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WO2025006547A2 - Variants de mrp chez brassica - Google Patents

Variants de mrp chez brassica Download PDF

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Publication number
WO2025006547A2
WO2025006547A2 PCT/US2024/035538 US2024035538W WO2025006547A2 WO 2025006547 A2 WO2025006547 A2 WO 2025006547A2 US 2024035538 W US2024035538 W US 2024035538W WO 2025006547 A2 WO2025006547 A2 WO 2025006547A2
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Prior art keywords
seq
mrp2
mrp
variant
variants
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WO2025006547A3 (fr
Inventor
Janel M BETTIS
Beverly KREJSA
Thomas G Patterson
Kevin G Ripp
Jinrui Shi
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Pioneer Hi Bred International Inc
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Pioneer Hi Bred International Inc
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
    • A01H6/202—Brassica napus [canola]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/10—Processes for modifying non-agronomic quality output traits, e.g. for industrial processing; Value added, non-agronomic traits

Definitions

  • sequence listing is submitted electronically as an xml- formatted sequence listing file named 96770-US-PSP ST26 created on June 29, 2023, and having a size of 267,241 bytes which is filed concurrently with the specification.
  • sequence listing comprised in this xml-formatted document is part of the specification and is herein incorporated by reference in its entirety.
  • the disclosures relates to phosphorous metabolism in Brassica, variants of MRP genes that reduce phytate and increase inorganic phosphate in Brassica grain and meal made thereof.
  • B. napus is one of the most important vegetable oilseed crops in the world, especially in China, Canada, the European Union and Australia.
  • Canola meal the fraction of the seed remaining after crushing and oil extraction, is approximately 55% of the volume of canola seed.
  • canola meal is rich in protein and capable of providing a substantial amount of energy when used in animal feed; it also includes levels of anti -nutritional factors such as glucosinolates, tannins, phytate (or phytic acid), and sinapine. Since meal comprises about half of the seed volume of canola, and the demand for canola/oilseed rape has risen and is expected to continue rising to meet demands for healthy cooking oils, biodiesel, and personal care products, there is a long-felt need to modify the compositional properties of canola meal and thereby increasing its nutritional value.
  • anti -nutritional factors such as glucosinolates, tannins, phytate (or phytic acid), and sinapine. Since meal comprises about half of the seed volume of canola, and the demand for canola/oilseed rape has risen and is expected to continue rising to meet demands for healthy cooking oils, biodiesel, and personal care products, there is a long-felt need to modify
  • Biosynthetic pathway genes involved in phytate production and transport have been identified in the model plant Arabidopsis and multiple crop plants including AtITPKI, AIITPK2, OsMIPS, OsIPK, OsMIK, 0sMRP5, OsPLDl, OsLPAl, ZmMRP4, BnPGK2, BnITPKI and BnITPK .
  • AtITPKI AtITPKI
  • AIITPK2 AtITPK2
  • OsMIPS OsMIPS
  • OsIPK OsMIK
  • 0sMRP5sMRP5 OsPLDl
  • OsLPAl OsLPAl
  • ZmMRP4 maize I pal gene
  • BnA9.MRP5 was associated with reduced phytate inasmuch as BnA9.MRP5 transcription levels were found to be “significantly elevated” relative to a high phytate variety. Liu et al. 2021 Authorea, DOI: 10.22541/au. 161165160.08519096/vl, see Abstract.
  • double mutants of BnMRP5 on chromosomes A10 and C09 showed “no significant differences in Pi” or inorganic phosphate. Sashidhar et al. 2020 Brontiers in Plant Science, 11 (Article 603): 1-10, at 1 and 5, 2 nd col.
  • compositions and methods are based, at least in part, on the surprising discovery that levels of inorganic phosphate in Brassica napus can be increased by a targeted alteration of the genomic sequences of MRP1 and MRP2 genes that encode for multidrug resistance-associated protein (MRP').
  • MRP multidrug resistance-associated protein
  • Brassica napus as used herein includes crop varieties of the species such as spring oilseed rape, winter oilseed rape, and low erucic cultivars of the foregoing which are called canola.
  • a method of increasing inorganic phosphate in Brassica napus plant, cell, seed, tissue or germplasm thereof that comprises introducing a targeted alteration to the sequence of one or more multidrug resistance-associated protein MRP) genes.
  • Targeted alterations can be made to an MRP 1 gene, to an MRP2 gene, or to genes of both MRP1 and MRP2 to thereby generate a Brassica napus plant, cell, seed, tissue or germplasm thereof that comprises one or more MRP variants which can provide an increased level of inorganic phosphate relative to the plant, seed, tissue or germplasm thereof prior to introducing the one or more MRP variants.
  • the method comprises introducing a targeted alteration of one or more alleles of MRP2 on chromosome A09 or C09. In another aspect, the method comprises introducing a targeted alteration of one or more alleles oiMRPl on chromosome A10 or C05. In yet another aspect, the method comprises introducing a targeted alteration of one or more alleles of MRP2 and a targeted alteration of one or more alleles of MRP1.
  • the types of targeted alterations that can be used to create MRP variants disclosed herein include nonsense mutations, missense mutations, and deletions that eliminate or reduce MRP gene function.
  • the targeted alteration can be a premature termination codon that reduces or eliminates expression of a full-length protein encoded by the altered MRP variant.
  • a targeted alteration that introduces a premature termination codon are shown in Table 2 for MRP2 on chromosome A09 (MRP2.A09) or on chromosome C09 (MRP2.C09), Table 3 for MRP1 on chromosome A10 (MRP1.A10) or on chromosome C05 (MRP2.C05), and Table 4 for MRP2.A09 or MRP2.C09.
  • Examples of a targeted alteration comprising a premature termination codon is shown in each of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO 27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:37, SEQ IDNO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43.
  • amethod ofincreasing inorganic phosphate in Brassica napus plant, cell, seed, tissue or germplasm thereof that comprises introducing a targeted alteration to the sequence of the multi drug resistance-associated protein (MRP) genes MRP 1 or MRP2 to generate a Brassica napus plant, cell, seed, tissue or germplasm thereof comprising double homozygous variants of MRP1, MRP2, or a combination thereof.
  • MRP multi drug resistance-associated protein
  • targeted alterations can be introduced to make Brassica napus plant, cell, seed, tissue or germplasm thereof comprising double homozygous knockouts of MRP2 (null for both MRP2.A09 and MRP2.C09).
  • targeted alterations can be introduced to make Brassica napus plant, cell, seed, tissue or germplasm thereof comprising double homozygous knockouts oiMRPl (null for both MRP1.A10 and MRP1 ,C05).
  • targeted alterations can be introduced to make Brassica napiis plant, cell, seed, tissue or germplasm thereof comprising double homozygous knockouts of MRP 2 (null for both MRP2.A09 and MRP2.C09) and homozygous knockout alleles of one MRP 1 gene (null for MRP1.A10 or MRP1.C05).
  • targeted alterations can be introduced to make Brassica napus plant, cell, seed, tissue or germplasm thereof comprising double homozygous knockouts of MRP1 (null for both MRP1.A10 and MRP1.C05) and homozygous knockout alleles of one MRP 2 gene (null for MRP2.A09 or MRP2.C09).
  • targeted alterations can be introduced to make Brassica napus plant, cell, seed, tissue or germplasm thereof comprising double homozygous knockouts of both MRP2 (null for both MRP2.A09 and MRP2.C09) and MRP1 (null for both MRP1.A10 and MRP1.C05).
  • MRP1 or MRP2 are shown in Table 2 for MRP2 on chromosome A09 (MRP2.A09) or on chromosome C09 (MRP2.C09), Table 3 for MRP1 on chromosome A10 (MRP1.A10) or on chromosome C05 (MRP1.C05), and Table 4 for MRP2.A09 or MRP2.C09.
  • targeted alterations that can be used are shown in each of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ IDNO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43.
  • the disclosed method comprises introducing a targeted alteration into each of two MRP genes of Brassica napus plant, cell, seed, tissue or germplasm thereof, thereby introducing two MRP variant alleles, which are disclosed by reference to the variant alleles disclosed in Table 2, Table 3, or Table 4 herein, such that the term “MRP2.A9 Variant” refers to MRP2.A9_vl, MRP2.A9_v2, MRP2.A9_v3, MRP2.A9_v4, or MRP2.A9_v5, MRP2.A9_v6, MRP2.A9_v7, MRP2.A9_v8; the term “MRP2.C9 Variant” refers to MRP2.C9_vl, MRP2.C9_v2, MRP2.C9_v3, MRP2.C9_v4, MRP2.C9_v5, MRP2.C9_v6, MRP2.C9_v7; the term “MRP1.A10 Variant” refers to MRPl.AlO
  • the method includes introducing (i) homozygous MRP2.A9_vl combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (ii) homozygous MRP2.A9_v2 combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (iii) homozygous MRP2.A9_v3 combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (iv) homozygous MRP2.A9 v4 combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (v) homozygous MRP2.A9_v5 combined with a heterozygous or homozygous MRP2.2.
