Cell wall polysaccharides from in vitro propagated Cereus hildmannianus K. Schum. plants.

Cereus hildmannianus is a cactus exhibiting morphological and physiological adaptation of its cladodes which ensuring growth in climatic and soil conditions unfavourable for many plant species. Currently, limited water resources and increasing demand for renewable energy make cacti a biomass source for the production of biofuels. Somaclones regenerated from callus in vitro can be a source of new raw material in useful plants. The objective of this work was to determine if the regenerated plants showing two different morphologies present polysaccharide composition different from the wild plant. Somaclones aqueous extraction shows the absence of soluble polysaccharides as mucilage. The alkaline extraction of in vivo cultivated plant showed the presence of starch, type I arabinogalactan, and arabinoxylan and the somaclones showed type I arabinogalactan and arabinoxylan in both morphologies. Hemicelluloses found in the somaclones are not different from in vivo cultivated plants, but somaclones not almost biosynthesize mucilage and starch.

Genetic variability among elite popcorn lines based on molecular and morphoagronomic characteristics.

Knowledge of genetic diversity among genotypes and relationships among elite lines is of great importance for the development of breeding programs. Therefore, the objective of this study was to evaluate genetic variability based on the morphoagronomic and molecular characterization of 18 elite popcorn (Zea mays var. everta) lines to be used by Universidade Estadual de Maringá breeding programs. We used 31 microsatellite primers (widely distributed in the genome), and 16 morphological descriptors (including the resistance to maize white spot, common rust, polysora rust of maize, cercospora and leaf blights). The molecular data revealed variability among the lines, which were divided into four groups that were partially concordant with unweighted pair group method with arithmetic mean (UPMGA) and Bayesian clusters. The lines G3, G4, G11, and G13 exhibited favorable morphological characters and low disease incidence rates. The four groups were confirmed using the Gower distance in the UPGMA cluster; however, there was no association with the dissimilarity patterns obtained using the molecular data. The absence of a correlation suggests that both characterizations (morphoagronomic and molecular) are important for discriminating among elite popcorn lines.

Changes on microsatellites of expressed sequence tag of sugarcane (Saccharum spp) during vegetative propagation.

The reduction in sugarcane productivity in subsequent cutting stages may be related to a gradual decrease of the allele number and mean observed heterozygosity (HO) in the sugarcane ratoon. This hypothesis was tested assessing the number of alleles and HO values in 10 expressed sequence tag microsatellites (Est-SSR loci) of the sugarcane varieties RB72454 and RB867515 in different cutting stages. Changes of allele numbers in samples of different cutting stages were observed in seven and six EstSSR loci of the RB72454 and RB867515 varieties, respectively. Reduction of allele numbers was observed in the samples collected in the fourth and sixth cutting stages of the RB72454 variety. In contrast, an increase of the allele numbers was detected in the samples collected on fourth, sixth, and seventh cutting stages of the RB867515 variety. Unchanged allele numbers were observed only in EstB41, EstC84, and EstB130 loci of the RB72454 variety, and EstB41, EstC67, EstA68, and EstB130 loci of the RB867515 variety. The variety RB867515 has lower polymorphism and values of HO than the RB72454 variety in different stages of cutting. At molecular level, in Est-SSR loci, the RB72454 variety showed higher changes in subsequent stages of cutting than RB867515. The similarities and divergences at molecular level between varieties RB72454 and RB867515 observed in the 10 Est-SSR loci during subsequent cutting stages can not explain the reduced productivity frequently observed after subsequent cutting stages but showed that phenotypic and physiological changes after each cutting stage are also accompanied by changes at genomic level.

Genetic divergence and admixture of ancestral genome groups in the sugarcane variety 'RB867515' (Saccharum spp).

