Identificazione di un QTL principale per resistenza a ruggine bruna sul cromosoma 7B di frumento duro

Leaf rust caused by Puccinia triticina is a serious disease of durum wheat (Triticum durum) worldwide. However, genetic and molecular mapping studies aimed at characterizing leaf rust resistance genes in durum wheat have been only recently undertaken. The Italian durum wheat cv. Creso shows a high level of resistance to P. triticina that has been considered durable and that appears to be due to a combination of a single dominant gene and one or more additional factors conferring partial resistance. In this study, the genetic basis of leaf rust resistance carried by Creso was investigated using 176 recombinant inbred lines (RILs) from the cross between the cv. Colosseo (C, leaf rust resistance donor) and Lloyd (L, susceptible parent). Colosseo is a cv. directly related to Creso with the leaf rust resistance phenotype inherited from Creso, and was considered as resistance donor because of its better adaptation to local (Emilia Romagna, Italy) cultivation environment. RILs have been artificially inoculated with a mixture of 16 Italian P. triticina isolates that were characterized for virulence to seedlings of 22 common wheat cv. Thatcher isolines each carrying a different leaf rust resistance gene, and for molecular genotypes at 15 simple sequence repeat (SSR) loci, in order to determine their specialization with regard to the host species. The characterization of the leaf rust isolates was conducted at the Cereal Disease Laboratory of the University of Minnesota (St. Paul, USA) (Chapter 2). A genetic linkage map was constructed using segregation data from the population of 176 RILs from the cross CL. A total of 662 loci, including 162 simple sequence repeats (SSRs) and 500 Diversity Arrays Technology markers (DArTs), were analyzed by means of the package EasyMap 0.1. The integrated SSR-DArT linkage map consisted of 554 loci (162 SSR and 392 DArT markers) grouped into 19 linkage blocks with an average marker density of 5.7 cM/marker. The final map spanned a total of 2022 cM, which correspond to a tetraploid genome (AABB) coverage of ca. 77% (Chapter 3). The RIL population was phenotyped for their resistance to leaf rust under artificial inoculation in 2006; the percentage of infected leaf area (LRS, leaf rust susceptibility) was evaluated at three stages through the disease developmental cycle and the area under disease progress curve (AUDPC) was then calculated. The response at the seedling stage (infection type, IT) was also investigated. QTL analysis was carried out by means of the Composite Interval Mapping method based on a selection of markers from the CL map. A major QTL (QLr.ubo-7B.2) for leaf rust resistance controlling both the seedling and the adult plant response, was mapped on the distal region of chromosome arm 7BL (deletion bin 7BL10-0.78-1.00), in a gene-dense region known to carry several genes/QTLs for resistance to rusts and other major cereal fungal diseases in wheat and barley. QLr.ubo-7B.2 was identified within a supporting interval of ca. 5 cM tightly associated with three SSR markers (Xbarc340.2, Xgwm146 e Xgwm344.2), and showed an R2 and an LOD peak value for the AUDPC equal to 72.9% an 44.5, respectively. Three additional minor QTLs were also detected (QLr.ubo-7B.1 on chr. 7BS; QLr.ubo-2A on chr. 2AL and QLr.ubo-3A on chr. 3AS) (Chapter 4). The presence of the major QTL (QLr.ubo-7B.2) was validated by a linkage disequilibrium (LD)-based test using field data from two different plant materials: i) a set of 62 advanced lines from multiple crosses involving Creso and his directly related resistance derivates Colosseo and Plinio, and ii) a panel of 164 elite durum wheat accessions representative of the major durum breeding program of the Mediterranean basin. Lines and accessions were phenotyped for leaf rust resistance under artificial inoculation in two different field trials carried out at Argelato (BO, Italy) in 2006 and 2007; the durum elite accessions were also evaluated in two additional field experiments in Obregon (Messico; 2007 and 2008) and in a green-house experiment (seedling resistance) at the Cereal Disease Laboratory (St. Paul, USA, 2008). The molecular characterization involved 14 SSR markers mapping on the 7BL chromosome region found to harbour the major QTL. Association analysis was then performed with a mixed-linear-model approach. Results confirmed the presence of a major QTL for leaf rust resistance, both at adult plant and at seedling stage, located between markers Xbarc340.2, Xgwm146 and Xgwm344.2, in an interval that coincides with the supporting interval (LOD-2) of QLr.ubo-7B.2 as resulted from the RIL QTL analysis. (Chapter 5). The identification and mapping of the major QTL associated to the durable leaf rust resistance carried by Creso, together with the identification of the associated SSR markers, will enhance the selection efficiency in durum wheat breeding programs (MAS, Marker Assisted Selection) and will accelerate the release of cvs. with durable resistance through marker-assisted pyramiding of the tagged resistance genes/QTLs most effective against wheat fungal pathogens.

