DNA barcode regions for differentiating Cattleya walkeriana and C. loddigesii / Regiões de DNA barcode para diferenciar Cattleya walkeriana e C. loddigesii

Growers appreciate Cattleya walkeriana and C. loddigesii due to striking shape and rarity. Thus, this study aimed to evaluate the feasibility of DNA barcode regions, namely ITS1, ITS2 and rpoC1, to discriminate between C. walkeriana and C. loddigesii species. DNA barcode regions were successfully amplified using primers designed to amplify plants. We also included sequences from public databases in order to test if these regions were able to discriminate C. walkeriana and C. loddigesii from other Cattleya species. These regions, and their combinations, demonstrated that the ITS1+ITS2 had the highest average interspecific distance (11.1%), followed by rpoC1 (1.06%). For species discrimination, ITS1+ITS2 provided the best results. The combined data set of ITS1+ITS2+rpoC1 also discriminated both species, but did not result in higher rates of discrimination. These results indicate that ITS region is the best option for molecular identification of these two species and from some other species of this genus.

As espécies Cattleya walkeriana e C. loddigesii são apreciadas pelos colecionadores devido às suas impressionantes forma e raridade. Este estudo teve como objetivo avaliar a viabilidade das regiões DNA barcode, ou seja, ITS1, ITS2 e rpoC1, para discriminar as espécies C. walkeriana e C. loddigesii. Regiões DNA barcode foram amplificadas com êxito utilizando os iniciadores desenhados para plantas. Nós também incluímos sequências de bases públicas de dados, a fim de testar se estas regiões foram capazes de discriminar C. walkeriana e C. loddigesii de outras espécies de Cattleya. Estas regiões e suas combinações demonstraram que o ITS1 + ITS2 teve a maior distância média interespecífica (11,1%), seguido por rpoC1 (1,06%). Para a discriminação das espécies, ITS1 + ITS2 proporcionaram os melhores resultados. Os dados combinados dos ITS1 + ITS2 + rpoC1 também discriminaram ambas as espécies, mas não resultaram em maiores taxas de discriminação. Estes resultados indicam que a região ITS é a melhor opção para a identificação molecular destas duas espécies e a partir de algumas outras espécies deste gênero.

Genome-wide analysis of EgEVE_1, a transcriptionally active endogenous viral element associated to small RNAs in Eucalyptus genomes

Abstract Endogenous viral elements (EVEs) are the result of heritable horizontal gene transfer from viruses to hosts. In the last years, several EVE integration events were reported in plants by the exponential availability of sequenced genomes. Eucalyptus grandis is a forest tree species with a sequenced genome that is poorly studied in terms of evolution and mobile genetic elements composition. Here we report the characterization of E. grandis endogenous viral element 1 (EgEVE_1), a transcriptionally active EVE with a size of 5,664 bp. Phylogenetic analysis and genomic distribution demonstrated that EgEVE_1 is a newly described member of the Caulimoviridae family, distinct from the recently characterized plant Florendoviruses. Genomic distribution of EgEVE_1 and Florendovirus is also distinct. EgEVE_1 qPCR quantification in Eucalyptus urophylla suggests that this genome has more EgEVE_1 copies than E. grandis. EgEVE_1 transcriptional activity was demonstrated by RT-qPCR in five Eucalyptus species and one intrageneric hybrid. We also identified that Eucalyptus EVEs can generate small RNAs (sRNAs),that might be involved in de novo DNA methylation and virus resistance. Our data suggest that EVE families in Eucalyptus have distinct properties, and we provide the first comparative analysis of EVEs in Eucalyptus genomes.

Variations in the sensitivity of different primers for detecting Wolbachia in Anastrepha (diptera: tephritidae)

Wolbachia are endosymbiont bacteria of the family Rickettsiacea that are widespread in invertebrates and occur between 20 percent and 60 percent of Neotropical insects. These bacteria are responsible for reproductive phenomena such as cytoplasmic incompatibility, male killing, feminization and parthenogenesis. Supergroups A and B of Wolbachia are common in insects and can be identified using primers for 16S rDNA, ftsZ and wsp; these primers vary in their ability to detect Wolbachia. The ftsZ primer was the first primer used to detect Wolbachia in Anastrepha fruit flies. The primers for 16S rDNA, ftsZ and wsp and the corresponding PCR conditions have been optimized to study the distribution of Wolbachia and their effect on the biology of Anastrepha in Brazil. In this work, we examined the ability of these primers to detect Wolbachia in Anastrepha populations from three regions in the State of São Paulo, southeastern Brazil. All of the samples were positive for Wolbachia supergroup A when screened with primers for 16S A rDNA and wsp A; the wsp B primer also gave a positive result, indicating cross-reactivity. The ftsZ primer showed a poor ability to detect Wolbachia in Anastrepha and generated false negatives in 44.9 percent of the samples. These findings indicate that reliable PCR detection of Wolbachia requires the use of primers for 16S rDNA and wsp to avoid cross-reactions and false negatives, and that the ftsZ primer needs to be redesigned to improve its selectivity.

