DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Restriction/Election
In response to the communication received on March 30th, 2026, from Seiko Okada, the election of Group I, claims 1-2, 7, 19-20, 28 and 30, without traversal, is acknowledged. Applicants have further elected the species of Group I of the QTL Gm09_1786061.
Examiner notes that Applicants have further elected “deletion markers comprising deletion of at least a portion of a gene encoding a peroxidase set forth as Glyma.09G022300” (see, Remarks filed 03/30/2026, page 1, ¶2), which appears to be a clause in claim 19. This was not required in the election requirement of Group I, where the election was required only in claims 1 and 30 (see, Requirement for Restriction filed 01/30/2026, page 3, ¶2, Group I Note)
For clarity and upon further consideration and as a courtesy to applicant, the species of Group I, the QTLs and high-protein molecular markers, are rejoined.
Priority
Applicant’s claim for the benefit of prior-filed provisional application nos. 63/294,603 and 63/295,606, filed December 29th and 31st, 2021, respectively, and PCT/IB2022/062882, filed December 29th, 2022, under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged.
Thus, the earliest possible priority for the instant application is December 29th, 2021
Information Disclosure Statement
The information disclosure statements (IDSs) submitted on 06/27/2024 and 10/11/2024 were considered, initialed, and attached hereto. A signed copy of the list of references cited is included with this Office Action.
Status of Claims
Claims 1-2, 7, 19-20, 28, 30-33, 35, 41, 44-45, 53-56, 62-63 filed June 20th, 2025 are pending.
Claims 31-33, 35, 41, 44-45, 53-56, 62-63 are withdrawn.
Claims 1-2, 7, 19-20, 28 and 30 are examined herein.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (see pg. 20, ln. 12, for example). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Interpretation
Claim 1 recites a method of producing a population of “high-protein” soybean plants. A "high-protein soybean plant” as used herein refers to a soybean plant or soybean seed having greater seed protein content than a reference sample of soybean plant or seed [pg. 16, lns. 4-6].
Claim 1 recites at least one “high-protein molecular marker.” As in the instant specification, this is a term used to denote a nucleic acid or amino acid sequence that is sufficiently unique to characterize a specific locus on the genome [pg. 14, lns. 11-13].
Claim 1 recites that the at least one high-protein molecular marker is within 20 centimorgans of one or more high-protein QTL. Although cM is a relative term for the frequency of recombination, this is taken to be equivalent to around 8,740 kb for the purpose of examination1.
Claim 20 recites an “expression QTL”. The instant specification defines an eQTL as a QTL that is associated with differential expression of a gene [pg. 29, lns. 1-8]. In specific embodiments, when a QTL is present in the genome, a gene associated with the eQTL is has reduced expression. For example, the presence of an eQTL can eliminate or substantially elimination expression of a gene. In some embodiments, a gene encoding a peroxidase comprises a high-protein eQTL. Further, the instant specification states that FIG. 2 shows that the expression of the peroxidase gene is associated with the deletion marker of Chr9:17866061, thereby demonstrating the status of the deletion QTL as an expression QTL (eQTL), indicating that only proximity and trait expression are needed to show that a QTL is an expression QTL [pg. 11, lns 7-9].
Improper Markush Group
Claims 1-2, 7, 19-20, 28, and 30 are rejected on the basis that it contains an improper Markush grouping of alternatives. See In re Harnisch, 631 F.2d 716, 721-22 (CCPA 1980) and Ex parte Hozumi, 3 USPQ2d 1059, 1060 (Bd. Pat. App. & Int. 1984). A Markush grouping is proper if the alternatives defined by the Markush group (i.e., alternatives from which a selection is to be made in the context of a combination or process, or alternative chemical compounds as a whole) share a “single structural similarity” and a common use. A Markush grouping meets these requirements in two situations. First, a Markush grouping is proper if the alternatives are all members of the same recognized physical or chemical class or the same art-recognized class, and are disclosed in the specification or known in the art to be functionally equivalent and have a common use. Second, where a Markush grouping describes alternative chemical compounds, whether by words or chemical formulas, and the alternatives do not belong to a recognized class as set forth above, the members of the Markush grouping may be considered to share a “single structural similarity” and common use where the alternatives share both a substantial structural feature and a common use that flows from the substantial structural feature. See MPEP § 2117.
The Markush grouping of the QTLs listed in claim 1 and claim 30 is improper because the alternatives defined by the Markush grouping do not share both a single structural similarity and a common use for the following reasons: the claims encompass QTLs on different chromosomes and/or in variable regions within the chromosomes. It appears that some QTLs are constitute proper groupings, such as Gm09_1765195-Gm09_1818440, which fall within the same region of the same chromosome, chromosome 9. However, there is no conserved structure among all of the claimed QTL groups as they are spread across the entire soybean genome. For example, a QTL on the 9th chromosome as compared to a QTL on the 20th chromosome would have different associated SNP markers and favorable alleles to indicate the gene of interest to confer the high-protein trait to the soybean population. This is shown in Table 5, which displays high-protein markers and favorable alleles. Further, Table 6 provides the top genomic regions (QTL) associated with high protein trait in soybean plants. 16 loci are shown with a number of markers in each distributed across the chromosomes, indicating the variability of the markers clustered in each QTL grouping.
