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DNA RESEARCH 4, 371-378 (1997) A Fine Physical Map of Arabidopsis thaliana Chromosome 5: Construction of a Sequence-ready Contig Map Hirokazu KOTANI,1'* Shusei SATO,1 Masanobu FUKAMI, 2 Tsutomu HOSOUCHI,1 Naomi NAKAZAKI,1 Satomi OKUMURA,1 Tsuyuko WADA, 1 You-Guang Liu,3 Daisuke SHIBATA,3 and Satoshi TABATA1 Kazusa DNA Research Institute, 1532-3 Yana, Kisarazu, Chiba 292, Japan,1 Chiba Prefectural Agricultural Experimental Station, Midori-ku, Chiba 266, Japan,2 and Mitsui Plant Biotechnology Research Institute, TCI D-21, Sengen 2-1-6, Tsukuba, Ibaraki 305, Japan3 (Received 25 November 1997) Abstract A fine physical map of Arabidopsis thaliana chromosome 5 was constructed by ordering the clones from YAC, PI, TAC and BAC libraries of the genome using the sequences of a variety of genetic and EST markers and terminal sequences of clones. The markers used were 88 genetic markers, 13 EST markers, 87 YAC end probes, 100 YAC subclone end probes, and 390 end probes of PI, TAC and BAC clones. The entire genome of chromosome 5, except for the centromeric and telomeric regions, was covered by two large contigs 11.6 Mb and 14.2 Mb long separated by the centromeric region. The minimum tiling path of the chromosome was constituted by a total of 430 PI, TAC and BAC clones. The map information is available at the Web site http://www.kazusa.or.jp/arabi/. Key words: Arabidopsis thaliana chromosome 5; physical map; contig map accurate physical map as well as a sequence-ready contig map which covers the entire chromosome is essential Arabidopsis thaliana has been adopted as an excel- to proceed with the sequencing project. In this paper, a lent model organism for genetic, developmental and fine physical map of the entire chromosome 5 was conmetabolic studies of higher plants due to its short life structed by ordering the clones from YAC, PI, TAC and cycle. This plant has recently been targeted for se- BAC libraries using the information on the sequences of quencing of its entire genome because of its smaller various genetic and EST (expression sequence tag) markgenome size (approximately 120 Mb) and its lower fre- ers, terminal sequences of clones and subclones. quency of repetitive sequences. Highly and moderately repetitive DNA sequences have each been estimated to 2. Materials and Methods share around 10% of the total genome.1'2 Although 3 4 genetic linkage and recombinant inbred (RI) maps 2.1. Libraries (http://nasc.nott.ac.uk/new_ri_map.html) have been reFive kinds of libraries constructed from the genome ported, the construction of accurate physical maps of of A. thaliana ecotype Columbia were used for the conthe chromosomes should be highly advantageous not only of the map: CIC YAC12 from Arabidopsis struction for the genomic sequencing, but also for map-based gene cloning.5"7 Based on this view, yeast artificial chromo- Biological Resource Center at Ohio State University, some (YAC)-based physical maps of chromosomes 28 and IGF BAC (Mozo, T. unpublished, http://194.94.225.1 and TAMU 49 of A. thaliana have recently been constructed. We /private_workgroups/pg_101/bac.html), 13 BAC libraries from Dr. Ian Bancroft at John Innes have focused our attention on sequencing chromosome 5, 14 and TAC (Liu, Y.-G., manuscript in Centre, and PI consistent with the international agreement of the Ara10 preparation) libraries from Mitsui Plant Biotechnology bidopsis Genome Initiative. Even though the progress of YAC ordering, which consists of 31 contigs of chro- Research Institute. The genomic coverage of these limosome 5, has been reported,11 the construction of an braries has been estimated to be equivalent to 3-10 times the genomic size. 1. * t Introduction Communicated by Mituru Takanami To whom correspondence should be addressed. Tel. +81-43852-3920, Fax. +81-438-52-3921, E-mail: [email protected] The nucleotide sequence data of MUB10-Sp6 will appear in the DDBJ, EMBL and GenBank nucleotide sequence databases under accession number AB008800. 