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 characteristies 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 population will not be uniform.

Corn ( Zea mays L.) 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 F¹ hybrids. 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: (1) DNA probes shown to reveal polymorphisms between two parent inbred lines and having known chromosomal locations, (2) statistical techniques that can find correlations between the inheritance of one or more DNA probes and the phenotype of the plants under investigation, and (3) 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 between agronomic traits and genetic markers to enhance plant breeding.

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. 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. Aoarose Gel Electrophoresis. 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, and 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 fragments 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 markers that are associated with a specific agronomic trait. 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: (1) DNA probes shown to reveal polymorphisms between two parent inbred lines and having known chromosomal locations, (2) statistical techniques that can find correlations between the inheritance of one or more DNA probes and the phenotype of the plants under investigation, and (3) 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 lodging, and stay green. A definition of each of these traits is as fol1 ows:

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.

GPU 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:

The highest maximum used is 86°F and the lowest minimum used is 50°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 maturity.

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

Root Lodging. The root lodging is the percentage of plants that do not root lodge; i.e., those that lean from the vertical axis at an approximately 30° angle or greater would be counted as root lodged.

Stalk Lodging. This is the percentage of plants that do not stalk lodge, i.e., 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 given restriction endonuclease. After the 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:

(a) in situ hybridization to metaphase chromosome spreads; or

(b) genetic linkage to any marker as already mapped to the region.

For method, (a), the new fragment need not be polymorphic, but for (b), polymorphisms must first be identified by comparing the restriction pattern of the genomic DNA at the new site in unrelated plants. In method (a), 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"). 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 (p<0.05), 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; - determining 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" 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 be working to develop a plant which is homozygous, i.e., 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 F₁ 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 an RFLP profile which precisely matches the ideal. However, one or more individuals will have a greater percentage of matches to the ideal than 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 AMP1, MDH2 and GLU1, or are probes for specific mutant genes which are well known and whose location has been identified, as mentioned above.

These Tables identify linkages observed in bulk F₄ 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 on visual grading of the shelled corn in terms of moldy and cracked kernels. 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, adjusted. 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.

Tabl e 1

List of genetic markers associated with quantitative traits evaluated in topcross progeny.

TRAIT=Yield, Bushels/Acre (BU ACR)

BNL6.20 PI020576 PI020558 PI020509

PI020508 PI020508 BNL5.37- BNL5.37-

BNL10.24 BNL6.16 BNL7.65 UMC015

PI02071 PI020589 PI020569 PI01537

PI020708 PI020684 PI020746 UMC116

UMC1I0 BNL8.32 BNL8.39 PI020593 PI020714

TRAIT= % Moisture (MST)

PI020640 BNL12.06 UMC061 UMC034

PI01012 PI0205 PI02017 BNL6.20

PI020569 PI01537 UMC116 BNL8.32

BNL10.39 UMC120 PI020714 UMC012

PI02052 PI020646 PI01513

TRAIT=Bulk Density , adjusted (TSTWTA)

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 SH1 WX1

PI02052 UMC081 CSS1 PI020626

PI02075 BNL3.04 PI065 GLU1

PI020646 PI01033 TRAIT=Yield/% moisture (YLDMST)

BNL12.06 PI020518 BNL8.29 UMC061

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 PI02026P

TRAIT=GDU Shed (GDUSHD)

PI01012 PIO205 PI02017 PI020576

PI020558 PI020509 PI020508 PI020508

BNL5.37- BNL5.37- BNL10.24 PI020521

PI01533 UMC999 PI020713 PI020725

BNL5.46 PI0612 PI01524 PI020589

BNL13.05 PI01040 BNL10.39 UMC120

PI020714 UMC012 WX1 PI02052

UMC081 PI020554 BNL10.13

TRAIT=Stay green (STAGRN)

PI020690 PI02017 BNL6.20 BNL5.37-

BNL10.24 UMC046 MDH2 PI020595

PI020599 PI020569 PI01537 PI020684

UMC116 BNL8.32 BNL10.39 UMC120

PI020714 UMC012 SH1 BNL3.04

TRAIT=Dropped Ears (DRPEAR)

