Genetic comparison of cultured and wild populations of the clam Coelomactra antiquata (Spengler) in China using AFLP markers

Aquaculture 271 (2007) 152 – 161 www.elsevier.com/locate/aqua-online

Genetic comparison of cultured and wild populations of the clam Coelomactra antiquata (Spengler) in China using AFLP markers Lingfeng Kong, Qi Li ⁎ Fisheries College, Ocean University of China, Qingdao 266003, China Received 25 March 2007; received in revised form 5 June 2007; accepted 9 June 2007

Abstract AFLP markers were used to investigate levels of genetic diversity within cultured populations of the clam Coelomactra antiquata and to compare them with the wild source populations. Seven pairs of primers generated 365 loci among 125 individuals in three cultured and three wild populations. High polymorphism at the AFLP markers was found within both cultured and wild C. antiquata populations. Although not statistically significant, reductions in the expected heterozygosity and percentage of polymorphic loci were observed in the cultured populations (2.8% and 8.3% reduction, respectively), and higher frequency of private alleles within the wild populations compared to the cultured populations indicated that rare alleles in some loci were lost in the cultured populations. Significant genetic differentiation was observed between the cultured populations, and between the cultured and wild populations. Northern populations were genetically distinct from southern populations (FST = 0.696–0.746). The results obtained in this study indicate that continued genetic monitoring of the cultured populations is warranted and the northern and southern populations of C. antiquata should be managed separately in hatchery practices for the preservation of genetic diversity in wild populations. © 2007 Elsevier B.V. All rights reserved. Keywords: AFLP; Coelomactra antiquata; Cultured populations; Genetic diversity

1. Introduction Coelomactra antiquata Spengler, a large benthic clam which is found in sandy habitats from the lower intertidal zone to 20 m depths, is an endemic along the coast of China and Japan (Wang, 1988). In China, the species is one of the valuable and important fisheries resources. Commercial size of the species (9 to 15 cm in shell length) is sold live for about US$15–20/kg in southern China markets. The production of the species was about 10 thousand tonnes per year in the early ⁎ Corresponding author. Tel.: +86 532 8203 1622; fax: +86 532 8289 4024. E-mail address: [email protected] (Q. Li). 0044-8486/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2007.06.007

1980s (Meng et al., 2005). Over-fishing, loss of habitat and pollution, however, have depleted present populations to a fraction of historical sizes (Qi et al., 1995; Wu et al., 2002). In 2004, the production of the species was less than 50 tonnes in China and may now be only 1% of that found ten years ago (Meng et al., 2005). Decline in the supply of wild caught C. antiquata has created an opportunity for the production of cultured clams and in the past three years; several commercial hatchery stocks are being developed in Shandong and Fujian Province, China (Liu et al., 2006). Aquaculture practices may inadvertently decrease the genetic variability present in farmed stocks by breeding related individuals or by the use of small numbers of parents as broodstock (Norris et al., 1999). This reduction

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in variability could possibly result in the loss of genetic variation for disease resistance and reduce a population's capability to adapt to new environments (Allendorf and Phelps, 1980). Therefore, for successful hatchery management, it is extremely important to monitor the genetic variability of hatchery stocks and determine how sufficient variation can be maintained during hatchery rearing (Beaumont and Hoare, 2003). In addition, understanding the genetic variation within hatchery stocks is needed for genetic enhancement programs aiming to avoid potential inbreeding, and random genetic drift (Mickett et al., 2003). Molecular markers have been very useful for analysis of genetic diversity. Among many types of molecular markers, we selected amplified fragment length polymorphism (AFLP) because it is easy, fast, inexpensive and robust. Although the dominant nature of AFLP markers was regarded as a potential drawback for population studies due to statistical complications, advances in procedures for the estimation of allele frequencies from dominant marker data have reduced these problems and increased the utility of AFLP analysis (Lynch and