Even given the broad interpretation of 20 cM (see, claim interpretation), wherein the high-protein molecular markers may be up to 8,740 kb from the QTL of interest, the soybean (Glycine max L. Merr.) genome is 1.1-1.15 Gb in size1. High-protein molecular markers associated with the high-protein QTL Gm03_45228377 would not reasonably be within 20 cM of Gm20_3814870, for example. Thus, the structure and the distribution across the chromosomes is too great to be considered of a single structural similarity.
To overcome this rejection, Applicant may set forth each alternative (or grouping of patentably indistinct alternatives) within an improper Markush grouping in a series of independent or dependent claims and/or present convincing arguments that the group members recited in the alternative within a single claim in fact share a single structural similarity as well as a common use.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Indefiniteness
Claims 1-2, 7, 19-20, 28, and 30 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The claims are broadly drawn to a method of producing a population of high-protein soybean plants or seeds comprising genotyping a first population of soybean plants for the presence of at least one high-protein molecular marker that is within 20 cM of one or more high-protein QTLs as listed in claim 1 and high-protein QTLs listed in Table 5.
Claim 1 is rendered indefinite for indirectly relying on Table 5 of the instant specification [pg. 67-69] to define the high protein QTLs. The claims should recite the actual QTLs and high-protein molecular markers rather than relying on Table 5 of the instant specification to define the metes and bounds of the high-protein QTLs.
In claims 1 and 30, the recitation of high QTLs appears to be defining the chromosomal positions in relation to the Williams 82 reference genome assembly. Applicant recites that “in specific embodiments, all chromosomal positions listed herein are identified relative to the reference genome published as the Williams 82 reference genome assembly (Wm82.a2.v1)”, which represents the species Glycine max [pg. 29, lns. 9-13]. Thus, the reference to the chromosomal positions relative to the Williams 82 reference genome assembly are merely referred to as exemplary. Even without the exemplary reference in the instant specification, the Wm82.a2.v1 is a relative external reference framework, as opposed to a fixed sequence or objectively structured reference for determining the location of the QTLs and SNPs. Other version of the Williams 82 reference genome assembly might become available in the future, during the life of a possibly granted patent .The claims do not make clear how the recited coordinates are to be determined if the reference assembly were to be annotated, or if the positions were to vary across soybean germplasm.
The instant specification additionally states that there is a wide variety of genetic diversity in the wild perennial soybeans, but that the soybeans of the claims may be for any member of the genus glycine, in particular Glycine arenaria, Glycine argyrea, Glycine canescens, Glycine clandestine, Glycine curvata, Glycine cyrtoloba, Glycine falcate, Glycine latifolia, Glycine latrobeana, Glycine max, Glycine microphylla, Glycine pescadrensis, Glycine pindanica, Glycine rubiginosa, Glycine soja, Glycine stenophita, Glycine tabacina and Glycine tomentella. As the claims are directed to producing a population of soybean plants, without explicitly defining the germplasm source, it is not clear that one of ordinary skill in the art would be able to locate the QTLs or high protein molecular markers in any soybean plant with simply the reference to the Wm82.a2.v1 assembly.
Further, the Williams 82 reference genome assembly is an external reference updated periodically that has already undergone several updates. For example, the Wm82.a4.v1 assembly differs from the Wm82.a2.v1 by the inclusion of more RNA evidence for the new gene models and the inclusion of optical mapping to resolve structural variations between other assemblies. The assembly was further enhanced by the use of long PACBIO reads2.
Claim 1 does not provide any specific high-protein molecular markers or denote the allele associated with the high-protein phenotype. Although claim 1 recites that the QTLs may be selected from Table 5, Table 5 is a list of high-protein molecular markers and favorable alleles. Table 5 appears to be the most common markers with favorable alleles that contribute to 8.1% protein in ultra-high protein lines when in combination [pg. 66, lns 4-7]. It is not clear if claim 1 intends to claim all the markers of Table 5 in combination or in the alternative, which may not lead to a high-protein line. It is not clear if Table 5 is intended to be the high-protein molecular markers within 20 cM of the QTLs or more QTLs that are within 20 cM from high-protein molecular markers.
The recitation of “selecting from the first population one or more soybean plants or seeds comprising one or more high-protein alleles having the one or more high-protein molecular markers” is unclear. The instant claims do not recite any high-protein alleles linked to the high-protein molecular markers within 20 cM of the high-protein. As such, it is not clear to one of ordinary skill in the art what the subject matter of the invention is without the specific favorable alleles for the high-protein molecular markers.
