# Patent

## Title
Brassica oleracea plants with improved nutritional value

## Abstract

The invention provides compositions and methods relating to the elevation of glucoraphanin compared to standard Brassica oleracea varieties. The invention also relates to the production of hybrid varieties having desired glucosinolate contents. The invention further provides plants, plant parts, and seeds comprising such traits and comprising a Myb28 allele from Brassica villosa that is not genetically linked to an ELONG allele from Brassica villosa.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. provisional application No. 61/700,762, filed Sep. 13, 2012, herein incorporated by reference in its entirety.


INCORPORATION OF SEQUENCE LISTING
The sequence listing that is contained in the file named “SEMB008US_ST25.txt”, which is 8 kilobytes as measured in Microsoft Windows operating system and was created on Aug. 28, 2013, is filed electronically herewith and incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the development and use of Brassica oleracea plants with a recombined chromosomal segment.
2. Description of Related Art
Glucosinolates are allelochemicals present in 16 families of plant species, especially in Brassicaceae, of which broccoli is a notable example. Although there are over 120 different glucosinolates identified in nature, closely related taxonomic groups typically contain only a small number of such compounds. Glucosinolates that are dominant in broccoli such as glucoraphanin and glucoiberin are derived biochemically from the amino acid methionine. In the glucosinolate pathway, methionine is converted to homo-methionine and dihomomethionine by the activity of the ELONG (elongation) locus by adding a single carbon unit to the tail each time. Homo-methionine is eventually converted to 3-methylthiopropyl glucosinolate (glucoiberin; “MSP”) while dihomo-methionine is converted to 4-methylthiobutyl glucosinolate (glucoraphanin; “MSB”). These glucosinolates (glucoiberin and glucoraphanin) are potent inducers of phase II detoxification enzymes, such as glutathione-S-transferase and quinone reductase, which promote the metabolism and excretion of potential carcinogens.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a Brassica oleracea plant comprising a Myb28 allele from Brassica villosa and lacking an ELONG allele from Brassica villosa genetically linked to said Myb28 allele, wherein the Myb28 allele confers elevated glucosinolates when compared to a plant that lacks the Myb28 allele.
In one embodiment, the plant is a broccoli plant. In other embodiments, the plant is inbred or hybrid. In another embodiment, the plant is homozygous for said Myb28 allele from Brassica villosa. In yet another embodiment, the plant is heterozygous for said Myb28 allele from Brassica villosa. In still another embodiment, the ELONG allele is from Brassica oleracea. 
In another aspect, the present invention provides a part of a plant of the invention. In some embodiments, the plant part may further be defined as a leaf, an ovule, a floret, pollen, a head, or a cell.
In yet another aspect, the present invention provides a seed that produces a plant of the invention.
In still another aspect, the present invention provides a Brassica oleracea plant comprising a chromosomal segment that comprises a Myb28 allele from Brassica villosa and lacking an ELONG allele from Brassica villosa genetically linked to said Myb28 allele, wherein the segment confers elevated glucosinolates relative to a plant lacking the Myb28 allele, and wherein a sample of seed comprising the chromosomal segment was deposited under ATCC Accession Number PTA-13165. In one embodiment, the invention provides a seed that produces such a plant. In another embodiment, the invention provides a plant part, wherein the part is a leaf, an ovule, a floret, pollen, a head, or a cell. In another embodiment the plant is a B. oleracea plant.
In another aspect of the present invention a recombined DNA segment comprising a Myb28 allele from Brassica villosa and an ELONG allele from Brassica oleracea is provided. In one embodiment, the DNA segment is further defined as comprised within a cell. In another embodiment, the DNA segment is further defined as comprised within a seed. In yet another embodiment, the DNA segment is further defined as comprised within a plant.
In another aspect, the present invention provides a method for obtaining a Brassica plant comprising a desired glucosinolate composition comprising: a) obtaining a Brassica plant heterozygous for a Myb28 allele from Brassica villosa that confers elevated glucosinolates and is genetically linked in the plant to an ELONG allele from Brassica villosa; (b) obtaining progeny of the plant; and (c) selecting at least a first progeny plant in which recombination has occurred such that the progeny comprises the Myb28 allele but not the ELONG allele from Brassica villosa, wherein the progeny plant possesses a desired glucosinolate composition as a result of the presence of the Myb28 allele but not the ELONG allele from Brassica villosa. 
In one embodiment, selection of the progeny plant comprises identifying a progeny plant that (1) comprises a genetic marker genetically linked to the Myb28 allele in Brassica villosa and/or lacks a genetic marker present at the corresponding locus in said Brassica plant, and (2) lacks a genetic marker genetically linked to the ELONG allele from Brassica villosa and/or comprises a genetic marker present at the corresponding locus from said Brassica plant.
In another embodiment, selection of the progeny plant comprises detecting a polymorphism that is found in the genome of said plant flanked by the complements of SEQ ID NO:1 and SEQ ID NO:2. In a further embodiment, such allele(s) are detected by a PCR-based method using oligonucleotide primer pair(s). In another embodiment, selection of the progeny plant comprises detecting a polymorphism in said progeny plant that is shown in FIG. 5. In a further embodiment, the Brassica plant may be a B. oleracea plant.
In yet a further aspect, the invention provides a plant produced by a method of the invention or a progeny thereof comprising the Myb28 allele but not the ELONG allele from Brassica villosa. In one embodiment, the invention provides a part of such a plant. In another embodiment, the part of the plant is selected from the group consisting of a cell, a seed, a root, a stem, a leaf, a head, a flower, and pollen.
In another aspect, the invention provides a method for producing a hybrid Brassica oleracea plant with elevated glucosinolate content comprising crossing a first Brassica oleracea parent plant with a second Brassica oleracea plant of a different genotype, wherein the first parent plant comprises a Myb28 allele from Brassica villosa that lacks an ELONG allele from Brassica villosa genetically linked to said Myb28 allele, wherein the Myb28 allele confers elevated glucosinolates relative to a plant lacking the Myb28 allele. In one embodiment, the method further comprises producing a plurality of hybrid Brassica oleracea plants comprising crossing the first Brassica oleracea parent plant with a plurality of second Brassica oleracea plants of different genotypes.
In still another aspect, the invention provides a method of producing a Brassica oleracea plant with a desired elevated glucosinolate content comprising introgressing into the plant a chromosomal segment comprising a Myb28 allele from Brassica villosa and lacking an ELONG allele from Brassica villosa genetically linked to said Myb28 allele, wherein segment confers a desired glucosinolate content relative to a plant lacking the segment, wherein a sample of seed comprising the chromosomal segment is deposited under ATCC Accession No. PTA-13165.
The term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value. The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and to “and/or.” When used in conjunction with the word “comprising” or other open language in the claims, the words “a” and “an” denote “one or more,” unless specifically noted. The terms “comprise,” “have” and “include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as “comprises,” “comprising,” “has,” “having,” “includes” and “including,” are also open-ended. For example, any method that “comprises,” “has” or “includes” one or more steps is not limited to possessing only those one or more steps and also covers other unlisted steps. Similarly, any plant that “comprises,” “has” or “includes” one or more traits is not limited to possessing only those one or more traits and covers other unlisted traits.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts the head from hybrid broccoli varieties Ironman (left) and RX05991199 (right). Varieties were grown in Summer at a spacing of 50×50 cm.
FIG. 2 shows a horizontal cross section of stems from hybrid broccoli varieties Ironman (left) and RX05991199 (right). Varieties were grown in Autumn at a spacing of 50×50 cm.
FIG. 3 shows a profile of the head and stem from hybrid broccoli varieties Ironman (left) and RX05991199 (right). Varieties were grown in Autumn at a spacing of 50×50 cm.
FIG. 4 shows assay results for different ELONG alleles with the QTL1-BoGLS-ELONG marker. The V allele is the B. villosa allele associated with Myb28, which results in high 3-MSP/4-MSB ratio and high total glucosinolates. The A, B and C alleles are examples of alleles found in broccoli without a B. villosa ELONG allele.
FIG. 5 shows an alignment between a consensus sequence of the Myb28 locus from B. villosa contained in broccoli variety FT69, and a consensus sequence of the corresponding locus from broccoli without increased level of glucosinolate, e.g. B. oleracea, (Oleracea) (SEQ ID NOs.:8-9).



