Genetic divergence of soybean genotypes in relation to grain components / Divergência genética de genótipos de soja quanto a componentes do grão

ABSTRACT: The objective of this paper was to estimate the genetic divergence among 49 soybean ( Glycine max L. Merrill .) genotypes to assist grain quality-focused breeding programs in the choice of progenitors. The genetic divergence was estimated using the Mahalanobis generalized distance from the percentages of protein, oil, and fatty acids oleic, linoleic and linolenic after cultivation of genotypes in different environments. Genotypes were grouped by agglomerative methods and the two and three-dimensional projections of the distance matrix were obtained. The average protein and oil contents in the four environments ranged from 34.25 to 45.18% and from 16.48 to 23.01%, respectively. The average contents of the fatty acids oleic, linoleic and linolenic ranged from 20.2 to 42.41%, from 44.17 to 63.18%, and from 5.89 to 10.39%, respectively. The genetic distances ranged from 0.11 to 251.02 and indicated genetic variability among the accessions. The most divergent pair of accessions was PI417360/CD01RR8384, followed by PI417360/B3PTA213-3-4 and PI417360/BARC-8. The most similar par of accessions was CS3032PTA276-1-2/CS3032PTA190-5-1, followed by UFV18/M-SOY8914 and BRSMG Garantia/CD983321RR. In this study we indicated as promising in terms of genetic variability the hybridizations involving BARC-8, CD2013PTA, CD01RR8384, CS303TNKCA, PI181544, and PI417360. Among these genotypes we can stand out BARC-8 and CD2013PTA, with protein contents above 43%, and CD01RR8384 and CS303TNKCA, with oil contents above 20%. The use of these genetically divergent genotypes and with high phenotypic means in future crosses should produce desirable recombinants for grain quality.

RESUMO: O objetivo deste trabalho foi estimar a divergência genética entre 49 genótipos de soja ( Glycinemax L. Merrill.), visando auxiliar programas de melhoramento voltados à qualidade do grão na escolha de progenitores. A divergência genética foi estimada por meio da distância generalizada de Mahalanobis a partir dos percentuais de proteína, óleo e ácidos graxos oleico, linoleico e linolênico após cultivo dos genótipos em diferentes ambientes. Os genótipos foram agrupados por métodos aglomerativos e as projeções bi e tridimensional da matriz de distância foram obtidas. Os teores médios de proteína e óleo nos quatro ambientes variaram de 34,25 a 45,18% e de 16,48 a 23,01%, respectivamente. Os teores médios dos ácidos graxos oleico, linoleico e linolênico variaram de 20,2 a 42,41%, de 44,17 a 63,18% e de 5,89 a 10,39%, respectivamente. As distâncias genéticas variaram de 0,11 a 251,02 e indicaram variabilidade genética entre os acessos. O par de acessos mais divergente foi PI417360/CD01RR8384, seguido por PI417360/B3PTA213-3-4 e PI417360/BARC-8. Já o par de acessos mais similar foi CS3032PTA276-1-2/CS3032PTA190-5-1, seguido por UFV18/M-SOY8914 e BRSMG Garantia/CD983321RR. Neste estudo, são indicadas como promissoras em termos de variabilidade genética as hibridações envolvendo BARC-8, CD2013PTA, CD01RR8384, CS303TNKCA, PI181544 e PI417360. Dentre esses genótipos, destacam-se BARC-8 e CD2013PTA, com teores proteicos acima de 43%, e CD01RR8384 e CS303TNKCA, com teores de óleo acima de 20%. A utilização desses genótipos geneticamente divergentes e com elevadas médias fenotípicas em futuros cruzamentos deverá produzir recombinantes desejáveis para a qualidade do grão.

