High resolution mapping of Dense spike-ar (dsp.ar) to the genetic centromere of barley chromosome 7H

Spike density in barley is under the control of several major genes, as documented previously by genetic analysis of a number of morphological mutants. One such class of mutants affects the rachis internode length leading to dense or compact spikes and the underlying genes were designated dense spike (dsp). We previously delimited two introgressed genomic segments on chromosome 3H (21 SNP loci, 35.5 cM) and 7H (17 SNP loci, 20.34 cM) in BW265, a BC7F3 nearly isogenic line (NIL) of cv. Bowman as potentially containing the dense spike mutant locus dsp.ar, by genotyping 1,536 single nucleotide polymorphism (SNP) markers in both BW265 and its recurrent parent. Here, the gene was allocated by high-resolution bi-parental mapping to a 0.37 cM interval between markers SC57808 (Hv_SPL14)-CAPSK06413 residing on the short and long arm at the genetic centromere of chromosome 7H, respectively. This region putatively contains more than 800 genes as deduced by comparison with the collinear regions of barley, rice, sorghum and Brachypodium, Classical map-based isolation of the gene dsp.ar thus will be complicated due to the infavorable relationship of genetic to physical distances at the target locus.

L-rhamnose-1-dehydrogenase gene and L-rhamnose catabolism in the yeast Pichia stipitis

The purpose of this work was to identify some of the genes of the catabolic route of L-rhamnose in the yeast Pichia stipitis. There are at least two distinctly different pathways for L-rhamnose catabolism. The one described in bacteria has phosphorylated intermediates and the enzymes and the genes of this route have been described. The pathway described in yeast does not have phosphorylated intermediates. The intermediates and the enzymes of this pathway are known but none of the genes have been identified. The work was started by purifying the L-rhamnose dehydrogenase, which oxidates L-rhamnose to rhamnonic acid-gamma-lactone. NAD is used as a cofactor in this reaction. A DEAE ion exchange column was used for purification. The active fraction was further purified using a non-denaturing PAGE and the active protein identified by zymogram staining. In the last step the protein was separated in a SDS-PAGE, the protein band trypsinated and analysed by MALDI-TOF MS. This resulted in the identification of the corresponding gene, RHA1, which was then, after a codon change, expressed in Saccharomyces cerevisiae. Also C- or N-terminal histidine tags were added but as the activity of the enzyme was lost or strongly reduced these were not used. The kinetic properties of the protein were analysed in the cell extract. Substrate specifity was tested with different sugars; L-rhamnose, L-lyxose and L-mannose were oxidated by the enzyme. Vmax values were 180 nkat/mg, 160 nkat/mg and 72 nkat/mg, respectively. The highest affinity was towards L-rhamnose, the Km value being 0.9 mM. Lower affinities were obtained with L-lyxose, Km 4.3 mM, and L-mannose Km 25 mM. Northern analysis was done to study the transcription of RHA1 with different carbon sources. Transcription was observed only on L-rhamnose suggesting that RHA1 expression is L-rhamnose induced. A RHA1 deletion cassette for P. stipitis was constructed but the cassette had integrated randomly and not targeted to delete the RHA1 gene. Enzyme assays for L-lactaldehyde dehydrogenase were done similarly to L-rhamnose dehydrogenase assays. NAD is used as a cofactor also in this reaction where L-lactaldehyde is oxidised to L-lactate. The observed enzyme activities were very low and the activity was lost during the purification procedures.

