Phylogénie moléculaire du genre Salix L. (Salicaceae) en Amérique du Nord

La culture de saules (Salix sp.) est une pratique courante en Europe et en Amérique du Nord pour produire de la biomasse végétale. Cependant, le développement d’outils moléculaires est très récent. De plus, la phylogénie des saules est incomplète. Il y a un manque d’information pour les programmes de sélection d'espèces indigènes et pour la compréhension de l’évolution du genre. Le genre Salix inclut 500 espèces réparties principalement dans les régions tempérées et boréo-arctique de l’hémisphère nord. Nous avons obtenu l’ensemble des espèces retrouvées naturellement en Amérique (121 indigènes et introduites). Dans un premier temps, nous avons développé de nouveaux outils moléculaires et méthodes : extraction d’ADN, marqueurs microsatellites et gènes nucléaires. Puis, nous avons séquencé deux gènes chloroplastiques (matK et rbcL) et la région ITS. Les analyses phylogénétiques ont été réalisées selon trois approches : parcimonie, maximum de vraisemblance et Bayésienne. L’arbre d’espèces obtenu a un fort support et divise le genre Salix en deux sous-genres, Salix et Vetrix. Seize espèces ont une position ambiguë. La diversité génétique du sous-genre Vetrix est plus faible. Une phylogénie moléculaire complète a été établie pour les espèces américaines. D’autres analyses et marqueurs sont nécessaires pour déterminer les relations phylogénétiques entre certaines espèces. Nous affirmons que le genre Salix est divisé en deux clades.

An enhanced microsatellite map of diploid Fragaria

A total of 45 microsatellites (SSRs) were developed for mapping in Fragaria. They included 31 newly isolated codominant genomic SSRs from F. nubicola and a further 14 SSRs, derived from an expressed sequence tagged library (EST-SSRs) of the cultivated strawberry, F. × ananassa. These, and an additional 64 previously characterised but unmapped SSRs and EST-SSRs, were scored in the diploid Fragaria interspecific F2 mapping population (FV×FN) derived from a cross between F. vesca 815 and F. nubicola 601. The cosegregation data of these 109 SSRs, and of 73 previously mapped molecular markers, were used to elaborate an enhanced linkage map. The map is composed of 182 molecular markers (175 microsatellites, six gene specific markers and one sequence-characterised amplified region) and spans 424 cM over seven linkage groups. The average marker spacing is 2.3 cM/marker and the map now contains just eight gaps longer than 10 cM. The transferability of the new SSR markers to the cultivated strawberry was demonstrated using eight cultivars. Because of the transferable nature of these markers, the map produced will provide a useful reference framework for the development of linkage maps of the cultivated strawberry and for the development of other key resources for Fragaria such as a physical map. In addition, the map now provides a framework upon which to place transferable markers, such as genes of known function, for comparative mapping purposes within Rosaceae.

Mapping quantitative trait loci for flag leave senescence as a yield determinant in winter wheat under optimal and drought stressed environments

The timing of flag leaf senescence (FLS) is an important determinant of yield under stress and optimal environments. A doubled haploid population derived from crossing the photo period-sensitive variety Beaver,with the photo period-insensitive variety Soissons, varied significantly for this trait, measured as the percent green flag leaf area remaining at 14 days and 35 days after anthesis. This trait also showed a significantly positive correlation with yield under variable environmental regimes. QTL analysis based on a genetic map derived from 48 doubled haploid lines using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers, revealed the genetic control of this trait. The coincidence of QTL for senescence on chromosomes 2B and 2D under drought-stressed and optimal environments, respectively, indicate a complex genetic mechanism of this trait involving the re-mobilisation of resources from the source to the sink during senescence.

