Distribution and amplification of interstitial telomeric sequences (ITSs) in Australian dragon lizards support frequent chromosome fusions in Iguania

Telomeric sequences are generally located at the ends of chromosomes; however, they can also be found in non-terminal chromosomal regions when they are known as interstitial telomeric sequences (ITSs). Distribution of ITSs across closely related and divergent species elucidates karyotype evolution and speciation as ITSs provide evolutionary evidence for chromosome fusion. In this study, we performed physical mapping of telomeric repeats by fluorescence in situ hybridisation (FISH) in seven Australian dragon lizards thought to represent derived karyotypes of squamate reptiles and a gecko lizard with considerably different karyotypic feature. Telomeric repeats were present at both ends of all chromosomes in all species, while varying numbers of ITSs were also found on microchromosomes and in pericentromeric or centromeric regions on macrochromosomes in five lizard species examined. This suggests that chromosomal rearrangements from ancestral squamate reptiles to Iguania occurred mainly by fusion between ancestral types of acrocentric chromosomes and/or between microchromosomes, leading to appearance of bi-armed macrochromosomes, and in the reduction of microchromosome numbers. These results support the previously proposed hypothesis of karyotype evolution in squamate reptiles. In addition, we observed the presence of telomeric sequences in the similar regions to heterochromatin of the W microchromosome in Pogona barbata and Doporiphora nobbi, while sex chromosomes for the two species contained part of the nucleolar organiser regions (NORs). This likely implies that these ITSs are a part of the satellite DNA and not relics of chromosome fusions. Amplification of telomeric repeats may have involved heterochromatinisation of sex-specific W chromosomes and play a role in the organisation of the nucleolus

Chromosomal localization of the 18S-28S and 5S rRNA genes and (TTAGGG)n sequences of butterfly lizards (Leiolepis belliana belliana and Leiolepis boehmei, Agamidae, Squamata)

Chromosomal mapping of the butterfly lizards Leiolepis belliana belliana and L. boehmei was done using the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences. The karyotype of L. b. belliana was 2n = 36, whereas that of L. boehmei was 2n = 34. The 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, while the 5S rRNA genes were found in the pericentromeric region of chromosome 6 in both species. Hybridization signals for the (TTAGGG)n sequence were observed at the telomeric ends of all chromosomes, as well as interstitially at the same position as the 18S-28S rRNA genes in L. boehmei. This finding suggests that in L. boehmei telomere-to-telomere fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located or, alternatively, at the secondary constriction of chromosome 1. The absence of telomeric sequence signals in chromosome 1 of L. b. belliana suggested that its chromosomes may have only a few copies of the (TTAGGG)n sequence or that there may have been a gradual loss of the repeat sequences during chromosomal evolution

Partial Amniote Sex Chromosomal Linkage Homologies Shared on Snake W Sex Chromosomes Support the Ancestral Super-Sex Chromosome Evolution in Amniotes

Squamate reptile chromosome 2 (SR2) is thought to be an important remnant of an ancestral amniote super-sex chromosome, but a recent study showed that the Siamese cobra W sex chromosome is also a part of this larger ancestral chromosome. To confirm the existence of an ancestral amniote super-sex chromosome and understand the mechanisms of amniote sex chromosome evolution, chromosome maps of two snake species [Russell’s viper: Daboia russelii (DRU) and the common tiger snake: Notechis scutatus (NSC)] were constructed using bacterial artificial chromosomes (BACs) derived from chicken and zebra finch libraries containing amniote sex chromosomal linkages. Sixteen BACs were mapped on the W sex chromosome of DRU and/or NSC, suggesting that these BACs contained a common genomic region shared with the W sex chromosome of these snakes. Two of the sixteen BACs were co-localized to DRU2 and NSC2, corresponding to SR2. Prediction of genomic content from all BACs mapped on snake W sex chromosomes revealed a large proportion of long interspersed nuclear element (LINE) and short interspersed nuclear element (SINE) retrotransposons. These results led us to predict that amplification of LINE and SINE may have occurred on snake W chromosomes during evolution. Genome compartmentalization, such as transposon amplification, might be the key factor influencing chromosome structure and differentiation. Multiple sequence alignments of all BACs mapped on snake W sex chromosomes did not reveal common sequences. Our findings indicate that the SR2 and snake W sex chromosomes may have been part of a larger ancestral amniote super-sex chromosome, and support the view of sex chromosome evolution as a colorful myriad of situations and trajectories in which many diverse processes are in action

