Worldwide HLA-E nucleotide and haplotype variability reveals a conserved gene for coding and 3' untranslated regions.

The human leukocyte antigen-E (HLA-E) locus is a human major histocompatibility complex (MHC) gene associated with immune-modulation and suppression of the immune response by the interaction with specific natural killer (NK) and T cell receptors (TCRs). It is considered one of the most conserved genes of the human MHC; however, this low nucleotide variability seems to be a consequence of the scarce number of studies focusing on this subject. In this manuscript we assessed the nucleotide variability at the HLA-E coding and 3' untranslated regions (3'UTRs) in Brazil and in the populations from the 1000Genomes Consortium. Twenty-eight variable sites arranged into 33 haplotypes were detected and most of these haplotypes (98.2%) are encoding one of the two HLA-E molecules found worldwide, E*01:01 and E*01:03. Moreover, three worldwide spread haplotypes, associated with the coding alleles E*01:01:01, E*01:03:01 and E*01:03:02, account for 85% of all HLA-E haplotypes, suggesting that they arose early before human speciation. In addition, the low nucleotide diversity found for the HLA-E coding and 3'UTR in worldwide populations suggests that the HLA-E gene is in fact a conserved gene, which might be a consequence of its key role in the modulation of the immune system.

Isolation and characterization of microsatellite markers in the armored catfish Hypostomus gymnorhynchus (Loricariidae).

We isolated and characterized 10 microsatellite loci in the armored catfish (Hypostomus gymnorhynchus, Loricariidae), using a genomic shotgun library to obtain the repetitive sequences. Twenty-four primers were designed and 14 individuals of H. gymnorhynchus from the Caiapó River, in central Brazil, were genotyped using these primers to analyze the polymorphism at each locus. All loci showed low polymorphism, with a low number of alleles per locus (1 or 2), except locus Hg_E19, which had 11 alleles. Expected heterozygosities for polymorphic loci ranged from 0.182 to 0.901. Combined paternity exclusion probability (0.857) was low and combined genetic identity (0.0026) was high, when we examined parentage. The low degree of polymorphism that we detected may be due to the small sample size and the small microsatellite size, despite the large motif size.

Development of microsatellite markers for Hoplias malabaricus (Erythrinidae).

We identified 14 microsatellite loci for the wolf fish, Hoplias malabaricus (Erythrinidae), from a genomic shotgun library. Twenty-five primers were designed, and 48 individuals of H. malabaricus from four localities of northwest Goiás, in central Brazil, were genotyped to characterize the polymorphism at each locus. Fourteen primers amplified clearly interpretable products using a single PCR protocol; six loci were polymorphic, but with a low number of alleles per locus (2 or 3). Expected heterozygosities for polymorphic loci ranged from 0.136 to 0.505. Combined paternity exclusion probability (0.638) was low and combined genetic identity (0.056) was high in studies of parentage. The low polymorphism may be due to the small microsatellite size and the large size of the motifs.

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.

Multiplexed systems of microsatellite markers for genetic analysis of mahogany, Swietenia macrophylla King (Meliaceae), a threatened neotropical timber species.

Mahogany (Swietenia macrophylla King [Meliaceae]) is the most valuable hardwood species in the neotropics. Its conservation status has been the subject of increasing concern due to overexploitation and habitat destruction. In this work we report the development and characterization of 10 highly variable microsatellite loci for S. macrophylla. Twenty-nine percent of the 126 sequenced mahogany clones yielded useful microsatellite loci. Three high-throughput genotyping systems were developed based on polymerase chain reaction (PCR) multiplexing of these mahogany loci. We identified a total of 158 alleles in 121 adult individuals of S. macrophylla, with an average of 15.8 alleles (range 11-25) per locus. All loci showed Mendelian inheritance in open-pollinated half-sib families. The mean expected heterozygosity was 0.84 and the mean observed heterozygosity was 0.73. The combined probability of identity-the probability that two individuals selected at random from a population would have identical genotypes--was 7.0 x 10(-15), and combined probability of paternity exclusion was 0.999998 overall loci. These microsatellite loci permit precise estimates of parameters such as gene flow, mating system, and paternity, thus providing important insights into the population genetics and conservation of S. macrophylla.

Cost-effective method to synthesize a fluorescent internal DNA standard for automated fragment sizing.

We describe a simple and cost-effective method for the synthesis of an internal fluorescently labeled DNA standard for fragment sizing using an automated DNA sequencer. A set of primer pairs labeled with ROX was developed to amplify 12 DNA fragments, 58-417 bp, derived from a conserved region of plant chloroplast DNA. These amplified fragments were mixed together, constituting a fluorescent internal DNA size marker. The precision of the size standard was evaluated by estimating the size of 20 alleles that were amplified at four dinucleotide microsatellite loci with the synthesized size standard and the commercial internal sizing standard, GeneScan Rox500. A number of intra-gel and inter-gel comparisons were run, and an analysis of variance was carried out. No significant difference was observed between the size estimates obtained with the synthesized DNA standard and the commercial standard. This facile and general PCR-based method for the synthesis of internal standards allows for significant savings in the implementation of large genotyping experiments using microsatellite or AFLP markers.

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.