Association study of resistance to Soilborne wheat mosaic virus in U.S. winter wheat

Soilborne wheat mosaic virus (SBWMV) is one of the most important winter wheat pathogens worldwide. To identify genes for resistance to the virus in U.S. winter wheat, association study was conducted using a selected panel of 205 elite experimental lines and cultivars from U.S. hard and soft winter wheat breeding programs. Virus symptoms were evaluated twice in virus-infected fields for the panel at Manhattan, KS in spring 2010 and 2011 and for a subpanel of 137 hard winter wheat accessions at Stillwater, OK in spring 2008. At the two locations, 69.8 and 79.5% of cultivars were resistant or moderately resistant to the disease, respectively. After 282 simple-sequence repeat markers covering all wheat chromosome arms were scanned for association in the panel, marker Xgwm469 on the long arm of chromosome 5D (5DL) showed a significant association with the disease rating. Three alleles (Xgwm469-165bp, -167bp, and -169bp) were associated with resistance and the null allele was associated with susceptibility. Correlations between the marker and the disease rating were highly significant (0.80 in Manhattan at P < 0.0001 and 0.63 in Stillwater at P < 0.0001). The alleles Xgwm469-165bp and Xgwm469-169bp were present mainly in the hard winter wheat group, whereas allele Xgwm469-167bp was predominant in the soft winter wheat. The 169 bp allele can be traced back to 'Newton', and the 165 bp allele to Aegilops tauschii. In addition, a novel locus on the short arm of chromosome 4D (4DS) was also identified to associate with the disease rating. Marker Xgwm469-5DL is closely linked to SBWMV resistance and highly polymorphic across the winter wheat accessions sampled in the study and, thus, should be useful in marker-assisted selection in U.S. winter wheat.

Clones Identification and Genetic Characterization of Garnacha Grapevine by Means of Different PCR-Derived Marker Systems

This study uses PCR-derived marker systems to investigate the extent and distribution of genetic variability of 53 Garnacha accessions coming from Italy, France and Spain. The samples studied include 28 Italian accessions (named Tocai rosso in Vicenza area; Alicante in Sicily and Elba island; Gamay perugino in Perugia province; Cannonau in Sardinia), 19 Spanish accessions of different types (named Garnacha tinta, Garnacha blanca, Garnacha peluda, Garnacha roja, Garnacha erguida, Garnacha roya) and 6 French accessions (named Grenache and Grenache noir). In order to verify the varietal identity of the samples, analyses based on 14 simple sequence repeat (SSR) loci were performed. The presence of an additional allele at ISV3 locus (151 bp) was found in four Tocai rosso accessions and in a Sardinian Cannonau clone, that are, incidentally, chimeras. In addition to microsatellite analysis, intravarietal variability study was performed using AFLP, SAMPL and M-AFLP molecular markers. AFLPs could discriminate among several Garnacha samples; SAMPLs allowed distinguishing few genotypes on the basis of their geographic origin, whereas M-AFLPs revealed plant-specific markers, differentiating all accessions. Italian samples showed the greatest variability among themselves, especially on the basis of their different provenance, while Spanish samples were the most similar, in spite of their morphological diversity.

Creation and Validation of the Spanish Durum Wheat Core Collection.

Spanish wheat (Triticum spp.) landraces have a considerable polymorphism, containing many unique alleles, relative to other collections. The existence of a core collection is a favored approach for breeders to efficiently explore novel variation and enhance the use of germplasm. In this study, the Spanish durum wheat (Triticum turgidum L.) core collection (CC) was created using a population structure–based method, grouping accessions by subspecies and allocating the number of genotypes among populations according to the diversity of simple sequence repeat (SSR) markers. The CC of 94 genotypes was established, which accounted for 17% of the accessions in the entire collection. An alternative core collection (CH), with the same number of genotypes per subspecies and maximizing the coverage of SSR alleles, was assembled with the Core Hunter software. The quality of both core collections was compared with a random core collection and evaluated using geographic, agromorphological, and molecular marker data not previously used in the selection of genotypes. Both core collections had a high genetic representativeness, which validated their sampling strategies. Geographic and agromorphological variation, phenotypic correlations, and gliadin alleles of the original collection were more accurately depicted by the CC. Diversity arrays technology (DArT) markers revealed that the CC included genotypes less similar than the CH. Although more SSR alleles were retained by the CH (94%) than by the CC (91%), the results showed that the CC was better than CH for breeding purposes.