2.2. DNA markers and PCR primers RFLP (restriction fragment length polymorphism) markers, expression sequence tags (EST) markers, A Fine Physical Map of Arabidopsis thaliana Chromosome 5 372 [Vol. 4, Table 1. DNA markers used in this work. Categoly RFLP markers EST Markers CAPS Markers SSLP Markers Cloned Genes Markers g3715, m447, m217, g3837, m224, pCIT1243, tt4, g5962, pCIT718, g4560, g4111, g6843, KG-8, KG-10, GRF3, m423, m442, cral, m331, m435, g4130, m558, g3844, g2455, m211, pCITdllO, g2368, CD3-42 1, mi97, miP5-3, mil74, mi322, mi438, mil38, mi90, mi433, mi219, mil25, mil37, mil94, mi83, mi61, miP5-8, mi69, mi70, mil84, mi335, mi323 T04154, T04657, T13673, T88208, T13672, T21894, T14190, T04366, T04492, R65089, T22529, T21228 T20526 CTR1, PAI2, ASA1, PAT1, CHS1, NIT4, PHYC, DFR, 17C2, 10A10, EG7F2, ASB2, LYF3 nga225, ngal51, ngalO6, ngal39, S0262, ngal58 pCA72, nga76, S0191, ngal29, CDPK9, nga249 HANKA PDC1 (U71121), PDC2 (U71122), UBQ3 (L5363),15 GLU1 (Y09667), AGL15 (U22528), GDH1 (U37771), ASP2 (U15033),16 TSB1 (M23872),17 AGL8 (U33473), GAS1 (U03773).18 BEL1 (U39944),19 CER3 (X95962)20 PAP1 (U48448) cleaved amplified polymorphic sequence (CAPS) markers, simple sequence length polymorphism (SSLP) and genes used for designation of polymerase chain reaction (PCR) primers and their sources are given in Table 1. Resouces Arabidopsis Biological Resource Center (Columbus, Ohaio) Mitsui Plant Biotechnology Research Institute (Tsukuba, Ibaraki) http://genome-www.stanford.edu /Arabidopsis/EST2CIC.html http://genome-www.stanford.edu /Arabidopsis/maps/C APS_Chr5.html http://cbil.humgen.upenn.edu/~atgc/ genetic-mapping/gen_maps.html Sequence Data are derived from the DDBJ, EMBL and GenBank. Accession numbers are indicated in parentheses probes, we isolated 109 YAC clones, 1130 PI clones, 748 TAC clones, and 130 BAC clones. By ordering the overlapping clones using PCR products which were amplified with primers designed from the end sequences of isolated clones, the physical map of chromosome 5 of A. thaliana was finally constructed (Fig. 1). As discussed in sec2.3. Screening and clone analysis tion 3.3, we tentatively postulated the gap sizes to be To construct a physical map by clone ordering, PCR 0.4 Mb, and in Fig. 1, the physical distance of the chroscreening of DNA pools was done to identify positive mosome is indicated by the number in of megabase pairs clones harbored in YAC, PI and TAC libraries. If posifrom the end of contig 1 containing the top marker of tive clones were not selected in the PI and TAC libraries, chromosome 5. YAC clones formed 11 contigs, and 9 the IGF and TAMU BAC libraries were screened. End seout of 10 gaps of the YAC contigs were closed by assigquences of positive clones were determined by amplification with cassette-ligated PCR21 followed by sequencing ment of PI, TAC and BAC clones. Finally, the physical with dye terminator cycle sequencing kit FS or dRho- map consisted of two contigs covering 11.6 Mb (contig damine dye terminator cycle sequencing kit (Perkin- 1) and 14.2 Mb (contig 2) of the chromosome 5 genome, Elmer-ABI, Foster City, CA). The primers designed from respectively. Since the identity of the overlapping regions the end sequences of positive clones were used to confirm among YAC clones was confirmed using PCR products the overlap between clones and also to select clones in amplified with the primers designed from the end regions libraries for genome walking. YAC subclones were pre- of the corresponding PI, TAC or BAC clones, we assume pared as follows; DNA fragments in YAC clones were iso- the estimation error for the contig sizes to be less than lated by PFG electrophoresis (CHEF Mapper, Bio-Rad, 5%. The total size of the two contigs becomes approxiCA), digested with Bglll or EcoT22I (TAKARA, Kyoto) mately 25.8 Mb. As described below, the genetic distance and ligated into pUCH8. After transformation, the se- between PDC3 and nga76 in contigl is 71.56 cM and the quences of YAC subclones were determined. Sequence physical distance is 11.0 Mb, so that contig 1 could cover data of ends and subclones were subjected to homology a region of about 75 cM. Also, the genetic distance besearch by the BLAST and FASTA software (GCG, Madi- tween PHYC (74.35 cM) and CD3-42 (135.21 cM) corresponds to 13.4 Mb, so that