Microsatellites within genes and ESTs of common carp and their applicability in silver crucian carp

Aquaculture 234 (2004) 85 – 98 www.elsevier.com/locate/aqua-online

Microsatellites within genes and ESTs of common carp and their applicability in silver crucian carp Gen Hua Yue a, Mei Yin Ho a, Laszlo Orban a,*, Johannes Komen b b

a Reproductive Genomics, Temasek Life Sciences Laboratory, Singapore 117604, Singapore Fish Culture and Fisheries Group, Department of Animal Sciences, Wageningen University, Wageningen, The Netherlands

Received 26 August 2003; received in revised form 23 December 2003; accepted 25 December 2003

Abstract Thirty-six new microsatellites were identified from common carp (Cyprinus carpio L.) by screening through genes found in GenBank, EST sequences from a testis cDNA library and a genomic DNA library enriched for CA repeats. Eleven of the twenty-eight microsatellites identified from genes and ESTs were AT repeats, suggesting their high abundance in the genome of common carp. Characterization of the 36 microsatellites on a panel of 18 unrelated common carp individuals revealed that all, except two, were polymorphic with an average allele number of 7.3/locus (range: 2 – 15 alleles/locus). The microsatellites located in genes and ESTs showed higher allele number than those eight which were isolated from a genomic DNA library (7.7/locus vs. 4.9/locus df = 32, P < 0.05). Cross-species amplification showed that 41.7% (15/34) of the primer pairs from common carp amplified specific and polymorphic PCR products in the silver crucian carp (Carassius auratus gibelio). Interestingly, the success rate of cross-species amplification was lower for microsatellites located within genes and ESTs, than for those presumably located in non-coding regions. These novel microsatellites will be very useful for genome mapping and population genetic studies in both species, as well as for studying reproductive strategies of the silver crucian cap. A set of 21 polymorphic microsatellites from our study and those of others were selected as a standardized marker set to be used in studies on genetic diversity of common carp. D 2004 Elsevier B.V. All rights reserved. Keywords: Common carp; Cyprinus carpio; Silver crucian carp; Carassius auratus gibelio; Microsatellite; Gynogenesis and mapping

* Corresponding author. Tel.: +65-6872-7413; fax: +65-6872-7007. E-mail address: [email protected] (L. Orban). 0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2003.12.021

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1. Introduction Common carp (Cyprinus carpio L.; Cyprinidae) apparently originated from eastern Asia, presumably from China, where many exotic varieties were bred. It was later introduced to Europe (Froufe et al., 2002), to North America (Eddy and Underhill, 1974; McCrimmon, 1968) and other continents. As a result, common carp can now be found in freshwaters worldwide, except for South America and Madagascar (Banarescu and Coad, 1991; Nelson, 1994; Hulata, 1995). Common carp is probably the most important freshwater food fish species in the world. By the beginning of the new millennium, the annual production of common carp reached at 15.6 million metric tons, exceeding that of the salmonids (2.3 million metric tons) several times (FAO, 2001). Ornamental varieties of the common carp, known as koi carps or fancy carps, with various forms and coloration have been bred since ancient times and are still popular today. Silver crucian carp (Carassius auratus gibelio) is also a cyprinid species with the ability to reproduce both by gynogenetic and gonochoristic mechanisms (Zhou et al., 2000). Triploid silver crucian carp females can be regarded as reproductive parasites, since they rely on sperm from other species (e.g. common carp) to initiate development of their eggs. The nucleus of the foreign sperm usually does not become part of the offspring’s genome (Yamashita et al., 1990), although there are observations for such occasional contribution (Jiang et al., 1983). The progeny of silver crucian carp are usually genetically identical to their mother, and the wild populations of this species consist of several clonal lines in Japan and China (Ohara et al., 1999; Yang et al., 2001). The species is becoming an important food fish in China and Japan (Ohara et al., 1999; Yang et al., 2001). Microsatellites are short tandem repeated DNA sequences with a length of 1 – 6 bp (Weber and May, 1989). Because of their high polymorphism, even distribution on genomes, ease of genotyping by using PCR and co-dominance, they have been the marker of choice for a number of studies including genome mapping, population and evolutionary genetics, and forensic studies (Goldstein and Schlotterer, 1999). For silver crucian carp, the first set of 15 microsatellites have been isolated by our lab, among which 11 amplified specific products from common carp DNA (Yue and Orban, 2002a). About 80 microsatellites have been isolated so far from the genomes of the common carp (Aliah et al., 1999; Crooijmans et al., 1997) and koi carp (David et al., 2001), several of them were used for studies on population structure of wild carp and cultured carp (Bartfai et al., 2003; David et al., 2001; Desvignes et al., 2001; Lehoczky et al., 2002; Tanck et al., 2000, 2001). However, results of studies generated by different sets of microsatellite markers are difficult to compare. Currently, linkage maps of the common carp genome are being constructed for Quantitative Trait Loci (QTL) mapping by several researcher groups (L. David, personal communication). However, due to shortage of polymorphic microsatellites, some studies had to rely on dominant markers generated by RAPD (Williams et al., 1990) and AFLP (Vos et al., 1995). Most of the microsatellites are type II markers for which no known function have been established (Weber, 1990). Type I markers are associated with genes of known functions and are more useful for comparative gene mapping to study genome evolution (Vignal et al., 2002). Once a QTL has been mapped in a species, conserved synteny of markers

