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

isozy es by electrophoresis, determining presence or absence of biochemical activity and detecting variabi¬ lity 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 localizatiαtn 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 monophyletisS. 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 mainta-ined 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 (cM) . 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

phenomenon. Nevertheless, conserved linkage groups allow predicting the location of markers among species. However, it was not long before the list of onogenic 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 monitored, 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

homology 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 beσins 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) i several I steps

(b)

B

The markers A,B,C and D comprise a linkage group (a), A represents sequences with homology to a clone 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 (b) . 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 containing a gene 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.

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 a phidiploid species with numerous subspecies and varieties serving as important sources of vegetable, oil and fodder throughout the world (Table 1) . 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_. nigra. B. oleracea and B_. campestris. may have evolved from a common pro¬ genitor (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

for 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).

Table 1

Genomic Classification of Selected Brassica

Genome

SDecies (p)* Variety descriptor Common Name

B. nigra (8) — bb Blackmustard

B. oleracea (9) cc "Cole crops" acephela cc.a Kales botrytis cc.b cauliflower capitata cc.c cabbage gemmifera cc.g brussels sprout gongylodes cc.go kohlrabi italica cc.i broccoli sabauda cc.s savoy sabellica cc.sa collards

B. camoestris (10) aa

(syn. rapa) chinen is aa.c pak choi oleifera aa.o turnip rape parachinensis aa.pa choy sum pekinensis aa.p petsai rapifera aa.r turnip

B. carinata (17) bbcc Ethiopian mustard

B. iuncea (18) aabb capitata aabb.c head mustard faciliflora aabb.f broccoli mustard oleifera aabb.o Indianmustard rugosa aabb.ru Leaf mustard spicea aabb.sp mustard

B. napus (19) aacc fodder rape oleifera aacc.o oil rape rapifera aacc.r rutabaga

* n is the haploid number of chromosomes

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 _f 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_ _s . campestris cross produced B^. iuncea. the B_j. nigra x J . oleracea cross produced Bj_ carinata and the remaining cross of B. oleracea x S _Λ . campestris produced J . napus (Vaughan, 1977; Prakash & Hinata, 1980; Quiros et al., 1988). Those interrelationships were proposed by ϋ in 1935 and confirmed most recently in analyses of chloroplast, mitochrondial and nuclear DNA sequences. Chloroplast DNA's of §j_ carinata and J__ nigra are virtually identical indicating that I . nigra is the likely maternal progenitor of J _. carinata. Similarly chloroplast DNA's of J _ iuncea and J . 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 re- synthesis, synthesis naturally or by in vitro techniques such as protoplast fusion of allotetraploid species (Williams & Hill, 1986; Tai & Ikonen, 1988). A novel JL. napus variety known as hakuran was produced in the laboratory by crossing Chinese cabbage (B. campestris pekinensis. aa.p) and cabbage (B. 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.

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 of 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 species- specific 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

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 (Williams, 1985) , and there is a conspicuous absence of a genetic map despite the presence of active breeding programs. Studies are 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.

SUKM&RY OF THE IflVEFTlQN

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 J . campestris. The resulting maps and markers will be useful for identifying and mapping important genes, for

organizing 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 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 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 _x and 10 F ₂ individuals.

Figure 4 is a diagram of a £. 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 species- specific clones. The clone hybridizes to accessions of B_. oleracea but not to those of B. campestris. No hybridization is observed in B. napus (Westar) .

Figure 8 depicts interspecific linkage group conservation between J$. oleracea and g. campestris. B. oleracea linkage groups are denoted as 1C through 9C and

B_. campestris linkage groups are denoted as 1A through 10A.

Figure 9 diagrams interspecific linkage group conservation between £. 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 £. 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 frequen¬ cies 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 self- replicating 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

approxi ation of distance between markers; the value can vary among species and between sexes, e.g. lcM is equivalent roughly to 139kb in Arabidopsis thaliana. 510kb in tomato, l,108kb in human and 2140kb in maize. Expressed Sequence: 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.

