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Analysis of genome survey sequences and SSR marker development for Siamese Mud Carp, Henicorhynchus siamensis, using 454 pyrosequencing.

Iranawati F, Jung H, Chand V, Hurwood DA, Mather PB - Int J Mol Sci (2012)

Bottom Line: Based on GSS-BLASTx results, 10.5% of contigs and 8.1% singletons possessed significant similarity (E value < 10(-5)) with the majority matching well to reported fish sequences.One thousand eight hundred and thirty seven sequences containing SSR motifs were identified, of which 422 qualified for primer design and eight polymorphic loci have been tested with average observed and expected heterozygosity estimated at 0.75 and 0.83, respectively.Regardless of their relative levels of polymorphism and heterozygosity, microsatellite loci developed here are suitable for further population genetic studies in H. siamensis and may also be applicable to other related taxa.

View Article: PubMed Central - PubMed

Affiliation: Earth, Environmental and Biological Sciences, Science and Engineering Faculty, Queensland University of Technology, Brisbane, Queensland 4000, Australia; E-Mails: v.chand@qut.edu.au (V.C.); d.hurwood@qut.edu.au (D.A.H.); p.mather@qut.edu.au (P.B.M.).

ABSTRACT
Siamese mud carp (Henichorynchus siamensis) is a freshwater teleost of high economic importance in the Mekong River Basin. However, genetic data relevant for delineating wild stocks for management purposes currently are limited for this species. Here, we used 454 pyrosequencing to generate a partial genome survey sequence (GSS) dataset to develop simple sequence repeat (SSR) markers from H. siamensis genomic DNA. Data generated included a total of 65,954 sequence reads with average length of 264 nucleotides, of which 2.79% contain SSR motifs. Based on GSS-BLASTx results, 10.5% of contigs and 8.1% singletons possessed significant similarity (E value < 10(-5)) with the majority matching well to reported fish sequences. KEGG analysis identified several metabolic pathways that provide insights into specific potential roles and functions of sequences involved in molecular processes in H. siamensis. Top protein domains detected included reverse transcriptase and the top putative functional transcript identified was an ORF2-encoded protein. One thousand eight hundred and thirty seven sequences containing SSR motifs were identified, of which 422 qualified for primer design and eight polymorphic loci have been tested with average observed and expected heterozygosity estimated at 0.75 and 0.83, respectively. Regardless of their relative levels of polymorphism and heterozygosity, microsatellite loci developed here are suitable for further population genetic studies in H. siamensis and may also be applicable to other related taxa.

No MeSH data available.


Related in: MedlinePlus

Top 25 hit species distribution based on BLASTx. E value cut-off is 10−5. Singleton (A) and contig (B). Bold text indicates teleosts.
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f2-ijms-13-10807: Top 25 hit species distribution based on BLASTx. E value cut-off is 10−5. Singleton (A) and contig (B). Bold text indicates teleosts.

Mentions: BLASTx searches of H. siamensis GSS sequences showed that 90 of the 857 (10.5%) contigs and 3751 of the 46,393 (8.1%) singletons possessed significant similarity (E value < 10−5) with proteins in the GenBank non-redundant (nr) database (Table S1). The majority of contigs (62%) and singletons (87%) matched well to reported fish sequences (Figures 2a,b), an outcome that agrees with previous fish studies [20,25,30,31]. Species most represented in the BLASTx searches included zebra fish (Danio rerio), spotted green puffer (Tetraodon nigroviridis), European seabass (Dicentrarchus labrax), carp (Cyprinus carpio), Torafugu (Takifugu rubripes), medaka (Oryzias latipes), Nile tilapia (Oreochromis niloticus), Atlantic salmon (Salmo salar), Florida lancelet (Branchiostoma floridae), and channel catfish (Ictalurus punctatus), largely due to their availability of their sequences in public databases. Sequence homologies are indicative of close evolutionary relationships of H. siamensis with other fish. While zebra fish (Danio rerio) produced the top hit number with H. siamensis, this does not necessarily imply that zebra fish is more closely related phylogenetically to H. siamensis than common carp (cyprinus carpio). The explanation may simply be that the sequence dataset available for zebra fish is greater than for common carp and that H. siamensis is close to other teleosts, in general. No GSSs identified here matched sequences published for H. siamensis in the database and this is most likely due to the low number of H. siamensis sequences currently available in the NCBI database (only a few mitochondrial DNA sequences were available [6,7]). The H. siamensis GSS sequences generated here will vastly expand the number of genes identified in this species. A significant number of H. siamensis GSSs did not possess coding sequences matching to any sequence in the GenBank database which is not surprising in uncharacterized GSS studies [21,25,29,31]. While most of the anonymous EST sequences probably span non-coding regions of gDNA, or are the result of assembly errors from homopolymers as reported in other EST and 454 pyrosequencing analyses [21,32,33], some may also constitute novel genes unique to this species or may not have been identified previously in teleosts.


