Combining the analyses of introgressive hybridisation and linkage mapping to investigate the genetic architecture of population divergence in the lake whitefish (Coregonus clupeaformis, Mitchill)

Adaptation and reproductive isolation, the engines of biological diversity, are still elusive when discussing the genetic bases of speciation. Namely, the number of genes and magnitude of selection acting positively or negatively on genomic traits implicated in speciation is contentious. Here, we describe the first steps of an ongoing research program aimed at understanding the genetic bases of population divergence and reproductive isolation in the lake whitefish (Coregonus clupeaformis). A preliminary linkage map originating from a hybrid cross between dwarf and normal ecotypes is presented, whereby some of the segregating AFLP markers were found to be conserved among natural populations. Maximum-likelihood was used to estimate hybrid indices from non-diagnostic markers at 998 AFLP loci. This allowed identification of the most likely candidate loci that have been under the influence of selection during the natural hybridisation of whitefish originating from different glacial races. As some of these loci could be identified on the linkage map, the possibility that selection of traits in natural populations may eventually be correlated to specific chromosomal regions was demonstrated. The future prospects and potential of these approaches to elucidate the genetic bases of adaptation and reproductive isolation among sympatric ecotypes of lake whitefish is discussed.

Assessment of SNPs for linkage mapping in Eucalyptus: construction of a consensus SNP/microsatellite map from two unrelated pedigrees

Financial support. Brazilian Ministry of Science and Technology (CNPq Grant 577047-2008-6), FAP-DF NEXTREE Grant 193.000.570/2009 and EMBRAPA Macroprogram 2 project grant 02.07.01.004.

Linkage mapping of ovine microphthalmia to chromosome 23, the sheep orthologue of human chromosome 18

PURPOSE: To characterize the phenotype and map the locus responsible for autosomal recessive inherited ovine microphthalmia (OMO) in sheep. METHODS: Microphthalmia-affected lambs and their available relatives were collected in a field, and experimental matings were performed to obtain affected and normal lambs for detailed necropsy and histologic examinations. The matings resulted in 18 sheep families with 48 cases of microphthalmia. A comparative candidate gene approach was used to map the disease locus within the sheep genome. Initially, 27 loci responsible for the microphthalmia-anophthalmia phenotypes in humans or mice were selected to test for comparative linkage. Fifty flanking markers that were predicted from comparative genomic analysis to be closely linked to these genes were tested for linkage to the disease locus. After observation of statistical evidence for linkage, a confirmatory fine mapping strategy was applied by further genotyping of 43 microsatellites. RESULTS: The clinical and pathologic examinations showed slightly variable expressivity of isolated bilateral microphthalmia. The anterior eye chamber was small or absent, and a white mass admixed with cystic spaces extended from the papilla to the anterior eye chamber, while no recognizable vitreous body or lens was found within the affected eyes. Significant linkage to a single candidate region was identified at sheep chromosome 23. Fine mapping and haplotype analysis assigned the candidate region to a critical interval of 12.4 cM. This ovine chromosome segment encompasses an ancestral chromosomal breakpoint corresponding to two orthologue segments of human chromosomes 18, short and long arms. For the examined animals, we excluded the complete coding region and adjacent intronic regions of ovine TGIF1 to harbor disease-causing mutations. CONCLUSIONS: This is the first genetic localization for hereditary ovine isolated microphthalmia. It seems unlikely that a mutation in the TGIF1 gene is responsible for this disorder. The studied sheep represent a valuable large animal model for similar human ocular phenotypes.

Mouse model for Usher syndrome: linkage mapping suggests homology to Usher type I reported at human chromosome 11p15.

Usher syndrome is a group of diseases with autosomal recessive inheritance, congenital hearing loss, and the development of retinitis pigmentosa, a progressive retinal degeneration characterized by night blindness and visual field loss over several decades. The causes of Usher syndrome are unknown and no animal models have been available for study. Four human gene sites have been reported, suggesting at least four separate forms of Usher syndrome. We report a mouse model of type I Usher syndrome, rd5, whose linkage on mouse chromosome 7 to Hbb and tub has homology to human Usher I reported on human chromosome 11p15. The electroretinogram in homozygous rd5/rd5 mouse is never normal with reduced amplitudes that extinguish by 6 months. Auditory-evoked response testing demonstrates increased hearing thresholds more than control at 3 weeks of about 30 decibels (dB) that worsen to about 45 dB by 6 months.

