Acta Herpetologica 14(2): 153-158, 2019 ISSN 1827-9635 (print) © Firenze University Press ISSN 1827-9643 (online) www.fupress.com/ah DOI: 10.13128/a_h-7755 AT-rich microsatellite loci development for Fejervarya multistriata by Illumina HiSeq sequencing Yan-Mei Wang, Jing-Yi Chen, Guo-Hua Ding*, Zhi-Hua Lin ADI, College of Ecology, Lishui University, Lishui 323000, Zhejiang, China. * Correspondence author. Email: guwoding@qq.com Submitted on 2018, 22th May; Revised on: 2019, 3rd June; Accepted on: 2019, 23rd August Editor: Emilio Sperone Abstract. In our study, a total of 2561 sequences that contained microsatellite loci were found potentially to be used for primer design. Furthermore, Illumina HiSeq sequencing technology identified trinucleotide repeats and AT-rich repeats with the the highest proportion in our genomic DNA sequence library of Fejervarya multistriata. Eighteen new microsatellite loci of F. multistriata were isolated and we characterize these loci genotyping 48 individuals sam- pled from 3 populations in Lishui City, Zhejiang Province, China. Seventeen loci were polymorphic, with the number of alleles ranging from 2 to 11 within each population. The polymorphic information content, observed and expected heterozygosity ranged 0-0.845, 0-1.0 and 0-0.871, respectively. None of the loci was observed in linkage disequilib- rium. One locus (FMA294) was deviated from Hardy-Winberg equilibrium in each population separately and com- bined. These informative microsatellite loci will be applicable for conservation genetic studies of F. multistriata across varying scales from inter-individual to inter-population. Keywords. Fejervarya multistriata, genome, microsatellite, next-generation sequencing, polymorphism. INTRODUCTION Microsatellite DNA loci, also known as simple sequence repeats, occur at thousands of locations within the eukaryotic genome, and are highly variable and suffi- cient in nuclear genome (Ellegren, 2004; Wei et al., 2015; Shao et al., 2017). Therefore, microsatellite DNA loci are widely applied as molecular markers in population genet- ics, species identifying, genetic breeding and genetic map (Selkoe and Toonen, 2006; Abe et al., 2012; Wambulwa et al., 2016; Soulard et al., 2017). Thanks to the development of next-generation sequencing technology, both through- put and efficiency of developing microsatellite DNA has increased with a decreased cost of sequencing process. In recent years, microsatellite DNA markers have been quickly developed in many species at a low cost when using the next-generation sequencing technology on Illu- mina HiSeq and Ion Torrent PGM platforms (Yang et al., 2012; Lü et al., 2013; Zhang et al., 2013; Sultana et al., 2014; Igawa et al., 2015; Song et al., 2017). Fejervarya multistriata (Anura: Dicroglossidae) is a species of frog, which is widely found in south of the Yellow River in China and some countries (regions) in Southeast Asia, such as northern India, Vietnam and Myanmar (AmphibiaChina, 2018). The conservation status of this species is listed as data deficient in IUCN (AmphibiaChina, 2018). This species prefers to inhab- it paddy field and still water and its ovulation remains active from April to September every year (Amphibi- aChina, 2018). As a dominant amphibian species, F. mul- tistriata plays an important role in farmland ecosystem, and its population density in field has decreased due to urbanization (Li et al., 2016). Meanwhile, environmen- tal degradation also threatens the survival of this species (Othman et al., 2009). In previous studies, mitochon- drial D-loop sequences were used as molecular mark- 154 Yan-Mei Wang et alii ers to study phylogeography of F. multistriata popula- tions (Zhong et al., 2008). Twenty-one microsatellite loci, mainly including dinucleotide repeats, had been isolated for the species, and only approximately 24% loci had AT repeats (Chen et al., 2018). Here, we sequentially devel- oped 18 new microsatellite DNA loci containing AT-rich repeats for F. multistriata by Illumina HiSeq sequencing. These new AT-rich