Progressive erosion of genetic and epigenetic variation in callus-derived cocoa (Theobroma cacao) plants

Relatively little is known about the timing of genetic and epigenetic forms of somaclonal variation arising from callus growth. We surveyed for both types of change in cocoa (Theobroma cacao) plants regenerated from calli of various ages, and also between tissues from the source trees. For genetic change, we used 15 single sequence repeat (SSR) markers from four source trees and from 233 regenerated plants. For epigenetic change, we used 386 methylation-sensitive amplified polymorphism (MSAP) markers on leaf and explant (staminode) DNA from two source trees and on leaf DNA from 114 regenerants. Genetic variation within source trees was limited to one slippage mutation in one leaf. Regenerants were far more variable, with 35% exhibiting at least one mutation. Genetic variation initially accumulated with culture age but subsequently declined. MSAP (epigenetic) profiles diverged between leaf and staminode samples from source trees. Multivariate analysis revealed that leaves from regenerants occupied intermediate eigenspace between leaves and staminodes of source plants but became progressively more similar to source tree leaves with culture age. Statistical analysis confirmed this rather counterintuitive finding that leaves of ‘late regenerants’ exhibited significantly less genetic and epigenetic divergence from source leaves than those exposed to short periods of callus growth.

A genetic linkage map of an apple rootstock progeny anchored to the Malus genome sequence

An apple rootstock progeny raised from the cross between the very dwarfing ‘M.27’ and the more vigorous ‘M.116’ (‘M.M.106’ × ‘M.27’) was used for the construction of a linkage map comprising a total of 324 loci: 252 previously mapped SSRs, 71 newly characterised or previously unmapped SSR loci (including 36 amplified by 33 out of the 35 novel markers reported here), and the self-incompatibility locus. The map spanned the 17 linkage groups (LG) expected for apple covering a genetic distance of 1,229.5 cM, an estimated 91% of the Malus genome. Linkage groups were well populated and, although marker density ranged from 2.3 to 6.2 cM/SSR, just 15 gaps of more than 15 cM were observed. Moreover, only 17.5% of markers displayed segregation distortion and, unsurprisingly in a semi-compatible backcross, distortion was particularly pronounced surrounding the self-incompatibility locus (S) at the bottom of LG17. DNA sequences of 273 SSR markers and the S locus, representing a total of 314 loci in this investigation, were used to anchor to the ‘Golden Delicious’ genome sequence. More than 260 of these loci were located on the expected pseudo-chromosome on the ‘Golden Delicious’ genome or on its homeologous pseudo-chromosome. In total, 282.4 Mbp of sequence from 142 genome sequence scaffolds of the Malus genome were anchored to the ‘M.27’ × ‘M.116’ map, providing an interface between the marker data and the underlying genome sequence. This will be exploited for the identification of genes responsible for traits of agronomic importance such as dwarfing and water use efficiency.

Development of a dense SNP-based linkage map of an apple rootstock progeny using the Malus Infinium whole genome genotyping array

Background A whole-genome genotyping array has previously been developed for Malus using SNP data from 28 Malus genotypes. This array offers the prospect of high throughput genotyping and linkage map development for any given Malus progeny. To test the applicability of the array for mapping in diverse Malus genotypes, we applied the array to the construction of a SNPbased linkage map of an apple rootstock progeny. Results Of the 7,867 Malus SNP markers on the array, 1,823 (23.2 %) were heterozygous in one of the two parents of the progeny, 1,007 (12.8 %) were heterozygous in both parental genotypes, whilst just 2.8 % of the 921 Pyrus SNPs were heterozygous. A linkage map spanning 1,282.2 cM was produced comprising 2,272 SNP markers, 306 SSR markers and the S-locus. The length of the M432 linkage map was increased by 52.7 cM with the addition of the SNP markers, whilst marker density increased from 3.8 cM/marker to 0.5 cM/marker. Just three regions in excess of 10 cM remain where no markers were mapped. We compared the positions of the mapped SNP markers on the M432 map with their predicted positions on the ‘Golden Delicious’ genome sequence. A total of 311 markers (13.7 % of all mapped markers) mapped to positions that conflicted with their predicted positions on the ‘Golden Delicious’ pseudo-chromosomes, indicating the presence of paralogous genomic regions or misassignments of genome sequence contigs during the assembly and anchoring of the genome sequence. Conclusions We incorporated data for the 2,272 SNP markers onto the map of the M432 progeny and have presented the most complete and saturated map of the full 17 linkage groups of M. pumila to date. The data were generated rapidly in a high-throughput semi-automated pipeline, permitting significant savings in time and cost over linkage map construction using microsatellites. The application of the array will permit linkage maps to be developed for QTL analyses in a cost-effective manner, and the identification of SNPs that have been assigned erroneous positions on the ‘Golden Delicious’ reference sequence will assist in the continued improvement of the genome sequence assembly for that variety.