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between species is very useful for finding candidate genes with influence on the QTL. However, type I markers are relatively rare and generally less polymorphic than type II markers. Detection of polymorphic microsatellites located within genes and ESTs provides a possibility to convert type II markers to type I markers (Liu et al., 1999). Here we report the identification of 36 novel microsatellites from common carp using three different methods, as well as their characterization and cross-species amplification in silver crucian carp.

2. Materials and methods 2.1. Searching for microsatellites in GenBank All common carp DNA sequences (ca. 2 Mb) except short sequence repeats (SSR) sequences present in the GenBank before 01. January 2002 were screened for microsatellites by using software Tandem Repeat Finder 2.2 (Benson, 1999) with following parameters: match: 2; mismatch: 7; indel: 7; PM: mini-score: 50; and max period size 500. Only di-, tri-, tetra-nucleotide repeats were targeted, since the mono-nucleotide repeats are not useful for mapping or population genetics due to difficulties with their genotyping. The output files were checked manually, and repeats with the same flanking regions were considered as a single locus. 2.2. Isolation of microsatellites from a genomic DNA library enriched for CA repeats A genomic DNA library enriched for CA repeats was constructed by a method originally described by Fischer and Bachmann (1998) with some modifications (Yue et al., 2000). The insert lengths of clones were examined by colony PCR using M13 – 20 and M13 reverse primers (Clontech, CA, USA). Clones with insert between 250 and 1000 bp were sequenced. Cleaned colony PCR products were used as templates for sequencing reactions. The sequencing reaction was carried out using the ABI Prism Big Dye terminator cycle sequencing kit (Applied Biosystems, CA, USA) with M13 – 20 or M13 reverse primer as described in the manufacturer’s manual. Sequencing was performed on the ABI 3700 (Applied Biosystems). Forward sequences and reverse sequences of each clone were aligned using software MegAlign from the DNASTAR software package (DNASTAR Madison, USA). 2.3. Detection of microsatellites in testis-derived EST sequences Subtracted testis cDNA libraries were constructed by using testis samples isolated from developing carp individuals at 70 and 100 days post-fertilization (dpf) by the use of the BD Clontech PCR-Selectk cDNA Subtraction Kit. Inserts from clones of the library were amplified by colony PCR and sequenced from either their 5Vor 3Vend (HMY, unpublished data). About 800 EST sequences were searched for microsatellites by using software Tandem Repeat Finder 2.2 (Benson, 1999). BLASTn (Altschul et al., 1990) search was carried out for EST sequences containing repeats to identify the genes. Matches were

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Table 1 Characterization of microsatellites in 18 common carp individuals Locus

Repeat

Location in genes Primer sequence (5V– 3V) (BLAST E Value)

