Enhancing the knowledge on the genetic basis of germination and heterotrophic growth at extreme t... more Enhancing the knowledge on the genetic basis of germination and heterotrophic growth at extreme temperatures is of major importance for improving crop establishment. A quantitative trait loci (QTL) analysis was carried out at sub- and supra-optimal temperatures at these early stages in the model Legume Medicago truncatula. On the basis of an ecophysiological model framework, two populations of recombinant inbred lines were chosen for the contrasting behaviours of parental lines: LR5 at sub-optimal temperatures (5 or 10°C) and LR4 at a supra-optimal temperature (20°C). Seed masses were measured in all lines. For LR5, germination rates and hypocotyl growth were measured by hand, whereas for LR4, imbibition and germination rates as well as early embryonic axis growth were measured using an automated image capture and analysis device. QTLs were found for all traits. The phenotyping framework we defined for measuring variables, distinguished stages and enabled identification of distinct ...
Ten populations of the model plant Arabidopsis thaliana were collected along a northsouth gradien... more Ten populations of the model plant Arabidopsis thaliana were collected along a northsouth gradient in Norway and screened for microsatellite polymorphisms in 25 loci and variability in quantitative traits. Overall, the average levels of genetic diversity were found to be relatively high in these populations, compared to previously published surveys of within population variability. Six of the populations were polymorphic at microsatellite loci, resulting in an overall proportion of polymorphic loci of 18%, and a relatively high gene diversity for a selfing species ( H E = 0.06). Of the overall variability, 12% was found within populations. Two of six polymorphic populations contained heterozygous individuals. Both F ST and phylogenetic analyses showed no correlation between geographical and genetic distances. Haplotypic diversity patterns suggested postglacial colonization of Scandinavia from a number of different sources. Heritable variation was observed for many of the studied quantitative traits, with all populations showing variability in at least some traits, even populations with no microsatellite variability. There was a positive association between variability in quantitative traits and microsatellites within populations. Several quantitative traits exhibited Q ST values significantly less than F ST , suggesting that selection may be acting to retard differentiation for these traits.
The genetic diversity in the red clover USA core collection was measured by RAPD and isozyme mark... more The genetic diversity in the red clover USA core collection was measured by RAPD and isozyme markers, comparing different approaches of isozyme data analysis and comparing the results of RAPD with those of isozymes. A total of 15 isozyme loci and 114 RAPD fragments were analyzed. Genetic diversity measured with RAPD and isozymes was high. The analyses of isozymes as binary data was highly correlated with the allele-frequency approach. There was no correlation between the distance matrices of different markers. Isozyme data grouped the accessions of red clover in four groups. The variation evidenced by AMOVA was higher among and within groups in the populations than at the intra-population level. Results showed the high diversity in the red clover core collection and indicated some populations that could be used in breeding programs of the crop in Brazil.
Red clover is one of the most utilized forage in agriculture and contains many of the isoflavones... more Red clover is one of the most utilized forage in agriculture and contains many of the isoflavones known for their human health benefits. The objectives of this study were: i) to quantify, using HPLC analysis, isoflavones in 77 accessions from the USDA core collection and a Brazilian line; ii) to verify possible relationships depending on their origin, improvement status or maturity type and; iii) to verify the seasonal variation. The isoflavone mean contents were 29.27 µg g -1 of dry material for daidzein, 163.69 µg g -1 for genistein, 11353.29 µg g -1 for formononetin and 6568.8 µg g -1 for biochanin A. Clustering was mainly influenced by the total amount of isoflavones and partially due to maturity type, improvement status and geographic origin. The seasonal evaluation demonstrated an increase of concentration during winter, and decrease during spring. These results highlighted accessions that can be used to develop new varieties with low or high isoflavones concentration.