microsatellite loci definitely would be useful in examining genetic diversity and protecting genetic resources of F. multistriata. MATERIAL AND METHODS Forty-eight F. multistriata individuals (n = 16 for each pop- ulation) used in this study were collected by hand and net from 3 localities in Lishui City, Zhejiang Province, China, which were Lanshantou (LST, 119.7607°E, 28.36366°N), Baimashan (BMS, 119.1337°E, 28.63823°N) and Jiulongshan (JLS, 118.8452°E, 28.39538°N), respectively. Our experimental procedures are compliant with current laws on animal welfare and research in China, which are also specifically approved by the Animal Research Ethics Committee of Lishui University (Permit No. AREC-LU 2017-04). Genomic DNA was extracted from toe muscle tissue of one male F. multistriata from LST population using the DNeasy Tissue Kit (Qiagen). The concentration of DNA sample was measured by using a spectrophotometer at 260 and 280 nm and DNA sample was quantified on an agarose gel. A 200-400 bp sequencing library was constructed according to the manufac- turer instructions (Illumina). This library was sequenced using an Illumina HiSeq 2500 Platform with RAD-Tag at Novogene Bioinformatics Technology Co., Ltd (Beijing, China, http:// www.novogene.com/). The microsatellite primer pairs of F. multistriata were designed using Primer Premier 3.0 software, which was used to check against potential primer dimers, hair- pin structures and the occurrence of mismatches. Parameters for designing the primers were set as follows: primer length ranged from 18 bp to 24 bp with 22 as the optimum; PCR prod- uct size ranged from100-280 bp; melting temperature ranged from 50 °C to 65 °C with 55 °C as the optimum annealing tem- perature; GC content ranged from 30% to 70% with 50% as the optimum. Finally, thirty newly designed primer pairs were selected to synthesize and initially screened for microsatellite loci using total genomic DNA isolated from 6 F. multistriata individuals collected from the LST population. PCR amplification reactions were performed using a ther- mal cycler (T100, Bio-Rad, USA). The total volume of each PCR mixture was 20 μL, containing 1 μL genomic DNA (100 ng/μL), 10 μL Premix Taq (TaKaRa, Japan), 1 μL of each primer (10 μM) and 6 μL double distilled H2O. The conditions of the PCR amplification were as follows: 95 °C for 5 min, then 35 cycles at 95 °C for 30 s, Ta (the optimal annealing temperatures, see Table 1) for 30 s, 72 °C for 30 s, and a further extension at 72 °C for 10 min. Twenty-six primer pairs were further selected due to successful amplification in the 6 individuals, and the forward primer was labeled with FAM or HEX fluorescent dye (Sangon Biotech Ltd. Co., Shanghai, China). The PCR products were genotyped on an ABI 3730 sequencer (Applied Biosystems) and following data were analyzed with GeneMarker v1.8 software. Population genetic parameters for polymorphic loci such as number of alleles (NA), observed heterozygosity (HO), expected heterozygosity (HE), polymorphic information content (PIC), P values in Hardy-Weinberg equilibrium (HWE) tests and linkage disequilibrium were calculated by Genepop 4.0 (Rousset, 2008), Cervus 2.0 (Marshall et al., 1998) and Fstat 2.9.3.2 (Goudet, 1995), respectively. RESULTS AND DISCUSSION Sequence data from Illunima HiSeq We obtained a total of 6970707900 bp and 23235693 reads in a single sequencing run on Illunima HiSe- qTM using RAD-Tag. The distribution frequency of read length for this species had a single peaks at approximately 125 bp. In a total of 307793 reads with more than 125 bp, 2561 reads contained microsatellite loci (0.83%). Compared to a traditional library-based approach such as magnetic beads enrichment (Guo et al. 2015; Chang et al. 2016), next-generation sequencing technol- ogy is a more powerful approach to develop microsatel- lite markers due to its efficiency and low cost. Sufficient microsatellite sequences can be constructed in a genomic DNA sequence library on Illunima HiSeqTM using RAD- Tag. This result agrees with recent studies