Mapping Toll-Like Receptor Signaling Pathway ...

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pathway genes in Zhikong scallop (Chlamys farreri) have mainly focused on sequence analysis and expression profiling, ..... aquaculture genome research.
J. Ocean Univ. China (Oceanic and Coastal Sea Research) DOI 10.1007/s11802-015-2643-8 ISSN 1672-5182, 2015 14 (6): 1075-1081 http://www.ouc.edu.cn/xbywb/ E-mail:[email protected]

Mapping Toll-Like Receptor Signaling Pathway Genes of Zhikong Scallop (Chlamys farreri) with FISH ZHAO Bosong, ZHAO Liang, LIAO Huan, CHENG Jie, LIAN Shanshan, LI Xuan, HUANG Xiaoting*, and BAO Zhenmin Key Laboratory of Marine Genetics and Breeding of Ministry of Education, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, P. R. China (Received April 4, 2014; revised August 21, 2014; accepted June 20, 2015) © Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2015 Abstract Toll-like receptor (TLR) signaling pathway plays a pivotal role in the innate immune system. Studies on TLR signaling pathway genes in Zhikong scallop (Chlamys farreri) have mainly focused on sequence analysis and expression profiling, no research has been carried out on their localization. The chromosomal position of TLR signaling pathway genes can be valuable for assemblying scallop genome and analysizing gene regulatory networks. In the present study, five key TLR signaling pathway genes (CfTLR, CfMyd88, CfTRAF6, CfNFκB, and CfIκB) containing bacterial artificial chromosomes (BACs) were isolated and physically mapped through fluorescence in situ hybridization on five non-homologous chromosome pairs, showing a similar distribution to another five model species. The isolation and mapping of these key immune genes of C. farreri will aid to the research on innate immunity, assignment of interested genes to chromosomes, and integration of physical, linkage and cytogenetic maps of this species. Key words

immunogenetics; Chlamys farreri; TLR signaling pathway; FISH

1 Introduction The innate immune system is the first-line defense for all living organisms, and it is almost the only path for invertebrates to cope with the invasion of microorganisms present in the environment (Wang et al., 2011). Innate immune responses are initiated by germline-encoded pattern-recognition receptors that recognize conserved motifs of pathogens termed pathogen-associated molecules (Meijer et al., 2004), such as lipopolysaccharides, β-1,3glucans and peptidoglycans (Janeway, 1989; Ashida et al., 1998; Hoffmann et al., 1999). Toll-like receptors (TLRs) are among the most extensively studied pattern-recognition receptors. TLRs act as signal transducers using adaptor proteins (MyD88, TIRAP, TRIF, and TRAM), making the common TLR signaling pathway function. TLR signaling pathway culminates in the activation of a variety of inducible transcriptional factors such as nuclear factor kappa B (NFκB) and interferon-regulatory factor, raising various downstream immunological responses to the invasion of pathogens (Kawai and Akira, 2010). Toll protein was first reported in Drosophila melanogaster (Belvin and Anderson, 1996), and it has now been identified in a wide range of species (Coscia et al., 2011). * Corresponding author. Tel: 0086-532-82031802 E-mail: [email protected]

Furthermore, the TLR gene family and their associating pathways are evolutionarily conservative from fly to humans (Roach et al., 2005; Hoffmann and Reichhart, 2002). Recent genomic analysis has detected a rich collection of TLR signaling pathway genes in non-mammalian organisms including marine invertebrates such as Ciona intestinalis (Sasaki et al., 2009), Strongylocentrotus purpuratus (Hibino et al., 2006), Tachypleus tridentatus (Inamori et al., 2004) and Crassostrea gigas (Zhang et al., 2011). The structures, expressions and possible signaling of these genes are well documented. Evidence shows that TLR signaling pathway genes are involved in the innate immune system of marine invertebrates (Coscia et al., 2011). Zhikong scallop, Chlamys farreri Jones et Preston, 1904, is one of the most important maricultured shellfish in northern China. Over the last decade, the population of C. farreri is lightened sharply due to various infections. A better understanding of the innate immune system of C. farreri would facilitate the control of infectious diseases. To date, most of the TLR signaling pathway genes have been found in C. farreri, which included CfTLR (Qiu et al., 2007a), CfMyd88 (Qiu et al., 2007b), CfTRAF6 (Qiu et al., 2009), CfNFκB, and CfIκB (Wang et al., 2011) with their sequence features characterized clearly. The transcripts of these genes are up-regulated after lipopolysaccharide stimulation and down-regulated once being RNA interferenced (Wang et al., 2011). A TLR signaling pathway exists in scallop, which may involve in immune signaling

