Search

US-20260125699-A1 - OVERCOMING SELF-INCOMPATIBILITY IN DIPLOID PLANTS FOR BREEDING AND PRODUCTION OF HYBRIDS THROUGH MODULATION OF HT

US20260125699A1US 20260125699 A1US20260125699 A1US 20260125699A1US-20260125699-A1

Abstract

The present invention relates to methods for controlling hybridization in plants and producing hybrid plants. The present invention also relates to nucleic acids encoding amino acid sequences for self-incompatibility (SI) proteins in plants, and the use thereof for the manipulation of SI, including seed production, in plants, particularly of the Solanaceae family. The present invention also relates to kits, compositions, constructs and vectors including such nucleic acids, and related polypeptides, regulatory elements and methods as well as resultant plant varieties developed through the use of self-pollination.

Inventors

  • David S. Douches
  • Felix E. Enciso-Rodriguez
  • Satya Swathi Nadakuduti
  • Norma C. Manrique-Carpintero

Assignees

  • BOARD OF TRUSTEES OF MICHIGAN STATE UNIVERSITY

Dates

Publication Date
20260507
Application Date
20251231

Claims (20)

  1. 1 . A genetically modified plant with modulated self-incompatibility when compared to a plant without such modification, said modification affecting the expression and/or activity of an HT gene and/or protein in said plant.
  2. 2 . A method for modulating self-incompatibility in a Solanaceae plant comprising the step of decreasing the expression and/or activity of an HT gene and/or protein in said plant or plant part, plant organ, or plant cell of said plant.
  3. 3 . The method of claim 2 , wherein said decreasing the expression and/or activity of the HT gene and/or protein comprises expressing in said plant, plant part, plant organ, or plant cell a nucleic acid comprising the following operably linked elements: a plant-expressible promoter, a nucleic acid which when transcribed results in decreased expression and/or activity of an endogenous HT gene and/or protein in said plant, plant part, plant organ, or plant cell.
  4. 4 . The method of claim 3 , wherein said nucleic acid encodes an RNA molecule comprising: (a) at least 20 consecutive nucleotides of the nucleotide sequence of an HT gene present in said Solanaceae plant; (b) at least 20 consecutive nucleotides of the complement of the nucleotide sequence of an endogenous HT gene present in said plant; or (c) a sense region comprising a nucleotide sequence of at least 20 consecutive nucleotides of the nucleotide sequence of an HT gene present in said plant and an antisense region comprising a nucleotide sequence of at least 20 consecutive nucleotides of the complement of the nucleotide sequence of said HT gene present in said plant, wherein said sense and antisense region are capable of forming a double stranded RNA region comprising said at 20 consecutive nucleotides.
  5. 5 . The method of claim 4 , wherein said RNA molecule comprises at least 20 consecutive nucleotides of a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, or 6 and/or the complement thereof, or the RNA molecule comprises at least 20 consecutive nucleotides of a nucleic acid encoding a protein comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 7 or 8, and/or the complement thereof.
  6. 6 . The method of claim 2 , wherein said decreasing the expression and/or activity of the HT gene and/or protein comprises introducing a mutation into an endogenous HT gene wherein the resulting mutated gene encodes an inactivated HT protein.
  7. 7 . The method of claim 6 , wherein the mutation is introduced by genome editing.
  8. 8 . The method of claim 7 , wherein said genome editing includes use of one or more of a CRISPR/Cas system, a Cre/Lox system, a TALEN system, a ZFN system, and homologous recombination.
  9. 9 . The method of claim 2 , wherein the HT gene is HT-A and/or HT-B.
  10. 10 . The method of claim 9 , wherein the expression and/or activity of both HT-A and HT-B are decreased.
  11. 11 . The method of claim 2 , wherein said Solanaceae plant is a potato plant.
  12. 12 . A plant, plant part, plant organ, plant cell, or seed of the plant of claim 1 .
  13. 13 . The plant of claim 12 which is a Solanaceae plant, plant part, plant organ, plant cell, or seed, wherein the expression and/or activity of an HT gene and/or protein has been modified as compared to a control plant.
  14. 14 . A Solanaceae plant comprising a mutant allele of an HT gene, said mutant allele resulting in a decrease of the expression and/or activity of the HT protein encoded by said gene compared to a plant of the same species not comprising said mutant allele.
  15. 15 . The plant of claim 14 , in which the self-incompatibility has been modulated compared to a plant of the same species not comprising said mutant allele.
  16. 16 . The plant of claim 14 , wherein the mutant allele is a knock-out allele of the endogenous HT gene.
  17. 17 . The plant of claim 14 , wherein the mutant allele comprises an insertion or deletion in the endogenous HT gene.
  18. 18 . The plant of claim 14 , wherein said HT protein comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 7 or 8, or wherein said HT protein is encoded by a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, or 6.
