Journal of Heredity Advance Access published November 25, 2014 Journal of Heredity doi:10.1093/jhered/esu071
© The American Genetic Association 2014. All rights reserved. For permissions, please e-mail: [email protected]
Microsatellite Marker Development by Multiplex Ion Torrent PGM Sequencing: a Case Study of the Endangered Odorrana narina Complex of Frogs Takeshi Igawa, Masafumi Nozawa, Mai Nagaoka, Shohei Komaki, Shohei Oumi, Tamotsu Fujii, and Masayuki Sumida
Address correspondence to Dr. Takeshi Igawa at the address above, or e-mail: [email protected]. Data deposited at Dryad: doi:http://dx.doi.org/doi:10.5061/dryad.9418j
The endangered Ryukyu tip-nosed frog Odorrana narina and its related species, Odorrana amamiensis, Odorrana supranarina, and Odorrana utsunomiyaorum, belong to the family Ranidae and are endemically distributed in Okinawa (O. narina), Amami and Tokunoshima (O. amamiensis), and Ishigaki and Iriomote (O. supranarina and O. utsunomiyaorum) Islands. Because of varying distribution patterns, this species complex is an intrinsic model for speciation and adaptation. For effective conservation and molecular ecological studies, further genetic information is needed. For rapid, cost-effective development of several microsatellite markers for these and 2 other species, we used nextgeneration sequencing technology of Ion Torrent PGM™. Distribution patterns of repeat motifs of microsatellite loci in these modern frog species (Neobatrachia) were similarly skewed. We isolated and characterized 20 new microsatellite loci of O. narina and validated cross-amplification in the threerelated species. Seventeen, 16, and 13 loci were cross-amplified in O. amamiensis, O. supranarina, and O. utsunomiyaorum, respectively, reflecting close genetic relationships between them. Mean number of alleles and expected heterozygosity of newly isolated loci varied depending on the size of each inhabited island. Our findings suggested the suitability of Ion Torrent PGM™ for microsatellite marker development. The new markers developed for the O. narina complex will be applicable in conservation genetics and molecular ecological studies. Subject areas: Conservation genetics and biodiversity; Population structure and phylogeography
Keywords: amphibian, anuran, endangered species, island species, next generation sequencing, Ryukyu Islands
Next-generation sequencing technologies were rapidly developed in the recent past, and their applications are widespread in various fields (Metzker 2010). This is particularly remarkable for evolutionary studies of nonexperimental organisms. One of their promising applications is the rapid identification of molecular makers, such as microsatellites and singlenucleotide polymorphisms, on the basis of genome-wide sequence data. For the development of microsatellite markers, a vast amount of data, including microsatellite regions along with adjacent regions, are needed to design primer pairs in order to amplify the loci. In this regard, sequencing using the Roche 454 platform is very popular because of its longer nucleotide length of each single read (Perry and Rowe 2010; Yoshikawa et al. 2012). However, the total cost and time associated with using Roche 454 are not reasonable when considering the amount of data generated [$22/Mb for 454 GS Jr. Titanium™ and $12/Mb for 454 FLX Titanium™ in 2013, updated values of Glenn (2011)]. In more recent years, Life Technologies’ new platform Ion Torrent has become available; it can achieve faster and higher throughput at a lower cost [$0.6/Mb for Ion PGM with a 318 chip in 2013, update of Glenn (2011)]. Although Ion Torrent has the disadvantage of affording a shorter mean read length, the range of read length
1
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From the Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8526, Japan (Igawa, Komaki, and Sumida); the Center for Information Biology, National Institute of Genetics, Mishima, Shizuoka, Japan (Nozawa); the Department of Genetics, The Graduate University of Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan (Nozawa); the Department of Health Sciences, Hiroshima Prefectural University, Hiroshima, Japan (Nagaoka and Fujii); and the Section of Agriculture and Forest, Amami City Government, Amami, Kagoshima, Japan (Oumi). Takeshi Igawa is now at the Division of Development Science, Department of Developmental Science, Graduate School of International Development and Cooperation, Hiroshima University, Higashi-Hiroshima 739-8529, Japan.
Journal of Heredity
Materials and Methods Samples and Genomic DNA Extraction We collected tissues of individuals from each single population of the 4 species of O. narina complex by clipping toe tips: 16 individuals of O. narina from Aha in the Okinawa Isl, 16 of O. amamiensis from Santaro in the Amami Isl, 10 of O. supranarina from Mt. Banna in the Ishigaki Isl, and 16 of O. utsunomiyaorum from Mt. Omoto in the Ishigaki Isl. Total genomic DNA from these individuals was extracted using the NucleoSpin® Tissue (Macherey & Nagel, Düren, Germany). One of the genomic DNA samples of O. narina was used for sequencing on Ion Torrent PGM™ after RNase A treatment. Genomic DNA of H. tigerinus and B. japonica was also extracted from liver tissues in the same manner (Sultana et al., in submission and Komaki et al. 2014). 2
Library Preparation and Sequencing on Ion Torrent PGM™ We performed sequencing using Ion Torrent PGM™ multiplexing with 2 other frog species, H. tigerinus and B. japonica, to rapidly obtain a large number of genomic sequences at a lower cost. Each genomic DNA library was constructed using the NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torrent (New England Biolabs, Beverly, MA); they were then combined after attachment of specific adapter oligonucleotides to each genomic library using the Ion Xpress Barcode Adaptors 1–16 Kit (Life Technologies, Carlsbad, CA). This multiplexed library was refined using the Ion OneTouch Template OT2 200 Kit (Life Technologies) and was sequenced on the PGM™ using the Ion 318 chip and Ion PGM Sequencing 200 Kit ver2 (Life Technologies). Mining of Microsatellite Loci and Primer Design Microsatellite regions were mined among reads with more than 150 bp using the Msatcommander ver 1.0.8 (Faircloth 2008). We did not use reads that were shorter than 150 bp to simplify the subsequent processes. The criterion used to detect the microsatellite region was set for di-, tri-, tetra-, penta-, and hexanucleotide motifs with a minimum of 8, 8, 6, 6, and 6 repeats, respectively, and primer pairs for the amplification of microsatellites were designed using Primer 3 ver. 2.2.3 (Rozen and Skaletsky 2000). PCR Amplification and Genotyping We selected a total of 85 microsatellite loci (45 of di-, 30 of tri-, and 10 of tetranucleotide motifs) in order from those with a larger number of repeats among each repeat motif. A bar-coded slit tag (BStag) was added at the 5′ end of the forward primer of each locus to enable the use of a postlabeling method for multiplexed and multicolored genotyping (Shimizu and Yano 2011). First, we tested the PCR amplification and polymorphism of all of the loci individually using the EmeraldAmp® MAX PCR Master Mix (Takara Bio, Otsu, Japan). The amplifications were carried out in a volume of 10 μL containing 5 μL of EmeraldAmp® MAX PCR Master Mix, 1 μL of 10 μM primer pairs, and 50 ng of genomic DNA. Thermal cycling was performed under the following conditions: 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s, followed by 8 cycles of 95 °C for 30 s and 49 °C for 30 s, and a final extension at 72 °C for 7 min. We labeled BStag primers with the following fluorescent dyes: F9GACFAM (5′-CTAGTATCAGGACGAC-3′), F9GTC-HEX (5′-CTAGTATGAGGACGTC-3′), F9TAC-NED (5′-CTA GTATCAGGACTAC-3′), F9GCC-PET (5′-CTAGTATTAG GACGCC-3′), F9CCG-FAM (5′-CTAGTATTAGGACC CG-3′), and F9AGG-HEX (5′-CTAGTATTAGGACAGG-3′). Amplified fragments were electrophoresed on 3130xl (Life Technologies) together with GeneScan™ LIZ 500® (Life Technologies) as an internal size standard and were genotyped using the GeneMapper® 4.0 (Life Technologies). Although using HEX label is deviated from the manufacturer’s standard
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is widely distributed, and a sufficient number of longer reads may be achieved by generating a larger amount of total data. In fact, Luo et al. (2012) reported the successful development of microsatellite markers for a fish species using Ion PGM™. Therefore, to test the applicability of Ion Torrent sequencing for the development of microsatellite markers in a more cost-effective manner, we used the Ion Torrent PGM™ sequencer to develop microsatellite makers for the Ryukyu tip-nosed frog Odorrana narina complex by multiplex sequencing using 2 other frog species, Hoplobatrachus tigerinus and Buergeria japonica. Odorrana narina and its related species, Odorrana amamiensis, Odorrana supranarina, and Odorrana utsunomiyaorum are moderate-sized frogs that belong to the family Ranidae and are endemically distributed in the Ryukyu Archipelago of Japan. Two of these species, O. narina and O. amamiensis, are allopatrically distributed on Okinawa Island (Isl) and Amami and Toku Isls, respectively. In contrast, O. supranarina and O. utsunomiyaorum are sympatrically distributed on Ishigaki and Iriomote Isls, respectively. Because their speciation and formation of present distribution pattern are affected by environmental factors and geographic formation of the Ryukyu Archipelago (Ota 1998, 2012; Matsui et al. 2005), O. narina complex is a promising model for molecular ecological studies, involving speciation and adaptation via interactions with the environment. In addition, because of their small original distribution range and recent habitat loss by deforestation, all species are declining in number and have been listed as class B1 endangered species in the IUCN Red List of Threatened Species (Kaneko and Matsui 2004). Therefore, highly polymorphic microsatellite markers that can be used to identify population structure and genetic differences between these populations are needed for evolutionary studies and conservation genetics. In this study, we report similar skewed frequency of repeat motifs in the 3 modern frog species (which belong to the suborder Neobatrachia) and the successful development of 20 polymorphic markers for O. narina and their crossamplification in related species. Our study described one of the most cost-effective methods for the development of new microsatellite markers using Ion Torrent PGM™.
