Comet and cytogenetic tests as tools for evaluating genomic instability in seeds of Oryza sativa L. and Phaseolus vulgaris L. from gene banks

Abstract This study aimed to assess the feasibility of comet and cytogenetic tests as tools for evaluating genomic instability in seeds of Oryza sativa L. (rice) and Phaseolus vulgaris (beans) L. from gene banks. Rice and beans were exposed to methyl methanesulfonate (MMS) as a reference DNA damaging agent. Seeds of two accessions of rice and beans were obtained from Embrapa Rice and Beans - Brazil. Seed groups were imbibed in three concentrations of MMS for three periods of time to carry out cytogenetic tests, and for one period for the comet test. At concentrations of 10 and 15 mg/L, MMS induced cytotoxic and/or mutagenic effects in the meristematic cells of roots from all the accessions of both species. In the comet test, MMS induced genotoxic effects at all the concentrations in the evaluated accessions of rice and beans, except in one accession of beans at the lowest concentration (5 mg/L). Both species showed sensitivity to MMS. The comet test can be proposed for the measurement of genomic instability in accessions of rice and beans in gene banks, as being more sensitive than the cytogenetic tests used.

Genetic diversity analysis in the section Caulorrhizae (genus Arachis) using microsatellite markers

Diversity in 26 microsatellite loci from section Caulorrhizae germplasm was evaluated by using 33 accessions of A. pintoi Krapov. & W.C. Gregory and ten accessions of Arachis repens Handro. Twenty loci proved to be polymorphic and a total of 196 alleles were detected with an average of 9.8 alleles per locus. The variability found in those loci was greater than the variability found using morphological characters, seed storage proteins and RAPD markers previously used in this germplasm. The high potential of these markers to detect species-specific alleles and discriminate among accessions was demonstrated. The set of microsatellite primer pairs developed by our group for A. pintoi are useful molecular tools for evaluating Section Caulorrhizae germplasm, as well as that of species belonging to other Arachis sections.

Genetic diversity in section Rhizomatosae of the genus Arachis (Fabaceae) based on microsatellite markers

The genus Arachis (Fabaceae) native to South America, contains 80 species divided into nine sections, three of which contain species of special economic importance such as the cultivated peanut (Arachis hypogaea), belonging to the section Arachis, and some perennial forage species from sections Caulorrhizae and Rhizomatosae. We used microsatellite markers to assay genetic variability among 77 accessions of four species from section Rhizomatosae, the diploid Arachis burkartii (2n = 2x = 20) and the tetraploid Arachis glabrata, Arachis pseudovillosa and Arachis nitida (2n = 4x = 40). A total of 249 alleles were found in the fifteen loci analyzed and a high degree of intra and interspecific polymorphism was detected. The lowest intraspecific variation occurred in Arachis burkartii, while the smallest estimated interspecific value was between A. nitida and A. pseudovillosa and the largest was between A. burkartii and A. nitida. High observed heterozygosity was detected in A. glabrata. The diploid accessions grouped in one cluster and the tetraploid accessions in another. It was possible to distinguish all 77 accessions and the genetic distance between accessions could not be correlated with geographic origin.

In silico analysis of phytohormone metabolism and communication pathways in citrus transcriptome

Plant hormones play a crucial role in integrating endogenous and exogenous signals and in determining developmental responses to form the plant body throughout its life cycle. In citrus species, several economically important processes are controlled by phytohormones, including seed germination, secondary growth, fruit abscission and ripening. Integrative genomics is a powerful tool for linking newly researched organisms, such as tropical woody species, to functional studies already carried out on established model organisms. Based on gene orthology analyses and expression patterns, we searched the Citrus Genome Sequencing Consortium (CitEST) database for Expressed Sequence Tags (EST) consensus sequences sharing similarity to known components of hormone metabolism and signaling pathways in model species. More than 600 homologs of functionally characterized hormone metabolism and signal transduction members from model species were identified in citrus, allowing us to propose a framework for phytohormone signaling mechanisms in citrus. A number of components from hormone-related metabolic pathways were absent in citrus, suggesting the presence of distinct metabolic pathways. Our results demonstrated the power of comparative genomics between model systems and economically important crop species to elucidate several aspects of plant physiology and metabolism.

Identifying water stress-response mechanisms in citrus by in silico transcriptome analysis

Water deficit is one of the most critical environmental stresses to which plants are submitted during their life cycle. The evolutionary and economic performance of the plant is affected directly by reducing its survival in the natural environment and its productivity in agriculture. Plants respond to water stress with biochemical and physiological modifications that may be involved in tolerance or adaptation mechanisms. A great number of genes have been identified as transcriptionally regulated for water deficit. EST sequencing projects provide a significant contribution to the discovery of expressed genes. The identification and determination of gene expression patterns is important not only to understand the molecular bases of plant responses but also to improve water stress tolerance. In our citrus transcriptome survey we have attempted to identify homologs to genes known to be induced and regulated under water stress conditions. We have identified 89 transcripts whose deduced amino acid sequences share similarities with proteins involved in uptake and transport of water and ion, 34 similar to components of the osmolyte metabolism, 67 involved in processes of membranes and proteins protection and 115 homologs of reactive oxygen species scavenger. Many drought-inducible genes identified are known to be regulated by development, salt, osmotic and low temperature. Their possible roles in specific or general mechanisms of water stress citrus responses are discussed.

