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SNP identification, verification, and utility for population genetics in a non-model genus.

Williams LM, Ma X, Boyko AR, Bustamante CD, Oleksiak MF - BMC Genet. (2010)

Bottom Line: In F. grandis, SNPs were less polymorphic but still established isolation by distance.Markers differentiated species and populations.In total, these approaches were used to quickly determine differences within the Fundulus genome and provide markers for population genetic studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149, USA.

ABSTRACT

Background: By targeting SNPs contained in both coding and non-coding areas of the genome, we are able to identify genetic differences and characterize genome-wide patterns of variation among individuals, populations and species. We investigated the utility of 454 sequencing and MassARRAY genotyping for population genetics in natural populations of the teleost, Fundulus heteroclitus as well as closely related Fundulus species (F. grandis, F. majalis and F. similis).

Results: We used 454 pyrosequencing and MassARRAY genotyping technology to identify and type 458 genome-wide SNPs and determine genetic differentiation within and between populations and species of Fundulus. Specifically, pyrosequencing identified 96 putative SNPs across coding and non-coding regions of the F. heteroclitus genome: 88.8% were verified as true SNPs with MassARRAY. Additionally, putative SNPs identified in F. heteroclitus EST sequences were verified in most (86.5%) F. heteroclitus individuals; fewer were genotyped in F. grandis (74.4%), F. majalis (72.9%), and F. similis (60.7%) individuals. SNPs were polymorphic and showed latitudinal clinal variation separating northern and southern populations and established isolation by distance in F. heteroclitus populations. In F. grandis, SNPs were less polymorphic but still established isolation by distance. Markers differentiated species and populations.

Conclusions: In total, these approaches were used to quickly determine differences within the Fundulus genome and provide markers for population genetic studies.

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Design of 454 pyrosequencing contig generated from the digestion of genomic DNA with restriction enzymes (EcoRI and BspEI), the addition of restriction site specific linkers, an individual barcode and a 454 amplicon adapter.
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Figure 2: Design of 454 pyrosequencing contig generated from the digestion of genomic DNA with restriction enzymes (EcoRI and BspEI), the addition of restriction site specific linkers, an individual barcode and a 454 amplicon adapter.

Mentions: Primers (Table 1) specific to the EcoRI restriction site were generated with the goals of labeling the DNA fragments from each individual with specific nucleotide barcodes [56] and preparing those samples for emulsion-based amplification. Starting at the 5' end, 19 nucleotides (Table 1) complementary to the primer on the DNA capture beads used in the emulsion PCR reaction [57] were synthesized (Integrated DNA Technologies). Following those nucleotides, each primer had a distinct 10 base pair barcode [56] used to identify individuals (ten primers in total). The final 19 base pairs of the primer were specific to the EcoRI adapter. The BspE1 primer (Table 1) started at its 5' end with 19 nucleotides (Table 1), which were complementary to the primer on the DNA capture beads followed by 18 base pairs specific to the BspE1 adapter (Figure 2). All primers were HPLC purified. Amplified selective fragments were diluted (1:10) and added to both EcoRI and BspE1 primers (Integrated DNA Technologies; 10 pmol) in a 25 μL volume. PCR conditions were 94°C for 10 sec, 50°C for 30 sec, and 72°C for one minute and were carried out for 30 cycles. PCR reactions were pooled into eight wells, where each of the ten distinct barcodes was represented only once in each of the pools. Each pool of PCR products was purified using QIAquick PCR Purification Kit (Qiagen, USA). PCR products were further purified with AMPure (Agencourt).


SNP identification, verification, and utility for population genetics in a non-model genus.

Williams LM, Ma X, Boyko AR, Bustamante CD, Oleksiak MF - BMC Genet. (2010)

Design of 454 pyrosequencing contig generated from the digestion of genomic DNA with restriction enzymes (EcoRI and BspEI), the addition of restriction site specific linkers, an individual barcode and a 454 amplicon adapter.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874759&req=5

Figure 2: Design of 454 pyrosequencing contig generated from the digestion of genomic DNA with restriction enzymes (EcoRI and BspEI), the addition of restriction site specific linkers, an individual barcode and a 454 amplicon adapter.
Mentions: Primers (Table 1) specific to the EcoRI restriction site were generated with the goals of labeling the DNA fragments from each individual with specific nucleotide barcodes [56] and preparing those samples for emulsion-based amplification. Starting at the 5' end, 19 nucleotides (Table 1) complementary to the primer on the DNA capture beads used in the emulsion PCR reaction [57] were synthesized (Integrated DNA Technologies). Following those nucleotides, each primer had a distinct 10 base pair barcode [56] used to identify individuals (ten primers in total). The final 19 base pairs of the primer were specific to the EcoRI adapter. The BspE1 primer (Table 1) started at its 5' end with 19 nucleotides (Table 1), which were complementary to the primer on the DNA capture beads followed by 18 base pairs specific to the BspE1 adapter (Figure 2). All primers were HPLC purified. Amplified selective fragments were diluted (1:10) and added to both EcoRI and BspE1 primers (Integrated DNA Technologies; 10 pmol) in a 25 μL volume. PCR conditions were 94°C for 10 sec, 50°C for 30 sec, and 72°C for one minute and were carried out for 30 cycles. PCR reactions were pooled into eight wells, where each of the ten distinct barcodes was represented only once in each of the pools. Each pool of PCR products was purified using QIAquick PCR Purification Kit (Qiagen, USA). PCR products were further purified with AMPure (Agencourt).

Bottom Line: In F. grandis, SNPs were less polymorphic but still established isolation by distance.Markers differentiated species and populations.In total, these approaches were used to quickly determine differences within the Fundulus genome and provide markers for population genetic studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33149, USA.

ABSTRACT

Background: By targeting SNPs contained in both coding and non-coding areas of the genome, we are able to identify genetic differences and characterize genome-wide patterns of variation among individuals, populations and species. We investigated the utility of 454 sequencing and MassARRAY genotyping for population genetics in natural populations of the teleost, Fundulus heteroclitus as well as closely related Fundulus species (F. grandis, F. majalis and F. similis).

Results: We used 454 pyrosequencing and MassARRAY genotyping technology to identify and type 458 genome-wide SNPs and determine genetic differentiation within and between populations and species of Fundulus. Specifically, pyrosequencing identified 96 putative SNPs across coding and non-coding regions of the F. heteroclitus genome: 88.8% were verified as true SNPs with MassARRAY. Additionally, putative SNPs identified in F. heteroclitus EST sequences were verified in most (86.5%) F. heteroclitus individuals; fewer were genotyped in F. grandis (74.4%), F. majalis (72.9%), and F. similis (60.7%) individuals. SNPs were polymorphic and showed latitudinal clinal variation separating northern and southern populations and established isolation by distance in F. heteroclitus populations. In F. grandis, SNPs were less polymorphic but still established isolation by distance. Markers differentiated species and populations.

Conclusions: In total, these approaches were used to quickly determine differences within the Fundulus genome and provide markers for population genetic studies.

Show MeSH