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Milligan, 1994; Krauss, 2000; Holsinger et al., 2002). In addition, because a large number of loci can be easily generated by AFLP, even a few individuals are sufficient for estimating reliable population genetic parameters, which is very important for over-exploited species since it is difficult to collect large sample in some sampling sites. AFLP markers have been used successfully to monitor genetic diversity in hatchery stocks for several fish and shellfish species (e.g. Mickett et al., 2003; Yue et al., 2004; Simmons et al., 2006; Yu and Chu, 2006). So, in the present study, we used seven AFLP primer pairs to estimate the level of genetic diversity within three hatchery strains and three corresponding wild populations of C. antiquata, and to compare the degree of genetic differentiation between them. 2. Materials and methods 2.1. Sample collection and DNA extraction Three wild populations from Jiaonan, Changle and Xiamen were surveyed in this study (Fig. 1). The samples

Fig. 1. Sample sites of wild (●) and cultured populations (○) of Coelomactra antiquata in China.

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of wild clam (shell length, 8.56 ± 1.42 cm) were collected from coastal waters in China between April and December 2005. Hatchery clams were collected from culture facilities in the same three localities in 2006. The three hatchery stocks were the first-generation offspring which were produced in 2005 using wild caught C. antiquata in corresponding localities. No detailed information was available on the exact number of broodstock and management practices of hatchery stocks. However, in Chinese hatcheries typically dozens of adult C. antiquata were used to produce artificial seeds. The adductor muscle was removed from fresh specimens and preserved at −80 °C until DNA preparation. Total genomic DNA was extracted from frozen adductor muscle tissue by a modification of the standard phenol–chloroform procedure previously described by Li et al. (2002). DNA extracts were purified using a UNIQ-10 spin Column DNA Purification Kit (Sangon Inc., Shanghai). DNA quality was assessed by running samples on 1% agarose gels and DNA concentration was measured with an Ultrospec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) for absorption at 260 nm.

and 8.5 μl of selective amplification solution (0.25 pmol of selective primers with additional three selective bases, 200 μm dNTPs, 1 × PCR buffer, 1.5 mM MgCl2 and 0.25 U Taq DNA polymerase). Selective amplification was performed with 2 min of denaturing at 94 °C, then 10 cycles of 20 s at 94 °C, 30 s at 66 °C, and 2 min at 72 °C, with a 1 °C decrease in the annealing temperature each cycle, followed by 20 cycles of 20 s at 94 °C, 30 s at 56 °C, and 2 min at 72 °C, with a final extension of 30 min at 60 °C. Seven selective primer combinations were chosen to generate all AFLP profiles: EcoRI-ACC + MseI-CAC, EcoRI-ACC + MseI-CCT, EcoRI-ACC + MseI-CTC, EcoRI-AGA + MseI-CTC, EcoRI-AGA + MseI-CTG, EcoRI-AGC + MseI-CCT and EcoRI-AGC + MseI-CTC. After amplification, each product was mixed with an equal volume of loading dye, then denatured at 95 °C for 5 min and placed immediately on ice. Five μl of each sample was run in a 6% 0.4 mm denaturing polyacrylamide gel (1 × TBE buffer) in a model S2001 sequencing gel electrophoresis apparatus (GibcoBRL). After electrophoresis, the amplification products were

2.2. AFLP analysis The AFLP analysis was performed essentially as described by Vos et al. (1995). Digestion of genomic DNA was processed in 10 μl of digestion solution containing 100 ng of genomic DNA, 3 U of EcoRI, 1 U of Tru1I (MseI), and 2 × Tango™ buffer (MBI Fermentas) at 37 °C for 3 h and then 65 °C for 3 h. After digestion, 10 μl of ligation solution including 5 pmol EcoRI adaptor, 50 pmol MseI adaptor, 1 U of T4 DNA ligase (MBI Fermentas), 5% polyethylene glycol 4000, and 1 × buffer was added. The reaction mixture was incubated at 16 °C overnight and then diluted 10-fold with TE0.1 (10 mM Tris–HCl, 0.1 mM EDTA, pH 8.0). Preselective amplification was performed in 10 μl of reaction mixture containing 2 μl of diluted ligation mixture and 8 μl of preselective amplification solution (0.25 pmol of preamplification primers with a single selective base, 200 μm dNTPs, 1 × PCR buffer, 1.5 mM MgCl2 and 0.25 U Taq DNA polymerase). After an initial denaturation at 72 °C for 2 min, 20 PCR cycles of 20 s at 94 °C, 30 s at 56 °C, and 2 min at 72 °C were performed; followed by a final 30-min extension at 60 °C. Five μl of preamplified products was dissolved in 95 μl of TE0.1 buffer. Selective amplification was also performed in a 10-μl volume with 1.5 μl of preselective amplification product

