USE OF ANTHOCYANIN GENES TO MAINTAIN MALE STERILE PLANTS

Abstract: A plant consisting essentially of cells which comprise in their genome a homozygous male-sterility genotype at a first genetic locus; and a color-linked restorer genotype at a second genetic locus, which is heterozygous (Rf/-) for a foreign DNA Rf. The foreign DNA Rf comprises: a) a fertility-restorer gene capable of preventing the phenotypic expression of the male-sterility genotype, and b) at least one anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesis in cells of seeds of the plant which is capable of producing anthocyanin at least in the seeds of the plant, so that anthocyanin production in the seeds is visible externally. Preferably, the anthocyanin regulatory gene is a shortened R, B or C1 gene or a continuation thereof. The invention also relates to DNA sequences encoding shortened R, B or C1 anthocyanin regulatory genes and to a process for maintaining a line of male-sterile plants which comprises crossing a male-sterile parent plant and a maintainer parent plant comprising homozygous male-sterility genotype and a restore genotype comprising fertility-restorer gene and an anthocyanin regulatory gene.

Description: /34634 USE OF ANTHOCYANIN GENES TO MAINTAIN MALE STERILE PLANTS The present invention relates to a method to maintain male-sterile plants that can be used for the production of hybrid seed of a plant crop species, to transgenic inbred plants that can be used in such process, and to chimeric genes that can be used to produce such transgenic inbred plants. Background to the Invention In many, if not most plant species, the development of hybrid cultivars is highly desired because of their generally increased productivity due to heterosis: the superiority of performance of hybrid individuals compared with their parents (see e.g. Fehr, 1987, Principles of cultivar development, Volume 1 : Theory and Technique, MacMillan Publishing Company, New York; Allard, 1960, Principles of Plant Breeding, John Wiley and Sons, Inc.). The development of hybrid cultivars of various plant species depends upon the capability of achieving essentially almost complete cross-pollination between parents. This is most simply achieved by rendering one of the parent lines male sterile (i.e. bringing them in a condition so that pollen is absent or nonfunctional) either manually, by removing the anthers, or genetically 5/34634 by using, in the one parent, cytoplasmic or nuclear genes that prevent anther and/or pollen development (for a review of the genetics of male sterility in plants see Kaul, 1988, 'Male Sterility in Higher Plants', Springer Verlag) . For hybrid plants where the seed is the harvested product (e.g. corn, oilseed rape) it is in most cases also necessary to ensure that fertility of the hybrid plants is fully restored. In systems in which the male sterility is under genetic control this requires the existence and use of genes that can restore male fertility. The development of hybrid cultivars is mainly dependent on the availability of suitable and effective sterility and restorer genes. Endogenous nuclear loci are known for most plant species that may contain genotypes which effect male sterility, and generally, such loci need to be homozygous for particular recessive alleles in order to result in a male-sterile phenotype. The presence of a dominant 'male fertile' allele at such loci results in male fertility. Recently it has been shown that male sterility can be induced in a plant by providing the genome of the plant with a chimeric male-sterility gene comprising a DNA sequence (or male-sterility DNA) coding, for example, for a cytotoxic product (such as an RNase) and under the control of a promoter which is predominantly active in selected tissue of the male reproductive organs. In this regard stamen-specific promoters, such as the promoter of the TA29 gene of Nicotiana tabacum, have been shown to be particularly useful for this purpose (Mariani et al., 1990, Nature 347:737, European patent publication ("EP") 0,344,029). By providing the nuclear genome of the plant with such a male-sterility gene, an artificial male- sterility locus is created containing the artificial male- sterility genotype that results in a male-sterile plant. In addition it has been shown that male fertility can be restored to the plant with a chimeric fertility- restorer gene comprising another DNA sequence (or fertility-restorer DNA) that codes, for example, for a protein that inhibits the activity of the cytotoxic product or otherwise prevents the cytotoxic product from being active in the plant cells (European patent publication "EP" 0,412,911). For example the barnase gene of Bacillus a yloliσuefaciens codes for an RNase, called barnase, which can be inhibited by a protein, barstar, that is encoded by the barstar gene of B. amyloliquefaciens. The barnase gene can be used for the construction of a sterility gene while the barstar gene can be used for the construction of a fertility-restorer gene. Experiments in different plant species, e.g. oilseed rape, have shown that a chimeric barstar gene can fully restore the male fertility of male sterile lines in which the male sterility was due to the presence of a chimeric barnase gene (EP 0,412,911, Mariani et al., 1991, Proceedings of the CCIRC Rapeseed Congress, July 9- 11, 1991, Saskatoon, Saskatchewan, Canada; Mariani et al., 1992, Nature 357:384). By coupling a marker gene, such as a dominant herbicide resistance gene (for example the bar gene coding for phosphinothricin acetyl transferase (PAT) that converts the herbicidal phosphinothricin to a non-toxic compound [De Block et al., 1987, EMBO J. 6:2513]), to the chimeric male- sterility and/or fertility-restorer gene, breeding systems can be implemented to select for uniform populations of male sterile plants (EP 0,344,029; EP 0,412,911) . The production of hybrid seed of any particular cultivar of a plant species requires the: 1) maintenance of small quantities of pure seed of each inbred parent, and 2) the preparation of larger quantities of seed of each inbred parent. Such larger quantities of seed would normally be obtained by several (usually two) seed multiplication rounds, starting from a small quantity of pure seed ("basic seed") and leading, in each multiplication round, to a larger quantity of pure seed of the inbred parent and then finally to a stock of seed of the inbred parent (the "parent seed" or "foundation seed") which is of sufficient quantity to be planted to produce the desired quantities of hybrid seed. Of course, in each seed multiplication round larger planting areas (fields) are required. In order to maintain and enlarge a small stock of seeds that can give rise to male-sterile plants it is necessary to cross the male sterile plants with normal pollen-producing parent plants. In the case in which the male-sterility is encoded in the nuclear genome, the offspring of such cross will in all cases be a mixture of male-sterile and male-fertile plants and the latter have to be removed from the former. With male-sterile plants containing an artificial male-sterility locus as described above, such removal can be facilitated by genetically linking the chimeric male sterility gene to a suitable marker gene, such as the bar gene, which allows the easy identification and removal of male-fertile plants (e.g. by spraying of an appropriate herbicide). However, even when suitable marker genes are linked to male-sterility genotypes, the maintenance of parent male- sterile plants still requires at each generation the removal from the field of a substantial number of plants. For instance in systems using a herbicide resistance gene (e.g. the bar gene) linked to a chimeric male-sterility gene, as outlined above, only half of the parent stock will result in male- sterile plants, thus requiring the removal of the male-fertile plants by herbicide spraying prior to flowering. In any given field, the removal of male-fertile plants effectively reduces the potential yield of hybrid seed or the potential yield of male-sterile plants during each round of seed multiplication for producing parent seed. In addition removal of the male- fertile plants may lead to irregular stands of the male-sterile plants. For these reasons removal of the male-fertile plants is economically unattractive for many important crop species such as corn and oilseed rape. Anthocyanins are pigments that are responsible for many of the red and blue colors in plants. The genetic basis of anthocyanin biosynthesis has been well characterized, particularly in corn, Petunia. and Antirrhinium (Dooner et al, 1991, Ann.Rev.Genet. 25:179- 199; Jayaram and Peterson, 1990, Plant Breeding Reviews 2:91-137; Coe, 1994, In 'The Maize Handbook', Freeling and Walbot, eds. Springer Verlag New York Inc., p. 279- 281) . In corn anthocyanin biosynthesis is apparently under control of 20 or more genes. The structural loci C2, Whp, Al, A2, Bzl, and Bz2 code for various enzymes involved in anthocyanin biosynthesis and at least 6 regulatory loci, acting upon the structural genes, have been identified in corn i.e. the R, B, Cl, PI, P and Vpl loci. The R locus has turned out to be a gene family (in corn located on chromosome 10) comprising at least three different genes i.e. R (which itself may comprise duplicate genes organized in a tandem array) , and the displaced duplicate genes R(Sn) and R(Lc) . R typically conditions pigmentation of the aleurone but various alleles are known to confer distinct patterns of pigmentation. R(Lc) is associated with unique pigmentation of leaves and R(Sn) with unique pigmentation of the scutellar node. One state of R is associated with pigmentation of the whole plant (R(P)) , while another is associated with pigmentation of the seeds (R(S)) . Alleles of the unlinked B locus (in corn located on chromosome 2) rarely condition pigmentation of the aleurone, but are frequently associated with pigmentation of mature plant parts. The B-peru allele however, pigments the aleurone (like R(S)) . Analysis at the molecular level has confirmed that the R and B loci are duplicate genes. in order that the R and B loci can color a particular tissue, the appropriate allele of Cl or PI loci also needs to be present. The Cl and Cl-S alleles, for instance, pigment the aleurone when combined with the suitable R or B allele. Alleles of the Cl locus have been cloned and sequenced. Of particular interest are Cl (Paz-Ares et al, 1987, EMBO J. 6:3553-3558) and Cl-S (Schleffer et al, 1994, Mol.Gen.Genet. 242:40-48). Analysis of the sequences revealed the presence of two introns in the coding region of the gene. The protein encoded by the Cl and Cl-S alleles shares homology with myb proto-oncogenes and is known to be a nuclear protein with DNA-binding capacity acting as transcriptional activators. The cDNA of the B-peru allele has also been analyzed and sequenced (Radicella et al, 1991, Plant Mol. Biol. 17:127-130). Genomic sequences of B-peru were also isolated and characterized based on the homology between R and B (Chandler et al., 1989, the Plant Cell 1:1175- 1183; Radicella et al. , 1992, Genes & Development 6:2152- 2164) . The tissue specificity of anthocyanin production of two different B alleles was shown to be due to differences in the promoter and untranslated leader sequences (Radicella et al, 1992, supra) . Various alleles of the R gene family have also been characterized at the molecular level, e.g. Lc (Ludwig et al, 1989, PNAS 86:7092-7096), R-nj , responsible for pigmentation of the crown of the kernel (Dellaporta et al, 1988, In "Chromosome Structure and Function,: Impact of New Concepts, 18th Stadeler Genetics Symposium, Gustafson and Appelε, eds. (New York, Plenum press, pp. 263-282)), Sn (Consonni ei al, 1992, Nucl. Acids. Res. 20:373), and R(S) (Perrot and Cone, 1989, Nucl. Acids. Res. 17:8003) . The proteins encoded by the B and R genes share homology with mvc proto-oncogenes and have characteristics of transcriptional activators. It has been shown that various structural and regulatory genes introduced in maize tissues by microprojectileε operate in a manner similar to the endogenous loci and can complement genotypes which are deficient in the introduced genes (Klein et al., 1989, PNAS 86:6681-6685; Goff et al., 1990, EMBO J. 9:2517- 2522) . The Lc gene was also used as a visible marker for plant transformation (Ludwig et al., 1990, Science 247:449- 450). Apart from the above other genes involved in anthocyanin biosynthesis have been cloned (Cone, 1994, In 'The Maize Handbook', Freeling and Walbot eds.. Springer Verlag New York Inc., p. 282-285). In Barley, Falk et al (1981, In Barley Genetics IV, proceedings of the 4th International Barley Genetics symposium, Edinburgh University press, Edinburgh, pp. 778-785) have reported the coupling of a male-sterile gene to a xenia-expressing shrunken endosperm gene which makes it possible to select seeds, before planting, that will produce male-sterile plants. Problems asociated with such proposal include complete linkage of the two genes (Stoskopf, 1993, Plant Breeding : Theory and Practice, Westview Press, Boulder, San Francisco, Oxford) . In sweetcorn, a genetic system to produce hybrid corn seeds without detasεling, which utilizes the closely linked genes y_ (white endosperm) and ms (male sterility) was suggested but was never used because of contamination from 5% recombination. Galinat (1975, J.Hered. 66:387- 388) described a two-step seed production scheme that resolved this problem by using electronic color sorters to separate yellow from white kernels . This approach has not been utilized commercially (Kankis and Davis, 1986, in « Breeding Vegetable Crops », the Avi Publishing Company Inc. Weεtport, Connecticut, U.S.A., p. 498). EP 0,198,288 and US Patent 4,717,219 describe methods for linking marker genes (which can be visible markers or dominant conditional markers) to endogenous nuclear loci containing nuclear male-sterility genotypes. EP 412,911 describeε foreign reεtorer genes (e.g. barstar coding region under control of a stamen-specific promoter) that are linked to marker genes, including herbicide resistance genes and genes coding for pigments (e.g. the Al gene) under control of a promoter which directs expresεion in specific cells, such as petal cells, leaf cellε or seed cells, preferably in the outer layer of the seed. Summary of the Invention The invention concerns a maintainer plant consisting essentially of cells which comprise in their genome: a homozygous male-sterility genotype at a first genetic locus; and a color-linked restorer genotype at a second genetic locus, which is heterozygous (Rf/-)for a foreign DNA Rf comprising: a) a fertility-restorer gene capable of preventing the phenotypic expression of said male-sterility genotype, and b) at least one anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesis in cells of seeds of said plant and which is capable of producing anthocyanin at least in the seeds of said plant, so that anthocyanin production in the seeds is visible externally. The invention also concerns an anthocyanin regulatory gene which is a shortened R, B or Cl gene or a combination of shortened R, B or Cl genes which is functional for conditioning and regulating anthocyanin production in the aleurone. 