Genetic dissection of grain beta-glucan and amylose content in barley (Hordeum vulgare L.)

High beta-glucan (BG) barleys (Hordeum vulgare L.) have major potential as food ingredients due to their well-known health benefits. Quantitative trait loci (QTL) associated with BG have been reported in traditional barley varieties with intermediate levels of BG, but no QTL studies have been reported in hull-less barley varieties with high BG levels. In this study, QTL analysis was performed to identify markers linked to high BG and amylose in the hull-less barley varieties Falcon (4–5 % BG) and Azhul (8–9 % BG) using a newly developed recombinant inbred line (RIL) mapping population. The population was grown over 3 years (2007–2009) at sites in Yuma, AZ, USA; Leeston, New Zealand; Aberdeen, ID, USA; and Tetonia, ID, USA. We identified 17 QTL associated with either BG or amylose content. QTL contributing to high BG were located on chromosomes 3H, 4H, 5H, 6H and 7H, while QTL contributing to amylose were located on chromosomes 1H, 5H and 7H. Additionally, we identified QTL affecting both BG and amylose content located on chromosomes 1H and 7H. Transgressive segregation was observed in some of the RILs and exceptions were discovered contradicting an inverse relationship between BG and amylose. This work will provide the basis for gene cloning and marker-assisted selection in combination with traditional field selection to improve barley breeding for high BG content.     

Genetic dissection of temperature-dependent sorghum growth during juvenile development

Key message

Promising genome regions for improving cold tolerance of sorghum were identified on chromosomes SBI-01, SBI-03, SBI-07, and SBI-10. Chlorophyll fluorescence had no major effect on growth rates at low temperatures.

Abstract

Developing fast growing sorghum seedlings is an important breeding goal for temperate climates since low springtime temperatures are resulting in a prolonged juvenile development. The adaptation of sorghum to tropical and subtropical highlands gives hint for certain genetic variation. The goals of the present study were to detect marker-trait associations for leaf and dry matter growth rate and for chlorophyll fluorescence and content (SPAD) in relation to temperature. A diversity set comprising 194 genotypes was tested in eight controlled environments with temperatures ranging from 9.4 to 20.8 °C. Significant marker-trait associations (p < 0.05) were identified for each individual temperature regime and on the parameters of regression analyses describing the responses of growth or chlorophyll related traits to temperatures. The diversity set was fingerprinted with 171 diversity array technology (DArT) and 31 simple-sequence repeat (SSR) markers. SSRs were used to analyze the population structure while association studies were performed on DArT markers. Promising marker-trait associations for growth rates in relation to temperature were detected on chromosomes SBI-01, SBI-03, SBI-07, and SBI-10. Many promising loci were also significantly associated to the results obtained in individual low-temperature environments. Marker-trait associations for chlorophyll content and fluorescence did occasionally co-locate to those for growth during juvenile development but there was no evidence supporting our hypothesis that seedling growth at low temperatures is largely influenced by SPAD or fluorescence.     

Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine

A genetic linkage map of grapevine was constructed using a pseudo-testcross strategy based upon 138 individuals derived from a cross of Vitis vinifera Cabernet Sauvignon × Vitis riparia Gloire de Montpellier. A total of 212 DNA markers including 199 single sequence repeats (SSRs), 11 single strand conformation polymorphisms (SSCPs) and two morphological markers were mapped onto 19 linkage groups (LG) which covered 1,249 cM with an average of 6.7 cM between markers. The position of SSR loci in the maps presented here is consistent with the genome sequence. Quantitative traits loci (QTLs) for several traits of inflorescence and flower morphology, and downy mildew resistance were investigated. Two novel QTLs for downy mildew resistance were mapped on linkage groups 9 and 12, they explain 26.0–34.4 and 28.9–31.5% of total variance, respectively. QTLs for inflorescence morphology with a large effect (14–70% of total variance explained) were detected close to the Sex locus on LG 2. The gene of the enzyme 1-aminocyclopropane-1-carboxylic acid synthase, involved in melon male organ development and located in the confidence interval of all QTLs detected on the LG 2, could be considered as a putative candidate gene for the control of sexual traits in grapevine. Co-localisations were found between four QTLs, detected on linkage groups 1, 14, 17 and 18, and the position of the floral organ development genes GIBBERELLIN INSENSITIVE1, FRUITFULL, LEAFY and AGAMOUS. Our results demonstrate that the sex determinism locus also determines both flower and inflorescence morphological traits.     

