Conferring cytoplasmic male sterility

Abstract

Provided herein are methods for conferring cytoplasmic male sterility (CMS) in plant lines. The methods comprise obtaining a first plant comprising a CMS cytoplasm, which is also a haploid inducer (CHIP), and hybridizing the first plant to a second plant comprising a desired nuclear genome (DIP). The CHIP further comprises a cenh3 mutation, and may contain an anthocyanin marker and a restorer factor. The method further comprises generating a progeny from the hybridization. The filial generation resulting from hybridization of the method is a haploid and comprises the CMS cytoplasm of the CHIP and the desired nuclear genome of the DIP. The progeny further lacks any anthocyanin marker or restorer factor.

Description

Technical Field

[0001] The subject matter disclosed in the present application generally relates to the field of plant breeding. More particularly, the subject matter relates to methods for conferring CMS (cytoplasmic male sterility) to plant lines. In addition, the subject matter disclosed in the present application relates to specific features of CMS inducing lines (e.g., CENH3 editing) used in transformation methods. In addition, the subject matter generally relates to haploid induction.

[0002] Sequence Listing

[0003] A sequence listing is attached to this application, which is named 82657-WO-REG-ORG-P-1.xml, created on May 5, 2023, and is approximately 187 kilobytes in size. The sequence listing is incorporated herein by reference in its entirety. The sequence listing is submitted with this application via EFS-Web and complies with 37 C.FR §1.824(a)(2)-(6) and (b). Background Art

[0004] In the production of corn hybrids, male sterile corn inbreds can be used to eliminate the need for emasculation, thereby significantly saving costs. The maternal genetic trait known as cytoplasmic male sterility (CMS) is a useful tool for effectively producing hybrid seeds. Three main types of CMS (CMS-T, CMS-S and CMS-C) have been identified. Nuclear genes known as fertility restorer lines (Rf) can overturn the male sterility effect of the cytoplasm. Lines with Rf genes produce functional pollen even if they still carry CMS cytoplasm. Known restorer loci Rf1 and Rf2 suppress the CMS phenotype of CMS-T lines, while the Rf3 gene restores the fertility of CMS-S lines. See Laughnan et al. Annu. Rev. Genet. [Genetics Yearbook] 1983. 17: 27-48. The restorer locus Rf4 of CMS-C is mapped to chromosome 8 reported by Sisco et al. in Crop Science [Crop Science] Vol. 31, No. 5, pp. 1263-1266, 1991. Kohls et al. reported the fine mapping of Rf4 at the 2010 Maize Genetics Conference. In addition, a patent application published as WO 2012047595 (incorporated herein by reference) reports the identification of genes and markers associated with Rf4. Breeders use the CMS system to produce hybrid seeds by developing female strains that carry CMS cytoplasm but lack restorer genes and developing male strains that carry appropriate restorer genes. The F1 hybrid seeds produced by the female strains carry CMS cytoplasm, but produce fertile plants due to the action of the nuclear restorer genes contributed by the father. For corn, cell type C is identified as a desired CMS system because it is generally stable in various environments and genetic backgrounds.

[0005] In order to form hybrid seeds, breeders hybridize inbred parent strains, one serves as male parent, and one serves as female parent. The process of developing substantially isozygous inbred parent strains usually requires hybrids to carry out selection and self-pollination (selfing) of multiple generations to become close to isozygous. This process is time-consuming and expensive. In order to shorten the time of developing isozygous inbreds in corn, rice, wheat, barley and other crops, breeders can choose to use haploid induction system to induce the haploid seeds of hybrid parents to produce. Then the chromosome of haploid plant is doubled, for example, by chromosome doubling agent such as colchicine, to form double haploid isozygous strains. In many plant species (such as sorghum, barley, wheat and other grasses), haploid induction has been observed.

[0006] In maize, haploid induction (HI) is associated with the genes MATRILINEAL, ig1, CENH3, and DMP. See generally WO 2017 / 087682 (incorporated herein by reference); T. Kelliher et al., MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction, Nature 542, 105-109 (2017); U.S. Pat. No. 7,439,416 (incorporated herein by reference); M Pollacsek, Management of the ig gene for haploid induction in maize, Agronomie, 12: 247-251 (1991); U.S. Pat. No. 8,618,354 (incorporated herein by reference); see also ABBritt and S. Kuppu, Cenh3: An Emerging Player in Haploid Induction Technology [Cenh3: An emerging player in haploid induction technology], Front. Plant Sci. [Plant Science Frontier] 7: 357 (2016) doi: 10.3389 / fpls.2016.00357; and CN 111763687B (incorporated herein by reference). Haploid induction lines can be established in various ways, such as by genome editing. As disclosed herein, for example, haploid induction lines can be generated by CENH3 mutations. CENH3 is a centromere-specific variant of HISTONE3 (H3) and is required for kinetochore nucleation and spindle attachment in mitosis and meiosis.

[0007] In the presently disclosed subject matter, CMS-C type lines with cenh3 mutations serve as haploid inducer lines in the CMS transformation method. Summary of the invention

[0008] Methods for converting corn lines with normal cell types into cytoplasmic male sterile (CMS) plants are beneficial for saving the cost and time of hybrid seed production. The current industry standard uses trait introgression to hybridize the superior lines in the female heterosis pool to become CMS cytoplasm. This article discloses a method for achieving this conversion to CMS cytoplasm without introgression and instead by a haploid induction process. This process results in 100% conversion of the nuclear genome to CMS cytoplasm in a single cross. In this method, the first plant (referred to herein as "CHIP") is CMS, and the first plant is heterozygous for a knockout mutation in CENH3 in its nuclear genome (i.e., one allele of CENH3 is wild type; "CENH3+ / -"), and is a male haploid induction line (i.e., it can produce male haploids). Optionally, CHIP can also contain restorer factors (e.g., Rf3, Rf4, Rf10, Rf11, etc.) in its nuclear genome, and if so, although it has a CMS cell type, it is still self-fertile. Alternatively, CHIP can also contain non-restorer alleles (e.g., rf3, rf4, rf10, rf11, rf12). If so, CHIP is male sterile and requires a maintainer line to continue breeding. The maintainer line is not CMS (i.e., has normal cytoplasm). In addition, the optional anthocyanin marker in CHIP is homozygous.

[0009] The second plant (referred to herein as "DIP") is not a haploid induction line (i.e., is homozygous wild type for CENH3 ("CENH3+ / +")), has a normal cytotype (i.e., non-cytoplasmic male sterility), and contains the desired nuclear genome to be converted to CMS cytoplasm. The DIP is the pollen donor.

[0010] CHIP is crossed with DIP pollen, and the offspring includes diploid progeny ("F1"), haploid progeny (containing only the nuclear genome of DIP), and aneuploid progeny. Only haploid progeny are desired, and will contain (i) a haploid nuclear genome (i.e., the desired genome) of DIP with one wild-type CENH3 allele ("CENH3+ / null"), and (ii) a CMS-C cell type, (iii) further lacking a restorer factor, and optionally (iv) a visual marker.

[0011] When used, visual markers can be used, such as anthocyanin markers (e.g., R1-nj or R1-scm2) to distinguish undesirable diploid F1 progeny from desired haploid progeny. If used, the marker is present in the haploid induction system genome (i.e., CHIP) in a homozygous state. Aneuploid is distinguished from true haploid progeny by using the genetic markers of the induction system parents, i.e., any presumed haploid showing female (induction system) parental markers (indicating CHIP nuclear DNA) will be discarded as aneuploid. Alternatively, haploid progeny can be distinguished from diploid and aneuploid by sequencing, selective markers, plant type, or ploidy level measurement.

[0012] Brief description of the sequences in the sequence listing

[0013] SEQ ID NO: 1 is the nucleotide sequence of gRNA140 carried by LbCas12a RNP in bioballistic bombardment of SYN-INBC34 inbred immature embryos of Example 1 and SYN-INBC34 x SYN-INBC34RS inbred immature embryos of Example 2, wherein gRNA140 targets the second exon of gene ID GRMZM2G158526.

[0014] SEQ ID NO: 2 is the nucleotide sequence of the primer used in TaqMan assay 3895.

[0015] SEQ ID NO: 3 is the nucleotide sequence of the primer used in TaqMan assay 3895.

[0016] SEQ ID NO:4 is the nucleotide sequence of the probe used in TaqMan assay 3895.

[0017] SEQ ID NO:5 is a nucleotide sequence representing a 19 base pair deletion in cenh3.

[0018] SEQ ID NO:6 is a nucleotide sequence representing a 10 base pair deletion in cenh3.

[0019] SEQ ID NO:7 is a partial nucleotide sequence of wild-type cenh3 (GRMZM2G158526).

[0020] SEQ ID NO:8 is a partial nucleotide sequence of a cenh3 10 base pair deletion mutant (509A115A).

[0021] SEQ ID NO:9 is a partial nucleotide sequence of a cenh3 19 base pair deletion mutant (509A151A).

[0022] SEQ ID NO: 10 is a partial amino acid sequence of the cenh3 allele (GRMZM2G158526).

[0023] SEQ ID NO: 11 is a partial amino acid sequence of the deduced amino acid sequence of the KD (Kelly Dawe, University of Georgia) cenh3 allele.

[0024] SEQ ID NO: 12 is a partial amino acid sequence of a cenh3 allele having a 10 base pair deletion.

[0025] SEQ ID NO: 13 is a partial amino acid sequence of a cenh3 allele having a 19 base pair deletion splice variant a.

[0026] SEQ ID NO: 14 is a partial amino acid sequence of a cenh3 allele having a 19 base pair deletion splice variant c.

[0027] SEQ ID NO: 15 is a partial amino acid sequence of a cenh3 allele having a 19 base pair deletion splice variant b.

[0028] SEQ ID NO: 16 is a partial amino acid sequence of a cenh3 allele having a 19 base pair deletion splice variant d.

[0029] SEQ ID NO: 17 is a partial amino acid sequence of a cenh3 allele having a 19 base pair deletion splice variant e.

[0030] SEQ ID NO: 18 is a partial amino acid sequence of a cenh3 allele having a 10 base pair deletion splice variant a.

[0031] SEQ ID NO: 19 is a partial amino acid sequence of a cenh3 allele having a 10 base pair deletion splice variant b.

[0032] SEQ ID NO: 20 is a partial amino acid sequence of a cenh3 allele having a 10 base pair deletion splice variant c.

[0033] SEQ ID NO:21 is a partial amino acid sequence of a cenh3 allele having a 10 base pair deletion splice variant d.

[0034] SEQ ID NO:22 is a partial amino acid sequence of a cenh3 allele having a 10 base pair deletion splice variant e.

[0035] SEQ ID NO: 23 is the nucleotide sequence of the forward primer used to measure SM0253EQ.

[0036] SEQ ID NO: 24 is the nucleotide sequence of the reverse primer used to measure SM0253EQ.

[0037] SEQ ID NO: 25 is the nucleotide sequence of a probe used to measure SM0253EQ.

[0038] SEQ ID NO: 26 is the nucleotide sequence of a probe used to measure SM0253EQ.

[0039] SEQ ID NO: 27 is the nucleotide sequence used to determine the target of SM0253EQ.

[0040] SEQ ID NO: 28 is the nucleotide sequence of the forward primer used for measuring SM0093B.

[0041] SEQ ID NO: 29 is the nucleotide sequence of the reverse primer used for measuring SM0093B.

[0042] SEQ ID NO: 30 is the nucleotide sequence of a probe used to measure SM0093B.

[0043] SEQ ID NO: 31 is the nucleotide sequence of a probe for measuring SM0093B.

[0044] SEQ ID NO:32 is the nucleotide sequence used to determine the target of SM0093B.

[0045] SEQ ID NO:33 is the nucleotide sequence of the forward primer used to measure SM0435A.

[0046] SEQ ID NO:34 is the nucleotide sequence of the reverse primer used to measure SM0435A.

[0047] SEQ ID NO: 35 is the nucleotide sequence of a probe for measuring SM0435A.

[0048] SEQ ID NO: 36 is the nucleotide sequence of a probe for measuring SM0435A.

[0049] SEQ ID NO:37 is the nucleotide sequence used to determine the target of SM0435A.

