MXPA97009730A - Use of the indeterminate gametophyte gene to improve the m - Google Patents

Abstract

The present invention relates to a methodology for checking the function of genes linking the selection of mutations in androgenetic haploids that are produced by fertilizing a maize plant carrying the undetermined gametophyte gene (gi) with the pollen obtained from a mutagenized plant. Changes in the phenotype of the hybrid progeny are then identified and characterized. A method is provided to direct the selection of androgenetic haploids

Description

USE OF THE GENE OF INDETERMINATE GAMETOPHYTES TO IMPROVE THE CORN BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to methods of checking the function of the gene in which mutations are selected in the androgenetic haploids that are produced as a result of the fertilization of a maize plant carrying the indeterminate gametophyte gene (ig) with the pollen obtained from a mutagenized plant. The present invention also relates to the identification of genes that control quantitative characters in which the undetermined gametophyte gene and the mutation are in different internal crosses. The present invention also relates to maize plants that carry the undetermined gametophyte gene that produces high frequency androgenetic haploids. The present invention also relates to a method for directly selecting androgenetic haploids produced from maize plants with indeterminate gametophyte. 2. Background Genes that affect a given trait are recognized by their mutant phenotypes and therefore methods that enhance the ability to observe those mutants will accelerate the discovery of the corresponding genes. Conventional diploid genetics requires that recessive mutations, which make up the vast majority of all mutations, be observed in the homozygous condition. Therefore, mutations that are carried within the zygote by one of the gametes can not be observed in the immediate diploid generation. The heterozygous individual must auto-cross or put through a series of crosses with his brothers to obtain the homozygous mutant. Typically, the plant breeder analyzes 20 progenies of each Fl individual to detect mutations that were transmitted to the Fl plant of one of their parents. In order to analyze the mutations in 1000 gametes, the cultivator usually examines 20 x 1000 or 20,000 progeny F2. Many experiments are impractical in view of the multiplier x 20 and the large number of plants that must be analyzed in F2. Some advantages associated with the use of haploids in a plant breeding program for crop improvement have been recognized and evidenced by Kasha et al., "Haploidy in Crop Improvement" in CYTOGENETICS OF CROP IMPROVEMENT 19-68 Macmillan 1983), or Nitzsche et al., "Haploids in Plant Breeding" in ADVANCES IN PLANT BREEDING 1-101 (Verlag Paul Paray 1977). Some of these advantages include the rapid production of homozygotes in lines of internal crosses through the haploidization and duplication of chromosomes, mutation studies to recover recessive mutations in a homozygous background, transfer of polyploid genes to diploid species, or incorporation of nuclei in foreign cytoplasm using androgenetic haploids. Coe, Amer. Natur. 93: 38 (1950) describes an internal cross line of lineage 6 that produces up to 3 percent haploids. Chalyc et al., J. Genet. & Breed. 47: 77-80 (1993) describes the production of maize haploids using the haptose inducing line ZMS (Zarodyshevy Marker Saratovsky). The ZMS was used as the paternal pollen and the haploids were produced at a frequency of 0.55 to 3.43 percent of maize plants observed. Kasha above, Nitzsche, supra, and Birchler, Practical Aspects of Haploid Production, in THE MAIZE HANDBOOK, Feeling and albot eds., Springer-Verlag, New York present reviews of known strategies for the production of haploids. The indeterminate maize gametophyte gene induces haploids of both male (androgenetic) and female (gynogenetic) origin. The indeterminate gametophyte gene was first described by Karmicle, Science 166: 1422-24 (1969), as arising spontaneously in the Wisconsin-23 very high internal cross strain (W23). The presence of indeterminate gametophyte increases the occurrence of paternal haploids from the spontaneous frequency of approximately 1 by 80, 000 at a frequency of 1 to 3 percent of corn plants observed. The indeterminate gametophyte gene is essential for the normal growth and development of the gametophyte and the loss of function of the undetermined gametophyte gene causes too many or too few nuclei to be produced. In the indeterminate gametophyte lines that develop megamethophytes, they are freed from their three normal mitotic divisions. Lin, Rev. Brasil, Biol. 4L3J_: 557-63 (1981), observed that the presence of indeterminate gametophyte allows the occurrence of a variable number of mitotic divisions and some of the degenerated nuclei. After fertilization of the megagametophyte, the sperm of the nuclei occasionally develop androgenetically as paternal haploid embryos. The embryonic development of sperm nuclei in maternal cytoplasm results in the formation of androgenetic haploids. Kermicle and collaborators, Maize Genet. Coop. Newsl. 54: 84-85 (1980), determined that the indeterminate gametophyte allele is placed on the long arm of chromosome 3 at 90 cM from the most distal locus in the short arm designated g. EPA 636,310 describes the production of haploids using undetermined gametophyte genes to analyze lethal conditional genes, markers that can be analyzed and selectable marker genes. The infrequent haploid occurrence of indeterminate gametophyte germplasm remains an obstacle to reliable identification and propagation of haploids. This obstacle is compounded by the difficulty of maintaining the lineage in a homozygous state (igig). In an attempt to increase the haploid frequency and globalize the utility of the system, Kindiger et al., Crop Science 33: 342-44 (1993), developed a tertiary trisomic lineage (A A B-A) using a simple translocation B-A designated TB-3Ld. The indeterminate gametophyte allele was placed in a 23 modified trisomic antecedent (B-A) the haploid frequency increased as much as 8 percent in some antecedents. The development of the undetermined trisomite tertiary indeterminate gametophyte lineage B-3Ld (Ig) also allows the rapid and successful development of male sterile cytoplasmic lineages (CMS) designed to carry indeterminate gametophyte in a homozygous condition. The trisomic method for increasing the frequency of indeterminate-induced haploid gametophyte plants suffers from several distinct disadvantages. The examination of the progeny must be carried out to maintain the translocation. In addition to this complexity, the haploid progeny must then be selected. To aid in the identification of maternal or paternal haploids, the undetermined gametophyte gene has been combined with rg (from colorless recessive seeds and green plants), and in separate lineages, with the dominant marker Rnj (grain, scutellum, plumule and seedlings). pigmented with purple) for the identification of haploids of paternal origin. For example, from crosses of indeterminate gametophytes Rnj with Ig rg pollen, haploids of paternal origin will have colorless scutellum and green seedlings. Typically, in maize, the Purple Embryo Marker (PEM) lineage of the genotype b pl A C RnJ: cudu pr pwr is used to detect haploids induced by indeterminate gametophytes. RnJ: cudu in combination with the dominant genes that condition the pigment genes A and C produce the red or purple pigmentation of aleurone, mainly in the crown portion of the grain, and a deep purple pigmentation in the embryo. For the detection of the Ig-induced androgenetic haploid, a PEM-ig lineage is used as the seed parent in the crosses with the donor line or the lineage of cultivators. The desired haploids have a white and colored aleurone embryo. A serious disadvantage of the PEM system is that igig females produce a high proportion of defective and small grains making it difficult to identify ig-induced haploids that have a white embryo and colored aleurone. Thus, there is a need for a strategy that facilitates the identification of androgenetic haploids among the progeny produced from crosses with indeterminate gametophyte plants.

