Genetics of barley hooded suppression

The molecular basis of the barley dominant Hooded (K) mutant is a duplication of 305 bp in intron IV of the homeobox gene Bkn3. A chemical mutagenesis screen was carried out to identify genetical factors that participate in Bkn3 intron-mediated gene regulation. Plants from recurrently mutagenized KK seeds were examined for the suppression of the hooded awn phenotype induced by the K allele and, in total, 41 suK (suppressor of K) recessive mutants were identified. Complementation tests established the existence of five suK loci, and alleles suKB-4, suKC-33, suKD-25, suKE-74, and suKF-76 were studied in detail. All K-suppressed mutants showed a short-awn phenotype. The suK loci have been mapped by bulked segregant analysis nested in a standard mapping procedure based on AFLP markers. K suppressor loci suKB, B, E, and F all map in a short interval of chromosome 7H, while the locus suKD is assigned to chromosome 5H. A complementation test between the four suK mutants mapping on chromosome 7H and the short-awn mutant lks2, located nearby, excluded the allelism between suK loci and lks2. The last experiment made clear that the short-awn phenotype of suK mutants is due to a specific dominant function of the K allele, a function that is independent from the control on hood formation. The suK loci are discussed as candidate participants in the regulation of Bkn3 expression.     

Characterization of non-dominant lethal mutations in the yeast plasma membrane H+-ATPase gene

Site-directed mutants of yeast ATPase were previously studied after introduction of mutant alleles into a yeast strain where these alleles were constitutively expressed while the expression of the wild-type chromosomal ATPase gene was turned off. As a functional H+ pump is essential, strong selective pressure leads to the accumulation of revertants during growth of cells harboring variants with low activity. Thus, constitutive expression of the mutant gene can select phenotypes which reflect events such as gene conversion or reversion. We have therefore re-evaluated the phenotypes of non-dominant lethal alleles in an alternative set of conditional expression systems. We show that eight of 11 previously described site-directed mutations behave as recessive lethal alleles.     

Characteristics and inheritance of radiation-induced mutations in barley II. Two short-awned mutations

Two mutations were obtained in a two-rowed, spring-type barley variety, Asahi No. 5. KM 114 was obtained in the X₂ population of X-irradiated dormant seeds (15 Kr) and KM 218 was found in X₂ of gamma-irradiated seeds (15 Kr). KM 114 had very short awns, short plant height, almost normal spike size, and narrow and thinner seeds with pointed tip. The mutant gene is Mendelian recessive. KM 218 plants were normal or slightly taller, had somewhat larger spike, with somewhat longer rachis internodes, medium-long awn, and slightly longer, narrower and thinner kernel than those of the parental variety. At maturity, grains are exposed in many seeds (semi-naked kernels). This mutant also has a conspicuous diagnostic trait; the abnormal development of rachilla into extra florets, some of which set seeds. This mutant gene is also Mendelian recessive. Genes for these two mutants are not allelic and are inherited independently as indicated in F₂ and F₃ analyses. Genes of these two mutations will serve as good marker genes in genetic and linkage studies in barley, because their character expression is consistent and the recessive homozygotes are viable and fertile.     

GRAIN LENGTH AND AWN 1 negatively regulates grain size in rice

Grain size is an important factor determining yield in rice. Here, we identified a recessive mutant gene, grain length and awn 1(gla1), which caused a significant increase in grain length and weight, and was associated with long awns. The gla1 mutation was mapped to a single-nucleotide polymorphism in a gene encoding a cytoplasmically-localized mitogen-activated protein kinase(MAPK) phosphatase. Overexpression of GLA1 caused a decrease in grain length, and the GLA1 protein interacted with Os MAPK6. These results suggest that GLA1 may serve as a negative regulator of the Os MAPKK4-Os MAPK6 cascade, controlling grain size via the dephosphorylation of Os MAPK6.     

Stomatal control of gas exchange in barley awns

The gas exchange of barley ears and awns was measured in the field using a gas analysis system and a diffusion porometer. Awn stomatal resistance decreased with increasing irradiance but to a smaller extent than leaf stomatal resistance. Measurements on ears immediately before and after successively removing awns showed that awn transpiration and photosynthesis were proportional to awn area and that awns accounted for 73% of transpiration by the ear. Although the maximum rates of photosynthesis of which awns were capable declined with age, awns accounted for 80–115% of the net CO₂ uptake of complete ears because the ears-less-awns could respire more CO₂ than they absorbed. Ear photosynthesis accounted for 52% of the weekly increment in ear dry weight after ear emergence, but 5 weeks later photosynthesis by the ear balanced respiration. Overall photosynthesis by the ear accounted for 35 % of its final weight. Differences in the light response curves of leaves and ears can be fully accounted for by the different relationships between stomatal resistance and irradiance of the two organs.     

