SOYBEAN EVENT DP-305423-1 AND COMPOSITIONS AND METHODS FOR THE IDENTIFICATION AND/OR DETECTION THEREOF

• US 20080312082A1
• Filed: 10/30/2007
• Published: 12/18/2008
• Est. Priority Date: 10/31/2006
• Status: Active Grant

• Current Assignee: Corteva Agriscience LLC (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Stecca, Kevin L., Meyer, Knut, Kinney, Anthony J.
• Granted Patent: US 8,609,935 B2
• Time in Patent Office: Days
• US Class Current: 504/139
• CPC Class Codes: C12N 15/8247 involving modified lipid metabolism, e.g. seed oil composition
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae

Abstract

Compositions and methods related to transgenic high oleic acid/ALS inhibitor-tolerant soybean plants are provided. Specifically, the present invention provides soybean plants having a DP-305423-1 event which imparts a high oleic acid phenotype and tolerance to at least one ALS-inhibiting herbicide. The soybean plant harboring the DP-305423-1 event comprises genomic/transgene junctions having at least the polynucleotide sequence of SEQ ID NO:8, 9, 14, 15, 20, 21, 83 or 84. The characterization of the genomic insertion site of the DP-305423-1 event provides for an enhanced breeding efficiency and enables the use of molecular markers to track the transgene insert in the breeding populations and progeny thereof. Various methods and compositions for the identification, detection, and use of the soybean DP-305423-1 events are provided.

Claims

1. An isolated polynucleotide comprising SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

25. A DNA expression construct comprising the isolated polynucleotide of claim 1 operably linked to at least one regulatory sequence.

2. A soybean plant comprising SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

3. A soybean seed comprising SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

26. Transgenic progeny plants obtained from the transgenic seed of claim 3.

4. A method for identifying a biological sample comprising:

a) contacting said biological sample with a first and a second primer;

b) amplifying a polynucleotide comprising any of SEQ ID NO;

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88; and

c) confirming said biological sample comprises a polynucleotide comprising any of SEQ ID NO;

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

5. The method of claim 4, further comprising detecting a polynucleotide comprising SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88 by hybridization to a probe, wherein said probe hybridizes under stringent hybridization conditions with a polynucleotide comprising SEQ ID NO;

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

6. The method of claim 4, wherein said first or said second primer comprises a fragment of a 5′

genomic region or a 3′

genomic region of SEQ ID NO;

5, 6, 7 or 82.

7. The method of claim 4, wherein said first or said second primer comprises a fragment of an insert region of SEQ ID NO:

5, 6, 7 or 82.

8. The method of claim 4, wherein said first or said second primer comprises at least 8 consecutive nucleotides of a 5′

genomic region or a 3′

genomic region of SEQ ID NO;

5, 6, 7 or 82.

9. The method of claim 4, wherein said first or said second primer comprises at least 8 consecutive nucleotides of an insert region of SEQ ID NO:

5, 6, 7 or 82.

10. The method of claim 4, wherein one of said first primer or said second primer comprises a fragment of a 5′

genomic region of SEQ ID NO;

5, 6, 7 or 82 and wherein the other of said first or said second primer comprises a fragment of a 3′

genomic region of SEQ ID NO;

5, 6, 7 or 82.

11. The method of claim 4, wherein said first or said second primer comprise SEQ ID NO:

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 93 or 94.

12. A method of detecting the presence of a polynucleotide comprising SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88 in a biological sample comprising DNA, comprising;

(a) extracting a DNA sample from said biological sample;

(b) contacting said DNA sample with at least one pair of DNA primer molecules selected from the group consisting of;

i) the sequences comprising SEQ ID NO;

26 and SEQ ID NO;

27;

ii) the sequences comprising SEQ ID NO;

29 and SEQ ID NO;

30;

iii) the sequences comprising SEQ ID NO;

31 and SEQ ID NO;

32;

iv) the sequences comprising SEQ ID NO;

33 and SEQ ID NO;

34;

v) the sequences comprising SEQ ID NO;

35 and SEQ ID NO;

36;

vi) the sequences comprising SEQ ID NO;

37 and SEQ ID NO;

38;

vii) the sequences comprising SEQ ID NO;

39 and SEQ ID NO;

40;

viii) the sequences comprising SEQ ID NO;

41 and SEQ ID NO;

42;

ix) the sequences comprising SEQ ID NO;

43 and SEQ ID NO;

44;

x) the sequences comprising SEQ ID NO;

45 and SEQ ID NO;

46;

xi) the sequences comprising SEQ ID NO;

47 and SEQ ID NO;

48;

xii) the sequences comprising SEQ ID NO;

47 and SEQ ID NO;

49;

xiii) the sequences comprising SEQ ID NO;