Igawa et al. • Microsatellite Marker for O. narina Complex Using Ion PGM
protocol of dye set G5 (FAM, VIC, NED, PET, and LIZ), we confirmed that HEX is consistently identified as VIC fluorescence in the resultant data with stability of peak sizing. We used 8 O. narina individuals for the validation of the polymorphism of the loci. For 20 developed primer pairs, 5 multiplexed PCR amplifications were performed using the KOD FX Neo (TOYOBO, Osaka, Japan) (Table 4). The amplifications were carried out in a volume of 10 μL containing 4.8 μL of 2× PCR Buffer for KOD FX Neo, 1.6 μL of 2 mM dNTPs, 2 pmole of the reverse primer, 0.5 pmole of both the tagged forward and the fluorescently labeled BStag primer for each target locus, 50 ng of genomic DNA, and 0.2 μL of KOD FX Neo. Thermal cycling was performed by changing the elongation temperature of the program mentioned above to 68 °C. Genotypic Analyses
Results and Discussion Sequence Data From Ion Torrent PGM™ We obtained a total of 482 328 312 bp and 3 519 683 reads in a single sequencing run on Ion Torrent PGM™ using a 318 chip (Table 1). To obtain a distribution frequency of read length, the single peaks at approximately 150 bp were observed in every species. In a total of 1 524 265 reads with more than 150 bp, 6820 reads contained more than one simple sequence repeat (SSR) (0.32–0.57% in each species). The traditional method for the development of SSR markers requires a long effort regarding the construction of SSR-enriched libraries or the screening of SSR loci by Southern hybridization. The use of Ion PGM™ with the 318 chip resolved this issue through the generation of a vast amount of data, even in a single multiplexed run of 3 species. In addition, the total cost and time involved in a shotgun sequencing of the 3 genomes were approximately $1200
Frequencies of Repeat Types and Motifs in 3 Frog Species The frequency of different repeat types (di-, tri-, tetra-, penta-, and hexanucleotide repeats) of microsatellites was basically similar among the 3 species and revealed a predominance of dinucleotide SSRs in all species (Table 2). However, the frequencies of the other repeat types were different between the species. The frequencies decreased with the increase in repeat unit length in O. narina. On the other hand, the frequency of tetranucleotide repeats was higher than that of trinucleotide repeats in both H. tigerinus and B. japonica and that of hexanucleotide repeats was higher than that of pentanucleotide repeats in B. japonica (Table 2). Although the 3 species belong to same suborder (Neobatrachia), the distribution patterns of repeat unit length were very different, which suggests that the accumulation ratio of each repeat type has changed randomly in each lineage, at least at the intergenus level. In contrast, the distribution pattern of repeat motifs for each repeat type (di-, tri-, and tetranucleotides) was almost identical among the 3 species, except the higher frequencies of AG/CT in B. japonica (Figure 1). In all species, AT, AAT/ ATT, and AGAT/ATCT repeats were most frequent in di-, tri-, and tetranucleotide repeats, respectively. A similar pattern has been reported in X. tropicalis; 64.72% of AT, 68.35% of AAT, and 74.50% of AGAT/ATCT (Xu et al. 2008) but not in the Japanese giant salamander (Andrias japonicus) (Ito et al. 2013). In contrast to the distribution pattern of repeat types, this concordant distribution pattern of motifs in each repeat type implies that the accumulation rates of repeat Table 2 Number and frequency of different repeat type microsatellites identified in Ion PGM™ reads more than 150 bp in 3 frog species Repeat type
O. narina
H. tigrinus
B. japonica
Dinucleotide (%) Trinucleotide (%) Tetranucleotide (%) Pentanucleotide (%) Hexanucleotide (%) Total
1257 (72.37) 252 (14.51) 214 (12.32) 9 (0.52) 5 (0.29) 1737
1344 (52.50) 463 (18.09) 589 (23.01) 161 (6.29) 3 (0.12) 2560
1543 (61.16) 180 (7.13) 775 (30.72) 10 (0.40) 15 (0.59) 2523
Table 1 Summary of Ion PGM™ sequencing of 3 frog species
Total no. of bases (Mbp) Total no. of reads Mean length of reads No. of reads (>150 bp) No. of reads (>150 bp) including SSR (%)
O. narina
H. tigrinus
B. japonica
Total
172.1 1 251 706 138 547 305 1736 (0.32)
169.0 1 217 280 139 537 649 2559 (0.48)
141.2 1 050 697 134 439 311 2522 (0.57)
482.3 3 519 683 137 1 524 265 6817 (0.45)
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First, we checked the existence of null alleles and genotyping errors in the resultant genotypic data using the Microchecker (Van Oosterhout et al. 2004). The number of alleles, observed and expected heterozygosity (HO and HE), and fixation index were calculated using the GenAlEx 6.5b3 (Peakall and Smouse 2006). Tests of deficiency in Hardy–Weinberg equilibrium (HWE) and of linkage disequilibrium (LD) were performed using GENEPOP 4.2 (Rousset 2008). Pairwise FST and Nei’s DA distance (Nei et al. 1983) were estimated among populations using POPTREE2 (Takezaki et al. 2010).
and 6 h, which lowers the cost and duration of the procedure drastically. Furthermore, while we used a method of 200-bp sequencing in this study, 400-bp sequencing has recently become available for Ion Torrent PGM, which will enable to develop microsatellite markers even more efficiently.