5/34634 12 The invention also includes a DNA such as a plaεmid comprising a fertility-restorer gene capable of preventing the phenotypic expression of a male-sterility genotype in a plant and at least one anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesis in cells of seeds of a plant and which is capable of producing anthocyanin at least in the seeds of a plant, so that anthocyanin production in the seeds is visible externally. Also within the scope of the invention is a proceεε to maintain a line of male-sterile plants, which comprises the following steps: i) crosεing: a) a male-εterile parent plant of said line having, in a firεt genetic locus, a homozygouε male- sterility genotype, and b) a maintainer parent plant of said line consiεting eεsentially of cells which comprise, stably intergrated in their nuclear genome: - a homozygous male-sterility genotype at a first genetic locus; and - a colored-linked restorer genotype at a second genetic locus, which is heterozygous for a foreign DNA comprising: i) a fertility-restorer gene capable of preventing the phenotypic expression of said male-sterility genotype, and ii) at least one anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesis in cells of seeds of said plant which is capable of producing anthocyanin at least in the seeds of said plant, so that anthocyanin production in the seeds is visible externally. ii) obtaining the seeds from said parent plants, and iii) separating on the basis of color, the seeds in which no anthocyanin is produced and which grow into male- sterile parent plants. Preferably, the genome of the male-sterile parent plant does not contain at least one anthocyanin regulatory gene necessary for the regulation of anthocyanin biosynthesis in seeds of this plant to produce externally visible anthocyanin in the seeds. In one embodiment of the invention, the genome of the male-sterile parent plant contains a first anthocyanin regulatory gene and the /34634 14 genome of the maintainer plant a second anthocyanin regulatory gene which, when present with the first anthocyanin regulatory gene in the genome of a plant, is capable of conditioning the production of externally visible anthocyanin in seeds. The invention also concerns a process to maintain a line of maintainer plants, which comprises the following steps: i) crossing: a) a male-sterile parent plant as described previously, and b) a maintainer parent plant as described previously, ii) obtaining the seeds from said male-sterile parent plant, and iii) separating on the basis of color, the seeds in which anthocyanin is produced and which grow into maintainer parent plantε. CONFIRMAΓION ' COPΓ The invention also relates to a kit for maintaining a line of male-sterile or maintainer plants, said kit comprising: a) a male-sterile parent plant of said line as described previously, having, in a first genetic locus, a homozygous male-sterility genotype and which is incapable of producing externally visible anthocyanin in seeds, and b) a maintainer parent plant of said line as described previously. Also within the scope of the invention is a process to maintain the kit described previously which comprises: - crosεing εaid male-sterile parent plant with said maintainer parent plant; - obtaining the seeds from said male-sterile parent plants and optionally the seeds from said maintainer parent plant in which no anthocyanin is produced; and - optionally growing said seeds into male-sterile parent plants and maintainer parent plants. As mentioned above, the present invention provides means to maintain a line of male-εterile plants, particularly corn or wheat plants. These means can be in /34634 16 the form of a process which comprises the following stepε: i) croεsing A) a first parent plant of said line, which is male-sterile, and which is genetically characterized by the absence of at least one anthocyanin regulatory gene thereby being incapable of producing anthocyanin in seeds, particularly in the aleurone layer, and also by having at a first genetic locus a homozygous ale- sterility genotype, and B) a second parent plant of said line, which is male-fertile, and which is genetically characterized by having at said first genetic locus, said homozygous male- sterility genotype, and at a separate second genetic locus the genotype Rf/-, whereby, Rf is a foreign chimeric DNA (the "color-linked reεtorer gene") stably integrated in the nuclear genome of said plant which comprises: a) a fertility-restorer gene that is capable of preventing the phenotypic expression, i.e. the male- sterility, of said male-sterility genotype. b) said at least one anthocyanin regulatory gene (the "color gene") involved in the regulation of the anthocyanin biosynthesis in cells of seeds of said cereal plant which is capable of producing anthocyanin at least in the seedε, particularly in the aleurone, of said cereal plant, ii) obtaining the εeedε from said first parent plants iii) separating, on the basis of color, the seeds in which no anthocyanin is produced and in which the genotype at said first genetic locus is said homozygouε male-sterility genotype and the genotype at said second genetic locuε iε -/-, and the seeds in which anthocyanin is produced and in which the genotype at said first genetic locus is εaid homozygouε male-εterility genotype and the genotype at said second genetic locus is Rf/-. Of particular interest in the invention is a second parent plant in which said at least one anthocyanin regulatory gene comprises a gene derived from a genomic clone of an R or B gene, particularly an R or B gene that conditions anthocyanin production in the aleurone, preferably the B-peru allele (e.g. the shortened B-peru gene in pCOL13) , and/or comprises a gene derived from a genomic clone of the Cl gene (e.g. the gene with the sequence of SEQ ID NO 1 or SEQ ID NO 5) or the Cl-S gene. The first genetic locus can be endogenous to plants of said line (in which case the homozygous male-sterility genotype will be m/m) , but is preferably a foreign locus with genotype S/S in which S is a foreign DNA which, when expressed in a plant is capable of rendering the plant male-εterile. A preferred foreign DNA comprises at least: si) a male-sterility DNA encoding a RNA, protein or polypeptide which, when produced or overproduced in a cell of the plant, significantly disturbε the metaboliεm, functioning and/or development of the cell, and, ε2)a εterility promoter capable of directing expreεεion of the male-sterility DNA εelectively in stamen cells, preferably tapetum cells, of the plant; the male- sterility DNA being in the same tranεcriptional unit as, and under the control of, the εterility promoter. In case such a foreign male-sterility genotype is used, the fertility-restorer gene in the foreign DNA Rf preferably comprises at least: al) a fertility-restorer DNA encoding a restorer RNA, protein or polypeptide which, when produced or overproduced in the same stamen cells as said male- sterility gene S, prevents the phenotypic expression of said foreign male-εterility genotype comprising S, and, a2) a restorer promoter capable of directing expression of the fertility-restorer DNA at least in the same stamen cells in which said male-sterility gene S is expressed, so that the phenotypic expression of said male-sterility gene iε prevented; the fertility- /34634 19 restorer DNA being in the same transcriptional unit as, and under the control of, the restorer promoter. In case of an endogenous male-εterility genotype which iε homozygous for the receεεive male-εterility allele m, the fertility reεtorer gene is preferably a DNA comprising the dominant allele M of said locus. The present invention also provides the novel foreign chimeric DNA Rf as used in the second parent plants, plasmids comprising these chimeric geneε, and host cells comprising these plasmids. The present invention also provides the shortened B- peru gene in pCOL13 (SEQ ID NO 6) and the shortened Cl gene, particularly the EcoRI-Sfil fragment of pCOL9 of SEQ ID NO 5. The present invention further provides plants the nuclear genome of which is transformed with the foreign chimeric DNA Rf, particularly the second parent plant. Detailed Description of the Invention A male-sterile plant iε a plant of a given plant species which iε male-sterile due to expresεion of a male-sterility genotype εuch as a foreign male-sterility genotype containing a male-sterility gene. A restorer plant iε a plant of the same plant species that contains within its genome at least one fertility-restorer gene that is able to restore the male fertility in those offεpring obtained from a croεε between a male-εterile plant and a reεtorer plant and containing both a male- sterility genotype and a fertility-restorer gene. A restored plant iε a plant of the same specieε that iε male- fertile and that contains within its genome a male- sterility genotype and a fertility-restorer gene. A line is the progeny of a given individual plant. A gene as used herein iε generally underεtood to compriεe at leaεt one coding region coding for an RNA, protein or polypeptide which iε operably linked to suitable promoter and 3' regulatory sequences. A structural gene is a gene whose product is a e.g. an enzyme, a structural protein, tRNA or rRNA. For example anthocyanin structural genes encode enzymes (e.g. chalcone synthase) directly involved in the biosynthesis of anthocyaninε in plant cellε. A regulatory gene is a gene which encodes a regulator protein which regulates the transcription of one or more structural genes. For example the R, B, and Cl genes are regulatory genes that regulate transcription of anthocyanin structural genes. For the purpose of this invention the expresεion of a gene, εuch aε a chimeric gene, will mean that the promoter of the gene directε tranεcription of a DNA into a mRNA which iε biologically active i.e. which is either capable of interacting with another RNA, or which is capable of being translated into a biologically active polypeptide or protein. The phenotype is the external appearance of the expression (or lack of expression) of a genotype i.e. of a gene or set of genes (e.g. male-εterility, εeed color, preεence of protein or RNA in specific plant tiεsues etc. ) As used herein, a genetic locus is the position of a given gene in the nuclear genome, i.e. in a particular chromosome, of a plant. Two loci can be on different chromosomes and will segregate independently. Two loci can be located on the same chromosome and are then generally considered as being linked (unless sufficient recombination can occur between them) . An endogenous locus is a locus which is naturally present in a plant. A foreign locus iε a locuε which iε formed in the plant becauεe of the introduction, by means of genetic transformation, of a foreign DNA. in diploid plants, as in any other diploid organisms, two copies of a gene are present at any autosomal locus. Any gene can be present in the nuclear genome in several variant states designated as alleles. If two identical alleles are present at a locus that locus is designated as being homozygous, if different alleles are present, the locuε is deεignated as being heterozygouε. The allelic composition of a locus, or a set of loci, is the genotype. Any allele at a locuε is generally represented by a separate symbol (e.g. M and m, S and -, representing the absence of the gene) . A foreign locus is generally characterized by the preεence and/or abεence of a foreign DNA. A heterozygouε genotype in which one allele corresponds to the absence of the foreign DNA is also designated as hemizygous (e.g. Rf/-) • A dominant allele iε generally represented by a capital letter and is uεually aεsociated with the presence of a biologically active gene product (e.g. a protein) and an observable phenotypic effect (e.g. R indicates the production of an active regulator protein and under appropriate conditionε anthocyanin production in a given tiεsue while r indicates that no active regulator protein is produced possibly leading to absence of anthocyanin production) . A plant can be genetically characterized by identification of the allelic state of at least one genetic locus. The genotype of any given locuε can be designated by the symbolε for the two alleleε that are present at the locus (e.g. M/m or m/m or S/-) . The genotype of two unlinked loci can be represented as a εequence of the genotype of each locuε (e.g. S/S,Rf/-) The nuclear male-sterility genotype as used in thiε invention refers to the genotype of at least one locus, preferably only one locus, in the nuclear genome of a plant (the "male-sterility locus") the allelic composition of which may result in male sterility in the plant. A male-εterility locuε may be endogenouε to the plant, but it iε generally preferred that it iε foreign to the plant. Foreign male-sterility loci are those in which the allele responεible for male sterility iε a foreign DNA sequence S (the "male-sterility gene") which when expressed in cells of the plant make the plant male- sterile without otherwise substantially affecting the growth and development of the plant. Such male-sterility gene preferably comprises at least: si) a male-sterility DNA encoding a sterility RNA, protein or polypeptide which, when produced or overproduced in a stamen cell of the plant, significantly disturbs the metabolism, functioning and/or development of the stamen cell, and, s2) a εterility promoter capable of directing expression of the male-sterility DNA selectively in stamen cells (e.g. anther cells or tapetum cells) of the plant; the male-sterility DNA being in the same transcriptional unit as, and under the control of, the sterility promoter. The male-sterility locus preferably also comprises in the same genetic locuε at least one first marker gene T which comprises at least: tl) a firεt marker DNA encoding a firεt marker RNA, protein or polypeptide which, when preεent at leaεt in a specific tissue or specific cells of the plant, renders the plant easily separable from other plants which do not contain the first marker RNA, protein or polypeptide encoded by the firεt marker DNA at least in the εpecific tiεsue or specific cells, and, t2) a firεt marker promoter capable of directing expression of the firεt marker DNA at least in the specific tissue or specific cells: the first marker DNA being in the same transcriptional unit as, and under the control of, the firεt marker promoter. Such male-εterility gene iε always a dominant allele at such a foreign male-sterility locus. The recessive allele corresponds to the absence of the male-sterility gene in the nuclear genome of the plant. Male-sterility DNAs and εterility promoters that can be used in the male-sterility genes in the first parent line of this invention have been described before (EP 0,344,029 and EP 0,412,911). For the purpose of this invention the expression of the male-sterility gene in a plant cell should be able to be inhibited or repressed for instance by means of expression of a suitable fertility-restorer gene in the same plant cell. In this regard a particular useful male-sterility DNA codes for barnase (Hartley, J.Mol. Biol. 1988 202:913) . The sterility promoter can be any promoter but it should at least be active in stamen cells, particularly tapetum cells. Particularly useful sterility promoters are promoters that are selectively active in stamen cells, such aε the tapetum-εpecific promoterε of the TA29 gene of Nicotiana tabacum (EP 0,344,029) which can be uεed in tobacco, oilseed rape, lettuce, cichory, corn, rice, wheat and other plant species; the PT72, the PT42 and PEl promoters from rice which can be used in rice, corn, wheat, and other plant species (WO 92/13956) ; the PCA55 promoter from corn which can be used in corn, rice, wheat and other plant species (WO 92/13957) ; and the A9 promoter of a tapetum- specific gene of Arabidopsis thaliana (Wyatt et al., 1992, Plant Mol. Biol. 19:611- 922) . However, the sterility promoter may also direct expresεion of the sterility DNA in cells outside the stamen; particularly if the effect of expression of the male-sterility DNA iε such that it will specifically disturb the metabolism, functioning and/or development of stamen cells so that no viable pollen is produced. One example of such a male-sterility DNA is the DNA coding for an antisense RNA which is complementary to the mRNA of the chalcone synthase gene (van der Meer et al (1992) The Plant Cell 4:253-262). In thiε reεpect a uεeful promoter iε the 35S promoter (εee EP 0,344,029), particularly a 35S promoter that is modified to have enhanced activity in tapetum cells as described by van der Meer et al (1992) The Plant Cell 4:253-262 (the "35S- tap promoter") . A preferred endogenouε male-εterility locuε is one in which a recessive allele (hereinafter designated as m) in homozygous condition (m/m) resultε in male εterility. At εuch loci male fertility is encoded by a corresponding dominant allele (M) . In many plant species such endogenous male- sterility loci are known (see Kaul, 1988, supra (in corn see also recent issues of Maize Genetics Cooperation Newsletter, published by Department of Agronomy and U.S. Department of Agriculture, University Of Missouri, Columbia, Missouri, U.S.A.). The DNA sequences in the nuclear genome of the plant corresponding to m and M alleles can be identified by gene tagging i.e. by insertional mutagenesis using transposons, or by meanε of T-DNA integration (see e.g. Wienand and Saedler, 1987, In 'Plant DNA Infectiouε Agentε 1 , Ed. by T.H.Hohn and J.Schell, Springer Verlag Wien New York, p. 205; Shepherd, 1988, In 'Plant Molecular Biology: a Practical Approach', IRL Press, p. 187; Teeri et al. , 1986, EMBO J. 5:1755). It will be evident that in the first and second parent plant of this invention S/S can be replaced by m/m without affecting the outcome of the proceεε. Indeed, one feature of the process of this invention iε that the male-εterility locus is homozygouε thuε allowing the use of 'recesεive' male-εterility alleles. Fertility-restorer DNAs that can be used in the fertility restorer gene in the second parent line of this invention have been described before (EP 0,412,911). In this regard, fertility-restorer