U.S. patent application number 16/676116 was filed with the patent office on 2020-02-27 for compositions and methods for identifying and selecting maize plants with resistance to northern leaf blight.
This patent application is currently assigned to PIONEER HI-BRED INTERNATIONAL, INC.. The applicant listed for this patent is PIONEER HI-BRED INTERNATIONAL, INC.. Invention is credited to ENRIQUE DOMINGO KREFF, GIRMA M TABOR.
Application Number | 20200063217 16/676116 |
Document ID | / |
Family ID | 53836251 |
Filed Date | 2020-02-27 |
United States Patent
Application |
20200063217 |
Kind Code |
A1 |
KREFF; ENRIQUE DOMINGO ; et
al. |
February 27, 2020 |
COMPOSITIONS AND METHODS FOR IDENTIFYING AND SELECTING MAIZE PLANTS
WITH RESISTANCE TO NORTHERN LEAF BLIGHT
Abstract
Compositions and methods useful in identifying and/or selecting
maize plants having resistance to northern leaf blight are provided
herein. The resistance may be newly conferred or enhanced relative
to a control plant. The methods use markers to identify, select
and/or construct resistant plants. Maize plants identified,
selected, and/or generated by the methods described herein are also
provided.
Inventors: |
KREFF; ENRIQUE DOMINGO;
(PERGAMINO, AR) ; TABOR; GIRMA M; (JOHNSTON,
IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PIONEER HI-BRED INTERNATIONAL, INC. |
Johnston |
IA |
US |
|
|
Assignee: |
PIONEER HI-BRED INTERNATIONAL,
INC.
JOHNSTON
IA
|
Family ID: |
53836251 |
Appl. No.: |
16/676116 |
Filed: |
November 6, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15502696 |
Feb 8, 2017 |
10513742 |
|
|
PCT/US2015/043529 |
Aug 4, 2015 |
|
|
|
16676116 |
|
|
|
|
62034806 |
Aug 8, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01J 47/026 20130101;
C12Q 1/6895 20130101; A01H 1/04 20130101; A01H 5/10 20130101; C12Q
2600/13 20130101 |
International
Class: |
C12Q 1/6895 20060101
C12Q001/6895; B01J 47/026 20060101 B01J047/026; A01H 5/10 20060101
A01H005/10; A01H 1/04 20060101 A01H001/04 |
Claims
1. A method of identifying a maize plant with northern leaf blight
resistance comprising: a. analyzing DNA of a maize plant for the
presence of a QTL allele associated with northern leaf blight
resistance, wherein said QTL allele is located within an interval
on chromosome 5 comprising and flanked by PHM18056 and PHM7958 and
said QTL allele comprises: i. a "G" at PZE-105068275; ii. an "A" at
PZE-105068432; iii. a "C" at PZE-105068572; iv. a "T" at SYN30642;
v. a "C" at PZE-105068746; vi. an "A" at PZE-105069095; vii. an "A"
at PZE-105069706; viii. a "T" at PZE-105069906; and ix. a "C" at
PZE-105070525; b. selecting said maize plant if said QTL allele is
detected.
2. The method of claim 1, wherein said QTL allele is located within
an interval on chromosome 5 defined by and including PZE-105068275
and PZE-105070525.
3. A method of introgressing a QTL allele associated with northern
leaf blight resistance into a maize plant said method comprising:
a. screening a population with at least one marker to determine if
one or more maize plants from the population comprises a QTL allele
associated with northern leaf blight resistance, wherein the QTL
allele comprises: i. a "G" at PZE-105068275; ii. an "A" at
PZE-105068432; iii. a "C" at PZE-105068572; iv. a "T" at SYN30642;
v. a "C" at PZE-105068746; vi. an "A" at PZE-105069095; vii. an "A"
at PZE-105069706; viii. a "T" at PZE-105069906; and ix. a "C" at
PZE-105070525; and b. selecting from said population a maize plant
comprising the QTL allele.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/502,696 filed Feb. 8, 2017, which claims the benefit from
International Application No. PCT/US2015/043529 filed Aug. 4, 2015,
which claims the benefit of U.S. Provisional Application No.
62/034,806, filed Aug. 8, 2014, the entire contents of each are
herein incorporated by reference.
FIELD
[0002] The field is related to plant breeding and methods of
identifying and selecting plants with resistance to northern leaf
blight.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0003] The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file named 20150715_BB1987PCT_SequenceListing_ST25.txt
created on Jul. 15, 2015 and having a size of 10 kilobytes and is
filed concurrently with the specification. The sequence listing
contained in this ASCII formatted document is part of the
specification and is herein incorporated by reference in its
entirety.
BACKGROUND
[0004] Northern leaf blight (NLB) caused by the fungus Setosphaeria
turcica (also known as Exserohilum turcicum or Helminthosporium
turcicum) is a major disease of maize in North America, South
America, Africa and Asia. Symptoms can range from cigar-shaped
lesions on the lower leaves to complete destruction of the foliage,
thereby reducing the amount of leaf surface area available for
photosynthesis which in turn impacts grain yield. Disease
management strategies include crop rotation, destruction of old
maize residues by tillage, and fungicide application, all of which
are aimed at reducing the fungal inoculum. However, the most
effective and most preferred method of control for northern leaf
blight is the planting of resistant hybrids.
[0005] Several varieties or races of Exserohilum turcicum are
present in nature, leaving growers with two hybrid options: partial
resistant hybrids, which offer low-level, broad spectrum protection
against multiple races, and race-specific resistant hybrids, which
protect against a specific race. Genetic sources of Exserohilum
turcicum have been described, and four Exserohilum turcicum
(previously called Helminthosporium turcicum) resistance loci have
been identified: Ht1, Ht2, Ht3, and Htn1. Gene Ht1 maps to the long
arm of chromosome 2 where it is closely linked to umc36 (Coe, E. H.
et al. (1988), Corn and Corn Improvement, 3rd edn., pp. 81-258),
sgcr506 (Gupta, M. et al. (1989) Maize Genet. Coop. Newsl. 63,
112), umc150B (Bentolila, S. et al. (1991) Theor. Appl. Genet.,
82:393-398), and pic18a (Collins et al. (1998) Molecular
Plant-Microbe Interactions, 11:968-978), and it is closely flanked
by umc22 and umc122 (Li et al. (1998) Hereditas, 129:101-106). Gene
Ht2 maps to the long arm of chromosome 8 in the umc48-umc89
interval (Zaitlin et al. (1992) Maize Genet. Coop. Newsl., 66,
69-70), and gene Ht3 maps to chromosome 7 near bn1g1666 (Van
Staden, D et al. (2001) Maize Genetics Conference Abstracts
43:P134). The Htn1 gene maps to chromosome 8, approximately 10 cM
distal to Ht2 and 0.8 cM distal to the RFLP marker umc117 (Simcox
and Bennetzen (1993) Maize Genet. Coop. Newl. 67, 118-119; Simcox
and Bennetzen (1993) Phytopathology, 83:1326-1330; Chung et al.
(2010) Theor App Gen Epub).
[0006] Since the QTL respond to different races and each QTL has a
variable effect on the northern leaf blight resistance trait, it is
desirable to identify new sources of genetic resistance that can be
combined with other known resistance loci to enhance overall
resistance to northern leaf blight.
SUMMARY
[0007] Compositions and methods for identifying and selecting maize
plants with enhanced resistance to northern leaf blight are
provided.
[0008] Methods for identifying maize plants with northern leaf
blight resistance are provided herein. The methods involve
analyzing DNA of a maize plant for the presence of a QTL allele
associated with northern leaf blight resistance and selecting maize
plants as having northern leaf blight resistance if the QTL allele
is detected. The QTL allele is located within an interval on
chromosome 5 comprising and flanked by PHM18056 and PHM7958 and may
comprise: a "G" at PZE-105068275; an "A" at PZE-105068432; a "C" at
PZE-105068572; a "T" at SYN30642; a "C" at PZE-105068746; an "A" at
PZE-105069095; an "A" at PZE-105069706; a "T" at PZE-105069906; and
a "C" at PZE-105070525. A subinterval of the interval in which the
QTL allele is located may be further defined by markers
PZE-105068275 and PZE-105070525.
[0009] Methods for introgressing a QTL allele associated with
northern leaf blight resistance into a maize plant are provided.
The methods involve screening a population with at least one marker
to determine if one or more maize plants from the population
comprises a QTL allele associated with northern leaf blight
resistance and selecting from the population one or more maize
plants that have the QTL allele. The QTL allele may comprise: a "G"
at PZE-105068275; an "A" at PZE-105068432; a "C" at PZE-105068572;
a "T" at SYN30642; a "C" at PZE-105068746; an "A" at PZE-105069095;
an "A" at PZE-105069706; a "T" at PZE-105069906; and a "C" at
PZE-105070525.
[0010] Plants identified, selected, or produced by the methods
described herein are also provided.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
[0011] The disclosure can be more fully understood from the
following detailed description and the accompanying drawings and
Sequence Listing which form a part of this application. The
Sequence Listing contains the one letter code for nucleotide
sequence characters and the three letter codes for amino acids as
defined in conformity with the IUPAC-IUBMB standards described in
Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical
Journal 219 (No. 2): 345-373 (1984), which are herein incorporated
by reference in their entirety. The symbols and format used for
nucleotide and amino acid sequence data comply with the rules set
forth in 37 C.F.R. .sctn.1.822.
[0012] FIG. 1 shows the diagram used as a guide to score northern
leaf blight infection.
[0013] SEQ ID NO:1 is the reference sequence for marker
PHM16750.
[0014] SEQ ID NO:2 is the reference sequence for marker
PHM15741.
[0015] SEQ ID NO:3 is the reference sequence for marker
PHM16854.
[0016] SEQ ID NO:4 is the reference sequence for marker
PHM3870.
[0017] SEQ ID NO:5 is the reference sequence for marker
PHM14018.
[0018] SEQ ID NO:6 is the reference sequence for marker
PHM18056.
[0019] SEQ ID NO:7 is the reference sequence for marker
PHM3467.
[0020] SEQ ID NO:8 is the reference sequence for marker
PHM7958.
[0021] SEQ ID NO:9 is the reference sequence for marker
PZE-105068275.
[0022] SEQ ID NO:10 is the reference sequence for marker
PZE-105068432.
[0023] SEQ ID NO:11 is the reference sequence for marker
PZE-105068572.
[0024] SEQ ID NO:12 is the reference sequence for marker
SYN30642.
[0025] SEQ ID NO:13 is the reference sequence for marker
PZE-105068746.
[0026] SEQ ID NO:14 is the reference sequence for marker
PZE-105069095.
[0027] SEQ ID NO:15 is the reference sequence for marker
PZE-105069706.
[0028] SEQ ID NO:16 is the reference sequence for marker
PZE-105069906.
[0029] SEQ ID NO:17 is the reference sequence for marker
PZE-105070525.
DETAILED DESCRIPTION
[0030] Maize marker loci that demonstrate statistically significant
co-segregation with the northern leaf blight resistance trait are
provided herein. Detection of these loci or additional linked loci
can be used in marker assisted selection as part of a maize
breeding program to produce maize plants that have resistance to
northern leaf blight, which is caused by the pathogen Exserohilum
turcicum.
[0031] Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation. Numeric ranges recited within the
specification are inclusive of the numbers defining the range and
include each integer or any non-integer fraction within the defined
range. Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the disclosure pertains. Methods
and materials similar or equivalent to those described herein can
be used in the practice for testing of the subject matter presented
herein. In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0032] The following definitions are provided as an aid to
understand the present disclosure.