Ecology and evolution of the diaspore "burial syndrome"

Hygroscopically active awns or "bristles" have long intrigued scientists. Experimental evidence shows that they are important for diaspore burial in the correct orientation, thereby increasing successful seed germination and seedling survival. Despite these ecological advantages, 38 of the 280 species of grasses in Danthonioideae lack awns. We provide the first study of awns in a phylogenetic context and show that although the awnless state has arisen ca. 25 times independently, the ecological disadvantage of not having an awn also applies in an evolutionary context. Only in Tribolium and Schismus have awnless ancestors diversified to form a clade of primarily awnless descendents. Several of the awnless species in these genera are annual and we find a significant correlation between the evolution of awns and the evolution of life history. A suite of other diaspore traits accompany the awned or awnless states. We interpret the awn as being the visible constituent of a compound "burial syndrome," the two ecological extremes of which may explain the correlation between awns and life history and provide an explanation why awnless species in Tribolium and Schismus persist.     

Structure and Photosynthetic Characteristics of Awns of Wheat and Barley

The surface and the cross section of awns of wheat and barley were examined by scanning electron microscopy,ultrastructure of cells were observed under a transmisson electron microscope and the photosynthetic rates were measured with an oxygen, electrode and infra-red CO2 analyser. The main results were as follows :The cross section of wheat awn appeared to be acutely trianglular whereas that of barley awn was obtusely triangular. There were rows of stomota on either side of epidermis in both wheat and barley awns. Under the stomatic band there were green tissues. The green cells in the awn were differentiated from the parenchyma cells . The mature green cells possessed papillae which were rich in chloroplasts and mitochondria. The tamella system in chloroplasts was well developed and contained many starch grains. There were three vascular bundles in each awn. The sheath cells near the green tissues contained chloroplasts. The photosynthate in the green cells might pass through the sheath cells and companion cells to sieve elements. The highest photosynthetic rate of the awn was seen at the flowering stage ,reaching about 20 μmol CO2·m-2·s-1. The light compensation point was 70—80 μE·m-2· s-1. The light saturation point was about 1500 μE·m-2·s-1. The CO2 compensation point was 50—60 ppm and the CO2 saturation point was about 900ppm . The photosynthetic rate and stomatal conductance were easily effected by CO2 concentration, light intensity and the duration of illumination . There was a positive correlation between the photosynthetic rate and the chloro-phyll content in the awns. The CO2-releasing rate in photorespiration of awn was about 4–5 μmol CO2·m-2·s-1.     

Allele-specific mitochondrial stress induced by Multiple Mitochondrial Dysfunctions Syndrome 1 pathogenic mutations modeled in Caenorhabditis elegans

Multiple Mitochondrial Dysfunctions Syndrome 1 (MMDS1) is a rare, autosomal recessive disorder caused by mutations in the NFU1 gene. NFU1 is responsible for delivery of iron-sulfur clusters (ISCs) to recipient proteins which require these metallic cofactors for their function. Pathogenic variants of NFU1 lead to dysfunction of its target proteins within mitochondria. To date, 20 NFU1 variants have been reported and the unique contributions of each variant to MMDS1 pathogenesis is unknown. Given that over half of MMDS1 individuals are compound heterozygous for different NFU1 variants, it is valuable to investigate individual variants in an isogenic background. In order to understand the shared and unique phenotypes of NFU1 variants, we used CRISPR/Cas9 gene editing to recreate exact patient variants of NFU1 in the orthologous gene, nfu-1 (formerly lpd-8), in C. elegans. Five mutant C. elegans alleles focused on the presumptive iron-sulfur cluster interaction domain were generated and analyzed for mitochondrial phenotypes including respiratory dysfunction and oxidative stress. Phenotypes were variable between the mutant nfu-1 alleles and generally presented as an allelic series indicating that not all variants have lost complete function. Furthermore, reactive iron within mitochondria was evident in some, but not all, nfu-1 mutants indicating that iron dyshomeostasis may contribute to disease pathogenesis in some MMDS1 individuals.     

