Biochem Genet (2011) 49:139–152 DOI 10.1007/s10528-010-9394-4
Identification, Inheritance, and Variation of Microsatellite Markers in the Black Scallop Mimachlamys varia Alberto Arias • Ruth Freire • Juan Pablo De La Roche • Guillermo Roma´n Josefina Me´ndez • Ana Insua
•
Received: 23 September 2009 / Accepted: 19 August 2010 / Published online: 26 November 2010 Ó Springer Science+Business Media, LLC 2010
Abstract Five polymorphic microsatellite loci were identified in the black scallop Mimachlamys varia after construction of a genomic library enriched for (GT)n. To examine the transmission pattern of microsatellite alleles, several families were created and genotypes scored for three loci. The expected Mendelian ratios were found in 12 of 14 segregations examined. Unexpected segregations may be explained by a genotyping error (allelic dropout), given that when a specific allele was treated as dominant, the phenotypic ratios conformed to Mendelian expectations. The five loci were also examined in two samples from the Spanish coast. The two localities displayed similar mean values for the number of alleles per locus (7.2–8.4), allelic richness (7.2–7.9), and observed (0.389–0.484) and expected heterozygosity (0.545–0.618). Significant Hardy–Weinberg deviations were observed at three loci, with heterozygote deficiency occurring in all cases. Global multilocus h value and allele frequencies at one locus revealed significant differentiation between the two localities. Keywords Mimachlamys varia Microsatellite markers Segregation analysis Genetic diversity Population differentiation
Introduction The black scallop Mimachlamys (Chlamys) varia (Linnaeus, 1758) is a bivalve of the family Pectinidae. Distributed from Norway to southern Spain, in the A. Arias R. Freire J. Me´ndez A. Insua (&) Departamento de Biologı´a Celular y Molecular, Universidade da Corun˜a, A Zapateira s/n, 15071 A Corun˜a, Spain e-mail:
[email protected] J. P. De La Roche G. Roma´n Instituto Espan˜ol de Oceanografı´a, Centro Oceanogra´fico de A Corun˜a, A Corun˜a, Spain
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Mediterranean, and along the coast of northwest Africa to near Senegal (Wagner 1991), it usually lives free or attached by the byssus to shells, stones, boulders, or rock faces on coarse sand or gravel substrates. It is a successive protandric hermaphrodite with a gender ratio related to age and size (the number of females is higher in older and larger scallops). This species is commercially exploited in France, but commercial fisheries are not developed in other European countries. In Spain, although there are no dense local populations, the samples caught with the queen scallop Aequipecten opercularis, a species of similar size, are commercialized. The spat of M. varia can be produced in hatcheries (Louro et al. 2003) and also captured from natural environments using collectors (Iglesias et al. 2008), making M. varia a good candidate to be exploited in aquaculture. Although M. varia is a resource of interest, there is little knowledge on the level of genetic diversity and population differentiation across its range of distribution. Early genetic studies were performed on a few samples from around the British Isles and involved allozyme loci, usually characterized by a low number of alleles per locus. Mathers (1975) examined the polymorphism at one locus, reporting no differences between samples from two localities on the west coast of Ireland but an unequal distribution of phenotypes between sublittoral and intertidal samples in one locality. In a sample from the Isle of Man, Beaumont and Beveridge (1984) investigated the genetic variation at 18 loci, and Gosling and Burnell (1988) reported significant differences in allelic and genotypic proportions of two loci between two samples from western and southern Ireland. Data from mitochondrial DNA (mtDNA) revealed a clear differentiation (FST = 0.266) between two samples from northwest and southern Spain (Ferna´ndez-Moreno et al. 2008), but markers based on nuclear gene introns (Arias et al. 2009) failed to detect such differentiation. Genetic studies in fisheries and aquaculture species require highly variable nuclear markers. Microsatellites, or simple sequence repeats (SSRs), are tandemly repeated motifs consisting of 1–6 bases that span less than a few hundred bases (Chambers and MacAvoy 2000). They are found in all genomes, mostly in noncoding regions, and are usually characterized by a high degree of polymorphism due to variation in the number of tandem repeats (Li et al. 2002). Typically, they represent selectively neutral markers, show a codominant mode of inheritance, can be easily amplified by PCR, and are highly reproducible. They have proven to be valuable for research in different areas, including genetic mapping, paternity and relatedness analysis, genetic dissection of complex traits, and population and conservation genetics (Chistiakov et al. 2006). The principal drawback of microsatellites is that usually they must be isolated and characterized in each species, given that the flanking sequences are not often conserved across species. Mendelian inheritance of microsatellite markers is usually assumed, but departures from the expected ratios are frequently observed in various bivalve species (McGoldrick et al. 2000; Reece et al. 2004; Zhan et al. 2007), disturbing genetic analyses. Incorrect parent assignation and erroneous individual relatedness in parentage analyses or overestimates of homozygotes, incorrect allele frequency estimates, and overestimates of inbreeding in population studies are some
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consequences of non-Mendelian segregation. Therefore, the Mendelian inheritance of the microsatellite markers should be verified before their use. The aims of this study were to isolate microsatellite sequences and identify polymorphic markers in M. varia, examining their transmission patterns in families to aid in the characterization of microsatellite markers and to facilitate genetic analysis in this species. In addition, the variation of the microsatellite markers identified was assessed in samples from northwest and southern Spain to provide new estimates of genetic diversity and population differentiation.