[0033] It is to be understood that the disclosure is not limited to
particular embodiments, which can, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting. As used in this specification and the appended claims,
terms in the singular and the singular forms "a", "an" and "the",
for example, include plural referents unless the content clearly
dictates otherwise. Thus, for example, reference to "plant", "the
plant" or "a plant" also includes a plurality of plants; also,
depending on the context, use of the term "plant" can also include
genetically similar or identical progeny of that plant; use of the
term "a nucleic acid" optionally includes, as a practical matter,
many copies of that nucleic acid molecule; similarly, the term
"probe" optionally (and typically) encompasses many similar or
identical probe molecules.
[0034] The term "allele" refers to one of two or more different
nucleotide sequences that occur at a specific locus.
[0035] "Allele frequency" refers to the frequency (proportion or
percentage) at which an allele is present at a locus within an
individual, within a line, or within a population of lines. For
example, for an allele "A", diploid individuals of genotype "AA",
"Aa", or "aa" have allele frequencies of 1.0, 0.5, or 0.0,
respectively. One can estimate the allele frequency within a line
by averaging the allele frequencies of a sample of individuals from
that line. Similarly, one can calculate the allele frequency within
a population of lines by averaging the allele frequencies of lines
that make up the population. For a population with a finite number
of individuals or lines, an allele frequency can be expressed as a
count of individuals or lines (or any other specified grouping)
containing the allele.
[0036] An "amplicon" is an amplified nucleic acid, e.g., a nucleic
acid that is produced by amplifying a template nucleic acid by any
available amplification method (e.g., PCR, LCR, transcription, or
the like).
[0037] The term "amplifying" in the context of nucleic acid
amplification is any process whereby additional copies of a
selected nucleic acid (or a transcribed form thereof) are produced.
Typical amplification methods include various polymerase based
replication methods, including the polymerase chain reaction (PCR),
ligase mediated methods such as the ligase chain reaction (LCR) and
RNA polymerase based amplification (e.g., by transcription)
methods.
[0038] The term "assemble" applies to BACs and their propensities
for coming together to form contiguous stretches of DNA. A BAC
"assembles" to a contig based on sequence alignment, if the BAC is
sequenced, or via the alignment of its BAC fingerprint to the
fingerprints of other BACs. Public assemblies can be found using
the Maize Genome Browser, which is publicly available on the
internet.
[0039] An allele is "associated with" a trait when it is part of or
linked to a DNA sequence or allele that affects the expression of
the trait. The presence of the allele is an indicator of how the
trait will be expressed.
[0040] A "BAC", or bacterial artificial chromosome, is a cloning
vector derived from the naturally occurring F factor of Escherichia
coli, which itself is a DNA element that can exist as a circular
plasmid or can be integrated into the bacterial chromosome. BACs
can accept large inserts of DNA sequence. In maize, a number of
BACs each containing a large insert of maize genomic DNA from maize
inbred line B73, have been assembled into contigs (overlapping
contiguous genetic fragments, or "contiguous DNA"), and this
assembly is available publicly on the internet.
[0041] A BAC fingerprint is a means of analyzing similarity between
several DNA samples based upon the presence or absence of specific
restriction sites (restriction sites being nucleotide sequences
recognized by enzymes that cut or "restrict" the DNA). Two or more
BAC samples are digested with the same set of restriction enzymes
and the sizes of the fragments formed are compared, usually using
gel separation.
[0042] "Backcrossing" refers to the process whereby hybrid progeny
are repeatedly crossed back to one of the parents. In a
backcrossing scheme, the "donor" parent refers to the parental
plant with the desired gene/genes, locus/loci, or specific
phenotype to be introgressed. The "recipient" parent (used one or
more times) or "recurrent" parent (used two or more times) refers
to the parental plant into which the gene or locus is being
introgressed. For example, see Ragot, M. et al. (1995)
Marker-assisted backcrossing: a practical example, in Techniques et
Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp.
45-56, and Openshaw et al., (1994) Marker-assisted Selection in
Backcross Breeding, Analysis of Molecular Marker Data, pp. 41-43.
The initial cross gives rise to the F1 generation; the term "BC1"
then refers to the second use of the recurrent parent, "BC2" refers
to the third use of the recurrent parent, and so on.
[0043] A centimorgan ("cM") is a unit of measure of recombination
frequency. One cM is equal to a 1% chance that a marker at one
genetic locus will be separated from a marker at a second locus due
to crossing over in a single generation.
[0044] As used herein, the term "chromosomal interval" designates a
contiguous linear span of genomic DNA that resides in planta on a
single chromosome. The genetic elements or genes located on a
single chromosomal interval are physically linked. The size of a
chromosomal interval is not particularly limited. In some aspects,
the genetic elements located within a single chromosomal interval
are genetically linked, typically with a genetic recombination
distance of, for example, less than or equal to 20 cM, or
alternatively, less than or equal to 10 cM. That is, two genetic
elements within a single chromosomal interval undergo recombination
at a frequency of less than or equal to 20% or 10%.
[0045] A "chromosome" is a single piece of coiled DNA containing
many genes that act and move as a unity during cell division and
therefore can be said to be linked. It can also be referred to as a
"linkage group".
[0046] The phrase "closely linked", in the present application,
means that recombination between two linked loci occurs with a
frequency of equal to or less than about 10% (i.e., are separated
on a genetic map by not more than 10 cM). Put another way, the
closely linked loci co-segregate at least 90% of the time. Marker
loci are especially useful with respect to the subject matter of
the current disclosure when they demonstrate a significant
probability of co-segregation (linkage) with a desired trait (e.g.,
resistance to northern leaf blight). Closely linked loci such as a
marker locus and a second locus can display an inter-locus
recombination frequency of 10% or less, preferably about 9% or
less, still more preferably about 8% or less, yet more preferably
about 7% or less, still more preferably about 6% or less, yet more
preferably about 5% or less, still more preferably about 4% or
less, yet more preferably about 3% or less, and still more
preferably about 2% or less. In highly preferred embodiments, the
relevant loci display a recombination a frequency of about 1% or
less, e.g., about 0.75% or less, more preferably about 0.5% or
less, or yet more preferably about 0.25% or less. Two loci that are
localized to the same chromosome, and at such a distance that
recombination between the two loci occurs at a frequency of less
than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.degree. A,
0.75%, 0.5%, 0.25%, or less) are also said to be "proximal to" each
other. In some cases, two different markers can have the same
genetic map coordinates. In that case, the two markers are in such
close proximity to each other that recombination occurs between
them with such low frequency that it is undetectable.
[0047] The term "complement" refers to a nucleotide sequence that
is complementary to a given nucleotide sequence, i.e. the sequences
are related by the Watson-Crick base-pairing rules.
[0048] The term "contiguous DNA" refers to an uninterrupted stretch
of genomic DNA represented by partially overlapping pieces or
contigs.
[0049] When referring to the relationship between two genetic
elements, such as a genetic element contributing to northern leaf
blight resistance and a proximal marker, "coupling" phase linkage
indicates the state where the "favorable" allele at the northern
leaf blight resistance locus is physically associated on the same
chromosome strand as the "favorable" allele of the respective
linked marker locus. In coupling phase, both favorable alleles are
inherited together by progeny that inherit that chromosome
strand.
[0050] The term "crossed" or "cross" refers to a sexual cross and
involved the fusion of two haploid gametes via pollination to
produce diploid progeny (e.g., cells, seeds or plants). The term
encompasses both the pollination of one plant by another and
selfing (or self-pollination, e.g., when the pollen and ovule are
from the same plant).
[0051] A plant referred to herein as "diploid" has two sets
(genomes) of chromosomes.
[0052] A plant referred to herein as a "doubled haploid" is
developed by doubling the haploid set of chromosomes (i.e., half
the normal number of chromosomes). A doubled haploid plant has two
identical sets of chromosomes, and all loci are considered
homozygous.
[0053] An "elite line" is any line that has resulted from breeding
and selection for superior agronomic performance.
[0054] An "exotic maize strain" or an "exotic maize germ plasm" is
a strain derived from a maize plant not belonging to an available
elite maize line or strain of germplasm. In the context of a cross
between two maize plants or strains of germplasm, an exotic germ
plasm is not closely related by descent to the elite germplasm with
which it is crossed. Most commonly, the exotic germplasm is not
derived from any known elite line of maize, but rather is selected
to introduce novel genetic elements (typically novel alleles) into
a breeding program.
[0055] A "favorable allele" is the allele at a particular locus
that confers, or contributes to, an agronomically desirable
phenotype, e.g., northern leaf blight resistance, and that allows
the identification of plants with that agronomically desirable
phenotype. A favorable allele of a marker is a marker allele that
segregates with the favorable phenotype.
[0056] "Fragment" is intended to mean a portion of a nucleotide
sequence. Fragments can be used as hybridization probes or PCR
primers using methods disclosed herein.
[0057] A "genetic map" is a description of genetic linkage
relationships among loci on one or more chromosomes (or linkage
groups) within a given species, generally depicted in a
diagrammatic or tabular form. For each genetic map, distances
between loci are measured by how frequently their alleles appear
together in a population (their recombination frequencies). Alleles
can be detected using DNA or protein markers, or observable
phenotypes. A genetic map is a product of the mapping population,
types of markers used, and the polymorphic potential of each marker
between different populations. Genetic distances between loci can
differ from one genetic map to another. However, information can be
correlated from one map to another using common markers. One of
ordinary skill in the art can use common marker positions to
identify positions of markers and other loci of interest on each
individual genetic map. The order of loci should not change between
maps, although frequently there are small changes in marker orders
due to e.g. markers detecting alternate duplicate loci in different
populations, differences in statistical approaches used to order
the markers, novel mutation or laboratory error.
[0058] A "genetic map location" is a location on a genetic map
relative to surrounding genetic markers on the same linkage group
where a specified marker can be found within a given species.
[0059] "Genetic mapping" is the process of defining the linkage
relationships of loci through the use of genetic markers,
populations segregating for the markers, and standard genetic
principles of recombination frequency.
[0060] "Genetic markers" are nucleic acids that are polymorphic in
a population and where the alleles of which can be detected and
distinguished by one or more analytic methods, e.g., RFLP, AFLP,
isozyme, SNP, SSR, and the like. The term also refers to nucleic
acid sequences complementary to the genomic sequences, such as
nucleic acids used as probes. Markers corresponding to genetic
polymorphisms between members of a population can be detected by
methods well-established in the art. These include, e.g., PCR-based
sequence specific amplification methods, detection of restriction
fragment length polymorphisms (RFLP), detection of isozyme markers,
detection of polynucleotide polymorphisms by allele specific
hybridization (ASH), detection of amplified variable sequences of
the plant genome, detection of self-sustained sequence replication,
detection of simple sequence repeats (SSRs), detection of single
nucleotide polymorphisms (SNPs), or detection of amplified fragment
length polymorphisms (AFLPs). Well established methods are also
know for the detection of expressed sequence tags (ESTs) and SSR
markers derived from EST sequences and randomly amplified
polymorphic DNA (RAPD).
[0061] "Genetic recombination frequency" is the frequency of a
crossing over event (recombination) between two genetic loci.
Recombination frequency can be observed by following the
segregation of markers and/or traits following meiosis.
[0062] "Genome" refers to the total DNA, or the entire set of
genes, carried by a chromosome or chromosome set.
[0063] The term "genotype" is the genetic constitution of an
individual (or group of individuals) at one or more genetic loci.
Genotype is defined by the allele(s) of one or more known loci that
the individual has inherited from its parents. The term genotype
can be used to refer to an individual's genetic constitution at a
single locus, at multiple loci, or, more generally, the term
genotype can be used to refer to an individual's genetic make-up
for all the genes in its genome.
