Mapping of quantitative trait loci for thermosensitive genic male sterility in indica rice

The objective of this work was to select and use microsatellite markers, to map genomic regions associated with the genetic control of thermosensitive genic male sterility (TGMS) in rice. An F2 population, derived from the cross between fertile and TGMS indica lines, was used to construct a microsatellite-based genetic map of rice. The TGMS phenotype showed a continuous variation in the segregant population. A low level of segregation distortion was detected in the F2 (14.65%), whose cause was found to be zygotic selection. There was no evidence suggesting a cause-effect relationship between zygotic selection and the control of TGMS in this cross. A linkage map comprising 1,213.3 cM was constructed based on the segregation data of the F2 population. Ninety-five out of 116 microsatellite polymorphic markers were assembled into 11 linkage groups, with an average of 12.77 cM between two adjacent marker loci. The phenotypic and genotypic data allowed for the identification of three new quantitative trait loci (QTL) for thermosensitive genic male sterility in indica rice. Two of the QTL were mapped on chromosomes that, so far, have not been associated with the genetic control of the TGMS trait (chromosomes 1 and 12). The third QTL was mapped on chromosome 7, where a TGMS locus (tms2) has recently been mapped. Allelic tests will have to be developed, in order to clarify if the two regions are the same or not.

Detection and mapping of a lethal locus in a eucalyptus hybrid population

The objective of this work was to verify the existence of a lethal locus in a eucalyptus hybrid population, and to quantify the segregation distortion in the linkage group 3 of the Eucalyptus genome. A E. grandis x E. urophylla hybrid population, which segregates for rust resistance, was genotyped with 19 microsatellite markers belonging to linkage group 3 of the Eucalyptus genome. To quantify the segregation distortion, maximum likelihood (ML) models, specific to outbreeding populations, were used. These models consider the observed marker genotypes and the lethal locus viability as parameters. The ML solutions were obtained using the expectation‑maximization algorithm. A lethal locus in the linkage group 3 was verified and mapped, with high confidence, between the microssatellites EMBRA 189 e EMBRA 122. This lethal locus causes an intense gametic selection from the male side. Its map position is 25 cM from the locus which controls the rust resistance in this population.

Microsatellite-dense genetic map: towards genome coverage in a tropical maize (Zea mays L.) population

Dense molecular genetic maps are used for an efficient quantitative trait loci (QTL) mapping and in the marker-assisted selection programs. A dense genetic map was generated with 139 microsatellite markers using 256 F2 plants generated by the crossing of two tropical maize inbred lines (L-02-03D and L-20-01F). This map presented 1,858.61 cM in length, where 10 linkage groups were found spanned, with an average interval of 13.47 cM between adjacent markers. Seventy seven percent of the maize genetic mapping bins were covered, which means an increase of 14% coverage in relation to the previous tropical maize maps. The results provide a more detailed and informative genetic map in a tropical maize population representing the first step to make possible the studies of genetic architecture to identify and map QTL and estimate their effects on the variation of quantitative traits, thus allowing the manipulation and use in tropical maize breeding programs.

An enhanced microsatellite map of diploid Fragaria

A total of 45 microsatellites (SSRs) were developed for mapping in Fragaria. They included 31 newly isolated codominant genomic SSRs from F. nubicola and a further 14 SSRs, derived from an expressed sequence tagged library (EST-SSRs) of the cultivated strawberry, F. × ananassa. These, and an additional 64 previously characterised but unmapped SSRs and EST-SSRs, were scored in the diploid Fragaria interspecific F2 mapping population (FV×FN) derived from a cross between F. vesca 815 and F. nubicola 601. The cosegregation data of these 109 SSRs, and of 73 previously mapped molecular markers, were used to elaborate an enhanced linkage map. The map is composed of 182 molecular markers (175 microsatellites, six gene specific markers and one sequence-characterised amplified region) and spans 424 cM over seven linkage groups. The average marker spacing is 2.3 cM/marker and the map now contains just eight gaps longer than 10 cM. The transferability of the new SSR markers to the cultivated strawberry was demonstrated using eight cultivars. Because of the transferable nature of these markers, the map produced will provide a useful reference framework for the development of linkage maps of the cultivated strawberry and for the development of other key resources for Fragaria such as a physical map. In addition, the map now provides a framework upon which to place transferable markers, such as genes of known function, for comparative mapping purposes within Rosaceae.