  • the disclosed method comprises introducing targeted alterations that generate a Brassica napus plant, cell, seed, tissue or germplasm thereof comprising (i) homozygous MRP2.C9_vl combined with a heterozygous or homozygous MRP2.A9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (ii) homozygous MRP2.C9_v2 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (iii) homozygous MRP2 C9_v3 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (iv) homozygous MRP2.C9_v4 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (ii) homozygous
  • the disclosed method comprises introducing targeted alterations that generate a Brassica napus plant, cell, seed, tissue or germplasm thereof comprising (i) homozygous MRP1 ,A10_vl combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.C05 Variant; (ii) homozygous MRPl.A10_v2 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.C05 Variant; or (iii) homozygous MRPl.A10_v3 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.C05 Variant.
  • a Brassica napus plant, cell, seed, tissue or germplasm thereof comprising (i) homozygous MRPl.C05_vl combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.A10 Variant; or homozygous MRPl.C05_v2 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.A10 Variant.
  • the Brassica napus plant, cell, seed, tissue or germplasm thereof that is altered can also further comprise an altered or variant gene that provides additional desirable meal quality.
  • the method can comprise introducing targeted alterations that generate one or more MRP variants in Brassica napus plant, cell, seed, tissue or germplasm thereof that further includes a targeted alteration, mutation or variant of one or more of LPA 1, TT2, TT8, DFR, F3H, ANR, LDOX, MYB28 or MAM1.
  • the desirable meal quality provided by LPA1 is reduced phytate and/or increased inorganic phosphate content.
  • Each of TT2, TT8, DFR and F3H, ANR, and LDOX can reduce fiber content.
  • Each of MYB28 and MAM1 can reduce glucosinolate content.
  • the method can also comprise introducing targeted alterations that generate one or more MRP variants in Brassica napus plant, cell, seed, tissue or germplasm thereof that provides increased Brassica napus meal protein and/or low fiber.
  • Such germplasm for example, are disclosed in US Patent Nos. 9,375,025 and 10,791,692; as well as International Patent Application Publication Nos. WO 2020/131600.
  • Brassica napus plant materials produced by targeted alteration or ethyl methanesulfonate (EMS) mutagenesis. Accordingly, provided herein is a Brassica napus plant, cell, seed, tissue or germplasm thereof that comprises one or more MRP variants, wherein the variants comprise a targeted alteration or an EMS mutant oiMRPl , MRP2, or both MRP1 and MRP2.
  • EMS ethyl methanesulfonate
  • MRP variants can comprise (i) a targeted alteration or EMS mutant of one or more alleles of MRP2 on chromosome A09 or C09, (ii) a targeted alteration or EMS mutant of one or more alleles oiMRPl on chromosome A10 or C05, or (iii) a combination of both (i) and (ii).
  • the types of targeted alterations and EMS mutants include nonsense mutations, missense mutations, and deletions that eliminate or reduce MRP gene function.
  • the targeted alteration can be a premature termination codon that reduces or eliminates expression of a full-length protein encoded by the altered MRP allele.
  • MRP2.A09 MRP2.A09
  • MRP2.C09 chromosome C09
  • Table 3 o MRPl on chromosome A10 MRP1.A10
  • MRP2.C05 chromosome C05
  • An example of an EMS mutant is shown in each of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID N0:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ IDNO:77, SEQ ID NO:78, SEQ IDNO:79, SEQ IDNO:80, SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83.
  • the disclosure provides Brassica napus plant, cell, seed, tissue or germplasm thereof comprising any combination of MRP variant alleles disclosed herein including (see definition above of terms “MRP2.A9 Variant”, “MRP2.C9 Variant,” “MRP1.A10 Variant”, or “MRP1.C05 Variant” and variants disclosed in Table 2, Table 3, or Table 4 herein): (i) homozygous MRP2.A9_vl combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (ii) homozygous MRP2.A9_v2 combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (iii) homozygous MRP2.A9_v3 combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.A10 Variant,
  • A9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant (xvi) homozygous MRPl.A10_vl combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1 C05 Variant; (xvii) homozygous MRPl .A10_v2 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.C05 Variant; or (xviii) homozygous MRPl.A10_v3 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.C05 Variant.
  • the Brassica napus plant, cell, seed, tissue or germplasm thereof comprises (i) homozygous MRP1 CO5_vl combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.A10 Variant; or homozygous MRPl.CO5_v2 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.A10 Variant.
  • the disclosure provides Brassica napus plant, cell, seed, tissue or germplasm thereof comprising any combination of MRP EMS mutants alleles disclosed herein.
  • MRP2.C9 EMS mutant refers to an MRP2 variant carrying the mutation shown in any one of SEQ ID NOs:58-67
  • MRP1.A10 EMS mutant refers to an MRP variant carrying the mutation shown in any one of SEQ ID NOs:68-74
  • MRP1.C5 EMS mutant refers to an MRP mutant carrying the mutation shown in any one of SEQ ID NOs:75-83.
  • Brassica napus plant, cell, seed, tissue or germplasm thereof that comprises: (1) any one or more of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO 61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ ID NO:77, SEQ IDNO:78, SEQ IDNO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83, (2) homozygous MRP
  • each of the foregoing Brassica napus plant, cell, seed, tissue or germplasm thereof comprising one or more MRP variants can further include a targeted alteration, mutation or variant of one or more of the following genes: LPA1, TT2, TT8, DFR, F3H, ANR, LDOX, MYB28 or MAM1, which can provide additional desirable meal quality.
  • each of the foregoing Brassica napus plant, cell, seed, tissue or germplasm thereof comprising one or more MRP variants can further include increased Brassica napus meal protein and/or low fiber.
  • Such germplasm for example, are disclosed in US Patent Nos. 9,375,025 and 10,791,692; as well as International Patent Application Publication Nos. WO 2020/131600.
  • Each of the Brassica napus plant, seed, tissue or germplasm thereof comprising one or more MRP variants disclosed herein can be used to produce oilseed or grain which is milled to produce meal, e.g., canola meal.
  • the meal produced from the disclosed Brassica napus oilseed comprises increased levels of inorganic phosphate relative to control seed or grain lacking the one or more MRP variants.
  • control refers to seed or grain that lacks the one or more MRP variants but is otherwise isogenic or substantially isogenic to the Brassica napus disclosed herein.
  • a method of producing high inorganic phosphate Brassica napus meal comprising providing or selecting any of the Brassica napus seed or grain disclosed herein that comprises one or more MRP variants, wherein the variants comprise a targeted alteration of MRP1, MRP2, or both MRP1 and MRP2 and the selected seed or grain comprise increased inorganic phosphate relative to control seed or grain lacking the one or more MRP variants.
  • the method then comprises milling the selected seed or grain to produce high inorganic phosphate Brassica napus meal.
  • the method can further include providing this high inorganic phosphate Brassica napus meal in feed to an animal, e.g., a monogastric animal.
  • Preferred monogastric animals include swine and chickens (e.g., egg-laying hens, broilers, pullets, etc.) as well as other poultry such as turkeys, ducks, geese, guinea fowl, etc.
  • Feed containing this high inorganic phosphate Brassica napus meal can also be fed to horses, rabbit, or any other commercially raised animal.
  • a method of producing high inorganic phosphate Brassica napus meal comprises milling Brassica napus seed or grain that comprises a targeted alteration to one or more alleles of MRP2 on chromosome A09 or C09 or a targeted alteration to one or more alleles of MRP1 on chromosome A10 or C05.
  • the types of targeted alterations include nonsense mutations, missense mutations, and deletions that eliminate or reduce MRP gene function.
  • the targeted alteration can be a premature termination codon that reduces or eliminates expression of a full-length protein encoded by the altered MRP allele.
  • MRP2.A09 MRP2.A09
  • MRP2.C09 chromosome C09
  • Table 3 MRP1 on chromosome A10
  • MRP2.C05 MRP2.C05
  • Table 4 for MRP2.A09 or MRP2.C09.
  • the method comprises milling seed or grain that comprises any combination of MRP variants disclosed herein (see definition above of terms “MRP2.A9 Variant”, “MRP2.C9 Variant,” “MRP1.A10 Variant”, or “MRP1.C05 Variant” and variants disclosed in Table 2, Table 3, or Table 4 herein): (i) homozygous MRP2.A9_vl combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (ii) homozygous MRP2.
  • A9_v2 combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant;
  • homozygous MRP2.A9_v5 combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant;
  • the Brassica napus seed or grain used to produce high inorganic phosphate meal comprises (i) homozygous MRPl.C05_vl combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.A10 Variant; or homozygous MRPl.CO5_v2 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.A10 Variant.
  • a method of producing high inorganic phosphate Brassica napus meal comprises milling Brassica napus seed or grain that comprises one or more mutant alleles of MRP2 on chromosome A09 or C09 or one or more mutant alleles of MRP1 on chromosome A10 or C05.
  • the method comprises milling seed or grain that comprises any combination o MRP EMS mutants disclosed herein (see definition above of terms “MRP2.C9 EMS mutant”, “MRP1.A10 EMS mutant”, and “MRP1.C5 EMS mutant” ) that comprises: (1) any one or more of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO 76, SEQ ID NO:77, SEQ ID NO:78,
  • a screening method for identifying Brassica napus plant, cell, seed, tissue or germplasm thereof comprising one or more MRP variants associated with increased inorganic phosphate level comprises providing a Brassica napus plant, cell, seed, tissue or germplasm thereof comprising one or more MRP variants disclosed herein (e.g., any of the foregoing disclosed examples of a Brassica napus plant, cell, seed, tissue or germplasm thereof comprising one or more MRP variants), obtaining a sample comprising nucleic acid from the plant, cell, seed, tissue or germplasm thereof, then screening the sample for any of the following: a) the one or more MRP variants or b) one or more marker alleles within 5 cM (of and genetically linked to the one or more of the MRP variants.
