Agrobacterium-mediated transformation of tomatillo (Physalis ixocarpa) and tissue specific and developmental expression of the CaMV 35S promoter in transgenic tomatillo plants

A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon expiants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed formation.     

Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles

Madagascar has become a model region for testing hypotheses of species diversification and biogeography, and many studies have focused on its diverse and highly endemic herpetofauna. Here we combine species distribution models of a near-complete set of species of reptiles and amphibians known from the island with body size data and a tabulation of herpetofaunal communities from field surveys, compiled up to 2008. Though taxonomic revisions and novel distributional records arose since compilation, we are confident that the data are appropriate for inferring and comparing biogeographic patterns among these groups of organisms. We observed species richness of both amphibians and reptiles was highest in the humid rainforest biome of eastern Madagascar, but reptiles also show areas of high richness in the dry and subarid western biomes. In several amphibian subclades, especially within the Mantellidae, species richness peaks in the central eastern geographic regions while in reptiles different subclades differ distinctly in their richness centers. A high proportion of clades and subclades of both amphibians and reptiles have a peak of local endemism in the topographically and bioclimatically diverse northern geographic regions. This northern area is roughly delimited by a diagonal spanning from 15.5°S on the east coast to ca. 15.0°S on the west coast. Amphibian diversity is highest at altitudes between 800–1200 m above sea-level whereas reptiles have their highest richness at low elevations, probably reflecting the comparatively large number of species specialized to the extended low-elevation areas in the dry and subarid biomes. We found that the range sizes of both amphibians and reptiles strongly correlated with body size, and differences between the two groups are explained by the larger body sizes of reptiles. However, snakes have larger range sizes than lizards which cannot be readily explained by their larger body sizes alone. Range filling, i.e., the amount of suitable habitat occupied by a species, is less expressed in amphibians than in reptiles, possibly reflecting their lower dispersal capacity. Taxonomic composition of communities assessed by field surveys is largely explained by bioclimatic regions, with communities from the dry and especially subarid biomes distinctly differing from humid and subhumid biomes.     

Biodiversity and Land uses at a regional scale: Is agriculture the biggest threat for reptile assemblages?

The human exploitation of land resources (land use) has been considered the major factor responsible for changes in biodiversity within terrestrial ecosystems given that it affects directly the distribution of the fauna. Reptiles are known to be particularly sensitive to habitat change due to their ecological constraints. Here, the impact of land use on reptile diversity was analysed, choosing Catalonia (NE Iberia) as a case study. This region provides a suitable scenario for such a biogeographical study since it harbours: 1) a rich reptile fauna; 2) a highly diverse environment showing strong variation in those variables usually shaping reptile distributions; and 3) good species distribution data. Potential species richness was calculated, using ecological modelling techniques (Ecological Niche Factor Analysis – ENFA). The subtraction of the observed from the potential species richness was the dependent variable in a backwards multiple linear regression, using land use variables. Agriculture was the land use with the strongest relation with the non-fulfilment of the potential species richness, indicating a trend towards a deficit of biodiversity. Deciduous forest was the only land use negatively related with the subtracted species richness. Results indicate a clear relationship between land use and biodiversity at a mesoscale. This finding represents an important baseline for conservation guidelines within the habitat change framework because it has been achieved at the same spatial scale of chorological studies and management policies.     

AnthropoAge,a novel approach to integrate body composition into the estimation of biological age

Aging is believed to occur across multiple domains, one of which is body composition; however, attempts to integrate it into biological age (BA) have been limited. Here, we consider the sex-dependent role of anthropometry for the prediction of 10-year all-cause mortality using data from 18,794 NHANES participants to generate and validate a new BA metric. Our data-driven approach pointed to sex-specific contributors for BA estimation: WHtR, arm and thigh circumferences for men; weight, WHtR, thigh circumference, subscapular and triceps skinfolds for women. We used these measurements to generate AnthropoAge, which predicted all-cause mortality (AUROC 0.876, 95%CI 0.864–0.887) and cause-specific mortality independently of ethnicity, sex, and comorbidities; AnthropoAge was a better predictor than PhenoAge for cerebrovascular, Alzheimer, and COPD mortality. A metric of age acceleration was also derived and used to assess sexual dimorphisms linked to accelerated aging, where women had an increase in overall body mass plus an important subcutaneous to visceral fat redistribution, and men displayed a marked decrease in fat and muscle mass. Finally, we showed that consideration of multiple BA metrics may identify unique aging trajectories with increased mortality (HR for multidomain acceleration 2.43, 95%CI 2.25–2.62) and comorbidity profiles. A simplified version of AnthropoAge (S-AnthropoAge) was generated using only BMI and WHtR, all results were preserved using this metric. In conclusion, AnthropoAge is a useful proxy of BA that captures cause-specific mortality and sex dimorphisms in body composition, and it could be used for future multidomain assessments of aging to better characterize the heterogeneity of this phenomenon.     