Conservación de plantas regeneradas in vitro y análisis de la variación somaclonal de Cinchona officinalis, Linneo

Cinchona officinalis (Rubiaceae), especie endémica del Valle de Loja, ubicado en la región sur del Ecuador, es un recurso forestal de importancia medicinal y ecológica, además la especie ha sido catalogada como planta nacional y es un ícono de la región sur por su aporte a la farmacopea mundial. Esta especie, entre los siglos XVII-XIX sufrió una gran presión en sus poblaciones debido a la extracción masiva de la corteza para la cura del paludismo. Aunque la actividad extractiva generó grandes ingresos a la Corona Española y a la región Sur del Ecuador, ésta fue poco o nada sustentable ecológicamente, provocando la desaparición de la especie en muchos sitios de la provincia, pues, en su momento, no se consideraron alternativas de recuperación de las poblaciones naturales. Actualmente la extracción y consumo de la corteza en la zona de origen es baja o nula, sin embargo esta zona enfrenta nuevas amenazas. La deforestación a causa de proyectos de desarrollo en infraestructuras, la práctica de actividades agrícolas y de ganadería, y los efectos del cambio climático han ocasionado, en estos últimos años, la fragmentación de los ecosistemas. La mayoría de los bosques del sur del Ecuador se han convertido en parches aislados (los bosques en los que se distribuye C. officinalis no son la excepción) siendo esta la principal causa para que la especie se encuentre en estado de amenaza. Los individuos de la especie tienen una alta capacidad de rebrote y producen semillas durante todo el año; sin embargo la capacidad germinativa y la tasa de sobrevivencia son bajas, además de estas dificultades la especie requiere de la asociación con otras especies vegetales para su desarrollo, lo cual ha limitado su distribución en pequeños parches aislados. Con esta problemática, la recuperación natural de las poblaciones es una necesidad evidente. Varios trabajos y esfuerzos previos se han realizado a nivel local: i. Identificación de la distribución actual y potencial; ii. Determinación de la fenología y fructificación iii. Programas de educación ambiental, iv. Análisis moleculares para determinar la diversidad genética. v. Ensayos de propagación vegetativa; y otras acciones de tipo cultural. No obstante, el estado de conservación y manejo de las poblaciones naturales no ha mejorado significativamente, siendo necesaria la aplicación de estrategias integradas de conservación in situ y ex situ, que permitan la recuperación y permanencia de las poblaciones naturales a largo plazo. El presente trabajo tiene como fin dar alternativas para el cultivo de tejidos in vitro de Cinchona officinalis centrados en la propagación masiva a partir de semillas, análisis de la fidelidad genética y alternativas de conservación de tejidos. Los objetivos específicos que se plantean son: i. Analizar el proceso de germinación y proliferación in vitro. ii. Evaluar la estabilidad genética en explantes cultivados in vitro, mediante marcadores ISSR. iii. Establecer protocolos de conservación in vitro mediante limitación del crecimiento y criopreservación de segmentos nodales y yemas. Los resultados más significativos de esta investigación fueron: i. El desarrollo de protocolos eficientes para mejorar los porcentajes de germinación y la proliferación de brotes en explantos cultivados in vitro. Para evaluar el efecto de los fenoles sobre la germinación, se determinó el contenido total de fenoles y el porcentaje de germinación en semillas de C. officinalis comparados con una especie de control, C. pubescens. Para inducir a proliferación, se utilizaron segmentos nodales de plántulas germinadas in vitro en medio Gamborg (1968) suplementado con diferentes combinaciones de reguladores de crecimiento (auxinas y citoquininas). Los resultados obtenidos sugieren que el contenido de compuestos fenólicos es alto en las semillas de C. officinalis en comparación con las semillas de C. pubescens. Estos fenoles pueden eliminarse con peróxido de hidrógeno o con lavados de agua para estimular la germinación. La formación de nuevos brotes y callos en la mayoría de las combinaciones de reguladores de crecimiento se observó en un período de 45 días. El mayor porcentaje de proliferación de brotes, formación de callos y presencia de brotes adventicios se obtuvo en medio Gamborg (B5) suplementado con 5.0 mg/l 6-bencil-aminopurina y 3.0 mg/l de ácido indol-3-butírico. ii. La evaluación de la fidelidad