Characterization of the Macropodus opercularis complete mitochondrial genome and family Channidae taxonomy using Illumina-based de novo transcriptome sequencing

Gene 559 (2015) 189–195

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Short communication

Characterization of the Macropodus opercularis complete mitochondrial genome and family Channidae taxonomy using Illumina-based de novo transcriptome sequencing Xidong Mu, Yi Liu, Mingxin Lai, Hongmei Song, Xuejie Wang, Yinchang Hu, Jianren Luo ⁎ Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Key Laboratory of Tropical & Subtropical Fishery Resource Application & Cultivation, Ministry of Agriculture, Guangzhou 510380, China

a r t i c l e

i n f o

Article history: Received 26 September 2014 Received in revised form 19 January 2015 Accepted 28 January 2015 Available online 29 January 2015 Keywords: Macropodus opercularis Mitochondrial genome Phylogeny De novo transcriptome sequencing

a b s t r a c t In this study, the complete mitochondrial genome of Macropodus opercularis was sequenced using Illuminabased de novo transcriptome technology and annotated using bioinformatic tools. The circular mitochondrial genome was 16,496 bp in length and contained two ribosomal RNAs, 13 protein-coding genes, 22 transfer RNA genes, and the control region. The gene composition and order were similar to suborder Anabantoidei. Phylogenetic analyses using concatenated amino acid and nucleotide sequences of the 13 protein-coding genes with two different methods (Neighbor-joining and Bayesian analysis) both highly supported the close relationship of M. opercularis to M. ocellatus, consistent with previous classifications based on morphological and molecular studies. Furthermore, family Channidae and Parachanna insignis were clustered in the same clade. Our results supported the inclusion of family Channidae in suborder Channoidei. The complete mitochondrial genome of M. opercularis will provide genetic markers for better understanding species identification, population genetics and phylogeographics of freshwater fishes. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Macropodus opercularis is a well-known freshwater fish with high ornamental value and belongs to the family Osphronemidae within the suborder Anabantoidei of order Perciformes (Pan et al., 1990; Nelson, 2006). In Western countries, it is one of the most popular ornamental fish, known as the paradise fish. It was once widely distributed in South China in paddies, streams and rivers, but populations have been massively reduced due to water pollution (Wang et al., 2009; Liu et al., 2011). Despite its status as a characteristic species with great conservation importance in China, few studies have been performed on the genetics and genomics of this species. Liu et al. (2011) used partial mitochondrial cytochrome b (Cyt b) to infer that there was high genetic diversity in five populations of M. opercularis from five provinces in China. Our group used the complete Cyt b gene and partial D-loop sequences to investigate the inter- and intra-species genetic diversity and phylogeography of genus Macropodus (Wang et al., 2011). These studies Abbreviations: NGS, next-generation sequencing; bp, base pair; PCR, polymerase chain reaction; tRNA, transfer RNA; rRNA, ribosomal RNA; CR, control region; ATP6 and ATP8, ATPase subunits 6 and 8; ND1–ND6 and ND4L, NADH dehydrogenase subunits 1–6 and 4 L; COX1–COX3, cytochrome c oxidase subunits 1–3; 16S and 12S, large and small subunits ribosomal RNA; Cyt b, cytochrome b. ⁎ Corresponding author at: Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, 1 Xingyu Road, Liwan District, Guangzhou, Guangdong Province, China. E-mail address: olfi[email protected] (J. Luo).

http://dx.doi.org/10.1016/j.gene.2015.01.056 0378-1119/© 2015 Elsevier B.V. All rights reserved.