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and activating downstream response and eliminating invading pathogens (Wang et al., 2011). In recent years, research on TLR signaling pathway genes of scallop has mainly focused on gene expression (Wang et al., 2011; Qiu et al., 2007a, b; Qiu et al., 2009). Physically mapping these genes is still unmentioned, although such mapping can help determine whether there are gene clusters in TLR signaling pathway, and how these genes are arranged on chromosomes. In species with complete genome sequence information, it is relatively easy to identify the physical location of genes through comparing sequences against a reference genome (Lorenzi et al., 2010). To scallop, however, the whole genome sequence is not available. Thus it is necessary to map genes with other methods such as fluorescence in situ hybridization (FISH). Recently, three bacterial artificial chromosome (BAC) libraries of C. farreri have been constructed based on different restriction enzymes (Zhao et al., 2013), providing researchers a convenience of physically mapping related genes on the chromosomes of C. farreri. In the present study, BAC clones containing five TLR signaling pathway genes (CfTLR, CfMyd88, CfTRAF6, CfNFκB and CfIκB) were screened from BAC libraries of C. farreri. The five genes were mapped to C. farreri chromosomes through FISH. It provided the first physical mapping of TLR signaling pathway genes in mollusk, aiding to better understanding this pathway and chromosomal assignment of gene sequences.

2 Materials and Methods 2.1 BAC Library Screening Primers for CfTLR, CfMyd88, CfTRAF6 and CfIκB were designed from their homologous cDNAs (Table 1) while those of CfNFκB were the published by Wang et al. (2011). Positive BAC clones were screened by four-dimensional, two-step PCR from the HindIII-BAC (BH) and BamHI-BAC (BB) libraries of C. farreri (Zhao et al., 2013). The PCR products of gene fragments were reconfirmed by sequencing (Zhao et al., 2012). Table 1 The primer sequences used for FISHing toll-like receptor signaling pathway genes GenBank accession No. DQ350772 DQ249918 DQ350773 – DQ852572

Gene

Primer sequence (5’–3’)

CGCAGAGCATGTTGGAGGATTC CCTTTACTCCCTCCTCCATCTTG TCCGTTGGACTCTTGTGGCG CfMyd88 ATGGCCGGAGAATAATCTGACC GAGAGTCTGGCTAGGCACGAAC CfTRAF6 GTCAAGCACGGTAAGCACGATAC CAGCATTCCAGGGGACAAGA CfNFκB TGAGGCTCGTAGGGTGTGTCTT TCACCGTTGTTGATTTGAGCG CfIκB GAAGCAGGTGTCGATTGTGAGG CfTLR

2.2 Preparation of Probe and C0t-1 DNA BAC DNA was isolated from 20 mL of overnight cul-

ture using a standard laboratory method (Sambrook et al., 1989). Approximately 1 μg of BAC DNA was labeled with nick translation kit (Roche, Basel, Switzerland) with digoxygenin-11-dUTP or biotin-16-dUTP according to the manufacturer’s instructions. Labeled probes were stored at −20℃. C0t-1 DNA and enriched repetitive DNA sequences were prepared according to the procedure described early (Hu et al., 2011).

