Sergio Delmar dos Anjos e Silva

Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses☆

The increasing demand of strawberry (Fragaria×ananassa Duch) fruits is associated mainly with their sensorial characteristics and the content of antioxidant compounds. Nevertheless, the strawberry production has been hampered due to its sensitivity to abiotic stresses. Therefore, to understand the molecular mechanisms highlighting stress response is of great importance to enable genetic engineering approaches aiming to improve strawberry tolerance. However, the study of expression of genes in strawberry requires the use of suitable reference genes. In the present study, seven traditional and novel candidate reference genes were evaluated for transcript normalization in fruits of ten strawberry cultivars and two abiotic stresses, using RefFinder, which integrates the four major currently available software programs: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. The results indicate that the expression stability is dependent on the experimental conditions. The candidate reference gene DBP (DNA binding protein) was considered the most suitable to normalize expression data in samples of strawberry cultivars and under drought stress condition, and the candidate reference gene HISTH4 (histone H4) was the most stable under osmotic stresses and salt stress. The traditional genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 18S (18S ribosomal RNA) were considered the most unstable genes in all conditions. The expression of phenylalanine ammonia lyase (PAL) and 9-cis epoxycarotenoid dioxygenase (NCED1) genes were used to further confirm the validated candidate reference genes, showing that the use of an inappropriate reference gene may induce erroneous results. This study is the first survey on the stability of reference genes in strawberry cultivars and osmotic stresses and provides guidelines to obtain more accurate RT-qPCR results for future breeding efforts.

Identifying MicroRNAs and Transcript Targets in Jatropha Seeds

MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs) as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs.

Carotenoid Biosynthetic and Catabolic Pathways: Gene Expression and Carotenoid Content in Grains of Maize Landraces

Plant carotenoids have been implicated in preventing several age-related diseases, and they also provide vitamin A precursors; therefore, increasing the content of carotenoids in maize grains is of great interest. It is not well understood, however, how the carotenoid biosynthetic pathway is regulated. Fortunately, the maize germplasm exhibits a high degree of genetic diversity that can be exploited for this purpose. Here, the accumulation of carotenoids and the expression of genes from carotenoid metabolic and catabolic pathways were investigated in several maize landraces. The carotenoid content in grains varied from 10.03, in the white variety MC5, to 61.50 μg·g−1, in the yellow-to-orange variety MC3, and the major carotenoids detected were lutein and zeaxanthin. PSY1 (phythoene synthase) expression showed a positive correlation with the total carotenoid content. Additionally, the PSY1 and HYD3 (ferredoxin-dependent di-iron monooxygenase) expression levels were positively correlated with β-cryptoxanthin and zeaxanthin, while CYP97C (cytochrome P450-type monooxygenase) expression did not correlate with any of the carotenoids. In contrast, ZmCCD1 (carotenoid dioxygenase) was more highly expressed at the beginning of grain development, as well as in the white variety, and its expression was inversely correlated with the accumulation of several carotenoids, suggesting that CCD1 is also an important enzyme to be considered when attempting to improve the carotenoid content in maize. The MC27 and MC1 varieties showed the highest HYD3/CYP97C ratios, suggesting that they are promising candidates for increasing the zeaxanthin content; in contrast, MC14 and MC7 showed low HYD3/CYP97C, suggesting that they may be useful in biofortification efforts aimed at promoting the accumulation of provitamin A. The results of this study demonstrate the use of maize germplasm to provide insight into the regulation of genes involved in the carotenoid pathway, which would thus better enable us to select promising varieties for biofortification efforts.

Caracterização de genótipos de mirtilo utilizando marcadores moleculares

The blueberry crop planting area is increasing in Brazil, especially in Temperate Climate Zones, generating demands relating to suitable cultivars adapted to regional climate and soil conditions. This work aimed to characterize blueberry genotypes from Embrapa Clima Temperado breeding program, using RAPD and SSR molecular markers. There were characterized 40 blueberry genotypes using RAPD and 8 cultivars using SSR molecular markers. The 9 RAPD primers generated 89 markers. The genetic similarity ranged from 64 to 89%. Through the average similarity (66%), it was possible to identify four groups. The three pairs of SSR primers generated 11 markers. The genetic similarity among cultivars ranged from 25 to 75%. With a similarity average of 42,4%, it was generated three groups. It was possible to define the pattern of the eight blueberry cultivars using only three pairs of SST primers, which shows the efficiency of SST technique when characterizing blueberry genotypes. These results reveal that both RAPD and SSR are efficient to characterize genotypes of this specie. However, SSR markers are more accurate and, therefore, recommended for use in breeding programs and cultivars identification.

Micronutrient and Functional Compounds Biofortification of Maize Grains

Maize, in addition to being the main staple food in many countries, is used in the production of hundreds of products. It is rich in compounds with potential benefits to health, such as carotenoids, phenolic compounds, vitamin E, and minerals that act as cofactors for antioxidant enzymes. Many of these compounds have been neglected thus far in the scientific literature. Nevertheless, deficiencies in the precursors of vitamin A and some minerals, such as iron and zinc, in maize, in association with the great genetic variability in its cultivars and our genomic, transcriptomic, and metabolomic knowledge of this species make targeted biofortification strategies for maize promising. This review discusses the potential of the main microconstituents found in maize with a focus on studies aimed at biofortification.