  • the method can then further comprise detecting the one or more (i) MRP variants or (ii) marker alleles in the sample to thereby identify the Brassica napus plant, cell, seed, tissue or germplasm thereof as having an MRP variant associated with increased inorganic phosphate level.
  • the method can additionally include selecting the Brassica napus plant, cell, seed, tissue or germplasm thereof identified as having an MRP variant or MRP EMS mutant associated with increased inorganic phosphate level.
  • the selected plant material can be used for breeding or trait introgression.
  • the method of identifying can include screening the sample for the presence of a marker allele linked to the MRP variant, e.g., by 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.9 cM, 0.8 cM, 0.7 cM, 0.6 cM, 0.5 cM, 0.4 cM, 0.3 cM, 0.2 cM, 0.1 cM, or less on a single meiosis- based genetic map, and associated.
  • a marker allele linked to the MRP variant e.g., by 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.9 cM, 0.8 cM, 0.7 cM, 0.6 cM, 0.5 cM, 0.4 cM, 0.3 cM, 0.2 cM, 0.1 cM, or less on a single meiosis- based genetic map, and associated.
  • the method can include detecting one or more targeted alteration to one or more alleles of MRP2 on chromosome A09 or C09 or a targeted alteration to one or more alleles of MRP1 on chromosome A10 or C05.
  • the types of targeted alterations include nonsense mutations, missense mutations, and deletions that eliminate or reduce MRP gene function (e.g., a premature termination codon that reduces or eliminates expression of a full-length protein encoded by the altered MRP allele).
  • MRP2.A09 MRP2.A09
  • MRP2.C09 chromosome C09
  • Table 3 MRP1 on chromosome A10
  • MRP2.C05 MRP2.C05
  • Table 4 for MRP2.A09 or MRP2.C09.
  • the method can include detecting a premature termination codon is shown in each of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO 31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43.
  • this method can include detecting any combination of MRP variants disclosed herein (see definition above of terms “MRP2.A9 Variant”, “MRP2.C9 Variant,” “MRP1.A10 Variant”, or “MRP1.C05 Variant” and variants disclosed in Table 2, Table 3, or Table 4 herein): (i) homozygous MRP2.A9_vl combined with a heterozygous or homozygous MRP2.C9 Variant, MRPl.AlO Variant, or MRP1.C05 Variant; (ii) homozygous MRP2.A9_v2 combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (iii) homozygous MRP2.A9_v3 combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (iv) homozygous MRP2.A9_v
  • A9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant (xvi) homozygous MRPl.A10_vl combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1 C05 Variant; (xvii) homozygous MRPl.A10_v2 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.C05 Variant; or (xviii) homozygous MRPl.A10_v3 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.C05 Variant.
  • the method can include detecting (i) homozygous MRPl.C05_vl combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP 1.Al 0 Variant; or homozygous MRP1 CO5_v2 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.A10 Variant.
  • the method of identifying can include screening the sample for the presence of a marker allele linked to an MRP EMS mutant, e.g., by 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.9 cM, 0.8 cM, 0.7 cM, 0.6 cM, 0.5 cM, 0.4 cM, 0.3 cM, 0.2 cM, 0.1 cM, or less on a single meiosis-based genetic map, and associated.
  • a marker allele linked to an MRP EMS mutant e.g., by 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.9 cM, 0.8 cM, 0.7 cM, 0.6 cM, 0.5 cM, 0.4 cM, 0.3 cM, 0.2 cM, 0.1 cM, or less on a single meiosis-based genetic map, and associated.
  • the method can include detecting one or more MRP EMS mutant of MRP2 on chromosome A09 or C09 or a MRP EMS mutant of MRP1 on chromosome A10 or C05.
  • the method can include can include screening the sample for the presence of a marker allele linked to one or more of SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO 67, SEQ ID NO 68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ IDNO:74, SEQ ID NO:75, SEQ IDNO:76, SEQ IDNO:77, S
  • this method can include detecting any combination of MRP EMS mutants disclosed herein (see definition above of terms “MRP2.A9 Variant”, “MRP2.C9 Variant,” “MRP1.A10 Variant”, or “MRP1.C05 Variant” and variants disclosed in Table 2, Table 3, or Table 4 herein), e.g., a combination of a (1) homozygous MRP2.C9 EMS mutant combined with heterozygous or homozygous MRP1.A10 EMS mutant or MRP1.C5 EMS mutant, (2) homozygous MRP1.A10 EMS mutant combined with heterozygous or homozygous MRP2.C9 EMS or MRP1.C5 EMS mutant, or (3) homozygous MRP1.C5 EMS mutant combined with heterozygous or homozygous MRP2.C9 EMS or MRP 1.
  • a method that includes crossing a Brassica napus plant disclosed herein comprising one or more MRP variants to a second plant that does not have the one or more MRP variants, thereby producing one or more progeny plants whose genome comprises the one or more MRP variants, i.e., any of the MRP variants or combinations disclosed herein and/or selected by the screening method disclosed herein.
  • the second plant is one of a plant line (a “recurrent parent line”) and the method further includes crossing the progeny plant with another plant of the recurrent parent line to produce a second- generation progeny whose genome comprises the one or more MRP variants.
  • the second-generation progeny can be crossed with the recurrent parent line to produce a third- generation progeny whose genome comprises the one or more MRP variants.
  • This process can be repeated three, four, five, six, seven, or more times, such that each subsequent generation progeny is crossed with the recurrent parent line, thereby introgressing the one or more MRP variants into the recurrent parent line.
  • a plant having the one or more MRP variants disclosed herein is crossed with a second plant to produce progeny plants.
  • the progeny plants are screened for the one or more MRP variants in accordance with the screening method disclosed herein.
  • screening includes obtaining a nucleic acid sample from each of the progeny plants and screening the sample for the presence of MRP variants; thereby identifying novel progeny plants comprising a one of the R genes disclosed herein.
  • an isolated recombinant nucleic acid comprising one or more of SEQ ID NOs: 13-43 or SEQ IDNOs:53-67.
  • the isolated recombinant nucleic acid is a gene editing construct comprising a guide RNA (e.g., any one or more of SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO: 19) for introducing an MRP variant disclosed herein.
  • a guide RNA e.g., any one or more of SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO: 19
  • a combination of primers for detecting an MRP variant disclosed herein e g., SEQ ID NO: 14 and SEQ ID NO:15; or SEQ ID NO: 17 and SEQ ID NO:18; or SEQ ID NO: 20 and SEQ ID NO:21.
  • a method of introducing an MRP variant into Brassica napus comprising delivering to a cell or tissue of the Brassica napus a Cas endonuclease and a guide RNA targeting a sequence (target site) of MRP2 gene on chromosome A09 or C09 or MRP1 gene on chromosome A10 or C05.
  • the endonuclease/guide RNA complex indues a targeted alteration at the target site which reduces or eliminates expression of the protein encoded by the targeted MRP 2 or MRP 1 gene, thereby introducing an MRP variant to the Brassica napus cell or tissue.
  • the method further comprises regenerating Brassica napus plant, cell, seed, tissue or germplasm thereof comprising the MRP variant.
  • the guide RNA can be any one or more of SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO: 19.
  • the MRP variant can be any MRP variant disclosed herein and shown in Table 2 for MRP2 on chromosome A09 (MRP2.A09) or on chromosome C09 (MRP2.C09), Table 3 for MRP1 on chromosome A10 (MRP1.A10) or on chromosome C05 (MRP2.C05), and Table 4 for MRP2.A09 or MRP2.C09.
  • the variant can include the premature termination codon shown in each of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43.
  • the method can include introducing any combination of MRP variant alleles disclosed herein (see definition above of terms “MRP2.A9 Variant”, “MRP2.C9 Variant,” “MRP1.A10 Variant”, or “MRP1.C05 Variant” and variants disclosed in Table 2, Table 3, or Table 4 herein): (i) homozygous MRP2.A9_vl combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (ii) homozygous MRP2.A9_v2 combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (iii) homozygous MRP2.A9_v3 combined with a heterozygous or homozygous MRP2.C9 Variant, MRP1.A10 Variant, or MRP1.C05 Variant; (iv) homozygous MRP2.A9_v
  • the method can include introducing (i) homozygous MRPl.C05_vl combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.A10 Variant; or homozygous MRPl.CO5_v2 combined with a heterozygous or homozygous MRP2.A9 Variant, MRP2.C9 Variant, or MRP1.A10 Variant.
  • a method of introducing an MRP variant into Brassica napus to generate any one of the mutations shown in Table 5 herein within SEQ ID NOs:53-83 comprising delivering to a cell or tissue of the Brassica napus a Cas endonuclease and a guide RNA targeting a sequence (target site) of MRP2 gene on chromosome A09 or C09 ox MRP 1 gene on chromosome A10 or C05.
