Consequences of introgression and gene flow on the genetic structure and diversity of Lima bean (Phaseolus lunatus L.) in its Mesoamerican diversity area.

We evaluated the role of gene flow and wild-crop introgression on the structure and genetic diversity of Lima bean (Phaseolus lunatus) in the Yucatan Peninsula, an important Mesoamerican diversity area for this crop, using a genotyping-by-sequencing approach (15,168 SNP markers) and two scales. At the local scale, STRUCTURE and NGSEP analyses showed predominantly crop-to-wild introgression, but also evidence of a bidirectional gene flow in the two wild-weedy-crop complexes studied (Itzinté and Dzitnup). The ABBA-BABA tests showed a higher introgression in Itzinté (the older complex) than in Dzitnup (the younger one); at the allelic level, the wild-crop introgression in Itzinté was similar in both directions, in Dzitnup it was higher from crop-to-wild; and at the chromosomal level, introgression in Itzinté was from wild-to-crop, whereas in Dzitnup it occured in the opposite direction. Also, we found H E values slightly higher in the domesticated accessions than in the wild ones, in both complexes (Itzinté: wild = 0.31, domesticated = 0.34; Dzinup: wild = 0.27, domesticated = 0.36), but %P and π estimators were higher in the wild accessions than in the domesticated ones. At a regional scale, STRUCTURE and MIGRATE showed a low gene flow, predominantly from crop-to-wild; and STRUCTURE, Neighbor-Joining and PCoA analyses indicated the existence of two wild groups and one domesticated group, with a marked genetic structure based in the existence of domesticated MI and wild MII gene pools. Also, at the regional scale, we found a higher genetic diversity in the wild accessions than in the domesticated ones, in all estimators used (e.g., H E = 0.27 and H E = 0.17, respectively). Our results indicate that gene flow and introgression are playing an important role at the local scale, but its consequences on the structure and genetic diversity of the Lima bean are not clearly reflected at the regional scale, where diversity patterns between wild and domesticated populations could be reflecting historical events.

Short-distance barriers affect genetic variability of Rhizophora mangle L. in the Yucatan Peninsula.

The environmental variability at local scale results in different physiognomic types of mangrove forest. However, this variability has never been considered in studies of mangrove genetic variability. This study analyzed the genetic and morphological variability and structure of Rhizophora mangle at regional and local scales in the Yucatan Peninsula. Thirteen mangrove populations (eight scrub and five tall), located in seven sites, were sampled, and their morphological variability and relationship with the availability of phosphorus and salinity were analyzed. The diversity and genetic structure were estimated at different hierarchical levels with nine microsatellites, also Bayesian inference and Principal Coordinates Analysis were used. We found a great morphological variability of R. mangle that responded to local environmental variability and not to the precipitation gradient of the peninsula. The genetic diversity found in the peninsula was greater than that reported for other populations in Mexico and was grouped into two regions: the Gulf of Mexico and the Caribbean Sea. At a local scale, tall and scrub mangroves had significant genetic differentiation suggesting that ecological barriers promote genetic differentiation within sites. These results need to be considered in future population genetic studies and for mangrove management and conservation.

Plant tissue culture and molecular markers.

Tissue culture can be used to propagate elite material or to generate new variability by employing somaclonal variation. Genetic stability of the process must be evaluated analyzing DNA profiles by the use of molecular markers. Several techniques have been reported for the screening of genetic variation on tissue culture derived material; however, a highly informative and good relation among the time-cost-information is obtained using Amplified Fragment Length Polymorphism (AFLP) in automatic sequencer. This technique involves a double-digestion of DNA with restriction enzymes, ligation of adapters at both extremities of the restriction fragments, and finally, selective polymerase chain reaction (PCR) amplification of the fragments. A semiautomatic process for the analysis could be used, but several considerations must be taken into account before such a use.