genes in which the fertility-restorer DNA encodes barstar (Hartley, J.Mol. Biol. 1988 202:913) are particularly useful to inhibit the expreεεion of a male-εterility DNA that encodes barnase. In this regard it is believed that a fertility- restorer DNA that codes for a mutant of the barstar protein, i.e. one in which the Cysteine reεidue at poεition 40 in the protein is replaced by serine (Hartley, 1989, TIBS 14:450), functions better in restoring the fertility in the restored plants of some specieε. In principle any promoter can be uεed as a restorer promoter in the fertility restorer gene in the second parent line of this invention. The only prerequisite is that such second parent plant, which contains both the color gene and the fertility-restorer gene, should be phenotypically normal and male-fertile. This requires that the restorer promoter in the fertility-restorer gene εhould be at least active in those cells of a plant of the same εpecies in which the sterility promoter of the corresponding male-sterility gene can direct expression of the male-sterility DNA. In this regard it will be preferred that the sterility promoter and the restorer promoter are the εame; they can for example be both stamen-specific promoters (e.g. the TA29 promoter or the CA55 promoter) or they can be both constitutive promoters (such as the 35S or 35S-tap promoter) . However, the sterility promoter may be active only in stamen cells while the reεtorer promoter is alεo active in other cellε. For instance, the sterility promoter can be a stamen-specific (such as the TA29 or CA55 promoter) while the restorer promoter is the 35S-tap promoter. When the male sterility to be reεtored iε due to the male- sterility genotype at an endogenouε male-sterility locuε being homozygouε for a recessive allele , it is preferred that the fertility-restorer gene is the dominant allele of that male- sterility locus, preferably under control of its own promoter. The DNA corresponding to such a dominant allele, including itε natural promoter can be iεolated from the nuclear genome of the plant by means of gene tagging aε described above. The nature of the color gene that iε used in the color- linked restorer gene in the second parent plant of this invention dependε upon the genotype of the untranεformed plantε of the same line. Preferably, only cereal plants with a genotype that does not condition externally visible anthocyanin production in seeds, particularly in the aleurone can be used to produce the εecond parent plantε. Theεe plants usually have a genotype in which no functional copy of a suitable regulatory gene such as the R or B gene, and/or the Cl gene, is present. In corn, for instance, all of the currently used inbred lines in the U.S.A. are r-r (pink anthers, leaf tips, plant base) or r-g (green) and most of these are cl and pi; at the B- locus the B-peru allele is very rare (Coe et al, 1988, In 'Corn and Corn Improvement', 3rd edition, G.F.Sprague and J.W. Dudley, eds. America Science of Agronomy, Inc. Publishers, Madison, Wisconsin, U.S.A.). The result is that no anthocyanins are produced in the aleurone of theεe lineε and that the kernelε are yellow. Thiε requireε that when these lines are transformed with a color-linked restorer gene, the color gene should consist of a functional R or B gene which conditions anthocyanin production in aleurone, and usually also a functional Cl gene capable of conditioning anthocyanin production in aleurone. A useful R or B gene is the B-peru gene, but of course also other R genes could be used such as the R(S) gene (Perrot and Cone, 1989, Nucl. Acids Res. 17:8003). In this regard a gene derived from genomic clones of the B-peru gene (Chandler et al, 1989, The Plant Cell 1:1175- 1183) is believed to be particularly useful. However the length of this genomic DNA (11 kbp) renders its practical manipulation and use for transformation by direct gene transfer, difficult, certainly in combination with other genes such as the restorer gene and the Cl gene. In one inventive aspect of this invention it was found that the B-peru gene could be considerably shortened while still retaining, under appropriate conditions, its capability of conditioning anthocyanin production in the aleurone of seeds of cereal plants such aε corn. A preferred εhortened B-peru gene iε that of Example 2.2 and which is contained in plasmid pCOL13 (deposited under accession number LMBP 3041) . A useful Cl gene iε the genomic clone aε described by Paz-Ares et al, 1987, EMBO J. 6:3553-3558. However the length of this genomic DNA (4 kbp) precludes its practical manipulation and use for tranεformation by direct gene tranεfer, certainly in combination with other geneε such as the restorer gene and the B-peru gene. Nevertheless other variants of the Cl gene can also be used. In this regard Scheffler et al, 1994, Mol.Gen.Genet. 242:40-48 have described the Cl-S allele which differs from the Cl allele of Paz-Ares et al, supra by a few nucleotides in the promoter region near the CAAT box and which is dominant to the wild-type allele (Cl) and shows enhanced pigmentation. The Cl-S gene can be easily used in this invention by appropriate changes in the Cl gene. For example the TGCAG at positions 935 to 939 in SEQ ID NO 1 (respectively at positions 884-888 in SEQ ID NO 5) can be easily changed to TTAGG yielding a Cl-S allele (reεpectively pC0L9S) . In one inventive aspect of this invention it was found that the Cl gene (and the Cl-S gene) could be considerably shortened while still retaining, under appropriate conditions, its capability of conditioning anthocyanin production in the aleurone of seeds of cereal plants such as corn. Preferred shortened Cl genes for instance are those of Example 2.1 such as comprised in pCOL9 which has the sequence of SEQ ID NO 5, particularly aε compriεed between the EcoRI and Sfil εiteε of pCOL9, and the corresponding εhortened Cl-S gene in pCOL9S. The transcribed region of the shortened B-peru and Cl geneε εtill contain some small introns which can also be deleted without affecting the function of the genes. It is also believed that the shortened B-peru and Cl genes can be somewhat further truncated at their 5* and 3* ends, without affecting their expression in aleurone. In particular it is believed that the sequence between positions 1 and 3272 of SEQ ID NO 6 can also be used as a suitable B-peru gene. It is also believed that this gene can still be truncated at its 3 ' end down to a position between nucleotides 2940 and 3000 of SEQ ID No. 6. Although the use of genomic sequences of the B-peru gene and the Cl gene, particularly the shortened B-peru and/or the εhortened Cl of Cl-S geneε, iε preferred, chimeric R, B, or Cl geneε can also be used. For instance a chimeric gene can be used which comprises the coding region (e.g. obtained from the cDNA) of any functional R or B gene (i.e. which conditions anthocyanin production anywhere in the plant) which is operably linked to the promoter region of a R or B gene which conditions anthocyanin production in the aleurone (such as R(S) or B- peru) . Since the presence of anthocyanin does not negatively affect growth, development and functioning of plant cells, a constitutive promoter (e.g. the 35S promoter) , or a promoter which directs expression at least in the aleurone can also be used in such a chimeric gene. In this regard the promoter of the Cl gene can also be used to direct expresεion of a DNA compriεing the coding region of εuitable R or B gene, particularly the B-peru gene. Similarly the coding region (e.g. obtained from cDNA) of the Cl gene can be operably linked to the promoter of a gene that directs expreεsion at least in the aleurone. In thiε regard, the promoter of the B-peru gene can alεo be used to direct expresεion of a DNA compriεing the coding region of a suitable Cl gene such as that of the Cl gene of SEQ ID No. 1 or of the Cl-S gene. In another inventive aspect of the invention it was found that the the promoters comprised in DNAs characterized by the εequenceε between poεitions 1 to 1077, particularly between positions 447 and 1077, quite particularly between positions 447 and 1061 of SEQ ID NO 1, between positions 396 and 1026 of SEQ ID NO 5, and between positions 1 to 575, particularly between position 1 to 188 of SEQ ID NO 6 are promoters that predominantly, if not selectively, direct expression of any DNA, preferably a heterologous DNA in the aleurone layer of the εeedε of plantε. Of course in those lines in which a functional Cl gene is already present in the genome the color gene can consiεt only of a εuitable functional R or B gene (or a chimeric alternative) . Alternatively if a line contains already a functional R or B gene which can condition anthocyanin production in the aleurone, but no functional Cl gene, only a functional Cl gene is required aε a color gene. It iε believed that the color geneε of thiε invention are eεpecially useful in cereal plants, and that they are of particular use in corn and wheat, and certainly in corn. For the purpoεeε of this invention it is preferred that, in the εecond parent plantε the "Rf" locuε and the male- εterility (e.g. "S") locuε are not linked and εegregate εeparately. In the εecond parent plant, the fertility reεtorer gene, the B-peru gene and the Cl gene are preferably cloεely linked. Thiε can of course be achieved by introducing these genes in the nuclear genome of the plants aε a εingle tranεforming foreign DNA (the Rf DNA) thus forming a foreign Rf locus. Alternatively, the fertility restorer gene and the color gene can be separately introduced by cotransformation which usually results in single locus insertions in the plant genome. The color-linked restorer gene Rf aε used in the second parent plant preferably also comprises at least c) a second marker gene which comprises at least: cl) a second marker DNA encoding a second marker RNA, protein or polypeptide which, when present at least in a specific tissue or specific cells of the plant, renderε the plant eaεily εeparable from other plantε which do not contain the second marker RNA, protein or polypeptide encoded by the εecond marker DNA at least in the specific tisεue or specific cellε, and, c2) a second marker promoter capable of directing expresεion of the second marker DNA at least in the specific tiεεue or specific cellε: the εecond marker DNA being in the εame transcriptional unit as, and under the control of, the εecond marker promoter. First and second marker DNAs and firεt and εecond marker promoterε that can be used in the firεt and εecond marker geneε of this invention are also well known (EP 0,344,029; EP 0,412,911). In thiε regard it is preferred that the first and second marker DNA are different, although the first and second marker promoter may be the same. Foreign DNA such as the fertility-restorer gene, the foreign male-sterility gene, the B-peru and the Cl genes, or the first or second marker gene preferably also are provided with suitable 3 ' transcription regulation sequences and polyadenylation signalε, downεtream (i.e. 3") from their coding εequence i.e. reεpectively the fertility-reεtorer DNA, the male-εterility DNA, the coding region of a color gene (such as a B-peru gene and/or a Cl gene) or the first or second marker DNA. In this regard either foreign or endogenous transcription 3• end formation and polyadenylation signals suitable for obtaining expresεion of the chimeric gene can be uεed. For example, the foreign 3' untranslated ends of genes, such as gene 7 (Velten and Schell (1985) Nucl. Acids Res. 13:6998), the octopine synthase gene (De Greve et al., 1982, J.Mol. Appl. Genet. 1:499; Gielen et al (1983) EMBO J. 3:835; Ingelbrecht et al., 1989, The Plant Cell 1:671) and the nopaline synthase gene of the T-DNA region of Agrobacterium tu efaciens Ti-plasmid (De Picker et al., 1982, J.Mol. Appl. Genet. 1:561), or the chalcon synthase gene (Sommer and Saedler, 1986, Mol.Gen.Genet. 202:429- 434) , or the CaMV 19S/35S transcription unit (Mogen et al., 1990, The Plant Cell 2:1261-1272) can be used. However, it is preferred that the color genes in thiε invention carry their endogenous transcription 3 ' end formation and polyadenylation εignalε. The fertility-reεtorer gene, the male-εterility gene, the color gene or the firεt or second marker gene in accordance with the present invention are generally foreign DNAs, preferably foreign chimeric DNA. In this regard "foreign" and "chimeric" with regard to such DNAs have the same meanings aε deεcribed in EP 0,344,029 and EP 0,412,911. The cell of a plant, particularly a plant capable of being infected with Agrobacterium such as most dicotyledonous plantε (e.g. Brassica napus) and some monocotyledonous plantε, can be transformed using a vector that iε a disarmed Ti-plasmid containing the male- sterility gene, the color linked restorer gene or both and carried by Agrobacterium. This tranεformation can be carried out uεing the procedures described, for example, in EP 0,116,718 and EP 0,270,822. Preferred Ti-plasmid vectors contain the foreign DNA between the border εequences, or at leaεt located to the left of the right border εequence, of the T-DNA of the Ti-plasmid. Of course, other types of vectors can be used to tranεform the plant cell, using procedures such as direct gene transfer (as described, for example, in EP 0,233,247), pollen mediated transformation (as described, for example, in EP 0,270,356, PCT patent publication "WO" 85/01856, and US patent 4,684,611), plant RNA virus- mediated tranεformation (aε described, for example, in EP 0,067,553 and US patent 4,407,956) and liposome-mediated tranεformation (as described, for example, in US patent 4,536,475). Cells of monocotyledonous plantε such as the major cerealε including corn, rice, wheat, barley, and rye, can be tranεformed (e.g. by electroporation) using wounded or enzyme-degraded intact tiεεueε capable of forming compact embryogenic calluε (εuch aε immature e bryoε in corn) , or the embryogenic callus (such as type I callus in corn) obtained thereof, as described in WO 92/09696. In case the plant to be tranεformed iε corn, other recently developed methodε can alεo be used such as, for example, the method described for certain lines of corn by Fromm et al., 1990, Bio/Technology 8:833; Gordon-Kamm et al. , 1990, Bio/Technology 2:603 and Gould et al., 1991, Plant Physiol. 95:426. In caεe the plant to be transformed iε rice, recently developed methods can also be used such aε, for example, the method deεcribed for certain lineε of rice by Shimamoto et al., 1989, Nature 338:274; Datta et al., 1990, Bio/Technology 8:736; and Hayaεhimoto et al., 1990, Plant Phyεiol. 