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Highly conserved linkage homology between birds and turtles: Bird and turtle chromosomes are precise counterparts of each other Yoichi Matsuda1,2*, Chizuko Nishida-Umehara1,2, Hiroshi Tarui3, Asato Kuroiwa1,2, Kazuhiko Yamada1, Taku Isobe1, Junko Ando1, Atushi Fujiwara4, Yukako Hirao3, Osamu Nishimura3, Junko Ishijima1, Akiko Hayashi5, Toshiyuki Saito5, Takahiro Murakami1, Yasunori Murakami6, Shigeru Kuratani6 & Kiyokazu Agata3 1Laboratory of Animal Cytogenetics, Division of Genome Dynamics, Creative Research Initiative BSousei^, Hokkaido University, North 10 West 8, Kita-ku, Sapporo 060-0810, Japan; Tel: +81-11-7062619; Fax: +81-11- 7366304; E-mail: yoimatsu@ees.hokudai.ac.jp; 2Division of Biological Sciences, Graduate School of Science, Hokkaido University, North 10 West 8, Kita-ku, Sapporo 060-0810, Japan; 3Laboratory for Evolutionary Regeneration Biology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan; 4Immunology Section, National Research Institute of Aquaculture, Fisheries Research Agency, Tamaki, Mie 519-0423, Japan; 5Transcriptome Profiling Group, Research Center for Radiation Safety, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan; 6Laboratory for Evolutionary Morphology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan Matsuda and Nishida-Umehara contributed equally to this work. * Correspondence Received 26 April 2005. Received in revised form and accepted for publication by Nobuo Takagi 9 June 2005 Key words: comparative mapping, evolution, linkage, sex chromosome, snake, turtle, Z chromosome Abstract The karyotypes of birds, turtles and snakes are characterized by two distinct chromosomal components, macrochromosomes and microchromosomes. This close karyological relationship between birds and reptiles has long been a topic of speculation among cytogeneticists and evolutionary biologists; however, there is scarcely any evidence for orthology at the molecular level. To define the conserved chromosome synteny among humans, chickens and reptiles and the process of genome evolution in the amniotes, we constructed comparative cytogenetic maps of the Chinese soft-shelled turtle (Pelodiscus sinensis) and the Japanese four-striped rat snake (Elaphe quadrivirgata) using cDNA clones of reptile functional genes. Homology between the turtle and chicken chromosomes is highly conserved, with the six largest chromosomes being almost equivalent to each other. On the other hand, homology to chicken chromosomes is lower in the snake than in the turtle. Turtle chromosome 6q and snake chromosome 2p represent conserved synteny with the chicken Z chromosome. These results suggest that the avian and turtle genomes have been well conserved during the evolution of the Arcosauria. The avian and snake sex Z chromosomes were derived from different autosomes in a common ancestor, indicating that the causative genes of sex determination may be different between birds and snakes. Introduction The karyotypes of birds, turtles and snakes are prin- cipally composed of two major chromosomal com- ponents, namely macrochromosomes and micro- chromosomes, which differ with respect to physical size, though the boundary between the two is not necessarily defined. Turtles have variable numbers of Chromosome Research (2005) 13:601–615 DOI : 10.1007/s10577-005-0986-5 # Springer 2005 chromosomes, ranging from 2n = 26 to 68 (Ayres et al. 1969, Bickham & Baker 1976, Bickham et al. 1983). The most common diploid number is around 50Y52 in Emydidae, including 12Y14 pairs of macro- chromosomes and 12Y14 pairs of microchromo- somes, and 66 in Trionycidae, including 8Y9 pairs of macrochromosomes and 24Y25 pairs of micro- chromosomes (Bickham & Baker 1976, Bickham et al. 1983). Similar karyotypes are also observed in birds. The bird karyotypes are remarkably uniform, and the modal number is around 80, which consists