Fig. 2. Representative AFLP gel showing the banding pattern in C. antiquata populations. The PCR products were amplified by E-ACC/ M-CCT primer pair and visualized by silver staining.

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detected by silver staining. A 10 bp DNA Ladder (Invitrogen) was used as a reference marker for allele size determination. 2.3. Genotyping and data analysis The silver stained AFLP bands in each gel were scored as present (1) or absent (0) and only distinct polymorphic bands were scored. AFLP fragments with the same electrophoretic mobility were assumed to be allelic and those with different mobility as nonallelic. To avoid any error bias, four individuals were replicated independently (starting from DNA extracts), revealing that the amplification patterns were highly reproducible. The number of shared multilocus AFLP patterns was evaluated with ARLEQUIN version 3.0 (Excoffier et al., 2005). We estimated the allele frequencies at each locus using a Bayesian method (Zhivotovsky, 1999) using

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AFLP-SURV 1.1 (Vekemans et al., 2002). Estimates of allele frequencies were used to calculate the percentage of polymorphic loci and expected heterozygosity (HE) to determine genetic diversity within populations. The average expected heterozygosity and percentage of polymorphic loci for cultured populations were tested against local wild populations. Significance was tested using the Wilcoxon matched-pairs signed-rank test (Zar, 1999) implemented with SPSS 14.0 software (SPSS Inc.). Pairwise estimates of FST among all populations were obtained using ARLEQUIN version 3.0 (Excoffier et al., 2005). The significance of these estimates was tested by comparing observed FST estimates with a null distribution created by 10,000 random permutations of the data set (Excoffier et al., 1992). Significance levels were adjusted for multiple tests using the sequential Bonferroni correction technique (Rice, 1989). Pairwise estimates of the F-statistic

Table 1 Average expected heterozygosity (HE, mean ± SE), percentage of polymorphic loci and number of polymorphic loci (before and after dash, respectively) and number of private alleles within each populations of C. antiquate Primer pairs

Populations (no. of samples analyzed in parentheses) Jiaonan wild (28) Jiaonan cultured (24) Changle wild (20) Changle cultured (20) Xiamen wild (13) Xiamen cultured (20)

HE E-ACC/M-CAC E-ACC/M-CCT E-ACC/M-CTC E-AGA/M-CTC E-AGA/M-CTG E-AGC/M-CCT E-AGC/M-CTC Overall

0.170 ± 0.023 0.163 ± 0.021 0.222 ± 0.032 0.195 ± 0.024 0.172 ± 0.021 0.184 ± 0.028 0.141 ± 0.024 0.179 ± 0.009

0.185 ± 0.024 0.162 ± 0.021 0.195 ± 0.028 0.192 ± 0.026 0.181 ± 0.022 0.180 ± 0.026 0.095 ± 0.022 0.173 ± 0.009

0.178 ± 0.022 0.169 ± 0.021 0.155 ± 0.026 0.217 ± 0.025 0.163 ± 0.023 0.168 ± 0.031 0.169 ± 0.025 0.176 ± 0.009

0.142 ± 0.018 0.185 ± 0.023 0.153 ± 0.027 0.206 ± 0.024 0.153 ± 0.023 0.169 ± 0.031 0.160 ± 0.023 0.169 ± 0.009