Examiner notes that claiming with specificity the location, SNP or marker, and favorable allele of the high-protein QTL, with reference to an objective fixed sequence or bounded structure may help to overcome this rejection. Examiner notes that claim 7 is rejected as above. Although it recites with specificity the deletion and oligonucleotide probe for the deletion marker are claimed, it does not indicate an associated QTL.
Claim 30 recites the method of claim 1 wherein the method further comprises determining the protein content of soybean plants or seeds, wherein the second population of soybean plants or seeds have an increased level of protein when compared to a second population of soybean plants or seeds lacking one or more high-protein QTLs selected from the listed group. It is unclear if the Applicant intends to claim that the second population of soybean plants or seeds lacks the high-protein molecular marker within 20 cM of the high-protein QTLs and thus has lower protein content than a population that has the marker or if the Applicant intends to claim that the second population entirely lacks the QTL. The instant disclosure identifies a deletion marker from positions 17866061-1786148 on chromosome 9, which appears to comprise several of the high protein QTLs [Example 2]. A population entirely lacking these QTLs would likely not have lower protein content as the deletion of the positions is the catalyst for higher protein content.
Clarification is requested.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Written Description
Claims 1-2, 7, 19-20, 28 and 30 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claims are broadly directed to a method of producing a population of high-protein soybean plants or seeds, which comprises genotyping a first population of soybean plants or seeds for the presence of at least one high-protein molecular marker that is within 20 centimorgans of one or more high-protein QTLs selected from a group of QTLs across the soybean genome, selecting a soybean plant that has one or more high protein alleles having one or more high-protein molecular markers, and producing progeny soybean plants or seeds that comprises the one or more high-protein alleles having one or more high-protein molecular markers and are high-protein soybean plants or seeds.
The Applicant describes:
Identifying SNP markers associated with high-protein phenotype in soybean seeds.
Identifying a deletion marker associated with high-protein phenotype in soybean seeds associated with a peroxidase gene having a deletion from positions 1786061-1786147 and/or 1786062-1786148 which corresponds to a portion of the 5’UTR, signal peptide, start site, and a portion of exon 1 within peroxidase gene, Glyma.09G022300.
Plants having the deletion have significantly decreased expression of the Glyma.09G022300 peroxidase and higher protein by 1.5% as compared to the wild-type.
A unique combination of the 78 favorable alleles that contributes to 8.1% protein in ultra-high protein lines [pg. 66, lns. 4-7; Fig. 4E].
The Applicant does not describe:
The production of a population of high-protein soybean plants or seeds with higher protein content than a line without the markers.
Selection of a single high-protein molecular marker within 20 cM from at least one high-protein QTL conferring the high-protein phenotype introduced to a second population of progeny wherein the second population are high-protein soybean plants.
That genotyping to any high-protein molecular marker within 20 cM of one or more high-protein QTLs and producing a second population from the selected first population would result in a high-protein progeny.
That a soybean population would have an increased level of protein when compared to a second population of soybean plants or seeds lacking any one or more of the high-protein QTLs as recited in claim 30.
The instant disclosure does not describe the production of a population of high-protein soybean plants or seeds with higher protein content than a line without the markers. The instant disclosure does not reduce to practice a realized method of producing of a second population of progeny, wherein the progeny comprises one or more high-protein alleles having the one or more high-protein molecular markers. The instant disclosure provides broad exemplary breeding for the production of high-protein soybean plants [pg. 52 ln. 14-pg. 54, ln. 31], but does not provide specific examples using the molecular markers or QTLs of the claimed invention. Thus, clear methodology on producing a population of progeny soybean plants or seeds comprising the high-protein alleles having the one or more high-protein molecular markers and being high-protein populations is not provided. A sufficient number of species was not provided to support the genus of second progeny soybean plants and seeds that are high-protein.
The instant disclosure does not describe selection of a single high-protein molecular marker within 20 cM from at least one high-protein QTL conferring the high-protein phenotype introduced to a second population of progeny wherein the second population are high-protein soybean plants. The instant disclosure does not provide sufficient species to reduce to practice the genus claim of only one of the many listed molecular markers of claim 1 providing the high-protein phenotype. The presence of one marker may not indicate that the progeny has high protein as compared to one without the singular marker. The instant disclosure states that the ultra-high-protein line had a unique combination of 78 SNP markers to result in 8.1% protein [Table 5], but does not reduce to practice a line with high-protein and only one high-protein molecular marker.