contig 2 could cover about son, WI) 64 cM. Therefore, the 25.8-Mb region of the two contigs cover a minimum of 139 cM which corresponds to more 3. Results and Discussion than 95% of chromosome 5 (140.23 cM). A minimal tiling path is constituted by 430 bacterial clones, as shown in 3.1. Construction of physical map Fig. 1. These clones should be suitable for the genomic Using the PCR primers from 88 genetically mapped sequencing of chromosome 5 22 ' 23 as well as for positional markers, 13 EST markers, 87 YAC end probes, 100 YAC cloning of genes. As a result of a BLAST search of the subclone probes, and 390 of PI, TAC and BAC end 3 i hi 12 10 I ;! II 11 14 20 22 24 Figure 1. The physical map of A. thaliana chromosome 5. The chromosome is covered by two large contigs (contig 1; blue bar, contig 2; green bar) of clones split by a gap presumably containing the centromere. The scale on the abscissa indicates the distance in megabase pairs from the end of contigl containing the top genetic marker of chromosome 5. Eighty-five genetic markers and 13 EST markers (vertically black letters) and 87 YAC end markers (vertically red letters) were assigned on the map by the PCR method using the clones from YAC, P I , TAC and BAC libraries. PDC3 and CERX (vertically green letters) are described in the text. Name and size of CIC YAC clones are indicated in wide open boxes and the chimeric YAC clones are shown by yellow boxes. Bacterial clones constituting the minimum tiling path are shown by open boxes (PI and TAC clones) and closed boxes (IGF and TAMU BAC clones) below the wide YAC boxes. The names of bacterial clones are indicated at the right side of boxes as PI (M—), TAC (K—), IGF BAC (F—) and TAMU BAC (T—). The red striped box shows the gap region. The vertically blue letters show the accession numbers of A. thaliana genes mapped on chromosome 5 by BLAST search using the end sequences of bacterial clones and subclone sequences of YAC clones. 26 No. 6] H. Kotani et al. Table 2. Genes of Arabidopsis thaliana mapped on chromosome 5. Accession No. X94248 X77199 U73526 U03990 U40566 X71787 U02565 X92393 AF014806 Y14199 Z68495 M55551 AF001415 U1651 Z49268 Z86095 M84698 Z15058 U33014 X70990 L23985 X99923 X79195 AB005889 U88087 U91414 S69727 U75194 U39782 L19354 D89342 U24177 U87794 U12126 D17582 U78866 X83380 D13044 X52629 U29699 Z34294 D21138 L16789 Z22596 X97323 S77849 U11254 Y08966 J04737 X90458 Z49859 U33757 X16432 X06477 Z18242 D86122 M84701 X99097 U18411 L29261 L40358 Y13180 Clone' MJE20T7 MHD9T7 MNC9SP6 MAN10SP6 MJP2SP6 MEO23T7 MYB9SP6 C7A7ET3F MZH1T7 K18J7L K21P8R K21N7L F2A6SP6 K6E24L K15A21L MQE23SP6 K14P17R K19B10R K13O21L K13O21R K17E15L K20K6L F12J1SP6 MJG5T7 C6D8BG7A C11F1LET6H MZD3T7 MKH4T7 MHK7T7 MPK23SP6 K11D4R K3J11L MSN3T7 MNI3T7 MWB16T7 K22K2R MIC6T7 C11F10BG5B MIM11T7 MPM10T7 MRK3T7 MRB17T7 K5P14 MTE17 MZI11SP6 MYN21T7 K16J12 MPL14 MNB21SP6 MZF3T7 MOM7T7 MTFi3T7 C9A1E12D MVE14SP6 K22G18L MVF19SP6 MQB2T7 MZL20SP6 K11D18L MPA24T7 MUB2SP6 K1L20L Map position (Mb) 0.3 0.6 0.9 1.9 2.2 3.0 3.43.7 3.8 3.9 4.5 5.4 5.5 5.9 5.9 6.9 7.2 7.2 7.4 7.5 7.6 7.9 9.5 9.9 10.0 12.3 12.7 14.4 15.2 15.4 15.7 17.6 18.7 19.0 19.7 19.9 20.0 20.3 20.9 21.0 21.1 21.2 21.3 21.5 21.6 21.8 22.0 22.2 22.3 23.0 23.1 2X2 23.5 23.9 24.0 24.1 24.3 24.7 25.3 25.4 25.5 25.6 a) See Fig. 1 legend. C— show YAC subclones Score Description 1095 A. thaliana mRNA for ferritin. 1121 A. thaliana hsc70-1 gene. 3000 A. thaliana B' regulatory subunlt of PP2A (AtB'alpha) mRNA ""36ro""""A7th^liar«Columbi7gamm 2346 A. thaliana ubiquiBn activating enzyme 2 (UBA2) gene 1264 A. thaliana AAP2 mRNA (or amino ackJ permease. 