Rapid genome wide mapping of phosphine resistance loci by a simple regional averaging analysis in the red flour beetle, Tribolium castaneum

Background Next-generation sequencing technology is an important tool for the rapid, genome-wide identification of genetic variations. However, it is difficult to resolve the ‘signal’ of variations of interest and the ‘noise’ of stochastic sequencing and bioinformatic errors in the large datasets that are generated. We report a simple approach to identify regional linkage to a trait that requires only two pools of DNA to be sequenced from progeny of a defined genetic cross (i.e. bulk segregant analysis) at low coverage (<10×) and without parentage assignment of individual SNPs. The analysis relies on regional averaging of pooled SNP frequencies to rapidly scan polymorphisms across the genome for differential regional homozygosity, which is then displayed graphically. Results Progeny from defined genetic crosses of Tribolium castaneum (F4 and F19) segregating for the phosphine resistance trait were exposed to phosphine to select for the resistance trait while the remainders were left unexposed. Next generation sequencing was then carried out on the genomic DNA from each pool of selected and unselected insects from each generation. The reads were mapped against the annotated T. castaneum genome from NCBI (v3.0) and analysed for SNP variations. Since it is difficult to accurately call individual SNP frequencies when the depth of sequence coverage is low, variant frequencies were averaged across larger regions. Results from regional SNP frequency averaging identified two loci, tc_rph1 on chromosome 8 and tc_rph2 on chromosome 9, which together are responsible for high level resistance. Identification of the two loci was possible with only 5-7× average coverage of the genome per dataset. These loci were subsequently confirmed by direct SNP marker analysis and fine-scale mapping. Individually, homozygosity of tc_rph1 or tc_rph2 results in only weak resistance to phosphine (estimated at up to 1.5-2.5× and 3-5× respectively), whereas in combination they interact synergistically to provide a high-level resistance >200×. The tc_rph2 resistance allele resulted in a significant fitness cost relative to the wild type allele in unselected beetles over eighteen generations. Conclusion We have validated the technique of linkage mapping by low-coverage sequencing of progeny from a simple genetic cross. The approach relied on regional averaging of SNP frequencies and was used to successfully identify candidate gene loci for phosphine resistance in T. castaneum. This is a relatively simple and rapid approach to identifying genomic regions associated with traits in defined genetic crosses that does not require any specialised statistical analysis.

Association mapping of resistance to Puccinia hordei in Australian barley breeding germplasm

Key message “To find stable resistance using association mapping tools, QTL with major and minor effects on leaf rust reactions were identified in barley breeding lines by assessing seedlings and adult plants.” Abstract Three hundred and sixty (360) elite barley (Hordeum vulgare L.) breeding lines from the Northern Region Barley Breeding Program in Australia were genotyped with 3,244 polymorphic diversity arrays technology markers and the results used to map quantitative trait loci (QTL) conferring a reaction to leaf rust (Puccinia hordei Otth). The F3:5 (Stage 2) lines were derived or sourced from different geographic origins or hubs of international barley breeding ventures representing two breeding cycles (2009 and 2011 trials) and were evaluated across eight environments for infection type at both seedling and adult plant stages. Association mapping was performed using mean scores for disease reaction, accounting for family effects using the eigenvalues from a matrix of genotype correlations. In this study, 15 QTL were detected; 5 QTL co-located with catalogued leaf rust resistance genes (Rph1, Rph3/19, Rph8/14/15, Rph20, Rph21), 6 QTL aligned with previously reported genomic regions and 4 QTL (3 on chromosome 1H and 1 on 7H) were novel. The adult plant resistance gene Rph20 was identified across the majority of environments and pathotypes. The QTL detected in this study offer opportunities for breeding for more durable resistance to leaf rust through pyramiding multiple genomic regions via marker-assisted selection.

