Triplophysa tenuis is distributed both in the Bosten lake and Tarim river systems in the south of Xinjiang and in the Shule river and Ruoshui river in the Hexi corridor of Gansu province (Zhu 1989). Although T. tenuis has not yet been classified as an endangered species, the fish species in Gansu province showed an obvious decreasing trend, especially the degradation of endemic fish (Wang et al. 2015). At present, the reports on T. tenuis are mainly in the aspect of biological characteristics (Chen et al. 2017, Yao et al. 2018). There are very few reports about molecular markers in this species. Genetic markers that are currently available for T. tenuis have mostly focused on mitochondrial DNA (Wang et al. 2015, Wang et al. 2016). Single-nucleotide polymorphism (SNP) markers are useful in population genetic studies because of their co-dominance, high levels of polymorphism, low cost and wide distribution (Vignal et al. 2002, Wang et al. 2015, Blanc-Jolivet et al. 2017). In the present study, we developed and characterized 45 SNP markers for the first time in T. tenuis. It is expected that these results will contribute to population conservation, genetic management and construction of genetic linkage map for this species.
In this study, restriction-site associated DNA sequencing (RAD-seq) was used to isolate and characterize SNP markers. A total of eighty potential SNP loci were selected for primer design using Primer 5.0. Fin clips of T. tenuis were collected from 33 individuals of the Shule river (Gansu, China). Genomic DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method (Doyle 1987) and was stored at -20°C.
PCR amplifications were performed in 30 µL volumes containing 1 µL of genomic DNA, 14 µL of Premix Taq (2×Taq Plus MasterMix, CWBIO), 1 µL of each gene primer and 13 µL of PCR-grade water. The PCR programme was 94°C for 5 min, then 32 cycles at 94°C for 30 s, annealing for 30 s (for annealing temperatures of each primer pair, Table 1), 72°C for 45 s, and one cycle of 72°C for 7 min for the final extension. Amplification products were sequenced using Sanger technology. The sequenced fragments were aligned using Vector NTI 10.3.0 (Invitrogen, Carlsbad, CA, USA) and the genotypes per locus were determined by BioEdit according to the peaks of each base. The minor allele frequency (MAF), observed heterozygosities (Ho), expected heterozygosities (He), polymorphism information content (PIC) and P value representing the deviations from Hardy–Weinberg equilibrium (HWE) were calculated using Cervus 3.0 (Kalinowski et al. 2007).
A total of 45 loci were found to be polymorphic and showed bi-allelic in 33 individuals of T. tenuis. The minor allele frequency ranged from 0.046 to 0.500 (Table 1). The observed heterozygosity varied from 0.061 to 0.667, while the expected heterozygosity ranged from 0.088 to 0.508. Polymorphic information content ranged from 0.083 to 0.375. Only three loci showed significant deviations from the HWE after Bonferroni correction. As far as we know, this is the first report of SNP identification in T. tenuis, which will be valuable tool for population conservation in this species.