In addition, the methodology that uses haploid androgenetic is needed to facilitate the characterization of gene functions, including the identification of genes labeled with transposons, enhancers, suppressors and genes that control quantitative characters. SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for directing the selection of androgenetic haploids in which the Rnj gene is only expressed in androgenetic haploids and therefore the androgenetic haploids are identified and selected as seeds with colored embryos. Another object of the present invention is to provide a method for testing the function of the genes in which the ig plant crosses as the female plant against a plant carrying transposon-induced mutations to facilitate the identification and characterization of the mutated gene in a background haploid Still another object of the present invention is to provide a method for identifying and characterizing enhancers and suppressors in which a population of mutant plants is produced which is (i) homozygous for a first mutation that causes a first mutant phenotype induced by chemical mutagenesis or the insertion of a first translocable element and (ii) carries a second mutation produced by the insertion in its genome of a second translocable element. The pollen from this population of mutant plants is used to pollinate plants capable of producing paternal haploid offspring to produce a plurality of haploid progeny plants Fl containing genetic material only from a population of mutant plants and which is analyzed to find a change in the first mutant phenotype. It is a further object of the present invention to provide a method of checking the function of genes to control quantitative characters in which a first internal cross is mutagenized and the pollen of this first mutagenized internal cross is used to fertilize an ig plant to produce haploid androgenetic. The haploids are then fertilized with pollen from a second cross to produce a seed that is isogenic. The seed is analyzed to see hybrid mutants. An object of the present invention is to provide a method for checking the function of genes, comprising the steps of selecting two parent plants, wherein the first plant is capable of producing paternal haploid offspring, and wherein the second plant as the parent male to produce a plurality of haploid progeny plants F? that contain genetic material only from the second plant; selecting the haploid offspring of the simultaneous presence of a translocable element and a mutant phenotype that differs from the paternal phenotype; and cloning DNA of the mutant haploid that is associated with the insertion of the transposon. Still another object of the present invention is to provide a method for checking the function of the genes comprising the steps of selecting two parental plants, wherein the first plant is capable of producing paternal haploid offspring, and wherein the second plant is (1) ) homozygous for a first mutation that causes a first mutant phenotype and is produced by chemical mutagenesis or insertion of a first translocable element and (2) carries a second mutation produced by the insertion within its genome of a second translocable element; cross the first floor as the female father with the second plant as male father to produce a plurality of haploid Fj plants that contain genetic material only from the second plant, - analyze the haploid offspring to see a plant that has a second characterized phenotype for a detectable change in the first mutant phenotype; and (d) cloning DNA from the mutant haploid that is associated with the insertion of the second transposon. A further object of the present invention is to provide a method for checking the function of the genes, which comprises the steps of selecting two parent plants, where the first plant is capable of producing paternal haploid offspring, and the second plant is a plant of first internal cross that carries at least one mutation produced by chemical mutagenesis or insertion within its genome of a translocable element, cross the first floor as the female father and the second floor as the male father to produce a plurality of plants of haploid progeny F? that contain genetic material only from the second plant; crossing the plurality of haploid progeny Fl as the female parent with a third plant that is a second internal cross to produce a plurality of progeny F2; analyze the plurality of progeny F2 to see a mutant phenotype; and that characterizes the mutant gene. Another object of the present invention is to provide a method for the identification and selection of androgenetic haploids, comprising the steps of selecting a first plant carrying the Ig and Idf genes and a second plant carrying the Rnj gene; cross the first floor as the female father with the second floor as the male father; and identify and select the haploid androgenetic progeny that has a colored embryo. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating the preferred embodiments of the invention, are given by way of illustration only. Undoubtedly, various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. DETAILED DESCRIPTION OF THE PREFERRED MODALITIES 1. Definitions In the description that follows, a number of terms is used extensively. The following definitions are provided to facilitate the understanding of the invention. A haploid plant has a single set (genome) of chromosomes and the reduced number of chromosomes (n) in the haploid plant is equal to that of the gamete. A diploid plant has two sets (genomes) of chromosomes and the number of chromosomes (2n) is equal to that in the zygote. A plant line is a group of individuals from a common ancestor and is a group defined more closely than a strain of a variety. Heterosis or hybrid vigor is the increased vigor, growth, size, product or function of a hybrid progeny over their parents that results from genetically crossing different organisms. A hybrid is the first generation of descendants of a cross between two individuals that differ in one or more genes.

