Diversity in bread and durum wheat stigma morphology and linkage of increased stigma length to dwarfing gene Rht14.

KEY MESSAGE: The dwarfing allele Rht14 of durum wheat associates with greater stigma length, an important trait for hybrid breeding, whilst major dwarfing alleles Rht-B1b and Rht-D1b showed little to no effect. Although much understudied in wheat, the stigma is a crucial component for attaining grain set, the fundamental basis for yield, particularly in hybrid production systems where successful grain set relies on wind-driven pollen dispersal by the male parent and effective pollen capture by the female parent. Females with long stigma that exsert early are thought to be advantageous. Using glasshouse-grown lines, we examined variation in Total Stigma Length (TSL) across diverse panels comprising 27 durum and 116 bread wheat genotypes. Contrasting genotypes were selected for population development and genetic analysis. Quantitative trait loci (QTL) analysis was performed on a durum F2 population and a bread wheat recombinant inbred line (RIL) population. Contrasting with studies of anther length, we found no large effect on TSL of the GA-insensitive semi-dwarfing genes Rht-B1 and Rht-D1 in either durum or bread wheat. However, in durum cultivar Italo, we identified a region on chromosome 6A which is robustly associated with larger TSL and contains the Rht14 allele for reduced plant height, a trait that is favourable for female line development in hybrid systems. This dual effect locus explained 25.2 and 19.2% of TSL phenotypic variation in experiments across two growing seasons, with preliminary results suggesting this locus may increase TSL when transferred to bread wheat. In a bread wheat, RIL population minor QTL on 1A and 2A was indicated, but the strongest association was with Ppd-B1. Methods developed here, and the identification of a TSL-enhancing locus provides advances and further opportunities in the study of wheat stigma.

Wheat ms5 male-sterility is induced by recessive homoeologous A and D genome non-specific lipid transfer proteins.

Nuclear male-sterile mutants with non-conditional, recessive and strictly monogenic inheritance are useful for both hybrid and conventional breeding systems, and have long been a research focus for many crops. In allohexaploid wheat, however, genic redundancy results in rarity of such mutants, with the ethyl methanesulfonate-induced mutant ms5 among the few reported to date. Here, we identify TaMs5 as a glycosylphosphatidylinositol-anchored lipid transfer protein required for normal pollen exine development, and by transgenic complementation demonstrate that TaMs5-A restores fertility to ms5. We show ms5 locates to a centromere-proximal interval and has a sterility inheritance pattern modulated by TaMs5-D but not TaMs5-B. We describe two allelic forms of TaMs5-D, one of which is non-functional and confers mono-factorial inheritance of sterility. The second form is functional but shows incomplete dominance. Consistent with reduced functionality, transcript abundance in developing anthers was found to be lower for TaMs5-D than TaMs5-A. At the 3B homoeolocus, we found only non-functional alleles among 178 diverse hexaploid and tetraploid wheats that include landraces and Triticum dicoccoides. Apparent ubiquity of non-functional TaMs5-B alleles suggests loss-of-function arose early in wheat evolution and, therefore, at most knockout of two homoeoloci is required for sterility. This work provides genetic information, resources and tools required for successful implementation of ms5 sterility in breeding systems for bread and durum wheats.

Molecular basis of adaptation to high soil boron in wheat landraces and elite cultivars.

Environmental constraints severely restrict crop yields in most production environments, and expanding the use of variation will underpin future progress in breeding. In semi-arid environments boron toxicity constrains productivity, and genetic improvement is the only effective strategy for addressing the problem. Wheat breeders have sought and used available genetic diversity from landraces to maintain yield in these environments; however, the identity of the genes at the major tolerance loci was unknown. Here we describe the identification of near-identical, root-specific boron transporter genes underlying the two major-effect quantitative trait loci for boron tolerance in wheat, Bo1 and Bo4 (ref. 2). We show that tolerance to a high concentration of boron is associated with multiple genomic changes including tetraploid introgression, dispersed gene duplication, and variation in gene structure and transcript level. An allelic series was identified from a panel of bread and durum wheat cultivars and landraces originating from diverse agronomic zones. Our results demonstrate that, during selection, breeders have matched functionally different boron tolerance alleles to specific environments. The characterization of boron tolerance in wheat illustrates the power of the new wheat genomic resources to define key adaptive processes that have underpinned crop improvement.

Diversity in boron toxicity tolerance of Australian barley (Hordeum vulgare L.) genotypes.

