AU631562B2 - Genetic linkages between agronomically important genes and restriction fragment length polymorphisms - Google Patents

Description

APPLN ID 64 2 8 PCT NUMBER PCT/US89/00709 OPI DATE 06/09/89 AOJP DATE 05/10/89

INTERNA

(51) International Patent Classification 4 C12N 15/00, C07H 15/12 A01H 1/00, 1/04 Al TREATY (PCT) (11) International Publication Number: WO 89/ 07647 (43) International Publication Date: 24 August 1989 (24.08.89) (21) International Application Number: PCT/US89/00709 (22) International Filing Date: 22 February 1989 (22.02.89) (31) Priority Application Numbers: 158,469 242,746 (32) Priority Dates: 22 February 1988 (22.02.88) 9 September 1988 (09.09.88) (33) Priority Country: US (71) Applicant: PIONEER HI-BRED INTERNATIONAL, INC. [US/US]; Capital Square, 400 Locust Street, Suite 700, Des Moines, IA 50309 (US).

(72) Inventors: GRANT, David 9600 Aurora Street, Urbandale, IA 50322 ALBERTSEN, Marc, Christian 1717 N.W. 70th Avenue, Ankeny, IA 50021 (US).

BEAVIS, William, Dale 6689 N.W. 51st, Johnston, IA 50131 FINCHER, Robert, Reid 614 60th Place, Des Moines, IA 50312 (US).

KUHN, William, Edward 4112 80th Street, Urbandale, IA 50322 SMITH, John, Stephen, Charles 506 4th Street, Box 404, Polk City, IA 50226 (US).

SMITH, Oscar, Samuel, Jr. 5325 N.W. 78th Court, Johnston, IA 50131 (US).

(74) Agents: GOLDSTEIN, Jorge, Saidman, Sterne, Kessler Goldstein, 1225 Connecticut Avenue, Suite 300, Washington, DC 20036 (US) et al.

(81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), FR (European patent), GB (European patent), IT (European patent), LU (European patent), NL (European patent), SE (European patent).

Published With international search report.

Before the expiration of the time limit for amending the claims and to be republished in the eent of the receipt of amendments.

(54) Title: GENETIC LINKAGES BETWEEN AGRONOMICALLY IMPORTANT GENES AND RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (57) Abstract The invention concerns methods for finding correlations between agronomically important genes in corn and restriction fragment length polymorphisms and DNA probes shown to reveal polymorphisms.

i i PCI'US89/00709 WO 89/07647 1 TITLE OF THE INVENTION GENETIC LINKAGES BETWEEN AGRONOMICALLY IMPORTANT GENES AND RESTRICTION FRAGMENT LENGTH POLYMORPHISMS This is a continuation-in-part of application Serial No. 158,469, filed February 22, 1988.

Field of the Invention This invention is in the field of genetic engineering and corn breeding. More specifically, the invention concerns methods for finding correlations between agronomically important genes in corn and restriction fragment length polymorphisms through the use of DNA probes that are shown to reveal polymorphisms.

Background of the Invention The goal of plant breeding is to combine in a single variety/hybrid various desirable traits of the parental lines and to exploit the heterosis exhibited by the cross of the parental lines.

For field crops, these traits may include resistance to diseases and insects, tolerance to heat and drought, reducing the time to crop maturity, greater yield, and better agronomic quality. With mechanical harvesting of many crops, uniformity of plant pCtfUS89/0709 WO 89/07647 -2characteristics such as germination and stand establishment, growth rate, maturity and fruit size are important.

Field crops are bred through techniques that use the plant's method of pollination. A plant is defined as self- pollinating if pollen from one flower is transferred to the same or another flower of the same plant. A plant is cross- pollinated if the pollen comes from a flower on a different plant.

Plants that have been self-pollinated and selected for type for many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny. A cross between two homozygous plants from differing backgrounds or two homozygous lines (inbred lines) produces a uniform population of hybrid plants that may be heterozygous for many gene loci. A cross of two plants, each heterozygous at a number of gene loci, will produce a population of hybrid plants. Each of these plants differ genetically and the p population will not be uniform.

Corn Zea mavs plants are bred by both self- pollination and cross-pollination techniques. Corn has male flowers, located on the tassel, and female flowers, located on the ear, on the same plant.

Natural pollination occurs in corn when wind blows pollen from the tassels to the silks that protrude from the tops of the incipient ears.

The development of corn hybrids requires the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. Pedigree breeding and recurrent selection breeding methods are used to develop inbred lines from breeding populations. Breeding programs are designed to combine desirable traits from two or more inbred lines or various broad-based sources into breeding pools from which new inbred lines are developed by selfing and selection of desirable phenotypes. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which have commercial potential as F 1 hybrids.

i~C IIL~6WU uui 7 WO 89/07647 -3- The identification of desirable agronomic traits has traditionally been done by phenotypic selection. It would be valuable to plant breeders to be able to identify genes affecting agronomic traits on the plant genome through the identification of linked genetic markers. Technology now provides a method for identifying genetic markers with potential application in plant breeding through the use of restriction fragment length polymorphisms (RFLPs). It is therefore of great importance to investigate the possibility of using genetic linkage analysis between DNA polymorphisms and traits of agronomic importance in order to identify agronomically important genes, to classify inbreds, hybrids and breeding populations according to their genes, and to then more effectively incorporate these genes into improved inbreds and hybrids.

Summary of the Invention The invention is based on the use of RFLPs to identify genetic linkages with agronomically important genes. This invention consists of three major parts: DNA probes shown to reveal polymorphisms between two parent inbred lines and having known chromosomal locations, statistical techniques that can find correlations between the inheritance of one or more DNA probes and the phenotype of the plants under investigation, and methods for using the identified genetic linkage between specific probes and genetic components of agronomically important traits in plant breeding.

In particular, this invention relates to a method for determining a particular trait in a maize plant which comprises analyzing each maize chromosome for DNA polymorphisms linked to a particular trait.

Any of a variety of RFLPs, probes and restriction enzymes can be used, as illustrated herein. The invention also relates to specific DNA probes that can be used in the method of this invention. The invention further relates to use of newly-identified relationships U1 3A between agronomic traits and genetic members to enhance breeding.

According to a first embodiment of this invention, there is provided a method of determining a correlation between a phenotypic trait in maize and a restriction fragment length polymorphism comprising: digesting genomic DNA from maize inbred B73 or maize inbred Mo17 with a restriction endonuclease that produces a restriction fragment length polymorphism digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step detecting said restriction fragment length polymorphism with a hybridization probe containing sequence information capable of hybridizing to and identifying said RFLP, thereby generating a restriction pattern; and correlating the presence or absence of said RFLP in said 15 digest with the respective presence or absence of said trait.

According to a second embodiment of this invention, there is provided a method of determining a correlation between a phenotypic trait in maize and a restriction fragment length polymorphism comprising: digesting genomic DNA from a maize plant that is a cross 20 between maize inbred B73 and maize inbred Mo17 with a restriction endonuclease that produces a restriction fragment length polymorphism digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step 25 detecting said restriction fragment length polymorphism with a hybridization probe containing sequence information capable of hybridizing to and identifying said RFLP, thereby generating a restriction pattern; and correlating the presence or absence of said RFLP in said 30 digest with the respective presence or absence of said trait.

According to a third embodiment of tlhis invention, there is provided a method of determining a correlation between a phenotypic trait in maize and a restriction fragment length polymorphism comprising: digesting genomic DNA from a maize plant with a restriction endonuclease that produces a restriction fragment length polymorphism digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step 3B detecting said restriction fragment length polymorphism with a hybridization probe made from maize inbred B73 DNA digested with restriction endonuclease Pst I, thereby generating a restriction pattern; and correlating the presence or absence of said RFLP in said digest with the respective presence or absence of said trait.

According to a fourth embodiment of this invention, there is provided a method for identifying and mapping quantitative trait loci (QTL) For a phenotypic trait in maize plant B73, Mo17 or a cross thereof by analyzing the chromosomes of said plants for DNA polymorphisms and linkage to said trait comprising: digesting genomic DNA from a maize plant with a restriction endonuclease that produces a restriction fragment length polymorphism digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step detecting said restriction fragment length polymorphism with a hybridization probe containing sequence information capable of hybridizing to and identifying said RFLP, therehy generating a restriction pattern; and i correlating the presence or absence of said RFLP in said digest with the respective presence or absence of said trait wherein said phenotypic trait is selected from adjusted yield, plant yield, plant S height, ear height, GDU shed, GDU silk, grain moisture, root lodging, stalk lodging, or stay green.

According to a fifth embodiment of this invention, there is I provided a restriction fragment length polymorphism probe that can detect a polymorphism in one or more maize plants wherein said polymorphism is linked to a phenotypic trait selected from adjusted yield, plant yield, ear height GDU shed, GDU silk, grain moisture, root lodging, stalk lodging, or stay green.

According to each of an eighth to a forty-seventh emodiment of this invention, there is provided a method for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with a phenotype herein defined, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers, herein defined, or a functionally equivalent genetic m arker; n 4CN/848Z c ~X1 3C determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile.

According to a forty-eighth embodiment of this invention, there is provided a restriction fragment length polymorphism probe that can detect a polymorphism in one or more maize plants wherein said polymorphism is linked to a phenotypic trait selected from the group consisting of adjusted bulk density, ratio of yield to percent moisture, dropped ears, statistically transformed dropped ears, grain quality, European corn borer second brood tolerance, statistically transformed root lodging, statistically transformed stalk lodging, weight of 100 kernels, cold test germination, soak test germination, early stand count, ear circumference, ear diameter, ear length, cob circumference, cob diameter, bare tip, kernel row length, kernel depth, kernels per kernel row, number of kernel rows per ear, and kernel width.

According to a forty-ninth embodiment of this invention, there is provided.a method of determining a correlation between a phenotypic trait in maize and a restriction fragment length polymorphism comprising: digesting genomic DNA from a maize plant with a restriction endonuclease that produces a restriction fragment length polymorphism digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step detecting said restriction fragment length polymorphism with a hybridization probe containing sequence information capable of hybridizing to and identifying said RFLP, thereby generating a restriction pattern; and correlating the presence or absence of said RFLP in said digest with the respective presence or absence of said trait; *i wherein said trait is selected from the group consisting of adjusted yield, plant yield, ear height, GDU shed, GDU silk, grain moisture, root lodging, stalk lodging, and stay green.

According to a fiftieth embodiment of this invention, there is A provided.a method of determining a correlation between a phenotypic trait in maize and a restriction fragment length polymorphism comprising: 1 1/848Z 7, 7 Fr 4 I j

I

I i I; i a~ 3Ddigesting genomic DNA from a maize plant with a restriction endonuclease that produces a restriction fragment length polymorphism digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step detecting said restriction fragment length polymorphism with a hybridization probe containing sequence information capable of i hybridizing to and identifying said RFLP, thereby generating a restriction pattern; and correlating the presence or absence of said RFLP in said digest with the respective presence or absence of said trait; 1] wherein said trait is selected from the group consisting of adjusted bulk density, ratio of yield to percent moisture, dropped ears, statistically transformed dropped ears, grain quality, European corn borer second brood ja tolerance, statistically transformed root lodging, statistically ;i transformed stalk lodging, weight of 100 kernels, cold test germination, soak test germination, early stand count, ear circumference, ear diameter, ear length, cob circumference, cob diameter, bare tip, kernel row length, kernel depth, kernels per kernel row, number of kernel rows ii .pei ear, and kernel width.

