A. P. de Souza

Analysis of genetic similarity detected by AFLP and coefficient of parentage among genotypes of sugar cane (Saccharum spp.)

Abstract Despite the economical importance of sugar cane, until the present-date no studies have been carried out to determine the correlation of the molecular-based genetic similarity (GS) and the coefficient of parentage (f)-estimates generated for cultivars. A comprehensive knowledge of the amount of genetic diversity in parental cultivars, could improve the effectiveness of breeding programmes. In this study, amplified fragment length polymorphism (AFLP) and pedigree data were used to investigate the genetic relationship in a group of 79 cultivars (interspecific hybrids), used as parents in one of the Brazilian breeding programmes, and four species of Saccharum (Saccharum sinense, Saccharum barberi and two of Saccharum officinarum) . The objectives of this study were to assess the level of genetic similarity among the sugar-cane cultivars and to investigate the correlation between the AFLP-based GSand f, based on pedigree information. Twenty one primer combinations were used to obtain the AFLP molecular markers, generating a total of 2,331 bands, of which 1,121 were polymorphic, with a polymorphism rate, on average, of 50% per primer combination. GSs were determined using Jaccard’s similarity coefficient, and a final dendrogram was constructed using an unweighted pair-group method using arithmetic average (UPGMA). AFLP-based GS ranged from 0.28 to 0.89, with a mean of 0.47, whereas f ranged from 0 to 0.503, with a mean of 0.057. Cluster analysis using GS divided the genotypes into related subgroups suggesting that there is important genetic relationship among the cultivars. AFLP-based GS and f were significantly correlated (r = 0.42, P < 0.001), thus the significance of this r value suggests that the AFLP data may help to more-accurately quantify the degree of relationship among sugar-cane cultivars.

Genetic distance of inbred lines and prediction of maize single-cross performance using RAPD markers

Abstract To evaluate the genetic diversity of 18 maize inbred lines, and to determine the correlation between genetic distance and single-cross hybrid performance, we have used random amplified polymorphic DNA (RAPD), a PCR-based technique. Eight of these lines came from a Thai synthetic population (BR-105), and the others derived from a Brazilian composite population (BR-106). Thirty two different primers were used giving a total of 325 reproducible amplification products, 262 of them being polymorphic. Genetic divergence was determinated using Jaccard’s similarity coefficient, and a final dendrogram was constructed using an unweighted pair-group method with arithmetical averages (UPGMA). Cluster analysis divided the samples into three distinct groups (GI, GII and GIII) that were confirmed by principal-coordinate analysis. The genetic distances (GD) were correlated with important agronomic traits for single-cross hybrids and heterosis. No correlation was found when group division was not considered, but significant correlations were detected between GI×GII and GI×GIII GDs with their respective single-cross hybrid grain-yield values. Three groups were identified; that is, the BR-106 population was divided in two different groups and the BR-105 population remained mostly as one group. The results indicated that RAPD can be used as a tool for determining the extent of genetic diversity among tropical maize inbred lines, for allocating genotypes into different groups, and also to aid in the choice of the superior crosses to be made among maize inbred lines, so reducing the number of crosses required under field evaluation.

Inhibition of human gingival gelatinases (MMP-2 and MMP-9) by metal salts.

OBJECTIVES The interaction between metal ions and the oral environment is a major subject matter in dental research. Matrix metalloproteinases (MMPs) have been implicated in several pathologic oral processes such as periodontal tissue destruction, root caries, tumour invasion and temporomandibular joint disorders. The aim of this work was to test the effect of Zn, Cu, Sn and Hg ions on the activity of the major gingival gelatinolytic MMPs. METHODS Gingival explants were cultured overnight in DMEM and the activity of secreted enzymes was analyzed by gelatin zymography in buffers containing different metal ion concentrations. The major gelatinolytic proteinases present in the conditioned media were characterized as MMP-2 and MMP-9 by immunoprecipitation with specific antibodies. The eletrophoretic bands were scanned and the transmittance values were analyzed with the Sigmagel software (Sigma). RESULTS ZnSO4 was a strong inhibitor of MMP-2 (I50 = 15 microM) and MMP-9 (I50 = 40 microM), whereas CuSO4, HgSO4 and SnCl2 showed less efficient inhibition potential. SIGNIFICANCE Our findings show that the activity of oral tissue MMPs may be modulated by metal ions present in the oral environment. Therefore, the accumulation of metals in connective tissue may interfere with the formation and resorption of the extracellular matrix components.

