Mapping MHC Genes in River Buffalo

The major histocompatibility complex (MHC) contains a set of genes necessary for antigen presentation in the immune system. This gene dense and polymorphic region of the mammalian genome is of considerable interest due to the role of MHC genes in immune function and animal health. Previous cytogenetic studies have indicated that the MHC in river buffalo resides on the short arm of chromosome 2 (BBU2). A 5000-rad radiation hybrid mapping panel was recently generated to enable construction of a whole genome map of river buffalo. To this and, the aims of this project were to elucidate the general organization of the MHC on BBU2, and to compare gene order within this region to the MHC in cattle. PCR primers were selected from the bovine gene map and used with the BBURH(5000) panel to map a set of ten MHC class 11 genes in river buffalo. Analysis indicates that these genes fall into two linkage groups, consistent with organization of the MHC in cattle. This comparison of buffalo and bovine MHC gene order provides the first insight into the organization of the MHC on river buffalo chromosome 2.

A first generation whole genome RH map of the river buffalo with comparison to domestic cattle

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative genomic analysis of plant-associated bacteria

This review deals with a comparative analysis of seven genome sequences from plant-associated bacteria. These are the genomes of Agrobacterium tumefaciens, Mesorhizobium loti, Sinorhizobium meliloti, Xanthomonas campestris pv campestris, Xanthomonas axonopodis pv citri, Xylella fastidiosa, and Ralstonia solanacearum. Genome structure and the metabolism pathways available highlight the compromise between the genome size and lifestyle. Despite the recognized importance of the type III secretion system in controlling host compatibility, its presence is not universal in all necrogenic pathogens. Hemolysins, hemagglutinins, and some adhesins, previously reported only for mammalian pathogens, are present in most organisms discussed. Different numbers and combinations of cell wall degrading enzymes and genes to overcome the oxidative burst generally induced by the plant host are characterized in these genomes. A total of 19 genes not involved in housekeeping functions were found common to all these bacteria.

Mapping QTLs for aluminum tolerance in maize

Aluminum toxicity is one of the major constraints for plant development in acid soils, limiting food production in many countries. Cultivars genetically adapted to acid soils may offer an environmental compatible solution, providing a sustainable agriculture system. The aim of this work was to identify genomic regions associated with Al tolerance in maize, and to quantify the genetic effects on the phenotypic variation. A population of 168 F-3:4 families derived from a cross between two contrasting maize inbred lines for Al tolerance was evaluated using the NSRL and RSRL parameters in nutrient solution containing toxic level of aluminum. Variance analyses indicated that the NSRL was the most reliable phenotypic index to measure Al tolerance in the population, being used for further QTL mapping analysis. RFLP and SSR markers were selected for bulked segregant analysis, and additional SSR markers, flanking the polymorphisms of interest, were chosen in order to saturate the putative target regions. Seven linkage groups were constructed using 17 RFLP and 34 SSR markers. Five QTLs were mapped on chromosomes 2, 6 and 8, explaining 60% of the phenotypic variation. QTL(4) and marker umc043 were located on chromosomes 8 and 5, close to genes encoding for enzymes involved in the organic acids synthesis pathways, a widely proposed mechanism for Al tolerance in plants. QTL(2) was mapped in the same region as Alm2, also associated with Al tolerance in maize. In addition, dominant and additive effects were important in the control of this trait in maize.

Comparative analyses of the complete genome sequences of Pierce's disease and citrus variegated chlorosis strains of Xylella fastidiosa

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X.fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X.fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X.fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X.fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.

Clarifications on breakpoints in HSAX and BTAX by comparative mapping of F9, HPRT, and XIST in cattle

The coagulation factor IX gene (179), the hypoxanthine phosphoribosyl transferase 1 gene (HPRT1), and the X-inactive specific transcript gene (XIST) were physically assigned in cattle to analyze chromosomal breakpoints on BTAX recently identified by radiation hybrid (RH) mapping experiments. Whereas the FISH assignment of XIST indicates a similar location on the q-arm of the human and cattle X chromosomes, the locus of HPRT1 supported the assumption of a chromosome rearrangement between the distal half of the q-arm of HSAX and the p-arm of BTAX identified by RH mapping. F9 previously located on the Cl-arm of BTAX was assigned to the p-arm of BTAX using RH mapping and FISH. The suggested new position of F9 close to HPRT I supports the homology between HSAXq and BTAXp. The F9 locus corresponds with the gene order found in the homologous human chromosome segment. XIST was assigned on BTAXq23, HPRT1 and F9 were mapped to BTAXp22, and the verification of the location of F9 in a 5000 rad cattle-hamster whole genome radiation hybrid panel linked the gene to markers URB10 and HPRT1. Copyright (C) 2003 S. Karger AG, Basel.

