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1 Mol Gen Genomics (2005) 274: DOI /s ORIGINAL PAPER J. Y. Park Æ D. H. Koo Æ C. P. Hong Æ S. J. Lee J. W. Jeon Æ S. H. Lee Æ P. Y. Yun Æ B. S. Park H. R. Kim Æ J. W. Bang Æ P. Plaha Æ I. Bancroft Y. P. Lim Physical mapping and microsynteny of Brassica rapa ssp. pekinensis genome corresponding to a 222 kbp gene-rich region of Arabidopsis chromosome 4 and partially duplicated on chromosome 5 Received: 7 January 2005 / Accepted: 5 August 2005 / Published online: 9 November 2005 Ó Springer-Verlag 2005 Abstract We constructed a bacterial artificial chromosome (BAC) library, designated as KBrH, from high molecular weight genomic DNA of Brassica rapa ssp. pekinensis (Chinese cabbage). This library, which was constructed using HindIII-cleaved genomic DNA, consists of 56,592 clones with average insert size of 115 kbp. Using a partially duplicated DNA sequence of Arabidopsis, represented by 19 and 9 predicted genes on chromosome 4 and 5, respectively, and BAC clones from the KBrH library, we studied conservation and microsynteny corresponding to the Arabidopsis regions in B. rapa ssp. pekinensis. The BAC contigs assembled according to the Arabidopsis homoeologues revealed triplication and Communicated by W.R. McCombie J. Y. Park Æ C. P. Hong Æ S. J. Lee Æ J. W. Jeon S. H. Lee Æ P. Y. Yun Æ Y. P. Lim (&) Department of Horticulture, Chungnam National University, Kung-Dong 220, Yusong-Gu, Daejeon , South Korea yplim@cnu.ac.kr Tel.: Fax: D. H. Koo Æ J. W. Bang Department of Biology, College of Natural Science, Chungnam National University, Kung-Dong 220, Yusong-Gu, Daejeon , South Korea B. S. Park National Institute of Agricultural Biotechnology, 250th, Seodun-dong, Gwonseon-gu Suwon Gyeonggi-do, , South Korea H. R. Kim Arizona Genomics Institute (AGI), University of Arizona, Tucson, AZ 85721, USA P. Plaha Advanced Centre of Hill Bioresources & Biotechnology, HP Agricultural University, Palampur , HP, India I. Bancroft Department of Crop Genetics, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK rearrangements in the Chinese cabbage. In general, collinearity of genes in the paralogous segments was maintained, but gene contents were highly variable with interstitial losses. We also used representative BAC clones, from the assembled contigs, as probes and hybridized them on mitotic (metaphase) and/or meiotic (leptotene/pachytene/metaphase I) chromosomes of Chinese cabbage using bicolor fluorescence in situ hybridization. The hybridization pattern physically identified the paralogous segments of the Arabidopsis homoeologues on B. rapa ssp. pekinensis chromosomes. The homoeologous segments corresponding to chromosome 4 of Arabidopsis were located on chromosomes 2, 8 and 7, whereas those of chromosome 5 were present on chromosomes 6, 1 and 4 of B. rapa ssp. pekinensis. Keywords Arabidopsis Æ Brassica rapa ssp. pekinensis Æ BAC Æ Genome evolution Æ FISH Introduction Genus Brassica is one of the core genera in the subtribe Brassicinae and includes a number of crops with wide adaptation under varied agroclimatic conditions. Of the six widely cultivated species of Brassica, B. rapa(aa, 2n=20), B. nigra (BB, 2n=16) and B. oleracea (CC, 2n=18), are monogenomic diploids. The remaining three species, namely, B. juncea (AABB, 2n=36), B. napus (AACC, 2n=38) and B. carinata (BBCC, 2n=34) are allotetraploids, evolved by hybridization between different monogenomic diploids (U 1935). Besides providing oil, vegetables and condiments, Brassica crops are important source of dietary fiber, vitamin C, and anticancer compounds (Fahey and Talalay 1995). Chinese cabbage (B. rapa ssp. pekinensis), used in the present study, is one of the most widely used vegetable crops in northeast Asia. In order to understand the complex eukaryotic genome, methods are needed that allow easy and efficient

