US010011828B2
`
`a2) United States Patent
`US 10,011,828 B2
`(0) Patent No.:
`Jul. 3, 2018
`(45) Date of Patent:
`Hannappel
`
`(54) NON-DISRUPTIVE DNA ISOLATION FROM
`CORN SEEDS
`
`(58) Field of Classification Search
`None
`See application file for complete search history.
`
`(71) Applicant: Syngenta Participations AG, Basel
`(CH)
`
`(56)
`
`(72)
`
`Inventor: Ulrich Stephan Hannappel, Slater, 1A
`(US)
`
`(73) Assignee: Syngenta Participations AG, Basel
`(CH)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`7,832,143 B2
`2006/0035227 Al*
`
`11/2010 Deppermannetal.
`2/2006 Minobe...........06.. C12Q 1/6895
`435/6.11
`
`FOREIGN PATENT DOCUMENTS
`
`(*) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`US.C. 154(b) by 127 days.
`
`WO
`WO
`
`2007103786 A2
`2011119763 Al
`
`9/2007
`9/2011
`
`(21) Appl. No.:
`
`—-14/890,955
`
`OTHER PUBLICATIONS
`
`Junior et al Chapter 18 “DNA Extraction from Seeds”. in Evolu-
`(22) PCT Filed:|May 28, 2014
`tionary Genetics and Molecular Biology. 2016.
`( M. Micic (ed.)
`(Springer Science + Business Media, New York, USA), p. 265-
`276.*
`Von Post et al. Euphytica. 2003. 130: 255-260.*
`International Search Report for International Patent Application No.
`PCT/EP2014/061036 dated Aug. 11, 2014.
`Wuet al., “Rapid and Reliable Purity Idnetification of Fl Hybrids
`of Maize (Zea may L.) Using SSR Marker,” Maize and Genomics
`and Genetics, 2010, vol. 1, pp. 1-4.
`
`(86) PCT No.:
`
`PCT/EP2014/061036
`
`§ 371 (€)(),
`(2) Date:
`
`Nov. 13, 2015
`
`(87) PCT Pub. No.: WO2014/195199
`
`PCT Pub. Date: Dec. 11, 2014
`
`(65)
`
`Prior Publication Data
`
`* cited by examiner
`
`US 2016/0115472 Al
`
`Apr. 28, 2016
`
`Related U.S. Application Data
`
`(60) Provisional application No. 61/830,242, filed on Jun.
`3, 2013.
`
`(51)
`
`(2006.01)
`(2018.01)
`(2006.01)
`(2018.01)
`
`Int. Cl.
`CI2N 15/10
`C120 168
`AOIH 1/04
`C120 1/6895
`(52) U.S. Cl.
`CPC veces CI2N 15/1003 (2013.01); AOLH 1/04
`(2013.01); C12Q 1/6895 (2013.01); C120
`2600/13 (2013.01); C12Q 2600/156 (2013.01);
`C120 2600/172 (2013.01)
`
`Primary Examiner — Carla J Myers
`(74) Attorney, Agent, or Firm Christopher Leming
`
`(57)
`
`ABSTRACT
`
`This invention relates to systems and process for isolating
`DNAfrom biological materials such as seeds while retaining
`a viable seed for further use. The seed from which the DNA
`is isolated remains viable and is used or discarded based on
`
`the DNAanalysis of the seed soak solution. The seed soak
`solution can have substantially all of the confounding mater-
`nal DNA from the seed eliminated from the seed soak
`solutions by employing intact seed pretreatments. This
`method is particularly useful for maize seed.