Table 1
Characterization of 45 SNPs in T. tenuis
Primer ID
|
Primer sequences (5′-3′)
|
Fragment size (bp)
|
Locus ID
|
SNP
type
|
SNP position
|
Ta (℃)
|
MAF
|
Ho
|
He
|
PIC
|
HWEP
|
TtSNP1
|
F:TCACAGACGTACATTTCCCATA
|
248
|
TtSNP1-1
|
C/T
|
128
|
48
|
0.152
|
0.182
|
0.261
|
0.224
|
NS
|
|
R:ATTCAAACACTGAGTGCTTTTT
|
|
TtSNP1-2
|
A/G
|
147
|
|
0.273
|
0.242
|
0.403
|
0.318
|
NS
|
TtSNP2
|
F:GCCTGTTTGTGTGTTTAACCTG
|
356
|
TtSNP2-1
|
C/G
|
177
|
51
|
0.182
|
0.303
|
0.302
|
0.253
|
NS
|
|
R:ctagaaacctgccagattcct
|
|
TtSNP2-2
|
A/T
|
197
|
|
0.197
|
0.333
|
0.321
|
0.266
|
NS
|
|
|
|
TtSNP2-3
|
G/T
|
253
|
|
0.485
|
0.667
|
0.507
|
0.375
|
NS
|
TtSNP3
|
F:CAATAGAAGTCAATGGGGATCG
|
340
|
TtSNP3-1
|
C/G
|
186
|
50
|
0.106
|
0.212
|
0.193
|
0.172
|
NS
|
|
R:ACAGACCACATAGATCATCCAT
|
|
TtSNP3-2
|
C/T
|
187
|
|
0.318
|
0.394
|
0.441
|
0.340
|
NS
|
|
|
|
TtSNP3-3
|
A/G
|
256
|
|
0.303
|
0.424
|
0.429
|
0.333
|
NS
|
|
|
|
TtSNP3-4
|
A/T
|
309
|
|
0.318
|
0.455
|
0.441
|
0.340
|
NS
|
|
|
|
TtSNP3-5
|
C/T
|
314
|
|
0.121
|
0.182
|
0.216
|
0.190
|
NS
|
TtSNP4
|
F:GCTTTCCTGTAGCTCAGTTGTA
|
371
|
TtSNP4-1
|
A/G
|
92
|
51
|
0.394
|
0.364
|
0.485
|
0.363
|
NS
|
|
R:ATAAGGGACTTTATTGCGTTCATGC
|
|
TtSNP4-2
|
A/T
|
93
|
|
0.424
|
0.364
|
0.496
|
0.369
|
NS
|
|
|
|
TtSNP4-3
|
G/T
|
162
|
|
0.091
|
0.061
|
0.168
|
0.152
|
*
|
|
|
|
TtSNP4-4
|
A/T
|
183
|
|
0.364
|
0.424
|
0.470
|
0.356
|
NS
|
|
|
|
TtSNP4-5
|
C/T
|
313
|
|
0.394
|
0.606
|
0.485
|
0.363
|
NS
|
|
|
|
TtSNP4-6
|
C/T
|
322
|
|
0.136
|
0.273
|
0.239
|
0.208
|
NS
|
|
|
|
TtSNP4-7
|
A/T
|
326
|
|
0.379
|
0.576
|
0.478
|
0.360
|
NS
|
TtSNP5
|
F:TTCCTAACCAGGTCAAAAGTAA
|
319
|
TtSNP5-1
|
A/G
|
103
|
48
|
0.273
|
0.121
|
0.403
|
0.318
|
**
|
|
R:AGCTTTGAAGCATTTACAGTCA
|
|
TtSNP5-2
|
A/G
|
155
|
|
0.485
|
0.364
|
0.507
|
0.375
|
NS
|
|
|
|
TtSNP5-3
|
A/G
|
251
|
|
0.046
|
0.091
|
0.088
|
0.083
|
NS
|
|
|
|
TtSNP5-4
|
A/T
|
278
|
|
0.227
|
0.333
|
0.357
|
0.290
|
NS
|
|
|
|
TtSNP5-5
|
A/G
|
285
|
|
0.197
|
0.333
|
0.321
|
0.266
|
NS
|
TtSNP6
|
F:CCCCATCCATTCAGCATCTCT
|
251
|
TtSNP6-1
|
C/G
|
212
|
51
|
0.212
|
0.303
|
0.339
|
0.278
|
NS
|
|
