Microsatellite markers in candidate genes for wood properties and its application in functional diversity assessment in Eucalyptus globulus

Background: Functional genetic markers have important implications for genetic analysis by providing direct estimation of functional diversity. Although high throughput sequencing techniques for functional diversity analysis are being developed nowadays, the use of already well established variable markers present in candidate genes is still an interesting alternative for mapping purposes and functional diversity studies. SSR markers are routinely used in most plant and animal breeding programs for many species including Eucalyptus . SSR markers derived from candidate genes (SSR-CG) can be used effectively in co-segregation studies and marker-assisted diversity management. Results: In the present study, eight new non reported SSRs were identified in seven candidate genes for wood properties in Eucalyptus globulus : cinnamoyl CoA reductase (CCR), homocysteine S-methyltransferase (HMT), shikimate kinase (SK), xyloglucan endotransglycosylase 2 (XTH2), cellulose synthase 3 (CesA3), glutathione S-transferase (GST) and the transcription factor LIM1. Microsatellites were located in promoters, introns and exons, being most of them CT dinucleotide repeats. Genetic diversity of these eight CG-derived SSR-markers was explored in 54 unrelated genotypes. Except for XTH2, high levels of polymorphism were detected: 93 alleles (mean of 13.1 sd 1.6 alleles per locus), a mean effective number of alleles (Ne) of 5.4 (sd 1.6), polymorphic information content values (PIC) from 0.617 to 0.855 and probability of Identity (PI) ranging from 0.030 to 0.151. Conclusions: This is the first report on the identification, characterization and diversity analysis of microsatellite markers located inside wood quality candidate genes (CG) from Eucalyptus globulus. This set of markers is then appropriate for characterizing genetic variation, with potential usefulness for quantitative trait loci (QTL) mapping in different eucalypts genetic pedigrees and other applications such as fingerprinting and marker assisted diversity management

Caracterización molecular y cariotípica de la variabilidad genética de germoplasma argentino de Solanum L.