showed that mtDNA sequences are suitable markers to infer genetic diversity and phylogeny, providing valuable information on the conservation of germplasm resources of genus Macropodus. However, although several species have been described within the genus Macropodus, the taxonomic classification in this genus of M. opercularis and other related species (M. opercularis, M. chinensis, M. hongkongensis, and M. erythropterus) based on morphological characteristics has been controversial for decades (Winstanley and Clements, 2008; Wang et al., 2011). Thus, resolution of the taxonomic position of the genus Macropodus using genetic markers is necessary. mtDNA has been widely used as a genetic marker for genetic structures, species identification, systematic analyses and phylogeography in organisms due to its patterns of maternal inheritance, rapid evolutionary rate and high mutation rate (Avise et al., 1987; Boore, 1999; Mu et al., 2012). Compared to partial mitochondrial sequences, such as Cytb and the D-loop, complete mitochondrial sequences may provide more insight and better resolution from higher-level groups down to closely related species (Velez-Zuazo and Agnarsson, 2011; Powell et al., 2013). For genus Macropodus, only one complete mitochondrial genome from M. ocellatus (GenBank: KJ813282) (Xu et al., 2014) has been sequenced and reported. In this study, we determined the complete sequences of the mitochondrial genomes of M. opercularis using next-generation sequencing and compared, in detail, the full sequences of four species belonging to the suborder Anabantoidei. We used 13 protein-

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Table 1 Nucleotide composition of the mitochondrial genomes of suborders Anabantoidei (Perciformes) and Channoidei. Subordera

Family

Genus

Species

T

C

A

G

AT%

AT skew

GC skew

Length

GenBank

Reference

Anabantoidei

Osphronemidae Osphronemidae Osphronemidae Anabantidae Helostomatidae Channidae Channidae Channidae Channidae Cichlidae

Macropodus Macropodus Colisa Anabas Helostoma Channa Channa Channa Parachanna Tilapia

Macropodus opercularis Macropodus ocellatus Colisa lalia Anabas testudineus Helostoma temminckii Channa argus Channa marulius Channa maculata Parachanna insignis Tilapia buttikoferi

29.6 29.8 30.2 26.2 25.8 24.6 24.4 24.6 24.9 25.6

24.7 24.3 24.5 28.4 29.4 31.1 31.3 31.1 31.2 30.8

30.9 30.4 29.8 29.6 28.6 28.2 28.4 28.2 28.0 27.4

14.8 15.5 15.5 15.7 16.2 16.2 15.9 16.1 15.9 16.2

60.5 60.2 60.1 55.8 54.4 52.8 52.8 52.8 52.9 53.0

0.02 0.01 −0.01 0.06 0.05 0.07 0.08 0.07 0.06 0.03

−0.25 −0.22 −0.23 −0.29 −0.29 −0.32 −0.33 −0.32 −0.32 −0.31

16,496 16,501 16,746 16,603 16,740 16,559 16,569 16,558 16,607 16,577

KM588227 KJ813282 NC_022479 KJ808811 AB861523 NC_015191 NC_022713 KC310861 NC_022480 KF866133

This study Xu et al. (2014) Miya et al. (2013) Zhao et al. (2014) Li et al., Unpublished Wang and Yang (2011) Singh et al. (2013) Wang et al. (2013) Miya et al. (2013) Mu et al. (2014)

Channoidei

Outgroup a

Data from Nelson (2006).

coding genes to reconstruct the phylogenetic relationships of the suborder Anabantoidei using Neighbor-joining (NJ) and Bayesian inferences (BI) analyses.

30–40 mg fin clip was collected and preserved in 95% ethanol at 4 °C. Total genomic DNA was extracted with the Tissue DNA Kit (OMEGA E.Z.N.A.) following the manufacturer's protocol.

2. Material and methods

2.2. Illumina library preparation and sequencing

2.1. Sample collection and DNA isolation

Paired-end libraries (500 bp) were prepared following the Illumina DNA manufacturer's instructions. The size-selected, adapter-modified DNA fragments were PCR-amplified using PE PCR primers and the following protocol: polymerase activation (98 °C for 2 min), followed by 10 cycles (denaturation at 98 °C for 30 s,

One adult M. opercularis (body weight: 21.6 g, body length: 6.5 cm) was obtained from the ornamental base, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, China. A

Fig. 1. Gene maps and organization of the complete mitochondrial genome of Macropodus opercularis. The outer circle represents the positive strand (H-strand), and the inner circle represents the negative strand (L-strand). The tRNA genes are named using single-letter amino acid abbreviations, as shown in the Table (right).aH and L indicate that the gene is encoded by the H or L strand. bIntergenic nucleotides. Numbers correspond to nucleotides separating a gene from one upstream; negative numbers indicate that adjacent genes overlap. cTA or T: Incomplete termination codon, which is probably extended by post-transcriptional adenylation.

