Transcript Analyzing Dallia pectoralis Population Genetics Using

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 Analysis
Sequencing Mitochondrial DNA
Microsatellites
Genotyping Microsatellites
454 sequencing
Developing 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
Collection Localities
North Slope: 2
Interior:
4
Coast:
8
Russia:
3
=17 Sampling sites
24
24 sampling locations
Interior
Coast
Russia
North Slope
Sequencing mtDNA
Coast
Galena
Coast
Naknek: 10
Galena: 17
Microsatellites
AGTA CCGTACTGAGCGTACTGAG AGTAGTAGTAGTAGT ACGGCAAATCGTATATTCGA GCG
Biparentally inherited
Mutation rate
Saturate the genome
ACACAC AC ACACACAC
Dinucleotide ACACACACACACACA
CGACG ACG ACGACGACG Trinucleotide ACGACGACGACGA
TACGT ACGT ACGTACGT Tetranucleotide ACGTACGTACG
Genotyping Microsatellites
Indiv idual
Galena 1
Galena 2
Goldstream v alley 85
Goldstream v alley 86
Iev ineem Riv er 150
Nov oe Chaplino 145
North Slope 78
North Slope 79
Number of alleles
Dpec 14
196
194
198
198
202
200
204
204
(2nd allele)
208
206
198
198
202
200
204
204
8
Dpec 31
245
245
245
245
245
245
245
245
(2nd allele)
247
247
245
245
245
245
245
245
2
Dinucleotide differences:
North Slope & Russia 1 repeat
North Slope & Interior 3 repeats
North Slope & Galena 5 repeats [at most]
Dpec C03
190
188
190
190
190
190
190
190
(2nd allele)
190
192
190
190
190
190
190
190
3
Developing Microsatellites Using 454 Sequencing
Initial study:
2009
13 polymorphic microsatellite loci
-66%
-33%
7,000
=10-100
microsatellites
microsatellites
Primer Design Process
1. Extract DNA from one individual
1. Fresh Fairbanks Fish
2. Check DNA quality
2. Nanodrop
3. Export to facility
3. Duke genome sequencing and
analysis facility
4. Filter sequence results
4. Msatcommander
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
Msatcommander search
6 repeats
12 repeats
247 microsatellites
65 Microsatellites
62 primers
10 primers
27 candidate primers
5 candidate primers
Locus Forward sequence
Reverse sequence
Mot if Number of repeat s Expect ed product size
B3 C Y D C A C T C G T C C A C A T C G T T A C A T
CG T C T G C C T G C C T T G T C T G
ACAG
6
123
C H 3 U 3 C A G T G G C C T T T G A A A C C A G GC A G T A A C A T G C A G C A G T C C GA C A T
7
170
BSN RK T C A C T C C C A C C T C T G T T C A G C T G T G T G C C G A G A C A A A G T C A A A C
6
235
C C 1 2 S C C G G C A G A G C C C T A G T A T T TA G G T C C G G C A T C T A G A G A C A T G T C
14
443
BWT JF C C C A A C A A C C T A C T C A T C T ATCACC C C G G A A T T A G T C T C G C G A C C T
6
145
C LL6 K GA G A C G C T T A GT T T G GC A A T G
T C C T C G T C C T C A C A T C A A C C T GC C
6
178
B4 Y 2 6 A G C G T A A T G G T T G A A A G G C GA T C C C G C A C C G A A A T T C A A GT G A G
7
288
C E RI K C A A T A C C G A G T C G A T G C G T GT T T G G A T G G G C T T T G C T T C C A C A G
11
422
B0 BO 1 G G T T G G C C C T T T C T A C T T C C G A T G T G A G C T C C T G G G T T T G T G G C