2 580 manipulation of nuclear DNA as large functional units for physical mapping, positional cloning and genome sequencing (Tanksley et al. 1995; Zhang et al. 1994). For this purpose, the bacterial artificial chromosome (BAC) cloning system is the most commonly used for large-insert DNA library (Shizua et al. 1992; Woo et al. 1994; Kim et al. 1996; Zimmer and Gibbins 1997; Tomkins et al. 1999). In contrast to yeast artificial chromosome (YAC), BAC insert DNA is easy to isolate and manipulate. In addition, the clones are highly stable and chimeras are either low or absent in BAC libraries (Shizua et al. 1992; Woo et al. 1994). Arabidopsis thaliana is ideally suited for comparative genomics because of its small genome size (125 Mbp), low amount of repetitive DNA, high gene density, and close relatedness with Brassica species. The 550 Mbp genome of the Chinese cabbage ( is over four times the size of the Arabidopsis genome (Arumuganathan and Earle 1991). Comparative genetic mapping in the Brassicaceae family has revealed collinear chromosome segments (Schmidt et al. 2001). The evidence for conserved linkage arrangements between Arabidopsis and Brassica was provided by mapping a small number of genes in both Arabidopsis and B. rapa (Teutonico and Osborn 1994). Extensive comparative genetic mapping experiments between Arabidopsis and B. oleracea corroborated the presence of collinear chromosome segments (Kowalski et al. 1994). At least 19 structural rearrangements were reported to differentiate B. oleracea and Arabidopsis chromosomes (Lan et al. 2000). Comparative analyses of small genomic regions in Brassica and Arabidopsis have indicated that Brassica genomes contain extensive duplication (Bohuon et al. 1998; Cavell et al. 1998; Lagercrantz et al. 1996; Scheffler et al. 1997). O Neill and Bancroft (2000) studied the microstructure of six regions of the genome of B. oleracea using assemblies of overlapping BIBAC clones. They confirmed genomes triplication in the regions studied. The results also showed extensive divergence of DNA sequences in the paralogous segments of B. oleracea and the corresponding homoeologous segments of Arabidopsis genome. The last few years have witnessed tremendous advances in the development of molecular tools allowing manipulation of large DNA fragments. Fluorescence in situ hybridization (FISH) is one of the strategies used for physical mapping. The mapping resolution, which was initially limited, has been improved by FISH to pachytene chromosomes (Zhong et al. 1996). Recently, the use of FISH to extended DNA fibers in plants (Fransz et al. 1996; Jackson et al. 2000) has further enhanced the resolution of physical mapping. In this technique, DNA fibers are released from the lysed nuclei, spread on the surface of microscope slide and hybridized using standard fluorescence protocol. Jackson et al. (2000) used FISH technique for comparative physical mapping of A. thaliana and B. rapa. A set of six BAC clones, representing a contiguous region of chromosome 2 of Arabidopsis, was mapped on chromosomes and DNA fibers of B. rapa. The DNA fragment, which had only one location in A. thaliana, hybridized to 4 6 B. rapa chromosomes. Here, we report the construction of a BAC library and its use in conjunction with FISH on mitotic/meiotic