93:857. The transformed cell can be regenerated into a mature plant and the resulting transformed plant can be used in a conventional breeding scheme to produce more transformed plants with the same characteristics or to introduce the male- sterility gene, the color-linked reεtorer gene (or both) , in other varieties of the same related plant species. Seeds obtained from the transformed plants contain the chimeric gene(s) of this invention as a stable genomic insert. Thus the male- εterility gene, or the color-linked restorer gene of this invention when introduced into a particular line of a plant species can always be introduced into any other line by backcroεεing. The firεt parent plant of thiε invention containε the male-εterility gene aε a εtable inεert in itε nuclear genome (i.e. it iε a male-εterile plant) . For the purpoεeε of thiε invention it is preferred that the first parent plant contains the male-εterility gene in homozygous condition so that it tranεmits the gene to all of its progeny. The εecond parent plant of thiε invention contains the male-sterility gene and the color-linked restorer gene aε stable insertε in itε nuclear genome (i.e. it iε a reεtored plant) . It is preferred that the male- sterility gene be in homozygous condition so that the εecond parent plant transmits the gene to all of its progeny and that the color-linked restorer gene be in heterozygous condition so that the second parent plant transmitε the gene to only half of its progeny. It iε preferred that the firεt and εecond parent plantε are produced from the same untranεformed line of a plant species, particularly from the same inbred line of that species. The first and second parent plants of this invention have the particular advantage that εeedε of such plantε can be maintained indefinitely, and can be amplified to any deεired amount (e.g. by continuouε crossing of the two plant lines) . The color genes of this invention can be used as marker gene in any situation in which it is worthwhile to detect the presence of a foreign DNA (i.e. a tranεgene) in seedε of a tranεformed plant in order to iεolate seeds which possess the foreign DNA. In this regard virtually any foreign DNA, particularly a chimeric gene can be linked to the color gene. Exampleε of such foreign DNAs are geneε coding for inεecticidal (e.g. from Bacilluε thuringienεiε) , fungicidal or nematocidal proteinε. Similarly the color- gene can be linked to a foreign DNA which iε the male- εterility gene as uεed in thiε invention. However, the color genes are believed to be of particular uεe in the proceεε of thiε invention in which they are preεent in a foreign DNA which compriεeε a fertility reεtorer gene (εuch aε the barεtar gene of Bacilluε amyloliguefaciens. under control of a stamen- specific promoter (such as PTA29) . In appropriate conditionε the uεe of the color genes allows the eaεy εeparation of harveεted seeds that will grow into male- sterile plants, and harvested seeds that will grow into male-fertile plants. In this regard the seedε are preferably harveεted from male-εterile plantε (the first parent plants) that are homozygouε at a male-εterility locuε (εuch aε a locuε compriεing the barnaεe gene under control of PTA29) and which have been pollinated by reεtorer plantε (the εecond parent plantε of this invention) which contain in their genome two unlinked gene loci one of which compriseε the εame male- sterility locuε which is homozygous for the same male-sterility gene while the other is a foreign locus which comprises an appropriate fertility restorer gene (i.e. whose expression will counteract the expresεion of the male- εterility gene) and alεo the color gene of this invention, particularly an R or B gene that is expressed in the aleurone and/or a Cl gene, preferably the B-peru and Cl gene (e.g. as deεcribed in the exampleε) . First and second parent plants can be esεentially produced aε deεcribed in the exampleε and aε summarized in Figure 1. In step 8 of Figure 1 it iε demonεtrated that the croεsing of the first and second parent plantε of thiε invention will give riεe in the progeny to about 50% new first parent (i.e. male- sterile) plantε and about 50% new εecond parent (i.e. male- fertile) plants and that these two types of plants can already be separated at the seed stage on the basis of color. Red kernels will grow into male-fertile plants while yellow kernels will grow into male-sterile plantε. Thus a line of male-sterile first parent plantε of thiε invention can be easily maintained by continued crossing with the second parent plants of this invention with, in each generation, harvesting the εeedε from the male-εterile plantε and separation of the yellow and red kernels. Of courεe in this way any desired amount of seed for foundation seed production of a particular line, such as an inbred line, can also be easily obtained. The red and yellow seedε harveεted from a cereal plant (e.g. the first parent plant of thiε invention) can be separated manually. However, such separation can also be effected mechanically. A color sorting machine for corn kernel and other granular products iε for instance available from Xeltron U.S. (Redmond, Washington, U.S.A.) Unlesε otherwise indicated all experimental procedures for manipulating recombinant DNA were carried out by the standardized procedures described in Sambrook et al., 1989, "Molecular Cloning: a Laboratory Manual", Cold Spring Harbor Laboratory, and Ausubel et al, 1994, "Current Protocols in Molecular Biology", John Wiley & Sons. The polymerase chain reactionε ("PCR") were used to clone and/or amplify DNA fragments. PCR with overlap extension was uεed in order to conεtruct chimeric geneε (Horton et al, 1989, Gene 77:61-68; Ho et al, 1989, Gene 77:51-59) . All PCR reactionε were performed under conventional conditionε uεing the Vent τ M polymeraεe (Cat. No. 254L - Biolabs New England, Beverley, MA 01915, U.S.A.) isolated from Thermococcus litoralis (Neuner et al., 1990, Arch.Microbiol. 153:205-207) . Oligonucleotides were designed according to known ruleε as outlined for example by Kramer and Fritz (1968, Methodε in Enzymology 154:350) , and εyntheεized by the phoεphoramidite method (Beaucage and Carutherε, 1981, Tetrahedron Letters 22:1859) on an applied Biosystemε 380A DNA synthesizer (Applied Biosystems B.V. , Maarssen, Netherlands) . In the following examples, reference will be made to the following sequence listing and figures: Sequence Listing SEQ ID NO 1 : sequence of Cl gene SEQ ID NO 2 : plasmid pTS256 SEQ ID NO 3 : EcoRI-Hindlll region of pTS200 comprising the chimeric gene PCA55-barstar-3 'nos (the omitted region of pTS200 is derived from pUC19. SEQ ID NO 4 : oligonucleotide 1 SEQ ID NO 5 : pCOL9 containing the shortened Cl gene as a EcoRI-Sfil fragment SEQ ID NO 6 : presumed sequence of the EcoRI- Hindlll region of pCOL13 containing the shortened B-peru gene (the rest of the plasmid is pUC19) . The stretch of N nucleotides corresponds to a region of approximate length which iε derived from the genomic clone of the B-peru gene but for 95/34634 44 which the sequence needs to be confirmed. SEQ ID NO 7 actual sequence of the EcoRl- Hindlll region of pC0L13 containing the shortened B-peru gene (the rest of the plasmid is pUC19) . Figures Figure 1 : Breeding εcheme to obtain the first and second parent plants of this invention Figure 2 : Schematic structure of pCOL25, pCOL26, pCOL27, pCOL28, pCOLlOO and pDEHO. Examples Example 1 : Construction of plasmids containing the male- sterility gene comprising the TA29 promoter and the barnase coding region Plasmids useful for transformation of corn plants and carrying a male-sterility gene and a selectable marker gene have been described in WO 92/09696 and WO 92/00275. Plasmid pVE107 contains the following chimeric genes: l) PTA29-barnase-3'nos, i.e. a DNA coding for barnase of Bacillus amyloliguefaciens (barnase) operably linked to the stamen-specific promoter of the TA29 gene of Nicotiana tabacum (PTA29) and the 3' regulatory sequence containing the polyadenylation signal of the nopaline synthase gene of Agrobacterium tumefaciens (3'nos), and 2) P35S-neo-3'ocs. i.e. the coding region of the gene of Tn5 of E.coli coding for neomycin phosphotransferase (neo) operably linked to the 35S promoter of Cauliflower Mosaic Virus (P35S) and the 3' regulatory sequence containing the polyadenylation signal of the octopine synthase gene of Agrobacterium tumefaciens (3Ocs). Plasmid pVE108 contains the following chimeric genes: 1) PTA29-barnase-3'nos, and 2) P35S-bar-3'nos, i.e. the gene of Streptomyces hygroscopicus (EP 242236) coding for phosphinothricin acetyl transferase (bar) operably linked to the P35S and 3'nos. PTA29-barnase-3'nos is an example of a foreign chimeric male-sterility gene (S) used in this invention. Example 2 : Construction of a plasmid containing the color-linked restorer gene 2.1. Obtaining a shortened functional Cl gene The Cl gene of maize was cloned from transposable- induced mutants and its sequence was reported (Paz-Ares, 1987, EMBO J. 6:3553-3558). This sequence is reproduced in SEQ ID NO. 1. Plasmid p36 (alternatively designated as pClLC5kb and further designated as plasmid pXX036) comprising a Cl genomic clone was obtained from Dr. H. Saedler and Dr. U. Wienand of the Max- Planck Institut fur Zύchtungsforschung, Kόln, Germany. pXX036 was digested with SnabI and Hindlll, filled-in with Klenow, and selfligated, yielding plasmid pCOL9. pCOL9 corresponds to pUC19 (Yanisch-Perron et al, 1985, Gene 33:103-119) which containε, between its EcoRI and modified Hindlll sites, the 2189 bp EcoRI-SnabI fragment (corresponding to the εequence between positions 448 and 2637 of SEQ ID NO 1) of pXX036. pXX036 was also digested with Sfil and Hindlll and treated with Klenow to make blunt ends. After ligation the plasmid in which the DNA downstream from the Sfil site was deleted was designated as pCOL12. The sequence TGCAG in pCOL9, corresponding to the sequence at positions 884 to 888 in SEQ ID NO 5, is changed to TTAGG, yielding pCOL9S which instead of a shortened Cl gene contains a shortened overexpressing Cl- S gene (Schleffer et al, 1994, Mol.Gen.Genet. 242:40-48). A similar change is introduced in pCOL12, yielding PCOL12S. 2.2. Obtaining a shortened functional B-peru gene Plasmid pBP2 (further designated as pXX004) is plasmid pTZ18U (Mead et al., 1986, Protein Engineering 1:67; U.S.Biochemical Corp.) containing the genomic clone of the B- peru gene. Plasmid p35SBPcDNA (further designated as pXX002) is plasmid pMF6 (Goff et al, 1990, EMBO J. 9:2517-2522) containing the cDNA corresponding to the B-peru gene. Both plasmids were obtained from Dr. V. Chandler of the University of Oregon, Oregon, U.S.A. A 2660 bp sequence of the genomic clone around the translation initiation codon was reported (EMBL/Genbank/DDBJ databases; locus name ZMBPERUA, Accession number X70791; see also Radicella et al, 1992, Genes & Development 6:2152-2164). The sequence of the B- peru cDNA was also reported (Radicella et al, 1991, Plant Mol. Biol. 17:127- 130). Substantial amounts of 5' and 3' flanking sequences were deleted from pXX004, and the MluI-MunI fragment in the coding region of the genomic clone was replaced by the 1615 bp Mlul- Muni fragment of the cDNA clone. The resulting plasmid waε designated as pC0L13 which was deposited at the Belgian Coordinated Collection of Microorganisms - LMBP Collection, Laboratory Molecular Biology, University of Ghent, K.L. Ledeganckstraat 35, B- 9000 Ghent, Belgium and was given the Accession Number LMBP 3041. A shortened but functional B-peru gene is contained in pC0L13 as an EcoRI-Sall fragment with an approximate length of 4 kbp (see SEQ ID NO 6) . 2.3. Combining the Cl and B-peru genes The Cl gene in pCOL9 and the B-peru gene in pCOL13 were then combined aε followε. The 4 kbp EcoRI-Sall fragment of pC0L13 waε introduced between the EcoRI and Sail εiteε of the vector pBluescript II SK(-) (Stratagene) , yielding #7 B SK(-) . pC0L9 was digested with Sfil, treated with Klenow to fill in protruding ends, and further digested with EcoRI. The 1978 bp Sfil(Klenow)/EcoRI was then introduced between the EcoRI and Smal sites of #7 B SK(-) , yielding #7 B+C SK(-) . Finally the Xhol site in the Cl sequence was removed as follows. The 950 bp EcoRI-SacII fragment of #7 B SK(-) (EcoRI site correεponding to the EcoRI site at position 1506 in SEQ ID NO 1; the SacII site from the pBluescript linker) was introduced between the EcoRI and SacII sites of the Phagescript Vector (Stratagene) to yield pC0L21. Single strandε of pC0L21 were prepared and hybridized to the following synthetic oligonucleotide 1 (SEQ ID No. 4) : 5--CGT TTC TCG AAT CCG ACG AGG-3' resulting in a silent change (CTCGAG -> CTCGAA) and removal of the Xhol site. The 710 Aatll-SacII fragment of #7 B SK(-) was then exchanged for the corresponding Aatll-SacII fragment of the mutated pCOL21, yielding pCOL23. pCOL23 was then linearized with SacII, treated with Klenow, and ligated to Xhol linker sequence (Stratagene) , yielding pCOL24. Using the same procedure as described above, the shortened Cl-S gene of pC0L9S is combined with the shortened B-peru gene of pCOL23, yielding plasmid pC0L24S. 2.4. Construction of vectors comprising the Cl and B-peru genes as well aε male-sterilitv gene and a selectable marker gene pTS256 is derived from pUC19 and contains the following two chimeric genes :1) P35S-bar-3'nos. and 2) PTA29-barstar- 3'nos, i.e. a DNA coding for barstar of Bacillus amyloliquefaciens (barstar or bar*) operably linked to PTA29 and 3'nos. The complete sequence of pTS256 is given in SEQ ID NO 2. pTS200 is derived from pUC19 and contains the following two chimeric genes : 1) P35S-bar-3'nos. and 2) PCA55-barstar- 3'nos, i.e. barstar operably linked to the εtamen-εpecific promoter PCA55 of Zea mays and 3'nos. The complete sequence of pTS200 is given in SEQ ID NO 3. pTS256 was modified by the inclusion of NotI linkers (Stratagene) in both the unique Sspl and Smal sites, yielding pTS256NN. The shorter BspEI-SacII fragment of PTS256NN was then replaced by the shorter BspEI-SacII fragment of pTS200, yielding pTS256+200. PTS256NN contains P35S-bar3'-nos and pTA29-barstar3'nos on a NotI cassette. pTS256NN+200 contains P35S-bar3'-nos and pCA55- barstar3'nos on a NotI cassette. The NotI cassette of pTS256NN was introduced in the NotI site of pCOL24, yielding pCOL25 and pCOL26 which CONFIRMATION ' COPY differ with respect to the orientation of the P35S-bar3'- noε gene with respect to the shortened Cl gene (Figure 2). The NotI cassette of pTS256NN+200 was introduced in the NotI site of pCOL24, yielding pCOL27 and pCOL28 which differ with respect to the orientation of the P35S-bar3'- nos gene with respect to the shortened Cl gene (Figure 2). Plasmids pCOL25, pCOL26, pCOL27 or pCOL28 contain a color- linked restorer gene Rf and a selectable marker gene (P35S-bar- 3'nos). Rf comprises the shortened Cl and B-peru genes and a chimeric barstar gene (either PTA29- barεtar-3'nos or PCA55- barstar-3'nos) . Plaεmidε pCOL25S, pCOL26S, pC0L27S or pCOL28S, containing the shortened Cl-S gene instead of the shortened Cl gene, are obtained in a similar way using pCOL24S instead of pC0L24. 2.5. Conεtruction of vectorε comprising the Cl and B-peru genes as well aε male-εterilitv gene Plasmid pTS59 can be obtained from plasmid pTS256 (of SEQ ID NO 2) by replacing the fragment extending from positions 1 to 1470 (compriεing the chimeric gene P35S- bar-3'nos) with the sequence TATGATA. Then NotI linkers (Stratagene) were introduced in the EcoRV and Smal sites of pTS59; yielding pTS59NN. Finally the NotI fragment comprising the chimeric gene PTA29-barstar-3 'nos was introduced in the NotI site of #7 B+C SK(-) , yielding pCOLlOO (the general structure of pCOLlOO and pDEllO is also presented in Figure 2) . 2.6. Expression of shortened Cl and B-peru in aleurone in corn seeds Dry seeds were incubated overnight in water at room temperature and were then peeled and sliced in half. Four to six half kernels were placed with the cut side on wet filter paper and were bombarded with tungsten particles (diameter 0.7 μm) which were coated with DNA. Particle bombardment was essentially carried out using the particle gun and procedures as described by Zumbrunn et al, 1989, Technique, 1:204-216. The tissue was placed at 10 cm from the stopping plate while a 100 μm mesh was placed at 5 cm from the stopping plate. - DNA of the following plasmids was used : - pXX002 : B-peru cDNA under control of the 35S promoter - pXX201 : Cl cDNA under control of the 35S promoter - pC0L13 : shortened B-peru gene as described in Example 2.2 - pC0L12 : shortened Cl gene as described in Example 2.1 - pCOLlOO : shortened B-peru and shortened Cl and PTA29- barεtar-3'noε aε deεcribed in Example 2.5. 