1* i r l -i.l riir;iin l -II- pCT/US89/00709 WO 89/07647 -4- Detailed Description of the Invention Definitions In the description and examples that follow, a number of terms are used herein. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided: Restriction Endonuclease. A restriction endonuclease or restriction enzyme is an enzyme that recognizes a specific base sequence in a double-stranded DNA molecule, and will cleave both strands of the DNA molecule at every place where this sequence appears.

Restriction Fragments. The DNA molecules produced by digestion with a restriction endonuclease are referred to as restriction fragments. Any given genome will be digested by a particular restriction endonuclease into a discrete set of restriction fragments.

4 The DNA fragments that result from restriction enzyme cutting are separated and displayed by electrophoresis through agarose gels.

Restriction Fragment Length Polymorphism (RFLP). The genomic DNA of two individuals in a species, for example, will differ in sequence at many sites. When these differences occur in the recognition site for a restriction endonuclease, the enzyme will not cleave the DNA molecule at that point. Likewise, a variation may introduce a recognition site where none exists in the other individual, causing the DNA to be cut by the restriction enzyme at that point. Because of this, digestion of the two individuals' DNA will produce fragments having different lengths. A polymorphism in the length of restriction fragments produced by digestion of the DNA of the two individuals will result.

WO 89/07647 PCUS8900709 Acarose Gel Electroohoresis. To detect a polymorphism in the length of restriction fragments, an analytical method for fractioning double-stranded DNA molecules on the basis of size is required. The most commonly used technique for achieving such fractionation is agarose gel electrophoresis. The rate at which DNA fragments move in such gels depends on their size; thus, the distances traveled decrease as the fragment lengths increase.

The DNA fragments fractionated by agarose gel electrophoresis can be visualized directly by a staining procedure if the number of fragments included in the pattern is small. However, most genomes, including the maize genome, contain far too many DNA sequences to produce a simple pattern of restriction fragments. In order to visualize a small subset of these fragments, a methodology referred to as the Southern hybridization procedure can be applied.

Southern Hybridization Procedure. The purpose of the Southern hybridization procedure, also referred to as Southern blotting, is to transfer physically DNA fractionated by agarose gel electrophoresis onto a support such as nylon membrane or nitrocellulose filter paper while retaining the relative positions of DNA fragments resulting from the fractionation procedure. The methodology used to accomplish the transfer from agarose gel to the support is to draw the DNA from the gel into the support by capillary action.

Nucleic Acid Hybridization. Nucleic acid hybridization is used to detected related DNA sequences by hybridization of single-stranded DNA on supports such as nylon membrane or nitrocellulose filter papers. Nucleic acid molecules that have complementary base sequences will reform the double- stranded structure if mixed in solution under the proper conditions. The double-stranded structure will be formed between two complementary single-stranded nucleic acids even if one is immobilized on a support. In the Southern hybridization procedure, the latter situation occurs. As noted previously, the maize genomic DNA is digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, converted to the single stranded form, WO 8907647 PCTUS89/00709 WO 89/07647 -6and transferred to the support, making it available for reannealing to the hybridization probe.

Hybridization Probe. To detect a particular DNA sequence in the Southern hybridization procedure, a labeled DNA molecule or hybridization probe is reacted to the fractionated DNA bound to a support such as nylon membrane or nitrocellulose filter paper. The areas on the filter that carry DNA sequences complementary to the labeled DNA probe become labeled themselves as a consequence of the reannealing reaction. The areas of the filter that exhibit such labeling can then be detected according to the type of label used.

The hybridization probe is generally produced by molecular cloning of a specific DNA sequence from the maize genome.

Description of the Preferred Embodiments This invention is based on the use of restriction fragment length polymorphism (RFLP) to identify genetic linkages to agronomically important genes. This invention consists of methods for locating agronomically important genes based on RFLPs and of novel DNA probes for use in the methods.

Specifically this invention is based on the identification of restriction fragjents from the ten chromosomes of maize that define genetic linkages between specific chromosomes and agronomically important traits using analysis techniques that can find correlations between the inheritance of one or more DNA sequences and the phenotype of the plant under investigation.

Identifying RFLPs involves the use of restriction endonucleases, DNA mapping, and cloned DNA probes. Restriction endonucleases cleave the genomic DNA molecules at specific sites. Cloned RFLPs are detected as the differences in the size of restriction fragments observed in Southern blotting experiments using cloned DNA probes free of repetitive sequences. Certain polymorphisms can thus be genetic marke's that are associated with a specific agronomic trait.

WO 89/07647 PCT/US89/00709 WO 89/07647 -7- Establishment of such an association permits the monitoring of heritable sequences of genomic DNA. Probes that can detect sequences associated with specific traits can therefore be derived from known gene loci or from anonymous DNA segments. RFLPs appear to be present in all regions of the maize genome, thus making it feasible to construct a detailed maize genetic linkage map and thereby localize agronomically important genes.

RFLPs that identify genetic characteristics are particularly.

useful in breeding programs in order to select for certain traits.

Other uses of RFLPs may include varietal identification, identification and mapping of quantitative trait loci (QTL), quantification of genetic diversity in a crop population, screening genetic resource strains, or populations for useful quantitative trait alleles and their marker-assisted introgression from the resource strain to a commercial variety, and marker-assisted early selection of recombinant inbred lines in plant pedigree breeding programs.

In accordance with the invention, RFLPs are used to identify genetic linkage to agronomically important genes. This invention consists of three major parts: DNA probes shown to reveal polymorphisms between two parent inbred lines and having known chromosomal locations, statistical techniques that can find correlations between the inheritance of one or more DNA probes and the phenotype of the plants under investigation, and use of the identified genetic linkages between specific probes and genetic components of agronomically important traits as an aid in selecting plants and populations in "classical" plant breeding based on Mendelian genetics.

More particularly, the invention comprises determining a particular trait in a maize plant by analyzing maize chromosomes for DNA polymorphisms and linkage to that trait. Specific traits determined include adjusted yield, plant yield, plant height, ear height, GDU shed, GDU silk, grain moisture, root lodging, stalk aL r WO 89/07647 pCT/US89/00709 -8lodging, and stay green. A definition of each of these traits is as follows: Adjusted Yield, Bushels/Acre. The yield in bushels/acre is the actual yield of the grain at harvest adjusted to 15.5% moisture.

Plant Yield. The plant yield is the field weight divided by the number of plants per plot.

Plant Height. This is a measure of the height of the hybrid from the ground to the tip of the tassel.

Ear Height. The ear height is a measure from the ground to the top ear node attachment.

GDU Shed. The GDU is the number of growing degree units (GDU) required for an inbred line or hybrid to shed pollen from the time of planting. Growing degree units are calculated by the Barger Method, where the heat units for a 24-hour period are: (Max. Min.) GDU 2 The highest maximum used is 860F and the lowest minimum used is 50 0

F.

For each hybrid it takes a certain number of GDUs to reach various stages of plant development. GDUs are a way of measuring plant A maturity.

Grain Moisture. The grain moisture is the percentage moisture of the grain at harvest.

Root Lodginq. The root lodging is the percentage of plants that do not root lodge; those that lean from the vertical axis at an approximately 300 angle or greater would be counted as root lodged.

Stalk Lodginq. This is the percentage of plants that do not stalk lodge, stalk breakage, as measured by either natural lodging or pushing the stalks and determining the percentage of plants that break off below the ear.

Stay Green. Stay green is the measure of plant health near the time of black layer formation (physiological maturity).

In general, to identify a polymorphism according to this invention, DNA is extracted from the plant cell and digested with a WO 89/07647 PCTUS89/00709 Wo 89/07647 -9given restriction endonuclease. After tne digest is obtained, and the same is separated by a standard technique such, as, for example, agarose gel electrophoresis, the separated bands are probed with a DNA fragment coding for the RFLP sequence.

Specific probes that may be used in this invention are discussed in the Examples detailed below. Other probes for the polymorphisms can be obtained.

For example, methods for generating additional new DNA fragments also linked with the gene for a particular trait are as follows: A first method is to test randomly chosen maize DNA fragments (either genomic or c-DNA clones) that map to the appropriate region of the maize genomic map. Such mapping can be achieved by two techniques: in situ hybridization to metaphase chromosome spreads; or genetic linkage to any marker as already mapped to the region.

For method, the new fragment need not be polymorphic, but for polymorphisms must first be identified by comparing the restriction pattern of the genomic DNA at the new site in unrelated plants. In method the mapped fragment must still be shown to detect a polymorphism in maize DNA. The polymorphism which represents a genetic marker can then be tested for genetic linkage with genes affecting agronomic traits or can be tested for linkage to other DNA probes, such as those described below.

Another method for obtaining DNA clones is to construct a library from maize DNA isolated from metaphase chromosomes that have been sorted on a fluorescence activated sorter. This method can sometimes yield purified chromosomes of 95% or greater purity.

A final method of obtaining new DNA probes from the region of the chromosome containing the agronomically important gene is to use any probes already mapped to the region in order to "fish out" adjacent overlapping pieces of DNA from genomic libraries (commonly called chromosome walking").

il i •I I pCTUS89/00709 WO 89/07647 CUS89/0709 In all the cases outlined above, a probe must ultimately be found to detect a polymorphism if it is to be useful for testing for the desired trait. The polymorphism must be found to be linked to genes affecting traits or to other useful markers in studies, or to be immediately adjacent to preexisting markers.

The particular probe can be of any desired sequence length as long as it is capable of identifying the polymorphism in the involved DNA region or locus. It can be a DNA fragment by itself, or be present in longer genetic sequences or fragments, or even in a plasmid or other appropriate vehicle. Labelling and hybridization conditions can be readily determined by those of skill in the art. Usually, the stringency is standard for unique sequence DNA from within the species.

A genetic linkage map was constructed from the data presented in the Examples by utilizing the algorithms described by E. Lander et al., Genomics, 1:174-181 (1987), herein incorporated by reference.

These genetic linkage groups were assigned to chromosome arms by (1) using as probes DNA sequences whose location was known, and/or (2) using as markers isozyme loci whose location was known.

By applying statistical analysis to maps, markers, and observed traits in field testing, a detailed table has now been constructed which associates specific genetic markers with the loci of genes which influence specific agronomic traits at a high degree of statistical significance and quantitates the amount of the variation in the observed trait which is accounted for by the genes associated with individual markers. As a result, this invention also provides methods for augmenting conventional plant breeding by identification of individual plants which have the desired genotype at a genetic marker locus associated with one of the foregoing traits, comprising the steps of constructing a preferred RFLP profile for genetic markers identified herein as being associated with the trait or traits in question; 2 WO 89/07647 PCTUS89/00709 WO 89/07647 -11determining the RFLP profiles of individuals in a segregating population with respect to the associated genetic markers; and selecting individuals from the population having RFLP profiles which most closely match the preferred profile.

The construction of the preferred RFLP profile is a matter within the skill of the typical plant breeder. For example, the breeder will select a particular allele which provides a desirable contribution in terms of ear circumference. The contribution viewed as "desirable" i will vary according to the objectives of the breeding program. In one instance, a large ear circumference be be desirable, while in another, a small ear circumference may be preferred. In yet another instance, the ear circumference per se may be unimportant, but the breeder may Sbe working to develop a plant which is homozygous, has the same contribution from both parents, with respect to that trait. In any event, it will be a simple matter for the breeder to prepare a list of desired traits and to construct a preferred RFLP profile for the ideal plant from that list and the genetic marker linkages identified herein.