[0064] "Germplasm" refers to genetic material of or from an
individual (e.g., a plant), a group of individuals (e.g., a plant
line, variety or family), or a clone derived from a line, variety,
species, or culture, or more generally, all individuals within a
species or for several species (e.g., maize germplasm collection or
Andean germplasm collection). The germplasm can be part of an
organism or cell, or can be separate from the organism or cell. In
general, germplasm provides genetic material with a specific
molecular makeup that provides a physical foundation for some or
all of the hereditary qualities of an organism or cell culture. As
used herein, germplasm includes cells, seed or tissues from which
new plants may be grown, or plant parts, such as leafs, stems,
pollen, or cells, which can be cultured into a whole plant.
[0065] A plant referred to as "haploid" has a single set (genome)
of chromosomes.
[0066] A "haplotype" is the genotype of an individual at a
plurality of genetic loci, i.e. a combination of alleles.
Typically, the genetic loci described by a haplotype are physically
and genetically linked, i.e., on the same chromosome segment. The
term "haplotype" can refer to alleles at a particular locus, or to
alleles at multiple loci along a chromosomal segment.
[0067] The term "heterogeneity" is used to indicate that
individuals within the group differ in genotype at one or more
specific loci.
[0068] The heterotic response of material, or "heterosis", can be
defined by performance which exceeds the average of the parents (or
high parent) when crossed to other dissimilar or unrelated
groups.
[0069] A "heterotic group" comprises a set of genotypes that
perform well when crossed with genotypes from a different heterotic
group (Hallauer et al. (1998) Corn breeding, p. 463-564. In G. F.
Sprague and J. W. Dudley (ed.) Corn and corn improvement). Inbred
lines are classified into heterotic groups, and are further
subdivided into families within a heterotic group, based on several
criteria such as pedigree, molecular marker-based associations, and
performance in hybrid combinations (Smith et al. (1990) Theor.
Appl. Gen. 80:833-840). The two most widely used heterotic groups
in the United States are referred to as "Iowa Stiff Stalk
Synthetic" (also referred to herein as "stiff stalk") and
"Lancaster or "Lancaster Sure Crop" (sometimes referred to as NSS,
or non-Stiff Stalk).
[0070] Some heterotic groups possess the traits needed to be a
female parent, and others, traits for a male parent. For example,
in maize, yield results from public inbreds released from a
population called BSSS (Iowa Stiff Stalk Synthetic population) has
resulted in these inbreds and their derivatives becoming the female
pool in the central Corn Belt. BSSS inbreds have been crossed with
other inbreds, e.g. SD 105 and Maiz Amargo, and this general group
of materials has become known as Stiff Stalk Synthetics (SSS) even
though not all of the inbreds are derived from the original BSSS
population (Mikel and Dudley (2006) Crop Sci: 46:1193-1205). By
default, all other inbreds that combine well with the SSS inbreds
have been assigned to the male pool, which for lack of a better
name has been designated as NSS, i.e. Non-Stiff Stalk. This group
includes several major heterotic groups such as Lancaster Surecrop,
lodent, and Leaming Corn.
[0071] An individual is "heterozygous" if more than one allele type
is present at a given locus (e.g., a diploid individual with one
copy each of two different alleles).
[0072] The term "homogeneity" indicates that members of a group
have the same genotype at one or more specific loci.
[0073] An individual is "homozygous" if the individual has only one
type of allele at a given locus (e.g., a diploid individual has a
copy of the same allele at a locus for each of two homologous
chromosomes).
[0074] The term "hybrid" refers to the progeny obtained between the
crossing of at least two genetically dissimilar parents.
[0075] "Hybridization" or "nucleic acid hybridization" refers to
the pairing of complementary RNA and DNA strands as well as the
pairing of complementary DNA single strands.
[0076] The term "hybridize" means to form base pairs between
complementary regions of nucleic acid strands.
[0077] An "IBM genetic map" can refer to any of following maps:
IBM, IBM2, IBM2 neighbors, IBM2 FPC0507, IBM2 2004 neighbors, IBM2
2005 neighbors, IBM2 2005 neighbors frame, IBM2 2008 neighbors,
IBM2 2008 neighbors frame, or the latest version on the maizeGDB
website. IBM genetic maps are based on a B73.times.Mo17 population
in which the progeny from the initial cross were random-mated for
multiple generations prior to constructing recombinant inbred lines
for mapping. Newer versions reflect the addition of genetic and BAC
mapped loci as well as enhanced map refinement due to the
incorporation of information obtained from other genetic maps or
physical maps, cleaned date, or the use of new algorithms.
[0078] The term "inbred" refers to a line that has been bred for
genetic homogeneity.
[0079] The term "indel" refers to an insertion or deletion, wherein
one line may be referred to as having an inserted nucleotide or
piece of DNA relative to a second line or the second line may be
referred to as having a deleted nucleotide or piece of DNA relative
to the first line.
[0080] The term "introgression" refers to the transmission of a
desired allele of a genetic locus from one genetic background to
another. For example, introgression of a desired allele at a
specified locus can be transmitted to at least one progeny via a
sexual cross between two parents of the same species, where at
least one of the parents has the desired allele in its genome.
Alternatively, for example, transmission of an allele can occur by
recombination between two donor genomes, e.g., in a fused
protoplast, where at least one of the donor protoplasts has the
desired allele in its genome. The desired allele can be, e.g.,
detected by a marker that is associated with a phenotype, at a QTL
(i.e. a QTL allele), a transgene, or the like. In any case,
offspring comprising the desired allele can be repeatedly
backcrossed to a line having a desired genetic background and
selected for the desired allele, to result in the allele becoming
fixed in a selected genetic background.
[0081] The process of "introgressing" is often referred to as
"backcrossing" when the process is repeated two or more times.
[0082] A "line" or "strain" is a group of individuals of identical
parentage that are generally inbred to some degree and that are
generally homozygous and homogeneous at most loci (isogenic or near
isogenic). A "subline" refers to an inbred subset of descendents
that are genetically distinct from other similarly inbred subsets
descended from the same progenitor.
[0083] As used herein, the term "linkage" is used to describe the
degree with which one marker locus is associated with another
marker locus or some other locus. The linkage relationship between
a molecular marker and a locus affecting a phenotype is given as a
"probability" or "adjusted probability". Linkage can be expressed
as a desired limit or range. For example, in some embodiments, any
marker is linked (genetically and physically) to any other marker
when the markers are separated by less than 50, 40, 30, 25, 20, or
15 map units (or cM) of a single meiosis map (a genetic map based
on a population that has undergone one round of meiosis, such as
e.g. an F2; the IBM2 maps consist of multiple meioses). In some
aspects, it is advantageous to define a bracketed range of linkage,
for example, between 10 and 20 cM, between 10 and 30 cM, or between
10 and 40 cM. The more closely a marker is linked to a second
locus, the better an indicator for the second locus that marker
becomes. Thus, "closely linked loci" such as a marker locus and a
second locus display an inter-locus recombination frequency of 10%
or less, preferably about 9% or less, still more preferably about
8% or less, yet more preferably about 7% or less, still more
preferably about 6% or less, yet more preferably about 5% or less,
still more preferably about 4% or less, yet more preferably about
3% or less, and still more preferably about 2% or less. In highly
preferred embodiments, the relevant loci display a recombination
frequency of about 1% or less, e.g., about 0.75% or less, more
preferably about 0.5% or less, or yet more preferably about 0.25%
or less. Two loci that are localized to the same chromosome, and at
such a distance that recombination between the two loci occurs at a
frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%,
2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be "in
proximity to" each other. Since one cM is the distance between two
markers that show a 1% recombination frequency, any marker is
closely linked (genetically and physically) to any other marker
that is in close proximity, e.g., at or less than 10 cM distant.
Two closely linked markers on the same chromosome can be positioned
9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or 0.25 cM or less from each
other.
[0084] The term "linkage disequilibrium" (or LD) refers to a
non-random segregation of genetic loci or traits (or both). In
either case, linkage disequilibrium implies that the relevant loci
are within sufficient physical proximity along a length of a
chromosome so that they segregate together with greater than random
(i.e., non-random) frequency. Markers that show linkage
disequilibrium are considered linked. Linked loci co-segregate more
than 50% of the time, e.g., from about 51% to about 100% of the
time. In other words, two markers that co-segregate have a
recombination frequency of less than 50% (and by definition, are
separated by less than 50 cM on the same linkage group.) As used
herein, linkage can be between two markers, or alternatively
between a marker and a locus affecting a phenotype. A marker locus
can be "associated with" (linked to) a trait. The degree of linkage
of a marker locus and a locus affecting a phenotypic trait is
measured, e.g., as a statistical probability of co-segregation of
that molecular marker with the phenotype (e.g., an F statistic or
LOD score).
[0085] Linkage disequilibrium is most commonly assessed using the
measure r.sup.2, which is calculated using the formula described by
Hill, W. G. and Robertson, A, Theor. Appl. Genet. 38:226-231(1968).
When r.sup.2=1, complete LD exists between the two marker loci,
meaning that the markers have not been separated by recombination
and have the same allele frequency. The r.sup.2 value will be
dependent on the population used. Values for r.sup.2 above 1/3
indicate sufficiently strong LD to be useful for mapping (Ardlie et
al., Nature Reviews Genetics 3:299-309 (2002)). Hence, alleles are
in linkage disequilibrium when r.sup.2 values between pairwise
marker loci are greater than or equal to 0.33, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, or 1.0.
[0086] As used herein, "linkage equilibrium" describes a situation
where two markers independently segregate, i.e., sort among progeny
randomly. Markers that show linkage equilibrium are considered
unlinked (whether or not they lie on the same chromosome).
[0087] A "locus" is a position on a chromosome, e.g. where a
nucleotide, gene, sequence, or marker is located.
[0088] The "logarithm of odds (LOD) value" or "LOD score" (Risch,
Science 255:803-804 (1992)) is used in genetic interval mapping to
describe the degree of linkage between two marker loci. A LOD score
of three between two markers indicates that linkage is 1000 times
more likely than no linkage, while a LOD score of two indicates
that linkage is 100 times more likely than no linkage. LOD scores
greater than or equal to two may be used to detect linkage. LOD
scores can also be used to show the strength of association between
marker loci and quantitative traits in "quantitative trait loci"
mapping. In this case, the LOD score's size is dependent on the
closeness of the marker locus to the locus affecting the
quantitative trait, as well as the size of the quantitative trait
effect.
[0089] "Maize" refers to a plant of the Zea mays L. ssp. mays and
is also known as "corn".
[0090] The term "maize plant" includes whole maize plants, maize
plant cells, maize plant protoplast, maize plant cell or maize
tissue culture from which maize plants can be regenerated, maize
plant calli, maize plant clumps and maize plant cells that are
intact in maize plants or parts of maize plants, such as maize
seeds, maize cobs, maize flowers, maize cotyledons, maize leaves,
maize stems, maize buds, maize roots, maize root tips and the
like.
[0091] A "marker" is a means of finding a position on a genetic or
physical map, or else linkages among markers and trait loci (loci
affecting traits). The position that the marker detects may be
known via detection of polymorphic alleles and their genetic
mapping, or else by hybridization, sequence match or amplification
of a sequence that has been physically mapped. A marker can be a
DNA marker (detects DNA polymorphisms), a protein (detects
variation at an encoded polypeptide), or a simply inherited
phenotype (such as the `waxy` phenotype). A DNA marker can be
developed from genomic nucleotide sequence or from expressed
nucleotide sequences (e.g., from a spliced RNA or a cDNA).