A genome-wide survey of R gene polymorphisms in Arabidopsis

We used polymorphism analysis to study the evolutionary dynamics of 27 disease resistance (R) genes by resequencing the leucine-rich repeat (LRR) region in 96 Arabidopsis thaliana accessions. We compared single nucleotide polymorphisms (SNPs) in these R genes to an empirical distribution of SNP in the same sample based on 876 fragments selected to sample the entire genome. LRR regions are highly polymorphic for protein variants but not for synonymous changes, suggesting that they generate many alleles maintained for short time periods. Recombination is also relatively common and important for generating protein variants. Although none of the genes is nearly as polymorphic as RPP13, a locus previously shown to have strong signatures of balancing selection, seven genes show weaker indications of balancing selection. Five R genes are relatively invariant, indicating young alleles, but all contain segregating protein variants. Polymorphism analysis in neighboring fragments yielded inconclusive evidence for recent selective sweeps at these loci. In addition, few alleles are candidates for rapid increases in frequency expected under directional selection. Haplotype sharing analysis revealed significant underrepresentation of R gene alleles with extended haplotypes compared with 1102 random genomic fragments. Lack of convincing evidence for directional selection or selective sweeps argues against an arms race driving R gene evolution. Instead, the data support transient or frequency-dependent selection maintaining protein variants at a locus for variable time periods.     

The amino-terminal domain of yeast U1-70K is necessary and sufficient for function.

The Saccharomyces cerevisiae SNP1 gene encodes a protein that shares 30% amino acid identity with the mammalian U1 small nuclear ribonucleoprotein particle protein 70K (U1-70K). We have demonstrated that yeast strains in which the SNP1 gene was disrupted are viable but exhibit greatly increased doubling times and severe temperature sensitivity. Furthermore, snp1-null strains are defective in pre-mRNA splicing. We have tested deletion alleles of SNP1 for their ability to complement these phenotypes. We found that the highly conserved RNA recognition motif consensus domain of Snp1 is not required for complementation of the snp1-null growth or splicing defects nor for the in vivo association with the U1 small nuclear ribonucleoprotein particle. However, the amino-terminal domain of Snp1, less strongly conserved, is necessary and sufficient for complementation.     

Applications of single nucleotide polymorphisms in crop genetics

The discovery of single nucleotide polymorphisms (SNPs) and insertions/deletions, which are the basis of most differences between alleles, has been simplified by recent developments in sequencing technology. SNP discovery in many crop species, such as corn and soybean, is relatively straightforward because of their high level of intraspecific nucleotide diversity, and the availability of many gene and expressed sequence tag (EST) sequences. For these species, direct readout of SNP haplotypes is possible. Haplotype-based analysis is more informative than analysis based on individual SNPs, and has more power in analyzing association with phenotypes. The elite germplasm of some crops may have been subjected to bottlenecks relatively recently, increasing the amount of linkage disequilibrium (LD) present and facilitating the association of SNP haplotypes at candidate gene loci with phenotypes. Whole-genome scans may help identify genome regions that are associated with interesting phenotypes if sufficient LD is present. Technological improvements make the use of SNP and indel markers attractive for high-throughput use in marker-assisted breeding, EST mapping and the integration of genetic and physical maps.     

Distribution of carbon-14 assimilated by wheat awns

The pattern of distribution of carbon assimilated by awns was investigated in two lines of Triticum aestivum. Single awns on basal florets of spikelets in the central part of the ear were dosed with ¹⁴CO₂. Five days after dosing, 99% of the carbon-14 recovered was in the spikelet bearing the awn. Of the carbon-14 exported from the treated awn 57% went to the grain of the first floret, 1% to the second, 28% to the third and 7% to the fourth.     

Positive Selection in East Asians for an EDAR Allele that Enhances NF-κB Activation

Genome-wide scans for positive selection in humans provide a promising approach to establish links between genetic variants and adaptive phenotypes. From this approach, lists of hundreds of candidate genomic regions for positive selection have been assembled. These candidate regions are expected to contain variants that contribute to adaptive phenotypes, but few of these regions have been associated with phenotypic effects. Here we present evidence that a derived nonsynonymous substitution (370A) in EDAR, a gene involved in ectodermal development, was driven to high frequency in East Asia by positive selection prior to 10,000 years ago. With an in vitro transfection assay, we demonstrate that 370A enhances NF-κB activity. Our results suggest that 370A is a positively selected functional genetic variant that underlies an adaptive human phenotype.     

