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Transcriptome sequencing of black grouse (Tetrao tetrix) for immune gene discovery and microsatellite development.

ABSTRACT: The black grouse (Tetrao tetrix) is a galliform bird species that is important for both ecological studies and conservation genetics. Here, we report the sequencing of the spleen transcriptome of black grouse using 454 GS FLX Titanium sequencing. We performed a large-scale gene discovery analysis with a focus on genes that might be related to fitness in this species and also identified a large set of microsatellites. In total, we obtained 182 179 quality-filtered sequencing reads that we assembled into 9035 contigs. Using these contigs and 15 794 length-filtered (greater than 200 bp) singletons, we identified 7762 transcripts that appear to be homologues of chicken genes. A specific BLAST search with an emphasis on immune genes found 308 homologous chicken genes that have immune function, including ten major histocompatibility complex-related genes located on chicken chromosome 16. We also identified 1300 expressed sequence tag microsatellites and were able to design suitable flanking primers for 526 of these. A preliminary test of the polymorphism of the microsatellites found 10 polymorphic microsatellites of the 102 tested. Genomic resources generated in this study should greatly benefit future ecological, evolutionary and conservation genetic studies on this species.

SUBMITTER: Wang B

PROVIDER: S-EPMC3376728 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Transcriptome sequencing of black grouse (Tetrao tetrix) for immune gene discovery and microsatellite development.

Wang Biao B Ekblom Robert R Castoe Todd A TA Jones Eleanor P EP Kozma Radoslav R Bongcam-Rudloff Erik E Pollock David D DD Höglund Jacob J

Open biology 20120401 4

The black grouse (Tetrao tetrix) is a galliform bird species that is important for both ecological studies and conservation genetics. Here, we report the sequencing of the spleen transcriptome of black grouse using 454 GS FLX Titanium sequencing. We performed a large-scale gene discovery analysis with a focus on genes that might be related to fitness in this species and also identified a large set of microsatellites. In total, we obtained 182 179 quality-filtered sequencing reads that we assembl ...[more]

PMID: 22724064

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Project description:BACKGROUND: The MHC, which is regarded as the most polymorphic region in the genomes of jawed vertebrates, plays a central role in the immune system by encoding various proteins involved in the immune response. The chicken MHC-B genomic region has a highly streamlined gene content compared to mammalian MHCs. Its core region includes genes encoding Class I and Class IIB molecules but is only ~92Kb in length. Sequences of other galliform MHCs show varying degrees of similarity as that of chicken. The black grouse (Tetrao tetrix) is a wild galliform bird species which is an important model in conservation genetics and ecology. We sequenced the black grouse core MHC-B region and combined this with available data from related species (chicken, turkey, gold pheasant and quail) to perform a comparative genomics study of the galliform MHC. This kind of analysis has previously been severely hampered by the lack of genomic information on avian MHC regions, and the galliformes is still the only bird lineage where such a comparison is possible. RESULTS: In this study, we present the complete genomic sequence of the MHC-B locus of black grouse, which is 88,390 bp long and contains 19 genes. It shows the same simplicity as, and almost perfect synteny with, the corresponding genomic region of chicken. We also use 454-transcriptome sequencing to verify expression in 17 of the black grouse MHC-B genes. Multiple sequence inversions of the TAPBP gene and TAP1-TAP2 gene block identify the recombination breakpoints near the BF and BLB genes. Some of the genes in the galliform MHC-B region also seem to have been affected by selective forces, as inferred from deviating phylogenetic signals and elevated rates of non-synonymous nucleotide substitutions. CONCLUSIONS: We conclude that there is large synteny between the MHC-B region of the black grouse and that of other galliform birds, but that some duplications and rearrangements have occurred within this lineage. The MHC-B sequence reported here will provide a valuable resource for future studies on the evolution of the avian MHC genes and on links between immunogenetics and ecology of black grouse.

