AU654467B2 - Brassica linkage map - Google Patents

Description

OPI DATE 05/11/90 F AOJP DATE 06/12/90 APPLN. ID 54383 PCT NUMBER PCT/US90/01848 .EATY (PCT)

INTERNATI

(51) International Patent Classification 5 C07H 21/04, C12Q 1/68 C12N 15/00 (11) International Publication Number: Al (43) International Publication Date: WO 90/12026 18 October 1990 (18.10.90)

S-

7 (21) International Application Number: (22) International Filing Date: PCT/US90/01848 3 April 1990 (03.04.90) Priority data: 332,323 3 April 1989 (03.04.89) US PohER toc-rbic IrEM1r\PtTM4r--, (Jc..

(71) Applicant: ETV F L•.-LATS I- RP&ORA BIUS/ US]; 417 Wakara Way, Salt Lake City, UT 84108 (US).

(72) Inventors: SLOCUM, Mary, S. 1140 East Herbert, Salt LAke City, UT 84105 OSBORN, Thomas, C.; 4053 Cherokee Drive, Madison, WI 53711 (US).

(74) Agents: HIGHET, David, W. et al.; Venable, Baetjer, Howard Civiletti, 1201 New York Avenue, Washington,DC 20005 (US).

(81) Designated States: AT (European patent), AU, BE (Euro.

pean patent), CA, CH (European patent), DE (European patent), DK (European patent), ES (European patent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), SE (European patent).

Published With international search report.

Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.

654467 (54)Title: BRASSICA LINKAGE MAP (57) Abstract Clones that hybridize to DNA of Brassica and of related genera such as Raphanus, use of said clones and the loci specified thereby are described. The clones and clone products thereof, through any of a variety of techniques, find utility in basic and applied research and in commercial applications. The utility of the disclosed clones are exemplified in, for example, varietal identification, gene mapping and gene isolation.

See back of page WO 90/12026 PCT/US90/0148 TITLE OF THE INVENTION BRASSICA LINKAGE MAP FIELD OF THE INVENTION The instant invention relates to a collection of clones and the loci specified thereby that can be used in a variety of Brassica and related genera such as Raphanus. The loci are mapped in Brassica genomes. The clones and the maps will find use particularly in selective breeding programs, for identification and as a first step in the isolation of genes of interest.

BACKGROUND OF THE INVENTION Gene mapping, once considered an arcane branch of biometry or cytogenetics, is fundamental to a thorough understanding of the genome. (For reasons detailed below, the name gene mapping is anachronistic. To maintain consistency with the literature, the name gene mapping is retained herein with the provision that gene refers to a nucleic acid fragment. Said fragment need not be transcribed.) Stated simply, gene mapping is the ordering of heritable markers on the chromosomes.

Historically, morphologic characters were scored within sibships to determine which of the characters were sorting independently. Those that failed to meet that criterion were assumed to be linked on a chromosome, i.e. those characters were more likely than not to be inherited together. But monogenic morphologic characters can be uncommon and informative crosses have to be ascertained or constructed.

The discovery of biochemical variants fueled a temporary resurgence in gene mapping. Common methods for detecting protein variation include detecting WO 90/12026 PCT/US90/01848 -2isozymes by electrophoresis, determining presence or absence of biochemical activity and detecting variability by immunologic means. Linkage of biochemical markers can be ascertained in family study.

Physical characteristics of the chromosomes themselves offer the possibility of assigning a marker or linkage group to a chromosome or chromosomal region.

Translocations, heterochromatin blocks, heterogeneous staining regions, inversions and the like allow regional localization of a marker or linkage group to say near the centromere, on the short arm or long arm, etc.

A significant advance to gene mapping was the use of parasexual methodologies. Somatic cell hybrids remove the constraints of ascertaining informative families and use of test crosses. An appropriately configured panel of cell lines permits highly probable assignment of markers or linkage groups to a chromosome or chromosomal region.

Another tool is comparative mapping. The apparent monophyletic origin of each of the plants and of the animals is suggested by the occurrence of conserved linkage groups among diverse species. For example, chromosomal segments carrying two or more markers are maintained throughout the genomes of mouse and man. In an analysis that considered homologous biochemical, morphologic and a small number of molecular markers in mouse and man, Nadeau Taylor (1984) found 83 homologous loci that define 46 homologous segments, in the two genomes. Of those 46 segments, 13 are shown to comprise segments with an average length of 8.1 1.6 centimorgans Another example is the fixation of genes on the eutherian X chromosome. Although it may be argued that convergent evolution has a role in the phenomenon of conserved linkage groups, it is equally likely that selection or monophyly is a cause of the WO 90/12026 PCT/US90/01848 -3phenomenon. Nevertheless, conserved linkage groups allow predicting the location of markers among species.

However, it was not long before the list of monogenic biochemically detectable variants was near exhaustion (not all proteins are polymorphic) and alternative technologies were sought to perpetuate the goal of saturating genomes with mapped markers. The development of recombinant DNA methodologies provided the opportunity of monitoring nucleic acid sequence variability. The redundancy of the genetic code and of the genome affords an almost unlimited resource of polymorphism. Furthermore, nucleic acid sequences in general are immune from extrinsic modification and the markers act as Mendelian codominants. In addition, because nucleic acids themselves are monitorei, it is unneccesary for a sequence to be transcribed or translated. Thus, sequences of no apparent function nevertheless can serve as markers. It is likely that nucleic acid polymorphism will enable saturation of linkage maps with informative markers.

Nucleic acid sequence variability is manifest commonly in the current technologies as differences in fragment lengths otherwise known as restriction fragment length polymorphism or RFLP. Nucleic acids from individuals are digested with a restriction endonuclease, separated electrophoretically, blotted onto a membrane, hybridized with labeled nucleic acids and samples compared. The digested target nucleic acids and the labeled nucleic acids may contain expressed or random sequences. RFLP's can result from base pair changes in an enzyme recognition site, rearrangements encompassing the site, or rearrangements localized between enzyme recognition sites. Variation can be observed also in the hybridization pattern or intensity of hybridization because of difference in the number of homologous sequences or in the degree of sequence ir4 WO 90/12026 PCT/US90/01848 -4homology between the hybridizing strands. RFLP's comprise a subset of total nucleic acid variability because not all sequences contain a known recognition site. Two other methods for detecting nucleic acid sequence variability are using allele-specific oligonucleotides and base sequencing.

A benefit of nucleic acid polymorphism is that it provides a key to mapping with a resolution of several million base pairs or less. That degree of resolution allows one to isolate and clone genes of interest. The isolation of genes without reference to a specific protein or without any reagents and functional assays useful in detecting said protein is termed reverse genetics. In one approach, a targeted gene is bracketed on each side with one, and preferably two markers and a directional chromosomal walk or jump begins with those markers to obtain clones carrying the sequences of interest. In that fashion, a desired nucleic acid fragment adjacent to or located more remotely from the marker is obtained. It is unnecessary for the desired fragment and marker to share sequence homology. The relationship is assured by a third fragment that shares sequence homology with the desired fragment and marker.

The concept is illustrated in the following diagram: A B C D 4 several 4 steps

A

1

B

2

C

3

D

4 WO 90/12026 P(7r/US90/018848 The -markers A,B,C and D comprise a linkage group A represents sequences with homology to a clon> of the instant invention and D represents sequences of a trait of interest. The region encompassing A,B,C and D, using several methods standard in the art, is fragmented so as to produce a series of overlapping fragments (numbered 1-4) as depicted in The degree of overlap can vary between adjacent fragments. Because 1 and 2 have substantial sequence homology by virtue of the shared sequences, they hybridize to each other. The same applies for 2 and 3. Thus, 1 and 3 are related because each hybridizes to 2 which shares sequences with 1 and 3. Carried a step further, fragment 4 carrying sequences of the trait of interest is obtained by virtue of sequences shared with 3. Therefore, 1,2,3 and 4 are related fragments. Alternatively, newer methods of pulsed field electrophoresis, e.g. CHEF and OFAGE, discriminate fragments of up to 10Mb. In that approach, a fragment containin a cene of interest and the marker is obtained from a gel, fractionated and the pieces cloned to form a mini-library. The key to the use of reverse genetics is a marker near the gene of interest.

At this juncture, it should be appreciated that any one polymorphic marker, whether detected at the morphologic, protein or nucleic acid level, in itself is useful for genetic analyses. For example, any one marker can be used to assess the organization of germplasm, as a tool in varietal protection, to evaluate levels of heterozygosity, to assist in the accelerated recovery of a donor parent genotype in a breeding program, to assess population diversity or taxonomic relationships, as a tool to enhance a selective breeding program for example by following an introgressed trait and to localize other genes of interest through linkage.

The analysis can be more discriminating if multiple unlinked markers are examined.

WO 90/12026 PC/US90/01848 -6- There are other circumstances, however, in which optimal usage of markers requires at least some knowledge of the linear arrangement of the markers in the genome. Examples of such situations include localizing genes contributing to a quantitative trait, mapping new markers, establishing predictive linkage associations for single and multigenic traits and any of the uses described above (and see Beckmann Soller, 1986). For example, nucleic acid polymorphism maps are being generated for maize (Helentjaris, 1987), tomato (Bernatzky Tanksley, 1986b) and lettuce (Landry et al., 1987); and genes of the tomato quantitative trait, soluble solids content, have been identified through linkage with cloned sequences by Osborn et al. (1987), Patterson et al. (1988) and Tanksley Hewitt (1988).

A saturated linkage map would be particularly valuable in the genus Brassica which includes both diploid and amphidiploid species with numerous subspecies and varieties serving as important sources of vegetable, oil and fodder throughout the world (Table A tremendous amount of morphologic and physiologic variability is evident not only between species of Brassica, but between and within subspecies as well.

The species Brassica oleracea, for example, includes vegetable crops such as broccoli, cabbage and cauliflower which vary greatly, particularly with respect to foliar organs and requirements for flowering.

The underlying genetic bases of the variability in the Brassica are not understood.

The diploid Brassica species, B. nira, B. oleracea and B. campestris, may have evolved from a common progenitor (see e.g. Attia Robbelen, 1986; Song et al., 1988a,b). Intragenomic chromosomal pairing in haploids and diploids has been interpreted to indicate that duplication is present within the Brassica genome (see WO 90/12026 PCT/US90/01848 -7for example, Armstrong Keller, 1981, 1982).

Recombination within or between genomes and subsequent functioning of duplication-deficiency gametes has been suggested as a possible mechanism for generating the morphologic and physiologic diversity observed in Brassica (Prakash, 1973).

WVO 90/12026 PCr/US90/01 848 -8- Table 1 Genomic Classification of Selected Brassica Genome A 4 nA IT.i- a 4 ai.mmn iMi m C J _u.Uj J. 1 4~rlll B. nigra (8) B. oleracea (9) acephela botrytis capitata gemmifera gongylodes italica sabauda sabellica cc cc. a cc.b CC. c cc. g cc. go cc. i CC. 1 cc. sa Black mustard "Cole crops" Kales cauliflower cabbage brussels sprout kohlrabi broccoli savoy collards B. campestris (syn. rapa) chinensis oleifera parachinensis pekinensis rapifera aa.c aa. o aa.pa aa.p aa* r bbcc B. carinata (17) B. iuncea (18) capitata faciliflora oleif era rugosa spicea oleifera rapifera aabb aabb. c aabb. f aabb. o aabb.ru aabb. sp aacc aacc.o aacc.r pak choi turnip rape choy sum petsai turnip Ethiopian mustard head mustard broccoli mustard Indian mustard Leaf mustard mustard fodder rape oil rape rutabaga B. napus (19) n is the haploid number of chromosomes WO 90/12026 P(3r/US90/01 848 -9- Classical analyses of hybrid and parent species relationships involve generally comparative morphology, cytogenetics, artificial hybridization and physical characterization of the genomes. More recently taxonomic relationships have been monitored by comparing the similarity of proteins or nucleic acids. With regard to Brassica, data support the suggestion that interspecific hybridization among the diploid species produced three amphidiploid varieties which are also important economic crops. The B. nigra x B. campestris cross produced B. juncea, the B. nigra x B. oleracea cross produced B. carinata and the remaining cross of B. oleracea x B. campestris produced B. napus (Vaughan, 1977; Prakash Hinata, 1980; Quiros et al., 1988).