  • the method can include generating several such variants such that the Cas endonuclease and guide RNA produce changes leading to a combination of the mutations shown in SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ IDNO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO
  • Fig. 1 is a bar graph showing levels of inorganic phosphate (Pi) in seed comprising double homozygous knockout of MRP2.A9/MRP2.C9 and control seed comprising respective wild type MRP 2 genes.
  • Fig. 2 is a bar graph showing Pi levels of seed comprising double homozygous knockout of MRP1.A10/MRP1.C05 and control seed comprising respective wild type MRP1 genes.
  • Fig. 3 is a bar graph showing Pi levels of seed comprising triple homozygous knockout alleles of MRP2.A9/MRP2.C9/MRP2.C9a and control seed comprising respective wild type MRP 2 genes.
  • Fig. 4 is a bar graph showing Pi levels of hybrid seed comprising double homozygous knockout of MRP2.A9/MRP2.C9 and control hybrid seed comprising respective wild type MRP 2 genes.
  • Fig. 5 is a bar graph showing Pi levels of inbred seed harvested from field trials in 2021, one set of seed comprised double homozygous knockout of MRP2.A9/MRP2.C9 and the control set hybrid seed comprised respective wild type MRP2 genes.
  • Fig. 6 is a bar graph showing Pi levels of seeds in the following lines harvested from field trials in 2022: double homozygous knockout of MRP 2 in G00010BC inbred and corresponding control (wild type MRP2 genes) G00010BC inbred; double homozygous knockout o MRP2 in G00555MC and corresponding control (wild type MRP2 genes) GOO555MC inbred; double homozygous knockout of MRP2 in second generation F2 hybrid seed and corresponding control (wild type MRP2 genes) hybrid.
  • Fig. 7 is a bar graph showing Pi levels of seed in MRP2/TT2 double knockout variants and corresponding control wild type plants.
  • Fig. 8 is a bar graph showing ADF levels of meal in MRP2/TT2 double knockout variants and corresponding control wild type plants.
  • Nucleic acid sequences listed in the accompanying sequence listing and referenced herein are shown using standard letter abbreviations for nucleotide bases. While only one strand of each nucleic acid sequence is shown, the complementary strand is understood to be included in any reference to the displayed strand. Sequence listings are described in the following Table 1 and in Table 5 herein.
  • a gene or allele is “associated with” a trait when it is part of or linked to a DNA sequence or allele that affects the expression of the trait.
  • the presence of the allele is an indicator of how the trait will be expressed.
  • B ackcrossing refers to the process whereby hybrid progeny are repeatedly crossed back to one of the parents.
  • the “donor” parent refers to the parental plant with the desired gene or locus to be introgressed.
  • the “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats loci refers to certain genetic loci encoding components of DNA cleavage systems, for example, used by bacterial and archaeal cells to destroy foreign DNA (Horvath and Barrangou, 2010, Science 327: 167-170; International Application Publication W02007/025097, published 01 March 2007).
  • a CRISPR locus can consist of a CRISPR array, comprising short direct repeats (CRISPR repeats) separated by short variable DNA sequences (called spacers), which can be flanked by diverse Cas (CRISPR-associated) genes.
  • Cas protein refers to a polypeptide encoded by a Cas (CRISPR-associated) gene.
  • a Cas protein includes but is not limited to: a Cas9 protein, a Cpfl (Casl2) protein, a C2cl protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas 5, Cas7, Cas8, CaslO, or combinations or complexes of these.
  • a Cas protein may be a “Cas endonuclease” or “Cas effector protein”, that when in complex with a suitable polynucleotide component, is capable of recognizing, binding to, and optionally nicking or cleaving all or part of a specific polynucleotide target sequence.
  • a Cas endonuclease described herein comprises one or more nuclease domains.
  • the endonucleases of the disclosure may include those having one or more RuvC nuclease domains.
  • a Cas protein is further defined as a functional fragment or functional variant of a native Cas protein, or a protein that shares at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a native Cas protein, and retains at least partial activity.
  • a “Cas endonuclease” may comprise domains that enable it to function as a double- strand-break-inducing agent.
  • a “Cas endonuclease” may also comprise one or more modifications or mutations that abolish or reduce its ability to cleave a double-strand polynucleotide (dCas).
  • the Cas endonuclease molecule may retain the ability to nick a single-strand polynucleotide (for example, a D10A mutation in a Cas9 endonuclease molecule) (nCas9).
  • crossed refers to a sexual cross and involved the fusion of two haploid gametes via pollination to produce diploid progeny (e.g., cells, seeds or plants).
  • diploid progeny e.g., cells, seeds or plants.
  • the term encompasses both the pollination of one plant by another and selfing (or self-pollination, e.g., when the pollen and ovule are from the same plant).
  • An “elite line” is any line that has resulted from breeding and selection for superior agronomic performance.
  • a “favorable allele” is the allele at a particular locus (a marker, a QTL, a gene etc.) that confers, or contributes to, an agronomically desirable phenotype, e.g., disease resistance, and that allows the identification of plants with that agronomically desirable phenotype.
  • a favorable allele of a marker is a marker allele that segregates with the favorable phenotype.
  • Gene includes a nucleic acid fragment that expresses a functional molecule such as, but not limited to, a specific protein, including regulatory sequences preceding (5’ non-coding sequences) and following (3’ non-coding sequences) the coding sequence, as well as intervening intron sequences.
  • “Native gene” refers to a gene as found in its natural endogenous location with its own regulatory sequences.
  • Genetic markers are nucleic acids that are polymorphic in a population and where the alleles of which can be detected and distinguished by one or more analytic methods, e.g., RFLP, AFLP, isozyme, SNP, SSR, and the like.
  • the term also refers to nucleic acid sequences complementary to the genomic sequences, such as nucleic acids used as probes. Markers corresponding to genetic polymorphisms between members of a population can be detected by methods well-established in the art.
  • PCR-based sequence specific amplification methods include, e.g., PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs).
  • ESTs expressed sequence tags
  • SSR markers derived from EST sequences and randomly amplified polymorphic DNA (RAPD).
  • germplasm refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture, or more generally, all individuals within a species or for several species (e.g., maize germplasm collection or Andean germplasm collection).
  • the germplasm can be part of an organism, cell, or can be separate from the organism or cell.
  • germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture.
  • germplasm includes cells, seed or tissues from which new plants may be grown, or plant parts, such as leaves, stems, pollen, or cells, that can be cultured into a whole plant.
  • the term “genome” as it applies to a prokaryotic and eukaryotic cell or organism cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondria, or plastid) of the cell.
  • genomic sequence or “genomic region” is a segment of a chromosome in the genome of a cell that is present on either side of the target site or, alternatively, also comprises the target site or a portion thereof.
  • endogenous genomic sequence refers to genomic sequence within a plant cell, (e.g. an endogenous genomic sequence of an MRP gene present within the genome of a Brassica plant cell).
  • a “genomic locus” as used herein refers to the genetic or physical location on a chromosome of a gene.
  • “gene” includes a nucleic acid fragment that expresses a functional molecule such as, but not limited to, a specific protein coding sequence and regulatory elements, such as those preceding (5’ non-coding sequences) and following (3’ non-coding sequences) the coding sequence.
  • genotype is the actual nucleic acid sequence at one or more loci in an individual plant.
  • phenotype means the detectable characteristics (e.g. increased free phosphate) of a cell or organism which can be influenced by genotype.
  • the term “guide polynucleotide”, relates to a polynucleotide sequence that can form a complex with a Cas endonuclease, including the Cas endonuclease described herein, and enables the Cas endonuclease to recognize, optionally bind to, and optionally cleave a DNA target site.
  • the guide polynucleotide sequence can be a RNA sequence, a DNA sequence, or a combination thereof (a RNA-DNA combination sequence).
  • single guide RNA and “sgRNA” are used interchangeably herein and relate to a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variable targeting domain (linked to a tracr mate sequence that hybridizes to a tracrRNA), fused to a tracrRNA (trans-activating CRISPR RNA).
  • CRISPR RNA crRNA
  • variable targeting domain linked to a tracr mate sequence that hybridizes to a tracrRNA
  • trans-activating CRISPR RNA trans-activating CRISPR RNA
  • the single guide RNA can comprise a crRNA or crRNA fragment and a tracrRNA or tracrRNA fragment of the type II CRISPR/Cas system that can form a complex with a type II Cas endonuclease, wherein said guide RNA/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, optionally bind to, and optionally nick or cleave (introduce a single or double-strand break) the DNA target site.
  • guide polynucleotide/Cas endonuclease complex As used herein, the terms “guide polynucleotide/Cas endonuclease complex”, “guide polynucleotide/Cas endonuclease system”, “ guide polynucleotide/Cas complex”, “guide polynucleotide/Cas system” and “guided Cas system” “Polynucleotide-guided endonuclease” , “PGEN” are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease, that are capable of forming a complex, wherein said guide polynucleotide/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce
  • a guide polynucleotide/Cas endonuclease complex herein can comprise Cas protein(s) and suitable polynucleotide component(s) of any of the known CRISPR systems (Horvath and Barrangou, 2010, Science 327:167-170; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13: 1-15; Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular Cell 60, 1-13).
  • a “haplotype” is the genotype of an individual at a plurality of genetic loci, i.e. a combination of alleles. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome segment.
  • heterogeneity is used to indicate that individuals within the group differ in genotype at one or more specific loci.