Natural variation of potato <Emphasis Type="Italic">allene oxide synthase 2</Emphasis> causes differential levels of jasmonates and pathogen resistance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Natural variation of plant pathogen resistance is often quantitative. This type of resistance can be genetically dissected in quantitative resistance loci (QRL). To unravel the molecular basis of QRL in potato (Solanum tuberosum), we employed the model plant Arabidopsis thaliana for functional analysis of natural variants of potato allene oxide synthase 2 (StAOS2). StAOS2 is a candidate gene for QRL on potato chromosome XI against the oömycete Phytophthora infestans causing late blight, and the bacterium Erwinia carotovora ssp. atroseptica causing stem black leg and tuber soft rot, both devastating diseases in potato cultivation. StAOS2 encodes a cytochrome P450 enzyme that is essential for biosynthesis of the defense signaling molecule jasmonic acid. Allele non-specific dsRNAi-mediated silencing of StAOS2 in potato drastically reduced jasmonic acid production and compromised quantitative late blight resistance. Five natural StAOS2 alleles were expressed in the null Arabidopsis aos mutant under control of the Arabidopsis AOS promoter and tested for differential complementation phenotypes. The aos mutant phenotypes evaluated were lack of jasmonates, male sterility and susceptibility to Erwinia carotovora ssp. carotovora. StAOS2 alleles that were associated with increased disease resistance in potato complemented all aos mutant phenotypes better than StAOS2 alleles associated with increased susceptibility. First structure models of ‘quantitative resistant’ versus ‘quantitative susceptible’ StAOS2 alleles suggested potential mechanisms for their differential activity. Our results demonstrate how a candidate gene approach in combination with using the homologous Arabidopsis mutant as functional reporter can help to dissect the molecular basis of complex traits in non model crop plants.     

Malate dehydrogenase types in the Asian-Pacific area,and a description of new phenotypes

A survey of more than 21 000 haemolysates from blood samples collected in various parts of south and southeast Asia, Australasia and the Western Pacific and examined in this laboratory has revealed several new alleles controlling variants of sMDH; in addition, further information has been provided on the distribution of sMDH3 in New Guinea. Two of the variant alleles, sMDH3 and sMDH6, achieve polymorphic frequency in various populations. sMDH3 is widely distributed in New Guinea, with highest frequencies in the Eastern Highlands. The pattern of its distribution suggests the mutant arose originally in a Papuan-speaking population. So far, sMDH6 has been detected only in Micronesians from a number of islands in the Carolines. A single example of another new variant, sMDH 5-1, and two examples of a slow variant, sMDH 7-1, were detected in samples from Iran and Singapore, respectively. No examples of mMDH variants were found in a total of 652 placental extracts from Papua New Guinea and Australia.     

Allelic Selection of Amplicons in Glioblastoma Revealed by Combining Somatic and Germline Analysis

Cancer is a disease driven by a combination of inherited risk alleles coupled with the acquisition of somatic mutations, including amplification and deletion of genomic DNA. Potential relationships between the inherited and somatic aspects of the disease have only rarely been examined on a genome-wide level. Applying a novel integrative analysis of SNP and copy number measurements, we queried the tumor and normal-tissue genomes of 178 glioblastoma patients from the Cancer Genome Atlas project for preferentially amplified alleles, under the hypothesis that oncogenic germline variants will be selectively amplified in the tumor environment. Selected alleles are revealed by allelic imbalance in amplification across samples. This general approach is based on genetic principles and provides a method for identifying important tumor-related alleles. We find that SNP alleles that are most significantly overrepresented in amplicons tend to occur in genes involved with regulation of kinase and transferase activity, and many of these genes are known contributors to gliomagenesis. The analysis also implicates variants in synapse genes. By incorporating gene expression data, we demonstrate synergy between preferential allelic amplification and expression in DOCK4 and EGFR. Our results support the notion that combining germline and tumor genetic data can identify regions relevant to cancer biology.     

Contrasting effects of ENU induced embryonic lethal mutations of the quaking gene.