Those interrelationships were proposed by U in 1935 and confirmed most recently in analyses of chloroplast, mitochrondial and nuclear DNA sequences. Chloroplast DNA's of B carinata and B. nigra are virtually identical indicating that B. nicra is the likely maternal progenitor of B. carinata. Similarly chloroplast DNA's of D. juncea and B. campestris are nearly identical suggesting that J. campestris is the likely maternal progenitor of that amphidiploid species (Erickson et al., 1983; Palmer et al., 1983).

The high degree of relatedness among the species facilitates artificial hybridization for the resynthesis, synthesis naturally or by in vitro techniques such as protoplast fusion of allotetraploid species (Williams Hill, 1986; Tai Ikonen, 1988). A novel B.

napus variety known as hakuran was produced in the laboratory by crossing chinese cabbage campestris pekinensis, aa.p) and cabbage oleracea capitata, cc.c) (discussed in Williams Hill, 1986). Hakuran serves as a vegetable and fodder crop. The relatedness of the Brassica varieties further suggests the use of markers of one variety in one or more other varieties.

WO 90/12026 PCr/US90/01 848 Conservation of sequences may extend to chromosomal segments and linkage groups thereby facilitating predictions of marker location between species. There are many feral forms of Brassica between species that are a valuable reservoir of genetic variability for introduction into the crop species.

Bonierbale et al. (1988) hybridized 135 tomato clones with potato DNA to determine the genomic relatedness of those species. Sequences homologous to nearly all of the tomato clones were found in the potato genome, and in the tomato the loci existed at similar copy number and mapped to similar linkage groups. For nine chromosomes the order of loci are identical in the two species. The linkage order of the remaining three chromosomes could be explained by a single paracentric inversion in each. But contrast the high degree 'f relatedness between potato and tomato with the low degree of relatedness between tomato and pepper, another member of the Solanaceae. Pepper has nearly four times as much DNA as tomato and the linkage groups of the two species are disparate and highly rearranged (Tanksley et al. 1988). Furthermore, most of the sequences in the tomato genome are rapidly evolving (Zamir Tanksley, 1988). Thus, most clones will be generally speciesspecific and will find little or no utility in related species.

Unlike tomato where the majority of cDNA clones correspond to single loci (Bernatzky Tanksley, 1986a), in several other plant species genetic analyses of enzyme systems have revealed that expression of multiple allelic forms is under duplicate or triplicate gene control, and the locations of corresponding multiple structural loci are known (McMillin Scandalios, 1980).

Although early breeding studies suggested that Brassica too contained numerous repeated gene families, actual presence of duplicated sequences within the Brassica 11 genome has not been demonstrated nor has the possible chromosomal organization of such duplicated sequences been described. The lack of evidence to support the theory of sequence duplication reflects how little is known about the genetics of Brassica. Inheritance patterns of very few Brassica genetic markers have been described in the literature (Willams, 1985), and there is a conspicuous absence of a genetic map despite the preserce of active breeding programs. Studies are '0 limited by the long generation time of the biennials, complex inheritance patterns of many traits and the difficulty in overcoming self-incompatibility. Thus, the selection and identification of improved varieties is limited by the lack of genetic information, such as the chromosomal location of markers and of loci controlling the expression of important traits.

Thus, the development of nucleic acid polymorphism markers in Brassica crops will facilitate genetic and evolutionary studies of this economically important genus and saturated Brassica linkage maps, heretofore undescribed, are crucial to the rapid genetic improvement of this economically important group of plants.

SUMMARY OF THE INVENTION According to a first aspect the present invention consists in an isolated, Brassica nucleic acid fragment which detects polymorphic Brassica DNA, said fragment being selected from the group consisting of a clone and the Brassica insert of said clone, wherein said clone is selected from the collection of clones listed in Table 3.

According to a second aspect the present invention consist in a Brassica nucleic acid fragment set which comprises at least two isolated Brassica fragments which detect polymorphic Brassica DNA, each of said fragments being selected from the group consisting of a clone selected from clones listed in Table 3 and the Brassica inserts of said clones.

o jI Ila According to a third aspect the present invention consists in a Brassica nucleic acid fragment set which comprises at least two isolated fragments which detect polymorphic Brassica DNA, each of said fragments being selected from the group consisting of a clone selected from the clones listed in Table 3 which map to Brassica oleracea and thc Brassica inserts of said clones, wherein when said fragments are of any one linkage group, their relative order on said linkage group is related to the order of loci as presented in the map of Figure 4 (hereinafter Map), the relative order of any two said loci of said set may be inverted in relation to the order presented in the Map and the arrangement of said loci of said set comprising 2 or more loci when considered from either direction is equivalent.

According to a fourth aspect the present ,invention consists in a Brassica nucleic acid fragment fill set which comprises at least two isolated Brassica fragments capable of detecting Brassica polymorphic DNA being selected from the group consisting of the collection of clones listed in Table 3 and the inserts of said clones, wherein said fragments are of any one linkage group, their relative order on said linkage group is related to the order of loci as presented in 25 the map of Figure 5 (hereinafter Map), the re 1 I ive order of any two said loci of said set may be -,iverted in relation to the order presented in said Map and the arrangement of said loci of said set comprising 2 or more loci when considered from either direction is equivalent.

According to a fifth aspect the present invention consists in a method for isolating, identifying or localizing a marker, trait, a gene contributing to a quantitative trait or nucleic acid fragment in Brassica using the nucleic fragment as described above.

I a i l lib According to a final aspect the present invention consists in a method for id, fying, distinguishing or tracing individuals, traits of said individuals or nucleic acid sequences of said individuals in Brassica using nucleic acid fragments as described above.

Linkage maps of Brassica have been developed by identifying and analyzing the coinheritance of nucleic acid polymorphism markers obtained from Brassica libraries within several segregating F 2 populations and determining the interrelationship of the markers by means of maximum likelihood analysis. Those markers comprise 9 linkage groups and encompass 818cM of the genome in B. oleracea and 10 linkage groups in B.

campestris. The resulting maps and markers will be useful for identifying and mapping important genes, for Rec'd PCT/PTO 2 9 M 199 01B 4 -12organizing and identifying varieties as part of crop improvement and protection program, as well as for studying the interrelationships and genome organization of Brassica varieties.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts a screening Southern blot, known colloquially as a garden blot, with DNA's representing B. oleracea, B. campestris and B. napus accessions.

Figure 2 depicts a secondary screening Southern blot, known colloquially as a salad blot, designed to show intraspecific and intrasubspecific variability, with DNA's representing B. oleracea and B. campestris accessions.

Figure 3 depicts a portion of a mapping blot containing DNA's of the two parents denoted P and W, an

F

1 and 10 F 2 individuals.

Figure 4 is a diagram of o B. oleracea map, vertical lines represent the linkage groups, loci are denoted to the right of the vertical and approximate distance between loci in cM is denoted to the left of the vertical. An area of uncertainty as to locus order is denoted with a secondary vertical line to the right of the loci designations (on 4C).

Figure 5 is a diagram of a B. campestris map in the same format as for the previous figure.

Figure 6 depicts a blot that is representative of the majority of the clones. EW4D12 hybridizes to a plurality of fragments. So far, it has been possible to identify and map three segregating loci from within the complex pattern.

Figure 7 depicts a blot of one of the few speciesspecific clones. The clone hybridizes to accessions of B. oleracea but not to those of B. campestris. No hybridization is observed in B. napus (Westar).

SUBSTITUTE SHEET WO 90/12026 rCr/US90/01 848 -13- Figure 8 depicts interspecific linkage group conservation between oleracea and B. campestris. B.

oleracea linkage groups are denoted as 1C through 9C and B. campestris linkage groups are denoted as 1A through Figure 9 diagrams interspecific linkage group conservation between R. oleracea and napus. B. napus linkage groups are denoted with an AC.

Figure 10 depicts intragenomic linkage group conservation in B. oleracea.

Figure 11 diagrams another f. oleracea map (that is under construction) using progeny from a cross (EW x CR7) different than that used in constructing the map in Figure 4 (P x WGA). Fewer markers have been placed on the EW x cR7 map. Note that the recombination frequencies differ in the two maps.

DETAILED DESCRIPTION OF THE INVENTION All of the terms in the specification and the claims are known to one skilled in the art.

Nevertheless in order to provide a clear and consistent understanding of the specification and the claims, including the scope given to such terms, the following definitions are provided: Accession: Synonymous with line or strain within a subspecies.

Allele: Any one of a series of alternative forms of a marker, trait or sequences at a locus.

Clone: Chimeric DNA molecule comprised of a biological vector, which can be viewed as a selfreplicating carrier, and a plant DNA insert.

cM: A relative measure for ordering nucleic acid fragments based on recombination frequency, which depends in part on sample size; provides an WO 90/12026 PCT~US90/0148 -14approximation of distance between markers; the value can vary among species and between sexes, e.g. IcM is equivalent roughly to 139kb in Arabidopsis thaliana, 510kb in tomato, 1,108kb in human and 2140kb in maize.

Expressed Secuence: Synonymous with expressed gene, a nucleic acid fragment that is transcribed; the RNA may or may not be translated into the corresponding protein.

Gene: As used herein, refers to a nucleic acid fragment.

Insert: Plant DNA ligated into a biological vector, such as a plasmid, virus or cosmid.

Linkage Group: Genes or loci or markers that are situated proximally in a chromosome. May be defined as loci that show less than 50% recombination or in another context as those loci that comprise a chromosome.

Locus: Position that a marker occupies in a chromosome; portion of a chromosome or linkage group that is defined functionally or descriptively (an example of a descriptive definition is a clone).

Map: Physical location of a nucleic acid fragment in the genome; the process of localizing a nucleic acid fragment in the genome which could involve linkage analysis, test crosses, somatic cell hybrids, sequencing and the like.

Marker: Any traceable polymorphism, character, trait, protein, gene, locus, nucleotide sequence and the like.

Polymorphism: Synonymous with variability, existing in more than one state or form; thus a monogenic character such as leaf shape may present with either round or ovoid leaves and DNA sequences from the same locus on the chromosomal homologs may vary at one or more nucleotides.

Probe: Any nucleotide segment capable of hybridizing to DNA sequences.

WO 90/12026 PCr/US90/01 848 uantitative Trait: Synonymous with polygenic trait, a phenotype whose expression depends on more than one gene; defined classically as a trait whose expression depends on three, and preferably more than three structural genes.

Random Sequence: A nucleic acid fragment that does not appear to or may not encode an RNA.

Related Fragments: As used herein, two nucleic acid fragments are related if each separately hybridizes to a third nucleic acid fragment having sequence homologies to the two fragments. The two nucleic acid fragments do not necessarily have to overlap.

Substantial Sequence Homolory: Substantial functional and/or structural equivalence between sequences of nuclectides. Functional and/or structural differences between sequences having substantial sequence homology will be de minimis.

Two/Three Point Analysis: Synonymous with two/three point testcross, following the inheritance of two or three markers in a sibship or family to determine whether or not they comprise or a part of a linkage group, to determine the linkage distance between the markers and in three point crosses to quantify single and double recombinants (double recombinants allow for the ordering of the loci).

The instant invention relates to clones, products of said clones, the ordered array of the loci specified thereby in Brassica genomes and uses of those clones and loci. The methods described in the specification are known in the art. Suitable methods may be found in Molecular Cloning (1982) by Maniatis et al., in selected volumes of Methods in Enzvmology and more specifically in Helentjaris et al., 1985, 1986 and Figdore et al., 1988.