BNL10.39 UMC081 BNL7.21B BNL10.13

TRAIT=Dropped Ears, Statistically transformed (FOGDE) P1020690

TRAIT=Ear height (EARHT)

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 PI01513

TRAIT=Plant height (PLTHT)

PI020690 PI020668 PI020518 PI020576

PI020511 PI020558 PI020509 PI020508

PI020508 BNL5.37- BNL5.37- BNL10.24

BNL6.16 PI020521 PI01533 UMC999

PI01080 BNL8.33 PI01040 BNL10.39

UMC120 UMC012 PI065 GLU1

PI020646 PI01513 UMC044

TRAIT=Grain quality (GRNOUL)

BNL12.06 BNL8.29 BNL6.20 UMC999

BNL6.29 PI020528 PI020599 BNL14.28

TRAIT=European Corn Borer Second Brood Tolerance (ECB2SC)

PI02017 PI020576 PI020509 PI020508

BNL5.37- PI020569 PI01537 PI020708

UMC116 BNL8.39 PI020593 BNL10.39

UMC120 PI020626 PI02075 TRAIT=Root lodging (RT LDG)

UMC094 PI020511 BNL5.37- BNL5.37-

BNL10.24 BNL6.16 UMC999 BNL8.23

BNL6.29 PI020527 PI020528 PI020854

PI02045 PI067 UMC065 MDH2

PI020595 UMC012 PI020562 PI020646

TRAIT=Root lodging, statistically transformed (FOGRTL)

UMC094 PI020576 PI020511 BNL5.37-

BNL5.37- BNL10.24 UMC999 UMC019

BNL6.29 PI020527 PI020528 PI020854

PI02045 PI067 UMC065 MDH2

PI020595 UMC012 PI020562

TRAIT=Stalk lodging (STKLDG)

PI020622 PI01537 BNL14.28 PI020562

UMC057 UMC044

TRAIT=Stalk lodging, statistically transformed (FOGSTL)

PI020537 PI01012 PI0205 E8

PI020558 PI020597 PI020622 PI0612

PI01524 UMC065 PI01537 UMC110

PI020626 PI02075 PI020646 PI01513

UMC057 BNL10.13 UMC044

Table 2

List of genetic markers associated with quantitative traits as identified in bulk f4 progeny

TRAIT=Yield, Bushels/Acre (BU_ACR) BNL5.59 BNL6.20 UMC042 PI020608

UMC043 PI020622 PI0612 PI01524

PI01518 PI020589 PI0105 SH1

PI01513

TRAIT=Plant yield (PLNTYLD)

PI020537 PI020518 BNL8.29 PI020557

BNL6.20 UMC043 PI020622 PI01524

PI01518 PI020589 PI020523 PI020569

PI0105 SH1 PI020626 PI020646

TRAIT=Weight of 100 kernels (CKWT)

PI020603 PI020689 PI020654 BNL5.59

PI020682 PI020674 PI020575 PI020644

BNL8.23 PI067 UMC065 UMC021

UMC110 PI020714 PI0105 PI020554

BNL14.28 UMC057 BNL10.13 UMC044

TRAIT=Dropped ears, statistically transformed (FOGDE) PI0205 PI02017 PI020558 PI020508

UMC066 UMC019 PI01518 PI020531

PI020527 PI020528 PI067 BNL8.39

PI020593

TRAIT=Cold test germination (CTST) UMC034 BNL8.33 PI01017 PI067

UMC065 UMC021 PI020569 PI01537

PI020708 PI020684 PI020746 UMCI16 BNL8.32 BNL8.39 SH1 PI065 GLU1 PI020646

TRAIT=Soak test germination (SKTST) PI020518 PI020511 PI020509 BNL8.33 PI020569 PI020708 PI020746 BNL9.11 PI02052 GLU1

TRAIT=Early stand count (ESTCNT) UMC061 PI020713 PI020725 PI020597

UMC042 BNL7.65 BNL8.33 PI01016

PI065

TRAIT=Ear circumference (EARCIR)