Full Length Research Paper Identification of species (meat and blood samples) using nested-PCR analysis of mitochondrial DNA

Crocodile meat product is an alternative protein source. Although, crocodile meat is more expensive, its taste is similar to that of chicken and fish. The authentication of commercial meat species is important for consumer’s confidence. In this study, sensitive and specific method multiplex nested-PCR was applied to identify commercial meat species. Dried blood was used as an alternative DNA source for detection. The detection sensitivity was enhanced by primers specifically designed to encompass the mitochondrial Cytochrome b and NADH dehydrogenase 5/6 genes. The specificity and sensitivity of multiplex PCR system were tested. Different lengths of specific nested-PCR products were detected to be 350, 570, 750 and 1000 bp for chicken, pig, cow, and crocodile, respectively. The system allowed detection with as little as 5 nanogram of DNA from either meat or blood sample. Detection sensitivity of individual species was improved, enabling the detection of DNA with as little as 1 picogram. Cross reaction was not detected among the tested species. It was shown that the multiplex-PCR assay enhanced the sensitivity of routine species identification and allowed the use of blood as an alternative DNA source for detection.Key words: Cytochrome b, NADH dehydrogenase, mitochondrial DNA, meat, blood, species identification, nested-PCR, crocodile

Identification of native Dendrobium species in Thailand by PCR-RFLP of rDNA-ITS and chloroplast DNA

ABSTRACT: The PCR-restriction fragment length polymorphism (PCR-RFLP) approach was successfully developed to identify 25 native Dendrobium species in Thailand. PCR-RFLP of the rDNA-ITS with six restriction enzymes and three chloroplast (cp) DNA regions with five primer-enzyme combinations produced 24 types of DNA patterns altogether. Twenty-three out of the 25 species determined in this study were found to belong to unique classes and were successfully differentiated. Two species, D. crumenatum and D. formosum, possessing the same DNA pattern, however, were identified after cutting the chloroplast DNA fragment amplified by psbC-trnS primer with MboI enzyme. An effective procedure for identifying each Dendrobium species was developed. PCR-RFLP of the rDNA-ITS with TaqI, which is the most informative enzyme, was used for the early detection of 16 Dendrobium species. To identify the remaining Dendrobium species, PCR-RFLP analysis was performed using one more primer-enzyme combination. Our study provides a rapid, simple, and reliable identification method for these Dendrobium species

Insights into avian molecular cytogenetics—with reptilian comparisons

In last 100 years or so, much information has been accumulated on avian karyology, genetics, physiology, biochemistry and evolution. The chicken genome project generated genomic resources used in comparative studies, elucidating fundamental evolutionary processes, much of it funded by the economic importance of domestic fowl (which are also excellent model species in many areas). Studying karyotypes and whole genome sequences revealed population processes, evolutionary biology, and genome function, uncovering the role of repetitive sequences, transposable elements and gene family expansion. Knowledge of the function of many genes and non-expressed or identified regulatory components is however still lacking. Birds (Aves) are diverse, have striking adaptations for flight, migration and survival and inhabit all continents most islands. They also have a unique karyotype with ~ 10 macrochromosomes and ~ 30 microchromosomes that are smaller than other reptiles. Classified into Palaeognathae and Neognathae they are evolutionarily close, and a subset of reptiles. Here we overview avian molecular cytogenetics with reptilian comparisons, shedding light on their karyotypes and genome structure features. We consider avian evolution, then avian (followed by reptilian) karyotypes and genomic features. We consider synteny disruptions, centromere repositioning, and repetitive elements before turning to comparative avian and reptilian genomics. In this context, we review comparative cytogenetics and genome mapping in birds as well as Z- and W-chromosomes and sex determination. Finally, we give examples of pivotal research areas in avian and reptilian cytogenomics, particularly physical mapping and map integration of sex chromosomal genes, comparative genomics of chicken, turkey and zebra finch, California condor cytogenomics as well as some peculiar cytogenetic and evolutionary examples. We conclude that comparative molecular studies and improving resources continually contribute to new approaches in population biology, developmental biology, physiology, disease ecology, systematics, evolution and phylogenetic systematics orientation. This also produces genetic mapping information for chromosomes active in rearrangements during the course of evolution. Further insights into mutation, selection and adaptation of vertebrate genomes will benefit from these studies including physical and online resources for the further elaboration of comparative genomics approaches for many fundamental biological questions