Rec'd PCT/PTO 2 9 MAY 1991 RCT/US 9 0 0 1 4 8 -16- The utilization of nucleic acid polymorphisms as genetic markers requires generally the development of a set of cloned sequences known commonly as a library or bank and then identification of a subset of clones which are of use in the varieties of interest. For example, as a source of cloned sequences, total genomic DNA extracted from lyophilized leaf tissue of "Early White" (EW) cauliflower o. botrvtis) was digested with the methylation sensitive restriction enzyme PstI and low molecular weight fragments cloned into the plasmid vector pUC19, as described previously (Figdore et al., 1988). Clones containing low copy number sequences were identified based on low signal strength following hybridization with "Early White" total genomic DNA.

Plasmid DNA extracted from individual clones was hybridized to lanes of EcoRI and HindIII digested genomic DNA extracted from a variety of different Brassica accessions (Table Other restriction endonucleases that commonly reveal polymorphism are BglII, EcoRV and SstI. The accessions screened varied but always included the following genotypes: "Wisconsin Golden Acres" and "Brunswick" (cabbages); "Packman", "CR7", and "CR8" (broccoli); "Early White" (cauliflower); "Westar" (oilseed rape); "Michihili" and "WR 70 Days" (pak choi); and "Spring Broccoli" (B.

utilis). An example of a typical screening Southern blot is shown in Figure 1.

14- WO 90/12026 WO 9012026PCT/US90/01848 -17- Table 2 Brassica Accessions Screened for Polymorphism pl. camnestris ssp. chinensis BR. oleracea ssp. italica 1 2.

3.

4.

6.

7.

8.

9.

Gai Choi Canton Dwarf Takii #1 Best Seed China Pak Choi Milky Way CC419 Hon Tsai Tsai Sai 1 2.

3.

4.

5.

6.

7.

8.

9.

11.

12.

13.

Atlantic Bonanza Surfer B19 Gem DiCicco Cruiser Green Top Premium crop B18 Packman 0SU CR-7 OSU CR-8 ssp. Dekinensis Michihi.i Dynasty Jade Pagoda Green Rocket Hakuran WR 70 Days ssp. capitata 1. Wisconsin Golden Acres 2. Brunswick ssp. botrytis 1. Early White ssp. nutijis 1. spring Broccoli ssp. oleifera 1. UCD 77-4 Turnip Rape ssp. campestris 1. Rapid-cycling ssp. oleracea 1. Rapid-cyling B. nanus ssp. oleifera Westar WO 90/12026 P~/US90/0148 -18- The potential usefulness of a clone is dependent on the degree of polymorphism among Brassica varieties of interest. A high level of polymorphism exists among and within Brassica crop species, particularly B. oleracea, B. campestris, and napus. (Wash conditions were 0.25 x SSC, 0.1% SDS at 60'C.) Variability is assessed at three taxonomic levels: among Brassica species, (2) among subspecies within species, and among accessions within subspecies. Differences in restriction fragment hybridization patterns for tested clones occur at frequencies of 95% among species, 79% among subspecies within species, and 70% within subspecies (Figure The high degree of polymorphism found even among closely related Brassica accessions indicates that the clones can be very useful tools in genetic, taxonomic, and evolutionary studies and in crop improvement and breeding programs of the Brassica.

The screening procedure is designed to identify those cloned sequences that reveal polymorphism between the parental material used in generating F 2 segregating populations among B. oleracea, B. campestris, B. napus and other species, as well as clones which are informative in a number of different varieties. That would permit comparative mapping and confer upon the clones utility in a variety of accessions. Gene maps were constructed in different species of Brassica with the set of informative clones described herein.

Some of the clones were mapped in the following illustrative manner. "Packman" o. italica) and "Wisconsin Golden Acres" o. capitata) were crossed and an F 2 segregating population was obtained by selfing a single F 1 plant. Genomic DNA was isolated, digested, separated, blotted and hybridized with labelled clones using standard procedures. Clones were hybridized to EcoRI-digested DNA of several "Packman" individuals; several "Wisconsin Golden Acres" individuals; and the WO 90/12026 PCT/US90/01 848 -19single "Packman" x "Wisconsin Golden Acres" (P x WGA) Fi individual which gave rise to the F 2 population used in the segregation analysis. Inclusion of a sample from the FI permitted identification of clones detecting segregating alleles that were heterozygous in the parentals. Such clones were then hybridized to DNA of each of the parents, the F, and each of 96 F 2 progeny described above (Figure Segregation data for the polymorphic fragments were analyzed by the method of maximum likelihood. Chi-square goodness of fit analyses to the expected 1:2:1 ratio were calculated for each of the loci.

Table 3 sets forth the locus, clone and map data in B. oleracea and B. campestris. A consistent, conventional method of identifying Brassica chromosomes does not exist. Chromosomes have been characterized cytologically to some degree, but no correlation exists between the cytological descriptions and genetic markers (Prakash Hinata, 1980). Thus, in the absence of conventional chromosome or linkage group nomenclature, numerical labels are assigned herein to the nine linkage groups found in B. olerac:ea, 1C through 9C, with C representing the traditional designation for the B.

oleracea genome (Figure For the B. campestris genome, the ten linkage groups are labelled as 1A through 10A (Figure In both species, the linkage groups are numbered arbitrarily. Multiple loci detected by a clone share a common locus name and are denoted individually by letter.

Although the strategy for obtaining clones favors unique or low copy number sequences, the majority of the clones, 93% of the clones from the "Early White" library, yielded complex hybridization patterns comprised of multiple segregating fragments. A typical blot is presented in Figure 6. The level of sequence complexity of the Brassica has not been encountered in 20 other plant species and in part provides an explanation for the scanty knowledge of Brassica genetics. It is true that duplicate loci exist in, for example, maize and lettuce, but in those species the majority of the loci are analyzable as single loci. Construction of linkage maps in Brassica presents a unique challenge because allelic forms must be perceived from within a multiplicity of hybridizing fragments. In the example of Figure 6 three loci discerned by EW4Dl2 were identified within a pattern comprised of more than 10 fragments.

A majority of the clones hybridize differentially to accessions representing different species and subspecies, reflecting deleted or derived sequences 4' 0resulting from evolutional processes. Figure 7 depicts a 15 clone that hybridizes only to B. oleracea accessions.

Alternatively, a few of the clones hybridize to a single locus in one accession and multiple loci in another. The species-specific clones are valuable for monitoring ,,genotypes in synthetic or natural hybridizations (see Schweizer et al. 1988).

The entire library of clones was introduced into E.

Coli and the transformed host was deposited on 213. March, 1989 with The American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, under accession number 67915 and the provisions of the Budapest Treaty.

44 Deposit is for the purpose of completeness but is not intended to limit the scope of the present invention to the materials deposited since the description as further illustrated by the examples enables fully the practice of the instant invention. Access to the cultures will be available during the pendency of the application to those determined by the Commissioner of Patents and Trademarks to be entitled thereto. All restrictions on availability of said culture to the public will be removed irrevocably upon the grant of the instant application and said culture will remain available

I'

21 during the term of said patent. The culture will be replaced should it become nonviable or be destroyed.

It is common general knowledge that the individual clones can be obtai-ed from the library of clones deposited with the ATCC using techniques conventionally utilized by persons of ordinary skill in the art. An example of such a process involves the following steps.

The library would be diluted in liquid medium and plated out on agar media by standard methods, to yield individual colonies each corresponding to one clone.

Since the original library consists of DNA fragments (the inserts) cloned into a common restriction site of the vector, one uses oligonucleotide primers with homology to vector sequences flanking the inserts, and with opposite orientation and complementarity to each other, to PCR-amplify the inserts directly from the colonies using the following approach. Individual colonies are placed in water in an Eppendorf tube, heated for 10 minutes at 95-100 0 C to lyse colonies, PCR reaction components are added, and reaction is run using standard methods and appropriate annealing temperatures for the specific primers used. Hundreds of these reactions can be run in a day, limited only by availability of the thermal cycler units. A sample of each amplified product is resolved on an agarose gel and the sizes of inserts determined. All clones having unique size inserts are set aside. All clones having identical insert sizes are digested with a common restriction enzyme(s) and rerun on a gel. Differences in digest patterns indicate unique clones and these are set aside as well; one of each of the clones having identical digest patterns is kept.

This way, the majority of unique clones from the library can be identified and isolated within about one to two weeks. If some clones can still not be recovered after screening through about 1,000 colonies, one can combine an aliquot of each of the unique clones, prepare labelled probes of the mixture and hybridize the colony lifts from 4,t 0eeee ••el eo 21a the entire library. Any colonies which do not 'light up' will represent new clones. DNA from these new clones can be isolated as above and shown to be unique. As a final verification, the individual clones could be hybridized to a Southern blot of genomic DNA, where a unique banding pattern is expected for each. This could be done on strip filters to permit many probes to be rapidly tested. This way all clones can be recovered.

tl o* *e TABLE 3 Brassica Clones

LOCUS

1 2A 2B 3 4A 4B 4C 6A 6B 7A 7B 7C

BA

8B 9A 9B 9C 1OA

IIA

11B lic 12A 12B 13 14 Clonie**** EWlDO7 EWIG0 3 EWIG03 EW2BOl EW2E07 EW2EO7 EW2EO7 EWlCO7 EWID02 EWID02 EWIF08 EWiFOB EWlFOB EW2AO 6 EW2AO6 EW3DO7 EW3 D07 EW3DO7 EW4DO4 EW4DO4 EW4DO6 EW4DO6 EW4DO6 EW3COB EW3CO8 EW4DO9 EW4EO5 B. oleracea Linkage Group 2C 9c

NYM

3C 5C

'C

NYM

ic

BC

7C 2C

NYM

NYM

2C

NYM

5C 3C 5C ic 4C iC 5C 3C 8c

NYM

4C ic B3. campestris Linkage Group

NYM*

6A 5A

NYM

5A 1A 3A 10A

NYM

NYM

IA

2A 3A 2A 2A 10A

NYM

NYM

4A

NYM

3A

NYM

NYM

BA

3A

NYM

lOA 1.25 .93 .93 1.15 NYD N YD

NYD

NYD

1.05 1.05 1.325 1.325 1.325

NYD

NYD

1.15 1.15 1.15 .92 .92 1.025 1.025 1.025 0.8

NYD

Insert Size TABLE 3 Cont'd Brassica Clones Locus 16 17 18A 18B 19A 19B 21A 21B 22A 22B 23 24A 24B 258 26A 26B 26C 27 28A 28B 29A 29B 29C Clone EW2CO8 EW2CO8 EW2EO9 EW3AO1 EW2FO2 EW2FO2 EW2AO7 EW2AO7 EW3 804 EW4AO5 EW4AO5 EW1A07 EW1A07 EW4GO8 EWID09 EWID09 EW4G1 EW4G11 EWlE04 EWIE04 EW1E04 EW2B12 EW2CO6 EW2CO6 EW2DO3 EW2DO3 EW2DO 3 EW5C11 B. oleracea Linkacre GrouD 6C

NYM

iC 6C iC 4C iC

NYM

7C 4C ic 5C

NYM

3C

BC

6C Ic 7C 4C

NYM

NYM

4C 8C 3C 3C iC

NYM

6C B. campestris Linkagre-Group 6A 1OA

IA

NYM

5A

IA

IA

7A 7A 1A

NYM

4A 3A 7A 9A 9A

NYM

10A 5A 5A 4A 3A 8A 3A 1A

SA

6A Insert Size NkYD

NYD

NYD

1.45 .77 .77 1.1 1.1 1.45 1.075 1.075 .72 1.45 1.45

NYD

NYD

1.175 1.175 1.175

NYD

NYD

NYD

1.75 1.75 1.75 .79 TABLE 3 Cont'd Brassica Clones

LOCUS

31lA 31lB 32A 32B 32C 33 34 3 6A 36B 37A 37B 38A 38B 38C 39A 39B 39C 41 42 43A 43B 43C 43D 43E clone EW5C12 EW5CI2 EW2EO5 EW2EO5 EW2EO5 EwiBlO EWIF02 EW2BIO EW2 F05 EW2FO5 EW5FO7 EW5FO7 EW5FO5 EW5FO5 EW5FO5 EW5FOB EW5FOB EW5FO B EW6AO4 EW6AO4 EW6AO4 EW6A09 EW6BO3 EW6 BO 7 EW6BO7 EW6BO7 EW6BO7 EW6BO7 B. oleracea Linkagre Group 3C 8C 2C

NYM

NYM

4C iC 6C

BC

3C 9c ic 3C

BC

5C 6C

SC

4C 2C 2C

BC

2C lC 3C 9c 1c 3C 7C B. campestris Linkcage GrouR 8A 3A 5A 7A 6A 4A 1A

NYM

BA

3A

IA

NYM

3A

IA

IA

SA

NYMd

NYM

2A 3A

NYM

9A 7A 7A

NYM

NYM

NYM

NYM

insert Size

NYD

NYD

.47 .47 .47

NYD

2.75 1.45 .73 .73

NYD

NYD

NYD

NYD

NYD

.96 .96 .96

NYD

NWD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

0 0

S.

