Claudio Brondani

Development, characterization and mapping of microsatellite markers in Eucalyptus grandis and E. urophylla

Abstract We report on the development, genetic characterization and linkage mapping of a battery of SSR (simple sequence repeat) loci in Eucalyptus grandis and E. urophylla. This study reveals the abundance of SSRs in Eucalyptus, the very high information content of these markers for mapping and individual identification, and demonstrates the feasibility of constructing a comprehensive microsatellite-based linkage map for Eucalyptus. Primer sequence for a set of 20 highly informative EMBRA (Eucalyptus microsatellites from Brazil) loci are made available together with their map position and estimates of the expected heterozygosity and allele size range in these two species. Using genomic library enrichment and anchored-PCR screening prior to sequencing, the efficiency of SSR marker locus development was 63% from sequencing data to operationally useful SSR loci. Absolute transportability between the two species and very high levels of allelic variability and expected heterozygosity (H) were seen at all SSR loci surveyed. The number of alleles per locus ranged from 9 to 26 with an average of 16.3±4.8. The average H of 15 loci was 0.86±0.04, 0.83±0.08 and 0.89±0.04, respectively, for E. urophylla, E. grandis and the combined two-species estimate. In the mapping analysis 16 out of 20 marker loci segregated in a fully informative configuration, allowing the determination of synteny of six homologous linkage groups between the two species. The availability of transportable, multiallelic, PCR-based co-dominant SSR loci represents a dramatic improvement in our ability to carry out detailed population genetic analysis and to search, understand, and manipulate allelic variation at QTLs (quantitative trait loci) in species of Eucalyptus.

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

Abstract.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 BC2F2 families of the interspecific cross Oryza sativa × 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.

A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus

BackgroundEucalypts are the most widely planted hardwood trees in the world occupying globally more than 18 million hectares as an important source of carbon neutral renewable energy and raw material for pulp, paper and solid wood. Quantitative Trait Loci (QTLs) in Eucalyptus have been localized on pedigree-specific RAPD or AFLP maps seriously limiting the value of such QTL mapping efforts for molecular breeding. The availability of a genus-wide genetic map with transferable microsatellite markers has become a must for the effective advancement of genomic undertakings. This report describes the development of a novel set of 230 EMBRA microsatellites, the construction of the first comprehensive microsatellite-based consensus linkage map for Eucalyptus and the consolidation of existing linkage information for other microsatellites and candidate genes mapped in other species of the genus.ResultsThe consensus map covers ~90% of the recombining genome of Eucalyptus, involves 234 mapped EMBRA loci on 11 linkage groups, an observed length of 1,568 cM and a mean distance between markers of 8.4 cM. A compilation of all microsatellite linkage information published in Eucalyptus allowed us to establish the homology among linkage groups between this consensus map and other maps published for E. globulus. Comparative mapping analyses also resulted in the linkage group assignment of other 41 microsatellites derived from other Eucalyptus species as well as candidate genes and QTLs for wood and flowering traits published in the literature. This report significantly increases the availability of microsatellite markers and mapping information for species of Eucalyptus and corroborates the high conservation of microsatellite flanking sequences and locus ordering between species of the genus.ConclusionThis work represents an important step forward for Eucalyptus comparative genomics, opening stimulating perspectives for evolutionary studies and molecular breeding applications. The generalized use of an increasingly larger set of interspecific transferable markers and consensus mapping information, will allow faster and more detailed investigations of QTL synteny among species, validation of expression-QTL across variable genetic backgrounds and positioning of a growing number of candidate genes co-localized with QTLs, to be tested in association mapping experiments.

Association mapping for yield and grain quality traits in rice (Oryza sativa L.)

Association analysis was applied to a panel of accessions of Embrapa Rice Core Collection (ERiCC) with 86 SSR and field data from two experiments. A clear subdivision between lowland and upland accessions was apparent, thereby indicating the presence of population structure. Thirty-two accessions with admixed ancestry were identified through structure analysis, these being discarded from association analysis, thus leaving 210 accessions subdivided into two panels. The association of yield and grain-quality traits with SSR was undertaken with a mixed linear model, with markers and subpopulation as fixed factors, and kinship matrix as a random factor. Eight markers from the two appraised panels showed significant association with four different traits, although only one (RM190) maintained the marker-trait association across years and cultivation. The significant association detected between amylose content and RM190 was in agreement with previous QTL analyses in the literature. Herein, the feasibility of undertaking association analysis in conjunction with germplasm characterization was demonstrated, even when considering low marker density. The high linkage disequilibrium expected in rice lines and cultivars facilitates the detection of marker-trait associations for implementing marker assisted selection, and the mining of alleles related to important traits in germplasm.

Determination of genetic variability of traditional varieties of Brazilian rice using microsatellite markers

The rice (Oryza sativa) breeding program of the Rice and Bean research center of the Brazilian agricultural company Empresa Brasileira de Pesquisa Agropecuaria (Embrapa) is well established and provides new cultivars every year to attend the demand for improved high yielding varieties with tolerance to biotic and abiotic stresses. However, the elite genitors used to compose new populations for selection are closely related, contributing to the yield plateau reached in the last 20 years. To overcome this limit, it is necessary to broaden the genetic basis of the cultivars using diverse germplasm such as wild relatives or traditional varieties, with the latter being more practical because they are more easily crossed with elite germplasm to accelerate the recovery of modern plant types in the breeding lines. The objective of our study was to characterize the allelic diversity of 192 traditional varieties of Brazilian rice using 12 simple sequence repeat (SSR or microsatellite) markers. The germplasm was divided into 39 groups by common name similarity. A total of 176 alleles were detected, 30 of which (from 23 accessions) were exclusive. The number of alleles per marker ranged from 6 to 22, with an average of 14.6 alleles per locus. We identified 16 accessions as a mixture of pure lines or heterozygous plants. Dendrogram analysis identified six clusters of identical accessions with different common names and just one cluster with identical accessions with the same common name, indicating that SSR markers are fundamental to determining the genetic relationship between landraces. A subset of 24 landraces, representatives of the 13 similarity groups plus the 11 accessions not grouped, was the most variable set of genotypes analyzed. These accessions can be used as genitors to increase the genetic variability available to rice breeding programs.