`
`6 Claims, 16 Drawing Sheets
`(16 of 16 Drawing Sheet(s) Filed in Color)
`
`Inari Exhibit 1072
`Inari Exhibit 1072
`Inari v. Pioneer
`Inari v. Pioneer
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`

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`U.S. Patent
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`Sheet 1 of 16
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`Sheet 2 of 16
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`Figure 2
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`Sheet 3 of 16
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`
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`Aibde¥Robottest
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`&
`43
` 10
`Skate X Rar test 4}
`
`Figure 3
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`

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`Sheet 4 of 16
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`US 10,011,828 B2
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` 45
`
`sa
`Sdlele % Pode test Ty
`
`BD
`
`we
`
`
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`Siete.¥Podetest
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`Bt
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`Figure 4
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`

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`Sheet 5 of 16
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`Afistic Liscrimination Plot
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`Stele X Robot teet 45
`
`A
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`Bika:¥GataLest2}
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`Figure 5
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`

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`Sheet 6 of 16
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`Allele X (Robot test 1)
`
`
`ao
`
`an
`
`
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`idle¥(Rubottest2)
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`Figure 6
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`

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`Sheet 7 of 16
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`
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`Riese¥Robottest3
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`Alkebs % Mudatest 1}
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`3B.
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`|
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`Figure 7
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`

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`Allele X (Robot test 14
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`a8
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`4.0
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`Figure 8
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`_fillelic Discrimination Plot.
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`Allalte¥(Rabottest2}
`
`
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`

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`Sheet 9 of 16
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`an Sieh: K (Rabidtest 4)
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`Figure 9
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`
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`Bilele¥Rabattast5
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`

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` Figure 10
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`Sheet 11 of 16
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`Figure 11
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`Sheet 12 of 16
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`
`
`Mieke¥abottent25
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`
`
`
`
`4
`$8
`Bkale X Robot test 4)
`
`8
`
`Figure 12
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`

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`Sheet 13 of 16
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`
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`Pele¥(Robottegt2)
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` 3.0
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`Sele X Moher tos 3}
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`Figure 13
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`Sheet 14 of 16
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`Marker
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`
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`Allslic Discriraination Pint
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`Sheet 15 of 16
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`BReticDiscriminationMat
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`Figure 15
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`

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`Sheet 16 of 16
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`Figure 16B
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`US 10,011,828 B2
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`1
`NON-DISRUPTIVE DNA ISOLATION FROM
`CORN SEEDS
`
`RELATED APPLICATION INFORMATION
`
`This application is a 371 of International Application No.
`PCT/EP2014/061036,filed 28 May 2014, which claims the
`benefit of U.S. Provisional Patent Application No. 61/830,
`242, filed 3 Jun. 2013, the contents of which are incorpo-
`rated by reference herein.
`
`FIELD OF THE INVENTION
`
`This invention relates to systems and process for isolating
`DNAfrom biological materials such as seeds while retaining
`a viable seed for further use. The seed from which the DNA
`is isolated remains viable and is used or discarded based on
`
`15
`
`the DNA analysis of the seed soak solution. The seed soak
`solution can have substantially all of the confounding mater-
`nal DNA from the seed eliminated from the seed soak
`
`20
`
`solutions by employing intact seed pretreatments. This
`methodis particularly useful for maize seed.
`
`BACKGROUND OF THE INVENTION
`
`Commercial seed industry research on plants results in
`genetic improvements in seed. A numberofdifferent pro-
`cesses can result in improved seeds, haploid/double haploid
`systems, traditional or genotype assisted breeding, mutation,
`transformation, etc. The improved seed when genetically
`stable is increased so that there is a large quantity of the
`improvedseed for sale to growers. In the process of increas-
`ing lines or finding and developing stable lines the seed is
`planted and harvested and seed is selected over several
`seasons. Until the plant has a fixed and stable inheritable
`desired improvement, not all seeds express the improve-
`ment. In the breeding process seeds without the improve-
`ment from the population are removedprior to planting the
`next generation of seed. A statistical samplings which are
`tested for determining if the improvementis in the seed is
`used when bulking the seed population. The results of the
`test determine if there is seed that should be removed
`
`because they lack the improvement. This process lacks
`efficiency because it often results in loss of some desired
`seed and some maintenance of some undesired seed.
`To overcomethe deficiencies of statistical sampling, the
`seed industry has developed systems and methods of non-
`destructively sampling material from seeds for testing. Prin-
`cipally, the goal of these methods is to remove a portion of
`a seed, the ‘seed chip’ which is used for testing. The ‘seed
`chip’ or seed sample, often has extracted DNA analyzed for
`the desired seed characteristics. Depending on the seed
`sample test results, the correlated viable seed is used for
`planting or discarded.