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Fig. 2. AT- and GC-skews and AT content (%) of the mitochondrial protein-encoding, ribosomal RNA genes and D-loop in the mitochondrial genome of Macropodus opercularis and four related species in suborder Anabantoidei.

annealing at 65 °C for 30 s, and extension at 72 °C for 60 s) with a final 4-min extension at 72 °C. DNA libraries were purified using magnetic beads and quantified by RT-PCR. The transcriptome was projected into a single Illumina lane (HiSeq2000) and sequenced using the 500-bp paired-end technique, with a 2*101-bp paired-end protocol.

Illumina paired-end reads were filtered on quality values, and the low quality bases (quality b 20, perror N 0.01) 5 nt upstream and 3 nt downstream were trimmed. Mitochondrial DNA reads were captured by blastx with mitochondrial protein sequences in the NR database. Reads were assembled to contigs and scaffolds with over-lap and

Fig. 3. Codon usage pattern of Macropodus opercularis mitochondrial genomes compared with four related species belonging to suborder Anabantoidei. Numbers on the Y-axis refer to the total number of codons. Codon families are provided on the X-axis.

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Table 2 The Relative Synonymous Codon Usage (RSCU) of 13 protein-coding genes in the Macropodus opercularis mitochondrial genome. Amino acid

Codon

Number

Frequency (%)

Amino acid

Codon

Number

Frequency (%)

Phe Phe Leu Leu Leu Leu Leu Leu IIe IIe Met Met Val Val Val Val Ser Ser Ser Ser Pro Pro Pro Pro Thr Thr Thr Thr Ala Ala Ala Ala

TTT TTC TTA TTG CTT CTC CTA CTG ATT ATC ATA ATG GTT GTC GTA GTG TCT TCC TCA TCG CCT CCC CCA CCG ACT ACC ACA ACG GCT GCC GCA GCG

167 92 139 68 115 63 79 42 166 62 84 56 46 22 54 15 93 68 77 31 102 85 87 13 80 62 101 26 54 58 56 4

4.4 2.42 3.66 1.79 3.03 1.66 2.08 1.11 4.37 1.63 2.21 1.48 1.21 0.58 1.42 0.4 2.45 1.79 2.03 0.82 2.69 2.24 2.29 0.34 2.11 1.63 2.66 0.69 1.42 1.53 1.48 0.11

Tyr Tyr Term Term His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Cys Cys Trp Trp Arg Arg Arg Arg Ser Ser Term Term Gly Gly Gly Gly

TAT TAC TAA TAG CAT CAC CAA CAG AAT AAC AAA AAG GAT GAC GAA GAG TGT TGC TGA TGG CGT CGC CGA CGG AGT AGC AGA AGG GGT GGC GGA GGG

117 87 75 54 70 59 70 32 94 85 89 22 39 28 51 27 23 31 59 31 26 21 32 17 38 47 24 39 36 29 44 32

3.08 2.29 1.98 1.42 1.84 1.55 1.84 0.84 2.48 2.24 2.35 0.58 1.03 0.74 1.34 0.71 0.61 0.82 1.55 0.82 0.69 0.55 0.84 0.45 1 1.24 0.63 1.03 0.95 0.76 1.16 0.84

Total number of codons is 3795. Term = Stop codon.