DETAILED DESCRIPTION OF THE INVENTION
The invention provides methods and compositions relating to plants, seeds and derivatives of Brassica oleracea plants comprising a new recombined introgression from Brassica villosa capable of conferring elevated 4-methylsulfinylbutyl glucosinolates (MSB), also known as glucoraphanin. It was also surprisingly found that plants comprising the introgression were capable of consistently producing hybrids that exhibited elevated glucoraphanin content relative to glucoiberin, while results were substantially more variable when using a parent line comprising the non-recombined introgression, such as in the case of the Myb28 donor parent of the hybrid PS05151639, which comprises Myb28 and ELONG alleles from Brassica villosa (US Patent Appln Pub No. 2011/0055945). The ability to produce multiple elite hybrid progeny with elevated glucoraphanin content and/or ratio of glucoraphanin to glucoiberin from a single inbred parent is significant in that the number of elite Brassica oleracea parent lines available to produce hybrid varieties is limited. The reduced introgression therefore substantially increases the utility of a given inbred and allows, for example, the production of multiple hybrids potentially adapted to different growing environments, end uses, or other criteria, each having a desired glucoraphanin content.
The new “reduced introgression” comprising a Myb28 locus from Brassica villosa and lacking the ELONG locus of Brassica villosa that has to date been genetically linked thereto is capable of consistently conferring in hybrids derived from a plant comprising the introgression elevated glucoraphanin relative to glucoiberin. One aspect of the current invention thus concerns methods for obtaining a Brassica oleracea plant comprising at least one such reduced introgression, wherein the resulting Brassica oleracea plant and/or progeny derived therefrom displays a desired glucoraphanin content relative to a control plant lacking the introgression. The invention thus provides plants possessing a desired glucoraphanin content conferred by a reduced introgression of the invention. In certain embodiments, methods for obtaining such plants comprise obtaining a Brassica oleracea plant heterozygous for a Myb28 allele from Brassica villosa that confers elevated glucosinolates and is genetically linked in the plant to an ELONG allele from Brassica villosa, obtaining progeny from such a plant, and selecting one or more such progeny plants wherein genetic recombination has occurred such that the progeny comprises an Myb28 allele from Brassica villosa, but does not comprise an ELONG allele from Brassica villosa. Such progeny or further progeny thereof may also possess a desired glucoraphanin content as a result of the presence of the Myb28 allele but not the ELONG allele from Brassica villosa. In particular embodiments, the method may comprise obtaining a progeny plant that comprises such allele(s) by identifying one or more genetic markers genetically linked to the Myb28 and/or ELONG allele(s). Identifying the genetic markers may comprise a phenotypic, a genetic, or a biochemical test, and may include screening a parent and/or progeny plant for the presence of, for instance, one or more allele described herein, including, for example, a Myb28 allele from B. villosa, an ELONG allele from B. villosa and an ELONG allele from B. oleracea. 
Certain traits such as a glucoraphanin content relative to glucoiberin content, or the unpredictability of the type of glucosinolates in hybrid progeny heterozygous for a Brassica villosa Myb28 allele, were found to co-locate with a glucosinolate trait in the Myb28 allele from Brassica villosa and ELONG allele from Brassica villosa introgressions. Thus, formation of a “reduced” introgression is understood to be caused by recombination event(s) in the vicinity of the Myb28 and ELONG QTL(s). Lines comprising a reduced introgression, i.e., which have undergone a recombination event close to the QTL having elevated glucosinolates may efficiently be screened by use of molecular and/or phenotypic markers. Thus, plant populations or progeny of such populations, segregating (i.e., heterozygous) with respect to the QTL specified by Myb28 and ELONG introgressions, may be screened for plants having a recombinant phenotype, e.g. elevated glucoraphanin levels relative to glucoiberin levels.
In other embodiments, a method of the invention may comprise identifying a Brassica oleracea plant comprising a Brassica villosa-derived reduced introgression, and comprising a meiotic recombination between Myb28 and ELONG alleles as described herein. In particular embodiments, identifying the introgression may comprise measuring glucoraphanin and/or glucoiberin using standard protocols. In certain embodiments, a plant of the invention comprising a reduced introgression as disclosed herein comprises an elevated average proportion of glucoraphanin relative to glucoiberin compared to a plant comprising Myb28 and ELONG alleles from Brassica villosa, or a plant lacking a Myb28 allele from Brassica villosa. In one embodiment, such a plant comprising an elevated average proportion of glucoraphanin relative to glucoiberin is an inbred line, and in another embodiment is defined as a F1 hybrid having as one or more parent a plant comprising a reduced introgression of the invention. In particular embodiments, a plant of the invention is provided comprising a ratio of glucoraphanin to glucoiberin of about 10:1, 12:1, 15:1, 18:1, 20:1, 23:1, 25:1, 28:1, 30:1, 35:1 and about 40:1. In one aspect, an increase in glucoraphanin content may be calculated in reference to a standard Brassica oleracea variety, such as the broccoli variety Ironman.
A. BREEDING OF BRASSICA OLERACEA LINES DISPLAYING ELEVATED GLUCORAPHANIN
One aspect of the current invention concerns methods for crossing a plant comprising a reduced Myb28/ELONG introgression provided herein with itself or a second plant and the seeds and plants produced by such methods. These methods can be used for production and propagation of cultivated Brassica oleracea plants displaying desired glucosinolate compositions, including MSB and/or MSP content. Yet further, the plants of the current invention having elevated glucosinolate content comprise improved nutritional value of the plant relative to plants without elevated glucosinolates. The methods also can be used to produce hybrid Brassica oleracea seeds and the plants grown therefrom. Hybrid seeds are produced by crossing such lines with a second Brassica oleracea parent line. The hybrids may be heterozygous or homozygous for the reduced introgression.
Brassica villosa is a wild species endemic to northwest and central Sicily, and thus a Myb28 allele could be obtained by one of skill in the art from a plant selected from the wild. Alternatively, Myb28 alleles are known in the art and may be obtained from other sources for use with the invention, including SNR 347 (FT69; referred to as 428-11-69 in Mithen et al., Theor Appl Genet, 106:727-734; 2003), BR384-014, SNP13 (580333), SNP88 (BRM51-1210), BR384-020, B1639 (ATCC Accession Number PTA-9676), BRM51-1162 (ATCC Accession Number PTA-9675) and RX 05991199 (ATCC Accession No. PTA-13165). In accordance with the invention, a plant provided herein will generally lack an ELONG allele from Brassica villosa genetically linked to the Myb28 allele. This can be achieved according to the invention through crossing a plant comprising Myb28 and ELONG alleles from Brassica villosa with Brassica plants not comprising the Myb28 and ELONG alleles from Brassica villosa, including standard Brassica oleracea varieties. This includes the many broccoli varieties well known in the art, among others.
The goal of vegetable breeding is to combine various desirable traits in a single variety/hybrid. Such desirable traits may include any trait deemed beneficial by a grower and/or consumer, including greater yield, resistance to insects or disease, tolerance to environmental stress, and nutritional value. Breeding techniques used in an attempt to obtain desired traits take advantage of a plant's method of pollination. There are two general methods of pollination: a plant self-pollinates if pollen from one flower is transferred to the same or another flower of the same plant. A plant cross-pollinates if pollen comes to it from a flower of a different plant.
The development of uniform varieties requires the development of homozygous inbred plants, the crossing of these inbred plants, and the evaluation of the crosses. Pedigree breeding and recurrent selection are examples of breeding methods that have been used to develop inbred plants from breeding populations. Those breeding methods combine the genetic backgrounds from two or more plants or various other broad-based sources into breeding pools from which new lines and hybrids derived therefrom are developed by selfing and selection of desired phenotypes.
In accordance with the invention, novel varieties may be created by crossing plants of the invention followed by generations of selection as desired and inbreeding for development of uniform lines. New varieties may also be created by crossing with any second plant. In selecting such a second plant to cross for the purpose of developing novel lines, it may be desired to choose those plants which either themselves exhibit one or more selected desirable characteristics or which exhibit the desired characteristic(s) when in hybrid combination. Once initial crosses have been made, inbreeding and selection take place to produce new varieties. For development of a uniform line, often five or more generations of selfing and selection are typically involved.
Uniform lines of new varieties may also be developed by way of doubled-haploids. This technique allows the creation of true breeding lines without the need for multiple generations of selfing and selection. In this manner true breeding lines can be produced in as little as one generation. Haploid embryos may be produced from microspores, pollen, anther cultures, or ovary cultures. The haploid embryos may then be doubled autonomously, or by chemical treatments (e.g. colchicine treatment). Alternatively, haploid embryos may be grown into haploid plants and treated to induce chromosome doubling. In either case, fertile homozygous plants are obtained. In accordance with the invention, any of such techniques may be used in connection with a plant of the present invention and progeny thereof to achieve a homozygous line.