An internal cross is a pure line that usually originates by self-pollination and selection. A quantitative character is a character that is influenced by a group of genes in different positions that are cumulative in their effect. A character is the expression of a gene as revealed in the phenotype. Phenotype is the physical or external appearance of an organism in contrast to its genetic constitution or genotype. An indeterminate g «» n? »M * t nfi jQ is An androqenético haploid arises when the maternal nucleus is eliminated or inactivated immediately after the fertilization of the egg cell and the haploid androgenetic haploid contains in its cells the set of chromosomes of the gamete male only A meioradora sequence is any of a class of DNA sequences that acts with cis that increases the transcription activity of a gene. A suppressor is any secondary mutation (mutation in second place) that restores totally or partially a lost function due to a primary mutation. A translocable genetic element or transposon is any of a class of diverse segments of DNA that can be inserted into non-homologous DNA (chromosomes, plasmids, virus DNA, mitochondria DNA and plastids) exit and relocate in a reaction that is independent of the general recombination function of the host. A structural gene is a DNA sequence that is transcribed into messenger RNA (mRNA) which then results in an amino acid sequence characteristic of a specific polypeptide. An isolated DNA molecule is a fragment of DNA that is not integrated into the genomic DNA of an organism. A cloning vector is a DNA molecule, such as a plasmid, cosmid or bacteriophage, which has the ability to replicate autonomously in a host cell. Cloning vectors typically contain one or a small amount of restriction endonuclease recognition sites in which foreign DNA sequences can be inserted in a determinable manner without loss of an essential biological function of the vector, as well as a marker gene that it is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide resistance to tetracycline or resistance to ampicillin. 2 . Overview The androgenetic haploids are made using maize plants with ig, according to the present invention. A dominant or recessive mutation is immediately revealed in the haploid generation. Isolated pollen from presumably mutant plants is used to polish female ig to recover mutant alleles. In order to examine 1000 gametes, only 1,000 paternal haploid plants are required in place of the 20,000 diploid plants that would be examined by conventional methods. The haploid androgenetic strategy of mutant selection significantly reduces the number of suspected mutants that have to be examined. As a result, it becomes possible to use biochemical assays for the detection of mutants in addition to the test to see changes in the visible phenotypes. An additional benefit is derived from the fact that a generation is saved by not having to auto-cross Fl plants. The gametes to be examined are obtained by mutagenizing pollen with EMS or taking pollen from plants mutagenized by transposon, such as Mutator plants. [insert *: cito?]. In the latter case, the mutants obtained can be used to clone mutated genes. Ig maize plants that produce androgenetic haploids have been identified at a frequency of about 1 to 3 percent of the total progeny. A method for directly selecting grains carrying androgenetic haploids is provided. 3. Checking the Function of the Genes by Transposon Labeling and Production of Androgenetic Haploids The identification of a restriction fragment that is secreted together with the mutant allele is a key step in the isolation of a gene by transposon labeling. Typically, a transposon insert event is presented in the progeny that contains the mutant allele, but is absent in its siblings. Conventional methods of analysis and selection of mutants by transposon require a cross-breeding of wild-type plants in the Fl and an examination of their progeny to identify the presumed mutants induced by transposon. Progeny F2 which are homozygous wild-type plants should not contain a transposon insert in the candidate DNA fragment associated with the mutant phenotype. Typically, the heterozygotes in F2 auto-cross to produce enough seeds to verify that the candidate DNA fragment in which the transposon has been inserted is co-segregated with the mutation. The autocross of the F2 plants still adds another generation to the analysis and requires the analysis of additional plants. The autocrossing of the F2 plants further complicates the analysis because the transposon copy number is likely to increase with autocrossing.