[0050] SEQ ID NO: 38 is the nucleotide sequence of the forward primer used for measuring SM1071CQ.

[0051] SEQ ID NO: 39 is the nucleotide sequence of the reverse primer used for measuring SM1071CQ.

[0052] SEQ ID NO: 40 is the nucleotide sequence of a probe for measuring SM1071CQ.

[0053] SEQ ID NO: 41 is the nucleotide sequence of a probe for measuring SM1071CQ.

[0054] SEQ ID NO:42 is the nucleotide sequence used to determine the target of SM1071CQ.

[0055] SEQ ID NO: 43 is the nucleotide sequence of the forward primer used for measuring SM1280CQ.

[0056] SEQ ID NO: 44 is the nucleotide sequence of the reverse primer used for measuring SM1280CQ.

[0057] SEQ ID NO: 45 is the nucleotide sequence of a probe for measuring SM1280CQ.

[0058] SEQ ID NO: 46 is the nucleotide sequence of a probe for measuring SM1280CQ.

[0059] SEQ ID NO: 47 is the nucleotide sequence used to determine the target of SM1280CQ.

[0060] SEQ ID NO:48 is the nucleotide sequence of the forward primer used for measuring SM1447AQ.

[0061] SEQ ID NO: 49 is the nucleotide sequence of the reverse primer used for measuring SM1447AQ.

[0062] SEQ ID NO: 50 is the nucleotide sequence of a probe for measuring SM1447AQ.

[0063] SEQ ID NO: 51 is the nucleotide sequence of a probe for measuring SM1447AQ.

[0064] SEQ ID NO:52 is the nucleotide sequence used to determine the target of SM1447AQ.

[0065] SEQ ID NO: 53 is the nucleotide sequence of the forward primer used for measuring SM1847AQ.

[0066] SEQ ID NO:54 is the nucleotide sequence of the reverse primer used for measuring SM1847AQ.

[0067] SEQ ID NO: 55 is the nucleotide sequence of a probe for measuring SM1847AQ.

[0068] SEQ ID NO: 56 is the nucleotide sequence of a probe for measuring SM1847AQ.

[0069] SEQ ID NO: 57 is the nucleotide sequence used to determine the target of SM1847AQ.

[0070] SEQ ID NO:58 is the nucleotide sequence of the forward primer used for measuring SM1286AQ.

[0071] SEQ ID NO:59 is the nucleotide sequence of the reverse primer used for measuring SM1286AQ.

[0072] SEQ ID NO: 60 is the nucleotide sequence of a probe for measuring SM1847AQ.

[0073] SEQ ID NO: 61 is the nucleotide sequence of a probe for measuring SM1847AQ.

[0074] SEQ ID NO:62 is the nucleotide sequence used to determine the target of SM1286AQ.

[0075] SEQ ID NO: 63 is the nucleotide sequence of the forward primer used for measuring SM2513.

[0076] SEQ ID NO: 64 is the nucleotide sequence of the reverse primer used for measuring SM2513.

[0077] SEQ ID NO: 65 is the nucleotide sequence of a probe for measuring SM2513.

[0078] SEQ ID NO:66 is the nucleotide sequence of a probe for measuring SM2513. SEQ ID NO:67 is the nucleotide sequence of a target for measuring SM2513. SEQ ID NO:68 is the nucleotide sequence of a forward primer for measuring SM2962. SEQ ID NO:69 is the nucleotide sequence of a reverse primer for measuring SM2962. SEQ ID NO:70 is the nucleotide sequence of a probe for measuring SM2962. SEQ ID NO:71 is the nucleotide sequence of a probe for measuring SM2962. SEQID NO:72 is the nucleotide sequence of a target for measuring SM2962. SEQ ID NO:73 is the nucleotide sequence of a forward primer for measuring SM2988. SEQ ID NO:74 is the nucleotide sequence of a reverse primer for measuring SM2988. SEQ ID NO:75 is the nucleotide sequence of a probe for measuring SM2988. SEQ ID NO:76 is the nucleotide sequence of a probe for measuring SM2988. SEQ ID NO:77 is a nucleotide sequence for determining a target of SM2988. SEQ ID NO:78 is a nucleotide sequence of a forward primer for determining SM3400. SEQ ID NO:79 is a nucleotide sequence of a reverse primer for determining SM3400. SEQ ID NO:80 is a nucleotide sequence of a probe for determining SM3400. SEQ ID NO:81 is a nucleotide sequence of a probe for determining SM3400. SEQ ID NO:82 is a nucleotide sequence for determining a target of SM3400. SEQ ID NO:83 is a nucleotide sequence of a forward primer for determining SM3747. SEQ ID NO:84 is a nucleotide sequence of a reverse primer for determining SM3747. SEQ ID NO:85 is a nucleotide sequence of a probe for determining SM3747. SEQ ID NO:86 is a nucleotide sequence of a probe for determining SM3747. SEQ ID NO:87 is a nucleotide sequence for determining a target of SM3747. SEQ ID NO:88 is the nucleotide sequence of a forward primer for measuring SM4788. SEQ ID NO:89 is the nucleotide sequence of a reverse primer for measuring SM4788. SEQ ID NO:90 is the nucleotide sequence of a probe for measuring SM4788. SEQ ID NO:91 is the nucleotide sequence of a probe for measuring SM4788. SEQ ID NO:92 is the nucleotide sequence of a target for measuring SM4788. SEQ ID NO:93 is the nucleotide sequence of a forward primer for measuring SM5515. SEQ ID NO:94 is the nucleotide sequence of a reverse primer for measuring SM5515.SEQ ID NO: 95 is the nucleotide sequence of a probe for measuring SM5515.

[0079] SEQ ID NO:96 is the nucleotide sequence of a probe for measuring SM5515.

[0080] SEQ ID NO: 97 is a nucleotide sequence used to determine the target of SM5515.

[0081] SEQ ID NOs: 98-100 are the nucleotide sequences of primers and probes used to assay PM1901 (wild-type ZmCENH3).

[0082] SEQ ID NOs: 101-103 are the nucleotide sequences of primers and probes used to assay PM1909 (a mutation comprising a 10 bp deletion in ZmCENH3).

[0083] SEQ ID NOs: 101, 102 and 104 are the nucleotide sequences of primers and probes used for determining PM1913 (a mutation comprising a 19 bp deletion in ZmCENH3).

[0084] SEQ ID NOs: 105-108 are the nucleotide sequences of primers and probes used to measure SM0576CQ.

[0085] SEQ ID NOs: 109-112 are the nucleotide sequences of primers and probes used to measure SM0956IQ.

[0086] SEQ ID NOs: 113-116 are the nucleotide sequences of primers and probes used for measuring SM2669.

[0087] SEQ ID NOs: 117-120 are the nucleotide sequences of primers and probes used for measuring SM2670.

[0088] SEQ ID NOs: 121-124 are the nucleotide sequences of primers and probes used for measuring SM2915.

[0089] SEQ ID NOs: 125-128 are the nucleotide sequences of primers and probes used for measuring SM2916.

[0090] SEQ ID NOs: 129-132 are the nucleotide sequences of primers and probes used for measuring SM6623.

[0091] SEQ ID NOs: 133-136 are the nucleotide sequences of primers and probes used for measuring SM8040.

[0092] SEQ ID NOs: 137-140 are the nucleotide sequences of primers and probes used for measuring SM8091.

[0093] SEQ ID NOs: 141-144 are the nucleotide sequences of primers and probes used for measuring SM2918.

[0094] SEQ ID NOs: 145-148 are the nucleotide sequences of primers and probes used for measuring SM4813.

[0095] SEQ ID NOs: 149-152 are the nucleotide sequences of primers and probes used for measuring SM2914.

[0096] SEQ ID NOs: 153-156 are the nucleotide sequences of primers and probes used for measuring SM4812.

[0097] SEQ ID NOs: 157-160 are the nucleotide sequences of primers and probes used to measure SM0954BQ.

[0098] SEQ ID NOs: 161-164 are the nucleotide sequences of primers and probes used for measuring SM6568.

[0099] SEQ ID NOs: 165-168 are the nucleotide sequences of primers and probes used to measure SM0953BQ.

[0100] SEQ ID NOs: 169-172 are the nucleotide sequences of primers and probes used for measuring SM7200.

[0101] SEQ ID NOs: 173-176 are the nucleotide sequences of primers and probes used for measuring SM5665.

[0102] SEQ ID NO: 177 is a partial nucleotide sequence of the cenh3 19 base pair deletion mutant from Table 1 (509A150A).

[0103] definition

[0104] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art. Reference to the technology employed herein is intended to refer to the technology commonly understood in the art, including variations of those technologies and / or alternatives to equivalent technologies that are well known to those of ordinary skill in the art. Although it is believed that the following terms can be well understood by those of ordinary skill in the art, the following definitions are stated to facilitate explanation of the subject matter disclosed in this application.

[0105] In accordance with long-standing patent law conventions, the terms "a / an" and "the" as used in this application (including the claims) mean "one or more". For example, the phrase "a / a cell" refers to one or more cells, and in some embodiments may refer to tissues and / or organs. Similarly, the phrase "at least one / a kind", when used herein to refer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 or more of the entity, including but not limited to all integer values ​​between 1 and 100 and integers greater than 100.

[0106] Unless otherwise indicated, all numbers expressing amounts of ingredients, reaction conditions, and the like used in the specification and claims are to be understood as modified in all instances by the term "about". As used herein, the term "about", when referring to a measurable value such as an amount of mass, weight, time, volume, concentration, or percentage, is meant to encompass variations of ±20% from the specified amount in some embodiments, ±10% from the specified amount in some embodiments, ±5% from the specified amount in some embodiments, ±1% from the specified amount in some embodiments, ±0.5% from the specified amount in some embodiments, and ±0.1% from the specified amount in some embodiments, as such variations are suitable for performing the disclosed methods and / or using the disclosed compositions, nucleic acids, polypeptides, and the like. Therefore, unless indicated to the contrary, the numerical parameters set forth in the specification and the appended claims are approximate values ​​that may vary depending upon the desired properties sought to be obtained by the subject matter disclosed herein.

[0107] As used herein, the term "allele" refers to a variant or alternative sequence form at a genetic locus. In a diploid, a single allele at each locus is inherited by the progeny from each parent, respectively. Although one of ordinary skill in the art understands that the alleles in any particular individual do not necessarily represent all the alleles present in the species, the two alleles present in a given locus in a diploid organism occupy corresponding positions on a pair of homologous chromosomes.

[0108] As used herein, the term "amplification" means using at least one nucleic acid molecule as a template to construct multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule. Amplification systems include polymerase chain reaction (PCR) systems, ligase chain reaction (LCR) systems, nucleic acid sequence-based amplification (NASBA, Cangene, Mississauga, Ontario), Q-β replicase systems, transcription-based amplification systems (TAS) and strand displacement amplification (SDA). See, for example, Diagnostic Molecular Microbiology: Principles and Applications, PERSING et al., American Society for Microbiology, Washington, DC, (1993). The product of amplification is referred to as "amplicon".

[0109] As used herein, the term "and / or," when used in the context of listing entities, refers to the entities either alone or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D (e.g., AB, AC, AD, BC, BD, CD, ABC, ABD, and BCD). In some embodiments, the one or more elements referred to by "and / or" may also be present alone in single or multiple occurrences in one or more combinations and / or one or more subcombinations.

[0110] The term "aneuploid" refers to a plant having an abnormal number of chromosomes in the haploid set.

[0111] As used herein, the term "backcrossing" or "backcrossed" is understood within the scope of the present invention to refer to a process in which hybrid progeny are repeatedly crossed back to one of the parents.

[0112] The terms "bombarding" and "bioballistic bombardment" refer to the process of accelerating particles toward a target biological sample (e.g., cells, tissues, etc.) to damage the cell membranes of cells in the target biological sample and / or to allow the particles to enter the target biological sample. Methods for bioballistic bombardment are known in the art (e.g., US 5,584,807) and are commercially available (e.g., helium-driven accelerators (PDS-1000 / He from BioRad)). TM)). The Bioballistic PDS-1000 Gene Gun (Bio-Rad, Hercules, CA) uses helium pressure to accelerate gold or tungsten microparticles coated with DNA toward target cells.