Materials and Methods Specimen Collection and DNA Extraction Samples of M. varia were collected from O Grove in northwest Spain and Fuengirola in southern Spain. Total genomic DNA was extracted from 30 mg of adductor muscle preserved in ethanol, according to Ferna´ndez-Tajes and Me´ndez (2007). Enriched Library Construction and Analysis A dot-blot analysis was performed to evaluate the relative richness of three microsatellite motifs (CT, GT, and GAA). Several concentrations of denatured genomic DNA (10, 20, 40, and 80 ng) were blotted onto Hybond-N? nylon membranes (Amersham Biosciences) and fixed by UV irradiation. The nylon membranes were incubated for 30 min at 37°C in 59 SSC, 0.02% SDS, 0.01% LSS, and 0.3% blocking reagent (Roche Applied Science). After the addition of the probe (20 pM), the hybridization proceeded for 3 h at 37°C. Then, membranes were washed twice for 15 min in 29 SSC, 0.1% SDS at room temperature, and 0.59 SSC, 0.1% SDS at 30°C. Hybridized probes were detected with the chemiluminescent substrate CSPD (Roche Applied Science), following the manufacturer’s procedure. The optical density of each point was determined using the software Leica Q-WIN 2.2 (Leica Imaging Systems), and relative richness was calculated, giving a value of 100 to the hybridization with the highest optical density. A genomic library enriched for the dinucleotide GT was constructed according to Billote et al. (1999). In brief, total genomic DNA was digested with RsaI restriction enzyme and the resulting fragments ligated to Rsa linkers (Edwards et al. 1996). To obtain more product and check the ligation, a PCR was carried out using one of the Rsa linkers as primer. Next, the fragments were selected using a 50 -biotinylated (GT)10 oligonucleotide probe and streptavidin MagneSphere paramagnetic particles (Promega). The enriched single-stranded DNA was amplified to obtain more product and double-stranded DNA. The PCR product was cloned into pCR 2.1-TOPO plasmids using the kit TOPO TA Cloning (Invitrogen). Recombinant clones were transferred to Hybond-N? nylon membranes (Amersham Biosciences) and screened by hybridization using an (AC)10 digoxigenin-labeled probe. The plasmid DNA of positive clones was extracted using the QIAprep Spin Miniprep Kit
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(Qiagen) and the sequence of the inserts determined at the Molecular Biology Unit of the University of A Corun˜a, Spain, using a CEQ 8000 (Beckman Coulter) or ABI Prism 3130xl (Applied Biosystems) automated sequencer. Sequences were analyzed for the presence of microsatellites using the program Tandem Repeats Finder (Benson 1999). A region was designated a microsatellite when it contained a minimum of four uninterrupted repeats for dinucleotides and three for larger motifs. Similarity between sequenced clones was checked by means of an all-against-all comparison using the local Blast tool of the program BioEdit version 7.0.9.0 (Hall 1999), selecting the option of filter sequences for lowcomplexity regions. Sequences containing microsatellites were also compared with those of public databases using the discontinuous megablast algorithm of the Blast tool (Altschul et al. 1997). Detection of Microsatellite Polymorphism Oligonucleotide primers flanking the microsatellite regions were designed using the program Primer3 (Rozen and Skaletsky 2000). Initial optimization reactions, when necessary, were carried out in a gradient thermal cycler. Approximately 50 ng of template DNA was used in a reaction volume of 12.5 ll containing 0.3 U Taq DNA polymerase in 19 reaction buffer (Roche Molecular Biochemicals), 0.2 mM each dNTP, 0.24 lM each primer, and MgCl2 ranging from 1.5 to 3.0 mM. The thermal cycler protocol consisted of an initial denaturation of 2 min at 95°C; followed by 35 cycles of 95°C for 45 s, 48–65°C for 45 s, and 72°C for 2 min; and a final extension of 72°C for 20 min. The PCR products were checked in 2% agarose or 6% polyacrylamide gels, followed by ethidium bromide or silver staining, respectively. Routine amplifications were performed with the MgCl2 concentration and annealing temperature determined for each microsatellite. The reverse primer was 50 labeled with one fluorescent phosphoramidite dye, 6-FAM or HEX, and the amplified products were run on an automated sequencer and analyzed using the GeneMapper version 3.7 software (Applied Biosystems). Crosses and Segregation Analysis Mimachlamys varia families were created with three females (A, B, and C) and six males (D, E, F, G, H, and I). Conditioning and stimulation of parents, fecundation, and larval incubation were performed according to Louro et al. (2003). The larvae were reared for 6 days and preserved in 95% ethanol at 4°C. DNA from parents was extracted as indicated above and that from individual larvae as described by Taris et al. (2005). PCR reactions were carried out as described above in a final volume of 25 ll with 2–5 ll of the larval DNA solution. Segregation analysis was carried out in crosses involving at least one heterozygote parent and larval progeny with successful microsatellite amplification. The goodness-of-fit of the progeny segregation ratios to the expected Mendelian ratios was tested using a binomial or multinomial exact test when the analysis involved two (e.g., 1:1) or more classes (e.g., 1:2:1). These procedures were carried out using tools available at http://www.quantitativeskills.com/sisa/index.htm.