`Efficiencies in producing the seed chips and viable
`chipped seeds have been the focus of the development of
`mechanical devices for automating the process. Two types of
`automated seed “chipping” devices have been developed.
`Each have a device for removing material from a seed, one
`uses a brocadeandthe other uses a laser. These devices have
`
`a seed conveyer for conveying the seed to a compartment in
`a seed tray; and a sample conveyor for conveying the
`material removed from the seed to a corresponding com-
`partment in a sample tray. The sample seed is used for DNA
`extraction which is tested. The viable seed is stored. And
`
`each process correlates the seed chips with the viable seed
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`2
`from which it was chipped. The chip is used for testing and
`the seed is discarded, stored or used for planting.
`In operation these sample tray and seed tray locations
`have to be associated so that when the seed samples are
`tested, the test results can be employed to select or sort the
`seeds. These devices are set upto test large quantities of seed
`which results in two sets of tray storage, two sets of tray
`documentation, and two sets of locations that must be
`coordinated so that the association between the two sets
`
`sample seed and viable untested seed is maintained.
`The prior art methods and devices for the automated
`system require two essential seed parts, the viable seed for
`planting and the seed sample, in other words, the chip for
`testing. The chip provided the seed DNA for testing.
`Although these different automated systems have different
`meansfor positioning seeds for cutting, for cutting the seed
`chip from the seed, for transporting seed and chip, etc., both
`of these systems are burdensome and complex. These seed
`chipping systems require a constant correlation to be kept
`between the two essential seed parts. The existing automated
`systemsrequire a tray of seed material anda tray of the seed
`chips.
`These systems require the use of two seed parts, each
`which are stored and handled separately but with a known
`association. This process requires that the corresponding
`chip and seed are correctly matched so that the test results
`for the chip will result in the removal of the correct unde-
`sired seed material. The present invention addresses the
`complexity of needing two essential seed parts and the need
`to track both the seed tray and the chip tray which cause a
`numberof the existing complexities of the prior inventions.
`
`SUMMARY OF THE INVENTION
`
`The present invention does not require two essential seed
`parts to be tracked. In one embodiment the methodof the
`present invention is developed for analyzing a population of
`seeds. In one embodiment, the seeds are corn seeds. The
`method has steps of exposing the seed endosperm, from
`individual seeds in a population of seeds while preserving
`germination viability of the seeds, and soaking the viable
`exposed seed in a non-disruptive DNAreleasing or isolating
`solution thereby forming a seed solution from each of the
`individual seeds in the population and analyzing the seed
`solution for the presence or absence of one or moretraits of
`interest.
`In one embodiment of the invention,
`the non-
`disruptive DNA releasing or isolating solution is an alkali
`solution. The alkali solution in some instances is sodium
`
`hydroxide, or potassium hydroxide,or other alkali solutions.
`Additionally, the method can also have a pretreatment
`step that decreases the maternal DNAthatis released into the
`seed solution. The pretreatment can be at least one soaking
`of the intact seed, prior to the exposure of the seed’s
`endosperm to encourage the release of the maternal DNA
`from the outer portion of the seed.
`In one embodiment, the pretreatment can be a pretreat-
`mentof the seed to inhibit the release of the maternal DNA.
`The pretreatment can be with an alkali solution or it can be
`a seed coating or spray on the outer portion of the seed. In
`one embodiment,
`the pretreatment can be spraying with
`metallic paint.
`The method can also include the step of using a prior art
`device to expose the seed endosperm. In one embodiment,
`the tip of the seed is removed. In one embodiment, the
`device used is a laser. In one embodiment, the device used
`
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`US 10,011,828 B2
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`3
`is a knife. In one embodiment, the device used is a razor
`blade. In one embodiment, the device used is an automated
`seed cutting system.
`The exposure can therefore be formed by the removal of
`a small portion of the seed, or by a tunnel through the seed’s
`endosperm, or by cutting a wedge portion out of the seed
`leaving a portion of the endosperm exposed. In one embodi-
`ment, floury endosperm is exposed.
