Isolation and characterization of sixteen microsatellite loci for fringe-lipped carp, Labeo fimbriatus S. Swain, S. P. Das, D. Bej, A. Patel, P. Jayasankar, P. C. Das & P. Das
Conservation Genetics Resources ISSN 1877-7252 Volume 4 Number 4 Conservation Genet Resour (2012) 4:913-915 DOI 10.1007/s12686-012-9672-z
1 23
Your article is protected by copyright and all rights are held exclusively by Springer Science+Business Media B.V.. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your work, please use the accepted author’s version for posting to your own website or your institution’s repository. You may further deposit the accepted author’s version on a funder’s repository at a funder’s request, provided it is not made publicly available until 12 months after publication.
1 23
Author's personal copy Conservation Genet Resour (2012) 4:913–915 DOI 10.1007/s12686-012-9672-z
TECHNICAL NOTE
Isolation and characterization of sixteen microsatellite loci for fringe-lipped carp, Labeo fimbriatus S. Swain • S. P. Das • D. Bej • A. Patel P. Jayasankar • P. C. Das • P. Das
•
Received: 3 May 2012 / Accepted: 14 May 2012 / Published online: 30 May 2012 Ó Springer Science+Business Media B.V. 2012
Abstract Labeo fimbriatus, the fringe-lipped peninsula carp, is a commercially important fish species next to Indian major carps. We isolated and characterized polymorphic microsatellite markers to be used as a tool for delineation of genetic stock of this species. Partial genomic libraries enriched for CT and GT repeat motifs generated 103 positive clones out of which 16 loci were found with flanking regions enough for designing primers. Thirty-two individuals of L. fimbriatus collected from wild were used to characterize the polymorphism. All the 16 loci were polymorphic with allele numbers ranging from 3 to 9. The observed and expected heterozygosity ranged from 0.093 to 0.833 and from 0.146 to 0.843, respectively. Nine loci were in agreement with Hardy–Weinberg equilibrium. No significant pair wise linkage disequilibrium was found among the loci. These markers would be very useful for characterization of natural populations of this species. Keywords Labeo fimbriatus Microsatellite marker Enriched library
Labeo fimbriatus, popularly known as podosi, is a native to lower peninsular region of India. It is distributed in the rivers and culture ponds of Pakistan, India, Nepal, Myanmar and Bangladesh (Molur 1998). Due to its good meat quality and taste, it fetches high market price and has been introduced recently as a potential culture species in
S. Swain S. P. Das D. Bej A. Patel P. Jayasankar P. C. Das P. Das (&) Fish Genetics and Biotechnology Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, Odisha, India e-mail:
[email protected]
species-diversification program for aquaculture in South Asia (Jena et al. 2011). The rivers Mahanadi, Godavari, Krishna, Kaveri and their tributaries form the principal habitats of this species. Anthropogenic intervention like overharvesting, poisoning of waters and construction of dams have made this fish vulnerable (Kowtal 1994). Polymorphic microsatellite markers are useful for answering ecological and population biology related questions (Balloux and Lugonae 2002). Recently, microsatellite markers have been developed for some Indian fishes such as rohu (Das et al. 2005; Patel et al. 2009), catla (McConnell et al. 2001) and chital (Punia et al. 2006). In the present investigation, we isolated and characterized a set of microsatellite loci in L. fimbriatus for stock identification in Indian river systems. Enriched partial genomic libraries for the repeat motifs (CT)15 and (GT)15 were constructed using DNA from one individual of L. fimbriatus (Bloor et al. 2001). In brief, genomic DNA (20 lg) was digested using restriction enzyme Sau3AI (NEW ENGLAND BIOLABS), and DNA fragments between 400 and 1,000 base pairs (bp) were excised from a 1.5 % agarose gel. Equal volumes of two oligonucleotides (200 pmol/lL) oligoA: 50 -GCGGTACCC GGGAAGCTTGG-30 and oligoB: 50 -P-ATCCCAAGCTT CCCGGGTACCGC-30 , (where p indicates phosphorylation) were mixed, denatured at 80 °C for 5 min and incubated at room temperature for 1 h to allow them to anneal and from adapters. DNA fragments were ligated to adapters and amplified by polymerase chain reaction (PCR) using specific primers. Genomic DNA fragments containing simple sequence repeats (SSR) sequences were captured by hybridization to only (CT)15 biotin-labeled probe. Captured fragments were ligated to pGEM-T easy vector (PROMEGA) and transformed into Escherichia coli (DH5a) competent cells by electroporation. Plasmid DNA was
123
Author's personal copy 914
Conservation Genet Resour (2012) 4:913–915
Table 1 Sixteen polymorphic microsatellite loci for Labeo fimbriatus Locus name
Gene bank accession no.