Backcrossing can also be used to improve an inbred plant. Backcrossing transfers a specific desirable trait, such as elevated glucoraphanin, from one inbred or non-inbred source to a variety that lacks that trait. This can be accomplished, for example, by first crossing a parent (A) (recurrent parent) to a donor inbred (non-recurrent parent), which carries the appropriate locus or loci for the trait in question. The progeny of this cross are then mated back to the recurrent parent (A) followed by selection in the resultant progeny for the desired trait to be transferred from the non-recurrent parent. After five or more backcross generations with selection for the desired trait, the progeny are heterozygous for loci controlling the characteristic being transferred, but are like the first parent for most or almost all other loci. The last backcross generation would be selfed to give pure breeding progeny for the trait being transferred.
The selection of a suitable recurrent parent is an important step for a successful backcrossing procedure. The goal of a backcross protocol is to alter or substitute a single trait or characteristic in the original variety. To accomplish this, a single locus of the recurrent variety is modified or substituted with the desired locus from the nonrecurrent parent, while retaining essentially all of the rest of the desired genetic, and therefore the desired physiological and morphological constitution of the original variety. The choice of the particular nonrecurrent parent will depend on the purpose of the backcross; one of the major purposes is to add some commercially desirable trait to the plant. The exact backcrossing protocol will depend on the characteristic or trait being altered to determine an appropriate testing protocol. Although backcrossing methods are simplified when the characteristic being transferred is a dominant allele, a recessive allele may also be transferred. It may be necessary to introduce a test of the progeny to determine if the desired characteristic has been successfully transferred.
Brassica oleracea varieties can also be developed from more than two parents. The technique, known as modified backcrossing, uses different recurrent parents during the backcrossing. Modified backcrossing may be used to replace the original recurrent parent with a variety having certain more desirable characteristics or multiple parents may be used to obtain different desirable characteristics from each.
Many single locus traits have been identified that are not regularly selected for in the development of a new inbred but that can be improved by backcrossing techniques. Single locus traits may or may not be transgenic; examples of these traits include, but are not limited to, male sterility, herbicide resistance, resistance to bacterial, fungal, or viral disease, insect resistance, restoration of male fertility, modified fatty acid or carbohydrate metabolism, and enhanced nutritional quality. These comprise genes generally inherited through the nucleus.
Direct selection may be applied where the single locus acts as a dominant trait. Selection of Brassica plants for breeding is not necessarily dependent on the phenotype of a plant and instead can be based on genetic investigations. For example, one can utilize a suitable genetic marker which is closely genetically linked to a trait of interest. One of these markers can be used to identify the presence or absence of a trait in the offspring of a particular cross, and can be used in selection of progeny for continued breeding. This technique is commonly referred to as marker assisted selection. Any other type of genetic marker or other assay which is able to identify the relative presence or absence of a trait of interest in a plant can also be useful for breeding purposes.
Procedures for marker assisted selection are of particular utility for introgression of given traits. Well known types of genetic markers that could be used in accordance with the invention include, but are not necessarily limited to, Simple Sequence Length Polymorphisms (SSLPs), Cleaved Amplified Polymorphic Sequences (CAPs), Randomly Amplified Polymorphic DNAs (RAPDs), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Amplified Fragment Length Polymorphisms (AFLPs), and Single Nucleotide Polymorphisms (SNPs).
B. PLANTS DERIVED FROM A PLANT OF THE INVENTION BY GENETIC ENGINEERING
Many useful traits that can be introduced by backcrossing, as well as directly into a plant, are those, which may be introduced by genetic transformation techniques. Genetic transformation may therefore be used to insert a selected transgene into a Brassica plant of the invention or may, alternatively, be used for the preparation of transgenes, which can be introduced by backcrossing. Methods for the transformation of plants, including Brassica, are well known to those of skill in the art.
Vectors used for the transformation of plant cells are not limited so long as the vector can express an inserted DNA in the cells. For example, vectors comprising promoters for constitutive gene expression in Brassica cells (e.g., cauliflower mosaic virus 35S promoter) and promoters inducible by exogenous stimuli can be used. Examples of suitable vectors include pBI binary vector. The “Brassica cell” into which the vector is to be introduced includes various forms of Brassica cells, such as cultured cell suspensions, protoplasts, leaf sections, and callus.
A vector can be introduced into Brassica cells by known methods, such as the polyethylene glycol method, polycation method, electroporation, Agrobacterium-mediated transfer, particle bombardment and direct DNA uptake by protoplasts.
To effect transformation by electroporation, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly. In this technique, one would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wound tissues in a controlled manner.
One efficient method for delivering transforming DNA segments to plant cells is microprojectile bombardment. In this method, particles are coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells to be bombarded can be positioned at an appropriate distance below the macroprojectile stopping plate. Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species.
Agrobacterium-mediated transfer is another widely applicable system for introducing gene loci into plant cells. An advantage of the technique is that DNA can be introduced into whole plant tissues, thereby bypassing the need for regeneration of an intact plant from a protoplast. Modern Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium (and other Rhizobia), allowing for convenient manipulations. Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. The vectors described have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes. Additionally, Agrobacterium containing both armed and disarmed Ti genes can be used for transformation.
In those plant strains where Agrobacterium-mediated transformation is efficient, it is the method of choice because of the facile and defined nature of the gene locus transfer. The use of Agrobacterium-mediated plant integrating vectors to introduce DNA into plant cells is well known in the art (U.S. Pat. No. 5,563,055). For example, U.S. Pat. No. 5,349,124 describes a method of transforming plant cells using Agrobacterium-mediated transformation. By inserting a chimeric gene having a DNA coding sequence encoding for the full-length B.t. toxin protein that expresses a protein toxic toward Lepidopteran larvae, this methodology resulted in plants having resistance to such insects.
A number of promoters have utility for plant gene expression for any gene of interest including but not limited to selectable markers, scorable markers, genes for pest tolerance, disease resistance, nutritional enhancements and any other gene of agronomic interest. Examples of constitutive promoters useful for Brassica plant gene expression include, but are not limited to, the cauliflower mosaic virus (CaMV) P-35S promoter, which confers constitutive, high-level expression in most plant tissues, including monocots; a tandemly duplicated version of the CaMV 35S promoter, the enhanced 35S promoter (P-e35S) the nopaline synthase promoter, the octopine synthase promoter; and the figwort mosaic virus (P-FMV) promoter as described in U.S. Pat. No. 5,378,619 and an enhanced version of the FMV promoter (P-eFMV) where the promoter sequence of P-FMV is duplicated in tandem, the cauliflower mosaic virus 19S promoter, a sugarcane bacilliform virus promoter, a commelina yellow mottle virus promoter, and other plant DNA virus promoters known to express in plant cells.
Exemplary nucleic acids which may be introduced to the plants of this invention include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical reproduction or breeding techniques. However, the term “exogenous” is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express. Thus, the term “exogenous” gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in the exogenous DNA can include DNA which is already present in the plant cell, DNA from another plant, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
Many hundreds if not thousands of different genes are known and could potentially be introduced into a Brassica plant according to the invention. Non-limiting examples of particular genes and corresponding phenotypes one may choose to introduce into a Brassica plant include one or more genes for insect tolerance, such as a Bacillus thuringiensis (B.t.) gene, pest tolerance such as genes for fungal disease control, herbicide tolerance such as genes conferring glyphosate tolerance, and genes for quality improvements such as yield, nutritional enhancements, environmental or stress tolerances, or any desirable changes in plant physiology, growth, development, morphology or plant product(s). For example, structural genes would include any gene that confers insect tolerance including but not limited to a Bacillus insect control protein gene as described in WO 99/31248, herein incorporated by reference in its entirety, U.S. Pat. No. 5,689,052, herein incorporated by reference in its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, herein incorporated by reference it their entirety. In another embodiment, the structural gene can confer tolerance to the herbicide glyphosate as conferred by genes including, but not limited to Agrobacterium strain CP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat. No. 5,633,435, herein incorporated by reference in its entirety, or glyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No. 5,463,175, herein incorporated by reference in its entirety.