High throughput, high resolution selection of polymorphic microsatellite loci for multiplex analysis

Background Large-scale genetic profiling, mapping and genetic association studies require access to a series of well-characterised and polymorphic microsatellite markers with distinct and broad allele ranges. Selection of complementary microsatellite markers with non-overlapping allele ranges has historically proved to be a bottleneck in the development of multiplex microsatellite assays. The characterisation process for each microsatellite locus can be laborious and costly given the need for numerous, locus-specific fluorescent primers. Results Here, we describe a simple and inexpensive approach to select useful microsatellite markers. The system is based on the pooling of multiple unlabelled PCR amplicons and their subsequent ligation into a standard cloning vector. A second round of amplification utilising generic labelled primers targeting the vector and unlabelled locus-specific primers targeting the microsatellite flanking region yield allelic profiles that are representative of all individuals contained within the pool. Suitability of various DNA pool sizes was then tested for this purpose. DNA template pools containing between 8 and 96 individuals were assessed for the determination of allele ranges of individual microsatellite markers across a broad population. This helped resolve the balance between using pools that are large enough to allow the detection of many alleles against the risk of including too many individuals in a pool such that rare alleles are over-diluted and so do not appear in the pooled microsatellite profile. Pools of DNA from 12 individuals allowed the reliable detection of all alleles present in the pool. Conclusion The use of generic vector-specific fluorescent primers and unlabelled locus-specific primers provides a high resolution, rapid and inexpensive approach for the selection of highly polymorphic microsatellite loci that possess non-overlapping allele ranges for use in large-scale multiplex assays.

Geographic Structure of Plasmodium vivax: Microsatellite Analysis of Parasite Populations from Sri Lanka, Myanmar, and Ethiopia

Genetic diversity and population structure of Plasmodium viva-V parasites call predict the origin and Spread of novel Variants Within a population enabling Population specific malaria control measures. We analyzed the genetic diversity and population Structure of 425 P. vivax isolates from Sri Lanka, Myanmar, and Ethiopia using 12 trinucleotide and tetranucleotide microsatellite markers. All three parasite populations were highly polymorphic with 3-44 alleles per locus. Approximately 65% were multiple-clone infections. Mean genetic diversity (H(E)) was 0.7517 in Ethiopia, 0.8450 in Myanmar, and 0.8610 in Sri Lanka. Significant linkage disequilibrium Was maintained. Population structure showed two clusters (Asian and African) according to geography and ancestry Strong clustering of outbreak isolates from Sri Lanka and Ethiopia was observed. Predictive power of ancestry using two-thirds of the isolates as a model identified 78.2% of isolates accurately as being African or Asian. Microsatellite analysis is a useful tool for mapping short-term outbreaks of malaria and for predicting ancestry.

Mapping of quantitative trait loci controlling tick [Riphicephalus (Boophilus) microplus] resistance on bovine chromosomes 5, 7 and 14

Differences in domestication and selection processes have contributed to considerable phenotypic and genotypic differences between Bos taurus and Bos indicus cattle breeds. of particular interest in tropical and subtropical production environments are those genetic differences between subspecies that underlie the phenotypic extremes in tolerance and susceptibility to parasite infection. In general, B. taurus cattle are more susceptible to ectoparasites than B. indicus cattle in tropical environments, and much of this difference is under genetic control. To identify genomic regions involved in tick resistance, we developed a B. taurus x B. indicus F-2 experimental population to map quantitative trait loci (QTL) for resistance to the Riphicephalus (Boophilus) microplus tick. About 300 individuals were measured for parasite load in two seasons (rainy and dry) and genotyped for 23 microsatellite markers covering chromosomes 5, 7 and 14. We mapped a suggestive chromosome-wide QTL for tick load in the rainy season (P < 0.05) on chromosome 5. For the dry season, suggestive (P < 0.10) chromosome-wide QTL were mapped on chromosomes 7 and 14. The additive effect of the QTL on chromosome 14 corresponds to 3.18% of the total observed phenotypic variance. Our QTL-mapping study has identified different genomic regions controlling tick resistance; these QTL were dependent upon the season in which the ticks were counted, suggesting that the QTL in question may depend on environmental factors.

