification and characterization of <Emphasis Type="Italic">Rht25</Emphasis>, a locus on chromosome arm 6AS affecting wheat plant height,heading time,and spike development

Key message

This study identified Rht25, a new plant height locus on wheat chromosome arm 6AS, and characterized its pleiotropic effects on important agronomic traits.

Abstract

Understanding genes regulating wheat plant height is important to optimize harvest index and maximize grain yield. In modern wheat varieties grown under high-input conditions, the gibberellin-insensitive semi-dwarfing alleles Rht-B1b and Rht-D1b have been used extensively to confer lodging tolerance and improve harvest index. However, negative pleiotropic effects of these alleles (e.g., poor seedling emergence and reduced biomass) can cause yield losses in hot and dry environments. As part of current efforts to diversify the dwarfing alleles used in wheat breeding, we identified a quantitative trait locus (QHt.ucw-6AS) affecting plant height in the proximal region of chromosome arm 6AS (<?0.4 cM from the centromere). Using a large segregating population (~?2800 gametes) and extensive progeny tests (70–93 plants per recombinant family), we mapped QHt.ucw-6AS as a Mendelian locus to a 0.2 cM interval (144.0–148.3 Mb, IWGSC Ref Seq v1.0) and show that it is different from Rht18. QHt.ucw-6AS is officially designated as Rht25, with Rht25a representing the height-increasing allele and Rht25b the dwarfing allele. The average dwarfing effect of Rht25b was found to be approximately half of the effect observed for Rht-B1b and Rht-D1b, and the effect is greater in the presence of the height-increasing Rht-B1a and Rht-D1a alleles than in the presence of the dwarfing alleles. Rht25b is gibberellin-sensitive and shows significant pleiotropic effects on coleoptile length, heading date, spike length, spikelet number, spikelet density, and grain weight. Rht25 represents a new alternative dwarfing locus that should be evaluated for its potential to improve wheat yield in different environments.

     

Control of late maturity alpha-amylase in wheat by the dwarfing gene Rht-D1b and genes on the 1B/1R translocation

The occurrence of late maturity alpha-amylase (LMA) was investigated using two doubled haploid wheat populations segregating for the dwarfing gene Rht-D1b and the 1B/1R translocation. Genotypes were assessed in the field and in controlled environments where a cold-shock treatment was used to induce LMA. Results from field-grown genotypes from the cross Spark × Rialto suggest that the absence of Rht-D1b or the presence of the 1B/1R translocation increases the expression of LMA.These two genetic factors were found to act independently and to have a positive interaction (complementary epistasis). In Option × Potent genotypes fixed for Rht-D1b, the 1B/1R effect was similar to that seen in the equivalent Spark × Rialto genotypes. Under controlled environment conditions, genotypes with the 1B/1R translocation showed a higher occurrence of LMA under both control and cold-shock conditions. 1B/1R was present in the majority of genotypes expressing LMA under control and cold-shock conditions. The results point to the novel finding that the 1B/1R translocation increases the expression of alpha-amylase in LMA-prone germplasm independently of effects of Rht-D1b, whereas previously it had been thought to act by a modification of the Rht-D1b effect.     

The 'Green Revolution' dwarfing genes play a role in disease resistance in Triticum aestivum and Hordeum vulgare

The Green Revolution dwarfing genes, Rht-B1b and Rht-D1b, encode mutant forms of DELLA proteins and are present in most modern wheat varieties. DELLA proteins have been implicated in the response to biotic stress in the model plant, Arabidopsis thaliana. Using defined wheat Rht near-isogenic lines and barley Sln1 gain of function (GoF) and loss of function (LoF) lines, the role of DELLA in response to biotic stress was investigated in pathosystems representing contrasting trophic styles (biotrophic, hemibiotrophic, and necrotrophic). GoF mutant alleles in wheat and barley confer a resistance trade-off with increased susceptibility to biotrophic pathogens and increased resistance to necrotrophic pathogens whilst the converse was conferred by a LoF mutant allele. The polyploid nature of the wheat genome buffered the effect of single Rht GoF mutations relative to barley (diploid), particularly in respect of increased susceptibility to biotrophic pathogens. A role for DELLA in controlling cell death responses is proposed. Similar to Arabidopsis, a resistance trade-off to pathogens with contrasting pathogenic lifestyles has been identified in monocotyledonous cereal species. Appreciation of the pleiotropic role of DELLA in biotic stress responses in cereals has implications for plant breeding.     