Alternatively, the DNA coding sequences can affect these phenotypes by encoding a non-translatable RNA molecule that causes the targeted inhibition of expression of an endogenous gene, for example via antisense- or cosuppression-mediated mechanisms. The RNA could also be a catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desired endogenous mRNA product. Thus, any gene which produces a protein or mRNA which expresses a phenotype or morphology change of interest is useful for the practice of the present invention.
D. DEFINITIONS
In the description and tables herein, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, the following definitions are provided:
Allele: Any of one or more alternative forms of a gene locus, all of which alleles relate to one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.
Backcrossing: A process in which a breeder repeatedly crosses hybrid progeny, for example a first generation hybrid (F1), back to one of the parents of the hybrid progeny. Backcrossing can be used to introduce one or more single locus conversions from one genetic background into another.
Cultivated Variety: A Brassica oleracea variety which is suitable for consumption and meets the requirements for commercial cultivation. An example is a broccoli variety. In addition to the plants themselves, and the parts thereof suitable for consumption, such as the heads or leaves, the invention comprises parts or derivatives of the plant suitable for propagation. Examples of parts suitable for propagation are organ tissues, such as leaves, stems, roots, shoots and the like, protoplasts, somatic embryos, anthers, petioles, cells in culture and the like. Derivatives suitable for propagation are for instance seeds. The plants according to the invention can be cultivated or propagated in the conventional manner but also by means of tissue culture techniques from plant parts.
Crossing: The mating of two parent plants.
Cross-pollination: Fertilization by the union of two gametes from different plants.
Diploid: A cell or organism having two sets of chromosomes.
Enzymes: Molecules which can act as catalysts in biological reactions.
F1 Hybrid: The first generation progeny of the cross of two nonisogenic plants.
Genotype: The genetic constitution of a cell or organism.
Haploid: A cell or organism having one set of the two sets of chromosomes in a diploid.
Linkage: A phenomenon wherein alleles on the same chromosome tend to segregate together more often than expected by chance if their transmission was independent.
Marker: A readily detectable phenotype, preferably inherited in co-dominant fashion (both alleles at a locus in a diploid heterozygote are readily detectable), with no environmental variance component, i.e., heritability of 1.
Phenotype: The detectable characteristics of a cell or organism, which characteristics are the manifestation of gene expression.
Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer to genetic loci that control to some degree numerically representable traits that are usually continuously distributed.
Recombination event is understood to mean a meiotic crossing-over.
Regeneration: The development of a plant from tissue culture.
Self-pollination: The transfer of pollen from the anther to the stigma of the same plant.
Single Locus Converted (Conversion) Plant: Plants which are developed by a plant breeding technique called backcrossing, wherein essentially all of the desired morphological and physiological characteristics of a broccoli variety are recovered in addition to the characteristics of the single locus transferred into the variety via the backcrossing technique and/or by genetic transformation.
Substantially Equivalent: A characteristic that, when compared, does not show a statistically significant difference (e.g., p=0.05) from the mean.
Tissue Culture: A composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant.
Transgene: A genetic locus comprising a sequence which has been introduced into the genome of a broccoli plant by transformation.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the invention, as limited only by the scope of the appended claims.
All references cited herein are hereby expressly incorporated herein by reference.
E. DEPOSIT INFORMATION
A deposit was made of at least 2500 seeds of broccoli hybrid RX 05991199, which comprises a reduced introgression comprising a Myb28 allele from Brassica villosa and an ELONG allele from Brassica oleracea, as described herein. The deposit was made with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209 USA. The deposit is assigned ATCC Accession No. PTA-13165. The date of deposit was Aug. 24, 2012. Access to the deposits will be available during the pendency of the application to persons entitled thereto upon request. The deposits will be maintained in the ATCC Depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent, whichever is longer, and will be replaced if nonviable during that period. Applicant does not waive any infringement of their rights granted under this patent or any other form of variety protection, including the Plant Variety Protection Act (7 U.S.C. 2321 et seq.).
EXAMPLES
Example 1
Development of Parent Lines with Improved MSB Profiles
One benefit of the current invention is that parent lines may be created comprising a reduced introgression of the invention, wherein the lines and hybrids derived therefrom exhibit an increased proportion of MSB/MSP and/or a greater stability of expression of MSB. The development of such a broccoli line comprising a Myb28/ELONG reduced introgression can be summarized as follows:
The line FT69, which has elevated levels of the phytochemical MSP (glucoiberin) as a result of the presence of Myb28 and ELONG loci from B. villosa (Mithen et al., 2003; Theor. Appl. Genet., 106:727-734), was crossed with a breeding line designated BR9. The resulting progeny was crossed with the male parent from Ironman (BRM 56-3905 SI). F1 progeny were grown in replications in a Wageningen selection trial. From plots designated 408, 409, 410, 411 and 412, 89 plants were selected that were analyzed for glucoiberin (MSP) and glucoraphanin (MSB). Six plants with the highest MSB were selected.
All 6 plants were selfed and backcrossed with the recurrent parent BRM 56-3905 SI. The 6 selfings and 6 BC1 were planted in replicated selection trials in Wageningen. Each BC1 (where BC=backcross) had 2 plots with 24 plants/plot. From these 12 plots, a total of 84 plants were selected that most resembled the recurrent parent. Heads from these selections were sent for MSP and MSB analyses. Seven plants with highest MSB were kept for further selfing and BC. All 7 BC1 plants were selfed and backcrossed with the recurrent parent (BRM 56-3905 SI). Selfings and 5 BC2 populations were sown and selected in replicated Wageningen Autumn selection trials. A total of 73 plants that resembled the recurrent parent most were selected and sent for MSP/MSB analyses. Eight BC2 plants with highest MSB levels were kept for further selfing and BC.
All 8 BC2 plants were selfed and backcrossed with the recurrent parent (BRM 56-3905 SI). Four BC3 were obtained and planted in 1-4 replications in Wageningen. 47 plants closest to the recurrent parent were sent for MSP/MSB analyses. 7 BC3 plants with highest MSB levels were kept for further selfing and BC. All 7 BC3 plants were selfed and backcrossed with the recurrent parent (BRM 56-3905 SI). 6 BC4 were obtained and these were planted in selection trials. 54 plants closest to the recurrent parent were sent for MSP/MSB analyses. 8 BC4 plants with the highest MSB levels were kept for further selfing and BC.
All 8 BC4 plants were selfed. 7 BC4 plants produced selfed seed and the BC4F2 were planted in unreplicated Wageningen selection trials. 37 BC4F2 plants closest to the recurrent parent were selected and these were sent for MSP/MSB analyses. 4 BC4F2 plants with highest MSB levels were kept for further selfing and BC.
All 4 BC4F2 plants were selfed. All 4 produced selfed seed and the 4 BC4F3 were planted in unreplicated selection trials. 12 BC4F3 plants closest to the recurrent parent were selected and sent for MSP/MSB analyses. 4 BC4F2 plants with the highest MSB levels were kept for further selfing and BC. All selected plants from plot 1169 (570114) had equally high MSB level of average 2.3 mmol/kg, or about 5× the level of the standard reference variety (General). This indicated that the source has alleles fixed for high MSB.
All 3 selections from 1169 (BC4F3) were selfed. All 3 produced seed and these were planted in selection trials. SNP13-580333 (1169-1, A75-1) was the most uniform on type and 5 plants were selected for MSB check. All 5 had equally high levels of MSB of about 3.2 mmol/kg. 1169-1, also designated BRM 53-3934 SI, was put on tissue culture for increase and use as a male parent to RX-1199 (RX 05991199). An analysis of BRM 53-3934 SI revealed that it contained the Myb28 reduced introgression, which results in a parent line with the ability to consistently produce progeny with glucosinolates comprising an increased proportion of MSB/MSP relative to plants having the B. villosa ELONG locus, as shown below.
Example 2
Analysis of Hybrid Broccoli Lines Having Elevated Glucoraphanin
Self and outcrosses were made using different standard broccoli lines as female parents as shown in Table 1 in crosses with the following: (1) FT69 (high glucoiberin line with ELONG and Myb28 from B. villosa), (2) SNP13-580333 (high glucoraphanin line with ELONG from B. oleracea and Myb28 from B. villosa, as described above), and (3) SNP88-BRM51-1210 (high glucoiberin line ELONG and Myb28 from B. villosa). As can be seen, regular broccoli lines have a relatively low amount of total glucosinolates. The ratio between MSB and MSP is particularly shown to be depending on the regular broccoli female line in the case of FT69 (428-11-69), which comprises ELONG and Myb28 alleles from B. villosa. In contrast, in all the crosses with SNP13-580333 most of the glucosinolates are glucoraphanin. This indicates the benefit of the reduced introgression in consistently producing hybrid varieties comprising elevated glucoraphanin in crosses with multiple different second parents.