N

0

N

C'

N

U,

'0 0

S.

0 TABLE 3 Cont'd Brassica Clones

LOCUS

44A 44B 46A 4 6B 47 48 4 9A 49B 51A 51B 52 53 54A 54B 54C 54D 56A 56B 56C 57A 57B 58 Clone EW5HO2 EW5HO2 EWD12 EWD12 EW5HO3 EW5HO3 EW5H06 EW5G04 EW6AO7 EW6AO7 EW2CO1 EW3GO9 EW3GO9 EW3GO9 EW3AO4 EWiHOl EW2BO9 EW2BO9 EW2BO9 EW2 B09 EW6BIO EW6BlO EW5GO9 EW5G09 EW5GO9 EW5FO9 EW5FO9 EW6CO5 B. oieracea Linkage Group 2C

SC

6C 5C 3C

NYM

4C 4C 2C

NYM

9c

NYM

NYM

NYM

7C

BC

9C

NYM

NYM

NYM

6C 5c 8C 3C 7C 4C 3C 7C B. camnpestris Linkage Group 6A

IA

5A

NYII

1A 8A 5A 4A 2A 9A 9A 6A 1A 6A

IA

BA

7A 6A 6A 6A 5A 4A

IA

4A NYZ4

NYM

NYM

5A Insert Size

NYD)

NYD

NYD

NYD

0.8

NYD

NYD

NYD

NYD

NYD

NYD

1.45

NYD

1.05 1.05 1.05 1.05 .96 .96 .66 .66 .66 1.2 1.2 TABLE 3 Cont'd Brassica Clones

LOCUS

59A 59B 61A 61B 62A 62B 62C 63 64 66A 66B 67A 67B 68 69A 69B 71 72A 72B 72C 73A 73B 74A 74B Clone EW6C11 EW6CIl EW6EO1 EW6CO9 EW6CO 9 EW6C12 EW6C12 EW6CI2 EW6DO B EW6EO 3 EW6FO2 EW6GI2 EW6G12 EW3CO1 EW3COl EW3CO4 EW2DO1 EW2DO1 EW3FO1 EW3FO1 EW4BO2 EW4 DI2 EW4DI2 EW4DI2 EW4EO9 EW4EO9 EW3H12 EW3H12 B. oleracea Linkage Group 4C NYI4 6C ic 6C 5c 6C 8C 5C ic Ic 3C 8C 5C 3C 8C 4C 3c ic 6C 4C 2C 2C 8C 6C 3C 6C iC B. campestris Linkage Group

SA

7A 1A 9A 2A 1A NYI4

NYM

3A 1A

NYI!

8A 3A

NYM

8A

NYM

NYM

1A

NYM

4A 2A

BA

NYM

6A

NYM

1A

NYM

Insert Size (kb,)1 1.3 1.3 1.7 68 .68

NYD

NYD

NYD

NYD

1.15 .66

NYD

NYD

1.35 1.35

NYD

NYD

NYD

NYD

1.1 1.1 1.1 .87 .87 1.3 1.3 TABLE 3 Cont'd Brass ica Clones Locus 7 6A 7 6B 77A 77B 78 79 8BB 81 82A 82B 83A 83B 83C 84 8 6A 86B 87A 87B 88 89 91A 91B Clone EW5CO5 EW5CO5 EW4FO4 EW4FO4 EW4HO5 EW4HO5 EW5BO2 EW4HO9 EW5B03 EW5B03 EWSE10 EW5FO2 EW5FO2 EW7CO8 EW7CO8 EW7CO8 EW5HO1 EW7C1O EW7BO2 EW7BO2 EW5AO1 EW5AOl EW5A12 EW7A1 1 EW7BO4 EW7BO4 EW7DO3 EW7DO3 B. oleracea Linkage GrouR ic

NYM

2C ic 9c 3C 5c 9c ic 3C 6C 8c 2C 2C 2C

NYM

ic 6C 5c ic 6C 5c 7C 4C 2C 2C 3C

NYM

P. campestris Linkage Group 8A 1OA

IA

3A 9A

NYM

NYM

7A

NYM

NYM

IA

NYM

NYM

2A 2A

IA

IA

9A

IA

NYM

9A

NYM

NYM

2A 1A 5A 9A Insert Size f(k) 1.35 1.35 .53 .53 1.2 1.2 1.025

NYD

NYD

.71 .66 .66

NYD

NYD

NYD

1.75 69 69 1.425 1.425 1.15 .87 .87 .87 .96 .96 0 '0 0

-S

0 t~J 0~~ cj~ ~0 0 0 cc TABLE 3 Conttd Brassica Clones

LOCUS

92A 92B 93A 93D3 93C 94 9 6A 968 96C 96D 97A 97B 98A 98B 9 9A 998 100 101 102A 102B 104 105SA 105B 106 107A 107B Clone EW7EO8 EW7EO8 EW7El2 EW7E12 EW7E12 EW7GO6 EW7GO7 EW7GO7 EWSAO1 EWSA01 EWBAO1 EW8AO1 EWA11 EWA11 EW8B07 EW8B07 EW5F03 EW5FO3 EW7H11 EW8E09 EWBF08 EWBF08 EW6F12 EW8C11 EWsCil EWBD06 EW8F06 EW8F06 B. oleracea Linkage Group 5C 3C Ic iC 7C

SC

3C

NYM

3C

NYM

NYM

NYM

5C iC 2C 3C iC

NYM

4C 9c 8C 9C

NYM

iC

NYM

9C 4C

NYM

P. campestris Linkage Group 9A

NYM

1A

IA

NYM

SA

10A 9A 6A 6A 5A 1OA 1A 5A 2A 6A 3A 8A

IA

NYM

3A

NYM

2A 1A 7A

NYM

7A 4A Insert Size 1.125 1.125 1.125 1. 1.15 .77 1.15 1.15 1.325 1.325 1.325 1.325 1.175 1.175 .96 .96 .87 .425 1.6 1.6 .66 .71 .71 1.1 1.2 1.2 TABLE 3 Cont'd Brassica Clones Locus 108 1 09A 109B 110 111 112 113 114 115 116 117 118 11 9A 119B 120 121 122 123 124A 124B 125A 125B 126 127 128 129 130 131lA Clone EW7EO1 EW8A06 EW8AO6 EW8AO9 EW8B11 EW5A09 EW5FO4 EW9AO7 EW9DO2 EW9AO8 EW9EO1 EW9AO6 EW9BO2 EW9BO2 EW9FO6 EW9 FO 2 EW9HO2 EW9DO6 EW9DO8 EW9DO8 EW9EO5 EW9EO5 EW9E1O EW9 FO 8 WG lAl 0 WG1AO1 WG2HO 5 WG2C1O B. oleracea Linkag~e Group 7C 9C 9c 6C 6C 8C 3C 9c 8C 6C 7C 5C 4C 2C 2C 7C 4C 3C 6C

NYM

3C iC 4C 7C 4C

IC

4C 9c B. cainpestris Linkagie Group

NYM

9A

NYM

NYM

9A

NYM

9A

IA

5A 7A 9A 5A

NYM

NYM

NYM

NYM

3A 1A 8A

NYM

NYM

NYM

NYM

4A

IA

8A 7A Insert Size .87 1.55 1.55

NYD

.79 68 1.25 1.1 .87 1.1 1.85 .76 1.1 1.1 1.35 2.2 1.6

NYD

.83 .83

NYD

NYD

1.7 1.8 1.8

NYD

TABLE 3 Cont'd Brass ica Clones

LOCUS

13 1B 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 14 7A 147B 148 149 150 151lA 151B 152 153A 153B 154A 154B Clone WG2 010 WGIAO07 WG1C10 WG2GO6 WG3H1 0 WG2HO4 WGSA02 WGIF04 WGIB07 WG2GO5 WG4A1 0 WG3E12 WG3 HO 1 WG5AO5 WG5BO 3 WG5BO 9 WG2 CO 7 WG2 C07 WG3 008 WG3D11 WGIE03 WG1E08 WG1EO B WG4H1O WR2 007 WR2 CO 7 WG2 GO 2 WG2 GO 2 B. oleracea Linkage Group 7C 7C 30 10 10 9C

NYM

2C

SC..

BC

IC

iC 8C 2C 60 9C 70 6C 7C 4C 4C

BC

HY!4 2C 9c 10 6C

NYM

B. campestris Linkage Group

NYM

NYM

3A

NYM

NYM

9A

IA

NYM

NYM

NYM

10A

NYM

8A 3A 9A

NYM

NYM

NYM

7A 4A

NYM

BA

1A

NYM

10A 9A 5A 9A Insert Size 1.475

NYD

NYD

1.8 1.6

NYD

1.3 1.55 1.2 1.875

NYD

1.4 1.55 .88 1.55 1.2 1.2

NYD

1.95

NYD

NYD

1.65 1.6 1.6

NYD

NYD

TABLE 3 Cont'd Brassica Clones

LOCUS

155 156 157 158A 158B 159 160 161 162 163 164 165 166 167 168 169 170 171 17 2A 172B3 173 174 175 176 178A 178B 179 180 Clone WRIG1 0 WG4AO 6 WG4DO8 WR2HO 9 WR2HO9 W112C02 WRIG07 WR2HO7 WR2AOB WR2BEiI WR2AO7 WR2 805 WR2HO 5 WR2BO2 WR2G1O WR2 FO 3 WRiGi 1 WR2EO 4 WG3FO4 WG3FO4 WG4CO9 WR2CO 1 WRlGO8 WG4CO7 WG5A0 7 WG 5A07 WG3 809 WG5A01 B. oleracea Linkag~e Group 8C 4C

C

2C

NYM

5C 7C 8C 7C 4C 6C 2C 2C 9c 8c 3C

BC

3C 9c 4C ic 5C 2C 6C 3C 3C 7C 4C B3. campestris Linikagre Group

BA

9A 7A 2A 2A

NYM

NYM

BA

NYM

7A IA 2A 8A 9A

BA

4A 8A

NYM

8A

NYM

NYM

5A

NYM

NYM

10A

NYM

NYM

NYM

Insert Size

NYD

1.2

NYD

NYD

1.6 .98 1.3 1.8 1.75

NYD

NYD

1.6 1.7

NYD

1.85 1.3 1.1 1.1 1.3 1.7 1.2

NYD

1.2 1.2 1.35 1.25 TABLE 3 Cont'd Brassica Clones

LOCUS

181 182 183 184 185 187 188 189 190OA 190B 191 192 193 19 4A 194B 195 196 197 198 199A 199 B 200QA 200B 200OC 201 2A 202B 203A Clone WG5E12 WG3 HI0 WRIE0 8 WR ID12 WR2AO1 WR2DlO WR2EO7 WR2EO9 WR2 FO 5 WR2FO5 WR2 FO 6 WR2BO9 WG4 EG7 WG3E 06 WG3 EO 6 EW8 Bi2 EW8CO4 EW8DO8 EW9AO9 EW9EO5 EW9EO5 EW8F03 EW8FO3 EW8 FO3 EW8E04 EW8E10 EW8E1O EW8 D1O B. oleracea Linkage Group 8C 2C 8C 6C 3C 3C iC 9C 4C