Project description:As many other wild living species, black grouse Tetrao tetrix has to cope with anthropogenic disturbances in many habitats. Impacts of tourism and outdoor recreation on grouse species Tetraoninae have been subject to several studies in mountainous habitats in Central Europe and the United Kingdom. The geographically isolated and critically endangered black grouse population of Lower Saxony (North Germany) has been consistently monitored but beyond that poorly studied. This also applies to the key habitats of the nature reserve Lüneburg Heath (Lüneburger Heide) which, in turn, serves as a recreational area as well. In this study, the impact of tourism activity on habitat use of black grouse was investigated using data of GPS-tracked individuals. Additionally, visitor numbers on public and (usually undisturbed) closed routes were monitored using infrared light barriers. The spatio-temporal distribution of locations and the recreational activity were evaluated by linear mixed-effects models. Tagged individuals avoided the vicinity of public routes and avoiding distances were directly related to intensity of human activity. There was no seasonal change of black grouse habitat use alongside public routes. However, towards closed routes, significantly higher distances appeared during peak phases of visitor numbers (August and September), implying temporary increased disturbance levels within a key refuge area. Diurnal adaptation of habitat use was strongly dependent on the route density within the home range. Individuals used the vicinity of public trails at night and dawn but evaded these habitats during peak human activity around noon and afternoon. Recreational disturbances appeared to significantly affect the effective habitat availability for black grouse in the nature reserve. Visual cover by vegetation, however, seemed to diminish negative effects emerging from hiking trails. This provides an effective protective measure which requires minimal effort for the local conservation management.

Project description:BACKGROUND: Amorphophallus is a genus of perennial plants widely distributed in the tropics or subtropics of West Africa and South Asia. Its corms contain a high level of water-soluble glucomannan; therefore, it has long been used as a medicinal herb and food source. Genetic studies of Amorphophallus have been hindered by a lack of genetic markers. A large number of molecular markers are required for genetic diversity study and improving disease resistance in Amorphophallus. Here, we report large scale of transcriptome sequencing of two species: Amorphophallus konjac and Amorphophallus bulbifer using deep sequencing technology, and microsatellite (SSR) markers were identified based on these transcriptome sequences. RESULTS: cDNAs of A. konjac and A. bulbifer were sequenced using Illumina HiSeq™ 2000 sequencing technology. A total of 135,822 non-redundant unigenes were assembled from about 9.66 gigabases, and 19,596 SSRs were identified in 16,027 non-redundant unigenes. Di-nucleotide SSRs were the most abundant motif (61.6%), followed by tri- (30.3%), tetra- (5.6%), penta- (1.5%), and hexa-nucleotides (1%) repeats. The top di- and tri-nucleotide repeat motifs included AG/CT (45.2%) and AGG/CCT (7.1%), respectively. A total of 10,754 primer pairs were designed for marker development. Of these, 320 primers were synthesized and used for validation of amplification and assessment of polymorphisms in 25 individual plants. The total of 275 primer pairs yielded PCR amplification products, of which 205 were polymorphic. The number of alleles ranged from 2 to 14 and the polymorphism information content valued ranged from 0.10 to 0.90. Genetic diversity analysis was done using 177 highly polymorphic SSR markers. A phenogram based on Jaccard's similarity coefficients was constructed, which showed a distinct cluster of 25 Amorphophallus individuals. CONCLUSION: A total of 10,754 SSR markers have been identified in Amorphophallus using transcriptome sequencing. One hundred and seventy-seven polymorphic markers were successfully validated in 25 individuals. The large number of genetic markers developed in the present study should contribute greatly to research into genetic diversity and germplasm characterization in Amorphophallus.

Dataset Information

Transcriptome sequencing of black grouse (Tetrao tetrix) for immune gene discovery and microsatellite development.

Publications

Transcriptome sequencing of black grouse (Tetrao tetrix) for immune gene discovery and microsatellite development.

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