[0113] As used herein, the term "bulk" refers to the process of increasing the number of seeds.

[0114] As used herein, the term "cDNA" refers to a single-stranded or double-stranded DNA that is complementary to and derived from mRNA.

[0115] As used herein, the term "CHIP" refers to one of the parents of the first hybridization in the method of the present invention. This parent has a heterozygous cenh3 mutation (C) in its nuclear genome, is a paternal haploid induction line (HI), and, as described above, is one of the parents (P). CHIP is female fertile and CMS male sterile. CHIP may optionally contain a homozygous restorer factor in its nuclear genome. CHIP may also optionally contain a homozygous anthocyanin marker in its nuclear genome.

[0116] The term "chromosome" as used herein is as recognized in the art and refers to a self-replicating genetic structure in the nucleus of a cell that contains the cell's DNA and has a linear array of genes.

[0117] The term "comprising" is synonymous with "including," "containing," and "characterized by," is inclusive or open-ended, and does not exclude additional, unrecited elements and / or method steps. "Comprising" is a term that means the specified elements and / or steps are present, but other elements and / or steps may be added and still fall within the scope of the relevant subject matter.

[0118] As used herein, the phrase "consisting of" excludes any elements, steps or ingredients not specifically recited. When the phrase "consisting of" appears in a clause of the body of a claim, rather than directly following the preamble, it limits only the elements set forth in the clause; other elements are not excluded from the claim as a whole.

[0119] As used herein, the phrase "consisting essentially of limits the scope of the associated disclosure or claim to the specified materials and / or steps, plus those that do not materially affect one or more basic and novel characteristics of the disclosed and / or claimed subject matter.

[0120] As used herein, the term "cell exchange" refers to the exchange of cytoplasm from one line to another (eg, "normal A" cytoplasm in a corn line is exchanged to another corn line that initially had "normal B" cytoplasm).

[0121] As used herein, the term "DIP" refers to one of the parents of the first cross in the method of the present invention. This parent contains the desired haploid nuclear genome ("desired parent" or "DIP"). The DIP is self-fertile (homozygous for wild-type CENH3) and has a normal cell type.

[0122] As used herein, a plant referred to as "haploid" has two complete sets of chromosomes (2n; one set from each parent).

[0123] As used herein, the term "superior line" or "inbred line" refers to any line resulting from breeding and selection for superior agronomic performance. A superior line has stable genetics, i.e., it is reasonably or nearly isogenic in its genome. In other words, a superior line is reasonably or nearly homozygous for all alleles in its genome.

[0124] As used herein, when used with a reference polynucleotide (such as a gene, ORF or part thereof, or transgenic of a plant), the term "expression" refers to a method in which the genetic information encoded in a gene is converted into RNA (e.g., mRNA, rRNA, tRNA or snRNA) by "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and is converted into a protein by "translation" of the mRNA in applicable cases (e.g., if the gene encodes a protein). Gene expression can be regulated at many stages of the method. For example, in the case of an antisense construct or a dsRNA construct, expression may refer only to the transcription of the antisense RNA or only to the transcription of the dsRNA. In an embodiment, "expression" refers to the transcription and stable accumulation of a sense (mRNA) or functional RNA. "Expression" may also refer to the production of a protein.

[0125] As used herein, the term "gene" refers to a unit of inheritance comprising a DNA sequence that occupies a specific location on a chromosome and contains the genetic instructions for a specific characteristic or trait in an organism.

[0126] As used herein, the term "genotype" refers to the genetic makeup of a cell or organism. "A group of genetically marked genotypes" of an individual include the specific alleles of one or more genetic marker loci present in the individual. As known in the art, genotypes can relate to a single locus or multiple loci, whether these loci are relevant or unrelated, and / or are linked or non-linked. In certain embodiments, the genotype of an individual relates to one or more relevant genes, because one or more of these genes participate in the expression of a target phenotype (e.g., quantitative traits as defined herein). Therefore, in certain embodiments, genotypes include the summation of one or more alleles present at one or more genetic loci of an individual intrinsic quantitative trait. In certain embodiments, genotypes are represented by haplotypes (defined below).

[0127] As used herein, the term "germplasm" refers to the totality of the genotype of a population or other individual group (e.g., species). The term "germplasm" may also refer to plant material; for example, a group of plants that serve as a repository for various alleles. The phrase "adapted germplasm" refers to plant material that has been confirmed to have genetic advantages; for example, for a given environment or geographic region, the phrases "unadapted germplasm," "original germplasm," and "exotic germplasm" refer to plant material with unknown or unconfirmed genetic value; for example, for a given environment or geographic region; like this, the phrase "unadapted germplasm" refers in some embodiments to plant material that does not belong to an established breeding population and has no known relationship with a member of an established breeding population.

[0128] As used herein, a plant referred to as a "haploid" has a single chromosome set (genome), and the number of chromosomes (1n) reduced in a haploid plant is equal to the number of chromosomes in a gamete. As used herein, a plant referred to as a "double haploid" is developed by doubling the haploid chromosome set (from 1n to 2n). Plants or seeds obtained from selfing to any number of generations of double haploid plants can still be identified as double haploid plants. Double haploid plants are considered to be homozygous plants. If a plant is fertile, even if the entire vegetative part of the plant is not composed of cells with a doubled chromosome set, the plant is considered to be a double haploid; that is, if a plant contains surviving gametes, even if it is mosaic, it will also be considered to be a double haploid.

[0129] As used herein, "Haploid BC1" refers to the progeny of a haploid plant that has been pollinated with its recurrent parent in order to perform backcrossing in the progeny. The haploid BC1 progeny comprises a diploid genome.

[0130] As used herein, haploid induction rate ("HIR") means the number of haploid kernels that survive pollination of an ear with pollen from a haploid induction line divided by the total number of kernels.

[0131] As used herein, when referring to a gene or nucleic acid, the term "heterologous" means that the gene encoding a factor is not in its natural environment (i.e., has been artificially altered). For example, a heterologous gene may include a gene introduced from one species into another species. A heterologous gene may also include a gene that is native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer polynucleotide, etc.). A heterologous gene may further comprise a plant gene polynucleotide comprising a cDNA form of a plant gene; the cDNA may be expressed in a sense direction (to produce mRNA) or an antisense direction (to produce an antisense RNA transcript complementary to an mRNA transcript). In one aspect of the invention, heterologous genes are distinguished from endogenous plant genes in that the heterologous gene polynucleotides are typically joined to polynucleotides containing regulatory elements such as promoters, these polynucleotides are not found naturally associated with the gene for the protein encoded by the heterologous gene or with plant gene polynucleotides in chromosomes, or these polynucleotides are associated with parts of chromosomes not found in nature (e.g., genes expressed in loci that do not normally express the gene). In addition, in embodiments, a "heterologous" polynucleotide is a polynucleotide that is not naturally associated with the host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring polynucleotide.

[0132] As used herein, the term "heterozygous" refers to a genetic condition that exists when different alleles are located at corresponding loci on homologous chromosomes.

[0133] As used herein, the term "homozygous" refers to a genetic condition that exists when identical alleles are located at corresponding loci on homologous chromosomes.

[0134] As used herein, the term "human-induced mutation" refers to any mutation that occurs due to direct or indirect human action. This term includes, but is not limited to, mutations obtained by any targeted mutagenesis method.

[0135] As used herein, the term "hybrid" refers to the offspring produced by hybridizing two genetically different parental plants. The resulting progeny of this hybridization is the "biparental" population. In the context of plant breeding, the terms "hybrid", "hybrid plant" and "hybrid progeny" refer to plants that are the offspring of genetically different parents produced by hybridizing plants of different strains or varieties or species, including but not limited to hybridization between two inbred strains (e.g., genetically heterozygous or mostly heterozygous individuals). The phrase "single cross F1 hybrid" refers to the F1 hybrid produced by the hybridization between two inbred strains.

[0136] The terms "identity" or "identical" in the context of two nucleic acid or amino acid sequences refers to the percentage of identical nucleotides or amino acids in the linear polynucleotide or amino acid sequence of a reference ("query") sequence (or its complementary strand) when the two sequences are fully aligned, such as compared to a test ("subject") sequence. Unless otherwise indicated, sequence identity as used herein refers to the value obtained by using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol. 48:443-453) implemented in the EMBOSS Needle alignment tool, using the default matrix file EBLOSUM62 (for proteins) and default parameters (gap open=10, gap extension=0.5, terminal gap penalty=false, terminal gap open=10, terminal gap extension=0.5) or DNAfull (for nucleic acids) and default parameters (gap open=10, gap extension=0.5, terminal gap penalty=false, terminal gap open=10, terminal gap extension=0.5); or any equivalent program thereof. EMBOSS Needle is available, for example, from EMBL-EBI, for example at the following website: ebi.ac.uk / Tools / psa / emboss_needle / and as described in the following publication: "The EMBL-EBI search and sequence analysis tools APIs in 2019." Madeira et al. Nucleic Acids Research, June 2019, 47(W1): W636-W641. As used herein, the term "equivalent program" refers to any sequence comparison program that, for any two sequences in question, generates an alignment with identical nucleotide or amino acid residue matches and identical percentages of sequence identity when compared to the corresponding alignment generated by EMBOSS Needle. In some embodiments, substantially identical nucleic acid or amino acid sequences can perform substantially the same function.

[0137] The phrase "inbred strain" refers to a population that is homozygous or nearly homozygous genetically. For example, an inbred strain can be obtained by several cycles of brother / sister breeding or self-fertilization. In certain embodiments, an inbred strain is pure bred for one or more phenotypic traits of interest. "Inbreeding", "inbred individuals" or "inbred progeny" are separate samples from an inbred strain. The term "inbreeding" refers to an individual or strain that is substantially homozygous.

[0138] As used herein, "introduction" refers to delivery, expression, administration, transportation, transfer, penetration or other similar terms to indicate the delivery of a desired nucleic acid or protein or a combination thereof to an object. For example, a nucleic acid encoding a site-directed nuclease and optionally at least one guide RNA can be introduced into a haploid embryo by haploid induction. Similarly, existing editing machinery (comprising a site-directed nuclease protein and optionally at least one guide RNA) can be introduced into a haploid embryo by administering a suitable cell-penetrating peptide.

[0139] As used herein, the terms "introgression," "introgressed," and "introgressing" refer to natural and artificial processes in which a genomic region of one species, variety, or cultivar is moved to the genome of another species, variety, or cultivar by crossing the other species, variety, or cultivar. The process can optionally be accomplished by backcrossing to the recurrent parent.

[0140] As used herein, when used in the context of nucleic acid molecules of the present invention or polynucleotides, the term "isolated" refers to polynucleotides that are identified and separated / separated in the context of chromosome polynucleotides in the corresponding source organism. If the isolated nucleic acid or polynucleotides do have naturally occurring counterparts, the isolated nucleic acid or polynucleotides are not nucleic acids that exist in their natural environment. In contrast, non-isolated nucleic acids are nucleic acids (such as DNA and RNA), which are found under the state of nature. For example, given polynucleotides (such as genes) are found on the host cell chromosome near adjacent genes. Isolated nucleic acid molecules can exist in single-stranded or double-stranded forms. Alternatively, it can include sense strands and antisense strands (that is, nucleic acid molecules can be double-stranded). In a preferred embodiment, nucleic acid molecules of the present invention are interpreted as isolated.

[0141] As used herein, the term "knockout mutation" refers to a gene mutation in which the expression of the gene is stopped or 'knocked out.' This mutation may include, but is not limited to, a mutation obtained by any targeted mutagenesis method.

[0142] As used herein, the term "locus" refers to a location on a chromosome in a given species (eg, the location of a gene, genetic marker, etc.).

[0143] As used herein, "maternal haploid inducer" refers to a line that produces pollen and, when crossed as a male, results in gynogenetic development of haploid seeds. "Paternal haploid inducer" refers to a line that, when used as a female in a cross, results in androgenetic development of haploid seeds. Haploid inducer plants can use any of these maternal or paternal mechanisms to derive haploids.