We analyzed 80 plants of the sugarcane (Saccharum spp) variety 'RB867515' in order to investigate its diversity and genetic structure at the molecular level. Four simple sequence repeat (SSR) loci (UGSM51, SMC1237, SEGMS1069, and UGSM38) and five expressed sequence tag (EST)-SSR loci (ESTA68, ESTB92, ESTB145, ESTC66, and ESTC84) were used as molecular markers. The polymorphic loci rate was 66.6%. A total of 17 alleles and an average of 1.88 alleles/locus were detected. The number of alleles in the EST-SSR loci was lower than the number of alleles in the SSRs of non-expressed loci. The mean observed heterozygosity among the nine SSR loci was 0.3291. Genetic structure analysis showed that 'RB867515' contains alleles from three ancestral groups (K = 3), but there is little admixing of alleles in the same plant (from 0.8 to 17.3%); only 1.88% of the plants shared alleles from two or three groups. ESTB92, ESTC84, and UGSM38 were monomorphic, but there was evidence of polymorphism in ESTA68, ESTB145, ESTC66, UGSM51, SMC1237, and SEGMS1069, indicating that 'RB867515' has variability at the molecular level and the potential to be used as a parent in breeding programs. The molecular variability observed in 'RB867515' indicates that the clone terminology that is used to identify this cultivar is inconsistent with the original meaning of "clone", which is defined as a sample of genetically identical plants.

Development of retrotransposon-based markers IRAP and REMAP for cassava (Manihot esculenta).

Retrotransposons are abundant in the genomes of plants. In the present study, inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) markers were developed for the cassava genome (Manihot esculenta Crantz). Four cassava cultivars (Fécula Branca, IPR-União, Olho Junto, and Tamboara, two samples per cultivar) were used to obtain IRAP and REMAP fingerprints. Twelve designed primers were amplified alone and in combinations. The 42 IRAP/REMAP primer combinations amplified 431 DNA segments (bands; markers) of which 36 (8.36%) were polymorphic. The largest number of informative markers (16) was detected using the primers AYF2 and AYF2xAYF4. The number of bands for each primer varied from 3 to 16, with an average of 10.26 amplified segments per primer. The size of the amplified products ranged between 100 and 7000 bp. The AYF2 primer generated the highest number of amplified segments and showed the highest number of polymorphic bands (68.75%). Two samples of each cassava cultivar were used to illustrate the usefulness and the polymorphism of IRAP/REMAP markers. IRAP and REMAP markers produced a high number of reproducible bands, and might be informative and reliable for investigation of genetic diversity and relationships among cassava cultivars.

Genetic structure of Pilosocereus gounellei (Cactaceae) as revealed by AFLP marker to guide proposals for improvement and restoration of degraded areas in Caatinga biome.

Amplified fragment length polymorphism (AFLP) analysis was used to evaluate DNA polymorphism in Pilosocereus gounellei with the aim of differentiating samples grown in different Brazilian semiarid regions. Seven primer pairs were used to amplify 703 AFLP markers, of which 700 (99.21%) markers were polymorphic. The percentage of polymorphic markers ranged from 95.3% for the primer combination E-AAG/M-CTT to 100% for E-ACC/M-CAT, E-ACC/M-CAA, E-AGC/M-CAG, E-ACT/M-CTA, and E-AGG/M-CTG. The largest number of informative markers (126) was detected using the primer combination E-AAC/M-CTA. Polymorphism of the amplified DNA fragments ranged from 72.55% (in sample from Piauí State) to 82.79% (in samples from Rio Grande Norte State), with an average of 75.39%. Despite the high genetic diversity of AFLP markers in xiquexique, analysis using the STRUCTURE software identified relatively homogeneous clusters of xiquexique from the same location, indicating a differentiation at the molecular level, among the plant samples from different regions of the Caatinga biome. The AFLP methodology identified genetically homogeneous and contrasting plants, as well as plants from different regions with common DNA markers. Seeds from such plants can be used for further propagation of plants for establishment of biodiversity conservation units and restoration of degraded areas of the Caatinga biome.

Genetic diversity of popcorn genotypes using molecular analysis.