paired-end relationships, super scaffold were built with the contigs and scaffolds, and gaps were filled with paired-end reads. Genome confirmation was necessary after assembly; the paired-end reads were mapped to the genome with 100% coverage and insert-size in accordance with the sequencing library. Coding genes were annotated with blastx using the “vertebrate mitochondrial code (transl_table = 5)”. Start codons and end codons were annotated using the vertebrate mitochondrial codon table, and blastp was used to confirm the length and start codon of protein sequences. 2.3. Sequence assembly and annotation Protein-coding regions were identified using BLAST (blastn, tblastx) searches of the NCBI databases, the MITOS WebServer BETA (http://bloodymary.bioinf.uni-leipzig.de/mitos/index.py) and comparisons with other sequences of Anabantoidei available in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). Putative tRNA genes were identified using the software ARWEN (http://130. 235.46.10/ARWEN/) (Laslett and Canback, 2008), combined with visual inspection of aligned mtDNA and tRNA genes. The predicted boundaries of rrnL and rrnS genes were identified by aligning them to published Anabantoidei mitochondrial rRNA genes (Miya et al., 2013; Zhao et al., 2014). The alignments were checked manually. 2.4. Alignment The complete mitochondrial genome sequence was aligned to available complete mt genome sequences of four Anabantoidei fish: Macropodus ocellatus (GenBank: KJ813282) (Xu et al., 2014), Colisa lalia (GenBank: NC_022479) (Miya et al., 2013), Anabas testudineus (GenBank: KJ808811) (Zhao et al., 2014), and Helostoma temminckii (GenBank: AB861523) (unpublished) using

Fig. 4. Control region (D-loop) of the Macropodus opercularis mitochondrial genome. (A) Structural elements found in the control region of M. operculari. The control region flanking genes tRNA-Pro (P) and tRNA-Phe (F) are represented in green boxes; ‘(TA)n’ (blue) indicates the microsatellite-like region; Poly T indicates a high T content region. (B) The D-loop sequence of M. operculari. (C) Putative stem-loop structure found in the D-loop.

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Fig. 5. The phylogenetic analyses investigated using Neighbor-joining (NJ) and Bayesian inferences (BI) analysis indicated evolutionary relationships among 9 taxa based on the nucleotide (A) and amino acid (B) alignments of 13 protein-coding genes. The tree topologies produced by NJ and BI analyses were equivalent. Bayesian posterior probability (black number) with 10,000 generations and bootstrap support values for NJ analyses (red number) with 1000 replicates are shown on the nodes. Tilapia buttikoferi (GenBank: KF866133) was used as the outgroup.

Seaview (Gouy et al., 2010). Codon usage and nucleotide composition statistics were computed using MEGA 6.06 (Tamura et al., 2011) and Microsoft Excel 2007. Strand asymmetry was calculated using the formulas: AT skew = [A − T] / [A + T] and GC skew = [G − C] / [G + C] (Perna and Kocher, 1995). The map of mitochondrial genome was visualized using the GenomeVx online tool (http://wolfe.ucd.ie/GenomeVx/) (Conant and Wolfe, 2008) with further manual correction.

2.5. Phylogenetic analyses The phylogenetics were analyzed based on the amino acid and nucleotide sequence data of 13 publically available PCGs (Table 1). Heterotilapia buttikoferi (GenBank: KF866133) was considered the outgroup in the present study. Neighbor-joining (NJ) and Bayesian inference (BI) were used to reconstruct phylogenetic relationships using 13 PCGs. NJ analysis was performed using MEGA 6.06 (Tamura et al., 2011). BI was performed on the combined database using MrBayes v.3.1.2 software (Ronquist and Huelsenbeck, 2003). Stationarity was considered to be reached when the average standard deviation of split frequencies was below 0.01. The bestfit models of evolution for the amino acid and nucleotide sequence datasets were selected using the Akaike Information Criterion (AIC) with jModeltest (Posada, 2008), and the resulting GTR + I + G (Ln = −68,146.89, AIC = 136,349.79) model was used to optimize the BI analysis. All phylogenetic trees were drawn using the Tree View program v.1.65 (Page, 1996).

3. Results and discussion 3.1. Illumina sequencing and assembly Using the Hiseq2500 plate platform, we generated ~ 905 million bases of transcriptome sequence data, containing 2,194,000 total reads and 21,940 reads from the mitochondrion (Table S1). A de novo assembly was first carried out using the trimmed reads from both elevations to generate reference contigs. Subsequently, the first assembled mitochondrial genomes were tested for completeness and exactness through paired-end reads mapping to the genome, resulting in 100% coverage and insert size in accordance with the sequencing library. Second, based on the sequences of the fragments, short-PCR primers were designed and employed to amplify the overlapping segments. Finally, we obtained the complete mitochondrial genome DNA sequence and deposited it into the GenBank database (accession number KM588227). 3.2. General features of the M. opercularis mitochondrial genome The complete mitochondrial genome of M. opercularis is 16,496 bp in size and shares the same 37 typical metazoan genes (13 protein-coding genes, 22 transfer RNA genes, and 2 ribosomal RNA genes) and a putative control region (Fig. 1). It possesses a gene order identical to mt genome belonging to the suborder Anabantoidei, including M. ocellatus (Xu et al., 2014), C. lalia (Miya et al., 2013), and A. testudineus (Zhao et al., 2014). Twelve protein-encoding genes (with the exception of ND6), two rRNAs, and 14 tRNAs (with the exception of tRNA-Gln,