- Quantitative 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 Secruence: 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 Secruence Homology: Substantial functional and/or structural equivalence between sequences of nucleotides. Functional and/or structural differences between sequences having substantial sequence homology will be <g 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 reco binants (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.

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 (JJ . o_j_ botrytis) was digested with the methylation sensitive restriction enzyme Pstl 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 Hindlll digested genomic DNA extracted from a variety of different Brassica accessions (Table 2) . Other restriction endonucleases that commonly reveal polymorphism are Bglll, EcoRV and Sstl. 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 garden blot is shown in Figure 1.

Table 2

Brassica Accessions Screened for Polvmorphism

1. campestris B. oleracea ssp. chinensis ssp. italica

1. Gai Choi 1. Atlantic

2. Canton Dwarf 2. Bonanza

3. Takii #1 3. Surfer

4. Best Seed 4. B19

5. China Pak Choi 5. Gem

6. Milky Way 6. DiCicco

7. CC419 7. Cruiser

8. Hon Tsai 8. Green Top

9. Tsai Sai 9. Premium Crop

10. B18 ssp. pekinensis 11. Packman

12. OSU CR-7

1. Michihili 13. OSU CR-8

2. Dynasty

3. Jade Pagoda ssp. capitata

4. CC25

5. Green Rocket 1. Wisconsin

6. Hakuran Golden Acres

7. WR 70 Days 2. Brunswick ssp. utilis ssp. botrvtis

1. Spring Broccoli 1. Early White ssp. oleifer ssp. oleracea

1. UCD 77-4 1. Rapid-cy1ing

Turnip Rape

__• napus ssp. campestris ssp. oleifera

1. Rapid-cycling 1. Westar

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 JL. oleracea. l_ _t . campestris. and Bj. napus. (Wash conditions were 0.25 x SSC, 0.1% SDS at 60°C.) Variability is assessed at three taxonomic levels: (1) among Brassica species, (2) among subspecies within species, and (3) 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 2) . 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 informa¬ tive 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" (J . &__ italica) and "Wisconsin Golden Acres" (B _j_ capitata) were crossed and an F ₂ segregating population was obtained by selfing a single F _x 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

single "Packman" x "Wisconsin Golden Acres" (P x WGA) F _λ individual which gave rise to the F ₂ population used in the segregation analysis. Inclusion of a sample from the F _x 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 _x and each of 96 F ₂ progeny described above (Figure 3) . 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 £. oleracea and B. campestyis. 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_. oleracea. 1C through 9C, with C representing the traditional designation for the B. oleracea genome (Figure 4) . For the B_ _s . campestris genome, the ten linkage groups are labelled as 1A through 10A (Figure 5) . 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

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 EW4D12 were identified within a pattern comprised of more than 10 fragments.

A minority of the clones hybridize differentially to accessions representing different species and subspecies, reflecting deleted or derived sequences resulting from evolutional processes. Figure 7 depicts a clone that hybridizes only to ]3. 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 e.g., Schweizer et al. 1988).

The clones have been deposited with The American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, under accession number 67915. 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

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

TABLE: 3

Brassica Clones

B. oleracea fi*. campestris Insert Size

Locus Clone**** Linkage Group Linkage Group (kb)