Microsatellite marker-mediated analysis of the EMBRAPA Rice Core Collection genetic diversity

The objectives of this study were to determine the genetic structure of 242 accessions from the EMBRAPA Rice Core Collection (ERiCC), to create a mini-core collection and to develop a multiplex panel of fluorescent labeled simple sequence repeats (SSRs). Eighty-six SSRs were used to identify 1,066 alleles, with an average number of 12.4 alleles/locus and average polymorphism information content (PIC)/locus of 0.75. A model-based clustering method recognized the structure of the accessions on two levels, according to their cultivation system and origin. The most divergent subgroup identified was the worldwide lowland accessions, with the highest values for gene diversity (0.75), average Rogers distance modified by Wright (0.80), average number of alleles/locus (11.7) and private alleles (132). A mini-core was assembled with the most divergent 24 lowland and upland accessions. This mini-core displayed an average distance of 0.86, an average number of alleles/locus of 8.4 and an average PIC/locus of 0.8. From the 86 SSRs, 24 were selected to compose six multiplex panels in order to optimize the process of rice genotyping. This set of markers distinguished all 242 accessions, and showed an average PIC of 0.80 and an average number of alleles/locus of 15.4, higher than the entire set of 86 SSRs. Since the heterogeneity found in lines and cultivars of ERiCC was higher than expected, it is necessary to analyze pooled DNA samples to get a better estimate of genetic variability. The SSR characterization of ERiCC clearly indicates that there is high genetic variability in rice accessions stored in genebanks worldwide which can be promptly explored by rice pre-breeding programs.

The characterization of a new set of EST-derived simple sequence repeat (SSR) markers as a resource for the genetic analysis of Phaseolus vulgaris

BackgroundOver recent years, a growing effort has been made to develop microsatellite markers for the genomic analysis of the common bean (Phaseolus vulgaris) to broaden the knowledge of the molecular genetic basis of this species. The availability of large sets of expressed sequence tags (ESTs) in public databases has given rise to an expedient approach for the identification of SSRs (Simple Sequence Repeats), specifically EST-derived SSRs. In the present work, a battery of new microsatellite markers was obtained from a search of the Phaseolus vulgaris EST database. The diversity, degree of transferability and polymorphism of these markers were tested.ResultsFrom 9,583 valid ESTs, 4,764 had microsatellite motifs, from which 377 were used to design primers, and 302 (80.11%) showed good amplification quality. To analyze transferability, a group of 167 SSRs were tested, and the results showed that they were 82% transferable across at least one species. The highest amplification rates were observed between the species from the Phaseolus (63.7%), Vigna (25.9%), Glycine (19.8%), Medicago (10.2%), Dipterix (6%) and Arachis (1.8%) genera. The average PIC (Polymorphism Information Content) varied from 0.53 for genomic SSRs to 0.47 for EST-SSRs, and the average number of alleles per locus was 4 and 3, respectively. Among the 315 newly tested SSRs in the BJ (BAT93 X Jalo EEP558) population, 24% (76) were polymorphic. The integration of these segregant loci into a framework map composed of 123 previously obtained SSR markers yielded a total of 199 segregant loci, of which 182 (91.5%) were mapped to 14 linkage groups, resulting in a map length of 1,157 cM.ConclusionsA total of 302 newly developed EST-SSR markers, showing good amplification quality, are available for the genetic analysis of Phaseolus vulgaris. These markers showed satisfactory rates of transferability, especially between species that have great economic and genomic values. Their diversity was comparable to genomic SSRs, and they were incorporated in the common bean reference genetic map, which constitutes an important contribution to and advance in Phaseolus vulgaris genomic research.

Genetic Structure of Wild Rice Oryza Glumaepatula Populations in Three Brazilian Biomes Using Microsatellite Markers

The existence of Oryza glumaepatula is threatened by devastation and, thus, the implementation of conservation strategies is extremely relevant. This study aimed to characterize the genetic variability and estimate population parameters of 30 O. glumaepatula populations from three Brazilian biomes using 10 microsatellite markers. The levels of allelic variability for the SSR loci presented a mean of 10.3 alleles per locus and a value of 0.10 for the average allelic frequency value. The expected total heterozygosity (He) ranged from 0.63 to 0.86. For the 30 populations tested, the mean observed (Ho) and expected heterozygosities (He) were 0.03 and 0.11within population, respectively, indicating an excess of homozygotes resulting from the preferentially self-pollinating reproduction habit. The estimated fixation index ( IS ) was 0.79 that differed significantly from zero, indicating high inbreeding within each O. glumaepatula population. The total inbreeding of the species (IT ) was 0.98 and the genetic diversity indexes among populations, ST and ST, were 0.85 and 0.90, respectively, indicating high genetic variability among them. Thus, especially for populations located in regions threatened with devastation, it is urgent that in situ preservation conditions should be created or that collections be made for ex situ preservation to prevent loss of the species genetic variability.

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.

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.