Genome-Wide Association Study Identifies Genomic Regions for Important Morpho-Agronomic Traits in Mesoamerican Common Bean.

The population growth trend in recent decades has resulted in continuing efforts to guarantee food security in which leguminous plants, such as the common bean (Phaseolus vulgaris L.), play a particularly important role as they are relatively cheap and have high nutritional value. To meet this demand for food, the main target for genetic improvement programs is to increase productivity, which is a complex quantitative trait influenced by many component traits. This research aims to identify Quantitative Trait Nucleotides (QTNs) associated with productivity and its components using multi-locus genome-wide association studies. Ten morpho-agronomic traits [plant height (PH), first pod insertion height (FPIH), number of nodules (NN), pod length (PL), total number of pods per plant (NPP), number of locules per pod (LP), number of seeds per pod (SP), total seed weight per plant (TSW), 100-seed weight (W100), and grain yield (YLD)] were evaluated in four environments for 178 Mesoamerican common bean domesticated accessions belonging to the Brazilian Diversity Panel. In order to identify stable QTNs, only those identified by multiple methods (mrMLM, FASTmrMLM, pLARmEB, and ISIS EM-BLASSO) or in multiple environments were selected. Among the identified QTNs, 64 were detected at least thrice by different methods or in different environments, and 39 showed significant phenotypic differences between their corresponding alleles. The alleles that positively increased the corresponding traits, except PH (for which lower values are desired), were considered favorable alleles. The most influenced trait by the accumulation of favorable alleles was PH, showing a 51.7% reduction, while NN, TSW, YLD, FPIH, and NPP increased between 18 and 34%. Identifying QTNs in several environments (four environments and overall adjusted mean) and by multiple methods reinforces the reliability of the associations obtained and the importance of conducting these studies in multiple environments. Using these QTNs through molecular techniques for genetic improvement, such as marker-assisted selection or genomic selection, can be a strategy to increase common bean production.

Genome-wide association study for grain mineral content in a Brazilian common bean diversity panel.

KEY MESSAGE: QTNs significantly associated to nine mineral content in grains of common bean were identified. The accumulation of favorable alleles was associated with a gradually increasing nutrient content in the grain. Biofortification is one of the strategies developed to address malnutrition in developing countries, the aim of which is to improve the nutritional content of crops. The common bean (Phaseolus vulgaris L.), a staple food in several African and Latin American countries, has excellent nutritional attributes and is considered a strong candidate for biofortification. The objective of this study was to identify genomic regions associated with nutritional content in common bean grains using 178 Mesoamerican accessions belonging to a Brazilian Diversity Panel (BDP) and 25,011 good-quality single nucleotide polymorphisms. The BDP was phenotyped in three environments for nine nutrients (phosphorus, potassium, calcium, magnesium, copper, manganese, sulfur, zinc, and iron) using four genome-wide association multi-locus methods. To obtain more accurate results, only quantitative trait nucleotides (QTNs) that showed repeatability (i.e., those detected at least twice using different methods or environments) were considered. Forty-eight QTNs detected for the nine minerals showed repeatability and were considered reliable. Pleiotropic QTNs and overlapping genomic regions surrounding the QTNs were identified, demonstrating the possible association between the deposition mechanisms of different nutrients in grains. The accumulation of favorable alleles in the same accession was associated with a gradually increasing nutrient content in the grain. The BDP proved to be a valuable source for association studies. The investigation of different methods and environments showed the reliability of markers associated with minerals. The loci identified in this study will potentially contribute to the improvement of Mesoamerican common beans, particularly carioca and black beans, the main groups consumed in Brazil.

Population structure, genetic diversity and genomic selection signatures among a Brazilian common bean germplasm.

Brazil is the world's largest producer of common bean. Knowledge of the genetic diversity and relatedness of accessions adapted to Brazilian conditions is of great importance for the conservation of germplasm and for directing breeding programs aimed at the development of new cultivars. In this context, the objective of this study was to analyze the genetic diversity, population structure, and linkage disequilibrium (LD) of a diversity panel consisting of 219 common bean accessions, most of which belonging to the Mesoamerican gene pool. Genotyping by sequencing (GBS) of these accessions allowed the identification of 49,817 SNPs with minor allele frequency > 0.05. Of these, 17,149 and 12,876 were exclusive to the Mesoamerican and Andean pools, respectively, and 11,805 SNPs could differentiate the two gene pools. Further the separation according to the gene pool, bayesian analysis of the population structure showed a subdivision of the Mesoamerican accessions based on the origin and color of the seed tegument. LD analysis revealed the occurrence of long linkage blocks and low LD decay with physical distance between SNPs (LD half decay in 249 kb, corrected for population structure and relatedness). The GBS technique could effectively characterize the Brazilian common bean germplasms, and the diversity panel used in this study may be of great use in future genome-wide association studies.