Multiple alleles of the quaking (qk) gene have a variety of phenotypes ranging in severity from early embryonic death to viable dysmyelination. A previous study identified a candidate gene, QKI, that contains an RNA-binding domain and encodes at least three protein isoforms (QKI-5, -6 and -7). We have determined the genomic structure of QKI, identifying an additional alternative end in cDNAs. Further we have examined the exons and splice sites for mutations in the lethal alleles qkl-1, qkkt1, qkk2, and qkkt3. The mutation in qkl-1 creates a splice site in the terminal exon of the QKI-6 isoform. Missense mutations in the KH domain and the QUA1 domains in qkk2 and qkkt3, respectively, indicate that these domains are of critical functional importance. Although homozygotes for each ENU induced allele die as embryos, their phenotypes as viable compound heterozygotes with qkv differ. Compound heterozygous qkv animals carrying qkkt1, qkk2, and qkkt3 all exhibit a permanent quaking phenotype similar to that of qkv/qkv animals, whereas qkv/qkl-1 animals exhibit only a transient quaking phenotype. The qkl-1 mutation eliminates the QKI-5 isoform, showing that this isoform plays a crucial role in embryonic survival. The transient quaking phenotype observed in qkv/qkl-1 mice indicates that the QKI-6 and QKI-7 isoforms function primarily during myelination, but that QKI-5 may have a concentration-dependent role in early myelination. This mutational analysis demonstrates the power of series of alleles to examine the function of complex loci and suggests that additional mutant alleles of quaking could reveal additional functions of this complex gene.     

Localized enhancement and repression of the activity of the TGF-beta family member, decapentaplegic, is necessary for dorsal-ventral pattern formation in the Drosophila embryo.

Seven zygotically active genes are required for normal patterning of the dorsal 40% of the Drosophila embryo. Among these genes, decapentaplegic (dpp) has the strongest mutant phenotype: in the absence of dpp, all cells in the dorsal and dorsolateral regions of the embryo adopt fates characteristic of more ventrally derived cells (Irish and Gelbart (1987) Genes Dev. 1, 868-879). Here we describe the phenotypes caused by alleles of another of this set of genes, tolloid, and show that tolloid is required for dorsal, but not dorsolateral, pattern. Extragenic suppressors of tolloid mutations were isolated that proved to be mutations that elevate dpp activity. We studied the relationship between tolloid and dpp by analyzing the phenotypes of tolloid embryos with elevated numbers of the dpp gene and found that doubling the dpp+ gene dosage completely suppressed weak tolloid mutants and partially suppressed the phenotypes of tolloid null mutants. We conclude that the function of tolloid is to increase dpp activity. We also examined the effect of doubling dpp+ gene dosage on the phenotypes caused by other mutations affecting dorsal development. Like tolloid, the phenotypes of mutant embryos lacking shrew gene function were suppressed by elevated dpp, indicating that shrew also acts upstream of dpp to increase dpp activity. In contrast, increasing the number of copies of the dpp gene enhanced the short gastrulation (sog) mutant phenotype, causing ventrolateral cells to adopt dorsal fates. This indicates that sog gene product normally blocks dpp activity ventrally. We propose that the tolloid, shrew and sog genes are required to generate a gradient of dpp activity, which directly specifies the pattern of the dorsal 40% of the embryo.     

Analysis of maxillopedia expression pattern and larval cuticular phenotype in wild-type and mutant tribolium

The Tribolium castaneum homeotic gene maxillopedia (mxp) is the ortholog of Drosophila proboscipedia (pb). Here we describe and classify available mxp alleles. Larvae lacking all mxp function die soon after hatching, exhibiting strong transformations of maxillary and labial palps to legs. Hypomorphic mxp alleles produce less severe transformations to leg. RNA interference with maxillopedia double-stranded RNA results in phenocopies of mxp mutant phenotypes ranging from partial to complete transformations. A number of gain-of-function (GOF) mxp alleles have been isolated based on transformations of adult antennae and/or legs toward palps. Finally, we have characterized the mxp expression pattern in wild-type and mutant embryos. In normal embryos, mxp is expressed in the maxillary and labial segments, whereas ectopic expression is observed in some GOF variants. Although mxp and Pb display very similar expression patterns, pb null embryos develop normally. The mxp mutant larval phenotype in Tribolium is consistent with the hypothesis that an ancestral pb-like gene had an embryonic function that was lost in the lineage leading to Drosophila.