Microsatellite DNA markers and uses thereof

Details Microsatellite DNA markers and uses thereof Microsatellite DNA markers and uses thereof

1999-01-19

2004-05-11

Whisenant, Ethan (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C436S094000, C536S023100, C536S024300, C536S024330

Reexamination Certificate

active

06733965

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to identification and isolation of the simple sequence repeat (SSR) loci in the higher eukaryotes, such as the plants, and particularly the pines. The SSR loci of the invention are particularly useful as genetic markers for genetic mapping, population genetics studies and inheritance studies in various plant breeding programs.
BACKGROUND OF THE INVENTION
Loblolly pine (
Pinus taeda
L.) is an important, experimental and commercial forest tree species native to the southeastern United States. Loblolly pine is planted extensively in the southeastern United States and to lesser degrees in other warm temperate regions of the world. In the United States, plantations are managed and utilized for a variety of products including raw materials (wood, fiber, and chemicals), ecosystem components (wildlife habitat and water and soil conservation), and recreational activities. Most of planting stock originates from production seed orchards established by various loblolly pine improvement programs. To date, such programs have completed one to three cycles of selection using progeny testing for parental selection and seed orchard development, and family and within-family testing and selection for population improvement. Loblolly pine breeding has various limitations, such as, long generation times to flower (>5 years) and harvest (>15 years), low tolerance to inbreeding, large size of individual trees, variable sites for testing and replanting, difficulty of vegetative propagation, low heritability of important traits, and uncertainty of trait values.
Marker-assisted selection (MAS) using DNA-based markers has much potential for improving the efficiency and effectivenes of tree breeding programs (O'Malley and McKeand 1994
For. Genet
. 1:207-218.). Important improvements afforded by MAS include reducing the time-to-selection and improving the accuracy of selection. An important goal of such research is to identify DNA markers or other measures that predict performance of mature trees. With this information, tree breeders could more confidently select trees at an early age, induce them to flower, and breed them to produce the next generation. In addition, selections made at an early age could be vegetatively propagated in mass using rooted cutting or tissue culture based technologies (Bradshaw and Foster 1992
Can. J. For. Res
., 22:1044-1049.). Vegetative propagation and deployment has the potential to greatly improve wood and fiber yield and quality by capturing within-family genetic variation and providing better performing varieites for plantation establishment.
Several of the fundamental limitations to MAS applications in loblolly pine (Strauss et al. 1992
Can. J. For. Res
., 22:1050-1061.) have been overcome in recent years. Most notably is the application of randomly-primed, PCR-based genetic markers (e.g., RAPD) to parent- or family-specific genome mapping (Tulsieram et al. 1992
, Biotechnology
, 10:686-690; Nelson et al 1994
Journal of Heredity
, 85:433-439; Plomion et al. 1996
Theor. Appl. Genet
., 93:1083-1089., Wilcox et al. 1996
Proc. Natl. Acad. Sci. USA
, 93:3859-3864.). Although family-specific mapping and MAS approaches have potential, these methods are limited to situations where small breeding (<10 parents) populations are maintained with progeny established in large-family (n>500) tests. In practice, however, most loblolly pine breeding programs do not fit this situation. More typical is large breeding populations, sometimes several populations per program, and always relatively small-family (n<150) progeny tests. In addition most programs now include many pedigrees of at least three-generations, with nearly mature third-generation trees in the field. Utilizing existing extensive pedigree and progeny test information is essential for developing better MAS technology and improving breeding programs.
Currently available marker systems are not optimal for detecting QTL variation across families and across multi-generation pedigrees. Reviews of current marker technologies and their limitations to use in QTL mapping and MAS is provided by Neale and Harry (1994
AgBiotech News Info
., 6:107N-114N.) and O'Malley and Whetten (1997
Molecular markers and forest trees. DNA Markers: Protocols, Application and Overviews
ed. G. Caetano-Anollés and P. M. Gresshoff. John Wiley and Sons, New York., 237-257.). Given a genome size of about 2000 cM(K) for loblolly pine, a large number of highly polymorphic, co-dominant genetic markers will be needed for genome mapping and QTL analyses (Echt and Nelson 1997
Theor. Appl. Genet
., 94:1031-1037.).
Accordingly, there is a need in the art for new genetic markers. In an effort to develop such markers for loblolly pine, the pines and the plants in general, the present inventors developed simple sequence repeat (SSR) markers described herein. The markers of the invention are also useful for other eukaryotic organisms.
SUMMARY OF THE INVENTION
Simple sequence repeats (SSRs), which are also known as microsatellite DNA repeats, have now been discovered in the pines and have been shown to exhibit length polymorphisms. These repeats represent an abundant pool of potential genetic markers.
Accordingly, in one aspect, the present invention relates to the plant SSR motifs, such as for example, di-, tri- and tetra-nucleotide repeated motifs.
In another aspect, the invention relates to the polynucleotides containing one or more such SSR motifs and the primers for the amplification of the fragments containing SSRs. The primers may be cloned polynucleotide fragments or chemically synthesized oligonucleotides, and contain at least a portion of the non-repeated, non-polymorphic sequence fang SSRs on either 5′ or 3′ end.
The present invention is also directed to a kit for the rapid analysis of one or more specific DNA polymorphisms of the type described in this application The kit includes oligodeoxynucleotide primers for the amplification of fragments containing one or more SSR sequences.
In a further aspect, the invention provides for a method of analyzing one or more specific SSR polymorphisms in an individual or a population, which involves amplification of small segment(s) of DNA containing the SSR and non-repeated flanking DNA by using the polymerase chain reaction, and sizing the resulting amplified DNA, preferably by electrophoresis on polyacrylamide gels.
In yet another aspect, the invention provides for a method of determining the sequence information necessary for primer production by isolation and sequencing of DNA fragments containing the SSRS, using hybridization of a synthetic, cloned, amplified or genomic probe, containing sequences substantially homologous to the SSR, to the DNA.
In a further aspect, the present invention is directed to a method for detecting the presence of a specific trait in a subject, such as a plant. The method includes isolating the genomic DNA from the subject individual and analyzing the genomic DNA with a polymorphic amplified DNA marker containing one or more SSR sequences.
In yet another aspect, the SSR markers of the invention are used in commercial plant breeding. Traits of economic importance in plant crops can be identified through linkage analysis using polymorphic DNA markers.
DETAILED DESCRIPTION OF THE INVENTION
All patents, patent applications and references cited in this specification are hereby incorporated herein by reference in their entirety. In case of any inconsistency, the present disclosure governs.
Definitions
The following terms and phrases are used throughout the specification with the following intended meanings.
The abbreviation “SSR” stands for a “simple sequence repeat” and refers to any short sequence, for example, a mono-, di-, tri-, or tetra-nucleotide that is repeated at least once in a particular nucleotide sequence. These sequences are also known in the art as “microsatellites.” A SSR can be represented by the general formula (N
1
N
2
. . . N
i
)
n
, wherein N represents nucleotides A, T, C or G, i rep

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