In the art, reliability of selection has been demonstrated to be much greater when flanking markers are used over single markers. For example, Akhtar, S. et al. (2010, “Marker assisted selection in rice,” Journal of Phytology, 2(10):66-81) teaches that the use of flanking or intragenic markers will greatly increase the reliability of the markers to predict phenotype, displaying that recombination may occur in approximately 5% of the progeny using a single marker within 5 cM of a target locus, while the change of recombination between two markers, each around 5 cM away from the target locus, is approximately 0.4% [pg. 69, col. 2, ¶1; Fig. 1]. Similarly, one or many QTLs can influence a trait or a phenotype, as taught by Abiola, O., et al. (2003, “Complex Trait Consortium. The nature and identification of quantitative trait loci: a community's view”, Nat Rev Genet. 4(11):911-6) [pg. 2, ¶2]. Abiola further teaches when more than one QTL affects a particular trait, each might have a different effect size and the effects of individual QTLs will vary from strong to weak [pg. 2, ¶4]. The size and nature of these effects can also be influenced by the genetic background (the total genotype of the individual) and interactions between QTLs are common. Therefore, the instant disclosure fails to provide written description to support the structure of a singular marker or a single QTL reliably being diagnostic without the additional markers.
The instant disclosure states that with the high-protein molecular marker being a deletion marker denoting a deletion of positions 1786061-1786147 and/or 1786062-1786148, protein content was increased 1.5% [pg. 3, lns. 19-22]. The deletion is partially within a peroxidase gene, leading to the protein increase. In this case, a deletion marker would be required and must comprise a deletion of at least a portion of a gene, while SNPs are required for other markers. For example, a SNP at high-protein QTL Gm09_1786061 would still be a high-protein related molecular marker at one of the listed QTLs, but would likely not provide sufficient structure to perform the function of increasing protein content through deletion of part of a peroxidase gene.
The instant disclosure does not describe that genotyping to any high-protein molecular marker within 20 cM of one or more high-protein QTLs and producing a second population from the selected first population would result in a high-protein progeny. Applicants do not claim the necessary alleles associated with the particular high-protein QTLs. The QTLs are defined by chromosome number and bp position, presumably the bp position on the chromosome within a reference genome. Applicants do not disclose a conserved structure responsible with respect to the loci and alleles as to accomplish the instantly claimed function of high-protein soybean plants. As QTLs are determined by unique genetic architecture and are generally specific to a species, population, and even environment, sufficient structure is not provided to demonstrate to one of ordinary skill in the art that the Applicant was in possession of the invention as currently claimed.
The claims are directed to any unspecified high-protein marker of any marker type, as long as the marker is associated with a high-protein trait. Within 20 cM of one or more high-protein QTL defines a range as large as 40 cM which may or may not encompass the undefined markers of interest. The disclosure reduces to practice several anchor markers with listed neighboring markers. The high-protein markers with defined distances range from 0.003 cM to 7.11 cM from the anchor position. Although cM is a relative term, based on the claim interpretation above, 20 cM from a QTL may be 8,740 kb. This leaves a range as wide as 17,480 kb around the listed QTLs. High-protein molecular markers within a range this great are not reduced to practice, as the Applicant has only identified the SNP markers associated with the high-protein phenotype as shown in Table 2. Further, as shown detailed above, a SNP marker at the high-protein molecular marker loci Gm09_1786061, the denoted deletion marker comprising a portion of the peroxidase gene, would likely not have the same function given the difference in structure. There is a lack of predictability in genotyping the high-protein molecular markers as no favorable alleles were provided indicating the high-protein phenotype.
The instant disclosure does not describe that a soybean population would have an increased level of protein when compared to a second population of soybean plants or seeds lacking any one or more of the high-protein QTLs as recited in claim 30. Claim 30 recites the method of claim 1 wherein the method further comprises determining the protein content of soybean plants or seeds, wherein the second population of soybean plants or seeds have an increased level of protein when compared to a second population of soybean plants or seeds lacking one or more high-protein QTLs selected from the listed group. The instant disclosure reduces to practice a deletion marker associated with the high-protein phenotype in soybean seeds [Example 2]. The instant disclosure states that a region associated with high protein was identified that is associated with a peroxidase gene. The region from positions 1786061-1786148 on chromosome 9 was identified as having a deletion from positions 1786061-1786147 and/or 1786062-1786148, which corresponds to a portion of the peroxidase gene. The instant disclosure specifies that plants having the deletion have significantly decreased expression of the peroxidase and therefore have 1.5% higher protein content [pg. 65, lns. 9-10; Table 3].
It stands to reason that a population with this deletion (i.e., lacking one or more of the high-protein QTLs such as Gm09_1786061) would not have lower protein content than one without the deletion because the instant disclosure states that deletion of these positions within the region would lead to higher protein content. Thus, a second population of soybean plants or seeds lacking any one or more of the high-protein QTLs would not necessarily have lower protein content than a population of soybean plants or seeds that have the high-protein QTL.