Desempenho agronômico e ganho genético pela seleção de pinhão‑manso em três regiões do Brasil

The objective of this study was to evaluate the agronomic performance and genetic gain by the selection of half-sib families of physic nut grown in three Brazilian regions. Based on previous phenotypic selection, three progeny tests were performed, in 2008, in the municipalities of Planaltina, DF, Nova Porteirinha, MG, and Pelotas, RS. A randomized complete block design was used, with three replicates, and five plants per plot. Randomly collected seeds from a population without selection were used as control. There was a significant interaction between the effects of genotypes and environments. Estimates of variance components and genetic parameters indicated that it is possible to obtain genetic gains from selection of the best families in the evaluated environments. In each environment, at least one family was selected with a higher performance than the control treatment. Mass selection in the three environments provided 72% of genetic gains. The agronomic performance had a high correlation coefficient between the environments of Planaltina and Nova Porteirinha, which did not occur in Pelotas. Genotype x environment interaction should be considered in the recommendations of physic nut genetic material for different environments.

Adaptabilidade e estabilidade de genótipos precoces de cana‑de‑açúcar no Rio Grande do Sul

O objetivo deste trabalho foi avaliar a adaptabilidade e estabilidade de genotipos de cana‑de‑acucar de maturacao precoce, quanto a produtividade de colmos e de Brix, no Estado do Rio Grande do Sul. Quinze genotipos de maturacao precoce foram avaliados nas safras 2009/2010 e 2010/2011, por meio da analise de efeitos principais aditivos e interacoes multiplicativas (AMMI). As interacoes genotipo x ambiente foram de alta significância e evidenciaram a alteracao de posicao na classificacao dos genotipos de um ambiente para outro. Novos genotipos superaram, em produtividade e estabilidade, o genotipo‑padrao precoce RB855156, embora este ainda se destaque pela produtividade, estabilidade moderada e ampla adaptabilidade. O genotipo RB966928 destacou-se pela riqueza e produtividade de Brix, estabilidade moderada e ampla adaptabilidade. Salto do Jacui, RS, e o ambiente mais indicado para testes preliminares de selecao de genotipos.

Capacidade combinatória de genótipos de milho para tolerância ao encharcamento do solo

In southern Brazil, corn could be an alternative for cultivation in flooded areas. However, the development of genotypes tolerant to flooding is fundamental to make this option economically feasible. The objective of this paper was to determine the general and specific combining ability and reciprocal effect for flooding tolerance in corn genotypes. The analysis was based on a diallel cross among four inbreds: two flooding tolerant (CT954280 and CT954330) and two sensitive inbreds (CT966200 and CT966208). The experiment was conducted in 2000 and 2001 under greenhouse conditions at Embrapa Clima Temperado, Pelotas, RS. Root dry matter, plant dry matter, plant high, root length, and leaf chlorophyll content were measured. Genetic variability among the inbreds was detected. Both additive and non-additive effects were important in the determination of the inheritance of flooding tolerance in corn. The correct choice of the female parent is fundamental for tolerant hybrids production, since maternal effects were relevant. Among the analyzed genotypes, the inbred CT954330 presented the highest GCA and may be indicated as a preferential progenitor in crossings that aim to increase flooding tolerance.

Genetic variability in maize and teosinte populations estimated by microsatellites markers

Wild species are important sources of genetic variability and may be exploited by breeding programs. Crosses between teosinte and maize occur freely and teosinte serves as genetic source of agronomic traits for introduction in maize. The objective of this study was to estimate genetic variability among and within maize and teosinte populations (Zea mays mexicana). Two sweet maize populations (BR400 and BR402), two common maize populations (Suwan and Pampa) and one teosinte population were analyzed using microsatellites markers. Results indicated that 64,5% of the variation was detected within the populations, suggesting the possibility of obtaining genetic progress by selection within each population. The analysis with 25 microsatellites loci enabled the identification of 92 alleles with a mean of 3.7 alleles per locus. The average Polymorphism Information Content (PIC) was 0.52. The percentage of polymorphic loci varied from 64% in the BR400 and Pampa populations to 80% in the teosinte population. The estimated genetic distance confirmed the genomic similarity of maize and teosinte.

Retardamento de colheita, método de secagem e qualidade de sementes de mamona

it is known that maxim seed quality is reached at physiological maturity, which is the point of maximum dry weight accumulation, seed vigor and germination. The high moisture content in seeds after their maturity until harvest can accelerate the seed deterioration process, due to high metabolic activity. The objective of this study was to evaluate harvest time with natural and artificial drying and castor seed quality. Seeds were used seeds from the second spike of two cultivars, Al Guarany 2002 and BRS 188 Paraguacu, produced at an experimental station of Embrapa Clima Temperado (Pelotas, RS). The experiment consisted of five treatments: premature harvest of castor seeds with four drying temperatures: 40 (standard), 60, 80 and 100oC, and harvest delayed for 25 days with natural drying on the mother plant (SNP). The premature harvest of castor seeds allied with increases in drying temperature significantly reduced their physiological quality, but the drying temperature 40oC provided better seed quality, while higher temperatures were damaging.