In accordance with the present invention, the presumably mutant plants in Fl are used to make paternal haploids. The heterozygous Fl progeny will segregate 1: 1 for the mutant phenotype, and the presence or absence of transposon can be easily determined. In addition, molecular analysis of paternal haploids is easier than that of F2 diploids. The copy number of the transposon in the paternal haploids is likely to be significantly lower than that of the progeny produced by autocrossing of heterozygotes in F2. The methodology of the present invention to check the function of the genes comprises the steps of (a) selecting two parent plants, wherein the first plant is capable of producing paternal haploid offspring, and wherein the second plant carries at least one mutation produced by the insertion within its genome of a translocable element, (b) crossing the first plant as the female father with the second plant as the male father to produce a plurality of haploid Fl progeny plants containing genetic material only from the second plant; (c) analyzing the haploid offspring to see the simultaneous presence of a translocable element and a mutant phenotype that differs from a paternal phenotype; and (d) cloning mutant haploid DNA associated with the transposon insertion. The first plant capable of producing paternal haploid offspring may be a maize plant carrying the ig gene and the translocable element may be a member of the Mutator family. Lineages of the Mutator element are well known to those skilled in the art, for example, S.P. Briggs, Curr. Top. Plant Biochem. Physiol. 6: 59 (1987). 4. Identification Improvers and Suppressors. Genes that interact directly or indirectly with each of the others can be identified by observing that a mutation in the gene reverses or increases the previously existing phenotype as a result of a mutation in another gene. These interactions are easily detected using haploids. Accordingly, the androgenetic haploids can be used to identify breeders and suppressors. In this method a population of mutant plants is produced which is (i) homozygous for a first mutation that causes a first mutant phenotype induced by chemical mutagenesis or the insertion of a first translocable element and (ii) carries a second mutation produced by the insertion in its genome a second translocable element that has a mutant phenotype of interest that is selected by means of a chemical mutagenesis or transposon. The pollen from this population of mutant plants is used to pollinate plants capable of producing paternal haploid offspring to produce a plurality of haploid progeny plants Fl containing genetic material from only one population of mutant plants.