Non-destructive staging of barley reproductive development for molecular analysis based upon external morphology

A prerequisite to study the molecular genetic pathways of pollen and anther development is an accurate staging system for reproductive development. However in barley, floret formation occurs mainly within the pseudostem, which makes the observation of floret development and access to the floret particularly difficult without dissecting the plant. Thus selecting stages for molecular analysis cannot be done non-destructively. A staging method has therefore been developed for barley in order to define the relationship between readily detectable growth points and reproductive development, to provide a clear key to enable accurate selection of reproductive material. Initial staging followed the traditional Zadoks decimal system, with minor adaptations to stages 31-34 and stage 37. The later stages, from 37 onward, were replaced by growth staging based upon the last flag elongation (LFE) and the position occupied by the spike within the pseudostem. Spike size could be readily predicted by using the staging system incorporating Zadoks stages 31-37, supplemented with substages and by LFE staging to improve accuracy. The different spike sizes, as well as the LFE stages, showed a clear relationship to events occurring within the anther, as confirmed by light microscopy of the anthers. The defined relationship between spike size and development to anther development now makes possible the accurate prediction of anther and pollen progression by external staging. This, therefore, provides a mechanism for non-destructive selection of material for analysis that is critical for the molecular characterization of genes in anther and pollen development.     

Genetic dissection of cytokinesis

Higher plants have evolved specific mechanisms for partitioning the cytoplasm of dividing cells. In the predominant mode of phragmoplast-assisted cytokinesis, a cell wall and flanking plasma membranes are made de novo from a transient membrane compartment, the cell plate, which in turn forms by vesicle fusion from the centre to the periphery of the dividing cell. Other modes of cytokinesis appear to occur in meiotic cells and developing gametophytes. Here we review recent progress in the analysis of plant cytokinesis, focusing on genetic studies in Arabidopsis which are beginning to identify structural and regulatory components of phragmoplast-assisted cytokinesis. Two classes of mutations have been described. In one class, the defects appear to be confined to cell plate formation, suggesting that the execution of cytokinesis is specifically affected. Mutations in the other class display more general defects in cell division. We also discuss possible roles of proteins that have been localised in cytokinetic cells but not characterised genetically. Finally, mutations affecting meiotic or gametophytic cell divisions suggest that mechanistically different modes of cytokinesis occur in higher plants.     

Genetic and physical mapping of barley telomeres

Barley (Hordeum vulgare L.) telomeres were investigated by means of pulsed field gel electrophoresis (PFGE) and in situ hybridization. In situ hybridization showed that a tandemly repeated satellite sequence has a subtelomeric location, and is present at thirteen of the fourteen chromosome ends. PFGE revealed that this satellite sequence is physically close to the telomeric repeat. Pulsed field gel electrophoresis was then used for segregation analysis and linkage mapping of several telomeric and satellite loci in a segregating doubled-haploid population. The telomeric repeat displayed a hypervariable segregation pattern with new alleles occurring in the progeny. Eight satellite and telomeric sites were mapped on an restriction fragment length polymorphism (RFLP)-map of barley, defining the ends of chromosome arms 1L, 2S, 3L, 4S, 4L, 5S and 6. One satellite locus mapped to an interstitial site on the long arm of chromosome 3. The pyhsical location of this locus was confirmed by in situ hybridization to wheat/barley addition line 3.     

Genetic dissection of neural circuits

Understanding the principles of information processing in neural circuits requires systematic characterization of the participating cell types and their connections, and the ability to measure and perturb their activity. Genetic approaches promise to bring experimental access to complex neural systems, including genetic stalwarts such as the fly and mouse, but also to nongenetic systems such as primates. Together with anatomical and physiological methods, cell-type-specific expression of protein markers and sensors and transducers will be critical to construct circuit diagrams and to measure the activity of genetically defined neurons. Inactivation and activation of genetically defined cell types will establish causal relationships between activity in specific groups of neurons, circuit function, and animal behavior. Genetic analysis thus promises to reveal the logic of the neural circuits in complex brains that guide behaviors. Here we review progress in the genetic analysis of neural circuits and discuss directions for future research and development.     

