INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Arabidopsis thaliana, a weed-like member of the Cruciferae family (tribe Sisymbrieae), offers many advantages for basic and applied plant research. These features include small stature, short life cycle, small genome size (2n=10, estimated physical genome size of 100-120 Mb), low frequency of repetitive sequences (~10% of the nuclear genome; Leutwiler et al. 1984), and prolific seed production. These features, combined with research of the past several decades yielding many mutants, efficient transformation systems, detailed genetic and physical maps, the availability of several P1, YAC, and BAC libraries, and 36,569 public ESTs (http://www.cbc.umn.edu/ResearchProjects/Arabidopsis), make A. thaliana an ideal model for further molecular and genetic study (Meyerowitz and Somerville 1994). A multinational genome research initiative aiming to completely sequence the Arabidopsis genome by year 2004 (The Multinational Science Steering Committee 1997) is ahead of schedule. Such an accomplishment will undoubtedly create new scientific challenges and opportunities. One of the core issues will be how to apply the information obtained from the Arabidopsis genome project to the improvement of the world's leading crops.

The genus Brassica (tribe Brassiceae), including many important crops, is in the same taxonomic family as Arabidopsis thaliana. Such a close relationship suggests that crop plants of the genus Brassica will be among the earliest beneficiaries of a complete sequence of Arabidopsis. Economically, Brassica can be loosely categorized into oilseed, vegetable, and condiment crops. Brassica campestris, Brassica juncea, Brassica napus, and Brassica carinata provide ~12% of the world-wide edible vegetable oil supplies (Labana and Gupta 1993) and generate >$8 billion market value in North America and Europe. Brassica oleracea and B. campestris, the so-called "Cole crops," comprise a large variety of vegetables in our daily diet. Many of these vegetables have extreme morphological characteristics of basic interest, such as the enlarged inflorescence of cauliflower (B. oleracea subsp. botrytis) and broccoli (B. oleracea subsp. italica); enlarged stem of kohlrabi (B. oleracea subsp. gongylodes) and marrowstem kale (B. oleracea subsp. medullosa); enlarged root of turnip (B. campestris subsp. rapifera); enlarged and twisted leaves of Pak-choi (B. campestris subsp. chinesis) and Chinese cabbage (B. campestris subsp. pekinesis); and enlarged single apical bud of cabbage (B. oleracea subsp. capitata) or many axillary buds of Brussels sprouts (B. oleracea subsp. gemmifera) (Kalloo and Bergh 1993). Notably, although Arabidopsis is considered a close relative to Brassica, none of these phenotypes occur in Arabidopsis to nearly the same degree. Finally, Brassica nigra is primarily used as a condiment (mustard seed).

Through cytological study, the species relationship of crop Brassicas was described by the "triangle of U" (U 1935). Three allotetraploids, B. juncea (2n=36, AABB), B. napus (2n=38, AACC), and B. carinata (2n=34, BBCC), originated through interspecific hybridization between different pairs of the three diploid species, B. nigra (2n=16, BB), B. oleracea (2n=18, CC), and B. campestris (2n=20, AA). Based on cytological examination and hybrid analysis, the haploid chromosome number of monogenomic species in the Brassiceae were found to range from 7 to 12 (Mizushima 1980). However, because of the available resolution of cytological techniques, detailed genomic relationships among monogenomic species were not fully revealed. Understanding the genomic relationship among monogenomic Brassica species will not only shed light on the evolution of the Brassica genome but also facilitate gene transfer among Brassica species. The rise of comparative mapping, the alignment of chromosomes based on common DNA markers, has provided the means to study in depth the parallels in genome structure and function of closely related species (Tanksley et al. 1988; Ahn and Tanksley 1993), and distantly related species (Paterson et al. 1996).

The present study aimed to better characterize the comparative genome organization of Brassica and Arabidopsis. Previous study of the genus Brassica showed that the proportion of low-copy DNA sequences was similar among diploid Brassica species, but a large number of rearrangements result in distinct chromosomal number and organization (Slocum et al. 1990; Landry et al. 1991, 1992; Song et al. 1991; Kianian and Quiros 1992; Lagercrantz and Lydiate 1996). Corresponding chromosomes in diploid and amphidiploid Brassica have been reported (Teutonico and Osborn 1994; Cheung et al. 1997a,b). Comparative mapping between Arabidopsis and Brassica revealed even more extensive chromosomal rearrangements (Kowalski et al. 1994a; Lagercrantz et al. 1996; Osborn et al. 1997). These studies, however, did not provide a complete scope of the genome comparison between Brassica and Arabidopsis because of the limited numbers of common markers. To address this issue, a larger number of markers were needed on the comparative maps. The present work, based on 186 corresponding loci detected, provides a much more detailed picture of the comparative genome organization of B. oleracea and A. thaliana. Furthermore, study of chromosomal duplication within the B. oleracea genome, based on 367 loci, illustrates some of the complexities that will be faced both in extrapolating Arabidopsis information to Brassica and in assembly of sequence-ready contigs for crop genomes.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

