# A Simple and Cost-Effective Method to Synthesize an Internal Size Standard Amenable to Use with a 5-Dye System

Stéphane Mauger, L Couceiro, M Valero

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Stéphane Mauger, L Couceiro, M Valero. A Simple and Cost-Effective Method to Synthesize an Internal Size Standard Amenable to Use with a 5-Dye System. Prime Research on Biotechnology (PRB), 2012, 2 (3), pp.40-46. hal-04208632

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6 **A Simple and Cost-Effective Method to Synthesize an Internal Size Standard**

7 **Amenable to Use with a 5-Dye System**

8

9 **Mauger, S.; Couceiro, L. and Valero, M.**

10 CNRS-UPMC, UMR7144, Équipe BEDIM, Station Biologique de Roscoff, Place

11 Georges Teissier, F-29682 Roscoff, France

12 <sup>1</sup> Author for correspondence: E-mail stephane.mauger@sb-roscoff.fr

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15 **Keywords:** 5-dye system, AFLPs, allele sizing, capillary electrophoresis, fluorescent

16 dye, internal size standard, microsatellites

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19 Abstract word count: 200

20 Body of the manuscript word count: 1886
21 ABSTRACT

22 Fluorescent end-labeling joint to capillary electrophoresis has become a common
23 practice for PCR fragment analysis. Besides its sensitivity/reproducibility, the major
24 advantage of this method is the possibility of detect simultaneously fragments
25 overlapping in size by the use of different labels. However, even thought multiplexing
26 approaches are usually presented as a way to reduce genotyping costs, the price of the
27 commercial size standards still represents a substantial deterrent. As an example, a new
28 generation of dye-systems allowing fragments labeled up to 5 different colors is now
29 commercially available but the price of the pertinent internal standard can easily
30 represent the 50% of the project budget. Here, we describe a simple and cost-effective
31 method to synthesize a fluorescently labeled size standard amenable to use with a 5-dye
32 system, the SM594. Its performance in fragment size determination is demonstrated
33 over a total of 63 alleles covering different molecular markers, species, and dyes. In
34 summary, allele calling using the SM594 and the equivalent commercial standard shows
35 identical results and maximum divergence between lectures from both standards never
36 exceed 0.64 bp. Since genotyping costs can be reduced by a 40 factor, the use of the
37 SM594 might allow researchers to address projects covering more
38 species/individuals/loci.
39 INTRODUCTION

40 Microsatellite markers are widely used in several research fields and they
41 usually represent the marker of choice for population genetic analysis (1). Reducing the
42 economic cost of microsatellite development/genotyping represents a technical
43 challenge, particularly in nonmodel organisms for which fewer resources are usually
44 available (2). In this regard, different authors have recently demonstrated the time- and
45 cost-effectiveness of next-generation sequencing technology to develop microsatellite
46 markers (3). By the contrary, and despite commonly used multiplexing assays,
47 genotyping costs continue still to be high or even limiting.

48 Since their description 20 years ago, the way that microsatellite alleles are
49 visualized/sized has changed according to technological advances. Allele sizing was
50 initially based on agarose gels. However, because microsatellite alleles can differ by as
51 little as 2 bp size, agarose gels were soon replaced by more accurate polyacrylamide gel
52 electrophoresis (PAGE). Semiautomated PAGE platforms and fluorescent dye
53 chemistry greatly improved microsatellite resolution/accuracy. Still, the major shift in
54 microsatellite genotyping arose with capillary electrophoresis (CE) technology which
55 allows higher sensitivity and reproducibility while reduces processing time.
56 Unfortunately, this increase in throughput genotyping rates also involved a considerable
57 increase of the running costs by the need to include a fluorescently labeled size standard
58 for each sample. In fact, the price of such size standards which are available from
59 relatively few vendors can in many cases exceed that of the genotyping assay itself (4).

60 In an attempt of lowering microsatellite genotyping costs, a handful of studies
61 have reported over the last decade the in-house production of fluorescently labeled size
62 standards (4-7). Although the cost-effectiveness of these methods has been clearly
established, two of them propose the use of specific DNA templates complicating their
use elsewhere (5,6). Moreover, all these protocols describe the synthesis of size
standards for a four-dye system. An advantage of a fluorescently-based detection system
is that DNA fragments overlapping in size range can be labeled with different dyes and
therefore be simultaneously detected in a single lane on a CE instrument. Thus,
although 4-dye technology has allowed a significant increase in throughput rates over
traditional methods, a third generation of dyes combining 5 different labels has been
introduced in the market in 2005.