50 and SEQ ID NO;

51;

xiv) the sequences comprising SEQ ID NO;

52 and SEQ ID NO;

53;

xv) the sequences comprising SEQ ID NO;

54 and SEQ ID NO;

49;

xvi) the sequences comprising SEQ ID NO;

55 and SEQ ID NO;

46;

xvii) the sequences comprising SEQ ID NO;

33 and SEQ ID NO;

56;

xviii) the sequences comprising SEQ ID NO;

57 and SEQ ID NO;

58;

xix) the sequences comprising SEQ ID NO;

59 and SEQ ID NO;

60;

xx) the sequences comprising SEQ ID NO;

61 and SEQ ID NO;

36;

xxi) the sequences comprising SEQ ID NO;

35 and SEQ ID NO;

62;

xxii) the sequences comprising SEQ ID NO;

37 and SEQ ID NO;

63;

xxiii) the sequences comprising SEQ ID NO;

64 and SEQ ID NO;

65;

xxiv) the sequences comprising SEQ ID NO;

66 and SEQ ID NO;

67;

xxv) the sequences comprising SEQ ID NO;

68 and SEQ ID NO;

69;

xxvi) the sequences comprising SEQ ID NO;

70 and SEQ ID NO;

71;

xxvii) the sequences comprising SEQ ID NO;

72 and SEQ ID NO;

73;

xxviii) the sequences comprising SEQ ID NO;

74 and SEQ ID NO;

75;

xxix) the sequences comprising SEQ ID NO;

76 and SEQ ID NO;

77;

xxx) the sequences comprising SEQ ID NO;

78 and SEQ ID NO;

79;

xxxi) the sequences comprising SEQ ID NO;

80 and SEQ ID NO;

81; and

xxxii) the sequences comprising SEQ ID NO;

89 and SEQ ID NO;

90;

(c) providing DNA amplification reaction conditions;

(d) performing said DNA amplification reaction, thereby producing a DNA amplicon molecule; and

(e) detecting said DNA amplicon molecule, wherein the detection of said DNA amplicon molecule in said DNA amplification reaction indicates the presence of a polynucleotide comprising SEQ ID NO;

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

13. A method of detecting the presence of SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88 in a biological sample, the method comprising;

(a) contacting the biological sample comprising DNA under stringent hybridization conditions with a polynucleotide probe wherein said probe hybridizes under stringent hybridization conditions with a polynucleotide comprising SEQ ID NO;

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88;

(b) detecting hybridization of the probe to the DNA.

14. The method of claim 13, wherein said sample comprises soybean tissue.

15. An isolated DNA primer comprising at least one sequence selected from the group consisting of SEQ ID NO:

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 93 or 94 or its complement.

16. A pair of DNA primers comprising a first DNA primer and a second DNA primer, wherein the DNA primers are of a sufficient length of contiguous nucleotides of SEQ ID NO:

5, 6, 7 or 82, to function as DNA primers diagnostic of DNA comprising SEQ ID NO;

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

17. A DNA probe wherein the DNA probe is of a sufficient length of contiguous nucleotides of SEQ ID NO:

5, 6, 7 or 82, to function as a DNA probe diagnostic of DNA comprising SEQ ID NO;

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

18. A method for screening seed for a polynucleotide comprising SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88, comprising;

a) contacting a sample comprising DNA from said seed with a first and a second DNA primer;

b) amplifying a polynucleotide comprising a polynucleotide comprising SEQ ID NO;

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88; and

c) detecting said amplified polynucleotide.

19. A method for screening seed for the presence of a polynucleotide comprising SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88 comprising;

(a) contacting a sample comprising DNA from said seed under stringent hybridization conditions with a polynucleotide probe that hybridizes under stringent hybridization conditions with a polynucleotide comprising SEQ ID NO;

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88; and

(b) detecting hybridization of the probe to the DNA.

20. A method of producing a high oleic acid and ALS inhibitor tolerant plant comprising breeding a plant comprising a polynucleotide comprising SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88, and selecting progeny by analyzing for progeny that comprise a polynucleotide comprising SEQ ID NO;

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

21. An isolated DNA sequence comprising at least one nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence set forth in SEQ ID NO;

26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 89, 90, 91, 92, 93 or 94; and

(b) a full-length complement of the nucleotide sequence of (a).

22. A pair of isolated DNA primer sequences, each comprising at least ten nucleotides and which when used together in a DNA amplification procedure will produce a DNA amplicon comprising a polynucleotide comprising SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

23. The pair of isolated DNA primer sequences of claim 22 wherein a first primer sequence is chosen from the group consisting of:

a) a 5′

genomic region of SEQ ID NO;

5, 6, 7 or 82; and

b) a 3′

genomic region of SEQ ID NO;

5, 6, 7 or 82; and

a second primer sequence is chosen from an insert region of SEQ ID NO;

5, 6, 7 or 82.