Size range

Cca01 Rhodopsin gene (type I) (AJ012013)a Cca02 c-myc gene-1 (D37887)a

(TC)13

5Vflanking region

233 – 259

(TA)20

3VUTR

Cca03 c-myc gene-1 (D37887)a

(TA)39

3VUTR

Cca04 Pit-1 gene (AF132287)a

(TA)24

3VUTR

Cca05 Pit-1 gene (AF132287)a

(TA)18

3rd intron

Cca06 c-myc gene-2 (D37888)a

(TA)26

Cca07 c-myc gene-2 (D37888)a Cca08 c-myc gene-2 (D37888)a

(GAA)3(GAG)6 (GAA)4 (TA)46

5Vflanking region 2nd exon

Cca09 c-myc gene-2 (D37888)a

(AGAC)8(AGAT)19

Cca10 c-myc gene-2 (D37888)a

(TA)30

Cca12 EST (AU183353)a

(GT)20

3Vflanking region 3Vflanking region 3Vflanking region Unknown

Cca13 EST (AU052096)a

(CT)18

Unknown

F: TAAGACATTTCAGTGGCTCAACAG R: TCAGATCAGATCACAAAGCAGAAT F: ATGCAGGGCTCATGTTGCTCATAG R: GCAGACAGACACGTTGCTCTCG F: TGTGTAATCTTGAATGCCTACTTG R: ATCCGATTTCTGTTCGTGATT F: ATCCCTTACCGCCCTGTGT R: AGCTGAAAAACGCTGTCACG F: AACAGTAAAGCCTCAGACACATTC R: GTCACTTTTTCCACAATTTTAAGC F: TTCTACAAGCATGGTTTCTACAGC R: TTTGCGTGCTAAAATTTGTCATA F: CCATTGCGCTGTAATATGAGGTTT R: CGCTTCAACACCAGGGGACTG F: TTTTGTAGACCTTCAGGAACAGAA R: TTGAATGCATTCCTATTTTCATAA F: AATGCCTATTCACATTATGAAAAT R: ACTAAGAATTTGCTATTAAACACTGG F: CAATAGGGCATATGTGCATGGT R: ATTCCTATTTGCATAATGTGAATATG F: ACGCGTCCGGCTGACATTAGAGC R: ACAACCCCCGATCCCCAACACA F: CCAGCAACAGACAGGAGGACA R: GAAAGAAAAACGTGATAAACTGA

Number Ta Ho of alleles (jC) 8

50

0.89

173 – 194 10

50

0.61

282 – 348 14

50

0.89

224 – 258 15

50

0.72

214 – 220

4

50

0.61

154 – 196 13

50

0.78

216 – 245

9

50

0.72

180 – 210

7

50

0.56

263 – 307

6

50

0.72

150/152

2

50

0.22

188 – 244 15

50

0.94

140 – 155

50

0.28

6

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Gene (Accession no.)

Cca14 EST (C88441)a

KIAA1058 protein (1e-08) 3VUTR

Cca16 CD45 (AB031424)a

(TG)32

3VUTR

Cca17 Rap1b (U53783)a

(AACC)6

3VUTR

Cca18 MHC class I antigen (AB018581)a Cca19 Thyrotropin beta subunit (AB003585)a Cca20 MHC class II beta chain (X95433)a Cca21 Gonadotropin beta subunit 2 (X59889)a Cca22 Urotensin II-gamma (M14088)a Cca23 Urotensin II-gamma (M14088)a Cca24 Gonadotropin type II (M37380)a Cca25 Class II TLA beta-1 (M37106)a

(CA)20

3VUTR

(TTTG)6

3VUTR

(AT)13

3VUTR

(TAA)17

5Vflanking region 3VUTR

(TC)21 (AC)32(AT)14 (GT)11

3VUTR

(AT)34

3VUTR

(AT)29

3VUTR

Cca28 EST (AU240319)a

(GACA)6(GATA)11 Unknown

Cca55 EST (testis cDNA library) (AY169245) Cca59 Genomic library (AY169246)

(TC)2(TG)2 (TC)3 TG(TC)3 (TC)11

C-type lectin (1e-25) Unknown

Cca65 Genomic library (AY169247)