Nicotiana tabacum as an experimental host for the study of plant-Xylella fastidiosa interactions

Xylella fastidiosa causes citrus variegated chlorosis (CVC). Information generated from the X. fastidiosa genome project is being used to study the underlying mechanisms responsible for pathogenicity. However, the lack of an experimental host other than citrus to study plant-X. fastidiosa interaction has been an obstacle to accelerated progress in this area. We present here results of three experiments that demonstrated that tobacco could be an important experimental host for X. fastidiosa. All tobacco plants inoculated with a citrus strain of X. fastidiosa expressed unequivocal symptoms, consisting of orange leaf lesions, approximately 2 months after injection of the pathogen. CVC symptoms were observed in citrus 3 to 6 months after inoculation. The pathogen was readily detected in symptomatic tobacco plants by polymerase chain reaction (PCR) and phase contrast microscopy. In addition, X. fastidiosa was reisolated on agar plates in 4 of 10 plants. Scanning electron microscopy analysis of cross sections of stems and petioles revealed the presence of rod shaped bacteria restricted to the xylem of inoculated plants. The cell size was within the limit typical of X. fastidiosa.

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

The plant energy-dissipating mitochondrial systems: Depicting the genomic structure and the expression profiles of the gene families of uncoupling protein and alternative oxidase in monocots and dicots

The simultaneous existence of alternative oxidases and uncoupling proteins in plants has raised the question as to why plants need two energy-dissipating systems with apparently similar physiological functions. A probably complete plant uncoupling protein gene family is described and the expression profiles of this family compared with the multigene family of alternative oxidases in Arabidopsis thaliana and sugarcane (Saccharum sp.) employed as dicot and monocot models, respectively. In total, six uncoupling protein genes, AtPUMP1-6, were recognized within the Arabidopsis genome and five (SsPUMP1-5) in a sugarcane EST database. The recombinant AtPUMP5 protein displayed similar biochemical properties as AtPUMP1. Sugarcane possessed four Arabidopsis AOx1-type orthologues (SsAOx1a-1d); no sugarcane orthologue corresponding to Arabidopsis AOx2-type genes was identified. Phylogenetic and expression analyses suggested that AtAOx1d does not belong to the AOx1-type family but forms a new (AOx3-type) family. Tissue-enriched expression profiling revealed that uncoupling protein genes were expressed more ubiquitously than the alternative oxidase genes. Distinct expression patterns among gene family members were observed between monocots and dicots and during chilling stress. These findings suggest that the members of each energy-dissipating system are subject to different cell or tissue/organ transcriptional regulation. As a result, plants may respond more flexibly to adverse biotic and abiotic conditions, in which oxidative stress is involved. © The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.

Characterization and genome organization of a repetitive element associated with the nucleolus organizer region in leporinuselongatus (anostomidae: Characiformes)

Chromosome mapping and studies of the genomic organization of repetitive DNA sequences provide valuable insights that enhance our evolutionary and structural understanding of these sequences, as well as identifying chromosomal rearrangements and sex determination. This study investigated the occurrence and organization of repetitive DNA sequences in Leporinus elongatus using restriction enzyme digestion and the mapping of sequences by chromosomal fluorescence in situ hybridization (FISH). A 378-bp fragment with a 54.2% GC content was isolated after digestion with the SmaI restriction enzyme. BLASTN search found no similarity with previously described sequences, so this repetitive sequence was named LeSmaI. FISH experiments were conducted using L. elongatus and other Anostomidae species, i.e. L. macrocephalus,L. obtusidens, L. striatus, L. lacustris, L. friderici, Schizodon borellii, S. isognathus, and Abramites hypselonotus which detected signals that were unique to male and female L. elongatus individuals. Double-FISH using LeSmaI and 18S rDNA showed that LeSmaI was located in a nucleolus organizer region (NOR) in the male and female metaphases of L. elongatus. This report also discusses the role of repetitive DNA associated with NORs in the diversification of Anostomidae species karyotypes. Copyright © 2012 S. Karger AG, Basel.