One of ordinary skill in the art would not recognize that Applicant was in possession of the necessary common attributes or features of the broadly claimed genus in view of the disclosed species. Therefore, given the lack of written description in the specification with regard to the structural and functional characteristics of the compositions used in the claimed methods, Applicant does not appear to have been in possession of the claimed genus at the time of filing. Examiner notes that providing the specific favorable alleles associated with the high-protein trait and defining the high-protein molecular markers with more specificity would potentially overcome this rejection.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-2 are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Zhao-ming, Q. et al., “A Molecular Marker Related to High Protein Content of Soybean and Method for Identifying soybean with High Protein Content, CN 1133223399 A, published 08/31/2021 (see, IDS filed 10/11/2024; translation included in file wrapper).
Claim 1 recites A method of producing a population of high-protein soybean plants or seeds, said method comprising: a) genotyping a first population of soybean plants or seeds for the presence of at least one high-protein molecular marker that is within 20 centimorgans of one or more high-protein Quantitative Trait Locus (QTLs) selected from the group listed and high protein QTLs listed in Table 5; b) selecting from the first population one or more soybean plants or seeds comprising one or more high-protein alleles having the one or more high-protein molecular markers; and c) producing a second population of progeny soybean plants or seeds from the selected one or more soybean plants or plants grown from the selected seeds, wherein the second population of progeny soybean plants or seeds comprises the one or more high-protein alleles having the one or more high-protein molecular markers, and wherein the second population of progeny soybean plants or seeds are high-protein soybean plants or seeds, thereby producing a population of high-protein soybean plants or seeds.
Claim 2 recites the method of claim 1, wherein said at least one high protein molecular marker is within 10 centimorgans of said one or more high protein QTLs wherein the one or more high-protein molecular markers confer a yield penalty of less than 5% under normal growing conditions; wherein genotyping comprises assaying a single nucleotide polymorphism (SNP) marker; wherein genotyping comprises assaying for a deletion marker; wherein genotyping comprises the use of an oligonucleotide probe; and/or wherein genotyping comprises detecting a haplotype.
Zhao-ming teaches molecular markers related to soybean high protein content (i.e., at least one high-protein molecular marker) and methods for identifying soybean plants with high protein content to filter high-quality soybean varieties comprising high protein content [Abstract]. The nucleotide locus corresponding to SNP1 is Gm01-50861576; the nucleotide site corresponding to SNP2 is Gm6_44869874 and the nucleotide site corresponding to SNP3 is Gm14 to 16525645 [pg. 3, ¶3-5]. Zhao-ming teaches using molecular marker closely linked with the gene determining the target trait of high protein, selecting the marker to select the trait of interest, and selecting a soybean variety of high protein. Zhao-ming teaches a method of identifying soybean plants with high protein content by extracting the DNA, using a primer of the SNP marker to detect the marker, for example the SNP2 molecular marker may be detected as GG genotype if the variety has high protein content, and AA genotype if the variety has low protein content (i.e., genotyping a first population of soybean plants or seeds for the presence of at least on high-protein molecular markers and selecting from a first population one or more soybean plants or seeds comprising on ore more high-protein alleles having the one or more high-protein molecular marker) [pg. 3, end half of page].
Zhao-ming teaches that the SNPs can greatly improve breeding efficiency by selecting varieties of high protein to improve other soybean varieties (i.e., producing a second population of progeny soybean plants or seeds from the selected soybean plant, wherein the second population has the high-protein allele and molecular marker and wherein the second population are high-protein soybean plants) [Abstract].
Zhao-ming teaches SNP2 with the nucleotide site of Gm6_44869874 (i.e., wherein genotyping comprises assaying for a SNP marker). SNP2 is within 20 cM from QTLs Gm06_46486319-Gm06_48368151 of the instant application, based on the structure of the SNP location and the broad interpretation of 20 cM detailed above (see, claim interpretation).
As the high-protein alleles and the high-protein molecular markers are undefined in claims 1 and 2 of the instant application, Zhao-ming renders the claims anticipated.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-2, 28 and 30 are rejected under 35 U.S.C. 103 as being unpatentable over Hwang, E. et al. (2014), “A genome-wide association study of seed protein and oil content in soybean”, BMC Genomics, 15:1, in view of Humbert, S. et al., “Genome Edited Fine Mapping and Causal Gene Identification”, International Publication No. WO 2020/081173 A1, published April 23, 2020 (see, IDS filed 10/11/2024).
Claim 1 recites a method of producing a population of high-protein soybean plants or seeds, said method comprising: a) genotyping a population of soybean for at least one high-protein molecular marker within 20 centimorgans of one or more high-protein QTLs selected from the listed group and high protein QTLs listed in Table 5; b) selecting one or more soybean plants or seeds comprising one or more high-protein alleles having the one or more high-protein molecular markers; and c) producing a second population of progeny soybean plants or seeds from the selected soybean plants or plants grown from the selected seeds, wherein the second population of progeny soybean plants or seeds comprises the one or more high-protein alleles having the one or more high-protein molecular markers, and wherein the second population of progeny soybean plants or seeds are high-protein soybean plants or seeds, thereby producing a population of high-protein soybean plants or seeds.