on other anu- ran species (Wei et al., 2015; Shao et al., 2017). Further- more, microsatellite obtained rate maybe higher on Ill- unima HiSeq platform (e.g., 0.83% in our study) than on Ion Torrent PGM platform (e.g., 0.32–0.57% in Igawa et al., 2015). Frequency and distribution of microsatellite loci in the genome The length of the microsatellite loci ranged from 12 to 33 bp (15.7 ± 5.2, mean ± SD). The microsatellite DNA loci included 5 motif types: dinucleotide repeats (36.20%), trinucleotide repeats (52.60%), tetranucleotide repeats (9.64%), pentanucleotide repeats (0.90%) and hexanucleotide repeats (0.66%) (Fig. 1A). The frequency distribution of the 5 motif types was different signifi- cantly (G-test, G = 3085.9, df = 4, P < 0.001). The motif repeat number of microsatellite loci ranged from 4 to 16, while 97.66% of the microsatellite loci had 4-12 motif repeats, and motifs with more than 12 repeats were only with a frequency of <1.0% (Fig. 1B). There were 4 dinu- cleotide motif types, and the main types were AC/GT (50.70%), AT (35.17%) and AG/CT (13.92%) (Fig. 1C), 155Microsatellite loci for F. multistriata respectively. There were 10 trinucleotide motif types, and the main types were AAT/ATT (60.65%) and AGG/CCT (13.51%) (Fig. 1C), respectively. There were 18 tetranu- cleotide motif types, and the main types were AAAT/ ATTT (43.32%), ACAT/ATGT (11.34%), AATT (10.12%) and AGAT/ATCT (10.12%) (Fig. 1C), respectively. There were 13 pentanucleotide motif types, and the main types were AAAAT/ATTTT (39.13%) and AAATT/AATTT (13.04%) (Fig. 1C), respectively. There were 6 hexanu- cleotide motif types, and the main types were AAAAAT/ ATTTTT (58.82%) and ACTCCG/CGGAGT (17.65%) (Fig. 1C), respectively. The frequency of different repeat types of microsatel- lites in F. multistriata was different from Xenopus tropi- calis (Xu et al., 2008), Odorrana narina, Hoplobatrachus tigerinus, and Buergeria japonica (Igawa et al., 2015). The dominant repeat type was trinucleotide in F. multistri- ata, but dinucleotide in the last 4 anuran species (Xu et al., 2008; Igawa et al., 2015). Such results may be related to the different next-generation sequencing platforms used in constructing sequence library. Since the library of microsatellite sequences was constructed by Illunima HiSeq platform for F. multistriata, however, by Ion Tor- rent PGM platform for O. narina, H. tigerinus and B. japonica (Igawa et al., 2015). In addition, the results sug- gested that the frequency of the repeat type changed ran- domly for each species and was species-specific. The fre- quencies decreased when the repeat unit length in each repeat type motif of F. multistriata increased (Fig. 1B), indicating that a relatively short repeat unit of microsat- ellites might be a main component in the genome of F. multistriata. Our finding suggested that the ratio of the repeat motifs with an AT content of approximately 56% in our F. multistriata was similar to other reported anuran species (e.g., X. tropicalis, Xu et al., 2008; O. narina, H. tigerinus and B. japonica, Igawa et al., 2015), suggesting that the AT content could be an important repeat unit in anurans. The dominant repeat motif in the trinucleotide type of F. multistriata, (AAT/ATT repeat) was similar to the other four reported anuran species (Xu et al., 2008; Igawa et al., 2015). However, other types of repeat motif were different among these species. For example, F. mul- tistriata had a higher frequency in AC/GT and AAAT/ ATTT repeats, but O. narina, H. tigerinus, B. japonica and X. tropicalis in AT and AGAT/ATCT repeats (Xu et al., 2008; Igawa et al., 2015). These results implied that the accumulation rates of repeat motifs were maintained in modern anurans, but skewed in a common ancestor (Igawa et al., 2015). Fig. 1. Characterization of microsatellite loci in Fejervarya multistriata genome. (A) distribution of different repeat motif types of microsat- ellite loci; (B) number of different repeat motifs; (C) frequency distribution of 5 different repeat types based on different motif types. Num- ber represent number of sequence. 