24. A method for controlling weeds in an area of cultivation comprising applying an effective amount of an ALS inhibitor to the area of cultivation comprising soybean plants comprising a polynucleotide comprising SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

32. A method of claim 24 further comprising applying an effective amount of at least one herbicide selected from the group consisting of inhibitors of Acetyl CoA carboxylase, inhibitors of Photosystem II, Photosystem I electron diverters, inhibitors of PPO, inhibitors of carotenoid biosynthesis, inhibitors of 4-HPPD, inhibitors of DHP, inhibitors of EPSP synthase, inhibitors of glutamine synthetase, inhibitors of DHP, inhibitors of microtubule assembly, inhibitors of mitosis/microtubule organization, inhibitors of cell division, inhibitors of cell wall synthesis, membrane disruptors, inhibitors of lipid synthesis by other than ACC inhibition, synthetic auxins, inhibitors of auxin transport, and other mechanism of action herbicides as described in Table 2.

27. A recombinant DNA construct comprising:

a first and second expression cassette, wherein said first expression cassette in operable linkage comprises;

(a) a soybean KTi3 promoter;

(b) a gm-fad2-1 fragment; and

(c) a soybean KTi3 transcriptional terminator; and

said second expression cassette comprising in operable linkage;

(i) a soybean SAMS promoter;

(ii) a soybean SAMS 5′

untranslated leader and intron;

(iii) a soybean gm-hra encoding DNA molecule; and

(iv) a soybean als transcriptional terminator.

