Genes Genet. Syst. (2009) 84, p. 417–424 A HindIII BAC library construction of martensii Karsch (Scorpiones:): An important genetic resource for comparative genomics and phylogenetic analysis

Songryong Li1,2, Yibao Ma1, Shenghun Jang1, Yingliang Wu1, Hui Liu1, Zhijian Cao1* and Wenxin Li1† 1State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, People’s Republic of 2Department of Zoology, Institute for Biology, Kim Hyong Jik University of Education, Pyongyang, Democratic People’s Republic of Korea

(Received 28 August 2009, accepted 22 December 2009)

Scorpions are “living but sophisticated fossils” that have changed little in their morphology since their first appearance over the past 450 million years ago. To provide a genetic resource for understanding the evolution of genome and the relationships between and other organisms, we first determined the genome size of the scorpion Karsch (about 600 Mbp) in the order Scorpiones and constructed a HindIII BAC library of the male scorpion M. martensii Karsch from China. The BAC library consists of a total of 46,080 clones with an average insert size of 100 kb, providing a 7.7-fold coverage of the scorpion haploid genome size of 600 Mbp as revealed in this study. High-density colony hybridization-based library screening was performed using 18S-5.8S-28S rRNA gene that is one of the most commonly used phylogenetic markers. Both library screening and PCR identification results revealed six positive BAC clones which were overlapped, and formed a contig of approximately 120 kb covering the rDNA. BAC DNA sequencing analysis determined the complete sequence of M. martensii Karsch rDNA unit that has a total length of 8779 bp, including 1813 bp 18s rDNA, 157 bp 5.8s rDNA, 3823 bp 28s rDNA, 530 bp ETS, 2168 bp ITS1 and 288 bp ITS2. Interestingly, some tandem repeats are present in the rRNA inter- genic sequence (IGS) and ITS1/2 regions. These results demonstrated that the BAC library of the scorpion M. martensii Karsch and the complete sequence of rDNA unit will provide important genetic resources and tools for comparative genomics and phylogenetic analysis.

Key words: Mesobuthus martensii Karsch, genome size, BAC library, rDNA, phylogeny

licerate phylogeny, and their local abundance and wide INTRODUCTION geographical distribution (David, 1990). Moreover, the The order Scorpiones, with sixteen living families and amazing suite of biochemical, behavioral and ecological about 1500 described species and subspecies, is a rather adaptations endues their continued success over the past small group of chelicerate (Prendini and 450 million years. Though these “living but sophisti- Wheeler, 2005; Soleglad and Fet, 2003). Nevertheless, cated fossils” have changed little in their morphology scorpions have received considerable attention through since their first appearance, their adaptations range from the years because of their medical importance, their superbly efficient behavioral repertoires to the maternal antiquity and obvious importance to the analysis of che- care of offspring, ultrasensitive tactile and visual fields, and complex venoms that are a precise mixture of differ- Edited by Norihiro Okada * Corresponding author. E-mail: [email protected] ent toxins, each with its own action (Goudet et al., 2002; † Corresponding author. E-mail: [email protected] Prendini et al., 2006). However, many taxa are poorly 418 S. LI et al. understood and the relationship of scorpions to other che- MATERIALS AND METHODS licerates remains a topic of controversy. In particular, the phylogenetic position of the Scorpiones, a key order in Determination of genome size by flow cytometry the phylogeny, is highly disputed (Jones et al., Adult specimens of the scorpion M. martensii Karsch 2007; Mallatt and Giribet, 2006; Murienne et al., 2008). were purchased from the Suizhou City in Central China’s Genome studies of scorpions will allow us to under- Hubei Province. The male reproductive organs and the stand the phylogeny of the order Scorpiones and to dive female ovaries were grinded by a pair of tweezers in ice- into the secrets of the “living fossils”. BAC library is a cold 1 × PBS, respectively. The mixture was filtered powerful tool for studying genomes. Since it was devel- through 40- to 100-μm nylon mesh, and then washed with oped by Shizuya et al. (1992), bacterial artificial chromo- ice-cold 1 × PBS twice by centrifugation (about 250 g for some system has become the preferred approach for 5 min) at 4°C. The cell density was adjusted approxi- constructing genomic library and been used to clone gene, mate 1 × 106 cells/ml using a haemacytometer. DNA con- map genome, and sequence BAC DNA, because of loading tent measurement was performed according to previously capacity of large fragments, high stability and convenient described methods (Vindelov et al., 1983; Wei et al., manipulation (Osoegawa et al., 2001). Bacterial artifi- 2003). The PI fluorescence intensity measurement was cial chromosome had already been used to construct performed with Phoenix Flow Systems (Beckman and genomic libraries from many to plants. How- Coulter). At least 10,000 cells were measured per indi- ever, no such large-insert genomic DNA library has been vidual sample. Nuclear DNA content of samples was reported to date in the order Scorpiones. Chinese scor- estimated using the DNA content of chicken blood cells pion M. martensii Karsch is widely distributed in China, (2.5 pg/nucleus) as the control (Dolezel et al., 2003). Korea and . It is also one of the important raw material used in traditional medicines for more than 1000 BAC library construction The scorpion BAC library years, especially for treating neural diseases such as apo- was constructed as described before with minor modifica- plexy, hemiplegia and facial paralysis (Shi and Zhang, tions (Osoegawa et al., 1998; Zhang, 2000). Briefly, 2005; Zhijian et al., 2006). genomic DNA was isolated from the cells of the testes, In this paper, we determined the genome size of the subjected to partial digestion with HindIII, size selected scorpion M. martensii Karsch, constructed its BAC on agarose gels by pulsed-field gel electrophoresis, and library, screened and obtained the complete sequence of ligated to the CopyControl pCC1BAC HindIII cloning- the ribosomal RNA gene unit of M. martensii Karsch that ready vector (Epicentre Biotechnologies, Madison, WI, was previously unknown in the order Scorpiones. Taken USA) as described (http://www.epibio.com/pdftechlit/ together, these results will provide important genetic 177pl074.pdf). Ligation products were transformed by resources and tools for comparative genomics and phylo- electroporation into the TransforMax Escherichia coli genetic analysis. EPI300 strain (Epicentre Biotechnologies). White colo-