Enhancing the knowledge on the genetic basis of germination and heterotrophic growth at extreme t... more Enhancing the knowledge on the genetic basis of germination and heterotrophic growth at extreme temperatures is of major importance for improving crop establishment. A quantitative trait loci (QTL) analysis was carried out at sub-and supra-optimal temperatures at these early stages in the model Legume Medicago truncatula. On the basis of an ecophysiological model framework, two populations of recombinant inbred lines were chosen for the contrasting behaviours of parental lines: LR5 at suboptimal temperatures (5 or 10°C) and LR4 at a supraoptimal temperature (20°C). Seed masses were measured in all lines. For LR5, germination rates and hypocotyl growth were measured by hand, whereas for LR4, imbibition and germination rates as well as early embryonic axis growth were measured using an automated image capture and analysis device. QTLs were found for all traits. The phenotyping framework we defined for measuring variables, distinguished stages and enabled identification of distinct QTLs for seed mass (chromosomes 1, 5, 7 and 8), imbibition (chromosome 4), germination (chromosomes 3, 5, 7 and 8) and heterotrophic growth (chromosomes 1, 2, 3 and 8). The three QTL identified for hypocotyl length at suboptimal temperature explained the largest part of the phenotypic variation (60% together). One digenic interaction was found for hypocotyl width at sub-optimal temperature and the loci involved were linked to additive QTLs for hypocotyl elongation at low temperature. Together with working on a model plant, this approach facilitated the identification of genes specific to each stage that could provide reliable markers for assisting selection and improving crop establishment. With this aim in view, an initial set of putative candidate genes was identified in the light of the role of abscissic acid/gibberellin balance in regulating germination at high temperatures (e.g. ABI4, ABI5), the molecular cascade in response to cold stress (e.g. CBF1, ICE1) and hypotheses on changes in cell elongation (e.g. GASA1, AtEXPA11) with changes in temperatures based on studies at the whole plant scale.
Plant Genetic Resources: characterization and utilization, 2004
The 17 Brazilian species of Adesmia DC were analysed, using 20 primers, with regard to their rand... more The 17 Brazilian species of Adesmia DC were analysed, using 20 primers, with regard to their randomly amplified polymorphic DNA (RAPD) patterns. From a total of 357 individuals analysed, the 20 primers produced 2249 fragments with molecular sizes ranging from 200 to 2700 bp, 56% of which were polymorphic. Average intra-population genetic similarity, estimated by Jaccard's coefficient, ranged from 0.35 in A. araujoi to 0.80 in A. punctata. Mean intra-specific genetic similarity varied greatly among species, ranging from 0.19 for A. tristis to 0.89 for A. arillata. Mean genetic similarity among the species, estimated by Dice's coefficient, was 0.56. RAPD markers were efficient at separating all the accessions analysed. The results obtained generally agreed with the partition of genetic variability expected according to the mode of reproduction.
Ten populations of the model plant Arabidopsis thaliana were collected along a northsouth gradien... more Ten populations of the model plant Arabidopsis thaliana were collected along a northsouth gradient in Norway and screened for microsatellite polymorphisms in 25 loci and variability in quantitative traits. Overall, the average levels of genetic diversity were found to be relatively high in these populations, compared to previously published surveys of within population variability. Six of the populations were polymorphic at microsatellite loci, resulting in an overall proportion of polymorphic loci of 18%, and a relatively high gene diversity for a selfing species ( H E = 0.06). Of the overall variability, 12% was found within populations. Two of six polymorphic populations contained heterozygous individuals. Both F ST and phylogenetic analyses showed no correlation between geographical and genetic distances. Haplotypic diversity patterns suggested postglacial colonization of Scandinavia from a number of different sources. Heritable variation was observed for many of the studied quantitative traits, with all populations showing variability in at least some traits, even populations with no microsatellite variability. There was a positive association between variability in quantitative traits and microsatellites within populations. Several quantitative traits exhibited Q ST values significantly less than F ST , suggesting that selection may be acting to retard differentiation for these traits.