`There is provided a method for analyzing a population of
`seeds: the step of pretreating at least once, an intact seed of
`the population forming an intact seed solution,
`thereby
`reducing the presence of maternal DNA from the outer
`portion of the exposed seed in the seed soak solution. Hence,
`in one embodiment, the amount of DNA released into the
`soaking solution can be reduced by a “two step” soaking
`approach. In one embodiment, the amount of pericarp (ma-
`ternal) DNA from the outer portion of the seed released into
`the second soaking solution is significantly lower. In one
`embodiment, only the allele present in the endosperm tissue
`is detectable in the second soak solution.
`
`The method for analyzing a population of seeds wherein
`the non-disruptive solution is a non-disruptive DNAreleas-
`ing or isolating solution. Such a non-disruptive DNAreleas-
`ing or isolating solutionis an alkali solution. The method can
`employ a sodium hydroxide solution. Alternative solutions
`include other alkali solutions such as potassium hydroxide,
`and the like. The seeds remain viable at all times.
`In one embodiment, the seeds are removedanddried after
`soaking in solution. In one embodiment, the seeds can then
`be stored for at least one week. In one embodiment, DNA
`which is released into the soaking solution can be concen-
`trated using isopropanol.
`In some embodiments the seed soak solution is used for
`
`determining the genotypic character or the phenotypic char-
`acter of the seeds in the population with the seed solution
`isolated from the exposed viable seed.
`The method includes diagnosing if one or more seeds
`from the population exhibits the presence or absence of the
`one or moretraits of interest; and (optionally) sorting seeds.
`The isolated DNA in the seed soak solution can be tested for
`
`one or moretraits of interest comprising a genetic marker, a
`single nucleotide polymorphism, a simple sequence repeat,
`a haplotype andthelike.
`In yet another embodiment the method for analyzing a
`population of seeds has one or more undesirable traits of
`interest as the traits of interest. In this embodiment the
`
`method has a step of sorting one or more seeds from the
`population of seeds based on the presence or absence of the
`one or more undesirable traits of interest, and discarding or
`retaining the sorted seeds.
`Another embodiment is a bulked population of viable
`seeds, wherein substantially all of the seeds in the population
`havea portion of exposed seed endosperm wherein substan-
`tially all of the seeds in the population have the presence or
`absenceofat least one targeted trait in common,and wherein
`the presence or absence of the at least one targeted trait in
`the seeds in the population is determined by analyzing the
`seed solution from the viable seed. Additionally this seed
`solution is not formed from a separate seed sample taken
`from the viable seed, but instead the seed solution is from the
`seed itself.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`The patentor application file contains at least one drawing
`executed in color. Copies of the patent or patent application
`
`4
`publication with color drawing(s) will be provided by the
`Office upon request and paymentof the necessary fee.
`FIG. 1: Depiction of a corn seed with the seed tip removed
`from seed like the 12 seeds in Example 1. The seed has been
`and soaked in NaOH. The tip (not pictured) has been
`discarded.
`FIG. 2: Depiction of plants which have germinated from
`the seeds of Example 1.
`FIG. 3: Allelic discrimination plot assay 1 of Example 1,
`DNAobtained from seeds. The arrow is marking the mis-
`matched sample. The *X’s in the upperleft are alleles Y (not
`undetermined) and ‘X’s in the lowerright are alleles X (not
`undetermined). The Y-axis reads “Allele Y (Robot test 2)”
`and the X-axis reads “Allele X (Robottest 1)” in the graph
`of FIG. 3, and in all the other figures with allelic discrimi-
`nation plots.
`FIG.4: Allelic discrimination plot assay 1, DNA extracted
`from leave tissue. The ‘X’s in the upperleft are alleles Y (not
`undetermined) and ‘X’s in the lowerright are alleles X (not
`undetermined).
`FIG. 5: Allelic discrimination plot, DNA obtained from
`soaking solution. Each sample tested with two DNA con-
`centrations.
`FIG. 6: Allelic discrimination plot assay 1, (blue dots:
`DNAobtained from first soaking solution, red dots: DNA
`obtained from second soaking solution, each sample tested
`with two DNA concentrations. Points on the plot to the left
`of the added dotted line correspond to blue dots. Points on
`the plot to the right ofthe added dotted line correspondto red
`dots.