TABLE 1







Levels of Glucoraphanin Resulting From Different Crosses











Female line (regular
Male self (self or crosses with different
Glucoiberin
Glucoraphanin



broccoli line)
high glucosinolates sources)
micromoles/gramDWt
micromoles/gramDWt
Ratio MSB/MSP














BR384-014
self
3.31
18.74
5.66


BR384-014
FT69 (428-11-69) (contains Myb28 and
57.47
15.12
0.26



ELONG from B. villosa)


BR384-014
SNP13-580333 (BRM 53-3934 SI)
3.77
40.14
10.65



(contains reduced introgression)


BR384-014
SNP88-BRM51-1210 (contains Myb28
28.71
5.85
0.20



and ELONG from B. villosa)


BR384-020
Self
1.83
18.24
9.97


BR384-020
FT69 (428-11-69)
36.72
10.00
0.27


BR384-020
SNP13-580333
2.34
26.29
11.23


BR384-020
SNP88-BRM51-1210
13.53
33.96
2.51


BR384-024
self
1.59
3.64
2.29


BR384-024
FT69 (428-11-69)
56.61
7.75
0.14


BR384-024
SNP13-580333
2.64
26.48
10.03


BR384-024
SNP88-BRM51-1210
22.11
3.24
0.15


BR384-025
self
1.43
2.67
1.87


BR384-025
FT69 (428-11-69)
39.66
6.39
0.16


BR384-025
SNP13-580333
3.52
27.55
7.83


BR384-025
SNP88-BRM51-1210
14.25
9.59
0.67


BR384-026
self
1.74
5.08
2.92


BR384-026
FT69 (428-11-69)
41.11
8.26
0.20


BR384-026
SNP13-580333
1.80
30.53
16.93


BR384-026
SNP88-BRM51-1210
19.52
2.89
0.15


BR384-027
self
2.06
13.52
6.55


BR384-027
FT69 (428-11-69)
9.32
47.34
5.08


BR384-027
SNP13-580333
2.60
27.47
10.57


BR384-027
SNP88-BRM51-1210
6.61
28.12
4.26


BR384-033
self
0.57
16.88
29.42


BR384-033
FT69 (428-11-69)
7.58
46.89
6.18


BR384-033
SNP13-580333
2.48
30.81
12.41


BR384-033
SNP88-BRM51-1210
5.23
28.75
5.49


BR384-040
self
0.91
14.03
15.46


BR384-040
FT69 (428-11-69)
4.77
45.61
9.56


BR384-040
SNP13-580333
1.56
16.02
10.27


BR384-040
SNP88-BRM51-1210
3.30
12.73
3.86


BR384-044
self
0.81
11.94
14.74


BR384-044
FT69 (428-11-69)
9.48
50.08
5.28


BR384-044
SNP13-580333
1.99
23.47
11.81


BR384-044
SNP88-BRM51-1210
5.19
27.95
5.38


BR384-048
FT69 (428-11-69)
62.53
5.85
0.09


BR384-048
SNP13-580333
3.02
27.95
9.25


BR384-048
SNP88-BRM51-1210
18.99
1.24
0.07


BR384-052
self
1.51
7.38
4.88


BR384-052
FT69 (428-11-69)
8.27
38.54
4.66


BR384-052
SNP13-580333
2.43
24.09
9.92


BR384-053
self
0.28
2.06
7.49


BR384-053
FT69 (428-11-69)
7.30
53.95
7.39


BR384-053
SNP13-580333
0.64
21.64
33.74


BR384-053
SNP88-BRM51-1210
3.39
14.50
4.27


BR384-057
self
1.43
3.79
2.64


BR384-057
FT69 (428-11-69)
38.32
8.11
0.21


BR384-057
SNP13-580333
4.13
32.20
7.80


BR384-057
SNP88-BRM51-1210
20.38
3.47
0.17


BR384-060
self
1.76
7.90
4.49


BR384-060
FT69 (428-11-69)
10.62
42.68
4.02


BR384-060
SNP13-580333
2.76
26.06
9.46


BR384-060
SNP88-BRM51-1210
5.46
23.19
4.25


BR384-061
self
0.99
15.47
15.65


BR384-061
FT69 (428-11-69)
8.03
49.14
6.12


BR384-061
SNP13-580333
2.58
35.61
13.82


BR384-061
SNP88-BRM51-1210
5.92
36.02
6.09


BR384-069
self
1.63
7.02
4.32


BR384-069
FT69 (428-11-69)
9.33
56.09
6.01


BR384-069
SNP13-580333
1.44
26.36
18.32


BR384-069
SNP88-BRM51-1210
4.60
29.96
6.51


BR384-078
self
1.23
7.59
6.15


BR384-078
FT69 (428-11-69)
10.54
36.99
3.51


BR384-078
SNP13-580333
2.75
24.94
9.06


BR384-078
SNP88-BRM51-1210
5.74
23.42
4.08


BR384-082
self
1.57
10.41
6.64


BR384-082
FT69 (428-11-69)
10.67
38.80
3.64


BR384-082
SNP13-580333
2.53
29.25
11.55


BR384-082
SNP88-BRM51-1210
6.38
31.80
4.98


BR384-083
self
1.31
8.44
6.45


BR384-083
FT69 (428-11-69)
9.33
39.32
4.21


BR384-083
SNP13-580333
2.23
22.39
10.04


BR384-083
SNP88-BRM51-1210
4.85
21.90
4.52


BR384-089
self
3.40
0.35
0.10


BR384-089
FT69 (428-11-69)
48.47
7.21
0.15


BR384-089
SNP13-580333
4.67
24.59
5.27


BR384-089
SNP88-BRM51-1210
19.12
1.98
0.10









Example 3
Development of Broccoli Hybrids
As explained above, one embodiment of the invention comprises producing broccoli hybrids wherein one or both parents of the hybrid comprise a Myb28 reduced introgression and, as a result, exhibit an elevated MSB content and/or more stable MSB content relative to hybrids lacking a parent comprising the introgression. One example of such a hybrid that was produced is the hybrid RX 05991199. This hybrid was created by crossing parents BRM 53-3934 SI and BRM 56-3907 CMS, typically with BRM 53-3934 SI as a male parent. The production of BRM 53-3934 SI is described in Example 1 above. As explained, this parent contains the Myb28 reduced introgression. The female parent BRM 56-3907 CMS is a known inbred that served as the parent of the commercial hybrid “Ironman.” BRM 56-3907 CMS is the subject of, and is described in, EU Plant Variety Rights Certificate #20341, granted Jun. 18, 2007.
A description of the physiological and morphological characteristics of broccoli hybrid RX 05991199 and the parent lines thereof is presented below.







TABLE 2







Physiological and Morphological Characteristics of Hybrid RX 05991199











Characteristic
RX05991199CMS
Ironman














A
Region of Adaptation
NW Europe
NW Europe


B
Maturity, Spring Planted



days from direct seeding to
no direct seeding
no direct seeding



50% harvest



days from transplanting to 50%
64
63



harvest



transplant date
10-May-10
10-May-10



length of harvest period in days
 8
 6



first harvest date
12-Jul-10
12-Jul-10



last harvest date
19-Jul-10
17-Jul-10



harvest season (main crop at
summer
summer



50% harvest)



time of harvest maturity (50%
medium (Sumosun)
medium (Sumosun)



of plants)
medium*8 (Coaster,
medium*8 (Coaster,



time of beginning of flowering
Cruiser)
Cruiser)



