The rep gene of Bean golden mosaic virus (BGMV) is essential for virus replication. A mutated rep gene with amino acid codon change in the putative nucleoside triphosphate (NTP) binding motif D262R was created. Phaseolus vulgaris transformation was achieved with a vector that contained the mutated rep and bar genes. A total of 17 initial (T 0) transformants were analyzed. One line (M1/4) showed tolerance to glufosinate ammonium and partial resistance to the virus, that is, disease incidence depended on inoculation level. The incidence of BGMV increased with the increasing number of viruliferous whiteflies per plant, both in the transgenic and in the control plants. However, the number of symptomless plants was significantly higher in the transgenic group. The line M1/4 was studied during several generations and presented stability in the transgene loci and virus resistance. Southern blot analysis with genomic DNA of eight generations led to an estimate of two copies of the rep gene integrated at the same locus. RT-PCR analysis revealed the presence of both bar and rep genes transcripts. The mutated REP protein was present in amounts detectable by Western blot analysis in transgenic plants.

Ten polymorphic microsatellite loci were isolated for Physalaemus cuvieri from a GA-CA enriched library. In 160 P. cuvieri individuals, the number of alleles per locus ranged to 2-9 and the expected heterozygosity ranged from 0.30 to 0.85. The primers were successfully cross-amplified in the congeneric species P. albonotatus, P. ephippifer and Physalaemus cf. cuvieri, suggesting that these loci are potentially useful for studies on population genetic structure of Physalaemus sp.

BackgroundForage grasses of the African genus Urochloa (syn. Brachiaria) are the basis of Brazilian beef production, and there is a strong demand for high quality, productive and adapted forage plants. Among the approximately 100 species of the genus Urochloa, Urochloa decumbens is one of the most important tropical forage grasses used for pastures due to several of its agronomic attributes. However, the level of understanding of these attributes and the tools with which to control them at the genetic level are limited, mainly due to the apomixis and ploidy level of this species. In this context, the present study aimed to identify and characterize molecular microsatellite markers of U. decumbens and to evaluate their cross-amplification in other Urochloa species.FindingsMicrosatellite loci were isolated from a previously constructed enriched library from one U. decumbens genotype. Specific primers were designed for one hundred thirteen loci, and ninety-three primer pairs successfully amplified microsatellite regions, yielding an average of 4.93 alleles per locus. The polymorphism information content (PIC) values of these loci ranged from 0.26 to 0.85 (average 0.68), and the associated discriminating power (DP) values ranged from 0.22 to 0.97 (average 0.77). Cross-amplification studies demonstrated the potential transferability of these microsatellites to four other Urochloa species. Structure analysis revealed the existence of three distinct groups, providing evidence in the allelic pool that U. decumbens is closely related to Urochloa ruziziensis and Urochloa brizantha. The genetic distance values determined using Jaccard’s coefficient ranged from 0.06 to 0.76.ConclusionsThe microsatellite markers identified in this study are the first set of molecular markers for U. decumbens species. Their availability will facilitate understanding the genetics of this and other Urochloa species and breeding them, and will be useful for germplasm characterization, linkage mapping and marker-assisted selection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-1967-9) contains supplementary material, which is available to authorized users.

Sugarcane (Saccharum spp.) is a monocotyledonous semi‐perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several limitations due to its complex polyploid and aneuploid genome. However, improvement by biotechnological engineering is currently the most promising alternative to introduce economically important traits. In this work, we present an improved protocol for Agrobacterium tumefaciens‐mediated transformation of commercial sugarcane hybrids using immature top stalk‐derived embryogenic callus cultures. The callus cultures are transformed with preconditioned A. tumefaciens carrying a binary vector that encodes expression cassettes for a gene of interest and the bialaphos resistance gene (bar confers resistance to glufosinate‐ammonium herbicide). This protocol has been used to successfully transform a commercial sugarcane cultivar, SP80‐3280, highlighting: (i) reduced recalcitrance and oxidation; (ii) high yield of embryogenic callus; (iii) improved selection; and (iv) shoot regeneration and rooting of the transformed plants. Altogether, these improvements generated a transformation efficiency of 2.2%. This protocol provides a reliable tool for a routine procedure for sugarcane improvement by genetic engineering. © 2017 by John Wiley & Sons, Inc.

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Background The production of glucose from cellulose requires cellulases, which are obtained from decomposing microorganisms such as fungi and bacteria. Among the cellulases, β-glucosidases convert cellobiose to glucose and have low concentration in commercial cocktails used for the production of second-generation (2G) ethanol. Genetic engineering can be used to produce recombinant β-glucosidases, and cyanobacteria may be interesting bioreactors. These photosynthetic microorganisms can be cultured using CO 2 emitted from the first-generation ethanol (1G) industry as a carbon source. In addition, vinasse, an effluent of 1G ethanol production, can be used as a source of nitrogen for cyanobacteria growth. Thus, photosynthetic bioreactors cannot only produce cellulases at a lower cost, but also reduce the environmental impact caused by residues of 1G ethanol production. Results In the present work, we produced a strain of Synechococcus elongatus capable of expressing high levels of a heterologous β-glucosidase from a microorganism from the Amazonian soil. For this, the pET system was cloned into cyanobacteria genome. This system uses a dedicated T7 RNA polymerase for the expression of the gene of interest under the control of a nickel-inducible promoter. The results showed that the pET system functions efficiently in S. elongatus , once nickel induced T7 RNA polymerase expression which, in turn, induced expression of the gene of the microbial β-glucosidase at high levels when compared with non-induced double transgenic strain. β-glucosidase activity was more than sevenfold higher in the transformed cyanobacteria than in the wild-type strain. Conclusions The T7 system promotes high expression levels of the cloned gene in S. elongatus , demonstrating that the arrangement in which an exclusive RNA polymerase is used for transcription of heterologous genes may contribute to high-level gene expression in cyanobacteria. This work was the first to demonstrate the use of cyanobacteria for the production of recombinant β-glucosidases. This strategy could be an alternative to reduce the release of 1G ethanol by-products such as CO 2 and vinasse, not only contributing to decrease the cost of β-glucosidase production, but also mitigating the environmental impacts of ethanol industrial plants.

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Macadamia is a nut tree native to Australian rainforest. Due to the small wild populations and the economic interest, studies assessing genetic information are fundamental for use and conservation of this species. Therefore, the present study aimed to assess the structure and genetic diversity of 28 cultivars of macadamia originated from United States, Australia and Brazil, introduced or developed by the Agronomic Institute's breeding program, using 29 new microsatellite loci. The microsatellite loci showed high genetic diversity (3.65 alleles per locus on average). Our results also suggested the existence of no genetic structure between cultivars, regardless of their geographic origin. The modified Rogers' distances between cultivars ranged from 0.227 to 0.671. Despite the lack of information about genealogy, the cultivars from this Brazilian germplasm showed moderate genetic diversity, so they can be used as parents in future crosses.