1076 A. thaliana Columbia 14-3-3-like protein 1 (AMI) mRNA 1517 A. thaliana mRNA for KNAT4 homeobox protein 1885 A. thaliana alpha-gjucosidase 1 (Aglui) gene 791 A. thaliana mRNA for MAP3K delta-1 protein kinase 2303 A. thaliana CHLH gene. 385 A. thaliana transcription factor (AGL2) mRNA 3332 A. thaliana 14-3-3-like protein GF14j>hi (GRF5) 510 A. thaliana syntaxln homologue mRNA 825 A. thaliana P2 mRNA for zeta-crystallin-like protein 512 A. thaliana mRNA for peptkfyl-prolyl cis-trans Isomerase 2487 A. thaliana alpha-5 tubulin (TUA5) gene 1276 A.thaliana of bcb gene encoding blue copper-binding protein 2592 A. thaliana polyubiquitin (ubq4) gene 1865 582 1051 2224 1275 1539 2878 915 710 778 1315 2170 774 2147 1428 690 2624 515 1209 3332 584 736 956 2500 1191 1312 711 676 2591 2704 2926 2716 A. thaliana gene for sucrose synthase A. thaliana protein kinase (TOUSLED) mRNA A. thaliana mRNA for male sterility 2-like protein A. thaliana TGG2 gene for myrosinase. A. thaliana mRNA for ATMYB4 A. thaliana LON protease homolog (LON_ARA) mRNA A. thaliana heteromeric acetyl-CoA carboxylase biotin carboxylase subunit (CAC2) gene A. thaliana light-regulated glutamine synthetase isoenzyme A. thaliana germin-like protein (GLP6) mRNA A. thaliana lysine and histidine specific transporter mRNA A. thaliana ubkjuitin conjugating enzyme (UBC4) gene A. thaliana DNA for luminal binding protein A. thaliana 3-ketoacyl-acyl carrier protein synthase I JKAS I) mRNA A. thaliana transcription factor inhibitor I kappa B homologjnimiJ gene A. thaliana clone SRP-54B signal recognition particle 54 kDa subunit (Srp-54) gene A. thaliana mRNA for ERD1 protein A. thaliana putative arginine-aspartate-rtch RNA binding protein genes A.thaliana mRNA for gibberellin 20-oxidase A. thaliana rd29A gene and rd29B gene A.thaliana gene for USB small nuclear RNA (snRNA) A. thaliana light-dependent NADPH:protochlorophyllide oxidoreductase A (PorA) mRNA A. thaliana (C1A) myosin heavy chain mRNA A. thaliana katC mRNA for heavy chain of kinesirvlike protein A. thaliana small Ras-like GTP-binding protein (Ran-1) mRNA A. thaliana protein phosphatase mRNA A.thaliana mRNA for outward rectifying potassium channel KCO1 HSP81 -3=heat-shock Protein [Arabidopsis thaliana=thale-cress, Genomic, 3094 ntj. A. thaliana Columbia eootype metallothioneiri (MT1 b) gene A. thaliana gene encoding ABI2 protein A. thaliana ATPase gene A. thaliana mRNA for cytochrome P450 A.thaliana mRNA for copper transporterprotein 1174 A"thaiiana ubiquran1 conjugating enzyme i E ^ i i i c ^ g e m 923 1266 2931 1226 2196 658 567 3266 1368 745 A. thaliana EF-1 alpha-A4 (NAEEFTU 4) gene A. thaliana U2 RNA gene (U2.7) A. thaliana calnexin homolog A. thaliana mRNA for Mei2-like protein A. thaliana bete-3 tubulin (TUB3) gene A. thaliana mRNA for peroxkjase ATP15a A. thaliana IAA9 (IAA9) gene A. thaliana 1 -amino-1 -cyclopropanecarboxylate synthase (ACS5) mRNA A. thaliana calmodulin-binding protein mRNA A. thaliana mRNA for proteasome subunit proSc 375 A Fine Physical Map of Arabidopsis thaliana Chromosome 5 [Vol. 4, the order of genetic markers well coincides with that of the physical map, but the ratio of physical and genetic distances between markers varied significantly along the chromosome. Within the range of this resolution, relatively cold spots of recombination are seen in the middle region of each contig (nga249 to g4560 and mi69 to LYF3). This pattern resembles that of chromosome 4 of A. thaliana, but contrasts with that of tomato and wheat chromosomes in which recombination is strongly suppressed over large intervals at the centromere; rather, maximum recombination occur in the proximal regions.9 The average values observed for contigs 1 and 2 are 154 Kb/cM and 209 Kb/cM, respectively. This difference may reflect the number and distribution of hot spots in each contig. In the classic genetic map,3 CER3 was mapped near the LYF3 marker which was mapped to the 24-Mb position of the physical map. However, the cloned gene (Accession No. X95962) reported as CER322 was mapped to the 0.5-Mb position of the physical map. According to the sequence data of one of the YAC subclones derived from CIC2B9, the sequence was identical to that of X95962. It is likely that the cloned CER3 gene may be one of the unmapped eceriferum genes in addition to CER1-8 : This gene is indicated as CERX in Fig. 1. Physical map Rl map Figure 2. The alignment of the physical and RI maps of chromosome 5 and the ratio of the physical (kb) and genetic (cM) scales along the RI map. Fifty-three out of 54 markers which have been mapped on the RI map were assigned on the physical map according to Fig. 1. The gray boxes separated by an open box at the left represent two contigs and the gap consisting the physical map (Mb) and the shaded box at the middle shows the RI map (cM). The ratio of the physical scale (kb) to the genetic scale (cM) was calculated from the physical and genetic distances. The average values for contigs 1 and 2 are indicated by vertical broken lines. end sequences used for contig construction, 62 genes indicated in Table 2 were mapped on chromosome 5, as shown in Fig. 1. 