of 7Y10 pairs of macrochromosomes, including ZW sex chromosomes, and a large number of micro- chromosomes, though diploid chromosome numbers range from 50 in the Falconidae to over 100 in the Rallidae and Ramphastidae (Takagi & Sasaki 1974, de Boer 1984, Belterman & de Boer 1984, Sasaki et al. 1984). The first three pairs of macrochromo- somes are outstandingly large, and the morphological similarities of the largest three chromosomes are shared by many of the species in diverse avian orders (Takagi & Sasaki 1974). Based on the comparison of G-banding patterns between bird and turtle chromo- somes, Takagi & Sasaki (1974) suggested that the largest three pairs might have been transmitted without many structural changes from a common ancestor of birds and turtles. The range of karyotypic variation is very narrow in snakes. The most com- mon diploid number of snakes is 2n = 36, which consists of eight pairs of macrochromosomes and ten pairs of microchromosomes (Bec¸ak et al. 1964, Bec¸ak & Bec¸ak 1969, Singh 1972). The close karyological relationship between birds and reptiles has long been a topic of speculation among cytogeneticists and evo- lutionary biologists; however, there is hardly any evidence to confirm this similarity at the molecular level. Graves & Shetty (2000) demonstrated by com- parative chromosome painting of the turtle (Chelodina longicollis) that chicken chromosome 4 painted the fourth largest pair of autosomes and the short arm of chromosome 7/8 in the turtle. The chicken Z chromo- some was equivalent to the fifth-largest autosomal pair of the turtle (Graves & Shetty 2001). These results suggest that chromosome homology might have been preserved between turtles and birds; however, gene- based conserved synteny between the two genera has not been verified by comparative gene mapping. As detailed physical and genetic linkage maps of the chicken have been constructed, extensive chromo- some homology between the chicken and human genomes (about 100 conserved syntenic segments) has been revealed (Groenen et al. 2000, Schmid et al. 2000). The comparative maps of functional genes between chicken and mammalian species provide new insights into the evolution of vertebrate genomes (Burt et al. 1995, 1999, Nanda et al. 1999, 2000, Groenen et al. 2000, Schmid et al. 2000, Burt 2002). This approach makes it possible to compare chro- mosomes between species belonging to different classes or phyla, but reptiles have not been the subject of comparative mapping because there are almost no DNA probes for functional genes in reptiles. Comparative mapping between birds and reptiles would provide more detailed information about the evolution of the amniotes, which has not been studied yet. Partial sequencing of a large number of cDNAs to develop expressed sequence tags (ESTs) facilitates gene discovery using the EST database (dbEST), and ESTs provide a ready source of DNA probes for comparative gene mapping between any species. Orthologues are homologous genes from different species that evolved from a common ancestral gene and normally retain the same function during evolution. The identification of orthologous genes from reptile EST clones facilitates the direct comparison of human, avian and reptilian genomes by comparative gene mapping. In this study we constructed cDNA libraries from the brain tissue and the 14-day-old whole embryos of the Chinese soft-shelled turtle and from the brain tissue of the Japanese four-striped rat snake. We iso- lated a large number of cDNA clones at random from the turtle and snake cDNA libraries, determined their partial sequences, and then searched for orthologues from the reptilian EST clones for comparative gene mapping. Here we address the relationships of genome organization between chicken and two reptilian species by constructing their comparative cytogenetic maps with the EST clones. Materials and methods