chromosomes and extended DNA fibers in studying microsynteny of a 222 kbp Arabidopsis segment of chromosome 4, which is partially duplicated on chromosome 5, with B. rapa ssp. pekinensis. Materials and methods BAC library construction and characterization Chinese cabbage (B. rapa ssp. pekinensis) inbred line Chiifu was used for the construction of a BAC library. We used protoplasts to isolate high molecular weight genomic DNA. For protoplast isolation, fresh leaves were surface sterilized with 1% sodium hypochlorite solution, cut in to long, narrow pieces and transferred to a protoplast isolation buffer (0.5 M sorbitol, 10 mm CaCl 2, ph 5.5) containing 3% cellulase R-10 (Yakult) and 0.4% macerozyme R-10 (Yakult), and incubated in petri dishes on a gyratory shaker (50 rpm) at room temperature in dark for 20 h. Protoplasts were checked under a microscope and incubation was stopped when about 70% of the leaves were lysed. The buffer containing protoplasts was filtered through a series of sieves from 200 to 60 lm pore. The filtered protoplasts were centrifuged for 10 min at 35 g using a swing bucket rotor at room temperature and resuspended in protoplast buffer. This step was repeated three times. Harvested protoplasts were embedded in 0.6% low-melting point agarose, pre-warmed to 45 C, and poured into plug moulds on ice (100 ll per plug). The plugs were transferred to lysis buffer (0.5 M EDTA, 1% sodium lauryl-sarcosine, 1 mg/ml proteinase K) at 50 C. Finally, the plugs were dialyzed and stored in TE buffer (10 mm Tris HCl, 10 mm EDTA, ph 8.0) at 4 C. The procedures for vector preparation, partial digestion of high molecular weight DNA, size selection, and ligation and transformation were the essentially the same as reported by Peterson et al. (2000). Southern blot analysis Standard alkaline method was used to prepare the DNA of BAC clones (Sambrook et al. 1989). The DNA was completely digested with NotI and separated by PFGE on 1% agarose gel in 0.5x TBE at 6 V/cm with a 5 15 s linear ramp time at 13 C for 14 h. For depurination, the gel was treated with 25 mm HCl for 10 min, denatured in 0.5 N NaOH and 1.5 M NaCl for 30 min, and neutralized in 0.5 M Tris HCl and 3 M NaCl for 30 min. The gel was blotted overnight to Hybond-N + membrane (Amersham) in 10x SSC by capillary method. Membrane was washed with 6x SSC and cross-linked by

3 581 UV. Chinese cabbage genomic DNA was labeled with 32 P using random labelling kit (Takara) (Tomkins et al. 1999). Hybridization was carried out at 65 C overnight. Stringent washing was performed with two changes each of 2x SSC and 0.5% SDS at room temperature and 0.2x SSC and 0.2% SDS at 65 C. Membrane was exposed to X-ray film at 70 C, and developed. High-density filter preparation and screening Three high density filters were made using a Q-bot (Genetix, Dorset, UK), each containing 18,432 double spotted clones to avoid false positives. The filters were incubated on Q-trays containing LB/agar with 25 lg/ml of chloramphenicol at 37 C. Fixation of the plasmid DNA onto the membrane was as per manufacturer s recommendation. Colony filters were processed and hybridized using standard protocol of Sambrook et al. (1989). For confirming chloroplast contamination, two chloroplast-specific genes of rice, rbcl (1,434 bp) and psba (1,062 bp), were used as probe and labeled with 32 P-dCTP. Hybridization was performed at 65 C in 360 mm sodium phosphate (dibasic) and 5 mm sodium phosphate (monobasic), 0.5 mm EDTA and 3.7% SDS overnight. Films were exposed for 12 days depending on the signal intensity. Contig