矮秆基因对小麦部分农艺性状的效应

以中国主要麦区的124份小麦品种为材料,利用分子标记和系谱分析相结合,对其按照所含的矮秆基因Rht-B1b、Rht-D1b和Rht8进行分类,结合田间株高、旗叶长、小穗数和穗粒数以及室内苗期根系长度等农艺形状的调查,分析不同矮秆基因对小麦农艺性状的效应.结果显示:(1)参试的124份小麦品种(系)中23份含有Rht-B1b,7份含有Rht-D1b,22份含有Rht8基因,34份同时含有Rht-B1b和Rht8,16份同时含有Rht-D1b和Rht8,可分为6组.(2)Rht-B1b和Rht-D1b在降低株高的同时也缩短了旗叶的长度和苗期叶长,Rht8对株高的影响较弱,对旗叶和苗期叶长的影响也较小;3个矮秆基因对苗期根系长度、小穗数没有显著影响;Rht-D1b和Rht8显著增加穗粒数.研究表明,矮秆基因Rht8对小麦株高以及其他农艺性状的影响均较小,但能够显著增加穗粒数,是小麦矮化育种中比较理想的矮秆基因.     

Genome-wide association study reveals genetic architecture of coleoptile length in wheat

Key message

Eight QTL for coleoptile length were identified in a genome-wide association study on a set of 893 wheat accessions, four of which are novel loci.

Abstract

Wheat cultivars with long coleoptiles are preferred in wheat-growing regions where deep planting is practiced. However, the wide use of gibberellic acid (GA)-insensitive dwarfing genes, Rht-B1b and Rht-D1b, makes it challenging to breed dwarf wheat cultivars with long coleoptiles. To understand the genetic basis of coleoptile length, we performed a genome-wide association study on a set of 893 landraces and historical cultivars using 5011 single nucleotide polymorphism (SNP) markers. Structure analysis revealed four subgroups in the association panel. Association analysis results suggested that Rht-B1b and Rht-D1b genes significantly reduced coleoptile length, and eight additional quantitative trait loci (QTL) for coleoptile length were also identified. These QTL explained 1.45–3.18 and 1.36–3.11% of the phenotypic variation in 2015 and 2016, respectively, and their allelic substitution effects ranged from 0.31 to 1.75 cm in 2015, and 0.63–1.55 cm in 2016. Of the eight QTL, QCL.stars-1BS1, QCL.stars-2DS1, QCL.stars-4BS2, and QCL.stars-5BL1 are likely novel loci for coleoptile length. The favorable alleles in each accession ranged from two to eight with an average of 5.8 at eight loci in the panel, and more favorable alleles were significantly associated with longer coleoptile, suggesting that QTL pyramiding is an effective approach to increase wheat coleoptile length.

     

Genetic characterization and mapping of the Rht-1 homoeologs and flanking sequences in wheat