Characterization and transferability of microsatellite markers of the cultivated peanut (Arachis hypogaea)

Background: the genus Arachis includes Arachis hypogaea (cultivated peanut) and wild species that are used in peanut breeding or as forage. Molecular markers have been employed in several studies of this genus, but microsatellite markers have only been used in few investigations. Microsatellites are very informative and are useful to assess genetic variability, analyze mating systems and in genetic mapping. The objectives of this study were to develop A. hypogaea microsatellite loci and to evaluate the transferability of these markers to other Arachis species.Results: Thirteen loci were isolated and characterized using 16 accessions of A. hypogaea. The level of variation found in A. hypogaea using microsatellites was higher than with other markers. Cross-transferability of the markers was also high. Sequencing of the fragments amplified using the primer pair AhII from 17 wild Arachis species showed that almost all wild species had similar repeated sequence to the one observed in A. hypogaea. Sequence data suggested that there is no correlation between taxonomic relationship of a wild species to A. hypogaea and the number of repeats found in its microsatellite loci.Conclusion: These results show that microsatellite primer pairs from A. hypogaea have multiple uses. A higher level of variation among A. hypogaea accessions can be detected using microsatellite markers in comparison to other markers, such as RFLP, RAPD and AFLP. The microsatellite primers of A. hypogaea showed a very high rate of transferability to other species of the genus. These primer pairs provide important tools to evaluate the genetic variability and to assess the mating system in Arachis species.

A microsatellite-based, gene-rich linkage map for the AA genome of Arachis (Fabaceae)

Cultivated peanut (Arachis hypogaea) is an important crop, widely grown in tropical and subtropical regions of the world. It is highly susceptible to several biotic and abiotic stresses to which wild species are resistant. As a first step towards the introgression of these resistance genes into cultivated peanut, a linkage map based on microsatellite markers was constructed, using an F-2 population obtained from a cross between two diploid wild species with AA genome (A. duranensis and A. stenosperma). A total of 271 new microsatellite markers were developed in the present study from SSR-enriched genomic libraries, expressed sequence tags (ESTs), and by data-mining sequences available in GenBank. of these, 66 were polymorphic for cultivated peanut. The 271 new markers plus another 162 published for peanut were screened against both progenitors and 204 of these (47.1%) were polymorphic, with 170 codominant and 34 dominant markers. The 80 codominant markers segregating 1:2:1 (P < 0.05) were initially used to establish the linkage groups. Distorted and dominant markers were subsequently included in the map. The resulting linkage map consists of 11 linkage groups covering 1,230.89 cM of total map distance, with an average distance of 7.24 cM between markers. This is the first microsatellite-based map published for Arachis, and the first map based on sequences that are all currently publicly available. Because most markers used were derived from ESTs and genomic libraries made using methylation-sensitive restriction enzymes, about one-third of the mapped markers are genic. Linkage group ordering is being validated in other mapping populations, with the aim of constructing a transferable reference map for Arachis.

