University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln

Faculty Papers and Publications in Science Animal Science Department

2006 Characterization of 35 Microsatellite Loci in the Pacific Lion-Paw ( subnodosus) and Their rC oss-Species Amplification in Four Other of the Pectinidae Family Ana M. Ibarra Centro de Investigaciones Biológicas del Noroeste S.C., [email protected]

Jessica Lynn Petersen University of Nebraska-Lincoln, [email protected]

Thomas R. Famula University of California - Davis

Bernie May University of California - Davis

Follow this and additional works at: http://digitalcommons.unl.edu/animalscifacpub Part of the and Fisheries Commons, Biodiversity Commons, Cellular and Molecular Physiology Commons, Marine Biology Commons, Molecular Genetics Commons, and the Zoology Commons

Ibarra, Ana M.; Petersen, Jessica Lynn; Famula, Thomas R.; and May, Bernie, "Characterization of 35 Microsatellite Loci in the Pacific Lion-Paw Scallop () and Their rC oss-Species Amplification in Four Other Scallops of the Pectinidae Family" (2006). Faculty Papers and Publications in Animal Science. 819. http://digitalcommons.unl.edu/animalscifacpub/819

This Article is brought to you for free and open access by the Animal Science Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Papers and Publications in Animal Science by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Published in Molecular Ecology Notes (2006) 6: 153-156. DOI: 10.1111/j.1471-8286.2005.01173.x. Copyright 2006, Wiley. Used by permission.

PRIMER NOTE Characterization of 35 Microsatellite Loci in the Pacific Lion-Paw Scallop (Nodipecten subnodosus) and Their Cross-Species Amplification in Four Other Scallops of the Pectinidae Family

Ana M. Ibarra,1 Jessica L. Petersen,2 Thomas R. Famula,2 and Bernie May2

1Aquaculture Genetics Laboratory, Centro de Investigaciones Biológicas del Noroeste S.C., La Paz B.C.S., México (Corresponding author: [email protected]) 2Department of Animal Science, University of California-Davis, Davis, California, USA Abstract Four microsatellite-enriched DNA libraries yielded 35 microsatellite loci from 100 primer pairs designed for Pacific lion-paw scallop,Nodipecten subnodosus. The number of alleles ranged from four to 28. Three of the 35 loci were not in Hardy–Weinberg equilibrium and linkage disequi- librium was found for one pair of loci. These microsatellites will be used to analyze the popula- tion structure of the species in Mexico’s Baja Peninsula to propose management strategies for scallop aquaculture development. Twenty-six primer pairs cross-amplified inNodipecten nodo- sus, whereas none (Argopecten ventricosus) or few cross-amplified in theArgopecten species. Keywords: Argopecten, Guerrero Negro — Ojo de Liebre lagoon, Microsatellite, Primers Article history: Received July 13, 2005; revision accepted August 25, 2005. The lion-paw scallop, Nodipecten (Lyropecten) subno- sequenced (n = 170) from this mixed DNA by Genetic dosus, is the largest scallop species found in the tropi- Identification Services (Chatsworth) according to Mer- cal waters of the East Pacific (Peña 2001). In Mexico, edith & May (2002) and Schwartz & May (2004). One it is harvested on the Pacific and Gulf of California hundred and four primers were designed using PRIM- sides of the Baja California Peninsula and on the coast ERSELECT 4.0 (DNA Star Inc.) at the GVL of Univer- of Sonora state. The largest population is found in the sity of California-Davis. lagoon of Ojo de Liebre, which is part of the Guerrero To determine if loci amplified in scallop or crayfish, Negro lagoon system on the Pacific side of the Baja primers were first tested on four scallops from the same Peninsula. A fundamental question for the optimal de- wild population. All 35 ‘positive’ primers for the scal- velopment of lion-paw scallop aquaculture is whether lop were screened on 30 individuals, including 25 from populations native to different lagoons are genetically Ojo de Liebre lagoon on the Pacific side of the Baja Cal- distinct. We isolated and characterized microsatellite ifornia Peninsula, and five from Bahia de La Paz, on loci that will allow for the analysis of population struc- the Gulf of California side of the peninsula. We further ture, from which optimum management strategies for tested these primers for cross-amplification of the mi- aquaculture ventures can be proposed. These markers crosatellite loci in five to six individuals of each of the will also allow for evaluation of optimum spawning following scallop species: Argopecten irradians (USA, strategies for hatcheries that minimize genetic drift and west Atlantic), Argopecten purpuratus (Chile, east Pa- inbreeding in this functional hermaphrodite species. cific), Argopecten ventricosus (Mexico, east Pacific) and Whole genomic DNA was extracted using QIAGEN’s (Venezuela, west Atlantic). DNA DNeasy Tissue Kit from adductor muscle of wild lion- was extracted from muscle tissue preserved in 70% paw scallops naturally settled at Bahia de La Paz. DNA alcohol (A. irradians and A. ventricosus), or from dried obtained from four scallops and 18 crayfish, the last adductor muscle tissue (A. purpuratus and N. nodosus). also being studied in the Genomic Variation Labora- Polymerase chain reaction (PCR) was performed us- tory (GVL), were mixed in equal concentrations prior ing 5 ng of genomic DNA, 1 × Taq DNA polymerase to library construction. Four libraries enriched for tet- buffer B, 2.0 mm MgCl2, 0.2 mm of each dNTP, 1 µm ra- and trinucleotide repeat motifs (TAGA)n, (TGAC)n, of each primer and 0.38 U Taq DNA polymerase (all