Claim 2 recites the method of claim 1, wherein said at least one high protein molecular marker is within 10 centimorgans of said one or more high protein QTLs wherein the one or more high-protein molecular markers confer a yield penalty of less than 5% under normal growing conditions; wherein genotyping comprises assaying a single nucleotide polymorphism (SNP) marker; wherein genotyping comprises assaying for a deletion marker; wherein genotyping comprises the use of an oligonucleotide probe; and/or wherein genotyping comprises detecting a haplotype.
Claim 28 recites the method of claim 1 wherein the resulting population of high-protein soybean plants or soybean seeds comprises at least 40% protein by weight; and/or wherein the second population of progeny soybean plants or seeds further comprise one or more allele associated with high yield.
Claim 30 recites the method of claim 1, wherein the method further comprises determining the protein content of the second population of soybean plants or seeds, wherein the second population of soybean plants or seeds have an increased level of protein when compared to a second population of soybean plants or seeds lacking one or more QTLs selected from the listed group.
Regarding claim 1, Hwang teaches a genome-wide association study of seed protein in soybean to detect the location of genes or QTLs controlling seed protein in 298 soybean germplasm accessions [Abstract]. A total of 55,159 SNPs were genotyped and high associations to seed protein were mapped providing more precise marker-assisted allele selection. Hwang teaches that many seed protein QTL have been reported at many positions across the 20 soybean chromosomes/linkage groups in numerous studies [pg. 7, col. 1, ¶1]. Hwang teaches BARC_1.01_Gm09_4921120_C_T, a SNP associated with higher seed protein [Table 3].
According to the claim interpretation detailed above, 20 cM is around 8,740 kb or 8,740,000 bp. Thus, as no other identifier is given in the instant application to specify the high-protein molecular marker (SEQ ID No. or allele), Gm09_4291120 of Hwang is taken to read on any of the QTLs of claim 1 found on chromosome 9 in the instant application (i.e., a high-protein molecular marker that is within 20 cM of one or more high-protein QTL selected from the group consisting of …Gm09_1786061…).
Regarding claim 2, Hwang teaches that Gm09_4921120 is a SNP marker associated with seed protein (i.e., wherein genotyping comprises assaying a SNP marker) [Table 3].
Regarding claim 28, Hwang teaches that the mean protein content associated with the SNP allele is 42.15% dry weight for allele C (i.e., wherein the resulting population of high-protein soybean plants or soybean seeds comprise at least42% protein by weight) [Table 3].
Hwang further teaches that the chromosome regions defined in the study can be used for analysis to identify the causal gene as well as to identify DNA markers that can be used in selection to alter soybean protein in a predictable matter [pg. 9, col. 1, ¶1.
Hwang does not explicitly teach a method of producing a population of high-protein soybean plants or seeds comprising genotyping for the high-protein molecular marker, selecting a plant or seed with one or more high-protein allele having said marker, and producing progeny that are high-protein soybean plants or seeds.
However, Humbert teaches fine mapping of soybean high protein QTLs. Humbert teaches a method for fine mapping a desired trait comprising introducing a site-specific modification in a genomic locus in a plant, screening for the modification, and screening for an increase or decrease of the modification [claim 1]. Humbert teaches that the deletion, insertion, or polymorphism is different than the endogenous genomic sequence allele or locus to exhibit the desired trait [pg. 3, lns. 14-18]. Humbert teaches that the locus may be a known QTL [claim 5], that the plant has increased soybean protein concentration [claim 9], and that the modification is a deletion or SNP [claim 11 and 13]. Several methods are available for SNP genotyping, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, minisequencing and coded spheres [pg. 24, lns. 23-26].
Humbert teaches that 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 [pg. 26, lns. 7-17]. Plants with increased or decreased phenotype of the desired trait can be selected for by detecting one or more marker alleles, and in addition, progeny plants derived from those plants can also be selected. Hence, a plant containing a desired genotype in a given chromosomal region is obtained and then crossed to another plant. The progeny of such a cross would then be evaluated genotypically using one or more markers and the progeny plants with the same genotype in a given chromosomal region would then be selected.
Given that Hwang teaches Gm09_4921120, a high protein SNP with a high-protein allele C which is interpreted to be within 20 cM from the QTLs listed in claim 1; and given that Humbert teaches a method for fine mapping the high protein content trait in soybean, including genotyping for a SNP marker, selecting for the marker by detecting the allele, and producing progeny with the desired trait, it would have been prima facie obvious to one of ordinary skill in the art at the time of filing to use the high-protein SNP of Hwang in the method of Humbert to genotype for the SNP associated with high protein content in soybean seeds, select a plant or seed comprising the high-protein allele having the high-protein molecular marker and produce a second population comprising the marker and the desired phenotype of high protein content.