A segregating population of plants is easy to obtain, and is typically found, for example, in the progeny of selfed F 1 hybrids of two different inbred lines. Once the population has been identified, RFLP profiles of each plant are performed using the well-known techniques described above.

Selection oF the individuals having RFLP profiles which most closely match the ideal profile simply involves comparison of each individual profile with the ideal profile. Some judgement may be required in this comparison, but the exercise of such judgment will be well within the skill of the typical plant breeder. For example, it will be extremely unlikely, and depending upon the population examined it may be genetically impossible, for any individual to have aq RFLP profile which precisely matches the ideal. However, one or more individuals will have a greater percentage of matches to the ideal pCTJUS89/00709 WO 89/07647 -12than the remainder of the population. Where two individuals have approximately equal matches to the ideal, the breeder will make tradeoffs among traits. One individual may offer the desired allele for ear circumference and kernel row length, but lack the desired allele for stay green. Another individual will offer the desired allele for ear circumference and stay green, but will lack the desired allele for kernel row length. Selection between the two will simply involve a decision by the breeder on whether it is preferable to have the allele for kernel row length or for stay green, and in fact such decisions will be a relatively trivial exercise compared to the judgements made in conventional plant breeding, which are based on less clear-cut information about the genetic makeup of the individuals. The breeder also has the option of proceeding to the next stage of breeding with both individuals.

The following tables identify specific trait-marker linkages for a number of agronomic traits commonly involved in plant breeding.

Markers identified by the prefix PIO- are available commercially from Pioneer Hi-Bred International, Des Moines, IA 50309. Markers identified by the prefixes BNL- and UMC- are publicly available markers which can be obtained from Brookhaven National Laboratory and the University of Missouri, respectively. The remainder of the markers are either published isozyme markers of known genetic location, such as AMPI, MDH2 and GLUI, or are probes fur specific mutant genes which are well known and whose location has been identified, as mentioned above.

These Tables identify linkages observed in bulk F 4 populations, which reflect inbred performance, and in topcross trials, which reflect performance in hybrids. As might be expected, some overlap is observed between the two sets of linkages.

Traits not self-explanatory and not identified elsewhere herein include: Grain quality. This is a subjective score based oi visual grading of the shelled corn in terms of moldy and cracked kernels.

I WO 89/07647 pCT/US89/00709 -13- Cold test germination. This is a percentage germination test performed in a cold chamber, and evaluates germination performance under adverse conditions.

Soak test germination. This is a test of percentage germination under ideal germination conditions, but after the seed has been soaked prior to planting.

Bulk density. adusted. This is a measure of weight per unit volume of bulk seed, adjusted to 15.5% moisture.

Dropped ears. This is a raw count of ears found on the ground per two-row test plot.

European corn borer second brood tolerance. This is a visual evaluation of plant damage caused by infestation by the second annual brood of the European corn borer caterpillar.

Plant yield. This is the yield of a test plot divided by the number of plants in the test plot.

Early stand count. This is a raw count of plants per test plot (or number of seeds planted) after emergence and before thinning.

Bare tip. Also called nose back, this is a visual grade based upon the amount of ear tip which is devoid of kernels.

"Statistically transformed" measurements involve the same physical measurements or evaluations as the raw scores, but the raw scores have then been put through a standard statistical transformation to fit them to a normal curve. Unless otherwise indicated, all values measured are averages over the individual ear, plant, test plot or larger field.

WO 89/07647 pTU8IOO -14- Table 1 List of genetic markers associated with quantitative traits evaluated in topcross progeny.

TRAIT=Yield. Bushels/Acre (BU ACR) BNL6. 20 PI020508 BNL1O.24 PI02071 PI020708 UNCilO P 10207 14 P1020576 P1020508 BNL6 .16 PI020589 PI020684 BNL8.32 PI020558 BNL5. 37- BNL7.65 PI020569 PI020746 BNL8.39 PI020509 BNL5.37- UMC015 P101537 UMC116 PI1020593 TRAIT= Moisture (MST) PI020640 PI01012 PI020569 BNL 10.39 P102052 BNL12.06 PI0205 PI01537 UNMC120 PI020646 UNMC061 PI02017 UMC 116 PI0207 14 PI015 13 UMC034 BNL6. BNL8.32 UMC012 '1 TRAIT=Bulk Density. adiusted (TSTWTA) PI020518 BNL6. 20 P1020508 P102071 PI020589 PI020708 UNCilO PI02052 PI02075 PI020646 PI01012 PI020511 PI020508 PI01025 PI102045 PI020684 BNL8.32 UNC081 BNL3.04 PI101033 PI0205 PI020558 BNL7.65 PI0612 PI020569 P 1020746 SH 1 csS1 PI065 P102017 PI020509 UNC015 PI01524 PI01537 UNC 116 wx 1 P1020626 G LU I PCr/US89/00709 WO 89/07647 TRAIT=Yield/% moisture (YLDMST) BNL12.06 PI020518 UMC034 PI01012 P1020509 PI020508 37- UMC019 UMC 043 PI020589 TRA IT=GDU PI01012 PI020558 BNL5.37- PI01533 BNL5.46 BNLI3 .05 PI020714 UMC081 BNL1O.24 BNL7 .65 PI020622 PI02026P BNL8. 29 PI020576 PI020508 BNL6.16 P102071 P10612 UMC061 P1020558 BNL5. 37- UMC066 BNL8.33 PI101524 Shed (GDUSHD) PI0205 PI020509 BNL5.37- UMC999 PI0612 PI01040 UMC012 PI020554 PI02017 PI020508 BNL 10.24 PI020713 PI01524 BNL1O.39 WX 1 BNL1O. 13 PI020576 PI020508 P 102052 1 PI020725 PI020589 UMC 120 PI02052 TRAIT=Stay green (STAGRN) PI020690 PI02017 BNL1O.24 UMC046 PI020599 PI020569 UMC116 BNL8.32 P1020714 UMC012 TRAIT=Dropped Ears (DRPEAR) BNL1O.39 UMC081 BNL6. 20 MDH2 PI01537 BNL1O.39

SHI

BNL7. 21 B BNL5. 37 P1020595 PI020684 UMC 120 BNL3.04 BNL1O.13 2 WO 8907647PCTfUS89/00709 -16- TRAIT=Dropped Ears, Statistically transformed (FOGDE) PI020690 TRAIT=Ear height (EARHT) PI020640 PI020558 BNL5.37- PI01533 PI020569 BNL 10.39 wx I UMC034 PI020509 BNL5.37- PI020713 BNL8.32 UMC 120 UMC 081 P1020576 P1020508 BNL1O. 24 PI020725 BNL8.39 P1020714 P1020646 PI020511 PI020508 PI020521 BNL8.33 PI020593 UMC012 PI01513 TRAIT=Plant height (PLTHT) PI020690 PI020511 PI020508 BNL6. 16 P101080 UMC 120 PI020646 PI020668 P1020558 BNL5.37- P1020521 BNL8.33 UMC012 PI01513 PI020518 P1020509 BNL5. 37- PI01533 PI101040 PI065 UMC044 PI020576 PI020508 BNL1O.24 UMC 999 BNL1O.39

GLUI

TRAIT=Grain quality (GRNQUL) BNL 2. 06 BNL6. 29 BNL8. 29 PI020528 BNL6. 20 PI020599 TRAIT=European Corn Borer Second Brood Tolerance (ECB2SC) PI02017 PI020576 PI020509 BNL5.37- PI020569 PI01537 UM C999 BNL 14 .28 PI020508 PI020708 BNLIO.39 UMC 116 UMC 120 BNL8.39 P 1020626 PI020593 PI02075 2 PCTJ[US89/00709 WO 89/07647 -17- TRAIT=Root UMC094 24 BNL6.29 PI02045 PI020595 lodgving (RT LDG) P1020511 BNL6.16 P1020527 PI067 UMC012 BNL5. 37- UMC 999 PI020528 UMC065 P1020562 BNL5. 37- BNL8. 23 P1020854 MOH 2 PI020646 BNL5.37- UMC019 P1020854 MDH 2 TRAIT=Root lodging, UMC094 BNL5.37- BNL6.29 PI02045 PI020595 *statistically transformed (FOGRTL) PI020576 PI020511 BNL1O.24 UMC999 PI020527 P1020528 PI067 UMC065 UMC012 PI020562 TRAIT=St~ PI020622 UMC 057 TRA1T=St~ PI020537 PI020558 PI01524 P1020626 UMC057 alk lodgiing (STKLDG) PI01537 UMC 044 BNL14. 28 alk lodgiing~, statistically transformed (FOGSTL) PI01012 PI0205 PI020597 PI020622 UMC065 P101537 PI02075 PI020646 BNLIO.13 UMC044 P1020562 E8 PI0612 UMC 110 P1015 13 PCr[US89/00709 WO 89/07647 18- Table 2 List of genetic markers associated with quantitative traits as identified in bulk f4 progeny TRATT=Yield. Bushels/Acre (BU ACR) BNL5.59 UMC043 PI01518 PI01513 BNL6.20 PI020622 PI020589 UMC042 P10612 PI0105 PI1020608 PI01524 SH 1 TRAIT=Plant yield (PLNTYLD) P1020537 BNL6. 20 P101518 PI0105 PI020518 UMC043 PI020589

SHI

BNL8. 29 P 1020622 PI020523 PI020626 PI020557 PI01524 PI1020569 PI020646 TRAIT=Weiciht of 100 kernels (CKWT) PI020603 PI020682 BNL8.23 UMC 110 BNL14.2R PI020689 PI020674 PI067 P1020714 UMC057 PI1020654 PI020575 UMC065 PI0105 BNL1O.13 BNL5. 59 PI020644 UMC021 P10205S4 UMC044 TRAIT=Dropped ears, statistically transformed (FOGDE) P102 05 UMC 066 PI1020527 P1020593 PI02017 UMC019 PI020528 PI020558 P101518 P1067 PI1020508 PI020531 BNL8.39 2 PC/US89/00709 WO 89/07647 -19-

