Isolation, characterization and predicted
genome locations of ruff (Philomachus
pugnax, AVES) microsatellite loci
Lindsay L. Farrell, Deborah A. Dawson,
Gavin J. Horsburgh, Terry Burke &
David B. Lank
Conservation Genetics Resources
ISSN 1877-7252
Conservation Genet Resour
DOI 10.1007/s12686-012-9639-0
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Author's personal copy
Conservation Genet Resour
DOI 10.1007/s12686-012-9639-0
TECHNICAL NOTE
Isolation, characterization and predicted genome locations of ruff
(Philomachus pugnax, AVES) microsatellite loci
Lindsay L. Farrell • Deborah A. Dawson
Gavin J. Horsburgh • Terry Burke •
David B. Lank
•
Received: 26 March 2012 / Accepted: 28 March 2012
Ó Springer Science+Business Media B.V. 2012
Abstract We identified 247 unique ruff (Philomachus
pugnax) microsatellite sequences. Primer sets were
designed from 102 selected loci and tested in 12–24 individuals from a captive population. Sequence homology was
used to assign locations in the chicken (Gallus gallus) and/
or zebra finch (Taeniopygia guttata) genome for the
majority of these loci. Fifty-two loci were found to be
polymorphic and 47 of these were typed in known families.
Forty-six loci displayed Mendelian inheritance including
Ppu058, which was confirmed to be Z-linked by the
complete absence of any heterozygous females.
Keywords Lek Male morphs Microsatellite Scolopacidae Shorebird Simple tandem repeat (STR)
We have isolated and characterized microsatellite markers
for the ruff (Philomachus pugnax). The ruff is a lekking
shorebird with an autosomal genetic polymorphism for
male mating behaviour (Lank et al. 1995) and belongs to
the Scolopacidae family. A proportion of the ruff microsatellites isolated will be of utility in closely related species
(Primmer et al. 1996), including other species of Scolopacidae (n = 88, Sibley and Monroe 1990). Within this
family, several species are of conservation concern,
including the critically endangered spoon-billed sandpiper
L. L. Farrell D. A. Dawson G. J. Horsburgh T. Burke
Department of Animal and Plant Sciences, University of
Sheffield, Western Bank, Alfred Denny Building, Sheffield S10
2TN, UK
L. L. Farrell (&) D. B. Lank
Department of Biological Sciences, Simon Fraser University,
Burnaby, BC V5A 1S6, Canada
e-mail: lfarrell@sfu.ca
(Eurynorhynchus pygmeus; IUCN Red List, Birdlife
International).
Blood was collected from captive ruff individuals at
Simon Fraser University, Canada (population maintained
by DBL since 1985), and stored in absolute ethanol.
Genomic DNA was extracted using ammonium acetate
(Nicholls et al. 2000).
Two ruff microsatellite-enriched libraries were created
following Armour et al. (1994). For both libraries, genomic
DNA from a single female ruff (Bird ID 6233) was
digested with MboI, size selected (178–856 bp) and enriched for (CA)n, (CAG)n, (GCC)n, (TTTC)n and their
complements. The first library was additionally enriched
for (AT)n. Clones were sequenced in both directions using
BigDye terminators and a consensus sequence created. We
used BlastN 2.2.4 software (Altschul et al. 1997) to check
for duplication of new and published ruff microsatellites
(n = 9 published, Thuman et al. 2002). In total, 247 unique
microsatellite sequences were identified and primers
designed for 102 loci using PRIMER3 (Rozen and Skaletsky 2000; Tables 1, 2). Primers were designed for just
three of the 104 sequences that contained only mononucleotide (A or T) repeats (Ppu017, Ppu033 and Ppu071).
One hundred and two markers were tested in 12–24
known-sex individuals using multiplex PCR. Each 2-ll PCR
contained approximately 10 ng of genomic DNA, 0.2 lM of
each primer and 1 ll Qiagen Multiplex PCR Mix (Qiagen
Inc.; Kenta et al. 2008). PCR amplification was performed
using a DNA Engine Tetrad 2 Thermal Cycler (MJ Research,
BioRad UK) with the profile: 15 min at 95 °C, followed by
35 cycles of 94 °C for 30 s, annealing temperature
(Tables 1, 2) for 90 s, 72 °C for 1 min, and a final step of
60 °C for 10 min. Products were loaded on an ABI3730
Genetic Analyzer (Applied Biosystems) using ROX500 size
standard and genotypes scored with GENEMAPPER v4.0
123
123
Ppu022
Ppu021
Ppu020
Ppu019
Ppu018
Ppu017
Ppu016
Ppu015
Ppu014
Ppu013
Ppu012
Ppu011
Ppu010
Ppu009
Ppu008
Ppu007
Ppu006
Ppu005
Ppu004
Ppu003
HE616911
Ppu001
24C08
HE616932
24C05
HE616931
24B05
HE616930
24A01
HE616929
6G09
HE616928
6F12
HE616927
5E02
HE616926
5B12b
HE616925
5A10
HE616924
4H10
HE616923
4G03
HE616922
4F08
HE616921
4D09
HE616920
4D05
HE616919
4D01
HE616918
4C09
HE616917
4A08
HE616916
3H04
HE616915
3E05
HE616914
3E04
HE616913
3C02
EMBL
acc. no.
