Recombinant inbred lines derived from wide crosses in Pisum.

Genomic resources are becoming available for Pisum but to link these to phenotypic diversity requires well marked populations segregating for relevant traits. Here we describe two such resources. Two recombinant inbred populations, derived from wide crosses in Pisum are described. One high resolution mapping population involves cv Caméor, for which the first pea whole genome assembly was obtained, crossed to JI0281, a basally divergent P. sativum sativum landrace from Ethiopia. The other is an inter sub-specific cross between P. s. sativum and the independently domesticated P. s. abyssinicum. The corresponding genetic maps provide information on chromosome level sequence assemblies and identify structural differences between the genomes of these two Pisum subspecies. In order to visualise chromosomal translocations that distinguish the mapping parents, we created a simplified version of Threadmapper to optimise it for interactive 3-dimensional display of multiple linkage groups. The genetic mapping of traits affecting seed coat roughness and colour, plant height, axil ring pigmentation, leaflet number and leaflet indentation enabled the definition of their corresponding genomic regions. The consequence of structural rearrangement for trait analysis is illustrated by leaf serration. These analyses pave the way for identification of the underlying genes and illustrate the utility of these publicly available resources. Segregating inbred populations derived from wide crosses in Pisum, together with the associated marker data, are made publicly available for trait dissection. Genetic analysis of these populations is informative about chromosome scale assemblies, structural diversity in the pea genome and has been useful for the fine mapping of several discrete and quantitative traits.

Development of new genetic resources for faba bean (Vicia faba L.) breeding through the discovery of gene-based SNP markers and the construction of a high-density consensus map.

Faba bean (Vicia faba L.) is a pulse crop of high nutritional value and high importance for sustainable agriculture and soil protection. With the objective of identifying gene-based SNPs, transcriptome sequencing was performed in order to reduce faba bean genome complexity. A set of 1,819 gene-based SNP markers polymorphic in three recombinant line populations was selected to enable the construction of a high-density consensus genetic map encompassing 1,728 markers well distributed in six linkage groups and spanning 1,547.71 cM with an average inter-marker distance of 0.89 cM. Orthology-based comparison of the faba bean consensus map with legume genome assemblies highlighted synteny patterns that partly reflected the phylogenetic relationships among species. Solid blocks of macrosynteny were observed between faba bean and the most closely-related sequenced legume species such as pea, barrel medic or chickpea. Numerous blocks could also be identified in more divergent species such as common bean or cowpea. The genetic tools developed in this work can be used in association mapping, genetic diversity, linkage disequilibrium or comparative genomics and provide a backbone for map-based cloning. This will make the identification of candidate genes of interest more efficient and will accelerate marker-assisted selection (MAS) and genomic-assisted breeding (GAB) in faba bean.

A role for an endosperm-localized subtilase in the control of seed size in legumes.

Here, we report a subtilase gene (SBT1.1) specifically expressed in the endosperm of Medicago truncatula and Pisum sativum seeds during development, which is located at a chromosomal position coinciding with a seed weight quantitative trait locus (QTL). Association studies between SBT1.1 polymorphisms and seed weights in ecotype collections provided further evidence for linkage disequilibrium between the SBT1.1 locus and a seed weight locus. To investigate the possible contribution of SBT1.1 to the control of seed weight, a search for TILLING (Targeting Induced Local Lesions in Genomes) mutants was performed. An inspection of seed phenotype revealed a decreased weight and area of the sbt1.1 mutant seeds, thus inferring a role of SBT1.1 in the control of seed size in the forage and grain legume species. Microscopic analyses of the embryo, representing the major part of the seed, revealed a reduced number of cells in the MtP330S mutant, but no significant variation in cell size. SBT1.1 is therefore most likely to be involved in the control of cotyledon cell number, rather than cell expansion, during seed development. This raises the hypothesis of a role of SBT1.1 in the regulation of seed size by providing molecules that can act as signals to control cell division within the embryo.

Genetic diversity in European Pisum germplasm collections.