In the present work, we describe a simple and cheap method to produce a
fluorescently labeled size standard (the SM594) amenable to use with 5-dye systems
and we show its accuracy and reproducibility in a range of different alleles and species.
Moreover, its suitability for other techniques that involve fragment separation as
Amplified Fragment Length Polymorphism (AFLP) is also demonstrated.

## MATERIAL & METHODS

In order to amplify known-length fragments using the universal cloning vector
pGEM®-3Z(+) (Promega Corporation, Madison, WI, USA) as DNA template, 19
primers were designed with the software Primer3 (8). The resulting primer set was
composed of a single forward primer with a 5'-ATTO 633 label and 18 reverse primers
(Table 1), and it amplified a total of 18 single-stranded, fluorescently labeled fragments
(theoretical/expected sizes: 60, 100, 140, 200, 214, 220, 240, 260, 314, 340, 380, 414,
420, 440, 480, 500, 520, and 600 bp) (Fig. 1).

Size standard synthesis was carried out by amplifying individually the 18
fragments. PCR reactions (50 µl) were identical for all fragments apart from the reverse
primer identity and they contained 1x GoTaq® Flexi buffer (Promega Corporation), 1
87 mM MgCl₂, 150 µM of each dNTP (Thermo Fisher Scientific Inc., Waltham, MA,
88 USA), 400 nM of each primer (Eurofins MWG Operon, Ebersberg, Germany), 1.75 U
89 GoTaq® Polymerase (Promega Corporation), and 20 ng of pGEM®-3Z(+). Likewise,
90 the same amplification profile was applied to all reactions and it included an initial
denaturing step at 94°C for 5 min, followed by 30 cycles at 94°C for 30s, 65°C for 30s,
91 and 72°C for 30s, and a final extension at 72°C for 1 hour (in order to allow a consistent
92 Taq polymerase terminal transferase activity and guarantee that all PCR products match
93 to the +A state). Finally, individual amplifications were tested on standard agarose gels
94 and equal volumes of each PCR product were combined into a single tube and stored at
95 -20°C until their use as size standard.
96

97 SM594 calibration and validation were performed as follow. First, actual size of
98 the synthesized fragments was estimated by co-electrophoresis with the commercial size
99 standard GeneScan™ 600LIZ® (Life Technologies Corporation, Carlsbad, CA, USA).
100 Briefly, 0.5 µl of SM594 were mixed with 0.5 µl of the commercial size standard and
101 11 µl of Hi-Di™ Formamide (Life Technologies Corporation), the obtained mixture
102 was run on a 3130XL Genetic Analyzer (Life Technologies Corporation), and
103 size/fluorescence intensity of each SM594 fragment was determined in the
104 GENEMAPPER version 4.0 (Life Technologies Corporation). Second, 21 microsatellite
105 loci from 4 different algae species: *Chondrus crispus* (9), *Gracilaria gracilis* (10,11),
106 *Lessonia nigrescens* (12), and *Sargasum muticum* (Daguin-Thiébaut C., unpublished
data), as well as 10 AFLP markers from the gastropod *Crepidula fornicata* (Riquet F.,
107 unpublished data), were amplified using conventional protocols and size estimation of
108 the 63 resulting alleles was done over 8 independent runs of a 3130XL Genetic
109 Analyzer using both the SM594 size standard and the commercial size standard
110 GeneScan™ 600LIZ®. Moreover, in order to check the reproducibility and consistency
111
112 of the obtained sizes, 8 additional runs using the SM594 as internal size standard were
113 repeated over a different 3130XL Genetic Analyzer instrument. Allele size comparisons
114 between different internal standards (i.e. GeneScan™ 600LIZ® vs. SM594) as well as
115 between different instruments (i.e. SM594-1 vs. SM594-2) were based on the range of
116 differences in size estimates and the mean size values. Highest divergence for allele size
117 among both treatments was also calculated.

## 118 RESULTS AND DISCUSSION

119 The designed primers amplified 18 unique PCR products (Fig. 1). Estimated
120 size of the synthesized fragments differed from theoretical/expected size by 3-7
121 nucleotides (Fig. 2), probably due to the influence of the ATTO 633 fluorescent label on
122 the electrophoretic mobility (13). However, those differences were stable over different
123 SM594 batches showing its reproducibility, stability and accuracy (mean sizes ±
124 standard deviation for the 18 fragments over 8 independent batches were 56.76±0.14,
125 95.42±0.10, 136.02±0.09, 195.44±0.11, 209.49±0.12, 215.46±0.10, 235.33±0.12,
126 255.38±0.10, 309.04±0.12, 335.40±0.07, 375.39±0.10, 409.31±0.09, 415.22±0.10,
127 434.40±0.11, 474.41±0.13, 494.48±0.08, 513.48±0.06, and 594.30±0.11). These results
128 show that the use of a universal cloning vector like the pGEM®-3Z(+) as DNA template
129 not only renders the protocol accessible to any interested laboratory but also guarantees
130 a robust amplification and avoids any subsequent purification step (6). Likewise,
131 differences in fluorescence intensity among fragments were low (at most 3-fold; Fig. 2)
132 and constant making unnecessary any further optimization (e.g. pooling unequal rates of
133 each fragment, diluting fragment pool to target an optimum peak height range) (6,7).