DNA Polymorphism

Table 1 summarizes the DNA polymorphism detected by 200 Arabidopsis EST clones and 123 Brassica PstI genomic clones. The relatively low level of polymorphism in the B. oleracea (RCB)×B. oleracea ssp. alboglabra var.Bugh Kana (BK) F₂ population was consistent with the origin of RCB from B. oleracea ssp. alboglabra types (Song and Osborn 1992). The chance of detecting polymorphic probes is low and similar in both Arabidopsis crosses. There is variation in polymorphism rate associated with different restriction enzymes in Brassica, but no particular pattern is clear. In Arabidopsis, the restriction enzyme CfoI consistently detects more polymorphism than the other restriction enzymes, in both populations.

Establishing Composite Linkage Maps

B. oleracea Linkage Maps

View larger version (45K): [in this window] [in a new window]   Figure 1   The assembly of the Brassica chromosome 1 composite map. Common loci (based on common restriction fragment sizes) were connected by solid lines, putatively homologous loci (with different restriction fragment sizes, but at corresponding sites) are connected by dashed lines. Filled circles placed on crossed lines indicate that respective orders of loci are statistically significantly different (LOD 2.0) in the respective maps, suggesting possible chromosomal rearrangements. Arrows indicate the inferred locations of unique loci in the consensus map.

A. thaliana Linkage Maps

Patterns of Correspondence of Brassica Chromosomes with One Another and with the Arabidopsis Chromosomes

Figure 2 illustrates and Table 4 summarizes the composite linkage map of B. oleracea. We developed a model for the comparative organization of the chromosomes of B. oleracea and A. thaliana that assumes duplication of most Brassica chromosomes and one-to-one correspondence of Brassica chromosomes with Arabidopsis chromosomes. The extent to which the observed data cannot be explained by this "null hypothesis," reflects the need for alternative hypotheses such as triplication of Brassica chromosomal segments or duplication of Arabidopsis chromosomal segments. The model was built based on the identification of SCEUS (smallest conserved evolutionary unit segments; O'Brien et al. 1993) of three or more loci that (1) maximize the number of corresponding DNA marker loci that are consistent with the model, (2) minimize the number of chromosomal rearrangements between duplicates (Brassica) or orthologs (Arabidopsis), (3) consider closely linked markers to be stronger evidence of synteny than distantly linked markers, and (4) consider a genetic distance of >5 cM to represent a true difference in locus order. This relatively large value was chosen to reflect not only the small size of the primary population but also the uncertainties associated with inference of loci mapped in other populations. Further constraints were imposed to evaluate the extent of duplication and triplication in Brassica. Specifically, possible regions of duplication along a chromosome were inferred first, in a manner that followed the above rules and did not allow different duplicated segments to overlap with each other by >5 cM (the threshold for inferring rearrangement). Finally, regions of possible triplication were inferred: These were allowed to overlap with duplicated segments but not with each other. From first principles, if the duplication process in Brassica were random (not associated with large chromosomal region), the duplication model would explain 12.5% of data (given that B. oleracea has nine chromosomes). The extent to which the model improves on this reflects the strength of evidence for duplication and triplication. By the same rationale, one-to-one correspondence of Brassica to Arabidopsis must account for significantly more than the random expectation of 25% of data to be meaningful. Higher levels of correspondence in small chromosomal regions may be suggestive of duplication of chromosomal segments.

View larger version (116K): [in this window] [in a new window]   Figure 2   Composite RFLP linkage map of Brassica olearacea RCB x GC, RCB x CAN, RCB x PK and RCB x BK F2 Populations. Filled circles next to the loci indicate homoeologous Brassica loci (chromosomes 1-9, near right) or homologous Arabidopsis loci (chromosomes 1-5, far right) detected by the same probe. Open circles indicate that no polymorphism was detected for homoeologous (Brassica) and homologous (Arabidopsis) loci. A letter "R" next to the probe name indicates that the probe hybridizes to a repetitive DNA sequence in Arabidopsis. Specific colors are assigned to each homoeologous and homologous chromosome. Markers included in the duplication (Brassica) or one-to-one (Arabidopsis) models are connected by colored columns. Open columns indicate possible triplicated (Brassica) or duplicated (Arabidopsis) regions.