28. A plant comprising the recombinant DNA construct of claim 27.

29. A plant of claim 28, wherein said plant is a soybean plant.

30. A seed comprising the recombinant DNA construct of claim 27.

31. A seed of claim 30, wherein said seed is a soybean seed.

33. A method for controlling weeds in an area of cultivation comprising applying an effective amount of at least one herbicide selected from the group consisting of acetochlor, acifluorfen and its sodium salt, aclonifen, acrolein (2-propenal), alachlor, alloxydim, ametryn, amicarbazone, amidosulfuron, aminopyralid, amitrole, ammonium sulfamate, anilofos, asulam, atrazine, azimsulfuron, beflubutamid, benazolin, benazolin-ethyl, bencarbazone, benfluralin, benfuresate, bensulfuron-methyl, bensulide, bentazone, benzobicyclon, benzofenap, bifenox, bilanafos, bispyribac and its sodium salt, bromacil, bromobutide, bromofenoxim, bromoxynil, bromoxynil octanoate, butachlor, butafenacil, butamifos, butralin, butroxydim, butylate, cafenstrole, carbetamide, carfentrazone-ethyl, catechin, chlomethoxyfen, chloramben, chlorbromuron, chlorflurenol-methyl, chloridazon, chlorimuron-ethyl, chlorotoluron, chlorpropham, chlorsulfuron, chlorthal-dimethyl, chlorthiamid, cinidon-ethyl, cinmethylin, cinosulfuron, clethodim, clodinafop-propargyl, clomazone, clomeprop, clopyralid, clopyralid-olamine, cloransulam-methyl, CUH-35 (2-methoxyethyl 2-[[[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)-oxy]-phenyl](3-fluoro-benzoyl)-amino]carbonyl]-1-cyclohexene-1-carboxylate), cumyluron, cyanazine, cycloate, cyclosulfamuron, cycloxydim, cyhalofop-butyl, 2,4-D and its butotyl, butyl, isoctyl and isopropyl esters and its dimethylammonium, diolamine and trolamine salts, daimuron, dalapon, dalapon-sodium, dazomet, 2,4-DB and its dimethylammonium, potassium and sodium salts, desmedipham, desmetryn, dicamba and its diglycolammonium, dimethylammonium, potassium and sodium salts, dichlobenil, dichlorprop, diclofop-methyl, diclosulam, difenzoquat metilsulfate, diflufenican, diflufenzopyr, dimefuron, dimepiperate, dimethachlor, dimethametryn, dimethenamid, dimethenamid-P, dimethipin, dimethylarsinic acid and its sodium salt, dinitramine, dinoterb, diphenamid, diquat dibromide, dithiopyr, diuron, DNOC, endothal, EPTC, esprocarb, ethalfluralin, ethametsulfuron-methyl, ethofumesate, ethoxyfen, ethoxysulfuron, etobenzanid, fenoxaprop-ethyl, fenoxaprop-P-ethyl, fentrazamide, fenuron, fenuron-TCA, flamprop-methyl, flamprop-M-isopropyl, flamprop-M-methyl, flazasulfuron, florasulam, fluazifop-butyl, fluazifop-P-butyl, flucarbazone, flucetosulfuron, fluchloralin, flufenacet, flufenpyr, flufenpyr-ethyl, flumetsulam, flumiclorac-pentyl, flumioxazin, fluometuron, fluoroglycofen-ethyl, flupyrsulfuron-methyl and its sodium salt, flurenol, flurenol-butyl, fluridone, fluorochloridone, fluoroxypyr, flurtamone, fluthiacet-methyl, fomesafen, foramsulfuron, fosamine-ammonium, glufosinate, glufosinate-ammonium, glyphosate and its salts such as ammonium, isopropylammonium, potassium, sodium (including sesquisodium) and trimesium (alternatively named sulfosate), halosulfuron-methyl, haloxyfop-etotyl, haloxyfop-methyl, hexazinone, HOK-201 (N-(2,4-difluorophenyl)-1,5-dihydro-N-(1-methylethyl)-5-oxo-1-[(tetrahydro-2H-pyran-2-yl)-methyl]-4H-1,2,4-triazole-4-carboxamide), imazamethabenz-methyl, imazamox, imazapic, imazapyr, imazaquin, imazaquin-ammonium, imazethapyr, imazethapyr-ammonium, imazosulfuron, indanofan, iodosulfuron-methyl, ioxynil, ioxynil octanoate, ioxynil-sodium, isoproturon, isouron, isoxaben, isoxaflutole, isoxachlortole, lactofen, lenacil, linuron, maleic hydrazide, MCPA and its salts (e.g., MCPA-dimethylammonium, MCPA-potassium and MCPA-sodium, esters (e.g., MCPA-2-ethylhexyl, MCPA-butotyl) and thioesters (e.g., MCPA-thioethyl), MCPB and its salts (e.g., MCPB-sodium) and esters (e.g., MCPB-ethyl), mecoprop, mecoprop-P, mefenacet, mefluidide, mesosulfuron-methyl, mesotrione, metam-sodium, metamifop, metamitron, metazachlor, methabenzthiazuron, methylarsonic acid and its calcium, monoammonium, monosodium and disodium salts, methyldymron, metobenzuron, metobromuron, metolachlor, S-metholachlor, metosulam, metoxuron, metribuzin, metsulfuron-methyl, molinate, monolinuron, naproanilide, napropamide, naptalam, neburon, nicosulfuron, norflurazon, orbencarb, oryzalin, oxadiargyl, oxadiazon, oxasulfuron, oxaziclomefone, oxyfluorfen, paraquat dichloride, pebulate, pelargonic acid, pendimethalin, penoxsulam, pentanochlor, pentoxazone, perfluidone, pethoxyamid, phenmedipham, picloram, picloram-potassium, picolinafen, pinoxaden, piperofos, pretilachlor, primisulfuron-methyl, prodiamine, profoxydim, prometon, prometryn, propachlor, propanil, propaquizafop, propazine, propham, propisochlor, propoxycarbazone, propyzamide, prosulfocarb, prosulfuron, pyraclonil, pyraflufen-ethyl, pyrasulfotole, pyrazogyl, pyrazolynate, pyrazoxyfen, pyrazosulfuron-ethyl, pyribenzoxim, pyributicarb, pyridate, pyriftalid, pyriminobac-methyl, pyrimisulfan, pyrithiobac, pyrithiobac-sodium, pyroxsulam, quinclorac, quinmerac, quinoclamine, quizalofop-ethyl, quizalofop-P-ethyl, quizalofop-P-tefuryl, rimsulfuron, sethoxydim, siduron, simazine, simetryn, sulcotrione, sulfentrazone, sulfometuron-methyl, sulfosulfuron, 2,3,6-TBA, TCA, TCA-sodium, tebutam, tebuthiuron, tefuryltrione, tembotrione, tepraloxydim, terbacil, terbumeton, terbuthylazine, terbutryn, thenylchlor, thiazopyr, thiencarbazone, thifensulfuron-methyl, thiobencarb, tiocarbazil, topramezone, tralkoxydim, tri-allate, triasulfuron, triaziflam, tribenuron-methyl, triclopyr, triclopyr-butotyl, triclopyr-triethylammonium, tridiphane, trietazine, trifloxysulfuron, trifluralin, triflusulfuron-methyl, tritosulfuron and vernolate to the area of cultivation comprising soybean plants comprising a polynucleotide comprising SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.

34. A method for controlling weeds in an area of cultivation comprising applying an effective amount of at least one sulfonylurea herbicide and at least one imidazolinone herbicide to the area of cultivation comprising soybean plants comprising a polynucleotide comprising SEQ ID NO:

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 82, 83, 84, 85, 86, 87 or 88.