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Methods and compositions for haploid mapping

Methods and compositions for haploid mapping description/claims

This application claims priority to U.S. Provisional Patent Application No. 60/966,706, filed Aug. 29, 2007 and incorporated herein by reference in its' entirety.

A sequence listing contained in the file named “46—25(54886—0001_US).txt” which is 2432225 bytes (measured in MS-Windows®), created on Aug. 21, 2008, and comprising 1,361 nucleotide sequences, is electronically filed herewith and is incorporated herein by reference in its entirety.

The present invention relates to the field of plant breeding. More specifically, the present invention includes a method of using haploid plants for genetic mapping of traits such as disease resistance. Further, the invention includes a method for breeding corn plants containing quantitative trait loci (QTL) that are associated with resistance to gray leaf spot (GLS), a fungal disease associated with Cercospora spp. The invention further includes a method for breeding corn plants containing QTL that are associated with Goss' Wilt, a bacterial disease associated with Clavibacter michiganense spp.

Plant breeding is greatly facilitated by the use of doubled haploid (DH) plants. The production of DH plants enables plant breeders to obtain inbred lines without multigenerational inbreeding, thus decreasing the time required to produce homozygous plants. DH plants provide an invaluable tool to plant breeders, particularly for generating inbred lines, QTL mapping, cytoplasmic conversions, and trait introgression. A great deal of time is spared as homozygous lines are essentially instantly generated, negating the need for multigenerational conventional inbreeding.

In particular, because DH plants are entirely homozygous, they are very amenable to quantitative genetics studies. Both additive variance and additive×additive genetic variances can be estimated from DH populations. Other applications include identification of epistasis and linkage effects. For plant breeders, DH populations have been particularly useful in QTL mapping, cytoplasmic conversions, and trait introgression. Moreover, there is value in testing and evaluating homozygous lines for plant breeding programs. All of the genetic variance is among progeny in a breeding cross, which improves selection gain.

Methods of utilizing haploids in genetic studies have been described in the art. A statistical method to utilize pooled haploid DNA to estimate parental linkage phase and to construct genetic linkage maps has been described (Gasbarra, D. et al., Genetics 172: 1325-1335 (2006)). An additional study has used the method of crossing haploid wheat plants with cultivars to map leaf rust resistance gene in wheat (Hiebert, C. et al., Theor Appl Genet 110: 1453-1457 (2005)). Haploid plants and SSR markers have been used in linkage map construction of cotton (Song, X. et al., Genome 48:378-392 (2005)). Further, AFLP marker ANALYSIS has been performed in monoploid potato (Varrieur, J., Thesis, AFLP Marker Analysis of Monoploid Potato (2002) To date a method of using haploid plants to genetically map loci associated with traits of interest is lacking. The present invention provides a method of using haploid plants to genetically map traits of interest.

Two diseases which cause significant damage to corn crops are Gray Leaf Spot (GLS) caused by the fungal pathogen Cercospora zeae-maydis (CZ) and Goss' Wilt caused by the bacterial pathogen Clavibacter michiganensis subsp. nebraskensis (CN). GLS is a global problem and, in addition to prevalence in Africa, Central America and South America, it has spread across most of the U.S. Corn Belt over the past 10-15 years. The fungus overwinters in field debris and requires moisture, usually in the form of heavy fogs, dews, or rains, to spread its spores and infect corn. Increasing pervasiveness has been linked to no-till practices which promote retention of fungi, such as CZ, in the soil (Paul et al., Phytopathology 95:388-396 (2005)). Symptoms include a rectangular necrotic lesion which can coalesce to larger affected regions and symptoms usually appear later in the growing season. GLS in corn elicits an increased allocation of plant resources to damaged leaf tissue, leading to elevated risk for root and stalk rots, which ultimately results in even greater crop losses (Ward et al. 1999; Saghai-Maroof et al., Theor. Appl. Genet. 93:539-546 (1996)). Yield-loss associated with GLS can be high if the symptoms are heavy and appear early, with reported losses exceeding 50% (Ward et al., 1999). Recent work has identified there are at least two sister species of CZ, as well as potentially other isolates of Cercospora, capable of causing GLS (Carson et al., Maydica 51:89-92 (2006); Carson et al, Plant Dis. 86:1088-109 (2002)). Genomic regions on maize Chromosomes 1, 2, 3, 4, 5, 6, 7, and 8 have been associated with GLS using RFLP, AFLP and SSR markers (U.S. Pat. No. 5,574,210; Lehmensiek, et al., TAG, (2001); Clements, et al. Phytopathology (2000); Gorden et al., Crop Science (2004); Bubeck, et al., Crop Science, (1993); Saghai-Maroof et al., Theor. Appl. Genet (1996)).

