Artur da Câmara Machado

Regeneration of transgenic plants of Prunus armeniaca containing the coat protein gene of Plum Pox Virus

SummaryA system was developed which allows the transfer of foreign genes into apricot cultivars. We report the transformation and regeneration of Prunus armeniaca plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene ß-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV). The marker gene GUS was used for optical evaluation of the efficiency of the transformation system. The coat protein gene of PPV was used to introduce coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area. This is the first report of the successful integration of a viral coat protein gene into a fruit tree species, opening a new perspective on the control of the disease.

Coat protein mediated resistance to Plum Pox Virus in Nicotiana clevelandii and N. benthamiana

Transgenic Nicotiana benthamiana and N. clevelandii plants expressing the coat protein of Plum Pox Virus under the control of the 35S promoter from Cauliflower Mosaic Virus were engineered by Agrobacterium tumefaciens mediated transformation. The phenomenon of virus resistance was observed at different levels when transgenic plants, expressing the coat protein and control plants were compared after challenge infection with Plum Pox Virus. N. clevelandii coat protein transgenic plants circumvent virus accumulation. After an initial increase in virus titer similar to the control plants, some coat protein expressing plants showed a reduced accumulation of virus and inhibition of the systemic spread, characterized by decrease of the virus titer and formation of new symptomless leaves. In other N. clevelandii coat protein expressing plants virus accumulation was inhibited and disease symptoms never appeared. N. benthamiana coat protein expressing plants were also protected. After a temporary virus accumulation, virus titer decreased without the appearance of symptoms with the exception of a few plants, which showed a delay of thirty days in the development of symptoms post challenge infection.

Coat protein-mediated protection against plum pox virus in herbaceous model plants and transformation of apricot and plum

The expression of the viral coat protein gene in transgenic plants has been shown to induce tolerance against virus infection (Beachy et al., 1990). Transgenic plants of Nicotiana clevelandii and Nicotiana benthamiana - herbaceous host plants for PPV - transformed with Agrobacterium strain LBA 4404 containing the plasmid pBinPPVm, regenerated on selection media containing kanamycin were tested for the expression of the PPV-coat protein gene by ELISA and immuno western blot. After rooting and acclimatisation plants were tested for the protection against PPV. Following the inoculation plants were investigated for symptom development and virus accumulation. Different lines were identified, according to the different reaction to the mechanical inoculation, ranging from a complete absence to a strong reduction of symptoms. There have not been many reports on transformation of trees in general, and in fruit trees particularly. It is obvious that the major obstacle is the regeneration of transformed plantlets. Attempts to improve crop plants by genetic engineering techniques will always depend very strongly on the availability of reliable protocols for transformation, selection and regeneration (Laimer et al., 1989, 1990). Different systems involving juvenile and adult plant material have been developed allowing the transfer of foreign genes into apricot and plum cultivars. We report the transformation and regeneration of Prunus armeniaca and Prunus domestica plants with Agrobacterium tumefaciens strain LBA 4404 containing various binary plasmids, pBinGUSint, carrying the marker gene β-glucuronidase (GUS) and pBinPPVm, carrying the coat protein gene of Plum Pox Virus (PPV), the causal agent of Sharka disease. The marker gene GUS was used for the optical evaluation of the efficiency of different transformation systems involving cotyledons of immature embryos as well as somatic embryos and leaf discs. The coat protein gene of PPV was used to introduce the coat protein mediated resistance against one of the most important pathogens of stone fruit trees in Europe and the whole Mediterranean area.

Diagnosis of Theileria equi infections in horses in the Azores using cELISA and nested PCR

Equine piroplasmosis is a tick-borne disease of equids that is often caused by the parasite Theileria equi. We applied competitive ELISA (cELISA) and nested PCR diagnostic methods to detect this parasite in horses by screening 162 samples from mainland Portugal where the parasite is endemic, and 143 from the Azores representing both native and imported horse populations. We found that 2.8% of the Azorean samples tested positive exclusively by cELISA, 1.4% tested positive only by nested PCR, and 9.1% tested positive using both tests. Samples from the native Terceira Pony population were negative for both tests. The parasite was more prevalent in samples from mainland Portugal when both test methods were considered (9.3% positive exclusively by cELISA, 1.9% positive exclusively by nested PCR, and 16.7% positive for both tests). To our knowledge, this is the first time that molecular techniques have been used to detect T. equi in the Azores and the first report of this parasite in the archipelago. Based on this study, it is clear that the import of horses into the Azores and the movement of horses between the islands must be controlled to reduce the risk of new infections, contributing to the protection of native horse populations such as the Terceira Pony population.