Diversity of nutritional content in seeds of Brazilian common bean germplasm.

Mineral deficiency is worldwide one of the major problems associated with human health, and biofortification through breeding is considered an important strategy to improve the nutritional content of staple food in countries that face this problem. The assessment of genetic variability for seed nutrient contents is a first step in the development of a biofortified crop. From the germplasm bank IDR-IAPAR-EMATER, a set of 1,512 common bean accessions, consisting of local and commercial varieties and improved lines, was analyzed. High variability among the accessions was observed for all evaluated nutrient contents (P, K, Ca, Mg, Cu, Zn, Mn, Fe and S and protein). In the mean, the contents of the carioca and black market groups (Mesoamerican gene pool), were around 7% higher for the minerals Ca, Cu, Mn and Fe and between 2-4% higher for P, K, Mg and Zn than in the other groups with Mesoamerican and Andean common bean. Few differences were observed among the Mesoamerican accessions that belong to the carioca and black commercial groups. Wide variability was observed among the evaluated genotypes, and the concentrations of the best accessions exceeded the overall mean by 14-28%. Due to the high variability in the evaluated accessions, these results may contribute to the selection of promising parents for the establishment of mating blocks. The nutritional contents of many of the improved lines evaluated in this study were higher than those of the commercial cultivars, indicating the possibility of developing new biofortified cultivars.

Development and characterization of microsatellite loci for Campomanesia xanthocarpa (Myrtaceae) and cross amplification in related species

Campomanesia xanthocarpa is a native tree, of common occurrence in almost all Brazilian Forest formations, which has its fruits and timber with high commercial value. Using an enriched genomic library we isolated and characterized microsatellite loci for C. xanthocarpa (Myrtaceae), in order to estimate genetic diversity parameters for this and related species. Twenty-eight microsatellite loci were identified and ten of them successfully amplified and showed polymorphism in a sample of 96 individuals, from four natural populations. The number of alleles per locus ranged from two to eight, and the observed and expected heterozygosities varied from 0.042 to 1.000 and from 0.294 to 0.855, respectively. These markers were tested and validated in two related species (C. eugenioides and C. guazumifolia). The microsatellite markers will be used in further studies of population genetics of C. xanthocarpa, in order to understand the genetic variability and to define the strategies needed for the conservation of the species.

Distinctness of Brazilian common bean cultivars with carioca and black grain by means of morphoagronomic and molecular descriptors.

Common bean (Phaseolus vulgaris L.) is one of the most important legumes for human consumption and is a staple food in the diet of the population of some countries of Latin America, Africa and Asia. The distinction between cultivars is based predominantly on morphological descriptors, which proved inefficient for the differentiation of some cultivars. This study had the objective of describing, distinguishing and evaluating the agronomic potential of 39 common bean cultivars of the carioca and black grain groups registered for cultivation in Brazil, based on 49 morphoagronomic descriptors and microsatellite (simple sequence repeat -SSR) markers. The morphoagronomic traits of each commercial group were characterized in four environments. Thirty-seven SSR markers were used for the molecular description. The morphological data, analyzed by the Shanonon-Weaver index, detected low variability among cultivars for qualitative data. On the other hand, the estimates of variance analysis, relative importance of the traits and hierarchical grouping analysis applied to the quantitative variables showed that the descriptors related to plant morphology were the most important for the carioca group, and those related to seed morphology were determining for the black group. The genetic parameters estimated for SSR markers by hierarchical and Bayesian cluster analysis identified 116 alleles, with 33 and 30 polymorphic loci and 24 and 22 private alleles for the carioca and black groups, respectively. The combined use of morphoagronomic and molecular descriptors improves the distinguishability of cultivars, contributing in a more efficient way to breeding and to the protection of cultivars.

Genetic variability of three natural populations of Maytenus aquifolium (Celesteraceae) from Telêmaco Borba, Paraná, Brazil

Three populations of Maytenus aquifolium from Monte Alegre farm, Telemaco Borba county, Paraná, Brazil were analyzed by RAPD markers. A total of 13 primers were applied wich yielded 283 well amplified markers in all the studied populations (Mortandade, Vila Preta and Trinita), producing different values of gene diversity and polymorphic loci. The analysis of molecular variance (AMOVA) indicated that 21.77 percent of the genetic variation was among the population. Pairwise F ST analysis showed that the most divergent populations were closer geographically, demonstrating that other factors such as different soil types could explain this variation. Bayesian analysis for K number of clusters and the Principal Coordinate indicated that these three populations were highly structured, corroborating the high values found for the F ST and indicating that for conservation purposes all populations should be maintained.