& clone
name
Locus
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Lib
2
2
1
1
11
11
–
3
2
2
–
4
1
1
2
2
24
–
3
3
3
3
3
3
4A
4
4
4
–
3
3
5
5
8
8
2
2
1
1
1A
1
CH
chr
ZF
chr
79772128
75106465
61096046
156510069
71134
19600964
–
84720681
71904153
61061152
–
52883524
65490386
166535076
22065196
19888521
3112345
–
18163845
18786830
56310381
51033809
49883043
57218799
2561871
6796049
29737180
23020195
–
76352543
76776326
16041469
17130526
19350034
22771586
92082626
89244831
13670975
122413141
50776302
52975585
Chicken
chr loc. T
–
Zebra
finch
chr loc. T
–
(ATAGAT)9
6-FAM
R: GGGAAACATCATGCAACAAC
F: TGAATGCATGAATTAGGTAGTGG
1.20e-37
8.90e-20
F: AAAGCTTGTAAGCTCTAAGCAATACC
6-FAM
R: AGGCTATTGACACTTCACAAAGG
(CTAT)12
3.90e-57
4.00e-81
R: GCGGTATTTCTGGCCTAGC
F: TCCTGTCCTGTCCTTGGAAC
HEX
3.10e-20
6.70e-25
R: GCTACTGGGTGCTGTTACTTCC
–
(GT)13
F: TAACCCACGAGTGGCTCTG
HEX
7.60e-10
(AC)11
R: AGAGATACAGTAAGCTTCGTATGACAGACAC
F: TGCCTTCTTACTTTCTCAATATTTGTGG
HEX
1.90e-28
4.90e-38
R: GTGCTACTGAAATCGTCTGATGTTGG
–
(AGAT)13
F: GTTGGCCTGGACTCCGTCTG
HEX
6.00e-06
(T)14
R: TCAAAGACTTCTGCAAAGTTATTCTTCTAAGC
HEX
F: TCAGGCAGTGGGACTAGATGATTG
(TC)12
1.10e-22
5.70e-13
R: TGACTTTGGAGGTTGTTACTTATTGTTGTC
F: GGTCCAGTTCTGTGTGCCAGTTT
HEX
1.70e-54
(AC)5
F: CAACCCCATCTCCTGGCTTTT
9.80e-27
6-FAM
R: TGCTCCATGGAATCAAACATGG
R: CAGCTCGGTACATTGGTGCTTG
(GT)10
HEX
1.20e-30
–
(AG)6
F: ACATGCTCCTCTTCCATTTGCAG
3.80e-63
1.40e-61
F: TGCAAGGCCAGGTAGAAACAAG
6-FAM
R: ACCAAGGCATTACTGTGTTGGAAG
(AC)6
4.90e-03
1.20e-32
R: TGGACTGAAGGTGACTATTCTGCTG
HEX
F: CGCACATCTGCTGTTGAGAAATC
(AC)5
3.40e-63
6.40e-43
R: GATGTGAACTACCTGCAAATCCACAG
F: GGGAGCTCAGGGATGCAGTG
HEX
F: TCTTTATGATGCTATTTGAGGGTTTGG
2.20e-37
(GT)12
F: GAAGTTCCTCTTACCAATTTGCTTGC
R: TGACCTGCTGGTACTCCACCAC
6.90e-17
HEX
6-FAM
R: CCTATTCATGTCTCCAAGTTCAATCC
R: AATGCCACTGCACCAGAAGTAGTC
(AC)12
(AC)12
6-FAM
3.70e-46
5.90e-64
–
(GT)5
F: GCCAGAGTAGCAACAGTCAGTGC
4.50e-52
4.00e-27
F: TGGAAGTGGAAGGAGGTCTGTG
6-FAM
R: TCCACTCAGGTGCAGGCTTC
(GT)9
8.10e-70
2.70e-53
F: GGAGCAATGTGATACCACTAAGGACTG
6-FAM
R: CTCCTGACCTCCACCGCAAC
(TC)5
4.40e-73
8.00e-108
F: TTGGCACCAATAGTTGCCTCA
6-FAM
R: CCTCTTCAGAGGAACAAAGCAAGA
(TCTA)10
9.50e-79
8.80e-84
R: AGCATGTAGTGCTTCAGTTATTTAGATGC
F: CAGGATTGCTTTGGCTGGAG
HEX
1.20e-27
(CTAT)11
F: ACCAGGCTTCTTCCCTCTGGA
5.60e-61
HEX
Primer sequence 50 –30
R: TGAAACTTCACATTTTGGGGATGA
(TAGA)12
1.00e-07
Fluoro
label
(F)
3.30e-27
Repeat
motif
E-value
in Gga
E-value
in ZF
722
791
367
407
536
414
584
693
431
542
495
371
530
436
427
577
534
480
646
337
494
Seq.
length
(bp)
Table 1 Characterization and predicted chromosome locations of 52 ruff (Philomachus pugnax) microsatellite loci
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
N
Y
Y
MI
58.83
59.02
59.70
59.92
59.82
60.09
60.19
59.84
63.51
63.74
65.42
65.16
65.15
64.74
64.80
64.51
65.07
64.61
64.86
65.17
63.55
63.69
63.99
64.29
65.06
65.55
63.91
64.36
64.68
64.41
63.66
63.20
63.80
63.90
65.42
65.13
62.9
60.8
63.18
63.13
64.67
64.65
Tm
(°C)
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
56
PCR
Ta
(°C)
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
24
n1
23
15
23
20
24
10
24
20
24
17
23
21
23
24
24
13
22
24
10
23
24
n2
7
5
7
5
8
2
6
3
6
2
2
3
5
12
3
2
2
2
3
4
4
A
297
319
246
150
250
223
299
248
201
230
219
222
371
226
300
293
250
227
230
274
197
Exp
allele
size
(bp)
°
267–302
(296, 296)
312–329
(321, 321)
245–302
(243, 245)
145–156
(151, 154)
242–270
(250, 258)
207–218
(227, 227)
212–227
NT
244–278
(247, 247)
194–204
(214, 220)
223–228
(229, 229)
214–222
NT
216–223
(223, 223)
221–233
NT
317–474
(436-436)
298–302
(299, 299)
281–290
(294, 294)
241–246
(245, 251)
216–220
(221, 221)
192–233
NT
367–373
(366, 371)
275–287
(275, 279)
Obs. allele
size
(ruff 6233)
& range
(bp) ¥
0.91
0.40
0.52
0.45
0.79
0.10
0.62
0.40
0.62
0.35
0.69
0.57
0.17
0.95
0.20
0.38
0.31
0.12
0.10
0.65
0.62
HO
0.80
0.70
0.44
0.69
0.78
0.52
0.62
0.51
0.72
0.47
0.48
0.51
0.71
0.89
0.19
0.40
0.48
0.11
0.59
0.70
0.57
HE
0.61
0.04§
1.00
0.02§
0.04
0.02§
0.05
0.65
0.19
0.50
0.19?
1.00
0.00§
1.00
1.00
1.00
0.06
1.00
0.11§
0.30
0.12
pHWE*
Author's personal copy
Conservation Genet Resour
Ppu046
Ppu043
Ppu042
Ppu041
Ppu040
Ppu039
Ppu038
Ppu037
Ppu036
Ppu035
Ppu034
Ppu033
Ppu032
Ppu031
Ppu030
Ppu029
Ppu028
Ppu027
Ppu025
Ppu024
HE616933
Ppu023
3C03
HE616956
5A12
HE616953
3A02
HE616952
6D06
HE616951
4G04
HE616950
3E11
HE616949
3D04
HE616948
6A11
HE616947
4A01
HE616946
4H11
HE616945
67A09
HE616944
65A09
HE616943
65A08
HE616942
64C01
HE616941
64A02
HE616940
63G02
HE616939
63D04
HE616938
63D02
HE616937
63C04
HE616935
63A05
HE616934
33C07
EMBL
acc. no.