Goss' Wilt is another disease of corn which has been identified throughout the U.S. Corn Belt, primarily in the western regions. Symptoms include leaf freckles which are small dark green to black water soaked spots and vascular wilt which results in loss of yield. Conservation tillage practices can increase pervasiveness because the bacterial pathogen Clavibacter michiganensis subsp. nebraskensis (CN) can overwinter in debris, particularly stalks, from infected corn plants (Bradbury, J. F. IMI description of Fungi and Bacteria, (1998)). A mapping study conducted by Rocheford et al., reported a genomic region on maize Chromosome 4 associated with Goss' Wilt (Rocheford, et al., Journal of Heredity 80(5), (1989)). Both GLS and Goss' Wilt are significant pathogens of corn, and a need exists for development of disease resistant lines.

Breeding for corn plants resistant to GLS and Goss' Wilt can be greatly facilitated by the use of marker-assisted selection. Of the classes of genetic markers, single nucleotide polymorphisms (SNPs) have characteristics which make them preferential to other genetic markers in detecting, selecting for, and introgressing disease resistance in a corn plant. SNPs are preferred because technologies are available for automated, high-throughput screening of SNP markers, which can decrease the time to select for and introgress disease resistance in corn plants. Further, SNP markers are ideal because the likelihood that a particular SNP allele is derived from independent origins in the extant population of a particular species is very low. As such, SNP markers are useful for tracking and assisting introgression of disease resistance alleles, particularly in the case of disease resistance haplotypes.

In certain embodiments, methods for the association of at least one genotype with at least one phenotype using a haploid plant comprising: a) assaying at least one genotype of at least one haploid plant with at least one genetic marker; and b) associating the at least one marker with at least one phenotypic trait are provided. In certain embodiments, the at least one genetic marker comprises a single nucleotide polymorphism (SNP), an insertion or deletion in DNA sequence (Indel), a simple sequence repeat of DNA sequence (SSR) a restriction fragment length polymorphism, a haplotype, or a tag SNP. In other embodiments, the methods can further comprise the step of using an association determined in step (b) to make a selection in a plant breeding program. In such embodiments comprising a selection, the selection can comprise any one or all of: 1) selecting among breeding populations based on the at least one genotype; 2) selecting progeny in one or more breeding populations based on the at least one genotype; 3) selecting among parental lines based on prediction of progeny performance; 4) selecting a line for advancement in a germplasm improvement activity based on the at least one genotype; and/or 5) selecting a line for advancement in a germplasm improvement activity where the germplasm improvement activity is selected from the group consisting of line development, variety development, hybrid development, transgenic event selection, making breeding crosses, testing and advancing a plant through self fertilization, purification of lines or sublines, using plants or parts thereof for transformation, using plants or parts thereof for candidates for expression constructs, and using plants or parts thereof for mutagenesis. In certain embodiments, the methods can further comprise the step of doubling at least one haploid plant selected in said breeding program to obtain a doubled haploid plant. In such embodiments where a doubled haploid plant is obtained, the doubled haploid plant can be used for introgression of the genotype of interest into at least a second plant for use in a plant breeding program. In certain embodiments, the haploid plant in step (a) is obtained from a haploid breeding population. In certain embodiments, the haploid plant or plants comprise an intact plant, a leaf, vascular tissue, flower, pod, root, stem, seed or portion thereof. In certain embodiments, the plants are selected from the group consisting of maize (Zea mays), soybean (Glycine max), cotton (Gossypium hirsutum), peanut (Arachis hypogaea), barley (Hordeum vulgare); oats (Avena sativa); orchard grass (Dactylis glomerata); rice (Oryza sativa, including indica and japonica varieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp); tall fescue (Festuca arundinacea); turfgrass species (e.g. species: Agrostis stolonifera, Poa pratensis, Stenotaphrum secundatum); wheat (Triticum aestivum), and alfalfa (Medicago sativa), members of the genus Brassica, carrot, cucumber, dry bean, eggplant, fennel, garden beans, gourd, leek, lettuce, melon, okra, onion, pea, pepper, pumpkin, radish, spinach, squash, sweet corn, tomato, watermelon, and ornamental plants. In certain embodiments, the haploid plant is a fruit, vegetable, tuber, or root crop. In certain embodiments, the trait is selected from the group consisting of herbicide tolerance, disease resistance, insect or pest resistance, altered fatty acid, protein or carbohydrate metabolism, increased grain yield, increased oil, enhanced nutritional content, increased growth rates, enhanced stress tolerance, preferred maturity, enhanced organoleptic properties, altered morphological characteristics, sterility, a trait for industrial use, and a trait for consumer appeal.