The distinctness of, and overlap between, pea genotypes held in several Pisum germplasm collections has been used to determine their relatedness and to test previous ideas about the genetic diversity of Pisum. Our characterisation of genetic diversity among 4,538 Pisum accessions held in 7 European Genebanks has identified sources of novel genetic variation, and both reinforces and refines previous interpretations of the overall structure of genetic diversity in Pisum. Molecular marker analysis was based upon the presence/absence of polymorphism of retrotransposon insertions scored by a high-throughput microarray and SSAP approaches. We conclude that the diversity of Pisum constitutes a broad continuum, with graded differentiation into sub-populations which display various degrees of distinctness. The most distinct genetic groups correspond to the named taxa while the cultivars and landraces of Pisum sativum can be divided into two broad types, one of which is strongly enriched for modern cultivars. The addition of germplasm sets from six European Genebanks, chosen to represent high diversity, to a single collection previously studied with these markers resulted in modest additions to the overall diversity observed, suggesting that the great majority of the total genetic diversity collected for the Pisum genus has now been described. Two interesting sources of novel genetic variation have been identified. Finally, we have proposed reference sets of core accessions with a range of sample sizes to represent Pisum diversity for the future study and exploitation by researchers and breeders.

The flowering locus Hr colocalizes with a major QTL affecting winter frost tolerance in Pisum sativum L.

An understanding of the genetic determinism of frost tolerance is a prerequisite for the development of frost tolerant cultivars for cold northern areas. In legumes, it is not known to which extent vernalization requirement or photoperiod responsiveness are necessary for the development of frost tolerance. In pea (Pisum sativum L.) however, the flowering locus Hr is suspected to influence winter frost tolerance by delaying floral initiation until after the main winter freezing periods have passed. The objective of this study was to dissect the genetic determinism of frost tolerance in pea by QTL analysis and to assess the genetic linkage between winter frost tolerance and the Hr locus. A population of 164 recombinant inbred lines (RILs), derived from the cross Champagne x Terese was evaluated both in the greenhouse and in field conditions to characterize the photoperiod response from which the allele at the Hr locus was inferred. In addition, the population was also assessed for winter frost tolerance in 11 field conditions. Six QTL were detected, among which three were consistent among the different experimental conditions, confirming an oligogenic determinism of frost tolerance in pea. The Hr locus was found to be the peak marker for the highest explanatory QTL of this study. This result supports the hypothesis of the prominent part played by the photoperiod responsiveness in the determinism of frost tolerance for this species. The consistency of three QTL makes these positions interesting targets for marker-assisted selection.

Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula.

The identification of the molecular polymorphisms giving rise to phenotypic trait variability-both quantitative and qualitative-is a major goal of the present agronomic research. Various approaches such as positional cloning or transposon tagging, as well as the candidate gene strategy have been used to discover the genes underlying this variation in plants. The construction of functional maps, i.e. composed of genes of known function, is an important component of the candidate gene approach. In the present paper we report the development of 63 single nucleotide polymorphism markers and 15 single-stranded conformation polymorphism markers for genes encoding enzymes mainly involved in primary metabolism, and their genetic mapping on a composite map using two pea recombinant inbred line populations. The complete genetic map covers 1,458 cM and comprises 363 loci, including a total of 111 gene-anchored markers: 77 gene-anchored markers described in this study, 7 microsatellites located in gene sequences, 16 flowering time genes, the Tri gene, 5 morphological markers, and 5 other genes. The mean spacing between adjacent markers is 4 cM and 90% of the markers are closer than 10 cM to their neighbours. We also report the genetic mapping of 21 of these genes in Medicago truncatula and add 41 new links between the pea and M. truncatula maps. We discuss the use of this new composite functional map for future candidate gene approaches in pea.

Microsatellite marker polymorphism and mapping in pea (Pisum sativum L.).

This paper aims at providing reliable and cost effective genotyping conditions, level of polymorphism in a range of genotypes and map position of newly developed microsatellite markers in order to promote broad application of these markers as a common set for genetic studies in pea. Optimal PCR conditions were determined for 340 microsatellite markers based on amplification in eight genotypes. Levels of polymorphism were determined for 309 of these markers. Compared to data obtained for other species, levels of polymorphism detected in a panel of eight genotypes were high with a mean number of 3.8 alleles per polymorphic locus and an average PIC value of 0.62, indicating that pea represents a rather polymorphic autogamous species. One of our main objectives was to locate a maximum number of microsatellite markers on the pea genetic map. Data obtained from three different crosses were used to build a composite genetic map of 1,430 cM (Haldane) comprising 239 microsatellite markers. These include 216 anonymous SSRs developed from enriched genomic libraries and 13 SSRs located in genes. The markers are quite evenly distributed throughout the seven linkage groups of the map, with 85% of intervals between the adjacent SSR markers being smaller than 10 cM. There was a good conservation of marker order and linkage group assignment across the three populations. In conclusion, we hope this report will promote wide application of these markers and will allow information obtained by different laboratories worldwide in diverse fields of pea genetics, such as QTL mapping studies and genetic resource surveys, to be easily aligned.