95/34634 52 After bombardment the tisεue was incubated for 2 days on wet filter paper at 27°C and was then checked for the presence of red spotε indicating anthocyanin production. Table 1 Note : + indicates that anthocyanin production was observed in at leaεt one experiment; - indicates that no anthocyanin production was obεerved, nt = not tested. The results for three public lines (H99, Pa91, B73) and 9 different, commercially important, proprietary inbred lines from various sources are shown in Table 1. The line c-ruq is a tester line which is homozygous for a Cl allele that is inactivated by insertion of a receptor for the regulator Uq (Cormack et al., 1988, Crop Sci. 28:941-944) . CONFIR ATION ' COPY All lines which were r and cl produced anthocyanin in the aleurone after introduction with both a functional B- peru and Cl gene. Lines which were R and cl produced anthocyanin upon introduction of a functional Cl gene. Lines which were r and Cl produced anthocyanin upon introduction of a functional B-peru gene. This proves that the B-peru and Cl gene are sufficient for anthocyanin production in most corn lines. From the data in Table 1 it is also evident that even the shortened B- peru and Cl genes are still functional and are capable of producing anthocyanin in aleurone of corn lines with suitable genotypes. Example 3 : Production of first parent corn plants by transformation of corn with the plasmids of example 1. Corn plants of line H99, transformed with a male- sterility gene comprising a DNA encoding barnase of Bacillus amyloliquefaciens under control of the promoter of the TA29 gene of Nicotiana tabacum have been described in WO 92/09696. The transformed plants were shown to be male-sterile. Example 4 : Production of second parent corn plants by transformation of corn with the plasmids of examples 2. Corn inbred lines H99 and Pa91 are transformed using the procedures as described in WO 92/09696 but using plasmids pCOL25, pCOL26, pC0L27 or pCOL28 of Example 2. Regenerated plantε are selected that are male fertile and in which the shortened Cl, the shortened B-peru gene, the P35S-bar-3'nos gene, and the PTA29- barstar-3'nos (or PCA55-barstar-3 'nos) are expressed. Alternatively the male-sterile plants of Example 3 (already containing the S gene) can be transformed with plasmids pCOL25, pCOL26, pCOL27 or pCOL28 of Example 2 on the condition that the S and Rf genes are linked to different selectable marker genes. Similarly, transformed corn plants are obtained using plasmidε pC0L25S, pCOL26S, pCOL27S or pCOL28S of Example 2. In an alternative set of experiments the second parent plants of this invention were obtained by transforming corn plants of line H99, Pa91, and (Pa91xH99)x H99 with two separate plasmids one of which contained the color linked restorer gene (pCOLlOO) , while the other contains an appropriate selectable marker gene such as a chimeric bar gene (pDEllO) (alternatively a chimeric neo gene may also be used) . pDEllO was described in WO 92/09696 and the construction of pCOLlOO was described in Example 2.5. In yet another set of experiments the second parent plants of this invention are obtained by transforming corn plants with a purified fragment of the plasmids of example 2.4. Such purified fragment is obtained by digestion of the plasmids of example 2.4 with Xhol and subsequent purification using conventional procedures such as gel filtration. Untransformed corn plants of lines H99 or Pa91 are detasseled and pollinated with pollen of the plants transformed with the Rf DNA. It is observed that the f gene segregates in a Mendelian way and that the seed that is harvested from theεe plants is colored and non-colored (yellow) in a 1:1 ratio. The red color of the seeds is correlated with the presence of the Rf gene. Example 5 : The production of the first and second parent plants of this invention. First parent plants and second parent plants (i.e. maintainer plants) according to the invention are produced along the lines set out in Figure 1. The male-sterile plants of step 1 are those produced in Example 1. The corn plants transformed with the color-linked restorer gene of step 2 are those produced in Example 4. A plant of Example 1 and a plant of Example 4 are crossed (Step 3) and the progeny plants with the genotype S/-, Rf/- are selected (Step 4) , e.g. by demonstrating the presence of both the S and Rf genes in the nuclear genome (e.g. by means of PCR) . The plantε selected in Step 4 are then crossed with the male-sterile plants with genotype S/- (Step 5) . The colored seedε (i.e. thoεe containing the Rf gene) are selected, grown into plants, and examined for the presence of both the S and Rf genes (e.g. by PCR) . The plants containing both the S and Rf geneε are selfed and the seeds of each plant are examined on seed color (red or yellow) . From the progeny of the selfings the non- colored seeds are grown into plants (step 6) . The progeny of the selfings in which all noncolored seeds grow into male-sterile plants are retained (Step 6) . These male- sterile plants are all homozygous for the S gene and are crossed with their fertile siblings (of genotype S/S,Rf/Rf or S/S,Rf/-) (Step 7) . For some crossings the seeds harvested from the male-sterile plantε are 50% colored and 50% non-colored (step 7) . The colored seeds all grow into fertile corn plants of genotype S/S,Rf/- which are the maintainer plants, or the second parent plants, of the present invention. The noncolored seeds all grow into male-sterile plants of the genotype S/S,-/- which are the first parent plants of this invention (Step 7). The first and second parent plants are crossed and the seeds harvested from the male-sterile plants are separated on the basis of color (Step 8) . All colored seeds grow again in second parent plants and all noncolored seeds grow in first parent plants, thereby establishing an easy maintenance of a pure male-sterile line of corn. If the plant DNA that is flanking the S gene in the plants of Example 1 has been characterized, the progeny of the cross in Step 5 with genotypes S/S,-/- and S/S,R/- can be easily identified by means of PCR using probes corresponding to the flanking plant DNA. In this way Step 6 can be skipped because the plants of Step 5 which grow from colored seeds (genotype S/S,Rf/-) can be crossed directly to plants with genotype S/S,-/- (as in Step 7). All publications cited in thiε application are hereby incorporated by reference. Example 6 : Maintainer plants containing a color-linked restorer gene compriεing the B-Peru coding region under control of the promoter of the Cl-S gene. Using conventional techniques a chimeric gene is inserted between the EcoRI and Hindlll sites of the polylinker of plasmid pUC19. The chimeric gene comprises the following elements in sequence: i) the promoter region of the Cl-S gene, i.e. the DNA fragment with the sequence of SEQ ID No. 1 from nucleotide positions 447 up to 1076 but containing at nucleotide positions 935-939 the sequence TTAGG instead of TGCAG. ii) a single C nucleotide iii) the coding region and 3'untranslated region of the B-peru gene, i.e. the DNA fragment with the sequence of SEQ ID No. 7 from nucleotide positions 576 up to 4137. This plasmid (designated as pLH52) , together with plasmid pC0L9S of Example 2 (comprising a Cl-S gene) and pTS256 of SEQ ID No. 2 (comprising the following chimeric genes: P35S-bar-3'nos and PTA29-barstar-3'nos) . is used to transform corn essentially as described in Example 4. The transformed plantε are then uεed to obtain second parent plantε aε described in Example 5. Example 7: Maintainer plants containing a color-linked restorer gene comprising the B-Peru coding region under control of the 35S promoter. Using conventional techniques a chimeric gene is inserted between the EcoRI and Hindlll sites of the polylinker of plasmid pUC19. the chimeric gene comprises the following elements in sequence: i) The promoter region of the 35S promoter, i.e. the DNA fragment of pDEllO which essentially has the sequence as described in SEQ ID No. 4 of WO 92/09696 (which is incorporated herein by reference) from nucleotide positions 396 up to 1779 ii) the coding region and 3'untranslated region of the B-peru gene, i.e. the DNA fragment with the sequence of SEQ ID No. 7 from nucleotide positions 576 up to 4137. This plasmid (designated aε pP35S-Bp) , together with plasmid pC0L9S of Example 2 (comprising a Cl-S gene) and pTS256 of SEQ ID No. 2 (comprising the following chimeric genes: P35S-bar-3'nos and PTA-29-barstar-3'nos) , is used to transform corn essentially as described in Example 4. The transformed plants are then used to obtain second parent plants as described in Example 5. Alternatively plaεmid p35SBperu aε described in Goff et al, 1990, EMBO 9:2517-2522 is used instead of pP35SBp. Example 8: Maintainer plants containing a color-linked restorer gene comprising the maize P gene coding region under the control of the promoter of the Cl-S gene. Using conventional techniques a chimeric gene is inserted in the EcoRI site of the polylinker of plasmid pUC19. The chimeric gene comprises the following elements in sequence: i) the promoter region of the Cl-S gene, i.e. the DNA fragment with the sequence of SEQ ID No. 1 for nucleotide positionε 447 up to 1076 but containing at nucleotide positions 935-939 the sequence TTAGG instead of TGCAG ; ii) a εingle C nucleotide; iii) a DNA sequence comprising the coding region and 3'end untranslated region of the maize P gene as described by Grotewold et al in 1991, PNAS 88:4587-4591 (nucleotideε 320-1517) . The maize P gene is an anthocyanin regulatory gene which specifies red phlobaphene pigmentation, a flavonoid pigment involved in the biosynthetic pathway of anthocyanin. In fact, the protein encoded by the P gene activates, among others, the Al gene required for both anthocyanin and phlobaphene pigmentation. Two cDNA clones have been isolated and sequenced by Grotewold et al and are described in the publication referred to above. It is the longer cDNA which is of particular interest for construction of this chimeric gene. However, alternatively, the coding region of the shorter transcript can also be used in this chimeric gene, as well aε the P gene leader sequence instead of the CI-S gene leader sequence. The P gene does not require a functional R or B gene to produce pigmentation. The visible pigment that is produced in the seeds of the maintainer plants is phlobaphene, a flavonoid pigment (like anthocyanin) directly involved in anthocyanin biosyntheεis. iv) a DNA fragment containing the polyadenylation signal of the nopaline synthase gene of Agrobacterium tumefaciens, i.e. the DNA fragment with the sequence of SEQ ID. No. 2 from nucleotide position 1600 up to nucleotide position 2909. The resulting plasmid (designated as pPCSl-P) , together with pTS256 of SEQ ID No. 2 is used to transform corn essentially as described in example 4. The transformed plants are then used to obtain second parent plants as described in example 5. Example 9: Maintainer plants containing a color-linked restorer gene comprising the B-peru coding region under the control of the B-peru promoter. Using conventional techniques a chimeric gene is inserted between the EcoRI and the Hindlll sites of the polylinker of plasmid pUC19. The chimeric gene comprises the following elements in sequence: i) the promoter of the B-peru gene, i.e. a 1952 bp DNA sequence as disclosed in the EMBL databank under accession number X70791; ii) the coding region and 3 'untranslated region of the B-peru gene, i.e. the DNA fragment with the sequence of SEQ ID No. 7 from nucleotide position 576 up to 4137. This plasmid (designated aspCOLll) , together with plasmid pCOL 9S of example 2 (comprising a Cl-S gene) and pTS256 of SEQ ID No. 2 (comprising the following chimeric genes: P35S-bar-3 "nos and PTA29-barεtar-3 *nos) is used to transform corn essentially as described in example 4. The transformed plants are then used to obtain second parent plants as described in example 5. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: PLANT GENETIC SYSTEMS N.V. (B) STREET: Jozef Plateaustraat 22 (C) CITY: Ghent (E) COUNTRY: Belgium (F) POSTAL CODE (ZIP) : 9000 (G) TELEPHONE: 32 9 235 84 11 (H) TELEFAX: 32 9 224 06 94 (I) TELEX: 11.361 Pgsgen (ii) TITLE OF INVENTION: Use of anthocyanin genes to maintain male-sterile plants (iii) NUMBER OF SEQUENCES: 7 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (vi) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/254,776 (B) FILING DATE: 06-JUN-1994 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4059 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: Cl gene of Zea mays (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:279..284 (D) OTHER INFORMATION: /label= Hpal (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 447..452 (D) OTHER INFORMATION: /label= EcoRI (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1735..1740 (D) OTHER INFORMATIO :/label= Aatll (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1505..1510 (D) OTHER INFORMATION:/label= EcoRI (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2081..2086 (D) OTHER INFORMATION:/label= Xhol (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2418..2430 (D) OTHER INFORMATION: /label= Sfil (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2669..2674 (D) OTHER INFORMATION:/label= SnaBI (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2634..2639 (D) OTHER INFORMATION: /label= SnaBI (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 3008..3013 (D) OTHER INFORMATION:/label= Hpal (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..1077 (D) OTHER INFORMATION:/label= PCI /note= "region containing promoter of Cl gene" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1078..2134 (D) OTHER INFORMATION: /label= Cl /note= "coding region of Cl gene" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2135..2430 (D) OTHER INFORMATIO :/label= 3'C1 /note= "region containing polyadenylation signal of Cl gene" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1033..1038 (D) OTHER INFORMATIO :/label= TATA-Box (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1061..1062 (D) OTHER INFORMATION: /label= transcript-init /note= "transcription initiation site" (ix) FEATURE: (A) NAME/KEY: intron (B) LOCATION: 1211..1299 (ix) FEATURE: (A) NAME/KEY: intron (B) LOCATION: 1430..1575 (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 935..939 (D) OTHER INFORMATION: /label= Cl-S /note= "TGCAG sequence (in Cl gene) which in the Cl-S sequence is changed to TTAGG" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TATCAACCTC CTGTGTTATT TTTAGTGACG GTTTCTTAAA AAACACCACT AGAAATCGTA 60 TTTTTATAGG TGGTTCCTTA AGAAAACTGC ATGCAGAAAT CCATGACGGT TTTCTTAAGG 120 AACCGTATGT AGAAATACGA TTTCTAGTGA CGATCTTCTT AAGGAAACCA CCACTAAAAA 180 TTATTTTTAT CCTTAATTTT CGAGTTTTTC AAACGATCTC GTATGATGAA ACCATCAAAA 240 TAAAAGTTGT ACATCTCTAA AAGTTATGAA AATTTGTAGT TAACAACTTT TTTATTTGAA 300 CTCATTTTGG TTCTCAAAAA TTGCATCTAA ATTTGTCAAA TTTAAAATTC AAATTTTCCA 360 AACGACCTCG GATGAAAAAA GTGTCAAAAT GAAAGTTGTA GAACTTCAAA AGTTATTCAA 420 CTTTGTAGTC GACTATCTTT TTATTTGAAT TCGCTTACGG TCTCAAACAA GCAATTTACA 480 CTCAGTTGGT TGTAATATGT GGACAATAAA ACTACAAACT AGACACAAAT CATACCATAG 540 ACGGAGTGGT AGCAGAGGGT ACGCGCGAGG GTGAGATAGA GGATTCTCCT AAAATAAATG 600 CACTTTAGAT GGGTAGGGTG GGGTGAGGCC TCTCCTAAAA TGAAACTCGT TTAATGTTTC 660 TAAAAATAGT TTTCACTGGT GATCCTTAGT TACTGGCATG TAAAAATGAT GATTTCTACT 720 GTCTCTCATA TGGACGGTTA TAAAAAATAC CATTATATTG AAAATAGGTC TCTGCTGCTA 780 CACTCGCCCT CATAGCAGAT CATGCATGCA CGCATCATTC GATCAGTTTT CGTTCTGATG 840 CAGTTTTCGA TAAATGCCAA TTTTTTAACT GCATACGTTG CCCTTGCTCA GCACCAGCAC 900 AGCAGTGTCG TGTCGTCCAT GCATGCACTT TAGGTGCAGT GCAGGGCCTC AACTCGGCCA 960 CGTAGTTAGC GCCACTGCTA CAGATCGAGG CACCGGTCAG CCGGCCACGC ACGTCGACCG 1020 CGCGCGTGCA TTTAAATACG CCGACGACGG AGCTTGATCG ACGAGAGAGC GAGCGCGATG 1080 GGGAGGAGGG CGTGTTGCGC GAAGGAAGGC GTTAAGAGAG GGGCGTGGAC GAGCAAGGAG 1140 GACGATGCCT TGGCCGCCTA CGTCAAGGCC CATGGCGAAG GCAAATGGAG GGAAGTGCCC 1200 CAGAAAGCCG GTAAAACTAG CTAGTCTTTT TATTTCATTT TGGGATCATA TATATACCCC 1260 CGAGGCAAGA CCGGAGGACG ATCACGTGTG TGGGTGCAGG TTTGCGTCGG TGCGGCAAGA 1320 GCTGCCGGCT GCGGTGGCTG AACTACCTCC GGCCCAACAT CAGGCGCGGC AACATCTCCT 1380 ACGACGAGGA GGATCTCATC ATCCGCCTCC ACAGGCTCCT CGGCAACAGG TCTGTGCAGT 1440 GGCCAGTGGT GGGCTAGCTT ATTACACGAG CTGACGACGA GGCGATCGAT CGAGCGTCTG 1500 CTGCGAATTC ATCTGTTCCG GTGTCGGCCG TGTGAGAGTG AGCTCATTCA TATGTACATG 1560 CGTGTTGGCG CGCAGGTGGT CGCTGATTGC AGGCAGGCTG CCTGGCCGAA CAGACAATGA 1620 AATCAAGAAC TACTGGAACA GCACGCTGGG CCGGAGGGCA GGCGCCGGCG CCGGCGCCGG 1680 CGGCAGCTGG GTCGTCGTCG CGCCGGACAC CGGCTCGCAC GCCACCCCGG CCGCGACGTC 1740 GGGCGCCTGC GAGACCGGCC AGAATAGCGC CGCTCATCGC GCGGACCCCG ACTCAGCCGG 1800 GACGACGACG ACCTCGGCGG CGGCGGTGTG GGCGCCCAAG GCCGTGCGGT GCACGGGCGG 1860 ACTCTTCTTC TTCCACCGGG ACACGACGCC GGCGCACGCG GGCGAGACGG CGACGCCAAT 1920 GGCCGGTGGA GGTGGAGGAG GAGGAGGAGA AGCAGGGTCG TCGGACGACT GCAGCTCGGC 1980 GGCGTCGGTA TCGCTTCGCG TCGGAAGCCA CGACGAGCCG TGCTTCTCCG GCGACGGTGA 2040 CGGCGACTGG ATGGACGACG TGAGGGCCCT GGCGTCGTTT CTCGAGTCCG ACGAGGACTG 2100 GCTCCGCTGT CAGACGGCCG GGCAGCTTGC GTAGACAACA AGTACACGTA TAGATGTCCA 2160 ATAAGCACGA GGCCCGCGAG CCCGGCACGA AGCCCGCTTT TTGGGCCCGG TCCGAGCCCG 2220 GCACGGCCCG GTTATATGCA GACCCGGGCC GGCCCGGCAC GAATAAGCGG GCCGGGCTCG 2280 GACAGGAAAT TAGGCACGGT GAGCTAGCCC GGCACGGCCC GTTTAGGTCT AAGCCCGTTA 2340 AGCCCGTTTT TTTACACTAA AACGTGCTTC TCGGCCCGCA TAGCCCGCTT CTCGGCCCGC 2400 TTTTTTCGTG CTAAACGGGC CGGCCCGGCC CGGTTTAGGC CCGTTGCGGG CCGGGCTCGG 2460 ACAGGAAATT GAGCCCGCGT GCTTAGCCGG CCCGGCCCGG TTTTTTAATC GTGCCTGGCG 2520 GGCCAGGCCC AAAACGGGCC GGGCTTCACC GGGCCCGGGC CGGACCGGGC CGGGCGGCCC 2580 GTTTGGACAT CTCTAAGTAC ACGTATGGAG GAGAATATAT ATATAGTCAT GCGTACGTAT 2640 AGATTTTTTC ATCCGATCCC AACAGAAATA CGTATGAAAA TGCTCTTCGT TCTTTTTCAT 2700 TTATCATATC TATACTATAC TTAAAACACC AGTTTCAACG GTCGTCATGC GTCATTTTTT 2760 TACAAATAAC CCCTCACAGC TATTTCAAAT TAATCCGCTG CACGTCTATA GATGCCAAAC 2820 GACGCCCAAC ACGGGCTAGA TGCACGCGGG CCACAACTAT GGCACAGGCA CGTCATGCCG 2880 GCCTGCTAAC TGTGTCGGGC TAGCCCGTTA GCCCGTCGAT CCATTTAATT AAATTAGCGT 2940 AACGACGCCC GACACGGGCT AGATGCACGT GGGCCACAAC TATGGCACAT GCACGTCATG 3000 CCGGCCTGTT AACTGTGTCG GGCCAGTCTG TTAGCCCATT GATCCATTTA ATTAAATCAG 3060 CGTAAAATGT TAAAAACGGT GCAGGAGGTG GGGTTCGAAC CCATACCCTG ATGGAAGAAG 3120 GGCGGGAGAC ACTGGGTGAA ACTGTCTAAC CAGTAGAATA TCTATCACGC TAAGATGTTT 3180 TTAATATTGA ATATAAATTG TATATAAGCA TATAAGTTTT TTTGTAAAAT AAAAAATAAT 3240 CGTGTCGGGC CGGGCCATCA CTACTGGCCG AGGCTACAAC CCAAGCACGA CACGACGTTC 3300 TTGGCTCTTG CAAGCATTAG GTCGTTTCTG AGACCATATT GGCGCAATGG ACTACATGAT 3360 ■GTTTGGGGTT GCTGAATTGA ATGGAGCAGC AATAATTTGT CACACTAACA GCAAAATGAA 3420 AGGTTATTTG TTGGTTTTAA ACGTTAGTAA TTGCTACGAA GTAGCATAAT TTATATGGAG 3480 CGCATCCAGT TTTTATTGAT GCCTGACTTT AGCAATCACT CCATATTTTG ATCTATCTTT 3540 TTTATAAGTT TGACTTCATG GGACTTATTT TAGAACTTGA TCTCACAAAC TTTCTCTTAT 3600 TTTGTCTCTA TATGATGAAA