An integrated Bayesian analysis of LOH and copy number data

Background  

Cancer and other disorders are due to genomic lesions. SNP-microarrays are able to measure simultaneously both genotype and copy number (CN) at several Single Nucleotide Polymorphisms (SNPs) along the genome. CN is defined as the number of DNA copies, and the normal is two, since we have two copies of each chromosome. The genotype of a SNP is the status given by the nucleotides (alleles) which are present on the two copies of DNA. It is defined homozygous or heterozygous if the two alleles are the same or if they differ, respectively. Loss of heterozygosity (LOH) is the loss of the heterozygous status due to genomic events.     

Recovery and characterization of a Citrus clementina Hort. ex Tan. 'Clemenules' haploid plant selected to establish the reference whole Citrus genome sequence

Background  

In recent years, the development of structural genomics has generated a growing interest in obtaining haploid plants. The use of homozygous lines presents a significant advantage for the accomplishment of sequencing projects. Commercial citrus species are characterized by high heterozygosity, making it difficult to assemble large genome sequences. Thus, the International Citrus Genomic Consortium (ICGC) decided to establish a reference whole citrus genome sequence from a homozygous plant. Due to the existence of important molecular resources and previous success in obtaining haploid clementine plants, haploid clementine was selected as the target for the implementation of the reference whole genome citrus sequence.     

A classification model for distinguishing copy number variants from cancer-related alterations

Background  

Both somatic copy number alterations (CNAs) and germline copy number variants (CNVs) that are prevalent in healthy individuals can appear as recurrent changes in comparative genomic hybridization (CGH) analyses of tumors. In order to identify important cancer genes CNAs and CNVs must be distinguished. Although the Database of Genomic Variants (DGV) contains a list of all known CNVs, there is no standard methodology to use the database effectively.     

Bayesian DNA copy number analysis

Background  

Some diseases, like tumors, can be related to chromosomal aberrations, leading to changes of DNA copy number. The copy number of an aberrant genome can be represented as a piecewise constant function, since it can exhibit regions of deletions or gains. Instead, in a healthy cell the copy number is two because we inherit one copy of each chromosome from each our parents.     

Extracting causal relations on HIV drug resistance from literature

Background  

In HIV treatment it is critical to have up-to-date resistance data of applicable drugs since HIV has a very high rate of mutation. These data are made available through scientific publications and must be extracted manually by experts in order to be used by virologists and medical doctors. Therefore there is an urgent need for a tool that partially automates this process and is able to retrieve relations between drugs and virus mutations from literature.     

Calcium-mediated perception and defense responses activated in plant cells by metabolite mixtures secreted by the biocontrol fungus <Emphasis Type="Italic">Trichoderma atroviride</Emphasis>

Background  

Calcium is commonly involved as intracellular messenger in the transduction by plants of a wide range of biotic stimuli, including signals from pathogenic and symbiotic fungi. Trichoderma spp. are largely used in the biological control of plant diseases caused by fungal phytopathogens and are able to colonize plant roots. Early molecular events underlying their association with plants are relatively unknown.     

Concurrent modifications in the three homeologs of <Emphasis Type="Italic">Ms45</Emphasis> gene with CRISPR-Cas9 lead to rapid generation of male sterile bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Key message

Hexaploid bread wheat is not readily amenable to traditional mutagenesis approaches. In this study, we show efficient utilization of CRISPR-Cas system and Next Generation Sequencing for mutant analysis in wheat.

Abstract

Identification and manipulation of male fertility genes in hexaploid bread wheat is important for understanding the molecular basis of pollen development and to obtain novel sources of nuclear genetic male sterility (NGMS). The maize Male sterile 45 (Ms45) gene encodes a strictosidine synthase-like enzyme and has been shown to be required for male fertility. To investigate the role of Ms45 gene in wheat, mutations in the A, B and D homeologs were produced using CRISPR-Cas9. A variety of mutations in the three homeologs were recovered, including a plant from two different genotypes each with mutations in all three homeologs. Genetic analysis of the mutations demonstrated that all three wheat Ms45 homeologs contribute to male fertility and that triple homozygous mutants are required to abort pollen development and achieve male sterility. Further, it was demonstrated that a wild-type copy of Ms45 gene from rice was able to restore fertility to these wheat mutant plants. Taken together, these observations provide insights into the conservation of MS45 function in a polyploid species. Ms45 based NGMS can be potentially utilized for a Seed Production Technology (SPT)-like hybrid seed production system in wheat.