(50% of plants with at least



10% flowers) **choice for



UPOV TG only


C
Seedling



cotyledon color
medium green
medium green



RHS color chart value for
147B
147B



seedling cotyledon color



cotyledon anthocyanin
intermediate
intermediate



hypocotyl anthocyanin
strong
strong


D
Plant



plant height in centimeters
56.3 cm
61.0 cm



from soil line to top of leaves



head height in centimeters from
33.7 cm
34.7 cm



soil line to top of leaves



height at harvest maturity
medium (Coaster)
medium (Coaster)



number of stems
one (Ramoso Calabrese,
one (Ramoso




Shogun)
Calabrese, Shogun)



branches
medium
few



habit
intermediate
intermediate



market class
fresh market
fresh market



life cycle
annual
annual



type of variety
first generation hybrid
first generation





hybrid


E
Leaves



outer leaves: number of leaves
14
17



per plant (at harvest)



outer leaves: width (at
22.6 cm
24.2 cm



midpoint of plant including



petiole)



leaf: width
medium (Buccaneer,
medium (Buccaneer,




Green Belt)
Green Belt)



outer leaves: length (at
48.7 cm
53.2 cm



midpoint of plant including



petiole)



leaf: length (including petiole)
medium (Brigadeer,
long (Green Duke,




Sumosun)
Laser)



outer leaves: petiole length
22.1 cm
24.9 cm



petiole: length
medium (Emperor,
long (Groene




Ramoso Calabrese)
Calabrese, Premium





Crop)



outer leaves: leaf ratio -
2:1
2:1



length/width



outer leaves: leaf attachment
petiolate
petiolate



outer leaves: wax presence
weak
intermediate



leaf: number of lobes
medium (Coaster,
medium (Coaster,




Topper)
Topper)



outer leaves: foliage color
grey green
grey green



(with wax, if present)



outer leaves: foliage color
189A
189A



(with wax, if present; RHS



color chart value)



leaf blade: color
grey green (Bishop)
grey green (Bishop)



leaf blade: intensity of color
dark
dark



leaf blade: anthocyanin
absent (Claudia,
absent (Claudia,



coloration
Embassy)
Embassy)



leaf blade: undulation of
weak (Beaufort, Early
weak (Beaufort,



margin
Pack, Laser, Paladin)
Early Pack, Laser,





Paladin)



leaf blade: dentation of margin
weak (Galaxy)
weak (Galaxy)



outer leaves: leaf shape
elliptic
elliptic



outer leaves: leaf base
blunt
blunt



outer leaves: leaf apex
blunt
blunt



outer leaves: leaf margins
slightly wavy
slightly wavy



outer leaves: leaf veins
intermediate
intermediate



outer leaves: midrib
slightly raised
not raised



leaf blade: blistering
medium (Medium Late
medium (Medium




145, Skiff)
Late 145, Skiff)



outer leaves: attitude (leaf
semi-erect (35-55
semi-erect (35-55



angle from ground)
degrees)
degrees)



leaf: attitude (at beginning of
semi-erect (Arcadia,
semi-erect



head formation)
Asti, Civet, Claudia)



outer leaves: torsion of leaf tip
none
none



outer leaves: profile of upper
planar
planar



side of leaf


F
Head



length of branching at base
very short (Viola)
very short (Viola)



(excluding stem)



diameter at widest point (at
15.2 cm
14.7 cm



market maturity)



depth (at market maturity)
9.7 cm
9.4 cm



weight, market trimmed (at
439.9 gm
400.3 gm



market maturity)



color
grey green (Brigadeer,
grey green




Galaxy)
(Brigadeer, Galaxy)



intensity of color
medium
medium



RHS color chart value for head
189B
189B



color



anthocyanin coloration
absent (Early White
present (Brigadeer,




Sprouting)
Shogun, Viola)



shape (at market maturity)
transverse elliptic
transverse elliptic




(Buccaneer, Futura)
(Buccaneer, Futura)



dome shape (at market
domed
domed



maturity)



size (at market maturity) for
medium** (Dundee,
medium** (Dundee,



US Exhibit C only **choice
Early Man)
Early Man)



compactness/firmness (at
medium (Late Corona)
short pedicels/tight/



market maturity)

firm (Captain)



surface knobbling (at market
fine (Apollo, Brigadeer)
medium (Southern



maturity)

Comet)



texture
medium (Clipper,
fine (Auriga, Bishop,




Coaster)
Green Top)



bead size (at market maturity)
medium
small



flower buds (at market
even in size
even in size



maturity)



anthocyanin coloration of leaf
absent
absent



axils (at market maturity)



anthocyanin coloration of leaf
absent
absent



veins (at market maturity)



anthocyanin coloration of leaf
absent
absent



blade (at market maturity)



anthocyanin coloration of
absent
absent



entire plant (at market



maturity)



anthocyanin coloration of leaf
absent (Claudia,
absent (Claudia,



petiole (at market maturity)
Embassy)
Embassy)



color of head leaves (at market
green
green



maturity)



RHS color chart value for the
N189B
N189B



color of head leaves



bracts
absent (Gem, Orion)
absent (Gem, Orion)



secondary heads (at market
combination, present**
present, combination



maturity)
(Marathon, Tribute, Late




Purple Sprouting)



prominence of secondary heads
intermediate (Citation)
weak



(at market maturity)



number of secondary heads (at
 4
 2



market maturity)


G
Flower



Color **choice for UPOV TG
yellow** (Brigadeer,
yellow** (Brigadeer,



only
Orion)
Orion)



intensity of yellow color
medium (Capitol,
medium (Capitol,




Corvet)
Corvet)



color
4B
4B



stalk color
green
green



RHS color chart value for
138B
138B



flower stalk color



male sterility
present (Chevalier,
present (Chevalier,




Montop)
Montop)





*These are typical values. Values may vary due to environment. Other values that are substantially equivalent are also within the scope of the invention.













TABLE 3







Physiological and Morphological Characteristics of Line BRM 53-3934 SI











Characteristic
BMR53-3934SI
Sibsey














A
Region of Adaptation
NW Europe
NW Europe


B
Maturity, Spring Planted



days from direct seeding to
no direct seeding
no direct seeding



50% harvest



days from transplanting to 50%
76
54



harvest



transplant date
10-May-10
10-May-10



length of harvest period in days
16
 5



first harvest date
17-Jul-10
1-Jul-10



last harvest date
1-Aug-10
5-Jul-10



harvest season (main crop at
summer
summer



50% harvest)



time of harvest maturity (50%
very late (Late Purple
very early



of plants)
Sprouting)
(Earlyman, Primor)



time of beginning of flowering
late** (Shogun, Viola)
early** (Clipper,



(50% of plants with at least

Southern Comet)



10% flowers) **choice for



UPOV TG only


C
Seedling



cotyledon color
medium green
medium green



RHS color chart value for
147B
147B



seedling cotyledon color



cotyledon anthocyanin
weak
intermediate



hypocotyl anthocyanin
weak
intermediate


D
Plant



plant height in centimeters
51.7 cm
55.0 cm



from soil line to top of leaves



head height in centimeters from
30.3 cm
50.0 cm



soil line to top of leaves



height at harvest maturity
medium (Coaster)
medium (Coaster)



number of stems
one (Ramoso Calabrese,
one (Ramoso




Shogun)
Calabrese, Shogun)



branches
medium
many



habit
compact
compact



market class
fresh market
fresh market



life cycle
annual
annual



type of variety
inbred
first generation





hybrid


E
Leaves



outer leaves: number of leaves
20
12



per plant (at harvest)



outer leaves: width (at
21.8 cm
18.9 cm



midpoint of plant including



petiole)



leaf: width
medium (Buccaneer,
narrow (Arcadia,




Green Belt)
Brigadeer)



outer leaves: length (at
45.3 cm
39.1 cm



midpoint of plant including



petiole)



leaf: length (including petiole)
medium (Brigadeer,
short (Dandy Early,




Sumosun)
Emperor)



outer leaves: petiole length
20.2 cm
16.0 cm



petiole: length
medium (Emperor,
short (High Sierra,




Ramoso Calabrese)
Padovano)



outer leaves: leaf ratio -
2:1
2:1



length/width



outer leaves: leaf attachment
petiolate
petiolate



outer leaves: wax presence
intermediate
intermediate



leaf: number of lobes
medium (Coaster,
medium (Coaster,




Topper)
Topper)



outer leaves: foliage color
blue green
grey green



(with wax, if present)



outer leaves: foliage color
N198A
189A



(with wax, if present; RHS



color chart value)



leaf blade: color
blue green (Citation,
grey green (Bishop)