Tropical maize germplasm: what can we say about its genetic diversity in the light of molecular markers?

Knowledge about genetic variability of a crop allows for more efficient and effective use of resources in plant improvement programs. The genetic variation within temperate maize has been studied extensively, but the levels and patterns of diversity in tropical maize are still not well understood. Brazilian maize germplasm represents a very important pool of genetic diversity due to many past introductions of exotic material. To improve our knowledge of the genetic diversity in tropical maize inbred lines, we fingerprinted 85 lines with 569 AFLP bands and 50 microsatellite loci. These markers revealed substantial variability among lines, with high rates of polymorphism. Cluster analysis was used to identify groups of related lines. Well-defined groups were not observed, indicating that the tropical maize studied is not as well organized as temperate maize. Three types of genetic distance measurements were applied (Jaccard’s coefficient, Modified Rogers’ distance and molecular coefficient of coancestry), and the values obtained with all of them indicated that the genetic similarities were small among the lines. The different coefficients did not substantially affect the results of cluster analysis, but marker types had a large effect on genetic similarity estimates. Regardless of genetic similarity coefficient used, estimates based on AFLPs were poorly correlated with those based on SSRs. Analyses using AFLP and SSR data together do not seem to be the most efficient manner of assessing variability in highly diverse materials because the result was similar to using AFLPs alone. It was seen that molecular markers can help to organize the genetic variability and expose useful diversity for breeding purposes.

Mapping QTLs for kernel oil content in a tropical maize population

Maize cultivars often have low kernel oil content. To increase the oil content, efficient maize breeding programs have to be developed, which require the knowledge of the inheritance of this trait. Thus, the objective of this research was to map quantitative trait locus (QTLs) and estimate their effects for kernel oil content in a tropical maize population. Two maize inbred lines, contrasting for kernel oil content, were used to develop an F2 population. Four hundred and eight F2 plants were self-pollinated, and their kernels (F2:3 progenies) were used for kernel oil evaluation. A genetic map with 75 microsatellites was developed, and the QTLs were mapped using the composite interval map (CIM); also, estimates of genetic and phenotypic variances, and heritability coefficient were computed. The map presented 10 linkage groups, spanned 1,438.6 cM in length with an average interval of 19.18 cM between adjacent markers. The kernel oil content averaged 58.40 g kg−1, and the broad-sense heritability was high (h2= 0.98). Thirteen QTLs were mapped, which were distributed into eight chromosomes, and explained 26.64% of the genetic variation. QTLs in chromosomes 1, 5, and 6 contributed the most for kernel oil content. Nine out of 13 QTLs with favorable alleles were from the parental inbred with the highest kernel oil content. The average level of dominance was partial, but gene action of the QTLs ranged from additive to overdominance. Eight out of 13 mapped QTLs were already reported for temperate maize populations.

Development of microsatellite markers for Brachiaria humidicola (Rendle) Schweick

We describe the first panel of nuclear simple sequence repeats (SSRs) loci for Brachiaria humidicola (Rendle) Schweick., a warmseason grass with facultative apomixis, variation in ploidy levels (6X–9X), and important forage grass species in the Tropics. Of 38 pairs of primers obtained by using an enriched-library methodology, 27 revealed polymorphism in 58 accessions of the Germplasm Collection of B. humidicola held at Embrapa Beef Cattle, Brazil. Eleven loci amplified in B. dictyoneura, a closely related species with unclear taxonomic boundaries with B. humidicola. Transferability to other three Brachiaria species was also evaluated. The developed microsatellites are potentially useful for genetic studies of B. humidicola, as well as phylogenetic evaluations, conservation and breeding applications.

Influence of MMP-8 promoter polymorphism in early osseointegrated implant failure.