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Population Genetic Analysis The number of alleles, the observed heterozygosity (Ho), and the unbiased expected heterozygosity (He) of Nei (1978) were obtained using Genetix software version 4.05 (Belkhir et al. 2004). Allelic richness per locus, population, and overall was computed with Fstat version 2.9.3 (Goudet 2001). Tests for agreement with Hardy– Weinberg equilibrium, allelic and genotypic differentiation, and linkage disequilibrium between pairs of loci within each population were carried out using Genepop version 3.4 (Raymond and Rousset 1995). Significance was determined by the Markov chain method using 10,000 dememorizations, 1,000 batches, and 5,000 iterations per batch, except when Hardy–Weinberg equilibrium was tested for samples with fewer than five alleles where the complete enumeration method (Louis and Dempster 1987) was used. The program MicroChecker (Van Oosterhout et al. 2004) was applied to examine the microsatellite data for evidence of null alleles, with frequency estimated following Brookfield (1996). Single locus and multilocus F-statistics were computed with Genetix for each sample and for all individual loci according to Weir and Cockerham (1984). Probability of significance of h values was determined by a nonparametric permutation approach (10,000 permutations). Since the h value is highly dependent on the level of genetic variation, making it difficult to interpret and compare the level of genetic differentiation between loci and studies, a standardized measure (h0 ) was calculated in an analogous way to Hedrick’s approach (2005). The standardized measure is obtained by dividing the observed value of h by the maximum value possible given the present within-population variance. This maximum value was calculated by recoding the data such that all populations only contain unique alleles. The standardized measure is interpreted as equal to zero when negative values of h are obtained. When multiple tests were performed, the significance values were adjusted by sequential Bonferroni correction (Rice 1989).
Results A dot-blot analysis carried out with three probes revealed that GT was the most abundant microsatellite motif (100), followed by CT (90.3) and GAA (46.5). Consequently, a GT-enriched library was constructed. This library consisted of 1,036 recombinant colonies, 509 of which showed positive hybridization with the (AC)10 probe. Of the 89 clones sequenced, 77 were unique, with an insert size of 214–878 bp. A total of 62 contained at least one microsatellite. In 11 clones, microsatellites were found within higher-order tandem repeats like minisatellites. Of the 21 primer sets designed, 5 (23%) yielded satisfactory amplifications with a polymorphic pattern that was interpretable in polyacrylamide gels (Table 1). The remaining primer sets (16) yielded nonscorable products due to excessive stutter, apparent amplification of multiple loci (more than two bands), and failure to amplify DNA from a large number of individuals, despite attempts at optimization. Polymorphic loci were tested for Mendelian inheritance, but two of them were excluded from the analysis because of their frequent amplification failure in larvae.
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Mva202
a
Classification following Chambers and MacAvoy (2000); P pure, IP interrupted pure
(GTTG)5 P
ACATTGTATCCACCGGGATG
CTGCAGAACACGGTCATGTAA
FM882165
Mva370
ACTGCACCTGCTGACCCTT CAGTGAACCCGTACCAATGA
P
FM882164
(GT)10
GCCTGTAAAAGGCACCTCTC
Mva302
P
CTTCGGTGTGGAAGCATTTT
CCTCCCGGTGTATGTGATTA
ACTTCGAAAGCGTTCACACA
ATGAGCGTGAACGTAAAACG
Primers (50 –30 )
GAGAAGCTCTCGCACCTGTC
(GT)8
IP
IP
Classa
FM882163
Mva215
FM882162
(GT)2AT(GT)GATTA(GT)6TT(GT)4GA(GT)16TTAGAGC(GT)2
(GT)GG(GT)2GG(GT)7TAT(GT)C(GT)AAGA(GT)2CT(GT)T(GT)
Mva191
FM882161
Repeat
Locus Acc. no.
