c Indian Academy of Sciences

ONLINE RESOURCES

Development and characterization of twenty-nine novel polymorphic microsatellite loci in the mandarin fish chuatsi

CHUN-MEI QU, XU-FANG LIANG∗, WEI HUANG, CHENG ZHAO, LIANG CAO, MIN YANG and CHANG-XU TIAN

College of Fisheries, Huazhong Agriculture University, Wuhan 430070, People’s Republic of China

[Qu C.-M., Liang X.-F., Huang W., Zhao C., Cao L., Yang M. and Tian C.-X. 2013 Development and characterization of twenty-nine novel polymorphic microsatellite loci in the mandarin fish Siniperca chuatsi. J. Genet. 92, e19–e23. Online only: http://www.ias.as.in/jgenet/ OnlineResources/92/e19.pdf]

Introduction expression is under the influence of two different and diver- gent genomes, gene expression in hybrids may reveal infor- Mandarin fish Siniperca chuatsi (Basilewsky), mainly dis- mation about interactions of two parental alleles and their tributed in the and Pearl rivers, is an important com- impacts. So, we hybridized S. chuatsi (♀)andS. scherzeri mercial freshwater fish in China (Liang 1996). It (♂), and de novo transcriptome sequencing for their F1 inter- has a fast growth rate, but is susceptible to diseases. Com- species hybrids was performed. Here, we describe the iso- pared with S. chuatsi, golden mandarin fish S. scherzeri lation and characterization of 29 novel polymorphic SSR (Steindachner) has great disease resistance, but grows slowly. markers for S. chuatsi from this transcriptome and test these Since the retained desired characteristics such as fast markers in S. scherzeri. growth rate and great disease resistance from their parents (Mi et al. 2009), breeding a disease-resistant and faster grow- ing strain has been attracting more and more attention from Materials and methods researchers. However, the research mainly focussed on the morphology of the hybrid (Zhao et al. 2008;Miet al. 2009), Using high-throughput Illumina RNA-seq (BGI, Guangdong, very little was known about its genetic variation. China), the transcriptome from RNA of F1 interspecies Microsatellites, also known as simple sequence repeats hybrids was analysed and 118,218 unigenes were obtained. (SSRs), have become a useful tool to assess genetic diversity This unigene set was used for mining genic-SSR mark- and develop molecular breeding technique in fish because ers using BatchPrimer3 v. 1.0 software and the parame- of their codominant nature and high allelic polymorphism ters were left at the default settings (You et al. 2008). A (Walter and Epperson 2001;Chenet al. 2005). Nevertheless, total of 22,418 SSRs were found. We selected a subset only a few microsatellite markers are available for S. chuatsi of 54 SSR markers from 52 unigene sequences for val- (Zhang et al. 2006; Kuang et al. 2007a, b, 2009;Liuet al. idation. All the relevant sequences have been deposited 2011;Quet al. 2012)andS. scherzeri (Qu et al. 2012;Yang in GenBank (JQ686834–JQ686884) (table 1). Primers for et al. 2012). The number of available SSRs is grossly inade- these SSR loci were designed using NCBI/Primer-BLAST quate for genetic and mapping studies. These research areas (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). have long suffered from one of the challenges of system- The polymorphism of microsatellite loci was evaluated on 39 atic biology studies, namely the lack of genomic resources individuals from a wild S. chuatsi population. Total genomic such as genome or transcriptome sequences. Hence, there is DNA was extracted from fin clips using the TIANamp a need to enhance such resources. With the advent of next- Genomic DNA Kit (Tiangen, Beijing, China) following generation sequencing technologies, transcriptome sequenc- the manufacturer’s instructions. Polymerase chain reaction ing is emerging as a rapid and efficient means for gene (PCR) amplification and SSR genotyping were performed discovery and genetic marker development. As hybrid gene using reagents and protocols described in our previous study (Qu et al. 2012). The genetic diversity indexes, such as the number of alleles (Na), expected (HE) and observed heterozygosities ∗ For correspondence. E-mail: xfl[email protected]. (HO) were calculated using POPGENE v. 3.2 (Yeh and Keywords. microsatellite; SSR; transcriptome; cross-species amplification; Siniperca chuatsi.

