M. E. Ferreira

Genetic diversity of peanut (Arachis hypogaea L.) and its wild relatives based on the analysis of hypervariable regions of the genome

BackgroundThe genus Arachis is native to a region that includes Central Brazil and neighboring countries. Little is known about the genetic variability of the Brazilian cultivated peanut (Arachis hypogaea, genome AABB) germplasm collection at the DNA level. The understanding of the genetic diversity of cultivated and wild species of peanut (Arachis spp.) is essential to develop strategies of collection, conservation and use of the germplasm in variety development. The identity of the ancestor progenitor species of cultivated peanut has also been of great interest. Several species have been suggested as putative AA and BB genome donors to allotetraploid A. hypogaea. Microsatellite or SSR (Simple Sequence Repeat) markers are co-dominant, multiallelic, and highly polymorphic genetic markers, appropriate for genetic diversity studies. Microsatellite markers may also, to some extent, support phylogenetic inferences. Here we report the use of a set of microsatellite markers, including newly developed ones, for phylogenetic inferences and the analysis of genetic variation of accessions of A. hypogea and its wild relatives.ResultsA total of 67 new microsatellite markers (mainly TTG motif) were developed for Arachis. Only three of these markers, however, were polymorphic in cultivated peanut. These three new markers plus five other markers characterized previously were evaluated for number of alleles per locus and gene diversity using 60 accessions of A. hypogaea. Genetic relationships among these 60 accessions and a sample of 36 wild accessions representative of section Arachis were estimated using allelic variation observed in a selected set of 12 SSR markers. Results showed that the Brazilian peanut germplasm collection has considerable levels of genetic diversity detected by SSR markers. Similarity groups for A. hypogaea accessions were established, which is a useful criteria for selecting parental plants for crop improvement. Microsatellite marker transferability was up to 76% for species of the section Arachis, but only 45% for species from the other eight Arachis sections tested. A new marker (Ah-041) presented a 100% transferability and could be used to classify the peanut accessions in AA and non-AA genome carriers.ConclusionThe level of polymorphism observed among accessions of A. hypogaea analyzed with newly developed microsatellite markers was low, corroborating the accumulated data which show that cultivated peanut presents a relatively reduced variation at the DNA level. A selected panel of SSR markers allowed the classification of A. hypogaea accessions into two major groups. The identification of similarity groups will be useful for the selection of parental plants to be used in breeding programs. Marker transferability is relatively high between accessions of section Arachis. The possibility of using microsatellite markers developed for one species in genetic evaluation of other species greatly reduces the cost of the analysis, since the development of microsatellite markers is still expensive and time consuming. The SSR markers developed in this study could be very useful for genetic analysis of wild species of Arachis, including comparative genome mapping, population genetic structure and phylogenetic inferences among species.

Development of microsatellite markers from an enriched genomic library for genetic analysis of melon (Cucumis melo L.)

BackgroundDespite the great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in melon (Cucumis melo L.) and cucurbit species. The development of microsatellite markers will have a major impact on genetic analysis and breeding of melon, especially on the generation of marker saturated genetic maps and implementation of marker assisted breeding programs. Genomic microsatellite enriched libraries can be an efficient alternative for marker development in such species.ResultsSeven hundred clones containing microsatellite sequences from a Tsp-AG/TC microsatellite enriched library were identified and one-hundred and forty-four primer pairs designed and synthesized. When 67 microsatellite markers were tested on a panel of melon and other cucurbit accessions, 65 revealed DNA polymorphisms among the melon accessions. For some cucurbit species, such as Cucumis sativus, up to 50% of the melon microsatellite markers could be readily used for DNA polymophism assessment, representing a significant reduction of marker development costs. A random sample of 25 microsatellite markers was extracted from the new microsatellite marker set and characterized on 40 accessions of melon, generating an allelic frequency database for the species. The average expected heterozygosity was 0.52, varying from 0.45 to 0.70, indicating that a small set of selected markers should be sufficient to solve questions regarding genotype identity and variety protection. Genetic distances based on microsatellite polymorphism were congruent with data obtained from RAPD marker analysis. Mapping analysis was initiated with 55 newly developed markers and most primers showed segregation according to Mendelian expectations. Linkage analysis detected linkage between 56% of the markers, distributed in nine linkage groups.ConclusionsGenomic library microsatellite enrichment is an efficient procedure for marker development in melon. One-hundred and forty-four new markers were developed from Tsp-AG/TC genomic library. This is the first reported attempt of successfully using enriched library for microsatellite marker development in the species. A sample of the microsatellite markers tested proved efficient for genetic analysis of melon, including genetic distance estimates and identity tests. Linkage analysis indicated that the markers developed are dispersed throughout the genome and should be very useful for genetic analysis of melon.