The haploids of Fl are analyzed to find descendants that exhibit a second mutant phenotype characterized by a detectable change in the first mutant phenotype. The DNA of mutant haploids associated with the transposon insertion is cloned and characterized. More specifically, a method for identifying breeders and suppressors comprises the steps of (a) selecting two parent plants, wherein the first plant is capable of producing paternal haploid offspring, and the second plant is (i) homozygous for a first mutation that causes a first mutant phenotype induced by chemical mutagenesis or insertion within its genome of a translocable element, and (ii) it carries a second mutation produced by the insertion within its genome of a second translocable element; (b) crossing the first plant and the second plant to produce a plurality of haploid Fl progeny plants containing genetic material only from the second plant; (c) analyzing the haploid offspring to see the presence of a second phenotype characterized by a detectable change in the first mutant phenotype; and (d) cloning the gene responsible for the change observed in the first mutant phenotype. The first plant capable of producing paternal haploid offspring may be a maize plant carrying the ig gene. The translocables elements useful for the labeling of the transposón, methods of gene identification by transposon labeling and maize lineages carrying the translocable elements are well known to those skilled in the art. See Dellaporta et al, "Gene Tagging with Ac / Ds Elements in Maize," THE MAIZE HANDBOOK, M. Freeling and V. albot (eds.), Springer-Verlag, New York, pages 219-233 (1994); K. Cone, "Transposon Tagging with Spm," THE MAIZE HANDBOOK, M. Freeling and V Walbot (eds.), Springer-Verlag, New York, pages 240-242 (1994) and Paul S. Chomet, "Transposon Tagging with Mutator, "THE MAIZE HANDBOOK, M-. Freeling and V. Walbot (eds.), Springer-Verlag, New York, pages 243-249 (1994). 5. Checking the Function of the Genes associated with quantitative characters. It has not been until recently that quantitative character analysis has been founded due to the difficulty to observe a gene that segregates among several that affect a given trait, and because these traits must be qualified in populations instead of in individuals to reduce variation phenotypic caused by the environment. Molecular markers and recombinant internal crosses have been used to address these issues but even these technical advances have serious limitations. Existing methods allow large regions of a chromosome to be associated with a trait. The number and nature of the corresponding genes within these regions generally can not be determined.