ObjectiveDental implants consist in the treatment of choice to replace tooth loss. The knowledge that implant loss tends to cluster in subsets of individuals may indicate that host immuneinflammatory response is influenced by genetic factors. In fact, genetic polymorphisms influence the osseointegration process. The objective of this study was investigate the possible relationship between C-799T polymorphism in matrix metalloproteinase 8 (MMP-8) gene and early implant failure in nonsmoker patients.Methods and materialsSubjects were divided into two groups: control group (100 patients with one or more healthy implants) and test group (80 patients that had suffered one or more early implant failures). Genomic DNA from oral mucosa was amplified by PCR and analyzed by restriction endonucleases. The significance of the differences in observed frequencies of polymorphisms was assessed by Chi-square.ResultsStatistical analysis shows that in the MMP-8 gene, the T allele in 76.25% in the test group and the T/T genotype, 63.75% in the same group, may predispose to early loss of implants osseointegrated.ConclusionThese results suggest that polymorphism in the promoter region of MMP-8 gene is associated with early implant failure. This polymorphism can be a genetic marker to risk of implant loss.Clinical relevanceThe determination of this genetic pattern in osseointegration would enable the identification of individuals at higher risk to loss implant. Thus, genetic markers will be identified, contributing to an appropriate preoperative selection and preparation of strategies for prevention and therapy individualized to modulate the genetic markers and increase the success rate of treatments.

Isolation and characterization of microsatellite markers for Brachiaria brizantha (Hochst. ex A. Rich.) Stap

The first set of nuclear simple sequence repeat (SSR) loci for Brachiaria brizantha (Hochst. ex A. Rich.) Stap is described. A microsatellite-enriched library was constructed and 19 loci were characterized. About 13 SSR loci were found to be polymorphic and across-taxa amplification tests showed that six of them can be transferred to four other species of Brachiaria. This new SSR resource will be a powerful tool for population genetic studies of B. brizantha, for interspecific genetic studies within the genus Brachiaria, for mapping and for marker assisted selection in breeding.

Isolation and characterization of microsatellite loci in tropical forage Stylosanthes capitata Vogel.

Stylosanthes capitata is an important tropical pasture legume. Knowledge of genetic diversity and structure of S. capitata populations is of great importance for the conservation and germplasm management of this species. Thus, eight microsatellite markers were developed from an S. capitata‐enriched library. They were characterized in 20 accessions from the germplasm collection of the Empresa Brasileira de Pesquisa Agropecuária (Embrapa). The observed and expected heterozygosities ranged from 0.16 to 0.85 and from 0.40 to 0.85, respectively. These microsatellites are the first set of molecular markers from this species and will contribute towards studies of genetic diversity, conservation and breeding of S. capitata.

DNA methylation levels of SOCS1 and LINE-1 in oral epithelial cells from aggressive periodontitis patients

OBJECTIVE DNA methylation has been shown to be critical in the regulation of inflammatory genes. Infections are able to trigger susceptibility to disease and it can be considered as potential epimutagenic factors in reshaping the epigenome. Therefore, what would be the DNA methylation status in cells present in an infected and inflamed oral environment? The aim was to verify the DNA methylation pattern in oral epithelium cells from aggressive periodontitis (AgP) patients in a specific gene involved in the inflammation control, as suppressor of cytokine signalling (SOCS)1 and in a broader way through long interspersed nuclear element (LINE)-1. DESIGN Genomic DNA from oral cells of 30 generalized AgP patients and 30 healthy patients were purified and modified by sodium bisulfite. DNA methylation patterns were analyzed using combined bisulfite restriction analysis (COBRA) for SOCS1 and LINE-1. RESULTS An overall scenario of demethylation was seen for both groups, whereas the healthy group presented a higher percentage of demethylation (p<0.001), also presenting the majority of total demethylated samples (83.3% versus 70.8% in the AgP group). Total LINE-1 methylation or at each specific loci presented significant differences amongst groups. CONCLUSION Epithelial cells, present in an infected and inflamed oral environment, show different DNA methylation status from those present in a healthy oral environment, regarding the SOCS1 and LINE-1. In addition, the investigation allows detecting alterations in the DNA in a non-limited manner, since the results observed might reflect a generalized condition of the oral epithelial cells, besides reflecting the condition of the gingival epithelium cells.