0.185 ± 0.022 0.188 ± 0.022 0.141 ± 0.024 0.189 ± 0.024 0.168 ± 0.025 0.198 ± 0.032 0.191 ± 0.025 0.183 ± 0.009

0.167 ± 0.023 0.169 ± 0.023 0.182 ± 0.030 0.194 ± 0.024 0.180 ± 0.025 0.181 ± 0.032 0.195 ± 0.027 0.181 ± 0.009

Polymorphic loci (%)/no. of polymorphic loci E-ACC/M-CAC 57.4/31 57.4/31 E-ACC/M-CCT 54.4/37 54.4/37 E-ACC/M-CTC 58.5/24 61.0/25 E-AGA/M-CTC 58.1/36 56.5/35 E-AGA/M-CTG 72.7/40 72.7/40 E-AGC/M-CCT 59.0/23 56.4/22 E-AGC/M-CTC 56.5/26 19.6/9 Total 59.5/217 54.5/199

64.8/35 60.3/41 53.7/22 67.7/42 65.0/36 51.3/20 56.5/26 60.8/222

63.0/34 54.4/37 53.7/22 45.2/28 60.0/33 51.3/20 58.7/27 55.1/201

59.3/32 57.4/39 51.2/21 67.7/42 60.0/33 53.8/21 58.7/27 58.9/215

51.9/28 45.6/31 53.7/22 66.1/41 61.8/34 48.7/19 54.3/25 54.8/200

No. of private alleles E-ACC/M-CAC 0 E-ACC/M-CCT 2 E-ACC/M-CTC 0 E-AGA/M-CTC 0 E-AGA/M-CTG 0 E-AGC/M-CCT 0 E-AGC/M-CTC 1 Total 3

1 3 0 0 2 1 1 8

1 1 0 0 1 1 0 4

4 8 0 1 1 1 0 15

1 1 1 0 1 0 1 5

0 1 0 0 0 0 0 1

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Table 2 Pairwise estimates of Nei's unbiased genetic distance (above diagonal) and FST (below diagonal) between all C. antiquate populations Population

Jiaonan wild

Jiaonan cultured

Changle wild

Changle cultured

Xiamen wild

Xiamen cultured

Jiaonan wild Jiaonan cultured Changle wild Changle cultured Xiamen wild Xiamen cultured

– 0.057 0.742 0.737 0.696 0.733

0.013 – 0.746 0.742 0.699 0.737

0.522 0.504 – 0.046 − 0.002 0.116

0.509 0.490 0.012 – 0.019 0.122

0.533 0.518 0.006 0.013 – 0.077

0.479 0.458 0.026 0.025 0.023 –

Significant FST values (P b 0.05/15) are in bold; 10,000 permutations were used to test the significance of FST estimates.

analogue θ B (Holsinger et al., 2002) were also obtained via a Bayesian approach that accounts for uncertainty in the magnitude of inbreeding within populations using the program HICKORY version 1.0.4 (Holsinger et al., 2002). Finally, Nei's unbiased genetic distances (Nei, 1978) between populations were calculated with the software package TFPGA (Miller, 1997). 3. Results 3.1. AFLP polymorphism and genetic variation within populations The seven AFLP selective primer combinations generated 365 unambiguous markers ranging in size from 80 to 300 bp (Fig. 2; Appendix A). Each individual tested had a unique AFLP band patterns, suggesting a high level of genetic variability. As shown in Table 1, HE values varied from 0.169 to 0.181 within cultured populations and ranged from 0.176 to 0.183 in the wild populations. The percentage of polymorphic loci ranged from 54.5% to 55.1% in cultured populations and varied from 58.9% to 60.8% in the wild populations. Between the cultured and local wild populations, there was no significant difference in the HE values (Wilcoxon matched-pairs signed-rank test, Jiaonan, Z = 0.845, P = 0.398; Changle, Z = 1.183, P = 0.237; Xiamen, Z = 0.169, P = 0.866) and the percentage of polymorphic loci (Wilcoxon matchedpairs signed-rank test, Jiaonan, Z = 1.095, P = 0.273; Changle, Z = 1.483, P = 0.138; Xiamen, Z = 1.521, P = 0.128). But as measured by the mean HE values and percentage of polymorphic loci, reductions in HE values and percentage of polymorphic loci were observed in the cultured populations (2.8% and 8.3% reduction, respectively). In addition, higher frequency of private alleles was found within the wild populations when compared to the cultured populations (26 vs. 10, Table 1).