[0144] As used herein, the term "maintainer line" refers to a plant line that is male fertile, comprises normal cytoplasm, is substantially genetically similar to a CMS plant line (e.g., isogenic), and is used to maintain a reserve of a CMS induction line. The maintainer line is preferably homozygous for non-restorer alleles (e.g., rf4) and R1 color markers (e.g., R1-nj or R1-SCM2). In addition, the maintainer line may comprise a CenH3 mutation. In this article, the maintainer line is used as a male for hybridization with a CMS line, which may or may not comprise a CenH3 mutation. As used herein, the term "normal cytoplasm" refers to a fertile cell type (as opposed to cytoplasmic male sterility). The maintainer line may be self-pollinated to increase its seed reserve.

[0145] As used herein, when identifying the presence / absence of the locus associated with HI, the term "molecular marker" can be used to refer to genetic markers as defined above, or its coded product (e.g., protein) as a reference point. Molecular markers can be derived from genomic nucleotide sequences or expressed nucleotide sequences (e.g., derived from RNA, cDNA, etc.). The term also refers to a nucleotide sequence complementary to a marker sequence or to a nucleotide sequence located on its flank, such as a nucleotide sequence used as a probe and / or primer that can amplify a marker sequence. When a nucleotide sequence is specifically hybridized in a solution, these nucleotide sequences are "complementary" (e.g., according to Watson-Crick base pairing principle). This term also refers to a genetic marker that does not exist to indicate a trait by being complementary to a marker sequence or to a nucleotide sequence located on its flank (e.g., a nucleotide sequence used as a probe and / or primer that can amplify a marker sequence).

[0146] As used herein, the terms "nucleotide sequence", "polynucleotide", "nucleic acid sequence", "nucleic acid molecule" and "nucleic acid fragment" refer to polymers of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural, and / or altered nucleotide bases. "Nucleotide" is a monomeric unit from which DNA or RNA polymers are constructed and consists of a purine or pyrimidine base, a pentose sugar and a phosphate group. Nucleotides (usually found in their 5'-monophosphate form) are represented by their single-letter names as follows: "A" represents adenylic acid or deoxyadenylic acid (for RNA or DNA, respectively), "C" represents cytidylic acid or deoxycytidylic acid, "G" represents guanylic acid or deoxyguanylic acid, "U" represents uridylic acid, "T" represents deoxythymidylic acid, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

[0147] The term "offspring" refers to any plant of the progeny of the nutrition or sexual reproduction of one or more parental plants or their descendants. For example, offspring plants can be obtained by cloning or selfing of parental plants or by the hybridization of two parental plants, and include selfing bodies and F1 or F2 or even more distant generations. F1 is the first generation offspring generated by parents (wherein at least one party is the donor used as proterties for the first time), and the offspring of the second generation (F2) or subsequent generations (F3, F4, etc.) are samples generated by the selfing of F1, F2, etc. Therefore, F1 can be the hybrid produced by the hybridization between two purebred parents (purebred is isozygous about proterties), and F2 can be the offspring produced by the self-pollination of this F1 hybrid.

[0148] The term "PCR (polymerase chain reaction)" is understood within the scope of the present invention to mean a method which generates relatively large amounts of specific regions of DNA, making possible different analyses based on those regions.

[0149] As used herein, the term "plant" may refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, unless otherwise indicated, the term "plant" may refer to any of the following: a whole plant, a plant component or organ (e.g., leaves, stems, roots, etc.), a plant tissue, a seed, and / or a plant cell.

[0150] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or as part of a higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.

[0151] As used herein, "preserved pollen" refers to pollen that has been manually collected and stored in some manner for future use (see US Application No. 63 / 289,299, incorporated herein by reference).

[0152] As used herein, the term "primer" refers to an oligonucleotide that is capable of annealing to a nucleic acid target (in some embodiments, specifically annealing to a nucleic acid target) when placed under conditions that induce synthesis of a primer extension product (e.g., in the presence of nucleotides and an agent for polymerization (e.g., a DNA polymerase) and at a suitable temperature and pH) to allow a DNA polymerase and / or reverse transcriptase to attach thereto, thereby serving as a starting point for DNA synthesis. In some embodiments, one or more primers are employed to amplify plant nucleic acids (e.g., using a polymerase chain reaction; PCR).

[0153] As used herein, the term "probe" refers to a nucleic acid (e.g., a single-stranded nucleic acid or a double-stranded or higher-order nucleic acid chain, or a subsequence thereof) that can form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence. Typically, the probe has a sufficient length to form a stable and sequence-specific duplex molecule with its complementary sequence, and this can be used in some embodiments to detect a target sequence present in multiple nucleic acids.

[0154] The term "plant cell culture" means a culture of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissue, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development.

[0155] "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.

[0156] A "plant organ" is a distinct and distinct structured and differentiated part of a plant, such as a root, stem, leaf, flower bud or embryo.

[0157] As used herein, "plant tissue" refers to a group of plant cells organized into structural and functional units. Included is any plant tissue in a plant or in culture. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into structural and / or functional units. The use of this term in conjunction with or alone any specific type of plant tissue as listed above or otherwise covered by this definition is not intended to exclude any other type of plant tissue.

[0158] The term "plant part" indicates a part of a plant, including single cells and cell tissues (e.g., intact plant cells in plants), cell clumps, and tissue cultures from which plants can be regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from: pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, buds, and seeds; and pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, buds, scions, rhizomes, seeds, protoplasts, callus, etc.

[0159] As used herein, the term "phenotype", "phenotypic traits" or "traits" refers to one or more traits of a plant or plant cell. Phenotype can be observed by the naked eye or by any other evaluation method known in the art (e.g., microscopy, biochemical analysis, or electromechanical determination). In some cases, the phenotype is directly controlled by a single gene or genetic locus (i.e., corresponding to a "single gene trait"). In the case of haploid induction, a color marker (e.g., R Navajo) and other markers (including a transgenic visualized by the presence or absence of color in the seed) are used to prove whether the seed is an induced haploid seed. Using RNavajo as a color marker and using transgenic as a means of detecting haploid seed induction on a female plant is well known in the art. In other cases, phenotype is the result of the interaction between several genes, and in some embodiments, it is also caused by plant and / or plant cell interacting with its environment.

[0160] As used herein, the term "population" means a genetically heterogeneous collection of plants sharing a common genetic derivation.

[0161] As used herein, the term "primer" refers to an oligonucleotide that is capable of annealing to a nucleic acid target (in some embodiments, specifically annealing to a nucleic acid target) when placed under conditions that induce synthesis of a primer extension product (e.g., in the presence of nucleotides and an agent for polymerization (e.g., a DNA polymerase) and at a suitable temperature and pH) to allow a DNA polymerase and / or reverse transcriptase to attach thereto, thereby serving as a starting point for DNA synthesis. In some embodiments, one or more primers are employed to amplify plant nucleic acids (e.g., using a polymerase chain reaction; PCR).

[0162] As used herein, the term "primer" refers to an oligonucleotide that, when placed under conditions of inducing the synthesis of a primer extension product (e.g., in the presence of nucleotides and reagents such as DNA polymerase for polymerization and at a suitable temperature and pH), can anneal to an amplification target to allow DNA polymerase to attach, thereby serving as a starting point for DNA synthesis. (Amplification) primers are preferably single-stranded to obtain maximum amplification efficiency. Preferably, primers are oligodeoxyribonucleotides. Primers are generally long enough to initiate the synthesis of extension products in the presence of reagents for polymerization. The exact length of a primer will depend on many factors, including the temperature and composition (A / T and G / C content) of the primer. A pair of bidirectional primers is composed of a forward primer and a reverse primer commonly used in the field of DNA amplification (such as in PCR amplification). It should be understood that, as used herein, "primer" may refer to more than one primer, particularly when there is some ambiguity in the information about one or more terminal sequences of a target region to be amplified. Therefore, "primer" includes a set of primer oligonucleotides containing sequences representing possible variations in the sequence, or includes nucleotides that allow typical base pairing. Oligonucleotide primers can be prepared by any suitable method. Methods for preparing oligonucleotides of specific sequences are known in the art, and include, for example, cloning and restriction of appropriate sequences and direct chemical synthesis. Chemical synthesis methods can include, for example, phosphodiester or triester methods, diethylaminophosphoryl ester methods, and solid phase support methods disclosed in, for example, US 4,458,066. If necessary, primers can be labeled by incorporating means that can be detected by, for example, spectral methods, fluorescent methods, photochemical methods, biochemical methods, immunochemical methods, or chemical methods. The template-dependent extension of one or more oligonucleotide primers is catalyzed by a polymerization agent in the presence of an appropriate amount of four deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, and dTTP; i.e., dNTP) or analogs, in a reaction medium (comprising appropriate salts, metal cations, and pH buffer systems). Suitable polymerization agents are enzymes known to catalyze primer and template-dependent DNA synthesis. Known DNA polymerases include, for example, E. coli DNA polymerase or its Klenow fragment, T4 DNA polymerase and Taq DNA polymerase. The reaction conditions for catalyzing DNA synthesis with these DNA polymerases are known in the art. The synthesized product is a double-stranded molecule consisting of a template strand and a primer extension strand, which includes a target sequence. These products in turn serve as templates for another round of replication. In the second round of replication, the primer extension strand of the first round of circulation is annealed with its complementary primer; synthesis produces a "short" product, which is combined at both the 5'-end and the 3'-end by the primer sequence or its complement. Repeated cycles of denaturation, primer annealing and extension result in an exponential accumulation of the target region defined by the primer. Sufficient cycles are performed to achieve the desired amount of polynucleotides containing the nucleic acid target region.The desired amount can vary and is determined by the function of the product polynucleotide. The PCR method is well described in the manual and is known to those skilled in the art. After amplification by PCR, the target polynucleotide can be detected by hybridization with a probe polynucleotide, which forms a stable hybrid with the polynucleotide of the target sequence under low, moderate or even highly stringent hybridization and washing conditions. If the probe is expected to be substantially fully complementary to the target sequence (i.e., about 99% or more), highly stringent conditions can be used. If it is expected that there are some mismatches, such as if the variant variety is expected to cause the probe to be incompletely complementary, the stringency of hybridization can be reduced. However, typically the conditions for excluding nonspecific / accidental binding are selected. Conditions that affect hybridization and are selected for nonspecific binding are known in the art and are described in, for example, Sambrook and Russell, 2001. Typically, lower salt concentrations and higher temperatures increase the stringency of hybridization conditions. "PCR primers" are preferably understood within the scope of the present invention to refer to relatively short fragments of single-stranded DNA used in the PCR amplification of a specific region of DNA.

[0163] As used herein, the term "probe" refers to a nucleic acid (e.g., a single-stranded nucleic acid or a double-stranded or higher-order nucleic acid chain, or a subsequence thereof) that can form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence. Typically, the probe has a sufficient length to form a stable and sequence-specific duplex molecule with its complementary sequence, and this can be used in some embodiments to detect a target sequence present in multiple nucleic acids.

[0164] The term "probe" refers to a single-stranded oligonucleotide that will form a hydrogen-bonded duplex with a substantially complementary oligonucleotide in a target nucleic acid analyte or a cDNA derivative thereof.

[0165] As used herein, the terms "label probe" and "probe" refer to nucleotide sequences or nucleic acid molecules that can be used to detect the presence or absence of a sequence within a larger sequence, for example, a nucleic acid probe that is complementary to all or part of a marker or marker locus by nucleic acid hybridization. Label probes comprising about 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more consecutive nucleotides can be used for nucleic acid hybridization.

[0166] The term "progeny" refers to one or more descendants of a particular cross. Typically, progeny are produced by breeding of two individuals, but some species (particularly some plants and hermaphroditic animals) can self-fertilize (i.e., the same plant serves as a donor of both male and female gametes). The one or more descendants can be, for example, F1, F2, or any subsequent generation.