I

.1' 1 TRAIT=Cold test germination (CTST) UMC034 BNL8.33 UMC065 UMC021 P1020708 P1020684 BNL8.32 BNL8.39 GLUl P1020646 TRAIT=Soak test germination (SKTST) PI020518 PI020511 PI020569 PI020708 P102052 GLUI TRAIT=Early stand count (ESTCNT) UMC061 PI020713 UMC042 BNL7.65 PI065 TRAIT=Ear circumference (EARCIR) P101017 PI020569 PI020746 SH 1 P1020509 PI1020746 P1020725 BNL8.33 BNL6.20 UMC 999 PI020597 UMC015 PI020622 PI020589 PI1020595 PI020708 UMC 120 P1067 P101537 UMC 116 P1065 BNL8.33 BNL9. 11 PI020597 PI01016 PI020508 PI020726 UMC042 P102071 PI0612 PI020531 P1020599 PI1020746 P 10105 P 102 0603 BNL5.37- UMC031 UMCO019 PI01025 PI01524 PI02026P PI020569 UMC 116 UMC081 P 1020640 BNL1O.24 BNL5.46 BNL7. 65 P1020608 PI101518 PI067 P101537 BNL1O.39 PI065 7 I'Cr[US89/00709 WO 89/07647 TRAIT=Ear diameter (EARDIA) P1020509 PI020508 BNL10.24 UMC999 PI02071 PI01025 PI01518 P1020595 PI01537 PI020708 UMCO81 TRAIT=Ear length (EARLGT) PI020508 PI020726 PI020622 PI020599 PI020746 BNL 15.45 UMCO062 P1020554 BNL5.37- UMC031 PI0612 PI020569 BNL8.39 UMC015 BNL8.39 P1020557 PI02071 PI02020 TRAIT=Cob PI020640 BNL6.20 PI020726 BNL15.45 BNL7.65 PI020608 PI01524 PI02026P P 1020726 PI020854 P101040 circumference (GOBCIR) BNL12.06 BNL5.37- UMC031 UMC042 UMC015 UMC043 P101518 UMC065 BNL8.29 UMC999 BNL5 .46 UMC066 PI02071 PI020622 PI020589 UMC081 PI020557 P 101080 P1020597 UMC019 PI101025 PI0612 P102053 1 PI065 TRAIT=Cob diameter (COBDIA) PI020640 UMC031 UMC042 UMC015 UMC043 PI01518 UMC065 MDH2 PI065 BNL12.06 BNL5.46 UMC066 PI02071 PI020622 PI020589 UMC021 P1020599 GLUl PI020713 PI020597 UMC019 PI01025 PI020531 UMC 046 BNL8.44 PI020646 PI020725 BNL15.45 BNL7.65 PI020608 PI101524 PI02026P UMC062 UMC081 pcTJUS89/00709 WO 89/07647 TRAIT=Bare PI020640 BNL1O.24 PI01025 PI020589 UMC065 BNL1O.39 UMC081 TRAIT=Kerne PI020518 UMC015 PI020527 PI01040 TRAIT=KernE PI020603 PI02017 PI020569 BNL8.39 tip (NOSBAC) P1020644 BNL15.45 P10612 PI020531 P1020569 UMC012

CSSI

I1 row leng~th (ROWLEN) BNL8.29 PI02071 UMC 065 SH 1 ~1 Depth (KDEPTH) PI020640 E8 PI01537 P1020661 UMC042 P 101524 PI02026P P1020708 WX 1 PI020554 PI020557 PI01524 UMC062 PI020661 PI020622 PI020708 BNL6.20 UMC031 PI01524 PI01014 cSsI P102017 UMC066 P101518 PI020854 UMC 116 PI02052 P102075 PI020726 PI020523 UMC 116 P101012 UMC021 UMC1 E8 UMC043 P101518 UMC062 P1020554 TRAIT=Kernels per kernel row (KERPRO) BNP'06 UMC061 PI020713 PI020725 P1020622 P10612 PI020589 UMC065 BNLIO.39 UMC081 PI020626 WO 89/07647 pCTJUS89/00709 -22- TRA1T=Number of kernel rows per ear (KERROW) PI020690 UMC999 PI020726 UMC031 BNL5.46 PI020597 UMC042 UMC019 BNL7.65 UMC015 PI02071 P101025 PI020608 PI0612 PI01524 P101518 PI020589 PI020531 PI020566 PI0~uC?gp PI02045 UMC065 UMC046 PI020569 PI01537 PI020708 P1020684 PI020746 UMC116 UMC1IO BNL8.32 BNL8.44 P10105 UMC057 UMC044 TRAIT=Kernel width (KWIDTH) BNL12.06 UMC023 P1020726 UMC015 PI02071 PI020608 P10612 PI01524 PI020589 P1020531 PI020566 PI02026P PI02045 UMC065 PI020569 PI01537 PI020708 PI020684 PI020746 UMC116 UMC11o BNL8.32 BNL8.44 UMC012 UMC057 BNLIO.13 UMC044 TRAIT=% Moisture (MST) UMC094 PI020537 PI020603 PI020689 PI020640 P1020575 PI02044 P1020661 PI020518 BNL8.29 PI020557 UMC061 UMC043 PI020622 P1020531 UMC021 PI01016 BNL8.32 P1020593 BNL1O.39 PI065 PI01513 UMC057 BNL1O.13 UMC044 r pCr[US89/ 00709 WO 89/07647 i Pt Ii

'I

~~1 TRAIT=Yield/% moisture (YLDMST) BNL12.06 BNL5.59 BNL6.16 UMC042 UMC043 P1020622 PI01518 P1020589 SH1 P1020646

TRAIT=GDU

P1020640 P10205 PI020713 UMC 066 UMC012 csS1 G LU 1 UMC057 Shed (GDUSHD) BNL12.06 BNL5.37- P1020725 PI020854 WX 1 PI020554 PI020646 BNLIO.13 UMC06 1 BNL7. 65 P10612 P1020527 PI01513 UMC034 BNL5.37- BNL5.46 BNL13. 05 P 102052 BNL8.17 PI01513 BNL6 PI020608 P101524 PI0105 PI01012 BNL 10.24 UMC042 BNL9.11 UN 0081 BNL7 .21B PI01033 TRAIT=GDU Silk (GDUSLK) P1020690 P1020713 UMC066 UMC012 PI02052 BNL8.17 BNL1O. 13 PI020537 P 102072 5 P101524 PI0105 UMC081 GLUl P1020640 BNL5 .46 P 1020854 SH 1 OSSi PI020646 BNL1O.24 UMC042 BNL13. WX 1 P10205 54 PI01513 TRA1T=Stay green (STAGRN) P 1020 640 P1020682 PI02044 BNL6.16 BNL12.06 PI020674 P1020661 PI01518 PI1020654 P 1020575 P 1020557 PI020531 BNL5 .59 PI020644 UMCO034 P1020 566 PCTIUS89/00709 WO 89/07647 -24- PI020527 wx I UMC044 BNL8.39 BNL8.17 BNL8.44 UMC057 P102020 BNLIO. 13 TRAIT=Plant height (PLTHT) BNL12.06 PI020576 BNL 10.24 BNL 10.39 PI02052 PI02075 UMC034 PI020508 PI020713 UMC 120 UMC 081

GLUI

PI01012 BNL5.37- PI020725 UMC0 12 PI020554 PI020646 PI0205 BNL5.37- PI020728 WX 1 P1020626 TRAIT=Ear height (EARHT) PI020640 PI01012 P1020508 BNLIO.24 PI020713 BNLIO.39 PI02052 BNL12 .06 PI0205 PI020508 BNL6.16 P1020725 UMC 120 UMC081 P102044 PI020576 BNL5. 37- PI020521 BNL6.29 UMC012 PI020554 UMCO034 PI020509 BNL5.37- UMC999 PI020728 WX 1 UMC057 TRAIT=European Corn Borer Second Brood Tolerance (ECB2SC) 59 E8 UMC042 UMC 065 PI020569 BNL1O.39 PI020644 BNL6. 16 PI020608 MDH2 P101537 UMC 120 P10205 PI020597 P10612 PI020595 P1020708 PI0105 BNL6. BNLI 5. PI02045 PI020599 UMC 110

A

27 PCTfUS89100709 WO 89/07647 TRAIT=Root lodg~ing~ (RT LMG UMC094 PI020537 P1020640 PI020518 P1020558 P1020509 BNL5.37- BNLIO.24 UMC031 UMC066 MDH2 PI020595 P1020603 PI020576 PI020508 PI020713 UMC019 UMC081 PI020689 P1020511 P1020508 P1020725 UMC065 icallv transformed (FOGRTL) PI020640 PI020654 PI020644 PI020576 PI020511 PI020558 PI020508 PI020508 BNLS.37- BNL1O.24 UMC019 BNL6.29 PI10205 18 PI020509 BNL5. 37- UMC 065 PI020581 UMC081 TRAIT=Stal k BNL6.20 PI020599 PI02075 lodging~ (STKLDG) PI020597 PI020728 UMC057 MDH2 UMC012 PI020595 PI0105 TRAIT=Stalk lodging. statistically transformed (FOGSTL) PI020682 BNL6.20 PI020599 BNL3.04 PI020661 UMC 042 BNL 13.05 PI020668 P1067 Pi0105 AM P1 P 1020595

SHI

TRAIT=Grai n 59 P1020661 P 101012 PI020509 P1020569 P1020746 quality (GRNQUL) PI020575 AM P1 P10205 PI102071 PI101537 UMC116 PI020644 P 1020557 PI02017 PI01025 P1020708 UMC 110 PI02044 UMC061 E8 BNL8.33 PI020684 BNL8.32 ~1 F WO 89/07647 BNL8. 39 BNL1O. 13 PCT/1JS89/00709 -26- PI020593 PI01033 UMC057 PCT/US89/00709 WO 89/07647 -27- Having now defined the invention, the same will be understood by means of specific examples which are, however, not intended to be limiting unless otherwise specified.

EXAMPLE I Determination of Agronomically Useful Phenotypes Development of Progenies for Field Testing The inbreds B73 and Mo17 were crossed to produce the F 1 hybrid designated B73/Mol7. Hybrid seed was planted, several plants selfed, and the seed bulked to produce the F2 generation designated B73/Mol7)X. 175 seeds from the F2 generation were planted in peat pots in a greenhouse. Seedlings were transplanted to the field at normal planting time. Each plant was self-pollinated using the usual procedures for pollination of corn to produce the F3 ears designated B73/Mol7)Xn where n ranged from I to 175 and represents the specific F3 ear. Ears were harvested, identified to plant number, and kept separate. Each ear therefore contained seed that would generate an F3 family.

V: A winter nursery was used to produce seed for field testing from the F3 ears. Twenty-four kernels from each of 112 F3 ears were planted in an isolated crossing block. Interplanted around those 112 rows were rows of Pioneer inbred V78. Tassels were removed by hand from every plant of the 112 F3 families. Thus, the ear of each F3 plant from the B73/Mo17 cross was pollinated by the inbred V78. For each of the 112 F3 families, each ear from the 24 plants were harvested, dried, shelled, and bulked together. These 112 entities of seed were considered as 112 F3 topcrosses to inbred V78.

In addition, a separate 24 kernels from each F3 ear were planted in 112 rows, one for each F3 family. Within each row, ten plants were self-pollinated to derive F4 ears. For each of the 112 F3 families, each of the self-pollinated ears were harvested, dried, shelled, and PCT/US89/00709 WO 89/07647 -28bulked together. These 112 entities of seed were considered as 112 F4 bulks.

Determination of Whole Plant Phenotype The 112 F3 topcrosses and 112 F4 bulks were evaluated in field performance tests (yield tests). The tests were conducted according to standard yield test procedures as used in the profession of agronomy and crop breeding.

Each of the 112 F3 topcrosses comprised an entry in a randomized complete block design. Check entries were added making a total entry list of 125. The experiment was grown in two replications at each of four locations in central Iowa. Each replication of an entry was planted in a two-row plot. Plots were 5.3 meters long with .76 meters between rows. Plots were overplanted and thinned to even and uniform stand of 50 plants per plot. These plants were allowed to grow to maturity and data were collected for various traits throughout the season.

The 112 F4 bulks were tested identically except there were three test locations instead of four. Two locations were in central Iowa, and the other was in central Indiana.

The following traits were measured on each plot. Each is considered an agronomic trait important in corn breeding.

TRAIT ABBREVIATION DESCRIPTION Adjusted Yield BUACR Field weight adjusted to 15.5% moisture expressed as bushels per acre.

