Downloaded from genome.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH A Physical Map of Chromosome 2 of Arabidopsis thaliana Eve Ann Zachgo, 2,4 Ming Li Wang, 1'2'4 Julia Dewdney, 1'2 David Bouchez, 3 Christine Carnilleri, 3 Stephen Belmonte, 2 Lu Huang, 2 Maureen Dolan, 2 and Howard M. Goodman 1'2'5 1Department of Genetics, Harvard Medical School and 2Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114; 3Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique (INRA), 78026 Versailles CEDEX, France A yeast artificial chromosome (YAC] physical map of chromosome 2 of Arabidopsis thaliana has been constructed by hybridization of 69 DNA markers and 61 YAC end probes to gridded arrays of YAC clones. Thirty-four YACs in four contigs define the chromosome. Complete closure of the map was not attained because some regions of the chromosome were repetitive or were not represented in the YAC library. Based on the sizes of the YACs and their coverage of the chromosome, the length of chromosome 2 is estimated to be at least 18 Mb. These data provide the means for immediately identifying the YACs containing a genetic locus mapped on Arabidopsis chromosome 2. The small flowering plant Arabidopsis thaliana is ters (Maluszynska and Heslop-Harrison 1991; A1- an excellent model system for metabolic, genetic, bini 1994; Copenhaver et al. 1995). We present and developmental studies in plants. Its haploid here a YAC contig physical map of chromosome nuclear genome is small (-100 Mb), consisting of 2 of A. thaliana. It is aligned to the genetic map five chromosomes with a low frequency of repet- (Lister and Dean 1993) and consists of four sets of itive DNA (Meyerowitz 1994). A classic genetic overlapping YAC clones covering 90% of the map (Koornneef et al. 1983) and maps based on chromosome. restriction fragment length polymorphisms (RFLPs) (Chang et al. 1988; Nam et al. 1989; Hauge et al. 1993; Lister and Dean 1993) have RESULTS AND DISCUSSION been published. A cosmid-based physical map containing -750 contigs obtained by DNA fin- The physical map of chromosome 2 of A. thaliana gerprinting (Hauge and Goodman 1992, shown in Figure 1 was obtained by the hybrid- AAtDB@http://probe.nalusda.gov:8300/) is avail- ization of 130 DNA probes to the CIC YAC library able, and -30% of the genome was covered by (Creusot et al. 1995). The entire CIC library (1152 anchoring the then available yeast artificial chro- clones) was grown in duplicate in a densely mosomes (YACs) to DNA-based genetic markers packed grid pattern on a single nylon filter the (Ward and Jen 1990; Grill and Somerville 1991; size of a microtiter plate. Dot colony hybridiza- Hwang et al. 1991). tion of radioactively labeled probes made from A more complete physical map of the Arabi- chromosome 2 markers to the grids allowed the dopsis genome would be highly advantageous for initial anchoring of YACs to chromosome 2. cloning genetic loci of interest, studying chromo- Many of the probes were RFLP-mapped genomic some structure, and sequencing the genome. Of cosmid clones from the fingerprinted cosmid the five A. thaliana chromosomes, chromosome 2 contig map. Supporting data were obtained by is one of the smallest (16%-17% of the genome) probing grids with additional cosmids from the and contains one of the two genomic rDNA clus- contig that contained the original RFLP-mapped clone. Other molecular markers were obtained 4These authors contributed equally to this work. from members of the Arabidopsis community. SCorresponding author. E-MAIL goodmanOfrodo.mgh.harvard.edu; FAX (617) 726- Positively hybridizing YAC clones identified 3535. on the grids were confirmed by hybridization of 6:19-25 ©1996 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/96 $5.00 GENOME RESEARCH~ 19 Downloaded from genome.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press ZACHGO ET AL. S,'Sl~~ O'Z~' :1~080 tl~" I1~~ o-z~,~ v 6.z'lS~JIO--"ll v ;~".J/.OI3-1 +;++ ~2N~8" in @ 11311310-I ~ N q. ~p ql, L'8£I- 8319013-I 9"/.