Genetic diversity within Pisum sativum using protein- and PCR-based markers.

A collection of 148 Pisum accessions, mostly from Western Europe, and including both primitive germplasm and cultivated types, was structured using 121 protein- and PCR-based markers. This molecular marker-based classification allowed us to trace back major lineages of pea breeding in Western Europe over the last decades, and to follow the main breeding objectives: increase of seed weight, introduction of the afila foliage type and white flowers, and improvement of frost tolerance for winter-sown peas. The classification was largely consistent with the available pedigree data, and clearly resolved the different main varietal types according to their end-uses (fodder, food and feed peas) from exotic types and wild forms. Fodder types were further separated into two sub-groups. Feed peas, corresponding to either spring-sown or winter-sown types, were also separated, with two apparently different gene pools for winter-sown peas. The garden pea group was the most difficult to structure, probably due to a continuum in breeding of feed peas from garden types. The classification also stressed the paradox between the narrowness of the genetic basis of recent cultivars and the very large diversity available within P. sativum. A sub-collection of 43 accessions representing 96% of the whole allelic variability is proposed as a starting point for the construction of a core collection.

Differential expression of two barley XET-related genes during coleoptile growth.

Plant cell elongation depends on the physical properties of the primary cell wall. Because xyloglucan endotransglycosylases (XETs) are enzymes that mediate cleavage and rejoining of the beta(1-4)-XG backbone of primary cell wall, they are potentially involved in cell elongation. In this paper, the growth of the barley coleoptile was related to the expression patterns of two genes from this family (hvEXT, hvXEB) in experiments where coleoptile elongation varied according to light/dark treatments in order to assess the potential role of these genes in cell elongation. In dark-grown and light-grown coleoptiles, growth rate variations were associated with altered levels of expression of hvEXT and hvXEB: they were higher in dark-grown than in light-grown seedlings, and decreased after 5 d in darkness, and after 4 d in continuous light. In 4-d-old seedlings, coleoptile elongation decreased significantly 4 h after the onset of a continuous white- light irradiation, and hvXEB and hvEXT mRNA levels decreased, respectively, 2 h and 4 h after the onset of white-light irradiation. Moreover, the distribution of hvXEB and hvEXT along the coleoptiles of 4-d-old dark-grown seedlings were different. Altogether, these results suggest a complex pattern of temporal and positional expression for the different genes of the XET-related family.

Molecular markers and protein quantities as genetic descriptors in maize. I. Genetic diversity among 21 inbred lines.

Twenty-one maize (Zea mays L.) inbred lines were analysed using isozyme electrophoresis, restriction fragment length polymorphism (RFLP), and two-dimensional electrophoresis of denatured proteins (2-D PAGE). Our goal was (1) to assess the genetic variability among these lines which are potential progenitors for the development of forage maize hybrids in Europe, and (2) to compare the relationship pattern revealed by the polymorphism at marker loci with the one derived from the amount of protein variability assessed by computer-assisted analysis of the 2-D electrophoregrams. Fourteen markers were obtained from isozyme polymorphism, 84 from the restriction fragment length polymorphism, and 70 from protein shifts revealed by 2-D PAGE. The Rogers' distance computed on the set of molecular markers was the most efficient to describe the pedigree relationships between lines. Quantitative protein data gave a picture of relationships between lines clearly different from the monogenic markers. When unrelated pairs of lines were considered, the Rogers' distance was weakly correlated to distances based on quantitative variations in the amount of protein which may be consistent with their polygenic control and the occurrence of gene interactions.

Analysis of scaling methods to minimize experimental variations in two-dimensional electrophoresis quantitative data: application to the comparison of maize inbred lines.