La papa cultivada se encuentra relacionada con un gran número de especies del género Solanum con las que frecuentemente se cruza. Por tal motivo, las aproximadamente 200 especies silvestres que producen tuberculo, constituyen un importante reservorio de genes para distintos propósitos de mejoramiento. Las mismas se encuentran distribuidas en un amplio rango de hábitats y nichos ecológicos, constituyendo el Noroeste argentino, una gran fuente de germoplasma. Es conocido que poseen gran variabilidad tanto dentro como entre especies. La información sobre el grado de relación genética que existe entre las distintas entradas, reviste gran interés para las aplicaciones al mejoramiento genético, organización de los bancos de germoplasma. identificación de cultivares, reducción del número de entradas necesarias para que alberguen en una pequeña muestra, la mayor variabilidad genetica posible, para el mejoramiento molecular, etc. La variabilidad genetica puede medirse utilizando distintas herramientas las que incluyen datos morfológicos, citológicos, bioquímicos y moleculares. El objetivo de este trabajo es evaluar la variabilidad genética de 67 entradas silvestres y cultivadas del género Solanum, coleccionadas en el Banco de Germoplasma de EEA INTA- Balcarce, aplicando metodologías citológicas y moleculares y contrastar los resultados obtenidos con la clasificación taxonómica y su hipótesis evolutiva. Se analiza la utilidad de estas herramientas y la información generada mediante los polimorfismos de restricción correspondientes a los ADNs ribosomales. Se evaluó mediante microdensitometría, el contenido de ADN de individuos pertenecientes a 14 especies silvestres del género Solanum con el fin de detectar diferencias en el valor C. A pesar de que la mayoria de las especies no mostró diferencias significativas entre ellas (dentro de los distintos grupos de ploidías), pequeñas diferencias fueron detectadas entre algunas de ellas. S. tarijense y S. kurzianum (2.3 pg) difirieron significativamente (0,1%) de S. Spegazzini, S. vernei, S. venturii y S. commersonii (1,9 pg); y S.. megistacrobolum (2,2 pg) difirió de S. spegazzinii (1,9 pg) dentro de los citotipos diploides. Entre los tetraploides el rango fue desde 3,6 pg (S. tuberosum ssp tuberosum cv. Baraka) hasta 4,7 pg (S. tuberosum ssp andigena y S. gourlayi) hallándose diferencias significativas al 0,1%. En cuanto a la variabilidad de los genes que codifican para el ARN ribosomal, se detectaron diferentes tamaños de las subunidades completas, tanto dentro como entre especies, y aún, dentro de una misma especie; sugiriendo una alta tasa de mutaciones localizada fundamentalmente el extremo 5' del espaciador externo. La pérdida del sitio EcoRI del extremo 3'de dicho espaciador en todas las entradas de S. venturii fue característica de dicha especie. El desarrollo de las nuevas metodologías ha expandido el rango de los ensayos para detectar polimorfismos de ADN con propósitos de "fingerprinting", distancias genéticas, etc. Se han desarrollado distintos marcadores moleculares (RFLP, AFLP, SSR etc) con el fin de estudiar las relaciones genéticas y se evaluó su utilidad en varios cultivos, incluidos la papa cultivada, S. tuberosum ssp tuberosumm. En este trabajo, se ensayaron 4 combinaciones de "primers" de AFLP, 15 sondas genómicas de RFLP y 7 pares de "primers" para SSR; para un grupo de 67 genotipos diploides y poliploides que pertenecen al mismo grupo de especies silvestres y cultivadas. Se compararon las asociaciones generadas mediante todas las técnicas y se las comparó con la clasificación taxonómica tradicional. Se calcularon los indices de contenido polimórfico promedios correspondiente a cada sistema (con valores aproximados de 0,25 para RFLP, 0,32 para AFLP y 0.58 para SSR), número de polimorfismos por ensayo y también se determinaron los alelos comunes entre las entradas diploides y poliploides, incluyendo al cultivar de papa. En términos generales, las relaciones entre las especies variaron según la metodología utilizada. Se observó una gran variabilidad dentro de especies, lo que dificultó el establecimiento de las relaciones entre especies. Como se esperaba, las entradas pertenecientes a una misma especie, en general se agruparon con mayor similitud que entre especies, independientemente de la técnica utilizada. También se detectaron asociaciones de individuos concordantes con la procedencia geográfica. Promediando todos los indices de similitud obtenidos con los tres métodos. la especie que tuvo menor variabilidad fue S. venturii (0,681) y la de mayor variabilidad S. gourlayii(0,411). Los mejores valores de correlaciones entre los distintos sistemas correspondieron a AFLP y SSR, en los dos grupos de ploidías, aunque los coeficientes de correlación entre las matrices de similitud obtenidas mediante las distintas herramientas no difirieron demasiado. Los valores de correlaciones en los citotipos diploides fueron desde 0,510 (RFLP vs SSR), 0,552 (RFLP vs AFLP), 0,640 (AFLP vs SSR). Considerando todos los citotipos independientemente de la ploidía, los valores oscilaron desde 0,441 (RFLP vs SSR), 0,505 (RFLP vs AFLP) a 0,629 (AFLP vs SSR). Debido a los altos valores obtenidos en los coeficientes de correlación cofenéticos (matrices de similitud vs. matrices cofenéticas, AFLP r=0.92; SSR r=0.85; RFLP r=0.78), las asociaciones entre los individuos pueden visualizarse directamente a partir del dendrograma. Los AFLP mostraron el mejor ajuste con la clasificación taxonómica, asociando dos especies que pertenecen a la misma serie Yungasensa (S. tarijense y S. chacoense), como así también agrupando más próximo al grupo de referencia externo tomate, a las entradas pertenecientes a S. commersonii, considerada como la especie más primitiva según la hipótesis propuesta por Hawkes, 1990. Esto quizás sea debido al gran número de bandas compartidas entre el germoplasma y a la alta cobertura genómica. En el caso de RFLP, se observaron algunos individuos dispersos, sugiriendo que sería necesario evaluar un mayor número de sondas. Los SSR en general se utilizan para obtener información dentro de especies. pero no entre especies. Pudo observarse que la mayoria de los loci evaluados produjeron amplificaciones, indicando la conservación de las regiones flanqueantes. Sin embargo, el número de repeticiones, no fue muy conservado entre las distintas especies, transformándolos en inapropiados para establecer relaciones entre especies, pero si muy útiles dentro de especies. El análisis reverso de los datos, permitió la detección de bandas caracteristicas de especies y también de individuos. Casi todas las especies pudieron discriminarse con estas bandas, y muchos de los individuos mostraron bandas únicas. Se evaluaron asimismo el número de bandas novedosas respecto del cultivar Huinkul MAG, en especies como en individuos particulares, pudiendo identificarse aquellas más distantes genéticamente, que serían potencialmente interesantes para aumentar las bases genéticas de los cultivares comerciales. También se determinó es grado de homocigosis de los distintos genotipos diploides, merced al uso de los marcadores de tipo codominantes (RFLP y SSR) mostrando un mínimo de homocigosis de 26% en S. goniocalyx megistacrolobum Oka 3787.Cultivated potato is related to a large number of Solanum sp and can easily be hybridized. This makes that the two hundred wild and cultivated tuber-bearing species of the genus Solanum form a valuable gene reservoir for different breeding purposes. They are distributed in a wide variety of habitats and niches (North-West Argentina is a source of a great number of these species), there is high genetic variation within and between species. Information on genetic relationships among accessions has several important applications for crop improvement: organizing germplasm banks, identifying cultivars, reducing the number of accessions needed to ensure a representative sample of broad range of genetic variability, molecular breeding, etc. Genetic variability can be assessed in different ways including the comparison of morphological, cytological, biochemical and molecular data. The objective of this work is to evaluate genetic variability of 67 wild and cultivated Solanum accessions belonging to the germplasm bank of EEA INTA Balcarce applying cytologic and molecular tools. The usefulness of this tools are compared with traditional taxonomy and its evolutive hypothesis. Ribosomal DNAcoding sequences are explored trough different restriction polymorphisms. Different subunits sizes were detected within and between species, and also in one specie, showing a high mutation rate mainly in the 5' end of the external spacer. All accessions of S. venturi showed the lost of an EcoRI restriction site located in the 3' end of the external intergenic spacer. DNA content of individuals belonging to 14 wild Solanum species was measured by microdensitometry to determine differences in their C value. Although most species did not show significant differences between them, slight differences could be detected between some of the species. Both S. tarijense and S. kurzianum (2.3 pg) differ from S. spegazzinii, S. vernei, S. venturii and S. commersonii (1,9 pg); and S. megistacrolobum (2,2 pg) differ from S. spegazzini (1,9 pg) at diploid level. Tetraploids ranged from 3.6 pg (S. tuberosum ssp tuberosum cv. Baraka) were found at 0.1% level. New technological developments have expanded the range of DNA polymorphism assays for genome fingerprinting, genetic relatedness, etc. Different molecular markers (RFLP, AFLP, SSR, etc.) has been developed to study genetic relationships and their usefulness was compared in many crop species including cultivated potato Solanum tuberosum ssp tuberosum In this work, 67 genotypes (diploids as well as tetraploids) belonging to the same group of wild and cultivated species, were assayed with, 15 RFLP genomic probes, 4 AFLP primers combinations and 7 microsatellite primers pairs. Associations generated by each technique were compared as well as how they fit with traditional taxonomy classification. Estimates of diversity indexes for each system (RFLP=O,25, O,32=AFLP and O,58=SSR), number of polymorphisms per assay as well as identification of common alleles between diploid and tetraploid accessions including cultivated potato were calculated. In general terms, quantification of relatedness between species varied depending on the technique applied. It was observed a very high level of variation within species, making more difficult to establish relationships between species. As expected, accessions of the same species grouped at a higher level of similarity than with the rest of the germplasm, independently of the molecular tool used for evaluation. Genotypes belonging to the same geographic origin, were also grouped. S. gourlayí showed to be the most variable specie calculated on the bases of the three methodologies with the with the smallest average similarity index (0,411) and the less variable was S. venturii with the highest average similarity index (0,681). Similarity indexes calculated on the bases of all three different methodologies were correlationed and r values were 0.510 (RFLP vs SSR), 0,552 (RFLP vs AFLP), 0,640 (AFLP vs SSR) for diploid species. Taking into account all citotypes, r values ranged from 0.441 (RFLP vs SSR), 0,505 (RFLP vs AFLP) a 0,629 (AFLP vs SSR). As similarity matrixes are comparable with cophenetic matrixes (AFLP r=0.92; SSR r=0.85; RFLP r=0.78), this allows us to roughly visualize the associations between accessions directly on dendograms. AFLP showed the best fit to taxonomic classification grouping two species (S. tarijense and S. chacoense) which belong to the same taxonomical serie Jungasensa, althogeter; also S. commersonii which is considered to be the most primitive taxon of this group (Hawkes. 1990) was grouped near the outgroup tomato. This might be due to the higher proportion of shared bands respect to the other and the relative better coverage of genomes. In the case of RFLP, some accessions were dispersed, suggesting that more probes need to be analyzed. SSR are usually used to obtain information within species and not between species, it was observed that the loci were conserved since amplification was possible in many of the genotypes analyzed. However, microsatellite loci showed very little conservation of alleles in different species (as expected for this very mutable loci) making them not appropriate for relatedness quantification when comparing different species while they keep their usefulness within species. Reverse analysis of data allowed the detection of specie specific bands as well as individuals ones. Nearly all of the species were able to be discriminated and most of single individuals had unique bands. Novel bands not found in potato cultivar Huinkul MAG were also computed in all species and in individuals allowing the knowledge of the more distant ones, which might be interesting for increasing genetic basis of commercial cultivars. Homozygous status of diploids genotypes were also estimated using codominant markers (RFLP and SSR) being of 36% for S. spegazzinii Oka 6108 and 64% for S. goniocalyx y S. megistacrolobums Oka 3787.Fil:Marcucci Poltri, Susana N.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Transferability of microsatellite markers located in candidate genes for wood properties between Eucalyptus species