BACKGROUND: Boron (B) is an important micronutrient for plant growth, but is toxic when levels are too high. This commonly occurs in environments with alkaline soils and relatively low rainfall, including many of the cereal growing regions of southern Australia. Four major genetic loci controlling tolerance to high soil B have been identified in the landrace barley, Sahara 3771. Genes underlying two of the loci encode the B transporters HvBot1 and HvNIP2;1. RESULTS: We investigated sequence and expression level diversity in HvBot1 and HvNIP2;1 across barley germplasm, and identified five novel coding sequence alleles for HvBot1. Lines were identified containing either single or multiple copies of the Sahara HvBot1 allele. We established that only the tandemly duplicated Sahara allele conferred B tolerance, and this duplicated allele was found only in a set of nine lines accessioned in Australian collections as Sahara 3763-3771. HvNIP2;1 coding sequences were highly conserved across barley germplasm. We identified the likely causative SNP in the 5'UTR of Sahara HvNIP2;1, and propose that the creation of a small upstream open reading frame interferes with HvNIP2;1 translation in Sahara 3771. Similar to HvBot1, the tolerant HvNIP2;1 allele was unique to the Sahara barley accessions. We identified a new source of the 2H B tolerance allele controlling leaf symptom development, in the landrace Ethiopia 756. CONCLUSIONS: Ethiopia 756, as well as the cultivar Sloop Vic which carries both the 2H and HvBot1 B tolerance alleles derived from Sahara 3771, may be valuable as alternative parents in breeding programs targeted to high soil B environments. There is significant diversity in B toxicity tolerance among contemporary Australian barley varieties but this is not related to variation at any of the four known B tolerance loci, indicating that novel, as yet undiscovered, sources of tolerance exist.

The scutellar vascular bundle-specific promoter of the wheat HD-Zip IV transcription factor shows similar spatial and temporal activity in transgenic wheat, barley and rice.

An HD-Zip IV gene from wheat, TaGL9, was isolated using a Y1H screen of a cDNA library prepared from developing wheat grain. TaGL9 has an amino acid sequence distinct from other reported members of the HD-Zip IV family. The 3' untranslated region of TaGL9 was used as a probe to isolate a genomic clone of the TaGL9 homologue from a BAC library prepared from Triticum durum L. cv. Langdon. The full-length gene containing a 3-kb-long promoter region was designated TdGL9H1. Spatial and temporal activity of TdGL9H1 was examined using promoter-GUS fusion constructs in transgenic wheat, barley and rice plants. Whole-mount and histochemical GUS staining patterns revealed grain-specific expression of TdGL9H1. GUS expression was initially observed between 3 and 8 days after pollination (DAP) in embryos at the globular stage and adjacent to the embryo fraction of the endosperm. Expression was strongest in the outer cell layer of the embryo. In developed wheat and barley embryos, strong activity of the promoter was only detected in the main vascular bundle of the scutellum, which is known to be responsible for the uptake of nutrients from the endosperm during germination and the endosperm-dependent phase of seedling development. Furthermore, this pattern of GUS staining was observed in dry seeds several weeks after harvesting but quickly disappeared during imbibition. The promoter of this gene could be a useful tool for engineering of early seedling vigour and protecting the endosperm to embryo axis pathway from pathogens during grain desiccation and storage.

A Major Locus on Wheat Chromosome 7B Associated With Late-Maturity α-Amylase Encodes a Putative ent-Copalyl Diphosphate Synthase.

Many wheat varieties have the potential to develop unacceptably high levels of α-amylase in the grains if exposed to a cool temperature shock or simply cool temperature during the early to middle stages of grain filling. This phenomenon is referred to as late maturity α-amylase (LMA). The enzyme persists in the grain until harvest and may result in wheat with a low Falling Number that does not meet receival and export specifications. Resistance to LMA is therefore a valuable target for wheat breeders and wheat industries in general. Genetic evidence implicating a locus on the long arm of chromosome 7B in variation in LMA phenotype was confirmed in this investigation. Through intensive fine-mapping an ent-copalyl diphosphate synthase (CPS), hitherto named LMA-1, was identified as the likely candidate gene associated with variation in LMA phenotype. Single Nucleotide Polymorphisms (SNPs) within the LMA-1 coding sequence of Chinese Spring, Maringa and Halberd result in either prematurely terminated or functionally altered proteins that are associated with useful levels of resistance to LMA. LMA-1 transcripts detected in de-embryonated grain tissue from around 15 days after anthesis, several days before the synthesis of α-amylase, were low in the resistant varieties Chinese Spring and Maringa compared with LMA susceptible genotype Spica. This was associated with a dramatic reduction in the concentrations of intermediates in the gibberellin biosynthesis pathway such as GA19, evidence that LMA-1 was functioning as CPS in the gibberellin biosynthesis pathway. A survey of a large collection of Australian and international wheat varieties distinguished 9 major haplotypes at the LMA-1 locus. Generally, within classes, there was notable variation for LMA phenotype and evidence for genotypes whose resistance is presumed to be due to genetic loci located elsewhere on the wheat genome. Further investigation is required to characterize the sequence of steps between LMA-1 and α-amylase synthesis as well as to gain a better understanding of the role and potential impact of other genetic loci. Diagnostic markers for sources of resistance and SNP variation reported in this study should assist breeders to deploy resistance associated with LMA-1 variants in breeding programs.