In this study, we analyzed dominant molecular markers to estimate the genetic divergence of 26 popcorn genotypes and evaluate whether using various dissimilarity coefficients with these dominant markers influences the results of cluster analysis. Fifteen random amplification of polymorphic DNA primers produced 157 amplified fragments, of which 65 were monomorphic and 92 were polymorphic. To calculate the genetic distances among the 26 genotypes, the complements of the Jaccard, Dice, and Rogers and Tanimoto similarity coefficients were used. A matrix of Dij values (dissimilarity matrix) was constructed, from which the genetic distances among genotypes were represented in a more simplified manner as a dendrogram generated using the unweighted pair-group method with arithmetic average. Clusters determined by molecular analysis generally did not group material from the same parental origin together. The largest genetic distance was between varieties 17 (UNB-2) and 18 (PA-091). In the identification of genotypes with the smallest genetic distance, the 3 coefficients showed no agreement. The 3 dissimilarity coefficients showed no major differences among their grouping patterns because agreement in determining the genotypes with large, medium, and small genetic distances was high. The largest genetic distances were observed for the Rogers and Tanimoto dissimilarity coefficient (0.74), followed by the Jaccard coefficient (0.65) and the Dice coefficient (0.48). The 3 coefficients showed similar estimations for the cophenetic correlation coefficient. Correlations among the matrices generated using the 3 coefficients were positive and had high magnitudes, reflecting strong agreement among the results obtained using the 3 evaluated dissimilarity coefficients.

Use of differential levels of mean observed heterozygosity in microsatellite loci of commercial varieties of sugarcane (Saccharum spp).

In this study, we measured the genetic diversity within and among a set of 9 commercial sugarcane varieties used for alcohol and sugar production using 17 microsatellite DNA markers. The UGSM148 and UGSM59 primers were monomorphic for all 74 sugarcane samples. The estimated proportion of simple sequence repeated (SSR) polymorphic loci was 88.23%; 17 alleles were detected. The mean gene diversity of all SSR loci was 0.7279. The highest observed heterozygosity (HO) value was found in the RB72454 variety, whereas the lowest HO value was recorded in the SP813250 variety. The SP813250, RB845210, and RB835054 sugarcane varieties were the most genetically uniform varieties. An extremely high level of population differentiation was detected in the varieties exhibiting similar agronomic characteristics. Analysis of the genetic structure of the 9 sugarcane varieties using SSR markers was especially important to identify SSR loci with high levels of heterozygosity and to identify varieties showing the highest levels of heterozygosity. The monomorphic primers may be used to evaluate the genetic stability of sugarcane during cycles of vegetative multiplication, i.e., propagation via rhizomes.

Genetic divergence among sweet corn lines estimated by microsatellite markers.

The purpose of this study was to analyze the genetic diversity of 15 sugary-1 sweet corn lines by microsatellite markers. One hundred pairs of simple sequence repeat primers that were mapped for field corn were tested. Of these primers, 15% were polymorphic, and all were selected for the evaluation. These primers identified a total of 39 alleles among the 15 loci that were evaluated. The number of alleles per locus in the genotypes ranged from 2 to 4, with an average of 2.60 alleles per locus; the highest number of alleles was observed at the loci Bnlg1083, Umc1241, and Umc1590. The occurrence of null alleles at locus Umc1363 was evident only in line DN44. The proportion of polymorphic loci was the highest in lines DN17.1 and DN6 (73.33%), whereas lines DN47, DN23, and DN28 were more monomorphic than other lines. The loci Bnlg1083 and Umc1506 were polymorphic in 8 and 7 lines, respectively, indicating that these loci might be effective and promising for the identification of polymorphism in other sweet corn lines. The genetic diversity calculated by Rogers' genetic distances indicated the lowest genetic similarity between lines DN9 and DN28 (0.7603) and the highest similarity between lines DN19 and DN6 (0.3724). The dendrogram obtained by the unweighted pair-group method based on arithmetic averages indicated the formation of 4 major groups, showing the crossing of the genotypes DN19 and DN6 with DN8 as a possible alternative for the expression of heterozygosis.