Brassica Chromosome 1

Brassica Chromosome 2

Brassica Chromosome 3

Brassica Chromosome 4

Brassica Chromosome 5

Brassica Chromosome 6

Brassica Chromosome 7

Brassica Chromosome 8

Brassica Chromosome 9

Patterns of Correspondence of Arabidopsis Chromosomes with the Brassica Chromosomes

The "one-to-one correspondence" model and duplication model were also tested on the Arabidopsis linkage map as well, which was illustrated in Figure 3 and summarized in Table 5.

View larger version (52K): [in this window] [in a new window]   Figure 3   Composite RFLP linkage map of Arabidopsis thaliana HM x WS and M13 x WS F2 populations. Markers designated "FQ" are anchor loci, common to both populations. Markers mapped in the HM x WS population are designated "Q". The remaining markers were mapped in M13 x WS only. The construction of the A. thaliana composite map was as reported previously (Kowalski et al. 1994a). It should be noted that integration of data from two populations tends to inflate recombinational distance due to unequal recombination between populations. The filled circles next to the loci indicate homoeologous loci detected by the same probe on the Brassica composite map. Open circles indicate that no polymorphism was detected for homoeologous loci in RCB x GC Brassica populations. The letter "R" next to a probe name indicates that it hybridizes to a repetitive DNA sequence in the Brassica genome. Specific colors are asigned to each homoeologous chromosome. Markers included in the one-to-one model for Arabidopsis-Brassica correspondence are connected by filled columns. Open columns indicate possible duplicated regions in Brassica.

Arabidopsis Chromosome 1

Arabidopsis Chromosome 2

Arabidopsis Chromosome 3

Arabidopsis Chromosome 4

Arabidopsis Chromosome 5

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

It is timely to consider the challenges and opportunities in extrapolating structural genomic information from Arabidopsis, the first plant for which the genome will be completely sequenced, to Brassica and other more distantly related plants.

Our model (Fig. 2) suggests that at least 22 chromosomal rearrangements differentiate the B. oleracea homologs from one another and at least 19 rearrangements differentiate A. thaliana from B. oleracea. In several instances the locations of chromosomal rearrangement breakpoints between Brassica homologs approximately match the locations of the breakpoints between Arabidopsis and Brassica. Some such instances include (1) Brassica chromosome 2, where the correspondence with Brassica chromosomes 2 and 8 breaks between EW7B04c and EW6G12a and the correspondence with Arabidopsis chromosomes 2 and 5 breaks between EW1F08 and EW2E05b; (2) Brassica chromosome 3, where the correspondence with Brassica chromosomes 1 and 8 breaks between EST130a and EW2D03a and the correspondence of Arabidopsis chromosomes 1 and 3 breaks between EW7D03y and EW2D03a; (3) Brassica chromosome 8, where the correspondence with Brassica chromosome 4 and 3 homologs breaks between EW5G04b and EST517d and the correspondence of Arabidopsis chromosomes 4 and 3 breaks between EST22a and EW8F03b; (4) Brassica chromosome 9, where the correspondence of Brassica chromosomes 9 and 1 breaks between K457b and EST517g and the correspondence of Arabidopsis chromosomes 1 and 5 breaks between EW1G03a and EST9a. Such rearrangement breakpoints that appear to be common to Brassica and Arabidopsis may reflect cases where both Arabidopsis and one Brassica homolog retain the chromosome organization of their common ancestor, whereas a duplicated Brassica homolog has undergone rearrangement. Similarly, chromosomal regions in which Arabidopsis gene order corresponds to one but not both Brassica homoeologs may reflect rearrangement of one Brassica homoeolog since duplication. For example, on Arabidopsis chromosome 5, the order for marker EW5D12, EST075, and EST150 is EST150-EST75-EW5D12, and on Brassica chromosome 4, it follows the same order, but on Brassica chromosome 1, the order changes to EST75-EST150-EW5D12.