assembly of homoeologous Brassica regions corresponding to the Arabidopsis chromosomal segments using gene-specific probes To identify homoeologous segments in Chinese cabbage, 19 gene-specific probes of Arabidopsis, representing a 222 kbp gene-region of chromosome 4 and partially duplicated on chromosome 5, were used as described by O Neill and Bancroft (2000). Briefly, the 19 genes were hybridized separately to three high-density BAC colony hybridization filters of the KBrH library using PCRgenerated gene-specific probes. Strongly hybridizing (positive) BAC clones common to 19 gene-specific probes were identified and collected into gene bins. DNAs from the clones in the gene-specific bins were digested with HindIII, fragments resolved in 1% agarose, and blotted onto nylon membranes. These filters were probed with the 19 gene-specific probes. Contigs were assembled by identifying clones common to two or more genes as well as their banding patterns. Separate contigs were assembled for the homoeologous regions of chromosomes 4 and 5 of Arabidopsis. FISH on chromosomes The FISH procedure applied to both mitotic and meiotic chromosomes was essentially the same as previously published protocols (Koo et al. 2002, 2004). The chromosomes showing hybridization signals were identified on the basis of certain landmarks following Koo et al. (2004). FISH on DNA fibers Leaf nuclei were prepared as described by Jackson et al. (1998). A suspension of nuclei was deposited at one end of a poly-l-lysine slide (Sigma) and air dried for 10 min. Eight microliters of STE lysis buffer was pipetted on top of the nuclei and the slide incubated at room temperature for 4 min. A clean cover-slip was used to slowly drag the contents along the slide. The preparation was air dried, fixed in 3:1 ethanol:glacial acetic acid for 2 min, and baked at 60 C for 30 min. The probe mixture was applied to the DNA fiber preparation, covered with a 22 mm 40 mm cover-slip and sealed with rubber cement. The slide was placed in an 80 C oven in direct contact with the heated surface for 3 min, transferred to a wet chamber, which had been prewarmed in an 80 C oven for 2 min, and transferred for overnight incubation at 37 C. Post-hybridization washing stringency was the same as for FISH on chromosome spreads. Signal detection was according to Koo et al. (2004). Results Construction and characterization of the KBrH BAC library The Chinese cabbage HindIII library, constructed in an inbred line Chiifu, consisting of 56,592 BAC clones, was designated as KBrH. About 2.5% of the clones (3 out of 122 randomly taken BAC clones) did not contain insert DNA. The remaining 119 clones had an average insert size of 115 kbp with a range from 25 to 225 kbp. Except for 2.2% pseudo-bac clones, an average insert size of BAC library is 126 kbp. Based on the 550 Mbp size of the Chinese cabbage genome ( the coverage of KBrH library is estimated to be about 11-fold. Of the 4,992 BAC clones analyzed with two chloroplast genes of rice, rbcl and psba, 183 and 155 clones, respectively, showed hybridization. Thus, on an average about 4% of the BAC clones consisted of chloroplast genome. Identification of BAC clones hybridizing to Arabidopsis gene-specific probes Strongly hybridized positive clones were identified for all the 19 gene-specific probes. A total of 324 clones showed hybridization with the known Arabidopsis gene probes and 72 loci were detected with average redundancy of 4.5 (supplemental Table 1). The number of loci, each containing the homologous Arabidopsis genes ranged from 1 to 8.

