Analyzing nuclear and mitochondrial genetic diversity in the Alaska Blackfish (Dallia pectoralis)

Rachel DeWilde University of Alaska Fairbanks EPSCoR All-Hands Meeting 2011

Overview

The Alaska Blackfish

Genetic AnalysisSequencing Mitochondrial DNA

MicrosatellitesGenotyping Microsatellites

454 sequencingDeveloping primer candidates

The Alaska Blackfish

Fresh water fish

Poor swimmers

Short life span

Prefer shallow vegetated habitats

Geographic Barriers

Brooks range

Yukon River

Tanana River

North Slope: 2

Interior: 4

Coast: 8

Russia: 3

=17 Sampling sites

Collection Localities

24

24 sampling locations

Galena

Interior

North Slope

Coast

Russia

Coast

Coast

Naknek: 10 Galena: 17

Sequencing mtDNA

Biparentally inherited

Mutation rate

Saturate the genome

AGTA CCGTACTGAGCGTACTGAG AGTAGTAGTAGTAGT ACGGCAAATCGTATATTCGA GCG

Microsatellites

ACACAC AC ACACACAC Dinucleotide ACACACACACACACA

ACGACG ACG ACGACGACG Trinucleotide ACGACGACGACGA

GTACGT ACGT ACGTACGT Tetranucleotide ACGTACGTACG

Individual Dpec 14 (2nd allele) Dpec 31 (2nd allele) Dpec C03 (2nd allele)Galena 1 196 208 245 247 190 190Galena 2 194 206 245 247 188 192Goldstream valley 85 198 198 245 245 190 190Goldstream valley 86 198 198 245 245 190 190Ievineem River 150 202 202 245 245 190 190Novoe Chaplino 145 200 200 245 245 190 190North Slope 78 204 204 245 245 190 190North Slope 79 204 204 245 245 190 190Number of alleles 8 2 3

Dinucleotide differences:

North Slope & Russia 1 repeat

North Slope & Interior 3 repeats

North Slope & Galena 5 repeats [at most]

Genotyping Microsatellites

Developing Microsatellites Using 454 Sequencing

7,000 microsatellites

Initial study:

2009

13 polymorphic microsatellite loci

-66% -33%=10-100

microsatellites

Primer Design Process

1. Extract DNA from one individual

2. Check DNA quality

3. Export to facility

4. Filter sequence results

1. Fresh Fairbanks Fish

2. Nanodrop

3. Duke genome sequencing and

analysis facility

4. Msatcommander

Msatcommander search

Duke Results

123,109 microsatellites

Microsatellite repeat Number of repeats Number of potential primers

Dinucleotide 12,100 4,600

Trinucleotide 320 100

Tetranucleotide 250 60

247 microsatellites

62 primers

27 candidate primers

65 Microsatellites

10 primers

5 candidate primers

6 repeats 12 repeats

27 six repeat primer candidatesand

5 twelve repeat primer candidates

Primer Forward primer sequence Reverse primer sequenceMotif Number of repeatsB74AX ATG CTG GTC AGG CTA AGA GG TTA AAT CGC TCT GCC CTG TG ACGC 13BWB5P CAA ATG GGA CCC TGT TGA CG ACA AGA GAA ATG AGC CAC CAT G ACAG 13CGIUZ GAG GTC CGG CAT CTA GAG AC ACT AAA GAC TTC CGT GGA GAG G ACAG 12CLC8X ACA AGA GAA ATG AGC CAC CAT G GAG GTC CGG CAT CTA GAG AC TGTC 12B2BSZ TTT GGA TGG GCT TTG CTT CC CCC TGA AAC GAG ACT TAC AGA C TGTC 14