Genetic dissection of retinoid dehydrogenases

Biochemical studies indicate that alcohol dehydrogenase (ADH) metabolizes retinol to retinal, and that aldehyde dehydrogenase (ALDH) metabolizes retinal to retinoic acid, a molecule essential for growth and development. Summarized herein are several genetic studies supporting in vivo functions for ADH and ALDH in retinoic acid synthesis. Gene targeting was used to create knockout mice for either Adh1 or Adh4. Both knockout mice were viable and fertile without obvious defects. However, when wild-type and Adh4 knockout mice were subjected to vitamin A deficiency during gestation, the survival rate at birth was 3.3-fold lower for Adh4 knockout mice. When adult mice were examined for production of retinoic acid following retinol administration, Adh1 knockout mice exhibited 10-fold lower retinoic acid levels in liver compared with wild-type, whereas Adh4 knockout mice differed from wild-type by less than 2-fold. Thus, Adh1 plays a major role in the metabolism of a large dose of retinol to retinoic acid in adults, whereas Adh4 plays a role in maintaining sufficient retinol metabolism for development during retinol deficiency. ALDHs were examined by overexpression studies in frog embryos. Injection of mRNAs for either mouse Raldh1 or Raldh2 stimulated retinoic acid synthesis in frog embryos at the blastula stage when retinoic acid is normally undetectable. Overexpression of human ALDH2, human ALDH3, and mouse Aldh-pb did not stimulate retinoic acid production. In addition, Raldh2 knockout mice exhibit embryonic lethality with defects in retinoid-dependent tissues. Overall, these studies provide genetic evidence that Adh1, Adh4, Raldh1, and Raldh2 encode retinoid dehydrogenases involved in retinoic acid synthesis in vivo.     

Genetic dissection of retinoid dehydrogenases

Biochemical studies indicate that alcohol dehydrogenase (ADH) metabolizes retinol to retinal, and that aldehyde dehydrogenase (ALDH) metabolizes retinal to retinoic acid, a molecule essential for growth and development. Summarized herein are several genetic studies supporting in vivo functions for ADH and ALDH in retinoic acid synthesis. Gene targeting was used to create knockout mice for either Adh1 or Adh4. Both knockout mice were viable and fertile without obvious defects. However, when wild-type and Adh4 knockout mice were subjected to vitamin A deficiency during gestation, the survival rate at birth was 3.3-fold lower for Adh4 knockout mice. When adult mice were examined for production of retinoic acid following retinol administration, Adh1 knockout mice exhibited 10-fold lower retinoic acid levels in liver compared with wild-type, whereas Adh4 knockout mice differed from wild-type by less than 2-fold. Thus, Adh1 plays a major role in the metabolism of a large dose of retinol to retinoic acid in adults, whereas Adh4 plays a role in maintaining sufficient retinol metabolism for development during retinol deficiency. ALDHs were examined by overexpression studies in frog embryos. Injection of mRNAs for either mouse Raldh1 or Raldh2 stimulated retinoic acid synthesis in frog embryos at the blastula stage when retinoic acid is normally undetectable. Overexpression of human ALDH2, human ALDH3, and mouse Aldh-pb did not stimulate retinoic acid production. In addition, Raldh2 knockout mice exhibit embryonic lethality with defects in retinoid-dependent tissues. Overall, these studies provide genetic evidence that Adh1, Adh4, Raldh1, and Raldh2 encode retinoid dehydrogenases involved in retinoic acid synthesis in vivo.     

Genetic dissection of familial Parkinson's disease

In the past few years, the genetic contribution to Parkinson's disease (PD) has gained major attention and has resulted in the identification of the first mutant gene, called α-synuclein, involved in the pathogenesis of autosomal-dominant PD. α-Synuclein is a major component of Lewy bodies, which are a neuropathological feature of PD. Furthermore, deletions in the parkin gene have been identified as the primary cause in rare forms of autosomal-recessive juvenile PD. The elucidation of polygenic changes in the dopamine pathway, mitochondrial dysfunction, and metabolism of xenobiotics is now technically possible by means of association and genotype studies. The increasing knowledge of the pathogenesis of PD at a molecular level will have important implications for the development of individual therapeutic strategies to prevent disease progression.     

Genetic dissection of centromere function.

A system to detect a minimal function of Saccharomyces cerevisiae centromeres in vivo has been developed. Centromere DNA mutants have been examined and found to be active in a plasmid copy number control assay in the absence of segregation. The experiments allow the identification of a minimal centromere unit, CDE III, independently of its ability to mediate chromosome segregation. Centromere-mediated plasmid copy number control correlates with the ability of CDE III to assemble a DNA-protein complex. Cells forced to maintain excess copies of CDE III exhibit increased loss of a nonessential artificial chromosome. Thus, segregationally impaired centromeres can have negative effects in trans on chromosome segregation. The use of a plasmid copy number control assay has allowed assembly steps preceding chromosome segregation to be defined.