Journal of Heredity 0.9 Frequencies of repeat mofs in each repeat type
O. narina 0.8
H. tigerinus
0.7
B. japonica
0.6
X. tropicalis (Xu et al. 2008)
0.5 0.4 0.3 0.2 0.1
Dinucleotide
/G TT /C TT A T A /A C C TT A /G G C T T A /A G G T C A /G G G CT A /C TC C T C /G C G AT /C G G A A A C A / G A A G TTT A A / CT A TT A T/ A C ATT C T A / G A C G GTT A / C A G C TT T A A / AG G TT C A A /G G G CTT A / A C G C TT T A A / AC TC TT A A / GA TG TT A C /C A A G T /C T TG T A A C A A A T / TT C A C C TG T A C / GG C T G A T / A C G G G C T A C /G C G G T T A C / AC TC G T A C / GA TG G A /C T G A AG T T A G /A TC C T T A G /A G G C C T A / G G GC G C T A / TC C C CC T A TC / GG G A /C T G A T G
A
A
A
C G
C A
A
G A
A
C
/G T /C T A T
0
Trinucleotide
Tetranucleotide
motifs was skewed in a common ancestor of anurans and is maintained in modern species. Screening of Polymorphic Loci in O. narina Among a total of 1737 SSR-containing reads, nonredundant primer pairs were designed for 185 reads (10.66%) (Table 3). Of these, a total of 85 primer sets was selected in order of decreasing repeat number in each repeat type and validated using PCR amplification and polymorphism. As a result, 20 primer pairs (i.e., loci) were stably amplified and exhibited polymorphism (23.53% of the loci tested). Among the different repeat types, tetranucleotide repeats had a slightly higher ratio of polymorphic loci (30.00 %, but not significant difference, P = 0.778, Fisher’s exact test) (Table 3). This was seemingly in agreement with the widely cited arguments that tetranucleotide loci have higher mutation rates (Weber and Wong 1993). However, we did not observe a larger number of alleles in tetranucleotide repeats compared with the other repeat types, as mentioned in the following section. Characteristics of the Microsatellite Loci Using the 20 polymorphic loci, we genotyped an O. narina population and tested their cross-amplification in populations of related species (O. amamiensis, O. supranarina, and (α = 0.0000954) were observed. In the O. narina population, the observed allelic diversity (NA) ranged from 3 to 16 alleles, and the HO and HE ranged from 0.200 to 0.938 and from 0.317 to 0.922, respectively (Table 4). The average number of alleles was 8.5, 7.6, and 4.6 in di-, tri-, and tetranucleotide repeats, respectively. Assuming a population without any gene flow and neutral loci without any linkage with genes under selection, the allelic diversity observed at a locus should reflect the mutation rate of the locus. Under this assumption, our results negate the general assumption of higher relative mutation rates in tetranucleotides and agree with the findings of a statistical study, which estimated that dinucleotides have mutation rates that are 1.5–2.0 times
4
Table 3 Summary of efficiency of primer development for microsatellite loci in O. narina Repeat type
No. of designed primer pair/ no. of SSR reads (>150 bp)
No. of polymorphic locus (= primer pair)/no. of tested primer pair
Dinucleotide (%) Trinucleotide (%) Tetranucleotide (%) Pentanucleotide (%) Hexanucleotide (%) Total (%)
110 / 1,257(8.75) 64/252 (25.40) 10/214(4.67) 1/9 (11.11) 0/5(0.00) 185/1736 (10.66)
11/45 (24.44) 6/30 (20.00) 3/10 (30.00) 0/0 0/0 20/85 (23.53)
higher than those of tetranucleotides and trinucleotides have mutation rate intermediate between dinucleotides and tetranucleotides (Chakraborty et al. 1997). In addition, we also assessed a positive correlation between the number of repeats and mutation rates in a similar manner, which was mentioned in a previous study (Schug et al. 1998); however, no obvious correlation was observed in our data. In cross-amplification tests, we verified the stable amplification of these newly developed loci in related species. As a result, 17, 16, and 13 loci were stably amplified and genotyped in O. amamiensis, O. supranarina, and O. utsunomiyaorum, respectively. The number of effective loci in each species appeared to correspond to the phylogenetic positions (Nishioka et al. 1987; Matsui et al. 2005) and divergence times of these 3 species against O. narina (1.7 Ma for O. amamiensis vs. O. narina, 3.5 Ma for R. supranarina vs. O. narina + O. amamiensis, and 6.6 Ma for O. utsunomiyaorum and other species; Matsui et al. 2005). This suggests that the success rate of cross-amplification simply depends on the nucleotide identity of priming regions that are disrupted by the accumulation of mutations after divergence. All genetic values of O. amamiensis (mean NA, HO, and HE of 7.5, 0.595, and 0.658, respectively) were similar to those of O. narina (mean NA, HO, and HE of 7.7, 0.679, and 0.709, respectively). In contrast, considerably smaller values
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Figure 1. Frequencies of repeat motifs in each repeat length in 3 frog species.
(AAT)12
AB900719
Onari104 AB900720 200–220
100–130
200–240
Size ranges (bp)
100–160
180–200
150–210
100–140
120–150
120–150
110–170
120–150
90–170
100–143
120–180
HEX
FAM
100–170
150–200
NED 100–150
FAM
NED 140–190
PET
HEX
HEX
HEX
PET
PET
HEX
NED 130–170
FAM
FAM
PET
NED 120–160
FAM
FAM
FAM
Dye
I
II
IV
V
II
II
III
IV
II
V
III
V
III
IV
IV
I
I
I
I
V
0.375
0.688
0.813
0.750
0.563
0.625
0.688
0.875
0.750
0.563
0.938
0.563
0.875
0.500
0.563
0.813
0.688
0.875
0.875
0.200
HO
7.7 0.679
4
4
10
12
7
7
4
13
5
6
15
4
9
7
6
16
5
6
11
3
NA
0.709
0.537
0.643
0.834
0.881
0.639
0.791
0.576
0.889
0.656
0.711
0.908
0.494
0.783
0.572
0.652
0.922
0.719
0.727
0.883
0.371
HE
Multiplex O. narina (N = 16)
0.688
0.462
0.563
0.875
—
0.625
0.250
0.875
0.750
0.538
1.000
0.500
0.563
0.063
0.438
0.875
0.375
0.688
—
—
HO
7.5 0.596
10
8
5
9
—
10
2
9
7
3
15
7
6
2
4
14
8
8
—
—
NA
O. amamiensis (N = 16)
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NA, number of alleles; HO, observed heterozygosity (in bold numbers, if values are significantly deviated from HWE); HE, expected heterozygosity.
Mean
Onari1711 AB900738 (AGAT)6
Onari1587 AB900737
(AT)11
Onari1567 AB900736 (ACT)9
Onari1533 AB900735
(AAT)9
Onari1458 AB900734 (AC)17
Onari1309 AB900733
(AT)10
Onari1112 AB900732 (AG)11
Onari1040 AB900731
(AG)9
Onari997 AB900730 (AAAT)6
Onari850 AB900729
(AC)10
Onari795 AB900728 (AG)14
Onari659 AB900727
(AAT)17
Onari538 AB900726 (AC)10
Onari504 AB900725
(AC)9
Onari429 AB900724 (AGG)9
Onari397 AB900723 (AC)10
Onari324 AB900722
(AAAC)6
Onari319 AB900721 (AC)12
F: F9CCG - AGGGCCCTGCTTAATTTGC R: AGCAGGGCCTCTTATTCCTC F: F9GAC - GATCCGGAGCCATAGAGACC R: ACACATCTACTCCCATAGGCAG F: F9CCG - ACCATTTCCTGTAGCATGTCC R: TCTTGCTTGTGTGATTGGCTC F: F9TAC - TGCACACTGATGCAAACTTTG R: CCTGCACACGTCTTAGATGC F: F9GCC - TTTGCGCAGAGTTCAAGAGG R: CAAGCTCCTCCATATCACCATC F: F9GAC - TGTGTGATCAGTGTCCTCGG R: TGATGGAGAGTGATGGTGCC F: F9GAC - ACTCCAAAGACATGCTGG R: GTTCAGGCTTGGACGTTGTG F: F9TAC - ATGCTTACCTGCCTGTTCTC R: TGAGTTGGGCTGTGTGAGG F: F9GTC - TACAAGAGCTGGGTGTCCTC R: CTGTGCACACTGTTGGCG F: F9GCC - CCGTGGGATGGGAAACTGG R: TAAGTGTGAGGAAGCCAGGC F: F9GCC - TGTCCTACATCAGTGCCTAGTG R: ATGCCTCACTCCCTCTGTTG F: F9GTC - ACTTGCTTTATAGTCCACAGGC R: AAAGTTCCAACAGCCAAGGG F: F9GTC - ACAAGCACTTACAATGCTCTG R: CTTGACTGCATCTGAGACATCC F: F9GTC - AGGGAGAAAGCCAACAGTG R: CAGGTCCCACTCTGCAAATC F: F9GCC - GGAATCGTTGTGGTGTGC R: AACCTCCGATAAACCGCTTC F: F9TAC - AGGCAGCACAAAGGGAATAC R: ACTCAATGCGTATGTTCTGGC F: F9CCG - CGACCTGCCGCAGTAAATAC R: GCGCTGTGTAAATTGATGGC F: F9TAC - AGGTCCAATGGTCAGGTGTC R: GCCTGTCACTTCCAACTCCC F: F9GAC - GTAACACACACTCGCTGCC R: AGACCTGTAACACGAGCCAC F: F9AGG - CACGTAGATGGACCATGCAC R: TCCTATCTCAACATGTGGTCAG
(AAT)8
Onari85
Primer sequence (5'-3')
Repeat motif
Accession no.