TTGTGTCATT TTATAATCTT TGTTCATTCA GTCAATCGTT 3660 GTGAACTCTC TTCTAATCAC TCACTTCATT AGTTGTGTTG TACCAAGACA TATTTGCATA 3720 GAGTAAACAA TAACATCAGT TAGCCAAATC AAAAAATATA TTATACAGAG AGCGGAGACA 3780 ATCAAATAAA AAATCTTGAA ATTTTTTTAA TGGATAGTTT ACGTGGGTAT TGTTGTAAGC 3840 CGTCGCAACG CACGGGCAAC CGACTAGTTT TAGTTTATAA ATTAATAAAC GTACGACAAA 3900 TATTAAGAAC GCCACCTTTC CATGCCTACG CGCGCGTGAG ACACGACCGG GGCACGTCAG 3960 ACGTGTGCCC CTGTTGTATA ATTTATTTAC TTTTTAATGA CTATGTGCTG TTGGTTGCCG 4020 TTGGCTTCAT CGTGTTCGTA GCCATGCATA AATCCAGCG 4059 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4896 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: plasmid pTS256, linearized at Hindlll (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: complement (39..317) (D) OTHER INFORMATION: /label= 3 'nos /note= "3' regulatory sequence containing the polyadenylation signal of the nopaline synthase gene of Agrobacterium T-DNA" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:complement (318..869) (D) OTHER INFORMATION: /label= bar /note= "coding region of bar gene of Streptomyces hygroscopicus" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATIONcomplement (870..1702) (D) OTHER INFORMATION: /label= P35S /note= "35S promoter of Cauliflower Mosaic Virus" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1740..2284 (D) OTHER INFORMATION: /label= PTA29 /note= "promoter of TA29 gene of Nicotiana tabacum" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2285..2557 (D) OTHER INFORMATION:/label= barstar /note= "coding region of barstar gene of Bacillusamyloliquefacien (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2558..2879 (D) OTHER INFORMATION: /label= 3'nos /note= "3 1 regulatory sequence containing the polyadenylation signal of the nopaline synthase gene of Agrobacterium T-DNA" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..38 (D) OTHER INFORMATIO :/label= pUC19 /note= "pUC19 derived sequence" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2880..4896 (D) OTHER INFORMATION:/label= pUC19 /note= "pUC19 derived sequence" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 3004..3009 (D) OTHER INFORMATIO :/label= EcoRI (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: AGCTTGCATG CCTGCAGGTC GACTCTAGAG GATCTTCCCG ATCTAGTAAC ATAGATGACA 60 CCGCGCGCGA TAATTTATCC TAGTTTGCGC GCTATATTTT GTTTTCTATC GCGTATTAAA 120 TGTATAATTG CGGGACTCTA ATCATAAAAA CCCATCTCAT AAATAACGTC ATGCATTACA 180 TGTTAATTAT TACATGCTTA ACGTAATTCA ACAGAAATTA TATGATAATC ATCGCAAGAC 240 CGGCAACAGG ATTCAATCTT AAGAAACTTT ATTGCCAAAT GTTTGAACGA TCTGCTTCGG 300 ATCCTAGACG CGTGAGATCA GATCTCGGTG ACGGGCAGGA CCGGACGGGG CGGTACCGGC 360 AGGCTGAAGT CCAGCTGCCA GAAACCCACG TCATGCCAGT TCCCGTGCTT GAAGCCGGCC 420 GCCCGCAGCA TGCCGCGGGG GGCATATCCG AGCGCCTCGT GCATGCGCAC GCTCGGGTCG 480 TTGGGCAGCC CGATGACAGC GACCACGCTC TTGAAGCCCT GTGCCTCCAG GGACTTCAGC 540 AGGTGGGTGT AGAGCGTGGA GCCCAGTCCC GTCCGCTGGT GGCGGGGGGA GACGTACACG 600 GTCGACTCGG CCGTCCAGTC GTAGGCGTTG CGTGCCTTCC AGGGGCCCGC GTAGGCGATG 660 CCGGCGACCT CGCCGTCCAC CTCGGCGACG AGCCAGGGAT AGCGCTCCCG CAGACGGACG 720 AGGTCGTCCG TCCACTCCTG CGGTTCCTGC GGCTCGGTAC GGAAGTTGAC CGTGCTTGTC 780 TCGATGTAGT GGTTGACGAT GGTGCAGACC GCCGGCATGT CCGCCTCGGT GGCACGGCGG 840 ATGTCGGCCG GGCGTCGTTC TGGGTCCATG GTTATAGAGA GAGAGATAGA TTTATAGAGA 900 GAGACTGGTG ATTTCAGCGT GTCCTCTCCA AATGAAATGA ACTTCCTTAT ATAGAGGAAG 960 GGTCTTGCGA AGGATAGTGG GATTGTGCGT CATCCCTTAC GTCAGTGGAG ATGTCACATC 1020 AATCCACTTG CTTTGAAGAC GTGGTTGGAA CGTCTTCTTT TTCCACGATG CTCCTCGTGG 1080 GTGGGGGTCC ATCTTTGGGA CCACTGTCGG CAGAGGCATC TTGAATGATA GCCTTTCCTT 1140 TATCGCAATG ATGGCATTTG TAGGAGCCAC CTTCCTTTTC TACTGTCCTT TCGATGAAGT 1200 GACAGATAGC TGGGCAATGG AATCCGAGGA GGTTTCCCGA AATTATCCTT TGTTGAAAAG 1260 TCTCAATAGC CCTTTGGTCT TCTGAGACTG TATCTTTGAC ATTTTTGGAG TAGACCAGAG 1320 TGTCGTGCTC CACCATGTTG ACGAAGATTT TCTTCTTGTC ATTGAGTCGT AAAAGACTCT 1380 GTATGAACTG TTCGCCAGTC TTCACGGCGA GTTCTGTTAG ATCCTCGATT TGAATCTTAG 1440 ACTCCATGCA TGGCCTTAGA TTCAGTAGGA ACTACCTTTT TAGAGACTCC AATCTCTATT 1500 ACTTGCCTTG GTTTATGAAG CAAGCCTTGA ATCGTCCATA CTGGAATAGT ACTTCTGATC 1560 TTGAGAAATA TGTCTTTCTC TGTGTTCTTG ATGCAATTAG TCCTGAATCT TTTGACTGCA 1620 TCTTTAACCT TCTTGGGAAG GTATTTGATC TCCTGGAGAT TGTTACTCGG GTAGATCGTC 1680 TTGATGAGAC CTGCTGCGTA GGAGCTTGCA TGCCTGCAGG TCGACTCTAG AGGATCCCCA 1740 TCTAGCTAAG TATAACTGGA TAATTTGCAT TAACAGATTG AATATAGTGC CAAACAAGAA 1800 GGGACAATTG ACTTGTCACT TTATGAAAGA TGATTCAAAC ATGATTTTTT ATGTACTAAT 1860 ATATACATCC TACTCGAATT AAAGCGACAT AGGCTCGAAG TATGCACATT TAGCAATGTA 1920 AATTAAATCA GTTTTTGAAT CAAGCTAAAA GCAGACTTGC ATAAGGTGGG TGGCTGGACT 1980 AGAATAAACA TCTTCTCTAG CACAGCTTCA TAATGTAATT TCCATAACTG AAATCAGGGT 2040 GAGACAAAAT TTTGGTACTT TTTCCTCACA CTAAGTCCAT GTTTGCAACA AATTAATACA 2100 TGAAACCTTA ATGTTACCCT CAGATTAGCC TGCTACTCCC CATTTTCCTC GAAATGCTCC 2160 AACAAAAGTT AGTTTTGCAA GTTGTTGTGT ATGTCTTGTG CTCTATATAT GCCCTTGTGG 2220 TGCAAGTGTA ACAGTACAAC ATCATCACTC AAATCAAAGT TTTTACTTAA AGAAATTAGC 2280 TACCATGAAA AAAGCAGTCA TTAACGGGGA ACAAATCAGA AGTATCAGCG ACCTCCACCA 2340 GACATTGAAA AAGGAGCTTG CCCTTCCGGA ATACTACGGT GAAAACCTGG ACGCTTTATG 2400 GGATTGTCTG ACCGGATGGG TGGAGTACCC GCTCGTTTTG GAATGGAGGC AGTTTGAACA 2460 AAGCAAGCAG CTGACTGAAA ATGGCGCCGA GAGTGTGCTT CAGGTTTTCC GTGAAGCGAA 2520 AGCGGAAGGC TGCGACATCA CCATCATACT TTCTTAATAC GATCAATGGG AGATGAACAA 2580 TATGGAAACA CAAACCCGCA AGCTTGGTCT AGAGGATCCG AAGCAGATCG TTCAAACATT 2640 TGGCAATAAA GTTTCTTAAG ATTGAATCCT GTTGCCGGTC TTGCGATGAT TATCATATAA 2700 TTTCTGTTGA ATTACGTTAA GCATGTAATA ATTAACATGT AATGCATGAC GTTATTTATG 2760 AGATGGGTTT TTATGATTAG AGTCCCGCAA TTATACATTT AATACGCGAT AGAAAACAAA 2820 ATATAGCGCG CAAACTAGGA TAAATTATCG CGCGCGGTGT CATCTATGTT ACTAGATCGG 2880 GAAGATCCCC GGGTACCGAG CTCGAATTCT GATCAGGCCA ACGCGCGGGG AGAGGCGGTT 2940 TGCGTATTGG GCGCTCTTCC GCTTCCTCGC TCACTGACTC GCTGCGCTCG GTCGTTCGGC 3000 TGCGGCGAGC GGTATCAGCT CACTCAAAGG CGGTAATACG GTTATCCACA GAATCAGGGG 3060 ATAACGCAGG AAAGAACATG TGAGCAAAAG GCCAGCAAAA GGCCAGGAAC CGTAAAAAGG 3120 CCGCGTTGCT GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC 3180 GCTCAAGTCA GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG 3240 GAAGCTCCCT CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT 3300 TTCTCCCTTC GGGAAGCGTG GCGCTTTCTC AATGCTCACG CTGTAGGTAT CTCAGTTCGG 3360 TGTAGGTCGT TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT 3420 GCGCCTTATC CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC 3480 TGGCAGCAGC CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT 3540 TCTTGAAGTG GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT ATCTGCGCTC 3600 TGCTGAAGCC AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA 3660 CCGCTGGTAG CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT 3720 CTCAAGAAGA TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC 3780 GTTAAGGGAT TTTGGTCATG AGACTCGAGC CAAAAAGGAT CTTCACCTAG ATCCTTTTAA 3840 ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG TCTGACAGTT 3900 ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT TCATCCATAG 3960 TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA TCTGGCCCCA 4020 GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA GCAATAAACC 4080 AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC TCCATCCAGT 4140 CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT TTGCGCAACG 4200 TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG GCTTCATTCA 4260 GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC AAAAAAGCGG 4320 TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG TTATCACTCA 4380 TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA TGCTTTTCTG 4440 TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA CCGAGTTGCT 4500 CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA AAAGTGCTCA 4560 TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG TTGAGATCCA 4620 GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT TTCACCAGCG 4680 TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA AGGGCGACAC 4740 GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT TATCAGGGTT 4800 ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA ATAGGGGTTC 4860 CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCA 4896 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3544 base pairs ' (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: EcoRI-Hindlll region of plasmid pTS200 (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 3227..3504 (D) OTHER INFORMATION:/label= 3 'nos /note= "3 1 regulatory sequence containing the polyadenylation signal of the nopaline synthase gene of Agrobacterium T-DNA" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2675..3226 (D) OTHER INFORMATION:/label= bar /note= "coding region of bar gene of Streptomyces hygroscopicus" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1841..2674 (D) OTHER INFORMATIO :/label= P35S /note= "35S promoter of Cauliflower Mosaic Virus" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATIONcomplement (626..1803) (D) OTHER INFORMATION:/label= PCA55 /note= "promoter of CA55 gene of Zea mays" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATIONcomplement (353..625) (D) OTHER INFORMATION:/label= barstar /note= "coding region of barstar gene of Bacillus amyloliquefaciens" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATIONcomplement (30..352) (D) OTHER INFORMATION:/label= 3'nos /note= "3' regulatory sequence containing the polyadenylation signal of the nopaline synthase gene of Agrobacterium T-DNA" (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..6 (D) OTHER INFORMATION:/label= EcoRI (ix) FEATURE: (A) NAME/KEY: - ,(B) LOCATION: 3539..3544 (D) OTHER INFORMATION: /label= Hindlll (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: GAATTCGAGC TCGGTACCCG GGGATCTTCC CGATCTAGTA ACATAGATGA CACCGCGCGC 60 GATAATTTAT CCTAGTTTGC GCGCTATATT TTGTTTTCTA TCGCGTATTA AATGTATAAT 120 TGCGGGACTC TAATCATAAA AACCCATCTC ATAAATAACG TCATGCATTA CATGTTAATT 180 ATTACATGCT TAACGTAATT CAACAGAAAT TATATGATAA TCATCGCAAG ACCGGCAACA 240 GGATTCAATC TTAAGAAACT TTATTGCCAA ATGTTTGAAC GATCTGCTTC GGATCCTCTA 300 GACCAAGCTT GCGGGTTTGT GTTTCCATAT TGTTCATCTC CCATTGATCG TATTAAGAAA 360 GTATGATGGT GATGTCGCAG CCTTCCGCTT TCGCTTCACG GAAAACCTGA AGCACACTCT 420 CGGCGCCATT TTCAGTCAGC TGCTTGCTTT GTTCAAACTG CCTCCATTCC AAAACGAGCG 480 GGTACTCCAC CCATCCGGTC AGAGAATCCC ATAAAGCGTC CAGGTTTTCA CCGTAGTATT 540 CCGGAAGGGC AAGCTCCTTT TTCAATGTCT GGTGGAGGTC GCTGATACTT CTGATTTGTT 600 CCCCGTTAAT GACTGCTTTT TTCATGGCTG CAGCTAGTTA GCTCGATGTA TCTTCTGTAT 660 ATGCAGTGCA GCTTCTGCGT TTTGGCTGCT TTGAGCTGTG AAATCTCGCT TTCCAGTCCC 720 TGCGTGTTTT ATAGTGCTGT ACGTTCGTGA TCGTGAGCAA ACAGGGCGTG CCTCAACTAC 780 TGGTTTGGTT GGGTGACAGG CGCCAACTAC GTGCTCGTAA CCGATCGAGT GAGCGTAATG 840 CAACATTTTT TCTTCTTCTC TCGCATTGGT TTCATCCAGC CAGGAGACCC GAATCGAATT 900 GAAATCACAA ATCTGAGGTA CAGTATTTTT ACAGTACCGT TCGTTCGAAG GTCTTCGACA 960 GGTCAAGGTA ACAAAATCAG TTTTAAATTG TTGTTTCAGA TCAAAGAAAA TTGAGATGAT 1020 CTGAAGGACT TGGACCTTCG TCCAATGAAA CACTTGGACT AATTAGAGGT GAATTGAAAG 1080 CAAGCAGATG CAACCGAAGG TGGTGAAAGT GGAGTTTCAG CATTGACGAC GAAAACCTTC 1140 GAACGGTATA AAAAAGAAGC CGCAATTAAA CGAAGATTTG CCAAAAAGAT GCATCAACCA 1200 AGGGAAGACG TGCATACATG TTTGATGAAA ACTCGTAAAA ACTGAAGTAC GATTCCCCAT 1260 TCCCCTCCTT TTCTCGTTTC TTTTAACTGA AGCAAAGAAT TTGTATGTAT TCCCTCCATT 1320 CCATATTCTA GGAGGTTTTG GCTTTTCATA CCCTCCTCCA TTTCAAATTA TTTGTCATAC 1380 ATTGAAGATA TACACCATTC TAATTTATAC TAAATTACAG CTTTTAGATA CATATATTTT 1440 ATTATACACT TAGATACGTA TTATATAAAA CACCTAATTT AAAATAAAAA ATTATATAAA 1500 AAGTGTATCT AAAAAATCAA AATACGACAT AATTTGAAAC GGAGGGGTAC TACTTATGCA 1560 AACCAATCGT GGTAACCCTA AACCCTATAT GAATGAGGCC ATGATTGTAA TGCACCGTCT 1620 GATTAACCAA GATATCAATG GTCAAAGATA TACATGATAC ATCCAAGTCA CAGCGAAGGC 1680 AAATGTGACA ACAGTTTTTT TTACCAGAGG GACAAGGGAG AATATCTATT CAGATGTCAA 1740 GTTCCCGTAT CACACTGCCA GGTCCTTACT CCAGACCATC TTCCGGCTCT ATTGATGCAT 1800 ACCAGGAATT GATCTAGAGT CGACCTGCAG GCATGCAAGC TCCTACGCAG CAGGTCTCAT 1860 CAAGACGATC TACCCGAGTA ACAATCTCCA GGAGATCAAA TACCTTCCCA AGAAGGTTAA 1920 AGATGCAGTC AAAAGATTCA GGACTAATTG CATCAAGAAC ACAGAGAAAG ACATATTTCT 1980 CAAGATCAGA AGTACTATTC CAGTATGGAC GATTCAAGGC TTGCTTCATA AACCAAGGCA 2040 AGTAATAGAG ATTGGAGTCT CTAAAAAGGT AGTTCCTACT GAATCTAAGG CCATGCATGG 2100 AGTCTAAGAT TCAAATCGAG GATCTAACAG AACTCGCCGT GAAGACTGGC GAACAGTTCA 2160 TACAGAGTCT TTTACGACTC AATGACAAGA AGAAAATCTT CGTCAACATG GTGGAGCACG 2220 ACACTCTGGT CTACTCCAAA AATGTCAAAG ATACAGTCTC AGAAGACCAA AGGGCTATTG 2280 AGACTTTTCA ACAAAGGATA ATTTCGGGAA ACCTCCTCGG ATTCCATTGC CCAGCTATCT 2340 GTCACTTCAT CGAAAGGACA GTAGAAAAGG AAGGTGGCTC CTACAAATGC CATCATTGCG 2400 ATAAAGGAAA GGCTATCATT CAAGATGCCT CTGCCGACAG TGGTCCCAAA GATGGACCCC 2460 CACCCACGAG GAGCATCGTG GAAAAAGAAG ACGTTCCAAC CACGTCTTCA AAGCAAGTGG 2520 ATTGATGTGA CATCTCCACT GACGTAAGGG ATGACGCACA ATCCCACTAT CCTTCGCAAG 2580 ACCCTTCCTC TATATAAGGA AGTTCATTTC ATTTGGAGAG GACACGCTGA AATCACCAGT 2640 CTCTCTCTAT AAATCTATCT CTCTCTCTAT AACCATGGAC CCAGAACGAC GCCCGGCCGA 2700 CATCCGCCGT GCCACCGAGG CGGACATGCC GGCGGTCTGC ACCATCGTCA ACCACTACAT 2760 CGAGACAAGC ACGGTCAACT TCCGTACCGA GCCGCAGGAA CCGCAGGAGT GGACGGACGA 2820 CCTCGTCCGT CTGCGGGAGC GCTATCCCTG GCTCGTCGCC GAGGTGGACG GCGAGGTCGC 2880 CGGCATCGCC TACGCGGGCC CCTGGAAGGC ACGCAACGCC TACGACTGGA CGGCCGAGTC 2940 GACCGTGTAC GTCTCCCCCC GCCACCAGCG GACGGGACTG GGCTCCACGC TCTACACCCA 3000 CCTGCTGAAG TCCCTGGAGG CACAGGGCTT CAAGAGCGTG GTCGCTGTCA TCGGGCTGCC 3060 CAACGACCCG AGCGTGCGCA TGCACGAGGC GCTCGGATAT GCCCCCCGCG GCATGCTGCG 3120 GGCGGCCGGC TTCAAGCACG GGAACTGGCA TGACGTGGGT TTCTGGCAGC TGGACTTCAG 3180 CCTGCCGGTA CCGCCCCGTC CGGTCCTGCC CGTCACCGAG ATCTGATCTC ACGCGTCTAG 3240 GATCCGAAGC AGATCGTTCA AACATTTGGC AATAAAGTTT CTTAAGATTG AATCCTGTTG 3300 CCGGTCTTGC GATGATTATC ATATAATTTC TGTTGAATTA CGTTAAGCAT GTAATAATTA 3360 ACATGTAATG CATGACGTTA TTTATGAGAT GGGTTTTTAT GATTAGAGTC CCGCAATTAT 3420 ACATTTAATA CGCGATAGAA AACAAAATAT AGCGCGCAAA CTAGGATAAA TTATCGCGCG 3480 CGGTGTCATC TATGTTACTA GATCGGGAAG ATCCTCTAGA GTCGACCTGC AGGCATGCAA 3540 GCTT 3544 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: oligonucleotide 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CGTTTCTCGA ATCCGACGAG G 21 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4824 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: circular (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: plasmid pCOL9 (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 396..401 (D) OTHER INFORMATION: /label= EcoRI (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2367..2379 (D) OTHER INFORMATION: /label= Sfil (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 884..888 (D) OTHER INFORMATION:/label= Cl-S /note= "TGCAG (in Cl) which in Cl-S allele is replaced with TTAGG" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TCGCGCGTTT CGGTGATGAC GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA 60 CAGCTTGTCT GTAAGCGGAT GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG 120 TTGGCGGGTG TCGGGGCTGG CTTAACTATG CGGCATCAGA GCAGATTGTA CTGAGAGTGC 180 ACCATATGCG GTGTGAAATA CCGCACAGAT GCGTAAGGAG AAAATACCGC ATCAGGCGCC 240 ATTCGCCATT CAGGCTGCGC AACTGTTGGG AAGGGCGATC GGTGCGGGCC TCTTCGCTAT 300 TACGCCAGCT GGCGAAAGGG GGATGTGCTG CAAGGCGATT AAGTTGGGTA ACGCCAGGGT 360 TTTCCCAGTC ACGACGTTGT AAAACGACGG CCAGTGAATT CGCTTACGGT CTCAAACAAG 420 CAATTTACAC TCAGTTGGTT GTAATATGTG GACAATAAAA CTACAAACTA GACACAAATC 480 ATACCATAGA CGGAGTGGTA GCAGAGGGTA CGCGCGAGGG TGAGATAGAG GATTCTCCTA 540 AAATAAATGC ACTTTAGATG GGTAGGGTGG GGTGAGGCCT CTCCTAAAAT GAAACTCGTT 600 TAATGTTTCT AAAAATAGTT TTCACTGGTG ATCCTTAGTT ACTGGCATGT AAAAATGATG 660 ATTTCTACTG TCTCTCATAT GGACGGTTAT AAAAAATACC ATTATATTGA AAATAGGTCT 720 CTGCTGCTAC ACTCGCCCTC ATAGCAGATC ATGCATGCAC GCATCATTCG ATCAGTTTTC 780 GTTCTGATGC AGTTTTCGAT AAATGCCAAT TTTTTAACTG CATACGTTGC CCTTGCTCAG 840 CACCAGCACA GCAGTGTCGT GTCGTCCATG CATGCACTTT AGGTGCAGTG CAGGGCCTCA 900 ACTCGGCCAC GTAGTTAGCG CCACTGCTAC AGATCGAGGC ACCGGTCAGC CGGCCACGCA 960 CGTCGACCGC GCGCGTGCAT TTAAATACGC CGACGACGGA GCTTGATCGA CGAGAGAGCG 1020 AGCGCGATGG GGAGGAGGGC GTGTTGCGCG AAGGAAGGCG TTAAGAGAGG GGCGTGGACG 1080 AGCAAGGAGG ACGATGCCTT GGCCGCCTAC GTCAAGGCCC ATGGCGAAGG CAAATGGAGG 1140 GAAGTGCCCC AGAAAGCCGG TAAAACTAGC TAGTCTTTTT ATTTCATTTT GGGATCATAT 1200 ATATACCCCC GAGGCAAGAC CGGAGGACGA TCACGTGTGT GGGTGCAGGT TTGCGTCGGT 1260 GCGGCAAGAG CTGCCGGCTG CGGTGGCTGA ACTACCTCCG GCCCAACATC AGGCGCGGCA 1320 ACATCTCCTA CGACGAGGAG GATCTCATCA TCCGCCTCCA CAGGCTCCTC GGCAACAGGT 1380 CTGTGCAGTG GCCAGTGGTG GGCTAGCTTA TTACACGAGC TGACGACGAG GCGATCGATC 1440 GAGCGTCTGC TGCGAATTCA TCTGTTCCGG TGTCGGCCGT GTGAGAGTGA GCTCATTCAT 1500 ATGTACATGC GTGTTGGCGC GCAGGTGGTC GCTGATTGCA GGCAGGCTGC CTGGCCGAAC 1560 AGACAATGAA ATCAAGAACT ACTGGAACAG CACGCTGGGC CGGAGGGCAG GCGCCGGCGC 1620 CGGCGCCGGC GGCAGCTGGG TCGTCGTCGC GCCGGACACC GGCTCGCACG CCACCCCGGC 1680 CGCGACGTCG GGCGCCTGCG AGACCGGCCA GAATAGCGCC GCTCATCGCG CGGACCCCGA 1740 CTCAGCCGGG ACGACGACGA CCTCGGCGGC GGCGGTGTGG GCGCCCAAGG CCGTGCGGTG 1800 CACGGGCGGA CTCTTCTTCT TCCACCGGGA CACGACGCCG GCGCACGCGG GCGAGACGGC 1860 GACGCCAATG GCCGGTGGAG GTGGAGGAGG AGGAGGAGAA GCAGGGTCGT CGGACGACTG 1920 CAGCTCGGCG GCGTCGGTAT CGCTTCGCGT CGGAAGCCAC GACGAGCCGT GCTTCTCCGG 