& clone
name
Locus
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
Lib
Table 1 continued
4
4
2
2
11
11
11
11
5
5
1A
1
2
2
26
26
10
10
3
3
10
10
8
8
2
2
13
13
2
2
10
10
1
1
7
7
–
1
–
13
7
7
CH
chr
ZF
chr
37989476
41459422
33590280
50681577
15017303
9474078
15593478
10001495
321716
2524315
49840927
52147383
101346526
98252724
1368934
2193405
895537
4306840
104595407
104574877
10268721
12101849
27910568
30604045
49636829
29908799
16024608
1071128
122215464
120354672
7745116
9185554
108220670
130142524
19299021
15230709
–
24109016
–
9775634
3954280
29587083
Chicken
chr loc. T
–
Zebra
finch
chr loc. T
–
6-FAM
R: TGACACACAGGTTTGGAA
F: TCGTCTGATTTGTATTGTTCTT
(GT)10
1.90e-138
2.20e-42
R: GACTTTGGTAGGTCTTCTGGTTCCTG
HEX
F: CTCTCCAGGTTGCCTAAAGTCTATGG
(GT)7
8.30e-54
2.10e-05
R: TTGTTCACATTGAGAAGTTGATGAC
HEX
F: TGGTCCCTTCCAACTCTAGAAA
(TTTC)48
1.0e-139
3.80e-07
R: AGCAGACCGGAGAAGCAACA
HEX
F: TGATTTTCCGAAACAAGTTTTAATCG
(AC)9
6.50e-56
1.90e-71
R: GGAACGATGTGGGTTACTTCCAG
HEX
F: CTCCTGGCTGCGTTGTTCTG
(GT)9
1.20e-59
1.90e-58
R: CTTGCCATTCAGGTTAAGTACACTTCC
6-FAM
F: GCAACTGCTGCACTCCCAAC
(TGAT)6
1.50e-73
1.30e-52
R: TTAATATGGCAGCCTTACCTAACGAAAC
HEX
F: CATGACTACCTATCGAATCCTCTTTGG
(GAAA)5
1.70e-42
4.10e-22
R: TCCATATTTATTACAGCCCAGAAGACC
HEX
F: CTCTTGTGGTACCTGGAAGAGGTG
(GT)6
1.10e-19
3.30e-50
R: TTTCCCAGCATGACATACATTGC
6-FAM
F: AGACCCGGGTGTTCAAGGTG
(GT)7
2.10e-33
1.20e-46
R: TTGAACGTTTGGGACAGGTGAC
6-FAM
F: AAGCTTGTCTTCTGGAATGAAGCAG
(GT)9
2.00e-14
7.90e-37
R: CCACCCTTCATATTGACTCG
HEX
F: CTCCATGGACCAGAAATGAG
(AAT)6
6.40e-37
3.20e-56
R: GATCATCTAGTCTAACCATCAACTCTG
HEX
F: AGTTGTTGAGGCACCATCC
(G)12
1.80e-07
9.00e-27
R: TAAGAGGTTGCCAGGTTGTG
6-FAM
F: CATTTCTTGTTGTGATTAATAGTCTCC
(AC)18
6.50e-10
1.70e-05
R: TGAGGACTGTGGTTTAAGAGC
6-FAM
F: TGATTCTTATTAGGATTATTTGATGC
(GT)10
2.30e-25
8.90e-41
R: CTCGTTGGTCATAATTTGAGG
HEX
F: CAGGCTTAACACTCTTTCTTCC
(GT)11
2.30e-77
1.30e-110
R: CTAACCTGGATGGCTGTTTG
HEX
F: AGGGTATTGTTGGAGAAATGG
(GT)10
1.50e-50
1.10e-62
R: GCACCAGAACTGCCACATAG
HEX
F: CCTGAACCATTAGTTTACTTGCTG
(TGAT)6
2.50e-12
2.30e-14
R: TCCTGTGAGCTGTTAATTCTGAG
F: TGTTAGCAGGCTGATGTGTG
6-FAM
1.20e-28
6.60e-43
R: GCATCACAAATGCAACTTCAG
–
(GAAA)8
6-FAM
F: GATCCAGACTGCCTAAACAGC
(AC)17
2.20e-18
F: GGAAACCTTCCCATCAACAG
R: GCCAGGGCAGAGACATAAAG
F: CAGCTATGAGGTTACCAGAGAGG
Primer sequence 50 –30
R: GAAGGGATGCATGGTTGG
HEX
6-FAM
Fluoro
label
(F)
–
1.80e-12
(TCTA)7
(AGAGAGGAG
AAGAGAGA)7
4.70e-13
5.80e-52
Repeat
motif
E-value
in Gga
E-value
in ZF
306
255
413
614
600
563
422
454
673
576
548
491
461
437
713
567
266
480
421
294
592
Seq.
length
(bp)
Y
N
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
MI
55.34
55.22
64.54
64.51
60.44
60.10
63.40
63.39
63.96
64.38
64.23
64.30
64.38
64.21
63.55
63.43
64.15
64.53
64.33
64.40
58.02
58.07
58.85
58.50
58.77
58.69
57.07
57.14
57.23
57.32
58.23
57.92
59.32
59.28
58.66
58.44
59.34
58.95
59.85
59.38
59.84
59.44
Tm
(°C)
56
64
60
63
64
64
64
63
64
56
56
56
56
56
56
56
56
56
56
56
56
PCR
Ta
(°C)
12
12
12
12
12
12
12
12
12
24
24
24
24
24
24
24
24
24
24
24
24
n1
12
12
11
12
12
12
12
12
12
22
15
24
19
17
21
18
24
24
14
17
21
n2
3
2
2
2
3
2
3
2
3
2
2
3
8
3
3
2
3
10
7
9
11
A
170
186
377
173
207
193
279
234
203
235
136
156
262
321
136
166
182
301
348
138
294
Exp
allele
size
(bp)
°
173–180
(177, 177)
185–187
NT
360–363
(127, 127)
172–174
(173, 173)
209–213
(207, 209)
190–194
(194, 194)
276–283
NT
235–237
(236, 236)
200–208
(200, 204)
178–214
NT
132–149
(132, 132)
135–162
NT
250–271
(258, 260)
320–325
(321, 321)
131–140
(136, 136)
168–170
(168, 168)
185–193
(185, 185)
277–320
NT
331–346
(332, 346)
122–161
(135, 157)
287–406
(287, 287)
Obs. allele
size
(ruff 6233)
& range
(bp) ¥
0.58
0.16
0.18
0.50
0.25
0.58
0.33
0.33
0.50
1.0
0.00
0.95
0.57
0.11
0.90
0.11
0.58
0.58
0.85
0.82
0.23
HO
0.62
0.15
0.17
0.46
0.23
0.50
0.30
0.29
0.42
0.51
0.12
0.55
0.77
0.11
0.62
0.35
0.58
0.72
0.87
0.86
0.78
HE
0.29
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
0.00§?