Loci
Table 4 Characterization of 20 microsatellite loci isolated from O. narina
0.659
0.791
0.760
0.604
0.836
—
0.795
0.219
0.826
0.695
0.500
0.906
0.694
0.711
0.061
0.568
0.906
0.609
0.713
—
—
HE
2.9
4
4
2
7
1
—
2
5
6
—
5
8
2
1
1
—
2
3
4
—
NA
0.338
0.500
0.700
0.700
0.889
0.000
—
0.300
0.700
0.300
—
0.700
0.600
0.100
0.000
0.000
—
0.600
0.300
0.375
—
HO
O. supranarina (N = 10)
0.369
0.715
0.570
0.495
0.796
0.000
—
0.375
0.680
0.730
—
0.715
0.850
0.095
0.000
0.000
—
0.420
0.405
0.539
—
HE
2.2
1
2
1
1
3
—
3
4
—
—
8
8
—
1
1
—
3
—
8
—
NA
0.225
0.000
0.438
0.000
0.000
0.625
—
0.200
0.875
—
—
0.692
0.667
—
0.000
0.000
—
0.313
—
0.700
—
HO
0.218
0.000
0.342
0.000
0.000
0.549
—
0.184
0.736
—
—
0.784
0.711
—
0.000
0.000
—
0.271
—
0.780
—
HE
O. utsunomiyaorum (N = 16)
Igawa et al. • Microsatellite Marker for O. narina Complex Using Ion PGM
5
Journal of Heredity
were obtained for O. supranarina (mean NA, HO, and HE of 2.9, 0.338, and 0.369, respectively) and O. utsunomiyaorum (mean NA, HO, and HE of 2.2, 0.225, and 0.218, respectively), with fixed alleles at some loci. Although additional samples and populations are required to reach definitive conclusions, the differences observed between the 4 species should reflect population sizes that were largely affected by the size of the inhabited islands. A pairwise FST and DA distance matrix among populations of the 4 species are shown in Supplementary Table S1. These distance values were substantially high between every combination of populations, ranging from 0.274 to 0.457, with a mean of 0.375, and from 0.618 to 0.881, with a mean of 0.743, respectively. However, the values obtained between O. narina and O. amamiensis were 0.274 for FST and 0.784 for DA, which were slightly lower compared with those of parapatrically dwelling Ishikawa’s frogs, O. ishikawae and O. splendida, which are also distributed endemically in Okinawa and Amami Isls, respectively (mean FST, 0.517 and mean DA, 0.934, based on 12 SSR markers; Igawa et al. 2011, 2013). In conclusion, we sequenced 3 frog species in a single multiplexed run of Ion PGM™ and found skewed distribution pattern of repeat motifs. Based on these data, we developed 20 polymorphic loci for the Ryukyu tip-nosed frog, O. narina. More than half of these were stably amplified and genotyped in three-related species. Multiplex sequencing on Ion PGM™ is rapid and one of the most cost-effective methods to develop microsatellite markers in nonexperimental animals. The polymorphism of the loci observed for each repeat type suggests that the higher mutation rates of dinucleotides compared with tetranucleotides is congruent with previous statistical studies. The genotypic data obtained for 4 species were considered to reflect the distribution area of the islands of origin. Therefore, these microsatellite markers have appropriate properties for use in future conservation and population genetic studies of this species group across varying scales, from the interpopulation to individual level.
Kaneko Y, Matsui M. 2004. Odorrana narina. In: IUCN Red List of Threatened Species. Version 2013.2. IUCN 2013.
Supplementary Material
Ota H. 1998. Geographic patterns of endemism and speciation in amphibians and reptiles of the Ryukyu Archipelago, Japan, with special reference to their paleogeographical implications. Res Popul Ecol. 40:189–204.
Funding Scientific Research (B) (24310173 to M.S.); Young Scientists (B) (23710282 to TI from the Ministry of Education, Culture, Sports, Science and Technology, Japan).
Acknowledgments We are grateful to the Boards of Education of Kagoshima Prefecture for allowing us to collect, for the purposes of this study, O. amamiensis samples that are protected by law. We also thank Chie Iwamoto, Nana Fukuda, Mai Fujimi, and Kanako Onizuka from the National Institute of Genetics for their help in the experiments.
6
Chakraborty R, Kimmel M, Stivers DN, Davison LJ, Deka R. 1997. Relative mutation rates at di-, tri-, and tetranucleotide microsatellite loci. Proc Natl Acad Sci U S A. 94:1041–1046. Faircloth BC. 2008. msatcommander: detection of microsatellite repeat arrays and automated, locus-specific primer design. Mol Ecol Resour. 8:92–94. Glenn TC. 2011. Field guide to next-generation DNA sequencers. Mol Ecol Resour. 11:759–769. Igawa T, Okuda M, Oumi S, Katsuren S, Kurabayashi A, Umino T, Sumida M. 2011. Isolation and characterization of twelve microsatellite loci of endangered Ishikawa’s frog (Odorrana ishikawae). Conserv Genet Resour. 3:421–424. Igawa T, Oumi S, Katsuren S, Sumida M. 2013. Population structure and landscape genetics of two endangered frog species of genus Odorrana: different scenarios on two islands. Heredity (Edinb). 110:46–56. Ito H, Adenyo C, Yoshikawa N, Murai M. 2013. Large scale development of microsatellite markers for wildlife and its application to conservation. Biol Sci. 64:159–167. (in Japanese)
Komaki S, Igawa T, Nozawa M, Lin SM, Oumi S, Sumida M. 2014. Development and characterization of 14 microsatellite markers for Buergeria japonica (Amphibia, Anura, Rhacophoridae). Genes Genet Syst. 89:35–39. Luo W, Nie Z, Zhan F, Wei J, Wang W, Gao Z. 2012. Rapid development of microsatellite markers for the endangered fish Schizothorax biddulphi (Günther) using next generation sequencing and cross-species amplification. Int J Mol Sci. 13:14946–14955. Matsui M, Shimada T, Ota H, Tanaka-Ueno T. 2005. Multiple invasions of the Ryukyu Archipelago by Oriental frogs of the subgenus Odorrana with phylogenetic reassessment of the related subgenera of the genus Rana. Mol Phylogenet Evol. 37:733–742. Metzker ML. 2010. Sequencing technologies - the next generation. Nat Rev Genet. 11:31–46. Nei M, Tajima F, Tateno Y. 1983. Accuracy of estimated phylogenetic trees from molecular data. II. Gene frequency data. J Mol Evol. 19:153–170. Nishioka M, Ueda H, Sumida M. 1987. Intraspecific differenciation of Rana narina elucidated by crossing experiments and electrophoretic analyses of enzymes and blood proteins. Sci Rep Lab Amphib Biol Hiroshima Univ. 9:261–303.