(TG)6(TATG)6

Unknown

Cca67 Genomic library (AY169248)

(CA)30

Unknown

F: GCAAAGTCCCATTCTACCCACTCA R: CTGCCACCTGCTGTTCATTCATAA F: CAGCCGCTGGATCCCAACTG R: TGCAGATGCGTAGCAATGTAAACC F: AATGTTTTCGCTAATTTGACACC R: ACAGCATCATTATACACCGATTCA F: CTTTTGCTCTGAGCCAGGTCTTGA R: CTCGCGACATTGGAAAGTGATGA F: ATAAAGATGAGACCAGATGAGTGT R: TAGAGCCATAATATAGATCAATCC F: CCTGACCCTGAAGAGAACAACTAC R: TGGCCTCATCAAAGACATCAAG F: GACACAAGGCTCTGACACATTTC R: TCTTCCCCTCTTTCTTCAACTGCC F: CTGGGTAAATGCATGCTTCAT R: CGTTGCCTTGCATAGTTGAA F: AGGGCTTTTATAGCTTTTCCTACC R: CAGCTTCTCTGTGGAGCAACTGT F: CACCGTGTTTAACCTGATTTACCA R: GCTTATTCCAGTCCTTCACACTCA F: AAATTTTCAAGACTGGGTGGTT R: ACAGCAAGATGACAAAATGAGTG F: TGAGAATGTTAACTGACCCTTTTG R: TCATTCCCTTTAGCTTCCCTGTC F: ATGACCGACAGACAGACAGACCTT R: CCATCCATCCGAATTCTGCTAA F: ATTCCTGGCTGATTTCAGTGAGAA R: AGACCACCCAAACCGACAGG F: TTTGCCAAATTTGCTACTGTTATG R: TTTGGCGAAAATTACTTCCAGA F: CAAGTGAGCGGGAGACAGAGA R: TCAGCGGTTAGGAGACAGTAGG F: GTAGCCCCAAAAGATGTAGCA R: TGGTCAAGTTCAGAGGCTGTAT

176 – 214

9

50

0.83

207 – 239 10

50

0.78

258 – 293 12

50

0.78

322 – 367

2

50

0.00

239

1

50

0.00

222 – 330 11

50

0.72

172 – 194

6

50

0.11

259 – 272

2

50

0.00

144 – 236 10

50

0.33

134 – 176

6

50

0.28

210 – 252 10

50

0.78

225 – 285

8

50

0.61

98 – 152

5

50

0.11

208 – 293

4

50

0.67

225 – 245

5

55

0.72

184 – 194

4

55

0.72

228 – 254

5

55

0.33

89

(continued on next page)

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Cca15 Taurine transporter (AB006986)a

(GT)7 TT(GT)24 (AT)8GT(AT)6

90

Table 1 (continued ) Locus

Gene (Accession no.)

Location in genes Primer sequence (5V– 3V)

Size range

Cca72 Genomic library (AY169249)

(GATA)9

Unknown

244 – 299

8

55

0.78

Cca80 EST (testis cDNA library) (AY169250) Cca84 EST (testis cDNA library) (AY169251) Cca85 Genomic library (AY169252)

(GAA)9

144 – 184

3

50

0.06

(CA)7

MetAP 2 (4e-15) Unknown

103 – 103

1

50

0.00

(AT)6(GT)11

Unknown

109 – 135

5

55

0.50

Cca86 Genomic library (AY169253)

(AC)10

Unknown

186 – 196

5

55

0.61

Cca90 Genomic library (AY169254)

(AC)12

Unknown

207 – 215

3

55

0.78

Cca91 Genomic library (AY169255)

(CT)6(GT)15

Unknown

252 – 260

4

55

0.61

Ta: annealing temperature; Ho: observed heterozygosity. a

Sequences retrieved from GenBank.