Inheritance and mapping of 11 avirulence genes in Phytophthora sojae

Two new crosses involving four races (races 7, 16, 17, and 25) of the soybean root and stem rot pathogen Phytophthora sojae were established (7/16 cross; 17/25 cross). An F-2 Population derived from each cross was used to determine the genetic basis of avirulence towards 11 different resistance genes in soybean. Avirulence was found to be dominant and determined by a single locus for Avr1b, 1d, 1k, 3b, 4, and 6, as expected for a simple gene-for-gene model. We also observed several cases of segregation, inconsistent with a single dominant gene being solely responsible for avirulence, which suggests that the genetic background of the different crosses can affect avirulence. Avr4 and 6 cosegregated in both the 7/16 and 17/25 crosses and, in the 7/16 cross, Avr1b and 1k were closely linked. Information from segregating RAPD, RFLP, and AFLP markers screened on F-2 progeny from the two new crosses and two crosses described previously (a total of 212 F-2 individuals, 53 from each cross) were used to construct an integrated genetic linkage map of P. sojae. This revised genetic linkage map consists of 386 markers comprising 35 RFLP, 236 RAPD, and 105 AFLP markers, as well as 10 avirulence genes. The map is composed of 21 major linkage groups and seven minor linkage groups covering a total map distance of 1640.4 cM. (C) 2002 Elsevier Science (USA). All rights reserved.

Fine mapping and replication of QTL in outbred chicken advanced intercross lines

Background: Linkage mapping is used to identify genomic regions affecting the expression of complex traits. However, when experimental crosses such as F2 populations or backcrosses are used to map regions containing a Quantitative Trait Locus (QTL), the size of the regions identified remains quite large, i.e. 10 or more Mb. Thus, other experimental strategies are needed to refine the QTL locations. Advanced Intercross Lines (AIL) are produced by repeated intercrossing of F2 animals and successive generations, which decrease linkage disequilibrium in a controlled manner. Although this approach is seen as promising, both to replicate QTL analyses and fine-map QTL, only a few AIL datasets, all originating from inbred founders, have been reported in the literature. Methods: We have produced a nine-generation AIL pedigree (n = 1529) from two outbred chicken lines divergently selected for body weight at eight weeks of age. All animals were weighed at eight weeks of age and genotyped for SNP located in nine genomic regions where significant or suggestive QTL had previously been detected in the F2 population. In parallel, we have developed a novel strategy to analyse the data that uses both genotype and pedigree information of all AIL individuals to replicate the detection of and fine-map QTL affecting juvenile body weight. Results: Five of the nine QTL detected with the original F2 population were confirmed and fine-mapped with the AIL, while for the remaining four, only suggestive evidence of their existence was obtained. All original QTL were confirmed as a single locus, except for one, which split into two linked QTL. Conclusions: Our results indicate that many of the QTL, which are genome-wide significant or suggestive in the analyses of large intercross populations, are true effects that can be replicated and fine-mapped using AIL. Key factors for success are the use of large populations and powerful statistical tools. Moreover, we believe that the statistical methods we have developed to efficiently study outbred AIL populations will increase the number of organisms for which in-depth complex traits can be analyzed.

A molecular linkage map for Drosophila mediopunctata confirms synteny with Drosophila melanogaster and suggests a region that controls the variation in the number of abdominal spots

The classic approach to gene discovery relies on the construction of linkage maps. We report the first molecular-based linkage map for Drosophila mediopunctata, a neotropical species of the tripunctata group. Eight hundred F2 individuals were genotyped at 49 microsatellite loci, resulting in a map that is approximate to 450 centimorgans long. Five linkage groups were detected, and the species' chromosomes were identified through cross-references to BLASTn searches and Muller elements. Strong synteny was observed when compared with the Drosophila melanogaster chromosome arms, but little conservation in the gene order was seen. The incorporation of morphological data corresponding to the number of central abdominal spots on the map was consistent with the expected location of a genomic region responsible for the phenotype on the second chromosome.

Genetic mapping of the belt pattern in Brown Swiss cattle to BTA3

The white belt pattern of Brown Swiss cattle is characterized by a lack of melanocytes in a stretch of skin around the midsection. This pattern is of variable width and sometimes the belt does not fully circle the body. To identify the gene responsible for this colour variation, we performed linkage mapping of the belted locus using six segregating half-sib families including 104 informative meioses for the belted character. The pedigree confirmed a monogenic autosomal dominant inheritance of the belted phenotype in Brown Swiss cattle. We performed a genome scan using 186 microsatellite markers in a subset of 88 animals of the six families. Linkage with the belt phenotype was detected at the telomeric region of BTA3. Fine-mapping and haplotype analysis using 19 additional markers in this region refined the critical region of the belted locus to a 922-kb interval on BTA3. As the corresponding human and mouse chromosome segments contain no obvious candidate gene for this coat colour trait, the mutation causing the belt pattern in the Brown Swiss cattle might help to identify an unknown gene influencing skin pigmentation.