The NPGS-USDA core collection with 85 accessions of red clover, an important forage species, is l... more The NPGS-USDA core collection with 85 accessions of red clover, an important forage species, is little described. The goal of the present study was to evaluate the diversity of a set of accessions from the core collection at the morphological and molecular level in order to extract some valuable accessions for Brazilian red clover breeding programs. Twentyone morphological traits, collected in field and greenhouse in South Brazil, and seven SSR markers were used to describe 57 accessions from the U.S. core collection and one population cultivated in Southern Brazil. Variation between accessions was large for most of the 21 morphological traits. A cluster analysis based on the morphological traits revealed five distinct clusters that separated the populations according to flowering earliness, as already described, but also according to persistency, growth habit and dry matter productivity. Over seven SSR loci, the number of alleles averaged 11.1 alleles per locus. Genetic diversity measured with SSR markers was high, with a mean expected heterozygosity of 0.86. An analysis of molecular variance revealed that the largest proportion of variation (83.6%) resided at the within population level. Although the molecular markers also separated accessions into five clusters, there was no coincidence between the composition of groups found with morphological and molecular data. Use of genetic diversity in breeding programs requires to use the most promising populations, to combine positive traits such as persistency and forage yield, and probably to use within population variation to detect valuable genotypes that could be used as parents of synthetic varieties.
Red clover (Trifolium pratense L.) is an important forage plant that contains the isoflavones dai... more Red clover (Trifolium pratense L.) is an important forage plant that contains the isoflavones daidzein, genistein, formononetin, and biochanin A. These compounds have been studied lately due to their human health benefits. The aim of this study was to develop and validate an HPLC method with simplified sample preparation to quantify daidzein, genistein, formononetin and biochanin A simultaneously in red clover leaves. The validation showed that the method is specific, accurate, precise and robust, not to mention that the sample preparation is easier and faster than those described earlier. The response was linear over a range of 0.01-0.2 lg mL À1 for daidzein, 0.05-0.5 lg mL À1 for genistein, 4-40 lg mL À1 for formononetin and 2-20 lg mL À1 for biochanin A. The range of recoveries was 85.6-101.0%. The RSD for intra-and inter-day precision were <2.54 and <7.22%, respectively. Five populations of red clover, from the National Plant Germplasm System-USDA were analyzed and the content of daidzein, genistein, formononetin and biochanin A ranged from 7. 87-91.31, 51.60-131.30, 6568.33-23461.82, to 2499.55-10337.33 lg g À1 of dried material, respectively.
Enhancing the knowledge on the genetic basis of germination and heterotrophic growth at extreme t... more Enhancing the knowledge on the genetic basis of germination and heterotrophic growth at extreme temperatures is of major importance for improving crop establishment. A quantitative trait loci (QTL) analysis was carried out at sub- and supra-optimal temperatures at these early stages in the model Legume Medicago truncatula. On the basis of an ecophysiological model framework, two populations of recombinant inbred lines were chosen for the contrasting behaviours of parental lines: LR5 at sub-optimal temperatures (5 or 10°C) and LR4 at a supra-optimal temperature (20°C). Seed masses were measured in all lines. For LR5, germination rates and hypocotyl growth were measured by hand, whereas for LR4, imbibition and germination rates as well as early embryonic axis growth were measured using an automated image capture and analysis device. QTLs were found for all traits. The phenotyping framework we defined for measuring variables, distinguished stages and enabled identification of distinct ...
Ten populations of the model plant Arabidopsis thaliana were collected along a northsouth gradien... more Ten populations of the model plant Arabidopsis thaliana were collected along a northsouth gradient in Norway and screened for microsatellite polymorphisms in 25 loci and variability in quantitative traits. Overall, the average levels of genetic diversity were found to be relatively high in these populations, compared to previously published surveys of within population variability. Six of the populations were polymorphic at microsatellite loci, resulting in an overall proportion of polymorphic loci of 18%, and a relatively high gene diversity for a selfing species ( H E = 0.06). Of the overall variability, 12% was found within populations. Two of six polymorphic populations contained heterozygous individuals. Both F ST and phylogenetic analyses showed no correlation between geographical and genetic distances. Haplotypic diversity patterns suggested postglacial colonization of Scandinavia from a number of different sources. Heritable variation was observed for many of the studied quantitative traits, with all populations showing variability in at least some traits, even populations with no microsatellite variability. There was a positive association between variability in quantitative traits and microsatellites within populations. Several quantitative traits exhibited Q ST values significantly less than F ST , suggesting that selection may be acting to retard differentiation for these traits.