0.04§
0.00?
0.04§
1.00
0.21?
0.34§
0.44
0.54§
0.72
0.38
0.00§
pHWE*
Author's personal copy
Conservation Genet Resour
123
123
5C04
HE616970
4F01
HE616969
4C06
HE616968
3F07
HE616967
4H06
HE616962
3F11
HE616961
6F01
HE616960
6B02
HE616959
5C08
HE616958
1
1
1
1
1
1
1
1
1
1
Lib
1
1
1
1
Z
Z
6
6
–
4
3
3
4
4
1
1
1A
1
6
6
CH
chr
ZF
chr
67624179
136476610
12955004
121754800
37214802
6052811
18076829
20079681
–
38001317
26505233
2063655
41075858
64774867
65490386
163634502
3982690
4758111
27151910
28674879
Chicken
chr loc. T
–
Zebra
finch
chr loc. T
–
1.80e-14
R: TTGTGATGCACAAGTCTTTCAAGG
F: TGAATGCAAATACAGCATCAGTGAG
6-FAM
R: CTGACTCATGATGCCTCATCTCG
(GT)7
2.60e-34
6.10e-27
HEX
F: TCTACTGAGCTCACAGAAACAAAGGAAC
(GT)8
2.20e-51
F: AGTAGCTGCCAATCCACAGG
6-FAM
R: TCTCCTGCTTGGCCTCTTT
(GT)14
1.30e-27
1.30e-45
R: CCTGCTGTGAAATCTACCCATCC
F: TGCAGTGCAATGTGTGTGACC
6-FAM
3.70e-21
8.50e-28
R: AATGCTTGTGGGTGGCAATG
–
(AG)8
F: TTGCACGTGTCCTTAGCTTGC
6-FAM
2.30e-02
(GT)6
R: GTTGGGTGTTCCTGCTGACG
HEX
F: CCCGTCAGCGAATATAAGAGCAG
(AC)10
1.60e-53
3.10e-41
F: TCAGCACTGAAACTGAGGAAATTATTG
6-FAM
R: GAGCATTCCTCCCGCTGTG
(AC)7
2.00e-58
4.20e-09
F: AACTTCAAAGACTTCTGCAAAGTTATTCTTC
6-FAM
R: TGAACTTACACTGGTGAACTAACTTTCTCTC
(AG)12
3.00e-08
2.10e-14
R: AACACACTGAGCGTCGTTTTATCA
F: TGCAGCATTCTTCGCAGCTA
HEX
2.70e-75
(TC)10
F: TGCAGCTTTAATTGCAACAGCTAATC
2.20e-70
HEX
Primer sequence 50 –30
R: AGCGCTCAGGTCTGAATGAGTTC
(TC)10
1.60e-103
Fluoro
label
(F)
1.60e-61
Repeat
motif
E-value
in Gga
E-value
in ZF
493
331
477
564
641
593
640
522
659
496
Seq.
length
(bp)
N
Y
Y
Y
N
N
N
Y
Y
Y
MI
63.60
63.51
64.42
64.36
60.08
60.28
64.07
64.28
64.11
63.67
64.02
64.26
64.26
63.80
63.48
63.61
63.09
63.22
64.35
64.66
Tm
(°C)
64
63
60
64
63
64
63
63
63
64
PCR
Ta
(°C)
12
12
12
12
12
12
12
12
12
12
n1
9
9
6 (F)
6 (M)
8
12
10
12
12
12
12
n2
2
2
6
6
8
3
3
4
10
4
3
A
214
263
224
360
238
198
276
389
225
291
Exp
allele
size
(bp)
°
213–214
NT
263–265
(262, 262)
221–233
(227, 227)
328–381
(329, 358)
238–242
NT
192–204
NT
225–275
NT
280–366
(368, 376)
223–231
(226, 230)
288–292
(290, 292)
Obs. allele
size
(ruff 6233)
& range
(bp) ¥
0.44
0.33
0.00
0.85
1.00
0.08
0.20
0.16
0.75
0.50
0.58
HO
0.36
0.50
0.00
0.87
0.90
0.23
0.24
0.23
0.87
0.53
0.62
HE
1.00?
0.49§
–
0.06
0.82
0.05§
0.04
0.13
0.05
0.45
0.21
pHWE*
Deficiency of heterozygotes observed;
?
excess of heterozygotes observed, determined by Chi squared test (two tailed p \ 0.05). Deficiencies and excesses of heterozygotes are likely attributable to non-random population structure caused by captive breeding
* HWE for all loci was assessed in 12 individuals, which may contain second order relatives (half-sibs and closer relatives were removed)
¥ the allele sizes presented in parentheses are those of the female ruff individual (Bird ID 6233) from which the genomic library was created, NT not tested
° Expected allele size based on the sequenced clone allele of the female ruff individual (Bird ID 6233) from which the genome library was created
MI Mendelian inheritance displayed when genotyped in a pedigree of 64 families, consisting of both parents and 5–33 offspring per family; Y yes, N no
pHWE calculated from a maximum of 12 individuals, using GENEPOP v.4.0 (Rousset 2008)
§
n1 Number of individuals tested in a captive population. n2 Number of individuals amplified and genotyped. A Number of alleles observed, M male, F female. HO observed heterozygosity (calculated from n, using CERVUS v3.0). HE expected heterozygosity (calculated from n, using CERVUS
v3.0), HWE Hardy–Weinberg equilibrium
T
– The location of each microsatellite sequence was assigned in the chicken (v 2.1, May 2006 ENSEMBL release) and zebra finch (December 2011 ENSEMBL Release 65) based on sequence homology (see Dawson et al. 2006, 2007). Of these 52 polymorphic loci characterized, 50 could be
assigned a location in the chicken genome and 46 in the zebra finch genome.
Ppu060
Ppu059
Ppu058 (Zlinked)
Ppu057
Ppu052
Ppu051
Ppu050
Ppu049
Ppu048
HE616957
Ppu047
3B01
EMBL
acc. no.