Table 1 Microsatellite markers identified in the black scallop
61
63
63
62
59
Annealing temp (°C)
2
1.5
2
3
3
MgCl2 (mM)
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Of the 640 individuals tested, 10 (1.56%) distributed in six families displayed genotypes that were not expected based on the parental genotypes (Table 2). In all cases, the unexpected genotypes corresponded to homozygotes for one allele present in one of the parents. To measure the goodness-of-fit for expected Mendelian segregation ratios, we ignored the unexpected genotypes (given their low frequency), assumed that they corresponded to a heterozygote genotype, and assumed the occurrence of null alleles in crosses involving a homozygote parent. Agreement with Mendelian expectations was observed in all cases, except at Mva215 in the family AH and Mva370 in the family CG and when null alleles were considered. In the family AH, a deficit of one homozygous class (121/121) and an excess of the other homozygous class (119/119) were observed. However, when allele 119 was scored as dominant over allele 121 and the classes 119/121 and 119/119 were pooled, the segregation did not significantly depart from expected Mendelian phenotypic ratios (P value = 0.745). In the family CG, a deficiency of genotypes containing allele 118, mainly the heterozygote 118/122, and an excess of the homozygous class (122/122) were observed. Similarly, when allele 122 was scored as dominant over allele 118 and the classes 118/122 and 122/122 were pooled, the phenotypic ratios conformed to Mendelian expectations (P value = 0.311). In the summary of genetic variation (Table 3), the number of alleles per locus ranged from 4 (Mva370) to 16 (Mva202). The loci displayed mostly alleles differing by multiples of the repeat motif; only Mva215 and Mva302 showed alleles with sizes outside the expected scale at low frequency (\0.05). Of the 50 alleles observed, nearly half (22) were private alleles, present in only one of the localities, with frequencies of 0.011–0.067 (mean = 0.019). Both samples displayed similar numbers of alleles for each locus, except at Mva202, where Fuengirola displayed 6 alleles more than O Grove. The mean number of alleles per locus was 7.2–8.4, and the mean allelic richness in each locality was 7.2–7.9. The highest values were observed in Fuengirola. The loci Mva202 and Mva302 showed the highest gene diversity (He C 0.765), and Mva191 showed the lowest (He = 0.338). The highest observed heterozygosity was found at Mva302 and the lowest at Mva191. The two localities showed similar heterozygosity values (He 0.545–0.618 and Ho 0.389–0.484). Of the 20 tests for linkage disequilibrium, one was significant at P \ 0.05, but none after sequential Bonferroni correction, indicating that the loci are not closely linked and that they can be treated as independent variables. Genotype proportions at loci Mva215 and Mva302 agreed with Hardy–Weinberg expectations in both samples, but those of the other loci did not conform after sequential Bonferroni correction, with heterozygote deficit occurring (f = 0.181–0.545). The estimation of f was positive for each locality-locus, except at Mva302, and also within localities across loci. The program MicroChecker did not detect any null alleles at the loci Mva215 and Mva302 in either locality and Mva370 in Fuengirola, but in the remaining locality-locus combinations their frequencies ranged from 0.107 at Mva191 to 0.239 at Mva202 in Fuengirola (Table 3). Given that the presence of null alleles at high frequencies ([0.2) led to a considerable overestimation of FST estimators (Chapuis and Estoup 2007), the locus Mva202 was removed from the subsequent analyses.
123
123
160/166
164/166
164/166
CG
CI
118/122
BG
BD
118/122
BD
160/166
118/122
BE
BH
118/122
CG
119/121
AF
118/122
119/121
AH
CH
121/121
BD
119/121
166/166
166/166
160/164
166/166
122/126
118/126
122/129
122/126
122/126
119/119
119/121
119/121
119/121
119/119, 119/121, 121/121
166/166, 164/166
164/166, 166/166
160/160, 160/164, 160/166, 164/166
160/166, 166/166
118/118, 118/122, 118/126, 122/122, 122/126
118/118, 118/122, 118/126, 122/126
118/122, 118/129, 122/122, 122/129
118/122, 118/126, 122/122, 122/126
118/122, 118/126, 122/122, 122/126, 118/118
119/119, 119/121, 121/121
119/119, 119/121, 121/121
119/119, 119/121, 121/121
119/119, 119/121, 121/121
22:19
10:17
9:8:13:8
23:21
1:8:12:11:12
10:10:10:9
9:23:18:16
7:12:29:15
15:13:13:12:1
13:12:1
21:17:6
1:21:11
5:30:24
22:30:1
Observed
1:1
1:1
1:1:1:1
1:1
0:1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1
1:1:1:1:0
1:1:0
1:2:1
0:1:1
0:1:1
1:1:0
Expected
0.755
0.248
0.66
0.341
0.817
1
0.096
0.001
0.962
1
0.004
0.110
0.497
0.332
I
P valuea
0.941
0.862
1
0.080
0.193
0.272
II
0.006
\0.001
\0.001
\0.001
III
P value of the binomial/multinomial exact tests: I unexpected genotypes were ignored, II unexpected genotypes were scored as heterozygous, III homozygous parent was considered heterozygous for a null allele
a
Mva302
Mva370
119/119
121/121
CH
Mva215
Genotypes
Dam
Sire
Genotype ratio of progeny
Parental genotype
BH
Family
Locus
Table 2 Segregation analysis of microsatellite alleles
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46
83
Fuengirola
Overall
0.573
0.609
0.522
0.781
0.777
0.749
0.501
0.553
0.432
0.765
0.592
122 (0.560)
122 (0.511)
122 (0.622)
166 (0.342)
160 (0.356)
166 (0.446)
119 (0.683)
119 (0.633)
119 (0.743)
124 (0.428)
0.807
0.685
0.618
3.694
3
4
10.01
9.579
10
6.728
7.407
7
11.53
124 (0.370)
124 (0.500)
0.338
0.343
0.336
He
Overall
118–130
118–126
118–130
131–174
158–174
131–174
106–127
113–127
106–125
108–162
8 12.91
134 (0.807)
134 (0.804)
134 (0.811)
AC (Freq)
Fuengirola
4 (1)
3
4 (1)
11 (2)
10 (1)
10 (1)
9 (3)
8 (2)
7 (1)
16 (10)
108–162
114–144
6.706
6.499
7
RS
0.545
83
Overall
8 (2)
14 (8)
120–142
120–142
126–138
R
O Grove
46
Fuengirola
82
Overall
37
45
O Grove
37
82
Overall
Fuengirola
45
O Grove
37
83