Primer reporte del empleo de marcadores ISTR en cactaceae (pilosocereus sp) / First report of the employment of ISTR markers in cactaceae (pilosocereus sp)

Pilosocereus sp es una especie en peligro crítico de extinción, la única población conocida se encuentra en una mina de mármol verde, hoy abandonada, en la que su explotación produjo la disminución del 80% de la población en 3 años; en la actualidad quedan 28 ejemplares, de ellos unos pocos son adultos, de los cuales solo dos producen frutos. Una de las etapas necesaria para su recuperación es la producción de plántulas para realizar el reforzamiento de la población natural. Como las plantas obtenidas serán plantadas en condiciones naturales, donde se enfrentarán a diversas situaciones ambientales, es conveniente realizar un estudio de diversidad genética. El objetivo de este trabajo fue estimar la variabilidad genética de plántulas de Pilosocereus sp empleando la técnica Inverse Sequence Tagged Repeat (ISTR). Se realizó la germinación in vitro de semillas y se determinó la variabilidad genética de las plántulas obtenidas. Con el análisis molecular se detectaron un total de 97 bandas, de ellas el 62,8% fueron polimórficas. El mayor porcentaje de bandas polimórficas (85,7%) se obtuvo con la combinación de oligonucleótidos F6/B6. Con las combinaciones de oligonucleótidos empleados se detectaron de 4 a 6 patrones de banda diferentes. La heterocigosidad media esperada fue de 0,39.

Pilosocereus sp is a species in critical extinction danger, the only known population is in a mine of green marble, abandoned today, but it exploitation produced the decrease of the population's 80% in 3 years, at the present time they are 28 individuals, of them some few ones are mature, of those which alone two produce fruits. One of the necessary stages for their recovery is the seedlings production to carry out the natural population's reinforcement. As the obtained plants they will be planted under natural conditions, where they will face diverse environmental situations, it is convenient to carry out a study of genetic diversity. The objective of this work was to estimate the genetic variability using the technical Inverse Sequence Tagged Repeat (ISTR). In vitro germination of seeds of Pilosocereus sp and the genetic variability of the obtained seedlings was determined. With the molecular analysis a total of 97 bands were detected, of them 62.8% were polymorphic. The biggest percentage of polymorphism (85.7%) was obtained with the primer combination F6/B6. With the primer combinations employed were detected from 4 to 6 different band patterns. The heterocigocity hoped was 0.39.

Microsatélites desarrollados en guayabo (Psidium guajava L) y su utilidad para evaluar diversidad en la familia myrtaceae / Microsatellites developed in guava (Psidium guajava L) and its utility to evaluate diversity in myrtaceae family

La familia Myrtaceae ha evolucionado desde las formas más primitivas en los bosques húmedos y lluviosos hasta formas especializadas en regiones semiáridas, muy secas y altamente influenciadas por los cambios estacionales. Aunque los botánicos han estado describiendo estas especies desde hace 200 años, la clasificación de las mismas no está completamente esclarecida. Las Secuencias Simples Repetidas (SSR), conocidas comúnmente como microsatélites, representan una porción significativa del genoma eucariótico y pueden ser de gran utilidad para estos fines. El objetivo del presente trabajo fue utilizar cebadores SSR, diseñados previamente para guayabo, en la identificación de accesiones y en el estudio de diversidad en Myrtaceae. Para este propósito se emplearon cuatro combinaciones de cebadores SSR siguiendo los protocolos establecidos en la literatura. Se realizó el análisis de los resultados atendiendo a la potencialidad de amplificación cruzada, la detección de diferentes alelos, la utilidad para la identificación de accesiones y el estudio de la diversidad y la determinación de las relaciones existentes entre las especies y cultivares en estudio. El alto nivel de polimorfismo detectado por los microsatélites evaluados, el cual se refleja en los valores de los índices relacionados con el nivel de polimorfismo y la capacidad de discriminación calculados, indica las potencialidades de los SSR para la identificación de accesiones en otros representantes de la familia Myrtaceae. Además, representan una herramienta de gran utilidad para estudios de diversidad en esta familia; así como para la estimación de las relaciones de parentesco entre los genotipos analizados, análisis taxonómico y de filogenia.

