Genetic diversity of high performance cultivars of upland and irrigated Brazilian rice.

The objective of this study was to analyze the diversity and discrimination of high-performance Brazilian rice cultivars using microsatellite markers. Twenty-nine rice cultivars belonging to EMBRAPA Arroz e Feijão germplasm bank in Brazil were genotyped by 24 SSR markers to establish their structure and genetic discrimination. It was demonstrated that the analyzed germplasm of rice presents an expressive and significant genetic diversity with low heterogeneity among the cultivars. All 29 cultivars were differentiated genetically, and were organized into two groups related to their upland and irrigated cultivation systems. These groups showed a high genetic differentiation, with greater diversity within the group that includes the cultivars for irrigated system. The genotyping data of these cultivars, with the morphological e phenotypical data, are valuable information to be used by rice breeding programs to develop new improved cultivars.

Expression of drought tolerance genes in tropical upland rice cultivars (Oryza sativa).

Gene expression related to drought response in the leaf tissues of two Brazilian upland cultivars, the drought-tolerant Douradão and the drought-sensitive Primavera, was analyzed. RNA-seq identified 27,618 transcripts in the Douradão cultivar, with 24,090 (87.2%) homologous to the rice database, and 27,221 transcripts in the Primavera cultivar, with 23,663 (86.9%) homologous to the rice database. Gene-expression analysis between control and water-deficient treatments revealed 493 and 1154 differentially expressed genes in Douradão and Primavera cultivars, respectively. Genes exclusively expressed under drought were identified for Douradão, including two genes of particular interest coding for the protein peroxidase precursor, which is involved in three distinct metabolic pathways. Comparisons between the two drought-exposed cultivars revealed 2314 genes were differentially expressed (978 upregulated, 1336 downregulated in Douradão). Six genes distributed across 4 different transcription factor families (bHLH, MYB, NAC, and WRKY) were identified, all of which were upregulated in Douradão compared to Primavera during drought. Most of the genes identified in Douradão activate metabolic pathways responsible for production of secondary metabolites and genes coding for enzymatically active signaling receptors. Quantitative PCR validation showed that most gene expression was in agreement with computational prediction of these transcripts. The transcripts identified here will define molecular markers for identification of Cis-acting elements to search for allelic variants of these genes through analysis of polymorphic SNPs in GenBank accessions of upland rice, aiming to develop cultivars with the best combination of these alleles, resulting in materials with high yield potential in the event of drought during the reproductive phase.

High-throughput transformation pipeline for a Brazilian japonica rice with bar gene selection.

The goal of this work was to establish a transformation pipeline for upland Curinga rice (Oryza sativa L. ssp. japonica) with bar gene selection employing bialaphos and phosphinothricin as selection agents. The following genes of interest: AtNCED3, Lsi1, GLU2, LEW2, PLD-alpha, DA1, TOR, AVP1, and Rubisco were cloned into the binary vector p7i2x-Ubi and were transferred into Agrobacterium strain EHA 105. Embryogenic calli derived from the mature embryos were transformed, and transgenic cells and shoots were selected on the medium supplemented with bialaphos or phosphinothricin (PPT) using a stepwise selection scheme. Molecular analyses were established using polymerase chain reaction and Southern blot for the bar gene and the NOS terminator. Overall, 273 putative transgenic plants were analyzed by Southern blot with 134 events identified. In total, 77 events had a single copy of the transgene integrated in the plant genome while 29 events had two copies. We tested backbone integration in 101 transgenic plants from all constructs and found 60 transgenic plants having no additional sequence integrated in the plant genome. The bar gene activity was evaluated by the chlorophenol red test and the leaf painting test using phosphinothricin with several transgenic plants. The majority of T0 plants carrying the single copy of transgene produced T1 seeds in the screen house.

Discrimination of common bean cultivars using multiplexed microsatellite markers.