NYM

8C 5C 3C 3C iC

NYM

NYM

4C

NYM

3C

NYM

8C 4C 3C ic 7C

NYM

3C B. campestris Linkage Group 8A

NYM

8 i

NYM

3A

BA

9A 1A 4A

NYM

NYM

N5M

NYM

10A 1A 9A

NYM

3A

NYM

NYM

1A 7A 6A 8A Insert Size

NYD)

NYD

NYD

1.35

NYD

NYD

1.3 1.4 1.4 1.15 1.525 1.1 0.9 0.9 .87

NYD

NYD

NYD

NYD

.69 .69 69 .63 .63 .63 .58 TABLE 3 Cont'd Brassica Clones Locus 203B 204 205 206 207 208 209 211lA 211B 212 213 214 300 302 303 304 305SA 305B 306 307 308 309 310 312 3 13A 313B 315 316 clone EW8D1O EW9BO5 EW9GO1 EW8BO5 EW8CO8 EWSB8 EW9CO7 EW8Ell EW8E 11 EW8Fl1 EW9AO5 EW9BO9 iWG4A07 WG5Co 6 WR2AO6 WR2AO5 WR2 806 WR2BO6 WR1H02 WRIG09 WG3AO 8 WR2 811 WRlBO9 WG2AO7 WG3C1O WG3C1O WG2EO8 WG2 004 B. oleracea Linkage Group 4C

NYM

NYM

NYM

9C 2C

NYM

5C 9C

SC

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYI4 I4YM

NYM

NYM

NYM

NYM

NYM

P. campestris Linkage Group

NYM*

BA

10A 1A 6A 1A 4A

NYM

6A 9A 6A 1A 5A 1A 6A 10A 3A 5A 8A 6A

NYM

7A 3A 10A 5A 9A 5A Insert Size .58

NYD

NYD

1.55 .82 1.1 1.3 1.55 1.3

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

TABLE 3 Cont'd Brassica. Clones Locus 317 318A 318B 319 320 321 322 323A 323B 325 32 6A 326EB 327 328 329 330 331 332 33 3A 333B 334 335 336 Clone WG2 BO 2 WG2 G12 WG2G12 WG3B11 WGlCO5 WG5FO8 WRIC0 4 WR2BI2 WR2 B12 WR2HO4 WR1EO 3 WRIE03 W'RA12 WR2 EO 2 WRiAl 0 WG2AO6 WG5G03 WR1BO 3 WG3E1 1 WG3E11 WG3 HO 9 WG5AO7 WG2HO 5 B. oleracea Linkage GrouR

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYN

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYM

NYM

B. campestris Linkage Group 9A IA 8A 4A 6A 8A 8A 4A 3A 4A A 3A 6A 1A 3A 9A 9A

SA

6A 6A 1A 10OA

SA

0:_ 0 Insert size

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

NYD

Not yet mapped, mapping in progress.

Not yet determined.

Tentative assignment Clone may hybridize to multiple loci and not all loci have necessarily been mapped.

WO 90/12026 PCT/US90/01848 The present invention is illustrated further by the following non-limiting examples. Unless indicated otherwise, the methods used are as described above.

EXAMPLE 1 Size-restricted (0.5-2.0kb) genomic libraries were prepared from lyophilized leaf tissue DNA digested with a methylation-sensitive restriction endonuclease. The eluted fragments were subcloned into a plasmid, commonly pUC19 or pTZ18R, which were used to transform hosts to antibiotic resistance. First, the colonies were screened with total genomic DNA to identify recombinants carrying highly repeated sequences. Those were not analyzed further. Several libraries were constructed and in each case, 75% or more of the clones did not contain highly repeated sequences. Recombinants carrying single or low copy number sequences were used as probe in garden and salad blots to determine whether the clones detected polymorphic loci, as described above. Clones that detected polymorphis loci were selected for hybridization to DNA from sibships in order to map the loci specified by the clones.

Each of the clones was used as probe to a panel of DNA's obtained from a population of F 2 plants (generally about 100 plants) obtained by selfing of a single F,.

The segregation of polymorphic fragments was determined among the panel of F 2 DNA's to produce a unique pattern corresponding to the inheritance of alleles carried at each of the loci among the mapping population. Greater than 94% of the loci presented with the expected 1:2:1 ratio of inheritance for codominant alleles.

Allele inheritance among the F 2 were analyzed in a computer to determine which of the loci were linked and to what degree. If patterns matched identically, it is possible to conclude that the clones are tightly linked, WO 90/12026 PCF/~US9 0/011848 -36i.e. separated by less than IcM or that they hybridize to the same locus. If two loci are 1cM apart in the genome, then the hybridization patterns for the two clones would be the same except for one difference, given that the mapping population comprised 100 F 2 samples. If patterns show no resemblance, the loci are either on different linkage groups or are separated by more than 50cM on one linkage group. Because such a large number of clones were analyzed, it is necessary to catalog and analyze the data using computerized statistical programs, such as maximum likelihood analysis. Briefly, the analysis involves comparing a pattern with each of the existing patterns.stored and computing a numerical value ranking based on the number of associations. The analysis then orders the loci into linkage groups in the most statistically supported fashion. The linkage groups comprise loci which had similar patterns of inheritance in the mapping population. An example of the data obtained following such an analysis is illustrated for seven loci of 8C in Table 4.

EXAMPLE 2 Using the clones of the instant invention it has been found that the genomes of B. oleracea, B. napus and B. campestris are highly homologous. Figdore et al.

(1988) shows the hybridization of some cauliflower clones in B. campestris varieties, among other subspecies of B. oleracea and in B. napus. A majority of the cauliflower clones hybridize to DNA of the accessions screened routinely showing the homology of sequences in those genomes with respect to the tested clones. Similarly, a majority of the clones obtained from a B. campestris library hybridize to B. campestris subspecies, B. oleracea and B. napus. The order of loci WO 90/12026 PC/US90/01848 -37in B. oleracea chromosomes is conserved to a high degree in B. campestris chromosomes. (Figure Because it is highly likely that B. naDus is a natural hybrid of B.

oleracea and B. campestris, it is not unexpected that the genomic organization of the three species is related. Early mapping studies in B. napus illustrate the :s3atedness (Figure Thus linkage relationships a-:e ~Antained in those species and enhances the probability that linkage of a marker to a desirable trait or quantitative trait locus in one species is maintained in others.

Table 4 Statistical Ordering of Seven Loci on 8C* Locus 72c 24a 31b 66b 28a 38b 36a 72c 4.5 5.2 5.7 6.4 16.2 17.4 24a 0.5 2.1 3.2 9.2 13.7 31b 1.6 2.7 8.9 13.2 66b 1.0 7.0 11.2 28a 7.5 10.1 38b 2.8 72c 24a 31b 66b 28a 38b 36a 8C (3) Maximum likelihood estimates of recombination frequency and inferred gene order. The values are expressed in cM. 4.3 was the largest standard error. In the diagram of 8C, that is not to scale, the loci are denoted above and the map distances are denoted parenthetically below. Three-point linkage analysis was used to confirm the linear order.

WO 90/12026 PC/US90/01i848 -38- An additional advantage of the instant invention is data regarding intragenomic redundancy. The clones reveal that many loci in the B. oleracea genome are present in more than one copy. The function of those low copy number loci is unknown and awaits more detailed molecular analysis such as sequencing. Nevertheless the map reveals regions of homology where linkage relationships are maintained within or between chromosomes, see for example the six loci maintained in register on linkage groups 3C and 8C (Figure 10). It follows that the rationale on the use of conserved linkages to predict the presence and location of loci expounded above can be applied here. Thus if a locus that contributes to a quantitative trait is found within a linkage group on one chromosome and the markers of said linkage group are duplicated and organized similarly on another region of the sace chromosome or on another chromosome, then there is an enhanced probability that a similar gene contributing to said quantitative trait is contained within the duplicated segment.

EXAMPLE 3 Clones that hybridize to polymorphic loci are valuable markers for identifying genes of interest.

Clones such as those of the instant invention may arise from an expressed gene or lie adjacent to an expressed gene, either a short or considerable distance away. A classic method for determining and subsequently quantifying the extent of linkage between two markers is to ascertain whether the markers are inherited together within a sibship segregating the markers. The closer the markers are on a chromosome the more likely the markers will be expressed either together or not at all in any one individual. If the markers are unlinked, either far apart on a large chromosome or on separate WO 90/12026 PCUS90/01 848 -39chromosomes, it is equally likely to find either, neither or both markers in any one individual of the segregating population. The minimum of 0% recombination occurs when the markers are linked tightly or identical, and the maximum of 50% recombination occurs when the markers assort independently. Values between 0% and recombination reflect partial linkage. The percent recombination is related to crossing over, the number and location of chiasmata between homologs at cell division. A near equivalent expression of percent recombination is the centimorgan Thus, one percent recombination between two markers is equivalent roughly to IcM and indicates statistically that the two markers were inherited concordantly in 99 of 100 offspring. Only once did a single crossover occur between the markers to place them on alternative segregating homologs at meiosis.

Prior to 1980 linkage analysis among higher organisms was restricted to the study of morphologic or protein variation. The number of scorable markers is limited. DNA polymorphism introduced a reservoir of markers that will likely permit saturation of the genome. By saturation, it is meant that scorable markers are at least 5cM, and preferably less than apart along the entire length of each chromosome. Thus, any new marker of interest will show linkage to known mapped loci with the percent recombination between the marker and any one locus decreasing with decreasing physical distance between the two.

The set of clones described herein cover a large portion of Brassica genomes. The average interval between two adjacent markers in B. oleracea is about 4cM (Table Thus, with a threshold established arbitrarily at 4cM, linkage of a new marker to one of the loci of the instant invention will be found 91% of the time. At that same threshold level, there are 17 WO 90/12026 PCr/US90101 t848 gaps in the map where linkage would be detectable with a recombination frequency that is greater than 4%.

However, because of the large number of mapped loci in the instant invention, the greatest recombination frequency that is required is only 9.5% (Table Thus for the largest gap, between clones 95 and 9b on linkage group 3C, a new marker situated midway between 95 and 9b would show 9.5% recombination with either locus. Given the large number of individuals that can be scored in a test cross, 10% recombination is a statistic that is readily resolved. Thus for all intents and purposes, any new marker that is situated somewhere between the most distal loci on the linkage groups 19 and 141 on 1C) can be identified and positioned by virtue of linkage to one or more of the loci described herein.

Table Linkage Group Information in Brassica oleracea Linkage Group ZcM Clones* Loci* Interval** 1 134 40 36 3.7 2 99 27 23 4.3 3 95 37 24 4 108 30 25 4.3 72 27 21 3.4 6 91 26 20 4.6 7 88 21 18 4.9 8 93 29 24 3.9 9 38 21 13 2.9 Total 818 258 204 Some loci are not distinguishable by recombination and are assigned to the same map position Expressed in cM WO 90/12026 PL~T/US90/01848 -41- Table 6 Gaps In Brassica oleracea Map Residual Linkage Gap Size* Group Flanking Loci 2 1 74b,61a 1 1 188,75 2 2 8,175 8 2 83a,90a 2 3 194a,80b 11 3 95,9b 4 4 33,100 4 47,59 2 5 78,67a 4 5 67a,97a 9 6 176,74a 4 6 145,24b 9 7 148,179 7 179,121 3 7 58,20 9 8 168,115 1 8 115,143 Size of nucleic acid fragment, expressed in cM, wherein sequences comprising said fragment are more than 4cM from a marker of the instant invention.