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tRNA-Ala, tRNA-Asn, tRNA-Cys, tRNA-Tyr, tRNA-Ser(S2), tRNA-Glu, and tRNA-Pro) are transcribed from the heavy (+) strand, as is the case with Anabantoidei species (Miya et al., 2013; Zhao et al., 2014; Xu et al., 2014). There is a major non-coding region (830 bp) that may contain the origin of replication. The nucleotide composition of the M. opercularis mt genome is biased towards A and T, with A (30.9%) being the most abundant nucleotide and G (14.8%) being the least abundant, in accordance with the mt genomes of other Anabantoidei species (Miya et al., 2013; Zhao et al., 2014) (Table 1). There is a small variation in overall A + T content, ranging from 54.4% (A. testudineus) to 60.5% (M. opercularis), with an average overall value equal to 58.2% in the suborder Anabantoidei. The A + T content is 60.5% for M. opercularis, higher than that of mt genomes of other Anabantoidei to date. The composition of the M. opercularis mtDNA sequence is skewed away from A in favor of T (AT skew = 0.02, GC skew = −0.25). The pattern of skew values of M. opercularis is similar to those observed for mtDNA sequences of other suborder Anabantoidei and Channoidei sequences, with the exception of C. lalia (Table 1). In the 13 protein-coding genes, the AT and GC skew values of ND6 genes in the suborder Anabantoidei are higher than those of other genes (Fig. 2; Table S2). There are several additional intergenic sequences up to 99 bp in length in 17 locations, ranging from 1 to 32 bp in M. opercularis (Fig. 1). The longest intergenic region is a 32bp region between tRNA-Asn and tRNA-Cys. 3.3. Protein-coding genes (PCGs) and codon usage patterns The complete mitochondrial genome of M. opercularis contains the 13 PCGs of metazoans with the typical order observed in fish. For comparison with C. lalia (Miya et al., 2013) and A. testudineus (Zhao et al., 2014), the predicted translation initiation and termination codons for the 13 protein-coding genes of the M. opercularis mitochondrial genome were analyzed. The most common initiation codon for M. opercularis is ATG (12 of 13 PCGs), followed by GTG (COX1 gene). Incomplete termination codons (T or TA) are found in 6 PCGs (ND2, COX2, COX3, ND3, ND4, and Cytb) in the M. opercularis mitochondrial genome (Fig. 1). These genes may be terminated by post-transcriptional polyadenylation by providing two adenosines (Ojala et al., 1980). Excluding the termination codons, a total of 3603 amino acids from protein-coding genes are encoded in the mitochondrial genome of M. opercularis. The codon in PCGs in the M. opercularis mitochondrial genome is summarized and compared with four Anabantoidei species in Fig. 3. The total number of amino acids, excluding the termination codons in the four mitochondrial genomes, shows low levels of variation, ranging from 3603 (M. opercularis) to 3722 (A. testudineus). The codon families exhibit very similar behavior among these four species. The most frequently used amino acid in suborder Anabantoidei is Leu, followed by Ser, Pro, and Thr. The Relative Synonymous Codon Usage (RSCU) of the 13 protein-coding genes in the M. opercularis mitochondrial genome (Table 2) exhibits extensive similarity with other Anabantoidei species. A significant bias for A + T rich codon usage is found, which plays a major role in the A + T bias of the entire M. opercularis mitochondrial genome. The most frequently used codon in M. opercularis is TTT (4.4%), followed by ATT (4.37%) and TTA (3.66%) (Fig. 2). 3.4. Transfer RNA genes, ribosomal RNA genes, and non-coding regions In total, the typical 22 tRNA genes with conventional secondary structures are found in the mitochondrial genomes of M. opercularis. Mitochondrial tRNA genes range in size from 67 to 74 nucleotides (Fig. 1), and the predicted secondary structures of 21 of these tRNAs had typical clover-leaf shapes with paired dihydrouridine (DHU) arms. tRNA-Ser (S1) contains a predicted secondary structure with the TΨC arm and loop, but lacks the DHU arm and loop (Fig. S1). Two ribosomal RNAs (12S rDNA and 16S rDNA) were identified on the H-strand in the M. opercularis mitochondrial genomes. These two