Differentiation in populations of the apple scab fungus Venturia inaequalis on cultivars in a mixed orchard remains over time

The ascomycete Venturia inaequalis causes annual epidemics of apple scab worldwide. Scab development is reduced in mixed cultivar orchards compared with monocultures. To use mixtures in commercial production, we need to understand how the population of scab changes in a mixed orchard and how likely a super race, with virulence factors overcoming multiple resistance factors in the mixed orchard, is to emerge and become dominant. We used short sequence repeat (SSR) markers to investigate the temporal change of scab populations in two mixed cultivar orchards in the UK to infer the likelihood of emergence of a scab super race. There were no significant differences between the populations at the two sampling times (six or seven years apart) in either of the two mixed orchards. In one of the orchards apple scab populations on different cultivars were significantly different and the differences did not diminish over time. These results suggest that it is not inevitable that a super race of V. inaequalis will become dominant during the lifetime of a commercial apple orchard.

Analysis of the genetic diversity and structure across a wide range of germplasm reveals prominent gene flow in apple at the European level

Abstract Background: The amount and structure of genetic diversity in dessert apple germplasm conserved at a European level is mostly unknown, since all diversity studies conducted in Europe until now have been performed on regional or national collections. Here, we applied a common set of 16 SSR markers to genotype more than 2,400 accessions across 14 collections representing three broad European geographic regions (North+East, West and South) with the aim to analyze the extent, distribution and structure of variation in the apple genetic resources in Europe. Results: A Bayesian model-based clustering approach showed that diversity was organized in three groups, although these were only moderately differentiated (FST=0.031). A nested Bayesian clustering approach allowed identification of subgroups which revealed internal patterns of substructure within the groups, allowing a finer delineation of the variation into eight subgroups (FST=0.044). The first level of stratification revealed an asymmetric division of the germplasm among the three groups, and a clear association was found with the geographical regions of origin of the cultivars. The substructure revealed clear partitioning of genetic groups among countries, but also interesting associations between subgroups and breeding purposes of recent cultivars or particular usage such as cider production. Additional parentage analyses allowed us to identify both putative parents of more than 40 old and/or local cultivars giving interesting insights in the pedigree of some emblematic cultivars. Conclusions: The variation found at group and sub-group levels may reflect a combination of historical processes of migration/selection and adaptive factors to diverse agricultural environments that, together with genetic drift, have resulted in extensive genetic variation but limited population structure. The European dessert apple germplasm represents an important source of genetic diversity with a strong historical and patrimonial value. The present work thus constitutes a decisive step in the field of conservation genetics. Moreover, the obtained data can be used for defining a European apple core collection useful for further identification of genomic regions associated with commercially important horticultural traits in apple through genome-wide association studies.

Caracterização morfológica, citogenética e molecular de uma população de tangerineiras híbridas de 'Clementina fina' (Citrus clementina Hort. ex Tan.) e 'Montenegrina' (Citrus deliciosa Ten.)

A produção citrícola se encontra dispersa por todos os continentes e no Brasil, os citros são a produção frutícola de maior volume de produção. A produção de citros de mesa, como as tangerinas, possibilita ao produtor obter maior valor pelo seu produto. O mercado consumidor é ávido por novas variedades e para tanto, um programa de melhoramento deve estar sempre em busca de genótipos que atendam ao mercado consumidor, bem como a cadeia produtiva. Na Estação Experimental Agronômica da Universidade Federal do Rio Grande do Sul, está localizada uma população de tangerineiras híbridas oriundas do cruzamento da tangerineira ‘Clementina Fina’ (Citrus clementina Hort. ex Tan.) e ‘Montenegrina’ (Citrus deliciosa Ten.) a qual foi caracterizada neste estudo, avaliando-se características morfológicas de acordo com os descritores propostos pelo International Board for Plant Genetic Resources, além da identificação da época de maturação, viabilidade de pólen, número cromossômico e caracterização molecular, utilizando marcadores do tipo microssatélites. Através da análise morfológica foi possível distinguir todas as 96 plantas avaliadas, porém não foi possível agrupar a F1 em grupos distintos de cada um dos genitores. A época de maturação de frutos das plantas se concentra entre a primeira quinzena de abril até a primeira quinzena de agosto. Todas as plantas analisadas apresentaram um alto grau de viabilidade de pólen, variando entre 79,04 e 98,08 %. Todas as plantas avaliadas são diplóides com um número cromossômico de 2n=18. Utilizando 12 pares de primers de microssatélites foi possível diferenciar 90 acessos do estudo, e agrupar a F1 em indivíduos mais próximos do genitor feminino e do genitor masculino. O PIC (Conteúdo de Informação de Polimorfismo) dos primers variou de 0,27 a 0,65. Não foi possível estabelecer uma relação entre a caracterização utilizando marcadores morfológicos e a caracterização utilizando marcadores moleculares.