1 EW1D07 2C NYM * 1.25

2A EW1G03 9C 6A .93 -

2B EW1G03 NYM 5A .93

3 EW2B01 3C NYM 1.15

4A EW2E07 5C 5A NYD **

4B EW2E07 1C 1A NYD

4C EW2E07 NYM 3A NYD

7A EW1F08 2C 1A 1.325

7B EW1F08 NYM 2A 1.325

7C EW1F08 NYM 3A 1.325

8A EW2A06 2C 2A NYD

8B EW2A06 NYM 2A NYD

9A EW3D07 5C 10A 1.15

9B EW3D07 3C NYM 1.15

9C EW3D07 5C NYM 1.15

10A EW4D04 1C 4A .92

10B EW4D04 4C NYM .92

11A EW4D06 1C 3A 1.025

11B EW4D06 5C NYM 1.025

11C . EW4D06 3C NYM 1.025

12A EW3C08 8C 8A 1.0

12B EW3C08 NYM 3A 1.0

13 EW4D09 4C NYM 0.8

14 EW4E05 1C 10A NYD

TABLE 3 - Cont'ii

Brassica Clones

TABLE 3 - Cont'd

Brassica Clones

B. oleracea B. campestris Insert size

Locus one Linkage Group Linkage Group H&l

TABLE 3 - Cont'd

Brassica Clones

B. oleracea

Locus B. campestris

Clone Insert si___> Linkage Group Linkage Group Xkbl

44A EW5H02 2C 6A

44B EW5H02 NYD

5C 1A

45A EWD12 NYD

6C 5A

45B 2.0

EWD12 5C NYM

46A EW5H03 2.0

3C 1A

46B EW5H03 NYD

NYM 8A

47 EW5H06 NYD

4C 5A

48 EW5G04 NYD

4C 4A

49A EW6A07 0.8

2C 2A

49B NYD

EW6A07 NYM 9A

50 EW2C01 NYD

9C 9A

51A EW3G09 NYD

NYM 6A

51B EW3G09 NYD

NYM 1A

51C EW3G09 NYD

NYM 6A

52 EW3A04 NYD

7C 1A

53 1.45

EW1H01 8C 8A

54A EW2B09 NYD

9C 7A

54B EW2B09 1.05

NYM 6A

54C EW2B09 1.05

NYM 6A

54D 1.05

EW2B09 NYM 6A

55A 1.05

EW6B10 6C 5A

55B .96

EW6B10 5C 4A

56A .96

EW5G09 8C 1A

56B .66

EW5G09 3C 4A

56C .66

EW5G09 7C NYM

57A .66

EW5F09 4C NYM

57B 1.2

EW5F09 3C NYM

58 1.2

EW6C05 7C 5A .85

TABLE 3 - Cont ' d

Brassica Clones

B. oleracea fi__ campestris Insert Size

Locus Clone Linkage Group l,inkaqe ( Sroup (Hb)

59A EW6C11 4C 5A 1.3

59B EW6C11 NYM 7A 1.3

60 EW6ED1 «6G 1A ? *** 1.7

61A EW6C09 1C 9A .68

61B EW6C09 6C 2A .68

62A EW6C12 5C 1A ? NYD

62B EW6C12 6C NYM NYD

62C EW6C12 8C NYM NYD

63 EW6D08 5C 3A NYD

64 EW6E03 1C 1A 1.15

65 EW6F02 1C NYM .66

66A EW6G12 3C 8A NYD

66B EW6G12 8C 3A NYD

67A EW3C01 5C 5A 1.35

67B EW3C01 3C NYM 1.35

68 EW3C04 8C 8A NYD

69A EW2D01 4C NYM NYD

69B EW2D01 5C NYM NYD

70A EW3F01 1C 1A 0.5

70B EW3F01 6C NYM 0.5

71 EW4B02 4C 4A NYD

72A EW4D12 2C 2A 1.1

72B EW4D12 2C 8A 1.1

72C EW4D12 8C NYM 1.1

73A EW4E09 6C 6A .87

73B EW4E09 3C NYM .87

TABLE 3 - Cont'd

Brassica Clones

B. oleracea

Locus B. camDestris

Linkage Insert Sly-p.

Group Linkage Group Ikbi

75A EW5C05 1C

75B 8A

EW5C05 NYM 1.35

76A 10A

EW4F04 2C 1.35

76B 1A

EW4F04 1C .53

77A 3A

EW4H05 9C .53

77B 9A

EW4H05 3C 1.2

78 NYM

EW5B02 5C 1.2

79 NYM

EW4H09 9C 1.0

80A 7A

EW5B03 1C 1.025

80B NYM

EW5B03 3C NYD

81 NYM

EW5E10 6C NYD

82A 1A ?