£ ZJ "8 I/) 0 13i~ I~13"I 01118313-'! ZJ O0 IP.l.lOd ............... ......i! 0 +++++~j~+L-" v 0 I~1 I-AHJ.d r',,I ~3Z~13113-1 0 IV~.OI:3--I 8"1£ I ,t I,V6010-1 v Z'6Z lad Z'9Z~ I"ZZ~ lllll 8 lZ u- Z'IZ~ 01--' "-I I ++Ira IIIIm ..... , q,2 m HIl'"" L'91 I~~~ o 84~43--1 o o £19¥ l'lPl L'ZI S'OI~ °il IlO/..OlO-1 -- 0 ~ 1,,4 1,,4 1.4 20 ~ GENOME RESEARCH Downloaded from genome.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press A. 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CJ (J (J (J GENOME RESEARCH @ 21 Downloaded from genome.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press ZACHGO ET AL. the probe to Southern blots of restriction en- (480-kb mean) and low incidence of chimeric zyme-digested yeast DNA preparations contain- clones in the CIC library (Creusot et al. 1995), as ing the YAC. Comparison of the hybridization well as to the high number and broad distribu- patterns of the YACs to Arabidopsis genomic DNA tion of markers on chromosome 2. verified that the colony hybridization signal re- Five regions of the map could not be closed. sulted from homology of the probe to unique As depicted in Figure 1, a gap exists to the right of Arabidopsis genomic restriction fragments and the last YAC clone (CIC6C3) and the telomere at not cross-hybridizing yeast sequences. the end of the long arm of the chromosome. On Complete YAC coverage of the chromosome the short arm of the chromosome, the gap be- was not achieved by anchoring YACs to the chro- tween the telomere at the left end and clone mosome 2 markers. Therefore, a modification of CIC7Cll contains a cluster of rDNA genes. the inverse polymerase chain reaction (IPCR) was CIC7Cll may actually be part of this cluster as it used to generate end probes from the ends of the contains rDNA (Creusot et al. 1995). Shown as anchored YACs. Radioactively labeled end probes broken line boxes in Figure 1, three internal gaps were hybridized to the grids, and positively hy- exist between mi310 and mi421 (gap 1), 17288 bridizing YAC colonies were confirmed by South- and R-CIC12G5 (gap 2), and L-CIC11C8 and ern blot analysis. End probes generated from the R-CIC2E5 (gap 3). These gaps could not be closed YACs enabled one or more steps to be taken to due to the absence of YACs containing these se- extend the anchored YAC contigs and close gaps quences in the library or the repetitive nature of between contigs. Fewer than four steps were some of the end probes (Fig. 1). The instability of needed to reach the next anchored YAC or YAC cloned repetitive sequences in yeast makes it dif- contig. We attribute this to the large insert size ficult to physically map such regions (Schmidt et Figure 1 A physical map of chromosome 2 of A. thaliana. One hundred and thirty DNA probes, hybridized to the CIC YAC library, are ordered along the abscissa. Fifty-eight probes, mapped to chromosome 2 by RFLP analysis, serve as contact points between the genetic and physical maps. The position of markers mapped to the recombinant inbred lines (Lister and Dean 1993) are indicated in cM above the marker names. When a set of probes hybridized to the same YACs, their order was listed by their positions in the genetic map (Lister and Dean 1993). If no genetic or auxilliary data (e.g., cosmid contig; Hauge and Goodman 1992) were available, the exact probe order was indeterminate and was assigned arbitrarily. A line drawn above a marker indicates a difference in marker order between the physical and genetic maps. Markers designated RI have been mapped by us to the recombinant inbred populations and are in correct relative position (with one exception, 15414, marked RI-?) to the markers on either side (no number is indicated as adding markers, and using a different mapping program generates different absolute positions; Hauge et al. 1993); F2 indicates a marker mapped on other mapping populations (Chang et al. 1988; Nam et al. 1989; Hauge et al. 1993) but not the RI lines.