Characterization of the wheat endosperm transfer cell-specific protein TaPR60.

The TaPR60 gene from bread wheat encodes a small cysteine-rich protein with a hydrophobic signal peptide, predicted to direct the TaPR60 protein to a secretory pathway. It was demonstrated by heterologous expression of recombinant TaPR60 protein that the signal peptide is recognized and cleaved in yeast cells. The full-length gene including promoter sequence of a TaPR60 orthologue was cloned from a BAC library of Triticum durum. A transcriptional promoter-GUS fusion was stably transformed into wheat, barley and rice. The strongest GUS expression in wheat and barley was found in the endosperm transfer cells, while in rice the promoter was active inside the starchy endosperm during the early stages of grain filling. The TaPR60 gene was also used as bait in a yeast two-hybrid screen. Five proteins were identified in the screen, and for some of these prey proteins, the interaction was confirmed by co-immunoprecipitation. The signal peptide binding proteins, TaUbiL1 and TaUbiL2, are homologues of animal proteins, which belong to proteolytic complexes, and therefore may be responsible for TaPR60 processing or degradation of the signal peptide. Other proteins that interact with TaPR60 may have a function in TaPR60 secretion or regulation of this process. Examination of a three dimensional model of TaPR60 suggested that this protein could be involved in binding of lipidic molecules.

Multiple genetic loci for zinc uptake and distribution in barley (Hordeum vulgare).

Micronutrient malnutrition, often called 'hidden hunger', affects over two billion people globally. This is particularly problematic in developing countries where widespread zinc (Zn) deficiency exists as a result of a predominantly plant-based diet. Furthermore, supplemental fertilizers are often unavailable or unaffordable in impoverished regions where soil infertility is common. Delivery of more Zn via food grains is theoretically possible through selective breeding strategies, but severe technical difficulties associated with trace element research have limited research on the underlying genetic components of Zn nutrition. Genetic dissection of Zn nutrition involved a pre-existing doubled haploid mapping population of barley (Hordeum vulgare). Association of mineral nutrient accumulation traits with regions of the barley genome was determined in two seasons of growth to maturity, using mapmanager qtx and QGene 4.0. Nine genetic loci segregating in the population associated clearly with measured traits, including five that contributed to grain Zn status. Pooling two-row doubled haploids by selecting the three most favourable alleles increased grain Zn content and concentration by an average of 53 and 75%, respectively. These results will inform breeding efforts for increased Zn density in the major food grain, wheat (Triticum aestivum), by enabling syntenic marker-assisted selection in conventional breeding programmes.

Genes and traits associated with chromosome 2H and 5H regions controlling sensitivity of reproductive tissues to frost in barley.

Frost at flowering can cause significant damage to cereal crops. QTL for low temperature tolerance in reproductive tissues (LTR tolerance) were previously described on barley 2HL and 5HL chromosome arms. With the aim of identifying potential LTR tolerance mechanisms, barley Amagi Nijo x WI2585 and Haruna Nijo x Galleon populations were examined for flowering time and spike morphology traits associated with the LTR tolerance loci. In spring-type progeny of both crosses, winter alleles at the Vrn-H1 vernalization response locus on 5H were linked in coupling with LTR tolerance and were unexpectedly associated with earlier flowering. In contrast, tolerance on 2HL was coupled with late flowering alleles at a locus we named Flt-2L. Both chromosome regions influenced chasmogamy/cleistogamy (open/closed florets), although tolerance was associated with cleistogamy at the 2HL locus and chasmogamy at the 5HL locus. LTR tolerance controlled by both loci was accompanied by shorter spikes, which were due to fewer florets per spike on 5HL, but shorter rachis internodes on 2HL. The Eps-2S locus also segregated in both crosses and influenced spike length and flowering time but not LTR tolerance. Thus, none of the traits was consistently correlated with LTR tolerance, suggesting that the tolerance may be due to some other visible trait or an intrinsic (biochemical) property. Winter alleles at the Vrn-H1 locus and short rachis internodes may be of potential use in barley breeding, as markers for selection of LTR tolerance at 5HL and 2HL loci, respectively.