EW5F02 8C .71

82B NYM

EW5F02 2C .66

83A NYM

EW7C08 2C .66

83B 2A

EW7C08 2C NYD

83C 2A

EW7C08 NYM NYD

84 1A

EW5H01 1C NYD

85 1A

EW7C10 6C .85

86A 9A

EW7B02 5C 1. 75

86B 1A

EW7B02 1C . 69

87A NYM

EW5A01 6C . 69

87B 9A

EW5A01 5C 1.425

88 NYM

EW5A12 7C 1. 425

89 5A

EW7A11 4C 1. 15

90A NYM

EW7B04 2C . 87

90B 2A

EW7B04 2C . 87

91A 1A

EW7D03 3C . 87

91B 5A

EW7D03 NYM . 96

9A . 96

TABLE 3 - Cont'd

Brassica Clones

B. oleracea B. campestris

Locus Insert Size Linkage Group Linkage Group i&bl

TABLE 3 - Cont ' d

Brassica Clones

B. oleracea B. campestris Insert Size

Locus Clone Linkage Group Linkage Group (kb)

108 EW7E01 7C NYM .87

109A EW8A06 9C 9A 1.55

109B EW8A06 9C NYM 1.55

110 EW8A09 6C NYM NYD

111 EW8B11 6C 9A .79

112 EW5A09 8C NYM .68

113 EW5F04 3C 3A 1.25

114 EW9A07 9C 9A 1.1

115 EW9D02 8C - 1A .87

116 EW9A08 6C 5A 1.1

117 EW9E01 7C 7A 1.85

118 EW9A06 5C 9A .76

119A EW9B02 4C 5A 1.1

119B EW9B02 2C NYM 1.1

120 EW9F06 2C NYM 1.35

121 EW9F02 7C NYM 2.2

122 EW9H02 4C NYM 1.6

123 EW9D06 3C 3A NYD

124A EW9D08 6C 1A ? .83

124B EW9D08 NYM 8A .83

125A EW9E05 3C NYM NYD

125B EW9E05 1C NYM NYD

126 EW9E10 4C NYM .85

127 EW9F08 7C NYM 1.7

128 WGlAiπ 4C 4A 1.8

129 WG1A0_. 1C 1A 1.8

130 WG2H05 4C 8A NYD

131A WG2C10 9C 7A 1.5

TABLE 3 - Cont'd

Brassica Clones

B. oleracea B. campestris

Locus Insert Size l nkage roup Linkage Group (kb)

TABLE 3 - Cont'd

Brassica Clones

B. oleracea

Locus B. campestris Insert size Linkage Group Linkage Group (kb)

TABLE 3 - Cont ' d

Brassica Clones

B. oleracea B. campestris Insert Size

Locus Clone Linkage Group Linkage Group (kb)

