Transcriptome analysis and SSR/SNP markers information of the blunt snout bream (Megalobrama amblycephala)

Abstract

Background: Blunt snout bream (Megalobrama amblycephala) is an herbivorous freshwater fish species native to China and has been recognized as a main aquaculture species in the Chinese freshwater polyculture system with high economic value. Right now, only limited EST resources were available for M. amblycephala. Recent advances in large-scale RNA sequencing provide a fast, cost-effective, and reliable approach to generate large expression datasets for functional genomic analysis, which is especially suitable for non-model species with un-sequenced genomes.

Methodology and principal findings: Using 454 pyrosequencing, a total of 1,409,706 high quality reads (total length 577 Mbp) were generated from the normalized cDNA of pooled M. amblycephala individuals. These sequences were assembled into 26,802 contigs and 73,675 singletons. After BLAST searches against the NCBI non-redundant (NR) and UniProt databases with an arbitrary expectation value of E(-10), over 40,000 unigenes were functionally annotated and classified using the FunCat functional annotation scheme. A comparative genomics approach revealed a substantial proportion of genes expressed in M. amblycephala tanscriptome to be shared across the genomes of zebrafish, medaka, tetraodon, fugu, stickleback, human, mouse, and chicken, and identified a substantial number of potentially novel M. amblycephala genes. A total number of 4,952 SSRs were found and 116 polymorphic loci have been characterized. A significant number of SNPs (25,697) and indels (23,287) were identified based on specific filter criteria in the M. amblycephala.

Conclusions: This study is the first comprehensive transcriptome analysis for a fish species belonging to the genus Megalobrama. These large EST resources are expected to be valuable for the development of molecular markers, construction of gene-based linkage map, and large-scale expression analysis of M. amblycephala, as well as comparative genome analysis for the genus Megalobrama fish species. The identified SSR and SNP markers will greatly benefit its breeding program and whole genome association studies.

Figure 1. Frequency distribution of 454 sequencing read lengths for M. amblycephala.

Figure 2. Overview of M. amblycephala transcriptome sequence length distributions for all contigs and singletons.

(A) Sequence length distribution for all contigs; (B) Sequence length distribution for all singletons.

Figure 3. Open reading frame (ORF) length distribution from all contigs of M. amblycephala.

Figure 4. Gene ontology assignments for M. amblycephala.

The annotated contigs and singletons from M. amblycephala 454 sequencing that matched various gene ontology (GO) categories.

Figure 5. Conservation of M. amblycephala gene identities with other species.

Number of M. amblycephala homologous genes identified from other species using BLASTX searches.

Figure 6. Classification of SNPs identified from 454 sequences of M. amblycephala.

Figure 7. Distribution of minor allele frequencies of filtered SNPs identified for M. amblycephala from 454 sequences.

The X-axis represents the number of SNPs with given minor allele frequency, while the Y-axis represents the SNP sequence derived minor allele frequency in percentage (>15%).