EXAMPLE 4 It is important to note that the order of the loci in the linkage groups is determined by a statistical method, maximum likelihood analysis, that estimates the recombination frequency between pairs of loci. In the absence of three-point linkage analysis, it is sometimes not possible to order the loci accurately, e.g. there are three possible linear orders for the three linked loci a,b and c; a-b-c, b-a-c or a-c-b. Thus, any two loci mapped by two-point analysis in one order may, upon increasing the sample size or conducting a three-point WO 90/12026 PCr/US90/01 948 -42analysis, be inverted. The maps presented in Figures 4, and 11 are based solely on successive two-point analyses. While the linkage data support the ordering of the loci, one must keep in mind the statistical limitations of the protocol used to establish the map.

Thus, any two markers may in reality be inverted.

Furthermore, in the absence of cytologic or anchoring landmarks, the orientation of the nine entire linkage groups is uncertain, i.e. it may be that the entire map of a linkage group is inverted. Nevertheless, that does not disrupt the overall relative order of the markers within each linkage group.

An example of discrepancies that can arise in an ordering of loci is alluded to in Paterson et al.

(1988). A map of loci in a cross of Lycopersicon esculentum and L. pennilli (E x P) ordered three pairs of linked loci as CD15-TG24 on chromosome 1, CD32B-TG63 on chromosome 10 and TG36-TG30 on chromosome 11 with the loci separated by 3, 9 and 6cM, respectively. However the order of each pair was reversed when the segregation data obtained from an L. esculentum by L. chmielewski (E x CL) cross was analyzed using a different computer linkage program. A reanalysis of the E x P data with the second program suggested that the inverse order for the first pair (TG24-CD15) is more likely in both E x P and E x CL. For the other pairs, the inverse order (TG63-CD32B and TG30-TG36) is more likely in E x CL by odds of 10':1 and 107:1, respectively, but the previous order is more likely in E x P by 11:1 and 8:1 odds, respectively. The problem is being remedied by studying a larger E x P population. The linear order of 64 other loci among the twelve chromosomes of tomato agreed in the two independent crosses using two different means of data analysis.

Asins Carbonell (1988) point out also thatstatistical methods based on the assumption of a WO 90/12026 PCr/US90/01 848 -43normal distribution and common variance (difference between means, one way ANOVA) and three-point analysis may be inadequate. The variability among genotypes may not be homogeneous in an F 2 population or in pooled backcrosses if linkage exists. The degree of dominance, linkage distance and heritability of the marker and gene of interest must be considered because linkage can contribute to variation.

In the map of B. campestris (Figure there are three regions where the exact order of loci is unclear (on linkage groups 1A and 8A, denoted by vertical lines to the right of the loci designations). The correct orders are being established by following the inheritance of the relevant markers in a larger number of progeny.

EXAMPLE A gene of interest can be isolated if the gene product or a scorable phenotype is known. But the biochemical bases for' many traits and disorders are unknown. The application of nucleic acid polymorphism to clone genes of interest in the absence of a phenotypic marker or identified gene product requires a cloned sequence that is linked to the gene of interest (Ruddle, 1984; Orkin, 1986). The limitation of that approach is technical, that is the physical distance between the marker and gene is the determinative factor.

The idea is that the investigator must walk along the chromosome using overlapping clones. Steinmetz et al.

(1982) used overlapping cosmids to map more than 200kb of contiguous DNA from mouse chromosome 17. In another example, about 200kb of contiguous DNA in the region of DXS164 was isolated by walking in a search for the human gene controlling Duchenne Muscular Dystrophy (DMD). A single clone, pERT87, was used to begin the walk. That WO 90/12026 PCT/US90/0 1848 -44clone was obtained from a library containing sequences from band p21 of the X chromosome where DMD had been mapped previously. pERT87 detected deletions in about of classical DMD patients. Family studies showed that the clone is tightly linked to expression of the disease. pERT87 allowed investigators to clone sequences at the DMD locus, to identify the transcript of that gene, to predict the sort of protein that could be encoded by that transcript and eventually to identify a protein, called dystrophin, that is likely to be the natural product of the DMD gene and may be involved in the pathogenesis associated with DMD (Kunkel et al., 1985; Koenig et al., 1987).

The advent of newer technologies such as jumping/hopping libraries and pulsed field gel electrophoresis enable identification of genes located much farther from a starting clone, i.e. the gene of interest need not be within a few hundred base pairs of the polymorphism but can be located IcM or more from the starting clone. It its essential to have a linked marker that is, according to the limits of the current technologies, within 5-10cM of the desired gene, for the linked marker initiates and enables the cloning of the desired gene. The desired gene is cloned primarily by its map position, proximity to a known marker and either presence of consensus sequences found commonly in expressed genes or a showing of the desired activity upon transformation.

In brief, chromosome hopping/jumping depends on the circularization of very large DNA fragments (Collins Weissman, 1984). All but the extreme ends of large DNA fragments is deleted and the ligated junction fragments are cloned. That process brings together sequences originally far apart in the chromosome. One end comprises known cloned sequences, i.e. sequences of the polymorphic locus, and the other end represents a new WO 90/12026 PCT/US90/01848 sequence located elsewhere on the chromosome. The library containing the junction fragments is screened with the clone that detects the polymorphic locus and the positives mapped to identify the new sequences.

With the technique of Collins and Weissman, jumps are generally of about 200kb. Thus, 5 consecutive jumps cover 1cM of human DNA, and it is not unrealistic to begin a jump to a gene of interest from a clone that is IcM, 5cM or even 10cM away.

Poutska et al. (1987) modifies the jumping method by not constructing the library with genomic DNA that is digested partially or completely with enzymes having common sites, but using DNA that is digested with enzymes that cut rarely such as NotI, NarI, BssHII, NruI, MluI, SStII or SfiI. The result is large DNA fragments that produce larger jumps. For example, a NotI library was used for jumping along human chromosome 4. Starting and end points of two identified clones spanning a jump of 350kb were positioned within an 850kb restriction map.

The other breakthrough for long range gene mapping and cloning is pulsed field gel electrophoresis (PFGE) which goes by a number of acronyms, CHEF, OFAGE, FIGE, ROGE, etc. The acronyms reflect the configuration and actuation of the electrodes. Basically, the electric field is not steady-state but instead pulses and/or inverts. That results in electric fields with alternating orientations which impose additional migrational constraints on DNA molecules distinct from the reptational migration found in the steady-state fields of standard electrophoresis. Generally, it is difficult to resolve fragments greater than 30kb in size using standard agarose gel electrophoresis. However, with PFGE one can resolve fragments in the 1000kb range.

Improvements will allow resolution of even larger fragments (the current upper size limit is 7-10Mb).

WO 90/12026 PCT/US90/01848 -46- Nevertheless, present methods permit the separation of mammalian DNA molecules that carry markers IcM and more apart. That enables construction of long range restriction maps and isolation of large segments of DNA that can be used for preparing mini-libraries (Anand et al., 1988).

Chromosome jumping and PFGE often come together when an investigator begins with one clone and seeks to move from that clone to nearby sequences of interest (Richards et al., 1988). The investigator confirms the direction of the jump and map of the region using PFGE.

Two other methods for isolating large segments of DNA are chromosome-mediated gene transfer and yeast artificial chromosomes (YAC). Currently there are a few shortcomings of the former technique. One is the need for a selectable marker in the region to be cloned.

Also molecular arrangements are known to occur during the procedure and there is little control of the size to be transferred. Nevertheless, from a few hundred kb to of DNA from the short arm of HSA11 have been transferred to a mouse cell line (Porteous et al., 1986). The technique will no doubt be improved and find more general usage.

Cloning into YACs is an alternative procedure which accommodates DNA fragments of several hundred kb up to 1Mb. A specialized vector contains a cloning site within a selectable marker, autonomous replication sequence, centromere, selectable markers on either side of the centromere and a pair of sequences that seed telomere formation in vivo. When recombinants are sized at 50kb or larger, the chimeric molecules, when transformed into yeast, are maintained stably as artificial chromosomes. The current limitations on the use of YACs is that inserts larger than 200kb tend not to be clonable and the overall cloning efficiency is low (Burke et al. 1987).

WO 90/12026 PCr/US90/01 848 -47- EXAMPIE 66 The value of a linkage map increases sigmoidally after the first few markers are fixed to specific regions of the linkage group. That occurs because gene mapping is an empirical endeavor whereby new markers are placed on the map relative to markers already fixed on the map by virtue of linkage with the known mapped markers. Markers with established positions in the linkage group are often called anchoring markers. Thus, each of the loci of the instant invention can be considered an anchoring marker because of its known map position in a linkage group.

When a new clone is obtained and is to be mapped, a subset of anchoring markers are selected for the initial mapping of the new locus. The subset of anchoring markers comprises loci that are present about apart for each linkage group. By comparing the segregation pattern of the new marker with that of the anchoring loci, the linkage group to which the new marker maps can be determined. Furthermore, one can discern roughly the region of the linkage group where the new locus is likely to be found. The next step is to then compare the segregation pattern of the newly localized marker with the patterns of other markers known to map in the region suspected of housing the marker. For example, on linkage group 9C one might select one anchoring locus located centrally to cover the entire group, such as 2 or 101, or one might select two loci such as 79 and 50. If a new clone is mapped provisionally to 9C by virtue of linkage to say locus the next step in fixing the new clone is to compare segregation patterns with 37a, 102b, 136, 146 and possibly 2. The comparison(s) showing the tightest linkage(s) would place the new clone adjacent to that locus or loci.

WO 90/12026 PCT/US90/01848 -48- Thus, anywhere from 15 to 35 or more loci can be selected to comprise a "quick screen" subset of clones for the rapid identification of provisional map position in a first step of fine scale mapping of a new clone.

An example of a "quick screen" subset is presented in Table 7.

0 Table 7 0 Ouick Screen Subset of Anchor Loci Linkage Group Clone EW2BO1 EWlDO2 EW3DO7 EW2EO9 EW3AO1 EW2FO2 EWlA07 EW4Gl1 EW2B12 EW5C12 EW6AO4 EW2BEQ9 EW6Cl 2 EW6G12 EW3CO1 EW4EQ 9 EW5B03 3C 4C 7C 8C 9A/C 18B 22 18A 25A 3 1A 40A/B 31lB 62C 66B 66A 67B 73B

SOB

62A 67A 62B 80A Table 7 -Continued Ouick screen Subset of Anchor Loci Linkage Group Clone iC 2C 3C 4C 5C 6C 7C 8C 9C EW7CO8 83A/B 84 EW7BO4 EW7EO8 92B 92A EW7El2 93A/B 93C EW7GO6 9 EWBA06 1 EW8Bll11 EW5AO9 112 EW5FO4 113 EW9DO2 115 EW9 EO 1 117 EW9BO2 119B 119A EW9DO6 123 125 WG1A1O 128 WR2 Co7 153 B 153A 0 Table 7 Continued Quick Screen Subset of Anchor Loci Linkage Group sc 6C Clone

WRIGII

WG3BO9 WR2 FO 5 EW8E1O 3C 4C 190 7C 8C 170 179 202 Rec'd PCT/PTO 2 9 MAY '1.

PCT/Us 9 0/ 01 4 8 -52- EXAMPLE 7 The present example demonstrates one utility of the clones of the instant application. .inety-six genotypes of Brassica oleracea that represented a diverse range of commercially available as well as proprietary germplasm and included 73 broccoli lines, 14 cauliflower lines and nine cabbage lines were studied. The lines were obtained from various seed companies, public and private breeders. The 73 broccoli lines included standard open pollinated cultivars as well as inbred parental lines and their corresponding F 1 hybrids. In the following discussion, the 96 accessions will be identified numerically as 1, 2, 96.