genética de los explantes obtenidos con distintas combinaciones de reguladores de crecimiento vegetal y diversos subcultivos. Se realizó el seguimiento a los explantes obtenidos de la fase anterior, determinando el índice de multiplicación y analizando la fidelidad genética de los tejidos obtenidos por las dos vías regenerativas: brotación directa y regeneración de brotes a partir de callos. Este análisis se realizó por amplificación mediante PCR de las secuencias ubicadas entre microsatélites-ISSR (Inter simple sequence repeat). El medio Gamborg (B5) con 3.0 mg/l de AIB y 5.0 mg/l de BAP usado como medio de inducción en la primera etapa de cultivo generó el mayor índice de proliferación (11.5). Un total de 13 marcadores ISSR fueron analizados, 6 de éstos fueron polimórficos. El mayor porcentaje de variación somaclonal fue inducido en presencia de 1.0 mg/l 2,4-D combinado con 0.2 mg/l Kin con un 1.8% en el segundo sub-cultivo de regeneración, la cual incrementó a 3.6% en el tercer sub-cultivo. Todas las combinaciones con presencia de 2,4-D produjeron la formación de callos y presentaron variación genética. Por su parte la fidelidad genética se mantuvo en los sistemas de propagación directa a través de la formación de brotes a partir de meristemos preformados. iii. El establecimiento de protocolos de conservación in vitro y crioconservación de segmentos nodales y yemas. Para la conservación limitando el crecimiento, se cultivaron segmentos nodales en los medios MS y B5 en tres concentraciones de sus componentes (25, 50 y 100%); y en medio B5 más agentes osmóticos como el manitol, sorbitol y sacarosa en diferentes concentraciones (2, 4 y 8%); los cultivos se mantuvieron por 12 meses sin subcultivos. Para el establecimiento de protocolos para la crioconservación (paralización del metabolismo) se usaron yemas axilares y apicales a las cuales se les aplicaron los métodos de encapsulación-deshidratación y vitrificación. La efectividad de los protocolos usados se determinó en función de la sobrevivencia, reducción del crecimiento y regeneración. Los resultados obtenidos en este apartado reflejan que un crecimiento limitado puede mantener tejidos durante 12 meses de almacenamiento, usando medio B5 más manitol entre 2 y 8%. En los protocolos de crioconservación, se obtuvo el mayor porcentaje de recuperación tras la congelación en NL en el tratamiento control seguido por el método crioprotector de encapsulación-deshidratación. Este trabajo brinda alternativas para la propagación de C. officinalis bajo condiciones in vitro, partiendo de material vegetal con alta diversidad genética. El material propagado puede ser fuente de germoplasma para la recuperación y reforzamiento de las poblaciones naturales así como una alternativa de producción para las comunidades locales debido a la demanda actual de corteza de la zona de origen para la elaboración de agua tónica. ABSTRACT Cinchona officinalis (Rubiaceae) is endemic to the Loja Valley, located in the southern area of Ecuador. The importance of this plant as medical and ecological resource is so great that it has been designated as the national flower and is an icon of the southern region for its contribution to the world pharmacopoeia. Between XVII-XIX centuries its population suffered great reduction due to massive harvesting of the bark to cure malaria. Although extraction activity generated large revenues to the Spanish Crown and the southern region of Ecuador, this was not ecologically sustainable, causing the disappearance of the species in many areas of the province, because during that time alternatives to prevent extinction and recover natural populations were not taken in account. Currently the extraction and consumption of bark in the area of origin is almost absent, but this species faces new threats. Deforestation due to infrastructure development, the practice of farming and ranching, and the effects of climate change had led to the fragmentation of ecosystems during the recent years. Most of the forests of southern Ecuador have become isolated patches, including those where C. officinalis is diffused. The lack of suitable habitat is today the main threat for the species. The species has a high capacity for regeneration and produces seeds throughout the year, but the germination rate is low and the growth is slow. In addition, the species requires the association with other plant species to develop. All these factors had limited its distribution to small isolated patches. The natural recovery of