(TACA)n and (ATC)n were constructed, screened and reagents from Promega), with a total PCR volume 154 Ib a r r a , Pe t e r s e n , Fa m u l a & Ma y i n Mo l e c u l a r Ec o l o g y No t e s (2006) 6 of 10 µL. PCR was carried out using an MJ Research = 5)

PTC-100 under the following conditions: 94 °C for 2.30 n min, 30 cycles at 94 °C for 30 s, 56 °C for 30 s (53 °C for cross-amplifications), 72 °C for 30 s, followed by 72 °C for 5 min, and held at 4 °C. For those primers 4 (237–247) 5 (204–218) 5 (180–300) 4 (287–298) 5 (220–300) 4 (128–138) 5 (200–242) Bahía de La Paz ( 4 (285–300) 3 (235–278) no. of alleles (size range) (size alleles of no. producing multiple-bands in the primary species (N. 8 (120–200) subnodosus), we performed touchdown PCR: 95 °C for

1 min followed by 30 cycles at 95 °C for 1 min, 67 °C (1) for 45 s with a 0.5 °C decrease each cycle, and 72 °C for 2 min, using the same reagents and equipment. The significance 0.7844 0.2937 0.6134 0.8284 0.6237 0.0013* 0.0000* P(HWE) & 0.6166 0.0069 amplified products were diluted 1:1 with 98% forma- 0.1053 mide loading buffer, denatured at 95 °C for 2 min and

E 0.86 0.66 0.92 0.54 0.80 0.74 H 0.91 0.54 0.67 chilled on ice for 2–3 min. Samples were separated on a 0.90 5% denaturing polyacrylamide gel at 50 W for 70 min.

O 0.86 0.80 0.95 0.58 0.91 0.95 0.48 0.65 H 0.50 Amplified products were visualized using the Sybr- 0.79 Green™-agarose overlay protocol (Rodzen et al. 1998) and scanned with a Molecular Dynamics FluorImager 595. Product sizes were estimated by comparison with a standard 400 bp ladder (The Gel Company). The primer sequences amplifying in N. subnodosus, size range (bp) 230–267 204–218 180–300 283–298 220–320 126–144 159–285 285–300 235–290 Gel estimated allele their polymorphism data and analyses for Hardy– 120–240 Weinberg equilibrium (HWE) and linkage disequilib- alleles 9 4 13 4 9 6 13 4 5 No. of rium (LD), are presented in Table 1. The repeat mo- 14

tifs are specific to the one sequenced clone for each 3

2

locus. Observed and expected heterozygosities were

9

9 6 11

calculated from the 25 individuals from Ojo de Liebre 11 (TCA) 5 5 12 (C) (TCG) 15 lagoon. For the five individuals from Bahía La Paz, 4 3

10 AAATCA (GTC) TA(TCA) (TCG) (TCG) TTA(TGA) N TAT(CAT)

we present number of alleles and their size ranges. 3 9 10 12 18 2 10 21 5 21

Deviations from HWE and LD were calculated using ) from Lagoon Ojo de Liebre (OL) within the Guerrero Negro lagoons system and Bahia de La Paz, Mexico. GenBank CAT(CGT) TCAATCATACA(TCA) TCG(TCA) CAA(TCA) (TGA) (TCA) (TCA) (CAT) (TCA) (ATG) (TCA) (TCA) (CAT) (ATC) gda program (Lewis & Zaykin 2001), and significance Repeat motif