A conceptual alternative to the study of natural variation is to recover mutants with alternating phenotypes by the character of interest. This form of approach has not been used due to the large number of plants that must be analyzed. With the present invention, however, parent haploids are used to eliminate the multiplier by which it is necessary to sample the F2. For example, a first internal cross can be mutagenized and used to make haploid parents. The haploids are then fertilized by pollen from a second internal cross. The seed produced in a given haploid parent is isogenic; therefore, a limited number of seeds of each plant can be analyzed to look for mutant hybrids. The first internal cross can be mutagenized by any conventional method such as chemical mutagenesis or transposon. In a preferred method, pollen from the first internal cross is mutagenized by treatment with a chemical mutagen and used to make androgenetic haploids using the mutagenized pollen in crosses with a maize plant carrying the Ig gene. In a particularly preferred method, the first internal cross is homozygous for "waxy" waxy (wx) while the second internal cross is WxWx. Plants that are Wx Wx or Wx wx can be easily distinguished from plants that are wx wx based on observable differences in the characteristics of the endosperm. The recessive wx grains exhibit a uniform opacity, similar to marble, and a hardness similar to that of normal grains except when combined with mealy mutants. Cut with a knife, the wx endosperm pieces leave an opaque, smooth surface evenly, while the normal endosperm (in the horn portions of the corn kernels for example) breaks unevenly and leaves an uneven surface , transparent. The starch on the cut surface of the non-waxy endosperm, whether stony, floury, opaque, glassy or brittle, will turn blue, rapidly turning black, with iodine (I2) in potassium iodide solution (KI). On the other hand, the homozygous wx (waxy) will turn reddish brown, soon turning dark brown, with iodine (I2) in potassium iodide solution (KI). The pollen of the first internal cross wx wx (internal cross A) is treated with a chemical mutagen and is used to polish maize that carries the ig gene to presumably produce androgenetic haploids, wx wx, mutants, (internal cross A '). The internal cross A 'is then fertilized with pollen taken from a second internal cross which is Wx Wx (internal cross B), and pollen from internal cross A at a ratio of 9: 1. All seeds produced in a given "fertilized" internal cross-breeding plant will be isogenic with respect to any mutation present in that internal cross-breeding plant A. According to the above, of every ten seeds produced in an internal cross-breeding plant A ' fertilized, nine seeds will be the result of the cross A 'x B and will be Wx wx, and one seed will be the result of the cross A' x A and will be wx wx, as soon as a mutant hybrid of the cross A 'x is identified B, the mutation can be obtained immediately in the background by selecting the wx wx seed produced in the same plant of internal cross A 'that gave rise to the Wx wx mutant of interest.The wx wx seed can be germinated and the plant used in crosses for genetic mapping and characterization of the mutant allele.This method can be used to check the function of the gene associated with a variety of different quantitative characteristics.The hybrid population can be analyzed to search for utations in the first internal cross that reduce hybrid vigor or growth under high densities of plants. Alternatively, the hybrid population can be analyzed to see mutations in the first plant that lead to increased tolerance to salt, flood tolerance, hybrid vigor or growth under high density conditions of plants. A method for checking genes that control quantitative traits thus comprises (a) selecting two parent plants, wherein the first plant is capable of producing paternal haploid descendants, and the second plant is a first-cross plant that carries at least one mutation produced by chemical mutagenesis or the insertion in its genome of a translocable element; (b) crossing the first and second plants to produce a plurality of haploid Fj progeny plants containing genetic material only from the second plant; (c) crossing the plurality of haploid Fj progeny as the female parent with a third plant that is a second internal cross to produce a plurality of progeny F2; (d) analyzing the plurality of progeny F2 to search for a mutant phenotype; and (e) characterizing the mutant gene. The method can be used to check the function of the gene associated with the control of heterosis or ability of a plant to grow efficiently at high plant density where the first plant capable of producing paternal haploid descendants is a maize plant carrying the gene ig. 6. Identification and Selection of Androgenetic Haploids A method for the direct selection of androgenetic haploids is provided. In this method the R1J gene is only expressed in androgenetic haploids and therefore the androgenetic haploids are identified and selected as seeds with colored embryos. The present method for the direct selection of androgenetic haploids overcomes the disadvantage of using R1J in the female for the selection of colorless haploids. Instead, a method is provided for selecting haploid embryos that have color. There is a high frequency of classification error when the absence of color is qualified due to the variation in the degree of synthesis of the pigment during development. For example, diploid grains may fail to produce detectable pigment depending on the environmental conditions and genetic background of the plant. In addition, the Rnj gene causes the pigment to be produced in a small part of the embryo that is the axis of the embryo and consequently the pigmentation is difficult to detect through the superimposed pericarp tissue. The plant from which the androgenetic haploid is going to be made homozygous for the Rnj gene. The female ig father who is able to produce androgenetic haploids becomes homozygous for the Idf or indeterminate-diffuse gene. The Idf gene suppresses the expression of the Rnj gene and the formation of the anthocyanin pigment that leads to the production of seeds with white embryos. A plant that is Rnj Rnj is crossed like the male father with a plant that carries the ig gene and is Idf Idf. Only the haploid androgenetic progeny will not carry the Idf gene and consequently only the haploid androgenetic progeny will have colored embryos. Therefore, it is possible to detect haploid progeny by looking for colored embryos. For example, the analysis process can be done mechanically and the colored embryos detected and selected by an electric eye that detects reflected light of certain wavelengths. A method for the identification and selection of androgenetic haploids comprises the steps of (a) selecting a first plant that carries the ig and is homozygous for the Idf gene and a second plant that is homozygous for the Rnj gene; (b) crossing the first floor as the female father with the second floor as the male father; and (c) identify and select the haploid androgenetic progeny that has a colored embryo. The present invention, thus generally described, will be more readily understood with reference to the following examples, which are provided by way of illustration and are not intended to limit the present invention.