     

A simple,rapid and systematic method for the developed GM rice analysis

We generated 383 independent transgenic lines that contained the PsGPD (Glyceraldehyde-3-Phosphate Dehydrogenase), ArCspA (Cold Shock Protein), BrTSR15 (Triple Stress Resistance 15) and BrTSR53 (Triple Stress Resistance 53) genes under the control of a constitutive (CaMV 35S) promoter to generate genetically modified (GM) rice. TaqMan copy number assay was performed to determine the copy numbers of inserted T-DNA. Flanking sequence tags (FSTs) were isolated from 203 single copy T-DNA lines of transgenic plants, and their sequences were mapped to the rice chromosomes. Of the 157 flanking sequence tags that were isolated from single copy lines, transgenes were found to be integrated into genic regions in 58 lines (36 %), whereas 97 lines (62 %) contained transgene insertions in intergenic regions. Approximately 27 putative homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assays. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNA from leaf tissue of homozygous T1 plants with a single copy and an intergenic insertion of T-DNA. The mRNA expression levels of the examined transgenic rice were significantly increased in all transgenic plants. In addition, myc-tagged 35S:BrTSR15 and 35S:BrTSR53 transgenic plants displayed higher levels of transgene protein. Using numerical data for the mass production of transgenic plants can reduce the time required to obtain a genetically modified plant. Moreover, the duration, cost, and efforts required for transformation can be deliberately predicted. These results may be useful for the large-scale production of transgenic plants or T-DNA inserted rice mutants.     

An algorithm for automatic evaluation of the spot quality in two-color DNA microarray experiments

Background  

Although DNA microarray technologies are very powerful for the simultaneous quantitative characterization of thousands of genes, the quality of the obtained experimental data is often far from ideal. The measured microarrays images represent a regular collection of spots, and the intensity of light at each spot is proportional to the DNA copy number or to the expression level of the gene whose DNA clone is spotted. Spot quality control is an essential part of microarray image analysis, which must be carried out at the level of individual spot identification. The problem is difficult to formalize due to the diversity of instrumental and biological factors that can influence the result.     

Integration of molecular biology tools for identifying promoters and genes abundantly expressed in flowers of Oncidium Gower Ramsey

Background  

Orchids comprise one of the largest families of flowering plants and generate commercially important flowers. However, model plants, such as Arabidopsis thaliana do not contain all plant genes, and agronomic and horticulturally important genera and species must be individually studied.     

Comparative transcriptional survey between laser-microdissected cells from laminar abscission zone and petiolar cortical tissue during ethylene-promoted abscission in citrus leaves

Background  

Abscission is the cell separation process by which plants are able to shed organs. It has a great impact on the yield of most crop plants. At the same time, the process itself also constitutes an excellent model to study cell separation processes, since it occurs in concrete areas known as abscission zones (AZs) which are composed of a specific cell type. However, molecular approaches are generally hampered by the limited area and cell number constituting the AZ. Therefore, detailed studies at the resolution of cell type are of great relevance in order to accurately describe the process and to identify potential candidate genes for biotechnological applications.     

Integrated analysis of DNA copy number and gene expression microarray data using gene sets

Background  

Genes that play an important role in tumorigenesis are expected to show association between DNA copy number and RNA expression. Optimal power to find such associations can only be achieved if analysing copy number and gene expression jointly. Furthermore, some copy number changes extend over larger chromosomal regions affecting the expression levels of multiple resident genes.     

Cold- and light-induced changes in the transcriptome of wheat leading to phase transition from vegetative to reproductive growth

Background  

For plants to flower at the appropriate time, they must be able to perceive and respond to various internal and external cues. Wheat is generally a long-day plant that will go through phase transition from vegetative to floral growth as days are lengthening in spring and early summer. In addition to this response to day-length, wheat cultivars may be classified as either winter or spring varieties depending on whether they require to be exposed to an extended period of cold in order to become competent to flower. Using a growth regime to mimic the conditions that occur during a typical winter in Britain, and a microarray approach to determine changes in gene expression over time, we have surveyed the genes of the major pathways involved in floral transition. We have paid particular attention to wheat orthologues and functional equivalents of genes involved in the phase transition in Arabidopsis. We also surveyed all the MADS-box genes that could be identified as such on the Affymetrix genechip wheat genome array.     