F: CAGGCCAGATCTATCATCATCAA R: CTGCTGTTGGATATGCACTACATC F: ACCGAACGGAGACTCTCACCTTCC R: CTCGCCATCTTCCTCTGCCTCCTC F: GTCGGCCAGCGCTGATGTGT R: CGAGCCGGAAGAGTTGAGTGATG F: GCTTCTTTCGAATAAGTAATTC R: TAAGTGCGCATCTGATGTAA F: GTATTCCTCTGCCTTTCCACAA R: CACTTCATGCACTCGTTCACC F: CCTCTGCCACAGTGCCAGTG R: AGGGCCACAGACAAGAGATACCA F: TATTTATCCATAAGCAGCCCTCAG R: TTCTTGCTCTGTATGCCTCAGTG

Number Ta Ho of alleles (jC) G.H. Yue et al. / Aquaculture 234 (2004) 85–98

Repeat

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considered to be significant only, when the smallest sum of the probability ( P) of the BLAST search was less then 0.0001. 2.4. Characterization of microsatellites and cross-species amplification Primers were designed for the flanking regions of each locus by using commercially available software PrimerSelect (DNASTAR Madison) in such a way that their annealing temperatures were as close to 50– 55 jC as possible. One primer of each pair was labeled with a fluorescent dye Hex or Fam. For characterization of microsatellites and crossspecies amplifications, fin clips were collected from 18 common carp individuals and 12 silver crucian carp individuals. The common carps originated from three different countries (14 from Hungary, 2 from China, and 2 from Israel), whereas the silver crucian carps were from Poland and China (3 from Poland, 3 from a fish farm in Jiangsu province, 3 from another fish farm in Zhejing province and 3 from the wild in Zhejing province). DNA samples were isolated from the fin clips using a traditional phenol – chloroform method (Blin and Stafford, 1976) or a simplified method developed in our lab (Yue and Orban, 2001). PCR amplification was conducted in 25 Al reaction volume; the master mix contained 1  PCR buffer (Finnzymes, Espoo, Finland) with 1.5 mM MgCl2, 0.2 AM of each primer, 200 AM dNTP, 40 ng genomic DNA as template and 0.6 U of DyNAzyme DNA polymerase (Finnzymes). The PCR program was: 2 min at 94 jC followed by 34 cycles of 94 jC for 30 s, annealing temperature (see Table 2) for 30 s and 72 jC for 30 s with a final extension at 72 jC for 5 min. Separation of PCR products was carried out by using an automated DNA sequencer ABI 377 (Applied Biosystems). Gels were analyzed by the use of GenScan and Genotyper supplied by Applied Biosystems. 2.5. Data analysis Allele number and observed heterozygosity were analyzed using software GDA (Lewis and Zaykin, 2000). Since silver crucian carp is a triploid gynogenetic species, we only looked at the observed proportion of tri-allelic and di-allelic heterozygotes. Association analysis and Student’s t-test were performed by using the options under Data analysis in Microsoft Excel 97.

3. Results 3.1. Detection of microsatellites A total of 28 microsatellite sequences were detected from the genomic DNA and EST sequences of the common carp deposited in GenBank before 01 January 2002. Twenty of them were di-nucleotide repeats, four tri-nucleotide repeats and four tetra-nucleotide repeats (Table 1). Most (24/28) of the microsatellites were located in the 5V UTR (5Vuntranslated region) or 3VUTR of genes. Out of the four remaining microsatellites, three (Cca12, Cca13 and Cca28) were detected in EST sequences deposited in GenBank, whereas Cca07 was located in the second exon of C-myc gene-2 (GenBank No. D37788;