Germanium as a tool to dissect boron toxicity effects in barley and wheat.

Tolerance to boron (B) toxicity in barley (Hordeum vulgare L.) is partially attributable to HvNIP2;1, an aquaporin with permeability to B, as well as to silicon, arsenic and germanium (Ge). In this study, we mapped leaf symptoms of Ge toxicity in a doubled-haploid barley population (Clipper×Sahara 3771). Two quantitative trait loci (QTL) associated with Ge toxicity symptoms were identified, located on Chromosomes 6H and 2H. These QTL co-located with two of four B toxicity tolerance loci previously mapped in the same population. The B toxicity tolerance gene underlying the 6H locus encodes HvNIP2;1, whereas the gene(s) and mechanisms underlying the 2H locus are as yet unknown. We provide examples of the application of Ge in studying specific aspects of B toxicity tolerance in plants, including screening of wheat (Triticum aestivum L.) and barley populations for altered function of HvNIP2;1 and related proteins. In particular, Ge may facilitate elucidation of the mechanism and gene(s) underlying the barley Chromosome 2H B tolerance locus.

A barley activation tagging system.

Activation tagging, as the result of random genomic insertion of either promoter or enhancer sequences, can produce novel, dominant mutations by over-expression of endogenous genes. This powerful genomics tool has been used extensively in dicot species such as Arabidopsis, while rice is the only cereal for which an equivalent system exists. In this study we describe an activation tagging system in barley based upon the maize Ac/Ds transposable element system. A modified Ds element (UbiDs) containing two maize polyubiquitin promoters, transposed in families derived from multiple independent UbiDs transformants and generated new Ds insertion events at frequencies ranging from 0% to 52% per family. The majority of transposed UbiDs elements activated high levels of adjacent flanking sequence transcription. Transposon-mediated expression was detected in all barley cell and tissue types analysed suggesting that this system is applicable to all aspects of plant development and biogenesis. In addition to transcriptional activation, this system is also capable of generating insertional knockout mutants and a UbiDs inactivated allele of the granule bound starch synthase I gene (waxy) was recovered that lead to reduced amylose accumulation. The recovery and analysis of dominant over-expression phenotypes generated by this system will provide a novel approach to understanding gene function in large cereal genomes where gene redundancy may mask conventional loss-of-function mutations.

Boron-toxicity tolerance in barley arising from efflux transporter amplification.

Both limiting and toxic soil concentrations of the essential micronutrient boron represent major limitations to crop production worldwide. We identified Bot1, a BOR1 ortholog, as the gene responsible for the superior boron-toxicity tolerance of the Algerian barley landrace Sahara 3771 (Sahara). Bot1 was located at the tolerance locus by high-resolution mapping. Compared to intolerant genotypes, Sahara contains about four times as many Bot1 gene copies, produces substantially more Bot1 transcript, and encodes a Bot1 protein with a higher capacity to provide tolerance in yeast. Bot1 transcript levels identified in barley tissues are consistent with a role in limiting the net entry of boron into the root and in the disposal of boron from leaves via hydathode guttation.

Construction of a genetic linkage map using MFLP and identification of molecular markers linked to domestication genes in narrow-leafed lupin (Lupinus angustifolius L.).

A mapping population of F(8)derived recombinant inbred lines (RILs) was established from a cross between a domesticated breeding line 83A:476 and a wild type P27255 in narrow-leaf lupin (Lupinus angustifolius L.). The parents together with the 89 RILs were subjected to DNA fingerprinting using microsatellite-anchored fragment length polymorphism (MFLP) to rapidly generate DNA markers to construct a linkage map. Five hundred and twenty two unique markers of which 21% were co-dominant, were generated and mapped. Phenotypic data for the domestication traits: mollis (soft seeds), leucospermus (white flower and seed colour); Lentus (reduced pod-shattering), iucundis (low alkaloid), Ku (early flowering) and moustache pattern on seed coats; were included. Three to 7 molecular markers were identified within 5 cM of each of these domestication genes. The anthracnose resistance gene Lanr1 was also mapped. Linkage groups were constructed using MapManager version QTXb20, resulting in 21 linkage groups consisting of 7 or more markers. The total map length was 1543 cM, with an average distance of 3.4 cM between adjacent markers. This is the first published map for a lupin species. The map can be exploited for marker assisted selection for genetic improvement in lupin breeding programs.