Molecular characterization of endophytes isolated from Saccharum spp based on esterase and ribosomal DNA (ITS1-5.8S-ITS2) analyses.

This study used esterases and ribosomal DNA (rDNA) markers to determine endophytic variability in order to better understand endophyte-host interactions. Polyacrylamide gel electrophoresis and esterase isoenzymes (EST; EC 3.1.1.3), with α-naphthyl acetate and ß-naphthyl acetate as substrates, were used to assess relationships among endophytes. ITS1-5.8S-ITS2 sequencing data were used as rDNA markers. Thirty-two esterases were obtained from 37 isolates of Saccharum spp, which clustered into five endophyte groups. Esterase EST-06 was observed with the highest frequency, being present in 22 of the 37 isolates analyzed, followed by esterase EST-11, which was present in 20 isolates. The esterases EST-10 and EST-14 were present in 19 isolates and EST-09 was present in 18 isolates. The esterase EST-01 was unique to isolate 33 and can, therefore, be used as a marker for this isolate. None of the esterases identified were common to all isolates tested. Similarly, phylogenetic analysis, based on rDNA sequence data, classified the isolates into 5 genus groups: 1) Curvularia with a 100% bootstrap value (BP), 2) Alternaria with 100% BP, 3) Epicoccum with 60% BP, 4) Phoma with 89% BP, and 5) Saccharicola with 100% BP. This polyphyletic analysis based on several markers, therefore, proved to be a valuable approach in determining the relationship between variation in endophytes and their associated host plants. Furthermore, both the esterase and rDNA analyses obtained similar results and were equally effective in resolving relationships.

Genetic diversity analysis of Capsicum spp germplasm bank accessions based on α/ß-esterase polymorphism.

Genetic diversity and structure were analyzed in 10 accessions belonging to Banco Ativo de Germoplasma de Capsicum located at Federal University of Piauí in northwestern Brazil that receives pepper samples grown in community gardens in various regions and Brazilian states. Selections were made from seeds of C. chinense (4 accessions), C. annuum (5 accessions), and C. baccatum (1 accession). Samples consisting of leaves were collected from 4-10 plants of each accession (a total of 85 plants). Native polyacrylamide gel electrophoresis was used to identify α- and ß-esterase polymorphisms. Polymorphism was clearly detected in 5 loci. Sixteen alleles were found at 5 α/ß-esterase loci of the three Capsicum species. In the C. chinense samples, the highest HO and HE values were 0.3625 and 0.4395, respectively, whereas in C. annuum samples, HO and HE values were 0.2980 and 0.3310, respectively; the estimated HO and HE values in C. chinense samples were higher than those detected in C. annuum samples. A deficit of homozygous individuals was found in C. chinense (FIS = -0.6978) and C. annuum (FIS = 0.7750). Genetic differentiation between C. chinense and C. annuum at these loci was high (FST = 0.1867) indicating that C. chinense and C. annuum are genetically structured species for α/ß- esterase isozymes. The esterase analysis showed high genetic diversity among the C. chinense and C. annuum samples and very high genetic differentiation (FST = 0.6321) among the C. chinense and C. annuum samples and the C. baccatum accession.

Selection of high heterozygosity popcorn varieties in Brazil based on SSR markers.

We analyzed genetic structure and diversity among eight populations of popcorn, using SSR loci as genetic markers. Our objectives were to select SSR loci that could be used to estimate genetic diversity within popcorn populations, and to analyze the genetic structure of promising populations with high levels of heterozygosity that could be used in breeding programs. Fifty-seven alleles (3.7 alleles per locus) were detected; the highest effective number of alleles (4.21) and the highest gene diversity (0.763) were found for the Umc2226 locus. A very high level of population differentiation was found (F(ST) = 0.3664), with F(ST) for each locus ranging from 0.1029 (Umc1664) to 0.6010 (Umc2350). This analysis allowed us to identify SSR loci with high levels of heterozygosity and heterozygous varieties, which could be selected for production of inbred lines and for developing new cultivars.