Ota H. 2012. Phylogeography of terrestrial biota and geohistory of the Ryukyu Archipelago, Japan -Hypothetical scenarios hitherto proposed and directions of future studies. Earth Mon. 34:427–436. (in Japanese) Peakall R, Smouse PE. 2006. Genalex 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes. 6:288–295. Perry JC, Rowe L. 2011. Rapid microsatellite development for water striders by next-generation sequencing. J Hered. 102:125–129. Rousset F. 2008. genepop’007: a complete re-implementation of the genepop software for Windows and Linux. Mol Ecol Resour. 8:103–106. Rozen S, Skaletsky H. 2000. Primer3 on the WWW for General Users and Biologist Programmers. In: Krawetz S, Missner S, editors. Bioinformatics Methods and Protocols; Methods in Molecular Biology Totowa (NJ): Humana Press. pp. 365–386. Schug MD, Hutter CM, Wetterstrand KA, Gaudette MS, Mackay TF, Aquadro CF. 1998. The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster. Mol Biol Evol. 15:1751–1760.
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Supplementary material can be found at http://www.jhered. oxfordjournals.org/.
References
Igawa et al. • Microsatellite Marker for O. narina Complex Using Ion PGM Shimizu T, Yano K. 2011. A post-labeling method for multiplexed and multicolored genotyping analysis of SSR, indel and SNP markers in single tube with bar-coded split tag (BStag). BMC Res Notes. 4:161.
Xu Z, Gutierrez L, Hitchens M, Scherer S, Sater AK, Wells DE. 2008. Distribution of polymorphic and non-polymorphic microsatellite repeats in Xenopus tropicalis. Bioinform Biol Insights. 2:157–169.
Takezaki N, Nei M, Tamura K. 2010. POPTREE2: Software for constructing population trees from allele frequency data and computing other population statistics with Windows interface. Mol Biol Evol. 27:747–752.
Yoshikawa N, Matsui M, Hayano A, Inoue-Murayama M. 2012. Development of microsatellite markers for the Japanese giant salamander (Andrias japonicus) through next-generation sequencing, and cross-amplification in its congener. Conserv Genet Resour. 4:971–974.
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.
Received March 17, 2014; First decision July 22, 2014; Accepted October 3, 2014
Weber JL, Wong C. 1993. Mutation of human short tandem repeats. Hum Mol Genet. 2:1123–1128.
Corresponding Editor: Andrew Crawford
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© The American Genetic Association 2014. All rights reserved. For permissions, please e-mail: [email protected]
Microsatellite Marker Development by Multiplex Ion Torrent PGM Sequencing: a Case Study of the Endangered Odorrana narina Complex of Frogs Takeshi Igawa, Masafumi Nozawa, Mai Nagaoka, Shohei Komaki, Shohei Oumi, Tamotsu Fujii, and Masayuki Sumida
Address correspondence to Dr. Takeshi Igawa at the address above, or e-mail: [email protected]. Data deposited at Dryad: doi:http://dx.doi.org/doi:10.5061/dryad.9418j
The endangered Ryukyu tip-nosed frog Odorrana narina and its related species, Odorrana amamiensis, Odorrana supranarina, and Odorrana utsunomiyaorum, belong to the family Ranidae and are endemically distributed in Okinawa (O. narina), Amami and Tokunoshima (O. amamiensis), and Ishigaki and Iriomote (O. supranarina and O. utsunomiyaorum) Islands. Because of varying distribution patterns, this species complex is an intrinsic model for speciation and adaptation. For effective conservation and molecular ecological studies, further genetic information is needed. For rapid, cost-effective development of several microsatellite markers for these and 2 other species, we used nextgeneration sequencing technology of Ion Torrent PGM™. Distribution patterns of repeat motifs of microsatellite loci in these modern frog species (Neobatrachia) were similarly skewed. We isolated and characterized 20 new microsatellite loci of O. narina and validated cross-amplification in the threerelated species. Seventeen, 16, and 13 loci were cross-amplified in O. amamiensis, O. supranarina, and O. utsunomiyaorum, respectively, reflecting close genetic relationships between them. Mean number of alleles and expected heterozygosity of newly isolated loci varied depending on the size of each inhabited island. Our findings suggested the suitability of Ion Torrent PGM™ for microsatellite marker development. The new markers developed for the O. narina complex will be applicable in conservation genetics and molecular ecological studies. Subject areas: Conservation genetics and biodiversity; Population structure and phylogeography
Keywords: amphibian, anuran, endangered species, island species, next generation sequencing, Ryukyu Islands
Next-generation sequencing technologies were rapidly developed in the recent past, and their applications are widespread in various fields (Metzker 2010). This is particularly remarkable for evolutionary studies of nonexperimental organisms. One of their promising applications is the rapid identification of molecular makers, such as microsatellites and singlenucleotide polymorphisms, on the basis of genome-wide sequence data. For the development of microsatellite markers, a vast amount of data, including microsatellite regions along with adjacent regions, are needed to design primer pairs in order to amplify the loci. In this regard, sequencing using the Roche 454 platform is very popular because of its longer nucleotide length of each single read (Perry and Rowe 2010; Yoshikawa et al. 2012). However, the total cost and time associated with using Roche 454 are not reasonable when considering the amount of data generated [$22/Mb for 454 GS Jr. Titanium™ and $12/Mb for 454 FLX Titanium™ in 2013, updated values of Glenn (2011)]. In more recent years, Life Technologies’ new platform Ion Torrent has become available; it can achieve faster and higher throughput at a lower cost [$0.6/Mb for Ion PGM with a 318 chip in 2013, update of Glenn (2011)]. Although Ion Torrent has the disadvantage of affording a shorter mean read length, the range of read length
1
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From the Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739-8526, Japan (Igawa, Komaki, and Sumida); the Center for Information Biology, National Institute of Genetics, Mishima, Shizuoka, Japan (Nozawa); the Department of Genetics, The Graduate University of Advanced Studies (SOKENDAI), Mishima, Shizuoka, Japan (Nozawa); the Department of Health Sciences, Hiroshima Prefectural University, Hiroshima, Japan (Nagaoka and Fujii); and the Section of Agriculture and Forest, Amami City Government, Amami, Kagoshima, Japan (Oumi). Takeshi Igawa is now at the Division of Development Science, Department of Developmental Science, Graduate School of International Development and Cooperation, Hiroshima University, Higashi-Hiroshima 739-8529, Japan.