Analysis of DNA polymorphisms allows for the genetic identification and precise discrimination of species with a narrow genetic base such as common bean. The primary objectives of the present study were to molecularly characterize commercial common bean varieties developed at various research institutions using microsatellite markers and to determine the degree of genetic diversity among the bean varieties analyzed. Fifty cultivars representing 12 grain classes and 64 genitors, i.e., accessions used to develop these cultivars, were characterized. Based on an analysis of 24 simple sequence repeats, the estimates for the average number of alleles and genetic diversity were 8.29 and 0.646, respectively. The combined probability of identity was estimated at 7.05 x 10(-17), indicating a high individual discriminatory power. Thirty-two percent of the cultivars exhibited heterogeneity for multiple loci that reflected either homozygosity for different alleles of a given locus in different individuals or heterozygosity for the locus. The average genetic diversity for the groups of cultivars and genitors was 0.605 and 0.660, respectively, with no genetic differentiation (FST) between these groups. Although similar estimates of expected heterozygosity were observed when the cultivars were grouped by release date, a greater number of private alleles was observed in the most recent cultivars. The genetic differentiation among cultivars originating from different institutions was not different from zero (FST = 0.01). The molecular profile database derived from these analyses may increase the statistical power of genetic estimates and may be incorporated into breeding programs for common bean. Furthermore, the profiles obtained for the different cultivars may be used as molecular descriptors to complement traditional descriptors used in distinctiveness, uniformity and stability tests, thereby improving the traceability of samples and their derivatives and helping to protect the intellectual property rights of breeders.

Molecular characterization of high performance inbred lines of Brazilian common beans.

The identification of germplasm genetic variability in breeding programs of the common bean (Phaseolus vulgaris) is essential for determining the potential of each combination of parent plants to obtain superior genotypes. The present study aimed to estimated the extent of genetic diversity in 172 lineages and cultivars of the common bean by integrating five tests of value for cultivation and use (VCU) that were conducted over the last eight years by the breeding program of Embrapa Arroz e Feijão in Brazil. Nine multilocus genotyping systems composed of 36 fluorescent microsatellite markers distributed across 11 different chromosomes of the common bean were used, of which 24 were polymorphic in all trials. One hundred and eighty-seven alleles were identified, with an average of 7.79 alleles per locus and an average gene diversity of 0.65. The combined probability of identity for all loci was 1.32 x 10(-16). Lineages that are more genetically divergent between the selection cycles were identified, allowing the breeding program to develop a crossbreed between elite genotypes with a low degree of genetic relatedness. HE values ranged from 0.31 to 0.63, with a large reduction in the genetic base over successive selection cycles. The test showed a significant degree of differentiation (FST = 0.159). Private alleles (26%) were identified and can be directly incorporated into the gene pool of cultivated germplasm, thereby contributing effectively to the expansion of genetic diversity in this bean-breeding program.

Backcross assisted by microsatellite markers in common bean.

The objectives of the present study were to monitor the effect of backcrossing through microsatellite markers and to compare different marker assisted selection strategies. Four populations were developed using donor parents resistant to the bean golden mosaic virus and, for all crosses, only individuals resistant to the bean golden mosaic virus were backcrossed. For crosses ARC100-4 x DOR303 and ARC100-4 x PHAS8328, assisted selection was carried out in the F(2) and F(2)BC(1) generations, while in the remaining crosses selection was performed only in the F(2)BC(1) generation. For the microsatellite analysis, in each generation, 20 markers were genotyped. The molecular data were analyzed using the NTSys program and the proportion of the recurrent genome introgressed was estimated, based on genotypical configuration of the segregant populations compared to the recurrent parents. The results indicate a higher efficiency in recovering the genotype of the elite genitor through the strategy of backcross assisted selection in the successive generations, and demonstrate a practical and useful application of molecular marker technology associated with bean breeding, to reduce the number of backcrosses and the time to recover the genome of the recurrent genitors.

Agronomic and molecular characterization of introgression lines from the interspecific cross Oryza sativa (BG90-2) x Oryza glumaepatula (RS-16).

The reduced genetic variability of modern rice varieties (Oryza sativa) is of concern because it reduces the possibilities of genetic gain in breeding programs. Introgression lines (ILs) containing genomic fragments from wild rice can be used to obtain new improved cultivars. The objective of the present study was to perform the agronomic and molecular characterizations of 35 BC2F8 ILs from the cross O. glumaepatula x O. sativa, aiming to select high-yielding ILs to be used in rice-breeding programs. All 35 ILs were field evaluated in the season 2002/2003 in three locations and the 15 best performing ones were evaluated in the season 2003/2004 in five locations. In 2003/2004, six ILs (CNAi 9934, CNAi 9931, CNAi 9930, CNAi 9935, CNAi 9936, and CNAi 9937) showed the highest yield means and were statistically superior to the controls Metica 1 and IRGA 417. Molecular characterization of the 35 ILs was performed with 92 microsatellite markers distributed on the 12 rice chromosomes and a simple regression quantitative trait locus analysis was performed using the phenotypic data from 2002/2003. The six high-yielding ILs showed a low proportion of wild fragment introgressions. A total of 14 molecular markers were associated with quantitative trait loci in the three locations. The six high-yielding ILs were incorporated in the Embrapa breeding program, and the line CNAi 9930 is recommended for cultivation due to additional advantages of good grain cooking and milling qualities and high yield stability. The O. glumaepatula-derived ILs proved to be a source of new alleles for the development of high-yielding rice cultivars.