Journal of Genetics Vol. 92, Online Resources e19 Chun-Mei Qu et al. * for a N P S. scherzeri PIC E H O H a N C) ◦ ( a T (bp) Size range )  –3  . S. chuatsi F: TTTTAAAGACGGGGCAGCGGR: ACCAACGTTTGGCGTAAAGC F: GGCACTTGTGTAAAGGTTGGATR:TAAATTAAGCCAGGAGCAAAGC 211–309F: AACTCCGCAACCTCATCTGG 252–321R:TCTGTAAAGTGCAGCCTCAGC 55.5F: AATGAGGCCCCTTTTTCCCC 59R:TCCTCTGAATCATCAGTGTGTGG 13 258–291F: AATCTATGCCTTGGCTTTTGG 0.7949R: 0.8615 CCGGACTATCAGTACCCCACT 5 192–286 0.8366 55.5F: CACGAACTACTCGCCCTCAC 1.000 0.2734R: TCTGTGGGAACCGTGGTGAT 174–265 0.6767F: 3 58 CAGGGAACGGGGAAAAAGCA 0.6090 0.5641R:AGAATTCCTCCGTACCTGTCTG 0.0000* 59 199–247 0.6550 12 F:GCATAAAATCTGAGGGGAGCGT 10 0.5739 222–277R: TTGGGCTCCACTGCCTGTAT 1.000 0.0357 58.5F: 206–228 GCTCCCTGCTTTTCCCTCTCA 8 5 0.8603R: GCTCCCTGCTTTTCCCTCTCA 0.1053 53.5 0.8396F:TCAAGGGAGTGTTTAGTTTAGTGT 7 0.1536 0.0088 55.5 0.2078 0.6316R: 0 ACTGGCACATTTGGTAGACAGG 226–276 6 132–186 0.1615 0.7895F: AGGCATTGTGTCACTTTGAGC 0.0526 4 0.7928R: 0.1522 TGCTGGTGTTACCCACCTGA 0.1500 0.0052 12 53.5 55.5 0.5935F: ACAACACCCGTCCCAATGTC 0.3872 0.0265R:GGGTGCTCACACTAGTAAGTGG 0.4027 182–254 0 6 0.0319 4F: AACGTCTCCGTTTCCATCAGG 10 1.000 1.000R: TCTGCTGCCCGAGAGACTTT 54.5 220–290 0.7256F: 0.7244 ACATGACAGCAGGCGACAAT 6 0.6849 0.5555R: 244–298 TTGCTGCTAGAAGAGATCCTCG 7 0.0602 5 0.0007* 54.5F: ACGGAGAGCAGAGTCTTACGTC 0.9744R: ACTCTCCAGCTGCCGTCACA 0.6613 263–392 54.5F: 5 0.5801 202–284 TATGAGCAGGTAGACGGCACA 0.0202 6 0.8718R: 8 TGCAGACCTGTCAGCTGTTTC 54.5 5 0.7483F: TCGGATGAACGCTTGTTGAGA 0.3500 54.5 0.6914 212–271R: GTGCCATAGAGCTCCGTTGT 0.3897 0.3517 9 0.4989 0 1.0000 8 270–318 0.3817 54.5 0.8846 1.0000 0.8483 0.8397 10 0.1891 5 0.8157 54.5 0.0132 1.0000 9 0.6415 5 0.6505 12 0.3684 0.0004* 0.6871 8 0.6208 0.0693 5 0 14 (CACT) 13 3 70 5 7 AA(AC) 6 7 N(AC) 9 (AAC) 3 13 10 AT(AC) 10 N(GAGG) 10 12 12 10 13 5 A(AC) 7 46 42 17 38 Characteristics of 29 polymorphic SSR markers of Accession Table 1. SC01 JQ686834 (TCA) SC02 JQ686835SC03 (GAT) JQ686836SC04 (CA) JQ686837SC05 (TGCT) JQ686838SC09 (ATAG) JQ686842SC10 (GCCTGA) JQ686843SC11 (TTTC) JQ686844SC12 (GAG) JQ686845SC13 (TG) JQ686846SC14 (AC) JQ686847SC15 (TAA) JQ686848SC16 (TAA) JQ686849SC23 (CTT) JQ686856SC24 (GT) JQ686857SC26 (CA) JQ686859SC28 (TGA) JQ686861 (GACA) Locus number Repeat motif Primer sequence (5