R:CTATTGCTAATTCGAGAGGGTG
|
|
|
|
|
|
|
|
|
|
|
TtSNP7
|
F:CAAAATGAAACCAAATCCTGCC
|
329
|
TtSNP7-1
|
A/G
|
254
|
49
|
0.091
|
0.182
|
0.168
|
0.152
|
NS
|
|
R:GAGGCAAATAAATTAGACCACAAAT
|
|
TtSNP7-2
|
A/T
|
281
|
|
0.349
|
0.515
|
0.461
|
0.351
|
NS
|
TtSNP8
|
F:GTCGTACCCCAAACAGTTCTG
|
324
|
TtSNP8-1
|
A/G
|
122
|
51
|
0.167
|
0.212
|
0.282
|
0.239
|
NS
|
|
R:AAGATAAGCAGAGAGAACAACAC
|
|
TtSNP8-2
|
C/T
|
142
|
|
0.409
|
0.576
|
0.491
|
0.367
|
NS
|
|
|
|
TtSNP8-3
|
C/T
|
153
|
|
0.303
|
0.364
|
0.429
|
0.333
|
NS
|
|
|
|
TtSNP8-4
|
A/T
|
156
|
|
0.061
|
0.121
|
0.116
|
0.107
|
NS
|
|
|
|
TtSNP8-5
|
C/T
|
193
|
|
0.333
|
0.364
|
0.451
|
0.346
|
NS
|
|
|
|
TtSNP8-6
|
C/T
|
197
|
|
0.303
|
0.364
|
0.429
|
0.333
|
NS
|
|
|
|
TtSNP8-7
|
A/G
|
224
|
|
0.091
|
0.182
|
0.168
|
0.152
|
NS
|
TtSNP9
|
F:CTTACCTCCTACTTCCTCG
|
339
|
TtSNP9-1
|
C/T
|
157
|
49
|
0.061
|
0.061
|
0.116
|
0.107
|
NS
|
|
R:TGTCCTGAAGTTTTGAGCGAA
|
|
TtSNP9-2
|
C/T
|
172
|
|
0.273
|
0.424
|
0.403
|
0.318
|
NS
|
|
|
|
TtSNP9-3
|
C/G
|
175
|
|
0.046
|
0.091
|
0.088
|
0.083
|
NS
|
|
|
|
TtSNP9-4
|
G/T
|
183
|
|
0.364
|
0.364
|
0.470
|
0.356
|
NS
|
|
|
|
TtSNP9-5
|
A/T
|
185
|
|
0.136
|
0.152
|
0.239
|
0.208
|
NS
|
|
|
|
TtSNP9-6
|
A/T
|
255
|
|
0.106
|
0.091
|
0.193
|
0.172
|
NS
|
TtSNP10
|
F:CTACACACTGTTTACCTGGCC
|
383
|
TtSNP10-1
|
A/T
|
126
|
53
|
0.091
|
0.182
|
0.168
|
0.152
|
NS
|
|
R:TAATCAGGGGCACTGTGGAAG
|
|
TtSNP10-2
|
A/C
|
132
|
|
0.197
|
0.091
|
0.321
|
0.266
|
**
|
|
|
|
TtSNP10-3
|
C/T
|
195
|
|
0.152
|
0.182
|
0.261
|
0.224
|
NS
|
|
|
|
TtSNP10-4
|
C/T
|
196
|
|
0.424
|
0.545
|
0.496
|
0.369
|
NS
|
|
|
|
TtSNP10-5
|
A/C
|
229
|
|
0.152
|
0.182
|
0.261
|
0.224
|
NS
|
|
|
|
TtSNP10-6
|
A/T
|
276
|
|
0.197
|
0.091
|
0.321
|
0.266
|
**
|
TtSNP11
|
F:TGTATTTGCTTATTTGACACTCAA
|
273
|
TtSNP11-1
|
A/T
|
218
|
48
|
0.500
|
0.636
|
0.508
|
0.375
|
NS
|
|
R:TTCTGATGGACTGAGAGATAGA
|
|
|
|
|
|
|
|
|
|
|
MAF minor allele frequency, Ho observed heterozygosity, He expected heterozygosity, PIC polymorphism information content, HWEP results for Hardy–Weinberg Equilibrium text, NS non-significant
*P < 0.05 **P < 0.01