Studies on locus content mapping.

Locus content maps are derived from monosomic or disomic chromosomes broken by radiation, shearing, or other clastogen, the fragments being distributed among clones by dilution or incorporation into the cells of another species and scored for segregation of markers. Locus content maps provide evidence about radiosensitivity of chromosome regions, support for order, and approximate location. Omission of the most aberrant and least informative clones increases efficiency of localization. Correct analysis must allow for preferential retention of certain sequences, monosomy or polysomy of donor chromosomes, and error filtration. Combination of these refinements extracts substantially more information from fewer clones. Because of unmodeled peculiarities in the data, the best analysis does not recover the physical map but roughly localizes markers that may be monomorphic and therefore unsuitable for linkage mapping. As with linkage for polymorphic loci, distance in the composite map should be confirmed by physical methods.

De Novo Assembly And Transcriptome Analysis Of The Rubber Tree (hevea Brasiliensis) And Snp Markers Development For Rubber Biosynthesis Pathways.

Hevea brasiliensis (Willd. Ex Adr. Juss.) Muell.-Arg. is the primary source of natural rubber that is native to the Amazon rainforest. The singular properties of natural rubber make it superior to and competitive with synthetic rubber for use in several applications. Here, we performed RNA sequencing (RNA-seq) of H. brasiliensis bark on the Illumina GAIIx platform, which generated 179,326,804 raw reads on the Illumina GAIIx platform. A total of 50,384 contigs that were over 400 bp in size were obtained and subjected to further analyses. A similarity search against the non-redundant (nr) protein database returned 32,018 (63%) positive BLASTx hits. The transcriptome analysis was annotated using the clusters of orthologous groups (COG), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam databases. A search for putative molecular marker was performed to identify simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). In total, 17,927 SSRs and 404,114 SNPs were detected. Finally, we selected sequences that were identified as belonging to the mevalonate (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways, which are involved in rubber biosynthesis, to validate the SNP markers. A total of 78 SNPs were validated in 36 genotypes of H. brasiliensis. This new dataset represents a powerful information source for rubber tree bark genes and will be an important tool for the development of microsatellites and SNP markers for use in future genetic analyses such as genetic linkage mapping, quantitative trait loci identification, investigations of linkage disequilibrium and marker-assisted selection.

Leaf-, Panel- And Latex-expressed Sequenced Tags From The Rubber Tree (hevea Brasiliensis) Under Cold-stressed And Suboptimal Growing Conditions: The Development Of Gene-targeted Functional Markers For Stress Response.

Hevea brasiliensis is a native species of the Amazon Basin of South America and the primary source of natural rubber worldwide. Due to the occurrence of South American Leaf Blight disease in this area, rubber plantations have been extended to suboptimal regions. Rubber tree breeding is time-consuming and expensive, but molecular markers can serve as a tool for early evaluation, thus reducing time and costs. In this work, we constructed six different cDNA libraries with the aim of developing gene-targeted molecular markers for the rubber tree. A total of 8,263 reads were assembled, generating 5,025 unigenes that were analyzed; 912 expressed sequence tags (ESTs) represented new transcripts, and two sequences were highly up-regulated by cold stress. These unigenes were scanned for microsatellite (SSR) regions and single nucleotide polymorphisms (SNPs). In total, 169 novel EST-SSR markers were developed; 138 loci were polymorphic in the rubber tree, and 98 % presented transferability to six other Hevea species. Locus duplication was observed in H. brasiliensis and other species. Additionally, 43 SNP markers in 13 sequences that showed similarity to proteins involved in stress response, latex biosynthesis and developmental processes were characterized. cDNA libraries are a rich source of SSR and SNP markers and enable the identification of new transcripts. The new markers developed here will be a valuable resource for linkage mapping, QTL identification and other studies in the rubber tree and can also be used to evaluate the genetic variability of other Hevea species, which are valuable assets in rubber tree breeding.

Microsatellite Markers For Urochloa Humidicola (poaceae) And Their Transferability To Other Urochloa Species.