Depending on the DNA marker technology, the marker will consist of
complementary primers flanking the locus and/or complementary
probes that hybridize to polymorphic alleles at the locus. A DNA
marker, or a genetic marker, can also be used to describe the gene,
DNA sequence or nucleotide on the chromosome itself (rather than
the components used to detect the gene or DNA sequence) and is
often used when that DNA marker is associated with a particular
trait in human genetics (e.g. a marker for breast cancer). The term
marker locus is the locus (gene, sequence or nucleotide) that the
marker detects.
[0092] Markers that detect genetic polymorphisms between members of
a population are well-established in the art. Markers can be
defined by the type of polymorphism that they detect and also the
marker technology used to detect the polymorphism. Marker types
include but are not limited to, e.g., detection of restriction
fragment length polymorphisms (RFLP), detection of isozyme markers,
randomly amplified polymorphic DNA (RAPD), amplified fragment
length polymorphisms (AFLPs), detection of simple sequence repeats
(SSRs), detection of amplified variable sequences of the plant
genome, detection of self-sustained sequence replication, or
detection of single nucleotide polymorphisms (SNPs). SNPs can be
detected e.g. via DNA sequencing, PCR-based sequence specific
amplification methods, detection of polynucleotide polymorphisms by
allele specific hybridization (ASH), dynamic allele-specific
hybridization (DASH), molecular beacons, microarray hybridization,
oligonucleotide ligase assays, Flap endonucleases, 5'
endonucleases, primer extension, single strand conformation
polymorphism (SSCP) or temperature gradient gel electrophoresis
(TGGE). DNA sequencing, such as the pyrosequencing technology has
the advantage of being able to detect a series of linked SNP
alleles that constitute a haplotype. Haplotypes tend to be more
informative (detect a higher level of polymorphism) than SNPs.
[0093] A "marker allele", alternatively an "allele of a marker
locus", can refer to one of a plurality of polymorphic nucleotide
sequences found at a marker locus in a population.
[0094] "Marker assisted selection" (of MAS) is a process by which
individual plants are selected based on marker genotypes.
[0095] "Marker assisted counter-selection" is a process by which
marker genotypes are used to identify plants that will not be
selected, allowing them to be removed from a breeding program or
planting.
[0096] A "marker haplotype" refers to a combination of alleles at a
marker locus.
[0097] A "marker locus" is a specific chromosome location in the
genome of a species where a specific marker can be found. A marker
locus can be used to track the presence of a second linked locus,
e.g., one that affects the expression of a phenotypic trait. For
example, a marker locus can be used to monitor segregation of
alleles at a genetically or physically linked locus.
[0098] A "marker probe" is a nucleic acid sequence or molecule that
can be used to identify the presence of a marker locus, e.g., a
nucleic acid probe that is complementary to a marker locus
sequence, through nucleic acid hybridization. Marker probes
comprising 30 or more contiguous nucleotides of the marker locus
("all or a portion" of the marker locus sequence) may be used for
nucleic acid hybridization. Alternatively, in some aspects, a
marker probe refers to a probe of any type that is able to
distinguish (i.e., genotype) the particular allele that is present
at a marker locus.
[0099] The term "molecular marker" may be used to refer to a
genetic marker, as defined above, or an encoded product thereof
(e.g., a protein) used as a point of reference when identifying a
linked locus. A marker can be derived from genomic nucleotide
sequences or from expressed nucleotide sequences (e.g., from a
spliced RNA, a cDNA, etc.), or from an encoded polypeptide. The
term also refers to nucleic acid sequences complementary to or
flanking the marker sequences, such as nucleic acids used as probes
or primer pairs capable of amplifying the marker sequence. A
"molecular marker probe" is a nucleic acid sequence or molecule
that can be used to identify the presence of a marker locus, e.g.,
a nucleic acid probe that is complementary to a marker locus
sequence. Alternatively, in some aspects, a marker probe refers to
a probe of any type that is able to distinguish (i.e., genotype)
the particular allele that is present at a marker locus. Nucleic
acids are "complementary" when they specifically hybridize in
solution, e.g., according to Watson-Crick base pairing rules. Some
of the markers described herein are also referred to as
hybridization markers when located on an indel region, such as the
non-collinear region described herein. This is because the
insertion region is, by definition, a polymorphism vis a vis a
plant without the insertion. Thus, the marker need only indicate
whether the indel region is present or absent. Any suitable marker
detection technology may be used to identify such a hybridization
marker, e.g. SNP technology is used in the examples provided
herein.
[0100] An allele "negatively" correlates with a trait when it is
linked to it and when presence of the allele is an indicator that a
desired trait or trait form will not occur in a plant comprising
the allele.
[0101] "Nucleotide sequence", "polynucleotide", "nucleic acid
sequence", and "nucleic acid fragment" are used interchangeably and
refer to a polymer of RNA or DNA that is single- or
double-stranded, optionally containing synthetic, non-natural or
altered nucleotide bases. A "nucleotide" is a monomeric unit from
which DNA or RNA polymers are constructed, and consist of a purine
or pyrimidine base, a pentose, and a phosphoric acid group.
Nucleotides (usually found in their 5'-monophosphate form) are
referred to by their single letter designation as follows: "A" for
adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" for
cytidylate or deoxycytidylate, "G" for guanylate or deoxyguanylate,
"U" for uridylate, "T" for deoxythymidylate, "R" for purines (A or
G), "Y" for pyrimidines (C or T), "K" for G or T, "H" for A or C or
T, "I" for inosine, and "N" for any nucleotide.
[0102] The term "phenotype", "phenotypic trait", or "trait" can
refer to the observable expression of a gene or series of genes.
The phenotype can be observable to the naked eye, or by any other
means of evaluation known in the art, e.g., weighing, counting,
measuring (length, width, angles, etc.), microscopy, biochemical
analysis, or an electromechanical assay. In some cases, a phenotype
is directly controlled by a single gene or genetic locus, i.e., a
"single gene trait" or a "simply inherited trait". In the absence
of large levels of environmental variation, single gene traits can
segregate in a population to give a "qualitative" or "discrete"
distribution, i.e. the phenotype falls into discrete classes. In
other cases, a phenotype is the result of several genes and can be
considered a "multigenic trait" or a "complex trait". Multigenic
traits segregate in a population to give a "quantitative" or
"continuous" distribution, i.e. the phenotype cannot be separated
into discrete classes. Both single gene and multigenic traits can
be affected by the environment in which they are being expressed,
but multigenic traits tend to have a larger environmental
component.
[0103] A "physical map" of the genome is a map showing the linear
order of identifiable landmarks (including genes, markers, etc.) on
chromosome DNA. However, in contrast to genetic maps, the distances
between landmarks are absolute (for example, measured in base pairs
or isolated and overlapping contiguous genetic fragments) and not
based on genetic recombination (that can vary in different
populations).
[0104] A "plant" can be a whole plant, any part thereof, or a cell
or tissue culture derived from a plant. Thus, the term "plant" can
refer to any of: whole plants, plant components or organs (e.g.,
leaves, stems, roots, etc.), plant tissues, seeds, plant cells,
and/or progeny of the same. A plant cell is a cell of a plant,
taken from a plant, or derived through culture from a cell taken
from a plant.
[0105] A maize plant "derived from an inbred in the Stiff Stalk
Synthetic population" may be a hybrid.
[0106] A "polymorphism" is a variation in the DNA between two or
more individuals within a population. A polymorphism preferably has
a frequency of at least 1% in a population. A useful polymorphism
can include a single nucleotide polymorphism (SNP), a simple
sequence repeat (SSR), or an insertion/deletion polymorphism, also
referred to herein as an "indel".
[0107] An allele "positively" correlates with a trait when it is
linked to it and when presence of the allele is an indicator that
the desired trait or trait form will occur in a plant comprising
the allele.
[0108] The "probability value" or "p-value" is the statistical
likelihood that the particular combination of a phenotype and the
presence or absence of a particular marker allele is random. Thus,
the lower the probability score, the greater the likelihood that a
locus and a phenotype are associated. The probability score can be
affected by the proximity of the first locus (usually a marker
locus) and the locus affecting the phenotype, plus the magnitude of
the phenotypic effect (the change in phenotype caused by an allele
substitution). In some aspects, the probability score is considered
"significant" or "nonsignificant". In some embodiments, a
probability score of 0.05 (p=0.05, or a 5% probability) of random
assortment is considered a significant indication of association.
However, an acceptable probability can be any probability of less
than 50% (p=0.5). For example, a significant probability can be
less than 0.25, less than 0.20, less than 0.15, less than 0.1, less
than 0.05, less than 0.01, or less than 0.001.
[0109] A "production marker" or "production SNP marker" is a marker
that has been developed for high-throughput purposes. Production
SNP markers are developed to detect specific polymorphisms and are
designed for use with a variety of chemistries and platforms. The
marker names used here begin with a PHM prefix to denote `Pioneer
Hi-Bred Marker`, followed by a number that is specific to the
sequence from which it was designed, followed by a "." or a "-" and
then a suffix that is specific to the DNA polymorphism. A marker
version can also follow (A, B, C etc.) that denotes the version of
the marker designed to that specific polymorphism.
[0110] The term "progeny" refers to the offspring generated from a
cross.
[0111] A "progeny plant" is a plant generated from a cross between
two plants.
[0112] The term "quantitative trait locus" or "QTL" refers to a
region of DNA that is associated with the differential expression
of a quantitative phenotypic trait in at least one genetic
background, e.g., in at least one breeding population. The region
of the QTL encompasses or is closely linked to the gene or genes
that affect the trait in question. An "allele of a QTL" (or "QTL
allele") can comprise multiple genes or other genetic factors
within a contiguous genomic region or linkage group. An allele of a
QTL can be defined by a haplotype within a specified window wherein
said window is a contiguous genomic region that can be defined, and
tracked, with a set of one or more polymorphic markers. The
haplotype is then defined by the unique fingerprint of alleles at
each marker within the specified window.
[0113] A "reference sequence" or a "consensus sequence" is a
defined sequence used as a basis for sequence comparison. The
reference sequence for a PHM marker is obtained by sequencing a
number of lines at the locus, aligning the nucleotide sequences in
a sequence alignment program (e.g. Sequencher), and then obtaining
the most common nucleotide sequence of the alignment. Polymorphisms
found among the individual sequences are annotated within the
consensus sequence. A reference sequence is not usually an exact
copy of any individual DNA sequence, but represents an amalgam of
available sequences and is useful for designing primers and probes
to polymorphisms within the sequence.
[0114] In "repulsion" phase linkage, the "favorable" allele at the
locus of interest is physically linked with an "unfavorable" allele
at the proximal marker locus, and the two "favorable" alleles are
not inherited together (i.e., the two loci are "out of phase" with
each other).
[0115] "Northern leaf blight" (NLB), sometimes referred to as
northern corn leaf blight (NCLB), is the disease caused by the
pathogen Exserohilum turcicum. The disease, characterized by
cigar-shaped lesions on leaf tissue, can have severe effects on
yield, particularly in tropical climates or during wet seasons in
temperate climates.
[0116] As used herein, "northern leaf blight resistance" refers to
enhanced resistance or tolerance to a fungal pathogen that causes
northern leaf blight when compared to a control plant. Effects may
vary from a slight increase in tolerance to the effects of the
fungal pathogen (e.g., partial inhibition) to total resistance such
that the plant is unaffected by the presence of the fungal
pathogen. An increased level of resistance against a particular
fungal pathogen or against a wider spectrum of fungal pathogens
constitutes "enhanced" or improved fungal resistance. The
embodiments of the disclosure will enhance or improve resistance to
the fungal pathogen that causes northern leaf blight, such that the
resistance of the plant to a fungal pathogen or pathogens will
increase. The term "enhance" refers to improve, increase, amplify,
multiply, elevate, raise, and the like.