populations is essential to face this problem. Several studies and previous efforts had been made at local level: i. Identification of current and potential distribution; ii. Phenology determination. iii. Environmental education programs, iv. Molecular analisis to determine the genetic diversity. v. Testing of vegetative propagation; and other actions of cultural nature. Despite these efforts, the state of conservation and management of natural populations has not improved significantly. Implementation of integrated in situ and ex situ conservation strategies for the recovery and permanence of long-term natural populations is still needed. This work aims to provide alternatives for in vitro culture of tissue of Cinchona officinalis focused on mass propagation from seeds, genetic fidelity analysis and tissue conservation alternatives. The specific aims are: i. Analyze the process of germination and proliferation in vitro. ii. To evaluate the genetic stability of the explants cultured in vitro by ISSR markers. iii. Establish protocols for in vitro conservation by limiting growth and cryopreservation of nodal segments and buds. The most significant results of this research were: i. The development of efficient protocols to improve germination rates and proliferation of buds in explants cultured in vitro. To study the effect of phenols on germination, the total phenolic content and percentage germination was measured in C. officinalis and in a control species, C. pubescens, for comparison. The content of phenolic compounds in C. officinalis seeds is higher than in C. pubescens. These phenols can be removed with hydrogen peroxide or water washes to stimulate germination. To analyze the regeneration, we used nodal explants from seedlings germinated in vitro on Gamborg medium (1968) supplemented with different combinations of growth regulators (auxins and cytokinins) to induce proliferation. The formation of new shoots and calluses was observed within a period of 45 days in most combinations of growth regulators. The highest percentage of shoot proliferation, callus formation and adventitious buds were obtained in B5 medium supplemented with 5.0 mg/l 6-benzyl-aminopurine and 3.0 mg/l indole-3-butyric acid. ii. Evaluating genetic fidelity explants obtained with various combinations of plant growth regulators and different subcultures. The genetic fidelity was analyzed in tissues obtained by the two regenerative pathways: direct sprouting and shoot regeneration from callus. This analysis was performed by PCR amplification of the sequences located between microsatellite-ISSR (Inter Simple Sequence Repeat). Among a total of 13 ISSR markers analyzed, 6 were polymorphic. The highest percentage of somaclonal variation was induced in the presence of 1.0 mg/l 2,4-D combined with 0.2 mg/l Kin with 1.8% in the second round of regeneration, and increased to 3.6% in the third round. The presence of 2,4-D induced genetic variation in all the combinations of growth regulators. Meanwhile genetic fidelity remained systems propagation through direct shoot formation from meristems preformed. iii. Establishing conservation protocols in vitro and cryoconservation of nodal segments and buds. For medium-term conservation (limited growth) nodal segments were cultured in MS and B5 media at three concentrations (25, 50 and 100%); we tested B5 medium with different concentrations of osmotic agents such as mannitol, sorbitol and sucrose (2, 4 and 8%); cultures were maintained for 12 months with regular subculturing. To establish protocols for cryoconservation (cessation of metabolism) different methods of encapsulation-dehydration and vitrification were applied to axillary and apical buds. The effectiveness of the used protocols is determined based on the survival, growth and regeneration success. The results show that these tissues can be maintained in storage for 12 months, using B5 medium plus mannitol between 2 and 8%. The cryoconservation protocol with highest percentage of recovery was obtained by contral treatment, followed by freezing in NL with encapsulation-dehydration method. This work provides alternatives for the propagation in vitro of C. officinalis, starting from plant material with high genetic diversity. The obtained material represents a source of germplasm to support the recovery and strengthening of natural populations as well as a creation of alternative sources for local communities due to the current demand of bark for the preparation of tonic water.