Overall
Fuengirola
46
Fuengirola
O Grove
37
O Grove
10 (6)
7 (3)
7 (3)
NA
0.442
0.484
0.389
0.410
0.500
0.297
0.817
0.867
0.757
0.451
0.489
0.405
0.349
0.370
0.324
0.181
0.196
0.162
Ho
0.096
0.117
0.062
0.539
0.545
0.530
0.471
0.433
0.521
0.248
0.218
0.288
0.284
0.181
0.433
-0.070
-0.120
-0.010
f
0.004**
0.007**
0.442
0.631
0.528
0.537
0.000**
0.000**
0.001**
0.000**
P valueb
0.144
0.239
0.210
0.107
0.127
Null allele frequency
Test for conformity to Hardy–Weinberg expectation
** Significant after sequential Bonferroni correction
b
N number of individuals, NA observed number of alleles (private alleles), R size range of alleles in base pairs, RS allelic richness based on a minimum sample size of 37 diploid individuals, AC (Freq) size in base pairs, with frequency of the most common allele in parentheses, He expected heterozygosity based on Nei’s unbiased estimate, Ho observed heterozygosity, f inbreeding coefficient estimated following Weir and Cockerham (1984)
a
All loci
Mva370
Mva302
Mva215
Mva202
37
O Grove
Mva191
N
Locality
Locus
Genetic variation statistica
Table 3 Summary of genetic variation by locus, population, and overall
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The global multilocus h value was significantly different from zero (h = 0.017, P value = 0.024), the standardized value being h0 = 0.038. Estimation of h per locus was significantly different from zero at Mva302 (h = 0.043, P value = 0.002). Pairwise tests for differentiation using allele and genotype frequencies were significant at P \ 0.05 for Mva302 and Mva370, the allele frequencies at Mva302 remaining significant after sequential Bonferroni correction.
Discussion The construction of a GT-enriched library allowed the isolation of the first microsatellite sequences of the scallop M. varia. Seventy percent of the sequences obtained contained microsatellites; the redundancy, frequently observed when enrichment methods are used (Zane et al. 2002), was moderate (13%), indicating that the procedure followed here was highly effective. It should be noted that in 18% of the clones, microsatellites were found within higher-order tandem repeats like minisatellites, revealing a close association between both types of sequences. In other scallop species, such as Mizuhopecten yessoensis (An et al. 2005) and Aequipecten opercularis (Arias et al. 2010), minisatellites have also been found in clones with microsatellites, but in these cases they are near or surrounding the microsatellite. The number of polymorphic microsatellite loci obtained with respect to the total sequences examined in Mimachlamys varia (5.62%) was within the range observed for other species where enriched libraries were examined; values obtained in other species were 4.55% in Mizuhopecten yessoensis (An et al. 2005), 6.02% in A. opercularis (Arias et al. 2010), and 15.79% in Pecten maximus (Watts et al. 2005). The abundance of microsatellites and their location in the genomes may explain the differences observed among species in the success of establishing reliable microsatellite markers (Megle´cz 2007). In M. varia and other scallops it is likely that the location of microsatellites within or near minisatellites is influencing the detection of usable markers. The linkage of microsatellites to other repetitive sequences reduces the availability of appropriate flanking regions for primer design and can also cause multiple locus amplification and unclear banding patterns. Segregation analysis in three microsatellite loci revealed Mendelian ratios, with a few exceptions. The incidence of segregation distortion observed here (14%) was similar to or lower than that reported by McGoldrick et al. (2000) and Launey and Hedgecock (2001) in Crassostrea gigas (37 and 21%, respectively) and Reece et al. (2004) in C. virginica (11%). Departures from Mendelian ratios have been largely attributed to null alleles and selection near microsatellite markers. Null alleles, which cannot be amplified by PCR because of mutations that occur at primer sites, have been often detected in bivalves. In C. gigas, 51% of the microsatellites tested for Mendelian segregation in three families displayed null alleles (Hedgecock et al. 2004), and unexpected progeny phenotypes were explained when null alleles were considered in 15 of 94 segregation ratios tested in C. gigas (Launey and Hedgecock 2001) or 2 of 15 in Argopecten irradians (Zhan et al. 2007). Underdominant selection patterns with a lower fitness for the heterozygote (McGoldrick et al. 2000)
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and selection acting on deleterious recessive mutations linked to some microsatellites (Launey and Hedgecock 2001) often appeared in families of C. gigas. In the case of M. varia, evidence of null alleles in the parents examined was not found; therefore, other causes should be involved. In the family AH, the deficit of one homozygous class and excess of the other homozygous class at the locus Mva215 could be initially attributed to a deleterious effect linked to the allele 121 transmitted by one of the parents. If this is the case, the family CH or AF should display a similar segregation distortion because they share a parent with the family AH, but this was not observed. In the family CG, underdominant selection could explain the segregation distortion at the locus Mva370, given the heterozygote deficiencies and homozygous excess observed. However, the segregation distortion observed in the two families can be related more probably to allelic dropout, a genotyping error consisting of the amplification of only one of the two alleles in a heterozygote (Miller and Waits 2003), frequently observed when the template DNA is low quality or a low quantity is used. Allelic dropout is usually explained as a stochastic sampling error; consequently, any of the two alleles could be amplified (Miller and Waits 2003). A nonrandom