<p><em>Aim of study:  </em>To analyze the feasibility of extrapolating conclusions on wood quality genetic control between different <em>Eucalyptus</em> species, particularly from species with better genomic information, to those less characterized. For this purpose, the first step is to analyze the conservation and cross-transferability of microsatellites markers (SSRs) located in candidate genes.</p><p><em>Area of study</em>: <em>Eucalyptus</em> species implanted in Argentina coming from different Australian origins.</p><p><em>Materials and methods: </em>Twelve validated and polymorphic SSRs in candidate genes (SSR-CGs) for wood quality in <em>E. globulus</em> were selected for cross species amplification in six species: <em>E. grandis, E. saligna, E. dunnii, E. viminalis, E. camaldulensis </em>and <em>E. tereticornis</em>.</p><p><em>Main results: </em>High cross-species transferability (92% to 100%) was found for the 12 polymorphic SSRs detected in <em>E. globulus.</em> These markers revealed allelic diversity in nine important candidate genes: cinnamoyl CoA reductase (CCR), cellulose synthase 3 (CesA3), the transcription factor LIM1, homocysteine S-methyltransferase (HMT), shikimate kinase (SK), xyloglucan endotransglycosylase 2 (XTH2), glutathione S-transferase (GST), glutamate decarboxylase (GAD) and peroxidase (PER).</p><p><em>Research highlights: </em>The<em> </em>markers described are potentially suitable for comparative QTL mapping, molecular marker assisted breeding (MAB) and for population genetic studies across different species within the subgenus <em>Symphyomyrtus</em>.</p><strong>Keywords</strong>: validation; cross-transferability; SSR; functional markers; eucalypts;<em> Symphyomyrtus.</em