Três populações de Maytenus aquifolium coletadas na fazenda Monte Alegre, localizada no município de Telêmaco Borba, Paraná, Brasil foram analisadas por marcadores de RAPD. Treze primers de RAPD foram amplificados produzindo 283 bandas com alta capacidade de repetição nas três populações estudadas (Mortandade, Trinita e Vila Preta), com as quais valores diferentes de diversidade gênica e locos poplimórficos foram calculados. Análise da variância molecular (AMOVA) mostrou que 21,77 por cento da variabilidade genética é encontrada entre populações. Análise do Fst entre pares de populações mostrou que as mais divergentes são geograficamente mais próximas, demonstrando que outros fatores tais como tipos distintos de solos podem explicar estas variações. Análise Bayesiana para número K de clusters e a Análise da Coordenada Principal mostraram que estas três populações estão altamente estruturadas, corroborando os altos valores encontrados para o Fst e indicando que para propósitos de conservação todas as populações devem ser mantidas.

Genetic analysis of species in the Genus Catasetum (ORCHIDACEAE) using RAPD Markers

In this work, RAPD molecular markers were used to access the genetic variability and to study the inter and intraespecifc relationship in a group of 37 species, including 56 individuals. A total of 15 RAPD primers were selected for DNA amplification. From a total of 221 bands analyzed, 209 (95 percent) were polymorphics. The level of interespecifc genetic similarity ranged from 37 percent between Catasetum complanatum and Catasetum laminatum to 83 percent between Catasetum triodon and Catasetum uncatum. The intraspecifc genetic similarity varied 88 percent for the individuals of Catasetum triodon to 93 percent between the individuals of Catasetum atratum and Catasetum macrocarpum. These results would contribute to understand the genetic relationship in Catasetum, to define the strategies to establish a germplasm core collection for the genus and to provide support for breeding programs.

Neste trabalho, marcadores moleculares de RAPD foram utilizados para acessar a variabilidade genética e estudar as relações interespecíficas e intraespecífica em um grupo de 37 espécies, compreendendo 56 plantas individuais. Um total de 15 primers foram selecionados para amplificação do DNA. De um total de 221 bandas analisadas, 209 (95 por cento) foram polimórficas. O nível de similaridade genética interespecífica variou de 37 por cento entre Catasetum complanatum e Catasetum laminatums a 83 por cento entre Catasetum triodon e Catasetum uncatum. A similaridade genética intraespecífica variou de 88 por cento entre os indivíduos de Catasetum triodon a 93 por cento entre os indivíduos de Catasetum atratum e Catasetum macrocarpum. Os resultados deste trabalho contribuem para o entendimento das relações interespecíficas no gênero Catasetum, para definir estratégias para o estabelecimento de um banco de germoplasma e para dar suporte a programas de melhoramento.

Progênies de Coffea arabica cv. IPR 100 com resistência ao nematóide Meloidogyne incognita raça 1 / Progenies of Coffea arabica cv. IPR 100 with resistance to nematode Meloidogyne incognita race 1

Objetivou-se, neste trabalho, identificar progênies de Coffea arabica cv. IPR 100 com resistência ao nematóide Meloidogyne incognita raça 1. O experimento foi conduzido em casa de vegetação (Londrina, PR, Brasil) no delineamento em blocos ao acaso com 3 repetições e parcelas com 15 plantas. Foram avaliadas seis progênies da cultivar IPR 100 e a cultivar Mundo Novo IAC 376-4 foi utilizada como padrão suscetível. Foram inoculados 500 ovos por planta, totalizando 7500 ovos por parcela de 150 cm². Foi avaliado o número de galhas e massas de ovos presentes nas raízes. As seis progênies da cultivar IPR 100 foram mais resistentes ao M.incognita raça 1 do que o padrão suscetível.

The objective of this study was to identify progenies of Coffea arabica cv. IPR 100 with resistance to nematode Meloidogyne incognita race 1. The experiment was performed in greenhouse (Londrina, PR, Brazil) with an experimental design of randomized blocks with 3 replications and 15 plants per plot. Six progenies of cultivar IPR 100 were evaluated, and Mundo Novo IAC 376-4 cultivar was used as susceptible control. Five hundred eggs per plant were inoculated, totalizing 7500 eggs per plot of 150 cm². The number of galls and egg masses in the roots were evaluated. All progenies of cultivar IPR 100 presented more resistance to M.incognita race 1 than the susceptible control.