Primer 50 –30
LF-1
JQ838157
F: GAAAGCTGCTCGTCCTTGAA
LF-3
JQ838158
F: GGGTGTGGGAGAGAAAGAGAG
Motif
No. of alleles
Allele size in (Bp)
Annealing temp
HO
HE
P value for HWE
(AG)8
6
192
60
0.666
0.802
0.052
(AG)52
6
190
62
0.555
0.798
0.062
(CT)12
9
227
61
0.607
0.743
0.000*
R: CTCGGGATGAGAGCAGAAAC R: GGAGTCTGACAAATGCAGCAAG LF-4
JQ838159
F: GGCCAGTGTGACACAAACA R: GTCCCGGAGTCTAAAGACGAAC
LF-5
JQ838160
F: TCTACTGTCCGCACAATGACC R: CCGAGAGACAGTGAGAGACAGA
(CT)8
3
201
59
0.458
0.403
0.612
LF-6
JQ838161
F: CCTTGAACAACTCCACCTCTCT
(CT)8
3
196
59
0.285
0.298
1.000
(AG)20
4
179
54
0.307
0.673
0.000*
(GT)6
5
216
59
0.833
0.765
0.520
(AG)27
5
223
60
0.666
0.718
0.288
(CT)10
4
155
61
0.718
0.570
0.016*
(GA)27
4
185
60
0.482
0.578
0.142
R: ACTACGACTCATCTCGGTGTGA LF-7
JQ838162
F: TAGCAGACCTGCACTGAGAAAG
LF-8
JQ838163
F: GTGAAGCAACGACTTCAGAGAG
R:TCTCCCTGGTCTTTTCCT R: CCAGAAGACCATAGCAACCAC LF-9
JQ838164
F: GAAGTGACGGTCGCTGTTTC R: GGGATTGTCTGTAGTCCGTAGG
LF-10
JQ838165
LF-11
JQ838166
F: CTCTCCCCCTCTCTCTGTTTTG R: CTTCTGTCAAGCCGTTTAGCAC F: TCTCAGTGGGTGTCATTACCTG R:CCCATCAAACCATCTCTCTAGC
LF-12a LF-12b
JQ838167
F: GTCACAGAGGAGGAGGAGAAAG
(CA)10
3
226
59
0.093
0.146
0.154
JQ838167
R: AGCTCGGCATGTTAGTCTCACT F: AGGAAACACAGTGAGTGCAGAG
(AG)64
6
206
60
0.571
0.829
0.000*
(CTG)4
5
186
60
0.451
0.750
0.000*
(AC)8
3
145
59
0.400
0.672
0.000*
(TC)10
6
216
60
0.806
0.803
0.009*
(GA)26
5
195
60
0.781
0.724
0.505
R: GAGAGAGCGATGCACAATCA LF-13
JQ838168
F: GCCTCATTCCCAGTTACATCAG R: TCCAGCAGAGAGAGAGAGAGAGA
LF-14
JQ838169
LF-15
JQ838170
F: AGGAGCCGAGTTCTCCTTAGAG R:TCTCACGCAGTCTCATGTAGTG F: ACACTCACACTCGCTCACTCAC R: CGGTGAATGCTGATGAACTG
LF-16
JQ838171
F: AACGTCACACATGCTCCTAGTC R: CTGCCCATGACACTGAAACTC
For each locus, loci name, genbank accession numbers, primer pair, repeat motif, number and size of alleles, annealing temp (C), observed (Ho) and expected (He) heterozygosity and p value for HWE are provided. * significant at a = 0.05
isolated from positive clones using QIAprep spin column (QIAGEN) and sequencing was performed using T7/sp6 primer in ABI 310 genetic analyser. To study polymorphism, 32 unrelated individuals collected from wild were genotyped. PCR reactions were carried out in 20 lL reaction volumes containing 19 Taq DNA polymerase reaction buffer (BANGLORE GENEI), 5 pmol of each primer (Table 1), 200 lM dNTPs, 0.25 units of Taq DNA polymerase and 20 ng genomic DNA as template. Amplification was performed in a GeneAmp 9700 thermocycler (APPLIED BIOSYSTEMS) programmed for initial denaturation of 5 min at 94 °C followed by 35 cycles of 94 °C for 45 s, 55° C for 1.5 min and 72 °C for 2 min and a
123
final extension at 72 °C for 7 min. Amplified products were dried in a vacuum concentrator (DNA plus, HETO HOLTEN), mixed with 7 lL of formamide loading dye, heat denatured at 95 °C for 5 min and then separated on 6 % denaturing polyacrylamide gel with 7.5 M urea and 19 TBE. Separation was performed in a vertical gel electrophoresis system (Hoffer, SE 600, AMERSHAM BIOCSIENCES) at a constant current of 25 W, and 55 °C for 2 h using U9174/HindIII (FINNZYMES) as size standard. Gels were silver stained and documented by visual counting. The number of alleles, observed heterozygosity (HO) and expected heterozygosity (HE) were determined by GDA software (Lewis and Zaykin 2001). Hardy–Weinberg
Author's personal copy Conservation Genet Resour (2012) 4:913–915