Fine mapping of qGW1, a major QTL for grain weight in sorghum

Key message We detected seven QTLs for 100-grain weight in sorghum using an F 2 population, and delimited qGW1 to a 101-kb region on the short arm of chromosome 1, which contained 13 putative genes. Abstract Sorghum is one of the most important cereal crops. Breeding high-yielding sorghum varieties will have a profound impact on global food security. Grain weight is an important component of grain yield. It is a quantitative trait controlled by multiple quantitative trait loci (QTLs); however, the genetic basis of grain weight in sorghum is not well understood. In the present study, using an F2 population derived from a cross between the grain sorghum variety SA2313 (Sorghum bicolor) and the Sudan-grass variety Hiro-1 (S. bicolor), we detected seven QTLs for 100-grain weight. One of them, qGW1, was detected consistently over 2 years and contributed between 20 and 40 % of the phenotypic variation across multiple genetic backgrounds. Using extreme recombinants from a fine-mapping F3 population, we delimited qGW1 to a 101-kb region on the short arm of chromosome 1, containing 13 predicted gene models, one of which was found to be under purifying selection during domestication. However, none of the grain size candidate genes shared sequence similarity with previously cloned grain weight-related genes from rice. This study will facilitate isolation of the gene underlying qGW1 and advance our understanding of the regulatory mechanisms of grain weight. SSR markers linked to the qGW1 locus can be used for improving sorghum grain yield through marker-assisted selection.

An extragenic suppressor of prp24-1 defines genetic interaction between PRP24 and PRP21 gene products of Saccharomyces cerevisiae

The temperature-sensitive prp24-1 mutation defines a gene product required for the first step in pre-mRNA splicing. PRP24 is probably a component of the U6 snRNP particle. We have applied genetic reversion analysis to identify proteins that interact with PRP24. Spontaneous revertants of the temperature-sensitive (ts) prp24-1 phenotype were analyzed for those that are due to extragenic suppression. We then extended our analysis to screen for suppressors that confer a distinct conditional phenotype. We have identified a temperature-sensitive extragenic suppressor, which was shown by genetic complementation analysis to be allelic to prp21-1. This suppressor, prp21-2, accumulates pre-mRNA at the non-permissive temperature, a phenotype similar to that of prp21-1. prp21-2 completely suppresses the splicing defect and restores in vivo levels of the U6 snRNA in the prp24-1 strain. Genetic analysis of the suppressor showed that prp21-2 is not a bypass suppressor of prp24-1. The suppression of prp24-1 by prp21-2 is gene specific and also allele specific with respect to both the loci. Genetic interactions with other components of the pre-spliceosome have also been studied. Our results indicate an interaction between PRP21, a component of the U2 snRNP, and PRP24, a component of the U6 snRNP. These results substantiate other data showing U2-U6 snRNA interactions.

Next-generation sequencing: A frameshift in skeletal dysplasia gene discovery

In the last decade, huge breakthroughs in genetics - driven by new technology and different statistical approaches - have resulted in a plethora of new disease genes identified for both common and rare diseases. Massive parallel sequencing, commonly known as next-generation sequencing, is the latest advance in genetics, and has already facilitated the discovery of the molecular cause of many monogenic disorders. This article describes this new technology and reviews how this approach has been used successfully in patients with skeletal dysplasias. Moreover, this article illustrates how the study of rare diseases can inform understanding and therapeutic developments for common diseases such as osteoporosis. © International Osteoporosis Foundation and National Osteoporosis Foundation 2013.