1980 CGACGGTGAC GGCGACTGGA TGGACGACGT GAGGGCCCTG GCGTCGTTTC TCGAGTCCGA 2040 CGAGGACTGG CTCCGCTGTC AGACGGCCGG GCAGCTTGCG TAGACAACAA GTACACGTAT 2100 AGATGTCCAA TAAGCACGAG GCCCGCGAGC CCGGCACGAA GCCCGCTTTT TGGGCCCGGT 2160 CCGAGCCCGG CACGGCCCGG TTATATGCAG ACCCGGGCCG GCCCGGCACG AATAAGCGGG 2220 CCGGGCTCGG ACAGGAAATT AGGCACGGTG AGCTAGCCCG GCACGGCCCG TTTAGGTCTA 2280 AGCCCGTTAA GCCCGTTTTT TTACACTAAA ACGTGCTTCT CGGCCCGCAT AGCCCGCTTC 2340 TCGGCCCGCT TTTTTCGTGC TAAACGGGCC GGCCCGGCCC GGTTTAGGCC CGTTGCGGGC 2400 CGGGCTCGGA CAGGAAATTG AGCCCGCGTG CTTAGCCGGC CCGGCCCGGT TTTTTAATCG 2460 TGCCTGGCGG GCCAGGCCCA AAACGGGCCG GGCTTCACCG GGCCCGGGCC GGACCGGGCC 2520 GGGCGGCCCG TTTGGACATC TCTAAGTACA CGTATGGAGG AGAATATATA TATAGTCATG 2580 CGTACAGCTT GGCGTAATCA TGGTCATAGC TGTTTCCTGT GTGAAATTGT TATCCGCTCA 2640 CAATTCCACA CAACATACGA GCCGGAAGCA TAAAGTGTAA AGCCTGGGGT GCCTAATGAG 2700 TGAGCTAACT CACATTAATT GCGTTGCGCT CACTGCCCGC TTTCCAGTCG GGAAACCTGT 2760 CGTGCCAGCT GCATTAATGA ATCGGCCAAC GCGCGGGGAG AGGCGGTTTG CGTATTGGGC 2820 GCTCTTCCGC TTCCTCGCTC ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG 2880 TATCAGCTCA CTCAAAGGCG GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA 2940 AGAACATGTG AGCAAAAGGC CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG 3000 CGTTTTTCCA TAGGCTCCGC CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA 3060 GGTGGCGAAA CCCGACAGGA CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG 3120 TGCGCTCTCC TGTTCCGACC CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG 3180 GAAGCGTGGC GCTTTCTCAA TGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC 3240 GCTCCAAGCT GGGCTGTGTG CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG 3300 GTAACTATCG TCTTGAGTCC AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA 3360 CTGGTAACAG GATTAGCAGA GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT 3420 GGCCTAACTA CGGCTACACT AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG 3480 TTACCTTCGG AAAAAGAGTT GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG 3540 GTGGTTTTTT TGTTTGCAAG CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC 3600 CTTTGATCTT TTCTACGGGG TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT 3660 TGGTCATGAG ATTATCAAAA AGGATCTTCA CCTAGATCCT TTTAAATTAA AAATGAAGTT 3720 TTAAATCAAT CTAAAGTATA TATGAGTAAA CTTGGTCTGA CAGTTACCAA TGCTTAATCA 3780 GTGAGGCACC TATCTCAGCG ATCTGTCTAT TTCGTTCATC CATAGTTGCC TGACTCCCCG 3840 TCGTGTAGAT AACTACGATA CGGGAGGGCT TACCATCTGG CCCCAGTGCT GCAATGATAC 3900 CGCGAGACCC ACGCTCACCG GCTCCAGATT TATCAGCAAT AAACCAGCCA GCCGGAAGGG 3960 CCGAGCGCAG AAGTGGTCCT GCAACTTTAT CCGCCTCCAT CCAGTCTATT AATTGTTGCC 4020 GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA ATAGTTTGCG CAACGTTGTT GCCATTGCTA 4080 CAGGCATCGT GGTGTCACGC TCGTCGTTTG GTATGGCTTC ATTCAGCTCC GGTTCCCAAC 4140 GATCAAGGCG AGTTACATGA TCCCCCATGT TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC 4200 CTCCGATCGT TGTCAGAAGT AAGTTGGCCG CAGTGTTATC ACTCATGGTT ATGGCAGCAC 4260 TGCATAATTC TCTTACTGTC ATGCCATCCG TAAGATGCTT TTCTGTGACT GGTGAGTACT 4320 CAACCAAGTC ATTCTGAGAA TAGTGTATGC GGCGACCGAG TTGCTCTTGC CCGGCGTCAA 4380 TACGGGATAA TACCGCGCCA CATAGCAGAA CTTTAAAAGT GCTCATCATT GGAAAACGTT 4440 CTTCGGGGCG AAAACTCTCA AGGATCTTAC CGCTGTTGAG ATCCAGTTCG ATGTAACCCA 4500 CTCGTGCACC CAACTGATCT TCAGCATCTT TTACTTTCAC CAGCGTTTCT GGGTGAGCAA 4560 AAACAGGAAG GCAAAATGCC GCAAAAAAGG GAATAAGGGC GACACGGAAA TGTTGAATAC 4620 TCATACTCTT CCTTTTTCAA TATTATTGAA GCATTTATCA GGGTTATTGT CTCATGAGCG 4680 GATACATATT TGAATGTATT TAGAAAAATA AACAAATAGG GGTTCCGCGC ACATTTCCCC 4740 GAAAAGTGCC ACCTGACGTC TAAGAAACCA TTATTATCAT GACATTAACC TATAAAAATA 4800 GGCGTATCAC GAGGCCCTTT CGTC 4824 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3915 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: EcoRI-Hindlll region of plasmid pCOL13 (ix) FEATURE: (A) NAME/KEY: prim_transcript (B) LOCATION: 188 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 188..212 (ix) FEATURE: (A) NAME/KEY: intron (B) LOCATION:213..556 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 557..718 (ix) FEATURE: (A) NAME/KEY: intron (B) LOCATION: 719..1224 ( ix ) FEATURE : (A) NAME/KEY: exon (B) LOCATION: 1225..2770 (D) OTHER INFORMATION:/codon_start= 2 /note= "exon containing 3' end coding region of B-peru gene" (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 576..718 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1225..2770 (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1268..2770 (D) OTHER INFORMATION:/note= "3' end of B-peru coding region which is derived from cDNA" (ix) FEATURE: (A) NAME/KEY: 3'UTR (B) LOCATION:2771..3272 (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:3273..3891 (D) OTHER INFORMATION: /label= 3 'region /note= "further 3' flanking region of B-peru gene. This region is only of approximate length and the sequence needs to be confirmed. " (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..6 (D) OTHER INFORMATION:/label= EcoRI (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 11..16 (D) OTHER INFORMATION: /label= Xbal (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:45..50 (D) OTHER INFORMATION:/label= Kpnl (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:265..270 (D) OTHER INFORMATION:/label= Hindlll (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:329..334 (D) OTHER INFORMATION:/label= Xbal (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 835..840 (D) OTHER INFORMATION: /label= BamHI (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1268..1273 (D) OTHER INFORMATION:/label= Mlul (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2787..2792 (D) OTHER INFORMATION: /label= Hindlll (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2883..2888 (D) OTHER INFORMATION: /label= Muni (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 2827..2832 (D) OTHER INFORMATION: /label= Hindlll (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 3892..3897 (D) OTHER INFORMATION:/label= Sail (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 3910..3915 (D) OTHER INFORMATION: /label= Hindlll (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 3892..3915 (D) OTHER INFORMATION:/label= polylinker /note= "part of polylinker of pUC19" ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GAATTCAGGT TCTAGACTAT TCTTGTGGCC TCGGGCGGAT GGCGGGTACC CATGTCTTCG 60 TTAGGCTTAT CTGACCGTGG AGATGAAATC TAACGGCTCA TAGAAATTAA ACTAACGTGG 120 ACACTCTGTC CTTGCTGTTT TGCTCCCTGC TCTTTATATA TAGAATGCCT GCTTGCATTG 180 CACCCGTACG TACAGCGTAG CGCGGAGTGG AGGTGAGCTC CTCCTCCGAT TCTTGCCTAA 240 TCTTTGGTCT TTGCACACGT ACGAAAGCTT TTTGCATTGT TTCGTTGCTT CTGGATGATC 300 AGTACTCTTA GATATTAAGC GATACCGATC TAGAATCGAG TTGTTGTACT CTCTCTGTCC 360 CTTTTGTGCA GCTATAACTA GCTAGGTTCC TTCGCATAGA GCCTCTCTAC AGAGTACAGA 420 CTAGCTAGCA GTGTCAGACA CGAAATGGAA ATGGTCACTT CCAAATTGCA CGAGCTGGAA 480 TTATATACTC TTCTGATCTT CTTCACCGTC TCTTTATAGC GTGATATGCG TTTCTGGCTT 540 CTTGCTTACG TGAAGGATTA TTAGTAAGGC GCGTGATGGC GCTCTCAGCT TCCCCGGCTC 600 AGGAAGAACT GCTGCAGCCT GCTGGGAGGC CGTTGAGGAA GCAGCTTGCT GCAGCCGCGA 660 GGAGCATCAA CTGGAGCTAT GCCCTCTTCT GGTCCATTTC AAGCACTCAA CGACCTCGGT 720 AAATGGAAGT CCTGATAATC TATAATTTGT CTGGCAGTTT TCTACAACTC TGGTGAATGA 780 TCGTCACTTC GTTTGCCTGA TACATACATA CATACATATG AAATAAAGAA AGTCGGATCC 840 CGTGATGCGA TTGTAGTTAT CGCTTTTCCG CAAAATGGTT GCTTTTTGAA TCTGCATTCG 900 TTTTTTTCCC ACATCTTCTT CCTTCTCGCG AGTAACGACA ACGCCACCCG CGCCGCCTGC 960 CGCCCATCGC CCCGCCTTGG CCGGCGAGAG CCTCAGCCTA TTACACCAGC GGCGACCTCT 1020 TTTCCCCTTC CTCTCACCGC CCTCGTGGCC GTGCTCTCCC CCGCTCTAAC CTGGTCTGGC 1080 CGCCTCCGCT GCCACCTGCT CCGGCGGCCT CACCCGCGTC TTTCTCGTCC CTACCCTCTC 1140 TGCCTCTGGG CGCATCATCA TCTGATATTC TGATGCAAAT AAAAAAGGTA TACCATATAA 1200 GGACAACAGA AAATATGGTT GCAGGGTGCT GACGTGGACG GACGGGTTCT ACAATGGCGA 1260 GGTGAAGACG CGTAAGATCT CCCACTCCGT GGAGCTGACA GCCGACCAGC TGCTCATGCA 1320 GAGGAGCGAG CAGCTCCGGG AGCTCTACGA GGCCCTCCGG TCCGGCGAGT GCGACCGCCG 1380 CGGCGCGCGG CCGGTGGGCT CGCTGTCGCC GGAGGACCTC GGGGACACCG AGTGGTACTA 1440 CGTGATCTGC ATGACCTACG CCTTCCTGCC GGGCCAAGGC TTGCCCGGCA GGAGTTCCGC 1500 GAGCAACGAG CATGTCTGGC TGTGCAACGC GCACCTCGCC GGCAGCAAGG ACTTCCCCCG 1560 CGCGCTCCTG GCCAAGAGCG CGTCCATTCA GACAATCGTC TGCATCCCGC TCATGGGTGG 1620 CGTGCTTGAG CTTGGTACTA CTGATAAGGT GCCGGAGGAC CCGGACTTGG TCAGCCGAGC 1680 AACCGTAGCA TTCTGGGAGC CGCAATGTCC GACATACTCG AAAGAGCCGA GCTCCAACCC 1740 GTCAGCATAC GAAACCGGGG AAGCCGCATA CATAGTCGTG TTGGAGGACC TCGATCACAA 1800 TGCCATGGAC ATGGAGACGG TGACTGCCGC CGCCGGGAGA CACGGAACCG GACAGGAGCT 1860 AGGAGAAGTC GAGAGCCCGT CAAATGCAAG CCTGGAGCAC ATCACCAAGG GGATCGACGA 1920 GTTCTACAGC CTCTGCGAGG AAATGGACGT GCAGCCGCTA GAGGATGCCT GGATAATGGA 1980 CGGGTCTAAT TTCGAAGTCC CGTCGTCAGC GCTCCCGGTG GATGGCTCAA GCGCACCCGC 2040 TGATGGTTCT CGCGCGACAA GTTTCGTGGT TTGGACGAGG TCATCGCACT CCTGCTCGGG 2100 TGAAGCGGCG GTGCCGGTCA TCGAAGAGCC GCAGAAATTG CTGAAGAAAG CGTTGGCCGG 2160 CGGCGGTGCT TGGGCGAACA CGAACTGCGG TGGCGGGGGC ACGACGGTAA CAGCCCAGGA 2220 AAACGGCGCC AAGAACCACG TCATGTCAGA GCGAAAGCGC CGGGAGAAGC TCAACGAGAT 2280 GTTCCTCGTT CTCAAGTCGT TGGTTCCCTC CATTCACAAG GTGGACAAAG CATCCATCCT 2340 CGCCGAAACG ATAGCCTATC TAAAGGAGCT TCAACGAAGG GTACAAGAAC TGGAATCCAG 2400 GAGGCAAGGT GGCAGTGGGT GTGTCAGCAA GAAAGTCTGT GTGGGCTCCA ACTCCAAGAG 2460 GAAGAGCCCA GAGTTCGCCG GTGGCGCGAA GGAGCACCCC TGGGTCCTCC CCATGGACGG 2520 CACCAGCAAC GTCACCGTCA CCGTCTCGGA CACGAACGTG CTCCTGGAGG TGCAATGCCG 2580 GTGGGAGAAG CTCCTGATGA CACGGGTGTT CGACGCCATC AAGAGCCTCC ATTTGGACGC 2640 TCTCTCGGTT CAGGCTTCGG CACCAGATGG CTTCATGAGG CTCAAGATAG GAGCTCAGTT 2700 TGCAGGCTCC GGCGCCGTCG TGCCCGGAAT GATCAGCCAA TCTCTTCGTA AAGCTATAGG 2760 GAAGCGATGA AAGGGCGCTA CATGTGAAGC TTAATTAATG GAAGCAAACT TGTATTTCTT 2820 GTGCAAAAGC TTACTATATA TTTCTGCAAA ACCTGGTGTG CCTTGTTTTG ATTTTCAGTC 2880 GCCAATTGTG CCTTTGTTTT TATCAAGTGA TGATCTACAC ATATATATAG GAATATTTGA 2940 AAAGAGCGAT GTCATAGGGT TTTTTTATTA CAAGGAACAA GTCTTTCACG TGCTGGCCTC 3000 ACAAATCCTA AGAGAAAATC TGCTCATTTT GATTGCGTTC CGCAACAACT CTGTAATCCA 3060 TATCCTATGT ATCCGATCAA CTAGTCGATA GCCTCCGTCC GCCACATCAT CATATATCTA 3120 TCTATGTGTG TCATCTGACA CATACTCCTC GCGTACTGTG CTGACATATG ATACTGACAC 3180 AGCATATATG CATGCACATC GTCACACGAC ATATATCTCG CTACTACACA GATATTGGAT 3240 ACGATACTAT ATAGCATCAT GCGTGCTGCG ATNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3300 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3360 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3420 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3480 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3540 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3600 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3660 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3720 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3780 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN 3840 NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NNNNNNNNNN NGTCGACCTG 3900 CAGGCATGCA AGCTT 3915 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4137 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: (A) ORGANISM: EcoRI-Hindlll region of plasmid pCOL13 (ix) FEATURE: (A) NAME/KEY: prim_transcript (B) LOCATION: 188 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 188..212 (ix) FEATURE: (A) NAME/KEY: intron (B) LOCATION:213..556 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 557..718 (ix) FEATURE: (A) NAME/KEY: intron (B) LOCATION: 719..1224 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1226..2771 (D) OTHER INFORMATION: /codon_start= 2 /note= "exon containing 3' end coding region of B-peru gene, this exon continues up to the polyadenylation site." (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 576..718 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1226..2771 (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1269..2771 (D) OTHER INFORMATION:/note= "fragment of B-peru coding region which is derived from cDNA" (ix) FEATURE: (A) NAME/KEY: 3'UTR (B) LOCATION:2772..4137 (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1..6 (D) OTHER INFORMATION:/label= EcoRI (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 11..16 (D) OTHER INFORMATION: /label= Xbal (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 45..50 (D) OTHER INFORMATIO :/label= Kpnl (i ) FEATURE: (A) NAME/KEY: - (B) LOCATION:265..270 (D) OTHER INFORMATION:/label= Hindlll (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 329..334 (D) OTHER INFORMATION:/label= Xbal (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 835..840 (D) OTHER INFORMATION:/label= BamHI (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 1269..1274 (D) OTHER INFORMATION:/label= Mlul (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 2788..2793 (D) OTHER INFORMATIO :/label= Hindlll (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2884..2889 (D) OTHER INFORMATION:/label= Muni (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:2828..2833 (D) OTHER INFORMATIO :/label= Hindlll (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 4114..4119 (D) OTHER INFORMATION:/label= Sail (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION:4132..4137 (D) OTHER INFORMATION:/label= Hindlll (ix) FEATURE: (A) NAME/KEY: - (B) LOCATION: 4114..4137 (D) OTHER INFORMATIO :/label= polylinker /note= "part of polylinker of pUC19" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GAATTCAGGT TCTAGACTAT TCTTGTGGCC TCGGGCGGAT GGCGGGTACC CATGTCTTCG 60 TTAGGCTTAT CTGACCGTGG AGATGAAATC TAACGGCTCA TAGAAATTAA ACTAACGTGG 120 ACACTCTGTC CTTGCTGTTT TGCTCCCTGC TCTTTATATA TAGAATGCCT GCTTGCATTG 180 CACCCGTACG TACAGCGTAG CGCGGAGTGG AGGTGAGCTC CTCCTCCGAT TCTTGCCTAA 240 TCTTTGGTCT TTGCACACGT ACGAAAGCTT TTTGCATTGT TTCGTTGCTT CTGGATGATC 300 AGTACTCTTA GATATTAAGC GATACCGATC TAGAATCGAG TTGTTGTACT CTCTCTGTCC 360 CTTTTGTGCA GCTATAACTA GCTAGGTTCC TTCGCATAGA GCCTCTCTAC AGAGTACAGA 420 CTAGCTAGCA GTGTCAGACA CGAAATGGAA ATGGTCACTT CCAAATTGCA CGAGCTGGAA 480 ■TTATATACTC TTCTGATCTT CTTCACCGTC TCTTTATAGC GTGATATGCG TTTCTGGCTT 540 CTTGCTTACG TGAAGGATTA TTAGTAAGGC GCGTGATGGC GCTCTCAGCT TCCCCGGCTC 600 AGGAAGAACT GCTGCAGCCT GCTGGGAGGC CGTTGAGGAA GCAGCTTGCT GCAGCCGCGA 660 GGAGCATCAA CTGGAGCTAT GCCCTCTTCT GGTCCATTTC AAGCACTCAA CGACCTCGGT 720 AAATGGAAGT CCTGATAATC TATAATTTGT CTGGCAGTTT TCTACAACTC TGGTGAATGA 780 TCGTCACTTC GTTTGCCTGA TACATACATA CATACATATG AAATAAAGAA AGTCGGATCC 840 CGTGATGCGA TTGTAGTTAT CGCTTTTCCG CAAAATGGTT GCTTTTTGAA TCTGCATTCG 900 TTTTTTTCCC ACATCTTCTT CCTTCTCGCG AGTAACGACA ACGCCACCGC GCGCCGCCTG 960 CCGCCCATCG CCCCGCCTTG GCCGGCGAGA GCCTCAGCCT ATTACACCAG CGGCGACCTC 1020 TTTTCCCCTT CCTCTCACCG CCCTCGTGGC CGTGCTCACC CCCGCTCTAA CCTGGTCTGG 1080 CCGCCTCCGC TGCCACCTGC TCCGGCGGCC TCACCCGCGT CTTTCTCGTC CCTACCCTCT 1140 CTGCCTCTGG GCGCATCATC ATCTGATATT CTGATGCAAA GAAAAAAGGT ATACCATATA 1200 AGGACAACAG AAAATATGGT TGCAGGGTGC TGACGTGGAC GGACGGGTTC TACAATGGCG 1260 AGGTGAAGAC GCGTAAGATC TCCCACTCCG TGGAGCTGAC AGCCGACCAG CTGCTCATGC 1320 AGAGGAGCGA GCAGCTCCGG GAGCTCTACG AGGCCCTCCG GTCCGGCGAG TGCGACCGCC 1380 GCGGCGCGCG GCCGGTGGGC TCGCTGTCGC CGGAGGACCT CGGGGACACC GAGTGGTACT 1440 ACGTGATCTG CATGACCTAC GCCTTCCTGC CGGGCCAAGG CTTGCCCGGC AGGAGTTCCG 1500 CGAGCAACGA GCATGTCTGG CTGTGCAACG CGCACCTCGC CGGCAGCAAG GACTTCCCCC 1560 GCGCGCTCCT GGCCAAGAGC GCGTCCATTC AGACAATCGT CTGCATCCCG CTCATGGGTG 1620 GCGTGCTTGA GCTTGGTACT ACTGATAAGG TGCCGGAGGA CCCGGACTTG GTCAGCCGAG 1680 CAACCGTAGC ATTCTGGGAG CCGCAATGTC CGACATACTC GAAAGAGCCG AGCTCCAACC 1740 CGTCAGCATA CGAAACCGGG GAAGCCGCAT ACATAGTCGT GTTGGAGGAC CTCGATCACA 1800 ATGCCATGGA CATGGAGACG GTGACTGCCG CCGCCGGGAG ACACGGAACC GGACAGGAGC 1860 TAGGAGAAGT CGAGAGCCCG TCAAATGCAA GCCTGGAGCA CATCACCAAG GGGATCGACG 1920 AGTTCTACAG CCTCTGCGAG GAAATGGACG TGCAGCCGCT AGAGGATGCC TGGATAATGG 1980 ACGGGTCTAA TTTCGAAGTC CCGTCGTCAG CGCTCCCGGT GGATGGCTCA AGCGCACCCG 2040 CTGATGGTTC TCGCGCGACA AGTTTCGTGG TTTGGACGAG GTCATCGCAC TCCTGCTCGG 2100 GTGAAGCGGC GGTGCCGGTC ATCGAAGAGC CGCAGAAATT GCTGAAGAAA GCGTTGGCCG 2160 GCGGCGGTGC TTGGGCGAAC ACGAACTGCG GTGGCGGGGG CACGACGGTA ACAGCCCAGG 2220 AAAACGGCGC CAAGAACCAC GTCATGTCAG AGCGAAAGCG CCGGGAGAAG CTCAACGAGA 2280 TGTTCCTCGT TCTCAAGTCG TTGGTTCCCT CCATTCACAA GGTGGACAAA GCATCCATCC 2340 TCGCCGAAAC GATAGCCTAT CTAAAGGAGC TTCAACGAAG GGTACAAGAA CTGGAATCCA 2400 GGAGGCAAGG TGGCAGTGGG TGTGTCAGCA AGAAAGTCTG TGTGGGCTCC AACTCCAAGA 2460 GGAAGAGCCC AGAGTTCGCC GGTGGCGCGA AGGAGCACCC CTGGGTCCTC CCCATGGACG 2520 GCACCAGCAA CGTCACCGTC ACCGTCTCGG ACACGAACGT GCTCCTGGAG GTGCAATGCC 2580 GGTGGGAGAA GCTCCTGATG ACACGGGTGT TCGACGCCAT CAAGAGCCTC CATTTGGACG 2640 CTCTCTCGGT TCAGGCTTCG GCACCAGATG GCTTCATGAG GCTCAAGATA GGAGCTCAGT 2700 TTGCAGGCTC CGGCGCCGTC GTGCCCGGAA TGATCAGCCA ATCTCTTCGT AAAGCTATAG 2760 GGAAGCGATG AAAGGGCGCT ACATGTGAAG CTTAATTAAT GGAAGCAAAC TTGTATTTCT 2820 TGTGCAAAAG CTTACTATAT ATTTCTGCAA AACCTGGTGT GCCTTGTTTT GATTTTCAGT 2880 CGCCAATTGT GCCTTTGTTT TTATCAAGTG ATGATCTACA CTATATATAT GGAATATTTG 2940 AAAAGAGCGA TGTCATAGGG TTTTTTTATT ACAAGGAACA AGTCTTTCAC GTGCTGGCCT 3000 CACAAATCCA AGAGAAAATC TGCTCATTTT GATTGGCTTC CGCAACAACT CTGTAATCCA 3060 TATCCTTTGT ATCCGATCAA CTATGATACC TCCTCCCCCA TCTCTTTTTT TTTTATCTGC 3120 ACAATCTTCT ATTCTACTAT AATGAAACAA TAGAGCCACT ACCGAATATT TCCTCAAAAA 3180 TGTACAACAA ACTAGGGTGG TCCAAACAAA TGCCTAGAGG AGCTAGATTC TCTTAAATTA 3240 GACATCGGTT TCTTTTATCT CTTCCAGAAG GGATAAAAGT ATGTGTTTAT GGTCTTCAGT 3300 AATACATTGT TCGTTTCTCA TAGTCAATTT AGAGGTGTTT AAATGTACTT GAACTAATAG 3360 TTAGTTGGTT TAAAAATTAC TATTAAAATT AGTTAGTTAA TAAATAGCTA GCTAAATATT 3420 AGCTAATTTG TCAAAAGTAG CTAATAGCTG AATTATTAGC TATATTGTTT TGATGTCTTC 3480 AGCTAATTTT AGCAGATCAT TATTAGTTCT AGTGTATCTA AACACACCCT TAGTCAAACA 3540 TGGTAAAAAA AAAGTTGATT CACTCATTGC TCATCGAAGA CGCAGATCAT GGCATCCCTC 3600 ACACGTTCTT CAGCCTACAC GGCACTTGCA TTGTAATTGC ATCTCATCTC ATCAACCCTT 3660 GTTGTGCATT ACTTGCCACA TGCGCCATCA ATTAACATTT TTTTGTCTCG TTCCTGAATT 3720 TCCTAACAAA TTTCATCAAA TGTACGCAGA GCTAAAGCTA GCTGTCGATG TCAGTTGACA 3780 GTTGACACCG ATGAATTTTA GAAAATTTAG TGTAAAGTAC TATTTATAAT GTTCATGACA 3840 CCCATATAAA ATATGTTGAC ACCGGCAAAC CTCAAGGCTA GCTTCGCCCC TGCCATCAAC 3900 CTTACATCTA CATTCACCAC GAGGTGTGCA CGGCCTAGGT TCGACTCCTA TGTCATGCCT 3960 TGCTATCTAC AGATTCAGCA AGTGTTGTGT TCCTTGTTGT CACAATCTAC CTTTATTATA 4020 AAATTGATGT CATATCATGC CAAACAACAA ATAATTAATA TCGTGTGAAA TTTGAATTTC 4080 TCTAACATGC TCAACCAACC TTACCCCTTC ACGGTCGACC TGCAGGCATG CAAGCTT 4137 SEQ . ID No . 