& clone
name
Locus
Table 1 continued
Author's personal copy
Conservation Genet Resour
Ppu104
Ppu101
Ppu095
Ppu092
Ppu086
Ppu083
Ppu079
Ppu071
Ppu070
Ppu064
Ppu056
Ppu055
Ppu054
Ppu053
Ppu045
Ppu044
Ppu026
HE616912
Ppu002
6H07
HE617014
5F03
HE617011
5C07
HE617005
6C11
HE617002
6B07
HE616996
4G10
HE616993
5G10B
HE616989
4G02
HE616981
6E05
HE616980
5H06
HE616974
66F02
HE616966
65H06
HE616965
65F04
HE616964
3H05
HE616963
4D02
HE616955
3E10
HE616954
63C05
HE616936
3D09
EMBL
acc. no. &
clone
name
Locus
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Lib
5
5
Z
Z
9
9
6
6
Z
Z
9
9
–
4
2
2
5
5
5
5
22
22
1
1
8
8
Z
Z
2
2
–
–
1A
1
–
CH
chr
ZF
chr
53923720
54678545
45558110
14416326
13825337
13341088
12562519
14776618
22314343
72933854
12079437
11757764
–
39272313
19900285
17900039
33970154
36514685
48965965
50276059
1590011
690245
85953917
196624265
11832348
15987120
59515109
32113785
141544149
140526702
–
–
36868616
39405325
–
Chicken
chr loc. T
–
Zebra
finch
chr loc. T
–
(GT)5
HEX
R: TGTGAAAGCCCTTGAAGGCATC
F: CCCAATGTGCAGAAACCCTCAC
6.40e-46
1.10e-70
F: TTCACTTACACTTGAGGATGGTTG
6-FAM
R: AAACTAAACTACTGCTGGGCATTG
(GT)5
6.10e-33
7.60e-82
F: GGTGTGTGTTTCTGTCTCCCTCT
6-FAM
R: ATGGGAGTTATGCACACATCAAA
(TC)5
1.70e-35
2.00e-71
F: AAAGGAGTTCCCTGGCACAGTTT
HEX
R: CGGCACAATTAATACTACCGGCTTA
(AT)5
1.30e-67
1.90e-55
F: TCTGCAGTTGGAAGGCTGTAA
6-FAM
R: TCTTGATGTTGCCAAAATATACATACA
(GT)5
2.90e-76
1.00e-81
R: TCCATCAAGAACTCATTAGCTTAGTAGCC
F: CTGGGTGTTGTGCCTTCAGC
6-FAM
7.10e-96
5.70e-92
R: GTTGGGCGGAGAATGGACTG
–
(GT)5
HEX
F: AGCTGAATGAACATCTGCATATTAGAACAC
(GA)7
2.10e-19
F: AAATCTGAACCAGAAATAATATAGTCAATCC
6-FAM
R: TGAAGTGTTCTCCACTTCTTTGATG
(T)10
1.20e-43
3.50e-61
F: TTCTCCTCACCCTCCTCTAGCATTTAG
6-FAM
R: GCGCATGGCTTTATACCATATTTCC
(AT)5
1.80e-44
5.70e-62
F: TGTTGCTGTTGTTGTCGTGGTC
6-FAM
R: GAGTGCTCTGCACCCTTCACC
(AC)5
5.20e-43
4.20e-135
F: CCTCTGGCAAATACTCAATGC
HEX
R: CACTGGAAAGGTCAGGAAGC
(AC)8
1.50e-06
1.00e-22
F: TGGAGCTTAACATCTACAAATGC
6-FAM
R: TTGGCTTTCTCTTATCCATCAC
(GAAA)14
7.90e-12
8.80e-13
F: GCACCGCAGAAGTTGATAAG
6-FAM
R: CTGAGGTGCTCATGGTTACAG
(GT)5
7.50e-48
2.00e-25
F: TTGGCACCAATAGTTGCCTCAT
6-FAM
R: CCTCTTCAGAGGAACAAAGCAAGA
(AC)10
9.00e-49
4.30e-84
F: TCATTTCTCCTCCTAACGCTGAAG
R: ACTGTCCTTGCATCTTCCTCTCC
HEX
7.10e-61
(AC)10
F: TCAGGCACATTACATTATTCACTATCTATG
R: GCTGCCACTTACTGAAATCTG
F: CTTTGTCATTATGTAGGGTCCTG
R: AACATACAACACCTCTCTCTCTTTCTTT
F: GAAGCTGAGATTCCCATATAATCACT
Primer sequence 50 –30
6.60e-70
HEX
6-FAM
HEX
Fluoro
label
(F)
R: CCACCCTCCTAGCAAACACC
(TGTT)6
(AC)10
(GAAA)44
Repeat
motif
–
–
5.40e-43
3.90e-19
–
E-value
in Gga
E-value
in ZF
471
367
504
606
524
562
324
585
401
357
305
375
505
407
561
543
414
538
Seq.