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 PI020531

PI02026P PI067 PI020595 PI020599

PI020569 PI01537 PI020708 PI020746

UMC116 BNL10.39 UMC120 PI0105

UMC081 PI065

TRAIT=Ear diameter (EARDIA)

PI020509 PI020508 PI020508 BNL5.37-

BNL10.24 UMC999 PI020726 UMC031

PI02071 PI01025 PI020622 PI0612

PI01518 PI020595 PI020599 PI020569

PI01537 PI020708 PI020746 BNL8.39 UMC081

TRAIT=Ear length (EARLGT)

PI020557 PIO20726 BNL15.45 UMC015

PI02071 PI020854 UMC062 BNL8.39

PI02020 PI01040 PI020554

TRAIT=Cob circumference (COBCIR)

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 PI01025

PI020608 UMC043 PI020622 PI0612

PI01524 PI01518 PI020589 PI020531

PI02026P UMC065 UMC081 PI065

TRAIT=Cob diameter (COBDIA)

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 PI020589 PI020531 PI02026P

UMC065 UMC021 UMC046 UMC062

MDH2 PI020599 BNL8.44 UMC081

PI065 GLU1 PI020646 TRAIT=Bare tip (NOSBAC)

PI020640 PI020644 PI020661 PI02017

BNL10.24 BNL15.45 UMC042 UMC066

PI01025 PI0612 PI01524 PI01518

PI020589 PI020531 PI02026P PI020854

UMC065 PI020569 PI020708 UMC116

BNL10.39 UMC012 WX1 PI02052

UMC081 CSS1 PI020554 PI02075

TRAIT=Kernel row length (ROWLEN)

PI020518 BNL8.29 PI020557 PI020726

UMC015 PI02071 PI01524 PIO20523

PI020527 UMC065 UMC062 UMC116

PI01040 SH1

TRAIT=Kernel Depth (KDEPTH)

PI020603 PI020640 PIO20661 PI01012

PI02017 E8 PI020622 UMC021

PI020569 PI01537 PI020708 UMC110

BNL8.39

TRAIT=Kernels per kernel row (KERPRO)

BNL12.06 UMC061 BNL6.20 E8

PI020713 PI020725 UMC031 UMC043

PIO20622 PI0612 PI01524 PI01518

PI020589 UMC065 PI01014 UMC062

BNL10.39 UMC081 CSS1 PI020554

PI020626 TRAIT=Number of kernel rows per ear (KERROW)

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 UMC110 BNL8.32 BNL8.44

PI0105 UMC057 UMC044

TRAIT=Kernel width (KWIDTH)

BNL12.06 UMC023 PI020726 UMC015

PI02071 PI020608 PI0612 PI01524

PI020589 PIO20531 PI020566 PI02026P

PI02045 UMC065 PI020569 PI01537

PI020708 PI020684 PI020746 UMC116

UMC110 BNL8.32 BNL8.44 UMC012

UMC057 BNL10.13 UMC044

TRAIT=% Moisture (MST)

UMC094 PI020537 PI020603 PI020689

PI020640 PI020575 PI02044 PI020661

PI020518 BNL8.29 PI020557 UMC061

UMC043 PI020622 PI020531 UMC021

PI0I016 BNL8.32 PI020593 BNL10.39

PI065 PI01513 UMC057 BNL10.13

UMC044

TRAIT=Yield/% moisture (YLDMST)

BNL12.06 BNL5.59 UMC061 BNL6. 2

BNL6.16 UMC042 BNL7.65 PI020608

UMC043 PI020622 PI0612 PI01524

PI01518 PI020589 PIO20527 PI0105 SH1 PI020646 PI01513

TRAIT=GDU Shed (GDUSHD)

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

CSS1 PI020554 BNL8.17 BNL7.21B

GLU1 PI020646 PI01513 PI01033

UMC057 BNL10.13

TRAIT=GDU Silk (GDUSLK)