pattern of amplification has also been reported (Soulsbury et al. 2007). Allele 119 at the locus Mva215 could be amplified preferentially over allele 121 in the family AH, and allele 122 at the locus Mva370 over allele 118 in the family CG. When this was taken into account and the dominance relationships of alleles were reconsidered, phenotype ratios conformed to Mendelian expectations, suggesting that a genotyping error, rather than a biological cause, explains the apparent distortion. Allelic dropout may also explain the unexpected genotypes appearing in other families at low frequency, since they always corresponded to homozygote genotypes, although contamination during larval manipulation cannot be ruled out. Why allelic dropout occurred in some crosses but not in others, even when identical genotypes or half-sib progenies were examined, is unclear, but PCR inhibitors coextracted by chance with the larval DNA are possibly involved. In other organisms, dilution of the template DNA prior to PCR amplification was useful to corroborate the existence of allelic dropout (Jeffery et al. 2001; Soulsbury et al. 2007), but the procedure followed for DNA extraction and PCR amplifications precluded this test in M. varia. As expected, given that microsatellites are characterized by a high mutation rate, all loci except Mva370 displayed a number of alleles higher than the 2–6 alleles reported for allozyme loci (Beaumont and Beveridge 1984) and the 2–3 alleles for intron-based nuclear markers (Arias et al. 2009). In terms of heterozygosity, the overall observed heterozygosity was also higher than that scored with allozyme loci (0.284) and intron-based nuclear markers (0.275). In the two localities, the levels of genetic variation revealed by the mean number of alleles per locus, the mean allelic richness, and observed and expected heterozygosity were similar, but Fuengirola always displayed the highest values for all parameters. Compared with other scallops, the overall observed heterozygosity of M. varia (0.442) was in line with that estimated for microsatellite loci in species such as Chamys nobilis (0.403, Hui et al. 2006) or Aequipecten opercularis (0.440, Arias et al. 2010) but clearly lower than that estimated in Mizuhopecten yessoensis (0.819, An et al. 2005) or Patinopecten caurinus (0.728, Elfstrom et al. 2005).
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Hardy–Weinberg equilibrium testing with the sequential Bonferroni correction revealed that the genotype proportions at two loci (Mva215 and Mva302) conformed to the expected frequencies, but those of the other three (Mva191, Mva202, and Mva370) showed significant deviations in the two localities. All deviations were associated with positive f values (0.181–0.530), indicating a heterozygote deficit, which is a very frequent occurrence in marine bivalve populations, including scallops (e.g., Benzie and Smith-Keune 2006; Hui et al. 2006). Various factors such as inbreeding, the Wahlund effect, and selection could explain heterozygote deficits, but the factor more frequently considered when microsatellite loci are examined is the presence of null alleles (e.g., Sobolewska and Beaumont 2005). Although evidence of null alleles was not found in the M. varia families examined, MicroChecker analysis indicated null alleles in all cases with significant deviation from Hardy–Weinberg equilibrium (except at the locus Mva370 in Fuengirola), suggesting that the presence of such alleles is a likely cause for most of the heterozygote deficits observed here. Allelic dropout cannot be ruled out, taking into account the segregation distortion observed in these families, although its effect could be more pronounced in larvae than in larger individuals where DNA of higher quality and quantity is usually obtained. The h value and one of the tests of allele differentiation revealed significant genetic differentiation between the two localities. The global h value (h = 0.017, P value = 0.024) was low, as usually occurs for microsatellite loci, because the high polymorphism can greatly reduce the estimates of FST values even in the absence of shared alleles (Balloux and Lugon-Moulin 2002). The detection of genetic differentiation is in line with the data provided by mtDNA (Ferna´ndezMoreno et al. 2008) but contrasts with those of intron polymorphism (Arias et al. 2009). The intron-based markers failed to detect the genetic differentiation observed here probably due to they displayed considerably lower levels of genetic diversity than did the microsatellites. Other scallops, such as Pecten maximus (Rı´os et al. 2002) and A. opercularis (Arias et al. 2010), collected at the same localities examined here, also showed low but significant population differentiation, suggesting that on the Spanish coast, M. varia could have a population structure similar to that of these species. In conclusion, this study reports the isolation of the first microsatellite sequences of M. varia and the identification of five polymorphic microsatellite markers. Furthermore, it reports the transmission patterns at three loci, mostly in agreement with a Mendelian inheritance model but also with segregation distortions due to selection near the microsatellite loci or more probably allelic dropout. New estimates of genetic diversity and genetic differentiation for two Spanish samples are also provided. The microsatellite markers identified will allow the undertaking of further genetic studies on this scallop and provide genetic information to assist the management and culture of this resource. Acknowledgments We thank Dr. Ricardo Pe´rez and Dr. Pedro Cruz for assisting in the microsatellite genotyping, Jose Garcı´a Gil for technical assistance in the laboratory, and anonymous reviewers for constructive comments. This study was supported by Ministerio de Ciencia y Tecnologı´a of Spain through project ACU01-022-C2-2.