7 acta g tacctg t cgc g cgcccat g cgcgcgtggcgtgcttctcgccctggtaactgttctcggcaaatga ctatttccaagtaaacatattcaatgatttt g ctattctta g caaagtaatttcacttggacttttgtgc caaaaacgcattg g aaaaaatctccttg g actccagcctaaggttgaaagtgtaaaaactgggaaaaatt at t gat g tttcgg g cagt t ac tt ggctat g taaattccataccttttcaaaatatcctaaacattctttt ctgtttctgcaacatacat g tttatcagttctggacctttgacgctacgaaagttcagtgagtattcagg ctttcgcaagtaaaaccta g aagtccaacggacattcattttagcgatcccatgtctttaggatgcactt gttatcggatgtctcctatgagacagaatgcacttgttatggtaactaaacaaaaaaatataatttaatt cgtgtgaaactttttcaaacctaccttccctgttcccggaggtccatatacccagacacctaatcgcttg cgcaatttagaagaaatcatgcgattatacgtcaaagggagctgaaatatcaagcaaaagaaaaggtcat cccacaaaagcccaaaactattgtagggaaaacacttgttttacctataattgagcgtcgtattggtgtt gctgatatttactgctaaaccaagtccaatttaccagaatagtatσtagaagaatccttttcacatcctc tagcccgccaacatcctaccatttgacattgagaactaaaaaacaaattgttcccagacgaaagctaaag tcgctttatacgattagctgcagtaggtgagcacgatctccgaacgctgggcatgacaαgaccatgatag acgacatggacattttgtcaaacacctgcatggcgtcaccagggaaaacaatccagcaggagagttggga gagagatggaaacaattaattatgcaaacacggaggagacacaatttgaagagtgttcgtacacctacgg caatcagcgaaacgatgagagagcataccaagctcgggtcgtcagacacgcggaggacggacggtggcac cgatggagatggagacagttgcgtgccgttttttgtggagggcttcgttggtgtcgggcgtcggcggagc etgaacgcggfcgggaagaagagcggcgtggtgggaagaagagcgacgtcaggttctagactattcttgtg gcctcgggcggatggcgggtacccatgtcttcgttaggcttatctgaccgtggagatgaaatctaacggc tca agaaattaaactaacgtggactcccagacgaaagctaaagtcgctttatacgattagctgcagtag gtgagcacgatctccgaacgctgggcatgacacgaccatgatagacgacatggacattttgtcaaacacc tgcatggcgtcaccagggaaaacaatccagcaggagagttgggagagagatggaaacaattaattatgca aacacggaggagacacaatttgaagagtgttcgtacacctacggcaatcagcgaaacgatgagagagcat accaagctcgggtcgtcagcaacgcggaggacggacggtggcaccgatggagatggagacagttgcgtgc cgttttttgtggagggcttcgttggtgtcgggcgtcggcggagcctgaacgσggtgggaagaagagcttc gtggtgggaagaagagcgacgaσaggttctagactattcttgtggcctcgggcggatggcgggtaccc&t gtcttcgttaggcttatctgaccgtggagatgaaatctaacggctcatagaaattaaactaacgtggaca ctctgtccttgctgttttgctccctgctctttatatatagaatgcctgcttgcattgcacσcgtacgtac agcgtagcgcggagtggaggtgagctcctcctccgattcttgcctaatctttggtctttgcacacgtacg aaagctttttgcattgtttcgttgcttctggatgatcagtactcttagatattaagcgataccgatctag aatcgagttgttgtactctctctgtcccttttgtgcagctataactagctaggttccttcgcatagagcc tctctacagagtacagactagctagcagtgtcagacacgaaatggaaatggtcacttccaaattgcacga gctggaattatatactcttctgatcttcttcaccgtctctttatagcgtgatatgcgtttctggcttctt gcttacgtgaaggattattagtaaggcgcgtgatggcgctctcagcttccccggctcaggaagaactgct gcagcctgctgggaggccgttgaggaagcagcttgctgcagccgcgaggagcatcaactggagctatgec ctcttctggtccatttcaagcactcaacgacctcggtaaatggaagtcctgataatctataatttgtctg gcagttttctacaactctggtgaatgatcgtcacttcgtttgcctgatacatacatacatacatatgaaa taaagaaagtcggatcccgtgatgcgattgtagttatcgcttttccgcaaaatggttgctttttgaatct gel

Claims: CLAIMS 1. A plant consisting essentially of cells which comprise in their genome: - a homozygous male-sterility genotype at a first genetic locus; and - a color-linked restorer genotype at a second genetic locus, which is heterozygous (Rf/-)for a foreign DNA Rf comprising: a) a fertility-restorer gene capable of preventing the phenotypic expression of said male-sterility genotype, and b) at least one anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesis in cells of seeds of said plant which is capable of producing anthocyanin at least in the seeds of said plant, so that anthocyanin production in the seeds is visible externally. 2. The plant of claim 1 in which said color gene iε capable of producing anthocyanin at least in the aleurone of the seeds of said plant. 3. The plant of claim 1, in which said first genetic locus is homozygous for a foreign RNA S (S/S) which comprises a male-sterility gene which when generated in cells of the plant renders the plant male-sterile without otherwise substantially affecting the growth and development of the plant. 4. The plant of claim 1, in which said first genetic locus is homozygous for a foreign DNA S (S/S) which comprises a male-sterility gene which comprises: si) a male-sterility DNA encoding a RNA, protein or polypeptide which, when produced or overproduced in a stamen cell of the plant, significantly disturbs the metabolism, functioning and/or development of said cell, and, s2) a sterility promoter capable of directing expression of the male-sterility DNA selectively in the stamen cells, preferably the tapetum cells, of the plant; the male-sterility DNA being in the same transcriptional unit as, and under the control of, the sterility promoter, and in which said fertility restorer gene in said second genetic locus comprises at least: al) a fertility-restorer DNA encoding a restorer RNA, protein or polypeptide which, when produced or overproduced in the same cell as said male-sterility gene S, prevents the phenotypic expression of S, and, a2) a restorer promoter capable of directing expression of the fertility-restorer DNA at least in the same cells in which said male-sterility gene is expressed, so that the phenotypic expression of said male-sterility gene is prevented; the fertility- restorer DNA being in the same transcriptional unit aε, and under the control of, the restorer promoter. 5. The plant of claim 1 in which said male-sterility DNA encodeε barnase and in which said fertility restorer DNA encodes barstar. 6. The plant of claim 1 in which the sterility promoter and/or the restorer promoter is selected from the group conεisting of PTA29, PCA55, PT72, PT42, and PEl. 7. The plant of claim 1 in which the homozygous male- steriity genotype iε endogenouε and is homozygous for a receεεive allele m (m/m) and in which the fertility restorer gene is the dominant allele M of εaid endogenouε male-εterility genotype. 8. The plant of claim 1, which iε a cereal plant which iε selected from the group consisting of corn, wheat, and rice. 9. The plant according to claim 1 wherein said anthocyanin regulatory gene is a εhortened R, B or Cl gene or a combination of εhortened R, B or Cl geneε which is functional for conditioning and regulating anthocyanin production in the aleurone. 10. The plant according to claim 9 wherein said anthocyanin regulatory gene is selected from the group consisting of a shortened Cl or Cl-S gene having a nucleotide sequence corresponding to the sequence between positionε 447 and 2418 of SEQ ID No. 1, a εhortened B- peru gene having a nucleotide εequence corresponding to the sequence between positionε 1 and 3272 of SEQ of ID NO. 6; and the Eco-Sall fragment having a length of about 4 aaa bp of pCOL13. 11. The plant according to claim 10 wherein said anthocyanin regulartory gene does not contain any introns. 12. The plant according to claim 9 wherein εaid anthocyanin regulatory gene compriεes a shortened Cl or Cl-S gene and a shortened B-peru gene. 13. The plant according to claim 9 wherein εaid anthocyanin regulatory gene is a chimaric DNA comprising a coding region of an R or B gene and/or Cl gene operably linked to an aleurone-specific promoter. 14. The plant according to claim 13 wherein said aleurone-εpecific promoter iε selected from the group consisting of: the sequence between positions 1 to 1077 or 447 to 1077 of SEQ ID No. 1, and the sequence between positions 1-575 of εequence ID No. 6. 15. The plant according to claim 14, wherein said aleurone-specific promoter is selected from the group consiεting of: the εequence between positions 1 to 1061 or 447 to 1061 of SEQ ID No. 1, and the sequence between positions 1 to 188 of SEQ ID No. 6. 16. A DNA comprising an anthocyanin regulatory gene which is a εhortened R, B or Cl gene or a combination of εhortened R, B or Cl geneε which is functional for conditioning and regulating anthocyanin production in the aleurone. 17. A DNA according to claim 16, which comprises a shortened Cl or Cl-S gene and a shortened B-peru gene. 18. A DNA according to claim 16, which compriεeε at leaεt one gene selected from the group consisting of a shortened B-peru gene having a nucleotide sequence corresponding to the sequence between positions 1 and 3272 of SEQ ID No. 6, a shortened B-peru gene which is the EcoRI-Sall fragment with a length of about 4 000 bp of pCOL13 and the shortened Cl or Cl-S gene having a nucleotide sequence corresponding to the sequence between poεitions 447 and 2418 SEQ ID No. 1. 19. The DNA of claim 18 in which said shortened B-peru, Cl or Cl-S gene is further characterized by not containing any intron. 20. A DNA according to claim 16, wherein said shortened Cl, Cl-S or B-peru genes are operably linked to an aleurone-specific promoter selected from the group consisting of: the sequence between positions 1 to 1077 or 447 to 1077 of SEQ ID No. 1 and the sequence between positionε 1-575 of ID No. 6. 21. A DNA according to claim 19, wherein εaid aleurone- εpecific promoter iε εelected from the group conεiεting of: the sequence between positionε 1 to 1061 or 447 and 1061 of SEQ ID No. 1 and the sequence between positions 1 to 188 of SEQ. ID No. 6. 22. A DNA according to claim 16 which further compriseε a fertility-reεtorer gene capable of preventing the phenotypic expreεεion of a male-εterioity genotype in a plant. 23. A DNA according to claim 22 wherein εaid fertility- reεtorer gene encodeε barεtar. 24. A DNA according to claim 23, wherein barεtar iε under the control of a promoter εelected from the group consisting of PTA29, PCA55, PT72, PT42 and PEl. 25. An aleurone-specific promoter selected from the group consiεting of: the εequence between positionis 1 to 1077 or 447 to 1077 of SEQ ID No. 1 and the sequence between positions 1-575 of SEQ ID No. 6. 26. An aleurone-specific promoter selected from the group consiεting of: the sequence between positions 1 to 1061 or 447 and 1061 of SEQ ID No. 1 and the sequence between positions 1 to 188 of SEQ ID No. 6. 27. A process to maintain a line of male-sterile plants, which compriseε the following εteps: i) crossing a) a male-εterile parent plant of said line having in a first genetic locus, a homozygous male-εterility genotype and b) a maintainer parent plant of εaid line conεiεting eεεentially of cells which comprise, stably intergrated in their nuclear genome: a homozygous male-sterility genotype at a first genetic locus; and - a colored-linked restorer genotype at a εecond genetic locus, which iε heterozygouε for a foreign DNA comprising: i) a fertility-restorer gene capable of preventing the phenotypic expresεion of εaid male-sterility genotype, and ii) at least one anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesis in cells of seeds of said plant which is capable of producing anthocyanin at least in the seeds of said plant, so that anthocyanin production in the εeeds is visible externally, obtaining the seeds from said parent plants, and iii) separating on the basiε of color, the seeds in which no anthocyanin is produced and which grow into male- sterile parent plants. 28. A procesε according to claim 27, wherein the genome of εaid male-εterile parent plant doeε not contain at least one anthocyanin regulatory gene neceεεary for the regulation of anthocyanin bioεyntheεiε in the εeedε of said plant to produce externally visible anthocyanin in said seedε. 29. The proceεε of claim 28, wherein the genome of aid male-εterile parent plant containε a firεt anthocyanin regulatory gene and the genome of εaid maintainer parent plant containε a εecond anthocyanin regulatory gene which, when preεent with εaid firεt anthocyanin regulatory gene in the genome of a plant is capable of conditioning the production of externally visible anthocyanin in seeds. 30. A procesε to maintain a line of maintainer plantε, which compriεeε the following steps: i) crosεing: a) a male-εterile parent plant of εaid line having, in a firεt genetic locuε, a homozygous male-sterility genotype, and b) a maintainer parent plant of said line consiεting eεεentially of cellε which compriεe, εtably integrated in their nuclear genome: a homozygous male-εterility genotype at a first genetic locus; and a colored-linked restorer genotype at a second genetic locuε, which is heterozygous for a foreign DNA comprising: i) a fertility-reεtorer gene capable of preventing the phenotypic expreεεion of said male- sterility genotype, and ii) at least one anthocyanin regulatory gene involved in the regulation of anthocyanin biosynthesiε in cellε of εeedε of εaid plant which is capable of producing anthocyanin at least in the εeeds of said plant, so that anthocyanin production in the εeedε iε viεible externally. ii) obtaining the seeds from said male-εterile parent plant, and iii) separating on the basiε of color, the εeedε in which anthocyanin iε produced and which grow into maintainer parent plants. 31. A process according to claim 30, wherein the genome of said male-sterile parent plant doeε not contain at leaεt one anthocyanin regulatory gene neceεεary for the regulation of anthocyanin biosynthesis in the seeds of said plant to produce externally visible anthocyanin in said seedε. 32. The process of claim 31, wherein the genome of said male-sterile parent plant containε a firεt anthocyanin regulatory gene and the genome of εaid maintainer parent plant containε a εecond anthocyanin regulatory gene which, when preεent with εaid first anthocyanin regulatory gene in the genome of a plant is capable of conditioning the production of externally visible anthocyanin in εeedε. 33. A kit for maintaining a line of male-εterile or maintainer plantε, said kit comprising: a) a male-εterile parent plant of εaid line having, in a firεt genetic locus, a homozygous male-sterility genotype, and b) a maintainer parent plant of said line conεiεting essentially of cells which comprise, integrated in their nuclear genome : a homozygous male-sterility genotype at a first genetic locus; and a colored-linked reεtorer genotype at a εecond genetic locuε, which iε heterozygouε for a foreign DNA compriεing: i) a fertility-reεtorer gene capable of prevening the phenotypic expression of said male-εterility genotype, and ii) at leaεt one anthocyanin regulatory gene involved in the regulation of anthocyanin bioεyntheεiε in cellε of seeds of said plant which iε capable of producing anthocyanin at leaεt in the seeds of said plant, εo that anthocyanin production in the εeeds is visible externally. 34. A proceεε according to claim 33, wherein the genome of said male-εterile parent plant does not contain at least one anthocyanin regulatory gene necessary for the regulation of anthocyanin biosynthesis in the seedε of said plant to produce externally visible anthocyanin in said seeds. 35. The process of claim 34, wherein the genome of said male-sterile parent plant containε a firεt anthocyanin regulatory gene and the genome of said maintainer parent plant containε a εecond anthocyanin regulatory gene which, when present with said first anthocyanin regulatory gene in the genome of a plant is capable fo conditioning the production of externally viεible anthocyanin in εeedε. 36. Process to maintain a kit according to claim 33 which comprises: - crosεing εaid male-εterile parent plant with εaid maintainer parent plant; obtaining the seedε from εaid male-sterile parent plants and optionally the εeedε from εaid maintainer parent plant in which no anthocyanin iε produced; and - optionally growing εaid εeedε into male-sterile parent plantε and maintainer parent plantε.

Inventors: KREBBERS ENNO, LEEMANS JAN, WILLIAMS MARK, KREBBERS, ENNO, LEEMANS, JAN, WILLIAMS, MARK
Applicants: KREBBERS ENNO, LEEMANS JAN, PLANT GENETIC SYSTEMS NV, WILLIAMS MARK, KREBBERS, ENNO, LEEMANS, JAN, PLANT GENETIC SYSTEMS, N.V., WILLIAMS, MARK
Application year: 1995
Main classification: C12N15
IPC codes: A01H1, A01H5, C07K14, C12N5, C12N15