  • homogeneity indicates that members of a group have the same genotype at one or more specific loci.
  • hybrid refers to the progeny obtained between the crossing of at least two genetically dissimilar parents.
  • inbred refers to a line that has been bred for genetic homogeneity.
  • inorganic phosphate and “free phosphate” are used interchangeably herein to mean forms of nutritional phosphate that can be absorbed by monogastric animals such as pigs and poultry.
  • introgression refers to the transmission of a desired allele of a genetic locus from one genetic background to another.
  • introgression of a desired R gene allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome.
  • transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome.
  • the desired allele can be, e.g., detected by a marker that is associated with a phenotype, at a QTL, a transgene, or the like.
  • Offspring comprising the desired allele may be repeatedly backcrossed to a line having a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background.
  • a “line” or “strain” is a group of individuals of identical parentage that are generally inbred to some degree and that are generally homozygous and homogeneous at most loci (isogenic or near isogenic).
  • a “subline” refers to an inbred subset of descendants that are genetically distinct from other similarly inbred subsets descended from the same progenitor.
  • plant material includes whole plants, plant cells, plant protoplast, plant cell or tissue culture from which plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants, or parts of plants, such as seeds, flowers, cotyledons, leaves, stems, buds, roots, root tips and the like.
  • a “modified plant” means any plant that has a genetic change due to human intervention.
  • a modified plant may have genetic changes introduced through plant transformation, genome editing, mutagenesis, or conventional plant breeding.
  • a “marker” is a means of finding a position on a genetic or physical map, or else linkages among markers and trait loci (loci affecting traits).
  • the position that the marker detects may be known via detection of polymorphic alleles and their genetic mapping, or else by hybridization, sequence match or amplification of a sequence that has been physically mapped.
  • a marker can be a DNA marker (detects DNA polymorphisms), a protein (detects variation at an encoded polypeptide), or a simply inherited phenotype (such as a low-erucic acid oil profile).
  • a DNA marker can be developed from genomic nucleotide sequence or from expressed nucleotide sequences (e.g., from a spliced RNA or a cDNA). Depending on the DNA marker technology, the marker may consist of primers complementary to sequence flanking the locus and/or probes that hybridize to polymorphic alleles at the locus.
  • a DNA marker, or a genetic marker may also be used to describe the gene, DNA sequence or nucleotide on the chromosome itself (rather than the components used to detect the gene or DNA sequence) and is often used when that DNA marker is associated with a particular trait in human genetics (e.g. a marker for breast cancer).
  • marker locus is the locus (gene, sequence or nucleotide) that the marker detects.
  • Markers can be defined by the type of polymorphism that they detect and also the marker technology used to detect the polymorphism. Marker types include but are not limited to, e.g., detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs), detection of simple sequence repeats (SSRs), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, or detection of single nucleotide polymorphisms (SNPs). SNPs can be detected e.g.
  • DNA sequencing via DNA sequencing, PCR-based sequence specific amplification methods, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), dynamic allele-specific hybridization (DASH), molecular beacons, microarray hybridization, oligonucleotide ligase assays, Flap endonucleases, 5’ endonucleases, primer extension, single strand conformation polymorphism (SSCP) or temperature gradient gel electrophoresis (TGGE).
  • DNA sequencing such as the pyrosequencing technology has the advantage of being able to detect a series of linked SNP alleles that constitute a haplotype. Haplotypes tend to be more informative (detect a higher level of polymorphism) than SNPs.
  • Marker assisted selection (of MAS) is a process by which individual plants are selected based on marker genotypes.
  • Marker assisted counter-selection is a process by which marker genotypes are used to identify plants that will not be selected, allowing them to be removed from a breeding program or planting.
  • a “marker haplotype” refers to a combination of alleles at a marker locus.
  • molecular marker may be used to refer to a genetic marker, as defined above, or an encoded product thereof (e.g., a protein) used as a point of reference when identifying a linked locus.
  • a molecular marker can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encoded polypeptide.
  • the term also refers to nucleic acid sequences complementary to or flanking the marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence.
  • a “molecular marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence.
  • a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus.
  • Nucleic acids are “complementary” when they specifically hybridize in solution. Some of the markers described herein are also referred to as hybridization markers when located on an indel region, such as the non-collinear region described herein.
  • the insertion region is, by definition, a polymorphism vis a vis a plant without the insertion.
  • the marker need only indicate whether the indel region is present or absent. Any suitable marker detection technology may be used to identify such a hybridization marker, e.g. SNP technology is used in the examples provided herein.
  • nucleic acid molecule is a polymeric form of nucleotides, which can include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above.
  • a nucleotide refers to a ribonucleotide, deoxynucleotide, or a modified form of either type of nucleotide.
  • a "nucleic acid molecule” as used herein is synonymous with “nucleic acid”, “nucleotide sequence”, “nucleic acid sequence", and “polynucleotide.” The term includes single- and double-stranded forms of DNA.
  • a nucleic acid molecule can include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
  • Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc ), charged linkages (e.g., phosphorothi oates, phosphorodithioates, etc ), pendent moieties (e.g., peptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoram
  • nucleic acid molecule also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations.
  • endogenous nucleic acid sequence refers to a nucleic acid sequence within a plant cell, (e.g. an endogenous allele of an IND gene present within the genome of a Brassica plant cell).
  • a “protospacer adjacent motif’ herein refers to a short nucleotide sequence adjacent to a target sequence (protospacer) that is recognized (targeted) by a guide polynucleotide/Cas endonuclease system described herein.
  • the Cas endonuclease may not successfully recognize a target DNA sequence if the target DNA sequence is not followed by a PAM sequence.
  • the sequence and length of a PAM herein can differ depending on the Cas protein or Cas protein complex used.
  • the PAM sequence can be of any length but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long.
  • plant material refers to any processed or unprocessed material derived, in whole or in part, from a plant.
  • a plant material may be a plant part, a seed, a fruit, a leaf, a root, a plant tissue, a plant tissue culture, a plant explant, or a plant cell.
  • a “polymorphism” is a variation in the DNA between two or more individuals within a population.
  • a polymorphism preferably has a frequency of at least 1% in a population.
  • a useful polymorphism can include a single nucleotide polymorphism (SNP), a simple sequence repeat (SSR), or an insertion/deletion polymorphism, also referred to herein as an “indel”.
  • “Phytate” and “phytic acid” are used interchangeably herein to mean forms of inositol polyphosphate (inositol hexakisphosphate, IP6, dihydrogenphosphate ester of inositol) which sequester their constituent phosphate and inhibit its absorption when consumed by monogastric animals such as pigs and poultry.
  • QTL quantitative trait locus
  • target site can be used interchangeably herein and refer to a polynucleotide sequence such as, but not limited to, a nucleotide sequence on a chromosome, episome, a locus, or any other DNA molecule in the genome (including chromosomal, chloroplastic, mitochondrial DNA, plasmid DNA) of a cell, at which a guide polynucleotide/Cas endonuclease complex can recognize, bind to, and optionally nick or cleave .
  • the target site can be an endogenous site in the genome of a cell, or alternatively, the target site can be heterologous to the cell and thereby not be naturally occurring in the genome of the cell, or the target site can be found in a heterologous genomic location compared to where it occurs in nature.
  • endogenous target sequence and “native target sequence” are used interchangeable herein to refer to a target sequence that is endogenous or native to the genome of a cell and is at the endogenous or native position of that target sequence in the genome of the cell.
  • a “targeted alteration” or “variant” is a gene (e.g., MRP gene) sequence that has been altered through human intervention.
  • Such an “altered” or “modified” gene has a sequence that differs from the sequence of the corresponding native or non-altered gene by at least one nucleotide (i) insertion (i.e., addition of a nucleotide in a sequence), (ii) deletion, (iii) substitution (i.e., replacement of at least one nucleotide), or (iv) a combination of the foregoing alterations.
  • An “altered” or “modified” plant is a plant comprising an altered gene sequence, e.g., a deletion.
  • a “targeted alteration” in a gene can be made by altering a target sequence within the target gene using any method known to one skilled in the art, including a method involving a guided Cas endonuclease system as disclosed herein.
  • a virus or vector “transforms” or “transduces” a cell when it transfers nucleic acid molecules into the cell.
  • a cell is “transformed” by a nucleic acid molecule transduced into the cell when the nucleic acid molecule becomes stably replicated by the cell, either by incorporation of the nucleic acid molecule into the cellular genome, or by episomal replication.
  • the term “transformation” encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell.
  • Examples include, but are not limited to, transfection with viral vectors, transformation with plasmid vectors, electroporation (Fromm et al., 1986, Nature 319:791-3), lipofection (Feigner et al., 1987, Proc. Natl. Acad. Sci. USA 84:7413-7), microinjection (Mueller et al., 1978, Cell 15:579-85), Agrobacteriu -mediated transfer (Fraley et al., 1983, Proc. Natl. Acad. Sci. USA 80:4803-7), direct DNA uptake, and microprojectile bombardment (Klein et al., 1987, Nature 327:70).
  • variants refer to substantially similar sequences.
  • a variant comprises a deletion and/or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide (e.g. an MRP variant disclosed herein).
  • a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively.