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References Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped Blast and PSI-Blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402 An HS, Park JY, Lee YG, Lee DS, Lee C (2005) Ten polymorphic microsatellite loci in the giant scallop (Mizuhopecten yessoensis). Mol Ecol Notes 5:806–808 Arias A, Freire R, Me´ndez J, Insua A (2009) Intron characterization and their potential as molecular markers for population studies in the scallops Aequipecten opercularis and Mimachlamys varia. Hereditas 142:46–57 Arias A, Freire R, Me´ndez J, Insua A (2010) Isolation and characterization of microsatellite markers in the queen scallop Aequipecten opercularis and their application to a population genetic study. Aquat Living Resour 23:199–207 Balloux F, Lugon-Moulin N (2002) The estimation of population differentiation with microsatellite markers. Mol Ecol 11:155–165 Beaumont AR, Beveridge CM (1984) Electrophoretic survey of genetic variation in Pecten maximus, Chlamys opercularis, Chlamys varia and Chlamys distorta from the Irish Sea. Mar Biol 81:299–306 Belkhir K, Borsa P, Chikhi L, Raufaste N, Bonhomme F (2004) Genetix 4.05, logiciel sous Windows pour la ge´ne´tique des populations. Laboratoire Ge´nome, Populations, Interactions, CNRS UMR 5171, Universite´ de Montpellier II, Montpellier (France). Available from http://www.genetix.univmontp2.fr/genetix/genetix.htm Benson G (1999) Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res 27:573–580 Benzie JAH, Smith-Keune C (2006) Microsatellite variation in Australian and Indonesian pearl oyster Pinctada maxima populations. Mar Ecol Prog Ser 314:197–211 Billote N, Lagoda PJL, Risterucci AM, Baurens FC (1999) Microsatellite-enriched libraries: applied methodology for the development of SSR markers in tropical crops. Fruits 54:277–288 Brookfield JF (1996) A simple new method for estimating null allele frequency from heterozygote deficiency. Mol Ecol 5:453–455 Chambers GK, MacAvoy ES (2000) Microsatellites: consensus and controversy. Comp Biochem Physiol B 126:455–476 Chapuis MP, Estoup A (2007) Microsatellite null alleles and estimation of population differentiation. Mol Biol Evol 24:621–631 Chistiakov DA, Hellemans B, Volckaert FAM (2006) Microsatellites and their genomic distribution, evolution, function and applications: a review with special reference to fish genetics. Aquaculture 255:1–29 Edwards KJ, Barker JHA, Daly A, Jones C, Karp A (1996) Microsatellite libraries enriched for several microsatellite sequences in plants. BioTechniques 20:758–760 Elfstrom CM, Smith CT, Jones KC, Seeb JE (2005) Characterization of 16 polymorphic microsatellite loci in weathervane scallop Patinopecten caurinus. Mol Ecol Notes 5:514–516 Ferna´ndez-Moreno M, Arias-Pe´rez A, Freire R, Me´ndez J (2008) Genetic analysis of Aequipecten opercularis and Mimachlamys varia (Bivalvia: Pectinidae) in several Atlantic and Mediterranean localities, revealed by mitochondrial PCR-RFLPs: a preliminary study. Aquac Res 39:474–481 Ferna´ndez-Tajes J, Me´ndez J (2007) Identification of the razor clam species Ensis arcuatus, E. siliqua, E. directus, E. macha, and Solen marginatus using PCR-RFLP analysis of the 5S rDNA region. J Agric Food Chem 55:7278–7282 Gosling EM, Burnell GM (1988) Evidence for selective mortality in Chlamys varia (L) transplant experiments. J Mar Biol Assoc UK 68:251–258 Goudet J (2001) Fstat, a program to estimate and test gene diversities and fixation indices (version 2.9.3). Institut d’Ecologie, Universite´ de Lausanne, Dorigny, Switzerland. Available from http://www2. unil.ch/popgen/softwares/fstat.htm Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98 Hedgecock D, Li G, Hubert S, Bucklin K, Ribes V (2004) Widespread null alleles and poor cross-species amplification of microsatellite DNA loci cloned from the Pacific oyster, Crassostrea gigas. J Shellfish Res 23:379–385 Hedrick PW (2005) A standardized genetic differentiation measure. Evolution 59:1633–1638
123
152
Biochem Genet (2011) 49:139–152
