BR112018006553B1 - RECOMBINANT NUCLEIC ACID AND METHOD FOR PRODUCING A TRANSGENIC CORN CELL - Google Patents

Abstract

RECOMBINANT NUCLEIC ACID AND METHOD FOR PRODUCING A TRANSGENIC CORN CELL. The present patent application relates to genomic loci of corn B chromosomes and methods of use for agronomic practices.

Description

CROSS REFERENCE TO RELATED ORDERS AND SEQUENCE LISTING INCORPORATION

[001] This application claims priority to Provisional Patent Application Serial No. US 62/236,709 filed on October 2, 2015, Provisional Patent Application Serial No. US 62/237,048, filed on October 5, 2015, and Provisional Patent Application Serial No. US 62/240,770, filed October 13, 2015, which are incorporated herein by reference in their entirety. The sequence listings contained in the files "P34350US00_SEQ.text" (3,202,544 bytes (measured on MS Windows operating system) created on October 2, 2015, filed with US Provisional Patent Application 62/236,709 on October 2, 2015 ), "P34350US01_SEQ. txt" (3,202,584 bytes (measured on MS Windows operating system) created on October 5, 2015, filed with US Provisional Patent Application 62/237,048 on October 5, 2015) and "P34350US02_SEQ. txt" (3,655,665 bytes (measured on MS Windows operating system) created on October 13, 2015, filed with US Provisional Patent Application 62/240,770, on October 13, 2015) are incorporated herein by reference in their entirety . A computer-readable form of a sequence listing is filed with this application via electronic submission and is incorporated into this application by reference in its entirety. The sequence listing is contained in the file called 61653_ANNIV_ST25.txt, which is 3,640,410 bytes in size (measured on the MS Windows operating system) and created on September 30, 2016.

BACKGROUND

[002] B chromosomes are supernumerary chromosomes that occur in many organisms and differ from standard nuclear chromosomes (A chromosomes) in that they rarely carry active genes. These chromosomes are not essential for the life of a species.

[003] The first transgenic plants were generated in the 1990s and were the result of the random insertion of transgenic DNA into nuclear A chromosomes (for example, Roundup Ready® soybeans). As more transgenic events conferring separate traits (e.g., insect resistance, drought tolerance, herbicide tolerance, quality traits) have been generated, there is a desire for plant breeders and farmers to have multiple traits in the plant. As the number of traits per plant increases, there is a corresponding increase in the resources and technical challenges required to generate a breeding stack of multiple traits present on the A chromosomes in elite germplasm. Additionally, with enlarged traces, there is a risk of linkage drag. In maize, trait stacking and reducing the risk of A chromosome linkage drag can be addressed by developing the next generation of transgenic traits on the B chromosome.

BRIEF DESCRIPTION OF FIGURES

[004] Figure 1. Image of a corn root tip cell with chromosomes stained with DAPI fluorescence stain. The arrows show the 20 B chromosomes in the nucleus of an individual plant cell. Image magnification is 40X.

[005] Figure 2. Mapping of the maize Chromosome B/Chromosome A translocation using B chromosome primers. Control PCR amplification regions are indicated: BCHR004 for proximal region; BCHR006 for a region of proximal euchromation; and BCHR007 for a distal region. The regions for PCR amplification primers identified from the unique B chromosome sequence are indicated as primers SEQ ID NO:889 + 890; SEQ ID NO:891 + 892; and SEQ ID NO:893 + 894. The abbreviations in the B chromosome schematic are as follows: CK indicates the centromere region of the B chromosome; PH indicates the region of proximal heterochromatin; PE1/PE2 indicates the region of proximal euchromatin regions; DH1-4 indicates the regions of distal heterochromatin regions; and DE indicates the distal euchromatin region.

SUMMARY

[006] Various embodiments refer to a recombinant nucleic acid comprising: a nucleic acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity for at least 0.25 Kb, 0.5 Kb, 0.75 Kb, 1 Kb, 1.25 Kb, 1.5 Kb, 1.75 Kb, 2 Kb, 2.25 Kb, 2.5 Kb, 2.75 Kb, 3 Kb, 3.25 Kb, 3.5 Kb, 3.75 Kb, 4 Kb , 4.25 Kb, 4.5 Kb, 4.75 Kb, or 5 Kb of a maize B chromosome sequence selected from the group consisting of SEQ ID NO: 1-126 and 128-888, and at least one DNA of interest, wherein at least one of the DNA of interest is integrated into the maize B chromosome sequence to produce the recombinant nucleic acid. In some embodiments, the DNA of interest is integrated into the double-strand break generated by one or more site-specific genome modification enzymes. In some embodiments, the DNA of interest encodes the site-specific genome modification enzyme. In some embodiments, the DNA of interest encodes one or more of an endonuclease, a recombinase, a transposase, a helicase, or any combination thereof. In some embodiments, the DNA of interest comprises one or more gene expression cassettes, wherein the gene expression cassettes are selected from the group comprising: an insecticide resistance gene expression cassette, herbicide tolerance gene expression cassette, nitrogen use efficiency gene expression cassette, a water use efficiency gene expression cassette, a nutritional quality gene expression cassette, a DNA binding gene expression cassette, a selectable marker gene expression cassette , an RNAi construct expression cassette, a site-specific genome modification enzyme gene expression cassette, or an expression cassette encoding one or more of a CRIPR-associated protein, a tracr RNA, and a guide RNA .