`FIG. 7: Allelic discrimination plot assay 1. Blue dots:
`DNAobtained from first soaking solution, red dots: DNA
`obtained from second soaking solution. Each sample tested
`with two DNA concentrations. Points on the plot to the left
`of the added dotted line correspond to blue dots. Points on
`the plot to the right of the added dotted line correspondto red
`dots.
`FIG. 8: Allelic discrimination plot, assay 1. DNA tem-
`plate: DNA obtained from the first soaking solution. Each
`sample tested with 2 DNA concentrations. The sample with
`circles and marked with the arrows are allelic mismatch
`
`between first and second soaking solution.
`FIG. 9: Allelic discrimination plot, assay 1. DNA tem-
`plate: DNA obtained from the second soaking solution. Each
`sample tested with 2 DNA concentrations. The sample with
`circle dots and marked with arrows are allelic mismatch
`between first and second soaking solution.
`FIG. 10: Depiction of seeds before planting.
`FIG. 11: Germinated seeds shown in FIG. 10.
`FIG. 12: Allelic discrimination plot, assay 1. DNA
`obtained from seeds (second soaking solution).
`FIG. 13 shows an allelic discrimination plot, assay 1,
`DNAextracted from leave tissue.
`FIG. 14: Allelic discrimination plot, assay 2, DNA
`obtained from seeds (second soaking solution).
`FIG. 15: Allelic discrimination plot, assay 2, DNA
`extracted from leave tissue.
`
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`FIGS. 16A & B: Depiction of the germinated seeds which
`were sprayed as a pretreatmentto lessen isolation of mater-
`nal DNA.
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`60
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`DETAILED DESCRIPTION OF THE
`INVENTION
`
`65
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`Seed breeding has been made more efficient by proce-
`dures that allow the plant breeder to more clearly understand
`the genetic material and the allelic variants that are present
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`US 10,011,828 B2
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`5
`or absent from breeding material. Genetic understanding of
`plants is gathered through analysis ofeither the plant’s DNA
`or the DNA of the seed from the plant. The seed isolated
`DNAcanbe used to screen seeds for the presence or absence
`of the desired characteristics for the breeding populations.
`The present invention is a method of isolating DNA from
`seeds, by soaking seeds in an alkali solution to solubilize
`DNA,primarily from an exposed inner seed portion of the
`floury endosperm into the seed soaking solution forming a
`seed solution and a viable soaked seed with some endosperm
`exposed. The seed solution is mainly comprised of seed
`DNAfrom the endosperm of the soaked seed. The soaked
`seed are dried and the seed remains viable.
`The seed solution will contain DNA. The DNAis
`
`obtained from the endosperm of the seed by soaking
`exposed seeds in an alkali solution, such as NaOH. Other
`alkali solutions can also be usedin this process. The simple
`test for use of other solutions is to determine if the seed is
`
`soakedin the solution is the DNA quality and quantity useful
`for the desired testing and is the viability of the seed
`retained.If the solution provides these two parameters then
`the solution can be used. In the present embodimentof the
`invention the NaOH concentration is kept
`low and the
`soaking time is kept short, then the viability of soaked seeds
`is maintained.
`
`The exposed seed is soaked and the seed solution will
`contain endosperm DNA and maternal DNA from theperi-
`carp. Many of the tests that use the seed solution do not
`require the presence of maternal DNA. The amount of
`pericarp DNA (maternal tissue) can be reduced by pre-
`soaking intact seeds in alkali before exposing parts of the
`endosperm tissue.
`Seed Priming
`Seed priming is a technique of controlled hydration
`(soaking in water or other solution) and drying that results
`in more rapid germination when the seeds are reimbibed.
`Seeds are soaked in a solution which disrupts cells and
`solubilizes DNA into the solution. Seeds are removed from
`the soaking solution, dried and stored. Solubilized DNA can
`be used to screen stored seeds for the presence ofallelic
`variants. Based on the screening results, stored seeds can be
`selected and planted.