genes are separated by tRNA-Val (V), a feature often found in animals, including Anabantoidei fishes. The sizes of the ribosomal RNAs (12S: 948 bp; 16S: 1673 bp) are within the range of sizes found in other fish. The A + T content of the ribosomal genes is 56.2% (12S) and 58.3% (16S), well within the range of AT content (54.8%–60.6%) observed in protein-coding genes in the M. opercularis mitochondrial genome and similar to the four species in suborder Anabantoidei (Fig. 3). The non-coding region of the M. opercularis mitochondrial genome comprises 2 major regions of more than 20 bp: a short non-coding region of 32 bp between tRNA-Asn and tRNA-Cys and a control region (D-loop) of 830 bp between tRNA-Glu and tRNA-Phe. Its A + T content (66.9%) is higher than the average value of the whole mitogenome (60.5%) and 13 PCGs (54.3%–60.6%) (Fig. 3; Table S1). Across the five Anabantoidei species, there is low variation, ranging from 816 bp (C. lalia) to 1035 bp (H. temminckii). The D-loop, which includes the origin sites for transcription and replication, is a common feature in most fish mitochondrial genomes (Taanman, 1999). Sbisà et al. (1997) proposed that the control region is characterized by discrete and conserved sequence blocks and possesses the typical tripartite with termination associated sequence (TAS), central, and conserved sequence block (CSB) domains. Based on the alignment of the D-loop sequences in M. opercularis and four Anabantoidei species, three domains (the termination associated sequence domain), TAS; the central conserved domains (CSB-E, CSB-D) and the conserved sequence block domains (CSB-1, CSB-2, CSB-3) were found, along with a poly-thymine (poly-T) sequence, an AT-rich region, and a stem-loop structure (Fig. 4). The conservative GGGGG-box was also found in the central domain of the Dloop region in M. opercularis and four other Anabantoidei species.

3.5. Phylogenetic relationships The phylogenetic trees inferred by different methods (NJ and Bayesian analyses: Fig. S2) using different building strategies were identical across the two datasets based on nucleotide and amino acid alignments of the 13 protein-coding genes, with high bootstrap values (Fig. 5). Both trees showed strong support for two clades, suborder Anabantoidei and suborder Channoidei, in agreement with a previous study based on morphological traits (Nelson, 2006). However, the taxonomic status of the family Channidae is still controversial. In Nelson (2006), the family Channidae was placed in the suborder Channoidei due to a series of morphological characteristics. In contrast, Pan et al. (1990) defined the family Channidae as belonging to the suborder Anabantoidei. Our results highly support that the family Channidae is a monophyletic group, consistent with the results of Nelson (2006). In conclusion, the present study determined complete mitochondrial genome sequences of M. opercularis (16,496 bp) using Illumina-based de novo transcriptome sequencing and investigated the phylogenetic relationships of suborder Anabantoidei using sequences from the protein coding genes. Here, our results revealed the taxonomic position of M. opercularis as belonging to suborder Anabantoidei and found that family Channidae should belong to suborder Channoidei, supporting previous classifications based on their morphology (Nelson, 2006). Further mitochondrial genome data would be useful for studying population genetics and phylogeographics within highly diversified freshwater fish, especially order Perciformes (~7800 species). Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.01.056.

Acknowledgments The authors appreciate to SCGene (Co., Ltd.) for technological support. This study was supported by the program of the National Science Infrastructure Platform of China (2015DKA30470) and the Guangdong Science Technology Project of China (2012A020602014).

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