In angiosperms, gametophytic apomixis (clonal reproduction through seeds) is strongly associated with polyploidy and hybridization. The trait is facultative and its expressivity is highly variable between genotypes. Here, we used an F1 progeny derived from diploid apomictic (aposporic) genotypes of Paspalum rufum and two F2 families, derived from F1 hybrids with different apospory expressivity (%AES), to analyze the influence of the environment and the transgenerational transmission of the trait. In addition, AFLP markers were developed in the F1 population to identify genomic regions associated with the %AES. Cytoembryological analyses showed that the %AES was significantly influenced by different environments, but remained stable across the years. F1 and F2 progenies showed a wide range of %AES variation, but most hybrids were not significantly different from the parental genotypes. Maternal and paternal genetic linkage maps were built covering the ten expected linkage groups (LG). A single-marker analysis detected at least one region of 5.7 cM on LG3 that was significantly associated with apospory expressivity. Our results underline the importance of environmental influence in modulating apospory expressivity and identified a genomic region associated with apospory expressivity at the diploid level.

Eragrostis curvula (Schrad.) Nees (weeping lovegrass) is an apomictic species native to Southern Africa that is used as forage grass in semiarid regions of Argentina. Apomixis is a mechanism for clonal propagation through seeds that involves the avoidance of meiosis to generate an unreduced embryo sac (apomeiosis), parthenogenesis, and viable endosperm formation in a fertilization-dependent or -independent manner. Here, we constructed the first saturated linkage map of tetraploid E. curvula using both traditional (AFLP and SSR) and high-throughput molecular markers (GBS-SNP) and identified the locus controlling diplospory. We also identified putative regulatory regions affecting the expressivity of this trait and syntenic relationships with genomes of other grass species. We obtained a tetraploid mapping population from a cross between a full sexual genotype (OTA-S) with a facultative apomictic individual of cv. Don Walter. Phenotypic characterization of F1 hybrids by cytoembryological analysis yielded a 1:1 ratio of apomictic vs. sexual plants (34:27, X 2 = 0.37), which agrees with the model of inheritance of a single dominant genetic factor. The final number of markers was 1,114 for OTA-S and 2,019 for Don Walter. These markers were distributed into 40 linkage groups per parental genotype, which is consistent with the number of E. curvula chromosomes (containing 2 to 123 markers per linkage group). The total length of the OTA-S map was 1,335 cM, with an average marker density of 1.22 cM per marker. The Don Walter map was 1,976.2 cM, with an average marker density of 0.98 cM/marker. The locus responsible for diplospory was mapped on Don Walter linkage group 3, with other 65 markers. QTL analyses of the expressivity of diplospory in the F1 hybrids revealed the presence of two main QTLs, located 3.27 and 15 cM from the diplospory locus. Both QTLs explained 28.6% of phenotypic variation. Syntenic analysis allowed us to establish the groups of homologs/homeologs for each linkage map. The genetic linkage map reported in this study, the first such map for E. curvula, is the most saturated map for the genus Eragrostis and one of the most saturated maps for a polyploid forage grass species.

Paspalum notatum Flügge is a subtropical grass native to South America, which includes sexual diploid and apomictic polyploid biotypes. In the past decade, a number of apomixis-associated genes were discovered in this species through genetic mapping and differential expression surveys. However, the scarce information on Paspalum sequences available in public databanks limited annotations and functional predictions for these candidates.

Fil: Stein, Juliana. Universidad Nacional de Rosario. Facultad de Ciencias Agrarias; Argentina

The “yerba mate” leaves are used to make an infusion named “mate tea”, which is deeply rooted in the historical, social and cultural tradition of South America. Its sustainable use is of strategic significance to several countries of this region, particularly Argentina, Brazil and Paraguay. Our objective was to establish a segregating population and build a framework genetic map, in order to contribute to the future identification of loci associated with desirable traits. Two genetically divergent genotypes with contrasting drought tolerance capacity were selected as female and male parents. A “pseudo-test cross” F 1 population of 700 individuals, with potential to be extended to 1900 individuals, was produced after controlled crossing followed by embryo rescue. A subset of 117 plants was used to produce a framework genetic map. Five AFLP primer combinations and 16 RAPD primers generated 119 informative markers, out of which 68.9% showed the expected Mendelian segregation values. Linkage analyses were carried out by using Joinmap 3.0. Markers originated from each progenitor were independently evaluated to generate a female map including 11 linkage groups and a male one with 16 linkage groups. Total genetic distances covered by the female and male maps were 223 cM and 678 cM, respectively. This work allowed the establishment of a yerba mate segregating population and the construction of a preliminary genetic map, which will be used as a framework for the future identification of markers linked to genes of interest.

