Biology subjects

Palacios, M. B.

Publications and source records attributed to Palacios, M. B..

2 recordsLinked to original sources

bioRxiv2026

Development of species-specific real-time PCR assays for the identification of five European Rhinolophus bats.

The detection and monitoring of bat species using non-invasive sampling and molecular techniques has become increasingly popular in recent years. In Europe, these approaches have been applied to identify horseshoe bats of the genus Rhinolophus, which includes five species: R. hipposideros, R. ferrumequinum, R. euryale, R. mehelyi and R. blasii. While species-specific real-time PCR assays exist for R. ferrumequinum and R. hipposideros, no unified panel of real-time PCR assays currently enables the identification of all five European Rhinolophus species from non-invasively collected samples. Here, we developed five species-specific real-time PCR assays, each targeting interspecies nucleotide variation within the mitochondrial cytochrome b gene. To enhance single-base discrimination, RNase H-dependent PCR (rhPCR) primers were employed, incorporating cleavable blocked primers that require perfect complementarity for extension. The assays were applied to droppings non-invasively collected from 18 caves and one church in Serbia and Romania. Of the 149 samples analysed, 131 (88%) yielded successful amplification of Rhinolophus DNA. Detection probabilities for the three species identified in the field ranged from 0.49 to 0.82. Occupancy estimates varied, with R. euryale showing the highest (0.86; UI: 0.69-0.97) and R. mehelyi the lowest (0.23; UI: 0.08-0.43). The assays were capable of detecting up to three species concurrently within a single pooled sample (approximately 15 droppings). These assays are especially valuable for detecting R. mehelyi, given its rarity and uncertain distribution, and offer a robust tool for monitoring Rhinolophus populations across Europe.

ecology↗

bioRxiv2024

A novel and efficient Apple Latent Spherical Virus-based gene silencing method for functional genomic studies in Chenopodium quinoa

Quinoa (Chenopodium quinoa Willd.), with its resilience in harsh environments and excellent nutritional value, has become crucial for global food security. Despite recent progress in genomic research, the inability to perform functional studies in quinoa due to the absence of transformation techniques remains a significant obstacle. In this work, we present the development of a novel Apple Latent Spherical Virus (ALSV)-mediated virus-induced gene silencing (VIGS) protocol that will allow to perform functional genomics studies in quinoa in a fast and simple way. The method was fine-tuned using ALSV plasmids that carry partial gene sequences of phytoene desaturase (PDS) from Nicotiana benthamiana and quinoa. The developed technique involves an initial inoculation in Nicotiana plants through agroinfiltration with Agrobacterium tumefaciens cultures carrying the different viral constructs. Viral extracts were prepared using local or systemic leaves, which were then used as inoculum to infect quinoa leaves through mechanical damage. The method was successfully tested in two contrasting quinoa varieties, although some differences were observed in infection phenotype and viral susceptibility. The effect of insertion sequence size in the viral vectors was also analyzed, resulting in differences in bleaching or chlorosis phenotype and impact on plant growth. The presence of the virus in infected plants was confirmed, and the reduction in PDS gene expression in silenced plants was verified. Because quinoa lacks stable transformation protocols, limiting heterologous expression assays, our ALSV-based VIGS protocol is very attractive for loss-of-function gene studies.

plant biology↗