Comparative analysis of genetic diversity among the maize inbred lines (Zea mays L.) obtained by RAPD and SSR markers

The RAPD and SSR markers were used to compare the genetic diversity among the 16 maize inbred lines. Twenty-two primers were used in the RAPD reactions, resulting in the amplification of 265 fragments, while 16 pairs of SSR primers resulted in 75 fragments. The similarity based on Dice coefficient for the RAPD ranged from 53 to 84 percent and for the SSR from 11 to 82 percent. The dendrogram obtained by the RAPD showed five groups, while dendrogram obtained by the SSR showed three groups and one isolated line. The association constructed from the markers and the principal coordinate’s analysis separated lines into two groups according to endosperm color, either orange or yellow. The RAPD were effective to validate pedigree data, while the SSR were effective to recognize the differences between the quantitative characters. Because they assess the distinct regions of the genome, the selection of one or other marker would depend on the characteristics of the material used and the objectives of the project.

RAPD e SSR foram utlizados para comparar a diversidade genética entre 16 linhagens de milho. Nas reações de RAPD foram utlizados 22 primers que resultaram na amplificação de 265 fragmentos, enquanto que 16 pares de primes de SSR resultaram em 75 fragmentos. A similaridade baseada no coeficiente de Dice variou de 53 por cento a 84 por cento para o RAPD; para o SSR variou de 11 por cento a 82 por cento. O dendrograma obtido a partir do RAPD mostrou 5 grupos enquanto que o dendrograma obtido a partir do SSR mostrou 3 grupos e uma linhagem isolada. A associação construída a partir dos marcadores e a análise de coordenadas principais separaram as linhagens em dois grupos de acordo coloração de endosperma alaranjado ou amarelo, os marcadores RAPD foram eficientes para a validação dos dados de pedigree enquanto os de microssatélites para reconhecerem diferenças entre caracteres quantitativos. Por acessarem regiões distintas do genoma a escolha de um ou outro marcador vai depender das características do material utilizado e dos objetivos do trabalho.

Genetic control of orange hilum corona of carioca beans (Phaseolus vulgaris)

The purpose of this research was to elucidate the genetic control of orange corona color in carioca common beans (Phaseolus vulgaris). We made four crosses between carioca group cultivars that differed in respect to the presence or absence of an orange hilum corona color. The F2, F3, F1BC11, F1BC21, F2BC11 and F2BC21 phenotypic segregations were evaluated with a chi-square test which fitted with the hypothesis that one gene with a dominant allele is responsible for the orange corona color. All generations resulting from the four different crosses showed segregation patterns which agreed with the expected proportions. Our results show that the dominant G allele controls orange corona color in the carioca bean group.

Genetic diversity among forty coffee varieties assessed by RAPD markers associated with restriction digestion

A variabilidade genética de 40 acessos de cafeeiros de fenótipo arabica foi obtida usando a técnica de RAPD associada a uma digestão prévia do DNA genômico com endonucleases. A variabilidade genética e a relação entre os accessos foram inicialmente avaliadas pela amplificação de 195 primers. Para incrementar a eficiência na detecção de polimorfismo, o DNA genômico de cada acessos foi submetido a digestão com endonucleases antes da PCR. Um total de 24 primers combinados com restrição do DNA gerou 318 bandas, das quais 266 (83,65%) foram polimórficas. A associação entre os 40 acessos foi estimada pelo método de clusters UPGMA, sendo os acessos agrupados de acordo com seu pedigree e aspectos agronômicos. Os resultados mostraram que o uso de enzimas de restrição antes da reação de amplificação pode ser considerada uma ferramenta eficiente para incrementar o número de bandas informativas, possibilitando a diferenciação entre os 40 acessos de C. arabica.

Genetic variability of pre and post-fragmentation cohorts of Aspidosperma polyneuron Muell. Arg. (Apocynaceae)

O método de RAPD foi usado para acessar a variabilidade genética em Aspisdosperma polyneuron, uma árvore tropical de vida longa e idade reprodutiva tardia, e muito importante na Floresta Atlântica. Amostras foram coletadas em seis fragmentos florestais na região da cidade de Londrina (Sul do Brasil), uma paisagem fragmentada na década de 30. O perfil de RAPD foi analisado em adultos (pré-fragmentação, >300 anos) e plântulas (pós-fragmentação, <<50 anos). Os resultados mostram uma queda no polimorfismo genético em gerações pós-fragmentação nos pequenos fragmentos e alta diversidade genética dentro das populações. A distribuição da diversidade genética sugere o estabelecimento dos fragmentos como reservas protegidas, e a transferência de plântulas entre os fragmentos para a conservação de A. polyneuron.