Agronomic and molecular characterization of introgression lines from the interspecific cross Oryza sativa (BG90-2) x Oryza glumaepatula (RS-16)

The reduced genetic variability of modern rice varieties (Oryza sativa) is of concern because it reduces the possibilities of genetic gain in breeding programs. Introgression lines (ILs) containing genomic fragments from wild rice can be used to obtain new improved cultivars. The objective of the present study was to perform the agronomic and molecular characterizations of 35 BC2F8 ILs from the cross O. glumaepatula x O. sativa, aiming to select high-yielding ILs to be used in rice-breeding programs. All 35 ILs were field evaluated in the season 2002/2003 in three locations and the 15 best performing ones were evaluated in the season 2003/2004 in five locations. In 2003/2004, six ILs (CNAi 9934, CNAi 9931, CNAi 9930, CNAi 9935, CNAi 9936, and CNAi 9937) showed the highest yield means and were statistically superior to the controls Metica 1 and IRGA 417. Molecular characterization of the 35 ILs was performed with 92 microsatellite markers distributed on the 12 rice chromosomes and a simple regression Oriza glumaepatula-derived introgression lines quantitative trait locus analysis was performed using the phenotypic data from 2002/2003. The six high-yielding ILs showed a low proportion of wild fragment introgressions. A total of 14 molecular markers were associated with quantitative trait loci in the three locations. The six high-yielding ILs were incorporated in the Embrapa breeding program, and the line CNAi 9930 is recommended for cultivation due to additional advantages of good grain cooking and milling qualities and high yield stability. The O. glumaepatula-derived ILs proved to be a source of new alleles for the development of high-yielding rice cultivars.

Genetic mapping of a new set of microsatellite markers in a reference common bean (Phaseolus vulgaris) population BAT93 x Jalo EEP558.

The present study describes a new set of 61 polymorphic microsatellite markers for beans and the construction of a genetic map using the BAT93 x Jalo EEP558 (BJ) population for the purpose of developing a reference linkage map for common bean (Phaseolus vulgaris). The main objectives were to integrate new microsatellites on the existing framework map of the BJ population, and to develop the first linkage map for the BJ population based exclusively on microsatellites. Of the total of 264 microsatellites evaluated for polymorphism, 42.8% showed polymorphism between the genitors. An integrated map was created totaling 199 mapped markers in 13 linkage groups, with an observed length of 1358 cM and a mean distance between markers of 7.23 cM. For the map constructed exclusively with microsatellites, 106 markers were placed in 12 groups with a total length of 606.8 cM and average distance of 6.8 cM. Linkage group designation and marker order for BM microsatellites generally agreed with previous mapping, while the new microsatellites were well distributed across the genome, corroborating the utility of the BJ population for a reference map. The extensive use of the microsatellites and the availability of a reference map can help in the development of other genetic maps for common bean through the transfer of information of marker order and linkage, which will allow comparative analysis and map integration, especially for future quantitative trait loci and association mapping studies.