length
(bp)
65.74
66.02
60.88
60.44
61.51
61.87
64.15
64.44
60.81
60.95
64.16
64.15
65.12
64.72
61.87
61.42
65.41
65.17
64.58
64.41
59.84
59.72
58.33
58.47
58.38
58.53
63.32
63.81
63.22
63.12
61.83
61.77
57.65
57.70
60.45
60.23
Tm
(°C)
56
60
56
56
60
60
56
56
56
56
56
56
56
56
63
61
56
56
PCR
Ta
(°C)
Table 2 Additional ruff microsatellite loci with assigned predicted genome locations but that were abandoned from further testing
24
6 (F)
6 (M)
24
24
6 (F)
6 (M)
12
24
24
24
12
24
24
24
12 (F)
12 (M)
12
12
24
24
n1
23
6
6
19
24
6
6
11
17
1
21
11
4
2
7
8
11
11
23
21
n2
1
1
1
1
1
1
1
1
1
1
1
1
2
2
3
1
1
1
1
1
11
A
246
205
247
280
196
212
245
241
198
248
143
336
284
233
326
193
294
384
Exp
allele
size
(bp)
¥
243
205
205
244
279
196
196
213
245
241
198
245
249–283
276–283
322–338
230
230–238
324
195
291
313–378
Obs
allele
size (bp)
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Very poor amplification
Very poor amplification
Very poor amplification
Very poor amplification
Monomorphic
Monomorphic
Monomorphic
Unreliable poor reverse
primer
Marker status
Author's personal copy
Conservation Genet Resour
123
123
Ppu121
Ppu107
Ppu093
Ppu085
Ppu080
Ppu068
Ppu067
Ppu062
Ppu242
Ppu221
Ppu199
Ppu181
Ppu172
Ppu164
Ppu148
Ppu140
Ppu138
HE617015
Ppu105
65E06
HE617031
6F04B
HE617017
5C11
HE617003
6A05
HE616995
6H10
HE616990
3E09
HE616978
4G09
HE616977
5E09
HE616972
67C06
HE617152
63C10
HE617131
67D10
HE617109
63D03
HE617091
66G11
HE617082
64B02
HE617074
64A03
HE617058
21A01
HE617050
36B04
HE617048
6H11
EMBL
acc. no. &
clone
name
Locus
Table 2 continued
1
1
1
1
1
1
1
1
2
1
2
1
2
2
2
2
2
1
Lib
6
6
1
1
3
3
2
–
1A
1
Z
Z
Z
–
9
9
1
1
26
26
10
10
Z
Z
3
3
1
1
12
12
18
18
5
5
–
22
CH
chr
ZF
chr
32662952
33702631
77944747
188022341
106323359
106292912
31937001
–
58008408
60285776
37374037
6212608
43145669
–
16851809
15940752
81416718
191524107
273083
1257327
18002891
19602417
7124071
42994287
110357795
110633741
51091377
178594445
13993026
13482713
9810117
5211094
2549845
4932892
–
1024052
Chicken
chr loc. T
–
Zebra
finch
chr loc. T
–
(GT)6
7.60e-12
HEX
R: ACGAGAGGCCAAATATGGTG
F: TTCCCTAACCAGCAATTAGCTATC
(AC)5
7.60e-25
1.80e-33
R: CACAGACACTTCTTTAACAGTTATTCCAACC
6-FAM
F: CTTTGGACATGCCAGCAGGA
(TTGTT)9
4.20e-09
3.40e-68
F: AGCCATTTGCTTAATCCTACCTTCTG
R: ATGGGAGGCCATGCAACAC
HEX
7.50e-40
(TAGA)6
F: TGCCCAGGGAAGTGGTTG
4.20e-60
HEX
R: AGTCCAACCATCAACCTAACTCTGAC
(GT)6
R: CCACAGCTCTGCCTCTGTTAATATG
6.90e-09
–
HEX
F: TTTCAGATGTGATTGGTGTTCAGC
(GA)7
6.90e-13
3.00e-19
R: GGCAGAAGCAAGAAACAACAGAAC
F: CTTGCCAAAGGGCAAGGTG
6-FAM
1.20e-32
(AGAT)11
F: CTGACTGCTGCCTGCCTCTC
4.20e-80
6-FAM
R: GGTGTCACCTCCTATACCTACATGCTC
(AGAAG)55
R: TGTGGAATTAAAGTTCAACCCATTAGC
3.00e-25
–
HEX
F: TCAGTGTTTCTACTTGGATGGAATGG
(AC)7
3.40e-98
1.40e-37
R: TTCTTCTGGAGCCCTCTCTG
6-FAM
F: GATCAGGTTGTGAGCAGCAG
(GT)7
7.30e-12
5.20e-18
R: TGCTCTGCACTTGTGTAACG
6-FAM
F: GCGAGACATCTGAATCTCCTC
(GT)8
2.10e-60
9.90e-22
F: TGCTGAAATCCATTCTACTAACG
HEX
R: CCTATTAATTGCACACAAATTGC
(GT)5
1.90e-35
7.10e-39
R: AGCTGCAATTGATGCCAGTG
6-FAM
F: TTGGAGGGTTCTTTGGGTTG
(GT)10
2.80e-41
7.20e-05
F: CAGGAAGAGGTGAGCTGGAG
HEX
R: CTAAGGCACACAGCTGAACG
(GA)6
2.10e-48
4.50e-52
R: CTGGAAAGGCTGAAATCCTG
6-FAM
F: CTGCTCATCTCTCCTTTCTTCC
(TC)5
5.20e-62
1.50e-69
R: TCAGGATGACTCCAGACGTG
6-FAM
F: TGGCTTGTGCAAAGTAGATTTC
(AC)7
1.20e-132
1.70e-106
F: TAGTAGCCAATCGGGTGAGC
HEX
R: AGCCAGCTGGAGTAGTGTGTG
(AC)6
7.00e-85
4.30e-105
F: TTGGATATTCTGCCGAGATG
R: GGGAAGCTTGGGATTCTATG
7.60e-42
1.00e-11
F: AAATTAGCATAAAGACGGAGAAGTTGC
Primer sequence 50 –30
R: GCCTTCTTGCTGTATTCAGGTGAG
HEX
6-FAM
Fluoro
label
(F)
–
(ATAG)4
Repeat
motif
E-value
in Gga
E-value
in ZF
593
316
813
450
570
669
606
400
323
468
557
677
305
609
756
713
531
304
Seq.
