Genome-wide association of barley plant growth under drought stress using a nested association mapping population.

BACKGROUND: Barley (Hordeum vulgare L.) is the fourth most important cereal crop worldwide. Barley production is compromised by many abiotic stresses including drought. Wild barley is a valuable source of alleles that can improve adaptation of cultivated barley to drought stress. RESULTS: In the present study, a nested association mapping population named HEB-25, consisting of 1420 BC1S3 lines that were developed by crossing 25 different wild barley accessions to the elite barley cultivar 'Barke', was evaluated under both control and drought-stressed conditions in the Australian Plant Phenomics Facility, University of Adelaide. Overall, 14 traits reflecting the performance of individual plants in each treatment were calculated from non-destructive imaging over time and destructive end-of-experiment measurements. For each trait, best linear unbiased estimators (BLUEs) were calculated and used for genome-wide association study (GWAS) analysis. Among the quantitative trait loci (QTL) identified for the 14 traits, many co-localise with known inflorescence and developmental genes. We identified a QTL on chromosome 4H where, under drought and control conditions, wild barley alleles increased biomass by 10 and 17% respectively compared to the Barke allele. CONCLUSIONS: Across all traits, QTL which increased phenotypic values were identified, providing a wider range of genetic diversity for the improvement of drought tolerance in barley.

Identification of proteins associated with malting quality in a subset of wild barley introgression lines.

Malted barley is an important ingredient used in the brewing and distilling industry worldwide. In this study, we used a proteomics approach to investigate the biochemical function of previously identified quantitative trait loci (QTLs) on barley chromosomes 1H and 4H that influence malting quality. Using a subset of barley introgression lines containing wild barley (Hordeum vulgare ssp. spontaneum) alleles at these QTLs, we validated that wild barley alleles at the chromosome 1H QTL reduced overall malting quality, whereas wild barley alleles at the chromosome 4H QTL improved the malting quality parameters α-amylase activity, VZ45, and Kolbach index compared to the control genotype Scarlett. 2DE was used to detect changes in protein expression during the first 72 h of micromalting associated with these QTLs. In total, 16 protein spots showed a significant change in expression between the introgression lines and Scarlett, of which 14 were successfully identified with MS. Notably, the wild barley alleles in the line containing the chromosome 4H QTL showed a sixfold increased expression of a limit dextrinase inhibitor. The possible role of the identified proteins in malting quality is discussed. The knowledge gained will assist ongoing research toward cloning the genes underlying these important QTL.

Accumulation of mutations in genes associated with sexual reproduction contributed to the domestication of a vegetatively propagated staple crop, enset.

Enset (Ensete ventricosum (Welw.) Cheesman) is a drought tolerant, vegetatively propagated crop that was domesticated in Ethiopia. It is a staple food for more than 20 million people in Ethiopia. Despite its current importance and immense potential, enset is among the most genetically understudied and underexploited food crops. We collected 230 enset wild and cultivated accessions across the main enset producing regions in Ethiopia and applied amplified fragment length polymorphism (AFLP) and genotype by sequencing (GBS) analyses to these accessions. Wild and cultivated accessions were clearly separated from each other, with 89 genes found to harbour SNPs that separated wild from cultivated accessions. Among these, 17 genes are thought to be involved in flower initiation and seed development. Among cultivated accessions, differentiation was mostly associated with geographical location and with proximity to wild populations. Our results indicate that vegetative propagation of elite clones has favoured capacity for vegetative growth at the expense of capacity for sexual reproduction. This is consistent with previous reports that cultivated enset tends to produce non-viable seeds and flowers less frequently than wild enset.

Variation among S-locus haplotypes and among stylar RNases in almond.

In many plant species, self-incompatibility systems limit self-pollination and mating among relatives. This helps maintain genetic diversity in natural populations but imposes constraints in agriculture and plant breeding. In almond [Prunus dulcis (Mill.) D.A. Webb], the specificity of self-incompatibility is mainly determined by stylar ribonuclease (S-RNase) and S-haplotype-specific F-box (SFB) proteins, both encoded within a complex locus, S. Prior to this research, a nearly complete sequence was available for one S-locus haplotype. Here, we report complete sequences for four haplotypes and partial sequences for 11 haplotypes. Haplotypes vary in sequences of genes (particularly S-RNase and SFB), distances between genes and numbers and positions of long terminal repeat transposons. Haplotype variation outside of the S-RNase and SFB genes may help maintain functionally important associations between S-RNase and SFB alleles. Fluorescence-based assays were developed to distinguish among some S-RNase alleles. With three-dimensional modelling of five S-RNase proteins, conserved active sites were identified and variation was observed in electrostatic potential and in the numbers, characteristics and positions of secondary structural elements, loop anchoring points and glycosylation sites. A hypervariable region on the protein surface and differences in the number, location and types of glycosylation sites may contribute to determining S-RNase specificity.

Barley yield formation under abiotic stress depends on the interplay between flowering time genes and environmental cues.