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(Zhang et al., 1995). Interestingly, four additional microsatellites (Cca06, Cca08, Cca09 and Cca10) were located in the UTRs of the same gene. Three microsatellites were found in the ESTs of the subtracted testis cDNA library of common carp; these were a CA-type, GAA-type and a compound type. Among the 31 microsatellites from expressed sequences, 11 were AT repeats, 5 TC repeats, 5 AC repeats and 10 other repeats. Their repeat number ranged from 6 (Cca17) to 46 (Cca08). Twentyeight of these thirty-one microsatellites had sufficiently long flanking sequences for primer design (Table 1). Eight microsatellites were obtained from the genomic library of common carp enriched for CA repeats. Seven of the microsatellites contained CA/GT repeats. Repeat length ranged from 6 to 30. Primers were designed for all of these eight microsatellites as well. 3.2. Characterization of microsatellites The characterization of microsatellites on 18 common carp individuals (from three different sources) revealed that all the 36 primers generated specific PCR products. Four primer pairs (Cca12, Cca15, Cca16 and Cca22) seemed to amplify products from two loci, only the highly amplified products were considered in the analysis. With the exception of Cca18 and Cca84, all markers were polymorphic. The average allele number of the 34 polymorphic microsatellites was 7.3/locus (range: 2 – 15). The average observed heterozygosity was 0.64, its value ranged from 0.11 to 0.94 (Table 1). The average allele number of the 28 microsatellites located within genes and ESTs was significantly higher than that of the 8 microsatellites isolated from a genomic DNA library (7.66 + 0.76 vs. 4.88 + 0.52, df = 32, P = 0.007).

Table 2 Polymorphism of cross-amplified common carp microsatellites in 12 silver crucian carp individuals Locus

Ta (jC)

Size range (bp)

Number of alleles

Proportion of heterozygosity (%) Tri-allelic

Di-allelic

Mono-allelic

Cca02 Cca04 Cca07 Cca12 Cca14 Cca15 Cca19 Cca22 Cca23 Cca55 Cca65 Cca67 Cca86 Cca90 Cca91

50 50 50 50 50 50 50 50 50 50 55 55 55 55 55

144 – 186 328 – 356 222 – 228 232 – 258 180 – 190 293 264 – 280 142 – 148 120 – 138 232 – 238 175 – 193 175 – 191 180 – 198 207 – 219 272 – 300

6 6 2 5 4 1 3 2 3 3 4 3 9 3 7

100 100 0 25 0 0 0 0 0 0 58 0 33 0 25

0 0 0 75 0 0 75 100 25 17 42 17 67 100 75

0 0 100 0 0 100 25 0 75 83 0 83 0 0 0

Ta: annealing temperature.

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3.3. Cross-amplification in silver crucian carp Cross-amplification of common carp microsatellites on silver crucian carp DNA showed that 15 (41.7%) of the 36 common carp primer pairs amplified specific PCR products in the silver crucian carp (Table 2). Five of the eight (62.8%) microsatellites from the common carp genomic library were conserved in the silver crucian carp, whereas the figure was only 35.7% (10/28) for microsatellites within genes and ESTs. The allele number at the 15 loci ranged from 1 to 9 with an average of 4.1/locus. Only locus Cca15 was monomorphic. At two loci (Cca02 and Cca04), all 12 individuals were tri-allelic heterozygotes. The observed heterozygosity (tri-, di-allelic heterozygosity) ranged from 0 to 100% with an average of 62.8%.

4. Discussion In this study, we have used three different methods to identify 36 microsatellites. The easiest way to obtain microsatellites is by screening DNA sequences deposited in GenBank. In previous studies, we have obtained polymorphic microsatellites from Asian seabass (Yue et al., 2001) and tilapias (Yue and Orban, 2002b) by using this method. However, for most commercially important fish species, there are a limited number of sequences available in public databases. The most productive method for the isolation of microsatellites is to search through the genomic DNA of the target species, however, traditional screening methods are rather time-consuming and costly. Currently, a number of simplified methods have been established (Zane et al., 2002). In these, target repeats (e.g. CA repeats, GC repeats) were first enriched and then cloned into vectors and sequenced. Most of these enrichment methods proved to be simple and cost-effective. Most of the microsatellites obtained through library screening are type II markers, which are not associated with functional genes. Type I markers are more useful for comparative mapping to study genomic evolution (Liu et al., 1999). Screening through cDNA libraries is an effective method for identifying polymorphic microsatellites within genes as shown by Liu et al. (1999) and by this publication. A third alternative for finding polymorphic microsatellites is cross-species amplification between genetically closely related species e.g. (Smith et al., 2000)). The successful rate of cross-species amplification decreases with the increase of phylogenetic distance between the species tested (Estoup et al., 1995), and seems to be locus-dependent (Peakall et al., 1998; Yue et al., 2003). CA-type repeats are the most abundant repeats in the majority of vertebrates (Toth et al., 2000), including mammals (Weber, 1990), and several fish species (e.g. Edwards et al., 1998; Lee and Kocher, 1996; Roest Crollius et al., 2000). On the other hand, AT-type repeats are the most frequent ones in yeast, exons of fungi and introns of C. elegans (Toth et al., 2000). In this study, we found that in the common carp, the most abundant repeats located within genes and ESTs were also AT repeats, followed by AC and GC repeats. Preliminary search for microsatellites in 100 Mb zebrafish genomic DNA sequence also found AT repeats being very abundant (unpublished data). It remains to be tested whether AT repeats are also so frequent in others species in the Cyprinidae family and whether their distribution is universal or preferential in expressed sequences.