Tratamento térmico adequado proporciona melhoria da qualidade nutricional de farinhas de soja elaboradas a partir de novos cultivares destinados à alimentação humana / Adequate heat treatment improves the nutritional quality of soybean flours from new cultivars for human consumption purpose

O efeito do calor seco sobre índices de urease, a solubilidade proteica e a desfosforilação de ácido fítico foram avaliados em grãos de diferentes cultivares de soja. Os grãos foram submetidos à temperatura de 130ºC, 150ºCe 170ºC por 30 minutos e, posteriormente, triturados. Foram realizadas as análises de urease, da solubilidade proteica e dos teores de hexafosfato, pentafosfato, tetrafosfato e trifosfato de inositol. O tratamento a 130ºC foi insuficiente para inativar a atividade da urease e a temperatura de 170°C ultrapassou o limite adequado; os valores desejáveis foram obtidas a 150°C. A solubilidade da proteína diferiu entre as temperaturas aplicadas (p< 0,05). O processamento térmico sob temperatura de 150°C indicou ser adequado por apresentar valores de solubilidade proteica entre 70% a 85%. O teor de hexafosfato de inositol foi 46% menor no cultivar convencional. Ocorreu redução média de 37,7% de IP6 no tratamento dos grãos de soja a 150ºC, quando comparado a de130ºC. O tratamento térmico a 150ºC por 30 minutos foi o melhor para processar a soja, proporcionar adequada solubilidade proteica na redução da atividade de urease, além de reduzir a concentração de inositóis hexa e pentafosfatos, sem afetar a concentração dos inositóis tetra e trifosfato.

The present work analyzed the effect of dry heat on grains from different soybean cultivars, and the indicesof urease, protein solubility and dephosphorylation of phytic acid were determined. The grains were exposedto temperatures at 130ºC, 150ºC and 170ºC for 30 minutes, and then powdered. Analyses of urease, proteinsolubility, and inositol hexaphosphate, pentaphosphate, tetrafhosphate and triphosphate were carried out.The treatment of grains at 130ºC was insufficient to inactivate the urease activity, while the temperatureat 170°C exceeded the appropriate limit; at 150°C desirable values were observed. The protein solubilitydiffered among the employed temperatures (p < 0.05). Heating processing at 130°C was not appropriate, butat 150°C indicated to be suitable temperature, as the protein solubility values were between 70% and 85%.Inositol hexaphosphate content was 46% lower in the conventional cultivar. An average reduction of 37.7%for the IP6 was observed in treating the soybean grains at 150ºC, in comparison with 130ºC. The heatingtreatment at 150ºC for 30 minutes was the most adequate for processing the soybean, and for providingappropriate protein solubility, reduced urease activity and reduced concentration of inositol penta- andhexaphosphates, without affecting the inositol tetra- and tri-phosphates concentrations.Key words. soybean, urease, protein solubility, phytic acid.

Método para mapeamento de locos controladores de características oligogênicas / Genetic mapping of oligogenic traits using maximum likehood functions

Características oligogênicas de distribuição discreta e expressão governada por poucos genes de maior efeito têm se mostrado importantes na condução dos programas de melhoramento, com destaque para a resposta de resistência das plantas às doenças. Métodos tradicionais de detecção de QTL's, que pressupõem normalidade e herança governada por múltiplos fatores, não deveriam ser utilizados para mapeamento dessas características de distribuição discreta e interação epistática predominante. O objetivo deste trabalho é avaliar os resultados de um método para mapeamento e detecção de locos controladores da expressão de características oligogênicas, OTL's (Oligogenic Trait Loci). Esse método, definido como MMCO (Método de Mapeamento de Características Oligogênicas), utiliza funções de verossimilhança para obtenção de estimativas de ligação fatorial entre locos marcadores e locos controladores de características oligogênicas. Os resultados indicam que o método foi adequado para detecção de OTL's em populações F2 relativamente pequenas, compostas por 200 indivíduos, e que a determinação a priori do padrão de herança é condição necessária para a utilização dessa estratégia, que se diferencia por atender as pressuposições de análise, não necessitar de informação prévia de ordenamento entre as marcas e por permitir a obtenção de estimativas a partir da informação contida em todas as classes genotípicas.

Oligogenic traits are distinguished by their heritage ruled by higher effect genes and by their importance for cultivated plants, with emphasis to the plant disease resistance inheritance. The qualitative nature and epistatic interaction of these traits results in a heritage pattern that should not be interpreted through traditional QTL detection strategies. The objective of this work was to propose a method for Oligogenic Traits Loci (OTL) detection. This method, defined as Oligogenic Trait Mapping Method (OTMM) uses maximum likehood probability functions to obtain adjusted "r" estimates that express the distance among the molecular markers and the OTL loci. The results show that the method was adequate for OTL detection even in relatively small F2 populations. The prior definition of the oligogenic heritage pattern is one the main requirements of this method that focus in the attainment of the analysis presumptions without previous markers order information.