2.3 Chromosome Preparation Chromosomes were prepared from trochophore larvae of C. farreri with the method described by Zhang et al. (2008). Trochophore larvae were treated with 0.01% colchicine for 2 h and then exposed to 0.075 mol L−1 of KCl for 30 min. Thereafter, the larvae were fixed three times, 15 min each, in Carnoy’s solution (methanol: glacial acetic acid, 3:1). The larvae were dissociated in 50% acetic acid, then dropped onto hot-wet slides and air dried. 2.4 FISH Analysis Chromosome slides were pretreated with 1.6% pepsin at 37℃ for 30 min and washed in 2x saline sodium citrate (SSC) for 5 min. Specimens were denatured in a mixture containing 70% formamide and 2x SSC at 75℃ for 2 min, followed by immediate dehydration in an ice-cold ethanol gradient (70%, 90%, and 100%; 5 min each) and air-drying. One microgram of labeled probe was mixed in a hybridization buffer of 50% deionized formamide and 2x SSC, plus 50 ng μL−1 C0t-1 DNA. For hybridization, the probe mixture was denatured at 75℃ for 5 min and preannealed at 37℃ for 30 min. Thereafter, each slide was covered with 20 μL of probe mixture and incubated for 16 h at 37℃ in a humid box. For double-color FISH, probes labeled with digoxigenin and biotin were mixed and incubated at 37℃. A series of washes was followed: 50% formamide and 2x SSC, 42℃, 5 min; 1x SSC, 42℃, 5 min; and 2x SSC at room temperature, 5 min. The probes were detected using anti-digoxigenin-rhodamine or/and fluorescein avidin D Cell Sorter Grade. Chromosomes were counterstained with 4’,6-diamidino-2-phenylindole or propidium iodide. Slides were viewed under an Eclipse-600 epifluorescence microscope equipped with a CCD camera. Pictures were merged and edited using LUCIA Cytogenetics and Photoshop CS3. For karyotype analysis, chromosomes were paired according to their morphology from 20 good metaphases. Short and long arms were measured to calculate the relative length and centrometric index in accordance with Levan et al. (1964).

3 Results 3.1 BAC Library Screening BAC libraries were screened by four-dimensional, two-step PCR of CfTLR, CfMyd88, CfTRAF6, CfNFκB and CfIκB. BAC clones yielded clear single DNA fragments and expected sizes were selected for further use.

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After PCR screening, all the five genes were found to be

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represented by at least one BAC clones each (Table 2).

Table 2 Positive bacterial artificial chromosome (BAC) clones containing Toll-like receptor signaling pathway genes identified from scallop BAC libraries through PCR screening Product size No. of positive (bp) BAC clones CfTLR 431 8 CfMyd88 312 6 CfTRAF6 396 8 CfNFκB 345 5 Gene

CfIκB

312

9

BAC code *

BH132D8, BH277F9, BH679A2, BB87B9 , BB124H11, BB264D3, BB290E1, BB325A5 BH89A3, BH254D8, BH794G3, BH925B2, BB26G9*, BB253F6 BH339D5, BH409H8*, BH650F4, BH861C2, BH1039B3, BH1241H2, BB194E2, BB305A3 BH291B10, BH473H6, BH802F5*, BH1167G4, BB45C12 BH222A5, BH327H7, BH520D10, BH729D3, BH953C3, BB30B3, BB215E9, BB275F7*, BB292G3

*

Note: BAC clones selected for FISH.

3.2 FISH Mapping For each gene, one representative clone was selected randomly for FISH (Table 2). FISH signals for individual positive BAC clones were analyzed in 20 metaphase chromosome spreads. All the five BAC clones were mapped to the corresponding chromosomes of C. farreri. The CfTLR-containing clone BB87B9 was hybridized to the telomeric region of the short arm on a pair of subtelocentric chromosomes (Fig.1a), and the CfNFκB-containing clone BH802F5 was mapped to a similar position on a pair of submetacentric chromosomes (Fig.1b). Probes derived from clones BB26G9 containing CfMyd88 (Fig.1c) and BH409H8 containing CfTRAF6 (Fig.1d) showed signals in the centromeric region of the long arm on a pair of submetacentric or subtelocentric chromosomes, respectively. The CfIκB-containing clone BB275F7 (Fig.1e) was mapped to the central section of the long arm in a pair of submetacentric chromosomes. In order to test whether all the screened BAC clones were located on the same pair of chromosomes, the representative clone was co-hybridized with each of the other clones through double-color FISH. For example (Fig.2), we co-hybridized BB26G9 with each of the other five CfMyd88-containing clones, i.e., BH89A3, BH254D8, BH794G3, BH925B2, and BB253F6. Co-localization of BB26G9 with each of the five clones was confirmed using probes capable of generating merged signals in each case. We concluded that all the six CfMyd88-containing clones were located at the same site in the genome. Similar conclusions were drawn from the study on the remaining four genes.