Impacts of population structure and analytical models in genome-wide association studies of complex traits in forest trees: a case study in Eucalyptus globulus.

The promise of association genetics to identify genes or genomic regions controlling complex traits has generated a flurry of interest. Such phenotype-genotype associations could be useful to accelerate tree breeding cycles, increase precision and selection intensity for late expressing, low heritability traits. However, the prospects of association genetics in highly heterozygous undomesticated forest trees can be severely impacted by the presence of cryptic population and pedigree structure. To investigate how to better account for this, we compared the GLM and five combinations of the Unified Mixed Model ( UMM ) on data of a low-density genome-wide association study for growth and wood property traits carried out in a Eucalyptus globulus population (n = 303) with 7,680 Diversity Array Technology (DArT) markers. Model comparisons were based on the degree of deviation from the uniform distribution and estimates of the mean square differences between the observed and expected p-values of all significant marker-trait associations detected. Our analysis revealed the presence of population and family structure. There was not a single best model for all traits. Striking differences in detection power and accuracy were observed among the different models especially when population structure was not accounted for. The UMM method was the best and produced superior results when compared to GLM for all traits. Following stringent correction for false discoveries, 18 marker-trait associations were detected, 16 for tree diameter growth and two for lignin monomer composition (S:G ratio), a key wood property trait. The two DArT markers associated with S:G ratio on chromosome 10, physically map within 1 Mbp of the ferulate 5-hydroxylase (F5H) gene, providing a putative independent validation of this marker-trait association. This study details the merit of collectively integrate population structure and relatedness in association analyses in undomesticated, highly heterozygous forest trees, and provides additional insights into the nature of complex quantitative traits in Eucalyptus

New validated Eucalyptus SSR markers located in candidate genes involved in growth and plant development

Aim of study: To validate and characterize new microsatellites or Simple Sequence Repeats (SSR) markers, located within genomic transcribed sequences related to growth and plant developmental traits, in Eucalyptus species. Area of study: Eucalyptus species from different Australian origins planted in Argentina. Materials and methods: In total, 134 SSR in 129 candidate genes (CG-SSR) involved in plant development were selected and physically mapped to the E. grandis reference genome by bioinformatic tools. Experimental validation and polymorphism analysis were performed on 48 individuals from E. grandis and interspecific hybrids (E. grandis x E. camaldulensis; E. grandis x E. tereticornis), E. globulus, E. maidenii, E. dunnii and E. benthamii. Main results: 131 out of 134 CG-SSR were mapped on the 11 chromosomes of E. grandis reference genome. Most of the 134 analyzed SSR (> 75%) were positively amplified and 39 were polymorphic in at least one species. A search of annotated genes within a 25 kbp up and downstream region of each SSR location retrieved 773 genes of interest. Research highlights: The new validated and characterized CG-SSR are potentially suitable for comparative QTL mapping, molecular marker-assisted breeding (MAB) and population genetic studies across different species within Symphyomyrtus subgenus

Mean squared differences (MSD) between observed and expected <i>p</i>-values for the six association genetics models applied in the study.

<p>The selected models are highlighted in bold (see text for models' and traits' abbreviations).</p

Plots of the observed vs. expected <i>p-values</i> for the six association genetics models studied for all traits.

<p>See text for models' and traits' abbreviation.</p