Pulse-field gel electrophoresis (PFGE) was used to determine the genome size and characterize karyotypic differences in isolates of the cacao biotype of Crinipellis perniciosa (C-biotype). The karyotype analysis of four isolates from Brazil revealed that this biotype could be divided into two genotypes: one presenting six chromosomal bands and the other presenting eight. The size of the chromosomes ranged from 2.7 to 5.3 Mb. The different genotypes correlate with telomere-based PCR analysis. The isolates with six chromosomal bands had two that appeared to be doublets, as shown by densitometric analysis, indicating that the haploid chromosome number for this biotype is eight. The size of the haploid genomes was estimated at approximately 30 Mb by both PFGE and Feulgen-image analysis. DNA hybridization revealed that the rDNA sequences are clustered on a single chromosome and these sequences were located on different chromosomes in an isolate dependent manner. This is the first report of genome size and chromosomal polymorphism for the C-biotype of C. perniciosa.
"Rincones et al. (2003, 2006) showed genotypic polymorphisms among different biotypes and among C-biotype isolates from different regions of southern Bahia by electrophoretic karyotype and microsatellite analysis. Furthermore, it has been postulated that the genome variability found in homothallic M. perniciosa may be due to transposable elements (TEs) and ectopic recombination guided by the numerous copies of these elements found in the genome (Rincones et al., 2003; Mondego et al., 2008). Transposable elements (TEs) can be defined as sequences of moderately repetitive DNA, characterized by the ability to move in the genome, from one place to another, regardless of the homology between the genomic region where it is inserted and the location to which it is transposed (Daboussi, 1997). "
[Show abstract][Hide abstract] ABSTRACT: Transposase sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative transposase sequence was analyzed in the genome of the phytopathogenic fungus Moniliophthora perniciosa, the causal agent of witches' broom disease of cocoa. Sequence comparisons of the predicted transposase peptide indicate a close relationship with the transposases from the elements of the Tc1-Mariner superfamily. The analysis of the distribution of transposase sequence was done by means of PCR and Southern blot techniques in different isolates of the fungus belonging to C-, L-, and S-biotypes and collected from various geographical areas. The distribution profile of the putative transposase sequence suggests the presence of polymorphic copies among the isolates from C-biotypes. The total DNA hybridization profile of each isolate was used to calculate genetic distance and group by the UPGMA method. C-biotype isolates colleted from of the Bahia showed two hybridization profiles for the transposase sequence. Thus the two different fingerprinting profiles for transposase sequence reported here by Southern analysis could also be correlated to the presence of two different genotypes in Bahia, Brazil.
"Author's personal copy genes associated with traits that may play a role in pathogenesis mechanisms (Mondego et al., 2008). Other complementary M. perniciosa genomic information previously available included: (i) analysis of the M. perniciosa karyotype (Rincones et al., 2003); (ii) analysis of genetic and chromosomal variation among 38 isolates of biotype-C, -S and -L, sampled from various regions of Brazil and Ecuador (Rincones et al., 2006); (iii) molecular epidemiological studies revealing that the fungus may be adapted to and overcome SCA6 resistance (Gramacho et al, 2008; Pires, 2003) and (iv) sequence of the mitochondria M. perniciosa genome (Formighieri et al., 2008). In parallel, a comparative transcription analysis between biotrophic and saprophytic M. perniciosa phases was conducted (Rincones et al., 2008). "
[Show abstract][Hide abstract] ABSTRACT: Cacao (Theobroma cacao L.) is one of the main tropical crops worldwide. It is cultivated primarily to provide cacao liquor, butter, and powder for the chocolate industry, essentially due to its flavour properties. Unfortunately, destructive and newly encountered diseases have frequently been the major factors that limit cacao production in nearly all producing countries. The primary challenge is to develop improved cacao tree cultivars with durable and sustainable resistance to these diseases that at the same time have high bean quality for chocolate production. To achieve this goal, the use of functional genomics can be a key step to speed the development of such cultivars. During the last 10years various functional genomics and some proteomic projects have been initiated, including expressed sequence tag and BAC libraries construction, cacao genome sequencing, expression studies of cacao tissues challenged with the main pathogens (Moniliophthora perniciosa, Phytophthora spp.) or subjected to other stress conditions (e.g. drought), and expression studies related to cacao quality flavour. Various others tools such as cacao in vitro culture, plant transformation or bioinformatics were developed to attend the necessity of cacao studies. Functional genomics research associated with genetics (molecular markers, maps and QTLs) will help to select new cacao varieties with agronomic characteristics demanded by farmers.
Advances in Botanical Research 01/2010; 55. DOI:10.1016/B978-0-12-380868-4.00003-X · 1.25 Impact Factor
"Due to the large error in this estimation we decided to assess the genome size using the genome estimation protocol established in the dog genome survey  (more details in Additional File 1). This analysis resulted in a genome size ranging from 38.7 to 39.0 Mbp, a value similar to the genome length of another fungus belonging the order Agaricales, C. cinerea (37.5 Mbp), and that agrees with the previous size estimated by Feulgen-image analysis . "
[Show abstract][Hide abstract] ABSTRACT: The basidiomycete fungus Moniliophthora perniciosa is the causal agent of Witches' Broom Disease (WBD) in cacao (Theobroma cacao). It is a hemibiotrophic pathogen that colonizes the apoplast of cacao's meristematic tissues as a biotrophic pathogen, switching to a saprotrophic lifestyle during later stages of infection. M. perniciosa, together with the related species M. roreri, are pathogens of aerial parts of the plant, an uncommon characteristic in the order Agaricales. A genome survey (1.9x coverage) of M. perniciosa was analyzed to evaluate the overall gene content of this phytopathogen.
Genes encoding proteins involved in retrotransposition, reactive oxygen species (ROS) resistance, drug efflux transport and cell wall degradation were identified. The great number of genes encoding cytochrome P450 monooxygenases (1.15% of gene models) indicates that M. perniciosa has a great potential for detoxification, production of toxins and hormones; which may confer a high adaptive ability to the fungus. We have also discovered new genes encoding putative secreted polypeptides rich in cysteine, as well as genes related to methylotrophy and plant hormone biosynthesis (gibberellin and auxin). Analysis of gene families indicated that M. perniciosa have similar amounts of carboxylesterases and repertoires of plant cell wall degrading enzymes as other hemibiotrophic fungi. In addition, an approach for normalization of gene family data using incomplete genome data was developed and applied in M. perniciosa genome survey.
This genome survey gives an overview of the M. perniciosa genome, and reveals that a significant portion is involved in stress adaptation and plant necrosis, two necessary characteristics for a hemibiotrophic fungus to fulfill its infection cycle. Our analysis provides new evidence revealing potential adaptive traits that may play major roles in the mechanisms of pathogenicity in the M. perniciosa/cacao pathosystem.
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