23 21 25 21 22 24 22 22 24 21 was evaluated with the Fisher’s exact test. Three loci n were not in HWE after applying a Bonferroni correc- + + + + + + TD tion, NsubA007, NsubA010 and NsubB215, although it PCR is not known if these deviations are due to null alleles Nodipecten subnodosus or nonrandom mating because of the self-fertilizing ca- pability for this functional hermaphrodite. Significant pairwise LD was found only between loci NsubA223 and NsubC262 following a test using genotypes to pre- vent within locus disequilibrium from affecting the significance and a Bonferroni correction for multiple comparisons (P = 0.0014). Results of cross-amplification of primers on the other

four species are in Table 2. Of the 35 primer pairs de- : CACAAAACAGAATGTGTGAAGA : GAATGCGACATAGACATCG : ACAGCGACCAGTGTAACG : TCGGTGATACAAGTTGAAGAGG : CGGCACATCATTGTTACTAAA : CGGCATCTGTACCTGACAC : CACCCGGAGCTTAAATATAAGG : GACAGGGGACCAGTCTACC : CTGTTGCTGTTCTCAGACAAC : CAATTTCCAGTGTGTTGTCAT : GAGGATGACAGTGTGAAGATG : TTTGACAGACATCCCTTCTTAC : CGACAAACATCCCCTCTT : AACGCTGTCACTGAAACAAAAG : TTTGACTTGAAAGACCATCTCT : GTTACGTGCGTGGACTGAA : CCTCCTCTCCACTTTCTTTCAC : TTTTGTGACGCACCAACTA : CCTGCTCTTTGAACTGCTAG : ACCTCATCTGGTGTTCAATTC Primer sequence (5'-3') veloped, nine (25%) resulted in no amplification for F F F F F F F F F F all the cross-amplified species. None of the primers amplified in A. ventricosus. Twenty-six (74%) of the 35 primer pairs that successfully amplified polymorphic GenBank Accession no. loci in N. subnodosus amplified in N. nodosus, the At- DQ108619 DQ108620 DQ108621 DQ108622 DQ108623 DQ108624 DQ108625 DQ108626 DQ108627 DQ108628 lantic species from the same genus. The next largest cross-amplification was seen forA. irradians (11 primer

pairs) and for A. purpuratus (seven primer pairs). Table 1 Characterization of 35 microsatellite loci in heterozygosities, expected and lion-paw observed (bp), range size scallop allele estimated gel alleles, ( of number genotyped, individuals of number PCR, touchdown using amplified loci motif, repeat sequences, primer nos, Accession and test for conformance to Hardy-Weinberg equilibrium the Ojo de Liebre lagoon. Allele number size ranges Bahía La Paz Locus NsubA001 R NsubA004 R NsubA005 R NsubA007 R NsubA010 R NsubA208 R NsubA214 R NsubA222 R NsubA223 R NsubA227 R Mi c r o s a t e l l i t e Lo c i i n Pa c ifi c Li o n p a w Sc a l l o p (No d i p e c t e n s u b n o d o s u s ) a n d Cr o s s -Sp e c i e s Am p l ifi c a t i o n i n Ot h e r Pe c t i n i d a e Sc a l l o p s 155 = 5) n 4 (145–160) 3 (78–90) 5 (200–225) 6 (110–155) 5 (142–158) 6 (142–358) 7 (170–290) 4 (168–217) 2 (195–202) 6 (240–278) 5 (270–295) 8 (255–300) 5 (240–290) 6 (147–210) 4 (242–280) Bahía de La Paz ( 6 (290–405) 8 (195–270) 5 (145–180) 4 (360–500) 3 (138–147) 4 (305–320) 6 (262–290) no. of alleles (size range) (size alleles of no. 4 (240–280) 4 (150–178) 9 (95–400)

(1) significance 0.0031 0.5216 0.4853 0.5319 0.0078 0.9794 0.4478 0.1219 0.6253 0.1025 0.5241 0.8634 0.0016 0.5334 0.3769 P(HWE) & 0.0084 0.2491 0.3788 0.5822 0.1647 0.2313 0.5650 0.6572 0.5375 0.0000*

E 0.85 0.72 0.78 0.92 0.78 0.93 0.89 0.93 0.84 0.85 0.83 0.89 0.76 H 0.89 0.83 0.89 0.88 0.75 0.96 0.57 0.81 0.87 0.87 0.72 0.97

O 0.71 0.86 0.71 1.00 0.88 1.00 0.94 0.74 0.95 1.00 0.77 0.84 0.60 0.90 1.00 0.70 H 0.81 0.86 0.96 0.48 0.88 0.95 0.95 0.73 0.80 size range (bp) 125–200 78–114 200–230 98–155 142–165 138–440 170–320 155–250 195–240 240–295 260–320 250–320 240–290 132–225 242–284 285–430 195–280 145–180 250–520 138–150 293–320 260–325 240–320 150–178 95–420 Gel estimated allele alleles 9 5 7 13 7 17 9 15 9 8 8 11 5 14 7 12 10 5 20 4 6 12 9 4 28 No. of