Esquire, Symphony)



leaf blade: intensity of color
medium
medium



leaf blade: anthocyanin
absent (Claudia,
absent (Claudia,



coloration
Embassy)
Embassy)



leaf blade: undulation of
weak (Beaufort, Early
strong (Aikido,



margin
Pack, Laser, Paladin)
Marathon, Samurai)



leaf blade: dentation of margin
weak (Galaxy)
weak (Galaxy)



outer leaves: leaf shape
elliptic
elliptic



outer leaves: leaf base
blunt
blunt



outer leaves: leaf apex
blunt
blunt



outer leaves: leaf margins
slightly wavy
very wavy



outer leaves: leaf veins
intermediate
thin



outer leaves: midrib
slightly raised
not raised



leaf blade: blistering
absent or very weak
absent or very weak




(Buccaneer, Colibri)
(Buccaneer, Colibri)



outer leaves: attitude (leaf
semi-erect (35-55
semi-erect (35-55



angle from ground)
degrees)
degrees)



leaf: attitude (at beginning of
semi-erect (Arcadia,
semi-erect



head formation)
Asti, Civet, Claudia)



outer leaves: torsion of leaf tip
none
weak



outer leaves: profile of upper
concave
convex



side of leaf


F
Head



length of branching at base
short (Brigadeer,
medium (Capitol,



(excluding stem)
Buccaneer Emperor)
Green Duke,





Perseus)



diameter at widest point (at
11.4 cm
13.7 cm



market maturity)



depth (at market maturity)
10.4 cm
10.9 cm



weight, market trimmed (at
164.3 gm
182.7 gm



market maturity)



color
light green; grey-green
grey-green




(Brigadeer, Galaxy)
(Brigadeer, Galaxy)



intensity of color
medium
medium



RHS color chart value for head
189A/144A
N189B



color



anthocyanin coloration
absent (Early White
absent (Early White




Sprouting)
Sprouting)



shape (at market maturity)
circular (Esquire)
transverse elliptic





(Buccaneer, Futura)



dome shape (at market
domed
domed



maturity)



size (at market maturity) for US
very small (Early Purple
small** (Orbit,



Exhibit C only **choice
Sprouting)
Scorpio)



compactness/firmness (at
long pedicels/loose
medium (Late



market maturity)
(Caravel)
Corona)



surface knobbling (at market
medium (Southern
fine (Apollo,



maturity)
Comet)
Brigadeer)



texture
fine (Auriga, Bishop,
fine (Auriga, Bishop,




Green Top); coarse
Green Top)




(Citation)



bead size (at market maturity)
small/medium/large
small



flower buds (at market
uneven in size (cateye)
even in size



maturity)



anthocyanin coloration of leaf
absent
absent



axils (at market maturity)



anthocyanin coloration of leaf
absent
absent



veins (at market maturity)



anthocyanin coloration of leaf
absent
absent



blade (at market maturity)



anthocyanin coloration of entire
absent
absent



plant (at market maturity)



anthocyanin coloration of leaf
absent (Claudia,
absent (Claudia,



petiole (at market maturity)
Embassy)
Embassy)



color of head leaves (at market
green
green



maturity)



RHS color chart value for the
N189B
189A



color of head leaves



bracts
present (Ramoso
absent (Gem, Orion)




Calabrese)



secondary heads (at market
basal, present**
combination



maturity)
(Marathon, Tribute, Late




Purple Sprouting)



prominence of secondary heads
intermediate (Citation)
intermediate



(at market maturity)

(Citation)



number of secondary heads (at
 3
 4



market maturity)


G
Flower



Color **choice for UPOV TG
yellow** (Brigadeer,
yellow** (Brigadeer,



only
Orion)
Orion)



intensity of yellow color
dark (Gem, Orion)
dark (Gem, Orion)



color
4A
4A



stalk color
green
green



RHS color chart value for
138B
138B



flower stalk color



male sterility
absent (Marathon)
present (Chevalier,





Montop)





*These are typical values. Values may vary due to environment. Other values that are substantially equivalent are also within the scope of the invention.






The MSB content of hybrid RX 05991199 relative to the hybrid “Heritage” was the subject of an objective analysis. The results of the analysis are presented below.







TABLE 4







Analysis of MSB Content of RX 05991199








Varieties
Least-Squares Means for MSB (micromoles/gm/FW)





RX 05991199
3.5833927


Heritage
1.6311887









A head-to-head analysis was also made of glucoraphanin (MSB) content of RX 05991199 relative to the hybrid Ironman in multiple locations and plantings. The results are presented below.