Functional analysis of the copy 1 of the fixNOQP operon of Ensifer meliloti under free‐living micro‐oxic and symbiotic conditions

Aim

In this work, phenotypic analyses of a Ensifer meliloti fixN1 mutant under free‐living and symbiotic conditions have been carried out.

Methods and Results

Ensifer meliloti fixN1 mutant showed a defect in growth as well as in TMPD‐dependent oxidase activity when cells were incubated under micro‐oxic conditions. Furthermore, haem c staining analyses of a fixN1 and a fixP1 mutant identified two membrane‐bound c‐type cytochromes of 27 and 32 kDa, present in microaerobically grown cells and in bacteroids, as the FixO and FixP components of the E. meliloti cbb₃ oxidase. Under symbiotic conditions, fixN1 mutant showed a clear nitrogen fixation defect in alfalfa plants that were grown in an N‐free nutrient solution during 3 weeks. However, in plants grown for a longer period, fixNOQP1 copy was not indispensable for symbiotic nitrogen fixation.

Conclusions

The copy 1 of the fixNOQP operon is involved in E. meliloti respiration and growth under micro‐oxic conditions as well as in the expression of the FixO and FixP components of the cbb₃ oxidase present in free‐living microaerobic cultures and in bacteroids. This copy is important for nitrogen fixation during the early steps of the symbiosis.

Significance and Impact of the Study

It is the first time that a functional analysis of the E. meliloti copy 1 of the fixNOQP operon is performed. In this work, the cytochromes c that constitute the cbb₃ oxidase operating in free‐living micro‐oxic cultures and in bacteroids of E. meliloti have been identified.     

A novel method for efficient and abundant production of Phytophthora brassicae zoospores on Brussels sprout leaf discs

Background  

Phytophthora species are notorious oomycete pathogens that cause diseases on a wide range of plants. Our understanding how these pathogens are able to infect their host plants will benefit greatly from information obtained from model systems representative for plant-Phytophthora interactions. One attractive model system is the interaction between Arabidopsis and Phytophthora brassicae. Under laboratory conditions, Arabidopsis can be easily infected with mycelial plugs as inoculum. In the disease cycle, however, sporangia or zoospores are the infectious propagules. Since the current P. brassicae zoospore isolation methods are generally regarded as inefficient, we aimed at developing an alternative method for obtaining high concentrations of P. brassicae zoospores.     

Differential proteomic analysis of Clostridium perfringens ATCC13124; identification of dominant, surface and structure associated proteins

Background  

Clostridium perfringens is a medically important clostridial pathogen causing diseases in man and animals. To invade, multiply and colonize tissues of the host, a pathogen must be able to evade host immune system, and obtain nutrients essential for growth. The factors involved in these complex processes are largely unknown and of crucial importance to understanding microbial pathogenesis. Many of the virulence determinants and putative vaccine candidates for bacterial pathogens are known to be surface localized.     

MCALIGN2: Faster, accurate global pairwise alignment of non-coding DNA sequences based on explicit models of indel evolution

Background  

Non-coding DNA sequences comprise a very large proportion of the total genomic content of mammals, most other vertebrates, many invertebrates, and most plants. Unraveling the functional significance of non-coding DNA depends on how well we are able to align non-coding DNA sequences. However, the alignment of non-coding DNA sequences is more difficult than aligning protein-coding sequences.     

Estimation of tumor heterogeneity using CGH array data

Background  

Array-based comparative genomic hybridization (CGH) is a commonly-used approach to detect DNA copy number variation in whole genome-wide screens. Several statistical methods have been proposed to define genomic segments with different copy numbers in cancer tumors. However, most tumors are heterogeneous and show variation in DNA copy numbers across tumor cells. The challenge is to reveal the copy number profiles of the subpopulations in a tumor and to estimate the percentage of each subpopulation.     

Efficiency clustering for low-density microarrays and its application to QPCR

Background  

Pathway-targeted or low-density arrays are used more and more frequently in biomedical research, particularly those arrays that are based on quantitative real-time PCR. Typical QPCR arrays contain 96-1024 primer pairs or probes, and they bring with it the promise of being able to reliably measure differences in target levels without the need to establish absolute standard curves for each and every target. To achieve reliable quantification all primer pairs or array probes must perform with the same efficiency.