The onset and extent of genomic instability in sporadic colorectal tumor progression

Cancer cell genomes contain alterations beyond known etiologic events, but their total number has been unknown at even the order of magnitude level. By sampling colorectal premalignant polyp and carcinoma cell genomes through use of the technique inter-(simple sequence repeat) PCR, we have found genomic alterations to be considerably more abundant than expected, with the mean number of genomic events per carcinoma cell totaling approximately 11,000. Colonic polyps early in the tumor progression pathway showed similar numbers of events. These results indicate that, as with certain hereditary cancer syndromes, genomic destabilization is an early step in sporadic tumor development. Together these results support the model of genomic instability being a cause rather than an effect of malignancy, facilitating vastly accelerated somatic cell evolution, with the observed orderly steps of the colon cancer progression pathway reflecting the consequences of natural selection.

Targeted development of informative microsatellite (SSR) markers

We describe a novel approach, selectively amplified microsatellite (SAM) analysis, for the targeted development of informative simple sequence repeat (SSR) markers. A modified selectively amplified microsatellite polymorphic loci assay is used to generate multi-locus SSR fingerprints that provide a source of polymorphic DNA markers (SAMs) for use in genetic studies. These polymorphisms capture the repeat length variation associated with SSRs and allow their chromosomal location to be determined prior to the expense of isolating and characterising individual loci. SAMs can then be converted to locus-specific SSR markers with the design and synthesis of a single primer specific to the conserved region flanking the repeat. This approach offers a cost-efficient and rapid method for developing SSR markers for predetermined chromosomal locations and of potential informativeness. The high recovery rate of useful SSR markers makes this strategy a valuable tool for population and genetic mapping studies. The utility of SAM analysis was demonstrated by the development of SSR markers in bread wheat.

A genetic linkage map in autotetraploid lucerne adapted to northern Australia, and use of the map to identify DNA markers linked to resistance to Phytophthora medicaginis

Phytophthora root rot, caused by Phytophthora medicaginis, is a major limitation to lucerne ( Medicago sativa L.) production in Australia and North America. Quantitative trait loci (QTLs) involved in resistance to P. medicaginis were identified in a lucerne backcross population of 120 individuals. A genetic linkage map was constructed for tetraploid lucerne using 50 RAPD ( randomly amplified polymorphic DNA), 104 AFLP (amplified fragment length polymorphism) markers, and one SSR ( simple sequence repeat or microsatellite) marker, which originated from the resistant parent (W116); 13 markers remain unlinked. The linkage map contains 18 linkage groups covering 2136.5 cM, with an average distance of 15.0 cM between markers. Four of the linkage groups contained only either 2 or 3 markers. Using duplex markers and repulsion phase linkages the map condensed to 7 homology groups and 2 unassigned linkage groups. Three regions located on linkage groups 2, 14, and 18, were identified as associated with root reaction and the QTLs explained 6 - 15% of the phenotypic variation. The research also indicates that different resistance QTLs are involved in conferring resistance in different organs. Two QTLs were identified as associated with disease resistance expressed after inoculation of detached leaves. The marker, W11-2 on group 18, identified as associated with root reaction, contributed 7% of the phenotypic variation in leaf response in our population. This marker appears to be linked to a QTL encoding a resistance factor contributing to both root and leaf reaction. One other QTL, not identified as associated with root reaction, was positioned on group 1 and contributed to 6% of the variation. This genetic linkage map provides an entry point for future molecular-based improvement of lucerne in Australia, and markers linked to the QTLs we have reported should be useful for marker-assisted selection for partial resistance to P. medicaginis in lucerne.

Transcriptome analysis of Neotyphodium and Epichloë grass endophytes

Large-scale gene discovery has been performed for the grass fungal endophytes Neotyphodium coenophialum, Neotyphodium lolii, and Epichloe festucae. The resulting sequences have been annotated by comparison with public DNA and protein sequence databases and using intermediate gene ontology annotation tools. Endophyte sequences have also been analysed for the presence of simple sequence repeat and single nucleotide polymorphism molecular genetic markers. Sequences and annotation are maintained within a MySQL database that may be queried using a custom web interface. Two cDNA-based microarrays have been generated from this genome resource, They permit the interrogation of 3806 Neotyphodium genes (Nchip (TM) rnicroarray), and 4195 Neotyphodium and 920 Epichloe genes (EndoChip (TM) microarray), respectively. These microarrays provide tools for high-throughput transcriptome analysis, including genome-specific gene expression studies, profiling of novel endophyte genes, and investigation of the host grass-symbiont interaction. Comparative transcriptome analysis in Neotyphodium and Epichloe was performed. (c) 2006 Elsevier

Mapping genes for resistance to net form of net blotch and strip rust in barley (Hordeum vulgare L.)