`Procedure
`Soaking seeds in a mild NaOH solution (2 steps):
`Step 1: Soaking seeds in NaOHto disrupt pericarp cells
`to release maternal DNA.
`(Soaking solution to be dis-
`carded).
`Top of the seeds is removed(e.g. razor blade, knife, laser
`etc.) to expose the floury endosperm.
`Step 2: Soaking seeds in NaOH to solubilize DNA
`(mainly) from the floury endosperm into the soaking solu-
`tion.
`After the soaking (with or without agitation, vortexing
`etc.) seeds are removed from the soaking solution, dried and
`stored. DNA from the soaking solution can be cleaned
`and/or concentrated to serve as templates for e.g. molecular
`markers.
`Noseed chips haveto be collected. The complexity of the
`two seed system is eliminated. The one seed system can be
`easily automated.
`The seed soak solution of the present disclosure is used
`for testing seed for a genetic or chemical trait while pre-
`serving the germination viability of the soaked seed. The
`results of the testing may be diagnostic for the trait, and
`allows the seeds to be sorted, screened, discarded or
`selected. The viable seed can be planted and new seeds can
`
`6
`be harvested from the plant and the seed soaking protocol
`can be performed on the new seed.
`inbred,
`One embodiment of the seed is a maize seed,
`hybrid, haploid, doubled haploid, transformed, mutatedetc.
`Any seed can be employed in the protocol if it remains
`viable after the exposure of the endosperm to the soaking
`protocol. The seed which can be used are grain seeds like
`maize, wheat, rice and the like, oilseeds, or vegetable, fruit
`or flowers seed that can maintain viability after soaking, and
`provide DNA quality and quantity needed for the testing.
`In addition, DNA in the seed soak solution may be
`amplified using suitable amplification method amplification
`is knownand products are commercially available for DNA
`amplification. The DNA is screened for genetic marker
`which maybe associated with selection of QTL, haplotypes,
`alleles, or genes. Genetic markers include butare notlimited
`to single nucleotide polymorphisms,
`simple sequence
`repeats and the like. Testing can employ DNA and RNA
`sequence material, promoters, genes, untranslated regions of
`genes, satellites, chips with markers, transcription profiles,
`methylation patterns, alleles and the like.
`Thetesting is often looking for agronomictraits like yield,
`emergence,
`lodging, height, maturity, disease resistance,
`pest resistance. This testing can also identify resistance or
`susceptibility to biotic and abiotic stresses, resistance to
`herbicides, and morphological characteristics. The testing
`can diagnose the presence or absenceofothertraits for food
`or industry uses. The testing can be used to detect the
`zygosity of the embryo,or the presence of a transgene. The
`testing can be usedfortrait introgression,or purity or ploidy
`testing.
`Selection of a breeding populations can be initiated based
`onthetesting results. After the seed solution is formed from
`the soaked seed, the soaked seed is dried. The dried soaked
`seeds can be bulked with all desired seed of the population
`planted in a breeding nursery or separated with the specific
`seeds being identified when planted. Seed selection and the
`numberof cycles of breeding depends on the trait and the
`breeding methods employed.If the seed being selected is an
`inbred then the steps of the breeding process may include
`crossing the selected seed with another inbred to form
`hybrid seed. Depending on the desired test the soaking
`protocol can be used on seeds with germplasm including
`hybrid seed as well. Maize seedis particularly useful for this
`soaking protocol.
`The identification of the tested viable seed allows for a
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`reduced use of time, energy and effort by the breeder.
`Additionally, it results in less land use or more populations
`on the same land.
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`The following examples serve to illustrate the invention
`but should not be regarded as limiting the scope of the
`invention.
`
`EXAMPLES
`
`Example 1
`
`Comparison of Allelic Profiles from Seed and Leaf
`DNA
`
`A total of 12 corn seeds had their tips removed with a
`razor blade to expose floury endosperm. The seeds were
`placed into 500 ul of 20 mM NaOH for 2 hours, with
`constant vortexing (FIG.1).
`After two hours, seeds were removed and dried at 30 C in
`an incubator, then stored at room temperature for one week.