  • yield refers to the productivity per unit area of a particular plant product of commercial value. Yield is affected by both genetic and environmental factors. “Agronomics,” “agronomic traits,” and “agronomic performance” refer to the traits (and underlying genetic elements) of a given plant variety that contribute to yield over the course of growing season. Individual agronomic traits include emergence vigor, vegetative vigor, stress tolerance, disease resistance or tolerance, herbicide resistance, branching, flowering, seed set, seed size, seed density, standability, threshability and the like. Yield can therefore be considered the final culmination of all agronomic traits.
  • the disclosed targeted alterations or variants of an MRP gene refer to human-made, intentionally produced and selected nucleic acid changes that can be generated by any known methods, including the use of targeted mutagenesis, Targeting Induced Local Lesions IN Genomes or TILLING (see e.g., McCallum et al., 2000, Nat Biotechnol 18:455-457), or the use of doublestrand-break inducing agents (DSB Agents).
  • Double-Strand-Break (DSB Agents) Double-Strand-Break (DSB Agents). Double-strand breaks can be induced by agents such as endonucleases that cleave the phosphodiester bond within a polynucleotide chain, can result in the induction of DNA repair mechanisms, including the non- homologous end-joining pathway, and homologous recombination. Endonucleases include a range of different enzymes, including restriction endonucleases (see e.g.
  • Any DSB or -nick or -modification inducing agent may be used for the methods described herein, including for example but not limited to: Cas endonucleases, recombinases, TALENs, zinc finger nucleases, restriction endonucleases, meganucleases, and deaminases.
  • Class I Cas endonucleases comprise multi -subunit effector complexes (Types I, III, and IV), while Class 2 systems comprise single protein effectors (Types II, V, and VI) (Makarova et al., 2015, Nature Reviews Microbiology 13: 1-15; Zetsche et al., 2015, Cell 163: 1-13; Shmakov et al., 2015, Molecular Cell 60, 1-13; Haft et al., 2005, Computational Biology, PLoS Comput Biol /(6): e60; and Koonin et al., 2017, Curr Opinion Microbiology 37:67-78).
  • the Cas endonuclease acts in complex with a guide RNA (gRNA) that directs the Cas endonuclease to cleave the DNA target to enable target recognition, binding, and cleavage by the Cas endonuclease.
  • the gRNA comprises a Cas endonuclease recognition (CER) domain that interacts with the Cas endonuclease, and a Variable Targeting (VT) domain that hybridizes to a nucleotide sequence in a target DNA.
  • CER Cas endonuclease recognition
  • VT Variable Targeting
  • the gRNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) to guide the Cas endonuclease to its DNA target.
  • the crRNA comprises a spacer region complementary to one strand of the double strand DNA target and a region that base pairs with the tracrRNA, forming an RNA duplex.
  • the Cas endonuclease-guide polynucleotide complex recognizes a short nucleotide sequence adjacent to the target sequence (protospacer), called a “protospacer adjacent motif’ (PAM).
  • PAM protospacer adjacent motif
  • Cas endonuclease examples include but are not limited to Cas9, Casl2f, Casl2a or Cpfl, and variants thereof (See e.g., US Patent No. 10,934,536 and International Application Publication WO 2022/082179).
  • Cas9 (formerly referred to as Cas5, Csnl, or Csxl2) is a Class 2 Type II Cas endonuclease (Makarova et al., 2015, Nature Reviews Microbiology 13: 1-15).
  • Cas9 and Casl2f a Cas-gRNA complex recognizes a 3’ PAM sequence at the target site, permitting the spacer of the guide RNA to invade the double-stranded DNA target, and, if sufficient homology between the spacer and protospacer exists, generate a DSB cleavage.
  • Cas9 endonucleases comprise RuvC and HNH domains that together produce DSBs, and separately can produce single strand breaks.
  • the DSB leaves a blunt end.
  • Cpfl is a Class 2 Type V Cas endonuclease, and comprises nuclease RuvC domain but lacks an HNH domain (Yamane et al., 2016, Cell 165:949-962). Casl2f can generate 5’ staggered overhangs at DSB sites (Karvelis et al., Nucl Acids Res 48(12): 5016-5023 ). Cpfl endonucleases create “sticky” overhang ends.
  • Some uses for Cas-gRNA systems at a genomic target site include but are not limited to insertions, deletions, substitutions, or modifications of one or more nucleotides at the target site; modifying or replacing nucleotide sequences of interest (such as a regulatory elements); insertion of polynucleotides of interest; gene dropout; gene knock-out; gene knock in; modification of splicing sites and/or introducing alternate splicing sites; modifications of nucleotide sequences encoding a protein of interest; amino acid and/or protein fusions; and gene silencing by expressing an inverted repeat into a gene of interest.
  • Genome editing using DSB-inducing agents has been described, for example in U.S. Patent Application No. 2015/0082478, US Patent No. 10,934,536, International Application Publication WO2015/026886 Al, International Application Publication W02016007347, International Application Publication WO201625131, and International Application Publication WO 2022/082179 all of which are incorporated by reference herein.
  • a targeted genomic modification is introduced in a B. napus plant cell, wherein the targeted modification includes a targeted alteration of the genomic sequence of an MRP gene in the B. napus plant cell.
  • the targeted genomic modification is induced by a DSB Agent, such as a CRISPR-associated (Cas) nuclease.
  • a Cas nuclease is introduced into the B. napus cell with a first and second guide RNAs as Cas-gRNA complexes that recognizes target sequences in the genome of the B. napus cell and is able to induce DSBs in the genomic sequence, e.g., thereby altering the endogenous target MRP gene.
  • the disclosed guide polynucleotides can be introduced into a cell with the disclosed DSB agents e.g., CRISPR-Cas endonucleases.
  • Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cells as well as plants and seeds produced by the methods described herein.
  • the cells are B. napus cells.
  • Vectors and constructs include circular plasmids, and linear polynucleotides, comprising a polynucleotide of interest and optionally other components including linkers, adapters, regulatory or analysis.
  • a recognition site and/or target site can be comprised within an intron, coding sequence, 5' UTRs, 3' UTRs, and/or regulatory regions.
  • the constructs of the disclosure comprise a promoter operably linked to a nucleotide sequence encoding a DSB Agent, such as a CAS nuclease (e g., gene encoding a Streptococcus pyrogenes Cas9 gene or Casl2f gene) and a promoter operably linked to a guide RNA of the present disclosure.
  • the promoter is capable of driving expression of an operably linked nucleotide sequence in a prokaryotic or eukaryotic cell/organism.
  • target specific guide RNAs are built as a fusion of CRISPR RNA (crRNA) fused to trans-activating CRISPR RNA (tracrRNA) of Streptococcus pyrogenes.
  • a guide RNA comprising SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 19, can be used to alter endogenous genomic MRP sequence in the B. napus plant cell.
  • the resulting targeted alterations can produce an MRP variant comprising a premature termination codon as shown in any of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ IDNO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO: 42, or SEQ ID NO: 43.
  • the isolated polynucleotides, constructs and vectors disclosed herein can comprise a selectable marker to identify or select for or against a molecule or a cell that comprises the construct or vector.
  • selectable markers include DsRed and Glyphosate N-Acetyltransferase (GAT) gene variant 4621 for herbicide resistance.
  • nucleic acid molecule refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be singlestranded. In some examples, the nucleic acid molecule can be double-stranded.
  • nucleic acid molecule e.g., RNA or DNA
  • isolated nucleic acid molecule e.g., RNA or DNA
  • recombinant nucleic acid molecule e.g., RNA or DNA
  • nucleic acid sequence e.g., RNA or DNA
  • an “isolated” or “recombinant” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • isolated or “recombinant” when used to refer to nucleic acid molecules excludes isolated chromosomes.
  • the recombinant nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences that naturally flank the MRP variant in the genome of the cell.
  • an isolated nucleic acid molecule comprising an MRP variant has one or more change in the nucleic acid sequence compared to the native or genomic nucleic acid sequence.
  • the change in the native or genomic nucleic acid sequence includes but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; changes in the nucleic acid sequence due to the amino acid substitution, insertion, deletion and/or addition compared to the native or genomic sequence; removal of one or more intron; deletion of one or more upstream or downstream regulatory regions; and deletion of the 5’ and/or 3’ untranslated region associated with the genomic nucleic acid sequence.
  • the nucleic acid molecule comprising one of SEQ ID NOs: 13-43 or SEQ ID NOs:53-67is non-genomic sequence.
  • polynucleotides comprising MRP variants disclosed herein are contemplated. Such polynucleotides are useful for production of encoded polypeptides in host cells when operably linked to a suitable promoter, transcription termination and/or polyadenylation sequences. Such polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that are MRP variants or related to MRP variants disclosed herein.
  • nucleic acid molecules comprising one or more of SEQ ID NOs: 13- 43 or SEQ ID NOs:58-67, and variants, fragments and complements thereof.
  • “Complement” is used herein to refer to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to thereby form a stable duplex.
  • a reverse complement is a complement formed by exchanging each A with T, T with A, C with G, and G with C in a sequence and then reversing the 5’ to 3’ order of the exchanged sequence, such that the reverse complement of 5’-ACCTGAG-3’ is 5’-CTCAGGT-3’.
  • Polynucleotide sequence variants is used herein to refer to a nucleic acid sequence that except for the degeneracy of the genetic code encodes the same polypeptide.