One would have been motivated to select the SNP on chromosome 9 of Hwang because Hwang specifies that the C allele at this position is associated with higher protein content, as well as higher oil content, and states that this is a QTL that should be of interest to those breeding for seed constituents [pg. 8, col. 2, ¶1]. One would have reasonable expectation of success in combining the teachings for a method of producing a soybean plant or seed with high-protein content as the SNP of Hwang was associated with 42.15% protein content (aligning with the instant application’s definition of high-protein soybean population) and Humbert teaches that 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 [pg. 26, lns. 7-10].
Hwang and Humbert do not explicitly teach that a second population of soybean plants with the high-protein QTLs as instantly claimed would have a higher protein content than a population lacking the high-protein QTLs. However, if a population were to lack high-protein QTLs on chromosome 9 and had lower protein content than one with the high-protein QLTs on chromosome 9, it would reasonably lack several high-protein alleles responsible for the high-protein phenotype. As Hwang specifies Gm09_4921120, a high protein SNP with a high-protein allele C which is interpreted to be within 20 cM from the QTLs listed in claim 1, a population lacking one or more unspecified high-protein QTLs on chromosome 9, as in the instantly claimed structure, may additionally lack Gm09_4921120 and thus have lower protein content, absent evidence to the contrary.
Claims 7, 19 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Hwang and Humbert as applied to claims 1-2, 28 and 30 above, and further in view of Gijzen, M., “Seed Coat Specific Nucleotide Sequence Encoding Peroxidase,” U.S. Patent No. US 7354390 B1, patented April 8, 2008.
Claim 7 recites the method of claim 2, wherein said oligonucleotide probe comprises SEQ ID NO: 4, and wherein said high-protein molecular marker is a deletion marker.
Claim 19 recites the method of claim 2, wherein said deletion marker comprises a deletion of at least a portion of a gene.
Claim 20 recites the method of claim 19, wherein said high-protein QTL is an expression QTL (eQTL); wherein said gene encodes a peroxidase; wherein said gene is Glyma.09G022300; and/or wherein said deletion is a deletion of positions GmO9 1786061-GmO9 1786147 or GmO9 1786062-GmO9 1786148.
As shown above, the combined teachings of Hwang and Humbert render obvious the method of producing a population of high-protein soybean plants or seeds comprising genotyping a population of soybean plants or seeds for the presence of at least one high-protein molecular marker that is within 20 cM of one or more high-protein QTL, selecting a soybean plant or seed with a high-protein allele having the molecular marker, and producing a high-protein soybean plant or seed.
Humbert teaches that the site-specific modifications may also include a deletion of a nucleotide, or of more than one nucleotide [claim 2; pg. 28, lns. 29-31]. The deletions may be encompassing the full region of interest or a subset of regions within the region of interest [pg. 58, lns. 24-29]. These smaller deletions may encompass targeted areas such as gene-rich regions, or regions containing clusters of disease resistance genes, or regions of major structural variation, or regions of higher gene expression. These deletions may be ranging from kbp to several Mbp. Humbert additionally teaches that 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 (i.e., wherein genotyping comprises the use of an oligonucleotide probe) [pg. 14, lns 10-12].
Hwang and Humbert do not teach that the deletion marker is detectable by an oligonucleotide probe comprising SEQ ID NO. 4 of the instant application, wherein said deletion marker comprises a deletion of at least a portion of a gene, and wherein said gene encodes a peroxidase.
However, Gijzen teaches a seed coat specific nucleotide sequence encoding peroxidase (i.e., wherein said gene encodes a peroxidase). Gijzen teaches that probes derived from the cDNA or genomic DNA can be used to detect polymorphisms that distinguish EpEp and epep genotypes (i.e., oligonucleotide probe) [Abstract]. The presence of a single dominant gene Ep causes a high seed coat peroxidase phenotype [¶5]. The peroxidase protein encoded by the Ep gene accumulates in the seed coat tissues [¶9]. Gijzen teaches an 87 bp deletion occurring in the ep allele that accounts for the drastically reduced amounts of peroxidase enzyme present in seed coats of epep plants since the deletion includes the translation start codon and the entire N-terminal signal sequence of the gene (i.e., wherein said deletion marker comprises a deletion of at least a portion of a gene) [¶39]. Gijzen teaches that seed-specific DNA regulatory regions can be used to control proteins that alter the nutritive protein value [¶49].