Locus Forward sequence Reverse sequence Motif Number of repeats Expected product sizeB3CYD CAC TCG TCC ACA TCG TTA CAC TGT CTG CCT GCC TTG TCT G ACAG 6 123CH3U3 CAG TGG CCT TTG AAA CCA GG CAG TAA CAT GCA GCA GTC CG ACAT 7 170BSNRK TCA CTC CCA CCT CTG TTC AG CTG TGT GCC GAG ACA AAG TC AAAC 6 235CC12S CCG GCA GAG CCC TAG TAT TT AGG TCC GGC ATC TAG AGA CA TGTC 14 443BWTJF CCC AAC AAC CTA CTC ATC TAC C TAC CCG GAA TTA GTC TCG CG ACCT 6 145CLL6K GAG ACG CTT AGT TTG GCA ATG TCC TCG TCC TCA CAT CAA CC TGCC 6 178B4Y26 AGC GTA ATG GTT GAA AGG CG ATC CCG CAC CGA AAT TCA AG TGAG 7 288CERIK CAA TAC CGA GTC GAT GCG TG TTT GGA TGG GCT TTG CTT CC ACAG 11 422B0BO1 GGT TGG CCC TTT CTA CTT CC GAT GTG AGC TCC TGG GTT TG TGGC 7 177B0H2X TCA TCA ATG GGG AGC TTT TC TTC TTC ACC CGG ACA CTT TC ACAG 7 176B6AO8 CCG GCA GAG CCC TAG TAT TT AGG TCC GGC ATC TAG AGA CA TGTC 12 426BUDDJ CAC TCG TCC ACA TCG TTA CAC TGT CTG CCT GCC TTG TCT G GTCT 6 123CF8Q9 GAA TGT GGT GAT GGA CTG GG TGT ACT TCT CAG CCA CCC AG ACAG 8 144B9618 TGT TAA GGT GCA TTG TCT GGC AGG AAC CCA GGA CAT CAC AC TGTA 8 120CEYSJ CTA AGG AGC AGA GGA CAG CC CCC ATC TAG TCC AGG CAG TG TTTA 6 174CD343 ACC ACT GTC AAT CAT GCA CTG CCT GCC TGA TTG TCT TAT GGC ACAG 6 294BQ0LT TCA TCA ATG GGG AGC TTT TC ATT TGT GTG TCA GGC TTG C ACAG 7 174BQ0ZY TGT TAA GGT GCA TTG TCT GGC AGG AAC CCA GGA CAT CAC AC ACAT 8 120B2BSZ TTT GGA TGG GCT TTG CTT CC CCC TGA AAC GAG ACT TAC AGA C TCTG 14 167BQO08 AAG ACG GAG GGC TTG AAA GG TAT CTG CAC TGT CCA GCC TC AAGG 6 118CF5PS TGT GTG CCC AGT CTC TCA C CCC TGA AAC GAG ACT TAC AGA C ACAG 11 406CB0CJ GCC CTG TCT GTT GGT TTC TG GAG AGA GGC GAT TGA TGG AC TGCC 8 131CE8X4 AAG AAA TGC AGC GGA ATC AC TGA AGG TCA AAT GCA CCA GA TGCG 6 165CJL5C GCC ATC ACA ATC GCT TCC TC TCC CAA CAT GCA CAA AGA CC TTTG 6 299CEGTV GAG AAG CAC AAA CCT CAG CC TGC CTC TGA CAT CTG GAA AC TCTG 7 190B0LK4 GTC GCT TCC GTT ACA GCA TT TTG CCC TCC ATT GTT CTA GG AGAT 6 191BXJ8S TCC TTT CAC ATC CAC CCA CC GAC GAA CAG ATA ACC GCG AC ACAG 6 144

12x trial

N Interior North Slope Coast Galena Interior North Slope Coast Galena N Interior North Slope

Interior North Slope Coast Galena NCoast Galena N Interior North Slope Coast Galena N

L

L

CLC8X B74AX

B2BSZ CGIUZ BWBSP

B2BSZ

Future Research

Galena sequencing

6 repeat microsatellites

12 repeat microsatellites

Candidate primer genotyping

Funded by: EPSCoR

Partial funding from:

The Center for Research

Services

INBREIDea Network for Biomedical Research

Hosted by: the Lopez lab

Mathew Campbell Mentor

Andres Lopez Advisor

Questions

Acknowledgements