[0117] A "topeross test" is a test performed by crossing each
individual (e.g. a selection, inbred line, clone or progeny
individual) with the same pollen parent or "tester", usually a
homozygous line.
[0118] The phrase "under stringent conditions" refers to conditions
under which a probe or polynucleotide will hybridize to a specific
nucleic acid sequence, typically in a complex mixture of nucleic
acids, but to essentially no other sequences. Stringent conditions
are sequence-dependent and will be different in different
circumstances.
[0119] Longer sequences hybridize specifically at higher
temperatures. Generally, stringent conditions are selected to be
about 5-10.degree. C. lower than the thermal melting point (Tm) for
the specific sequence at a defined ionic strength pH. The Tm is the
temperature (under defined ionic strength, pH, and nucleic acid
concentration) at which 50% of the probes complementary to the
target hybridize to the target sequence at equilibrium (as the
target sequences are present in excess, at Tm, 50% of the probes
are occupied at equilibrium). Stringent conditions will be those in
which the salt concentration is less than about 1.0 M sodium ion,
typically about 0.01 to 1.0 M sodium ion concentration (or other
salts) at pH 7.0 to 8.3, and the temperature is at least about
30.degree. C. for short probes (e.g., 10 to 50 nucleotides) and at
least about 60.degree. C. for long probes (e.g., greater than 50
nucleotides). Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide. For selective
or specific hybridization, a positive signal is at least two times
background, preferably 10 times background hybridization. Exemplary
stringent hybridization conditions are often: 50% formamide,
5.times. SC, and 1% SDS, incubating at 42.degree. C., or, 5.times.
SC, 1.degree. A SDS, incubating at 65.degree. C., with wash in
0.2.times. SSC, and 0.1.degree. A SDS at 65.degree. C. For PCR, a
temperature of about 36.degree. C. is typical for low stringency
amplification, although annealing temperatures may vary between
about 32.degree. C. and 48.degree. C., depending on primer length.
Additional guidelines for determining hybridization parameters are
provided in numerous references.
[0120] An "unfavorable allele" of a marker is a marker allele that
segregates with the unfavorable plant phenotype, therefore
providing the benefit of identifying plants that can be removed
from a breeding program or planting.
[0121] The term "yield" refers to the productivity per unit area of
a particular plant product of commercial value. For example, yield
of maize is commonly measured in bushels of seed per acre or metric
tons of seed per hectare per season. Yield is affected by both
genetic and environmental factors. "Agronomics", "agronomic
traits", and "agronomic performance" refer to the traits (and
underlying genetic elements) of a given plant variety that
contribute to yield over the course of growing season. Individual
agronomic traits include emergence vigor, vegetative vigor, stress
tolerance, disease resistance or tolerance, herbicide resistance,
branching, flowering, seed set, seed size, seed density,
standability, threshability and the like. Yield is, therefore, the
final culmination of all agronomic traits.
[0122] Sequence alignments and percent identity calculations may be
determined using a variety of comparison methods designed to detect
homologous sequences including, but not limited to, the
MEGALIGN.RTM. program of the LASERGENE.RTM. bioinformatics
computing suite (DNASTAR.RTM. Inc., Madison, Wis.). Unless stated
otherwise, multiple alignment of the sequences provided herein were
performed using the CLUSTAL V method of alignment (Higgins and
Sharp, CABIOS. 5:151 153 (1989)) with the default parameters (GAP
PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise
alignments and calculation of percent identity of protein sequences
using the CLUSTAL V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5
and DIAGONALS SAVED=5. For nucleic acids these parameters are
KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After
alignment of the sequences, using the CLUSTAL V program, it is
possible to obtain "percent identity" and "divergence" values by
viewing the "sequence distances" table on the same program; unless
stated otherwise, percent identities and divergences provided and
claimed herein were calculated in this manner.
[0123] Standard recombinant DNA and molecular cloning techniques
used herein are well known in the art and are described more fully
in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning:
A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold
Spring Harbor, 1989 (hereinafter "Sambrook").
Genetic Mapping--Identification of Genetic Loci Associated with
Enhanced Resistance to Helminthosporium turcicum
[0124] It has been recognized for quite some time that specific
genetic loci correlating with particular phenotypes, such as
resistance to northern leaf blight, can be mapped in an organism's
genome. The plant breeder can advantageously use the genetic loci
(i.e. molecular markers) to identify desired individuals by
detecting alleles at the loci that show a statistically significant
probability of co-segregation with a desired phenotype, manifested
as linkage disequilibrium. By identifying a molecular marker or
clusters of molecular markers that co-segregate with a trait of
interest, a breeder is able to rapidly select a desired phenotype
by selecting for the proper molecular marker allele (a process
called marker-assisted selection, or MAS). Such markers could also
be used by breeders to design genotypes in silico and to practice
whole genome selection.
[0125] A variety of methods well known in the art are available for
detecting molecular markers or clusters of molecular markers that
co-segregate with a trait of interest, such as resistance to
northern leaf blight. The basic idea underlying these methods is
the detection of markers, for which alternative genotypes (or
alleles) have significantly different average phenotypes. Thus, one
makes a comparison among marker loci of the magnitude of difference
among alternative genotypes (or alleles) or the level of
significance of that difference. Trait genes are inferred to be
located nearest the marker(s) that have the greatest associated
genotypic difference.
[0126] Two such methods used to detect trait loci of interest are:
1) Population-based association analysis and 2) Traditional linkage
analysis. In a population-based association analysis, lines are
obtained from pre-existing populations with multiple founders, e.g.
elite breeding lines. Population-based association analyses rely on
the decay of linkage disequilibrium (LD) and the idea that in an
unstructured population, only correlations between genes
controlling a trait of interest and markers closely linked to those
genes will remain after so many generations of random mating. In
reality, most pre-existing populations have population
substructure. Thus, the use of a structured association approach
helps to control population structure by allocating individuals to
populations using data obtained from markers randomly distributed
across the genome, thereby minimizing disequilibrium due to
population structure within the individual populations (also called
subpopulations). The phenotypic values are compared to the
genotypes (alleles) at each marker locus for each line in the
subpopulation. A significant marker-trait association indicates the
close proximity between the marker locus and one or more genetic
loci that are involved in the expression of that trait.
[0127] The same principles underlie traditional linkage analysis;
however, LD is generated by creating a population from a small
number of founders. The founders are selected to maximize the level
of polymorphism within the constructed population, and polymorphic
sites are assessed for their level of cosegregation with a given
phenotype. A number of statistical methods have been used to
identify significant marker-trait associations. One such method is
an interval mapping approach (Lander and Botstein, Genetics
121:185-199 (1989), in which each of many positions along a genetic
map (say at 1 cM intervals) is tested for the likelihood that a
gene controlling a trait of interest is located at that position.
The genotype/phenotype data are used to calculate for each test
position a LOD score (log of likelihood ratio). When the LOD score
exceeds a threshold value, there is significant evidence for the
location of a gene controlling the trait of interest at that
position on the genetic map (which will fall between two particular
marker loci).
[0128] Molecular marker loci that demonstrate statistically
significant co-segregation with resistance to northern leaf blight,
as determined by association mapping and traditional linkage
mapping techniques, are provided herein. Detection of these marker
loci or additional linked marker loci can be used in
marker-assisted maize breeding programs to produce plants with
enhanced resistance to northern leaf blight or to eliminate plants
that do not have enhanced resistance to northern leaf blight from
breeding programs or planting.
Markers Associated with Resistance to Northern Leaf Blight
[0129] Methods involving detecting the presence of one or more
marker alleles (at one or more marker loci) associated with
enhanced resistance to northern leaf blight in the germplasm of the
maize plant are provided herein. The maize plant can be a hybrid or
inbred.
[0130] The marker locus can be selected from any of the marker loci
provided herein including but not limited to: PHM16750, PHM15741,
PHM16854, PHM3870, PHM14018, PHM18056, PHM3467, PHM7958,
PZE-105068275, PZE-105068432, PZE-105068572, SYN30642,
PZE-105068746, PZE-105069095, PZE-105069706, PZE-105069906, and
PZE-105070525; as well as any other marker linked to these markers
(linked markers can be determined from the MaizeGDB resource).
[0131] A common measure of linkage is the frequency with which
traits cosegregate. This can be expressed as a percentage of
cosegregation (recombination frequency) or in centiMorgans (cM).
The cM is a unit of measure of genetic recombination frequency. One
cM is equal to a 1% chance that a trait at one genetic locus will
be separated from a trait at another locus due to crossing over in
a single generation (meaning the traits segregate together 99% of
the time). Because chromosomal distance is approximately
proportional to the frequency of crossing over events between
traits, there is an approximate physical distance that correlates
with recombination frequency.
[0132] Marker loci are themselves traits and can be assessed
according to standard linkage analysis by tracking the marker loci
during segregation. Thus, one cM is equal to a 1% chance that a
marker locus will be separated from another locus, due to crossing
over in a single generation.
[0133] The closer a marker is to a gene controlling a trait of
interest, the more effective and advantageous that marker is as an
indicator for the desired trait. Closely linked loci display an
inter-locus cross-over frequency of about 10% or less, preferably
about 9% or less, still more preferably about 8% or less, yet more
preferably about 7% or less, still more preferably about 6% or
less, yet more preferably about 5% or less, still more preferably
about 4% or less, yet more preferably about 3% or less, and still
more preferably about 2% or less. In highly preferred embodiments,
the relevant loci (e.g., a marker locus and a target locus) display
a recombination frequency of about 1% or less, e.g., about 0.75% or
less, more preferably about 0.5% or less, or yet more preferably
about 0.25% or less. Thus, the loci are about 10 cM, 9 cM, 8 cM, 7
cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM
or less apart. Put another way, two loci that are localized to the
same chromosome, and at such a distance that recombination between
the two loci occurs at a frequency of less than 10% (e.g., about
9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less)
are said to be "proximal to" each other.
[0134] Although particular marker alleles can show co-segregation
with the northern leaf blight resistance phenotype, it is important
to note that the marker locus is not necessarily responsible for
the expression of the northern leaf blight resistance phenotype.
For example, it is not a requirement that the marker polynucleotide
sequence be part of a gene that imparts enhanced northern leaf
blight resistance (for example, be part of the gene open reading
frame). The association between a specific marker allele and the
enhanced northern leaf blight resistance phenotype is due to the
original "coupling" linkage phase between the marker allele and the
allele in the ancestral maize line from which the allele
originated. Eventually, with repeated recombination, crossing over
events between the marker and genetic locus can change this
orientation. For this reason, the favorable marker allele may
change depending on the linkage phase that exists within the
resistant parent used to create segregating populations. This does
not change the fact that the marker can be used to monitor
segregation of the phenotype. It only changes which marker allele
is considered favorable in a given segregating population.
Chromosomal Intervals
[0135] Chromosomal intervals that correlate with northern leaf
blight resistance are provided. A variety of methods well known in
the art are available for identifying chromosomal intervals. The
boundaries of such chromosomal intervals are drawn to encompass
markers that will be linked to the gene controlling the trait of
interest. In other words, the chromosomal interval is drawn such
that any marker that lies within that interval (including the
terminal markers that define the boundaries of the interval) can be
used as a marker for northern leaf blight resistance. Each interval
comprises at least one QTL, and furthermore, may indeed comprise
more than one QTL. Close proximity of multiple QTL in the same
interval may obfuscate the correlation of a particular marker with
a particular QTL, as one marker may demonstrate linkage to more
than one QTL. Conversely, e.g., if two markers in close proximity
show co-segregation with the desired phenotypic trait, it is
sometimes unclear if each of those markers identifies the same QTL
or two different QTL. Regardless, knowledge of how many QTL are in
a particular interval is not necessary to make or practice the
subject material presented herein.