Analysis of genetic variability of South American wild rice populations (Oryza glumaepatula) with isozymes and RAPD markers

The knowledge of population structure and genetic diversity of wild relatives of rice is needed to investigate their evolutionary history and potential use in breeding programs. Very little is known about the wild rice species (Oryza spp.), particularly those that are native to South America. A study using isozyme and RAPD markers was conducted to estimate the level of genetic diversity of four South American wild rice populations (Oryza glumaepatula) recently collected in the Amazon forest and western Brazil rivers. F‐statistics and genetic diversity parameters calculated from isozyme and RAPD markers indicated high values for inbreeding coefficients and differentiation among the four populations. In agreement with this, a pattern of greater variation between than within populations was observed with both types of markers. These findings were corroborated by an AMOVA analysis, which indicated that a large portion of the total genetic variation was attributed to regional divergence. The partition of the AMOVA analysis among populations showed that most of the genetic diversity was due to differences among populations. This distribution pattern of genetic variation of O. glumaepatula populations is in agreement with the expectation for an autogamous species and provides important baseline data for conservation and collection strategies for this species.

Identification of drought-responsive genes in roots of upland rice (Oryza sativa L)

BackgroundRice (Oryza sativa L.) germplasm represents an extraordinary source of genes that control traits of agronomic importance such as drought tolerance. This diversity is the basis for the development of new cultivars better adapted to water restriction conditions, in particular for upland rice, which is grown under rainfall. The analyses of subtractive cDNA libraries and differential protein expression of drought tolerant and susceptible genotypes can contribute to the understanding of the genetic control of water use efficiency in rice.ResultsTwo subtractive libraries were constructed using cDNA of drought susceptible and tolerant genotypes submitted to stress against cDNA of well-watered plants. In silico analysis revealed 463 reads, which were grouped into 282 clusters. Several genes expressed exclusively in the tolerant or susceptible genotypes were identified. Additionally, proteome analysis of roots from stressed plants was performed and 22 proteins putatively associated to drought tolerance were identified by mass spectrometry.ConclusionSeveral genes and proteins involved in drought-response, as well as genes with no described homologs were identified. Genes exclusively expressed in the tolerant genotype were, in general, related to maintenance of turgor and cell integrity. In contrast, in the susceptible genotype, expression of genes involved in protection against cell damage was not detected. Several protein families identified in the proteomic analysis were not detected in the cDNA analysis. There is an indication that the mechanisms of susceptibility to drought in upland rice are similar to those of lowland varieties.

RAPD linkage mapping of the shell thickness locus in oil palm (Elaeis guineensis Jacq.).

Abstract Shell thickness is an important trait in oil palm breeding programs and is the basis for the classification of the varieties of oil palm into the types dura, tenera and pisifera. This trait seems to be controlled by a single locus, with two alleles (sh+ and sh−) showing codominant expression. Two single-tree linkage maps were constructed for a maternal tenera (sh+sh−) palm and for a paternal pisifera (sh−sh−) palm using the pseudo-testcross mapping strategy in combination with RAPD markers through the analysis of an F1 tenera×pisifera progeny. A total of 308 arbitrary primers were screened in a sample of eight F1 plants and 121 markers were detected in a testcross configuration. An average of 1.66 polymorphic marker per selected primer were identified in this cross. At LOD 5.0 (with some few exceptions) and θ=0.25 the maternal tenera map included a total of 48 markers distributed in 12 linkage groups or pairs of markers (449.3 cM) while the paternal pisifera map included 42 markers distributed in 15 linkage groups or pairs of markers (399.7 cM). We used RAPD and bulked segregant analysis (BSA) to identify markers more tightly linked to the sh+ locus. A total of 174 new primers not previously used in the linkage analysis were screened using bulks of DNA extracted from plants selected for the contrasting shell-thickness phenotypes. Two RAPD markers (R11–1282 and T19–1046) were identified to be linked on both sides of the sh+ locus on linkage group 4. The estimated map distances from sh+ to R11–1282 and to T19–1046 were 17.5 cM and 23.9 cM, respectively. The results demonstrate the usefulness of RAPD markers and the pseudo-testcross mapping strategy for developing genetic linkage information, and constitute an important step towards early marker-assisted selection for shell thickness in oil palm.

A set of multiplex panels of microsatellite markers for rapid molecular characterization of rice accessions

BackgroundThis study aimed to analyze the efficiency of three new microsatellite multiplex panels, which were designed to evaluate a total of 16 loci of the rice genome, based on single PCR reactions of each panel. A sample of 548 accessions of traditional upland rice landraces collected in Brazil in the last 25 years was genotyped, a database of allelic frequencies was established, estimates of genetic parameters were performed and analysis of genetic structure of the collection was developed.ResultsThe three panels yielded a combined matching probability of 6.4 × 10-21, polymorphism information content (PIC) of 0.637, and a combined power of exclusion greater than 99.99%. A few samples presented a genetic background of indica rice. The 16 SSR loci produced a total of 229 alleles. Gene diversity values averaged 0.667, and PIC values averaged 0.637. Genetic structure analysis of the collection using a Bayesian approach detected three possible major clusters, with an overall FST value of 0.177. Important inputs on the knowledge about upland rice germplasm differentiations which happened in Brazil in the last few centuries were also achieved and are discussed.ConclusionThe three multiplex panels described here represent a powerful tool for rice genetic analysis, offering a rapid and efficient option for rice germplasm characterization. The data gathered demonstrates the feasibility of genotyping extensive germplasm collections using panels of multiplexed microsatellite markers. It contributes to the advancement of research on large scale characterization and management of germplasm banks, as well as identification, protection and assessments of genetic relationship of rice germplasm.