134 The comparison of allele sizes using both the SM594 and the GeneScan™
135 600LIZ® corroborated the reproducibility and accuracy of the in-house synthesized
standard (Table 2). Differences in size estimates over 8 independent runs for 53 microsatellite alleles ranged from 0.07 to 0.64 bp and from 0.06 to 0.51 bp for the SM594 and the GeneScan™ 600LIZ®, respectively. The range of differences in size estimates was also similar for both internal standards in the case of the 10 AFLP markers (from 0.05 to 0.43 bp and from 0.08 to 0.23 bp for the SM594 and the GeneScan™ 600LIZ®, respectively). By the other hand, although statistical differences between mean sizes obtained with both standards were found in 40 alleles, maximum divergence never exceed 0.64 bp guaranteeing their right calling (6,7). In fact, the highest difference among independent lectures of the same allele was lower than 0.5 bp in the 90% of the cases. Briefly, 4 out of the 6 alleles showing maximum divergence values ≥ 0.5 bp were found in *G. gracilis* (Gg121-205, 2CT-230, 2CT-203, and Gg173-170) while the 2 remaining ones belonged to *L. nigrescens* (Less1T3-138 and Less1T3-141). However, the range of maximum divergence values was quite similar among the 5 species: 0.16-0.44 for *C. crispus*, 0.20-0.64 for *G. gracilis*, 0.28-0.46 for *C. fornicata*, 0.27-0.52 for *L. nigrescens*, and 0.14-0.37 for *S. muticum*. Likewise, any relationship could be established between maximum divergence values and fluorescent dye (0.24-0.50 for 6-FAM, 0.16-0.44 for VIC, 0.20-0.52 for NED, and 0.14-0.64 for PET).

Finally, a similar range of differences in size estimates was also observed when samples were genotyped on a different Genetic Analyzer (from 0.06 to 0.42 bp for microsatellite alleles and from 0.23 to 0.38 for AFLP markers) and, as it was the case for the comparisons between both standards, the highest difference between lectures obtained from different instruments never exceed 0.64 bp (Table 2).

Unlike genotyping using an equivalent commercial size standard (i.e. an internal size standard amenable to use with a 5-dye system), allele sizing with the SM594 can be easily afforded by any laboratory furnished with basic molecular equipment. First, their
synthesis is a simple and fast procedure; in fact, any person familiarized with the PCR
technique can produce large volumes of this size standard in a few hours. Second, cost
analysis reveals that genotyping is 40 times more economical when comparing with an
equivalent commercial size (estimated price includes primer and cloning vector
synthesis, PCRs and working hours). In fact, the economic expenditure needed to
acquire a batch of the commercial size standard GeneScan™ 600LIZ® (approximately
800 samples) allows to buy an amount of primers and cloning vector enough to
 synthesize 110 SM594 batches (approximately 990000 samples). This reduction of
genotyping costs is agree with previous reports of in-house synthesized size standards
(from 10 to 128 times more economical, 4-7). However, the economic advantage of the
SM594 might go further since it can be used to size fragments labeled with up to 4
different colors and, therefore, it increases multiplexing options.

In conclusion, the present work describes a simple, robust and cheap procedure
to synthesize a fluorescently labeled size standard amenable to use with a 5-dye system.
Accuracy in fragment size determination comparable to that obtained using the
equivalent commercial size standard is demonstrated in a wide range of microsatellite
loci covering different motifs, labels, and species but also in AFLP markers. Since the
use of this size standard reduces genotyping costs by a 40 factor, it should allow
researchers to address projects covering a larger number of species/individuals/loci.