The genetic diversity in the red clover USA core collection was measured by RAPD and isozyme mark... more The genetic diversity in the red clover USA core collection was measured by RAPD and isozyme markers, comparing different approaches of isozyme data analysis and comparing the results of RAPD with those of isozymes. A total of 15 isozyme loci and 114 RAPD fragments were analyzed. Genetic diversity measured with RAPD and isozymes was high. The analyses of isozymes as binary data was highly correlated with the allele-frequency approach. There was no correlation between the distance matrices of different markers. Isozyme data grouped the accessions of red clover in four groups. The variation evidenced by AMOVA was higher among and within groups in the populations than at the intra-population level. Results showed the high diversity in the red clover core collection and indicated some populations that could be used in breeding programs of the crop in Brazil.
Red clover is one of the most utilized forage in agriculture and contains many of the isoflavones... more Red clover is one of the most utilized forage in agriculture and contains many of the isoflavones known for their human health benefits. The objectives of this study were: i) to quantify, using HPLC analysis, isoflavones in 77 accessions from the USDA core collection and a Brazilian line; ii) to verify possible relationships depending on their origin, improvement status or maturity type and; iii) to verify the seasonal variation. The isoflavone mean contents were 29.27 µg g -1 of dry material for daidzein, 163.69 µg g -1 for genistein, 11353.29 µg g -1 for formononetin and 6568.8 µg g -1 for biochanin A. Clustering was mainly influenced by the total amount of isoflavones and partially due to maturity type, improvement status and geographic origin. The seasonal evaluation demonstrated an increase of concentration during winter, and decrease during spring. These results highlighted accessions that can be used to develop new varieties with low or high isoflavones concentration.
Enhancing the knowledge on the genetic basis of germination and heterotrophic growth at extreme t... more Enhancing the knowledge on the genetic basis of germination and heterotrophic growth at extreme temperatures is of major importance for improving crop establishment. A quantitative trait loci (QTL) analysis was carried out at sub-and supra-optimal temperatures at these early stages in the model Legume Medicago truncatula. On the basis of an ecophysiological model framework, two populations of recombinant inbred lines were chosen for the contrasting behaviours of parental lines: LR5 at suboptimal temperatures (5 or 10°C) and LR4 at a supraoptimal temperature (20°C). Seed masses were measured in all lines. For LR5, germination rates and hypocotyl growth were measured by hand, whereas for LR4, imbibition and germination rates as well as early embryonic axis growth were measured using an automated image capture and analysis device. QTLs were found for all traits. The phenotyping framework we defined for measuring variables, distinguished stages and enabled identification of distinct QTLs for seed mass (chromosomes 1, 5, 7 and 8), imbibition (chromosome 4), germination (chromosomes 3, 5, 7 and 8) and heterotrophic growth (chromosomes 1, 2, 3 and 8). The three QTL identified for hypocotyl length at suboptimal temperature explained the largest part of the phenotypic variation (60% together). One digenic interaction was found for hypocotyl width at sub-optimal temperature and the loci involved were linked to additive QTLs for hypocotyl elongation at low temperature. Together with working on a model plant, this approach facilitated the identification of genes specific to each stage that could provide reliable markers for assisting selection and improving crop establishment. With this aim in view, an initial set of putative candidate genes was identified in the light of the role of abscissic acid/gibberellin balance in regulating germination at high temperatures (e.g. ABI4, ABI5), the molecular cascade in response to cold stress (e.g. CBF1, ICE1) and hypotheses on changes in cell elongation (e.g. GASA1, AtEXPA11) with changes in temperatures based on studies at the whole plant scale.
Plant Genetic Resources: characterization and utilization, 2004
The 17 Brazilian species of Adesmia DC were analysed, using 20 primers, with regard to their rand... more The 17 Brazilian species of Adesmia DC were analysed, using 20 primers, with regard to their randomly amplified polymorphic DNA (RAPD) patterns. From a total of 357 individuals analysed, the 20 primers produced 2249 fragments with molecular sizes ranging from 200 to 2700 bp, 56% of which were polymorphic. Average intra-population genetic similarity, estimated by Jaccard's coefficient, ranged from 0.35 in A. araujoi to 0.80 in A. punctata. Mean intra-specific genetic similarity varied greatly among species, ranging from 0.19 for A. tristis to 0.89 for A. arillata. Mean genetic similarity among the species, estimated by Dice's coefficient, was 0.56. RAPD markers were efficient at separating all the accessions analysed. The results obtained generally agreed with the partition of genetic variability expected according to the mode of reproduction.