4 582 Contigs of homoeologous Brassica regions corresponding to the chromosomal segments of Arabidopsis The hybridization pattern of the HindIII digested BAC clones, hybridized with gene specific probes is given in supplemental Table 2. Contigs were assembled from clones, which were common to two or more genes and their hybridization banding patterns. Using this approach, seven contigs (A to G) were assembled (Fig. 1a, b) corresponding to the two homoeologous regions of the Arabidopsis genome. For homoeologous region of B. rapa corresponding to the 19 Arabidopsis genes located on chromosome 4, four contigs (D to G) could be assembled. Contig D spanned the entire region covering all the 19 genes for which hybridization probes were developed, but homologues of only nine of these were detected in the BAC clones representing the contig. Contig E spanned a region containing nine genes in Arabidopsis with homologues detected for five of these in the BAC clones. Contig F, which also spanned a region containing nine genes in Arabidopsis, contained homologues of eight. Contigs G spanned a region containing nine genes, and had homologues for five of these. One gene for which hybridization probe was developed, At4g17700, detected no homologues in any of the contigs. A comparison of the homoeologous region of B. rapa ssp. pekinensis, corresponding to nine genes of Arabidopsis chromosome 5, revealed three contigs (A to C). Contig A consisted of six BAC clones, with homologues detected for four of the corresponding nine genes in Fig. 1 BAC clone contigs assembled for regions homoeologous to Arabidopsis chromosome segments. Contigs are shown for the regions homoeologous to chromosome 4(a) and chromosome 5 (b) segments. Closed circle: genespecific probe hybridized to BAC clone; Open circle: no hybridization

5 583 Fig. 2 Fiber-FISH using BAC clones representing assembled contigs on extended DNA fibers of B. rapa ssp. pekinensis. Hybridization pattern of BAC clones showing overlap regions: 77A05 (red) and 93K03 (green) of contig B (a); 43E02 (red) and 77G24 (green) of contig D (b); 48O11 (red) and 56L03 (green) of contig F (c). Bar represents 10 lm (1 lm=2.87 kbp) Arabidopsis. BAC clone 87J23, though did not show overlapping with the remaining clones, yet based on the restriction pattern and overlapping of DNA of 26D17 and 87J23 clones revealed by fiber FISH, the latter was included in this contig. Contig B spanned a region containing seven genes in Arabidopsis, and had homologues of four of these. Contig C, spanned over eight genes and showed hybridization with all these genes but At4g The contigs assembled were further confirmed by physically mapping the overlapping BAC clones as probes by FISH on extended DNA fibers of B. rapa ssp. pekinensis (Fig. 2a c). Physical location of contigs on B. rapa ssp. pekinensis chromosomes using FISH The BAC clone pairs belonging to seven contigs were used as probes for bicolor FISH on the mitotic/meiotic chromosomes of B. rapa ssp. pekinensis. FISH signals of the pairs of BAC clones belonging to different contigs on B. rapa ssp. pekinensis chromosomes are shown in Fig. 3a f. The ideogram showing the location of different contigs as revealed by FISH on different chromosomes of B. rapa ssp. pekinensis is given in Fig. 4. Contigs D and E were physically located on the long arm of chromosomes 2 and 8, respectively. However, contigs F and G were located on the long arm of chromosome 7, but physically separated. The contigs A, B and C were physically located onto chromosomes 6, 1 and 4, respectively. Discussion In the last few years, physical mapping of the genome of crop plants has been greatly facilitated due to advances in techniques allowing generation and manipulation of large insert DNA clones. We constructed a Chinese cabbage HindIII BAC library, designated as KBrH. In the BAC libraries reported in rice (Wang et al. 1995), human (Kim et al. 1996), soybean (Tomkins et al. 1999) and B. oleracea (O Neill and Bancroft 2000), the