Urochloa humidicola is a warm-season grass commonly used as forage in the tropics and is recognized for its tolerance to seasonal flooding. This grass is an important forage species for the Cerrado and Amazon regions of Brazil. U. humidicola is a polyploid species with variable ploidy (6X-9X) and facultative apomixis with high phenotypic plasticity. However, this apomixis and ploidy, as well as the limited knowledge of the genetic basis of the germplasm collection, have constrained genetic breeding activities, yet microsatellite markers may enable a better understanding of the species' genetic composition. This study aimed to develop and characterize new polymorphic microsatellite molecular markers in U. humidicola and to evaluate their transferability to other Urochloa species. A set of microsatellite markers for U. humidicola was identified from two new enriched genomic DNA libraries: the first library was constructed from a single sexual genotype and the second from a pool of eight apomictic genotypes selected on the basis of previous results. Of the 114 loci developed, 72 primer pairs presented a good amplification product, and 64 were polymorphic among the 34 genotypes tested. The number of bands per simple sequence repeat (SSR) locus ranged from 1 to 29, with a mean of 9.6 bands per locus. The mean polymorphism information content (PIC) of all loci was 0.77, and the mean discrimination power (DP) was 0.87. STRUCTURE analysis revealed differences among U. humidicola accessions, hybrids, and other Urochloa accessions. The transferability of these microsatellites was evaluated in four species of the genus, U. brizantha, U. decumbens, U. ruziziensis, and U. dictyoneura, and the percentage of transferability ranged from 58.33% to 69.44% depending on the species. This work reports new polymorphic microsatellite markers for U. humidicola that can be used for breeding programs of this and other Urochloa species, including genetic linkage mapping, quantitative trait loci identification, and marker-assisted selection.

A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance.

We report the study of a large American family displaying autosomal dominant retinitis pigmentosa with reduced penetrance, a form of hereditary retinal degeneration. Although the inheritance pattern and previous linkage mapping pointed to the involvement of the PRPF31 gene, extensive screening of all its exons and their boundaries failed in the past to reveal any mutation. In this work, we sequenced the entire PRPF31 genomic region by both the classical Sanger method and ultrahigh throughput (UHT) sequencing. Among the many variants identified, a single-base substitution (c.1374+654C>G) located deep within intron 13 and inside a repetitive DNA element was common to all patients and obligate asymptomatic carriers. This change created a new splice donor site leading to the synthesis of two mutant PRPF31 isoforms, degraded by nonsense-mediated mRNA decay. As a consequence, amounts of PRPF31 mRNA derived from the mutant allele were very reduced, with no evidence of mutant proteins being synthesized. Our results indicate that c.1374+654C>G causes retinitis pigmentosa via haploinsufficiency, similar to the vast majority of PRPF31 mutations described so far. We discuss the potential of UHT sequencing technologies in mutation screening and the continued identification of pathogenic splicing mutations buried deep within intronic regions.

Stronger transferability but lower variability in transcriptomic- than in anonymous microsatellites: evidence from Hylid frogs.

A simple way to quickly optimize microsatellites in nonmodel organisms is to reuse loci available in closely related taxa; however, this approach can be limited by the stochastic and low cross-amplification success experienced in some groups (e.g. amphibians). An efficient alternative is to develop loci from transcriptome sequences. Transcriptomic microsatellites have been found to vary in their levels of cross-species amplification and variability, but this has to date never been tested in amphibians. Here, we compare the patterns of cross-amplification and levels of polymorphism of 18 published anonymous microsatellites isolated from genomic DNA vs. 17 loci derived from a transcriptome, across nine species of tree frogs (Hyla arborea and Hyla cinerea group). We established a clear negative relationship between divergence time and amplification success, which was much steeper for anonymous than transcriptomic markers, with half-lives (time at which 50% of the markers still amplify) of 1.1 and 37 My, respectively. Transcriptomic markers are significantly less polymorphic than anonymous loci, but remain variable across diverged taxa. We conclude that the exploitation of amphibian transcriptomes for developing microsatellites seems an optimal approach for multispecies surveys (e.g. analyses of hybrid zones, comparative linkage mapping), whereas anonymous microsatellites may be more informative for fine-scale analyses of intraspecific variation. Moreover, our results confirm the pattern that microsatellite cross-amplification is greatly variable among amphibians and should be assessed independently within target lineages. Finally, we provide a bank of microsatellites for Palaearctic tree frogs (so far only available for H. arborea), which will be useful for conservation and evolutionary studies in this radiation.