The analysis of two-dimensional (2-D) electrophoresis quantitative data from a design involving 21 maize genotypes revealed a significant experimental variation. In order to minimize this variation, we investigated the possible causes and found that it was essentially due to global effects, affecting all the spots in a gel in a similar way, and occurring during the 2-D run/staining procedure. Three scaling methods to discard these experimental variations were analyzed: the linear scaling method, a method based on principal component analysis, and a combined method that unites the advantages of both of the former. Comparing these three methods, we found that they led to consistent results with regard to the factor under study, i.e. the genetic factor in our case. However, the combined scaling method was the most efficient in reducing experimental variations.

Isquenia intestinal en la cirugia re / Intestinal ischemia in the repairing surgery of the abdominal aorta

La isquemia intestinal es una complicacion grave de la cirugia reconstructiva de la aorta abdominal, que involucra generalmente el colon izquierdo, mas raramente el intestino delgado o ambos simultaneamente. Es mas frecuente como complicacion de la aneurismectomia. La incidencia de esta complicacion se ve del 0.2 al 10% de los casos. Los autores lal observaron 7 veces en 216 intervenciones de reconstruccion aortica (3.24%). La mortalidad global de la colitis isquemica es del 40%, cuando la isquemia es transmural la mortalidad del 90%. De los 7 casos a los que hacen referencia los autores 5 fallecieron (71,42%). Los autores discuten los distintos factores que pueden condicionar la instalacion de una isquemia intestinal. Se discute la prevencion de esta complicacion por medidas que deben ser tomadas en el pre e intraoperatorio. Se destacan los elementos clinicos que permitem realizar el diagnostico de isquemia constituida, asi como las medidas paraclinicas que la certificaram.

El tratamiento quirurgico de los aneurismas de aorta ascendente asociados a valvulopatia aortica. / The surgical treatment of ascending aortic aneurysms associated with aortic valvulopathies

Se presenta la experiencia del Departamento de Cirugia Cardiaca del Hospital de Clinicas con pacientes portadores de aneurisma da aorta ascendente e insuficiencia aortica. Un paciente fue explorado quirurgicamente y fallecio en forma brusca mientras esperaba su reoperacion. Un paciente fue sometido a reemplazo valvular y aneurismorrafia; falecio en el postoperatorio por deterioro marcado de la funcion miocardica. Un paciente fue operado reemplazandose la valvula aortica y el aneurisma de la aorta ascendente en su sector supracoronario, protegiendo con cardiplejia: la evolucion inmediata y alejada fue buena.Se considera que estos pacientes son de tratamiento quirurgico en la actualidad en nuestro medio. La tecnica quirurgica dependera del compromiso de la pared aortica en cada caso. Las protesis valvulares seran mecanicas, dado el alto indice de disfuncion que se han detectado con algunas valvulas biologicas en nuestro medio. Se recomienda el control periodico con aortografia, para detectar precozmente complicaciones locales postoperatorias

El clampeo supradiafragmatico en los aneurismas de aorta abdominales rotos. / Supradiaphragmatic clamping of ruptured abdominal aorta aneurysms

Los autores preconizan el clampeo aortico supradiafragmatico por toracotomia anterolateral izquierda a nivel del 7o. y 8o. intercostal en los aneurismas de aorta abdominal rotos, para poder controlar la hemorragia y el consiguiente colapso con muerte subita en el momento del abordaje abdominal. Relatan un caso que fuera operado 44 dias antes por una hemorragia digestiva por ulcus duodenal, habiendo sido comprobado en esa oportunidad un aneurisma de aorta subrenal y habiendo sido inclusive planteada como causa de esa hemorragia una complicacion del aneurisma confirmandose la no participacion del mismo en el accidente rojo, los autores plantean el tratamiento simultaneo de ambos patologias. Por ultimo preconizan la via de abordaje transversa bitransrectal extendida hacia ambos lados sobre los musculos anchos del abdomen supra o infraumbilical, a ras del ombligo, con lo que se logra un buen dominio de la aorta infrarenal

Oclusion de colon por calculo biliar. / Colonic occlusion by biliary calculi

Se presenta un caso de oclusion de colon descendente por calculo biliar. Se destacan las dimensiones habituales del calculo y la no existencia de patologia previa en el colon