Plant DNA was isolated from lyophilized leaf tissue.

Standard procedures for DNA isolation, restriction endonuclease digestion, electrophoresis, blotting, hybridization and autoradiography were used.

The clones hybridized to multiple loci resulting in complex banding patterns. For each clone-enzyme combination, restriction fragments across all accessions were assigned numbers 2, 3, n) according to decreasing molecular weight. A total of 61 fragments were identified in 15 clone-enzyme combinations. Each polymorphic fragment was treated as a unit character, and each genotype was scored for the presence or absence of a fragment.

The coefficient of relatedness (CR) between each pair of genotypes is calculated by dividing the difference of all concordant polymorphisms and all discordant polymorphisms by the total number of polymorphisms evaluated. For example, in a comparison WO 90/12026 P~/US90/01848 -53of genotypes 10 and 11, of the 61 total fragments, one was deleted because genotype 10 had a missing value for the particular restriction endonuclease utilized. Of the remaining 60 fragments, in 28 cases both genotypes lacked a particular fragment, in 12 cases genotype 11 had a fragment which genotype 10 lacked, in 8 cases genotype 10 had a fragment which genotype 11 lacked, and in 12 cases both genotypes 10 and 11 had the same fragment. The distribution can be arranged into a 2x2 table: Genotype 0 1 Total Genotype 11 0 1 28 8 12 12 36 24 Total 40 20 The coefficient of relatedness is: CR (40-20)/60 0.333 For illustration, Table 8 sets forth coefficients of relatedness for genotypes 1-12. Note that identity of the genotypes is manifest as a CR value of 1.00.

Table 9 sets forth coefficients of relatedness for genotypes 6, 10-12, 26-29, 31-34 and 41.

The average CR for the complete 96x96 matrix is 0.25. That value suggests the sample of 96 cultivated broccoli, cauliflower and cabbage are related. A high level of relatedness among genotypes 28, 33 and 29 was suggested by the analysis (Accessions 28 and 33 are inbreds and 29 a hybrid, all of the same species). A feature of the pertinent CR values is that genotypes 28 and 33 are highly related to genotype 29, 0.770 and 0.700 respectively, whereas the CR between 28 and 33 is smaller, 0.533. (A parent is generally more closely related to its progeny than to the other parent. Hence, WO 90/12026 PC/US90/01848 -54larger CR values are expected between a parent and its progeny than between parents.) Thus, genotypes 28 and 33 might be the parents of hybrid 29.

In an attempt to confirm the suggested relationship, genotypes 28 and 33 were "crossed" in a simulation to produce a hypothetical 28x33 hybrid. A simple algorithm used was for the simulation: 1 x 1 1 1xl=l 1 x 1 0 x 0= 0 The algorithm can be interpreted in terms of the inheritance of polymorphism, if either parent has a variant then the fragment will be observed in their progeny, and only if both parents lack a variant will it not be observed in their progeny. The algorithm is predictive if the observed polymorphism represents an inbred homozygous parent, an "AA" or "aa" genotype. If the polymorphism represents an allele of a heterozygous parent, then the probability of that allele being transmitted to any one progeny is 1/2. The probability of that allele being observed in a sibship increases with the number of progeny (l-(1/2 where n the number of progeny sampled). Thus, although heterozygosity in the parents can be a potential source of error in the algorithm, the error can be minimized by sampling several individuals from each entry.

The polymorphism inheritance patterns for genotypes 28, 33, 29 and the hypothetical 28x33 hybrid are presented in Table 10. The total number of concordant and discordant entries for 29 and 28x33 were arranged into a 2x2 contingency table and the coefficient of relatedness was calculated as 0.934: WO 90/12026 PCT/US90/01848 Hypothetical 28x33 Hybrid Accession 29 0 1 0 26 1 1 1 33

TOTAL

27 34 61 TOTAL That value corresponds to only two discordant values out of a total of 61, and was as high as the largest CR value calculated for the 96x96 matrix. Thus, it is highly likely that genotypes 28 and 33 are the inbred parents of hybrid 29.

WO 90/12026 WO 9012026PCI'/US90/01848 -56- Coefficients of Relatedness for Genotvmes 1-12 Genotype 2 3 1 .000 0.333 0.367 0.533 0.400 0.333 0.467 0.367 0.233 0.567 0.633 0.333 O0333 1.000 0.443 0.267 0.607 0.541 0.200 0.367 0.377 0.233 0.443 0.410 7 8 0.367 0.443 1.000 0.433 0.443 0.705 0.233 0.267 0.475 0.333 0.410 0.574 9 0.233 0.377 0.475 0.300 0.443 0.639 0.433 0.333 1.000 0.333 0.344 0.508 4 0.533 0.267 0.433 1.000 0.533 0.4 67 0.400 0.300 0.300 0.300 0.500 0.333 10 0.567 0.233 0.333 0.300 0.433 0.300 0.233 0.267 1.333 1.000 0.333 0.367 5 0.400 0.607 0.443 0.533 1.000 0.410 0.200 0.233 0.443 0.433 0.443 0.279 6 0.333 0.541 0.705 0.467 0.410 1.000 0.333 0.500 0.639 0.300 0.443 0.672 11 12 0.467 0.200 0.233 0.400 0.200 0.333 1.000 0.767 0.433 0.233 0.500 0.400 0.367 0.367 0.267 0.300 0.233 0.500 0.767 1.000 0.333 0.267 0.600 0.500 0.633 0.443 0.410 0.500 0.443 0.443 0.500 0.600 0.344 0.333 1.000 0.443 0.333 0.410 0.574 0.333 0.279 0.672 0.400 0.500 0.508 0.367 0.443 1.000 WO 90/12026 WO 9012026PCT/US9O/0 1848 -57- Coefficients of Relatedness for Selected Genotypes Genotype 1.000 0.300 0.433 0.672 0.738 0.738 0.410 0.377 0.367 0.467 0.400 0.377 0.705 28 0.410 0.300 0.443 0.410 0.475 0.541 1.000 0.770 0.300 0.200 0.533 0.180 0.574 10 0.300 1.000 0.333 0.367 0.433 0.433 0.300 0.333 0.200 0.167 0.433 0.267 0.400 11 0.433 0.333 1.000 0.443 0.311 0.311 0.443 0.541 0.467 0.300 0.500 0.410 0.344 12 0.672 0.367 0.433 1.000 0.672 0.738 0.410 0.443 0.433 0.467 0.400 0.377 0.639 26 0.738 0.433 0.311 0.672 1.000 0.934 0.475 0.508 0.300 0.267 0.600 0.115 0.500 27 0.738 0.433 0.311 0.738 0.934 1.000 0.541 0.508 0.367 0.333 0.533 0.180 0.902 29 31 32 33 34 41 0.377 0.333 0.541 0.443 0.508 0.508 0.770 1.000 0.467 0.300 0.700 0.279 0.541 0.367 0.200 0.467 0.433 0.300 0.367 0.300 0.467 1.000 0.767 0.433 0.533 0.333 0.467 0.167 0.300 0.467 0.267 0.333 0.200 0.300 0.767 1.000 0.333 0.767 0.300 0.400 0.433 0.500 0.400 0.600 0.533 0.533 0.700 0.433 0.333 1.000 0.300 0.567 0.377 0.267 0.410 0.377 0.115 0.180 0.180 0.279 0.533 0.767 0.300 1.000 0.148 0.705 0.400 0.344 0.639 0.836 0.902 0.574 0.541 0.333 0.300 0.567 0.148 1.000 WO 90/12026 PCT/US90/01848 -58- TABLE Polymorphism Distribution in 28, 33. 29 and 28x33 Marker 28 3 29 28X33 1 0 0 0 0 2 0 1 1 1 3 1 0 1 1 4 1 1 1 1 0 0 0 0 6 0 0 0 0 7 0 1 1 1 8 1 1 1 1 9 0 0 1 0 1 0 1 1 11 1 1 1 1 12 1 0 1 1 13 0 1 1 1 14 1 0 1 1 1 1 1 1 16 0 0 0 0 17 0 0 0 0 18 1 1 1 1 19 0 0 0 0 0 0 0 0 21 1 1 1 1 22 1 1 1 1 23 0 1 1 1 24 0 0 0 0 0 1 1 1 26 0 1 1 1 27 0 0 0 0 28 0 0 0 0 29 1 1 1 1 0 0 0 0 31 0 0 0 0 32 0 0 0 0 33 0 0 0 0 34 1 1 1 1 1 1 1 1 36 0 0 0 0 37 0 0 0 0 38 1 1 1 1 39 1 1 1 1 1 1 1 41 1 1 1 1 42 1 0 1 1 43 0 1 1 1 44 0 0 0 0 1 1 1 1 46 0 0 0 0 47 0 0 0 0 WO 90/12026 P~C/US90/01 848 -59- TABLE 10 (Cont'd) OBS 83_ E29 H2833 48 0 0 0 0 49 0 0 0 0 1 0 1 1 51 1 0 1 1 52 0 0 0 0 53 0 0 0 0 54 0 0 0 0 1 1 1 1 56 0 0 0 0 57 1 1 1 1 58 0 1 0 1 59 1 1 1 1 1 1 1 1 61 0 0 0 0 Could not be scored.

EXAMPLE 8 The clones of the instant invention can be used in alternative methodologies of detecting nucleic acid sequence polymorphism for distinguishing varieties and other uses detailed above. The polymerase chain reaction (PCR) enables amplification of specific target genomic) sequences contained between oligonucleotide primers. The reaction occurs in primer excess and involves repeated cycles of hybridizationsynthesis-denaturation. When a clone detects polymorphism that arises by variation in the length of alleles due to insertion or variable numbers of repeats), then the PCR can be used directly to compare individuals such as by visualizing fragments in ethidium bromide-stained gels. If, however, the polymorphism involves changes in single nucleotides then the PCR must be combined with subsequent steps. One such combination method is called oligomer restriction which requires the WO 90/12026 P&Tr/US90/01 848 mutation to occur within a restriction endonuclease recognition site. A fragment containing said restriction site is amplified by the PCR. The resulting mixture is then hybridized with a third labelled oligonucleotide that contains said restriction site. In the wild type allele, hybridization of the labelled oligo will be exact generating the restriction site. In a mutant that contains one or more base changes in the restriction site, the pairing of the oligo to the genomic sequence will be imperfect and the restriction site not generated. Thus the alleles are distinguished by the resultant reaction products following digestion of said fragment with the appropriate restriction endonuclease. It is possible to forego the PCR and distinguish alleles by virtue of hybridization fidelity between the labelled oligo and the genomic sequence however use of oligos as probe requires stringent control of the hybridization and wash conditions.

Although more labor-intensive and time-consuming, the actual base sequences of the variants can be determined and compared (Higuchi et al., 1988).

EXAMPLE 9 In Brassica, there has been relatively little genetic description of traits of interest such as morphologic characteristics, disease resistance, etc.

In many of the studies conducted to date, complex patterns of inheritance have been observed. The inheritance patterns and genetic bases for complex expression can be dissected into individual components by identifying and analyzing linkage associations with molecular markers. Statistical tests, such as analysis of variance, can be used to determine whether loci and specific alleles are associated significantly with expression of the trait of interest. Once markers WO 90/12026 PCTUS90/01 848 -61associated with the trait of interest have been identified, they can be used to measure indirectly the effects of individual genes and the environment on expression of the trait dominance, additivity, epistasis, genotype by environment effects). Models which incorporate the genetic contribution of numerous loci to trait expression can be developed and used to aid in the selection and fixation of the trait, or importantly, simultaneous selection for multiple traits of interest.