Development and validation of microsatellite markers for Brachiaria ruziziensis obtained by partial genome assembly of Illumina single-end reads

BackgroundBrachiaria ruziziensis is one of the most important forage species planted in the tropics. The application of genomic tools to aid the selection of superior genotypes can provide support to B. ruziziensis breeding programs. However, there is a complete lack of information about the B. ruziziensis genome. Also, the availability of genomic tools, such as molecular markers, to support B. ruziziensis breeding programs is rather limited. Recently, next-generation sequencing technologies have been applied to generate sequence data for the identification of microsatellite regions and primer design. In this study, we present a first validated set of SSR markers for Brachiaria ruziziensis, selected from a de novo partial genome assembly of single-end Illumina reads.ResultsA total of 85,567 perfect microsatellite loci were detected in contigs with a minimum 10X coverage. We selected a set of 500 microsatellite loci identified in contigs with minimum 100X coverage for primer design and synthesis, and tested a subset of 269 primer pairs, 198 of which were polymorphic on 11 representative B. ruziziensis accessions. Descriptive statistics for these primer pairs are presented, as well as estimates of marker transferability to other relevant brachiaria species. Finally, a set of 11 multiplex panels containing the 30 most informative markers was validated and proposed for B. ruziziensis genetic analysis.ConclusionsWe show that the detection and development of microsatellite markers from genome assembled Illumina single-end DNA sequences is highly efficient. The developed markers are readily suitable for genetic analysis and marker assisted selection of Brachiaria ruziziensis. The use of this approach for microsatellite marker development is promising for species with limited genomic information, whose breeding programs would benefit from the use of genomic tools. To our knowledge, this is the first set of microsatellite markers developed for this important species.

Genetic diversity of Brazilian oil palm (Elaeis oleifera H.B.K.) germplasm collected in the Amazon Forest.

Elaeis oleifera or ‘caiaué’, a close relative of oil palm (E. guineensis), has some agronomic traits of great interest for the oil palm genetic breeding such as slow growth, oil quality (mostly unsaturated) and disease resistance. An analysis of a Brazilian oil palm germplasm collection was carried out using RAPD (Random Amplified Polymorphic DNA) markers with the objective of understanding the genetic variation of ‘caiaué’ accessions collected in the Amazon Forest in the last two decades. A sample of 175 accessions obtained along the Amazon River Basin was analyzed and compared to 17 accessions of oil palm from Africa. Ninety-six RAPD markers were used in the analysis, of which fourteen were shown to be specific to oil palm, while twelve were specific to ‘caiaué’. Results showed that the Brazilian ‘caiaué’ accessions studied have moderate levels of genetic diversity as compared to oil palm accessions. The data allowed the establishment of similarity groups for ‘caiaué’ accessions, which is useful for selecting parental plants for population breeding. Cluster analysis showed that, in general, genetic similarities are not correlated to geographical distances, but consistent with geographical dispersal along the Amazon River network. AMOVA showed that most of the genetic variation is found within populations, as expected for anallogamous and long-lived perennial species. The study provides important information to define strategies for future collection expeditions, for germplasm conservation and for the use of E. oleifera in breeding programs.

Development of microsatellite markers for the genetic analysis of Magnaporthe grisea.

An AG microsatellite-enriched genomic DNA library was constructed for Magnaporthe grisea (anamorph Pyricularia grisea), the causal agent of rice blast. Seventy-two DNA clones containing microsatellite repeats were isolated and sequenced in order to develop a series of new PCR-based molecular markers to be used in genetic studies of the fungus. Twenty-four of these clones were selected to design primer pairs for the PCR amplification of microsatellite alleles. Single spore cultures of M. grisea isolated from rice and wheat in Brazil, Colombia and China were genotyped at three microsatellite loci. Isolates from southern Brazil were predominantly monomorphic at the tested SSR loci, indicating a low level of genetic variability in these samples. However, seven alleles were observed at the MGM-1 locus in isolates from Central Brazil and at least nine alleles were detected at the same locus in a sample of Colombian isolates. Polymorphism analysis at SSR loci is a simple and direct approach for estimating the genetic diversity of M. grisea isolates and a powerful tool for studying M. grisea genetics.

Embrapa's contribution to the development of new plant varieties and their impact on Brazilian agriculture

Plant breeding programs conducted by Embrapaand partners have significantly contributed to major qualitative and quantitative advances achieved by the Brazilian agriculture over the last 40 years. In this article, an overview of the diversity of crop species and the multiple goals established by Embrapas plant breeding programs is presented, highlighting some of the main contributions to the agricultural sector. The economic, social. and environmental impacts of the major cultivars released by Embrapa are reviewed and an analysis of the present and future role of the institution in cultivar improvement for tropical and subtropical regions is provided. Risks, opportunities and challenges of this endeavor are discussed, considering the recent market changes and innovations observed in the seed industry in Brazil.