Chromosome mapping of repetitive sequences in Anostomidae species: Implications for genomic and sex chromosome evolution

Background: Members of the Anostomidae family provide an interesting model system for the study of the influence of repetitive elements on genome composition, mainly because they possess numerous heterochromatic segments and a peculiar system of female heterogamety that is restricted to a few species of the Leporinus genus. The aim of this study was to isolate and identify important new repetitive DNA elements in Anostomidae through restriction enzyme digestion, followed by cloning, characterisation and chromosome mapping of this fragment. To identify repetitive elements in other Leporinus species and expand on studies of repetitive elements in Anostomidae, hybridisation experiments were also performed using previously described probes of LeSpeI repetitive elements. Results: The 628-base pair (bp) LeSpeII fragment was hybridised to metaphase cells of L. elongatus individuals as well as those of L. macrocephalus, L. obtusidens, L. striatus, L. lacustris, L. friderici, Schizodon borellii and S. isognathus. In L. elongatus, both male and female cells contained small clusters of LeSpeII repetitive elements dispersed on all of the chromosomes, with enrichment near most of the terminal portions of the chromosomes. In the female sex chromosomes of L. elongatus (Z2,Z2/W1W 2), however, this repeated element was absent. In the remaining species, a dispersed pattern of hybridisation was observed on all chromosomes irrespective of whether or not they were sex chromosomes. The repetitive element LeSpeI produced positive hybridisations signals only in L. elongatus, L. macrocephalus and L. obtusidens, i.e., species with differentiated sex chromosomes. In the remaining species, the LeSpeI element did not produce hybridisation signals. Conclusions: Results are discussed in terms of the effects of repetitive sequences on the differentiation of the Anostomidae genome, especially with respect to sex chromosome evolution. LeSpeII showed hybridisation patterns typical of Long Interspersed Elements (LINEs). The differential distribution of this element may be linked to sex chromosome differentiation in L. elongatus species. The relationship between sex chromosome specificity and the LeSpeI element is confirmed in the species L. elongatus, L. macrocephalus and L. obtusidens. © 2012 da Silva et al.; licensee BioMed Central Ltd.

Cytogenetic mapping of H1 histone and ribosomal RNA genes in hybrids between catfish species Pseudoplatystoma corruscans and Pseudoplatystoma reticulatum

A physical chromosome mapping of the H1 histone and 5S and 18S ribosomal RNA (rRNA) genes was performed in interspecific hybrids of Pseudoplatystoma corruscans and P. reticulatum. The results showed that 5S rRNA clusters were located in the terminal region of 2 chromosomes. H1 histone and 18S ribosomal genes were co-localized in the terminal portion of 2 chromosomes (distinct from the chromosomes bearing 5S clusters). These results represent the first report of association between H1 histone and 18S genes in fish genomes. The chromosome clustering of ribosomal and histone genes was already reported for different organisms and suggests a possible selective pressure for the maintenance of this association. © 2012 S. Karger AG, Basel.

Screening of genomic libraries

Microsatellites, or simple sequence repeats (SSRs), have proven to be an important molecular marker in plant genetics and breeding research. The main strategies to obtain these markers can be through genomic DNA and from expressed sequence tags (ESTs) from mRNA/cDNA libraries. Genetic studies using microsatellite markers have increased rapidly because they can be highly polymorphic, codominant markers and they show heterozygous conserved sequences. Here, we describe a methodology to obtain microsatellite using the enrichment library of DNA genomic sequences. This method is highly efficient to development microsatellite markers especially in plants that do not have available ESTs or genome databases. This methodology has been used to enrich SSR marker libraries in Citrus spp., an important tool to genotype germplasm, to select zygotic hybrids, and to saturate genetic maps in breeding programs. © Springer Science+Business Media, LLC 2013.