180 ACKNOWLEDGMENTS

The authors are deeply indebted to Daguin-Thiébaut C. and Riquet F. for providing S. muticum and C. fornicata samples. Funding for this work was provided by XXX; L.C. acknowledges a postdoctoral fellowship from Secretaría de Estado de Universidades e Investigación, Ministerio de Educación.
185 COMPETING INTERESTS STATEMENT

186 The authors declare no competing interests.
<table><caption>188 Table 1. Primer sequences for SM594 synthesis.</caption><thead><tr><th>Primer name</th><th>Direction</th><th>Primer Sequence (5' → 3')</th></tr></thead><tbody><tr><td>SMF*</td><td>Forward</td><td>CGCTAGGGCGCTGGCAAGTG</td></tr><tr><td>57R</td><td>Reverse</td><td>AGCGCGGCGGGTGTGGTG</td></tr><tr><td>95R</td><td>Reverse</td><td>GCCTGAATGGCGAATGGACGC</td></tr><tr><td>136R</td><td>Reverse</td><td>GAAGAGGCCCGCACCGATCGC</td></tr><tr><td>195R</td><td>Reverse</td><td>GGCGTTACCCAACTTAATCGCCTT</td></tr><tr><td>209R</td><td>Reverse</td><td>ACTGGGAAAACCCTGGCGTTACCC</td></tr><tr><td>215R</td><td>Reverse</td><td>GTCGTGACTGGGAAAACCCTGGC</td></tr><tr><td>235R</td><td>Reverse</td><td>ACTGGCCGTCGTTTTACAACGTC</td></tr><tr><td>255R</td><td>Reverse</td><td>AGTGAGTCGTATTACAATTCACTGGCCG</td></tr><tr><td>309R</td><td>Reverse</td><td>GCCTGCAGGTCGACTCTAGAGGAT</td></tr><tr><td>335R</td><td>Reverse</td><td>ACACTATAGAATACTCAAGCTTGCATGC</td></tr><tr><td>375R</td><td>Reverse</td><td>GAAACAGCTATGACCATGATTACGC</td></tr><tr><td>409R</td><td>Reverse</td><td>TGTGGAATTGTGAGCGGATAACAATTTC</td></tr><tr><td>415R</td><td>Reverse</td><td>ATGTTGTGTGGAATTGTGAGCGGATAAC</td></tr><tr><td>434R</td><td>Reverse</td><td>ACTTTATGCTTCCGGCTCGTATGTTG</td></tr><tr><td>474R</td><td>Reverse</td><td>AATGTGAGTTAGCTCACTCATTAGGC</td></tr><tr><td>494R</td><td>Reverse</td><td>GCAGTGAGCGCAACGCAATTAAT</td></tr><tr><td>513R</td><td>Reverse</td><td>GTTTCCCGACTGGAAAGCGGGC</td></tr><tr><td>594R</td><td>Reverse</td><td>GAGGAAGCGGAAGAGCGCCC</td></tr></tbody><tfoot><tr><td colspan="3">*SMF has a 5'ATTO 633 label.</td></tr></tfoot></table>
<table><caption>Table 2. Microsatellite/AFLP allele sizing using a commercial size standard (GeneScan™ 600LIZ®) and the in-house synthesized size standard SM594.</caption><thead><tr><th rowspan="2">Species</th><th rowspan="2">Locus<sup>b</sup></th><th rowspan="2">Allele</th><th colspan="2">GeneScan™ 600LIZ®</th><th colspan="2">SM594-GA1<sup>a</sup></th><th colspan="2">SM594-GA2<sup>a</sup></th><th rowspan="2">MDM<sup>c</sup></th><th rowspan="2">MDS<sup>d</sup></th></tr><tr><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th></tr></thead><tbody><tr><td rowspan="8">C. crispus</td><td rowspan="2">Chc40<sup>6-FAM</sup></td><td>160</td><td>160.33</td><td>0.06</td><td><strong>160.35</strong></td><td>0.07</td><td>160.22</td><td>0.09</td><td>0.25</td><td>0.36</td></tr><tr><td>176</td><td>175.69</td><td>0.04</td><td><strong>175.64</strong></td><td>0.09</td><td><strong>175.61</strong></td><td>0.09</td><td>0.24</td><td>0.29</td></tr><tr><td rowspan="2">Chc24<sup>VIC</sup></td><td>175</td><td>174.84</td><td>0.04</td><td>174.74</td><td>0.05</td><td>174.63</td><td>0.10</td><td>0.25</td><td>0.33</td></tr><tr><td>219</td><td>218.82</td><td>0.05</td><td>218.58</td><td>0.09</td><td><strong>218.62</strong></td><td>0.09</td><td>0.38</td><td>0.30</td></tr><tr><td