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The observed heterozygosity of our microsatellites was higher than those in freshwater fish species and lower than those in anadromous and marine fish species (De Woody and Avise, 2000). An interesting finding of this study is that microsatellites from genes and ESTs showed higher allele number than the ones isolated from genomic DNA library. There is indirect (for review, see Kashi et al., 1997) and direct evidence (Streelman and Kocher, 2002), that microsatellites located in regulatory regions of genes can have a functional importance, which might explain the higher allele number for gene-related repeats. Whether this tendency would hold for a larger number of microsatellites in common carp needs to be determined, since the number of loci tested here was limited to 36 and in other studies genomic DNA libraries were screened with CA repeats. Nonetheless, as most of the microsatellites reported in this paper were located in genes and ESTs, they will be very useful for mapping these genes and ESTs in linkage maps and comparative mapping. The Cca07 microsatellite may be of particular interest, because the change of repeat length will change the amino acid sequence. Microsatellites have been used extensively for genetic diversity and population structure studies in many species. For genetic diversity studies on common carp, various laboratories used different sets of microsatellites (Bartfai et al., 2003; David et al., 2001; Desvignes et al., 2001; Lehoczky et al., 2002; Tanck et al., 2000), which makes the comparison of their results difficult. Based on 91 common carp microsatellites characterized (Aliah et al., 1999; Crooijmans et al., 1997; David et al., 2001) and on those 10 silver crucian carp microsatellites, which cross-amplify from the carp genome (Yue and Orban, 2002a), as well as the 36 ones in this study, it is now possible to select a set of highly polymorphic, easily scoreable microsatellites for genetic diversity and population structure studies. By taking the polymorphism of the markers, ease of their amplification, their previous usage into account and positions of some microsatellites in the linkage map (Sun and Liang, in press), we would like to propose the standardized use of 21 microsatellites1 (Table 3) for population genetic studies in common carp (and possibly other cyprinids as well). Such standardized microsatellite sets have been successfully implemented for the studies of farm mammals, simplifying the comparisons among studies performed by different labs (Laval et al., 2000). Fourteen of common carp microsatellites primer pairs cross-amplified polymorphic products from silver crucian carp DNA. Together with the 15 polymorphic microsatellite markers isolated earlier from silver crucian carp (Yue and Orban, 2002a), they provide a suitable number of markers applicable for studies on genetic diversity, identification of clonal lines and reproductive strategies of C. auratus gibelio. One surprising finding of the present study is that the successful rate of cross-species amplification was lower for microsatellites located within genes and ESTs, suggesting that these microsatellites evolved at a quicker rate than those located in other genomic regions. The reasons for this remain to be studied.

1 Due to the current status of the genetic map for common carp, the mapping positions could only be determined only for three markers (data not shown).