Table 1. Data on species for phylogenetic analysis

Classification Species GenBank accession number Buthida Buthidae Mesobuthus martensii FJ948787* Androctonus australis X77908 Centruroides hentzi AF062948 Chaerilida Chaerilidae Chaerilus sp. AY371538 Pseudochactida pseudochactidae Pseudochactas ovchinnikovi AY368258 Iurida Chactidae Belisarius xambeui AF005442 Scorpionidae Pandinus imperator AY210831 Vaejovidae Paruroctonus mesaensis AF062950 Euscorpiidae Euscorpius italicus AY371537 Caraboctonidae Hadrurus arizonensis AF062949 *In this study. The first BAC library of the order Scorpiones 419 nies were picked and transferred into 384-well plates for minescent Hybridization and Detection Kit (Pierce). growth, storage, and replication. High-density filters representing the whole library were prepared on 30 pieces BAC contig and sequence analysis of rRNA gene of nylon membranes, with each membrane harboring the The positive BAC DNAs were purified using the BAC96 BAC clones corresponding to four 384-well plates and two Miniprep Kit (Millipore, USA). The primers for BAC replications per BAC clone. Estimation of the average end-sequencing were designed according to the flanking insert size and a stability test of the BAC library were region of the HindIII cloning site of pCC1BAC conducted as described by Shizuya et al. (1992).

BAC library screening The R1, R2 and R3 probes were designed according to the complete sequence of Pandinus imperator (Scorpiones; Scorpionidae) 5.8S ribo- somal RNA gene (GenBank accession no, AY210830), the partial sequence of P. imperator 28S ribosomal RNA gene (GenBank accession no, AY210830), and the partial sequence of M. martensii Karsch 18S rRNA gene (GenBank accession no, AB008465), respectively. Primers used for R1 are 5’-GGCTGTACTCCCAAACAACCCGACT-3’ and 5’-CCTGTCTGAGGGTCGGACGAATAAC-3’. Prim- ers used for R2 are 5’-CGCGAGACCCGACACTACCGT-3’ and 5’-ACCGCGAAAGCGGGGCCTAT-3’. Primers used for R3 are 5’-GGCAGTCCGGGAAACAAAGT-3’ and 5’- CCTACGGAAACCTTGTTACGACTT-3’. Probes were labeled with Biotin-11-dUTP by random prime labeling method as following the procedure described by North2South Biotin Random Prime Labeling Kit (Pierce). The hybridization procedures were performed following methods described by a North2South Chemilu-

Fig. 2. Analysis of the insert size from the scorpion M. martensii Karsch BAC library. A: Analysis of the insert size of 30 random BAC clones from the scorpion M. martensii Karsch BAC library by pulsed-field gel electrophoresis (PFGE). The DNA inserts were released by digesting with NotI enzyme and separated by PFGE. The 8.1 kb common band is pCC1BAC (HindIII) cloning vector. Molecular weight size marker is PFGE lambda DNA ladder (M). B: Insert size distribution of 200 clones randomly Fig. 1. Comparison of the DNA contents of the male scorpion selected from the scorpion BAC library. The average insert size cells and the chicken erythrocyte cells. of the library was estimated to be 100.45 kb.