  • nucleotide Constructs are not intended to limit the disclosure to constructs comprising DNA.
  • Polynucleotide constructs particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed in the methods disclosed herein.
  • the isolated polynucleotide constructs, nucleic acids, and nucleotide sequences disclosed herein additionally encompass all complementary forms (e.g., the reverse complement) of each sequence disclosed for such a construct.
  • polynucleotide constructs and nucleotide sequences disclosed herein can encompass any such constructs, molecules, and sequences suitable for use in a method for transforming plant material disclosed herein. Such constructs can include naturally occurring molecules and/or synthetic analogues.
  • the disclosed nucleotide constructs, nucleic acids, and nucleotide sequences also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.
  • Transformed organisms disclosed herein include plant cells, bacteria, yeast, baculovirus, protozoa, nematodes and algae.
  • the transformed organism comprises a disclosed sequence (e.g., as part of a construct, expression cassette, or vector comprising the nucleotide sequence disclosed herein which are associated with increased disease resistance.
  • the disclosed sequences can be used in constructs for expression in the organism of interest.
  • Constructs can include 5’ and 3’; regulatory sequences operably linked to an R gene sequence, variant or fragment disclosed herein.
  • operably linked refers to a functional linkage between a promoter and/or a regulatory sequence and a second sequence, wherein the promoter and/or regulatory sequence initiates, mediates, and/or affects transcription of the DNA sequence corresponding to the second sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary, to join two protein coding regions in the same reading frame.
  • the construct may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs.
  • Such a DNA construct is provided with a plurality of restriction sites for insertion of the polypeptide gene sequence of the disclosure to be under the transcriptional regulation of the regulatory regions.
  • the DNA construct may additionally contain selectable marker genes.
  • the DNA construct will generally include in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (e.g., a promoter), a DNA sequence of the embodiments, and a transcriptional and translational termination region (e.g., termination region) functional in the organism serving as a host.
  • the transcriptional initiation region e.g., the promoter
  • the transcriptional initiation region may be native, analogous, foreign or heterologous to the host organism and/or to the sequence of the embodiments.
  • the promoter or regulatory sequence may be the natural sequence or alternatively a synthetic sequence.
  • the term “foreign” as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced.
  • heterologous in reference to a sequence means a sequence that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Where the promoter is a native or natural sequence, the expression of the operably linked sequence is altered from the wild-type expression, which results in an alteration in phenotype.
  • the DNA construct comprises a polynucleotide comprising one or more of SEQ ID NOs: 13-43 or SEQ ID NOs:58-67 or a fragment or variant thereof.
  • a DNA construct may also include a transcriptional enhancer sequence.
  • An “enhancer” refers to a DNA sequence which can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter.
  • Various enhancers include, for example, introns with gene expression enhancing properties in plants (US Patent Application Publication Number 2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie et al. 1989 Molecular Biology of RNA ed.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host or may be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host or any combination thereof).
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. 1991 Mol. Gen. Genet. 262: 141-144; Proudfoot 1991 Cell 64:671-674; Sanfacon et al. 1991 Genes Dev. 5: 141-149; Mogen et al. 1990 Plant Cell 2: 1261-1272; Munroe et al. 1990 Gene 91 : 151-158; Ballas et al. 1989 Nucleic Acids Res. 17:7891-7903 and Joshi et al. 1987 Nucleic Acid Res. 15:9627-9639.
  • a nucleic acid may be optimized for increased expression in the host organism.
  • the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri 1990 Plant Physiol. 92: 1-11 for a discussion of host-preferred usage.
  • nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. 1989 Nucleic Acids Res. 17:477-498).
  • the plantpreferred for a particular amino acid may be derived from known gene sequences from plants.
  • Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exonintron splice site signals, transposon-like repeats, and other well -characterized sequences that may be deleterious to gene expression.
  • the GC content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell.
  • host cell refers to a cell which contains a vector and supports the replication and/or expression of the expression vector is intended. Host cells may be prokaryotic cells such as E.
  • coli or eukaryotic cells such as yeast, insect, amphibian or mammalian cells or monocotyledonous or dicotyledonous plant cells.
  • An example of a monocotyledonous host cell is a maize host cell.
  • the sequence is modified to avoid predicted hairpin secondary mRNA structures.
  • the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • a number of promoters can be used in the practice of the embodiments.
  • the promoters can be selected based on the desired outcome.
  • the nucleic acids can be combined with constitutive, tissue-preferred, inducible
  • Plant Transformation Any suitable techniques known in the art for introduction of transgenes into plants may be used to produce a transformed plant or plant cell disclosed herein. Suitable methods for transformation of plants may include virtually any method by which DNA can be introduced into a cell, such as: by electroporation as illustrated in U.S. Patent No. 5,384,253; by microprojectile bombardment, as illustrated in U.S. Patent Nos. 5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865; by Agrobacterium- mediated transformation as illustrated in U.S. Patent Nos.
  • Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay may be cultured in media that supports regeneration of plants.
  • any suitable plant tissue culture media may be modified by including further substances, such as growth regulators.
  • Tissue may be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration (e.g., at least 2 weeks), then transferred to media conducive to shoot formation. Cultures are transferred periodically until sufficient shoot formation has occurred. Once shoots are formed, they are transferred to media conducive to root formation. Once sufficient roots are formed, plants can be transferred to soil for further growth and maturity.
  • the alteration (e.g., introduction of a stop codon, mutation or deletion) of an endogenous gene (e.g., MRP1 orMRP2) in regenerating plants can be confirmed by one or more assays, for example, a molecular biological assay, such as Southern blotting, Northern blotting, or PCR; a biochemical assay, such as detecting the absence of a protein product by immunoassay (ELISA or Western blot) or by screening for reduced enzymatic function; plant part assays, such as leaf or root assays; and analysis of the phenotype of the whole regenerated plant.
  • a molecular biological assay such as Southern blotting, Northern blotting, or PCR
  • a biochemical assay such as detecting the absence of a protein product by immunoassay (ELISA or Western blot) or by screening for reduced enzymatic function
  • plant part assays such as leaf or root assays
  • MRP variant-containing plants are generated.
  • a plant comprising an altered endogenous genomic sequence containing one or more of the following MRP variants: SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, or SEQ ID NO:43.
  • the modified B. napus plant comprises double homozygous variants of MRP1, MRP2, or both MRP1 and MRP2.
  • “Stable transformation” as used herein means that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” as used herein means that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant. “Plant” as used herein refers to whole plants, plant organs (e g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells and pollen).
  • Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. 1986 Proc. Natl. Acad. Sci. USA 83:5602- 5606), Agrobacterium-mediated transformation (US Patent Numbers 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. 1984 EMBO J.
  • Marker assisted selection Molecular markers can be used in a variety of plant breeding applications (e.g. see Staub et al. 1996 Hortscience 31 :729-741; Tanksley 1983 Plant Molecular Biology Reporter. 1 :3-8).
  • One of the main areas of interest is to increase the efficiency of backcrossing and introgressing genes using marker-assisted selection (MAS).
  • MAS marker-assisted selection
  • a molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is particularly true where the phenotype is hard to assay. Since DNA marker assays are less laborious and take up less physical space than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line. The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives.
  • flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed.
  • a marker is located within the gene itself, so that recombination cannot occur between the marker and the gene.
  • the methods targeted alteration disclosed herein produce a marker that can be used to identify the MRP variant.
  • FLP markers refer to fragment length polymorphisms that are in many ways similar to SSR markers, except that the region amplified by the primers is not typically a highly repetitive region.
  • SNP markers detect single base pair nucleotide substitutions, which can be assayed at an even higher level of throughput than SSRs, in so-called “ultra-high-throughput” fashion, as SNPs do not require large amounts of DNA and automation of the assay may be straight-forward. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS.
  • SNP genotyping including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, mini sequencing, and coded spheres. Such methods have been reviewed in: Gut 2001 Hum Mulat 17: 475-492; Shi 2001 Clin Chem T.
  • ESTs expressed sequence tags
  • RAPD randomly amplified polymorphic DNA
  • Isozyme profiles and linked morphological characteristics can, in some cases, also be indirectly used as markers. Even though they do not directly detect DNA differences, they are often influenced by specific genetic differences. However, markers that detect DNA variation are far more numerous and polymorphic than isozyme or morphological markers (Tanksley 1983 Plant Molecular Biology Reporter 1 : 3-8).
  • Example 1 Gene-edited A7RP2 knockouts.
  • CRISPR-Cas gene editing system was used to edit MRP 2 genes in Pioneer Brassica napus inbred line G00010BC, which contains two MRP2 genes.
  • MRP2 gene is located on chromosome A9 (MRP2.A9) (genomic: SEQ ID NO: 1, coding: SEQ ID NO:2, or encoding a. a.: SEQ ID NO:3) and another one is located on chromosome C9 (MRP2.C9) (genomic: SEQ ID N0:4, coding: SEQ ID NO: 5, or encoding a. a.: SEQ ID NO:6).
  • a guide RNA (SEQ ID NO: 13) was designed to target the second exon of MRP2 and induce a double stranded cut in the MRP 2 gene, leaving two free chromosomal ends.