Plant Yield PLNTYLD Field weight divided by the number of plants per plot.

Plant Height PLTHT From ground to tip of tassel.

Ear Height EARHT From ground to top ear node.

pWO 89/07647 CT/US89/00709 WO 89/0747 -29- GDU Shed GDUSHD Accumulated heat units to the day that of plants in a plot were shedding pollen.

GDU Silk GDUSLK Accumulated heat units to the day that of plants in a plot had silks emerged at least one inch.

Grain Moisture MST Percent moisture in grain.

Root Lodging RTLDG Number of plants per plot leaning from vertical more than Stalk Lodging STKLDG Number of plants with stalks broken below the ear at harvest.

Stay green STAGRN Relative amount of green leaf tissue remaining at physiological maturity.

The data from plots of F3 topcrosses and F4 bulks were made available for statistical analyses in October 1987. Data were collected on the progenies rather than the original, individual F2 plants because of the well-established principle that the heritability of these complex traits is very low when measured on a single plant basis. Each trait is probably governed by more than one gene, and expression is affected by environmental conditions. Thus, by testing F3 topcrosses and F4 bulks in replicated trials, a more accurate measure of phenotype was obtained. These measures a phenotype of F3 and F4 progeny were considered accurate estimates of the phenotype of each 112 F2 plants from which they were derived.

EXAMPLE 2 Identification of Informative Probes Maize DNA Isolations Total DNA can be isolated from various maize tissues (leaves, seedlings, etc.) by any one of several standard methods (for example see Maniatis et al Molecular Cloning. A Laboratory Manual (1982); Dillon et al., Recombinant DNA Methodology (John Wiley Sons 1985).

E

WO 89/07647 PCT/US89/007 0 9 Southern Hybridization Agarose gel electrophoresis of restriction enzyme- generated fragments of maize DNA, transfer of the DNA to nylon membranes, and hybridization with a radioactively labeled probe can be done by any of several standard methods (for example, see Maniatis et al., Molecular Cloning, A Laboratory Manual).

Identification of Informative DNA Probes Total DNA from the maize inbred B73 was purified and digested to completion with the restriction enzyme Pst I and electrophoresed on an agarose gel. Fragments from the size classes 600-1,000 base pairs 1,000-1,500 bp, 1,500-2,000 bp, and 2,000-2,500 bp were ligated into one of several E. coli vectors at the Pst I site and transformed into one of several laboratory strains of E. coli using standard conditions (for example, see Maniatis et al., Molecular Cloning, A Laboratory Manual). Colonies containing plasmids with single inserts were identified by plasmid minipreps and agarose gel electrophoresis.

Plasmid DNA from each previously characterized colony was purified, digested to completion with Pst I, and the two fragments (maize DNA insert and cloning vector) separated by agarose gel electrophoresis. Isolated maize insert DNA, still in the agarose plug, was radioactively labeled with P 32 by either nick translation or random priming.

Southern hybridization was done using labeled maize insert DNA as probe and total cell DNA from various maize inbreds digested to completion with one or more restriction enzymes as target. After washing the membrane to remove any nonhybridized probe, the membranes were subjected to autoradiography.

Probes were selected for further consideration if 1-3 autoradiographic bands were observed for at least one restriction enzyme, and band patterns were different between at least two maize inbreds.

1 WO 89/07647 PCT/US89/00709 -31- EXAMPLE 3 Determination of Linkage Among RFLP Marker Loci Individual probes were hybridized to DNAs prepared from each of the 112 F2 plants described above. Each plant was scored for its allelic composition at the locus defined by the probe. Scores were A (only band(s) contributed by B73 present), B (only band(s) contributed by Mo17 present) or H (bands from both parents present). A genetic linkage map was constructed from these data by using the algorithms described by Lander et al. (1987), supra.

These genetic linkage groups were assigned to chromosome arms by using as probes DNA sequences whose location was known, and/or (2) using as markers isozyme loci whose location was known.

EXAMPLE 4 Determination of Correlations between Phenotypic Expression of Traits and Specific Probes Assessment of the Quality of Quantitative Trait Data Obtained in the Field and Determination of Quantitative Trait Data to Group for Investigation Linkaqes between RFLP Marker Loci and Quantitative Trait Loci Analysis of variance due to environments, genotypes, and genotype by environment interactions were determined for all quantitative traits. A maximum/minimum test for heterogeneity of variances among locations was used to make sure that data from all locations had similar variability. Data from traits which showed significant variability among genotypes and were highly heritable 0.65) were kept for analyses of associations with RFLP marker loci. Because all traits showed some genotype by environment interactions, it was decided to investigate relationships between probes and quantitative traits by location.

i!

I

1 S89/07647 PCTUS89/00709 -32- Linear and Non-linear Relationships between Quantitative Traits and RFLP Marker Loci Were Used to Detect Linkaqes between Marker Loci and Quantitative Trait Loci (QTLs).

Data from each test were analyzed using a two-factor analysis of variance for each pair-wise combination of quantitative trait and marker locus; where factors were the marker locus and environment.

Data from traits which showed no significant locus by environment interactions, as judged by an F-test, were investigated across environments. Data from traits which showed significant locus by environment interactions were investigated by environment. An F-test 4 was used to determine if significant variability in the expression of a trait was associated with differences in genotypes of a RFLP marker locus. The marker locus is considered linked to a QTL if there is a significant F-value for the variability at the marker locus and either the linear or non-linear orthogonal contrasts associated with the marker.

Based upon the above criteria, loci involved in the expression of grain moisture, adjusted yield, stalk lodging, root lodging, plant height, ear height, plant yield, GDU shed, GDU silk, and stay green in F3 top crosses (TC) and F4 bulk tests (PS) are found to be linked with the indicated mapped probes (Table The numbers in each TC or PS column give the percent of the total variation for that trait expressed in the population associated with a particular RFLP locus.

Only values significant at the 0.05 level are shown. CM refers to spacing between the probes in centi-Morgans.

1 Having now fully described the invention, it will be understood that the same can be carried out within a broad and equivalent range of probes, conditions, enzymes, detection techniques, and the like without affecting the spirit or scope of the invention or of any embodiment herein.

C C C 4 0 4 S. C 0 0** .4 4 C S C 4** 0 4 5 S C C C C C

A

cM PROBE BUACR TC PS

PLNTYLD

TC PS B73/Mo 17

PLTHT

rC PS Tabl e 3 RFLP Linkage Data EARHT GDUSHD TC PS TC PS AMP 1 PI020668 PI020661 PI02044 PI020575 PI020644 PI020674 PI020682 PI020 654 2 2 4 3 2 2 Chromosome 1 GDUSLK MST TC PS TC PS 2.3 0.3 2.9 0.7 5.9 0.4 7.3 0.6 4.0 0.4 2.6

RTLDG

TC PS STK LOG TC PS 2 2 2

STAGRN

TC PS 2 2 3 3 4 8 2 6 6 2 2 1 4 3 3 2 3 3 4 cM PROBE BUACR TC PS PI01012 3 573/Mo 17 PLNIYL0 PLTHT TC PS TC PS RFLP Linkage Data EARHT GOUSHO TC PS TC PS Chromosome 2 GDUSLK MST TC PS TC PS RTLDG STKL0G TC PS TC PS

STAGRN

TC PS PI0205 P102017 3 5 5 5 1 2 3 4 4 I I TCW/846Z ~pr S S S

S

S 5 *5 **S

S

S S S S S S S S*S S S S S S S S S S S**

-U,

cm PROBE BUACR TC PS P1020599 2 6 P1020595 1 3 MDH2 14 PI01014 1 22 PI01016 873/Mo 17 PLNTYLO PLTHT TC PS TC PS Table 3 (Continued) RFLP Linkage Data Chromosome 6 EARHT GDUSHO GDUSLK MST TC PS TC PS TC PS TC PS

RTLDG

TC PS 4 5 7

STKLDG

TC PS

STAGRN

TC PS 4 2 4 2 cM PROBE BUACR TC PS P10105 3 12 PI02052 11 PI020554 61 PI020562

PLNTYLO

TC PS 1 11 B73/Mo17

PLTHT

TC PS 3 3 RFLP Linkage Data Chromosome 9 EARHT GOUSHO GDUSLK MST TC PS TC PS TC PS TC PS 2 11 1.0 6 4 10 11 0.7 7 4 9 11

RTLDG

TC PS

STKLDG

TC PS 6

STAGRN

IC PS 4 TCW/M4Z S S

S

Tal 3 (Contnued S C PS C S CP cm PROBE 8UACR TC PS

PLNTYLO

TC PS B73/Mol7 P LTHT TC PS

RTLDG

1TC PS

STKLOG

rC PS

STAGRN

TC PS E8 PIOH206 PI020576 PI020511 PI01080 PI020521 PI01533 2 3 2 3 3 6 8 5 3 3 2 8 10 4 7 4 6 4 4 4 0.3 3 7 12 6 3 1 5 2 11 3 1 4 4 7 3 3 4 cm PROBE BUACR TC PS PI020589 PI01518 PI0612 PI01524 PI020622 PI020531 PI0H2026 PI020566 PI020527 PI020528 PI067 2 4 5 1 4 5 1 6 B73/Mo 17 PLNTYLO PLTHT TC PS TC PS 1 4 51 1 3 5 1 5 RFLP Linkage Data EARHT GOUSHO TC PS TC PS Chromosome GDUSLK MST C PS TC PS RTLDG STKLOG TC PS TC PS

STAGRN

TC PS 3 4 2 5 5 4 3.1 0.3 4.3 0.4 2.8 0.3 4.2 0.4 4.4 2 2 2 6 12 3 6 2.5 6 2 1 2 TCW/846Z S S S S S S S S S S S S* S 55 S

S

S S S S S S S S S S S S S S S S S S S *SS cm PROBE BUACR TC PS P LN

TC

Table 3 (Continued) B73/Mol7 RFLP Linkage Data C TYLD PLTHT EARHT GDUSHD GD[ PS TC PS TC PS TC PS TC 3 8 5 2 4 4 3 4 3 5 2 6 3 2 8 3 3 3 3 6 P101 033 GLUl PI020646 PI065 ~romosome JSLK MST PS TC PS 4 0.3 4 5 0.44 7 0.5 4 4 0.4 5

RTLDG

TC PS

STKLDG

TC PS

STAGRN

TC PS .4 .4 .8 cm PROBE BUACR TC PS PI020558 3 4 PI020509 3 PI020508B 4 3 PI020508E 2 B73/Mol7 RFLP Linkage Data Chromosome XI PLNTYLD PLTHT EARHT GIJUSHO GDUSLK MST TC PS TC PS TC PS TC PS TC PS TC PS 2 3 1 5 2 2 2 5 2 7 5 4 3 4 2 9 5 6 3 0.7 2.3 2 4 2 8 4 6

RTLDG

TC PS 3 9

STKLDG

TC PS

STAGRN

TC PS TCW/846Z 41! a a a a a a. a a a a a a a a a a. a a a a cm PROBE BUACR 1-C PS PI020746 2 4 PI020684 2 3 PI020708 3 3 PI01537 4 14 PI020569 7 PI020581 873/Mo 17 PLNTYLD PLTHT TC PS TC PS Table 3 (Continued) RFLP Linkage Data Chromosome X2 EARHT GDUSHD GOUSLK MST TC PS TC PS TC PS TC PS