TABLE 5







Analysis of Glucoraphanin Content of RX 05991199 Relative to Hybrid











Sub-
Plant-
calculated in μmol per gDW












Variety
Country
Region
ing
Glucoiberin
Glucoraphanin















Ironman
Italy
South
A
2.1
3.9


RX 05991199
Italy
South
A
5.0
20.0


RX 05991199
Italy
South
A
1.5
6.7


Ironman
Spain
South
B
2.1
3.1


RX 05991199
Spain
South
B
3.5
13.4


Ironman
Italy
South
C
3.2
9.2


Ironman
Italy
South
C
3.6
9.1


Ironman
Italy
South
C
3.4
10.3


Ironman
Italy
South
C
3.3
10.8


RX 05991199
Italy
South
C
4.7
20.7


RX 05991199
Italy
South
C
5.4
22.3


RX 05991199
Italy
South
C
5.5
26.0


RX 05991199
Italy
South
C
4.7
22.9


Ironman
Spain
South
D
1.9
5.5


Ironman
Spain
South
D
2.8
9.6


Ironman
Spain
South
D
2.4
5.9


RX 05991199
Spain
South
D
5.1
21.7


RX 05991199
Spain
South
D
5.5
24.8


RX 05991199
Spain
South
D
5.1
25.7


Ironman
UK
North
A
2.5
9.8


Ironman
UK
North
A
3.1
12.8


RX 05991199
UK
North
A
3.8
21.3


RX 05991199
UK
North
A
3.4
24.4


Ironman
UK
North
B
0.9
7.1


Ironman
UK
North
B
1.9
12.1


RX 05991199
UK
North
B
2.4
11.9


RX 05991199
UK
North
B
3.0
16.0


Ironman
UK
North
C
1.8
5.8


Ironman
UK
North
C
1.9
5.8


RX 05991199
UK
North
C
3.1
14.1


RX 05991199
UK
North
C
3.3
15.1


Ironman
UK
North
D
1.1
6.2


Ironman
UK
North
D
0.9
6.2


RX 05991199
UK
North
D
3.2
17.2


RX 05991199
UK
North
D
3.1
16.6


ironman
Italy
South
F
0.9
5.1


ironman
Italy
South
F
1.0
5.3


RX 05991199
Italy
South
F
3.8
24.5


RX 05991199
Italy
South
F
3.6
25.1


ironman
Italy
South
A
0.9
4.3


ironman
Italy
South
A
1.0
5.1


RX 05991199
Italy
South
A
4.6
23.5


RX 05991199
Italy
South
A
4.0
22.4


ironman
Spain
South
B
1.5
6.0


ironman
Spain
South
B
2.4
11.2


RX 05991199
Spain
South
B
4.0
20.7


RX 05991199
Spain
South
B
4.4
22.1


ironman
Spain
South
C
1.9
8.3


ironman
Spain
South
C
1.7
8.8


RX 05991199
Spain
South
C
4.1
24.3


RX 05991199
Spain
South
C
4.3
22.4


Ironman
Spain
South
D
1.2
5.5


Ironman
Spain
South
D
0.9
4.9


RX 05991199
Spain
South
D
3.5
19.0


RX 05991199
Spain
South
D
2.8
17.5


Ironman
Italy
South
E
0.9
7.6


Ironman
Italy
South
E
0.8
6.7


RX 05991199
Italy
South
E
3.0
24.4


RX 05991199
Italy
South
E
1.8
23.3


Ironman
Spain
South
F
0.8
5.0


Ironman
Spain
South
F
0.6
3.8


RX 05991199
Spain
South
F
2.9
17.1


RX 05991199
Spain
South
F
3.0
19.3


Ironman
Spain
South
G
1.0
6.3


Ironman
Spain
South
G
0.9
6.2


RX 05991199
Spain
South
G
3.4
20.8


RX 05991199
Spain
South
G
3.0
19.9


Ironman
Spain
South
H
2.5
14.7


Ironman
Spain
South
H
2.3
14.1


RX 05991199
Spain
South
H
3.6
21.9


RX 05991199
Spain
South
H
4.0
23.0


Ironman
UK
North
B
0.9
6.2


Ironman
UK
North
B
1.0
6.1


RX 05991199
UK
North
B
4.2
20.9


RX 05991199
UK
North
B
3.6
18.6


Ironman
UK
North
C
2.1
6.3


Ironman
UK
North
C
1.9
6.4


RX 05991199
UK
North
C
4.7
15.1


RX 05991199
UK
North
C
4.4
15.5


Ironman
UK
North
D
1.7
11.3


Ironman
UK
North
D
1.1
6.3


RX 05991199
UK
North
D
4.1
22.0


RX 05991199
UK
North
D
2.7
14.6


Ironman
UK
North
E
2.1
11.9


RX 05991199
UK
North
E
5.3
24.2


Ironman
UK
North
F
1.2
8.0


Ironman
UK
North
F
1.5
8.7


RX 05991199
UK
North
F
4.2
19.9


RX 05991199
UK
North
F
4.5
21.9


Ironman
UK
North
G
1.0
6.1


Ironman
UK
North
G
1.0
6.7


RX 05991199
UK
North
G
2.7
16.8


RX 05991199
UK
North
G
2.8
17.7


Ironman
UK
North
H
1.0
6.7


Ironman
UK
North
H
0.8
5.8


RX 05991199
UK
North
H
3.4
19.2


RX 05991199
UK
North
H
3.9
23.1


Ironman
Spain
South
A
0.6
4.0


Ironman
Spain
South
A
0.9
4.2


Ironman
Spain
South
A
0.8
3.7


RX 05991199
Spain
South
A
2.6
13.3


RX 05991199
Spain
South
A
2.7
14.1


RX 05991199
Spain
South
A
2.8
13.4


Ironman
Italy
South
B
1.4
7.3


Ironman
Italy
South
B
1.2
8.2


RX 05991199
Italy
South
B
4.9
26.6


RX 05991199
Italy
South
B
3.5
18.7


Ironman
Spain
South
C
1.1
5.9


Ironman
Spain
South
C
1.0
4.8


Ironman
Spain
South
C
1.0
5.5


RX 05991199
Spain
South
C
3.3
16.0


RX 05991199
Spain
South
C
2.7
13.0


RX 05991199
Spain
South
C
2.9
11.7


Ironman
Italy
South
D
1.1
5.9


Ironman
Italy
South
D
1.1
5.3


RX 05991199
Italy
South
D
2.9
16.0


RX 05991199
Italy
South
D
3.9
21.4


Ironman
Spain
South
E
0.9
4.3


Ironman
Spain
South
E
1.0
3.9


Ironman
Spain
South
E
1.1
4.8


RX 05991199
Spain
South
E
3.6
21.2


RX 05991199
Spain
South
E
2.2
16.5


RX 05991199
Spain
South
E
2.5
15.5


Ironman
Spain
South
F
1.1
4.6


Ironman
Spain
South
F
0.8
4.4


Ironman
Spain
South
F
1.0
4.5


RX 05991199
Spain
South
F
1.5
9.7


RX 05991199
Spain
South
F
2.3
15.4


RX 05991199
Spain
South
F
2.4
15.3


Ironman
Spain
South
G
1.2
4.4


Ironman
Spain
South
G
1.4
5.6


Ironman
Spain
South
G
1.0
3.4


RX 05991199
Spain
South
G
2.9
16.7


RX 05991199
Spain
South
G
2.8
18.7


RX 05991199
Spain
South
G
3.0
15.9


Ironman
UK
North
L
0.7
8.2


Ironman
UK
North
L
0.8
9.8


Ironman
UK
North
L
0.8
10.3


RX 05991199
UK
North
L
1.6
15.0


RX 05991199
UK
North
L
1.9
17.1


RX 05991199
UK
North
L
1.9
18.1


Ironman
UK
North
M
0.2
4.9


Ironman
UK
North
M
0.3
4.5


Ironman
UK
North
M
0.3
5.2


RX 05991199
UK
North
M
1.1
8.5


RX 05991199
UK
North
M
1.6
9.8


RX 05991199
UK
North
M
1.3
10.0









Example 4
Use of Genetic Markers to Identify and Track Reduced Introgressions
Genetic marker assays were developed to genotype for Myb28 and ELONG alleles. The assays thus permit identification of a reduced introgression in accordance with the invention as well as marker assisted introduction of the reduced introgression into any other genotype.
A. Markers for Detection of ELONG
A marker designated QTL1-BoGLS-ELONG was developed and permits detection of the presence or absence of a B. villosa ELONG allele. This marker can be detected using the primer pair AF399834F2: 5′-cggattttcaaattttctcg-3′ (SEQ ID NO:1) and AF399834R2: 5′-atttcgcatgaccactaggc-3′ (SEQ ID NO:2). To detect the marker, plates were loaded with 20 ng DNA template (sample) in a 2 μL volume. 34, master mix (0.437 μL water, 2.5 μL Q PCR(ROX) mix, 0.063 uL assay mix) was added to each well for a final volume of 5 μL. PCR conditions were as follows: 95° C. for 15 min then 40 cycles of 95° C. for 15 sec, 60° C. for 1 min. FIG. 4 shows results of an assay for different ELONG alleles using the marker. The V allele is indicative of the B. villosa ELONG allele, while A, B and C alleles are examples of alleles found in broccoli without a B. villosa ELONG allele.
B. Marker for the Detection of Myb28
The identification of genetic polymorphisms in Myb28 alleles and their use as genetic markers is described in U.S. Provisional Appln. Ser. No. 61/700,731, filed concurrently herewith, the disclosure of which is incorporated herein by reference in its entirety. Specifically, sequence alignments were described therein between B. oleracea alleles and the corresponding Myb28 sequences to identify polymorphisms that can be used for marker based selection for a Myb28 allele of choice. The results are shown in FIG. 5, which is an alignment between a consensus sequence of the Myb28 locus from B. villosa contained in broccoli variety FT69, and a consensus sequence of the corresponding locus from broccoli without increased level of glucosinolate, e.g. B. oleracea, (Oleracea). Shown are 26 polymorphisms (e.g. single feature polymorphisms (SFPs)—of which there are 16 SNPs and 10 indels) detected in a sequence with a total length of 2202 bp. Any of these or other identified polymorphisms may be used as genetic markers for the presence or absence of a desired Myb28, including from B. oleracea or B. villosa. 
A TaqMan assay (NBOLI009111370) was designed based on one of the sequence polymorphisms identified, as follows:







NBOLI009111370 sequence (SEQ ID NO: 3):


GACCACCTAAAGACAAGAATAGTGAAAGAGATAAGATGGAAGACCAAAGT


 


TAATCAAATTTATTTTGAAGCTTTT[C/T]TATGGAATAGAGACTAAAAT


 


GATGTGTGCTATTGCAATTTTTAGTCACATATTGCTAATCAAACACATAT


 


TTTGCATCAGAGAATTGTCAAATACATGAAAAAAATAAAGAATAATTTTT


 


Forward primer (SEQ ID NO: 4):


GTGAAAGAGATAAGATGGAAGACCAAAGT


 


Reverse primer(SEQ ID NO: 5):


GTGACTAAAAATTGCAATAGCACACATCA


 


Vic probe (SEQ ID NO: 6):


CTATTCCATAGAAAAGC


 


Fam probe (SEQ ID NO: 7):


CTATTCCATAAAAAAGC






The assay was carried out using standard procedures as follows: Load plates with 20 ng DNA template in 5 μL volume. Add 10 μL master mix (2 parts each of 1×PCR mix, 0.437 μL water, 2.5 μL Q PCR (ROX) mix, 0.063 μL assay mix, 2 μL primers at 5 ng/μL) to each well for a final volume of 15 μL.

PCR conditions are as follows: 50° C. for 2 min followed by 95° C. for 2 min then 40 cycles of 95° C. for 15 sec, 60° C. for 1 min.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

## Claims

1. A Brassica oleracea plant comprising a chromosomal segment that comprises a Myb28 allele from Brassica villosa and lacking an ELONG allele from Brassica villosa genetically linked to said Myb28 allele, wherein the segment confers elevated glucosinolates relative to a Brassica oleracea plant lacking the Myb28 allele, wherein a sample of seed comprising the chromosomal segment was deposited under ATCC Accession Number PTA-13165.

2. The plant of claim 1, wherein the plant is a broccoli plant.

3. The plant of claim 1, wherein the plant is inbred.

4. The plant of claim 1, wherein the plant is hybrid.

5. The plant of claim 1, wherein the plant is homozygous for said Myb28 allele from Brassica villosa.

6. The plant of claim 1, wherein the plant is heterozygous for said Myb28 allele from Brassica villosa.

7. The plant of claim 1, further comprising an ELONG allele from Brassica oleracea.

8. A seed that produces the plant of claim 1.

9. A plant part of the plant of claim 1, wherein the plant part comprises the Myb28 allele but not the ELONG allele from Brassica villlosa.

10. The plant part of claim 9, wherein the part is a leaf, an ovule, a floret, pollen, a head, or a cell, and wherein the part comprises the Myb28 allele but not the ELONG allele from Brassica villlosa.

11. A progeny of the plant of claim 1, wherein said progeny comprises said Myb28 allele but not the ELONG allele from Brassica villosa.

12. A part of the progeny of claim 11 selected from the group consisting of a cell, a seed, a root, a stem, a leaf, a head, a flower, and pollen, wherein said part comprises said Myb28 allele but not the ELONG allele from Brassica villosa.