Estrutura genética de populações de melhoramento de Pinus caribaea var. Hondurensis por meio de marcadores microssatélites

O Pinus caribaea var. hondurensis Barret & Golfari tem elevada importância como espécie comercialmente plantada; aproximadamente 1,8 milhões de hectares estão ocupados por plantios desta espécie no Brasil. O trabalho teve como objetivo verificar por meio de marcadores microssatélites a variabilidade genética em Pinus caribaea var. hondurensis, bem como sua manutenção durante o processo de melhoramento genético, dentro de uma população- base de melhoramento, uma população de matrizes selecionadas e uma população melhorada F1. Para a realização das análises foi necessária a transferência de primers desenvolvidos para locos microssatélites de outras espécies do gênero. Dos 20 pares de primers testados, 8 foram transferidos para a espécie (RPS 25b, RPS 150, PSM 2, PR 4.6, PtTX 2037, PtTX 3029, RPTest 01 e RPTest 09). Verificou-se a existência de endogamia entre e dentro das populações estudadas, e o maior valor observado entre as populações foi F ST = 0,0213 (população base e F1). A heterozigosidade média observada e a heterozigosidade esperada na população-base foram, respectivamente, H0 = 0,2469 e He = 0,2489. A maior distância genética (D = 0,0119) foi observada entre as populações-base e a população melhorada F1. Através da distância genética entre as matrizes, foram indicados 10 cruzamentos potenciais entre as matrizes mais contrastantes, almejando a obtenção de vigor de híbrido nas progênies obtidas a partir destes cruzamentos.

caracterização in situ e estrutura genética de populações de Gossypium mustelinum

Gossypium mustelinum Miers ex Watt is the only cotton species native from Brazil. It is endemic of the semi-arid region from North-east of the country, where it occur near from resilient water sources. The threats to the in situ conservation of the populations are caused by human interference in its habitat, mainly by excessive cattle graze and deforestation. Establish efficient strategies of in situ conservation depend on the accomplishment of a diagnosis of how the specie is found in its natural environment, and the knowledge about the genetic structure of the populations. The objectives of this work were i) to determine the in situ conditions of two populations present in rivers from basin of Rio Paraguaçu at the Bahia State, ii) to evaluate the structure and genetic variability presented in both populations, iii) to establish in situ and ex situ conservation strategies. It were realized collection in november 2007, when was realized in situ characterization of G. mustelinum. SSR markers were used for analyze 218 genotypes deriving from two populations of the G. mustelinum, localized at Tocó river and the Capivara river. The allelic frequencies, the heterozigosity and the F statics were estimated. All the plants were classified as wild and natives, and there was no evidence of the use the plants or its parts. The populations showed different conservation conditions in situ. Few plantlets were found in sites with excessive cattle feed, an indication that the damages in young plants should be high enough to compromise the renovation of the populations. On the other hand, populations were well preserved when the anthropic damages was low or inexistent. The 14 SSR primer pairs amplified 17 loci with a medium number of 5 alleles per locus (a total of 85 alleles). The high level of endogamy estimated (FIS=0,808) and the low observed heterozygosity (H0=0,093) were indicatives that the populations reproduce mainly by selfing, geitonogamy and crosses between related individuals. The genetic diversity was high (HE=0,482) and the differentiation between the populations was very high (FST=0,328). At least two sites from both populations of G. mustelinum must be preserved to achieve suitable in situ conservation. Actions that preserve the gallery forest and keep the cattle away should implemented, and could be as simple as erecting a fence. It is not possible anticipated if the in situ preservation will be possible. Therefore collections and ex situ preservation of representative specimens are essential to conserve the genetic diversity of native G. mustelinum