The introgression of Reduced height (Rht)-B1b and Rht-D1b into bread wheat (Triticum aestivum) varieties beginning in the 1960s led to improved lodging resistance and yield, providing a major contribution to the ‘green revolution’. Although wheat Rht-1 and surrounding sequence is available, the genetic composition of this region has not been examined in a homoeologous series. To determine this, three Rht-1-containing bacterial artificial chromosome (BAC) sequences derived from the A, B, and D genomes of the bread wheat variety Chinese Spring (CS) were fully assembled and analyzed. This revealed that Rht-1 and two upstream genes were highly conserved among the homoeologs. In contrast, transposable elements (TEs) were not conserved among homoeologs with the exception of intronic miniature inverted-repeat TEs (MITEs). In relation to the Triticum urartu ancestral line, CS-A genic sequences were highly conserved and several colinear TEs were present. Comparative analysis of the CS wheat BAC sequences with assembled Poaceae genomes showed gene synteny and amino acid sequences were well preserved. Further 5′ and 3′ of the wheat BAC sequences, a high degree of gene colinearity is present among the assembled Poaceae genomes. In the 20 kb of sequence flanking Rht-1, five conserved non-coding sequences (CNSs) were present among the CS wheat homoeologs and among all the Poaceae members examined. Rht-A1 was mapped to the long arm of chromosome 4 and three closely flanking genetic markers were identified. The tools developed herein will enable detailed studies of Rht-1 and linked genes that affect abiotic and biotic stress response in wheat.     

Characterization of the first dominant dwarf maize mutant carrying a single amino acid insertion in the VHYNP domain of the <Emphasis Type="Italic">dwarf8</Emphasis> gene

The “green revolution” involving mainly wheat and rice was based on the use by breeders of semidominant mutations involved in the signal transduction pathway of Gibberellin (GA). In particular, mutations in the Reduced height (Rht) gene of wheat have been used to reduce plant height and consequently to avoid storm damage and lodging. These genes have been cloned and they encode for DELLA proteins which contain an N-terminal DELLA and a VHYNP domain essential for GA-dependent degradation of these proteins. In maize several mutations have been isolated which affect gibberellin biosynthesis and perception and in particular, mutations in Dwarf8 (D8) gene cause a severe dwarfing phenotype. D8 gene has been identified as an orthologue of Rht (Reduced height), Slr1(Slender rice 1) and Gibberellic Acid Insensitive (GAI) genes, this latter is a negative regulator of GA response in Arabidopsis. In this work, for the first time, we isolated and characterized a single amino acid insertion in the VHYNP domain of D8 maize gene causing the appearance of a dominant dwarf mutation. This spontaneous mutation, named D8-1023, showed a phenotype which is less severe in comparison with the other D8 mutants previously isolated which have modifications in the DELLA domain. This mutant appears to be an useful tool either to study the mechanism of GA-modulated growth in plants or to lower the height of maize tropical germplasm for breeding purposes.     

Arabidopsis DELLA Protein Degradation Is Controlled by a Type-One Protein Phosphatase,TOPP4

Gibberellins (GAs) are a class of important phytohormones regulating a variety of physiological processes during normal plant growth and development. One of the major events during GA-mediated growth is the degradation of DELLA proteins, key negative regulators of GA signaling pathway. The stability of DELLA proteins is thought to be controlled by protein phosphorylation and dephosphorylation. Up to date, no phosphatase involved in this process has been identified. We have identified a dwarfed dominant-negative Arabidopsis mutant, named topp4-1. Reduced expression of TOPP4 using an artificial microRNA strategy also resulted in a dwarfed phenotype. Genetic and biochemical analyses indicated that TOPP4 regulates GA signal transduction mainly via promoting DELLA protein degradation. The severely dwarfed topp4-1 phenotypes were partially rescued by the DELLA deficient mutants rga-t2 and gai-t6, suggesting that the DELLA proteins RGA and GAI are required for the biological function of TOPP4. Both RGA and GAI were greatly accumulated in topp4-1 but significantly decreased in 35S-TOPP4 transgenic plants compared to wild-type plants. Further analyses demonstrated that TOPP4 is able to directly bind and dephosphorylate RGA and GAI, confirming that the TOPP4-controlled phosphorylation status of DELLAs is associated with their stability. These studies provide direct evidence for a crucial role of protein dephosphorylation mediated by TOPP4 in the GA signaling pathway.