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`DNAwhich wasreleased into soaking solution was concen-
`trated via isopropanol precipitation.
`Seeds were planted into single pots in the greenhouse. All
`seeds germinated and produced plants (FIG. 2). At the 3
`leaves stage, leaf tissue was harvested for DNA isolation.
`DNAfrom soaking solution and from leaf tissue was used to
`run molecular markers (Taqman assays). Allelic calls from
`seed DNA and leaf DNA were compared.
`Results:
`
`Assay 1: Allele calls from seed and leaf DNA matched for
`all 12 plants (FIG.4).
`Assay 2: Allele calls from seed and leaf DNA matched for
`11 plants (FIG.3),
`One mismatch was observed in which the locus was
`
`called heterozygous for seed DNA, homozygous for leaf
`DNA
`
`Explanation: Allelic difference between endosperm tissue
`and pericarp tissue (maternal tissue) DNA.
`Conclusions: DNA can be obtained from an alkali solu-
`tion in which seeds were soaked; seeds can be dried and
`stored; seeds are viable; and pericarp DNA might cause
`mismatches between allelic calls from seeds and plants.
`
`Example 2
`
`Release of Pericarp DNA into Soaking Solution
`
`The purpose of the next experiment was to show that a
`significant amount of DNAis released from pericarp tissue
`into the NaOH soaking solution.
`12 Corn seeds(tip not removed, i.e. endosperm tissue not
`exposed) were placed in 500 ul 20 mM NaOH.
`The tubes were agitated for 2 hours at room temperature;
`After two hours, seeds were removed from the soaking
`solution and released DNA wasconcentrated via iso-
`
`propanol concentration; and
`DNA obtained from soaking solution was used to run
`assay 1
`The results are shown in FIG. 5, which showsanallelic
`discrimination plot of DNA obtained from soaking solution.
`Each sample was tested with two DNA concentrations.
`The conclusion from this experiment was that a signifi-
`cant amount of DNA from pericarp tissue is released into the
`soaking solution.
`
`Example 3
`
`Reduction in Amountof Isolated Pericarp DNA
`
`The purpose of the next experiment wasto test a method
`which should reduce the amountof isolated pericarp DNA.
`12 corn seeds (tips not removed, i.e. endosperm tissue not
`exposed) were placed in 500 ul of 20 mM NaOH;
`The tubes were agitated for 1 hour at room temperature;
`After 1 hour seeds were removed into a second tube
`
`containing 500 ul of 20 mM NaOHandagitated for 1
`hour;
`After 1 hour, the seeds were removed from the second
`tube;
`Released DNA from the first and second soaking was
`concentrated via isopropanolprecipitation.
`Results
`
`FIG. 6 showsthe results of the experiment of Example 3.
`It showsthe allelic discrimination plot assay 1, (blue dots:
`DNA obtained from first soaking solution, red dots: DNA
`
`8
`obtained from second soaking solution) Each sample tested
`with two DNAconcentrations.
`
`Example 4
`
`Further Reduction in Pericarp DNA
`
`The purpose of the next experiment was the sameas for
`experiment 3 but with modifying the soaking procedure to
`reduce isolated pericarp DNA further.
`12 Cornseeds(tips not removed,i.e. endosperm tissue not
`exposed) were placed in 500 ul of 20 mM NaOH;
`Tubes were agitated for 1.5 hours at room temperature;
`After 1.5 hours seeds were removed into a second tube
`
`containing 500 ul of 10 mM NaOHandagitated for 0.5
`hours;
`After 1 hour seeds were removed from the secondtube;
`Released DNA from the first and second soaking was
`concentrated via isopropanol precipitation.
`The results of experiment 4 are shown in FIG. 7, which
`shows an allelic discrimination plot assay 1. Blue dots
`indicate DNA obtained from first soaking solution. The red
`dots indicate DNA obtained from second soaking solution.
`Each sample tested with two DNA concentrations.
`The conclusion of experiments 3 & 4 are that the amount
`of DNAreleasedinto the soaking solution can be reduced by
`a “two-step” soaking approach
`
`Example 5
`
`Increased Efficiency of Two-Step Soaking Process
`
`The purpose of the next experiment was to measure the
`effect of the 2 step soaking approach on DNAisolation from
`seeds on predictive accuracy.