Esterase polymorphisms for analysis of genetic diversity and structure of soybean (Glycine max) cultivars.

We used native polyacrylamide gel electrophoresis to identify polymorphism levels in α- and ß-esterase loci from leaf tissues of Brazilian soybean cultivars for the analysis of population genetic diversity and structure, and to investigate relationships between conventional and genetically modified cultivars. The cultivars included lines developed by a soybean-grower cooperative (CD), by EMBRAPA (BR), and "Roundup Ready" (RR) cultivars. Esterase isozymes recorded with α-naphthyl acetate and ß-naphthyl acetate were produced from 14 loci. Two to three allelic variants were detected in leaves from 420 plants of 21 CD, BR, and RR cultivars at Est-1, Est-2, Est-3, Est-5, and Est-14 loci. The estimated proportion of polymorphic loci in CD cultivars was 21.4%, and in BR and RR cultivars it was 28.6%. High and low H(O) and H(E) values were observed within CD and BR cultivars and a very high cultivar differentiation level was evident in the plants of the 21 CD, BR, and RR cultivars (F(ST) = 0.3865). A low level of differentiation (F(ST) = 0.0289) was detected between conventional and RR cultivars. Plants from cultivar BR37 had the highest level of genetic differentiation compared to the other cultivars. The genetic basis of BR cultivars (0.5538-0.9748) was found to be broader than the genetic basis of CD cultivars (0.7058 for CD205 and CD209 and 0.9995 for CD205 and CD208). Higher genetic identity was detected between plants of CD and CDRR cultivars (I = 0.9816). Understanding the genetic structure of these populations can help provide specific culture strategies for each cultivar, depending on its level of heterozygosity.

Genetic diversity in somatic mutants of grape (Vitis vinifera) cultivar Italia based on random amplified polymorphic DNA.

Random amplified polymorphic DNA (RAPD) markers were used to detect polymorphism and to examine relationships among four table grape clones from northwestern Paraná, in southern Brazil. The 10 primers used for RAPD fingerprints generated 126 reproducible fragments, of which 63, 68, 76, and 72 were polymorphic in cultivars Italia, Rubi, Benitaka, and Brasil, respectively. Among the primers, OPP-08 generated the highest number of fragments, whereas OPE-15 was the most efficient for discriminating polymorphic fragments. The distribution of the clones by cluster analysis indicated that there were no differences in RAPD markers between the colored mutant and the original clone (cultivar Italia), supporting the hypothesis that the non-colored and the colored mutant are the same cultivar. However, we found high levels of polymorphism within and between the cultivars Italia, Rubi, Benitaka, and Brasil (65.1%), contrary to a previous hypothesis that the four clones are genetically uniform. This confirmed our expectation of genetic variation among the clones and within each clone. We conclude that the primers are useful for analyzing the development of the genetic diversity within each of these clones.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean.

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Differential expression for tissue-specific isozymes in three species of Hypostomus Lacépède, 1803 (Teleostei: Loricariidae).

The expression patterns of 14 enzymatic systems in skeletal muscle, liver and heart tissues of three species of Hypostomus from the Iguaçu River basin (Brazil) were investigated. Although the patterns were similar for the three species, different tissues showed differential expressions, and the data showed that differential tissue expressions of isoperoxidases may be due to preferential combination or association of polypeptide subunits. The detected patterns for SOD isozymes showed that the quaternary structures of these enzymes were in disagreement with the subunit number reported for the majority of other vertebrate groups. Tissue-specific restriction on heterotetramer formation also were described in LDH and MDHP isozymes. Thus, these tissue-specific gene expression character in the species of Hypostomus have the greatest potential to be recognized and applied in systematic studies among species of Hypostomus.