3.2. Genetic differentiation among populations Estimates of the Bayesian F-statistic analogue θB (Holsinger et al. 2002) and FST were similar for all pairwise comparisons; we present only FST estimates for simplicity. Genetic differentiation across all populations as a whole was highly significant (FST = 0.624, P b 0.001), and only 1 of 15 pairwise estimates of FST between populations was not significant (Changle wild and Xiamen wild, Table 2). Most genetic differentiation was between northern populations in Jiaonan and southern populations in Changle and Xiamen (FST range: 0.696 to 0.746). In concordance with FST values, the pairwise Nei's (1978) genetic distance values were low within the northern and southern populations (0.006 to 0.026), but high between the northern and southern populations (0.458 to 0.533) (Table 2). 4. Discussion The present study is a first attempt to compare genetic variation in cultured and wild populations of C. antiquata using AFLPs, and our results demonstrate that the large amount of markers generated by this technique provide enough resolution to reveal a large amount of genetic variation and genetic differentiation between cultured and wild populations. Both cultured and wild populations were characterized by high level of genetic diversity, since each of the 125 individuals tested displayed a unique AFLP band pattern. In aquaculture, there are major concerns that genetic variability will be lost in hatchery stocks because in the long-term the population depends on sufficient genetic variation to maintain evolutionary potential (Frankham et al., 2002). Indeed, many studies have demonstrated reduced genetic variability in hatchery populations compared to wild populations (e.g. Hedgecock and Sly, 1990; Clifford et al., 1998; Sekino et al., 2002; Li et al., 2004). In contrast, some studies show no significant difference in genetic

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variability between hatchery and wild populations (Palm et al., 2003; Yu and Chu, 2006). In the present study, although not statistically significant, reductions in average HE values and percentage of polymorphic loci were observed in the cultured populations (Table 1). Moreover, a higher frequency of private alleles was seen in wild populations compared to the cultured ones, suggesting that rare alleles at some loci were lost in the cultured populations. Loss of rare alleles in cultured populations is a common phenomenon that has been observed in many cultured fish and mollusk species (Evans et al., 2004; Yu and Guo, 2004; Pampoulie et al., 2006; Yu and Chu, 2006), and which is generally viewed as a more meaningful measure of genetic variability than heterozygosity because the latter tends to be an insensitive indicator of substantial genetic change occurring in the first generation of cultured populations (Pampoulie et al., 2006). The large number of AFLP markers represent whole genome polymorphisms, and loss of rare alleles may signify loss of variation at coding regions which ultimately could have implications for commercially important traits such as growth and disease resistance (Pampoulie et al., 2006; Evans et al., 2004). Therefore, loss of rare alleles is more serious than loss of heterozygosity and should not be ignored in management practices. In aquaculture strains, genetic variability is positively correlated with the number of broodstock used to produce each strain (Allendorf and Ryman, 1987). The reductions in HE values, the percentage of polymorphic loci and number of rare alleles observed here are probably caused by the limited number of founder stock used in the process of crossing breeders obtained from wild. In this study, as indicated by the pairwise FST analysis, significant genetic differentiation was detected between cultured populations, and between cultured and wild populations. Similar findings have been reported for cultured molluscs, e.g. Pacific abalone (Li et al., 2004), eastern oyster (Yu and Guo, 2004), Pacific oyster (Li et al., 2006), and pearl oyster (Yu and Chu, 2006). The significant genetic differentiation between cultured populations and between cultured and wild populations is most likely due to random drift, bottleneck effect, and artificial selection during the production of a new generation (Pampoulie et al., 2006). In addition, consistent with our previous study using allozyme and morphometric analyses (Kong et al., 2007), significantly high genetic differentiation (FST = 0.696–0.746) was found between the northern populations (Jiaonan) and the southern populations (Changle and Xiamen). The marked genetic differentiation might be due to restricted gene flow caused by the freshwater outflow of Yangtze