A dihaploid mapping population comprising 65 lines was developed between barley parent varieties Tallon and Kaputar and used to construct a genetic linkage map. This map, comprising 195 amplified fragment length polymorphism and 38 simple sequence repeat markers, was used to identify markers linked to the net form of net blotch (Pyrenophora teres f.sp. teres) and to stripe rust (Puccinia striiformis f.sp. hordei) in barley. The population was screened with five pathotypes of net blotch at the seedling stage in the glasshouse and subjected to a natural inoculation in Hermitage, Queensland. Stripe rust screening was conducted at the adult plant stage in Toluca, Mexico. Analyses of the markers were performed using Mapmanager and Qgene software. One region on chromosome 6H was highly significantly associated with resistance to the net blotch (R2 = 79%). This association was consistent for all pathotypes studied. One region on chromosome 5H was found to be highly significantly associated with resistance to stripe rust (R2= 65%). There are a number of very closely linked markers showing strong associations in these regions, and these markers present an opportunity for marker assisted selection of these traits in barley breeding programs.

Molecular phylogenetics of the palm tribe Geonomeae, and differentiation of Geonoma macrostachys western Amazonian varieties

Phylogenetic analyses were performed on six genera and 46 species of the Neotropical palm tribe Geonomeae. The analyses were based on two low copy nuclear DNA sequences from the genes encoding phosphoribulokinase and RNA polymerase II. The basal node of the tribe was polytomous. Pholidostachys formed a monophyletic group. The currently accepted genera Calyptronoma and Calyptrogyne formed a well-supported clade with Calyptronoma resolved as paraphyletic to Calyptrogyne. Geonoma formed a strongly supported monophyletic group consisting of two main clades. ^ An evaluation of the genetic distinctness between Geonoma macrostachys varieties at a local and regional scale using inter-simple sequence repeat (ISSR) markers was performed. Clustering, ordination, and AMOVA suggested a lack of genetic distinctness between varieties at the regional level. A hierarchical AMOVA revealed that the genetic diversity mainly lies among the four localities sampled. A significant genetic differentiation between sympatric varieties occurred in one locality only. The current taxonomy of G. macrostachys, which recognizes only one species, was therefore supported. ^ The preferred habitat of sympatric G. macrostachys varieties with respect to edaphic, topographic, and light factors in three Peruvian lowland forests was studied. The two varieties were mostly encountered in different physiographically defined habitats, with variety acaulis occurring more often in floodplain forest and variety macrostachys in the tierra firme. Comparison of means tests revealed that nine to eleven of the 16 environmental variables were significantly different between varieties. Edaphic factors, mainly soil texture and K content, were better contributors than light conditions to distinguish the habitats occupied by the two varieties in all three study sites. It is concluded that habitat differentiation plays a role in the coexistence of these closely related species taxa. ^

Herança genética e marcadores moleculares associados à resistência de Euphorbia heterophylla L. aos herbicidas inibidores da ALS e PROTOX

This study aimed to assess the genetic inheritance, determine the better DNA isolation protocol for this species and to identify molecular markers associated with the Wild Poinsettia (Euphorbia heterophylla L.) resistance ALS- and PROTOX- inhibiting herbicides and. The genetic inheritance of resistance was determined from crosses between E. heterophylla biotypes susceptible (S) and resistant (R), backcrosses and F2 generation. The complete dominance of resistance was confirmed with dose response curves. Ten adjusted methods for DNA isolation described in the literature were tested. The specific primers for ALS and PROTOX genes were designed from the consensus DNA sequence of these genes, obtained by aligning the gene sequences of the species Manihot esculenta and Ricinus communis L. Additionally, it was assessed the transferability of twenty SSR (simple sequence repeat) markers designed for Manihot esculenta, because among the species of Euphorbiaceae with more developed SSRs markers, because it is the closest relative phylogenetic species of E. heterophylla. Regarding genetic inheritance, the frequencies observed in the F1, F2, RCs and RCr did not differ significantly from the expected frequencies for a trait controlled by two dominant genes for multiple resistance and a single dominant gene for simple resistance to ALS- and PROTOX-inhibiting herbicides. The similar levels of resistance to dosage up to 2000 g i.a. ha-1 of fomesafen and dosage up to 800 g i.a. ha-1 of imazethapyr observed in F1 (heterozygous) and homozygous R biotype confirm the complete dominance of resistance to PROTOX- and ALS-inhibiting herbicides, respectively. The 0.2%BME protocol allowed the isolation of 7,083 ng μL-1 DNA, significantly (P=0.05) higher than other methods. Co-isolation of phenolic compounds was observed in FENOL and 3%BME+TB methods, but the addition of polyvinylpyrrolidone (PVP40) in the protocol extraction buffer 3%BME+TA solved this problem. The primers designed for ALS and PROTOX genes amplified but not showed no visible polymorphism in agarose gel between the S and R biotypes of E. heterophylla. Regarding the SSR transferability, ten markers were transferred to E. heterophylla, however, these six primers showed polymorphism among S and R biotypes.