Example 1 Detection and Isolation of Genes that Contribute to Heterosis To isolate mutations in genes that contribute to heterosis, the pollen of the AM21WX mutant waxy internal cross was treated with the ethylmethane sulfonate mutagene (EMS) using methods well known to the skilled artisan. . See Neuffer and collaborators. , Maydica 22: 21 (1977) or G.F. Sprague and J. W. Dudley, eds., CORN AND CORN IMPROVEMENT, American Society of Agronomy, Madison, 3a. edition (1988). The mutagenized pollen was used to fertilize eggs of igigWxWx mutant plants. Grains carrying paternal haploid embryos were selected based on the color of the embryo and planted. The haploids were fertilized with a mixture of pollen from the internal crosses HD93 and AM21WX. The waxy grains produced by the AM21WX pollen fertilization were selected and separated. The non-waxy, glassy grains produced by fertilization with HD93 pollen were selected and cultured for the selection of mutants. Observations were made to identify the progeny obtained from non-waxy grains that carry mutations that affect the heterosis as the yield and the time required for maturation. Mutations that are inherited by the haploid embryo that affects the heterosis of the HD93 / AM21WX are recovered from the waxy seeds produced in the haploid plant. The serous seeds are the internal cross AM21WX / AM21WX that are heterozygous for the mutation. The inheritance of the mutant allele can be followed, repeating the heterosis test. If the mutation causes a phenotype that can be directly labeled in mutant plants, then the gene is cloned by transposon labeling methods.