Efeito do inibidor de protease Kunitz sobre níveis de lipoxigenases em sementes de soja / Effect of protease inhibitor on the levels of lipoxygenase in soybean seeds

As lipoxigenases (LOX) são responsáveis pelo "beany flavor", sabor característico da soja [Glycine max. (L.) Merrill], que é o maior obstáculo ao consumo da soja nos países ocidentais. O inibidor de tripsina Kunitz (KTI) é responsável por 80 por cento da inibição da atividade tríptica, além de provocar hiperplasia pancreática em animais monogástricos. Essas duas características são muito importantes em programas de melhoramento que visam à melhoria da qualidade da soja. Objetivou-se, neste trabalho verificar o efeito do inibidor de protease (KTI), sobre níveis de LOX em sementes de soja, nos seguintes genótipos: KTI+/LOX+, KTI+/LOX-, KTI-/LOX+ e KTI-/LOX-. As LOXs foram identificadas por teste colorimétrico, atividade enzimática e eletroforese em gel de poliacrilamida. O teor de proteína foi quantificado pelo método do ácido bicinconínico para determinação da atividade específica de LOX. Observou-se que, para o genótipo KTI+/LOX+, a atividade específica de LOX 1 foi alta, variando de 177,92 a 206,94 UA/mg de proteína e para LOX 3 variou de 17,90 a 19,85 UA/mg de proteína; para KTI+/LOX- e KTI-/LOX- não foi detectada atividade de LOX e que para o genótipo KTI-/LOX+ a atividade específica para LOX 1 variou de 117,24 a 124,64 UA/mg de proteína, e de 17,35 a 20,29 UA/mg de proteína para LOX 3. Houve uma redução de 40 por cento na atividade específica de LOX1 e de 1 por cento para LOX 3, no genótipo KTI-/LOX+, quando comparados ao KTI+/LOX+. Esses resultados comprovam uma associação negativa entre o inibidor de tripsina Kunitz e a atividade de lipoxigenases.

Lipoxygenases (LOX) are responsible for the beany flavor, which is the most important obstacle for the consumption of soybean [Glycine max. (L.) Merrill] products in Western countries. The Kunitz trypsin inhibitor (KTI) is responsible for 80 percent of the inhibition of tryptic activity in soybean seeds, and besides it can cause pancreatic hyperplasia in monogastric animals. These two traits are very important in soybean breeding programs that aim the improvement of soy protein quality. The objective of this study was to verify the influence of KTI on LOX levels in soybean seeds by analyzing the following genotypes: KTI+/LOX+, KTI+/LOX-, KTI-/LOX+ and KTI-/LOX-. LOX 1 and LOX 3 were identified by colorimetric, activity and electrophoretic assays. Protein content was determined by the bicinchoninic method for determination of LOX specific activity. For genotype KTI+/LOX+, LOX 1 specific activity was high varying from 177.92 to 206.94 AU/mg of protein and LOX 3 activity varied from 17.90 to 19.85 AU/mg of protein. No LOX activity was detected in genotypes KTI+/LOX- and KTI-/LOX-. For genotype KTI-/LOX+, LOX 1 activity varied from 117.24 to 124.64 AU/mg of protein, and the activity of LOX 3 from 17.35 to 20.29 AU/mg of protein. There was a reduction of 40 percent on LOX 1 specific activity and 1 percent for LOX 3 in the KTI-/LOX+ genotype in comparison to genotype KTI+/LOX+. These results indicate an inverse relationship between the presence of the Kunitz trypsin inhibitor in soybean seeds and lipoxygenase activity.

Simulation of population size and genome saturation level for genetic mapping of recombinant inbred lines (RILs)

Various population sizes and number of markers have been used to obtain genetic maps. However, the precise number of individuals and markers needed for obtaining reliable maps is not known. We used data simulation to determine the influence of population size, the effect of the degree of marker saturation of the genome, and the number of individuals required for mapping of recombinant inbred lines (RILs). Three genomes with 11 linkage groups were generated with saturation levels of 5, 10 and 20 cM. For each saturation level populations were generated with 50, 100, 154, 200, 300, 500 and 800 individuals with 100 replications for each population size. A total of 2100 populations was generated and mapped. Small marker numbers and small population sizes produced maps with more than 11 linkage groups. As population size and marker saturation increased, marker inversion and non-linked markers decreased, moreover, between-marker distance estimates were improved. In this study, a minimum size of 200, 300 and 500 individuals were necessary for obtaining reliable maps when they were evaluated over the saturation levels of 5, 10 and 20 cM, respectively.