After karyotyping, the means and standard deviations of the relative length and centromeric index were calculated for chromosome pairs with signals (Table 3). The CfIκB-containing chromosomes has a smaller relative length while the remaining four chromosomes with signals were considerably larger than the largest metacentric chromosome. The results indicated that clone BB275F7 containing CfIκB was localized to a different pair of chromosomes from the other clones, BB87B9, BB26G9, BH409H8, and BH802F5. Co-hybridization was necessary to estimate whether the latter four BAC clones were located on different pairs of chromosomes separately. However, signals of probes derived from each clone were weakened when all these 4 clones were co-hybridized in one experiment. Thus, two BAC clones were assigned to similar chromosomes to confirm their chromosomal assignments by double-color FISH. Clone BB87B9 containing CfTLR was labeled with biotin and BH409H8 containing CfTRAF6 with digoxigenin. Results showed that the two probes were localized to two different subtelocentric chromosome pairs (Fig.3a). Then, the other two BAC clones BB26G9 (digoxigenin) and BH802F5 (biotin) were co-hybridized, and signals were observed on two non-homologous submetacentric chromosome pairs (Fig.3b). The locations of the four BACs obtained from double-color FISH were consistent with the results of one-color FISH. All these available data indicated that the five BAC clones, which contained CfTLR, CfMyd88, CfTRAF6, CfNFκB and CfIκB, respectively, were located in five non-homologous chromosome pairs of C. farreri.

Fig.1 FISH mapping of bacterial artificial chromosome clones containing CfTLR(a), CfNFκB(b), CfIκB (c), CfMyd88(d), and CfTRAF6 (e) from Chlamys farreri. Inset at top right for each probe corresponds to one chromosomal location showing the labeled chromosome adjacent to the largest metacentric chromosome. Scale bars = 5 μm.

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Fig.2 Double-color FISH showing 6 CfMyd88-containing bacterial artificial chromosome clones co-localized on the Chlamys farreri genome. Red, green, and blue channels were recorded separately and then merged. Red signals indicate localization of clone BB26G9 first mapped using single-color FISH, and green signals indicate clones BH89A3, BH254D8, BH794G3, BH925B2, and BB253F6. Signals are indicated by arrows in merged images. Table 3 Measurements (X ± S.D) and classification of Toll-like receptor pathway genes containing chromosomes and the largest metacentric chromosome from metaphases of Chlamys farreri Chromosome pair CfTLR containingCfMyd88 containingCfTRAF6 containingCfNFκB containingCfIκB containingThe largest metacentric chromosome

Relative length

Centromeric index

Type

5.93 ± 0.36 6.27 ± 0.34 5.76 ± 0.42 6.72 ± 0.52 5.03 ± 0.39 5.65 ± 0.48

23.70 ± 0.56 27.93 ± 0.79 21.01 ± 0.58 33.44 ± 0.73 28.90 ± 0.96 44.64 ± 0.45

st sm st sm sm m

Fig.3 Co-hybridization of Toll-like receptor pathway genes. (a) Bacterial artificial chromosome clones containing CfTLR and CfTRAF6; and (b) clones containing CfNFκB and CfMyd88. Scale bars = 5 μm.

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4 Discussion An understanding of immune components that underpine host resistance to pathogens is a key step towards elucidating immune mechanisms in scallop. A large number of immune components are known in scallop (Su et al., 2004; Gao et al., 2007; Wang et al., 2007; Yu et al., 2007; Zhang et al., 2007). Most of these components have been characterized and analyzed regarding gene function, but few have been physically mapped to chromosomes. In the present study, we used FISH to map five immune genes functioning in C. farreri TLR signaling pathway in order to study their chromosomal locations. The results showed that each gene occupied a single position on a chromosome pair. Early studies have shown that genes with similar expression patterns tend to cluster more frequently than those with different expression patterns (Liu and Han, 2009; Chen et al., 2010). In Arabidopsis and human, there is about 43% and 65% of analyzed pathways showing significant physical clustering of genes across the genome, respectively (Lee and Sonnhammer, 2003). Immune genes in Drosophila are highly concentrated on chromosome 2, clustered in regions of high recombination rates (Wegner, 2008), which may be a fast and effective way to control expression of genes. As to scallop, the lack of whole genome data limits the research of immune genes. Recently, 2 lipopolysaccharide and beta-1,3-glucan binding protein genes and 3 membrane transport genes have been shown clustered in 2 scallop BAC clones (Zhao et al., 2012), there may exist groups of functionally related genes that are linked, which could cluster in scallop.