RTLDG

TC PS

STKLOG

TC PS

STAGRN

TC PS 2 3 2 1 3 4 0.5 0.4 3.3 0.6 2.5 1.2 3 2.1 2 4 2 2 cM PROBE BUACR TC PS PI020690 18 PI020537 4 PI020603 3 PI020689 16 PI020640

I

PINTYLD

TC PS B73/Mol7

PLTHT

TC PS 2 RFLP Linkage Data EARHI GDUSHO TC PS TC PS 2 Chromosome X3 GDUSLK MST TC PS TC PS 4 5 3.3 4 7.1 4 6 1.0 11.9

RTLDG

TC PS 5

STKLDG

TC PS

STAGRN

1-C PS 3 2 2 2 5 TCW/846Z

Claims (9)

1. A method of determining a correlation between a phenotypic trait in maize and a restriction fragment length polymorphism comprising: digesting genomic DNA from maize inbred B73 or maize Inbred Mo17 with a restriction endonuclease that produces a restriction fragment length polymorphism digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step detecting said restriction fragment length polymorphism with a hybridization probe containing sequence information capable of hybridizing to and identifying said RFLP, thereby generating a restriction pattern; and correlating the presence or absence of said RFLP in said digest with the respective presence or absence of said trait. 15 2. A method of determining a correlation between a phenotypic trait in maize and a restriction fragment length polymorphism comprising: o digesting genomic DNA from a maize plant that is a cross between maize inbred B73 and maize inbred Mo17 with a restriction S endonuclease that produces a restriction fragment length polymorphism 20 digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step detecting said restriction fragment length polymorphism with a hybridization probe containing sequence information capable of 25 hybridizing to and identifying said RFLP, thereby generating a restriction pattern; and correlating the presence or absence of said RFLP in said digest with the respective presence or absence of said trait.

3. A method of determining a correlation between a phenotypic 30 trait in maize and a restriction fragment length polymorphism comprising: digesting genomic DNA from a maize plant with a restriction endonuclease that produces a restriction fragment length polymorphism digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step detecting said restriction fragment length polymorphism with a hybridization probe made from maize inbred B73 DNA digested with V z restriction endonuclease Pst I, thereby generating a restriction pattern; ~E CWI/848Z i i WO 89/07647 PCT/US89/00709 determining the RFLP profiles of individual corn plants in a n 39 and correlating the presence or absence of said RFLP in said digest with the respective presence or absence of said trait.

4. The method of any one of claims 1 to 3 wherein said trait is selected from adjusted yield, plant yield, plant height, ear height, GDU shed, GDU silk, grain moisture, root lodging, stalk lodging or stay green. The method of claim 1 or claim 2 wherein said hybridization probe is a genomic DNA probe made from maize inbred B73 DNA digested with restriction endonuclease Pst I.

6. A method for identifying and mapping quantitative trait loci (QTL) for a phenotypic trait in maize plant B73, Mol7 or a cross thereof by analyzing the chromosomes of said plants for DNA polymorphisms and linkage to said trait comprising: digesting genomic DNA from a maize plant with a restriction endonuclease that produces a restriction fragment length polymorphism digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step detecting said restriction fragment length polymorphism with a hybridization probe containing sequence information capable of hybridizing to and identifying said RFLP, thereby generating a restriction pattern; and correlating the presence or absence of said RFLP in said digest with the respective presence or absence of said trait wherein said 25 phenotypic trait is selected from adjusted yield, plant yield, plant height, ear height, GDU shed, GDU silk, grain moisture, root lodging, stalk lodging, or stay green.

7. A restriction fragment length polymorphism probe that can detect a polymorphism in one or more maize plants wherein said 30 polymorphism is linked to a phenotypic trait selected from adjusted yield, plant yield, ear height, GDU shed, GDU silk, grain moisture, root lodging, stalk lodging, or stay green.

8. The probe of claim 7 wherein said maize plant is inbred maize B73, Mol7 or a cross thereof.

9. A method for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with yield in bushels per acre, comprising the steps of \constructing a preferred RFLP profile with respect to one or more CW/848Z 40 genetic markers selected from BNL5.59, BNL6.20, UMC042, PI020608, UMC043, PI020622, PI0612, PI01524, PI01518, PI020589, PI0105, SH1, and PI01513, or a functionally equivalent genetic marker; determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with plant yield, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020537, PI020518, BNL8.29, PI020557, BNL6.20, UMC043, PI020622, PI01524, PI01518, PI020589, PI020523, P1020569, PI0105, SH1, PI020626, and PI020646, or a functionally equivalent genetic marker; determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. e *o L 848 8 WO 89/07647 PCT/US89/00709 A method acc:rding to al m 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with the weight of 100 kernels, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020603, PI020689, PI020654, BNL5.59, PI020682, PI020674, PI020575, PI020644, BNL8.23, PI067, UMC065, UMC021,. UMC110, PI020714, PI0105, PI020554, BNL14.28, UMC057, BNL10.13, and UMC044 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method ba-e;rig for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with statistically transformed dropped ear count, comprising the steps of constructing a prefer'ed RFLP profile with respect to one or more genetic markers selected from PI0205, PI02017, PI020558, PI020508, UMC066, UMC019, P 0 1 5 18 PI020531, PI020527 PI020528, or- o :lve-^\na ecO\ Ce orV-e .C PI067, BNL8.39, and PI020593) determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. 3 "4 A method -leimM t- e-.oam- for identifying individual corn plants which have the desired genotype at a genetic marker locus 1 _I PCT/US89/00709 WO 89/07647 associated in inbred performance with cold test germination, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from UMC034, BNL8.33, PI01017, PI067, UMC065, UMC021 PI020569, PI01537 PI020708, P,I020684 PI020746, UMC116, BNL8.32, BNL8.39, SHT, PI065, GLUI, and PI020646 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method se -4a t 4 9-lw4m-9 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with soak test germination, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers .selected from PI020518,. PI020511, PI020509, or- 7" vz=-V\o C\^vo.^t A cVeuv\.e \or\4 -le-c BNL8.33, PI020569, PI020708, PI020746, BNL9.11, PI02052, and GLU; determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. \S -46 A method zcc::rdin to Claim 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with early stand count, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected fro UMC061, PI020713, PI020725 PI020597, UMC042, BNL7.65~ BNL8.33, PI01016, and PI065; S8 CT/US89/00709 WO 89/07647 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. I A method a.creding to-- Cl-4if-9- for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with ear circumferenc, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020603, PI020640, BNL6.20, PI020508, BNL5.37-, BNL10.24, UMC999, PI020726, UMC031, BNL5.46, PI020597, UMC042, UMC019, BNL7.65, UMC015, PI02071, PI01025, PI020608, PI020622, PI0612, PI01524, PI01518, PI020589, P1020531, PI02026P, PI067, PI020595, PI020599, PI020569, PI01537, PI020708, PI020746, UMC116, BNL10.39, UMC120, PI0105, UMC081, and PI065; determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. j 1~ A method according- to Cl'.am for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with ear diameter, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020508, PI020509, BNL5.37-, BNL10.24, UMC999, PI020726, UMC031, PI02071, PI01025, P1020622, PIO612. PL1518, PI020595 PI020599, PI020569, PI01537, PI020708, PI020746, BNL8.39, and UMC08; c@.Atq1 N i PCTJIS89/00709 WO 89/0764 Y

93-9- determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. I A method ,according to Claim 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with ear length, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020557, PI020726, BNL15.45, UMC015, EP02071,. PI020854, UMC062, BNL8.39, PI02020, PI01040, and PI020554t determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method according- to Cl aim- for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with cob circumference, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020640, BNL12.06, BNL8.29, PI020557, BNL6.20, BNL5.37-, UMC999, PI01080, PI020726, UMC031, BNL5.46, PI020597, BNL15.45, UMC042, UMC066, UMC019, BNL7.65, UMC015, PI02071, PIOl125, PI020608, UMC043 PI020622, PI0612 PI01524, PI01 18, PI020589, PI020531 1I02026P, UMC065, UMC081, and PI065) determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and WO 89/07647 PCT/US89/00709 selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method according to -laim- 9- for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with cob diameter, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020640, BNL12.06, PI020713, PI020725, UMC031, BNL5.46, PI020597, BNL15.45, UMC042, UMC066, UMC019, BNL7.65, UMC015, PI02071, PI01025, PI020608, UMC043, PI020622, PI0612, PI01524, PI01518, P1020589, PI020531, PI02026P, UMC065, UMC021, UMC046, UMC062,, MDH2, PI02Q599, BNL8.44, UMC081, PI065, GLU1, and PI020646; determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. 2-t A method accsrding t- Clalm 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with bare tip, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from P1020640, PI020644, P1020661, PI02017, BNL10.24, BNL15.45, UMC042, UMC066, P101025, PI0612, PI01524, PI01518, P1020589, P1020531, PI02026P, PI020854, UMC065, PI020569, PI020708, UMC116, BNL10.39, UMC012, WX1, P102052, UMC081, CSS1, and PI02055j determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and PCT/US89/00709 WO 89/07647 selecting individual p lants which have RFLP profiles which most closely match the preferred profile. 23- A method -ae.rd.. to Cr1i 0- for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with kernel row length, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020518, BNL8.29, PI020557, PI020726. UMC015, .PI02071, PI01524 PI0205231 PI020527, UMC065, UMC062, UMC116, PI01040, and SH1 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. -s 4-4 A method -according to Clam- for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with kernel depth, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020603, PI020640, PI020661, PI01012, PD2017, E8, PI020622, UMC021, PI020569, PI01537, PI020708, UMC110, and BNL8.3 determining the RFLP profiles of individual corn plants in a segregating population of corn--plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. 25- A method according to Claim 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus 7 PCT/US89/00709 WO 89/07647 associated in inbred performance with kernels per kernel row, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from BNL12.06, UMC061, BNL6.20, E8, PI020713, PI020725, UMC031, UMC043, PI020622, PI0612, PI01524, PI01518, PI020589, UMC065, PI01014, UMC062, BNL10.39, UMC081, CSS1, 0~ 0. F fr~cz- CC Ijo r e ic-. e PI020554, and PI020626; determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method -c ordn g t- 4-a-m-9 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with number of kernel rows per ear, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020690, UMC999, PI020726, UMC031, BNL5.46, PI020597, UMC042, UMC019, BNL7.65, UMC015, PI02071, PI01025, PI020608, PI0612, PI01524, PI01518, PI020589, PI020531, PI020566, PI02026P, PI02045, UMC065, UMC046, .PI020569, PI01537, PI020708, PI020684,_PI020746, UMC116, UMC11O, BNL8.32 BNL8.44, PI0105, UMC057, and UMC044 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method according to Claim 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus PCT/US89/00709 WO 89/07647 4-a- associated in inbred performance with kernel width, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from BNL12.06, UMC023, PI020726, UMC015, PI02071, PI020608, PI0612, PI01524, PI020589, PI020531, PI020566, PI02026P, PI02045, UMC065, PI020569, PI01537, PI020708, PI020684, PI020746, UMC116, UMC110, BNL8.32, BNL8.44, UMC012, UMC057 BNL10.13, o. \y a-c a fv'\Loxize< !I and UMC044 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. 7 8- A method according toG aim S-for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with percent moisture at harvest, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from UMC094, PI020537, PI020603, PI020689, PI020640, P1020575, PI02044, PI020661, PI020518, BNL8.29, PI020557, UMC061, UMC043, PI020622, PI020531, UMC021, PI01016, V BNL8.32, PI020593, P"LO1.39, PI065, PI01513, UMC057 BNL10.13, and UMC044/ determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. lS, 2P-. A method acco:ding to for identifying individual Scorn plants which have the desired genotype at a genetic marker locus PCT/US89/0070 9 WO 89/07647 associated in inbred performance with the ratio of yield in bushels per acre to percent moisture at harvest, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from BNL1-:06, BNL5.59, UMC061, BNL6.20, BNL6.16, UMC042, BNL7 65, PI02068 UMC043, 91020622, PI0612, PI01524 PI01518, PI020589, P1020527, PI0105 SHI, PI020646, and PI01513_ determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. j, A method a ~ein-g to Claim for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with GDU shed, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020640, BNL12.06, UMC034, PI01012, PI0205, BNL5.37-, BNL5.37-, BNL10.24, PI020713, PI020725, BNL5.46, UMC042, UMC066, Pi020854, BNL13.05, BNL9.11, UMC012, WX1, PI02052, UMC081, CSS, PI020554, BNL8.17, BNL7.21B, GLU1, P1020646, PI01513, ec. Vo.^c'van' ^^U\-alce-t Cteee {^a\Cer PI01033, UMC057, and BNI 10.1 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method -acc-eedi-ng- t4-Claim 0 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with GDU silk, comprising the steps PCT/US89/00709 WO 89/07647 constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020690, P1020537, PI020640, BNL10.24, PI020713, PI020725, BNL5.46, UMC042, UMC066, PI01524, P1020854, BNL13.05, UMC012, PI0105, SHI, WX1, PI02052, UMC081, CSS1, PI020554, BNL8.17, GLUI, PI020646, PI01513, and BNL10.13 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method -a.cording to Claim 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with stay green, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020640, BNL12.06, PI020654, BNL5.59, PI020682, PI020674, PI020575, PI020644, PI02044, P1020661, PI020557, UMC034, BNL6.16, PI01518 PI020531, PI20566, PI02Q527, BNL8.39, BNL8.44, PI02020, WX1, BNL8.17, UMT057, BNL10.13, and UMC044 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. 3 A method accerding to Claitr- 9- for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with plant height, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from BNL12.06, UMC034, PI01012, PI0205, PI020576, PI020508, BNL5.37-, BNL5.37-, BNL10.24, PI020713, PI020725, WO 89/07647 PCT/US89/00709 WO 89/07647 I 4 6 PI020728, BNL10.39, UMC120, UMC012, WX1, PI02052, UMC081, PI020554, PI020626, PI02075, GLU1, and PI020646; determining the RFLP profiles of individual corn plants in a 1 segregating population of corn plants with respect to the selected l genetic marker(s); and P selecting individual plants which have RFLP profiles which most i closely match the preferred profile. !4: I S34-. A method a-e-rding to Clai for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with ear height, comprising the steps of I constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020640, BNL12.06, PI02044, UMC034, i PI01012, PI0205, PI020576, PI020509, PI020508, PI020508, BNL5.37-, BNL5.37-, BNL10.24, BNL6.16, PI020521, UMC999, PI020713, PI020725, I BNL6.29, PI020728, BNLIO.39, UMC120, UMC012, WX1, PI02052, UMC081, j 1 j~ o^ tur~oC o.a ecc y Ae5etL <^c;-Le i PI020554, and UMC057; 1 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. S+ A method caoording to Claim 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus I associated in inbred performance with European corn borer second brood tolerance, comprising the steps of Sconstructing a preferred RFLP profile with respect to one or more ai genetic markers selected from BNL5.59, PI020644, PI0205, BNL6.20, E8, BNL6.16, PI020597, BNL15.45, UMC042, PI020608, PI0612, PI02045, UMC065, MDH2, PI020595, PI020599, PI020569, PI01537, PI020708, UMC110, BNL10.39, UMC120, and PI0105, WO 89/07647 PCT/US89/00709 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile.

