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Development and Characterization of 29 Polymorphic Development and characterization of 29 polymorphic microsatellite loci of Megalobrama pellegrini by next- generation sequencing technology and cross-species amplification in related species Wen Song 1 , Dongmei Zhu 1 , Yefeng Lv 1 , Weimin Wang Corresp. 1 1 Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricutural University, Wuhan, China Corresponding Author: Weimin Wang Email address: [email protected] Megalobrama pellegrini is one of the economically important freshwater fish in China. Here, we developed 29 polymorphic microsatellite loci of M. pellegrini. The number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (HO), expected heterozygosity (HE) and polymorphic information content (PIC) ranged from 3 to 11 (mean±SD 5.4828±1.9571), 2.8708 to 9.6257 (mean±SD 5.0865±1.6681), 0.4333 to 0.9333 (mean±SD 0.7874±0.1213), 0.6627 to 0.9113 (mean±SD 0.7946±0.0751) and 0.5785 to 0.8868 (mean±SD 0.7439±0.0950), respectively. Cross-species amplification was successful at most loci for related species such as M. amblycephala, M. hoffmanni, M. skolkovii and Parabramis pekinensis. The transferability rate of the 29 polymorphic microsatellite markers in M. amblycephala, M. hoffmanni, M. skolkovii and P. pekinensis were 96.55%, 86.21%, 86.21% and 75.86%, respectively. These polymorphic microsatellites are not only useful in genetic study and conservation of M. pellegrini, but also an effective tool for identifying the related species. We could use 5 microsatellite markers (HHF-63, HHF-104, HHF-113, HHF-148, HHF-163) to distinguish the 5 species. PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2490v1 | CC BY 4.0 Open Access | rec: 30 Sep 2016, publ: 30 Sep 2016 1 Development and characterization of 29 polymorphic microsatellite loci of 2 Megalobrama pellegrini by next-generation sequencing technology and cross-species 3 amplification in related species 4 Wen Song, Dongmei Zhu,Yefeng Lv and Weimin Wang 5 Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of 6 Education, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, College of 7 Fisheries, Huazhong Agricultural University, Wuhan 430070, China 8 Corresponding Author: Weimin Wang 9 [email protected] PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2490v1 | CC BY 4.0 Open Access | rec: 30 Sep 2016, publ: 30 Sep 2016 10 Abstract 11 Megalobrama pellegrini is one of the economically important freshwater fish in China. Here, 12 we developed 29 polymorphic microsatellite loci of M. pellegrini. The number of alleles (Na), 13 effective number of alleles (Ne), observed heterozygosity (HO), expected heterozygosity (HE) 14 and polymorphic information content (PIC) ranged from 3 to 11 (mean±SD 5.4828±1.9571), 15 2.8708 to 9.6257 (mean±SD 5.0865±1.6681), 0.4333 to 0.9333 (mean±SD 0.7874±0.1213), 16 0.6627 to 0.9113 (mean±SD 0.7946±0.0751) and 0.5785 to 0.8868 (mean±SD 17 0.7439±0.0950), respectively. Cross-species amplification was successful at most loci for 18 related species such as M. amblycephala, M. hoffmanni, M. skolkovii and Parabramis 19 pekinensis. The transferability rate of the 29 polymorphic microsatellite markers in M. 20 amblycephala, M. hoffmanni, M. skolkovii and P. pekinensis were 96.55%, 86.21%, 86.21% 21 and 75.86%, respectively. These polymorphic microsatellites are not only useful in genetic 22 study and conservation of M. pellegrini, but also an effective tool for identifying the related 23 species. We could use 5 microsatellite markers (HHF-63, HHF-104, HHF-113, HHF-148, 24 HHF-163) to distinguish the 5 species. 25 Key words: Megalobrama pellegrini, Megalobrama hoffmanni, Megalobrama skolkovii, 26 Megalobrama amblycephala, Parabramis pekinensis, Microsatellite, Polymorphism 27 Introduction 28 Microsatellites, also known as simple sequence repeats, have been extensively used as 29 molecular markers due to their abundance and high degree of polymorphism (Ellegren, 2004; 30 Saenz-Agudelo et al., 2015; Camargo et al., 2015). With the development of next-generation 31 sequencing technology, the sequencing throughput has dramatically increased and the costs 32 have decreased. Microsatellites can be developed quickly and at low cost for most species 33 using next-generation sequencing in recent years (Gardner et al., 2011; Berman, Austin & 34 Miller, 2014; Plough & Marko, 2014). Microsatellites are currently the most widely used 35 molecular markers in genetic breeding, genome mapping, gene location, species identifying 36 and so on. 37 Megalobrama pellegrini belongs to the genus Megalobrama that also includes M. 38 amblycephala, M. hoffmanni and M. skolkovii, and is one of the most important economic PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2490v1 | CC BY 4.0 Open Access | rec: 30 Sep 2016, publ: 30 Sep 2016 39 freshwater fish in China (Wu, 1964; Luo, 1990; Chen, 1998). The species of the genus 40 Megalobrama are extremely