16 9

11

13 5 15 TGA

2 (AGA)

4

2

11 8 4 14 10 8

6

(GTC) 3 4 6 3

11 6 (ATAC) 6 (ATC) (ATG) (TGGA) (TCA) N CA(TACA) 9

(TGA) (GATA) 7 18 (TAGA) N 8 TGGTA(TGTA) 8 4

ATGTCTGACC (ATCT) (TATC) 16 10 8 12 13 4 12 12 4

7 8 7 4 4 4

N AT(GAT) TCTCTTAAA N T(ATC) N G(CAT) 5 11 7 5 4 4 8 13 6 4 9 9 (TCC) 6 A(CGA) (TCA) N (ATAC) TACG(TACA) CA(TACA) (TATC) (TGGA) CATA(GATA) (TAGA) Repeat motif (TCA) (TCA) (TCA) (TGTA) (TACA) (GTCT) (GAT) (CAT) (TCA) (GATA) (TATC) (ACG) (TGA) (TCA) (TCA) (ATC) (ATGT) (CATA) (ATAC) (TACA)4CA(TACA) (TGTC) (GACA) (TATC) (TGTC)

23 n 25 21 21 22 19 18 25 22 22 21 22 19 24 24 25 20 22 20 25 22 21 25 24 23 PCR TD + + + + + + + + + + +

: GGCTGTAAATCTCACCAAATG : GAGGAAGATGATGATGATGATT : CTGTTTACTTTCGATGCAAGTT : TGCTGGTCGAGTTCTTAAAGAG : CACACGGAACATCAACTTAACC : TAATGCTAATACGGCACCAGT : GCCCCATCGTAACAATCT : CGTTTATACAGGCATGTTTCTG : CCTGGTAGGTGAATCACACTAA : AATGGGTTGATGATGATTGTC : ATAGCGTAAAGTGTGCAACGT : TTCCATTCCGTTTACACAGATA : TGGAAGTACAGAAAGGAATGTC : TATTCCTTTCTCGCCCATAT : ATGGATTTGACATCATCACAAC : ACCATTGACCAGGCTACAT : GCTTCAGCTTAACGATTGAAAC : TGTGAAAGACAGAGTTATGATCGAG : TTATGGCCGGAAAGTATCAATCAG : CAGCATAAACCCTACTCATGGT : GTCGTTAAAGGTTGGTGTTG : TTACCTGTAGGTGTGGAGACAC : CTTGGCACCATATTCGGAC : GCATTGTCAACTGAATTAGCA : CGGTGATTACACAAATACACC : TCTCCCTGGAATGATAAAGG : CTGGGAATTTGTAGCTTTACC : GTGGCAAGTCTTATTTGGTAGA : TTTTATCGAAATGTCGGATGTC : CGCAGGAAACGATCATTTAGTA : TGCCTATTTCGATAACTGATTG : CCGCTGAAAATCTCTCCT : ACGAAAAGTTGAAATCGTGATC : ATCACGAGTCAAAACAGTATCG : GCCAAATTATGAGACATCCAC : GAACTTGGTGCAGTTATCTTCA : GGCATGTGAACTACATTTCAAT : GAGAGGTGAAGTGAAAGTGAAG : CCTGACTTATCCTGTCATTTCA : GTCCTTTGGTCTTGGTTACTG : ACAAGCGAATACAACGACA : CACACGATTCACTTTCCTATTG : CACCTACCTCTAGGTGTGGAAG : CCCCAAAACACAATGCAGAACAAA : GTCAAAAGGTTGGCAGTCTAAG : CCGCAAATATCATTTATCTCG : ACCTGAAGCAAAGAAAGAAAGA : TTCAAACGCACGTCATTTG : CCGCAAATATCATTTATCTCG : AGGCTGGGGTCACTATCA Primer sequence (5’-3’) F F F F F F F F F F F F F F F F F F F F F F F F F < 0.05). P DQ108646 DQ108647 DQ108648 DQ108649 DQ108650 DQ108651 DQ108652 GenBank Accession no. DQ108640 DQ108641 DQ108642 DQ108643 DQ108644 DQ108645 DQ108653 DQ108629 DQ108630 DQ108631 DQ108632 DQ108633 DQ108634 DQ108635 DQ108636 DQ108637 DQ108638 DQ108639