3.2. Comparison of genetic and physical maps A comparison of genetic and physical maps is shown in Fig. 2. Fifty-four of the genetic distances have been derived from up to 100 lines of a recombinant inbred mapping population.4 Therefore, the two maps can be compared directly. Except for the markers mi90 and GRF3, In the RI map, PDC1 has been assigned as the top marker of chromosome 5. Using the sequence of the PDC1 gene registered in DNA databases (Accession No. U71121), we isolated two YAC clones (CIC11D9 and CIC6A9) and several PI and TAC clones. However, clones CIC11D9 and CIC6A9 have already been mapped on contig IV of chromosome 4.9 A PI clone, MJO17, containing the PDC1 gene also has the ATH1 gene24 which is used as a characterization marker of chromosome 4 and has also been mapped on CIC11D9 and CIC6A9.9 Therefore, we concluded that the PDC1 gene is located on chromosome 4 near the ATH1 locus. In a BLAST search of the end sequences of PI and TAC clones, the end sequence (Accession No. AB008800) of the Sp6 side of MUB10, which is mapped on the left end of CIC2B9, has a high similarity for the PDC1 gene. So, we concluded the top end (0 cM) of the RI map of chromosome 5 was the Sp6 side of MUB10 and is termed PDC3 (see Fig. 1). STL has been assigned as the bottom marker of the chromosome 5 RI map,4 but we did not succeed so far in isolating clones containing the STL marker because of the lack of a suitable probe. It is necessary to fill in the gap presumably encompassing the centromere and the telomeric 5-cM region between CD3-42 and STL for the complete coverage of the genetic map of chromosome 5. 3.3. Comparison of cytogenetic and physical maps Due to the lack of suitable genetic markers, the centromere was not mapped on the physical map in the present study. It seems likely, however, that the cen- No. 6] H. Kotani et al. tromeric region of chromosome 5 should be positiond in the gap region between contigs 1 and 2, because any end sequences of mapped clones did not contain the sequences similar to the tandemly repeated 180-bp sequence contained in plasmid pALl. 25 This repeated DNA sequence is present in arrays of over 50 kb, has been shown to locate in the heterochromatin moiety surrounding the centromere26'27 and to hybridize equally to both sides of the centromere on all five chromosome pairs.28 Thus, we assume that the centromere is present in the gap between the two contigs. So far now, this gap could not be closed due to the absence of YACs containing end probes of contigs in the YAC library. Such repetitive sequences in yeast cells have been reported to be unstable,12'28 and this may account for the difficulty isolating YAC clones covering the centromere. The genetic distance between nga76 and PHYC containing the gap is only 2.8 cM and 1.3 Mb of that region has already been covered. Since the average value estimated from the two contigs is 0.18 Mb/cM, we assume that the gap region is a few hundred kilo bases unless recombination is strongly suppressed at the gap. The physical distances of telomeres to the ends of contigs have also not been estimated. For the construction of the complete physical map, we have been continuing genomic walking using various libraries. Acknowledgments: We are grateful to Drs. S. Choi and R. Wing of Texas A & M University, T. Altman of Max-Plank Institute and I. Bancroft of John Innes Centre for providing the TAMU and IGF BAC libraries. We thank E. Mitsui for her excellent technical assistance and Dr. T. Hashimoto (Nara Institute of Science and Technology) for providing unpublished informations about the ordering of mi69-mi70 region. This work was supported by the Kazusa DNA Research Institute Foundation. 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