The "yerba mate" leaves are used to make an infusion named "mate tea", which is deeply rooted in the historical, social and cultural tradition of South America. Its sustainable use is of strategic significance to several countries of this region, particularly Argentina, Brazil and Paraguay. Our objective was to establish a segregating population and build a framework genetic map, in order to contribute to the future identification of loci associated with desirable traits. Two genetically divergent genotypes with contrasting drought tolerance capacity were selected as female and male parents. A "pseudo-test cross" F-1 population of 700 individuals, with potential to be extended to 1900 individuals, was produced after controlled crossing followed by embryo rescue. A subset of 117 plants was used to produce a framework genetic map. Five AFLP primer combinations and 16 RAPD primers generated 119 informative markers, out of which 68.9% showed the expected Mendelian segregation values. Linkage analyses were carried out by using Joinmap 3.0. Markers originated from each progenitor were independently evaluated to generate a female map including 11 linkage groups and a male one with 16 linkage groups. Total genetic distances covered by the female and male maps were 223 cM and 678 cM, respectively. This work allowed the establishment of a yerba mate segregating population and the construction of a preliminary genetic map, which will be used as a framework for the future identification of markers linked to genes of interest.

Tetraploid (2n = 4x = 40) races of Paspalum notatum Flüggé are important natural forage grasses for the tropical and subtropical areas of the Americas. Almost all natural accessions reproduce by obligate aposporous apomixis. Previous work on the species allowed the identification of several molecular markers completely linked to apospory, one component of apomictic reproduction. Moreover, after a fingerprinting characterization of a germplasm collection, 11 amplified fragment length polymorphism (AFLP) markers exclusive to apomictic accessions were detected. The objectives of this work were (1) to validate the presence of molecular markers linked to apospory in tetraploid races of different geographic origins, (2) to determine if markers specific to apomictic accessions were associated with the mode of reproduction, and (3) to develop single-locus markers of apospory that can be used for marker-assisted selection. Thirteen natural apomictic accessions were analyzed. Moreover, the parental plants Q4188 (non-aposporous) and Q4117 (aposporous) and 44 F1 progenies (36 non-aposporous, 8 aposporous) derived from them were used as a validation population. Nine markers [two random amplification of polymorphic DNA (RAPD) and seven AFLP] 100% linked to apospory in Q4117 were tested. Amplification reactions with the corresponding primers showed that all markers were present in the 13 aposporous (apomictic) accessions, but were absent in the non-aposporous controls. On the other hand, linkage analysis of the 11 AFLP markers specific to the apomictic accessions showed that all of them were linked in coupling to apospory (r = 0.00, LOD 13.245). Based on one AFLP (E36M37c), two sequence characterized amplification region (SCAR) markers (SPNA1 and SPNA2) co-segregating with the trait and present in the 13 apomictic accessions were developed. The presence of markers associated with apospory was conserved among tetraploid accessions of different geographic origins. Moreover, the single-locus markers SPNA1 and SPNA2 could be used for routine marker-assisted selection in hybrid populations segregating for apospory and to facilitate the isolation of apospory-related genes.

We analyzed anaphase I configurations and pollen viability in aposporous and sexual tetraploid (2n = 4x = 40) cytotypes of Paspalum notatum. Five natural aposporous accessions and three experimentally obtained sexual individuals were used. In addition, 16 (8 aposporous and 8 sexual) F-1 hybrids, previously classified by their mode of reproduction, were analyzed. Cytogenetic observations revealed normal and abnormal anaphase I configurations in both aposporous and sexual genotypes. Anaphase I abnormalities were mainly laggard chromosomes, chromatin bridges, and micronuclei. On average, 44.36 % of aposporous meiocytes and 29.66 % of sexual ones showed abnormal anaphase I configurations. The total numbers of normal and abnormal anaphase I were highly significantly different between aposporous and sexual strains. The pollen viability test indicated that aposporous individuals had significantly more non-viable pollen than sexual ones; a positive correlation (r = 0.71; r (2) = 0.50) between the variables was detected. Analysis of aposporous and sexual hybrids confirmed differences in the numbers of normal and abnormal anaphase I patterns in the aposporous and sexual parents. However, similar proportions of viable pollen were produced by both groups of hybrids. In this case, the variables were not correlated (r = 0.23; r (2) = 0.05). Data from this study indicated that aposporous strains had a genetic rearrangement affecting meiosis that was absent in the experimentally obtained sexual individuals and that it was transmitted to the progeny. The possible association between meiotic abnormalities and the inheritance of apospory is discussed.