Myrtaceae family has evolved from primitive forms in rainy and humid forest to specialized forms in semiarid and very dry regions, highly influenced by seasonal changes. Although botanist have been describing Myrtaceae family species over 200 years, classification is far from be completely clarify. Simple Sequences Repeats (SSR), usually known as microsatellites, represent a significant portion of eukaryotic genome and can be of great utility for these purposes. The objective of the present work was to utilize SSR primers, previously designed in guava, for accessions identification and diversity studies of other Myrtaceae members. For this purpose, four SSR primer combinations were utilized following the protocols established in the literature. The results were analyzed attending to cross amplification potentiality, detection of different alleles, utility for accessions identification and diversity studies and relationships determination between the studied species and cultivars. The high level of polymorphism detected by the evaluated microsatellites, which is reflected in the values of each index related with polymorphism and calculated discriminating capacity, indicates the potentials of SSR for accessions identification in other Myrtaceae family members. Also, represents a great utility tool for diversity studies in this family, as well as for the estimation of parentage relationship between the genotypes investigated and the taxonomic and phylogenetic analysis.

Caracterización de accesiones de papaya (Carica papaya L)a través de marcadores AFLP en Cuba / Characterising Cuban papaya accessions (Carica papaya L) by AFLP markers

Los marcadores moleculares son herramientas valiosas en los estudios genéticos en plantas, y están siendo empleados exitosamente en programas de mejoramiento principalmente en la elección de progenitores y en la selección. El polimorfismo observado mediante la técnica molecular AFLP (Amplified Fragment Length Polymorphism) ha sido de utilidad para estudios de diversidad genética en frutales. En el presente trabajo se realizó la caracterización molecular de 12 accesiones de papaya (Carica papaya L.) del banco de germoplasma del Instituto de Investigaciones en Fruticultura Tropical (IIFT), empleando la técnica AFLP. Se evaluaronseis combinaciones de iniciadores para la amplificación selectiva, las cuales amplificaron un total de 431 bandascon 73,3% de polimorfismo. El número total de patrones de bandas identificados fue igual en todas las combinaciones utilizadas, con un porcentaje de identificación alto, lo que sugiere que dichas combinaciones pudieran ser empleadas para estudios de variabilidad genética en papaya. En general, los resultados presentados demuestran que existe diversidad genética entre las accesiones evaluadas, lo cual constituye un reflejo del origen que presentan los genotipos analizados a partir de la introducción de materiales foráneos y la polinización abierta de un grupo de materiales selectos. Por tanto, se recomienda retomar la prospección y selecciónde accesiones locales, así como la introducción de nuevos genotipos foráneos, como dos vías fundamentalespara aumentar la diversidad genética presente en el banco de germoplasma de papaya de Cuba.

Molecular markers are valuable tools for genetic studies in plants and they are often used successfully in genetic breeding, mainly for choosing progenitors and selection. Polymorphism observed by amplified fragment length polymorphism (AFLP) has been useful for genetic diversity studies in fruit trees. Twelve papaya accessions from the Tropical Fruit Crop Research Institute germplasm bank were molecularly characterised by AFLP. 431 bands having 73.3% polymorphism were obtained using 6 primer combinations. The total number of band patterns identified was the same in all combinations assayed with a high percentage of identification, suggesting that such primer combinations could be used for genetic variability studies in papaya.The results demonstrated genetic diversity among the papaya accessions evaluated, indicating the origin ofthe analysed genotypes from exogenous material and open pollination of a selected group of material. It is thus recommended that local accessions and their selection be monitored as well as the introduction of new foreign genotypes as two ways of increasing the genetic diversity of the Cuban papaya germplasm bank.

Isolation of cDNA encoding the catalytic site of phosphatidylinositol-specific phospholipase C from Coffea arabica L.: Recombinant expression and peptide purification.

A cDNA encoding the catalytic site of a phosphatidylinositol-specific phospholipase C (PI-PLC) was isolated from Coffea arabica suspension cells. The cDNA (designated CaPLC) encodes a polypeptide of 308 amino acids, containing the catalytic X and Y domains, and has 99% identity to the soybean gene. Recombinant CaPLC protein was expressed in Escherichia coli, purified, and used to produce a polyclonal antibody. The peptide has a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analyses. Immunoblots revealed the presence of PLC-like proteins in the tissues of different plant species.