Genetic mapping of a new set of microsatellite markers in a reference common bean (Phaseolus vulgaris) population BAT93 x Jalo EEP558

The present study describes a new set of 61 polymorphic microsatellite markers for beans and the construction of a genetic map using the BAT93 x Jalo EEP558 (BJ) population for the purpose of developing a reference linkage map for common bean (Phaseolus vulgaris). The main objectives were to integrate new microsatellites on the existing framework map of the BJ population, and to develop the first linkage map for the BJ population based exclusively on microsatellites. Of the total of 264 microsatellites evaluated for polymorphism, 42.8% showed polymorphism between the genitors. An integrated map was created totaling 199 mapped markers in 13 linkage groups, with an observed length of 1358 cM and a mean distance between markers of 7.23 cM. For the map constructed exclusively with microsatellites, 106 markers were placed in 12 groups with a total length of 606.8 cM and average distance of 6.8 cM. Linkage group designation and marker order for BM microsatellites generally agreed with previous mapping, while the new microsatellites were well distributed across the genome, corroborating the utility of the BJ population for a reference map. The extensive use of the microsatellites and the availability of a reference map can help in the development of other genetic maps for common bean through the transfer of information of marker order and linkage, which will allow comparative analysis and map integration, especially for future quantitative trait loci and association mapping studies.

Towards a genus-wide reference linkage map for Eucalyptus based exclusively on highly informative microsatellite markers.

A novel set of 50 highly polymorphic microsatellite markers were developed and mapped on existing RAPD framework maps of Eucalyptus grandis and E. urophylla. Together with the twenty previously developed microsatellite markers, these were used to align the existing maps for the two most commercially important Eucalyptus species in the tropics. Sixty-three microsatellite markers were placed on the E. grandis map in 11 linkage groups, and 53 on the E. urophylla map distributed in 10 linkage groups. Approximately 66% of the microsatellite markers segregated in a fully informative fashion, allowing the establishment of colinear syntenic linkage groups between the two maps. The 50 new microsatellite markers were highly informative, with an average of 14 alleles per locus, and average expected heterozygosity between 0.82 and 0.87. Furthermore, within the subgenus Symphyomyrtus, to which the vast majority of commercially important Eucalyptus species belong, these markers display on average 90% transportability. This set of 70 mapped microsatellite markers represents a significant step toward the development of a genus-wide reference linkage map for Eucalyptus. These highly multiallelic and transportable markers constitute a powerful tool for QTL discovery and validation, and can be used in directed searches for QTL allele variation across Eucalyptus pedigrees.

QTL mapping and introgression of yield-related traits from Oryza glumaepatula to cultivated rice ( Oryza sativa) using microsatellite markers.

Rice ( Oryza sativa) cultivar development currently faces the task of overcoming yield plateaus, which is difficult due to the narrow genetic base of breeding programs. Oryza glumaepatula is a diploid wild relative of cultivated rice, native to Central and South America, and is therefore a potential source of alleles of agronomic importance to rice breeding programs. We studied 11 agronomic traits in BC(2)F(2) families of the interspecific cross Oryza sativa x O. glumaepatula. Transgressive lines which are almost isogenic to the elite recurrent O. sativa parent were identified for most of these traits. Quantitative trait locus (QTL) analysis was performed by single-point and interval mapping using a molecular map based on 157 microsatellite and STS markers. Marker regions accounting for 14.5 to 72.9% of a phenotypic variation trait were identified in 9 of the 12 rice chromosomes. Positive QTL effects from O. glumaepatula were observed in chromosomal regions associated with tillering and panicle-number traits.

Development and mapping of Oryza glumaepatula-derived microsatellite markers in the interspecific cross Oryza glumaepatula x O. sativa.

Wild germplasm of domesticated crops is a source of genetic variation little utilized in breeding programs. Interspecific crosses can potentially uncover novel gene combinations that can be important for quantitative trait analysis. The combined use of wide crosses and genetic maps of chromosomal regions associated with quantitative traits can be used to broaden the genetic basis of rice breeding programs. Oryza glumaepatula is a diploid (AA genome) wild rice species native from South and Central America. A genetic map was constructed with 162 PCR-based markers (155 microsatellite and 7 STS markers) using a backcross population derived from the cross O. glumaepatula, accession RS-16 from the Brazilian Amazon Region x O. sativa BG-90-2, an elite rice inbred line. The map included 47 new SSR markers developed from an O. glumaepatula genomic library enriched for AG/TC sequences. All SSR markers were able to amplify the O. sativa genome, indicating a high degree of SSR flanking region conservation between O. glumaepatula and O. sativa species. The map covered 1500.4 cM, with an average of one marker every 10 cM. Despite some chromosomes being more densely mapped, the overall coverage was similar to other maps developed for rice. The advantage to construct a SSR-based map is to permit the combination of the speed of the PCR reaction, and the codominant nature of the SSR marker, facilitating the QTL analysis and marker assisted selection for rice breeding programs.