rowspan="2">Chc31<sup>NED</sup></td><td>210</td><td>210.09</td><td>0.06</td><td>209.81</td><td>0.06</td><td>209.89</td><td>0.06</td><td>0.44</td><td>0.25</td></tr><tr><td>216</td><td>215.86</td><td>0.06</td><td>215.64</td><td>0.05</td><td><strong>215.68</strong></td><td>0.05</td><td>0.39</td><td>0.19</td></tr><tr><td rowspan="2">Chc03<sup>6-FAM</sup></td><td>248</td><td>247.40</td><td>0.02</td><td>247.27</td><td>0.05</td><td>247.16</td><td>0.09</td><td>0.24</td><td>0.35</td></tr><tr><td>250</td><td>250.55</td><td>0.05</td><td>250.40</td><td>0.07</td><td><strong>250.33</strong></td><td>0.06</td><td>0.37</td><td>0.23</td></tr><tr><td rowspan="2">Chc02<sup>VIC</sup></td><td>250</td><td>249.75</td><td>0.03</td><td>249.59</td><td>0.08</td><td>249.47</td><td>0.07</td><td>0.28</td><td>0.31</td></tr><tr><td>276</td><td>276.26</td><td>0.06</td><td><strong>276.26</strong></td><td>0.02</td><td><strong>275.27</strong></td><td>0.07</td><td>0.16</td><td>0.20</td></tr></tbody></table>
<table><tr><td></td><td>279</td><td>279.21</td><td>0.03</td><td><strong>279.21</strong></td><td>0.08</td><td><strong>279.17</strong></td><td>0.07</td><td>0.17</td><td>0.23</td></tr><tr><td>Chc04<sup>PET</sup></td><td>290</td><td>290.42</td><td>0.06</td><td><strong>290.43</strong></td><td>0.09</td><td><strong>290.45</strong></td><td>0.06</td><td>0.33</td><td>0.33</td></tr><tr><td></td><td>294</td><td>294.24</td><td>0.05</td><td><strong>294.27</strong></td><td>0.05</td><td><strong>294.22</strong></td><td>0.09</td><td>0.19</td><td>0.24</td></tr><tr><td><strong>G. gracilis</strong></td><td>162</td><td>162.11</td><td>0.03</td><td><strong>162.09</strong></td><td>0.10</td><td>161.86</td><td>0.05</td><td>0.29</td><td>0.40</td></tr><tr><td></td><td>170</td><td>170.25</td><td>0.04</td><td><strong>170.24</strong></td><td>0.18</td><td>169.95</td><td>0.05</td><td>0.50</td><td>0.64</td></tr><tr><td>Gg155<sup>PET</sup></td><td>201</td><td>200.76</td><td>0.06</td><td>200.51</td><td>0.08</td><td><strong>200.53</strong></td><td>0.04</td><td>0.40</td><td>0.24</td></tr><tr><td></td><td>203</td><td>202.68</td><td>0.05</td><td>202.49</td><td>0.12</td><td><strong>202.41</strong></td><td>0.06</td><td>0.36</td><td>0.27</td></tr><tr><td>2CT<sup>NED</sup></td><td>171</td><td>171.11</td><td>0.06</td><td><strong>171.09</strong></td><td>0.07</td><td>171.22</td><td>0.10</td><td>0.20</td><td>0.34</td></tr><tr><td></td><td>193</td><td>192.79</td><td>0.12</td><td>192.61</td><td>0.06</td><td>192.50</td><td>0.04</td><td>0.39</td><td>0.25</td></tr><tr><td></td><td>199</td><td>198.70</td><td>0.13</td><td>198.56</td><td>0.13</td><td><strong>198.51</strong></td><td>0.03</td><td>0.44</td><td>0.32</td></tr><tr><td></td><td>201</td><td>200.63</td><td>0.14</td><td>200.34</td><td>0.05</td><td><strong>200.38</strong></td><td>0.03</td><td>0.48</td><td>0.16</td></tr><tr><td></td><td>203</td><td>202.58</td><td>0.14</td><td>202.33</td><td>0.08</td><td><strong>202.32</strong></td><td>0.03</td><td>0.51</td><td>0.20</td></tr><tr><td></td><td>219</td><td>218.45</td><td>0.13</td><td><strong>218.36</strong></td><td>0.06</td><td>218.26</td><td>0.05</td><td>0.44</td><td>0.18</td></tr><tr><td></td><td>228</td><td>228.24</td><td>0.15</td><td><strong>228.21</strong></td><td>0.10</td><td><strong>228.22</strong></td><td>0.09</td><td>0.44</td><td>0.31</td></tr><tr><td></td><td>230</td><td>230.14</td><td>0.18</td><td><strong>230.16</strong></td><td>0.05</td><td>230.21</td><td>0.05</td><td>0.51</td><td>0.15</td></tr></table>