Characterization and genetic diversity analysis of cotton cultivars using microsatellites

Genetic diversity and the relationship between varieties are of great importance for cotton breeding. Our work was designed to estimate the informativeness of the cotton (Gossypium hirsutum L.) simple sequence repeat (SSR) microsatellite locus and to estimate the genetic distance between 53 cotton cultivars as well as to select a set of SSR primers able to differentiate between the 53 cotton cultivars studied. After extracting DNA from the 53 cultivars and characterized it using 31 pairs of SSR primers we obtained a total of 66 alleles with an average of 2.13 alleles per SSR locus and values of polymorphism information content (PIC) varying from 0.18 to 0.62, the dissimilarity coefficient varying from zero to 0.41. Statistical analysis using the unweighted pair-group method using arithmetic average (UPGMA) revealed seven subgroups which were consistent with the genealogical information available for some of the cultivars. The SSR genetic profile obtained for each of the cultivars made it possible to discriminate 52 of the 53 cultivars. This study of the genetic diversity of cotton cultivars with SSR markers support the need to introduce new alleles into the gene pool of the breeding cultivars.

Multivariate analysis of combining ability for soybean resistance to Cercospora sojina Hara

Seven soybean cultivars (Bossier, Cristalina, Davis, Kent, Lincoln, Paraná and Uberaba), with different levels of resistance to Cercospora sojina, race 04, were crossed according to a diallel design, with no reciprocals, to determine the general and the specific combining abilities for the resistance. The evaluations of the reaction to the disease were performed 20 days after the inoculation of the fungus on the most infected leaflet of the plant, in the parents and in the F1 hybrids. To quantify the resistance, the following characteristics were evaluated: infection degree (ID); number of lesions per leaflet (NLL); lesion mean diameter (LMD); lesioned leaf area (LLA); percentage of lesioned leaf area (PLLA); number of lesions per square centimeter (NLC) and disease index (DI). The relative importance of each characteristic was evaluated by the canonical variables analysis and the LLA and NLL characteristics were eliminated from the multivariate function. With the remaining five characteristics, a multivariate index was created using the first canonical vector, which was submitted to the diallel analysis, according to Griffings fixed model, method 2. The most important characters to discriminate resistant from susceptible soybean plants to C. sojina were: ID, LMD, NLC, DI and PLLA. Cristalina, Davis and Uberaba cultivars are the best ones among those tested that can be recommended as parents in soybean breeding programs seeking resistance to Cercospora sojina. The additive, dominant and epistatic genetic effects were important for the expression of the resistance, although the additive genetic effect was the most important component.

Genetic distance and its association with heterosis in cacao

The efficiency of cacao breeding program can be increased by choosing superior crosses to be made between divergent clones. We assessed the genetic distance among five clones with RAPD data (genetic distance - GD) and with yield component data (Mahalanobis distance - MD). The clones were evaluated in a diallel, during five years, for five yield components. A total of 130 RAPD bands were scored. GD and MD were used to determine the correlation between genetic distances among clones and the performance of their hybrids. The correlation between GD and MD was 0.67 (P=0.03). Both distances were related to heterotic performance of hybrids for wet seed weight/plant and wet seed weight/fruit. The average hybrid performance for the same two yield components was correlated with only MD. Hence, genetic distances measured by RAPD and yield components can be used as a guide to the choice of the superior crosses

Biometrical analyses of linolenic acid content of soybean seeds

The genetic reduction of linolenic acid levels increases the quality and stability of soybean oil. The objective of this study was to determine the inheritance and evaluate the nature and magnitude of gene effects on soybean seed linolenic acid level. Means and variances of F-1, F-2, and F-3 generations were made from the cross between accession BARC-12 (low linolenic acid content) and the commercial Brazilian cultivar CAC-1 (normal linolenic acid content). The results demonstrated that linolenic acid content in soybean is under the genetic control of a small number of genes. The additive model explained the means for the three generations and for the parents. Non-allelic gene interactions had little effect on the determination of genotypic values for the individuals. The generation means and population variation analyses demonstrated that the dominance deviations contribute little to the trait. These results showed that backcross breeding programs can be used to introduce the low linolenic acid content trait into soybean seeds, since it is possible to identify with very high accuracy the desired genotypes in segregating populations