Positional cloning and pathway analysis of the asthma susceptibility gene, NPSR1

In the present study, we identified a novel asthma susceptibility gene, NPSR1 (neuropeptide S receptor 1) on chromosome 7p14.3 by the positional cloning strategy. An earlier significant linkage mapping result among Finnish Kainuu asthma families was confirmed in two independent cohorts: in asthma families from Quebec, Canada and in allergy families from North Karelia, Finland. The linkage region was narrowed down to a 133-kb segment by a hierarchial genotyping method. The observed 77-kb haplotype block showed 7 haplotypes and a similar risk and nonrisk pattern in all three populations studied. All seven haplotypes occur in all three populations at frequences > 2%. Significant elevated relative risks were detected for elevated total IgE (immunoglobulin E) or asthma. Risk effects of the gene variants varied from 1.4 to 2.5. NPSR1 belongs to the G protein-coupled receptor (GPCR) family with a topology of seven transmembrane domains. NPSR1 has 9 exons, with the two main transcripts, A and B, encoding proteins of 371 and 377 amino acids, respectively. We detected a low but ubiquitous expression level of NPSR1-B in various tissues and endogenous cell lines while NPSR1-A has a more restricted expression pattern. Both isoforms were expressed in the lung epithelium. We observed aberrant expression levels of NPSR1-B in smooth muscle in asthmatic bronchi as compared to healthy. In an experimental mouse model, the induced lung inflammation resulted in elevated Npsr1 levels. Furthermore, we demonstrated that the activation of NPSR1 with its endogenous agonist, neuropeptide S (NPS), resulted in a significant inhibition of the growth of NPSR1-A overexpressing stable cell lines (NPSR1-A cells). To determine which target genes were regulated by the NPS-NPSR1 pathway, NPSR1-A cells were stimulated with NPS, and differentially expressed genes were identified using the Affymetrix HGU133Plus2 GeneChip. A total of 104 genes were found significantly up-regulated and 42 down-regulated 6 h after NPS administration. The up-regulated genes included many neuronal genes and some putative susceptibility genes for respiratory disorders. By Gene Ontology enrichment analysis, the biological process terms, cell proliferation, morphogenesis and immune response were among the most altered. The expression of four up-regulated genes, matrix metallopeptidase 10 (MMP10), INHBA (activin A), interleukin 8 (IL8) and EPH receptor A2 (EPHA2), were verified and confirmed by quantitative reverse-transcriptase-PCR. In conclusion, we identified a novel asthma susceptibility gene, NPSR1, on chromosome 7p14.3. NPS-NPSR1 represents a novel pathway that regulates cell proliferation and immune responses, and thus may have functional relevance in the pathogenesis of asthma.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

Purpose: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease.

Physical mapping of the genomic DNA of the Oryctes rhinoceros baculovirus, KI

A non-occluded baculovirus, OBV-KI has been isolated from the insect pest, Oryctes rhinoceros. The viral genome is estimated to be 123 kb, with a G + C content of 43 mol% and no detectible methylated bases. A restriction map of the OBV-KI genome for BamHI, EcoRI, HindIII, PstI, SalI and XbaI has been constructed.

Multiple molecular alterations in phosphodegrons 1-3 within AAV2 capsid demonstrates higher hepatic gene transfer efficiency

The success of AAV2 mediated hepatic gene transfer in human trials for diseases such as hemophilia has been hampered by a combination of low transduction efficiency and a robust immune response directed against these vectors. We have previously shown that AAV2 is targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal degradation machinery and modification of the serine(S)/threonine(T) kinase and lysine(K) targets on AAV capsid is beneficial. Thus targeted single mutations of S/T>A(S489A, S498A, T251A) and K>R (K532R) improved the efficiency of gene transfer in vivo as compared to wild type (WT)-AAV2 vectors (∼6-14 fold). In the present study, we evaluated if combined alteration of the phosphodegrons (PD), which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, improves further the gene transfer efficiency. Thus, we generated four multiple mutant vectors (PD: 1+3, S489A+K532R, PD: 1+3, S489A+K532R together with T251 residue which did not lie in any of the phosphodegrons but had shown increased transduction efficiency compared to the WT-AAV2 vector (∼6 fold) and was also conserved in 9 out of 10 AAV serotypes (AAV 1 to 10), PD: 1+3, S489A+K532R+S498A and a fourth combination of PD: 3, K532R+T251. We then evaluated them in vitro and in vivo and compared their gene transfer efficiency with either the WT-AAV2 or the best single mutant S489A-AAV2 vector. The novel multiple mutations on the AAV2 capsid did not affect the overall vector packaging efficiency. All the multiple AAV2 mutants showed superior transduction efficiency in HeLa cells in vitro when compared to either the WT (62-72% Vs 21%) or the single mutant S489A (62-72% Vs 50%) AAV2 vectors as demonstrated by FACS analysis (Fig. 1A). On hepatic gene transfer with 5x10^10 vgs per animal in C57BL/6 mice, all the multiple mutants showed increased transgene expression compared to either the WT-AAV2 (∼15-23 fold) or the S489A single mutant vector (∼2-3 fold) (Fig.1B and C). These novel multiple mutant AAV2 vectors also showed higher vector copy number in murine hepatocytes 4 weeks post transduction, as compared to the WT-AAV2 (∼5-6 Vs 1.4 vector copies/diploid genome) and further higher when compared to the single mutant S489A(∼5-6 fold Vs 3.8 fold) (Fig.1D). Further ongoing studies will demonstrate the therapeutic benefit of one or more of the multiple mutants vectors in preclinical models of hemophilia.