Journal of Heredity
Materials and Methods Samples and Genomic DNA Extraction We collected tissues of individuals from each single population of the 4 species of O. narina complex by clipping toe tips: 16 individuals of O. narina from Aha in the Okinawa Isl, 16 of O. amamiensis from Santaro in the Amami Isl, 10 of O. supranarina from Mt. Banna in the Ishigaki Isl, and 16 of O. utsunomiyaorum from Mt. Omoto in the Ishigaki Isl. Total genomic DNA from these individuals was extracted using the NucleoSpin® Tissue (Macherey & Nagel, Düren, Germany). One of the genomic DNA samples of O. narina was used for sequencing on Ion Torrent PGM™ after RNase A treatment. Genomic DNA of H. tigerinus and B. japonica was also extracted from liver tissues in the same manner (Sultana et al., in submission and Komaki et al. 2014). 2
Library Preparation and Sequencing on Ion Torrent PGM™ We performed sequencing using Ion Torrent PGM™ multiplexing with 2 other frog species, H. tigerinus and B. japonica, to rapidly obtain a large number of genomic sequences at a lower cost. Each genomic DNA library was constructed using the NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torrent (New England Biolabs, Beverly, MA); they were then combined after attachment of specific adapter oligonucleotides to each genomic library using the Ion Xpress Barcode Adaptors 1–16 Kit (Life Technologies, Carlsbad, CA). This multiplexed library was refined using the Ion OneTouch Template OT2 200 Kit (Life Technologies) and was sequenced on the PGM™ using the Ion 318 chip and Ion PGM Sequencing 200 Kit ver2 (Life Technologies). Mining of Microsatellite Loci and Primer Design Microsatellite regions were mined among reads with more than 150 bp using the Msatcommander ver 1.0.8 (Faircloth 2008). We did not use reads that were shorter than 150 bp to simplify the subsequent processes. The criterion used to detect the microsatellite region was set for di-, tri-, tetra-, penta-, and hexanucleotide motifs with a minimum of 8, 8, 6, 6, and 6 repeats, respectively, and primer pairs for the amplification of microsatellites were designed using Primer 3 ver. 2.2.3 (Rozen and Skaletsky 2000). PCR Amplification and Genotyping We selected a total of 85 microsatellite loci (45 of di-, 30 of tri-, and 10 of tetranucleotide motifs) in order from those with a larger number of repeats among each repeat motif. A bar-coded slit tag (BStag) was added at the 5′ end of the forward primer of each locus to enable the use of a postlabeling method for multiplexed and multicolored genotyping (Shimizu and Yano 2011). First, we tested the PCR amplification and polymorphism of all of the loci individually using the EmeraldAmp® MAX PCR Master Mix (Takara Bio, Otsu, Japan). The amplifications were carried out in a volume of 10 μL containing 5 μL of EmeraldAmp® MAX PCR Master Mix, 1 μL of 10 μM primer pairs, and 50 ng of genomic DNA. Thermal cycling was performed under the following conditions: 94 °C for 3 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s, followed by 8 cycles of 95 °C for 30 s and 49 °C for 30 s, and a final extension at 72 °C for 7 min. We labeled BStag primers with the following fluorescent dyes: F9GACFAM (5′-CTAGTATCAGGACGAC-3′), F9GTC-HEX (5′-CTAGTATGAGGACGTC-3′), F9TAC-NED (5′-CTA GTATCAGGACTAC-3′), F9GCC-PET (5′-CTAGTATTAG GACGCC-3′), F9CCG-FAM (5′-CTAGTATTAGGACC CG-3′), and F9AGG-HEX (5′-CTAGTATTAGGACAGG-3′). Amplified fragments were electrophoresed on 3130xl (Life Technologies) together with GeneScan™ LIZ 500® (Life Technologies) as an internal size standard and were genotyped using the GeneMapper® 4.0 (Life Technologies). Although using HEX label is deviated from the manufacturer’s standard
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is widely distributed, and a sufficient number of longer reads may be achieved by generating a larger amount of total data. In fact, Luo et al. (2012) reported the successful development of microsatellite markers for a fish species using Ion PGM™. Therefore, to test the applicability of Ion Torrent sequencing for the development of microsatellite markers in a more cost-effective manner, we used the Ion Torrent PGM™ sequencer to develop microsatellite makers for the Ryukyu tip-nosed frog Odorrana narina complex by multiplex sequencing using 2 other frog species, Hoplobatrachus tigerinus and Buergeria japonica. Odorrana narina and its related species, Odorrana amamiensis, Odorrana supranarina, and Odorrana utsunomiyaorum are moderate-sized frogs that belong to the family Ranidae and are endemically distributed in the Ryukyu Archipelago of Japan. Two of these species, O. narina and O. amamiensis, are allopatrically distributed on Okinawa Island (Isl) and Amami and Toku Isls, respectively. In contrast, O. supranarina and O. utsunomiyaorum are sympatrically distributed on Ishigaki and Iriomote Isls, respectively. Because their speciation and formation of present distribution pattern are affected by environmental factors and geographic formation of the Ryukyu Archipelago (Ota 1998, 2012; Matsui et al. 2005), O. narina complex is a promising model for molecular ecological studies, involving speciation and adaptation via interactions with the environment. In addition, because of their small original distribution range and recent habitat loss by deforestation, all species are declining in number and have been listed as class B1 endangered species in the IUCN Red List of Threatened Species (Kaneko and Matsui 2004). Therefore, highly polymorphic microsatellite markers that can be used to identify population structure and genetic differences between these populations are needed for evolutionary studies and conservation genetics. In this study, we report similar skewed frequency of repeat motifs in the 3 modern frog species (which belong to the suborder Neobatrachia) and the successful development of 20 polymorphic markers for O. narina and their crossamplification in related species. Our study described one of the most cost-effective methods for the development of new microsatellite markers using Ion Torrent PGM™.
Igawa et al. • Microsatellite Marker for O. narina Complex Using Ion PGM
protocol of dye set G5 (FAM, VIC, NED, PET, and LIZ), we confirmed that HEX is consistently identified as VIC fluorescence in the resultant data with stability of peak sizing. We used 8 O. narina individuals for the validation of the polymorphism of the loci. For 20 developed primer pairs, 5 multiplexed PCR amplifications were performed using the KOD FX Neo (TOYOBO, Osaka, Japan) (Table 4). The amplifications were carried out in a volume of 10 μL containing 4.8 μL of 2× PCR Buffer for KOD FX Neo, 1.6 μL of 2 mM dNTPs, 2 pmole of the reverse primer, 0.5 pmole of both the tagged forward and the fluorescently labeled BStag primer for each target locus, 50 ng of genomic DNA, and 0.2 μL of KOD FX Neo. Thermal cycling was performed by changing the elongation temperature of the program mentioned above to 68 °C. Genotypic Analyses
Results and Discussion Sequence Data From Ion Torrent PGM™ We obtained a total of 482 328 312 bp and 3 519 683 reads in a single sequencing run on Ion Torrent PGM™ using a 318 chip (Table 1). To obtain a distribution frequency of read length, the single peaks at approximately 150 bp were observed in every species. In a total of 1 524 265 reads with more than 150 bp, 6820 reads contained more than one simple sequence repeat (SSR) (0.32–0.57% in each species). The traditional method for the development of SSR markers requires a long effort regarding the construction of SSR-enriched libraries or the screening of SSR loci by Southern hybridization. The use of Ion PGM™ with the 318 chip resolved this issue through the generation of a vast amount of data, even in a single multiplexed run of 3 species. In addition, the total cost and time involved in a shotgun sequencing of the 3 genomes were approximately $1200
Frequencies of Repeat Types and Motifs in 3 Frog Species The frequency of different repeat types (di-, tri-, tetra-, penta-, and hexanucleotide repeats) of microsatellites was basically similar among the 3 species and revealed a predominance of dinucleotide SSRs in all species (Table 2). However, the frequencies of the other repeat types were different between the species. The frequencies decreased with the increase in repeat unit length in O. narina. On the other hand, the frequency of tetranucleotide repeats was higher than that of trinucleotide repeats in both H. tigerinus and B. japonica and that of hexanucleotide repeats was higher than that of pentanucleotide repeats in B. japonica (Table 2). Although the 3 species belong to same suborder (Neobatrachia), the distribution patterns of repeat unit length were very different, which suggests that the accumulation ratio of each repeat type has changed randomly in each lineage, at least at the intergenus level. In contrast, the distribution pattern of repeat motifs for each repeat type (di-, tri-, and tetranucleotides) was almost identical among the 3 species, except the higher frequencies of AG/CT in B. japonica (Figure 1). In all species, AT, AAT/ ATT, and AGAT/ATCT repeats were most frequent in di-, tri-, and tetranucleotide repeats, respectively. A similar pattern has been reported in X. tropicalis; 64.72% of AT, 68.35% of AAT, and 74.50% of AGAT/ATCT (Xu et al. 2008) but not in the Japanese giant salamander (Andrias japonicus) (Ito et al. 2013). In contrast to the distribution pattern of repeat types, this concordant distribution pattern of motifs in each repeat type implies that the accumulation rates of repeat Table 2 Number and frequency of different repeat type microsatellites identified in Ion PGM™ reads more than 150 bp in 3 frog species Repeat type
O. narina
H. tigrinus
B. japonica
Dinucleotide (%) Trinucleotide (%) Tetranucleotide (%) Pentanucleotide (%) Hexanucleotide (%) Total
1257 (72.37) 252 (14.51) 214 (12.32) 9 (0.52) 5 (0.29) 1737
1344 (52.50) 463 (18.09) 589 (23.01) 161 (6.29) 3 (0.12) 2560
1543 (61.16) 180 (7.13) 775 (30.72) 10 (0.40) 15 (0.59) 2523
Table 1 Summary of Ion PGM™ sequencing of 3 frog species
Total no. of bases (Mbp) Total no. of reads Mean length of reads No. of reads (>150 bp) No. of reads (>150 bp) including SSR (%)
O. narina
H. tigrinus
B. japonica
Total
172.1 1 251 706 138 547 305 1736 (0.32)
169.0 1 217 280 139 537 649 2559 (0.48)
141.2 1 050 697 134 439 311 2522 (0.57)
482.3 3 519 683 137 1 524 265 6817 (0.45)
3
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First, we checked the existence of null alleles and genotyping errors in the resultant genotypic data using the Microchecker (Van Oosterhout et al. 2004). The number of alleles, observed and expected heterozygosity (HO and HE), and fixation index were calculated using the GenAlEx 6.5b3 (Peakall and Smouse 2006). Tests of deficiency in Hardy–Weinberg equilibrium (HWE) and of linkage disequilibrium (LD) were performed using GENEPOP 4.2 (Rousset 2008). Pairwise FST and Nei’s DA distance (Nei et al. 1983) were estimated among populations using POPTREE2 (Takezaki et al. 2010).