length
(bp)
59.96
60.02
64.61
65.04
64.27
63.93
63.02
63.12
63.49
63.36
63.96
63.57
63.79
64.27
64.28
64.50
59.67
59.58
58.65
58.98
58.63
58.47
62.35
62.15
59.66
60.13
59.81
59.60
59.82
59.41
60.52
60.24
58.60
58.67
63.56
63.14
Tm
(°C)
56
56
60
60
56
56
56
60
56
56
56
56
56
56
56
56
56
60
PCR
Ta
(°C)
24
24
12
12
24
12 (F)
12 (M)
12 (F)
12 (M)
12
24
24
24
12 (F)
12 (M)
24
24
24
24
24
12
n1
0
0
0
0
0
0
0
0
5
18
4
4
23
21
23
23
15
10
n2
–
–
–
–
–
–
–
–
1
1
1
0
1
1
1
1
1
1
1
A
256
215
230
203
447
263
509
243
315
356
148
–
262
155
329
340
256
257
249
Exp
allele
size
(bp)
¥
–
–
–
–
–
–
–
–
126
357
148
–
265
155
331
341
255
284
251
Obs
allele
size
(bp)
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Monomorphic
Marker status
Author's personal copy
Conservation Genet Resour
67A12
HE617150
66F08
HE617146
64E01
HE617094
67G04
HE617083
66E10
HE617080
65H12
HE617078
67F11
HE617073
67B07
HE617071
66A03
HE617065
66A01
HE617064
65C11
HE617060
66C09
HE617044
66E12
HE617039
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Lib
1A
–
–
3
1A
1
1A
1
2
2
2
2
1
1
4
4
4
4
2
2
15
15
6
6
3
3
1A
1
CH
chr
ZF
chr
48527357
–
–
97005426
6252519
7368828
56094038
58206176
39737798
70828859
92057916
89221409
112179239
104968881
12229126
32471914
68748861
91803651
147316308
146180882
994445
3838997
6728242
2107306
31383766
31663341
61734639
73188968
Chicken
chr loc. T
–
Zebra
finch
chr loc. T
–
6.20e-23
–
(GT)10
6-FAM
6-FAM
R: TCTCATGTACTGTGATGTCTACTGTG
F: CCCAGGCTAGGAGCTTTG
R: CCAAGACATGCACTGTGTCC
F: AACTCACCATATGTGCCAAGG
(GT)6
4.00e-29
–
R: AAACTGTAAGTTATTCCAAAGGAACC
HEX
F: GGGTTACAGCTGTTTGTATTTCC
(GT)6
1.30e-09
7.00e-13
R: AGAGTATGCTGGGCTTGTGG
6-FAM
F: ACTTGGGCAGAACCAGAATG
(GA)8
1.50e-39
2.30e-80
R: AACTTTCCAACTGTTTCAGTTCC
HEX
F: AAATATGTGAAATTGGTCCAACAG
(CTTTCT)8
7.30e-27
2.10e-80
R: TCCAGGGTTACAAACAAGAGC
6-FAM
F: TCAGCATTGCCTTTACCTTTC
(CTTC)10
1.40e-56
1.10e-68
R: AATAGCAAGCTGCATCACAAAG
6-FAM
F: GCCAGTGAGTAGTGGGTTCC
(CA)9
7.50e-27
9.90e-18
R: TTTCTCTTCCTGCTAAATCCAAC
6-FAM
F: ACTGATGGCAGAGCAGATTG
(CA)8
7.90e-44
4.00e-86
R: GCTCCAAGCTCTTAGTCATGG
HEX
F: GATCTAAGTTTCCTGGGATTGC
(CA)10
7.00e-18
8.00e-30
R: GAGGTTTATGGTAATGAAGAAGTTGC
HEX
F: ATCACACGTGCACCAAACAC
(CA)5
5.00e-28
3.30e-42
R: CTGGCACTAATGTGTAGTCGTG
HEX
F: GATCGGAGCCTTCTTACCTG
(CA)6
3.70e-39
9.40e-23
R: GCTGGAGGGAAGCTTGTTC
HEX
F: TTGTGTCATTTGGATGCCTTAG
(AG)6
6.80e-54
8.60e-34
R: GCTCTCCTTGTGCTTGTTTG
F: ACACCCACTATTTGTGAAGAGC
HEX
2.10e-47
(AC)7
F: TCTATGCCTTAAATCCTATTTACAAAC
1.40e-36
HEX
Primer sequence 50 –30
R: CAGCACTGTCATTGTTCTGC
(AC)5
5.70e-30
Fluoro
label
(F)
4.60e-28
Repeat
motif
E-value
in Gga
E-value
in ZF
338
398
206
507
856
721
441
621
326
561
307
808
460
401
Seq.
length
(bp)
58.41
58.54
60.16
59.87
59.35
58.99
60.28
60.11
58.73
59.21
59.23
59.35
59.56
59.58
58.94
58.99
59.09
59.09
60.59
60.50
58.39
58.89
59.94
60.00
58.65
58.25
57.98
57.68
Tm
(°C)
56
56
56
56
56
56
56
56
56
56
56
56
56
56
PCR
Ta
(°C)
24
24
24
24
24
24
24
24
24
24
24
24
24
24
n1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
n2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
A
259
263
132
319
175
358
284
287
154
131
232
166
159
199
Exp
allele
size
(bp)
¥
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Obs
allele
size
(bp)
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Failed to amplify
Marker status
¥ Expected allele size based on the sequenced clone allele of the female ruff individual (Bird ID 6233) from which the genomic library was created
n1 Number of individuals tested in a captive population. n2 Number of individuals amplified and genotyped. A Number of alleles observed, M male, F female
T
– The location of each microsatellite sequence was assigned in the chicken (v 2.1, May 2006 ENSEMBL release) and zebra finch (December 2011 ENSEMBL Release 65) based on sequence homology (see Dawson et al. 2006, 2007). Of these 50
additional loci tested, 45 could be assigned a location in the chicken genome and 46 in the zebra finch genome.
Ppu240
Ppu236
Ppu184
Ppu173
Ppu170
Ppu168
Ppu163
Ppu161
Ppu155
Ppu154
Ppu150
Ppu134
Ppu129
HE617035
Ppu125
65F08
EMBL
acc. no. &
clone
name
Locus
Table 2 continued
Author's personal copy
Conservation Genet Resour
123
Author's personal copy
Conservation Genet Resour
Gga1
Tgu1A
Gga2
Tgu2
Gga3
Ppu048*
Ppu184
Ppu048*
Ppu184
Ppu025
Ppu026
Ppu240
Ppu039*
Ppu001
Ppu173
Ppu080
Ppu125
Ppu026
Ppu039*
Ppu001
Ppu173
Ppu080
Ppu125
Ppu059*
Ppu003
Ppu163
Ppu059*
Ppu003
Ppu028
Ppu060*
Tgu3
Gga4
Ppu010
Ppu010
Ppu071
Ppu015
Ppu032
Ppu071
Ppu015
Ppu085*
Ppu043*
Ppu170
Ppu032
Ppu043*
Ppu018
Ppu170
Ppu022
Ppu129
Ppu013
Ppu051*
Ppu129
Ppu012
Ppu011
Ppu011
Ppu012
Ppu013
Ppu009
Ppu161
Ppu079
Ppu046*
Ppu161
Ppu017
Ppu050*
Ppu168
Ppu004
Ppu038*
Ppu168
Ppu004
Ppu038*
Ppu030
Ppu030
Ppu045
Ppu154
Ppu045
Ppu154
Ppu007
Ppu007
Ppu019
Ppu236
Ppu035
Ppu093*
Ppu172
Tgu5
Ppu040*
Ppu138
Ppu040*
Ppu138
Ppu006
Ppu006
Ppu070
Ppu070
Ppu064*
Ppu104
Ppu064*
Ppu104
Ppu009
Ppu046*
Ppu050*
Ppu018
Ppu022
Gga5
Tgu4A
Ppu051*
Ppu155
Ppu052*
Ppu035
Ppu093*
Ppu172
Ppu155
Ppu044
Ppu164
Ppu021
Ppu016 Ppu049*
Ppu060*
Ppu107
Ppu242
Ppu055
Ppu021
Ppu049*
Ppu016
Ppu164
Ppu107
Ppu242
Ppu055
Ppu028
Ppu163
Gga6
Ppu134
Ppu092
Ppu057*
Ppu047*
Ppu121
Gga11
Tgu6
Gga7
Ppu134
Ppu092
Ppu057*
Ppu047*
Ppu121
Gga22
Ppu056
Ppu105*
Ppu042*
Ppu041*
Tgu22
Ppu056
Gga9
Ppu027
Ppu023
Ppu148
Tgu24
Ppu014
Tgu13
Gga13
Gga15
Ppu031
Ppu024
Ppu148
Ppu083*
Ppu095
Ppu062*
Gga10
Tgu10
Ppu036*
Ppu029
Ppu034
Ppu199
Ppu036*
Ppu029
Ppu034
Ppu199
Ppu033