Chromosomal organization and phylogenetic relationships in Hypochaeris species (Asteraceae) from Brazil

The association of cytogenetic and molecular techniques has contributed to the analysis of chromosome organization and phylogeny in plants. The fluorochrome GC-specific CMA3, fluorescent in situ hybridization (FISH) and RAPD (Random Amplified Polymorphic DNA) markers were used to investigate chromosome structure and genetic relationships in Hypochaeris (Asteraceae). Seven species native to South America, and two species introduced from Europe (H. glabra and Hypochaeris sp) were studied. FISH with rDNA probes identified one or two loci of 18S-5.8S-25S rDNA in the South American Hypochaeris species and one locus in the European species. Only one 5S rDNA locus was seen in all species studied. Blocks of GC-rich heterochromatin (CMA-positive bands) associated to 18S-5.8S-25SrDNA loci were detected in all species investigated. Co-location of 5S rDNA and CMA bands was also observed, except for three South American species and Hypochaeris sp. In two South American species, additional CMA bands not related to rDNA were observed on the long arm of chromosome 2, near to the centromere. Hypochaeris glabra exhibited additional CMA-positive signals distributed at pericentromeric regions, on the short arms of all chromosomes. A total of 122 RAPD markers were used to determine the genetic relationships among species. The level of polymorphism was very high, revealing two genetic groups comprising the South American and the European species, thus supporting a previous hypothesis of monophyly of the South American Hypochaeris species. The coefficients of genetic similarity between European and South American species were 0.35, on average. Polymorphism was also high within the two groups. The genetic associations observed with RAPD markers were consistent with chromosome characteristics. Species carrying similar distribution of 45S rDNA loci and CMA-positive signals were included in the same group revealed by RAPDs. Cytogenetic and molecular data support the view that not only chromosome rearrangements, but also changes in DNA sequence took place during the diversification of the South American Hypochaeris species.

Genetic polymorphism among 14 elite Coffea arabica L. cultivars using RAPD markers associated with restriction digestion

Knowledge of the genetic variability among genotypes is important for the transfer of useful genes and to maximize the use of available germplasm resources. This study was carried out to assess the genetic variability of 14 elite Coffea arabica cultivars using random amplified polymorphic DNA (RAPD) associated with a prior digestion of genomic DNA with restriction endonucleases. The accessions were obtained from the Coffea collection maintained at the Instituto Agronomico do Parana (IAPAR), located in Londrina, Parana, Brazil. Twenty-four informative RAPD primers, used in association with restriction enzymes, yielded 330 reproducible and scorable DNA bands, of which 224 (68 per cent) were polymorphic. The amplified products were used to estimate the genetic variability using Dice's similarity coefficient. The data matrix was converted to a dendrogram and a three-dimensional plot using principal coordinate analysis. The accessions studied were separated into clusters in a manner that was consistent with the known pedigree. The associations obtained in the dendrogram and in the principal coordinate analysis plot suggest the probable origin of the Kattimor cultivar. The RAPD technique associated with restriction digestion was proved to be a useful tool for genetic characterization of C. arabica genotypes making an important contribution to the application of molecular markers to coffee breeding

Assessment of genetic variability within and among coffee progenies and cultivars using RAPD markers

The RAPD technique associated with restriction digestion of genomic DNA was used to assess the genetic variability within and among nine populations of Coffea arabica, including six progenies belonging to the Sarchimor germplasm, the progeny PR 77054-40-10 (Catuaí Vermelho IAC 81 x Icatu), and two commercial cultivars (IAPAR 59 and Catuaí Vermelho IAC-81). These populations were evaluated using analysis of molecular variance (AMOVA), genetic similarity among progenies, and percentage of polymorphic loci. A total of 99 RAPD markers were evaluated of which 67 (67.67 percent) were polymorphic. AMOVA showed that 38.5 percent and 61.5 percent of the genetic variation was distributed among and within populations, respectively. The fixation index (F ST) of the genotypes was 0.385. The mean genetic variability estimated within populations ranged from 15.58 (IAPAR 59) to 8.27 (Catuaí Vermelho IAC 81). A distinct level of genetic variability was revealed for each of the coffee progenies and varieties studied. The methodology used in this investigation was useful to determine the genetic variability within and among C. arabica L. populations providing significant information for coffee breeding