Comparative Organization of Brassica Homoeologous Chromosomes

The Brassica chromosomal duplication model explains 41% of the duplicated restriction fragment length polymorphism (RFLP) loci we mapped (Table 4). If there were no pattern to duplication, then the duplication would be expected to account for <12.5% (1 out of 8) of data, because there are nine pairs of chromosomes in B. oleracea. Our data clearly indicate that duplication has involved large chromosome segments in Brassica. In a similar manner, if triplication accounts for more than an additional 14.3% (1 out of 7) of data in Brassica, then it would be more common than expected to occur at random. Based on our model, triplication of Brassica chromosomal segments best explains 18% of the data, which is nominally greater than the expected value (14.3%). Although the case for triplication is much weaker than for duplication, the clustering of triplicated loci into linked groups does tend to support prior suggestions based on smaller numbers of probes and isolated genomic regions (Kowalski et al. 1994a; Lagercrantz et al. 1996; Osborn et al. 1997) that some regions of the genome of B. oleracea (as well as B. rapa and B. nigra) may be triplicated. A fundamental problem in the use of genetic mapping data to evaluate duplication (and triplication) of chromatin is the need to detect DNA polymorphism. The assembly of physical maps for the Brassica genomes will alleviate this limitation but will require new methodology to efficiently determine the locus-specificity of BACs (or other large DNA clones) that hybridize to duplicated (or triplicated) probes.

Alignment of Brassica and Arabidopsis Chromosomes

The Brassica/Arabidopsis one-to-one correspondence model explain 57% of our observed data (Table 4). If the genomes of Brassica and Arabidopsis were randomly arranged with respect to one another, then one-to-one correspondence would account for < 20% (1 out of 5) of data in Arabidopsis. Our data clearly indicate extensive synteny of Brassica and Arabidopsis.

A total of 31 pairs of duplicated loci, including 20 pairs reported here for the first time (Table 3), mapped to A. thaliana, accounting for 23.7% of the loci detected. These duplicated loci expand on the earlier suggestion (Kowalski et al. 1994a) that part of the A. thaliana genome may have undergone ancient duplication. These ancient duplications could complicate contig-map construction and also could reduce the subset of Arabidopsis genes that are susceptible to "knockout" experiments (Sundaresan et al. 1995; Kempin et al. 1997). Notably, an intrachromosomal duplication appears to occur in A. thaliana chromosome 1 (Fig. 4).

View larger version (35K): [in this window] [in a new window]   Figure 4   Intrachromosomal duplication of Arabidopsis chromosome 1, and an possible more-than-triplicated region of Brassica chromosome 1 and 9. Solid lines connect homoeologous loci (based on different restriction fragment sizes) located on the same chromosome. Dashed lines connect homoeologous loci located on different chromosomes.

Intrachromosomal duplication was observed in chromosomes 1, 2, and 9 of B. oleracea (Fig. 5). Two independent studies on the genome of B. nigra reveal similar patterns on chromosome 5 (Truco and Quiros 1994) and chromosome 6 (Lagercrantz and Lydiate 1996), suggesting that such intrachromosomal duplication might be common in Brassica. If such intrachromosomal duplications preceded the duplication/triplication of the ancestral B. oleracea genome, then even higher levels of duplication might be expected in modern B. oleracea. In our study, five probes did detect more than three segregating loci in B. oleracea, including EW4D04 (chromosomes 1, 2, 4, and 8), EW8A06 (chromosomes 1, 4, 5, and 7), EST55 (chromosomes 1, 2, 4, and 6), EST453 (chromosomes 1, 4, 5, 6, and 9), and EST517 (chromosomes 1, 6, 8, and 9). Although we cannot rule out the possibility that some of these more-than-triplicated loci might be the consequence of other duplication mechanisms, segments of Brassica chromosomes 1 and 9 did suggest the existence of such high-order chromosome segmental duplication (Fig. 5). More probes mapped in this region should provide further evidence.

View larger version (23K): [in this window] [in a new window]   Figure 5   Intrachromosomal duplication detected by three or more duplicate loci in Brassica.

