Final Activity Report Summary - MARBLED GENE (Identification of genes involved in colour pattern formation and growth regulation in brown and marble trout)
The proposed project represents the initial phase of a research work that aims to identify candidate genes involved in marble colour pattern formation in marble trout (Soca river, Slovenia). Marble trout (S. trutta marmoratus), one of the most phenotypically distinct evolutionary lineages of S. trutta, sometimes referred as a separate species (S. marmoratus), was chosen as a model due to:
(i) it's highly distinct morphological characters (marbled colour pattern, growth), and
(ii) due to the availability of the material from F1 and F2 generations of the cross between the marble and the brown trout.
Species-specific colours and patterns on animal body are determined primarily by neural-crest-derived pigment cells in the skin. However, even closely related species display widely differing patterns. Mutations of orthologous genes were often found to produce a similar pigmentation phenotype in many animal species. Furthermore, using comparative approach, important genes implicated in other species skin colour determination were found through characterization of the model species mutant allele.
Within the first year of a project, different approaches were undertaken to achieve the main goal of the project and can be divided in three sections:
I. Publicly available databases with determined genomic / cDNA sequences, together with the relatively high DNA sequence identity observed even among distantly related taxa, enable an alternative approach for characterising genes in non-model species, i.e. comparative genomics approach in combination with candidate gene approach. The first part of this project made use of this approach, and can be divided in two parts:
1.) Sequence characterisation of Salmo salar skin cDNA library and determination of transcript specifically expressed in skin
Transcripts expressed specifically in skin tissue of S. salar were identified based on differential screening of available skin cDNA library against cDNA library of other 15 S. salar tissues, cDNA libraries available at http://www.salmongenome.no/cgi-bin/sgp.cgi. Among 200 blasted sequences, 45 have shown high similarities with genes, characterised particularly in zebrafish (Danio rerio) and were according to predicted protein function (cell-cell, protein-protein interactions, filament proteins, etc.) chosen for further analyses. We predicted exon-intron boundaries and designed primers to amplify genomic DNA and cDNA isolated from skin. Screening included samples from different phylogenetic lineages of Salmo trutta and from 4-8 populations of pure Salmo marmoratus from river Soca. We optimized PCR protocol for 10 different fragments and with sequence analyses we located SNPs differentiating S. marmoratus from S. trutta in eight of the fragments. These regions will be included in SNP genotyping of F2 and F3 generation of hybrids between S. marmoratus x S. trutta.
2.) Identification of genes known to be involved in skin pattern formation in zebrafish
Genes known to be involved in skin pigmentation in zebrafish were selected from Zebrafish database http://zfin.org/cgi-bin/webdriver?MIval=aa-ZDB_home.apg. Primers were designed for 21 genomic regions of Salmo and till now PCR was optimized for 10 of them. Based on sequence alignment, we located SNPs differentiating S. marmoratus from S. trutta in seven of the fragments. These regions will be included in SNP genotyping of F2 and F3 generation of hybrids between S. marmoratus x S. trutta.
II. Construction of suppression subtractive hybridisation (SSH) library from skin tissue
Subtractive cDNA library was produced from mRNA isolated from skin. Cloning the enriched subtracted cDNA produced over 300 clones. In order to confirm positive clones and to eliminate false positives, differential screening will be performed.
(i) it's highly distinct morphological characters (marbled colour pattern, growth), and
(ii) due to the availability of the material from F1 and F2 generations of the cross between the marble and the brown trout.
Species-specific colours and patterns on animal body are determined primarily by neural-crest-derived pigment cells in the skin. However, even closely related species display widely differing patterns. Mutations of orthologous genes were often found to produce a similar pigmentation phenotype in many animal species. Furthermore, using comparative approach, important genes implicated in other species skin colour determination were found through characterization of the model species mutant allele.
Within the first year of a project, different approaches were undertaken to achieve the main goal of the project and can be divided in three sections:
I. Publicly available databases with determined genomic / cDNA sequences, together with the relatively high DNA sequence identity observed even among distantly related taxa, enable an alternative approach for characterising genes in non-model species, i.e. comparative genomics approach in combination with candidate gene approach. The first part of this project made use of this approach, and can be divided in two parts:
1.) Sequence characterisation of Salmo salar skin cDNA library and determination of transcript specifically expressed in skin
Transcripts expressed specifically in skin tissue of S. salar were identified based on differential screening of available skin cDNA library against cDNA library of other 15 S. salar tissues, cDNA libraries available at http://www.salmongenome.no/cgi-bin/sgp.cgi. Among 200 blasted sequences, 45 have shown high similarities with genes, characterised particularly in zebrafish (Danio rerio) and were according to predicted protein function (cell-cell, protein-protein interactions, filament proteins, etc.) chosen for further analyses. We predicted exon-intron boundaries and designed primers to amplify genomic DNA and cDNA isolated from skin. Screening included samples from different phylogenetic lineages of Salmo trutta and from 4-8 populations of pure Salmo marmoratus from river Soca. We optimized PCR protocol for 10 different fragments and with sequence analyses we located SNPs differentiating S. marmoratus from S. trutta in eight of the fragments. These regions will be included in SNP genotyping of F2 and F3 generation of hybrids between S. marmoratus x S. trutta.
2.) Identification of genes known to be involved in skin pattern formation in zebrafish
Genes known to be involved in skin pigmentation in zebrafish were selected from Zebrafish database http://zfin.org/cgi-bin/webdriver?MIval=aa-ZDB_home.apg. Primers were designed for 21 genomic regions of Salmo and till now PCR was optimized for 10 of them. Based on sequence alignment, we located SNPs differentiating S. marmoratus from S. trutta in seven of the fragments. These regions will be included in SNP genotyping of F2 and F3 generation of hybrids between S. marmoratus x S. trutta.
II. Construction of suppression subtractive hybridisation (SSH) library from skin tissue
Subtractive cDNA library was produced from mRNA isolated from skin. Cloning the enriched subtracted cDNA produced over 300 clones. In order to confirm positive clones and to eliminate false positives, differential screening will be performed.