Ppu033
Tgu12
Tgu9
Ppu083*
Ppu095
Ppu062*
Ppu054
Ppu005
Ppu054
Ppu005
Ppu020
Ppu042*
Ppu041*
Ppu020
Tgu8
Gga8
Ppu023
Ppu027
Gga12
Tgu11
Tgu7
Tgu15
Ppu150
Ppu150
Gga18
Ppu140
Tgu18
Ppu140
Ppu031
Gga26
Ppu221
Ppu037*
Tgu26
Ppu221
GgaZ
Ppu058*
Ppu068
Ppu101*
TguZ
Ppu181
Ppu086*
Ppu053
Ppu181
Ppu058*
Ppu068
Ppu067
Ppu101*
Ppu053
Ppu086*
Fig. 1 Chromosomal locations in the chicken and zebra finch
genomes of 102 ruff microsatellite loci. Locations were assigned as
in Table 1. Gga, chicken (Gallus gallus) chromosome; Tgu, zebra
finch (Taeniopygia guttata) chromosome; Underlined loci are polymorphic, loci listed in bold were monomorphic (*polymorphism
assessed in 12 individuals; all other loci were tested in 24
individuals); Loci listed in italics failed to amplify a product or
amplified a stutter/unreliable product (Table 2) and the loci in plain
text have not been tested for amplification or polymorphism
software (Applied Biosystems). Observed and expected
heterozygosities were calculated using CERVUS v3.0
(Kalinowski et al. 2007; Table 1). Deviations from
Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium were assessed using GENEPOP v.4.0 (Rousset
2008). All loci were assessed for Hardy–Weinberg equilibrium and linkage disequilibrium in captive individuals,
selected to avoid parent–offspring relationships and full
and half sibs. Polymorphic loci were suspected of Z-linkage
if no females were heterozygous. Mendelian inheritance
was assessed in 64 families, consisting of both parents and
5–33 offspring per family, comprising 381 known-sex
individuals. Any Z-linked loci were tested for HWE in males
only.
Of the 102 markers tested, 23 were monomorphic, 27
failed to amplify or amplified a stuttery/unreliable product,
and 52 were polymorphic (Tables 1, 2). Five polymorphic
autosomal loci (Ppu001, Ppu003, Ppu009, Ppu010 and
Ppu016) amplified a product [70 bp larger than the cloned
sequence but were all inherited in a Mendelian fashion
(Table 1). In total, 47 of the 52 polymorphic loci were
checked for Mendelian inheritance, which was confirmed
for all except Ppu002 (Tables 1, 2).
A high proportion of sequences (85 %) could be
assigned locations in both the zebra finch and chicken
genomes based on sequence homology (following Dawson
et al. 2006, 2007). Of the 102 newly-isolated ruff microsatellites, 95 could be assigned to a chromosome location
123
Author's personal copy
Conservation Genet Resour
in the chicken, 92 in the zebra finch and 87 in both chicken
and zebra finch (Fig. 1). Only two loci could not be
assigned a chromosomal location in either chicken or zebra
finch (Ppu002 and Ppu008, Tables 1, 2). There was no
statistical difference in the number of sequences that could
be assigned a location to each genome (Fisher’s two-tailed
Exact test p = 0.66). One locus (Ppu058) assigned to both
the zebra finch and chicken Z chromosomes (Fig. 1), was
homozygous in all of the 127 females genotyped, but
heterozygous in 27 of the 140 males typed, confirming
its sex-linked status (Fisher’s two-tailed Exact test
p \ 0.001).
Twelve autosomal loci, assessed in 12 individuals, showed
significant deviation from Hardy–Weinberg equilibrium
(p \ 0.05) or a deficiency of heterozygotes (Table 1). Three
groups of loci showed evidence of linkage disequilibrium
(p \ 0.05; Ppu010–Ppu016, Ppu036–Ppu038–Ppu041–
Ppu042–Ppu051–Ppu052, Ppu037–Ppu040; Table 1), however following FDR correction (Benjamini and Hochberg
1995) no p-values were significant. All loci in each group were
assigned to different chromosomes, except Ppu041–Ppu042,
which are closely neighbouring on chromosome 11 in chickens and zebra finches, and therefore may be physically linked
(Table 1, Fig. 1). The deviation from Hardy–Weinberg
equilibrium and linkage disequilibrium displayed by some
groups of loci is probably due to the non-random structure of
our captive population. However, 46 of the 47 loci checked
displayed a pattern consistent with Mendelian inheritance
(Tables 1, 2).
The microsatellite loci developed during this study are
suitable for the analysis of parentage and population
structure and will be used to construct a linkage map for the
ruff. The utility of these loci in other shorebird species can
be predicted based on the BLAST E-value recorded from a
comparison of their sequences similarity with the chicken
genome (Küpper et al. 2008, but see also Dawson et al.
2010). The sequences of three loci (Ppu004, Ppu005 and
Ppu030) displayed particularly high sequence homology to
both the genetically distant chicken and zebra finch
(BLAST E-values \ E-70; Table 1). These loci are therefore expected to amplify in a wide range of species,
including those of conservation interest. Additionally, if
required, these specific loci would be the most suitable to
develop into conserved markers, which would further
enhance their cross-species utility (Dawson et al. 2010).
Any primer sets designed as a consensus among all three
species (ruff, zebra finch and chicken) are expected to
display the highest cross-species utility.
Acknowledgments This work was performed at the NERC Biomolecular Analysis Facility–Sheffield supported by the UK Natural
Environmental Research Council. The captive ruff colony was supported by the Natural Sciences and Engineering Research Council of
Canada (NSERC; to DBL) and LLF was supported by an NSERC
studentship.
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