and 6 h, which lowers the cost and duration of the procedure drastically. Furthermore, while we used a method of 200-bp sequencing in this study, 400-bp sequencing has recently become available for Ion Torrent PGM, which will enable to develop microsatellite markers even more efficiently.
Journal of Heredity 0.9 Frequencies of repeat mofs in each repeat type
O. narina 0.8
H. tigerinus
0.7
B. japonica
0.6
X. tropicalis (Xu et al. 2008)
0.5 0.4 0.3 0.2 0.1
Dinucleotide
/G TT /C TT A T A /A C C TT A /G G C T T A /A G G T C A /G G G CT A /C TC C T C /G C G AT /C G G A A A C A / G A A G TTT A A / CT A TT A T/ A C ATT C T A / G A C G GTT A / C A G C TT T A A / AG G TT C A A /G G G CTT A / A C G C TT T A A / AC TC TT A A / GA TG TT A C /C A A G T /C T TG T A A C A A A T / TT C A C C TG T A C / GG C T G A T / A C G G G C T A C /G C G G T T A C / AC TC G T A C / GA TG G A /C T G A AG T T A G /A TC C T T A G /A G G C C T A / G G GC G C T A / TC C C CC T A TC / GG G A /C T G A T G
A
A
A
C G
C A
A
G A
A
C
/G T /C T A T
0
Trinucleotide
Tetranucleotide
motifs was skewed in a common ancestor of anurans and is maintained in modern species. Screening of Polymorphic Loci in O. narina Among a total of 1737 SSR-containing reads, nonredundant primer pairs were designed for 185 reads (10.66%) (Table 3). Of these, a total of 85 primer sets was selected in order of decreasing repeat number in each repeat type and validated using PCR amplification and polymorphism. As a result, 20 primer pairs (i.e., loci) were stably amplified and exhibited polymorphism (23.53% of the loci tested). Among the different repeat types, tetranucleotide repeats had a slightly higher ratio of polymorphic loci (30.00 %, but not significant difference, P = 0.778, Fisher’s exact test) (Table 3). This was seemingly in agreement with the widely cited arguments that tetranucleotide loci have higher mutation rates (Weber and Wong 1993). However, we did not observe a larger number of alleles in tetranucleotide repeats compared with the other repeat types, as mentioned in the following section. Characteristics of the Microsatellite Loci Using the 20 polymorphic loci, we genotyped an O. narina population and tested their cross-amplification in populations of related species (O. amamiensis, O. supranarina, and (α = 0.0000954) were observed. In the O. narina population, the observed allelic diversity (NA) ranged from 3 to 16 alleles, and the HO and HE ranged from 0.200 to 0.938 and from 0.317 to 0.922, respectively (Table 4). The average number of alleles was 8.5, 7.6, and 4.6 in di-, tri-, and tetranucleotide repeats, respectively. Assuming a population without any gene flow and neutral loci without any linkage with genes under selection, the allelic diversity observed at a locus should reflect the mutation rate of the locus. Under this assumption, our results negate the general assumption of higher relative mutation rates in tetranucleotides and agree with the findings of a statistical study, which estimated that dinucleotides have mutation rates that are 1.5–2.0 times
4
Table 3 Summary of efficiency of primer development for microsatellite loci in O. narina Repeat type
No. of designed primer pair/ no. of SSR reads (>150 bp)
No. of polymorphic locus (= primer pair)/no. of tested primer pair
Dinucleotide (%) Trinucleotide (%) Tetranucleotide (%) Pentanucleotide (%) Hexanucleotide (%) Total (%)
110 / 1,257(8.75) 64/252 (25.40) 10/214(4.67) 1/9 (11.11) 0/5(0.00) 185/1736 (10.66)
11/45 (24.44) 6/30 (20.00) 3/10 (30.00) 0/0 0/0 20/85 (23.53)
higher than those of tetranucleotides and trinucleotides have mutation rate intermediate between dinucleotides and tetranucleotides (Chakraborty et al. 1997). In addition, we also assessed a positive correlation between the number of repeats and mutation rates in a similar manner, which was mentioned in a previous study (Schug et al. 1998); however, no obvious correlation was observed in our data. In cross-amplification tests, we verified the stable amplification of these newly developed loci in related species. As a result, 17, 16, and 13 loci were stably amplified and genotyped in O. amamiensis, O. supranarina, and O. utsunomiyaorum, respectively. The number of effective loci in each species appeared to correspond to the phylogenetic positions (Nishioka et al. 1987; Matsui et al. 2005) and divergence times of these 3 species against O. narina (1.7 Ma for O. amamiensis vs. O. narina, 3.5 Ma for R. supranarina vs. O. narina + O. amamiensis, and 6.6 Ma for O. utsunomiyaorum and other species; Matsui et al. 2005). This suggests that the success rate of cross-amplification simply depends on the nucleotide identity of priming regions that are disrupted by the accumulation of mutations after divergence. All genetic values of O. amamiensis (mean NA, HO, and HE of 7.5, 0.595, and 0.658, respectively) were similar to those of O. narina (mean NA, HO, and HE of 7.7, 0.679, and 0.709, respectively). In contrast, considerably smaller values
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Figure 1. Frequencies of repeat motifs in each repeat length in 3 frog species.
(AAT)12
AB900719
Onari104 AB900720 200–220
100–130
200–240
Size ranges (bp)
100–160
180–200
150–210
100–140
120–150
120–150
110–170
120–150
90–170
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HEX
FAM
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NED 100–150
FAM
NED 140–190
PET
HEX
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PET
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HEX
NED 130–170
FAM
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PET
NED 120–160
FAM
FAM
FAM
Dye
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II
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V
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I
I
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V
0.375
0.688
0.813
0.750
0.563
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0.688
0.875
0.750
0.563
0.938
0.563
0.875
0.500
0.563
0.813
0.688
0.875
0.875
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HO
7.7 0.679
4
4
10
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7
7
4
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5
6
15
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9
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6
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6
11
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NA
0.709
0.537
0.643
0.834
0.881
0.639
0.791
0.576
0.889
0.656
0.711
0.908
0.494
0.783
0.572
0.652
0.922
0.719
0.727
0.883
0.371
HE
Multiplex O. narina (N = 16)
0.688
0.462
0.563
0.875
—
0.625
0.250
0.875
0.750
0.538
1.000
0.500
0.563
0.063
0.438
0.875
0.375
0.688
—
—
HO
7.5 0.596
10
8
5
9
—
10
2
9
7
3
15
7
6
2
4
14
8
8
—
—
NA
O. amamiensis (N = 16)
Downloaded from http://jhered.oxfordjournals.org/ at Seton Hall on December 1, 2014
NA, number of alleles; HO, observed heterozygosity (in bold numbers, if values are significantly deviated from HWE); HE, expected heterozygosity.