In previous studies we reported the identification of several AFLP, RAPD and RFLP molecular markers linked to apospory in Paspalum notatum. The objective of this work was to sequence these markers, obtain their flanking regions by chromosome walking and perform an in silico mapping analysis in rice and maize. The methylation status of two apospory-related sequences was also assessed using methylation-sensitive RFLP experiments. Fourteen molecular markers were analyzed and several protein-coding sequences were identified. Copy number estimates and RFLP linkage analysis showed that the sequence PnMAI3 displayed 2-4 copies per genome and linkage to apospory. Extension of this marker by chromosome walking revealed an additional protein-coding sequence mapping in silico in the apospory-syntenic regions of rice and maize. Approximately 5 kb corresponding to different markers were characterized through the global sequencing procedure. A more refined analysis based on sequence information indicated synteny with segments of chromosomes 2 and 12 of rice and chromosomes 3 and 5 of maize. Two loci associated with apomixis locus were tested in methylation-sensitive RFLP experiments using genomic DNA extracted from leaves. Although both target sequences were methylated no methylation polymorphisms associated with the mode of reproduction were detected.

En estudios previos informamos la identificacion de varios marcadores moleculares de AFLP, RAPD y RFLP completamente ligados al locus que controla la aposporia en Paspalum notatum. El objetivo de este trabajo fue caracterizar las secuencias de estos marcadores, estimar el numero de copias de los mismos en el genoma, obtener las regiones flanqueantes de algunos de ellos por medio de estrategias de caminata cromosomal y realizar un analisis de mapeo in silico sobre arroz y maiz. Asimismo, el estado de metilacion de dos secuencias asociadas a este locus fue determinado mediante experimentos RFLP utilizando enzimas sensibles a metilacion. Como material vegetal se emplearon los genotipos tetraploides de P. notatum Q4188 (sexual), Q4117 (apomictico) y una poblacion segregante por el modo de reproduccion derivada del cruzamiento entre ambos. Los marcadores moleculares de AFLP y RAPD ligados a la apomixis fueron ensayados sobre los genotipos parentales y su progenie a fin de confirmar su cosegracion con el caracter aposporia. Las bandas correspondientes a cada uno fueron aisladas a partir del genotipo apomictico (Q4117) y clonadas en el sistema pGEM-Teasy (Promega) y secuenciadas por Macrogen (Corea). Aproximadamente 5 kb correspondiente a diferentes marcadores fueron caracterizados por este procedimiento. El analisis de 14 secuencias de este tipo revelo que la mayoria de las secuencias no presento similitudes significativas en la bases de datos. Solo dos fragmentos correspondieron a ESTs expresados en Panicum virgatum. La estimacion del numero de copias mostro la presencia de elementos repetitivos como tambien de copia simple. En particular los fragmentos derivados de los marcadores PnMAC5 y PnMAI3 mostraron 1 y 2-4 copias por genoma, respectivamente. El marcador PnMAC5 resulto monomorfico entre los progenitores de la poblacion de mapeo, mientras que PnMAI3 mostro un fragmento ligado a la aposporia. La extension de este marcador por caminata cromosomal posibilito la identificacion de secuencias adicionales, una de las cuales correspondio a una proteina del tipo AMT-70. El analisis de mapeo in silico determino que las secuencias asociadas al locus aposporia en P. notatum se localizan principalmente en los cromosomas 2 y 12 de arroz y 3 y 5 de maiz. Dos loci asociados a la apomixis fueron analizados en experimentos RFLP utilizando enzimas sensibles a metilacion. Los resultados experimentales mostraron que ambas secuencias se encuentran metiladas en los genotipos sexuales y apomicticos y no fue posible detectar polimorfismos de metilacion asociados con el modo de reproduccion.