Table 2. Flow cytometry analysis of M. martensii Karsch against a chicken erythrocyte standard (2C = 2.5 pg)

sex Female male Numbers of samples 12 10 DNA content of each sample 1.05, 1.09, 1.12, 1.16, 1.18, 1.19, 1.02, 1.07, 1.14, 1.17, 1.19, (pg/nucleus) 1.20, 1.21, 1.24, 1.26, 1.28, 1.31 1.21, 1.24, 1.25, 1.27, 1.31 Mean DNA conent (pg/nucleus)* 1.190 ± 0.067 1.187 ± 0.073 *Data are presented as mean ± standard deviation. 420 S. LI et al.

Vector. The primers for each end of the positive BAC deposited in GenBank under the accession number clones were designed to assemble rRNA gene contig (data FJ948787. not shown). 52F9 BAC clone contained M. martensii Karsch rRNA gene was sequenced by Beijing Genomics Phylogenetic analysis of rRNA gene The 18S rDNA Institute (BGI), China. The analysis of M. martensii sequences used for alignment and phylogeny analysis Karsch rDNA sequence was performed with Gene were obtained through NCBI ENTREZ (http:// Runner, and GenBank NCBI database (http://www.ncbi. www.ncbi.nlm.nih.gov/entrez/) (Table 1). The alignment nlm.nih.gov/blast). The complete ribosomal DNA was performed by Clustal_X 1.83 software followed by sequence of M. martensii Karsch reported here has been manual adjustment (Thompson et al., 1997), and viewed by the software Jalview (Waterhouse et al., 2009). Phy- logeny analysis was carried out with Neighbor joining (NJ) and Maximum Parsimony (MP) methods imple- mented in MEGA3.1 (Kumar et al., 2004).

RESULT AND DISCUSSION Genome size of the scorpion M. martensii Karsch Flow cytometrical analysis of the gonadal cell suspensions without chicken erythrocyte reference cells gave fluores-

Table 3. The lengths and GC contents of ETS and ITS regions, 18S, 5.8S, and 28S rRNA genes

Section Length (bp) GC (%) Total 8779 54.62 EST 530 56.03 18S 1813 49.53 ITS1 2168 54.94 5.8S 157 57.96 Fig. 3. Six individual positive BAC clones were digested with ITS2 288 51.39 NotI enzyme. The result indicates that all BAC clones were (excluding 26G17) overlapped with each other. 28S 3823 56.77

Fig. 4. Map of the BAC contigs covering the region of the scorpion 18S-5.8S-28S rRNA gene. The contig was constructed based on analysis of HindIII and NotI digestion, BAC end-sequencing, PCR identification, and finally complete DNA-sequencing of 52F9 positive BAC clone. The locations of the 18S, 5.8S and 28S rRNA coding sequences are indicated below the map of BAC clone 52F9. The arrow shows the transcription initia- tion site (TIS). The first BAC library of the order Scorpiones 421 cence histograms showing three peaks (corresponding to the scorpion M. martensii Karsch is very small, e. g., C- C, 2C and 4C DNA content nuclei) in mature testes, or values of the spiders set range from 0.74 pg to 5.7 pg two main peaks (2C and 4C) in immature or spent (Gregory, 2005; Gregory and Shorthouse, 2003). gonads. Simultaneously, flow cytometrical analysis of the ovarian cell suspensions gave fluorescence histograms BAC library construction and characterization showing the main peak (2C) and the weak peak The BAC library of the scorpion M. martensii Karsch has (4C). The fluorescence peak position of diploid (2C) cells 46,080 clones assembled into 120 384-well plates. To for the scorpion M. martensii Karsch was therefore determine the average insert size of the library, DNAs determined. Subsequently, the corresponding sample from 200 random BAC clones were isolated and digested with reference cells was run and the mean values of the with NotI, and then fractionated by PFGE (Fig. 2A). The diploid peak of the scorpion and the chicken (internal statistic results showed that the average insert size of the standard) were recorded (Fig. 1). Estimated values of BAC library was estimated to be 100 kb with clones rang- average diploid (2C) DNA content are summarized in ing from 80 to 120 kb (92.5%), and the percentage of Table 2. As shown in Fig. 1 and Table 2, the mean DNA empty recombinant clones was 3.0% (Fig. 2B). This BAC content of scorpion was 1.19 pg/nucleus. Moreover, t- library represented about 7.7-fold genome coverage, cal- tests of the average values showed that there was no a culated from the estimated insert size and the genome significant difference between the male and female cells size of the scorpion M. martensii Karsch. The library of of DNA content (P > 0.05). Therefore, the genome size of the scorpion M. martensii Karsch is the first BAC library the scorpion M. martensii Karsch was estimated to be in the order Scorpiones, providing useful resources for approximate 600 Mbp. Although the chromosome num- further genomic and genetic studies. bers of several species in the order Scorpiones are known (Moustafa et al., 2005), the genome size of any scorpion BAC library screening with rDNAs To further char- species has not been reported previously. Compared acterize the quality of the library and facilitate its appli- with that of the other arthropods, the C-value 1.19 pg of cation, we screened this BAC library using 18S-5.8S-28S

