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