Through comparative mapping, many powerful tools already created for Arabidopsis can now be applied to Brassica. For example, Arabidopsis cDNA sequences may be used to isolate homologous genes in Brassica, Arabidopsis BAC/YAC contigs may be used in Brassica for map-based cloning, and Arabidopsis high-resolution maps may help to resolve clustered markers in Brassica (Liu et al. 1996). Arabidopsis genomic tools may guide the isolation of Brassica alleles conferring unique phenotypes. Brassica and Arabidopsis may have diverged as little as 10 mya (Muller 1981), suggesting that ~90% of chromosomal segments <5 cM may remain colinear (Paterson et al. 1996). A comparative map with a density of <5 cM/marker makes it relatively easy to evaluate correspondence of Brassica quantitative trait loci (QTLs) to Arabidopsis mutations or candidate genes. Furthermore, a comparative map of B. oleracea (CC genome) and A. thaliana can be extended to an amphidiploid species of Brassica such as B. napus (AACC genome), where genome complexity is redoubled.

Genetic linkage maps based on ESTs (Berry et al. 1995) enable one to use sequence information to screen for conservation with distantly related taxa. For example, disease-resistance-like ESTs could be potentially useful in locating disease-resistance loci in a specifically designed segregating population other than Arabidopsis (Botella et al. 1997). Also, through selection of highly conserved ESTs, comparative organization of the chromosomes of even distantly related species such as Arabidopsis, Gossypium (cotton), and Sorghum can be studied using the same probes (Paterson et al. 1996). Thus, a cross-genome comparative map based on a common set of ESTs may eventually provide a direct comparison of macro- and microcolinearity across various species. The combination of ESTs and DNA microarray technology (Winzeler et al. 1998) could accelerate this process. Furthermore, mapping the common set of ESTs to Arabidopsis megabase DNA libraries (Schmidt et al. 1995; Zachgo et al. 1996; Agyare et al. 1997) will extend the Arabidopsis physical map and DNA contigs to other plants. Thus, using Arabidopsis contigs to assist map-based cloning in cotton, sorghum, or other genomes may be more feasible. Such a cross-genome framework and toolbox could profoundly affect future genome sequencing projects in related taxa. It is of interest not only to elucidate the portions of genome that are conserved (common) among various species but also the portions that are divergent among species. Thus, the priority of subsequent crop genome sequencing projects might be focused on genomic regions that are poorly conserved, so that scarce financial resources are used more efficiently.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Plant Materials

Two A. thaliana F₂ populations were used in this study: A. thaliana ecotype Wassilewskija (WS)×mutant stock M13 (Liu et al. 1996) and WS×Hannover/Münden (HM) (Kowalski et al. 1994b). Subsets of 78 individuals from each population were used for mapping Arabidopsis ESTs. Four B. oleracea F₂ mapping populations were used in this experiment: RCB (self-compatible)×B. oleracea var. Green Comet (USDA collection, accession no. G30771, from North America), RCB×B. oleracea var. Cantanese (USDA collection, accession no. PI462224, originally from Italy), RCB×B. oleracea var.Pusa Katki (USDA collection, accession no. PI274783, originally from India), and RCB×B. oleracea var.Bugh Kana (USDA collection, accession no. PI249556, originally from Thailand), composed of 56, 247, 250, and 246 individuals, respectively. A. thaliana seed were obtained from the Arabidopsis Biological Resources Center at Ohio State University, directed by Dr. R.L. Scholl. Rapid-cycling Brassica was from the Crucifer Genetics Cooperative, Madison, WI. Seed and pollen of other B. oleracea varieties were generously provided by Dr. J. McFerson and Dr. S. Kresovich, then at USDA-ARS, Geneva, NY.

Genotyping

DNA extraction, electrophoresis, Southern blotting and autoradiography were as described previously (Kowalski et al. 1994a). A total of 113 Brassica PstI genomic clones ("EW," "WG," and "WR," from Pioneer HiBred), 35 Arabidopsis genomic clones ("M," from Dr. E. Meyerowitz, Caltech), 23 Arabidopsis anonymous cDNA clones ("AC," "ATEX," and "TCH"), four cloned RAPD-PCR products ("R;" unpubl.), 198 Arabidopsis EST clones ("EST," from Dr. R.L. Scholl, the Arabidopsis Biological Resources Center, Ohio State University), and 19 putatively embryo-specific Arabidopsis EST clones ("AHD," "AKJ," "AKN," "Cla," "d2P," "FLS," "HD," "HMG," "K," "S," and "Seed," from Dr. Terry L. Thomas, Texas A&M University) were used in this study.

Data Analysis

RFLP linkage maps were constructed using MapMaker (Lander et al. 1987). Linkage groups were built at threshold of lod (logarithm of odds)=2.1 for A. thaliana and lod=2.5 for B. oleracea. Genetic distances (in centiMorgans) were calculated using the Kosambi mapping function.