Ten populations of the model plant Arabidopsis thaliana were collected along a northsouth gradien... more Ten populations of the model plant Arabidopsis thaliana were collected along a northsouth gradient in Norway and screened for microsatellite polymorphisms in 25 loci and variability in quantitative traits. Overall, the average levels of genetic diversity were found to be relatively high in these populations, compared to previously published surveys of within population variability. Six of the populations were polymorphic at microsatellite loci, resulting in an overall proportion of polymorphic loci of 18%, and a relatively high gene diversity for a selfing species ( H E = 0.06). Of the overall variability, 12% was found within populations. Two of six polymorphic populations contained heterozygous individuals. Both F ST and phylogenetic analyses showed no correlation between geographical and genetic distances. Haplotypic diversity patterns suggested postglacial colonization of Scandinavia from a number of different sources. Heritable variation was observed for many of the studied quantitative traits, with all populations showing variability in at least some traits, even populations with no microsatellite variability. There was a positive association between variability in quantitative traits and microsatellites within populations. Several quantitative traits exhibited Q ST values significantly less than F ST , suggesting that selection may be acting to retard differentiation for these traits.
The NPGS-USDA core collection with 85 accessions of red clover, an important forage species, is l... more The NPGS-USDA core collection with 85 accessions of red clover, an important forage species, is little described. The goal of the present study was to evaluate the diversity of a set of accessions from the core collection at the morphological and molecular level in order to extract some valuable accessions for Brazilian red clover breeding programs. Twentyone morphological traits, collected in field and greenhouse in South Brazil, and seven SSR markers were used to describe 57 accessions from the U.S. core collection and one population cultivated in Southern Brazil. Variation between accessions was large for most of the 21 morphological traits. A cluster analysis based on the morphological traits revealed five distinct clusters that separated the populations according to flowering earliness, as already described, but also according to persistency, growth habit and dry matter productivity. Over seven SSR loci, the number of alleles averaged 11.1 alleles per locus. Genetic diversity measured with SSR markers was high, with a mean expected heterozygosity of 0.86. An analysis of molecular variance revealed that the largest proportion of variation (83.6%) resided at the within population level. Although the molecular markers also separated accessions into five clusters, there was no coincidence between the composition of groups found with morphological and molecular data. Use of genetic diversity in breeding programs requires to use the most promising populations, to combine positive traits such as persistency and forage yield, and probably to use within population variation to detect valuable genotypes that could be used as parents of synthetic varieties.
Red clover (Trifolium pratense L.) is an important forage plant that contains the isoflavones dai... more Red clover (Trifolium pratense L.) is an important forage plant that contains the isoflavones daidzein, genistein, formononetin, and biochanin A. These compounds have been studied lately due to their human health benefits. The aim of this study was to develop and validate an HPLC method with simplified sample preparation to quantify daidzein, genistein, formononetin and biochanin A simultaneously in red clover leaves. The validation showed that the method is specific, accurate, precise and robust, not to mention that the sample preparation is easier and faster than those described earlier. The response was linear over a range of 0.01-0.2 lg mL À1 for daidzein, 0.05-0.5 lg mL À1 for genistein, 4-40 lg mL À1 for formononetin and 2-20 lg mL À1 for biochanin A. The range of recoveries was 85.6-101.0%. The RSD for intra-and inter-day precision were <2.54 and <7.22%, respectively. Five populations of red clover, from the National Plant Germplasm System-USDA were analyzed and the content of daidzein, genistein, formononetin and biochanin A ranged from 7. 87-91.31, 51.60-131.30, 6568.33-23461.82, to 2499.55-10337.33 lg g À1 of dried material, respectively.
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Papers by Paula Dias