Genetic diversity of an Azorean endemic and endangered plant species inferred from inter-simple sequence repeat markers.

Picconia azorica is an endangered endemic species of the Azores whose hard and high density wood is very appreciated for the production of toys, agricultural tools, furniture and religious statuary. Its renewed economic interest represents a good opportunity for establishing conservation programmes. To contribute with information useful for the decision making we performed the genetic analysis of 230 samples from 11 populations collected in three Azorean islands. The majority of the genetic variability was found within populations and no genetic structure was detected between populations and between islands, indicating that the oceanic barriers do not greatly affect gene flow.

Rhizogenesis in stem discs of Malus pumila rootstock M9 “Jork”: I. Hormonal and environmental effects on root induction and callus formation

To establish a protocol suited for the molecular characterization of root induction the influences of explant position, etiolation state and orientation as well as temperature and light intensity on root and callus formation were investigated. Depending on these factors stem discs of Malus incubated on indole-3-butyric acid containing medium and subsequently on hormone-free medium regenerated roots and callus of “filamentous” and “smooth” texture to a varying extent. Concentration and incubation duration of indole-3butyric acid strongly affected rooting and the production of “smooth” callus. Moreover “smooth” callus was profuse at the low light levels applied during rooting. Rooting efficiency decreased and “filamentous” callus increased between 19°C and 25°C. Explants close to the shoot apex displayed reduced rooting efficiency and profuse “filamentous” callus. There was a strong effect of explant orientation on root and “filamentous” callus formation.

Morphological and genetic characterization of an emerging Azorean horse breed: the Terceira Pony

The Terceira Pony is a horse indigenous to Terceira Island in the Azores. These horses were very important during the colonization of the island. Due to their very balanced proportions and correct gaits, and with an average withers height of 1.28 m, the Terceira Pony is often confused with a miniature pure-bred Lusitano. This population was officially recognized as the fourth Portuguese equine breed by the national authorities in January, 2014. The aim of this study was to analyze the morphology and the genetic diversity by means of microsatellite markers of this emerging horse breed. The biometric data consisted of 28 body measurements and nine angles from 30 animals (11 sires, 19 dams). The Terceira Pony is now a recognized horse breed and is gaining in popularity amongst breeders and the younger riding classes. The information obtained from this study will be very useful for conservation and management purposes, including maximizing the breed’s genetic diversity, and solidifying the desirable phenotypic traits.

Development of a high-density, 2M SNP genotyping array and 670k SNP imputation array for the domestic horse

Background To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. Results Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of ~5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix) a set of ~2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of ~670 thousand SNPs (MNEc670k), was designed for genotype imputation. Conclusions Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.Background: To date, genome-scale analyses in the domestic horse have been limited by suboptimal single nucleotide polymorphism (SNP) density and uneven genomic coverage of the current SNP genotyping arrays. The recent availability of whole genome sequences has created the opportunity to develop a next generation, high-density equine SNP array. Results: Using whole genome sequence from 153 individuals representing 24 distinct breeds collated by the equine genomics community, we cataloged over 23 million de novo discovered genetic variants. Leveraging genotype data from individuals with both whole genome sequence, and genotypes from lower-density, legacy SNP arrays, a subset of ~5 million high-quality, high-density array candidate SNPs were selected based on breed representation and uniform spacing across the genome. Considering probe design recommendations from a commercial vendor (Affymetrix, now Thermo Fisher Scientific) a set of ~2 million SNPs were selected for a next-generation high-density SNP chip (MNEc2M). Genotype data were generated using the MNEc2M array from a cohort of 332 horses from 20 breeds and a lower-density array, consisting of ~670 thousand SNPs (MNEc670k), was designed for genotype imputation. Conclusions: Here, we document the steps taken to design both the MNEc2M and MNEc670k arrays, report genomic and technical properties of these genotyping platforms, and demonstrate the imputation capabilities of these tools for the domestic horse.