Chromosome Evolution in African Cichlid Fish: Contributions from the Physical Mapping of Repeated DNAs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 7 and comparative mapping to the cattle and human genomes

A preliminary radiation hybrid (RH) map containing 50 loci on chromosome 7 of the domestic river buffalo Bubalus bubalis (BBU; 2n = 50) was constructed based on a comparative mapping approach. The RH map of BBU7 includes thirty-seven gene markers and thirteen microsatellites. All loci have been previously assigned to Bos taurus (BTA) chromosome BTA6, which is known for its association with several economically important milk production traits in cattle. The map consists of two linkage groups spanning a total length of 627.9 cR(5,000). Comparative analysis of the BBU7 RH 5,000 map with BTA6 in cattle gave new evidence for strong similarity between the two chromosomes over their entire length and exposed minor differences in locus order. Comparison of the BBU7 RH 5,000 map with the Homo sapiens (HSA) genome revealed similarity with a large chromosome segment of HSA4. Comparative analysis of loci in both species revealed more variability than previously known in gene order and several chromosome rearrangements including centromere relocation. The data obtained in our study define the evolutionarily conserved segment on BBU7 and HSA4 to be between 3.5 megabases (Mb) and 115.8 Mb in the HSA4 (genome build 36) DNA sequence. Copyright (c) 2008 S. Karger AG, Basel.

Microsatellite loci isolation from river buffalo using enriched partial genomic libraries

The extensive use of buffalo in agriculture, especially in developing countries, begs for genetic resources to evaluate and improve traits important to local and regional economies. Brazil presents the largest water buffalo populations in the New World, with 1 1 million heads including swamp and river types. To design rational breeding strategies for optimum utilization and conservation of available genetic variability in the Brazilian buffalo's population, it is essential to understand their genetic architecture and relationship among various breeds. This depends, in part, on the knowledge of their genetic structure based on molecular markers like microsatellites. In the present study, we developed six enriched partial genomic libraries for river buffalo using selective hybridization methods. Genomic DNA was hybridized with six different arrays of repeat motif, 5' biotinylated - (CA)(15), (CT)(15), (AGG)(8), (GAAA)(8), (GATA)(8), (AAAAC)(8) - and bound to streptavidin coated beads. The cloning process generated a total of 1920 recombinant clones. Up to date, 487 were directly sequenced for the presence of repeats, from which 13 have been positive for presence of repeats as follows: 9 for di-nucleotide repeats, 3 for tri-nucleotide repeats and 1 for tetra-nucleotide repeat. PCR primer pairs for the isolated microsatellites are under construction to determine optimum annealing temperature. These microsatellites will be useful for studies involving phylogenetic relationships, genome mapping and genetic diversity analysis within buffalo populations worldwide.

Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel and preliminary RH mapping of chromosomes 3 and 10

The buffalo (Bubalus bubalis) not only is a useful source of milk, it also provides meat and works as a natural source of labor and biogas. To establish a project for buffalo genome mapping a 5,000-rad whole genome radiation hybrid panel was constructed for river buffalo and used to build preliminary RH maps from two chromosomes (BBU 3 and BBU10). The preliminary maps contain 66 markers, including coding genes, cattle ESTs and microsatellite loci. The RH maps presented here are the starting point for mapping additional loci, in particular, genes and expressed sequence tags that will allow detailed comparative maps between buffalo, cattle and other species to be constructed. A large quantity of DNA has been prepared from the cell lines forming the RH panel reported here and will be made publicly available to the international community both for the study of chromosome evolution and for the improvement of traits important to the role of buffalo in animal agriculture.

Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel and preliminary RH mapping of chromosomes 3 and 10

The buffalo (Bubalus bubalis) is a source of milk and meat, and also serves as a draft animal. In this study, a 5000-rad whole-genome radiation hybrid (RH) panel for river buffalo was constructed and used to build preliminary RH maps for BBU3 and BBU10 chromosomes. The preliminary maps contain 66 markers, including coding genes, cattle expressed sequence tags (ESTs) and microsatellite loci. The RH maps presented here are the starting point for mapping additional loci that will allow detailed comparative maps between buffalo, cattle and other species whose genomes may be mapped in the future. A large quantity of DNA has been prepared from the cell lines forming the river buffalo RH panel and will be made publicly available to the international community both for the study of chromosome evolution and for the improvement of traits important to the role of buffalo in animal agriculture.

Genotypic characterization of microsatellite markers in broiler and layer selected chicken lines and their reciprocal F1s

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)