Relatorio e synopse dos trabalhos da Camara dos Srs. Deputados : na 1ª e 2ª sessão do anno de 1879

Sinopse dos trabalhos da Câmara dos Deputados, em 1879.

Relatorio e synopse dos trabalhos da Camara dos Srs. Deputados : na 1ª e 2ª sessão do anno de 1877 : acompanhada de differentes documentos, e mappas estatisticos, organisados na Secretaria da mesma Camara

Sinopse dos trabalhos da Câmara dos Deputados, em 1877.

Estudo do polimorfismo Ser49Gly do gene do receptor beta adrenérgico 1 (β-adr 1) na população do estado do Rio de Janeiro, Brasil estratificada por cor da pele e ancestralidade genômica

As doenças cardiovasculares possuem a maior taxa de óbitos no mundo, e notavelmente nos últimos anos as pesquisas genéticas sobre as mesmas estão baseadas em estudos de associação, no qual o gene suspeito que esteja em maior frequência entre os pacientes passa a ser considerado um possível fator causal. Os polimorfismos genéticos que ocorrem no receptor beta-adrenérgico podem resultar em mudanças significativas na função do receptor, podendo acarretar fisiopatologias. Neste trabalho, o objetivo foi estimar a diversidade e a frequência do polimorfismo Ser49Gly do gene do receptor beta-adrenérgico 1 a partir de uma amostra de 188 indivíduos da população do Estado do Rio de Janeiro. As frequências também foram analisadas a partir da estratificação da amostra por critério fenotípico em função do padrão de cor da pele em (negros e não negros) ou ancestralidade genética em (afrodescendente e não afrodescendente), definida através da informação dos marcadores de ancestralidade Indels e SNP de cromossomo Y, para avaliar se os padrões de ancestralidade ou cor da pele são fundamentais para a diferenciação e distanciamento genético. Fragmentos de interesse foram amplificados por PCR (reação de cadeia de polimerase) com primers específicos para o marcador Ser49Gly e as reações de genotipagem foram realizadas com enzimas de restrição Eco0109I. Os valores da heterozigosidade variaram entre 0,25-0,50 e 0,20-0,41 nos grupos estratificados por ancestralidade e cor da pele, respectivamente. No que diz respeito à análise do equilíbrio de Hardy-Weinberg, não houve um desvio significativo na distribuição do marcador nas amostras gerais do Estado do Rio de Janeiro, ou mesmo nas amostras estratificadas. A distribuição dos alelos na amostra dos 188 indivíduos da população geral do Rio de Janeiro (AC_RJ) mostrou uma frequência de 80,30% e 19,70% para o alelo selvagem e mutado Ser49Gly, respectivamente. A comparação das análises sobre a distribuição das frequências alélicas para este marcador mostrou a ocorrência de diferenças significativas na distribuição das frequências alélicas entre negros e não negros e afrodescendentes e não afrodescendentes. A diferença significativa observada entre os negros e afrodescendentes, foi em menor grau de distanciamento. A informação obtida em relação à ancestralidade foi crucial para a obtenção dos dados sobre o aumento da variável mutada do polimorfismo Ser49Gly nas populações negras e afrodescendentes do Estado Rio de Janeiro. Tal evidência, em combinação com estudos clínicos podem contribuir para uma análise pormenorizada do padrão de susceptibilidade à doença em questão, em falhas do mecanismo deste receptor.