181 WG5E12 8C 8A NYD

182 WG3H10 2C NYM NYD

183 WR1E08 8C 8A NYD

184 WR1D12 6C NYM 2.0

185 WR2A01 3C 3A 1.35

187 WR2D10 3C 8A NYD

188 WR2E07 1C 5A NYD

189 WR2E09 9C 9A 1.3 1

19OA WR2F05 4C 1A 1.4

190B WR2F05 NYM 4A 1

1.4

191 WR2F06 8C NYM 1.15

192 WR2B09 5C NYM 1.525

193 WG4E07 3C 5A 1.1

194A WG3E06 3C NYM 0.9

194B WG3E06 1C NYM 0.9

195 EW8B12 NYM 10A .87

196 EW8C04 NYM 1A NYD

197 EW8D08 4C 9A .95

198 EW9A09 NYM 5A NYD

199A EW9E05 3C 5A NYD

199B EW9E05 NYM NYM NYD

200A EW8F03 8C 3A .69

200B EW8F03 4C NYM .69

200C EW8F03 3C NYM .69

201 EW8E04 1C 1A .63

202A EW8E10 7C 7A .63

202B EW8E10 NYM 6A .63

203A EW8D10 3C 8A .58

TABLE 3 - Cont'd

Brassica Clones

B. oleracea

Locus B. campestris Insert Size Linkage Group Linkage Group (kbi

203B EW8D10 4C

204 NYM*

EW9B05 .58

NYM

205 8A

EW9G01 NYD

NYM

206 10A

EW8B05 NYD

NYM

207 1A

EW8C08 1.55

9C

208 6A

EW8B08 .82

2C

209 1A

EW9C07 1.1

NYM 4A

211A EW8E11 1.3

5C

211B 5A

EW8E11 1.1

9C

212 NYM

EW8F11 1.1

5C

213 6A

EW9A05 1.55

NYM 9A

214 EW9B09 1.3

NYM

300 6A

WG4A07 NYD

NYM

302 1A ?

WG5C06 NYD

NYM

303 5A

WR2A06 NYD

NYM

304 1A

WR2A05 NYD

NYM 6A

305A WR2B06 NYD

NYM

305B 10A

WR2B06 NYD

NYM

306 3A

WR1H02 NYD

NYM

307 5A

WR1G09 NYD

NYM 8A

308 WG3A08 NYD

NYM 6A

309 WR2B11 NYD

NYM NYM

310 WR1B09 NYD

NYM 7A

312 WG2A07 NYD

NYM 3A

313A WG3C10 NYD

NYM 10A

313B WG3C10 NYD

NYM 5A

315 WG2E08 NYD

NYM 9A

316 WG2D04 NYD

NYM 5A NYD

TABLE 3 - Cont'd

Brassica Clones

B. oleracea fi__ campestris Insert Size

Locus ClPne Linkage Group Linkage Group (kb)

317 WG2B02 NYM 9A NYD

318A WG2G12 NYM 1A ? NYD

318B WG2G12 NYM 8A NYD

319 WG3B11 NYM 4A NYD

320 WG1C05 NYM 6A NYD

321 WG5F08 NYM 8A NYD

322 WR1C04 NYM 8A NYD

323A WR2B12 NYM 4A NYD

323B WR2B12 NYM 3A NYD

325 WR2H04 NYM 4A NYD

326A WR1E03 NYM 8A ? NYD

326B WR1E03 NYM 3A NYD

327 WR1A12 NYM 6A NYD

328 WR2E02 NYM 1A ? NYD

329 WR1A10 NYM 3A NYD

330 WG2A06 NYM 9A ? NYD

331 WG5G03 NYM 9A NYD

332 WR1B03 NYM 8A NYD

333A WG3E11 NYM 6A NYD

333B WG3E11 NYM 6A NYD

334 WG3H09 NYM 1A NYD

335 WG5A07 NYM 10A NYD

336 WG2H05 NYM 8A NYD

* Not yet mapped. mapping in progress.

** Not yet determined.

*** Tent :ative assigiment

-fc*** Clone may hybridize to multiple loci and not all loci have necessarily been mapped.

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 polymorphic 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 _x . 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.

i.e- separated by less than lcM or that they hybridize to the same locus. If two loci are lcM 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 Js. oleracea. B. napus and B. campestris are highly homologous. Figdore et al. (1988) shows the hybridization of some cauliflower clones in JL_ campestris varieties, among other subspecies of £_. oleracea and in J _ 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 JL. campestris library hybridize to B___ campestris subspecies, B_ oleracea and fi _t napus. The order of loci

in fii. oleracea chromosomes is conserved to a high degree in S _Λ . campestris chromosomes. (Figure 8) . Because it is highly likely that B. napus is a natural hybrid of B. oleracea and fi. campestris. it is not unexpected that the genomic organization of the three species is related. Early mapping studies in B. napus illustrate the relatedness (Figure 9) . Thus linkage relationships are maintained 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 ^{m mm} ^ 2 . 8

72C 24a 31b 66b 28a 38b 36a

8C -+— ._+ +_.