`12 Seeds (floury endosperm not exposed) were soaked in
`20 mM NaOH for 1.5 hours (vortexing) at room
`temperature;
`Seeds were removed from the soaking solution, briefly
`dried and the floury endosperm of each seed was
`exposed by removing the outer seed tissue of the seed
`tip with a razor blade. Seeds were then placed into a
`second tube containing 500 ul 10 mM NaOH and
`vortexed at room temperature for 0.5 hours;
`Seeds were removed, briefly rinsed in 0.5 mM Tris and
`water, dried overnight at 30 C in an incubator and then
`stored at room temperature for one week;
`DNAreleased into the first and second soaking solution
`was concentrated via isopropanol precipitation.
`Results are shown in FIG. 8 and FIG.9.
`
`FIG. 8 is showing an allelic discrimination plot, assay 1.
`The DNA template used was DNA obtained from the first
`soaking solution. Each sample tested with 2 DNA concen-
`trations. The sample with circles and marked with the arrows
`are allelic mismatch betweenfirst and second soaking solu-
`tion. FIG. 9 is showing an allelic discrimination plot, assay
`1. DNA template: DNA obtained from the second soaking
`solution. Each sample tested with 2 DNA concentrations.
`The sample with circle dots and marked with arrows are
`allelic mismatch betweenfirst and second soaking solution.
`Conclusion from experiment 5: Additional allele from
`pericarp tissue which is not present in endosperm tissue is
`released into the soaking solution. Amountof pericarp DNA
`released into soaking solution 2 is significantly lower, only
`the allele present in the endosperm tissue is detected.
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`Example 6
`
`Comparison of Allelic Calls from Seed DNA and
`Leaf Tissue DNA
`
`Purpose: Comparing allelic calls from DNA obtained
`from seeds (2 step soaking approach) and the DNAisolated
`from leaf tissue of germinated seeds.
`12 Seeds from experiment 5 were planted into single pots
`in a green house. The soaked and dried seeds without
`the seed tops exposing the endosperm. See FIG. 10. All
`seeds germinated. See FIG. 11.
`Results FIG. 12 shows an allelic discrimination plot,
`assay 1. DNA obtained from seeds (second soaking solu-
`tion). And FIG. 13 shows an allelic discrimination plot,
`assay 1, DNA extracted from leave tissue.
`FIG. 14 showsan allelic discrimination plot, assay 2,
`DNA obtained from seeds (second soaking solution). And
`FIG. 15 showsanallelic discrimination plot, assay 2, DNA
`extracted from leaf tissue.
`Final Results: Allelic calls match of seed DNA and leaf
`
`DNA match for both assays tested.
`Conclusion from experiment 6: Allelic calls of plants can
`be accurately predicted by soaking seeds in alkali before
`planting.
`
`Example 7
`
`Germination Capabilities of Pretreated Seed
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`To prevent release of pericarp DNA into soaking solution
`the seed without
`the endosperm being exposed can be
`pretreated. One pretreatment is by spraying seeds with a
`solution before seed cutting and soaking. Below is an
`example: seeds were sprayed with magnetic paint before
`removing the top of seeds. Sprayed seeds did germinate in
`paper towel.
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`The invention claimed is:
`1. A method for analyzing a population of seeds, the
`method comprising:
`(a) exposing, the seed endosperm from individual seeds in
`a population of seeds while preserving germination
`viability of the seeds, thereby obtaining at least one
`viable seed with exposed endosperm;
`(b) soaking the at least one viable seed with exposed
`endosperm in a non-disruptive DNA-releasing alkali
`solution thereby forming a seed soak solution compris-
`ing DNA; and
`(c) analyzing the seed soak solution for the presence or
`absence of DNAindicative of the presence or absence
`of one or moretraits of interest.
`2. The methodfor analyzing a population of seeds accord-
`ing to claim 1, comprising prior to step (a): the step of
`pretreating, at least once, individual seeds in the population
`of seeds to reduce the presence o