Gijzen teaches SEQ ID NO. 1, the cDNA encoding soybean seed coat peroxidase, and SEQ ID NO. 2, the genomic sequence. SEQ ID NO. 20 of Gijzen consists of nucleotides from SEQ ID NO. 2, but with the 87 bp deletion [¶7]. The OX347(Ep) sequence is defined by nucleotides 1513-1621 of SEQ ID NO. 2; OX342(ep) sequence defined by SEQ ID NO. 20 (nucleotides 1513-1624 of SEQ ID NO:2 but with deletion of nucleotides 1524-1610). SEQ ID NO. 20 is a 95% match to the instant SEQ ID NO. 4, the oligonucleotide probe for the deletion (see, alignment below). SEQ ID NO. 20 is missing the G nucleotide in position 1 to be a 100% match to SEQ ID NO. 4 of the instant application, however, position 1512 of SEQ ID NO. 2 (one nucleotide before the start of SEQ ID NO. 20) is a G nucleotide. Thus, the deletion may be detected with SEQ ID NO. 20 of Gijzen or NO. 4 of the instant application (i.e., wherein said oligonucleotide probe comprises SEQ ID NO. 4, and wherein the said high-protein molecular marker is a deletion marker).
Query Match 95.0%; Score 19; Length 22;
Best Local Similarity 100.0%;
Matches 19; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 2 TAAAATCATATCAGCTTAC 20
|||||||||||||||||||
Db 1 TAAAATCATATCAGCTTAC 19
Gijzen does not explicitly teach the location of the peroxidase gene, thus making it challenging to determine proximity to the high-protein QTLs. However, upon performance of a sequence search of SEQ ID NO. 2 of Gijzen in NCBI Blast, mRNA of Glycine max peroxidase (Ep) NM_001251386.1 specifies that the Ep peroxidase is found on chromosome 9 of Glycine max. Given that the genotyping of the instant application includes the use of an oligonucleotide probe with full identity to SEQ ID NO. 4, which is identical to SEQ ID NO. 20 (apart from one nucleotide) of Gijzen and given that the deletion of part of a peroxidase gene is found in chromosome 9, it appears that being within 20 cM of one or the high-protein QTLs is an inherent property of the deletion based on the structure, absent evidence to the contrary.
Given that Hwang and Humbert teach a method of producing a population of high-protein soybean plants or seeds comprising genotyping a population of soybean plants or seeds for the presence of at least one high-protein molecular marker that is within 20 cM of one or more high-protein QTL, selecting a soybean plant or seed with a high-protein allele having the molecular marker, and producing a high-protein soybean plant or seed; and given that Gijzen teaches a high-protein molecular marker that is a deletion with an oligonucleotide probe for deletion detection, and given that Gijzen teaches that the gene with a partial deletion encodes a peroxidase, the instant invention would have been prima facie obvious to one of ordinary skill in the art at the time of filing. One of ordinary skill in the art would have readily substituted the deletion marker in the teachings of Hwang and Humbert to generate a method of producing a population of high-protein soybean plants or seeds comprising genotyping a population for the presence of a deletion high-protein molecular marker with oligonucleotide probe SEQ ID NO. 20 of Gijzen, the deletion marker being for an 87 bp deletion occurring in the ep allele and deleting a portion of a peroxidase gene, accounting for reduced amounts of peroxidase enzyme present in seed.
The method further comprises selecting for the population with the deletion and further producing a second population that includes the allele and high-protein molecular deletion marker, as well as high protein over a control plant. One would have been motivated to use the deletion marker of Gijzen as Gijzen teaches that modification of peroxidase genes can contribute to overall nutritive protein in soybean plants. One would have reasonable expectation of success and predictability in selecting for the high-protein molecular marker as Gijzen teaches that the probes derived from the cDNA or genomic DNA can be used to detect polymorphisms that distinguish EpEp and epep genotypes, or the phenotype for the high protein trait. Humbert additionally teaches that the modification that passes the desired genotype may be a deletion and that 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.
Thus, the combined teachings of Hwang, Humbert and Gijzen render obvious the claimed invention.
Conclusion
No claims allowed.
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/EMILY K JOHNSON/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
1 Shultz, J. et al. (2006, “The Soybean Genome Database (SoyGD): a browser for display of duplicated, polyploid, regions and sequence tagged sites on the integrated physical and genetic maps of Glycine max”, Nucleic Acid Research, 34) teaches Genetic maps represent chromosomes as linkage groups with features (loci and markers) at centimorgan (cM) positions [pg. D760, col. 2, ¶1]. Physical maps represent chromosomes as contiguous stretches of DNA sequence with features at base pair positions. SoyGD 20 assumes that the 2512 cM genetic map of soybean encompasses its entire genome (1.1 Gb). The cM positions on the genetic map were converted to base pair positions on Gbrowse such that 1 cM is equivalent to 437 kb (i.e., 1 cM = 437 kb; 20 cM = 8,740 kb).
2 SoyBase, Genome Assembly Page, https://www.soybase.org/resources/genome_info/
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