Development, characterization and use of genomic SSR markers for assessment of genetic diversity in some Saudi date palm ( Phoenix dactylifera L.) cultivars

Background: The present study was undertaken towards the development of SSR markers and assessing genetic relationships among 32 date palm ( Phoenix dactylifera L.) representing common cultivars grown in different geographical regions in Saudi Arabia. Results: Ninety-three novel simple sequence repeat markers were developed and screened for their ability to detect polymorphism in date palm. Around 71% of genomic SSRs were dinucleotide, 25% tri, 3% tetra and 1% penta nucleotide motives. Twenty-two primers generated a total of 91 alleles with a mean of 4.14 alleles per locus and 100% polymorphism percentage. A 0.595 average polymorphic information content and 0.662 primer discrimination power values were recorded. The expected and observed heterozygosities were 0.676 and 0.763 respectively. Pair-wise similarity values ranged from 0.06 to 0.89 and the overall cultivars averaged 0.41. The UPGMA cluster analysis recovered by principal coordinate analysis illustrated that cultivars tend to group according to their class of maturity, region of cultivation, and fruit color. Analysis of molecular variations (AMOVA) revealed that genetic variation among and within cultivars were 27% and 73%, respectively according to geographical distribution of cultivars. Conclusions: The developed microsatellite markers are additional values to date palm characterization tools that can be used by researchers in population genetics, cultivar identification as well as genetic resource exploration and management. The tested cultivars exhibited a significant amount of genetic diversity and could be suitable for successful breeding program. Genomic sequences generated from this study are available at the National Center for Biotechnology Information (NCBI), Sequence Read Archive (Accession numbers. LIBGSS_039019).

Genetic diversity assessment of the almond (Prunus dulcis (Mill.) DA Webb) traditional germplasm of Algarve, Portugal, using molecular markers

In this study, 123 almond (Prunus dulcis (Mill.) D. A. Webb) trees identified among traditional orchards in the Algarve region and 53 trees of the local field collection managed by the regional office of the Portuguese Ministry of Agriculture (DRAALG) were assessed using isozyme, inter- single sequence repeat and simple sequence repeat or microsatellite techniques for the evaluation of genetic diversity and genetic relatedness and identification of new accessions for the field collection. The isozyme analysis allowed the distribution of the 176 plants into 13 different classes of enzyme similarity, while the use of DNA markers increased the distribution of the analysed trees among 140 discriminating DNA patterns. Multiple cases of homonymy and synonymy were identified in the local germplasm. Some traditional varieties, such as Lourencinha, appeared to be relatively homogeneous, while other local denominations, e.g. Galamba, included diverse genotypes. Of the 13 commercial varieties analysed in this study, 11 assembled in one major cluster clearly differentiated from the majority of the local genotypes. These results reinforced the perception that the Algarve traditional germplasm constitutes an important repository of genetic diversity, eventually carrying alleles of high agricultural interest such as the recently identified Phomopsis resistance in the traditional variety Barrinho Grado.

Genetic diversity assessment of the almond (Prunus dulcis (Mill.) DA Webb) traditional germplasm of Algarve, Portugal, using molecular markers

In this study, 123 almond (Prunus dulcis (Mill.) D. A. Webb) trees identified among traditional orchards in the Algarve region and 53 trees of the local field collection managed by the regional office of the Portuguese Ministry of Agriculture (DRAALG) were assessed using isozyme, inter- single sequence repeat and simple sequence repeat or microsatellite techniques for the evaluation of genetic diversity and genetic relatedness and identification of new accessions for the field collection. The isozyme analysis allowed the distribution of the 176 plants into 13 different classes of enzyme similarity, while the use of DNA markers increased the distribution of the analysed trees among 140 discriminating DNA patterns. Multiple cases of homonymy and synonymy were identified in the local germplasm. Some traditional varieties, such as Lourencinha, appeared to be relatively homogeneous, while other local denominations, e.g. Galamba, included diverse genotypes. Of the 13 commercial varieties analysed in this study, 11 assembled in one major cluster clearly differentiated from the majority of the local genotypes. These results reinforced the perception that the Algarve traditional germplasm constitutes an important repository of genetic diversity, eventually carrying alleles of high agricultural interest such as the recently identified Phomopsis resistance in the traditional variety Barrinho Grado.