Apomixis is a route of asexual reproduction through seeds, that progresses in the absence of meiosis and fertilization to generate maternal clonal progenies. Gametophytic apomicts are usually polyploid and probably arose from sexual ancestors through a limited number of mutations in the female reproductive pathway. A differential display analysis was carried out on immature inflorescences of sexual and apomictic tetraploid genotypes of Paspalum notatum, in order to identify genes associated with the emergence of apospory. Analysis of ∼10,000 transcripts led to the identification of 94 high-quality differentially expressed sequences. Assembling analysis, plus validation, rendered 65 candidate unigenes, organized as 14 contigs and 51 singletons. Thirty-four unigenes were isolated from apomictic plants and 31 from sexual ones. A total of 45 (69.2%) unigenes were functionally categorized. While several of the differentially expressed sequences appeared to be components of an extracellular receptor kinase (ERK) signal transduction cascade, others seemed to participate in a variety of central cellular processes like cell-cycle control, protein turnover, intercellular signalling, transposon activity, transcriptional regulation and endoplasmic reticulum-mediated biosynthesis. In silico mapping revealed that a particular group of five genes silenced in apomictic plants clustered in a rice genomic area syntenic with the region governing apospory in Paspalum notatum and Brachiaria brizantha. Two of these genes mapped within the set of apo-homologues in P. notatum. Four genes previously reported to be controlled by ploidy were identified among those expressed differentially between apomictic and sexual plants. In situ hybridization experiments were performed for selected clones.

Rapid sequence rearrangements along with alterations in cytosine-directed methylation patterns follow allopolyploid formation in several plant species. The occurrence of similar changes is yet to be examined in detail during the generation of autopolyploids. The objective of this work was to investigate the presence of nascent variation in genome sequence after autopolyploidization in two Paspalum species. The increments of ploidy level were produced either by hybridization involving unreduced gametes or colchicine treatment. Genetic modifications affected 15–23% and 9.55% of the genomic loci in Paspalum rufum and Paspalum notatum, respectively. In all cases the frequency of band loss from the progenitors was significantly higher than that of novel band gain. In random amplified polymorphic DNA (RAPD) experiments the majority of polymorphisms were detected by particular sets of primers, suggesting that repetitive domains were preferentially targeted. Several P. notatum polymorphic fragments were cloned, sequenced and used as probes in genomic hybridization analyses. Some of them corresponded to interspersed middle-repetitive sequences with unknown function. Methylation-sensitive genomic hybridization showed that sequence modifications occurred invariably in cytosine-methylated regions, which remained methylated after polyploidization.

Summary Cutaneous metastases from internal malignancies or primary skin cancers are uncommon, and a zosteriform pattern is very rare. In order to elucidate the characteristics of zosteriform metastatic skin cancer we reviewed 38 cases from the medical literature and 1 case seen in our clinic. Methods: The age and sex of each patient, location of the primary tumor, pathology of primary and metastatic lesions, location of the skin cancer and presence of pain were determined for the 39 cases. Results: The most frequent site of the primary tumor was the breast (7 cases) and skin squamous all carcinoma (6 cases). The most common site of the skin metastases was the chest wall (21 cases). Conclusion: This review highlights the importance of including cutaneous metastases within the differential diagnosis of zosteriform eruptions. Skin metastases should be considered and a skin biopsy is necessary to confirm the diagnosis. We think that this pattern of metastatic disease is more fre- quent than is indicated in the literature.

Paspalum notatum Flügge is a warm-season forage grass with mainly diploid (2n = 20) and autotetraploid (2n = 40) representatives. Diploid races reproduce sexually and require crosspollination due to a self-incompatible mating system, while autotetraploids reproduce by aposporous apomixis. The objectives of this work were to develop a genetic linkage map of Paspalum notatum Flügge at the tetraploid level, identify the linkage/s group/s associated with apomixis and carry out a general characterization of its mode of inheritance. A pseudo test-cross F 1 family of 113 individuals segregating for the mode of reproduction was obtained by crossing a synthetic completely sexual tetraploid plant (Q4188) as female parent with a natural aposporous individual (Q4117) as pollen donor. Map construction was based on single-dose markers (SDAFs) segregating from both parents. Two linkage maps (female and male) were constructed. Within each map, homologous groups were assembled by detecting repulsion-phase linked SDAFs. Putative Q4188 and Q4117 homolog groups were identified by mapping shared single dose markers (BSDF). The Q4188 map consisted of 263 markers distributed on 26 co-segregation groups over a total genetic distance of 1.590.6 cM, while the Q4117 map contained 216 loci dispersed on 39 co-segregation groups along 2.265.7 cM, giving an estimated genome coverage of 88% and 83%, respectively. Seven and 12 putative homologous chromosomes were detected within Q4188 and Q4117 maps, respectively. Afterward, ten female and male homologous chromosomes were identified by mapping BSDFs. In the Q4117 map, a single linkage group was associated with apospory. It was characterized by restriction in recombination and preferential chromosome pairing. A BPSD marker mapping within this group allowed the detection of the female homolog and the putative four male groups of the set carrying apospory.