156 Yan-Mei Wang et alii Characterization of microsatellite loci Twenty-six primer pairs were used to successfully amplify genomic DNA of F. multistriata from LST popu- lation. Of the 26 pairs, 18 pairs generated target bands, and the other 8 pairs generated non-target bands. Finally, a total of 18 primer pairs were characterized, of which 17 were polymorphic. The genomic sequences containing a microsatellite locus, which were used to design these primers, were deposited in GenBank (accession num- ber: MG744293–MG744310). The information of primer sequences, repeat motifs, Ta, NA, PIC and heterozygosity for each locus were shown in Table 1. The Na, PIC and heterozygosities (HO and HE) ranged from 1 to 11 (4.815 ± 2. 699, mean ± SD), 0 to 0.845 (0.549 ± 0.240, mean ± SD), 0 to 1.0 (0.452 ± 0.246, mean ± SD), 0 to 0.871 (0.571 ± 0.237, mean ± SD) within each population, respectively. No significant linkage disequilibrium was observed after Bonferroni correction for multiple tests (all P > 0.05). Of the 18 loci, one (FMA294) deviated sig- nificantly from HWE testing each population separately and combined (all P < 0.05; Table 1), which indicated that null alleles may be present in this locus (Song et al., 2017). The locus was assessed to contain moderately high Table 1. Characterization of 18 microsatellite DNA markers developed for F. multistriata. Size range: size range of fragment; bp: base pair; Ta,: annealing temperature of primer pairs; Na: number of alleles; Ho: observed heterozygosity; HE: expected heterozygosity; HWE: Hardy- Weinberg equilibrium; PIC: polymorphic information content; bold: significant deviation from HWE after Bonferroni correction (P < 0.05). Locus (GenBank #) Primer sequences (5’-3’) Repeat motif Ta (°C) Size range (bp) NA Ho HE PHWE PIC FMA102 MG744293 F: GCACTGTAGAGCACTGGATTC R: GAGCGTCATAGGGGTCAAATAG (TA)16 53 129-219 13 0.4792 0.7592 1.000 0.72 FMA349 MG744294 F: CACTCATGTTATCACTCTACTCTC R: CCTCCTACCTCTTGTACTAAATTG (TAT)11 53 156-237 11 0.3333 0.8145 1.000 0.782 FMA117 MG744295 F: ACTTGAGTCTATTCTATTCTGCTG R: ACTGCTGCTCTGATCTCTATG (ATA)7 53 149-158 4 0.4167 0.5893 0.996 0.495 FMA402 MG744296 F: AGACATTACCTTAAAGCCATAGTG R: CTTCTGACATGACCTGTTCTTC (AGAT)6 53 189-201 4 0.4583 0.6090 0.975 0.538 FMA041 MG744297 F: CCAGGAGGATTCTAGTGACAG R: ATGAAGGCAAGAGCAATGTAC (AT)12 53 152-192 12 0.3958 0.8169 1.000 0.789 FMA466 MG744298 F: GGTGCCACTGTCTTAACTATCC R: AGTCCAATCAAGTCCAATTCAAAC (TTTTTA)4 53 199-205 2 0.3125 0.4086 0.975 0.323 FMA294 MG744299 F: GTCCTCCTACCTCTTGTACTG R: CGAATGAGAACCTTCACAGAC (ATA)10 53 187-238 6 0.9375 0.6515 < 0.01 0.586 FMA302 MG744300 F: TCCGACCTCTTGAAACTGTATTG R: AGGATCACCACTAGGAGCATC (ATA)10 53 205-259 13 0.6042 0.8680 1.000 0.845 FMA355 MG744301 F: TATGACCACAGTCTAGCATCC R: CTCCAGTAGTTATCACCTTCTTG (AAAT)5 58 158 1 – – na 0 FMA188 MG744302 F: CCTCTTGTGTTGGTGTATTTCTG R: TTATGCTTGTGTTCTGGTCATTC (AAT)8 63 209-215 3 0.6042 0.5340 0.170 0.434 FMA231 MG744303 F: GCTGCTGCATGATAGTGTCTC R: TGATGTCTGATGGTCGTCCTG (TAT)8 53 155-185 10 0.7917 0.8353 0.992 0.805 FMA072 MG744304 F: TGCAGTAGACATCGGAGTTG R: GCCTCTCTCATCTTATTAAGTGG (TA)13 53 209-237 10 0.6875 0.7934 0.998 0.757 FMA140 MG744305 F: TTCATTGTGCCAAGTGTAACG R: TAACAAAGAGGTCATCACTAATCC (ATT)7 63 153-165 3 0.1875 0.2254 0.927 0.206 FMA403 MG744306 F: GCGTGGATCGTTATTGAAGTG R: GGTGACCTAATGTGAAATTCCTG (ATTT)6 63 193-197 2 0.2708 0.2945 0.858 0.249 FMA116 MG744307 F: CTCCCTAACTATTGTAAAGCACTG R: ATTATAGATGGAAGCAACAGGAAC (ATA)7 55 165-195 8 0.5208 0.7825 1.000 0.743 FMA399 MG744308 F: TTCAGGCTACAGGCATTACAG R: ATAAGGGTGTTCTGCTAAATCAAG (AATA)6 55 168-228 4 0.2708 0.4476 1.000 0.416 FMA269 MG744309 F: AATGCTTGCAGAACTATTCACAC R: TACGGCGGTCCTAAGATGG (TAT)9 55 177-192 6 0.2500 0.6732 1.000 0.616 FMA139 MG744310 F: GATTGATGGATTGATGATGGACTG R: AATGTTCAAGATGGACGAATTACC (ATG)7 55 177-189 5 0.6042 0.6535 0.813 0.584 157Microsatellite loci for F. multistriata polymorphism degree when the value of PIC was larger than 0.5 (Song et al., 2017). 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