Mapping QTLs for aluminum tolerance in maize

Aluminum toxicity is one of the major constraints for plant development in acid soils, limiting food production in many countries. Cultivars genetically adapted to acid soils may offer an environmental compatible solution, providing a sustainable agriculture system. The aim of this work was to identify genomic regions associated with Al tolerance in maize, and to quantify the genetic effects on the phenotypic variation. A population of 168 F-3:4 families derived from a cross between two contrasting maize inbred lines for Al tolerance was evaluated using the NSRL and RSRL parameters in nutrient solution containing toxic level of aluminum. Variance analyses indicated that the NSRL was the most reliable phenotypic index to measure Al tolerance in the population, being used for further QTL mapping analysis. RFLP and SSR markers were selected for bulked segregant analysis, and additional SSR markers, flanking the polymorphisms of interest, were chosen in order to saturate the putative target regions. Seven linkage groups were constructed using 17 RFLP and 34 SSR markers. Five QTLs were mapped on chromosomes 2, 6 and 8, explaining 60% of the phenotypic variation. QTL(4) and marker umc043 were located on chromosomes 8 and 5, close to genes encoding for enzymes involved in the organic acids synthesis pathways, a widely proposed mechanism for Al tolerance in plants. QTL(2) was mapped in the same region as Alm2, also associated with Al tolerance in maize. In addition, dominant and additive effects were important in the control of this trait in maize.

Molecular mapping of two loci that confer resistance to Asian rust in soybean

Asian soybean rust (ASR) is caused by the fungal pathogen Phakopsora pachyrhizi Sydow & Sydow. It was first identified in Brazil in 2001 and quickly infected soybean areas in several countries in South America. Primary efforts to combat this disease must involve the development of resistant cultivars. Four distinct genes that confer resistance against ASR have been reported: Rpp1, Rpp2, Rpp3, and Rpp4. However, no cultivar carrying any of those resistance loci has been released. The main objective of this study was to genetically map Rpp2 and Rpp4 resistance genes. Two F(2:3) populations, derived from the crosses between the resistant lines PI 230970 (Rpp2), PI 459025 (Rpp4) and the susceptible cultivar BRS 184, were used in this study. The mapping populations and parental lines were inoculated with a field isolate of P. pachyrhizi and evaluated for lesion type as resistant (RB lesions) or susceptible (TAN lesions). The mapping populations were screened with SSR markers, using the bulk segregant analysis (BSA) to expedite the identification of linked markers. Both resistance genes showed an expected segregation ratio for a dominant trait. This study allowed mapping Rpp2 and Rpp4 loci on the linkage groups J and G, respectively. The associated markers will be of great value on marker assisted selection for this trait.

The population genetic structure of Rhizoctonia solani AG-3PT from potato in the Colombian Andes

The soilborne fungus Rhizoctonia solani anastomosis group 3 (AG-3PT) is a globally important potato pathogen. However, little is known about the population genetic processes affecting field populations of R. solani AG-3PT, especially in the South American Colombian Andes, which is near the center of diversity of the two most common groups of cultivated potato, Solanum tuberosum and S. phureja. We analyzed the genetic structure of 15 populations of R. solani AG-3PT infecting potato in Colombia using 11 simple-sequence repeat (SSR) markers. In total, 288 different multilocus genotypes were identified among 349 fungal isolates. Clonal fractions within field populations were 7 to 33%. R ST statistics indicated a very low level of population differentiation overall, consistent with high contemporary gene flow, though moderate differentiation was found for the most distant southern populations. Genotype flow was also detected, with the most common genotype found widely distributed among field populations. All populations showed evidence of a mixed reproductive mode, including both asexual and sexual reproduction, but two populations displayed evidence of inbreeding. © 2013 The American Phytopathological Society.

Diversidade fenotípica e molecular de cultivares brasileiras de soja portadoras de gene RR

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV