ABSTRACT
Las tortugas marinas (Cheloniidae) son un grupo de siete especies originadas en el cretaceo. Analisis de secuencias parciales de DNA mitocondrial han revelado inconsistencias filogeneticas dentro de este grupo de quelonios. Sin embargo, estos marcadores mitocondriales han permitido entender y dilucidar la composicion de las poblaciones en areas de forrajeo, habitos reproductivos, inferencias de patrones de migracion y tambien definir las unidades de manejo en el mundo, con el fin de proponer planes de manejo y conservacion. El objetivo de este estudio fue evaluar la posicion de la tortuga carey E. imbricata dentro de la familia Cheloniidae y la filogenia de las tortugas marinas utilizando genes mitocondriales codificantes de proteinas, genes ribosomicos y el genoma mitocondrial completo de la tortuga carey anidante del Caribe colombiano, al compararlo con las otras seis especies de tortugas marinas disponibles en GenBank. Se utilizaron cuatro metodos de inferencias filogeneticas: Neighbor-Joining (NJ), Maxima Verosimilitud (ML), Maxima Parsimonia (MP) e Inferencia Bayesiana (IB). Los arboles NJ, ML, MP e IB mostraron que ND2, COX1, 16S ARNr, ND5, 12S ARNr, ND4, COX3 y ND1 son los marcadores que presentan una mejor resolucion filogenetica con sustentos bootstrap entre 89,0% y 99,98%. Los genes ATP6, ATP8, COX2, ND3, ND4L y ND5 presentaron politomias y establecieron relaciones filogeneticas equivocadas. El analisis con el mitogenoma completo presento arboles altamente sustentados (bootstrap de 98,0%) en comparacion con el analisis con marcadores individuales. Los arboles obtenidos con el gen ND2 e IB resolvieron con buen sustento las relaciones evolutivas entre las especies comparadas, consolidandose la posicion de E. imbricata dentro de la tribu Carettini con probabilidad posterior de 0,98-1,0. Los marcadores ND2, ND5, ND4, COX3 y ND1 no han sido utilizados en trabajos previos y representan una nueva alternativa para explicar la filogenia en este grupo de reptiles marinos. En el presente caso utilizando mitogenomas completos se obtuvieron arboles robustos y altamente sustentados.
The sea turtles (Cheloniidae) are a group of seven species of cretaceous origin. Analyses of partial mitochondrial sequences have revealed phylogenetic inconsistences within this group. Nevertheless, these mitochondrial markers have allowed us to understand, explain and clarify population composition in areas of foraging, reproductive habits, inferences of migration patterns and, also, to define management units in the world, in order to trace conservation and monitoring plans. In this study, four methods were evaluated and compared for phylogenetic inference (Neighbor-Joining-NJ, Maximum Likelihood-ML, Maximum Parsimony-MP and Bayesian inference-BI) by using coding genes, ribosomal genes and full mitogenomes of the hawksbill, E. imbricata, and other six species of sea turtles obtained from GenBank. The sequences were analyzed independently and jointly to identify the method and marker that better explain the phylogenetic relationships among this group of reptiles. The NJ, ML, MP and BI trees showed that ND2, COX1, 16S rRNA, ND5, 12S rRNA, ND4 and COX3 are the markers that give phylogenetic trees with better resolution and support, with bootstrap values ranging from 89.0% to 99.98%. ATP6, ATP8, COX2, ND1, ND3, ND5 and ND4L genes presented polytomies. The analysis with full mitogenome often provides highly supported trees (bootstrap 98.0%) compared with single marker analysis. Trees obtained with the BI method and the ND2 gene is the one that better resolved the evolutionary relationships among the species, consolidating the position of E. imbricata within the Carettini tribe with a value of posterior probability of 0.98-1.0. The markers ND2, ND4, ND5 and COIII, not used in previous works, represent a new alternative to explain the phylogeny in this group of marine reptiles. In the present study, a complete mitogenome analysis produced robust and highly supported trees.