[0167] As used herein, the terms "progeny" and "progeny plants" refer to plants generated from one or more parent plants by vegetative propagation or sexual reproduction. In the haploid induction mediated by unisexual reproduction, the haploid embryo on the mother contains female chromosomes and excludes male chromosomes-so it is not the progeny of the male haploid-induced strain. Haploid corn seeds typically still have normal triploid endosperm containing male chromosome sets. Edited haploid progeny and subsequent edited double haploid plants and subsequent seeds are not the only desired progeny. There are also seeds from the haploid induction line itself, and subsequent plants and seed progeny of haploid induction plants that often carry Cas9 transgenes. Haploid seeds and haploid induction line (self-pollination induced) seeds can be progeny. Progeny plants can be obtained by cloning a single parent plant or selfing a single parent plant, or by hybridizing two or more parent plants. For example, progeny plant can be obtained by cloning or selfing of a parental plant or by hybridization of two parental plants, and includes selfing body and F1 or F2 or even further generations. F1 is the first generation progeny generated from two parents (at least one of the two parents is used as the donor of proterties for the first time), and the progeny of the second generation (F2) or subsequent generations (F3, F4, etc.) is the sample generated from the selfing, mutual crossing, backcrossing and / or other hybridization of F1, F2, etc. Therefore, F1 can be (and in some embodiments is) the hybrid produced from the hybridization between two purebred parents (that is, the purebred parents are all isozygous about the purpose proterties or their alleles respectively), and F2 can be (and in some embodiments is) the progeny produced from the self-pollination of F1 hybrid.

[0168] The terms "R1-nj" and "R1-SCM2" refer to the R1-Navajo and R1-SCM2 anthocyanin markers. These visual markers can be used to distinguish haploids from diploids (or aneuploids). As described herein, the color of haploid plants is identified as cream, while the color of diploids is purple.

[0169] As used herein, the term "regeneration" and grammatical variations thereof refer to the generation of plants from tissue culture.

[0170] As used herein, "restorer factor" or "fertility restorer" or "Rf" or "restorer allele" refers to one or more genes in a plant that restore fertility to a male sterile plant. Examples of restorer factor genes include, but are not limited to, Rf3, Rf4, Rf10, Rf11, and Rf12. A plant may be heterozygous or homozygous for one or more restorer factor genes. For example, a plant may contain Rf4 as well as Rf11, but may also be rf10. The other plant in the cross may be rf4, but may also be Rf11 and Rf10. Therefore, each plant may contain and lack restorer alleles and non-restorer alleles. Therefore, a CMS plant comprising at least one restorer allele will still be male fertile.

[0171] Non-restorer alleles (also referred to as "rf") refer to one or more genes in a plant that will not restore fertility to a male sterile plant. Examples of non-restorer alleles include, but are not limited to, rf3, rf4, rf10, rf11, and rf12. If a plant has CMS, a plant that is homozygous for a non-restorer allele will be male sterile.

[0172] As used herein, "spontaneous chromosome doubling" ("SCD") or "spontaneous haploid genome doubling" or "haploid male fertility" or "spontaneous genome doubling" are used interchangeably to describe the doubling of a haploid genome in the absence of any intervention. SCD allows for correct meiotic reduction of chromosomes and subsequent formation of mature pollen. In the present disclosure, SCD is calculated by dividing the number of fertile haploid plants by the total number of plants.

[0173] As used herein, "spontaneously doubled haploid plant" refers to a plant whose florets have undergone spontaneous doubling. Other tissues of the spontaneously doubled haploid plant may retain their haploid state (e.g., roots, leaves, stems).

[0174] As used herein, the term "targeted mutagenesis" or "mutagenesis strategy" refers to any mutagenesis method that results in the intentional mutagenesis of a selected gene. Targeted mutagenesis includes CRISPR, TILLING, TALEN methods and other methods that have not yet been discovered but can be used to achieve the same results. As used herein, the term "targeted mutagenesis" or "mutagenesis strategy" refers to any mutagenesis method that results in the intentional mutagenesis of a selected gene. Targeted mutagenesis includes CRISPR, TILLING, TALEN methods and other methods that have not yet been discovered but can be used to achieve the same results.

[0175] As used herein, the term "trait" refers to a target phenotype, a gene that contributes to the target phenotype, and a nucleic acid sequence associated with the gene that contributes to the target phenotype. For example, "HI trait" refers to a haploid-induced phenotype, and a gene that contributes to haploid induction (e.g., mat1 in maize or Os03g27610 in rice) and a nucleic acid sequence (e.g., a gene product associated with HI) associated with the presence or absence of the haploid-induced phenotype.

[0176] As used herein, the term "transfection" refers to the introduction of a nucleic acid into a cell.

[0177] As used herein, the term "transgenic" refers to a nucleic acid molecule introduced into an organism or one or more of its ancestors by some form of artificial transfer technology. Therefore, artificial transfer technology establishes a "transgenic organism" or "transgenic cell". It should be understood that artificial transfer technology can occur in an ancestral organism (or a cell therein and / or a cell that can develop into an ancestral organism), and even if one or more natural and / or assisted breeding results in the presence of an artificially transferred nucleic acid molecule in a progeny individual, any progeny individual with an artificially transferred nucleic acid molecule or a fragment thereof is still considered to be transgenic. DETAILED DESCRIPTION

[0178] The present invention describes a method for conferring cytoplasmic male sterility (CMS) to a plant line. The method includes obtaining a first plant comprising a CMS cytoplasm as a haploid induction line (CHIP), obtaining a second plant comprising a desired nuclear genome (DIP), and hybridizing CHIP with DIP and generating progeny by the hybridization. The resulting progeny comprises CMS cytoplasm and a desired nuclear genome from CHIP and DIP, respectively. In an embodiment, the CMS cytoplasm is selected from the group consisting of: CMS-C, CMS-S, and CMS-T. In one embodiment, the CMS cytoplasm is CMS-C. In one embodiment, CHIP is female fertile and CMS male sterile. In another embodiment, CHIP is female fertile and CMS male fertile. CHIP is a male haploid induction line and comprises a cenh3 mutation. In one embodiment, the cenh3 mutation is a knockout mutation. In one embodiment, the cenh3 knockout mutation is obtained by gene editing. In another embodiment, the cenh3 knockout mutation comprises SEQ ID NO:5 or SEQ ID NO:6. In another embodiment, the cenh3 knockout mutation is heterozygous. In yet another embodiment, CRISPR-Cas12a is used to edit the cenh3 mutation. In one embodiment, CRISPR-Cas12a is selected from the group consisting of: AsCas12a, LbCas12a and FnCas12a, MbCas12a and Mb2Cas12a. In one embodiment, CRISPR-Cas12a is LbCas12a.

[0179] In yet another embodiment, CHIP further comprises anthocyanin markers. Anthocyanin markers are selected from the group consisting of R1-navajo and R1-SCM2. In one embodiment, the anthocyanin marker is R1-navajo; and in another embodiment, the anthocyanin marker is R1-SCM2. The anthocyanin marker is homozygous. In yet another embodiment, CHIP further comprises a restorer allele, wherein the restorer allele is selected from the group consisting of: Rf3, Rf4, Rf11, Rf10, and Rf12. In an embodiment, the restorer allele is Rf4. In another embodiment, the restorer allele is homozygous. In another embodiment, CHIP comprises a non-restorer allele, wherein the non-restorer allele is selected from the group consisting of: rf3, rf4, rf10, rf11, and rf12. The non-restorer allele is rf4 and is homozygous.

[0180] In an embodiment, the CHIP comprises a cenh3 mutation, an R1-navajo marker, and a restorer allele of a restorer factor 4 gene. In another embodiment, the CHIP comprises a cenh3 mutation, an R1-SCM2 marker, and a restorer allele of a restorer factor 4 gene. In one embodiment, the CHIP comprises a cenh3 mutation, an R1-navajo marker, and a non-restorer allele of a restorer factor 4 gene; and in another embodiment, the CHIP comprises a cenh3 mutation, an R1-SCM2 marker, and a non-restorer allele of a restorer factor 4 gene. In yet another embodiment, the CHIP is selected from the group consisting of: corn, wheat, rice, sunflower, tomato, barley, brassicas, cucumber, and watermelon. In another embodiment, the CHIP is corn.

[0181] In an embodiment, in the hybridization of CHIP and DIP, DIP is a pollen donor. DIP can be homozygous or heterozygous for non-restorer alleles. In another embodiment, DIP is homozygous for non-restorer alleles, wherein non-restorer alleles are selected from the group consisting of: rf3, rf4, rf10, rf11 and rf12. In one embodiment, non-restorer alleles are rf4. In an embodiment, DIP is selected from the group consisting of: corn, wheat, rice, and sunflower, tomato, barley, Brassica, cucumber and watermelon. In one embodiment, DIP is corn.

[0182] In yet another embodiment, disclosed herein is a plant produced by the above method, wherein the plant is a CMS haploid plant. In an embodiment, the CMS haploid plant comprises the CMS cytoplasm of CHIP and the nuclear genome of DIP, while lacking anthocyanin markers, restorer alleles and cenh3 knockout mutations. In another embodiment, the CMS haploid plant is treated with a doubling agent. In one embodiment, the doubling agent is selected from the group consisting of: colchicine, natol, dithiopyr, trifluralin, nitrous oxide, or another known anti-microtubule agent. In another embodiment, the doubling agent is colchicine. In yet another embodiment, the CMS haploid plant is pollinated with pollen from DIP. In another embodiment, the CMS haploid plant is pollinated with preserved pollen. In yet another embodiment, the CMS haploid plant is confirmed by genotyping or other molecular analysis.

[0183] Examples Example 1: Establishment of CMS haploid inducible line (CHIP) with R1-nj color markers.

[0184] 1. To select effective gRNAs for gene editing, we transfected etiolated maize protoplasts with LbCas12a-crRNARNP complexes with various candidate gRNAs. The crRNA scaffold for LbCas12a is based on the CRISPR-LbCpf1 system. Protoplasts were isolated from etiolated maize leaves grown in dark conditions as described (Sheen, 1991). Protoplast transfection was performed as described (Sant'Ana et al., 2020) with some modifications. Each transfection reaction consisted of 5 x 10 5 Protoplasts were composed of 1000 cells / mL and incubated with PEG solution (40% PEG-4000, 0.6M mannitol, 100mM CaCl2) for 15 min. After termination with W5 solution (154mM NaCl, 125mM CaCl2, 5mM KCl, and 2mM MES (pH 5.7)), the transfected protoplasts were resuspended in 300 μl W1 solution (0.6M mannitol, 4mM MES (pH 5.7), 4mM KCl), transferred to a 96-well transparent bottom microplate, and incubated in the dark at 28 ° C without shaking for 2 days. DNA was isolated from transfected protoplasts 2 days later, and gene editing efficiency was analyzed by PCR amplification followed by restriction endonuclease I (NEB) digestion of the amplicon.

[0185] 2. Using the gRNA140 (targeting gene ID GRMZM2G158526 second exon) with sequence CAGGTGGTGCGAGTACCTCGGCG (SEQ ID NO: 1) selected as described above, and the DNA vector 26258 (see Table 16) carrying PMI selective markers, SYN-INBC34 inbred immature embryos were subjected to biological ballistic bombardment. In order to generate LbCas12a-crRNA RNP complexes, 0.3nmol of Cas12 protein and 0.3nmol of crRNA were mixed with a total volume of 11 μl, and incubated at room temperature for 10 minutes. For single RNP delivery, RNP was applied to 0.6 μm gold particles (Bio-Rad, the U.S.) as follows: 100 μl of gold particles (10 mg / ml aqueous suspension) and 20 μl of glycogen (20 mg / ml) were added to premixed RNP, gently mixed, and then incubated on ice for 10 minutes. For the co-delivery of RNP and DNA, RNP and DNA carrier plasmid 26258 were coated onto gold particles as follows: 100 μl of gold particles (10 mg / ml aqueous suspension) and 20 μl of glycogen (20 mg / ml) were added to the premixed RNP and DNA carrier, gently mixed, and incubated on ice for 10 minutes. The gold particles coated with RNP / DNA were centrifuged at 8,000 g for 40 seconds, and the supernatant was removed. The precipitate was resuspended with 30 μl of sterile water by brief ultrasonic treatment, and then spread on a large carrier plate (10 μl each), followed by air drying in a laminar flow hood (2-4 h).

[0186] 3. Immature embryos were separated from the harvested spikes about 9-11 days after pollination and pre-cultured on an infiltration medium for 1-3 days. The pre-cultured embryos were then bombarded with LbCas12a-RNP complexes and the above-mentioned DNA using the Bio-Rad PDS-1000HeTM bioballistic particle delivery system. The bombarded embryos were incubated in callus induction medium. The induced callus was then moved to mannose selection medium. Mannose-resistant callus was transferred to a regeneration medium to induce bud formation. The buds were then subcultured to a rooting medium. Using the previously described real-time quantitative polymerase chain reaction (qPCR) TaqMan R method, leaf samples were harvested from rooted plants for TaqMan R assays to detect mutations in the target site.