Specimen Adult females and embryos of the Chinese soft- shelled turtle (Pelodiscus sinensis, Trionychidae, Testudinata) were purchased from a breeding farm in Japan, and used for constructing cDNA libraries and chromosome preparations. Wild individuals of 602 Y. Matsuda et al. the Japanese four-striped rat snake (Elaphe quad- rivirgata, Colubridae, Ophidia) were captured in the field in Japan and used for the experiments. Construction of cDNA libraries, DNA sequencing and database analysis The sources of RNA used for constructing cDNA libraries were the brain tissue of an adult female and whole 14-day embryos for the turtle, and the brain tissues of eighteen adult male and female individuals for the snake. Poly (A) mRNAs were isolated from the fresh tissues, and cloned into the l uni-ZAP vector (Stratagene) using standard protocols. Lambda uni-ZAP clones were converted into pBluescript SK (+) clones, and transformed into XL1-Blue bacterial cells (Stratagene). Colonies were randomly picked and transferred into 96-well plates using the FQ_ Pix (GENETIX). The clones were grown overnight, and the plasmid DNAs were prepared using MultiScreen- NA and FB plates (Millipore, Bedford, MA). Sequencing reactions were performed with dideoxy dye-labelled terminator using SK primer according to the manufacturer’s protocol (Applied Biosystems), and the nucleotide sequences were determined using an ABI PRISM3700 DNA Analyzer (Applied Bio- systems). The nucleotide sequence comparisons versus the National Center for Biotechnology Infor- mation (NCBI) database were performed using the Blast X program. Individual ESTs were translated in all reading frames and compared against the NCBI Fnon-redundant_ nucleotide and/or peptide sequence database (http://www.ncbi.nlm.nih.gov/blast/Blast. cgi?). All the EST clones mapped to turtle and snake chromosomes were deposited in DNA Data Bank of Japan (DDBJ; http://www.ddbj.nig.ac.jp/Welcome. html). Cell culture, chromosome preparation and FISH Preparation of R-banded chromosomes and fluores- cence in-situ hybridization (FISH) were performed as described previously (Matsuda & Chapman 1995, Suzuki et al. 1999). The fibroblast cells collected from the embryos of the turtle and the heart tissues of the female snakes were cultured in 199 medium supplemented with 15% fetal bovine serum at 26-C in 5% CO2 5-Bromodeoxyuridine (BrdU) was incor- porated during the late replication stage for differen- tial staining. The cells were harvested after colcemid treatment for 1 h, suspended in 0.075 mol/L KCl, fixed in 3:1 methanol:acetic acid three times, then dropped on glass slides and air-dried. R-banded chromosomes were obtained by exposure of chromo- some slides to UV light after staining with Hoechst 33258. Slides were kept at j80-C until use. The cDNA fragments amplified from the EST clones by PCR were used as probes for FISH mapping. The inserts were amplified using Insert Check Ready Blue (Toyobo) which included univer- sal M13 P7 and M13 P8 primers. DNA amplification was performed in a total reaction volume of 100 ml containing 100 ng of plasmid as a template. PCR products were electrophoresed on a 1% agarose gel, recovered using Sprec-DNA Recovery Filter Tubes (Takara Biomedical), and purified according to the manufacturer’s instructions. The DNA probes were labelled by nick translation with biotin-16-dUTP (Roche Diagnostics) using a standard protocol. The hybridized cDNA probes were reacted with goat anti-biotin antibodies (Vector Laboratories), and then stained with fluoresceinated donkey anti-goat IgG (Nordic Immunology). The slides were stained with 0.50 mg/ml propidium iodide for observation. FISH images were observed under a Nikon fluorescence microscope using Nikon filter sets B-2A and UV-2A. Kodak Ektachrome ASA100 films were used for microphotography. Molecular cloning of reptilian homologues of chicken Z-linked genes The turtle