In most cases, the molecular markers are associated with genes involved in trait expression, rather than being directly equivalent to such genes. Evaluation of the molecular markers, therefore, typically provides an indirect means of measuring the effects of the genes of interest. However, this is often an advantage since a single marker or clone can be used for identifying numerous alleles which give varying levels of trait expression. There will be some chance for recombination between the molecular markers and genes. The confounding effects of this are reduced by the availability and analysis of numerous molecular marker loci, such that increasingly tighter linkage associations are identified and markers flanking each gene of interest are available to discern potential recombination events which disrupt the desired linkage associations between the markers and genes of interest.

Molecular markers have been used to identify regions of the genome involved in a wide variety of traits, including maturity, head morphology, head extrusion, head color, leaf angle, leaf texture and leaf shape.

"Early White" cauliflower (Bf 2. botrvtis) was crossed with "OSU CR-7" broccoli o. italica) to generate F, plants segregating for many traits. Eight F, plants were scored for phenotype and evaluated with 58 mapped clones of the instant invention (Figure 11). Those plants were WO 90/12026 PCT/ US90/01t848 -62self- pollinated to produce 180 F 2 progeny that were similarly scored and evaluated. Associations between markers and traits of interest were evaluated using homogeneity chi-square tests as well as one-way analysis of variance (ANOVA). Markers linked to genes with significant effect on head color and head morphology are shown in Tables 11 and 12. Linkage associations between the markers and traits are being confirmed by evaluating the linkage associations in the F 3 generation.

Thirteen loci were found to be statistically associated with genes controlling head color, with individual loci accounting for between 3.1% and 11.5% of the variability in color, which ranged between green and white. Observed variation was statistically significant at the 0.01 confidence level or at the 0.05 confidence level Eight loci were found to be associated with differences in head morphology, ranging from the short, compact, round, smooth dome of cauliflower to the branched, knuckled, exerted head of broccoli.

Individual loci account for between 3.34% and 19.55% of the variability in head morphology. Levels of statistical significance are as indicated above.

EXAMPLE A comparison of B. oleracea linkage maps generated in different segregating populations illustrates how linkage distances between markers may differ among populations. As an example, the approximate linkage distance between markers 23 and 96 on 3C is 14 cM in the "Packman" x "Wisconsin Golden Acres" population and 26 cM in the "Early White" x "OSU CR-7" population.

Variation in linkage distance can reflect overall difference in recombination and difference in the number of individuals studied, but more likely reflect genomic WO 90/12026 P(r/U90/01 848 -63rearrangements amongst accessions. Linkage group 3C in "Wisconsin Golden Acres" is nullisomic for loci 43b and 57b. Such a condition could result in suppression of recombination. That suggestion is supported by the frequent observation of clusters of loci amidst mapped null loci. The map positions of the loci that comprise the clusters cannot be distinguished based on recombination events.

It will be appreciated that the methods and compositions of the present invention are capable of being incorporated in the form of a variety of embodiments, only a few of which have been illustrated and described herein. The invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within the scope.

WO 90/12026 WO 9012026PCFr/US90/0 1848 -64- TABlL II Loci Linked to Genes Controlling Head Color Determined by ANOVA Lo-s CHROMOSOME, F VALUE 16 iC 3.1 2.40* 34 iC 9.2 4.63** iC 3.6 2.85* 96A 3C 7.5 6.30** 96B iC 10.4 6.35** 2 9C 11.5 9.78** 37A 9C 9.5 8.03** 79 9C 9.9 4.60* 47 4C 5.1 4.16* 59 4C 11.2 9.28** 114 9c 6.4 5.12* 118 5C 3.9 3.14* 126 4C 3.9 2.84* WO 90/12026 WO 9012026PCI'/US90/0 1848 TABLE 12 Loci Linked to Genes Controlling Head Morphology Determined by ANOVA LOCUS CHROMOSOM 34 96A 2 37A 7 S 59 114 145 f, 6.3 7.9 31.8 8.2 7.6 6.1 F VALUE 3.34* 5.25** 11. 67** 6.52** 19. 6.61** 7.60* 4. 86** WO 90/12026 PCT/US90/01848 -66-

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Claims (51)

1. An isolated, Brassica nucleic acid fragment which detects polymorphic Brassica DNA, said fragment being selected from the group consisting of a clone and the Brassica insert of said clone, wherein said clone is selected from the collection of clones listed in Table 3.

2. The nucleic acid fragment of claim 1, wherein said fragment is a clone selected from the collection of clones listed in Table 3.

3. A Brassica nucleic acid fragment set which comprises at least two isolated Brassica fragments which detect polymorphic Brassica DNA, each of said fragments being selected from the group consisting of a clone selected from clones listed in Table 3 and the Brassica inserts of said clones.

4. The nucleic acid fragment set of claim 3, wherein said fragments are clones selected from the collection of clones listed in Table 3. S, 5. The nucleic acid fragment set of claim 3, wherein said fragments map to the same linkage group.

6. The nucleic acid fragment set of claim 3, wherein said fragments map to the same linkage group and are more than 30cM apart from each other.

7. The nucleic acid fragment set of claim 3, wherein ft ft 25 said fragments map to the same linkage group and are from about 20cM to about 30 cM apart from each other.

8. The nucleic acid fragment set of claim 3, wherein said fragments map to the same linkage group and are from about 10cM to about 20cM apart from each other.

9. The nucleic acid fragment set of claim 3, wherein said fragments map to the same linkage group and are from about 5cM to about 10cM apart from each other. The nucleic acid fragment set of claim 3, wherein said fragments map to the same linkage group and are from about IcM to about 5cM apart from each other. 72

11. The nucleic acid fragment set of claim 6, wherein said fragments are clones selected from the collection of clones listed in Table 3.

12. A Brassica nucleiu acid fragment set which comprises at least two isolated fragments which detect polymorphic Brassica DNA, each of said fragments being selected from the group consisting of a clone selected from the clones listed in Table 3 which map to Brassica oleracea and the Brassica inserts of said clones, wherein when said fragments are of any one linkage group, their relative order on said linkage group is related to the order of loci as presented in the map of Figure 4 (hereinafter Map), the relative order of any two said loci of said set may be inverted in relation to the order S* 15 presented in the Map and the arrangement of said loci of said set comprising 2 or more loci when considered from ,either direction is equivalent.

13. The nucleic acid fragment set of claim 8, wherein said fragments are clones selected from the collection of i 6 clones listed in Table 3.

14. The nucleic acid fragment set of claim 12, wherein said fragments map to the same linkage group and are located within 10cM of each other. The nucleic acid fragment set of claim 14, wherein said fragments map to the same linkage group and are located within 5cM of each other.

16. The nucleic acid fragment set of claim 12, wherein the relative order of said fragments is as presented in the Map.

17. The nucleic acid fragment set of claim 16, wherein said fragments map to linkage group 1C.

18. The nucleic acid fragment set of claim 16, wherein said fragments map to linkage group 2C.

19. The nucleic acid fragment set of claim 16, wherein said fragments map to linkage group 3C. The nucleic acid fragment set of claim 16, wherein said fragments map to linkage group 4C. 73

21. The nucleic acid fragment set said fragments map to linkage group

22. The nucleic acid fragment set said fragments map to linkage group

23. The nucleic acid fragment set said fragments map to linkage group

24. The nucleic acid fragment set said fragments map to linkage group The nucleic acid fragment set said fragments map to linkage group

26. The nucleic acid fragment set of claim 16, of claim 19, 6C. of claim 16, 7C. of claim 16, 8C. of claim 16, 9C. of claim 16, wherein wherein wherein wherein wherein wherein I I I I I said fragments are selected from the clones of Table 3 which map to linkage groups IC, 2C, 3C, 4C, 5C, 6C, 7C, 8C or 9C.

27. A Brassica nucleic acid fragment set which comprises at least two isolated Brassica fragments capable of detecting Brassica polymorphic DNA being selected from the group consisting of the collection of clones listed in Table 3 and the inserts .o said clones, 20 wherein said fragments are of any one linkage group, their relative order on said linkage group is related to the order of loci as presented in the map of Figure (hereinafter Map), the relative order of any two said loci of said set may be inverted in relation to the order presented in said Map and the arraneenent of said loci of said set comprising 2 or more loci when considered from either direction is equivalent.

28. The nucleic acid fragment set of claim 27, wherein said fragments map to the same linkage group and are located within 10cM of each other.

29. The nucleic acid fragment set of claim 27, wherein said fragments map to the same linkage group and are located within 5cM of each other. The nucleic acid fragment set of claim 27, wherein said fragments map to linkage group 2A.

31. The nucleic acid fragments set of claim 27, wherein said fragments map to linkage group 3A. 74

32. The nucleic acid fragment set of claim 27, wherein said fragments map to linkage group 4A.

33. The nucleic acid fragment set of claim 27, wherein said fragments map to linkage group

34. The nucleic acid fragment set of claim 27, wherein said fragments map to linkage group 6A. The nucleic acid fragment set of claim 27, wherein said fragments map to linkage group 7A.

36. The nucleic acid fragment set of claim 27, wherein said fragments map to linkage group 8A.

37. The nucleic acid fragment set of claim 27, wherein said fragments map to linkage group 9A.

38. The nucleic acid fragment set of claim 27, wherein said fragments map to linkage group 15 39. The nucleic acid fragment set of claim 27, wherein said fragments are selected from the clones of Table 3 which map to linkage groups 1A, 2A, 3A, 4A, 5A, 6A, 7A, 8A, 9A or A method for isolating, identifying or localizing a marker, trait, a gene contributing to a quantitative trait or nucleic acid fragment in Brassica using the nucleic acid fragment set of claim 3.

41. The method of claim 40, wherein said set is 4 selected from the list of clones of Table 3.

42. The method of claim 40, wherein said marker, trait, quantitative trait gene or fragment is located within of a locus of Table 3.

43. The method of claim 40, wherein said marker, trait, quantitative trait gene or fragment is located within of a locus of Table 3.

44. The method of claim 40, wherein said marker, trait, quantitative trait gene or fragment is located within 3cM of a locus of Table 3. The method of claim 40, wherein said marker, trait, quantitative trait gene or fragment is located within 1cM of a locus of Table 3. Y )4 L 75

46. The method of claim 40, Brassica oleracea.

47. The method of claim 40, Brassica campestris.

48. The method of claim 40, Brassica nigra.

49. The method of claim 40, Brassica napus. The method of claim 40, Brassica carinata.

51. The method of claim 40, Brassica juncea.

52. The method of claim 40, linkage analysis.

53. The method of claim 40, chromosome walking or hopping

54. The method of claim 40, wherein said Brassica is wherein said Brassica is wherein said Brassica is wherein said Brassica is wherein said Brassica is wherein said Brassica is that uses the technique of that uses the technique of or jumping. that uses the technique of r I i I I ill r r I I I II r r I I r r r pulsed field gel electrophoresis.

55. The method -f claim 40, that uses the technique of amplification by the polymerase chain reaction.

56. The method of claim 40, that uses the technique of hybridization with allele-specific oligonucleotides.

57. A method for identifying, distinguishing or tracing individuals, traits of said individuals or nucleic acid sequences of said individuals in Brassica using nucleic acid fragments of claim 1.

58. The method of claim 57, wherein said fragment is selected from the list of clones of Table 3.

59. The method of claim 57, that uses the technique of filter hybridization or "Southern" blot. The method of claim 57, that uses the technique of dot or slot blot.

61. The method of claim 57, that uses the technique of pulsed filed gel electrophoresis. 76

62. The method of claim 57, that uses the technique of amplification by the polymerase chain reaction.

63. The method of claim 57, that uses the technique of hybridization with allele-specific oligonucleotides. DATED this 12th day of SEPTEMBER, 1994 PIONEER HI-BRED INTERNATIONAL, INC. Attorney: IAN T. ERNST Fellow Institute of Patent Attorneys of Australia of SHELSTON WATERS