[0187] 4. We used TaqMan assay 3895 with primers TCCTTGTTCCGTCTTTTGCAG (SEQ ID NO: 2) and AAGGCAAAAGGAGGGAACTGAT (SEQ ID NO: 3) and probe TACCTCGGCGACGCC (SEQ ID NO: 4) and unambiguously identified CenH3-het edited plants by PCR sequencing for frameshift alleles. SYN-INBC34 is rf4 / rf4, does not have R-nj markers, and is not CMS. The following steps were performed to bring the edited CenH3 allele into a genetic background including homozygous R1-nj, CMS, and homozygous Rf4.

[0188] 5. The TO events were grown to maturity and self-pollinated and / or outcrossed as males to CMS-C material SYN-INB77M-CMS (rf4 / rf4 and male sterile). T1 generation plants from TO selfing were also identified as heterozygous for frameshift mutations in the CenH3 coding sequence (from here on referred to as CenH3[+ / -] plants) by TaqMan assay and PCR sequencing, and grown to maturity to cross with CMS, thereby generating more CMS-Cenh3 edited seeds.

[0189] a. After crossing T0 or T1 CenH3 mutant pollen to SYN-INB77M-CMS, in the F1 generation, we identified plants carrying a 10-base deletion in heterozygous CENH3 by TaqMan assay and Sanger sequencing. This plant was male sterile and was pollinated by pollen from a RWKS plant line that was homozygous for R-nj and Rf4.

[0190] b. T1 generation (non-CMS) CenH3(+ / -) mutant plants were also intercrossed with RWKS to generate F1 with CenH3 and R-nj markers.

[0191] c. All seeds from the CMS CenH3(+ / -) x RWKS cross in step 5b were red or purple, indicating that the R1-nj marker was present in at least a heterozygous condition. These seeds were planted, and the resulting plants were genotyped for CenH3(+ / -) and Rf4(+ / -) zygosity by TaqMan assay, and then self-pollinated (they were fertile due to the Rf4 marker). Some plants were also backcrossed with seeds carrying CenH3(+ / -) and R-nj from the F1 progeny of the CenH3(+ / -) x RWKS cross in step 5b.

[0192] d. Plant the purple seeds obtained from the cross of step 5c and select for CenH3 (+ / -) heterozygous, R-nj homozygous and CMS cytoplasm by TaqMan and sequencing. These inducer materials are easily increased by selfing. If the Rf4 marker is not present (i.e., the inducer plants are rf4 / rf4), crosses with sibling plants or maintainer plants carrying the R-nj marker and optionally the mutant allele of CenH3 (+ / + or + / -) can also be used to increase seeds.

[0193] e. This induced line is then used as a CMS donor line for one-step transformation. This material is used as female and crossbred with pollen from any line that wishes to be directly converted to CMS cytoplasm. After crossbred, haploids are color sorted as mature dry seeds with cream embryos (diploid hybrids have purple embryos). The haploid seeds may or may not then be chemically treated to induce genome doubling before planting in soil and grown to maturity where they are then crossbred with recurrent parent pollen.

[0194] Table 1. Nucleotide alignment of CENH3 deletion mutant alleles compared to wild type (SEQ ID NO:7) (509A115A is represented by SEQ ID NO:8; 509A151A is represented by SEQ ID NO:9, and 509A150A is represented by SEQ ID NO:177)

[0195] GRMZM2G158526CTCTCCGACCCTGGTGCTAAGCACGTTCCTTGTTCCGTCTTTTGCAGGTGGTGCG509A115A CTCTCCGACCCTGGTGCTAAGCACGTTCCTTGTTCCGTCTTTTGCAGGTGGTGCG 509A150A CTCTCCGACCCTGGTGCTAANCACGTTCCTTGTTCCGTCTTTTGCAGGTGGTGCG 509A151A CTCTCCGACCCTGGTGCTAAGCACGTTCCTTGTTCCGTCTTTTGCAGGTGGTGCG GRMZM2G158526AGTACCTCGGCGACGCCGGTGAGCGCGTGCGTGCGGGGATCAGTTCCCTCCTTTT 509A115A AG----------ACGCCGGTGAGCGCGTGCGTGCGGGGATCAGTTCCCTCCTTTT 509A150A AG-------------------AGCGCGTGCGTGCGGGGATCAGTTCCCTCCTTTT 509A151A -------------------TGAGCGCGTGCGTGCGGGGATCAGTTCCCTCCTTTT GRMZM2G158526GCCTTTTTTTGTTGGGCTGCTCTTACTTGCTTGCAAGCTGTTTGATGGAATGCAG509A115A GCCTTTTTT-GT-GGGCTGCTCTTACTTGCTTGCAAGCTGTTTGATGGAATGCAG 509A150AGCCTTTTTT-GT-GGGCTGCTCTTACTTGCTTGCAAGCTGTTTGATGGAATGCAG 509A151A GCCTTTTTT-GT-GGGCTGCTCTTACTTGCTTGCAAGCTGTTTGATGGAATGCAG GRMZM2G158526GAAAGGGCTGCTGGGACCGGGGGAAG

[0196] 509A115A GAAAGGGCTGCTGGGACCGGGGGAAG

[0197] 509A150A GAAAGGGCTGCTGGGACCGGGGGAAG

[0198] 509A151A GAAAGGGGCTGCTGGGACCGGGGGAAG

[0199] Table 2. Partial alignment of the deduced amino acid sequences of CENH3 deletion mutant alleles (GRMZM2G158526WT is represented by SEQ ID NO: 10). 509A115A (10 bp deletion SEQ ID NO: 12) is completely present in exon 2, so the derived splicing remains in wild type. For 509A151A and 509A150A (19 bp deletion), we generated all possible splice variants (SEQ ID NO: 13-22) depending on the GT donor site for intron splicing. The deduced amino acid sequence of the KD allele (Kelly Dawe, University of Georgia) is also shown (SEQ ID NO: 11).

[0200] GRMZM2G158526 MARTKHQAVRKTAEKPKKKLQFERSGGASTSATP--------ERAAG---

[0201] CENH3_KD MARTKHQAVRKTAEKPKKKLQFERSGGASTSATP--------ERAAG---

[0202] 509A115A_Transcript_ MARTKHQAVRKTAEKPKKKLQFERSGGARRR-------------------

[0203] 509A151A_transcript_a MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPVSA-------------

[0204] 509A151A_transcript_b MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPVSAC----ERAAG---

[0205] 509A151A_transcript_c MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPVSACVRGSERAAG---

[0206] 509A151A_Transcript_d MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPVSACVRGSVPSFCLFL

[0207] 509A151A_Transcript_e MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPVSACVRGSVPSFCLFL

[0208] 509A150A_transcript_a MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPVSA-------------

[0209] 509A150A_transcript_b MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPVSAC----ERAAG---

[0210] 509A150A_transcript_c MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPVSACVRGSERAAG---

[0211] 509A150A_Transcript_d MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPVSACVRGSVPSFCLFL

[0212] 509A150A_Transcript_e MARTKHQAVRKTAEKPKKKLQFERSGGASTSATPVSACVRGSVPSFCLFL

[0213] GRMZM2G158526 TG---------GRAASGGDSVKKTKPRHRWRPGTVALREIRKYQKSTEPL

[0214] CENH3_KD TG---------GRAASGGDSVKKTKPRHRWRPGL*RCGRSGSTRSPLNRS

[0215] 509A115A_Transcript_ KG---LL----GPGEERRLEVTQLRRRNHATAGGQGL*RCGRSGSTRSPL

[0216] 509A151A_transcript_a KG---LL----GPGEERRLEVTQLRRRNHATAGGQGL*RCGRSGSTRSPL

[0217] 509A151A_transcript_b TG---------GRAASGGDSVKKTKPRHRWRPGTVALREIRKYQKSTEPL

[0218] 509A151A_transcript_c TG---------GRAASGGDSVKKTKPRHRWRPGTVALREIRKYQKSTEPL

[0219] 509A151A_transcript_d KG---LL----GPGEERRLEVTQLRRRNHATAGGQGL*RCGRSGSTRSPL

[0220] 509A151A_transcript_e LGCSYLLAS*KGCWDRGKSGVWR*LS*EDETTPPLAARDCSAAGDQEVPE

[0221] 509A150A_transcript_a KG---LL----GPGEERRLEVTQLRRRNHATAGGQGL*RCGRSGSTRSPL

[0222] 509A150A_transcript_b TG---------GRAASGGDSVKKTKPRHRWRPGTVALREIRKYQKSTEPL

[0223] 509A150A_transcript_c TG---------GRAASGGDSVKKTKPRHRWRPGTVALREIRKYQKSTEPL

[0224] 509A150A_transcript_d KG---LL----GPGEERRLEVTQLRRRNHATAGGQGL*RCGRSGSTRSPL

[0225] 509A150A_transcript_e LGCSYLLAS*KGCWDRGKSGVWR*LS*EDETTPPLAARDCSAAGDQEVPE

[0226] GRMZM2G158526 IPFAPF-VRVVR---ELTNFVTN--GKVERYTAEALLALQE

[0227] CENH3_KD *GS*PIS*QTGK*SAIPQKPSLRC—K—S---PLRLSSVW--

[0228] 509A115A_Transcript_ NRSSPLRLSSVW---*GS*PIS*QTGK*SAIPQKPSLRCK-

[0229] 509A151A_transcript_a NRSSPLRLSSVW---*GS*PIS*QTGK*SAIPQKPSLRCK-

[0230] 509A151A_transcript_b IPFAPF-VRVVR---ELTNFVTN--GKVERYTAEALLALQE

[0231] 509A151A_transcript_c IPFAPF-VRVVR---ELTNFVTN--GKVERYTAEALLALQE

[0232] 509A151A_transcript_d NRSSPLRLSSVW---*GS*PIS*QTGK*SAIPQKPSLRCK-

[0233] 509A151A_transcript_e VH*TAHPLCAFRPCGEGVNQFRNKRESRALYRRSPPCAAR-

[0234] 509A150A_transcript_a NRSSPLRLSSVW---*GS*PIS*QTGK*SAIPQKPSLRCK-

[0235] 509A150A_transcript_b IPFAPF-VRVVR---ELTNFVTN--GKVERYTAEALLALQE

[0236] 509A150A_transcript_c IPFAPF-VRVVR---ELTNFVTN--GKVERYTAEALLALQE

[0237] 509A150A_transcript_d NRSSPLRLSSVW---*GS*PIS*QTGK*SAIPQKPSLRCK-

[0238] 509A150A_transcript_e VH*TAHPLCAFRPCGEGVNQFRNKRESRALYRRSPPCAAR-

[0239] Example 2: Establishment of CMS haploid inducible line with R1-SCM2 color marker ("CHIP").

[0240] 1. To select effective gRNAs for gene editing, we transfected etiolated maize protoplasts with LbCas12a-crRNARNP complexes with various candidate gRNAs. The crRNA scaffold for LbCas12a is based on the CRISPR-LbCpf1 system. Protoplasts were isolated from etiolated maize leaves grown in dark conditions as described (Sheen, 1991). Protoplast transfection was performed as described (Sant'Ana et al., 2020) with some modifications. Each transfection reaction consisted of 5 x 10 5 Protoplasts were composed of 1000 cells / mL and incubated with PEG solution (40% PEG-4000, 0.6M mannitol, 100mM CaCl2) for 15 minutes. After termination with W5 solution (154mM NaCl, 125mM CaCl2, 5mM KCl, and 2mM MES (pH 5.7)), the transfected protoplasts were resuspended in 300μl W1 solution (0.6M mannitol, 4mM MES (pH 5.7), 4mM KCl), transferred to a 96-well transparent bottom microplate, and incubated in the dark at 28°C for 2 days without shaking. DNA was isolated from transfected protoplasts 2 days later, and gene editing efficiency was analyzed by PCR amplification followed by restriction endonuclease I (NEB) digestion of the amplicon.