For comparison analysis, the distributions of TLR signaling pathway genes in five model species (Table 4) were obtained from the NCBI database (NCBI Map Viewer, http://www.ncbi.nlm.nih.gov/mapview/). In Mus musculus, all the five TLR signaling pathway genes locate on five non-homologous chromosome pairs. However, there are different distribution patterns in the remaining four species. In Caenorhabditis elegan, there are two TLR components (Tol-1 and IκB-1) co-locate on chromosome 1, 9.2 Mb apart. In Drosophila melanogaster, cactus and dorsal, which are homologous with IκB and NFκB, respectively, are co-located on the long arm of chromosome 2, 1.1 Mb apart, while MyD88 is located on the other arm of chromosome 2. In Danio rerio, NFκB3 spaces out TRAF6 49.5 Mb apart on chromosome 7. In Homo sapiens, TRAF6 is located on the short arm of chromosome 11, while NFκB3 is located on the long arm of the same chromosome. In summary, the candidate immune genes TLR, MyD88, TRAF6, IκB and NFκB are distantly linked in the latter four species. As to C. farreri, the five immune genes studied located in five non-homologous chromosome pairs, indicating that the TLR pathway may not show significant clustering as in Mus musculus. These TLR signaling pathway genes were significantly more distant than other functionally related genes, such as lipopolysaccharide and beta-1,3glucan binding protein genes and membrane transport genes. However, the non-clustering of these genes possibly has little effect on the immune response. In D. melanogaster, there is no significant difference in gene expression between clustered and non-clustered immune genes (Wegner, 2008). Here, the co-expression of TLR signaling pathway genes in C. farreri may not act in a distance-dependent way.

Table 4 Chromosomal localization of Toll-like receptor signal pathway genes in five model organisms Organism

TLR

Caenorhabditis Tol-1(171635*), elegan (2n = 12) Chromosome 1

MyD88 Unknown

TRAF6 TRF-1(176767*), Chromosome 3

IκB IκB-1(172817*), Chromosome 1

NFκB Unkown

Drosophila melanogaster (2n = 8)

Cactus (34969*), Toll-6 (39663*), MyD88 (35956*), TRAF6 (31746*), Chromosome 2L, Chromosome 3L, 71C2 Chromosome 2R, C5 Chromosome X, 7D16 35F1

Dorsal (35047*), Chromosome 2L, 36C8-C9

Danio rerio (2n = 50)

TLR-21 (402884*), Chromosome 16

MyD88 (403145*), Chromosome 24

TRAF6 (554561*), Chromosome 7

Unknown

NFκB3 (425099*), Chromosome 7

Mus musculus (2n = 40)

TLR-4 (21898*), Chromosome 4, C1

MyD88 (17874*) Chromosome 9, F3

TRAF6 (22034*), Chromosome 2, E2

NFκBI (18035*), Chromosome 12, C1-C3

NFκB3 (19697*), Chromosome 19, B1-B3

Homo sapiens TLR-2 (7097*), MyD88 (4615*), TRAF6 (7189*), (2n = 46) Chromosome 4, q32 Chromosome 3, p22 Chromosome 11, p12 Note: * Gene ID in NCBI GENE database.

FISH is a powerful tool significantly contributing to aquaculture genome research. FISH mapping of multicopy genes and repetitive elements has been frequently reported in scallop. Huang et al. (2007) mapped ribosomal DNA and (TTAGGG)n telomeric sequence to chromosomes in Patinopecten yessoensis. Zhang et al. (2007) detected histone H3 gene sites by FISH in four scallops, C. farreri, P. yessoensis, C. nobilis, and Argopecten irradians. All these results have led to research

IκBA (4792*), NFκB3 (5970*), Chromosome 14, q13 Chromosome 11, q13

advance on bivalve evolution and facilitated chromosome identification. However, there is a limited range of probes derived from multi-copy genes and repetitive elements. Mapping of large-insert clones will extend the application of FISH. Nine P1 clones were mapped in the eastern oyster, Crassostrea virginica, identifying seven chromosomes (Wang et al., 2005). In C. farreri, Zhang et al. (2008) identified eight of nineteen chromosomes by co-hybridizing eight fosmid clones. In the present study,

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we anchored five BAC clones with immune genes to five non-homologous chromosome pairs. These results will provide useful probes for chromosome identification of C. farreri.

Acknowledgements We thank Xunshan Aquatic Product Group Co., Ltd. (Rongcheng, China) for scallop samples. This research was financially supported by the National Natural Science Foundation of China (31270047), the National High Tech R&D Program (2012AA10A410), the National Basic Research Program of China (2010CB126402), and the National Key Technology R&D Program of China (2011BAD45B01 and 2011BAD13B05).