Heterologous microsatellite primer pairs informative for the whole genus Arachis

The genus Arachis currently comprises 69 described species, some of which have potential and real value as human and animal foods. These Arachis species have been collected and maintained in germplasm banks to provide material that can be used as sources of genes in breeding programs and for the selection of new cultivars. One of the principal objectives of germplasm conservation is the evaluation of genetic variability, which is best conducted using molecular markers. We investigated the use of heterologous primers to amplify microsatellite loci that could be used to evaluate genetic variability in Arachis germplasm. Fifteen microsatellite primer pairs were tested in 76 accessions of 34 species from the nine Arachis sections. The data indicated that heterologous primers were very useful in Arachis since they had high transferability among the species (91 percent) and allowed the amplification of very polymorphic putative loci, which allowed both the characterization of most accessions and to make inferences about the mating systems of some species analyzed. Our data also revealed that the germplasm analyzed showed high variability, even when represented by few accessions.

Sequence characterization of coding regions of the myostatin gene ( GDF8 ) from Brazilian Murrah buffaloes ( Bubalus bubalis ) and comparison with the Bos taurus sequence

Within about 30 years the Brazilian buffalo (Bubalus bubalis) herd will reach approximately 50 million head as a result of the great adaptive capacity of these animals to tropical climates, together with the good productive and reproductive potential which make these animals an important animal protein source for poor and developing countries. The myostatin gene (GDF8) is important in the physiology of stock animals because its product produces a direct effect on muscle development and consequently also on meat production. The myostatin sequence is known in several mammalian species and shows a high degree of amino acid sequence conservation, although the presence of non-silent and silent changes in the coding sequences and several alterations in the introns and untranslated regions have been identified. The objective of our work was to characterize the myostatin coding regions of B. bubalis (Murrah breed) and to compare them with the Bos taurus regions looking for variations in nucleotide and protein sequences. In this way, we were able to identify 12 variations at DNA level and five alterations on the presumed myostatin protein sequence as compared to non double-muscled bovine sequences.

In silico evaluation of the Eucalyptus transcriptome

The expressed sequence tags (ESTs) produced in the Forests project provide an invaluable opportunity to assess the Eucalyptus transcriptome. Besides providing information on the different proteins produced by this plant, it is possible to infer gene expression profiles because non-normalized cDNA libraries were used. The EST frequency from any gene is correlated to the transcript levels in the tissues from which the cDNA libraries were constructed. The goal of this work was to identify Eucalyptus genes that showed either differential expression pattern or were ubiquitously expressed in the tissues sampled in the Forests project. Six robust statistical tests and very restrictive rules were applied to gain confidence in the in silico data aiming to avoid false positives. Several genes with interesting expression profiles were identified and some of them were validated by RT-PCR

Effects of CSN3 and LGB gene polymorphisms on production traits in beef cattle

The objective of the present study was to estimate the allele and genotype frequencies of the CSN3/HinfI and LGB/HaeIII gene polymorphisms in beef cattle belonging to different genetic groups, and to determine the effects of these polymorphisms on growth and carcass traits in these animals, which are submitted to an intensive production model. Genotyping was performed on 79 Nelore, 30 Canchim (5/8 Charolais + 3/8 Zebu) and 275 crossbred cattle originating from the crosses of Simmental (n = 30) and Angus (n = 245) sires with Nelore females. Body weight, weight gain, dressing percentage, longissimus dorsi area and backfat thickness were fitted using the GLM procedure, and least square means of the genotypes were compared by the F test. The results showed that the CSN3/HinfI and LGB/HaeIII polymorphisms did not have any effect on growth or carcass traits (p > 0.05).

Genetic relationships among Arachis species based on AFLP

Amplified Fragment Length Polymorphism (AFLP) was used to establish the genetic relationships among 20 species from seven of the nine sections of genus Arachis. The level of polymorphism among nine accessions of the cultivated peanut, A. hypogaea L., was also evaluated. Three combinations of primers were used to amplify the AFLPs. The fragments were separated in 6 por cento denaturing acrylamide gels. A total of 408 fragments were analyzed. An average of 135.3 fragments per primer combination were scored, and the largest number of fragments was 169 using primer combination Eco RI - ACC / Mse I - CTG, while the lowest was 108, with Eco RI - ACT / Mse I - CTT. In general, the genetic relationships established using AFLPs agreed with the classification established using morphology and crossability data. The results indicated that AFLPs are good markers for establishing the relationships among Arachis species. The polymorphism detected in A. hypogaea by this method was higher than the one found with other markers, like RAPDs and RFLPs. However, our data suggest that the polymorphism detected be using AFLP with only three primer combinations is still too low to be used for any kind of genetic study in this species