<table><tr><td>Gg216<sup>6-FAM</sup></td><td>166</td><td>165.80</td><td>0.04</td><td><strong>165.73</strong></td><td>0.10</td><td>165.55</td><td>0.09</td><td>0.31</td><td>0.44</td></tr><tr><td>Gg182<sup>VIC</sup></td><td>121</td><td>121.17</td><td>0.05</td><td>121.05</td><td>0.07</td><td>120.88</td><td>0.06</td><td>0.32</td><td>0.31</td></tr><tr><td></td><td>131</td><td>130.64</td><td>0.07</td><td><strong>130.69</strong></td><td>0.08</td><td>130.59</td><td>0.07</td><td>0.38</td><td>0.40</td></tr><tr><td></td><td>135</td><td>134.44</td><td>0.12</td><td>134.60</td><td>0.07</td><td>134.49</td><td>0.06</td><td>0.36</td><td>0.31</td></tr><tr><td>Gg121<sup>PET</sup></td><td>203</td><td>203.65</td><td>0.02</td><td>203.45</td><td>0.13</td><td><strong>203.46</strong></td><td>0.04</td><td>0.4</td><td>0.37</td></tr><tr><td></td><td>205</td><td>205.59</td><td>0.04</td><td><strong>205.54</strong></td><td>0.19</td><td><strong>205.43</strong></td><td>0.03</td><td>0.64</td><td>0.64</td></tr><tr><td>Gg202<sup>NED</sup></td><td>168</td><td>166.99</td><td>0.05</td><td><strong>166.91</strong></td><td>0.12</td><td>166.75</td><td>0.11</td><td>0.35</td><td>0.40</td></tr><tr><td>C. fornicata</td><td>E14M1<sup>6-FAM</sup></td><td>157</td><td>157.50</td><td>0.04</td><td>157.36</td><td>0.12</td><td><strong>157.46</strong></td><td>0.11</td><td>0.33</td><td>0.41</td></tr><tr><td></td><td>225</td><td>224.82</td><td>0.05</td><td>224.65</td><td>0.14</td><td><strong>224.55</strong></td><td>0.12</td><td>0.41</td><td>0.48</td></tr><tr><td></td><td>383</td><td>383.37</td><td>0.04</td><td>383.17</td><td>0.12</td><td><strong>383.11</strong></td><td>0.08</td><td>0.46</td><td>0.43</td></tr><tr><td>E15M2<sup>VIC</sup></td><td>114</td><td>113.99</td><td>0.03</td><td>113.67</td><td>0.02</td><td><strong>113.71</strong></td><td>0.08</td><td>0.35</td><td>0.24</td></tr><tr><td></td><td>149</td><td>148.45</td><td>0.04</td><td><strong>148.49</strong></td><td>0.12</td><td><strong>148.56</strong></td><td>0.12</td><td>0.32</td><td>0.38</td></tr><tr><td></td><td>310</td><td>310.41</td><td>0.08</td><td><strong>310.34</strong></td><td>0.11</td><td><strong>310.43</strong></td><td>0.10</td><td>0.36</td><td>0.43</td></tr><tr><td>E2M11<sup>PET</sup></td><td>154</td><td>154.17</td><td>0.03</td><td><strong>154.10</strong></td><td>0.10</td><td><strong>154.19</strong></td><td>0.12</td><td>0.28</td><td>0.4</td></tr><tr><td>E1M12<sup>NED</sup></td><td>228</td><td>228.32</td><td>0.03</td><td>228.10</td><td>0.10</td><td><strong>228.12</strong></td><td>0.12</td><td>0.39</td><td>0.39</td></tr></table>
<table><tbody><tr><td></td><td>281</td><td>281.59</td><td>0.05</td><td><strong>281.47</strong></td><td>0.14</td><td><strong>281.48</strong></td><td>0.11</td><td>0.42</td><td>0.42</td></tr><tr><td></td><td>319</td><td>319.11</td><td>0.05</td><td>318.92</td><td>0.05</td><td><strong>318.90</strong></td><td>0.10</td><td>0.33</td><td>0.29</td></tr><tr><td