Snake W Sex Chromosome: The Shadow of Ancestral Amniote Super-Sex Chromosome

Heteromorphic sex chromosomes, particularly the ZZ/ZW sex chromosome system of birds and some reptiles, undergo evolutionary dynamics distinct from those of autosomes. The W sex chromosome is a unique karyological member of this heteromorphic pair, which has been extensively studied in snakes to explore the origin, evolution, and genetic diversity of amniote sex chromosomes. The snake W sex chromosome offers a fascinating model system to elucidate ancestral trajectories that have resulted in genetic divergence of amniote sex chromosomes. Although the principal mechanism driving evolution of the amniote sex chromosome remains obscure, an emerging hypothesis, supported by studies of W sex chromosomes of squamate reptiles and snakes, suggests that sex chromosomes share varied genomic blocks across several amniote lineages. This implies the possible split of an ancestral super-sex chromosome via chromosomal rearrangements. We review the major findings pertaining to sex chromosomal profiles in amniotes and discuss the evolution of an ancestral super-sex chromosome by collating recent evidence sourced mainly from the snake W sex chromosome analysis. We highlight the role of repeat-mediated sex chromosome conformation and present a genomic landscape of snake Z and W chromosomes, which reveals the relative abundance of major repeats, and identifies the expansion of certain transposable elements. The latest revolution in chromosomics, i.e., complete telomere-to-telomere assembly, offers mechanistic insights into the evolutionary origin of sex chromosomes

Ad hoc Joint FAO/WHO Expert Consultation on Risk Assessment of Food Allergens Part 2: Review and establish threshold levels in foods of the priority allergens

The main purpose of this second meeting was to establish threshold levels in foods of the priority allergens. Based on the defined approach, the Expert Committee discussed and agreed on the safety objective, which could be described as “to minimise, to a point where further refinement does not meaningfully reduce health impact, the probability of any clinically relevant objective allergic response, as defined by dose distribution modelling of minimum eliciting doses (MEDs) and supported by data regarding severity of symptoms in the likely range of envisioned Reference Doses (RfD)”. The Committee further identified several important considerations to guide decision-making. These included a clear definition of criteria to be met by quantitative data on which reference doses (RfD) are based, supporting data on health manifestations (severity) at the proposed RfD, quality, quantity, availability and accessibility of data (for priority allergens), as well as how to deal with priority allergens for which information supporting one or more of those considerations was lacking.El objetivo principal de esta segunda reunión fue establecer niveles umbral en los alimentos de los alérgenos prioritarios. Sobre la base del enfoque definido, el Comité de Expertos discutió y acordó el objetivo de seguridad, que podría describirse como “minimizar, hasta un punto en el que un mayor refinamiento no reduzca significativamente el impacto en la salud, la probabilidad de cualquier respuesta alérgica objetiva clínicamente