Los tissue microarrays (TMAs) hacen posible agrupar más de 1.000 tumores en una sola laminilla, y ser utilizados para el análisis de numerosos marcadores de interés diagnóstico, pronóstico o de ayuda en la decisión terapéutica y selección de estudios moleculares. Así hace posible la detección y estudio de DNA y RNA (por hibridación in situ o FISH) y proteínas (inmunohistoquímica).

Although the causes of novel variation in polyploids are not well understood, they could involve changes in gene expression through dosage-regulation, altered regulatory interactions and rapid genetic/epigenetic changes. The objective of this work was to contribute to the elucidation of the patterns and mechanisms of gene regulation during the early stages of polyploid formation. The genome-wide expression of ∼10,000 genes was monitored by using differential display transcript profiling on flowers of a diploid genotype and a newly-formed autotetraploid derivative. Differential expression of 64 clones was validated by reverse-Northern blot. Sequence analysis showed that 42 were homologous to 26 distinct genes of known function while the remaining 22 were classified as novel sequences. Genes detected were involved in processes of DNA repair, chromatin structure modification, regulation of transcription, proteolysis, protein folding, carbohydrate and lipid metabolism and signal transduction. Our results show evidence for gene expression alterations occurring immediately after polyploidization in Paspalum.

The objective of this work was to determine the type of inheritance (disomic/polysomic) in tetraploid (2 n=4 x=40) Paspalum notatum and investigate the transmission pattern of the chromosome region associated with apospory. An F(1) family segregating for the reproductive mode (aposporous vs non-aposporous) was generated by crossing a tetraploid sexual plant as female parent with an apomictic individual as pollen donor. Pollen mother cells from both parental plants were examined to ascertain chromosome-pairing behavior at meiosis. The high rate of quadrivalent chromosome associations indicated an autotetraploid origin of the species, although bivalent pairing and occasional univalents were detected. The observation of a lagging bivalent, a bridge of chromatin, or two aligned laggards in the aposporous parent suggested a chromosome inversion in this strain. Segregation ratios of AFLP markers and the proportion of linkages in repulsion versus coupling phase denoted tetrasomic inheritance, but markers displaying disomic ratios were also observed. Preferential chromosome pairing (disomic inheritance) in the chromosome segment related to apospory was detected. The possible relationship between a chromosome rearrangement and the inheritance of apospory is discussed.

Tetraploid Paspalum notatum (bahiagrass) is a valuable forage grass with aposporous apomictic reproduction. In a previous study, we showed that apospory in bahiagrass is under the control of a single dominant gene with a distorted segregation ratio. The objective of this work was to identify molecular markers linked to apospory in tetraploid P. notatum and establish a preliminary syntenic relationship with the genomic region associated with apospory in P. simplex. A F-1 population of 290 individuals, segregating for apospory, was generated after crossing a completely sexual plant (Q4188) with a natural aposporous apomictic plant (Q4117). The whole progeny was classified as sexual or aposporous by embryo sacs analysis. A bulked segregant analysis was carried out to identify molecular markers co-segregating with apospory. Four hundred RAPD primers, 30 AFLP primers combinations and 85 RFLP clones were screened using DNA from both parental genotypes and aposporous and sexual bulks. Linkage analysis was performed with cytological and genetic information from the complete progeny. Cytoembryological analysis showed 219 sexual and 71 aposporous F1 individuals. Seven different molecular markers (2 RAPD, 4 AFLP and 1 RFLP) were found to be completely linked to apospory. The RFLP probe C1069, mapping to the telomeric region of the long arm of rice chromosome 12, was one of the molecular markers completely linked to apospory in P. notatum. This marker had been previously associated with apospory in P. simplex. A preliminary map of the chromosome region carrying the apospory locus was constructed.