(5) (1) (2) (1) (8) (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.

- 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 same 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

chromosomes, 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 50% 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 (cM) . Thus, one percent recombination between two markers is equivalent roughly to lcM 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 5cM 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 J . oleracea is about 4cM (Table 5) . 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

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 6). 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 (e.g., 19 and 141 on IC) can be identified and positioned by virtue of linkage to one or more of the loci described herein.

Table 5

Linkage Group Information in Brassica oleracea

Linkage

Group ΣcM # Clones* # Loci* In (terval**

1 134 40 36 3.7

2 99 27 23 4.3

3 95 37 24 4.0

4 108 30 25 4.3

5 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 11 2.9

Total 818 258 204 4.0

* Some loci are not distinguishable by recombination and are assigned to the same map position ** Expressed in cM

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

5 4 47,59

2 5 78,67a

4 5 67a,97a

9 6 176,74a

4 6 145,24b

9 7 148,179

5 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

analysis, be inverted. The maps presented in Figures 4, 5 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 Lvcopersicon esculentum and £. pennilli (E x P) ordered three pairs of linked loci as CD15-TG24 on chromosome 1, CD32B-TG63 on chromosome IQ 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 - 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 10 ⁷ :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

normal 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 £. campestris (Figure 5) , 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 5

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

clone 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 55 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 lcM or more from the starting clone. It is 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

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 lcM of human DNA, and it is not unrealistic to begin a jump to a gene of interest from a clone that is lcM, 5cM or even lOcM 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 Notl, Narl, BssHII, Nrul, Mlul, SStll or Sfil. The result is large DNA fragments that produce larger jumps. For example, a Notl 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 100Okb range. Improvements will allow resolution of even larger fragments (the current upper size limit is 7-10Mb) .

-46-

Nevertheless, present methods permit the separation of mammalian DNA molecules that carry markers lcM 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 50Mb 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 cloneible and the overall cloning efficiency is low (Burke et al. 1987)-.

EX E 6

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 30cM 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 50, 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.

- Thus, anywhere from 15 to 35 or more loci can be selected to comprise a "quick screen" subset of clones for thie rapid identification of provisional map position in a first step of fine scale mapping of a new clone. An exεunple of a "quick screen" subset is presented in Table 7.

Table 7

Quick Screen Subset of Anchor Loci Linkage Group

Clone IC 2C 3C 4C 5C 6C 7C 8C 9C

EW2B01 3 EW1D02 EW3D07 9B 9A/C EW4E05 14 EW2E09 16 EW3A01 17 EW2F02 18A 18B EW1A07 22 EW4G11 25A 25B EW2B12 27 EW5C11 30 EW5C12 31A 31B EW6A04 40A/B 40C EW2B09 54 EW6C12 62A 62B 62C EW6G12 66A 66B EW3C01 67B 67A

Table 7 - Continued Quick Screen Subset of Anchor Loci

Linkage Group

Clone IC 2C 3C 4C 5C 6C 7C 8C 9C

EW7C08 83A/B

EW5H01 84

EW7B04 90A/B

EW7E08 92B 92A

EW7E12 93A/B 93C I V l

EW7G06 94 O

EW8A06 109A/B I

EW8B11 111

EW5A09 112

EW5F04 113

EW9D02 115

EW9E01 117

EW9B02 119B 119A

EW9D06 123

EW9E05 125

WG1A10 128

WR2C07 153B 153A

Table 7 - Continued

Quick Screen Subset of Anchor Loci

Linkage Group

Clone IC 2C 3C 4C 5C 6C 7C 8C 9C

WR1G11 170

WG3B09 179

WR2F05 190

EW8E10 202

I

EXAMPLE 7

The present example demonstrates one utility of the clones of the instant application. Further details of this example and other utilities are disclosed in application S.N. (to be assigned), entitled "Method for Determining Relatedness of Genotypes", filed concur¬ rently herewith. Ninety-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 _x 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 (1, 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

of 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 10 0 1 Total

0 28 8 36 Genotype 11

1 12 12 24

Total 40 20 60

The coefficient of relatedness is:

CR = ((28+12)-(8+12))/(28+8+12+12) = (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,

larger 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 1 0 = 1 0 X 0 = 0

The algorithm can be interpreted in terms of the inheritance of polymorphism, i.e., 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, e.g., an "AA" or "aa" genotype. If the polymorphism represents an allele of a heterozygous parent, e.g., "Aa," 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-(l/2 ⁿ ) , where n = ithe 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:

TOTAL 27

34

TOTAL 27 34 61

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.

8

Coefficients of Relatedness for Genotypes 1-12

Genotype

1 1.000 0.333 0.367 0.533 0.400 0.333

2 0. 333 1. 000 0.443 0.267 0. 607 0.541

3 0.367 0.443 1. 000 0.433 0.443 0.705

4 0.533 0.267 0.433 1. 000 0.533 0.467

5 0.400 0.607 0.443 0.533 1.000 0.410

6 0.333 0.541 0.705 0.467 0.410 1. 000

7 0.467 0.200 0.233 0.400 0.200 0. 333

8 0.367 0.367 0.267 0.300 0.233 0.500

9 0.233 0.377 0.475 0.300 0.443 0. 639

10 0.567 0.233 0.333 0.300 0.433 0.300

11 0. 633 0.443 0.410 0.500 0.443 0.443

12 0.333 0.410 0.574 0.333 0.279 0. 672

10 11 12

1 0.467 0.367 0.233 0.567 0.633 0.333

2 0.200 0.367 0.377 0.233 0.443 0.410

3 0.233 0.267 0.475 0.333 0.410 0.574

4 0.400 0. 300 0.300 0.300 0.500 0. 333

5 0.200 0.233 0.443 0.433 0.443 0.279

6 0.333 0.500 0.639 0.300 0.443 0.672

7 1.000 0.767 0.433 0.233 0.500 0.400

8 0.767 1.000 0.333 0.267 0. 600 0.500

9 0.433 0.333 1.000 1.333 0. 344 0.508

10 0.233 0.267 0.333 1. 000 0.333 0.367

11 0.500 0.600 0.344 0.333 1.000 0.443

12 0.400 0.500 0.508 0.367 0.443 1. 000

TABLE 9

Coefficients of Relatedness for Selected Genotypes

Genotype 10 11 12 26 27

6 1.000 0.300 0.433 0.672 0.738 0.738 10 0.300 1.000 0.333 0.367 0.433 0.433 11 0.433 0.333 1.000 0.433 0.311 0.311 12 0.672 0.367 0.443 1.000 0.672 0.738 26 0.738 0.433 0.311 0.672 1.000 0.934 27 0.738 0.433 0.311 0.738 0.934 1.000 28 0.410 0.300 0.443 0.410 0.475 0.541 29 0.377 0.333 0.541 0.443 0.508 0.508 31 0.367 0.200 0.467 0.433 0.300 0.367 32 0.467 0.167 0.300 0.467 0.267 0.333 33 0.400 0.433 0.500 0.400 0.600 0.533 34 0.377 0.267 0.410 0.377 0.115 0.180 41 0.705 0.400 0.344 0.639 0.500 0.902