References Ashida, M., and Brey, P. T., 1998. Recent advances on the research of the insect prophenoloxidase cascade. In: Molecular Mechanisms of Immune Responses in Insects. Chapman and Hall, London, 135-172. Belvin, M. P., and Anderson, K. V., 1996. A conserved signaling pathway: The Drosophila toll-dorsal pathway. Annual Reviews Cell and Developmental Biology, 12: 393-416. Chen, W., Meaux, J., and Lercher, M. J., 2010. Co-expression of neighbouring genes in Arabidopsis: Separating chromatin effects from direct interactions. BioMed Central Genomics, 11: 178. Coscia, M. R., Giacomelli, S., and Oreste, U., 2011. Toll-like receptors: An overview from invertebrates to vertebrates. Invertebrate Survival Journal, 8: 210-226. Gao, Q., Song, L., Ni, D., Wu, L., Zhang, H., and Chang, Y., 2007. cDNA cloning and mRNA expression of heat shock protein 90 gene in the haemocytes of Zhikong scallop Chlamys farreri. Comparative Biochemistry and Physiology Part B, 147: 704-715. Hibino, T., Loza-Coll, M., Messier, C., Majeske, A. J., Cohen, A. H., Terwilliger, D. P., Buckley, K. M., Brockton, V., Nair, S. V., Berney, K., Fugmann, S. D., Anderson, M. K., Pancer, Z., Cameron, R. A., Smith, L. C., and Rast, J. P., 2006. The immune gene repertoire encoded in the purple sea urchin genome. Developmental Biology, 300: 349-365. Hoffmann, J. A., Kafatos, F. C., Janeway, C. A., and Ezekowitz, R. A., 1999. Phylogenetic perspectives in innate immunity. Science, 284: 1313-1318. Hoffmann, J. A., and Reichhart, J. M., 2002. Drosophila innate immunity: An evolutionary perspective. Nature Immunology, 3: 121-126. Huang, X., Hu, X., Hu, J., Zhang, L., Wang, S., Lu, W., and Bao, Z., 2007. Mapping of ribosomal DNA and (TTAGGG)n telomeric sequence by FISH in Patinopecten yessoensis. Journal of Molluscan Studies, 73: 393-398. Hu, L., Shang, W., Sun, Y., Wang, S., Ren, X., Huang, X., and Bao, Z., 2011. Comparative cytogenetics analysis of Chlamys farreri, Patinopecten yessoensis, and Argopecten irradians with C0t-1DNA by fluorescence in situ hybridization. Evidence-Based Complementary and Alternative Medicine, 2011: 785831. Inamori, K., Ariki, S., and Kawabata, S., 2004. A Toll-like receptor in horseshoe crabs. Immunological Reviews, 198: 106115.