relevante, como definido por el modelo de distribución de dosis de dosis mínimas provocadoras (MED) y respaldado por datos sobre la gravedad de los síntomas en el rango probable de dosis de referencia previstas (RfD) ”. El Comité identificó además varias consideraciones importantes para orientar la toma de decisiones. Estos incluyeron una definición clara de los criterios que deben cumplir los datos cuantitativos en los que se basan las dosis de referencia (RfD), datos de apoyo sobre manifestaciones de salud (gravedad) en la RfD propuesta, calidad, cantidad, disponibilidad y accesibilidad de los datos (para alérgenos prioritarios). , así como cómo tratar los alérgenos prioritarios para los que faltaba información que respaldara una o más de esas consideraciones.Instituto de Investigación de Tecnología de AlimentosFil: Baumert, Joseph. Universidad de Nebraska-Lincoln. Departamento de Ciencia y Tecnología de Alimentos; Estados UnidosFil: Brooke-Taylor, Simon. Brooke-Taylor & Co. Consultor australiano de regulación alimentaria y análisis de riesgos (Pty Ltd); Australia.Fil: Crevel, René W.R. René Crevel Consulting Limited; Reino Unido.Fil: Houben, Geert F. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido.Fil: Jackson, Lauren. Administración de Alimentos y Medicamentos de los Estados Unidos. División de Ciencia y Tecnología del Procesamiento de Alimentos. Ingeniería de Procesos; Estados UnidosFil: Kyriakidis, Symeon. Laboratorio Estatal de Química General (GCSL).Autoridad Independiente de Ingresos Públicos (IAPR); Grecia.Fil: La Vieille, Sébastien. Universidad Laval. Departamento de Ciencias de los Alimentos; Canadá.Fil: Lee, N Alice. Universidad de Nueva Gales del Sur. Escuela de Química e Ingeniería. Ciencia e ingeniería de los alimentos; Australia.Fil: López, María Cristina. Universidad Nacional de San Martín. Ingeniería de Alimentos; Argentina.Fil: Luccioli, Stefano. Administración de Alimentos y Medicamentos de los Estados Unidos. Centro de Seguridad Alimentaria y Nutrición Aplicada; Estados UnidosFil: O’Mahony, Patrick. Autoridad de Seguridad Alimentaria de Irlanda; Irlanda.Fil: O’Mahony, Patrick. Universidad College Dublin; Irlanda.Fil: Polenta, Gustavo Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Investigación Tecnología de Alimentos; Argentina.Fil: Polenta, Gustavo Alberto. Instituto de Ciencia y Tecnología de los Sistemas Alimentarios Sustentables (ICyTeSAS) UEDD INTA-CONICET; Argentina.Fil: Pöpping, Bert. Food Consulting Strategically (FOCO); Alemania.Fil: Pöpping, Bert. Comités de Normalización ISO - CEN. Grupo de trabajo CEN Alérgenos Alimentarios (CEN TC 275 WG 12).); Alemania.Fil: Remington, Benjamin C. Remington Consulting Group B.V.; Holanda.Fil: Remington, Benjamin C. Universidad de Nebraska–Lincoln. Programa de Recursos e Investigación de Alergias Alimentarias. Estados UnidosFil: Srikulnath, Sirinrat. Universidad de Kasetsart (UKaset). Instituto de Investigación y Desarrollo de Productos Alimentarios. Centro de Servicio de Aseguramiento de la Calidad de los Alimentos. Unidad de Alérgenos Alimentarios; Tailandia.Fil: Taylor, Stephen L. Universidad de Nebraska-Lincoln. Departamento de Ciencia y Tecnología de Alimentos; Estados UnidosFil: Turner, Paul J. Colegio Imperial de Ciencia, Tecnología y Medicina. Alergia e Inmunología Pediátricas; Inglaterra