and snake homologues of the chicken Z-linked genes, DMRT1, ACO1 and CHD1, were molecularly cloned by RT-PCR. Total RNAs were extracted from testes of the turtle and the snake using Trizol (Invitrogen). The cDNAs were synthesized using SuperScript II Rnase H(j) Reverse Transcrip- tase (Invitrogen).Various sets of PCR primers were synthesized based on the conserved regions of the three genes. The degenerate primer pairs used in the RT-PCR reactions were as follows: Primers for DMRT1: F1, 50-GCA GCG GGT GAT GGC NGC NCA GGT-30; R1, 50-GCC AGA ATC TTG ACT GCT GGG YGG YGA-30. Primers for ACO1:F1, 50- GAC AGY TTR CAR AAG AAT CAR GAY-30; R1, 50-CCY TTR AAT CCT TGC TTN GYT CC-30; F2, 50-GTG CTC ACY RTN ACN AAG CAC CT-30; R2, 50-AGG TCT CCC TGN GTD ATN GCY TC-30. Primers for CHD1:F1, 50-CTC CAG AAG ATG Comparative chromosome maps of reptiles 603 TGG AAT ATT ATA AYT GC-30; R1, 50-TAT TGT TTT NCC NAG NCC CAT TTC A-30; F2, 50-TGG TGC AAA GGN AAT AGT TGY ATH C-30; R2, 50- AGY TCY TTG TGN AGR CTT GCA TAA CC-30; F3, 50-TGT AAC CAT TGC TAC CTC ATT AAR CC-30; R3, 50-AGA TCA TTY TGT GGA TTC CAR TCN GAA TCR-30. Amplification of the fragments was achieved using the Ex Taq system (Takara Biomedical). The PCR conditions were an initial denaturation at 94-C for 2 min, followed by 35 cycles of 94-C for 30 s, 60-C for 30 s and 72-C for 30 s; and finally 72-C for 5 min. The PCR products with more than one band were separately isolated and subcloned using the pGEM-T Easy Vector System (Promega). Figure 1. Giemsa-stained karyotypes of chicken (2n = 78) (a), the Chinese soft-shelled turtle (2n = 66) (b) and the Japanese four-striped rat snake (2n = 36) (c). 604 Y. Matsuda et al. Table 1. List of 59 EST clones mapped to Chinese soft-shelled turtle chromosomes. Closed boxes indicate conserved syntenies between chicken and the Chinese soft-shelled turtle, which are equivalent between the two species. Gene symbola Insert length (kb) Sequence length (bp) E-value Chromosome location in human Chromosome location in the turtle Chromosome location in chicken Accession Number PECI 2.0 809 e-105 6p24.3 1p AU312267 NAV3 1.2 1033 e-123 12q14.3 1p 1 AU312263 NAPILI 2.0 811 2e-90 12q21.1 1p 1 AU312281 TRA1 2.5 531 2e-84 12q24.2-q24.3 1p 1 AU312248 RPL3 1.6 498 1e-95 22q13 1p 1 AU312265 MAP3K7IP1 1.0 561 3e-78 22q13.1 1p 1 AU312271 DPT 1.2 680 1e-79 1q12-q23 1q AU312278 RPL8 1.0 824 e-148 8q24.3 1q AU312288 USP5 1.2 800 e-106 12p13 1q 1 AU312276 PPP1CC 2.5 519 3e-64 12q24.1-q24.2 1q 15 AU312244 ZNF294 2.0 543 4e-71 21q22.11 1q 1 AU312299 C21orf33 2.5 655 e-113 21q22.3 1q 1 AU312295 EIF2S3 2.0 658 e-134 Xp22.2-p22.1 1q 1 AU312268 ARF1 2.0 649 2e-96 1q42 2p 2 AU312289 LAMR1 1.0 829 e-129 3p21.3 2p AU312259 GARS 2.0 802 e-162 7p15 2p 2 AU312286 BAZIB 2.0 756 3e-97 7q11.23 2q 19 AU312277 NSMAF 4.0 521 5e-97 8q12-q13 2q 2 AU312241 EIF3S6 1.6 548 e-110 8q22-q23 2q 2 AU312274 KIAA0153 1.8 809 7e-49 22q13.31 2q AU312266 NVL 2.0 794 e-101 1q41-q42.2 3p 3 AU312294 EPHX1 1.7 737 4e-79 1q42.1 3p 3 AU312282 XPO1 2.3 573 e-124 2p16 3p 3 AU312293 RNASEH1 3.0 520 5e-70 2p25 3q 3 AU312243 ARG1 1.2 760 5e-87 6q23 3q AU312300 UCHL1 3.0 532 4e-77 4p14 4q 4 AU312247 PAPSS1 1.5 810 e-166 4q24 4q 4 AU312290 HMGB2 1.8 901 2e-90 4q31 4q 4 AU312262 FAT 2.1 527 e-100 4q34-q35 4q 4 AU312273 C14orf166 1.0 687 4e-95 14q22.1 5q 5 AU312301 EIF2S1 1.5 461 6e-73 14q24.1 5q 5 AU312298 COQ6 1.0 925 9e-66 14q24.2 5q 5 AU312260 EIF2B2 2.1 839 e-108 14q24.3 5q 5 AU312297 ACTC 1.5 739 e-148 15q11-q14 5q 5 AU312292 CLTA 1.0 799 3e-96 9p13 6p Z AU312285 CHD1 2.0 798 5e-92 5q15-q21 6q AU312270 ALDH7A1 1.7 616 2e-84 5q31 6q Unknown AU312269 FBP1 1.5 732 e-134 9q22.3 6q Z AU312291 CDK9 1.5 275 5e-45 9q34.1 6q AU312239 SIAT8C 1.4 420 1e-86 18q21.31 6q AU312252 SLC20A1 3.0 740 2e-48 2q11-q14 micro AU312245 SCG2 2.3 636 2e-53 2q35-q36 micro 9 AU312275 RASA2 3.0 561 3e-67 3q22-q23 micro 9 AU312254 PLD1 3.0 517 e-102 3q26 micro 9 AU312251 