346. A method -aecprding to Claim 90 for identifying individual corn plants which have the desired genotype at a genetic marker locus I associated in inbred performance with root lodging, comprising the steps of I constructing a preferred RFLP profile with respect to one or more genetic markers selected from UMC094, PI020537, PI020603, PI020689, PI020640, PI020518, PI020576, PI020511, PI020558, PI020509, PI020508, PI020508, BNL5.37-, BNL10.24, PI020713, PI020725, UMC031, UMC066, UMC019, UMC065, MDH2, PI020595, and UMC081; determining the RFLP profiles of individual corn plants in a Ssegregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method -ccording to Claim for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with statistically transformed root lodging score, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020640, PI020654, PI020644, PI020518, PI020576, PI020511 PI020558 PI020509, PI020508, PI020508, BN5.37-, BNL5.37-, BNL10.24, UMC019, BNL6.29, UMC065, PI020581, and UMC081- determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and I PCT/JS89/00709 WO 89/07647 |i selecting individual plants which have RFLP profiles which most 4 closely match the preferred profile. l 3r: A method eeeor dng-t Cleaim for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in inbred performance with stalk lodging, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from BNL6.20, P1020597 .MDH2, PI020595, PI020599, PI020728, UMC012, PI0105, PI02075, and UMC05 7 determining the RFLP profiles of individual corn plants in a I segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. s A method -afetd-i-qg-- to Clim 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated 'in inbred performance with statistically transformed stalk lodging score, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from P1020682, PI020661, PI020668, AMP1, BNL6.20, UMC042,,PI067, P1020595, PI020599, BNL13.05, PI0105, SH1, and BNL3.04 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. -4 A method acrding t Claim 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus PCT/US89/00709 WO 89/07647 T associated in inbred performance with grain quality, comprising the steps of i constructing a preferred RFLP profile with respect to one or more genetic markers selected from BNL5.59, PI020575, PI020644, PI02044, PI020661, AMP1, PI020557, UMCO61, PI01012, PI0205, PI02017, E8, PI020509, PI02071, PI01025, BNL8.33, P1020569, PIO1537, PI020708, PI020684, PIQ20746, UMC116, UMC110, BNL8.32, BNL8.39, PI020593, PI01033, UMC057, and BNL.10.13 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. So4 A method according to Claim 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with yield in bushels per acre, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from BNL6.20, PI020576, PI020558, PI020509, PI020508, PI020508, BNL5.37-, BNL5.37-, BNL10.24, BNL6.16, BNL7.65, UMC015, PI02071, PI020589 PI020569 PIQ1537, PI020708, PI020684, PI020746, UMC116, UMC110, BNL8.32, BNL8.39, PI020593, and PI020714/ determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. L, A method according to Clair m 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with Moisture, comprising the steps PCT/US89/00709 WO 89/07647 constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020640, BNL12.06, UMC061, UMC034, PI01012, PI0205, PI02017 BNL6.20, PI020569. PI01537, UMC116, BNL8.32, I o- o (Fu.cf-N oYNvo^.os e \s>Ac .j 5 cee.e\f- VW^-o BNL10.39, UMC120, PI020714, UMC012, PI02052, PI020646, and PI01513 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method -accrd ing to Claim 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with Bulk Density, adjusted, comprising the steps of Sconstructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020518, PI01012, PI0205, PI02017, BNL6.20, PI020511, PI020558, PI020509, PI020508, PI020508, BNL7.65, UMC015, PI02071, PI01025, PI0612, PI01524, PI020589, PI02045, PI020569, PI01537, PI020708, PI020684, PI020746, UMC116, UMC110, BNL8.32, SHI, WX1, PI02052, UMC081, CSS1, PI020626, PI02075, BNL3.04, i o r 0 S o\eA_=i, AAS-t_' PI065, GLU1, PI020646, and PT01033 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have-RFLP profiles which most closely match the preferred profile. L453 W4. A method accrd-ee to Claii for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with Yield to moisture ratio, comprising the steps of constructing a preierred RFLP profile with respect to one or more genetic markers selected from BNL12.06, PI020518, BNL8.29, UMC061, PCT/US89/00709 WO 89/07647 UMC034, PI01012, PI020576, PI020558, PI020509, PI020508, PI020508, BNL5.37-, BNL5.37-, BNL10.24, BNL6.16, UMC066, UMC019, BNL7.65, PI02071, BNL8.33, UMC043, PI020622, PI0612, PI01524, PI020589, and PI02026P; determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method aeeerdii t-e-Claim i for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with GDU Shed, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI01012, PI0205, PI02017, PI020576, PI020558, PI020509, PI020508, PI020508, BNL5.37-, BNL5.37-, BNL10.24, PI020521, PI01533, UMC999, P1020713, PI020725, BNL5.46, PI0612, PI01524, PI020589, BN13.05, PI01040 BNL10.39, UMC120, PI020714, UMC012, WX1, PI02052, UMC081, PI020554, and BNL10.134 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. 4 46r. A method acc-nording t -Claim f for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with Stay green, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020690, PI02017, BNL6.20, BNL5.37-, BNL10.24, UMC046, MDH2, PI020595, PI020599, PI020569, PI01537, U ixL1A 3Gr 0I s r i r 7 PCT/US89/00709 WO 89/07647 PI020684 UMC116, BNL8.32, BNL10.39, UMC120, PIO?0714, UMC012, SH1, and BNL3.04 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. I L, A method .a.cording te Claim 9- for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with Dropped Ears, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers .selected from BNL10.39, UMC081, BNL7.21B, and BNL10.13, 1 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. i LF7 -48. A method -a-~odi t m a. for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with Dropped Ears, Statistically transformed, comprising the steps of constructing a preferred RFLP, profile with respect to the genetic marker PI020690; determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the genetic marker; and selecting individual plants which have RFLP profiles which most closely match the preferred profile. WO 89/07647 .PCT/US89/00709 WO 89/07647 4g. -494 A method s-eae l)-i for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with Ear height, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020640, UMC034, PI020576, PI020511, PI020558, PI020509, PI020508, PI020508, BNL5.37-, BNL5.37-, BNL10.24, PI020521, PI01533, PI020713, PI020725, BNL8.33, PI020569, BNL8.32, BNL8.39, PI020593, .BNL10.39, UMC120, PI020714 UMC012, WX1 UMC081 PI020646, and PI01513 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. L4 A method a cording to C11aim for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with Plant height, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020690, PI020668, PI020518, PI020576, PI020511, PI020558, PI020509, PI020508, PI020508, BNL5.37-, BNL5.37-. BNL10.24, BNL6.16, PI020521, PI01533, UMC999, PI01080, BNL8.31 PI01040, BNL10.39, UMC120, UMC012, PI065, GLU1, PI020646, PI01513, and UMCT04j4" F' o y oe oeec o-eX determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. WO 89/07647 PCUS89/00709 SO A method ccarding to C laim for iuentifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with Grain quality, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from BNL12.06, BNL8.29, BNL6.20, UMC999, BNL6.29, PI020528, PI020599, and BNL14.28A determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. A method -a.:rding to Claim-- 9- for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with European Corn Borer Second Brood Tolerance, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI02017, PI020576, PI020509, PI020508, BNL5.37-, PI020569, PI01537, PI020708 UMC116, BNL8.39, PI020593, BNL10.39, UMC120, PI020626, and PIu0 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. S2-653-. A method -a:cording tc Clai 9- for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with Root lodging, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from UMC094, PI020511, BNL5.37-, BNL5.37-, iii. I- i. Li i ~lil_. iiil*iIII~UL. WO89/07647 PCT/US89/00709 WO 89/07647 BNL10.24, BNL6.16, UMC999, BNL8.23, BNL6.29, PI020527, P1020528, PI020854, PI02045, PI067, UMC065, MDH2, PI020595, UMC012, P1020562, and PI020646 determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. S2 A method according to Claim 9 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with Root lodging, statistically transformed, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from UMC094, PI020576, PI020511, BNL5.37-, BNL5.37-, BNL10.24, UMC999, UMC019, BNL6.29, PI020527, PI020528, PI020854, PI02045, PI067, UMC065, DH2, PI020595, UMC012, and PI02056;, determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. SL f -Sf. A method -acr ding to lim 0 for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in hybrid performance with Stalk lodging, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from P1020622 PI01537, BNL14.28, PI020562 3 or c \o j ecu\vja-V2\M= c.ee-cAt o ie UMC057, and UMC044 determining the RFLP profiles of individual corn plants in a S89/ 7 PC/US89/00709 WO 89/07647 segregating population of corn plants with respect to the selected genetic marker(s); and Sselecting individual plants which have RFLP profiles which most closely match the preferred profile. A method according to Claim 9- for identifying individual corn plants which have the desired genotype at a genetic marker locus associated in _hybrid performance with Stalk lodging, statistically transformed, comprising the steps of constructing a preferred RFLP profile with respect to one or more genetic markers selected from PI020537, PI01012, PI0205, E8, PI020558, PI020597, PI020622, PI0612, P01524, UMC065, PI01537, UMC110, PI020626, PI02075, PI020646, PI01513, JMC057, BNL10.13, and UMC044_ determining the RFLP profiles of individual corn plants in a segregating population of corn plants with respect to the selected genetic marker(s); and selecting individual plants which have RFLP profiles which most closely match the preferred profile. I ,t In\r 56. A method for identifying and mapping quantitative trait loci (QTL) for a phenotypic trait in maize plant B73, Mo17 or a cross thereof by analyzing the chromosomes of said plants for DNA polymorphisms and linkage to said trait, which method is substantially as hereinbefore described with reference to Example 3. 