Journal of Genetics Vol. 92, Online Resources e20 Siniperca chuatsi microsatellites * for a N P S. scherzeri PIC E H O H 0.0017). The last column shows the number of alleles per < P a N C) ◦ ( a T , observed and expected heterozygosities; PIC, polymorphism information content; E H / O H (bp) )  , number of alleles; a –3 N  , annealing temperature; a T F: TGCCGTTGGAGGAAGTCAGAR: CGGTTGTGCCTGGAGGTATC F: ATGACTCTGGCACCAACGCR:AACAACAGCTAACTGAACACAAG F: AATTAGCATGAGCCAGGGACC 246–277R: GCAGCCTTGAACACACTTGA F: CTGCACACAAACCATCAGACTR: GGGAGACACCTGCTCTTACT 222–257 54.5 180–253F: GTGATTGTCTTGCTCCACCTGR:ATCCTTCCATAGTGTTCTACAGT 298–420F: 55 CAGACCAAAGGACTGCACTG 4 55R: AGGGGCTGGGTTGGCATTAT 0.0500F: AGATTAGGCCTGCAGGTGAC 210–323 55R:ACACACATTTCTTACAGTTTGGT 0.2731 6F: AGAGGGGCCTTGAATCTGGG 7 205–265 0.1960R: 0.9744 TCTGGCTTCAGCCAAACGTG 55 1.0000F: 0.0007* TTGGTGGTGGCTGATCCCAGG 9 175–251 0.7989R: GACGGGCAGAAAACATCCTCC 0.7949 55 1.0000F:CTGTGCTTCGGAAGTTGAATGG 0.7556 204–307 0.7744 8R:ACCTTTACAGTTTCAGTGTCCATGT 0.8137 55 0.0040 8 F: 0.3233 GAATGCCATGTGTCGTTGCG 1.0000 99–106 0.8563 7R: CCGGCCTGACATCCCTAAGA 55 249–320 0.7639F: GTCGGACTTGGTTCAGCTACA 0.0490 1.0000 8R: GTCCTACTCTACCTGCCCCG 0.7564 11 55 0.7816 11 55.5 0.9744 10 0.0955 165–211 0.7352 0.7602 1.0000 224–276 0 0.0505 2 6 0.7092 0.8492 55.5 0.4000 13 1.0000 0.0032 0.7816 55.5 0.4923 0.6730 0.0821 5 11 0.3690 0.6051 6 0.5128 13 0.0391 0.0000* 0.9744 0.7746 10 0.6980 0.7260 0.6318 0.0255 0 8 0.0658 9 10 8 10 22 27 19 19 35 27 25 25 27 19 .) . Continued Accession Size range ( S. scherzeri of 0 indicates no amplification or smear; a , the test for deviation from Hardy–Weinberg expectation; *, significant deviation from HWE after Bonferroni correction ( N Table 1. SC29 JQ686862 (AGTGT) SC36 JQ686868SC38 JQ686870 (GT) SC41 JQ686873 (TG) SC43 JQ686875 (TG) SC44 JQ686876 (TG) SC45 JQ686877 (CA) SC46 JQ686878 (TG) SC49 JQ686880 (CA) SC51 JQ686882 (CTC) SC52 JQ686883 (CA) SC53 JQ686884 (TG) (CA) P loci for Locus number Repeat motif Primer sequence (5 *

Journal of Genetics Vol. 92, Online Resources e21 Chun-Mei Qu et al.