6 584 Fig. 3 FISH mapping of BAC clones in B. rapa ssp. pekinensis. Hybridization signals of pairs of BAC clones: a 26D17 (red) and 30H05 (green) of contig A on pachytene bivalent; b 20J11 (red) and 93K03 (green) of contig B on mitotic metaphase chromosomes; c 139H08 (red) and 43N20 (green) of contig C on meiotic metaphase I chromosome; d 59M07 (red) and 46G16 (green) of contig D on pachytene bivalent; e 138O03 (red) and 25G02 (green) of contig E on leptotene chromosomes; f 48O11 (red) and 24B21 (green) of contigs F and G, respectively on pachytene bivalent percentage of clones having no insert was 7, 18, 4, and 15, respectively. However, in the B. rapa ssp. pekinensis library under report, only 2.5% of the clones had no insert. On the other hand, about 4% of the BAC clones of KBrH library had chloroplast genome. This contamination of the library by chloroplast genome is quite high in comparison to 0.3% in rice (Wang et al. 1995) and 1.85% in soybean (Tomkins et al. 1999). Since Chinese cabbage high molecular weight DNA was isolated from protoplast, the frequency of clones containing organelle DNA is expected to be higher than in libraries constructed from DNA extracted from nuclei. This warrants the need to devise ways to minimise the contamination of BAC library by chloroplast genome. The 11-fold coverage of KBrH library makes it suitable for physical mapping and even genome sequencing. All the 19 predicted genes of Arabidopsis, representing a 222 kbp segment of chromosome 4 and partially duplicated on chromosome 5, when hybridized with KBrH BAC clones, identified the counterparts in B. rapa

7 585 Fig. 4 Physical location of BAC contigs on different chromosomes of B. rapa ssp. pekinensis ssp. pekinensis. This affirms the earlier reports on gene order conservation in Arabidopsis and Brassica species (Lagercrantz et al. 1996; Lagercrantz 1998; Sadowski and Quiros 1998; O Neill and Bancroft 2000; Gao et al. 2004). FISH using pairs of overlapping BAC clones of different contigs as probes on extended DNA fibers of B. rapa ssp. pekinensis confirmed the contigs assembled by analyzing clones common to two or more genes and their hybridization banding pattern. All the genes, except At4g17700, were represented in the contigs assembled. However, in B. oleracea, At4g17750 was also absent from the contigs assembled (O Neill and Bancroft 2000). The assembled contigs showed not only conserved synteny but also conserved linkage. The collinearity of the genes was maintained in the paralogous segments of Chinese cabbage, indicating a common ancestry of the duplicated regions. However, gene contents were highly variable. Transposition and/or deletion events with insertion elsewhere in the genome might have led to high level of gene content variation. FISH is a useful tool for physical mapping of the plant genomes. Recently, the availability of karyotype of B. rapa ssp. pekinensis (Koo et al. 2004) has made it possible to assign BAC clones to individual chromosome and its arm based on morphometric parameters and distribution of 5S and 45S rdna, and repetitive sequence C11-350H. Following them, contigs A, B and C were assigned to chromosome 6, 1 and 4, respectively. Contig D, spanning over the entire region involving 19 genes, was located on chromosome 2 of B. rapa ssp. pekinensis. Contigs E and F spanning over nine genes each, represented nearly a proximal half of the Arabidopsis chromosomal segment probed. Contig G corresponded to the distal 112 kbp region of the Arabidopsis chromosome segment. Contigs F and G were present on the long arm of the chromosome 7 of B. rapa, but physically separated. Most likely, contigs F and G originally belonged to the same duplicated region. During the process of evolution, chromosome breakage within this region followed by insertion of an interstitial fragment appears to have occurred, leading to disruption of the original contiguous structure. Thus, the homoeologous segments corresponding to the two regions of Arabidopsis were spread across six chromosomes of B. rapa ssp. pekinensis, indicating the occurrence of chromosomal rearrangements during evolution. In FISH analysis, BAC clones showed hybridization signals at one location (to which the BAC clones belonged) and not in the paralogous segments of B. rapa ssp. pekinensis, indicating a high level of DNA sequence divergence. Quiros et al. (2001) sequenced and analyzed the B. oleracea region corresponding to the ABI1-Rps2- Ck1 segment of chromosome 4 of A. thaliana. They observed high levels of sequence identity for the coding sequences of all the genes studied. However, in general, spacers were larger in B. oleracea and the promoters were found to be poorly conserved. Such intergenic variation coupled with variation in the gene contents might be responsible for high sequence variation in the paralogous segments. O Neill and Bancroft (2000) studied the same two regions of Arabidopsis in B. oleracea by assembling contigs (A to G) as in the present study. Therefore, it is important to compare the contigs assembled in the two