57. A restriction fragment length polymorphism probe that can detect a polymorphism in one or more maize plants wherein said i polymorphism is linked to a phenotypic trait selected from adjusted yield, plant yield, plant height, ear height GDU shed, GDU silk, grain moisture, root lodging, stalk lodging, or stay green, which probe is ;I substantially as hereinbefore described with reference to Example 2. I 58. A method of determining a correlation between a phenotypic trait in maize and a restriction fragment length polymorphism comprising: digesting genomic DNA from maize inbred B73 or maize inbred I 15 Mo17 with a restriction endonuclease that produces a restriction fragment j length polymorphism digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step 4 detecting said restriction length polymorphism with a restriction length polymorphism probe according to claim 57, thereby i i' generating a restriction pattern; and correlating the presence or absence of said RFLP in said I digest with the respective presence or absence of said trait. 59. A restriction fragment length polymorphism probe that can 25 detect a polymorphism in one or more maize plants wherein said polymorphism is linked to a phenotypic trait selected from the group consisting of adjusted bulk density, ratio of yield to percent moisture, dropped ears, statistically transformed dropped ears, grain quality, European corn borer second brood tolerance, statistically transformed 30 root lodging, statistically transformed stalk lodging, weight of 100 kernels, cold test germination, soak test germination, early stand count, ear circumference, ear diameter, ear length, cob circumference, cob diameter, bare tip, kernel row length, kernel depth, kernels per kernel row, number of kernel rows per ear, and kernel width. 60. A method of determining a correlation between a phenotypic trait in maize and a restriction fragment length polymorphism comprising: yP4i/ digesting genomic DNA from a maize plant with a restriction P endonuclease that produces a restriction fragment length 848Z -63- polymorphism digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step detecting said restriction fragment length polymorphism with a hybridization probe containing sequence information capable of hybridizing to and identifying said RFLP, thereby generating a restriction pattern; and correlating the presence or absence of said RFLP in said digest with the respective presence or absence of said trait; wherein said trait is selected from the group consisting of adjusted yield, plant yield, ear height, GDU shed, GDU silk, grain moisture, root lodging, stalk lodging, and stay green. 61. A method of determining a correlation between a phenotypic trait in maize and a restriction fragment length polymorphism comprising: digesting genomic DNA from a maize plant with a restriction endonuclease that produces a restriction fragment length polymorphism digestion pattern that is associated with said trait; separating the fragments obtained from said digestion in step detecting said restriction fragment length polymorphism with a hybridization probe containing sequence information capable of hybridizing to and identifying said RFLP, thereby generating a restriction pattern; and correlating the presence or absence of said RFLP in said digest with the respective presence or absence of said trait; wherein said trait is selected from the group consisting of adjusted bulk density, ratio of yield to percent moisture, dropped ears, statistically transformed dropped ears, grain quality, European corn borer second brood tolerance, statistically transformed root lodging, statistically :S S S. S 1/848Z I transformed stalk lodging, weight of 100 kernels, cold test germination, soak test germination, early stand count, ear circumference, ear diameter, ear length, cob circumference, cob diameter, bare tip, kernel row length, kernel depth, kernels per kernel row, number of kernel rows per ear, and kernel width. DATED this TWENTY-EIGHTH day of AUGUST 1992 Pioneer Hi-Bred International, Inc. Patent Attorneys for the Applicant SPRUSON FERGUSON i I t [i I I ?-48 I N1* 87 2 INTERNATIONAL SEARCH REPORT International Apolication No .T 3' 1, CLASSIFICATION OF SUBJECT MATTER (it several classification sym~ols apply, mdicate all) Accoidnq to international Patent Classification IIPC) or to tboth National Classification and IPC 11 FIELDS SEARCHED Minimum Documentation Searched7 Documentation Searched other than Minimum Documentation to the, Eatqlnt tha! such Documents aeIncluded in the Fields Searched B I 1 7E- 1939 File iBiosis, z-'9 Autiomated P:atenc. Systemn iFile USPATI, 1975-1989)1. SEE ATTACHMENT. 1t1, DOCUMENTS CONSIDERED TO BE RELEVANT B Category I- Citation of Document. 11 with indication, where appropriate, of the relevant p ausages 12 Relevant to Claim No. 13 T-rends in Geneti cs. Lne 3z, issued Itgt4st 1~-f i98)7, CAmmster-dam. Nethe'r lands) Hdelent.iarls I s& A' 1-4enetic 1ii~ map fit-r ie based on pages 217-221, e pa~ge 2271 in p a r t i C i1l 1 'ar FHi ant Mo 1 CuLl ar B icil1ogy, v:l utne 5Y iss.-71-ed 2 li 1935, t:The Hagute, Nethe-rlands.t Helentj1ari1:sj et al. iRestriction -fragment polymnorphismn piibes *or plant diyersity and there development as toicils fo-r applied plant bzreeding," 'cle piages 1CYD-113. Crop science, Volumfe 27 issued July/-Auigust 1 -56 13D37, (Mada-son, USA) Nienhi-tis et al." iiF'esticticn -fragment length po-lymil_-rph3ism *.tnalysis iof loci assoCate-d ywiih n e t r~esistanCe in tomatci. "see Pages 797-iSCI. *Special categories of cited documents: 10 later document pubtished alter the international filing date document defining the general state of the art which is not or priority date and not in conflict with the application but consdere tobe o paticuar elevncecited to understand the principle or theory underlying the consdere tobe o paticuar elevnceinvention earlier document but published on or fter the international 'XI document of particular relevance: the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claimls) or involve an inventive step whi ch is cited to establish the publication date of another document of particular relevance: the claimed invention citation or other special reason las specified) cannot be considered Is involve an inventive step when the document referring to an oral disclosure, use, exhibition or documeni is combined with one or (nore other such docu- other means ments, such comoination being obvious to a person skiilled P'document published prior to the international filing date but in the art. later than the priority date claimed &'document member of the same patent family IV. CERTIFICATION Date. f 4 te AFqual C~rryalBtion of the International Search Date of Mailing of this International Search Report IntrntioalSerchngAutorty12 JUN 1989 IntrntioalSerchngAutortySignature of Authoried Ofc e,1 1) i Si- /UjS GARY ENZION, N Form PCTASA210 Ucc5ind the iv.l I." I~ .l I PCT/US89/U0709 Attachment To Form PCT/ISA/210 II. FIELDS SEARCHED Dialog Information Services Inc., (File Agri). Search Terms: Plant Breeding, RFLP, Restriction length fragment poly- morphism, correlation, maize, corn, zea, dropped ear count, cold germination, soaK germination, early stand count, ear circumference, ear diameter, ear length, yield, correlated response, cob circumference, cob diameter, bare tip, kernel row length, Kernel depth, kernel per row, percent moisture, GDU shed, stay green, European corn borer second brood, root lodging, stalk lodging, inventor's names. I International Application No. PCT/US89/007)09 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET Y Crop Science, Volume 27, issued July-Au.aust 1-56 1937 (Madison, USA) Stuber et al., "Molecula marker-f-cilitated investigations of quantitative ilo ci in maize. II. Factors influencing yield and its component traits," See pages -39-649. Y Theoretical Applied Genetics, Volume 67, 1-56 issued 1983. (New York, USA) Soiller et al., "Genetic polymorphism in varietal identifi- cation and genetic improvement," see pages .27-30, and 32. V OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE I This international search report has not been established in resoect of certain claims under Article 17(2) for the following reasons: I I Claim numbers because they relate to subject matter not required to be searched by this Authority, namely: 2.1] Claim numbers because they relate to parts of the International application that do not comply with the prescribed require- ments to such an extent that no meaningful international search can be carried out l3, specifically: 3. Claim numbers because they are dependent claims not drafted in accordance with :he second and third sentences of PCT Rule 6.4(a). VI.1 OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING This international Searching Authority found multiple inventions in this international application as follows: 1.1 As all required additional search lees were timely paid by the applicant, this International search report covers all searchable claims of the international application. As only some of the required additional search lees were timely paid by the applicant, this international search report covers only those claims of the International application for which fees were paid, specifically claims: 3.1I No required additional search fees were timely paid by the applicant, Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claim numbers: 4.d As all searchable claims could be searched without efort lustilying an additional fee, the International Searching Authority did not invite payment of any additional lee. Remark on Protest F The additional search fees were accompanied by applicant's protest. No protest accompanied the payment of additional search lees. Form PCTSSA210 (sipplOminu sh 1 (a2) 11-87) International Application No. C/S91179 111. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category *i Ctialion ot Docunment, with indication, where appropriate, of the relevant passages IRelevant to Claim No L. n ar y e 1 l't5e1rhl1-1ids -i nu appI Ic -TrI on s 0:T restriction lengthi fragment polyflorph..B-- m analysis tici plants. in Driuening et a~l. eds., Tailoiring genes fo--r crop improivement. An agri ctit -tiral kperspectivL e, published 11=1/, Press, (New York:, USA). See pages :25-44 in particular pag-=tes 34-35. Burr et al., The application of restriction fragment length polymorphism to plant reeding in Setklow et al. Genetic Engineering, Principles an-d MetHi. ublish( 1983, by Plenum Press (New York, USA), see pages 51-56. 1 ZI 1-56 Form PCTtSA21O (Oxva grow) 14M7