similar in morphological traits (Song et al., 2013). Parabramis 41 pekinensis is similar in morphological traits with the species of the genus Megalobrama (Cai 42 et al., 2001). M. pellegrini distributes in the upstream area of Yangtze River that differs from 43 the other species of the genus Megalobrama (Chen, 1998). Due to habitat changes and human 44 factors such as pollution and overfishing, populations of M. pellegrini have been decreasing 45 rapidly across their original distribution (Chen, 2002; Xie, 2003; Gao et al., 2010). Since the 46 21st century, the natural resources and population of M. pellegrini have obviously declined 47 (Li et al., 2005; Gao et al., 2009). M. pellegrini is hard to find in large tributaries of Yangtze 48 River, and they only distribute in some small flow tributaries. M. pellegrini has become an 49 endangered species, so that it is in need of protection (Wang et al., 2012). But there are only a 50 few articles about M. pellegrini, and only one article is about microsatellite markers of M. 51 pellegrini (Wang et al., 2012). Here, we developed 29 polymorphic microsatellite loci for M. 52 pellegrini by next-generation sequencing technology. These polymorphic microsatellite 53 markers will be useful in protecting genetic resources and molecular breeding of this species. 54 They also offers the potential to identify species of M. pellegrini, M. hoffmanni, M. skolkovii, 55 M. amblycephala and P. pekinensis. 56 Materials & methods 57 Aquaculture materials and genomic DNA extraction 58 The sampling positions of the 5 species were listed in Table 1. 16 individuals of the 59 population of M. pellegrini were used for the initial screening of microsatellite loci. The DNA 60 of the M. pellegrini was extracted using the CTAB method (Cota-Sánchez, Remarchuk & 61 Ubayasena, 2006). The concentration of each DNA sample was measured using a 62 spectrophotometer at 260 and 280 nm and each DNA sample was quantified on an agarose 63 gel. Genome sequencing of M. pellegrini was performed on an Illumina HiSeq 2000TM by 64 Biomarker Technologies (Beijing, China). The procedure was performed in accordance with 65 the standard Illumina protocol, including sample preparation and sequencing as follows: 66 quantified DNA was extracted and DNA fragments were obtained using ultrasound, which 67 was purified using the QIA quick PCR kit. End repair was performed with poly-A on the 3’ PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2490v1 | CC BY 4.0 Open Access | rec: 30 Sep 2016, publ: 30 Sep 2016 68 ends, then the adaptors were ligated, clusters were generated, agarose gel electrophoresis was 69 used to select fragments, and PCR amplification was performed. Sequencing was performed 70 by establishing a library with Illumina HiSeq 2000TM. The short reads were aligned using the 71 Burrows-Wheeeler transformation (Li & Durbin, 2010). The microsatellite primer pairs of M. 72 pellegrini were designed using Primer Premier 6.0 software, which was used to check against 73 potential primer dimers, hairpin structures and the occurrence of mismatches. The specificity 74 of primer pairs were checked by comparing their sequences with the genomic sequences. 75 Only those microsatellite loci that contained motifs 4 to 6 nucleotides in size were selected to 76 synthesize. The minimum motif repeat was defined as 5 for hexanucleotides, 10 for 77 pentanucleotides and 20 for tetranucleotides. Parameters for designing the primers were set as 78 follows: primer length ranged from 20 bases to 28 bases with 24 as the optimum; PCR 79 product size ranged from 160-300 bp; melting temperature ranged from 55℃ to 63℃ with 59℃ 80 as the optimum annealing temperature; GC content ranged from 40% to 70% with 50% as the 81 optimum. At last, total of 192 primer pairs were chosen to synthesize. 82 PCR amplification and microsatellite loci analysis 83 These primer pairs, which were successfully amplified and found to be polymorphic in initial 84 screening tests, were then tested in 30 individuals. The PCR amplification reactions were 85 performed using a thermal cycler (Eppendof, Germany). The PCR was performed in a final 86 volume of 20 μL. The PCR mixture was as follows: 2 μl genomic DNA (100 ng/μl), 10 μl 87 PCR mix (TaKaRa, Japan), 1 μl of each primer (10 μM) and 6 μl ddH2O. The PCR conditions 88 was as follows: 95℃ for 5 min, 40 cycles with 95℃ denaturation for 30s, 72℃ annealing for 89 45s, and 72℃ elongation for 10 min，followed by 4℃ for the end. Each effective PCR 90 product was validated in 30 individuals of M. pellegrini and analyzed using QIAxcel 91 Advanced (Qiagen, Germany). Population genetic parameters for polymorphic loci such as 92 number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (HO), 93 expected heterozygosity (HE), polymorphic information content (PIC), P values in 94 Hardy-Weinberg equilibrium (HWE) tests were calculated by GENEPOP 4.0 (Rousset, 2007).
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