HNRPD 1.5 504 2e-87 4q21.1-q21.2 micro AU312284 CTNNA1 4.5 520 5e-88 5q31 micro 13 AU312240 SKP1A 1.8 815 8e-95 5q31 micro AU312280 SPARC 2.0 513 2e-65 5q31.3-q32 micro 13 AU312255 CSNK1A1 2.0 449 5e-73 5q32 micro 13 AU312296 GTF2I 2.0 761 1e-90 7q11.23 micro 19 AU312279 PTN 2.2 516 4e-71 7q33-q34 micro 1 AU312250 LHX2 1.5 710 e-129 9q33-34.1 micro 17 AU312297 COX15 1.2 519 3e-81 10q24 micro 6 AU312249 KARS 1.5 516 e-106 16q23-q24 micro 11 AU312242 Comparative chromosome maps of reptiles 605 The 50-UTR of the DMRT1 gene was amplified using the 50 RACE system version 2.0 (Invitrogen). The two pairs of primers for the ACO1 gene, F1/R1 and F2/R2, amplified 794 bp and 797 bp products, respec- tively. The three pairs of primers for the CHD1 gene, F1/R1, F2/R2, and F3/R3, amplified 443 bp, 584 bp and 401 bp products, respectively. The nucleotide sequences of the cDNA clones were determined using an ABI PRISM3100 DNA Analyzer (Applied Biosystems) after performing the sequencing reaction with dideoxy dye-labelled terminator using SK primer according to the manufacturer’s protocol. Results The chromosome number of the Chinese soft-shelled turtle was 66 with nine pairs of macrochromosomes and 24 pairs of microchromosomes, which was quite similar to that of the chicken (Figure 1a, b). The present study confirmed the previous data reported by Sato & Ota (2001). The Japanese four-striped rat snake had 2n = 36, with eight pairs of macro- chromosomes, including differentiated Z and W chromosomes, and 10 pairs of microchromosomes (Figure 1c). The submetacentric W chromosomes might have resulted from a pericentric inversion of the metacentric Z chromosome followed by partial deletion. We isolated 382 and 1150 non-redundant EST clones from the cDNA libraries constructed from the adult brain and the 14-day-old embryos of the Chinese soft-shelled turtle, respectively. Two thou- sand and ninety-seven non-redundant ESTs were also isolated from the brain cDNA library of the Japanese four-striped rat snake. EST clones with Blast X scores less than 1ej45 were classified as putative reptile homologues of human genes in this study. Fifty-nine turtle and 52 snake homologues of human orthologous genes were carefully selected by elimi- nating family genes (Tables 1 and 2), and cytogenet- ically localized to chromosomes by FISH (Figure 2). Forty turtle homologues were specifically local- ized to the six largest pairs of macrochromosomes, and the remaining 19 homologue clones were local- ized to chromosomes smaller than chromosome 6 (the microchromosomes) (Table 1). Ten conserved segments, to which two or more genes were mapped, were identified between the human chromosomes and turtle chromosomes. Chromosome homologies between chicken and the turtle were examined using the current information on the humanYchicken comparative map (Schmid et al. 2000, Burt 2002) (Figure 3). Twelve out of 13 clones located on the Chinese soft-shelled turtle chromosome 1 (Pelodis- cus sinensis chromosome: PSI) were localized to seven regions homologous to human chromosomes (Homo sapiens chromosome: HSA) 1q, 6p, 12p, 12q, 21q, 22q and Xp, where conserved synteny has been also identified in chicken chromosome 1 (Gallus gallus chromosome: GGA). Seven genes on PSI2 were localized to regions homologous to HSA1q, 3p, 7p, 7q, 8q and 22q, which are orthologous to GGA2. Five genes on PSI3 were localized to the conserved regions of GGA3 homologous to HSA1q, 2p and 6q. Four genes on PSI4 and five genes on PSI5 were localized to regions conserved between GGA4 and HSA4p and 4q, and between GGA5 and HSA14q and 15q, respectively. The locations of the turtle homo- logues on chicken chromosomes were searched using the annotation database of the first draft chicken genome assembly, Ensembl Chicken Web Server (URL: http://www.ensembl.org/Gallus_gallus/) (Inter- national Chicken Genome Sequencing Consortium Table 1.