28 29 31 32 33 34 41

6 0.410 0.377 .0.367 0.467 0.400 0.377 0.705

10 0.300 0.333 0.200 0.167 0.433 0.267 0.400

11 0.443 0.541 0.467 0.300 0.500 0.410 0.344

12 0.410 0.443 0.433 0.467 0.400 0.377 0.639

26 0.475 0.508 0.300 0.267 0.600 0.115 0.836

27 0.541 0.508 0.367 0.333 0.533 0.180 0.902

28 1.000 0.770 0.300 0.200 0.533 0.180 0.574

29 0.770 1.000 0.467 0.300 0.700 0.279 0.541

31 0.300 0.467 1.000 0.767 0.433 0.533 0.333

32 0.200 0.300 0.767 1.000 0.333 0.767 0.300

33 0.533 0.700 0.433 0.333 1.000 0.300 0.567

34 0.180 0.279 0.533 0.767 0.300 1.000 0.148

41 0.574 0.541 0.333 0.300 0.567 0.148 1.000

TABLE 10

Polymorphism Distribution in 28. 33. 29 and 28X33

Marker 28. 22 21 28X33

1 0 0 0 0

2 0 1 1 1

3 1 O i l

4 1 1 1 1

5 0 0 0 0

6 0 0 0 0

7 0 1 1 1

8 1 1 1 1

9 0 0 1 0

10 1 0 1 1

11 1 1 1 1

12 1 0 1 1

13 0 1 1 1

14 1 O i l

15 1 1 1 1

16 0 0 0 0

17 0 0 0 0

18 1 1 1 1

19 0 0 0 0

20 0 0 0 0

21 1 1 1 1

22 1 1 1 1

23 0 1 1 1

24 0 0 0 0

25 0 1 1 1

26 0 1 1 1

27 0 0 0 0

28 0 0 0 0

29 1 1 1 1

30 0 0 0 0

31 0 0 0 0

32 0 0 0 0

33 0 0 0 0

34 1 1 1 1

35 1 1 1 1

36 0 0 0 0

37 0 0 0 0

38 1 1 1 1

39 1 1 1 1

40 1 * 1 1

41 1 1 1 1

42 1 O i l

43 0 1 1 1

44 0 0 0 0

45 1 1 1 1

46 0 0 0 0

47 0 0 0 0

TABLE 10 (Cont 'd)

OBS E28 E33 £29 H2833

48 0 0 0 0

49 0 0 0 0

50 1 0 1 1

51 1 0 1 1

52 0 0 0 0

53 0 0 0 0

54 0 0 0 0

55 1 1 1 1

56 0 0 0 0

57 1 1 1 1

58 0 1 0 1

59 1 1 1 1

60 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 (e.g., genomic) sequences contained between oligonucleotide primers. The reaction occurs in primer excess and involves repeated cycles of hybridization- synthesis-denaturation. When a clone detects polymorphism that arises by variation in the length of alleles (e.g., 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

mutatiόn 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

associated 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 (i.e. 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 (JL. Q. botrvtis. was crossed with "OSU CR-7" broccoli (J . Q. italica) to generate F _x plants segregating for many traits. Eight F _x plants were scored for phenotype and evaluated with 58 mapped clones of the instant invention (Figure 11) . Those plants were

self- 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 10

A comparison of JJ. 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

rearrangements 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.

TΛSLE 11

Loci Linked to Genes Controlling Head Color Determined by ANOVA

OWS CHROMOSOME Δ F VMffE

6 IC 3.1 2.40* 4 IC 9.2 4.63** A IC 3.6 2.85* 6A 3C 7.5 6.30** 6B IC 10.4 6.35**

9C 11.5 9.78** 7A 9C 9.5 8.03** 9 ^* 9C 9.9 4.60* 7 4C 5.1 4.16* 9 4C 11.2 9.28** 14 9C 6.4 5.12* 18 5C 3.9 3.14* 26 4C 3.9 2.84*

TABLE 12

Loci Linked to Genes Controlling

LOCUS CHROMOSOME £_ F VALUE

34 IC 6.8 3.34*

96A 3C 6.3 5.25**

2 9C 13.3 11.67**

37A 9 7.9 6.52**

79 9C 31.8 19.55**

59 4C 8.2 6.61**

114 9C 7.6 7.60*

145 6C 6.1 4.86**

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