In vitro chili pepper biotechnology

Summary Chili pepper is an important horticultural crop that can surely benefit from plant biotechnology. However, although it is a Solanaceous member, developments in plant cell, tissue, and organ culture, as well as on plant genetic transformation, have lagged far behind those achieved for other members of the same family, such as tobacco (Nicotiana tabacum), tomato (Lycopersicon esculentum), and potato (Solanum tuberosum), species frequently used as model systems because of their facility to regenerate organs and eventually whole plants in vitro, and also for their ability to be genetically engineered by the currently available transformation methods. Capsicum members have been shown to be recalcitrant to differentiation and plant regeneration under in vitro conditions, which in turn makes it very difficult or inefficient to apply recombinant DNA technologies via genetic transformation aimed at genetic improvement against pests and diseases. Some approaches, however, have made possible the regeneration of chili pepper plants from in vitro-cultured cells, tissues, and organs through organogenesis or embryogenesis. Anther culture has been successfully applied to obtain haploid and doubledhaploid plants. Organogenic systems have been used for in vitro micropropagation as well as for genetic transformation. Application of both tissue culture and genetic transformation techniques have led to the development of chili pepper plants more resistant to at least one type of virus. Cell and tissue cultures have been applied successfully to the selection of variant cells exhibiting increased resistance to abiotic stresses, but no plants exhibiting the selected traits have been regenerated. Production of capsaicinoids, the hot principle of chili pepper fruits, by cells and callus tissues has been another area of intense research. The advances, limitations, and applications of chili pepper biotechnology are discussed.     

Protein turnover dynamics suggest a diffusion-to-capture mechanism for peri-basal body recruitment and retention of intraflagellar transport proteins

Intraflagellar transport (IFT) is essential for construction and maintenance of cilia. IFT proteins concentrate at the basal body where they are thought to assemble into trains and bind cargoes for transport. To study the mechanisms of IFT recruitment to this peri-basal body pool, we quantified protein dynamics of eight IFT proteins, as well as five other basal body localizing proteins using fluorescence recovery after photobleaching in vertebrate multiciliated cells. We found that members of the IFT-A and IFT-B protein complexes show distinct turnover kinetics from other basal body components. Additionally, known IFT subcomplexes displayed shared dynamics, suggesting shared basal body recruitment and/or retention mechanisms. Finally, we evaluated the mechanisms of basal body recruitment by depolymerizing cytosolic MTs, which suggested that IFT proteins are recruited to basal bodies through a diffusion-to-capture mechanism. Our survey of IFT protein dynamics provides new insights into IFT recruitment to basal bodies, a crucial step in ciliogenesis and ciliary signaling.     

Melon Fruits: Genetic Diversity,Physiology, and Biotechnology Features

Among Cucurbitaceae, Cucumis melo is one of the most important cultivated cucurbits. They are grown primarily for their fruit, which generally have a sweet aromatic flavor, with great diversity and size (50 g to 15 kg), flesh color (orange, green, white, and pink), rind color (green, yellow, white, orange, red, and gray), form (round, flat, and elongated), and dimension (4 to 200 cm). C. melo can be broken down into seven distinct types based on the previously discussed variations in the species. The melon fruits can be either climacteric or nonclimacteric, and as such, fruit can adhere to the stem or have an abscission layer where they will fall from the plant naturally at maturity. Traditional plant breeding of melons has been done for 100 years wherein plants were primarily developed as open-pollinated cultivars. More recently, in the past 30 years, melon improvement has been done by more traditional hybridization techniques. An improvement in germplasm is relatively slow and is limited by a restricted gene pool. Strong sexual incompatibility at the interspecific and intergeneric levels has restricted rapid development of new cultivars with high levels of disease resistance, insect resistance, flavor, and sweetness. In order to increase the rate and diversity of new traits in melon it would be advantageous to introduce new genes needed to enhance both melon productivity and melon fruit quality. This requires plant tissue and plant transformation techniques to introduce new or foreign genes into C. melo germplasm. In order to achieve a successful commercial application from biotechnology, a competent plant regeneration system of in vitro cultures for melon is required. More than 40 in vitro melon regeneration programs have been reported; however, regeneration of the various melon types has been highly variable and in some cases impossible. The reasons for this are still unknown, but this plays a heavy negative role on trying to use plant transformation technology to improve melon germplasm. In vitro manipulation of melon is difficult; genotypic responses to the culture method (i.e., organogenesis, somatic embryogenesis, etc.) as well as conditions for environmental and hormonal requirements for plant growth and regeneration continue to be poorly understood for developing simple in vitro procedures to culture and transform all C. melo genotypes. In many cases, this has to be done on an individual line basis. The present paper describes the various research findings related to successful approaches to plant regeneration and transgenic transformation of C. melo. It also describes potential improvement of melon to improve fruit quality characteristics and postharvest handling. Despite more than 140 transgenic melon field trials in the United States in 1996, there are still no commercial transgenic melon cultivars on the market. This may be a combination of technical or performance factors, intellectual property rights concerns, and, most likely, a lack of public acceptance. Regardless, the future for improvement of melon germplasm is bright when considering the knowledge base for both techniques and gene pools potentially useable for melon improvement.     

An improved method for isolating RNA from dehydrated and nondehydrated chili pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) plant tissues

High-quality RNA is important in studying gene expression. This report describes an improved method for isolating intact purified RNA from dehydrated organs of chili pepper plants. Common RNA extraction protocols have produced poor yields because dehydrated leaves accumulate polysaccharides and RNases. Our protocol is based on a guanidine thiocyanate extraction combined with additional purification steps using butanol and the ionic detergent CTAB (cetyltrimethylammonium bromide). Using this protocol, RNA yields ranged from 40–70 μg of total RNA per 200 mg of fresh tissue. This method can be adapted to large-scale isolations, allowing the recovery of larger amounts of intact RNA (up to 250 μg per gram of fresh tissue).     

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone

Background

Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone.

Results

We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9?=?CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines.

Conclusions

Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).