[0136] An interval on chromosome 5 containing one or more QTL
associated with northern leaf blight resistance may be defined by
and includes: PHM18056 and PHM7958. The interval may further be
refined to a chromosomal interval defined by and including
PZE-105068275 and PZE-105070525, which represents a subinterval of
the chromosomal interval defined by and including PHM18056 and
PHM7958. Any marker located within any of these intervals finds use
as a marker for northern leaf blight resistance in maize.
[0137] Chromosomal intervals can also be defined by markers that
are linked to (show linkage disequilibrium with) a marker of
interest, and r.sup.2 is a common measure of linkage disequilibrium
(LD) in the context of association studies. If the r.sup.2 value of
LD between any marker locus identified herein and another marker
within the chromosome 5 interval (also described herein) is greater
than 1/3 (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)),
the loci are linked.
Marker Alleles and Haplotype Combinations
[0138] A haplotype, or a combination of alleles at one or more
marker loci, can represent the genetic signature of a QTL allele.
Any of the marker alleles described herein could be used alone or
in combination to identify and select maize plants with enhanced
northern leaf blight by identifying a haplotype representative of a
QTL allele as could any marker allele in linkage disequilibrium
with the marker alleles described herein. The marker alleles
representative of the QTL allele may include: a "G" at
PZE-105068275; an "A" at PZE-105068432; a "C" at PZE-105068572; a
"T" at SYN30642; a "C" at PZE-105068746; an "A" at PZE-105069095;
an "A" at PZE-105069706; a "T" at PZE-105069906; and/or a "C" at
PZE-105070525.
[0139] Methods for identifying maize plants with northern leaf
blight resistance are provided herein. The methods involve
analyzing DNA of a maize plant for the presence of a QTL allele
associated with northern leaf blight resistance and selecting maize
plants as having northern leaf blight resistance if the QTL allele
is detected.
[0140] The QTL allele may comprise any of the following marker
alleles alone or in combination: a "G" at PZE-105068275; an "A" at
PZE-105068432; a "C" at PZE-105068572; a "T" at SYN30642; a "C" at
PZE-105068746; an "A" at PZE-105069095; an "A" at PZE-105069706; a
"T" at PZE-105069906; and a "C" at PZE-105070525.
[0141] In one aspect, the QTL allele is located on chromosome 5 in
a chromosomal interval defined by and including PHM18056 and
PHM7958. In another aspect, the QTL allele is located on chromosome
5 in a chromosomal interval defined by and including PZE-105068275
and PZE-105070525, which is a subinterval of the PHM18056 and
PHM7958 interval.
[0142] The skilled artisan would expect that there are additional
polymorphic sites at marker loci in and around the chromosome 5
markers identified herein, wherein one or more polymorphic sites is
in linkage disequilibrium (LD) with one or more of the polymorphic
sites in the representative haplotype. Two particular alleles at
different polymorphic sites are said to be in LD if the presence of
the allele at one of the sites tends to predict the presence of the
allele at the other site on the same chromosome (Stevens, Mol.
Diag. 4:309-17 (1999)). Detecting the presence of a QTL allele does
not in any way mean that the QTL allele can only be defined by the
haplotype comprising: a "G" at PZE-105068275; an "A" at
PZE-105068432; a "C" at PZE-105068572; a "T" at SYN30642; a "C" at
PZE-105068746; an "A" at PZE-105069095; an "A" at PZE-105069706; a
"T" at PZE-105069906; and a "C" at PZE-105070525. Rather, the
presence of the QTL allele can be detected using any of the marker
alleles defined herein alone or in combination and/or any other
marker allele within the specified chromosomal interval that is in
linkage disequilibrium with any of the marker alleles defined
herein.
Marker-Assisted Selection (MAS)
[0143] Molecular markers can be used in a variety of plant breeding
applications (e.g. see Staub et al. (1996) Hortscience 31: 729-741;
Tanksley (1983) Plant Molecular Biology Reporter. 1: 3-8). One of
the main areas of interest is to increase the efficiency of
backcrossing and introgressing genes using marker-assisted
selection (MAS). A molecular marker that demonstrates linkage with
a locus affecting a desired phenotypic trait provides a useful tool
for the selection of the trait in a plant population. This is
particularly true where the phenotype is hard to assay, e.g. many
disease resistance traits, or, occurs at a late stage in plant
development, e.g. kernel characteristics. Since DNA marker assays
are less laborious and take up less physical space than field
phenotyping, much larger populations can be assayed, increasing the
chances of finding a recombinant with the target segment from the
donor line moved to the recipient line. The closer the linkage, the
more useful the marker, as recombination is less likely to occur
between the marker and the gene causing the trait, which can result
in false positives. Having flanking markers decreases the chances
that false positive selection will occur as a double recombination
event would be needed. The ideal situation is to have a marker in
the gene itself, so that recombination cannot occur between the
marker and the gene. Such a marker is called a `perfect
marker`.
[0144] When a gene is introgressed by MAS, it is not only the gene
that is introduced but also the flanking regions (Gepts. (2002).
Crop Sci; 42: 1780-1790). This is referred to as "linkage drag." In
the case where the donor plant is highly unrelated to the recipient
plant, these flanking regions carry additional genes that may code
for agronomically undesirable traits. This "linkage drag" may also
result in reduced yield or other negative agronomic characteristics
even after multiple cycles of backcrossing into the elite maize
line. This is also sometimes referred to as "yield drag." The size
of the flanking region can be decreased by additional backcrossing,
although this is not always successful, as breeders do not have
control over the size of the region or the recombination
breakpoints (Young et al. (1998) Genetics 120:579-585). In
classical breeding it is usually only by chance that recombinations
are selected that contribute to a reduction in the size of the
donor segment (Tanksley et al. (1989). Biotechnology 7: 257-264).
Even after 20 backcrosses in backcrosses of this type, one may
expect to find a sizeable piece of the donor chromosome still
linked to the gene being selected. With markers however, it is
possible to select those rare individuals that have experienced
recombination near the gene of interest. In 150 backcross plants,
there is a 95% chance that at least one plant will have experienced
a crossover within 1 cM of the gene, based on a single meiosis map
distance. Markers will allow unequivocal identification of those
individuals. With one additional backcross of 300 plants, there
would be a 95% chance of a crossover within 1 cM single meiosis map
distance of the other side of the gene, generating a segment around
the target gene of less than 2 cM based on a single meiosis map
distance. This can be accomplished in two generations with markers,
while it would have required on average 100 generations without
markers (See Tanksley et al., supra). When the exact location of a
gene is known, flanking markers surrounding the gene can be
utilized to select for recombinations in different population
sizes. For example, in smaller population sizes, recombinations may
be expected further away from the gene, so more distal flanking
markers would be required to detect the recombination.
[0145] The availability of integrated linkage maps of the maize
genome containing increasing densities of public maize markers has
facilitated maize genetic mapping and MAS. See, e.g. the IBM2
Neighbors maps, which are available online on the MaizeGDB
website.
[0146] The key components to the implementation of MAS are: (i)
Defining the population within which the marker-trait association
will be determined, which can be a segregating population, or a
random or structured population; (ii) monitoring the segregation or
association of polymorphic markers relative to the trait, and
determining linkage or association using statistical methods; (iii)
defining a set of desirable markers based on the results of the
statistical analysis, and (iv) the use and/or extrapolation of this
information to the current set of breeding germplasm to enable
marker-based selection decisions to be made. The markers described
in this disclosure, as well as other marker types such as SSRs and
FLPs, can be used in marker-assisted selection protocols.
[0147] In general, MAS for the purposes described herein uses
polymorphic markers that have been identified as having a
significant likelihood of co-segregation with northern leaf blight
resistance. Such markers are presumed to map near a gene or genes
that give the plant its northern leaf blight resistance phenotype,
and are considered indicators for the desired trait, or markers.
Plants are tested for the presence of a desired allele in the
marker, and plants containing a desired genotype at one or more
loci are expected to transfer the desired genotype, along with a
desired phenotype, to their progeny.
[0148] Markers were identified from both linkage mapping and
association analysis as being associated with resistance to
northern leaf blight. Reference sequences for each of the markers
are represented by SEQ ID NOs:1-17. The SNPs could be used alone or
in combination (i.e. a SNP haplotype) to select for a favorable QTL
allele associated with resistance to northern leaf blight.
[0149] Methods for introgressing a QTL allele associated with
northern leaf blight resistance into a maize plant are provided
herein. The methods involve screening a population with at least
one marker to determine if one or more maize plants from the
population comprises a QTL allele associated with northern leaf
blight resistance and selecting from the population one or more
maize plants that have the QTL allele. The QTL allele may comprise:
a "G" at PZE-105068275; an "A" at PZE-105068432; a "C" at
PZE-105068572; a "T" at SYN30642; a "C" at PZE-105068746; an "A" at
PZE-105069095; an "A" at PZE-105069706; a "T" at PZE-105069906; and
a "C" at PZE-105070525.
[0150] The skilled artisan would expect that there might be
additional polymorphic sites at marker loci in and around the
chromosome 5 markers identified herein, wherein one or more
polymorphic sites is in linkage disequilibrium (LD) with an allele
at one or more of the polymorphic sites in the haplotype and thus
could be used in a marker assisted selection program to introgress
a QTL allele of interest. Two particular alleles at different
polymorphic sites are said to be in LD if the presence of the
allele at one of the sites tends to predict the presence of the
allele at the other site on the same chromosome (Stevens, Mol.
Diag. 4:309-17 (1999)). The marker loci can be located within 5 cM,
2 cM, or 1 cM (on a single meiosis based genetic map) of the
resistance to northern leaf blight QTL.
[0151] The skilled artisan would also understand that allelic
frequency (and hence, haplotype frequency) can differ from one
germplasm pool to another. Germplasm pools vary due to maturity
differences, heterotic groupings, geographical distribution, etc.
As a result, SNPs and other polymorphisms may not be informative in
some germplasm pools.
Plant Compositions
[0152] Maize plants identified, selected, and/or generated by any
of the methods described above are also of interest.
Seed Treatments
[0153] To protect and to enhance yield production and trait
technologies, seed treatment options can provide additional crop
plan flexibility and cost effective control against insects, weeds
and diseases, thereby further enhancing the methods and
compositions described herein. Seed material can be treated,
typically surface treated, with a composition comprising
combinations of chemical or biological herbicides, herbicide
safeners, insecticides, fungicides, germination inhibitors and
enhancers, nutrients, plant growth regulators and activators,
bactericides, nematicides, avicides and/or molluscicides. These
compounds are typically formulated together with further carriers,
surfactants or application-promoting adjuvants customarily employed
in the art of formulation. The coatings may be applied by
impregnating propagation material with a liquid formulation or by
coating with a combined wet or dry formulation. Examples of the
various types of compounds that may be used as seed treatments are
provided in The Pesticide Manual: A World Compendium, C. D. S.
Tomlin Ed., Published by the British Crop Production Council, which
is hereby incorporated by reference.