8 586 monogenomic Brassica species (Fig. 5). Contig A of B. rapa ssp. pekinensis had 4 genes, whereas B. oleracea had one additional gene, At4g Contig B had four and seven genes in B. rapa ssp. pekinensis and B. oleracea, respectively. Three genes, At4g17480, At4g17570 and At4g17610, present in the latter species, were absent in B. rapa ssp. pekinensis. Contigs C and D in both the species had the same gene order and content. In contig E, two genes (At4g17300 and At4g17440), present in B. rapa ssp. pekinensis, were absent in B. oleracea. On the other hand, two genes (At4g17570 and At4g17650) of B. oleracea were absent in B. rapa. In B. oleracea, gene At4g17280 was present downstream of its original location in contig E. In contig F, an inversion involving three genes, At4g17480, At4g17460 and At4g17440, was observed in B. oleracea and gene At4g17650 was present upstream. In contig G, gene At4g17730 was common in B. rapa and B. oleracea. Moreover, in the latter species, gene At4g17650 was present in contig F. The latter situation indicates that these two contigs originally belonged to one region of B. oleracea and the same may be true in B. rapa ssp. pekinensis. A comparison of the microstructure of the two regions of Arabidopsis in B. rapa ssp. pekinensis revealed conservation of 42 (63%) genes in collinear manner, whereas 25 interspersed genes were lost. Using the same regions of Arabidopsis, O Neill and Bancroft (2000) reported the conservation of 68% genes in B. oleracea. A comparison of the sequence of a BAC clone of B. oleracea with the corresponding region in Arabidopsis revealed conservation of 57% genes in the former species (Gao et al. 2004). Ostensibly, changes in the microstructure of these two species occurred after their divergence from a common ancestor. Combined over all the contigs, five genes, At4g17380, At4g17650, At4g17750, At4g17760 and At4g17800 had only one locus, whereas At4g17700 was absent in B. rapa ssp. pekinensis. However, in B. oleracea, of these genes, At4g17380 had single copy; At4g17750 was absent; and At4g17670, At4g17760 and At4g17800 had 2 copies each (O Neill and Bancroft 2000). The remaining genes in both the species had two to five copies. It appears that the loss of genes after duplication did not occur at random during the process of evolution. Complete understanding of the fine structure and functions of the different genes involved in this study will provide an insight as to why some genes were lost whereas others were retained. Of the 19 Arabidopsis genes probed in Chinese cabbage, At4g17700 could not be assigned to Fig. 5 Comparison of microstructure of the two regions of Arabidopsis on the basis of contigs assembled in B. rapa (this study) and B. oleracea (O Neill and Bancroft 2000)

9 587 any of the contigs assembled, although the gene hybridized with 5 BAC clones including 47J18. FISH using this BAC clone showed hybridization signals on the long arm of chromosome 6 (data not shown), indicating the rearrangement of chromosomal segment containing this gene in Chinese cabbage. In B. oleracea also, this gene showed hybridization with BAC clones, which was not a part of the assembled contigs (O Neill and Bancroft 2000). The polyploidy model provides a good evidence of the role of gene and genome duplications in evolution. The genomes of many flowering plants have undergone one or more rounds of duplication. Analysis of genome sequence of A. thaliana shows large scale gene duplications (Blanc et al. 2003). Even the genome of Saccharomyces cerevisae has been reported to be evolved from ancient whole-genome duplication from Kluyveromyces waltii (Kellis et al. 2004). The duplicated regions have further triplicated in the monogenomic Brassica species (Kowalski et al. 1994; Lagercrantz 1998; O Neill and Bancroft 2000), substantiating the view that diploid Brassica species descended from a hexaploid ancestor (Lagercrantz and Lydiate 1996; Lagercrantz 1998). Our results show the presence of three copies of each of the two genomic regions of A. thaliana. However, there was a large variation in the gene repertoire of the paralogous segments. The occurrence of chromosomal rearrangements in Brassica species (Kowalski et al. 1994; Lagercrantz and Lydiate 1996) might be responsible for the tandem/dispersed gene losses coupled with gross as well as localized genic rearrangements as observed in the present study. The understanding of comparative relationship between Arabidopsis and Brassica genomes will have a great bearing in the improvement of the latter crops. 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