RAPD and SCAR markers linked to resistance to frogeye leaf spot in soybean

The soybean (Glycine max (L.) Merrill) frogeye leaf spot is caused by the fungus Cercospora sojina Hara and is a widespread disease in Brazil and other countries, causing severe losses in grain yield and also affecting seed quality. The availability of DNA markers linked to genes for resistance to this disease would accelerate breeding programs, particularly when other traits are also being evaluated. Bulked segregant analysis was applied to 3 F2 populations derived from crosses between the resistant cultivars Parana, Cristalina and Uberaba, and the susceptible cultivar Bossier. In the cross 'Parana' x 'Bossier', 2 RAPD markers were identified, CSOPA1800C and CSOPA21,250C, located at 4.4 1.8 centiMorgans (cM) and 3.4 1.7 cM respectively from the resistance locus. DNA fragments of similar molecular weight were observed in the population derived from the cross 'Cristalina' x 'Bossier' at 2.3 1.2 and 4.7 1.5 cM from the resistance locus, respectively. In the offspring of the cross 'Uberaba' x 'Bossier', a DNA fragment corresponding to marker CSOPA1800C was detected at 5.6 2.1 cM from the resistance locus. Although marker CSOPA21,250C was not observed in this population, an additional marker was detected (CSOUB11,100C) at 6.7 2.2 cM from the resistance locus. The 1,250 bp fragment of CSOPA21,250C was cloned and converted into a SCAR marker, which amplified a single fragment whose size corresponded to the cloned segment of the crosses involving cultivars Cristalina and Parana. Markers CSOPA1800C, CSOPA21,250C and CSOUB11,100C were mapped to soybean linkage group J with the aid of known SSRs linked to the Rcs3 locus, indicating that the RAPD and SCAR markers identified in our research also tag this resistance gene

Use of the random amplified polymorphic DNA technique to characterize soybean (Glycine max (L.) Merrill)genotypes

We have started a breeding program to genetically eliminate the lipoxygenase isozymes (LOX) from soybean seeds. These enzymes are believed to be the main cause of off-flavors in soybean products. LOX are present in the seed as three isozymes encoded by three different genes, which are inherited in a simple mendelian fashion. Mutants lacking each one of these isozymes have been identified in the world germplasm. To introduce these null alleles into the Brazilian variety Cristalina, three mutant progenitors were chosen: PI 408.251 (LOX1 minus), PI 86.023 (LOX2 minus), and Ichigowase (LOX3 minus). The random amplified polymorphic DNA (RAPD) technique was used to charactetize these progenitors, as well as lines lacking LOX1 (CR1), LOX3 (CR3), LOX1 and 3 (CR1,3), and LOX2 and 3 (UFV 91-263, UFV 91-401 and UFV 91-717). The results enabled us to establish the fingerprint of each genotype and the genetic distances among them.

ELISA na determinaçäo quantitativa e análise genética nas lipoxigenases L1 e L2 em soja / ELISA in the quantification and genetic analysis of lipoxygenases L1 and L2 from soybeans

A soybean breeding program has been undertaken at the Federal University of Viçosa aiming to reduce lipoxygenase lelvels ib the seeds of commercial varieties. A direct ELISA (Enzyme Linked Immunosorbent Assay) procedure has been developed to quantify lipoxygenases L1 and L2 in soybean seeds. Polyclonal antibodies were reised in rabbits against pure L1 and L2 and purified further by affinity chromatography. The best conditions to perform the analaysis were determined as foloow: coating antibody dilution 1:100, crude soybean protein extract 1:2 x 105 and conjugate 1:100 for both L1 and L2 isozymes. The method proved to be sensible enough to identify homozygous and heterozygous genotypes in the breeding program performing single non destructive analysis of seeds or even cotileldones right after germination. Besides, L1 was quantified in about 120 soybean Brazilian cultivars an L2 in some of them. The results showed a significant variability in the level of lipoxygenase-1 among the cultivars analysed. With respect to L2, it was found that the level of this isozyme was significantly greater in the seeds than observed for L1, in spite of its lower "in vitro" specific activity