7
177
B0 H 2 X T C A T C A A T G G G G A G C T T T T C T T C T T C A C C C G G A C A C T T T C A C A G
7
176
B6 A O 8 C C G G C A G A G C C C T A G T A T T TA G G T C C G G C A T C T A G A G A C A T G T C
12
426
BU D D J C A C T C G T C C A C A T C G T T A C A T
CG T C T G C C T G C C T T G T C T G
GT C T
6
123
C F8 Q 9 G A A T G T G G T G A T G G A C T G G GT G T A C T T C T C A G C C A C C C A G A C A G
8
144
B9 6 1 8 T G T T A A G G T G C A T T G T C T G G CA G G A A C C C A G G A C A T C A C A CT G T A
8
120
C E Y SJ C T A A G G A G C A G A G G A C A G C CC C C A T C T A G T C C A G G C A G T G T T T A
6
174
C D 3 4 3 A C C A C T G T C A A T C A T G C A C TC
GC T G C C T G A T T G T C T T A T G G CA C A G
6
294
BQ 0 L T T C A T C A A T G G G G A G C T T T T C A T T T G T G T G T C A G G C T T G C
ACAG
7
174
BQ 0 ZY T G T T A A G G T G C A T T G T C T G G CA G G A A C C C A G G A C A T C A C A CA C A T
8
120
B2 BSZ T T T G G A T G G G C T T T G C T T C C C C C T G A A A C G A G A C T T A C A G T
A CCT G
14
167
BQ O 0 8 A A G A C G G A G G G C T T G A A A G GT A T C T G C A C T G T C C A G C C T C A A G G
6
118
C F5 P S T G T G T G C C C A G T C T C T C A C C C C T G A A A C G A G A C T T A C A G A CCA G
11
406
C B0 C J G C C C T G T C T G T T G G T T T C T G G A G A G A G G C G A T T G A T G G A C T G C C
8
131
C E 8 X4 A A G A A A T G C A G C G G A A T C A CT G A A G G T C A A A T G C A C C A G A T G C G
6
165
C JL 5 C G C C A T C A C A A T C G C T T C C T CT C C C A A C A T G C A C A A A G A C CT T T G
6
299
C E G T V G A G A A G C A C A A A C C T C A G C CT G C C T C T G A C A T C T G G A A A C T C T G
7
190
B0 L K 4 G T C G C T T C C G T T A C A G C A T T T T G C C C T C C A T T G T T C T A G G A G A T
6
191
BXJ 8 S T C C T T T C A C A T C C A C C C A C CG A C G A A C A G A T A A C C G C G A CA C A G
6
144
27 six repeat primer candidates
and
5 twelve repeat primer candidates
Prim er
B74AX
BWB5P
C GIUZ
C LC 8X
B2BSZ
F orward primer sequence
Reverse prim er sequence Motif Num ber of repeats
ATG CTG GTC AGG CTA AGA GG
TTA AAT CGC TCT GCC CTG TG
A C GC
13
CAA ATG GGA CCC TGT TGA CG
ACA AGA GAA ATG AGC CAC CAT G
ACAG
13
GAG GTC CGG CAT CTA GAG AC
ACT AAA GAC TTC CGT GGA GAG G
ACAG
12
ACA AGA GAA ATG AGC CAC CAT G
GAG GTC CGG CAT CTA GAG AC
T GT C
12
TTT GGA TGG GCT TTG CTT CC
CCC TGA AAC GAG ACT TAC AGA C
T GT C
14
N Interior North Slope Coast
Galena
Interior North Slope Coast
Galena
N
Interior North Slope
L
CLC8X
B74AX
B2BSZ
12x trial
B2BSZ
Coast
Galena
BWBSP
CGIUZ
N
Interior North Slope Coast
Galena
N
Interior North Slope Coast Galena N
L
Future Research
Galena sequencing
6 repeat microsatellites
12 repeat microsatellites
Candidate primer genotyping
Funded by:
EPSCoR
Acknowledgements
Hosted by:
the Lopez lab
Mathew Campbell
Partial funding from:
 Mentor
The Center for Research
Services
INBRE
Andres Lopez
IDea Network for Biomedical Research
 Advisor
Questions