Since the dawn of agriculture, crop yield has always been impaired through abiotic stresses. In a field trial across five locations worldwide, we tested three abiotic stresses, nitrogen deficiency, drought and salinity, using HEB-YIELD, a selected subset of the wild barley nested association mapping population HEB-25. We show that barley flowering time genes Ppd-H1, Sdw1, Vrn-H1 and Vrn-H3 exert pleiotropic effects on plant development and grain yield. Under field conditions, these effects are strongly influenced by environmental cues like day length and temperature. For example, in Al-Karak, Jordan, the day length-sensitive wild barley allele of Ppd-H1 was associated with an increase of grain yield by up to 30% compared to the insensitive elite barley allele. The observed yield increase is accompanied by pleiotropic effects of Ppd-H1 resulting in shorter life cycle, extended grain filling period and increased grain size. Our study indicates that the adequate timing of plant development is crucial to maximize yield formation under harsh environmental conditions. We provide evidence that wild barley alleles, introgressed into elite barley cultivars, can be utilized to support grain yield formation. The presented knowledge may be transferred to related crop species like wheat and rice securing the rising global food demand for cereals.

Genotyping by Sequencing in Almond: SNP Discovery, Linkage Mapping, and Marker Design.

In crop plant genetics, linkage maps provide the basis for the mapping of loci that affect important traits and for the selection of markers to be applied in crop improvement. In outcrossing species such as almond (Prunus dulcis Mill. D. A. Webb), application of a double pseudotestcross mapping approach to the F1 progeny of a biparental cross leads to the construction of a linkage map for each parent. Here, we report on the application of genotyping by sequencing to discover and map single nucleotide polymorphisms in the almond cultivars "Nonpareil" and "Lauranne." Allele-specific marker assays were developed for 309 tag pairs. Application of these assays to 231 Nonpareil × Lauranne F1 progeny provided robust linkage maps for each parent. Analysis of phenotypic data for shell hardness demonstrated the utility of these maps for quantitative trait locus mapping. Comparison of these maps to the peach genome assembly confirmed high synteny and collinearity between the peach and almond genomes. The marker assays were applied to progeny from several other Nonpareil crosses, providing the basis for a composite linkage map of Nonpareil. Applications of the assays to a panel of almond clones and a panel of rootstocks used for almond production demonstrated the broad applicability of the markers and provide subsets of markers that could be used to discriminate among accessions. The sequence-based linkage maps and single nucleotide polymorphism assays presented here could be useful resources for the genetic analysis and genetic improvement of almond.

QTL controlling grain filling under terminal drought stress in a set of wild barley introgression lines.

Drought is a major abiotic stress impeding the yield of cereal crops globally. Particularly in Mediterranean environments, water becomes a limiting factor during the reproductive developmental stage, causing yield losses. The wild progenitor of cultivated barley Hordeum vulgare ssp spontaneum (Hsp) is a potentially useful source of drought tolerance alleles. Wild barley introgression lines like the S42IL library may facilitate the introduction of favorable exotic alleles into breeding material. The complete set of 83 S42ILs was genotyped with the barley 9k iSelect platform in order to complete genetic information obtained in previous studies. The new map comprises 2,487 SNPs, spanning 989.8 cM and covering 94.5% of the Hsp genome. Extent and positions of introgressions were confirmed and new information for ten additional S42ILs was collected. A subset of 49 S42ILs was evaluated for drought response in four greenhouse experiments. Plants were grown under well-watered conditions until ten days post anthesis. Subsequently drought treatment was applied by reducing the available water. Several morphological and harvest parameters were evaluated. Under drought treatment, trait performance was reduced. However, there was no interaction effect between genotype and treatment, indicating that genotypes, which performed best under control treatment, also performed best under drought treatment. In total, 40 QTL for seven traits were detected in this study. For instance, favorable Hsp effects were found for thousand grain weight (TGW) and number of grains per ear under drought stress. In particular, line S42IL-121 is a promising candidate for breeding improved malting cultivars, displaying a TGW, which was increased by 17% under terminal drought stress due to the presence of an unknown wild barley QTL allele on chromosome 4H. The introgression line showed a similar advantage in previous field experiments and in greenhouse experiments under early drought stress. We, thus, recommend using S42IL-121 in barley breeding programs to enhance terminal drought tolerance.

Water uptake in barley grain: Physiology; genetics and industrial applications.

Water uptake by mature barley grains initiates germination and is the first stage in the malting process. Here we have investigated the effects of starchy endosperm cell wall thickness on water uptake, together with the effects of varying amounts of the wall polysaccharide, (1,3;1,4)-ß-glucan. In the latter case, we examined mutant barley lines from a mutant library and transgenic barley lines in which the (1,3;1,4)-ß-glucan synthase gene, HvCslF6, was down-regulated by RNA interference. Neither cell wall thickness nor the levels of grain (1,3;1,4)-ß-glucan were significantly correlated with water uptake but are likely to influence modification during malting. However, when a barley mapping population was phenotyped for rate of water uptake into grain, quantitative trait locus (QTL) analysis identified specific regions of chromosomes 4H, 5H and 7H that accounted for approximately 17%, 18% and 11%, respectively, of the phenotypic variation. These data indicate that variation in water uptake rates by elite malting cultivars of barley is genetically controlled and a number of candidate genes that might control the trait were identified under the QTL. The genomics data raise the possibility that the genetic variation in water uptake rates might be exploited by breeders for the benefit of the malting and brewing industries.