  19. 19 . The plant of claim 14 , wherein the plant comprises a mutant allele of HT-A and HT-B.
  20. 20 . The plant of claim 14 , which is a potato plant.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation application of U.S. National Phase application Ser No. 17/310,979, filed Sep. 2, 2021, which application claims priority to PCT/US2020/021672, filed Mar. 9, 2020, which application claims priority to provisional application U.S. Ser. No. 62/815,689, filed Mar. 8, 2019, which are incorporated herein by reference in their entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is herein incorporated by reference in its entirety. Said XML copy, created on Dec. 31, 2025, is named “P12873US02.xml” and is 20,071 bytes in size. FIELD OF THE INVENTION The invention relates generally to the field of molecular biology and plant genetics. BACKGROUND OF THE INVENTION Potato (Solanum tuberosum L) is a global food security crop and is the third most important food crop after rice and wheat. Improvement of cultivated potato (2n=4x=48) is difficult because tetra ploidy and heterozygosity hinder the fixation of desirable alleles in new cultivars. Research is in progress to reinvent potato as a diploid F1 hybrid (2n=2x=24). Inbred potatoes will not only accelerate the generation of new varieties with favorable allele combinations targeting yield, tuber quality, and disease resistance traits but will also aid in understanding the genetics underlying these traits. Diploid potatoes, however, are mostly self-incompatible, and the few lines that are self-compatible exhibit unreliable expression of this trait in succeeding generations. The gametophytic self-incompatibility (GSI) system present in diploid potatoes poses a great limitation in diploid breeding programs, causing rejection of self-pollen through the S-RNase-mediated degradation of pollen RNA, a conserved phenomenon in Solanaceae. Potato breeding programs rely on the discovery and introgression of genes of interest, however GSI severely limits the ability to develop inbred lines used to improve traits in traditional breeding schemes. SUMMARY OF THE INVENTION Applicants have surprisingly found that modulation of HT activity can be used to develop self-compatible diploid potatoes and other Solanaceae species. The invention provides methods for creating hybrid plants and inbred lines by modulating HT genes and plants produced by such methods for breeding and agronomic trait improvement and varietal development. The present invention provides a method for genetic modification of plants to reduce or eliminate self-incompatibility. Using the methods and materials of the present invention, self-incompatibility may be induced, increased, decreased, repressed or otherwise altered, in a transformed plant relative to an untransformed control plant, for example by incorporating additional copies of a sense nucleic acid of the present invention, preferably to overexpress the polypeptide or in sense suppression. They may be decreased or otherwise altered, for example by incorporating an antisense nucleic acid of the present invention or preferably by chromosomal insertion or deletion of sequences through the use of gene editing techniques. In a further aspect, the present invention provides a method for altering the self-incompatibility status of a plant, said method including identifying a gene encoding an HT polypeptide which is active in the self-incompatibility pathway of the plant and up-regulating or down-regulating expression of said gene to repress or induce the self-incompatibility mechanism in said plant. Preferably said gene is a nucleic acid according to the present invention. Preferably the plant is as hereinbefore described. The up-regulation or down-regulation may be carried out by methods known to those skilled in the art. For example, a gene may be upregulated by incorporating additional copies of a sense copy of the gene. A gene may be down-regulated, for example, by incorporating an antisense nucleic acid, a frame-shifted or otherwise modified sense copy of the gene, or nucleic acid encoding interfering RNA (RNAi). Up or down regulation may also be achieved through the use of transcription activator-like effector nucleases or zinc-finger nucleases, mediating cleavage of specific target sites in the nucleic acid, leading to micro-deletions and insertions within the endogenous nucleic acid sequence. One of several genome modification techniques such as genome editing technologies, including but not limited to RTDS, TALEN, CRISPR-Cas9, CRISPR-Cmsl, ARCUS or base editing, chemical mutagenesis, RNAi, antisense, etc. may be used to introduce a deletion, insertion, or substitution in an HT gene to lead to the reduction or elimination of the HT activity, thereby reducing the level of functionally active endogenous HT and reducing and/or preventing activation of self-incompatibility. Applicants have a targeted knock-out of HT genes in self-incompatibility potato lines using Clustered Regularly Interspaced Short Palindrom