[0241] 2. Using gRNA140 (sequence CAGGTGGTGCGAGTACCTCGGCG, SEQ ID NO: 1) compounded with the second exon of targeting gene ID GRMZM2G158526 LbCas12a and DNA vector 26258 (Table 16) (both carry PMI selective markers) SYN-INBC34 x SYN-INBC34RS is separated from immature embryos (labeled as heterozygous for R1-SCM2) Bioballistic bombardment. In order to generate Cas12a-crRNA RNP complexes, 0.3nmol of Cas12 protein and 0.3nmol of crRNA are mixed with a total volume of 11 μl and incubated at room temperature for 10 minutes. For independent RNP delivery, RNP is coated on 0.6 μm gold particles (Bio-Rad, USA) as follows: 100 μl gold particles (10 mg / ml water suspension) and 20 μl glycogen (20 mg / ml) are added to the premixed RNP, gently mixed, and incubated on ice for 10 minutes. For the co-delivery of RNP and DNA, RNP and DNA carrier plasmid 26258 are coated on gold particles as follows: 100 μl gold particles (10 mg / ml water suspension) and 20 μl glycogen (20 mg / ml) are added to the premixed RNP and DNA carrier, gently mixed, and incubated on ice for 10 minutes. The gold particles coated with RNP / DNA are centrifuged at 8,000 g for 40 s, and the supernatant is removed. The pellet was resuspended with 30 μl of sterile water by brief sonication and spread on macrocarrier plates (10 μl each) and then air-dried in a laminar flow hood (2-4 h).

[0242] 3. Immature embryos were separated from the harvested spikes about 9-11 days after pollination and pre-cultured on an infiltration medium for 1-3 days. The pre-cultured embryos were bombarded with LbCas12a-RNP complexes and the above-mentioned DNA using the Bio-Rad PDS-1000HeTM bioballistic particle delivery system. The bombarded embryos were then incubated in callus induction medium. The induced callus was moved to mannose selection medium. Mannose-resistant callus was transferred to a regeneration medium to induce bud formation. The buds were then subcultured onto a rooting medium. Using the previously described real-time quantitative polymerase chain reaction (qPCR) TaqMan R method, leaf samples were harvested from rooted plants for TaqMan R assays to detect mutations in the target site.

[0243] 4. We used TaqMan assay 3895 with primers TCCTTGTTCCGTCTTTTGCAG (SEQ ID NO: 2) and AAGGCAAAAGGAGGGAACTGAT (SEQ ID NO: 3) and probe TACCTCGGCGACGCC (SEQ ID NO: 4) and unambiguously identified CenH3-het edited plants by PCR sequencing for the frameshift allele. The following steps were performed to bring the edited CenH3 allele into a genetic background containing homozygous R1-SCM2, CMS and homozygous Rf4.

[0244] 5. The T0 events were grown to maturity and selfed. Purple seeded T1 progeny were planted and CenH3(+ / -) plants were identified by TaqMan and PCR sequencing, grown to maturity for selfing and crossing to ears of the SYN-INB77M-CMS line carrying the maintainer alleles rf4 and rf11.

[0245] 6. After pollination with a plant carrying the 19 base pair deletions of the R1-SCM2 marker and sequence TACCTCGGCGACGCCGGTG (SEQ ID NO:5), two SYN-INB77M-CMS spikes were obtained. The progeny seeds to be planted were selected again for purple seed color. For isozygous R1-SCM2 R1 marker and for 19bp deletion, the seedlings were genotyped and further selected for heterozygous TaqMan assays with specificity for this cenh3 mutant allele. If these plants are male fertile, they are selfed. If these plants are male sterile, they are hybridized as females with non-CMS T2 plants with R1-SCM2 and CenH3 (+ / -).

[0246] Table 3. List of markers used to identify the desired genotype of the R1 color marker and R1-color suppressor gene.

[0247]

[0248] Table 4. List of markers used to identify cytoplasmic or mitochondrial genome type.

[0249]

[0250] Table 5. List of markers used to identify the allele type of restorer genes.

[0251]

[0252] Table 6. RT-PCR sequences used for CENH3 assay.

[0253]

[0254] 7. Genotyping the next generation for homozygosity of R1-SCM2, presence of CMS and heterozygous knockout CenH3 (+ / -) configuration. This particular combination is one of the ideal inducer genotypes that can be used for CMS cell exchange. These inducer materials are easily increased by selfing. However, if the strain is male sterile (i.e., Rf4 marker is absent and inducer plants are rf4 / rf4), these inducers can also be crossed as females with maintainer pollen to increase seeds, which are homozygous for mutant alleles of R1-SCM2 or R-nj markers and optionally CenH3 (+ / + or + / -).

[0255] 8. This induced line is then used as a CMS donor line for one-step transformation. This material is used as female and is hybridized with pollen from any line that wishes to be directly converted into CMS cytoplasm. After hybridization, the haploids are color sorted between 10 and 25 days after in vitro pollination (we select cream embryos after 24 hours of color induction in an incubator). Haploid seeds may or may not be chemically treated to induce genome doubling, and simply transplanted into the soil to grow them for further hybridization with the recurrent parent. Similarly, fertile induced line plants can be maintained by selfing, and sterile induced lines can be hybridized with non-CMS (i.e., normal cytoplasm) maintenance lines carrying R1-SCM2 (or R-nj) color markers. Optionally, the maintenance line can have non-restorer alleles of rf4 and rf11 and CenH3 WT or CenH3 knockout (mutant) alleles.

[0256] Example 3. Proof of concept of cell exchange in maize (normal A cell exchange)

[0257] By obtaining genome-edited material from the Kelly Dawe laboratory of the Department of Plant Biology at the University of Georgia and crossing it with a small group of corn lines to generate haploids, cell exchange using the CenH3 (+ / -) induction line in corn was demonstrated. The haploids were then doubled and selfed to establish DH1 seeds. The cytoplasm of the material obtained from Dr. Dawe was "Standard A", a cytoplasm known to be common to transformable corn genetic backgrounds and characterized by the characteristic genotype of the SM2914 marker. In contrast, the strain known to be selected for cell exchange to this normal A is a normal B, which is not a transformable background and is characterized by the different characteristic genotypes of the SM2914 marker.

[0258] We have genotyped the alleles edited for CenH3 from plants of Dr. Dawe, selected CenH3 heterozygous (+ / -) CenH3 heterozygous (+ / -) plants, and crossed them as females with seven normal B Syngenta males. We harvested these spikes after 16 days of pollination, and separated the embryos into germination medium containing colchicine in a petri dish for 24 hours to induce genome doubling. Then we moved the embryos to the recovery medium in the plant culture tray (phytatray), and the plants were grown to the two-leaf stage. Then, we sampled leaves from the plants and extracted DNA to evaluate with a small group of TaqMan endpoint markers, which were polymorphic between normal A haploid induction lines and normal B male parents. These markers cover all 10 chromosomes and are used to select plants that only carry chromosomes from pollen donors.

[0259] Table 7. Marker panels used to identify haploid plants

[0260]

[0261] We identified "paternal haploids" by being "homozygous" for the paternal marker, and sorted out plants that were heterozygous for all nuclear DNA markers in the panel from those plants that were genotyped as hybrids. Table 8 below shows a summary of haploid induction rates based on this data. Several plants were also tested by ploidy analysis to determine if they were indeed haploid. See Figures 3a (diploid control) and 3b (haploid).

[0262] Table 8. Summary of haploid induction rates of haploid paternal offspring.

[0263]

[0264] Table 9. Cell exchange proof of concept and efficiency testing in different maize germplasms.

[0265] Confirmation of cytoplasmic exchange in doubled haploids

[0266]

[0267]

[0268]

[0269]

[0270] The data show that we recovered 3% to 10% haploids from the induction line crosses. The haploids were grown to maturity and self-pollinated (recall that they had their genomes doubled by colchicine treatment, so it is more reasonable to say that they are doubled haploids). The seed numbers shown in Table 10 below were obtained on the self-pollinated ears.

[0271] Table 10. Seeds produced by selfing of doubled haploids that underwent cytoplasmic transformation

[0272]

[0273]

[0274] Importantly, both the doubled haploid and DH1 generation progeny were genotyped and shown to have cytoplasmic markers associated with normal A, but not normal B. This clearly indicates that the cell exchange concept works effectively using the CenH3+ / - induced line. This concept is easily applied to exchange male sterile cytoplasm (shown in the following example).

[0275] Example 4: Exchange of the recurrent parent and the R1-SCM2 marker using CMS cells of the heterozygous CENH3 line.

[0276] Step 1. We selected thirteen DIP corn lines (9 field corn lines and 4 sweet corn lines) for conversion to CMS. These lines have the non-fertility restorer genotypes rf4 and rf11. DIP corn lines can also be called recurrent parents.

[0277] Step 2. We planted seeds of two transformed line materials, one segregating for a 19 bp deletion in CENH3 (SEQ ID NO: 9) and the other segregating for a 10 bp deletion in CENH3 (SEQ ID NO: 8). Both types of materials segregated 1:1 between wild type and heterozygous and carried the R1-SCM2 allele (an anthocyanin marker expressed in the scutellum of the embryo).

[0278] Step 3. We identified plants heterozygous for the cenh3 mutant from these lines (3 assays total) using TaqMan assays specific for the wild type and both deletion alleles and grew them to maturity. These resulting lines are the transformed plants, ie, CHIP plants.

[0279] Table 11. Assays used to identify cenh3 heterozygous plants, cytoplasmic type (mitochondrial genome), and desired alleles at R1, C1, Rf4, and Rf11.

[0280]

[0281]

[0282]

[0283]

[0284] Step 4. We pollinated the transformed line spikes from CHIP plants with pollen from the recurrent parent (DIP) plant and harvested spikes for embryo extraction 16-19 days after pollination. The extracted embryos (i.e., F1 generation) were placed on a petri dish containing: 40 ml Murashige and Skoog medium (MS) medium containing 0.5 mg / ml colchicine or the same MS medium without colchicine (generally see Maluszynski et al., ed., Doubled Haploid Production in Crop Plants: A Manual [Double haploid generation in crop plants: Manual] (2003); also see WO 2002 / 085104, incorporated herein by reference). At 28°C, the plate was placed in a Percival growth chamber under continuous light and 123 μmol / m.sec for 16-24 hours to allow the embryo to express the color from the dominant R1-SCM2 allele.

[0285] Step 5. After 16 to 24 hours, white embryos (i.e., those lacking R1-SCM2 expression) were transferred to plant culture trays containing 100 ml of germination medium and placed in a growth chamber at 28° C. with 16 hours of light (118 μmol / m.sec) and 8 hours of darkness at 24° C. The germination medium formulation contained MS salts, vitamins, and inositol (see generally Murashige and Skoog, A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures, Physiologia Plantarum 15:473-497 (1962)), with 0.5 ml / liter of plant preservative mixture (PPM, Plant Cell Technology) added.

[0286] Step 6. After about 10 days, we transplanted the surviving seedlings into 2.5-inch pots containing soil and placed them in a hardening room under the following environmental conditions: day temperature - 80°F, night temperature - 72°F, humidity - 55%, photoperiod - 12 hours, light intensity - about 1200 μmol, CO2 - 400 ppm.

[0287] Step 7. Seedlings were sampled approximately 5 days after transplantation and genotyped with markers covering all 10 maize chromosomes. A subset of 144 plants that were found to be homozygous for all markers was selected for doubling (Table 13). We also tested them for cytoplasmic type by two markers of CMS cytoplasm (SM2915 and SM2916).

[0288] Table 12. Number of F1 embryos extracted, seedlings genotyped, and haploid seedlings.

[0289]

[0290]

[0291] Table 13. Number of haploids doubled per treatment per line (colchicine vs. no colchicine).

[0292] strain Colchicine No colchicine total Strain 1 3 16 19 Strain 2 7 11 18 Strain 3 4 6 10 Strain 4 2 10 12 Strain 5 0 7 7 Strain 6 0 6 6 Strain 7 1 16 17 Strain 8 5 2 7 Strain 9 0 10 10 Strain 10 7 9 16 Strain 11 0 13 13 Strain 12 0 3 3 Strain 13 0 6 6

[0293] Step 8. We calculated the haploid recovery rate (HRR) as the ratio of the number of confirmed haploid seedlings to the number of extracted embryos. Plants with the CENH3 mutation (10 bp deletion or 19 bp deletion) in the heterozygous state had very similar HRR (Table 14).