expectation for genotype frequency and pair wise linkage disequilibrium (LD) test were carried out using the same software. Pairwise LD was tested using allele frequencies for loci that were in HWE and genotypic frequencies were used to calculate LD of loci that deviated from HWE to prevent interference from within-locus disequilibrium. General measures of diversity and GenBank accession number of 16 polymorphic loci are given in Table 1. After screening two enriched libraries, 103 positive clones were subjected to sequencing. However, 78 clones (75.72 %) were sequenced completely and remaining 25 clones (24.27 %) could not be sequenced due to high GC content or long repeat motifs. Thirtyfour sequences did not have enough flanking regions for primer designing and 28 clones were found to be redundant. Consequently, 16 sequences were used for designing primers using BatchPrimer3 (You et al. 2008). All the 16 markers were found to be polymorphic and number of alleles per locus ranged from 3 to 9. Observed and expected heterozygosity ranged from 0.093 to 0.833 and from 0.146 to 0.843, respectively. Nine loci (P [ 0.05) were in agreement with Hardy– Weinberg Equilibrium (HWE) and seven loci deviated from it. Presence of null alleles or population admixtures may have caused these seven loci to deviate from HWE. No significant pairwise linkage disequilibrium was found among the loci except between locus Lf-3 and Lf-9 (P \ 0.05). Present investigation showed an average observed heterozygosity of 0.542 and no significant LD among most of the loci. Hence, these loci could be useful in describing genetic stocks of L. fimbriatus for informed management practice. Acknowledgments The work presented here was supported by Indian Council of Agricultural Research, New Delhi. We are thankful to Director, CIFA for providing laboratory facilities to work. We are also thankful to Dr. S. Ayyappan, Secretary DARE and Director General, ICAR for his support and encouragement. Help and support provided by Vijayawada and Bangalore centers of CIFA during samples collection are duly acknowledged.
915
References Balloux F, Lugonae MN (2002) The estimation of population differentiation with microsatellite markers. Mol Ecol 11: 155–165 Bloor PA, Barker FS, Watts PC, Noyes HA, Kemp SJ (2001). Microsatellite libraries enrichment. Protocol available at: http://www. Genomics. liv. ac. uk/animal/research/microsatellite Das P, Barat A, Meher PK, Ray PP, Majumdar D (2005) Isolation and characterization of polymorphic microsatellites in Labeo rohita and their cross species amplification in related species. Mol Ecol Notes 5:231–233 Jena J, Das PC, Mitra G, Patro B, Mohanta D, Mishra B (2011) Evaluation of growth performance of Labeo fimbriatus (Bloch), L. gonius (Hamilton) and Puntius gonionotus (Bleeker) in polyculture with Labeo rohita (Hamilton) during fingerlings rearing at varied densities. Aq. Aquaculture 319:493–496 Kowtal GV (1994) Endangered, vulnerable and rare/food fishes of the east coast river system. Nature conservators, muzaffarnagar (India) 1:57–61 Lewis PO, Zaykin D (2001) Genetic data analysis: Computer program for analysis of allelic data, Ver 1.0. Free program distributed by the authors over the internet available at http://hydrodyction. eeb.uconn.edu/people/plewis/software.php McConnell SKJ, Leamon J, Skibinski DOF, Mair GC (2001) Microsatellite markers from the Indian major carp species, Catla catla. Mol Ecol Notes 1:115–116 Molur S, Walker S (1998) Report of the workshop on conservation assessment and management plan for freshwater fishes of India, Zoo Outreach Organisation, Conservation Breeding Specialist Group, India Coimbatore, India. 156 p report Patel A, Das P, Swain SK, Meher PK, Jayasankar P, Sarangi N (2009) Development of 21 new microsatellite markers in Labeo rohita (rohu). Anim Genet 40:253–254 Punia P, Gupta HS, Singh RK, Mahindra V, Lal KK, Chauhan VS, Lakra WS (2006) Polymorphic microsatellite markers isolated from partially enriched genomic library of Chitala chitala. Mol Ecol Notes 6:1263–1265 You F, Huo N, Gu Y, Luo M, Ma Y, Hane D, Lazo G, Dvorak J, Anderson O (2008) BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinformatics 9:253
123