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Table 3 A list of 21 microsatellites recommend for genetic diversity studies of common carp Locus

Motif

(CA)na

Primer (5V– 3V)

F: TCCAAGTCAFTTTAATCACCG R: GGGAAGCGTTGACAACAAGC MFW6 (CA)na F: ACCTGATCAATCCCTGGCTC R: GGGAAGCGTTGACAACAAGC MFW7 (CA)na F: TACTTTGCTCAGGACGGATGC R: ATCACCTGCACATGGCCACTC MFW9 (CA)na F: GATCTGCAAGCATATCTGTCG R: ATCTGAACCTGCAGCTCCTC MFW13 (CA)na F: ATGATGAGAACATTGTTTACAG R: TGAGAGAACAATGTGGATGAC MFW16 (CA)na F: GTCCATTGTGTCAAGATAGAG R: TCTTCATTTCAGGCTGCAAAG MFW26 (CA)na F: CCCTGAGATAGAAACCACTG R: CACCATGCTTGGATGCAAAAG CCA30 (GT)18 F: CTGCCTTCTTCTACTCTACAC (GA)20 R: TTGCCTCTAAGCTTGATTTT Koi29 – 30 (TGGT)6 F: CTGACCCTGAAGAGAACAAC R: GCCTCATCAAAGACATCAAG Koi57 – 58 (CT)21 F: TGTCCTTTATTGCTCAGAAC R: CCACCACATTCATCACAT Koi69 – 70 (GT)13 F: GTGATAGGTTTAGGTGTAGG R: TTTGTTATTTTTTGATTACTT Koi89 – 90 (CT)20 F: CTTCAGACAACCCCAATA R: ACACATTAGAGCCGAAAGAG Koi105 – 106 (CA)34 F: AGTCCAAGCGGGTGAATA R: TGTTTCTGCCCTGCTCTG Koi115 – 116 (GT)11 F: GAGGAAATGATGGAATAAAT R: TAAGAGGGTTTTGTAGTGTA J58 (CA)14 F: GCGGTCCTGCCTCAAAGTA R: GAACCCTAAAGGCGACATCAA Cca02 (TA)20 F: ATGCAGGGCTCATGTTGCTCATAG R: GCAGACAGACACGTTGCTCTCG Cca04 (TA)24 F: ATCCCTTACCGCCCTGTGT R: AGCTGAAAAACGCTGTCACG Cca06 (TA)26 F: TTCTACAAGCATGGTTTCTACAGC R: TTTGCGTGCTAAAATTTGTCATA Cca24 (AT)34 F: AAATTTTCAAGACTGGGTGGTT R: ACAGCAAGATGACAAAATGAGTG Cca67 (CA)30 F: GTAGCCCCAAAAGATGTAGCA R: TGGTCAAGTTCAGAGGCTGTAT Cca72 (GATA)9 F: CAGGCCAGATCTATCATCATCAA R: CTGCTGTTGGATATGCACTACATC MFW4

Ta (jC)

Mg2 + Number Size (mM) of range alleles (bp)

55

1.5

7

138 – 152 A, B, C

50 – 55 1.5

5

144 – 152 A, E, F 174 – 269 A, B, C, D, E, F 87 – 135 A, C, E, F, 174 – 196 A, C, D, F 115 – 181 A, D, F

Reference

55

1.5

16

55

1.5

8

50 – 55 1.5

11

55

1.5

17

55

1.5

15

50

1.5

9

122 – 150 A, C, D, E 260 – 318 B, G

50

1.5

5

247b

B

52

1.5

4

231b

B

50

1.5

4

186b

B

52

1.5

4

194b

B

50

1.5

5

192b

B

46 – 55 1.5

6

245b

B

55

1.5

4

124 – 172 H

50

1.5

10

50

1.5

15

50

1.5

13

50

1.5

10

55

1.5

5

55

1.5

8

173 – 194 This study 224 – 258 This study 154 – 196 This study 210 – 252 This study 228 – 254 This study 244 – 299 This study

A: (Crooijmans et al., 1997), B: (David et al., 2001), C: (Bartfai et al., 2003), D: (Desvignes et al., 2001), E: (Tanck et al., 2000), F: (Lehoczky et al., 2002), G: (Aliah et al., 1999), and H: (Yue and Orban, 2002a). a No detailed information was given in the paper (Crooijmans et al., 1997). b No size range was given in the literature (David et al., 2001).

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Acknowledgements This study was supported from internal research funds by Temasek Life Sciences Laboratory. We would like to thank Drs. Lior David and Pawel Brzuzan for supplying fin clip samples or DNA samples.

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