Boyle 1997). Deviations from Hardy–Weinberg equilib- 23 of these were polymorphic (table 1). These results were rium (HWE) for each locus, and genotypic linkage dis- anticipated because the transcriptome was from F1 inter- equilibrium (LD) between all loci were tested using the species hybrids of S. chuatsi and S. scherzeri. It may reveal online version of GENEPOP (http://genepop.curtin.edu.au/) information about interactions of two parental alleles and (Raymond and Rousset 1995). All results were adjusted their impacts. Another explanation is that these EST-SSRs for multiple simultaneous comparisons using a sequential are derived from transcribed regions of the DNA that gener- Bonferroni correction (Rice 1989). ally are more conserved across species, therefore such SSR markers may have a higher rate of transferability than SSRs derived from nontranscribed regions (Barbara et al. 2007). Results and discussion The high rate of successful cross-species amplification indi- cated substantial cross-species transferability of these and In total, 29 of the 54 loci showed moderate to high levels of probably other EST-SSR markers developed from this tran- polymorphism. The remaining 25 loci were either monomor- scriptome. Further, the markers described here may be suit- phic or failed to amplify. Characteristics of these 29 loci are able for assessments of genetic diversity and population given in table 1. The number of alleles per locus ranged from structure in the Siniperca . 2 to 13, with an average of 6.3 alleles per locus. The observed In conclusion, a total of 29 polymorphic microsatellite heterozygosity (H ) varied from 0.0500 to 1.0000 (average O markers were developed using transcriptome sequencing. We 0.7500), while the expected heterozygosity (H ) varied from E only tested a small subset of the SSR loci identified in this 0.1522 to 0.8846 (average 0.6659). Five loci (SC02, SC12, transcriptome. Be that as it may, high levels of polymor- SC26, SC29 and SC51) showed significant deviation from phism and high rate of successful cross-species amplification the HWE after Bonferroni correction (adjusted P = 0.0017). indicated that the transcriptome of F interspecies hybrids It may be the result of nonrandom mating, genetic bottle- 1 could be a rich source of candidate molecular markers for necks and a large heterozygote excess. The presence of null the Siniperca genus. These novel markers will facilitate alleles was checked by Micro-Checker v. 2.2.3 software (Van further studies on genetic diversity evaluation, conserva- Oosterhout et al. 2004), but no evidence for allelic dropout tion genetics, the construction of high-density linkage map was found in these loci. No significant LD was detected and molecular-marker-assisted breeding of S. chuatsi and its across all loci following Bonferroni correction (adjusted P = related species. 0.00013). To determine the function of genes associated with the Acknowledgements SSR markers, BLASTx searches were conducted for all uni- genes with polymorphic SSRs using cut-off E-value <1.00E- This work was financially supported by the National Natural Sci- 10 (Sha et al. 2011). Of the 29 SSR-containing unigenes, ence Foundation of China (31172420), the National Basic Research six had significant similarity to known genes, including Program of China (2009CB118702) and the Fundamental Research mediator of RNA polymerase II transcription subunit 30, Funds for the Central Universities (2010PY010, 2011PY030). We are very grateful to Ashley Flecher for her help in editing the transcription elongation factor B polypeptide 3, lysosomal manuscript. protective protein, transcription initiation factor IIA sub- unit 2, metabotropic glutamate receptor 5 and double C2- References like-domain-containing protein beta-like isoform 2 (table 2). These markers will be useful for molecular marker-assisted Barbara T., Palma-Silva C., Paggi G. M., Bered F., Fay M. F. and breeding. Lexer C. 2007 Cross-species transfer of nuclear microsatellite To evaluate cross-species amplification, we tested the 29 markers: potential and limitations. Mol. Ecol. 16, 3759–3767. Chen S. L., Liu Y. G., Xu M. Y. and Li J. 2005 Isolation and primer pairs described here in a related species, S. scherzeri. characterization of polymorphic microsatellite loci from an EST- Thirty individuals were screened. Of the 29 SSR makers, 27 library of red sea bream (Chrysophrys major) and cross-species primer pairs successfully cross-amplified in S. scherzeri and amplification. Mol. Ecol. Notes 5, 215–217.

Table 2. Annotation of gene-associated SSRs by BLASTx analysis.

Locus BLASTx similarity match E value Species Accession number

SC3-2 Mediator of RNA polymerase II transcription subunit 30 1E-45 Salmo salar ACM08366.1 SC9 Transcription elongation factor B polypeptide 3 2E-43 Danio rerio NP_998620.1 SC29 PREDICTED: Lysosomal protective protein 0 D. rerio XP001331905.3 SC36 Transcription initiation factor IIA subunit 2 3E-45 Esox lucius ACO13378.1 SC46 Metabotropic glutamate receptor 5 1E-90 Cricetulus griseus EGW01814.1 SC51 PREDICTED: Double C2-like domain-containing 0 Oreochromis niloticus XP_003454262.1 protein beta-like isoform2

Journal of Genetics Vol. 92, Online Resources e22 Siniperca chuatsi microsatellites

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Received 22 October 2012, in revised form 31 December 2012; accepted 2 January 2013 Published on the Web: 4 April 2013

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