[0154] Some seed treatments that may be used on crop seed include,
but are not limited to, one or more of abscisic acid,
acibenzolar-S-methyl, avermectin, amitrol, azaconazole,
azospirillum, azadirachtin, azoxystrobin, bacillus spp. (including
one or more of cereus, firmus, megaterium, pumilis, sphaericus,
subtilis and/or thuringiensis), bradyrhizobium spp. (including one
or more of betae, canariense, elkanii, iriomotense, japonicum,
liaonigense, pachyrhizi and/or yuanmingense), captan, carboxin,
chitosan, clothianidin, copper, cyazypyr, difenoconazole,
etidiazole, fipronil, fludioxonil, fluquinconazole, flurazole,
fluxofenim, harpin protein, imazalil, imidacloprid, ipconazole,
isoflavenoids, lipo-chitooligosaccharide, mancozeb, manganese,
maneb, mefenoxam, metalaxyl, metconazole, PCNB, penflufen,
penicillium, penthiopyrad, permethrine, picoxystrobin,
prothioconazole, pyraclostrobin, rynaxypyr, S-metolachlor, saponin,
sedaxane, TCMTB, tebuconazole, thiabendazole, thiamethoxam,
thiocarb, thiram, tolclofos-methyl, triadimenol, trichoderma,
trifloxystrobin, triticonazole and/or zinc. PCNB seed coat refers
to EPA registration number 00293500419, containing quintozen and
terrazole. TCMTB refers to 2-(thiocyanomethylthio)
benzothiazole.
[0155] Seeds that produce plants with specific traits (such as
resistance to northern leaf blight) may be tested to determine
which seed treatment options and application rates may complement
such plants in order to enhance yield. Further, the good root
establishment and early emergence that results from the proper use
of a seed treatment may result in more efficient nitrogen use, a
better ability to withstand resistance to northern leaf blight and
an overall increase in yield potential of a plant or plants
containing a certain trait when combined with a seed treatment.
EXAMPLES
[0156] The following examples are offered to illustrate, but not to
limit, the claimed subject matter. It is understood that the
examples and embodiments described herein are for illustrative
purposes only and that persons skilled in the art will recognize
various reagents or parameters that can be altered without
departing from the scope of the appended claims.
Example 1
Phenotyping of Northern Leaf Blight Infection
[0157] Maize plants can be evaluated for Northern Leaf Blight (NLB)
on a 1 (highly susceptible) to 9 (highly resistant) scale, where
scores of 1-3 indicate "susceptible", scores of 4-6 indicate
"intermediate", and scores of 7-9 indicate "resistant". The scoring
diagram in FIG. 1 can be used as a guide, with an emphasis placed
on lesions above the ear. The lesions can be verified as being
caused by northern leaf blight infection by checking that the
lesions are cigar or boat-shaped with smooth sides and/or by
sending a sample to a diagnostic lab to confirm the identity of the
pathogen.
[0158] At two to four weeks after flowering, scores can be obtained
from a few known susceptible lines and then compared to their
historical scores. If the known susceptible lines rate at least two
scores higher than their historical scores, scoring of the lines in
the test set can be delayed, thereby allowing the disease to
advance to a standard state of infection. The scoring period can
only be extended until prior to plant senescence. Thus, if the
scores are still too high after 4-5 weeks, the disease pressure is
insufficient for effective scoring.
[0159] If scores from the known susceptible lines do correlate with
their historical scores in the time period from 2-4 weeks after
flowering until prior to plant senescence, the test lines can be
scored on a plot basis using the scoring diagrams in FIG. 1 as a
guide.
Example 2
Association Mapping Analysis
[0160] An association mapping strategy was undertaken to identify
maize genetic markers associated with resistance to Northern Leaf
Blight.
[0161] A collection of maize lines was analyzed using ILLUMINA.RTM.
SNP Genotyping (a 1536-plex assay). SNP variation was used to
generate specific haplotypes across inbreds for regions of the
genome. This data was used for identifying associations between
alleles and northern leaf blight resistance at the genome
level.
[0162] Resistance scores and genotypic information were
incorporated into an association mapping analysis. A
structure-based association analysis was conducted using standard
association mapping methods where the population structure is
controlled using marker data. Two chromosome 5 markers, PHM16750
and PHM15741, were significantly associated with the northern leaf
blight resistance trait in a non-Stiff Stalk subpopulation. In
addition, three chromosome 5 markers, PHM16854, PHM3870, and
PHM14018, were significantly associated with the northern leaf
blight resistance trait in a tropical subpopulation. Table 1
provides marker information for the chromosome 5 markers that
demonstrated linkage disequilibrium with the northern leaf blight
phenotype using the structured association mapping method.
TABLE-US-00001 TABLE 1 Maize markers significantly associated with
resistance to northern leaf blight infection in structured
association analysis Single meiosis IBM2 based genetic genetic map
map Marker Reference position position Name sequence Subpop P-value
(cM) (cM) PHM16750 SEQ ID NSS 5.60E-04 96.3 N/A NO: 1 PHM15741 SEQ
ID NSS 6.60E-04 96.7 289.3 NO: 2 PHM16854 SEQ ID Tropical 0.0038
86.8 257.8 NO: 3 PHM3870 SEQ ID Tropical 0.0046 91.7 271.5 NO: 4
PHM14018 SEQ ID Tropical 0.0047 96.1 289.3 NO: 5
[0163] In addition, an association analysis was performed on a set
of Argentine inbreds. This association analysis also identified
significant marker trait associations for subpopulation 2 in the
same interval of chromosome 5 (Table 2).
TABLE-US-00002 TABLE 2 Maize markers significantly associated with
resistance to northern leaf blight infection in an association
analysis performed on a set of Argentine inbreds Single meiosis
based IBM2 genetic Marker Reference genetic map map Name sequence
P-value position (cM) position (cM) PHM18056 SEQ ID 9.80E-05 87.1
N/A NO: 6 PHM3467 SEQ ID 2.26E-04 90 N/A NO: 7 PHM7958 SEQ ID
5.20E-05 105.2 307 NO: 8
[0164] The statistical probabilities that the marker allele and
phenotype are segregating independently are reflected in the
association mapping adjusted probability values in Tables 1 and 2,
which is a probability (P) derived from analysis of association
between genotype and phenotype. The lower the probability value,
the more significant is the association between the marker genotype
at that locus and the northern leaf blight infection tolerance
phenotype.
[0165] Note. The results shown in Tables 1 and 2 are based on two
independent sets of data. Table 2 shows the results from a single
set of data from Argentine inbreds.
Example 3
QTL Mapping Using Double Haploid Breeding Populations
[0166] A QTL interval mapping analysis was undertaken to identify
chromosome intervals and markers associated with northern leaf
blight resistance using a population of 186 doubled haploids
generated by a cross between PHBNB and PHFHH. Line PHBNB has
greater resistance to northern leaf blight infection than line
PHFHH. The doubled haploid lines generated from the cross were
phenotyped under natural northern leaf blight infection in a single
growing season and in two locations. Maize doubled haploid progeny
were genotyped using a set of 768 SNPs distributed in the maize
genome.
[0167] A significant peak was identified on chromosome 5, between
90 to 100 cM on the internally derived single meiosis based genetic
map, indicating that the region houses one or more QTL associated
with resistance to northern leaf blight. Using interval mapping
analysis, a number of markers showed association with the phenotype
at a confidence level of p <0.05. This finding concurred with
the other Examples, showing that the different approaches identify
the same region. Table 3 shows the genetic effects for the QTL on
chromosome 5, position 90-100 cM.
TABLE-US-00003 TABLE 3 Haplotypic effects for PHBNB .times. PHFHH
cross Haplotype Average of Chr5 90-100 cM NLFBLT n Favorable 5.78
138 (from PHBNB) Unfavorable 4.98 86 (from PHFHH)
EXAMPLE 4
High-Resolution Gene Mapping and Near Isogenic Lines and
Hybrids
[0168] High-resolution gene mapping by progeny testing of
homozygous recombinant plants was undertaken to further refine the
northern leaf blight resistance QTL. A mapping population was
created from the cross of PH890 RC1 and inbred PHBNB. Another
population for fine mapping was created from the cross of inbreds
PHFHH and PHBD6. The PH890 RC1.times.PHBNB population consisted of
94 BC.sub.5F.sub.3 families generated by selfing and fixing
selected recombinant BC.sub.5 plants from a total of approximately
3000 BC.sub.5 plants harbouring a heterozygous fragment at the
region from 90 to 105 cM on chromosome 5. This strategy permitted
coverage with recombinants of the whole QTL region. The
PHFHH.times.PHBD6 population consisted of 37 BC.sub.4F.sub.3
families generated by selfing and fixing selected recombinant
BC.sub.4 plants from a total of approximately 3000 BC.sub.4 plants
harbouring a heterozygous fragment at the region from 90 to 105 cM
on chromosome 5.
[0169] BC.sub.5F.sub.3 and BC.sub.4F.sub.3 near-isogenic lines
(NIL) harbouring allelic variation at the region of the preferred
markers were generated by marker assisted selection for both
crosses. The NILs were generated by introgressing the QTL region
from PHBNB or PHBD6 into recurrent parents, cleaning the genetic
background, and selecting specific recombinants at the region of
the preferred markers. By selfing individual
BC.sub.4F.sub.2/BC.sub.5F.sub.2 plants harbouring a heterozygous
fragment at the region of the preferred markers, negative and
positive near-isogenic lines were derived, and the QTL was treated
as a single Mendelian factor.
Phenotypic Scoring
[0170] Phenotypic scoring of each of the different families from
the cross involving PH890 RC1.times.PHBNB and from
PHFHH.times.PHBD6 cross, the parents of the crosses and the
generated NILs, was based on sets of phenotypic data collected from
the field (field experiments under natural infection; three
locations) obtained in one crop season.
Maize Genotyping
[0171] Maize BC.sub.5F.sub.3 progeny from the PH890 RC1.times.PHBNB
cross and BC.sub.4F.sub.3 progeny from PHFHH.times.PHBD6, the
parents of the crosses, and the generated NILs were genotyped using
polymorphic SNPs at the QTL region on chromosome 5.
[0172] Windows QTL Cartographer was used for both the marker
regression analysis and QTL interval mapping. LOD scores (logarithm
of the odds ratio) were estimated across the target regions
according the standard QTL mapping procedures.
[0173] Mean scores were used in QTL interval mapping. The LOD
threshold was 2.5. A confidence interval was estimated for each
QTL. As these populations were generated by marker assisted
selection (not random events of recombination), marker regression
analysis was considered as powerful as interval mapping
analysis.
Near Isogenic Lines and Fine Mapping
[0174] The near isogenic genetic materials harbouring allelic
variation at the region of preferred markers (93.3-96.8 cM on the
proprietary single meiosis based genetic map) showed a significant
difference in their response to the disease in both Argentina and
USA. Additional marker loci were evaluated in an increased number
of individuals in an effort to further narrow the QTL region. The
region housing the QTL was further refined to a region between and
including markers PZE-105068275 (reference sequence is represented
by SEQ ID NO:9) and PZE-105070525 (reference sequence is
represented by SEQ ID NO:17), which are located at 96.7 and 97.5
cM, respectively.
SNP Haplotype
[0175] Table 4 shows the genotype of SNPs at the region of
preferred markers for the favorable resistant haplotype.
TABLE-US-00004 TABLE 4 SNP Haplotypes Genetic Map Ref SNP Marker
Position SNP Seq Position PZE-105068275 96.67 G SEQ 51 ID NO: 9
PZE-105068432 96.72 A SEQ 51 ID NO: 10 PZE-105068572 96.78 C SEQ 51
ID NO: 11 SYN30642 96.79 T SEQ 61 ID NO: 12 PZE-105068746 96.84 C
SEQ 51 ID NO: 13 PZE-105069095 97.02 A SEQ 51 ID NO: 14
PZE-105069706 97.31 A SEQ 51 ID NO: 15 PZE-105069906 97.39 T SEQ 51
ID NO: 16 PZE-105070525 97.45 C SEQ 51 ID NO: 17
[0176] This present study has identified chromosome intervals and
individual markers that correlate with northern leaf blight
resistance. Markers that lie within these intervals are useful for
use in MAS, as well as other purposes.