ABSTRACT
El ajo (Allium sativum L) se reproduce vegetativamente utilizando bulbillos, condición que favorece la propagación de enfermedades, especialmente bacterias, hongos y virus que afectan la calidad y el rendimiento del cultivo. Por este motivo se implementó la identificación molecular por RT-PCR de los potyvirus LYSV y OYDV en el sistema de producción de semilla limpia de ajo en tres clones nacionales. En la fase de producción de semilla limpia mediante micropropagación, se estandarizó el establecimiento de meristemos de ajo. La presencia de potyvirus se analizó en 586 plántulas mediante ELISA y en 70 por RT-PCR. Para la RT-PCR se extrajo ARN a partir de microbulbillos y hojas de plántulas, obteniéndose 1.7 a 226 ng/microlitro de ARN y se sintetizó entre 35 a 50 ng de cADN. Los resultados obtenidos mostraron que el protocolo de desinfección produjo una viabilidad del 73.6%. El análisis ELISA presentó un saneamiento del 96.1% de las plántulas a potyvirus, mientras que con RT-PCR se identificó la presencia de LYSV en el 8.6% de las muestras evaluadas. El virus del enanismo amarrillo de la cebolla (OYDV) no fue detectado en ninguna de las muestras. Los resultados muestran que el cultivo in vitro de meristemos de ajo es una excelente alternativa para la producción de semilla, mostrando un 92% de eficiencia. Además, validan el diagnóstico eficiente del potyvirus LYSV en hojas y microbulbillos de ajo.
Garlic (Allium sativum L), reproduces vegetatively using bulbils, condition that favors the spread of diseases, especially bacteria, fungi and viruses, which affect the quality and crop yield. For this reason, the molecular identification by RT-PCR of potyvirus: LYSV and OYDV in the production system of clean seed garlic of three national clones were implemented. In the production phase of clean seed was establishing garlic meristems micropropagation. Potyvirus presence in 586 seedlings was analyzed by ELISA and for RT-PCR in 70. RNA was extracted from leaves and small bulbs, yielding 1.7 to 226 ng/μl, and with this RNA, between 35 to 50 ng of cDNA. The results showed that the disinfection protocol produced a 73.6% viability of plants. ELISA analysis showed 96% sanitation of seedling to potyvirus, whereas, Leek Yellow Strip Virus, LYSV was identified in 8.6% of samples used RT-PCR methodology. Onion yellow dwarf virus (OYDV) was not detected in any sample. The results show that the in vitro culture of meristem of garlic, is an excellent alternative for seed production, showing a 92% efficiency. Moreover, efficient diagnostics of LYSV potyvirus was validated in leaves and small bulbs of garlic.
ABSTRACT
Hawksbill sea turtles Eretmochelys imbricata are found extensively around the world, including the Atlantic, Pacific, and Indian Oceans; the Persian Gulf, and the Red and Mediterranean Seas. Populations of this species are affected by international trafficking of their shields, meat, and eggs, making it a critically endangered animal. We determined the haplotypes of 17 hawksbill foraging turtles of Islas del Rosario (Bolivar) and of the nesting beach Don Diego (Magdalena) in the Colombian Caribbean based on amplification and sequencing of the mitochondrial gene cytochrome oxidase c subunit I (COI). We identified 5 haplotypes, including EI-A1 previously reported in Puerto Rico, which was similar to 10 of the study samples. To our knowledge, the remaining 4 haplotypes have not been described. Samples EICOI11 and EICOI3 showed 0.2% divergence from EI-A1, by a single nucleotide change, and were classified as the EI-A2 haplotype. EICOI6, EICOI14, and EICOI12 samples showed 0.2% divergence from EI-A1 and 0.3% divergence from EI-A2 and were classified as EI-A3 haplotype. Samples EICOI16 and EICOI15 presented 5 nucleotide changes each and were classified as 2 different haplotypes, EI-A4 and EI-A5, respectively. The last 2 haplotypes had higher nucleotide diversity (K2P=1.7%) than that by the first 3 haplotypes. EI-A1 and EI-A2 occurred in nesting individuals, and EI-A2, EI-A3, EI-A4, and EI-A5 occurred in foraging individuals. The description of the haplotypes may be associated with reproductive migrations or foraging and could support the hypothesis of natal homing. Furthermore, they can be used in phylogeographic studies.