Fig. 5. DNA sequence of the intergenic region of the M. martensii Karsch rRNA unit. The TIS (+1) is underlined and denoted by an arrow. The locations of the 308 bp and 551 bp IGSRE are shown, indicating the same positions by an asterisk. 422 S. LI et al. rDNA as probe. The probe pool was made by mixing 5.8S, dIII BAC library used 18S, 5.8S, and 28S rRNA coding 28S and 18S partial rDNA sequences of M. martensii sequences as probes, respectively. Sequence analysis of Karsch. High-density nylon membranes of the whole this clone showed that 52F9 only contains one of the BAC library were hybridized using the probe pool and 426 rDNA repeat unit because of rDNA repeat unit near to positive BAC clones were obtained. These positive BACs the terminus of this clone. This is the first report of the were picked from the BAC library and transferred onto complete sequence of the rDNA unit in the order nylon membrane filter with low density. Low-density Scorpiones. The rRNA gene of M. martensii Karsch is nylon filter was hybridized with each individual probe to fragmented into ETS, 18S, ITS1, 5.8S, ITS2, and 28S, get the positive BACs corresponding to each probe. which shares the same gene organization and structure Almost positive BAC clones were overlapped with each with the other animals. Sequence analysis of 52F9 clone other as revealed by restriction enzyme analysis with revealed an 8779 bp rDNA unit of M. martensii Karsch, HindIII (not shown) and NotI (Fig. 3), PCR analysis, and (1813 bp 18S rDNA, 157 bp 5.8S rDNA, 3823 bp 28S DNA sequencing. Especially, the results by BAC end- rDNA, 530 bp ETS, 2168 bp ITS1 and 288 bp ITS2) and sequencing and PCR analyses showed that the six posi- a GC content of 54.53% in the coding sequences and tive BAC clones (605, 7F12, 7H11, 26F11, 52M15 and 54.62% in the complete rDNA unit (Table 3). The 52F9) cover a genomic region of ~120 kb and contain sequence of M. martensii Karsch rRNA gene shares high rDNAs (Fig. 4). Screening the library with the 18S-5.8S- homology with the previously reported rRNA genes from 28S rDNA identified a total of 426 positive clones, repre- the other species of Scorpiones (Soleglad and Fet, 2003). senting 0.92% of the library clones. Unfortunately, the complete sequence of rRNA gene has never been reported in the order Scorpiones. Sequence analysis of ribosomal RNA gene A geno- The transcription initiation site (TIS) was predicted mic BAC clone, 52F9, was isolated by screening the Hin- using Neural Network Promoter Prediction software

Fig. 6. DNA sequence of ITS1, 5.8S, and ITS2 regions of the M. martensii Karsch rRNA unit. The locations of the 242 bp, 61 bp repeats in ITS1 and the 61 bp repeat in ITS2 are shown, indicating the same positions by an asterisk. It is also indicated 5.8S rRNA gene, 18S rRNA 3’-ending and 28S rRNA 5’-ending. The first BAC library of the order Scorpiones 423