  • the plant’ s repair mechanisms can attempt to repair the double-strand DNA break by non-homologous end joining (“NHEJ”), which can result in nucleotide insertions and deletions (Indels) at the cleavage site.
  • NHEJ non-homologous end joining
  • Indels nucleotide insertions and deletions
  • Tl seed was planted in growth chamber and seedlings were genotyped with the same NGS sequencing assay to identify MRP2. A9 and MRP2.C9 single homozygous and double homozygous plants as well as wildtype (WT) segregants. Because T-DNA from the plasmid was incorporated into the genome of TO plants, PCR assays were used to screen for T1 plants (“clean” T1 plants) that were null homozygous segregants for the desired MRP2 variants and that were free of genomeediting reagent plasmids. Selected clean T1 plants were self-pollinated to generate T2 seeds.
  • the free phosphorous inorganic phosphate (Pi) content of T2 seeds was evaluated both in whole seeds or in ground, defatted meal made from the seeds.
  • the method used for Pi determination was a modified version of the methods described in Chen et. al. 1956, Analytical Chemistry, 28: 1756-1758.
  • MRP2.A9 single homozygotes and MRP2.C9 single homozygotes were each found to have higher Pi than WT in T2 seeds. However, levels were not as high as desired.
  • Double homozygous knockouts (null for both MRP2.A9 and MRP2.C9) had higher Pi than single homozygotes. See Fig.
  • Example 2 Gene-edited A7 7N knockouts.
  • CRISPR-Cas gene editing system was used to edit MRP1 genes in Pioneer Brassica napus inbred line G00010BC, which also contains two MRP1 genes.
  • MRP1 gene is located on chromosome A10 (MRP1.A10) (genomic: SEQ ID NO:7, coding: SEQ ID NO:8, or encoding a. a.: SEQ ID NO:9) and another one is located chromosome C5 (MRP1.C5) (genomic: SEQ ID NO: 10, coding: SEQ ID NO: 11, or encoding a. a. : SEQ ID NO: 12).
  • a guide RNA (SEQ ID NO: 19) was designed to target an exon of MRP1 gene.
  • Transformation of CRISPR-Cas gene editing system was done as in Example 1. Regenerated plantlets were screened using a NGS sequencing assay to obtain the knockout variants identified herein.
  • Table 3 shows variants generated in the MRP1 genes using the indicated gRNA sequence. In Table 3, a period indicates the double-stranded break point that was targeted; underlined residues represent nucleotide insertions; dashes represent nucleotide deletions.
  • T1 plants identified as positive TO knockout plants were self-pollinated to produce T1 seed.
  • T1 plants were genotyped as described in Example 1 to identify MRP1.A10 and MRP1.C05 single homozygous knockout and double homozygous knockout plants as well as wildtype (WT) segregants.
  • WT wildtype
  • Free phosphorous (Pi) content of T2 seeds was evaluated as described in Example 1.
  • Double homozygous knockouts (null for both MRP1.A10 and MRP1.C05) contained 18.3 pmoles Pi per gram of seed, which is higher than wild type content of 10.0 pmoles Pi per gram of seed. See Fig. 2.
  • Example 3 Effect of MRP gene knockouts on germination.
  • T2 seeds were placed in a row between two layers of filter papers wetted with deionized water. Filter papers were rolled inside a piece of waxed paper and set vertically in a beaker containing 1 inch (2.5 cm) of deionized water. The beaker was covered with plastic wrap to prevent evaporation and placed at 25°C in the dark. Germination was scored 5 days after seeding. MRP2 double homozygous seeds had the same germination frequency as WT segregants and the WT inbred G00010BC.
  • Example 4 Phenotype of MRP gene knockouts in hybrid plants.
  • G00555MC Male inbred G00555MC has an MRP2 gene on chromosome A9 (MRP2.A9) and two MRP2 genes on chromosome C9 (MRP2.C9a and MRP2.C9b).
  • MRP2.A9 MRP2 gene on chromosome A9
  • MRP2.C9a and MRP2.C9b MRP2 genes on chromosome C9
  • G00555MC was performed using the gRNA BNA-MRP2-C2 (SEQ ID NO: 16).
  • Transformation of CRISPR-Cas gene editing system was done as in Example 1. Regenerated plantlets were screened using a NGS sequencing assay to obtain the knockout variants identified herein.
  • Table 4 shows various mutations generated in the MRP2 gene variants using the indicated gRNA sequence. In Table 4, a period indicates the double-stranded break point that was targeted using gRNA BNA-MRP2-C2 (SEQ ID NO: 16); underlined residues represent nucleotide insertions; dashes represent nucleotide deletions.
  • MRP2.C9a MRP2.C9 v3, MRP2.C9 v4, MRP2.C9 v5, MRP2.C9 v6; and MRP2.C9 v7; and in MRP2.C9b: MRP2.C9_v3 and MRP2.C9_v4.
  • Triple homozygous knockout plants (homozygous null for MRP2.A9, MRP2.C9a, and MRP2.C9b) were selected using a NGS sequencing assay as described in Example 1. Free or inorganic phosphate (Pi) content of the triple homozygous knockout was about 72.5 pmole/g of seed as compared to a wild type control that had Pi content of 22.3 pmole/g of seed. See Fig. 3.
  • the gene-edited female inbred G00010BC was converted to male sterile by carrying the pollen of G00010BC-MRP2 to the cytoplasmic male-sterile G00010FC, followed by two additional generations of crossing and selection for homozygous MRP2 knockouts.
  • Hybrid Fl seeds were produced in growth chamber and F2 seeds were generated for seed composition analysis.
  • the Pi content of the MRP2 hybrid was 65.1 pmole/g of seed as compared to wild type level of 6.8 pmole/g seed. See Fig. 4.
  • NIR near infrared
  • Pi content of MRP2 double knockout inbred G00010BC was 45.2 pmole/g seed as compared to 9.2 pmole/g seed in WT control in 2021. See Fig. 5.
  • the MRP2 knockouts had Pi contents of 34.9 pmole/g seed of G00010BC and 38.8 pmole/g seed of G00555MC, respectively. Wild type inbreds had Pi contents of 5.3 and 8.9 pmole/g seed. MRP2 hybrid F2 seeds had Pi content of 35.2 pmole/g seed; while the WT hybrid had 5.9 pmole/g seed.
  • Example 6 MRP knockout trait interactions with other grain traits.
  • A7/?/ J -conferred high seed Pi trait was tested for compatibility with other grain traits such as low fiber and low glucosinolates.
  • MRP2 knockout plants were crossed with a line having low-ADF (low acid detergent fiber) caused by TT8 loss-of-function variants generated by gene editing.
  • Homozygous MRP2 plants, TT8 plants, MRP2/TT8 stacked plants, and wild type plants were identified in the F2 population. Seed composition analysis showed that the Pi content in MRP2/TT8 stacks was similar to that in MRP2 plants and was higher than TT8 and wild type.
  • MRP2 and TT2 genes were knocked out in the same plants, both the high-Pi and low-ADF phenotype was observed in MRP2/TT2 plants. See Fig. 7 showing that seed phosphate levels in the MRP2/TT2 double knockouts were much higher than wild type (WT) plants; see also Fig. 8 showing that ADF levels c MRP2 TT2 were significantly reduced relative to WT plants.
  • Example 7 MRP 2 m MRPl Knockout Mutants generated by EMS mutagenesis
  • Ethyl methanesulfonate (EMS) mutagenesis population was developed. Dry seeds of canola inbred BC were treated with 0.3%, 0.5% and 0.8% of EMS, rinsed with distilled water, and the treated seeds (Ml) were air-dried. Ml seeds were planted in the field and M2 seeds were harvested from 3,200 individual Ml plants. Three seeds from each M2 were planted out in greenhouse and 7,000 M2 plants were obtained. Leaf samples were taken from individual M2 plants for DNA extraction and M3 seeds were harvested. Of the 7,000 M2 plants, the genome of 550 lines were sequenced.
  • MRP2 and MRP1 knockout mutants included nonsense mutation and splicing site mutations.
  • Table 5 shows the name for each MRP2 and MRP1 knockout mutants obtained, and describes the mutation: indicating chromosome, nucleotide alteration and position within the indicated sequence listing, as well as the altered encoded amino acid alteration.
  • MRP2 C9-m6 plant were crossed, Fl was self-pollinated, and F2 plants were genotyped.
  • the MRP2 double knockout homozygous plants were identified.

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Abstract

L'invention concerne des plantes, des cellules, des tissus et un germoplasme de celles-ci comprenant une ou plusieurs altérations ciblées des séquences génomiques des gènes MRP1 ou MRP2 qui codent pour une protéine associée à une résistance pléiotrope (MRP), ainsi que de la farine fabriquée à partir de telles matières végétales. L'invention concerne également des procédés et des compositions pour fabriquer des variants de gènes MRP1 et MRP2 modifiés ; des procédés de reproduction et des procédés d'identification et de sélection de matières végétales présentant les variants MRP1 et MRP2 divulgués.
PCT/US2024/035538 2023-06-30 2024-06-26 Variants de mrp chez brassica Pending WO2025006547A2 (fr)

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WO2025137127A1 (fr) * 2023-12-19 2025-06-26 Pioneer Hi-Bred International, Inc. Compositions de dfr et de f3h et procédés permettant d'obtenir une plus faible teneur en fibres dans des brassica

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