EXAMPLE 2 Ig-induced Haploids and Identification of Transposon-Tagged Genes A diverse collection of heterozygous lines, active by Mutator was used to polish Igig plants. Five thousand grains bearing paternal haploid embryos were selected. Grain plants were analyzed for loss of disease resistance to maize pathogens such as Fusarium monili forme, Cochliobalus carbonum, Erwinia stewartii or corn dwarf mosaic virus. Alternatively, the loss of function of the specific voltage gene was sought as the defense genes to pathogens or peroxidase. The mutant plants are polinated by a non-mutant internal cross to recover the mutant allele. The heterozygous progeny are cultured and self-pollinated or cross with an igig line to produce progeny in which the segregating mutation can be observed. DNA from the segregating progeny is examined by Southern blot analysis using Mu-specific hybridization probes to identify a Mu element that co-segregates with each labeled mutant site. You should examine as many different segregated cross lines as possible. In addition, it is useful to examine the population using several different restriction enzymes since it can obscure segregating fragments by other elements homologous to Mu. DNA from parental lines is included in Southern blot analysis because a cosegregant fragment should not be present in the father plant. As soon as a cosegregant fragment is identified, additional analyzes should be carried out with a larger and different population group. As soon as a cosegregant band is identified, cloning or polymerase chain reaction is used to obtain single flanking sequence. This flanking probe is then used to prove that the site is responsible for the mutant phenotype. For example, the identification of DNA rearrangements, insertions, or deletions at the allele site generated independently demonstrates that the clone is, or is very close to, the relevant site. Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is not limited. It will occur to those of ordinary skill in the art that various modifications can be made to the embodiments described and that these modifications are intended to be within the scope of the present invention., which is defined with the following claims. All the publications and patent applications mentioned in this specification indicate the level of skill of the technicians for which they are interested. All publications and patent applications are hereby incorporated by reference to the same extent as if each publication or individual patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims (8)

1. A method to check the function of the gene, comprising the steps of: (a) selecting two parental maize plants, where the first maize plant is capable of producing paternal haploid offspring and carries the undetermined gametophyte gene (ig), and wherein the second corn plant carries at least one mutation produced by the insertion in its genome of a translocable element; (b) crossing the first plant as a female parent with the second plant as the male parent to produce a plurality of F ^ haploid progeny plants containing genetic material only from the second plant; (c) analyzing the haploid offspring to see the simultaneous presence of a translocable element and a mutant phenotype that differs from a parent phenotype; and (d) cloning DNA from said mutant haploid that is associated with the insertion of the transposon.

2. A method according to claim 1, wherein the translocable element is a member of the Mutator family.

3. A method to check the function of the gene, comprising the steps of: (a) selecting two parental maize plants, where the first maize plant is capable of producing paternal haploid offspring and carries the undetermined gametophyte gene, and where the second corn plant is (i) homozygous for a first mutation that causes a first mutant phenotype and is produced by chemical mutagenesis or insertion of a first translocable element and (ii) carries a second mutation produced by the insertion into its genome of a second translocable element; (b) crossing the first plant as the female father with the second plant as the male father to produce a plurality of haploid Fj progeny plants containing genetic material only from the second plant; (c) analyzing the haploid offspring to look for a plant exhibiting a second mutant phenotype characterized by a detectable change in the first mutant phenotype; and (d) cloning DNA from the mutant haploid that is associated with the insertion of the second transposon.

4. A method for testing the function of the gene, comprising the steps of: (a) selecting two parental maize plants, wherein the first maize plant is capable of producing paternal haploid offspring and carries the indeterminate gametophyte gene, and the second maize plant is a plant of first internal cross that carries at least one mutation produced by chemical mutagenesis or the insertion in its genome of a translocable element, (b) crossing the first plant as the female father and the second plant as the male father to produce a plurality of progeny plants F? haploid containing genetic material only from the second plant; (c) crossing the plurality of haploid Fj progeny as the female parent with a third plant that is a second internal cross to produce a plurality of progeny F2; (d) analyzing the plurality of progeny F2 to search for a mutant phenotype; and (e) characterizing the mutant gene.

5. A method for the identification and selection of androgenetic corn haploids, comprising the steps of: (a) selecting a first plant that carries the genes of indeterminate gametophytes and the indeterminate-diffuse (Idf) and a second plant that carries a colored embryo gene; (b) crossing the first floor as the female father with the second floor as the male father; and (c) identify and select the haploid androgenetic progeny that has a colored embryo.

6. The method according to claim 5, wherein the colored embryo gene is the Rnj gene. The method according to any one of claims 1, 3, or 4 wherein the first plant carries the Idf gene and the second plant carries a colored embryo gene. The method according to claim 7, wherein the colored embryo gene is the Rnj gene.