Analysis of genome survey sequences and SSR marker development for Siamese Mud Carp, Henicorhynchus siamensis, using 454 pyrosequencing.

Iranawati F, Jung H, Chand V, Hurwood DA, Mather PB - Int J Mol Sci (2012)

Top 25 hit species distribution based on BLASTx. E value cut-off is 10−5. Singleton (A) and contig (B). Bold text indicates teleosts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472715&req=5

f2-ijms-13-10807: Top 25 hit species distribution based on BLASTx. E value cut-off is 10−5. Singleton (A) and contig (B). Bold text indicates teleosts.
Mentions: BLASTx searches of H. siamensis GSS sequences showed that 90 of the 857 (10.5%) contigs and 3751 of the 46,393 (8.1%) singletons possessed significant similarity (E value < 10−5) with proteins in the GenBank non-redundant (nr) database (Table S1). The majority of contigs (62%) and singletons (87%) matched well to reported fish sequences (Figures 2a,b), an outcome that agrees with previous fish studies [20,25,30,31]. Species most represented in the BLASTx searches included zebra fish (Danio rerio), spotted green puffer (Tetraodon nigroviridis), European seabass (Dicentrarchus labrax), carp (Cyprinus carpio), Torafugu (Takifugu rubripes), medaka (Oryzias latipes), Nile tilapia (Oreochromis niloticus), Atlantic salmon (Salmo salar), Florida lancelet (Branchiostoma floridae), and channel catfish (Ictalurus punctatus), largely due to their availability of their sequences in public databases. Sequence homologies are indicative of close evolutionary relationships of H. siamensis with other fish. While zebra fish (Danio rerio) produced the top hit number with H. siamensis, this does not necessarily imply that zebra fish is more closely related phylogenetically to H. siamensis than common carp (cyprinus carpio). The explanation may simply be that the sequence dataset available for zebra fish is greater than for common carp and that H. siamensis is close to other teleosts, in general. No GSSs identified here matched sequences published for H. siamensis in the database and this is most likely due to the low number of H. siamensis sequences currently available in the NCBI database (only a few mitochondrial DNA sequences were available [6,7]). The H. siamensis GSS sequences generated here will vastly expand the number of genes identified in this species. A significant number of H. siamensis GSSs did not possess coding sequences matching to any sequence in the GenBank database which is not surprising in uncharacterized GSS studies [21,25,29,31]. While most of the anonymous EST sequences probably span non-coding regions of gDNA, or are the result of assembly errors from homopolymers as reported in other EST and 454 pyrosequencing analyses [21,32,33], some may also constitute novel genes unique to this species or may not have been identified previously in teleosts.

Bottom Line: Based on GSS-BLASTx results, 10.5% of contigs and 8.1% singletons possessed significant similarity (E value < 10(-5)) with the majority matching well to reported fish sequences.One thousand eight hundred and thirty seven sequences containing SSR motifs were identified, of which 422 qualified for primer design and eight polymorphic loci have been tested with average observed and expected heterozygosity estimated at 0.75 and 0.83, respectively.Regardless of their relative levels of polymorphism and heterozygosity, microsatellite loci developed here are suitable for further population genetic studies in H. siamensis and may also be applicable to other related taxa.

View Article: PubMed Central - PubMed

Affiliation: Earth, Environmental and Biological Sciences, Science and Engineering Faculty, Queensland University of Technology, Brisbane, Queensland 4000, Australia; E-Mails: v.chand@qut.edu.au (V.C.); d.hurwood@qut.edu.au (D.A.H.); p.mather@qut.edu.au (P.B.M.).

ABSTRACT
Siamese mud carp (Henichorynchus siamensis) is a freshwater teleost of high economic importance in the Mekong River Basin. However, genetic data relevant for delineating wild stocks for management purposes currently are limited for this species. Here, we used 454 pyrosequencing to generate a partial genome survey sequence (GSS) dataset to develop simple sequence repeat (SSR) markers from H. siamensis genomic DNA. Data generated included a total of 65,954 sequence reads with average length of 264 nucleotides, of which 2.79% contain SSR motifs. Based on GSS-BLASTx results, 10.5% of contigs and 8.1% singletons possessed significant similarity (E value < 10(-5)) with the majority matching well to reported fish sequences. KEGG analysis identified several metabolic pathways that provide insights into specific potential roles and functions of sequences involved in molecular processes in H. siamensis. Top protein domains detected included reverse transcriptase and the top putative functional transcript identified was an ORF2-encoded protein. One thousand eight hundred and thirty seven sequences containing SSR motifs were identified, of which 422 qualified for primer design and eight polymorphic loci have been tested with average observed and expected heterozygosity estimated at 0.75 and 0.83, respectively. Regardless of their relative levels of polymorphism and heterozygosity, microsatellite loci developed here are suitable for further population genetic studies in H. siamensis and may also be applicable to other related taxa.

No MeSH data available.


Related in: MedlinePlus