Sequence CWU 1
1
171692DNAartificialPHM16750 Reference sequence 1ccccagcaac
tctttttatg acatgctgag agatctcgtg ggcttaacag atgtcacggg 60tatgactatt
atcagatggt cttacctttt ttttaggttg tataataaat cttgcaacta
120aaactggatg cctttctgac aagtgaaaag tgaaactgta cctagtttta
atcttcaatc 180gtgtctcttc taatcggtat gtgatatgtg cataacattt
atttttgcag tatgaaatac 240tgaataatct tcagaaagcg cagaagtcct
ttcatataga aaccgtacct ccattgaaac 300ttgcaccgcc agctatatac
catgagaaga tacagaagag gaccacttcg gttggtgaaa 360ctagcaaaca
ttctgttaga agtcagaaac cccagaaaat ttggcaaatg aaggagaaaa
420aatcaaaaga ggctgggagt catcatccac aaaagtcaag ttttctacgt
gagttcatat 480tccagtcact gacatcatag gcattaattt cactgtgctg
attcaattgt ggctttctgt 540ttgcctgctt acttcctttg atcctctgca
ggttaataga agaaaagatt gctgatttgt 600taagtatggt atgtagttca
agctttacgc cttcagtaat cttgcttgta tcgtgtggtt 660ttaaaatatg
cgatattttg ggaaaatgtc tt 6922654DNAartificialPHM15741 Reference
sequence 2cccggcccaa tttttaaacc ccgcgacttt tttaaacccc ctgctgtcag
cgcagcagca 60gcaggtggcg ggcctgtcca agttctgccg ctgctaccgg aactgctaca
ccgactgcag 120gaagtccacg ggccgctacc cctgcaacgc caactgcttt
caggactgca tcaacgggat 180gctgccgccg gctccggcgg aggtcgtcgt
ccccgccgac tgccgcgaca tctgcctcat 240gggcttctgc ggctccatgg
agatcgccgg tgacggtgag gccagctagc tagctaactt 300ctggacaata
atctgataga caggcatata tgcatgcatg catacttcag ttcgatagta
360taacctttcg atcatagatg aatgaaatct gactgcttcc tgtgaattaa
tgttgtattt 420cagccgtggc tggggatgcc gaggcgtgtg tggctgactg
caccaagaac ctcggtgcct 480ttgcaccaag tgcagccaag acgatcaact
gaagcatgca ttagggcccg ttcgcttgta 540caggattaaa ccggaattcg
ttccagctca tcaaaatcta tataaattaa agaagtaatc 600cggttaggaa
ttaattcgaa gctccaatcc ctaaaaaccg attagggccy tatg
6543502DNAartificialPHM16854 Reference sequence 3ccgagatctc
aagaatgaat cgaatctcag aatgacatca gggtaaagca gaccttgacg 60ccttgggtgt
ctcggtgatg ttggatggta ctgcatccgc tcaatcctgt gggctgttga
120ctatgagctg cctgagaccg tgatcgctca ccgccatcct gtcaagaacc
aggctggcgt 180gctccttgct tgcggtgcga ccttgtactg ggcagatggc
aagactgcaa acttcaactg 240ctcgtttctt gctaacctcg cctttgacgt
gaccgtctat ggcacaaacg gcactctcca 300tgtcactgac ctggtcattc
cgtacgaaga gagctctgcg gagttcagtg tggcctcgaa 360gtcaagcttc
gtcaaaccca ccatcggatg ggatccattg ccagagaagc atgttgttac
420tactgatctg ccacaggagg cactcatgat ccaggaattc acaaggcttg
tgcagaacgt 480atggtcatag ctgctctttc cc 5024400DNAartificialPHM3870
Reference sequence 4aamcacaaga cttgacacga agctgcagct gtctccgtgg
caaccaaaga tgtttgaggc 60aatgtgaaaa gagccatgac cggaggctac gtaggccaac
actatttcca aagatgcttg 120gcttgatgtg atggcttcaa gccagcgggc
ccatgtatca gcaccagcgc gcccaggttt 180ggaggatttt tgtggattta
gggcgcttct ggaagcaaag gtcaaatcgg tcgttgttga 240taatgtgatg
tactccctca gtctttttat ttttcatgtt ttagtataaa aatgaactaa
300caaacgacaa atattcgata acgaggtagt atctgtttgc cctgtgacgt
gtttaaaacc 360ggtgaatttg gggatatgga tggtcatagc tgttccttcc
4005683DNAartificialPHM14018 Reference sequence 5agcagccccc
atctttggac cggagtgtgg cgcttctcat cctgatactc ttggtgtgta 60cagcaacctt
gctgcaatct atgatgctat gggaaggtat gtgttgtatc ctgttcttct
120gcaaccttca gtcattatcc agaatccatg ctcatatgtc atatctctgt
ctgtgatgca 180attacatact tctagtctgt agtacagatg gatggtgtaa
acaaccacac atttaggatc 240tctaggtgtt attgatatgc cctaaggtat
atctgtatgt gcctcatcat ttacaacatt 300aagaactagg agagtaagaa
ggggcgctga gattttttcc tgctatctga agtggtaaat 360ttgttagatc
taattttttg ctgaatggct gtgggggttt gtgggttctg gatggttctg
420tgggttaagc tccttcttta gattatcctg tgtaagaaaa ttgctttgat
tttgattagg 480agaaactagt acctagattg gttgatgagt tacttaccag
atgatcattt ctggcatgca 540atgtcatgat ctatggtcca tggtgtaatt
gcccacatgt tctgtaccca actttggtac 600agttgcccat ttattcttgc
tttccaaact agagagtcct atgcccattt atcgatcctt 660taaagcttcg
tgctctctga agt 6836563DNAartificialPHM18056 Reference sequence
6agaaaaggcc aagtcacaaa gcttggtcga agcgctgatc ggccaaggcg cgcggcagat
60cgcgctgacc ggggcgccgc cggtggggtg cgtgccctcg cagcggcgca tcgccggcgg
120ggttcggatg cagtgcgcca cggaccgcaa ccagctggcg ctcttgttca
accggaagct 180gagcctggag gtggccaagc tgtccgggaa gtaccgcggc
gtcaacatct tctacgtcga 240cctctactcc gtcctcgccg acgtggtcca
gcgctaccag gctctcgggt tcaaggacgg 300caaggacgcc tgctgcggct
acgtcggcct ggcggtgggc ccgctctgca acatcggcag 360ccgtacctgc
ccggacccct ccaagtacgt gttctgggac agctaccacc ccaccgagag
420ggcgtacaag ctcatgatgg acgacttcct cacaagatac atgagataca
tacactagct 480agctagagct agtcgatccg ccatgttaat ttgcgcttta
attatagctt ttgctagccg 540ctcgctaata agggaattaa aat
5637475DNAartificialPHM3467 Reference sequence 7acgggacaaa
tcccagtcac aacagccggt gcgcgcgcga gagggatagc cgattatcat 60caagctctga
gctagcagag aagatgagcg catcgtcgtc catgaccaag aaggcgtcgt
120cgttcgtggt ggcggcgagc atgagcgcgg tggaggcgct caaggaccag
gcggggctgt 180gccggtggga ctacgcgctc cgctccctct acaaccgcgc
ggccgccgcc aataaggtcg 240tcgccggccg cgccgtcccg ttgtcgttgt
cgtcgtccca aaccgcgggt ggcagtggca 300gcgccgcggc cgccggtagg
gccgccaggc ccaggcgatc ggaggaggag aagatgcaca 360aggcgtacca
cctcgtctgc tggggcccta actaattgtt aggggcattc tcttattaat
420tactccttta tacacgctgt ccgattcctc cacttttttt taatttacgt taata
4758674DNAartificialPHM7958 Reference sequence 8tctcttctca
tttttgctka ttgyrtttcg gtatttccat ttttattact tctatggggt 60aggtcccagt
cacgacgagg tggtggtgga agagacgctc tgcttgtcgc agagccatgc
120cttcaaaggc gtgtgcctca gcaacaccaa ctgcgacaac gtatgcaaga
cggagaagtt 180cacaggcggc gagtgcaaga tggacggcgt catgcgcaag
tgctactgca agaaggtctg 240ctagggcatg accggcagca agccccagcc
gtacggctgg ttgatccggt tgcacactgc 300acagctcgtt tgggcacgcg
gtcatgttcc ggcttctcgg ctttatttat ttcttctttg 360ttataataaa
tagactctgt tagtcacgtg cgttttagtc tgggtcgtac gttattaatt
420ctctagtgta ttgtatttgc gcaacgcgcg ctgtacttaa cgtagccagc
attattcgcg 480taaaatgtaa taaaatctag ggactaaaca ttagtctcta
gaaacgagag acccccttat 540ttcttctttg ttataataaa tagactctgt
tagtcacgtg cgttttagtc tgggttgtac 600gttattaatt ctctagtgta
ttgtatttgc gcaacgcgct gtacttaacg tagccatgat 660tcgcgtgcta cctt
6749101DNAartificialPZE-105068275 Reference sequence 9tctcctcctg
gacaaacact ctgaattctg ttgtgtacac ggtgactact rctacattgt 60caggtaatcc
ggcaggttgt tggggcgcgc ccgacaaccc g
10110101DNAartificialPZE-105068432 Reference sequence 10aaaagagtct
atattcgagg cacctaaaat acaacagctt gatagctgcg rtttgctgcg 60actttaatct
agttgtagtt gtgttttcta cattatttta t
10111101DNAartificialPZE-105068572 Reference sequence 11caagcacgag
cacacaaagt aggagcatag caatgtacca agcaacaaaa yccttggcat 60gaagcaagca
cttgctgcca ggtagtaccc tatgtatgtg c 10112121DNAartificialSYN30642
Reference sequence 12gaccctttgg tcattggtaa agacactggg gaaaagccat
ggaacaagtg tccctactac 60ycccaaggtc aatccagtta tcgctccaat gatcacaagt
gacttcaata gcatccttgc 120c 12113101DNAartificialPZE-105068746
Reference sequence 13gcgaccagaa ctcggaagtc ggaacggcgc ggcggcggct
agctgatcaa yctcacaagc 60aaagctagtg ataagagagc cagagataaa ttaatcaggc
c 10114101DNAartificialPZE-105069095 Reference sequence
14tggatttcct ggatataaga atgaaaggaa gccatggccc acgaaacacg mgtccatccc
60tctctccggc ccggcccaca aactcacctc gtcatctccc c
10115101DNAartificialPZE-105069706 Reference sequence 15gttgactctg
ttttggccaa gtacctcgcg gcactatgca agagtgggaa matggaggcg 60gcatgcgaac
tgcctcatgt agcaagcagc aagagccatg t
10116101DNAartificialPZE-105069906 Reference sequence 16tctacctttg
aggcaaaacc tccaaggtca tggaagcttg cagctcctcg kcgtagaaag 60acacataaac
ctgatcaagc cttggttgtc cacaacctgg a
10117101DNAartificialPZE-105070525 Reference sequence 17cactatcctc
cgctccggtg agtagagcca actcgcctcc ttggatacct ytgggtaaat 60ggcagcgtgg
gctgcttcga aacgagctgg ttttgctaat a 101
* * * * *