Meristem micropropagation of cassava (Manihot esculenta) evokes genome-wide changes in DNA methylation.

There is great interest in the phenotypic, genetic and epigenetic changes associated with plant in vitro culture known as somaclonal variation. In vitro propagation systems that are based on the use of microcuttings or meristem cultures are considered analogous to clonal cuttings and so widely viewed to be largely free from such somaclonal effects. In this study, we surveyed for epigenetic changes during propagation by meristem culture and by field cuttings in five cassava (Manihot esculenta) cultivars. Principal Co-ordinate Analysis of profiles generated by methylation-sensitive amplified polymorphism revealed clear divergence between samples taken from field-grown cuttings and those recovered from meristem culture. There was also good separation between the tissues of field samples but this effect was less distinct among the meristem culture materials. Application of methylation-sensitive Genotype by sequencing identified 105 candidate epimarks that distinguish between field cutting and meristem culture samples. Cross referencing the sequences of these epimarks to the draft cassava genome revealed 102 sites associated with genes whose homologs have been implicated in a range of fundamental biological processes including cell differentiation, development, sugar metabolism, DNA methylation, stress response, photosynthesis, and transposon activation. We explore the relevance of these findings for the selection of micropropagation systems for use on this and other crops.

High-Resolution Genotyping of Wild Barley Introgression Lines and Fine-Mapping of the Threshability Locus thresh-1 Using the Illumina GoldenGate Assay.

Genetically well-characterized mapping populations are a key tool for rapid and precise localization of quantitative trait loci (QTL) and subsequent identification of the underlying genes. In this study, a set of 73 introgression lines (S42ILs) originating from a cross between the spring barley cultivar Scarlett (Hordeum vulgare ssp. vulgare) and the wild barley accession ISR42-8 (H. v. ssp. spontaneum) was subjected to high-resolution genotyping with an Illumina 1536-SNP array. The array enabled a precise localization of the wild barley introgressions in the elite barley background. Based on 636 informative SNPs, the S42IL set represents 87.3% of the wild barley genome, where each line contains on average 3.3% of the donor genome. Furthermore, segregating high-resolution mapping populations (S42IL-HRs) were developed for 70 S42ILs in order to facilitate QTL fine-mapping and cloning. As a case study, we used the developed genetic resources to rapidly identify and fine-map the novel locus thresh-1 on chromosome 1H that controls grain threshability. Here, the recessive wild barley allele confers a difficult to thresh phenotype, suggesting that thresh-1 played an important role during barley domestication. Using a S42IL-HR population, thresh-1 was fine-mapped within a 4.3cM interval that was predicted to contain candidate genes involved in regulation of plant cell wall composition. The set of wild barley introgression lines and derived high-resolution populations are ideal tools to speed up the process of mapping and further dissecting QTL, which ultimately clears the way for isolating the genes behind QTL effects.

Comparative mapping of a QTL controlling black point formation in barley.

The dark discoloration of the embryo end of barley grain (known as black point) is a physiological disorder and the discovery of a quantitative trait locus (QTL) on 2H confirms this trait is controlled genetically. The mechanisms underlying black point tolerance can now be dissected through identification of candidate genes. Comparisons between the QTL identified on chromosomes 2H of barley and 2B of wheat suggest that they are in similar positions near the centromere. In silico analysis, using rice, identified genes residing on two comparative chromosomes (4 and 7) of the rice genome. Analysis of the 12.6 Mb region revealed 1928 unique annotations classified into 11 functional categories. Expressed sequence tags (ESTs) with high sequence similarity to enzymes proposed to be involved in black point formation were used to develop restriction fragment length polymorphisms (RFLPs). To ensure an even coverage of markers across the QTL, RFLP markers were also developed from other ESTs. Mapping of these markers has reduced the QTL region from 28 to 18 cM. This study has identified candidate genes for the control of black point formation and paves the way for future research to develop black point resistant barley cultivars.

A novel late embryogenesis abundant protein and peroxidase associated with black point in barley grains.

Black point of barley grain is a disorder characterised by a brown-black discolouration at the embryo end of the grain. Black point is undesirable to the malting industry and results in significant economic loss annually. To identify proteins associated with barley black point we utilised a proteomic approach with 2-DE to compare proteins from whole grain samples of black pointed and healthy grain. From this comparison two condition-specific proteins were identified: a novel 75 kDa late embryogenesis abundant (LEA) protein and a barley grain peroxidase 1 (BP1) that were specifically more abundant in healthy grain and black pointed grain, respectively. Although LEA protein was less abundant in black pointed grain, LEA gene expression was greater suggesting protein degradation had possibly occurred in black pointed grain. Similarly, the increase in BP1 in black pointed grain could not be explained by gene expression. Western blot analysis also revealed that the identified LEA protein is biotinylated in vivo. The role that each of these proteins might have in black point development is discussed.