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River and upwelling of Zhejiang province between the northern and southern populations (see details in Kong et al., 2007). In contrast, no significant genetic differentiation was found between the two southern wild populations. This could be the result of persistent gene flow during the two weeks of planktonic life of C. antiquata larvae in the sampling areas (Liu and Xie, 2003). In conclusion, genetic comparison of cultured strains with local wild populations is timely with respect to the rapid increase of C. antiquata breeding practices and information obtained in this study will give a better insight in the efficiency of hatchery programs in conserving the genetic variability of natural populations within the farmed strains. Considering the reduced genetic variability in the firstgeneration cultured populations, genetic changes in cultured populations should be closely monitored in the future using AFLP or polymorphic microsatellites to maintain diversity for potential restocking in areas of depletion. Although the breeding and farming practices can restore the resource in a short time, it also has the potential risk of leading to alteration in the genetic structure of wild populations. Introgression of farmed and wild population has been reported in several commercially exploited fish species (Clifford et al., 1998; McGinnity et al., 2003; Alarcón et al., 2004) as well as molluscs (Gaffney et al., 1996; Arnaud-Haond et al., 2004), and it can result in lowered fitness in wild populations (McGinnity et al., 2003). Therefore, we propose that future farming programs should avoid farming in areas where local spawning occurs, and possible signs of introgression between cultured and wild populations should be examined. Because of significant genetic differentiation between northern and southern populations, cultured populations should be managed separately, and any translocation between the two areas should be avoided. Acknowledgements We would like to thank to Mr. Zhaoxing Qiu from Shandong Mariculture Research Institute, and Mr. Jianxin Zhu from Yellow Sea Fisheries Research Institute, for providing the samples of C. antiquata. We are also grateful to three anonymous reviewers for improving the manuscript. The study was supported by grants from the National High Technology Research and Development Program (2006AA10A409) and the National Natural Science Foundation of China (30571442).

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Appendix A. Locus information for 365 AFLP markers used in population analysis. The locus code follows the commonly accepted protocol, with the first 2 characters referring to the primer pair and the last 3 digits indicating fragment size.

Locus

Primer set

Code

Locus

Primer set

Code

Locus

Primer set

Code

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 139 140 141 142 143 144 145 146

E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC

c1f081 c1f082 c1f087 c1f088 c1f092 c1f093 c1f101 c1f102 c1f104 c1f109 c1f115 c1f119 c1f121 c1f125 c1f127 c1f128 c1f135 c1f137 c1f142 c1f143 c1f148 c1f152 c1f154 c1f157 c1f158 c1f159 c1f163 c1f164 c1f166 c1f167 c1f168 c1f169 c1f173 c1f177 c1f184 c1f190 c1f196 c1f197 c1f198 c1f199 c1f202 c1f210 c1f218 c1f221 c1f224 c1f236 c3f138 c3f140 c3f141 c3f147 c3f149 c3f150 c3f153 c3f154

47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 185 186 187 188 189 190 191 192

E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CAC E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC

c1f248 c1f260 c1f274 c1f275 c1f285 c1f290 c1f295 c1f300 c2f080 c2f082 c2f083 c2f084 c2f085 c2f086 c2f087 c2f088 c2f089 c2f090 c2f091 c2f092 c2f102 c2f103 c2f105 c2f106 c2f107 c2f108 c2f111 c2f112 c2f113 c2f114 c2f115 c2f116 c2f118 c2f119 c2f120 c2f122 c2f124 c2f127 c2f132 c2f133 c2f134 c2f135 c2f136 c2f138 c2f142 c2f144 d3f118 d3f119 d3f120 d3f122 d3f124 d3f128 d3f130 d3f131