Hui M, Bao Z, Zhan A, Hu X, Lu W, Chang D, Hu J (2006) Ten polymorphic dinucleotide microsatellite markers of the noble scallop Chlamys nobilis. Mol Ecol Notes 6:1033–1035 Iglesias P, Louro A, Roma´n G (2008) How does reproduction and recruitment patterns match? The case of Chlamys varia in Rı´a de Betanzos-Sada, NW Spain. 2nd Mollusc Physiology Conference. September 1–4, Brest, France. Book of abstracts, p 119 Jeffery KJ, Keller LF, Arcese P, Bruford MW (2001) The development of microsatellite loci in the song sparrow, Melospiza melodia (Aves) and genotyping errors associated with good quality DNA. Mol Ecol Notes 1:11–13 Launey S, Hedgecock D (2001) High genetic load in the Pacific oyster Crassostrea gigas. Genetics 159:255–265 Li YC, Korol AB, Fahima T, Beiles A, Nevo E (2002) Microsatellites: genomic distribution, putative functions and mutational mechanisms: a review. Mol Ecol 11:2453–2465 Louis EJ, Dempster ER (1987) An exact test for Hardy–Weinberg and multiple alleles. Biometrics 43:805–811 Louro A, Roche DL, Campos MJ, Roma´n G (2003) Hatchery rearing of the black scallop, Chlamys varia (L.). J Shellfish Res 22:95–99 Mathers NF (1975) Environmental variability at the phosphoglucose isomerase locus in the genus Chlamys. Biochem Syst Ecol 3:123–127 McGoldrick DJ, Hedgecock D, English LJ, Baoprasertkul P, Ward RD (2000) The transmission of microsatellite alleles in Australian and North American stocks of the Pacific oyster (Crassostrea gigas): selection and null alleles. J Shellfish Res 19:779–788 Megle´cz E (2007) Microfamily (version 1): a computer program for detecting flanking-region similarities among different microsatellite loci. Mol Ecol Notes 7:18–20 Miller CR, Waits LP (2003) The history of effective population size and genetic diversity in the Yellowstone grizzly (Ursus arctos): implications for conservation. Proc Natl Acad Sci USA 100:4334–4339 Nei M (1978) Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics 89:583–590 Raymond M, Rousset F (1995) Genepop (version 1.2): population genetics software for exact tests and ecumenicism. J Hered 86:248–249 Reece KS, Ribeiro WL, Gaffney PM, Carnegie RB, Allen SK Jr (2004) Microsatellite marker development and analysis in the eastern oyster (Crassostrea virginica): confirmation of null alleles and non-Mendelian segregation ratios. J Hered 95:346–352 Rice WR (1989) Analyzing tables of statistical tests. Evolution 43:223–225 Rı´os C, Sanz S, Saavedra C, Pena JB (2002) Allozyme variation in populations of scallops, Pecten jacobaeus (L.) and P. maximus (L.) (Bivalvia: Pectinidae), across the Almeria-Oran front. J Exp Mar Biol Ecol 267:223–244 Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, NJ, pp 365–386 Sobolewska H, Beaumont AR (2005) Genetic variation at microsatellite loci in northern populations of the European flat oyster (Ostrea edulis). J Mar Biol Assoc UK 85:955–960 Soulsbury CD, Iossa G, Edwards KJ, Baker PJ, Harris S (2007) Allelic dropout from a high-quality DNA source. Conserv Genet 8:733–738 Taris N, Baron S, Sharbel TF, Sauvage C, Boudry P (2005) A combined microsatellite multiplexing and boiling DNA extraction method for high-throughput parentage analyses in the Pacific oyster (Crassostrea gigas). Aquac Res 36:516–518 Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) MicroChecker: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4:535–538 Wagner HP (1991) Review of the European Pectinidae. Vita Marina 41:1–48 Watts PC, Mallanaphy WJ, McCarthy C, Beukers-Stewart BD, Mosley MWJ, Brand AR, Saccheri IJ (2005) Polymorphic microsatellite loci isolated from the great scallop, Pecten maximus (Bivalvia: Pectinidae). Mol Ecol Notes 5:902–904 Weir BS, Cockerham CC (1984) Estimating F-statistics for the analysis of population structure. Evolution 38:1358–1370 Zane L, Bargelloni L, Patarnello T (2002) Strategies for microsatellite isolation: a review. Mol Ecol 11:1–16 Zhan AB, Bao ZM, Hui M, Wang ML, Zhao HB, Lu W, Hu XL, Hu JJ (2007) Inheritance pattern of ESTSSRs in self-fertilized larvae of the bay scallop Argopecten irradians. Ann Zool Fenn 44:259–268
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