rowspan="2"><strong>L. nigrescens</strong></td><td>Les1P16<sup>6-FAM</sup></td><td>307</td><td>307.24</td><td>0.05</td><td>307.35</td><td>0.09</td><td>307.50</td><td>0.10</td><td>0.35</td><td>0.36</td></tr><tr><td></td><td>309</td><td>309.23</td><td>0.06</td><td>309.34</td><td>0.08</td><td>309.47</td><td>0.08</td><td>0.35</td><td>0.30</td></tr><tr><td></td><td></td><td>326</td><td>326.47</td><td>0.13</td><td>326.21</td><td>0.09</td><td>326.34</td><td>0.10</td><td>0.48</td><td>0.34</td></tr><tr><td></td><td></td><td>328</td><td>328.52</td><td>0.10</td><td>328.32</td><td>0.10</td><td><strong>328.36</strong></td><td>0.08</td><td>0.48</td><td>0.3</td></tr><tr><td rowspan="2">Les2D25<sup>VIC</sup></td><td></td><td>222</td><td>221.77</td><td>0.08</td><td>221.51</td><td>0.06</td><td>221.60</td><td>0.07</td><td>0.44</td><td>0.25</td></tr><tr><td></td><td>226</td><td>225.61</td><td>0.08</td><td>225.42</td><td>0.10</td><td>225.54</td><td>0.08</td><td>0.39</td><td>0.30</td></tr><tr><td></td><td></td><td>228</td><td>227.47</td><td>0.05</td><td><strong>227.43</strong></td><td>0.09</td><td><strong>227.51</strong></td><td>0.10</td><td>0.27</td><td>0.36</td></tr><tr><td></td><td></td><td>232</td><td>231.40</td><td>0.07</td><td>231.26</td><td>0.06</td><td>231.40</td><td>0.11</td><td>0.29</td><td>0.35</td></tr><tr><td></td><td></td><td>234</td><td>233.37</td><td>0.09</td><td>233.25</td><td>0.08</td><td>233.39</td><td>0.09</td><td>0.40</td><td>0.43</td></tr><tr><td rowspan="2">Les2D26<sup>PET</sup></td><td></td><td>135</td><td>134.81</td><td>0.06</td><td>134.95</td><td>0.10</td><td>135.04</td><td>0.07</td><td>0.37</td><td>0.29</td></tr><tr><td></td><td>139</td><td>138.72</td><td>0.05</td><td>138.86</td><td>0.12</td><td>138.96</td><td>0.06</td><td>0.41</td><td>0.34</td></tr><tr><td rowspan="2">Les1T3<sup>NED</sup></td><td></td><td>122</td><td>122.21</td><td>0.06</td><td>121.95</td><td>0.08</td><td>122.07</td><td>0.11</td><td>0.49</td><td>0.43</td></tr><tr><td></td><td>135</td><td>134.76</td><td>0.06</td><td>135.03</td><td>0.05</td><td><strong>135.05</strong></td><td>0.06</td><td>0.46</td><td>0.20</td></tr></tbody></table>
<table><tbody><tr><td></td><td>138</td><td>137.63</td><td>0.18</td><td>137.90</td><td>0.09</td><td><strong>137.93</strong></td><td>0.06</td><td>0.52</td><td>0.28</td></tr><tr><td></td><td>141</td><td>140.50</td><td>0.11</td><td>140.73</td><td>0.07</td><td><strong>140.78</strong></td><td>0.07</td><td>0.50</td><td>0.28</td></tr><tr><td></td><td>152</td><td>152.08</td><td>0.10</td><td>152.31</td><td>0.07</td><td><strong>152.31</strong></td><td>0.06</td><td>0.49</td><td>0.23</td></tr><tr><td rowspan="3"><strong>S. muticum</strong></td><td>D10<sup>6-FAM</sup></td><td>212</td><td>211.99</td><td>0.07</td><td>211.78</td><td>0.13</td><td><strong>211.75</strong></td><td>0.11</td><td>0.37</td><td>0.37</td></tr><tr><td></td><td>213</td><td>212.97</td><td>0.07</td><td>212.82</td><td>0.12</td><td><strong>212.76</strong></td><td>0.15</td><td>0.32</td><td>0.42</td></tr><tr><td>C15<sup>VIC</sup></td><td>87</td><td>87.43</td><td>0.03</td><td><strong>87.42</strong></td><td>0.11</td><td>87.29</td><td>0.11</td><td>0.32</td><td>0.52</td></tr><tr><td></td><td>D33<sup>PET</sup></td><td>289</td><td>288.84</td><td>0.04</td><td><strong>288.83</strong></td><td>0.05</td><td>288.72</td><td>0.09</td><td>0.14</td><td>0.31</td></tr><tr><td></td><td>T29<sup>NED</sup></td><td>175</td><td>175.49</td><td>0.05</td><td>175.35</td><td>0.07</td><td><strong>175.28</strong></td><td>0.09</td><td>0.30</td><td>0.37</td></tr></tbody></table>