Janeway, J. C., 1989. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harbor Symposia on Quantitative Biology. 54: 1-13. Kawai, T., and Akira, S., 2010. The role of pattern-recognition receptors in innate immunity: Update on Toll-like receptors. Nature Immunology, 11: 373-384. Lee, J. M., and Sonnhammer, E. L., 2003. Genomic gene clustering analysis of pathways in eukaryotes. Genome Research, 13: 875-882. Levan, A., Fredga, K., and Sandberg, A. A., 1964. Nomenclature for centrometric position on chromosomes. Hereditas, 52: 201-220. Liu, X., and Han, B., 2009. Evolutionary conservation of neighboring gene pairs in plants. Gene, 437: 71-79. Lorenzi, L., Molteni, L., and Parma, P., 2010. FISH mapping in cattle (Bos taurus L.) is not yet out of fashion. Journal of Applied Genetics, 51: 497-499. Meijer, A. H., Gabby Krens, S. F., Medina Rodriguez, I. A., He, S., Bitter, W., Ewa Snaar-Jagalska, B., and Spaink, H. P., 2004. Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish. Molecular Immunology, 40: 773-783. Qiu, L., Song, L., Xu, W., Ni, D., and Yu, Y., 2007a. Molecular cloning and expression of a Toll receptor gene homologue from Zhikong scallop, Chlamys farreri. Fish and Shellfish Immunology, 22: 451-466. Qiu, L., Song, L., Yu, Y., Xu, W., Ni, D., and Zhang, Q., 2007b. Identification and characterization of a myeloid differentiation factor 88 (MyD88) cDNA from Zhikong scallop Chlamys farreri. Fish and Shellfish Immunology, 23: 614-623. Qiu, L., Song, L., Yu, Y., Zhao, J., Wang, L., and Zhang, Q., 2009. Identification and expression of TRAF6 (TNF receptor-associated factor 6) gene in Zhikong scallop Chlamys farreri. Fish and Shellfish Immunology, 26: 359-367. Roach, J. C., Glusman, G., Rowen, L., Kaur, A., Purcell, M. K., Smith, K. D., Hood, L. E., and Aderem, A., 2005. The evolution of vertebrate Toll-like receptors. Proceedings of the National Academy of Sciences, 102: 9577-9582. Sambrook, J., Fritsch, E. F., and Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. 2nd edition. Cold Spring Harbor Laboratory, New York, 1659pp. Sasaki, N., Ogasawara, M., Sekiguchi, T., Kusumoto, S., and Satake, H., 2009. Toll-like receptors of the ascidian, Ciona intestinalis: Prototypes with hybrid functionalities of vertebrate Toll-like receptors. Journal of Biological Chemistry, 284: 27336-27343. Su, J., Song, L., Xu, W., Wu, L., Li, H., and Xiang, J., 2004. cDNA cloning and mRNA expression of the lipopolysaccharide- and beta-1,3-glucan-binding protein gene from scallop Chlamys farreri. Aquaculture, 239: 69-80. Wang, H., Song, L., Li, C., Zhao, J., Zhang, H., Ni, D., and Xu, W., 2007, Cloning and characterization of a novel C-type lectin from Zhikong scallop Chlamys farreri. Molecular Immunology, 44: 722-731. Wang, M., Yang, J., Zhou, Z., Qiu, L., Wang, L., Zhang, H., Gao, Y., Wang, X., Zhang, L., Zhao, J., and Song, L., 2011. A primitive Toll-like receptor signaling pathway in mollusk Zhikong scallop Chlamys farreri. Developmental and Comparative Immunology, 35: 511-520. Wang, Y., Xu, Z., Pierce, J. C., and Guo, X., 2005. Characterization of Eastern oyster (Crassostrea virginica Gmelin) chromosomes by fluorescence in situ hybridization with bacteriophage P1 clones. Marine Biotechnology, 7: 207-214. Wegner, K. M., 2008. Clustering of Drosophila melanogaster

ZHAO et al. / J. Ocean Univ. China (Oceanic and Coastal Sea Research) 2015 14: 1075-1081 immune genes in interplay with recombination rate. PLoS ONE, 3: e2835. Yu, Y., Qiu, L., Song, L., Zhao, J., Ni, D., Zhang, Y., and Xu, W., 2007. Molecular cloning and characterization of a putative lipopolysaccharide-induced TNF-a factor (LITAF) gene homologue from Zhikong scallop Chlamys farreri. Fish and Shellfish Immunology, 23: 419-429. Zhang, H., Song, L., Li, C., Zhao, J., Wang, H., Gao, Q., and Xu, W., 2007. Molecular cloning and characterization of a thioester-containing protein from Zhikong scallop Chlamys farreri. Molecular Immunology, 44: 3492-3500. Zhang, L., Bao, Z., Wang, S., Huang, X., and Hu, J., 2007. Chromosome rearrangements in Pectinidae (Bivalvia: Pteriomorphia) implied based on chromosomal localization of histone H3 gene in four scallops. Genetica, 130: 193-198.

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Zhang, L., Bao, Z., Wang, S., Hu, X., and Hu, J., 2008. FISH mapping and identification of Zhikong scallop (Chlamys farreri) chromosomes. Marine Biotechnology, 10: 151-157. Zhang, L., Li, L., and Zhang, G., 2011. A Crassostrea gigas Toll-like receptor and comparative analysis of TLR pathway in invertebrates. Fish and Shellfish Immunology, 30: 653-660. Zhao, B., Cheng, J., Chen, L., Yu, N., Huang, X., and Bao, Z., 2013. Construction of three bacterial artificial chromosome (BAC) libraries for Zhikong scallop (Chlamys farreri). Periodical of Ocean University of China, 43: 57-63. Zhao, C., Zhang, T., Zhang, X., Hu, S., and Xiang, J., 2012. Sequencing and analysis of four BAC clones containing innate immune genes from the Zhikong scallop (Chlamys farreri). Gene, 502: 9-15. (Edited by Qiu Yantao)