Ad hoc Joint FAO/WHO Expert Consultation on Risk Assessment of Food Allergens Part 1: Review and validation of Codex priority allergen list through risk assessment

The objectives of the meeting is to see whether the published criteria (FAO/WHO, 2000) for assessing additions and exclusions to the list is still current and appropriate. The Expert Committee determined that only foods or ingredients that cause immune-mediated hypersensitivities such as IgE-mediated food allergies and coeliac disease should be included on the list of foods and ingredients included in section 4.2.1.4 of the GSLPF. Thus, it was recommended that foods or ingredients such as lactose, sulphite, and food additives which cause food intolerances rather than immune-mediated responses, should be excluded from this list. The Committee identified prevalence of the immune-mediated hypersensitivity to a specific food, severity (i.e. proportion of severe objective reactions to a food/ingredient such as anaphylaxis), and the potency of food/ingredient (i.e. the amount of the food/ingredient required to cause objective symptoms) as the three key criteria that should be used to establish the priority allergen list. Subgroups of the Expert Committee were established to review the literature on the prevalence, severity and potency of immune-mediated hypersensitivity of each food currently on the GSLPF list (cereals containing gluten and products of these; crustacea and products of these; eggs and egg products; fish and fish products; peanuts, soybeans and products of these; milk and milk products; tree nuts and nut products; ), as well as other foods found on priority allergen lists established in individual countries or regions (e.g. mollusks, mustard, celery, sesame, buckwheat, lupin, and others).Los objetivos de la reunión son ver si los criterios publicados (FAO / OMS, 2000) para evaluar las adiciones y exclusiones a la lista siguen vigentes y son apropiados. El Comité de Expertos determinó que solo los alimentos o ingredientes que causan hipersensibilidades inmunomediadas, como las alergias alimentarias mediadas por IgE y la enfermedad celíaca, deben incluirse en la lista de alimentos e ingredientes incluidos en la sección 4.2.1.4 de la GSLPF. Por lo tanto, se recomendó que se excluyeran de esta lista alimentos o ingredientes como lactosa, sulfito y aditivos alimentarios que causan intolerancias alimentarias en lugar de respuestas inmunomediadas. El Comité identificó la prevalencia de la hipersensibilidad inmunomediada a un alimento específico, la gravedad (es decir, la proporción de reacciones objetivas graves a un alimento / ingrediente como la anafilaxia) y la potencia del alimento / ingrediente (es decir, la cantidad de alimento / ingrediente requerida causar síntomas objetivos) como los tres criterios clave que deben utilizarse para establecer la lista de alérgenos prioritarios. Se establecieron subgrupos del Comité de Expertos para revisar la literatura sobre la prevalencia, severidad y potencia de la hipersensibilidad inmunomediada de cada alimento actualmente en la lista GSLPF (cereales que contienen gluten y productos de estos; crustáceos y productos de estos; huevos y productos de huevo ; pescado y productos de pescado; cacahuetes, soja y productos de estos; leche y productos lácteos; frutos secos y productos de frutos secos;), así como otros alimentos que se encuentran en las listas de alérgenos prioritarios establecidas en países o regiones individuales (por ejemplo, moluscos, mostaza, apio , sésamo, alforfón, altramuz y otros).Instituto de Investigación de Tecnología de AlimentosFil: Baumert, Joseph. Universidad de Nebraska-Lincoln. Departamento de Ciencia y Tecnología de Alimentos; Estados UnidosFil: Brooke-Taylor, Simon. Imperial College London. Instituto Nacional del Corazón y los Pulmones; Reino Unido.Fil: Chen, Hongbing. Nanchang Universidad. Instituto Conjunto de Investigación Chino-Alemán; China.Fil: Crevel, René W.R. René Crevel Consulting Limited; Reino Unido.Fil: Geert Houben. Organización para la Investigación Científica Aplicada TNO; Países Bajos.Fil: Jackson, Lauren. División de Ciencia y Tecnología del Procesamiento de Alimentos. Ingeniería de Procesos de la Administración de Alimentos y Medicamentos de los EE. UU. (FDA); Estados Unidos de América.Fil: Kyriakidis, Symeon. Laboratorio Estatal de Química General (GCSL). Autoridad Independiente de Ingresos Públicos (IAPR); Grecia.Fil: La Vieille, Sébastien. Universidad Laval. Departamento de Ciencias de los Alimentos; Canadá.Fil: Lee, N Alice. Universidad de Nueva Gales del Sur . Escuela de Química e Ingeniería. Ciencia e ingeniería de los alimentos; Australia.Fil: López, María Cristina. Universidad Nacional de San Martín. Ingeniería de Alimentos; Argentina.Fil: Luccioli, Stefano. Administración de Alimentos y Medicamentos de los Estados Unidos. Centro de Seguridad Alimentaria y Nutrición Aplicada; Estados UnidosFil: O’Mahony, Patrick. Universidad College Dublin; Irlanda.Fil: O’Mahony, Patrick. Autoridad de Seguridad Alimentaria de Irlanda; Irlanda.Fil: Polenta, Gustavo Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Investigación Tecnología de Alimentos; Argentina.Fil: Polenta, Gustavo Alberto. Instituto de Ciencia y Tecnología de los Sistemas Alimentarios Sustentables (ICyTeSAS) UEDD INTA-CONICET; Argentina.Fil: Pöpping, Bert. Food Consulting Strategically (FOCO); Alemania.Fil: Pöpping, Bert. Comités de Normalización ISO - CEN. Grupo de trabajo CEN Alérgenos Alimentarios (CEN TC 275 WG 12).); Alemania.Fil: Remington, Benjamin C. Remington Consulting Group B.V.; Holanda.Fil: Remington, Benjamin C. Universidad de Nebraska–Lincoln. Programa de Recursos e Investigación de Alergias Alimentarias. Estados UnidosFil: Södergren, Eva. ThermoFisher Scientific; Suecia.Fil: Srikulnath, Sirinrat. Universidad de Kasetsart (UKaset). Instituto de Investigación y Desarrollo de Productos Alimentarios. Centro de Servicio de Aseguramiento de la Calidad de los Alimentos. Unidad de Alérgenos Alimentarios; Tailandia.Fil: Taylor, Stephen L. Universidad de Nebraska-Lincoln. Departamento de Ciencia y Tecnología de Alimentos; Estados UnidosFil: Turner, Paul J. Universidad de Sídney; Australia.Fil: Turner, Paul J. Colegio Imperial de Ciencia, Tecnología y Medicina. Alergia e Inmunología Pediátricas; Inglaterra