a Allele size estimation was carried out on two different 3130XL Genetic Analyzer instruments (GA1 and GA2) using the SM594. Values in bold for GA1 denote mean sizes that are not statistically significant from those obtained with GeneScan™ 600LIZ; values in bold for GA2 denote mean sizes that are not statistically significant from those obtained using also the SM594 but on a different 3130XL Genetic Analyzer instrument (GA1).

b Fluorescent label for each locus is indicated in the right superscript position.

c Maximum difference among allele size estimates using the commercial size standard GeneScan™ 600LIZ and the in-house synthesized standard SM594.

d Maximum difference among allele size estimates obtained from two different 3130X Genetic Analyzer instruments using the in-house synthesized standard SM594.
191 FIGURE & LEGENDS

<figure><img src="image_2.png" /></figure>

192

193 Figure 1. (A) Map of the universal cloning vector pGEM®-3Z(+) showing the position
194 of the 19 primers (white and grey arrows represent forward and reverse primers
195 respectively). (B) Agarose gel of the 18 amplified PCR products; lane M corresponds to
196 the commercial molecular weight standard 50 bp DNA Ladder (New England BioLabs
197 Inc., Ipswich, MA, USA).

198
<figure><img src="image_3.png" /><figcaption>Figure 2. Chromatogram of the SM594 size standard on a 3130XL Genetic Analyzer.</figcaption></figure>

Fragment size (bp) is indicated above each peak and it was estimated by co-electrophoresis with the commercial size standard GeneScan™ 600LIZ®.
203 REFERENCES

204 1.Ellegren, H. 2004. Microsatellites: simple sequences with complex evolution. Nature
205 Reviews Genetics 5:435-445.

206 2.Lepais, O. and C.F.E. Bacles. 2011. Comparison of random and SSR enriched
207 shotgun pyrosequencing for microsatellite discovery and single multiplex PCR
208 optimization in *Acacia harpophylla* F. Muell. Ex Benth. Molecular Ecology
209 Resources 11:711-724.

210 3.Abdelkrim, J., B.C. Robertson, J.A.L. Stanton, and N.J. Gemmell. 2009. Fast,
211 cost-effective development of species-specific microsatellite markers by
212 genomic sequencing. BioTechniques 46:185-192.

213 4.DeWoody, J.A., J. Schupp, L. Kenefic, J. Busch, L. Murfitt, and P. Keim. 2004.
214 Universal method for producing ROX-labeled size standards suitable for
215 automated genotyping. BioTechniques 37:348-352.

216 5.Ueno, S., Y. Tsumura, and I. Washitani. 2003. Cost-effective method to synthesize
217 fluorescently labeled DNA size standards using cloned AFLP fragments.
218 BioTechniques 34:1146-1148.

219 6.Brondani, R.P.V. and G. D. 1993. Cost-effective method to synthesize a fluorescent
220 internal DNA standard for automated fragment sizing. BioTechniques 31:793-
221 800.

222 7.Symonds, V.V. and A.M. Lloyd. 2004. A simple and inexpensive method for
223 producing fluorescently labelled size standard. Molecular Ecology Notes 4:768-
224 771.

225 8.Rozen, S. and H. Skaletsky. 2000. Primer3 on the WWW for general users and for
226 biologist programmers. Methods Mol Biol 132:365-386.
227 9.Krueger-Hadfield, S.A., J. Collén, C. Daguin-Thiébaut, and M. Valero. 2011.

228 Genetic population structure and mating system in *Chondrus crispus*

229 (Rhodophyta). Journal of Phycology 47:440-450.

230 10.Luo, H., M. Mörchen, C.R. Engel, C. Destombe, J.T. Epplen, C. Epplen, P.

231 Saumitou-Laprade, and M. Valero. 1999. Characterization of microsatellite

232 markers in the red alga *Gracilaria gracilis*. Molecular Ecology 8:700-702.

233 11.Wattier, R., J.F. Dallas, C. Destombe, P. Saumitou-Laprade, and M. Valero.

234 1997. Single locus microsatellites in Gracilariales (Rhodophyta): high level of

235 genetic variability within *Gracilaria gracilis* and conservation in related species.

236 Journal of Phycology 33:868-880.

237 12.Faugeron, S., D. Veliz, G. Peralta, J. Tapia, F. Tellier, C. Billot, and E.

238 Martinez. 2009. Development and characterization of nine polymorphic

239 microsatellite markers in the Chilean kelp *Lessonia nigrescens*. Molecular

240 Ecology Resources 9:937-939.

241 13.Tu, O., T. Knott, M. Marsh, K. Bechtol, D. Harris, D. Barker, and J. Bashkin.

242 1998. The influence of fluorescent dye structure on the electrophoretic mobility

243 of end-labeled DNA. Nucleic Acids Research 26:2797-2802.