(http://www.fruitfly.org/seq_tools/promoter.html) and WWW Promoter Scan software (http://www-bimas.cit. nih.gov/molbio/proscan/). The predicted result showed that the TIS (+ 1) corresponds to a base C at the 530 nt upstream of the 18S rRNA gene (Figs. 5 and 6). The rRNA gene promoter has been characterized in several organisms, ranging from protozoa to vertebrates. There is little sequence similarity between the ribosomal pro- moters from different species, and only closely related organisms show some degree of sequence conservation (Mallatt et al., 2004). Interestingly, 308 bp and 551 bp tandem repeat elements are present in IGS of M. martensii Karsch rRNA gene (Fig. 5). A 308 bp beginning at -2168 nt and ending at -1243 nt upstream of TIS repeats three times, while a 551 bp from -1173 nt to -71 nt upstream of TIS repeats twice. The rRNA genes of yeasts and multi- cellular eukaryotes are organized in tandem arrays where the genes are separated by intergenic spacers composed of repetitive sequence elements, such as ~5 copies of a 63 bp repeat in Leishmania major, ~8 copies of a 172 bp repeat in Trypanosoma cruzi (Martinez-Calvillo et al., 2001), ~8 copies of 35 bp, ~5 copies of 100 bp and ~20 cop- ies of 60/81 bp repeats in Xenopus (Robinett et al., 1997), ~6 copies of 6–500 bp repeats in Drosophila (Stage and Eickbush, 2007). Although the sequences of the inter- genic spacers are not conserved among different eukary- Fig. 7. Cladograms (A; NJ tree, B; MP tree) showing phylogeny of ten taxa based on 18S rDNA sequences. Numbers represent otes, the presence and analogous arrangement of bootstrap percentages. The topologies were tested using boot- repetitive enhancer elements, promoter duplications, and strap analysis (10000 replicates). terminator sequences in Xenopus, Drosophila, and mice suggest that there is a functional conservation of inter- genic spacer elements across distantly related species ogy of the tree by NJ method was similar to that by MP (Robinett et al., 1997; Stage and Eickbush, 2007). method. Tree topologies showed that parvorder Iurida Interestingly, ITS1 has four 242 bp repeats and two was clearly a sister group to the other three parvorders 61 bp repeats. Surprisingly, the sequence like 61 bp tan- (clade Buthida + Chaerilida + Pseudochactida), a result dem repeat element in ITS1 is also present in ITS2 (Fig. was disagreed with previously described (Prendini and 6). The internal transcribed spacer of rDNA repeat unit Wheeler, 2005; Soleglad and Fet, 2003). So resolution of is one of the most commonly applied phylogenetic mark- this outstanding question would require sequence infor- ers (Keller et al., 2009; Schultz et al., 2006). However, mation from more species and more sophisticated meth- the comparison between the other scorpions and arthro- ods of phylogeny reconstruction. pods showed that the ITS1 and ITS2 sequences are highly In conclusion, we first reported the scorpion M. martensii variable. Comparative sequence analysis of rRNA unit Karsch BAC library in the order Scorpiones that would be from different organisms has shown that sequence simi- important genetic resource for phylogenetic analysis, larities can be found in related groups and the sequence comparative genomics and toxicological research of of ITSs region can only be used for phylogenetic recon- scorpion. We also determined the complete sequence of structions at a low taxonomic level. The phylogenetic rDNA unit that would provide not only further insight trees resulting from NJ and MP analyses based on 18S into the genomic organization of rRNA gene in scorpion rDNA sequences, with Calocheiridius cf. termitophilus species, but also important informative genetic marker (Arachnida: Pseudoscorpiones, GenBank accession no. for phylogeny reconstruction of the order Scorpiones. AY859559) as the outgroup species, are presented in Fig. 7. The 18S rDNA analysis included ten taxa from seven The authors thank Prof. Wang Yaping and Liao Lanjie in Lab- families: Buthidae, Caraboctonidae, Chaerilidae, Euscor- oratory of Freshwater Ecology and Biotechnology, Institute of piidae, Pseudochactidae, Scorpionidae, Vaejovidae. Of Hydrobiology, Chinese Academy of Sciences, and Prof. Ma Lunlin, and Miss Liu Ka in Institute of Genetics and Develop- total 885 characters, 714 characters were constant, 155 mental Biology, China Academy of Sciences for technical advice variable characters were parsimony-uninformative, and and assistance. This study was supported by grants from the 49 characters were parsimony-informative. The topol- National Natural Sciences Foundation of China to Li Wenxin 424 S. LI et al.

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