(2)

Table 1 Continued Locus NsubA229 R NsubA231 R NsubA235 R NsubA238 R NsubA243 R NsubA245 R NsubA249 R NsubA261 R NsubA262 R NsubA266 R NsubA274 R NsubB007 R NsubB210 R NsubB215 R NsubB235 R NsubB252 R NsubB278 R NsubC020 R NsubC023 R NsubC205 R NsubC218 R NsubC250 R NsubC261 R NsubC262 R NsubC275 R + Best amplification — resolution by using TD PCR. *Statistically significant ( (1) After standard Bonferroni correction. (2) Forward primer within minisatellite; amplification at 3 other size ranges (c. 130–150, 200–220 & 250–270). 156 Ib a r r a , Pe t e r s e n , Fa m u l a & Ma y i n Mo l e c u l a r Ec o l o g y No t e s (2006) 6

Table 2 Cross-species amplification results (number of alleles per locus with size range (bp)) in parentheses of 35 microsatellite loci for the scal- lop family Pectinidae, genus Argopecten (A. irradians, A. purpuratus, A. ventricosus) and Nodipecten (N. nodosus)

A. irradians (n = 6) A. purpuratus (n = 5) A. ventricosus (n = 6) N. nodosus (n = 6) Locus ID USA — Atlantic Chile — Pacific Mexico — Pacific Venezuela —Atlantic

NsubA001 U — — U (140, 215) NsubA004 — — — 2 (215–235) NsubA005 — — — 3 (180–200) NsubA007 U U — 6 (160–220) NsubA010 U (115, 300) — — 4 (120–135) NsubA208 — 3 (280–300) NsubA214 — — — — NsubA222 — — — — NsubA223 U — — 7 (90–120) NsubA227 — — — U (200–240) NsubA229 — — — — NsubA231 4 (300–400) U (340, 460) — 5 (140–150) NsubA235 — 6 (200–300) — 7 (200–235) NsubA238 — — — U (120–140) NsubA243 — — — U (120–160) NsubA245 — — — — NsubA249 — — — 2 (250–270) NsubA261 — — — 7 (220–300) NsubA262 — U — 5 (280–340) NsubA266 — — — — NsubA274 U — — 3 (260–280) NsubB007 — — — — NsubB210 — — — — NsubB215 — — — 5 (100–300) NsubB235 — — — U NsubB252 U — — 2 (140–160) NsubB278 — — — — NsubC020 — — — 2 (160–200) NsubC023 — — — — NsubC205 2 (> 400) U — 3 (170–210) NsubC218 U U — 4 (290–320) NsubC250 2 (300–340) — — 4 (210–240) NsubC261 — — — 3 (140–240) NsubC262 — — — 6 (150–250) NsubC275 U U — U Total no. of amplified loci 11 7 0 26

U, indicates amplification but unclear; ‘—’ indicates no amplification; (num-num) indicates range; (num, num) indicates when two bp ranges amplified. Species, sample size (n), and origin — distribution.

Acknowledgements Meredith, E.P., and B. May (2002). Microsatellite loci in Lahontan tui chub, Gila bicolor obesa, and their utiliza- UCMEXUS-CONACYT supported this research tion in other chub species. Molecular Ecology Notes 2: through grant (CN-04–61; B. May — A.M. Ibarra) and a 156-158. sabbatical fellowship (A.M. Ibarra). Samples from other Peña, J.B. (2001). Taxonomía, morfología, distribución species were donated by Amy Wilbur (Argopecten irradi- y hábitat de los pectínidos iberoamericanos. In: Los ans), Iker Uriarte (Argopecten purpuratus) and Cesar Lo- Moluscos Pectínidos de Iberoamérica: Ciencia y Acuicul- deiros (Nodipecten nodosus). Melinda Baerwald and Rick tura (edited by A.N. Maeda-Martínez), Chapter 1: Topinka reviewed and commented on the manuscript. 1-25. Ed. Limusa, México. Rodzen, J.R., J.J. Agresti, G.J. Tranah, and B.P. May References (1989). Agarose overlays allow simplified staining of Lewis, P.O., and D. Zaykin (2001). GENETIC DATA polyacrylamide gels. BioTechniques 25: 584. ANALYSIS: computer program for the analysis of al- Schwartz, R.S., and B. May (2004). Characterization of lelic data, version 1.1. Free program distributed by microsatellite loci in Sacramento perch (Archoplites the authors over the Internet from http://hydrod- interruptus). Molecular Ecology Notes 4: 694-697. ictyon.eeb.uconn.edu/people/plewis/software.php (accessed July 2005).