In certain embodiments, methods for identifying an association of a plant genotype with one or more traits of interest comprising: a) screening a plurality of haploid plants displaying heritable variation for at least one trait wherein the heritable variation is linked to at least one genotype; and b) associating at least one genotype of at least one haploid plant to at least one trait are provided. In certain embodiments, the genotype comprises a genetic marker. In certain embodiments, the genetic marker comprises a single nucleotide polymorphism (SNP), an insertion or deletion in DNA sequence (Indel), a simple sequence repeat of DNA sequence (SSR) a restriction fragment length polymorphism, a haplotype, or a tag SNP. In certain embodiments, the methods can further comprising the step of using an association determined in step (b) to make a selection in a plant breeding program. In such embodiments comprising a selection, the selection can comprise any one or all of: 1) selecting among breeding populations based on the at least one genotype; 2) selecting progeny in one or more breeding populations based on the at least one genotype; 3) selecting among parental lines based on prediction of progeny performance; 4) selecting a line for advancement in a germplasm improvement activity based on the at least one genotype; and/or 5) selecting a line for advancement in a germplasm improvement activity where the germplasm improvement activity is selected from the group consisting of line development, variety development, hybrid development, transgenic event selection, making breeding crosses, testing and advancing a plant through self fertilization, purification of lines or sublines, using plants or parts thereof for transformation, using plants or parts thereof for candidates for expression constructs, and using plants or parts thereof for mutagenesis. In certain embodiments, the methods can further comprise the step of doubling at least one haploid plant selected in the breeding program to obtain a doubled haploid plant. In certain embodiments, the doubled haploid plant is used for introgressing the genotype of interest into a plant for use in a plant breeding program. In certain embodiments, the haploid plant or plants comprise an intact plant, a leaf, vascular tissue, flower, pod, root, stem, seed or portion thereof. In certain embodiments, the plants are selected from the group consisting of maize (Zea mays), soybean (Glycine max), cotton (Gossypium hirsutum), peanut (Arachis hypogaea), barley (Hordeum vulgare); oats (Avena sativa); orchard grass (Dactylis glomerata); rice (Oryza sativa, including indica and japonica varieties); sorghum (Sorghum bicolor); sugar cane (Saccharum sp); tall fescue (Festuca arundinacea); turfgrass species (e.g. species: Agrostis stolonifera, Poa pratensis, Stenotaphrum secundatum); wheat (Triticum aestivum), and alfalfa (Medicago sativa), members of the genus Brassica, carrot, cucumber, dry bean, eggplant, fennel, garden beans, gourd, leek, lettuce, melon, okra, onion, pea, pepper, pumpkin, radish, spinach, squash, sweet corn, tomato, watermelon, and ornamental plants. In certain embodiments, the haploid plant is a fruit, vegetable, tuber, or root crop. In certain embodiments, the trait is selected from the group consisting of herbicide tolerance, disease resistance, insect or pest resistance, altered fatty acid, protein or carbohydrate metabolism, increased grain yield, increased oil, enhanced nutritional content, increased growth rates, enhanced stress tolerance, preferred maturity, enhanced organoleptic properties, altered morphological characteristics, sterility, a trait for industrial use, and a trait for consumer appeal.

In certain embodiments, methods for the association of at least one phenotype with at least one genetic marker using a haploid plant comprising: a) assaying at least one phenotype of at least one haploid plant with at least one phenotypic marker to determine the presence or absence of said phenotype; and b) associating the presence or absence of said phenotype with at least one genetic marker are provided. In certain embodiments of the methods, the haploid plant is obtained from a haploid breeding population. In certain embodiments of the methods, the at least one genetic marker can comprise a single nucleotide polymorphism (SNP), an insertion or deletion in DNA sequence (Indel), a simple sequence repeat of DNA sequence (SSR) a restriction fragment length polymorphism, a haplotype, or a tag SNP. In certain embodiments of the methods, the at least one phenotypic marker can comprise at least one of a transcriptional profile, a metabolic profile, a nutrient composition profile, a protein expression profile, protein composition, protein levels, oil composition, oil levels, carbohydrate composition, carbohydrate levels, fatty acid composition, fatty acid levels, amino acid composition, amino acid levels, biopolymers, pharmaceuticals, starch composition, starch levels, fermentable starch, fermentation yield, fermentation efficiency, energy yield, secondary compounds, metabolites, morphological characteristics, or an agronomic characteristic. In certain embodiments of these methods, the methods can further comprising the step of using an association determined in step (b) to make a selection in a plant breeding program. In certain embodiments comprising a selection, the selection can comprise any one or all of: 1) selecting among breeding populations based on the at least one genotype; 2) selecting progeny in one or more breeding populations based on the at least one genotype; 3) selecting among parental lines based on prediction of progeny performance; 4) selecting a line for advancement in a germplasm improvement activity based on the at least one genotype; and/or 5) selecting a line for advancement in a germplasm improvement activity where the germplasm improvement activity is selected from the group consisting of line development, variety development, hybrid development, transgenic event selection, making breeding crosses, testing and advancing a plant through self fertilization, purification of lines or sublines, using plants or parts thereof for transformation, using plants or parts thereof for candidates for expression constructs, and using plants or parts thereof for mutagenesis. In certain embodiments of these methods, the methods can further comprise the step of doubling at least one haploid plant selected in said breeding program to obtain a doubled haploid plant. In certain embodiments comprising obtainment of a doubled haploid plant, the doubled haploid plant is used for introgression of the genotype of interest into at least a second plant for use in a plant breeding program.

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