[0294] Table 14. Mean values ​​of haploid recoveries for all F1 families for the two CENH3 mutations tested.

[0295] CENH3 mutation Average HRR N 509A115A (10 bp deletion) 6.7 48 509A151A (19 bp deletion) 5.3 13

[0296] All haploids were pollinated with pollen from the recurrent parent plant to generate the haploid BC1 generation.

[0297] Table 15. Doubled haploid seeds produced by line and treatment (colchicine vs. no colchicine)

[0298] strain Colchicine No colchicine total Strain 1 449 134 583 Strain 2 33 2 35 Strain 3 411 46 457 Strain 4 10 79 89 Strain 5 NA 6 6 Strain 6 NA 17 17 Strain 7 2 45 47 Strain 8 192 12 204 Strain 9 NA 15 15 Strain 10 33 3 36 Strain 11 NA 0 0 Strain 12 NA 5 5 Strain 13 NA 1 1

[0299] After pollination, the ears are allowed to dry. Another round of embryo rescue and pollination by the recurrent parent will be completed to expand the DH seed stock.

[0300] Example 5. General method for converting non-CMS recurrent parent lines into CMS lines.

[0301] Step 1. Select the DIPs to be converted to CMS. The lines selected need to be homozygous (ideally) or heterozygous for rf4 (a recessive allele that confers male sterility when combined with CMS cytoplasm). These lines are the rf4 recurrent parents.

[0302] Step 2. CHIP is grown, and if necessary, each plant is genotyped for CenH3 and for the markers of CMS, Rf4 and R1 loci. Plants heterozygous for CenH3 knockout alleles are used to exchange CMS cells. In any induction line population, there will be many plants that are homozygous WT for CenH3 gene. These plants are not induction lines and must be sorted out. The CHIP plant selected is optionally R1-SCM2 or R1-nj homozygous. In an ideal one-step cell exchange method, one of these two alleles has been fixed in the strain. Induction lines can be increased by self-pollination (if they are male fertile) or by hybridizing with the pollen of the maintenance line (if they are male sterile). Maintenance lines will have R1-SCM2 or R1-nj color markers so that they are fixed in the induction line. The maintainer line can have non-restorer alleles of rf4 and rf11 and either a CenH3 WT or a CenH3 knockout (mutant) allele.

[0303] Step 3. Cross DIP as male (pollen donor) to CHIP.

[0304] Step 4. If the R1-nj marker is used, the resulting seeds are grown to maturity, dried and harvested, and sorted for haploids (cream-colored embryos) and then planted. In contrast, if the R1-SCM2 marker is used, the resulting spike is harvested between ten and twenty-five days after pollination. The embryos from the grains are separated and incubated in an appropriate medium (referred to as embryo rescue medium) suitable for maintaining embryo viability. In one embodiment, the rescue medium for haploid induction rate (HIR) determination comprises 4.43 grams of Murashige and Skoog basal medium containing vitamins, 30 grams of sucrose, and 70 mg of salicylic acid. The embryos in the rescue medium are placed under conditions that allow the expression of color indicator genes (e.g., R1-SCM2). In an exemplary embodiment, the embryos are placed under 22°C-31°C 100-400 micromolar light for 16-24 hours until some embryos become purple due to the expression of the R1-SCM2 gene (see, for example, the scheme described in WO 2015 / 104358). Purple (diploid) and cream (haploid) embryos can be counted from each ear. The frequency of haploid (called HIR or haploid induction rate) can be determined according to the number of haploids in the total embryo. Optionally, colchicine is used to process to induce genome doubling at a certain time point during this process. Generally referring to Maluszynski et al., ed., Doubled Haploid Production in Crop Plants:A Manual [Double haploid production in crop plants: manual] (2003). Also referring to WO 2002 / 085104, which is incorporated herein by reference. In one embodiment, colchicine is co-applied in the above-mentioned rescue medium.

[0305] Step 5. Plant the DIP seed so that it will be successfully bred (pollen shedding occurs while the ear is in a matable state, silking, on the progeny CMS-transformed haploid plants) and used as a pollen donor for haploid plants when they bloom. However, optionally, the pollen stored or preserved here can also be simply used as a donor for flowering haploid plants.

[0306] Step 6. haploid plantlets are sampled and genotyped. The plant carrying the mark of CMS cytoplasm and the genotype of the father for other determinations is confirmed as the father's haploid. At the minimum, the genotyping for CenH3 gene is performed on haploid, and haploid contains wild-type (unedited) allele. If tested, haploid will have rf4 allele and any other allele from DIP (father) genome. Whether they have been treated with doubling agent, due to CMS cytoplasm and rf4 allele, these haploids are expected to be male sterile. In other words, by genotyping, if the genotyping result is that they do not carry the CenH3 allele of editing, and they have CMS cytoplasm genotype mark and all other nuclear markers are shown as DIP parent (rather than CHIP) naming, then it is optionally possible to infer that haploid plant (sorted by embryo color) is confirmed as the haploid through cell exchange by genotyping.

[0307] Step 7. pollinate haploid plants with DIP parent pollen. If haploid is not treated with chemical doubling agents, any seed set (implied female fertility) will be the result of the natural biological process (known to be common in corn germplasm) of spontaneous doubling of female inflorescence (ear). Treating embryos with chemical doubling agents can improve the seed set of ears by generating double haploid sectors. In general, it is not desirable to be bound by theory, and it is expected that the CMS cell exchange approach can be carried out with or without the doubling step in the case of almost any corn germplasm, because the haploid ear will have some ovules or embryo sacs that spontaneously double, and those ovules or embryo sacs can be fertilized by recurrent parent pollen (e.g., backcrossing), to establish pure haploid BC1 seeds. Haploid BC1 is a hybrid between recurrent parent and haploid genome, and if there is any variation in the parental line (i.e., if the recurrent parent is not a fixed inbred line), the variation in the different cell exchange lines from this process may be obvious.

[0308] Step 8. After maturity and drying, harvest one or more ears of the haploid plants that have been pollinated, and plant the resulting seeds next to the DIP parent (which will again serve as a pollen donor). Cross again to expand the seed quantity of the CMS line. In this generation, the CMS line can be genotyped again to confirm the state and purity of the transformation and verify the absence of CHIP nuclear DNA (including Rf4 and CenH3 mutant markers).

[0309] Step 9. When it is desired to produce hybrid seed, the CMS cell exchange line can be planted as a female next to the male line. The female CMS line should be male sterile so that it can be easily crossed with pollen donors in the hybrid production field without much human intervention. Table 16. Construct annotations.

[0310] Construct 26258:

[0311]

[0312]

Claims

1. A method for conferring cytoplasmic male sterility (CMS) to a plant line, the method comprising a. obtaining a first plant comprising CMS cytoplasm, wherein the first plant is a haploid induced line (CHIP); b. obtaining a second plant comprising the desired nuclear genome (DIP); and c. hybridizing said first plant with said second plant; and d. generating progeny from the resulting hybrid plants; i. wherein said progeny comprises said CMS cytoplasm and said desired nuclear genome.

2. The method of claim 1, wherein the CMS cytoplasm is selected from the group consisting of: CMS-C, CMS-S and CMS-T.

3. The method of claim 2, wherein the CMS cytoplasm is CMS-C.

4. The method of claim 1, wherein the CHIP is female fertile and the CMS is male fertile.

5. The method of claim 1, wherein the CHIP is female fertile and the CMS is male sterile.

6. The method of claim 4, wherein the CHIP is a paternal haploid induction line.

7. The method of claim 6, wherein the CHIP comprises a cenh3 mutation.

8. The method of claim 7, wherein the cenh3 mutation is a knockout mutation.

9. The method of claim 8, wherein the cenh3 knockout mutation is obtained by gene editing.

10. The method of claim 9, wherein the cenh3 knockout mutation comprises SEQ ID NO:5 or SEQ ID NO:

6.

11. The method of claim 10, wherein the cenh3 knockout mutation is heterozygous.

12. The method of claim 11, wherein the cenh3 knockout mutation is edited using CRISPR-Cas12a.

13. The method of claim 12, wherein the CRISPR-Cas12a is selected from the group consisting of AsCas12a, LbCas12a, FnCas12a, MbCas12a, Mb2Cas12a, etc.

14. The method of claim 13, wherein the CRISPR-Cas12a is LbCas12a.

15. The method of claim 7, wherein the CHIP further comprises an anthocyanin tag.

16. The method of claim 15, wherein the anthocyanin marker is selected from the group consisting of R1-navajo and R1-SCM2.

17. The method of claim 16, wherein the anthocyanin marker is R1-navajo.

18. The method of claim 16, wherein the anthocyanin marker is R1-SCM2.

19. The method of claim 16, wherein the anthocyanin marker is homozygous.

20. The method of claim 7, wherein the CHIP further comprises a restorer allele.

21. The method of claim 20, wherein the restorer allele is selected from the group consisting of: Rf3, Rf4, Rf10, Rf11 and Rf12.

22. The method of claim 5, wherein the CHIP comprises a non-restorer allele, wherein the non-restorer allele is selected from the group consisting of: rf3, rf4, rf10, rf11, and rf12.

23. The method of claim 21, wherein the restorer allele is Rf4.

24. The method of claim 22, wherein the non-restorer allele is rf4.

25. The method of claim 23, wherein the restorer allele is homozygous.

26. The method of claim 24, wherein the non-restorer allele is homozygous.

27. The method of claim 1, wherein the CHIP comprises a cenh3 mutation, an R1-navajo marker, and a restorer allele of a restorer factor 4 gene.

28. The method of claim 1, wherein the CHIP comprises a cenh3 mutation, an R1-SCM2 marker, and a restorer allele of a restorer factor 4 gene.

29. The method of claim 1, wherein the CHIP comprises a cenh3 mutation, an R1-navajo marker, and a non-restorer allele of a restorer factor 4 gene.

30. The method of claim 1, wherein the CHIP comprises a cenh3 mutation, an R1-SCM2 marker, and a non-restorer allele of a restorer factor 4 gene.

31. The method of claim 1, wherein the CHIP is selected from the group consisting of corn, wheat, rice, sunflower, tomato, barley, Brassica, cucumber, and watermelon.

32. The method of claim 31 , wherein the CHIP is corn.

33. The method of claim 1, wherein in the cross of step c, the DIP is a pollen donor.

34. The DIP of claim 1, wherein the DIP is homozygous or heterozygous for a non-restorer allele.

35. The DIP of claim 34, wherein the DIP is homozygous for a non-restorer allele.

36. The restorer factor of claim 35, wherein the non-restorer allele is selected from the group consisting of rf3, rf4, rf10, rf11 and rf12.

37. A restorer factor as described in claim 36, wherein the non-restorer allele is rf4.

38. The method of claim 1, wherein the DIP is selected from the group consisting of corn, wheat, rice, and sunflower, tomato, barley, Brassica, cucumber, and watermelon.

39. The method of claim 38, wherein the DIP is corn.

40. A plant produced by the method of claim 1, wherein the plant is a CMS haploid plant.

41. The CMS haploid plant of claim 40, wherein the CMS haploid plant comprises the CMS cytoplasm of the CHIP and the nuclear genome of the DIP.

42. The CMS haploid plant of claim 41, wherein the CMS haploid plant lacks anthocyanin markers, restorer alleles and a cenh3 knockout mutation.

43. The CMS haploid plant of claim 42, wherein said CMS haploid plant is treated with a doubling agent.

44. The doubling agent of claim 43, wherein the doubling agent is selected from the group consisting of colchicine, nafate, dithiopyr, trifluralin, nitrous oxide, or another known anti-microtubule agent.

45. The doubler of claim 44, wherein the doubler is colchicine.

46. ​​The CMS haploid plant of claim 42, wherein said CMS haploid plant is pollinated with pollen from said DIP.

47. The CMS haploid plant of claim 42, wherein the CMS haploid plant is pollinated with preserved pollen.

48. The CMS haploid plant of claim 40, wherein said CMS haploid plant is confirmed by genotyping or other molecular analysis.

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