PI020690 PI020537 PI020640 BNL10.24

PI020713 PI020725 BNL5.46 UMC042

UMC066 PI01524 PI020854 BNL13.05

UMC012 PI0105 SH1 WX1

PI02052 UMC081 CSS1 PI020554

BNL8.17 GLU1 PI020646 PI01513

BNL10.13

TRAIT=Stay oreen (STAGRN)

PI020640 BNL12.06 PI020654 BNL5.59

PI020682 PI020674 PI020575 PI020644

PI02044 PI020661 PI020557 UMC034

BNL6.16 PI01518 PI020531 PI020566 PI020527 BNL8.39 BNL8.44 PI02020

WX1 BNL8.17 UMC057 BNL10.13

UMC044

TRAIT=Plant heioht (PLTHT)

BNL12.06 UMC034 PI01012 PI0205

PI020576 PI020508 BNL5.37- BNL5.37-

BNL10.24 PI020713 PI020725 PI020728

BNL10.39 UMC120 UMC012 WX1

PI02052 UMC081 PI020554 PI020626

PI02075 GLU1 PI020646

TRAIT=Ear height (EARHT)

PI020640 BNL12.06 PI02044 UMC034

PI01012 PI0205 PI020576 PI020509

PI020508 PI020508 BNL5.37- BNL5.37-

BNL10.24 BNL6.16 PI020521 UMC999

PI020713 PI020725 BNL6.29 PI020728

BNL10.39 UMC120 UMC012 WX1

PI02052 UMC081 PI020554 UMC057

TRAIT=European Corn Borer Second Brood Tolerance (ECB2SC)

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 PI0105

TRAIT=Root lodging (RT_LDG)

UMC094 PI020537 PI020603 PI020689

PI020640 PI020518 PI020576 PI020511

PI020558 PI020509 PI020508 PI020508

BNL5.37- BNL10.24 PI020713 PI020725

UMC031 UMC066 UMC019 UMC065

MDH2 PI020595 UMC081

TRAIT=Root lodging, statistically transformed (FOGRTL)

PI020640 PI020654 PI020644 PI020518

PI020576 PI020511 PI020558 PI020509

PI020508 PI020508 BNL5.37- BNL5.37-

BNL10.24 UMC019 BNL6.29 UMC065

PI020581 UMC081

TRAIT=Stalk lodqing (STKLDG)

BNL6.20 PI020597 MDH2 PI020595

PI020599 PI020728 UMC012 PI0105

PI02075 UMC057

TRAIT=Stalk lodging, statistically transformed (FOGSTL)

PI020682 PI020661 PI020668 AMP1

BNL6.20 UMC042 PI067 PI020595

PI020599 BNL13.05 PI0105 SH1

BNL3.04

TRAIT=Grain quality (GRNOUL)

BNL5.59 PI020575 PI020644 PI02044

PI020661 AMP1 PI020557 UMC061

PI01012 PI0205 PI02017 E8

PI020509 PI02071 PI01025 BNL8.33

PI020569 PI01537 PI020708 PI020684

PI020746 UMC116 UMC110 BNL8.32 BNL8.39 PI020593 PI01033 UMC057

BNL10.13

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 1 Determination of Agronomically Useful Phenotypes

Development of Progenies for Field Testing

The inbreds B73 and Mo17 were crossed to produce the F₁ hybrid designated B73/Mol7. Hybrid seed was planted, several plants selfed, and the seed bulked to produce the F2 generation designated B73/Mo17)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/Mo17)Xn where n ranged from 1 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.

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 bulked 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. GDU Shed GDUSHD Accumulated heat units to the day that 40% of plants in a plot were shedding pollen.

GDU Silk GDUSLK Accumulated heat units to the day that 50% 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 30º.

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). 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 (bp), 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 P³² 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) 1-3 autoradiographic bands were observed for at least one restriction enzyme, and (2) band patterns were different between at least two maize inbreds. 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 Mol7 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 (1) 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 Linkages 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. Linear and Non-linear Relationships between Quantitative Traits and RFLP Marker Loci Were Used to Detect Linkages 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 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 3). 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.

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.