93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 231 232 233 234 235 236 237 238

E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CCT E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG

c2f147 c2f148 c2f151 c2f153 c2f156 c2f157 c2f164 c2f169 c2f170 c2f171 c2f172 c2f175 c2f177 c2f180 c2f181 c2f184 c2f185 c2f188 c2f189 c2f192 c2f197 c2f198 c2f200 c2f208 c2f230 c2f265 c2f269 c2f278 c2f286 c2f296 c3f081 c3f087 c3f088 c3f089 c3f094 c3f097 c3f098 c3f099 c3f100 c3f109 c3f110 c3f119 c3f131 c3f132 c3f134 c3f136 d4f089 d4f090 d4f091 d4f092 d4f094 d4f096 d4f098 d4f099

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Appendix A (continued) Locus

Primer set

Code

Locus

Primer set

Code

Locus

Primer set

Code

147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295

E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-ACC/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT

c3f155 c3f157 c3f166 c3f172 c3f175 c3f187 c3f188 c3f197 c3f198 c3f217 c3f221 c3f223 c3f224 c3f225 c3f247 c3f248 c3f263 d3f080 d3f081 d3f083 d3f084 d3f085 d3f086 d3f088 d3f089 d3f090 d3f094 d3f096 d3f097 d3f099 d3f100 d3f101 d3f105 d3f108 d3f109 d3f113 d3f115 d3f116 d4f196 d4f266 d4f269 d4f280 e2f081 e2f085 e2f086 e2f087 e2f091 e2f092 e2f095 e2f098 e2f099 e2f100 e2f102 e2f103 e2f105 e2f106 e2f109

193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325

E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTC E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC

d3f133 d3f134 d3f138 d3f139 d3f152 d3f154 d3f158 d3f160 d3f162 d3f166 d3f169 d3f172 d3f175 d3f178 d3f180 d3f181 d3f190 d3f191 d3f198 d3f199 d3f203 d3f206 d3f217 d3f218 d3f227 d3f235 d3f239 d3f249 d3f250 d3f273 d3f290 d3f298 d3f300 d4f080 d4f084 d4f085 d4f086 d4f088 e2f163 e2f164 e2f170 e2f180 e2f194 e2f195 e2f208 e2f210 e2f220 e2f225 e2f233 e2f248 e2f263 e3f086 e3f087 e3f089 e3f093 e3f094 e3f097

239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355

E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGA/M-CTG E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC

d4f100 d4f102 d4f104 d4f108 d4f109 d4f111 d4f112 d4f113 d4f114 d4f121 d4f122 d4f123 d4f124 d4f125 d4f127 d4f128 d4f129 d4f130 d4f132 d4f135 d4f137 d4f140 d4f146 d4f147 d4f148 d4f153 d4f154 d4f156 d4f160 d4f163 d4f166 d4f169 d4f170 d4f173 d4f177 d4f183 d4f186 d4f193 e3f130 e3f132 e3f135 e3f137 e3f145 e3f151 e3f152 e3f156 e3f161 e3f162 e3f171 e3f173 e3f174 e3f178 e3f179 e3f184 e3f185 e3f194 e3f195

(continued on next page)

160

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Appendix A (continued) Locus

Primer set

Code

Locus

Primer set

Code

Locus

Primer set

Code

296 297 298 299 300 301 302 303 304 305 306

E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT E-AGC/M-CCT

e2f118 e2f119 e2f129 e2f133 e2f139 e2f140 e2f141 e2f150 e2f153 e2f159 e2f161

326 327 328 329 330 331 332 333 334 335 336

E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC

e3f098 e3f099 e3f101 e3f102 e3f105 e3f108 e3f109 e3f110 e3f114 e3f116 e3f125

356 357 358 359 360 361 362 363 364 365

E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC E-AGC/M-CTC

e3f209 e3f211 e3f213 e3f214 e3f217 e3f222 e3f240 e3f246 e3f255 e3f260

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