Mean
Onari1711 AB900738 (AGAT)6
Onari1587 AB900737
(AT)11
Onari1567 AB900736 (ACT)9
Onari1533 AB900735
(AAT)9
Onari1458 AB900734 (AC)17
Onari1309 AB900733
(AT)10
Onari1112 AB900732 (AG)11
Onari1040 AB900731
(AG)9
Onari997 AB900730 (AAAT)6
Onari850 AB900729
(AC)10
Onari795 AB900728 (AG)14
Onari659 AB900727
(AAT)17
Onari538 AB900726 (AC)10
Onari504 AB900725
(AC)9
Onari429 AB900724 (AGG)9
Onari397 AB900723 (AC)10
Onari324 AB900722
(AAAC)6
Onari319 AB900721 (AC)12
F: F9CCG - AGGGCCCTGCTTAATTTGC R: AGCAGGGCCTCTTATTCCTC F: F9GAC - GATCCGGAGCCATAGAGACC R: ACACATCTACTCCCATAGGCAG F: F9CCG - ACCATTTCCTGTAGCATGTCC R: TCTTGCTTGTGTGATTGGCTC F: F9TAC - TGCACACTGATGCAAACTTTG R: CCTGCACACGTCTTAGATGC F: F9GCC - TTTGCGCAGAGTTCAAGAGG R: CAAGCTCCTCCATATCACCATC F: F9GAC - TGTGTGATCAGTGTCCTCGG R: TGATGGAGAGTGATGGTGCC F: F9GAC - ACTCCAAAGACATGCTGG R: GTTCAGGCTTGGACGTTGTG F: F9TAC - ATGCTTACCTGCCTGTTCTC R: TGAGTTGGGCTGTGTGAGG F: F9GTC - TACAAGAGCTGGGTGTCCTC R: CTGTGCACACTGTTGGCG F: F9GCC - CCGTGGGATGGGAAACTGG R: TAAGTGTGAGGAAGCCAGGC F: F9GCC - TGTCCTACATCAGTGCCTAGTG R: ATGCCTCACTCCCTCTGTTG F: F9GTC - ACTTGCTTTATAGTCCACAGGC R: AAAGTTCCAACAGCCAAGGG F: F9GTC - ACAAGCACTTACAATGCTCTG R: CTTGACTGCATCTGAGACATCC F: F9GTC - AGGGAGAAAGCCAACAGTG R: CAGGTCCCACTCTGCAAATC F: F9GCC - GGAATCGTTGTGGTGTGC R: AACCTCCGATAAACCGCTTC F: F9TAC - AGGCAGCACAAAGGGAATAC R: ACTCAATGCGTATGTTCTGGC F: F9CCG - CGACCTGCCGCAGTAAATAC R: GCGCTGTGTAAATTGATGGC F: F9TAC - AGGTCCAATGGTCAGGTGTC R: GCCTGTCACTTCCAACTCCC F: F9GAC - GTAACACACACTCGCTGCC R: AGACCTGTAACACGAGCCAC F: F9AGG - CACGTAGATGGACCATGCAC R: TCCTATCTCAACATGTGGTCAG
(AAT)8
Onari85
Primer sequence (5'-3')
Repeat motif
Accession no.
Loci
Table 4 Characterization of 20 microsatellite loci isolated from O. narina
0.659
0.791
0.760
0.604
0.836
—
0.795
0.219
0.826
0.695
0.500
0.906
0.694
0.711
0.061
0.568
0.906
0.609
0.713
—
—
HE
2.9
4
4
2
7
1
—
2
5
6
—
5
8
2
1
1
—
2
3
4
—
NA
0.338
0.500
0.700
0.700
0.889
0.000
—
0.300
0.700
0.300
—
0.700
0.600
0.100
0.000
0.000
—
0.600
0.300
0.375
—
HO
O. supranarina (N = 10)
0.369
0.715
0.570
0.495
0.796
0.000
—
0.375
0.680
0.730
—
0.715
0.850
0.095
0.000
0.000
—
0.420
0.405
0.539
—
HE
2.2
1
2
1
1
3
—
3
4
—
—
8
8
—
1
1
—
3
—
8
—
NA
0.225
0.000
0.438
0.000
0.000
0.625
—
0.200
0.875
—
—
0.692
0.667
—
0.000
0.000
—
0.313
—
0.700
—
HO
0.218
0.000
0.342
0.000
0.000
0.549
—
0.184
0.736
—
—
0.784
0.711
—
0.000
0.000
—
0.271
—
0.780
—
HE
O. utsunomiyaorum (N = 16)
Igawa et al. • Microsatellite Marker for O. narina Complex Using Ion PGM
5
Journal of Heredity
were obtained for O. supranarina (mean NA, HO, and HE of 2.9, 0.338, and 0.369, respectively) and O. utsunomiyaorum (mean NA, HO, and HE of 2.2, 0.225, and 0.218, respectively), with fixed alleles at some loci. Although additional samples and populations are required to reach definitive conclusions, the differences observed between the 4 species should reflect population sizes that were largely affected by the size of the inhabited islands. A pairwise FST and DA distance matrix among populations of the 4 species are shown in Supplementary Table S1. These distance values were substantially high between every combination of populations, ranging from 0.274 to 0.457, with a mean of 0.375, and from 0.618 to 0.881, with a mean of 0.743, respectively. However, the values obtained between O. narina and O. amamiensis were 0.274 for FST and 0.784 for DA, which were slightly lower compared with those of parapatrically dwelling Ishikawa’s frogs, O. ishikawae and O. splendida, which are also distributed endemically in Okinawa and Amami Isls, respectively (mean FST, 0.517 and mean DA, 0.934, based on 12 SSR markers; Igawa et al. 2011, 2013). In conclusion, we sequenced 3 frog species in a single multiplexed run of Ion PGM™ and found skewed distribution pattern of repeat motifs. Based on these data, we developed 20 polymorphic loci for the Ryukyu tip-nosed frog, O. narina. More than half of these were stably amplified and genotyped in three-related species. Multiplex sequencing on Ion PGM™ is rapid and one of the most cost-effective methods to develop microsatellite markers in nonexperimental animals. The polymorphism of the loci observed for each repeat type suggests that the higher mutation rates of dinucleotides compared with tetranucleotides is congruent with previous statistical studies. The genotypic data obtained for 4 species were considered to reflect the distribution area of the islands of origin. Therefore, these microsatellite markers have appropriate properties for use in future conservation and population genetic studies of this species group across varying scales, from the interpopulation to individual level.
Kaneko Y, Matsui M. 2004. Odorrana narina. In: IUCN Red List of Threatened Species. Version 2013.2. IUCN 2013.
Supplementary Material
Ota H. 1998. Geographic patterns of endemism and speciation in amphibians and reptiles of the Ryukyu Archipelago, Japan, with special reference to their paleogeographical implications. Res Popul Ecol. 40:189–204.
Funding Scientific Research (B) (24310173 to M.S.); Young Scientists (B) (23710282 to TI from the Ministry of Education, Culture, Sports, Science and Technology, Japan).
Acknowledgments We are grateful to the Boards of Education of Kagoshima Prefecture for allowing us to collect, for the purposes of this study, O. amamiensis samples that are protected by law. We also thank Chie Iwamoto, Nana Fukuda, Mai Fujimi, and Kanako Onizuka from the National Institute of Genetics for their help in the experiments.
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Received March 17, 2014; First decision July 22, 2014; Accepted October 3, 2014
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Corresponding Editor: Andrew Crawford
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