Boron transport in Eucalyptus. 2. Identification in silico of a putative boron transporter for xylem loading in eucalypt

Boron (B) is a low mobility plant micronutrient whose molecular mechanisms of absorption and translocation are still controversial. Many factors are involved in tolerance to Boron excess or deficiency. Recently, the first protein linked to boron transport in biological systems, BOR1, was characterized in Arabidopsis thaliana. This protein is involved in boron xylem loading and is similar to bicarbonate transporters found in animals. There are indications that BOR1 is a member of a conserved protein family in plants. In this work, FORESTS database was used to identify sequences similar to this protein family, looking for a probable BOR1 homolog in eucalypt. We found five consensus sequences similar to BOR1; three of them were then used in multiple alignment analysis. Based on amino acid similarity and in silico expression patterns, a consensus sequence was identified as a candidate BOR1 homolog, helping deeper experimental assays that could identify the function of this protein family in Eucalyptus

Eucalyptus ESTs associated with resistance to herbicide inhibitors of aromatic and branched-chain amino acid synthesis

Herbicides inhibit enzymatic systems of plants. Acetolactate synthase (ALS, EC = 4.1.3.18) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) are key enzymes for herbicide action. Hundreds of compounds inhibit ALS. This enzyme is highly variable, enabling the selective control of weeds in a number of crops. Glyphosate, the only commercial herbicide inhibiting EPSPS is widely used for non-selective control of weeds in many crops. Recently, transgenic crops resistant to glyphosate were developed and have been used by farmers. The aim of this study was the data mining of eucalypt expressed sequence tags (ESTs) in the FORESTs Genome Project database (https://forests.esalq.usp.br) related to these enzymes. Representative amino acid sequences from the NCBI database associated with ALS and EPSPS were blasted with ESTs from the FORESTs database using the tBLASTx option of the blast tool. The best blasting reads and clusters from FORESTs, represented as nucleotide sequences, were blasted back with the NCBI database to evaluate the level of similarity with available sequences from different species. One and seven clusters were identified as showing high similarity with EPSPS and ALS sequences from the literature, respectively. The alignment of EPSPS sequences allowed the identification of conserved regions that can be used to design specific primers for additional sequencings

Eucalyptus ESTs corresponding to the enzyme glutamine synthetase and the protein D1, sites of action of herbicides that cause oxidative stress

This work was aimed at locating Eucalyptus ESTs corresponding to the GS enzyme (Glutamine Synthetase, EC = 6.3.1.2) and to the D1 protein, which are directly related to resistance to herbicides that promote oxidative stress. Glutamine Synthetase corresponds to the site of action of the herbicide glufosinate. Herbicides that belong to groups such as ureas, uracils, triazines and triazinones act on the D1-Qb complex (receptor of electrons from the Photosystem II) by inactivating it. The clusters EGEQRT3302E01.g, EGEQRT3001F12.b; EGEZLV1203B04.g; EGBGFB1211H06.g and EGEZLV1205F09.g enclosed complete sequences (with 356 amino acids) of the Glutamine Synthetase enzyme. The cluster EGEQSL1054G06.g is a consensus of four reads and enclosed a complete sequence of D1 Protein (with 353 amino acids). The comparison of the sequences of Protein D1 from different species showed that the substitutions of serine (S) by glycine (G) or serine (S) by threonine (T) at the position 264 could produce plants resistant to herbicides that act on electron flow on Photosystem II. The sequence of amino acids corresponding to the cluster EGEQSL1054G06.g had a serine in position 264 indicating sensitivity of the Eucalyptus plants to herbicides that act on this site

Eucalyptus ESTs corresponding to the protoporphyrinogen IX oxidase enzyme related to the synthesis of heme, chlorophyll, and to the action of herbicides

This work was aimed at locating Eucalyptus ESTs corresponding to the PROTOX or PPO enzyme (Protoporphyrinogen IX oxidase, E.C. 1.3.3.4) directly related to resistance to herbicides that promote oxidative stress, changing the functionality of this enzyme. PROTOX, which is the site of action of diphenyl-ether (oxyfluorfen, lactofen, fomesafen), oxadiazole (oxadiazon and oxadiargyl), and aryl triazolinone (sulfentrazone and carfentrazone) herbicides, acts on the synthesis route of porphyrins which is associated with the production of chlorophyll a, catalases, and peroxidases. One cluster and one single read were located, with e-values better than e-70, associated to PROTOX. The alignment results between amino acid sequences indicated that this enzyme is adequately represented in the ESTs database of the FORESTs project

Acid phosphatase polymorphism within and among populations of Cauliflower (Brassica oleracea var botrytis)

Eighty-one lines of cauliflower (Brassica oleracea var. botrytis) from 12 populations used to produce commercial hybrids in Brazil were screened for polymorphism in the acid phosphatase system, in order to evaluate the usefulness of this marker for the determination of the parental contamination level in hybrid seeds. Little polymorphism was detected in the examined lines, but the system appeared to be very useful for hybrid identification, since the only condition required was polymorphism between the two parental lines. If the analyzed lines were used for hybrid production, 8.4 percent and 12.3 percent of the possible crosses would result in hybrids which can be positively identified using the APS-1 and B1 loci, respectively. If only one plant of each homozygous type (SS or FF) was analyzed in each population, 41 percent and 50 percent of the possible crosses would result in hybrids which can be positively identified using the APS-1 and B1 loci, respectively