[007] Various embodiments relate to a corn plant comprising a recombinant nucleic acid comprising: a nucleic acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity for at least 0.25 Kb, 0.5 Kb, 0.75 Kb, 1 Kb, 1.25 Kb, 1.5 Kb, 1.75 Kb, 2 Kb, 2.25 Kb, 2.5 Kb, 2.75 Kb, 3 Kb, 3.25 Kb, 3.5 Kb, 3, 75 Kb, 4 Kb, 4.25 Kb, 4.5 Kb, 4.75 Kb, or 5 Kb of a corn B chromosome sequence selected from the group consisting of SEQ ID NO: 1-126 and 128-888, and at least one DNA of interest, wherein at least one of the DNA of interest is integrated into the maize B chromosome sequence to produce the recombinant nucleic acid. In some embodiments, the DNA of interest is integrated into the double-strand break generated by one or more site-specific genome modification enzymes. In some embodiments, the DNA of interest encodes the site-specific genome modification enzyme. In some embodiments, the DNA of interest encodes one or more of an endonuclease, a recombinase, a transposase, a helicase, or any combination thereof. In some embodiments, the DNA of interest comprises one or more gene expression cassettes, wherein the gene expression cassettes are selected from the group comprising: an insecticide resistance gene expression cassette, herbicide tolerance gene expression cassette, nitrogen use efficiency gene expression cassette, a water use efficiency gene expression cassette, a nutritional quality gene expression cassette, a DNA binding gene expression cassette, a selectable marker gene expression cassette , an RNAi construct expression cassette, a site-specific genome modification enzyme gene expression cassette, or an expression cassette encoding one or more of a CRIPR-associated protein, a tracr RNA, and a guide RNA .

[008] Various embodiments refer to a recombinant B chromosome comprising: a nucleic acid sequence with at least 90%, at least 91%, at least 92%, at least 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity for at least 0.25 Kb, 0.5 Kb, 0.75 Kb, 1 Kb, 1.25 Kb, 1.5 Kb, 1.75 Kb, 2 Kb, 2.25 Kb, 2.5 Kb, 2.75 Kb, 3 Kb, 3.25 Kb, 3.5 Kb, 3.75 Kb, 4 Kb , 4.25 Kb, 4.5 Kb, 4.75 Kb, or 5 Kb of a maize B chromosome sequence selected from the group consisting of SEQ ID NO: 1-126 and 128-888, and at least one DNA of interest, wherein at least one of the DNA of interest is integrated into the maize B chromosome sequence to produce the recombinant nucleic acid. In some embodiments, the DNA of interest is integrated into the double-strand break generated by one or more site-specific genome modification enzymes. In some embodiments, the DNA of interest encodes the site-specific genome modification enzyme. In some embodiments, the DNA of interest encodes one or more of an endonuclease, a recombinase, a transposase, a helicase, or any combination thereof. In some embodiments, the DNA of interest comprises one or more gene expression cassettes, wherein the gene expression cassettes are selected from the group comprising: an insecticide resistance gene expression cassette, herbicide tolerance gene expression cassette, nitrogen use efficiency gene expression cassette, a water use efficiency gene expression cassette, a nutritional quality gene expression cassette, a DNA binding gene expression cassette, a selectable marker gene expression cassette , an RNAi construct expression cassette, a site-specific genome modification enzyme gene expression cassette, or an expression cassette encoding one or more of a CRIPR-associated protein, a tracr RNA, and a guide RNA .

[009] Various embodiments refer to a method of manufacturing a corn cell comprising at least one DNA of interest integrated into a B chromosome by selecting at least one target having at least 90%, at least 91%, at least 92%, at least 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity for at least 0.25 Kb, 0 .5 Kb, 0.75 Kb, 1 Kb, 1.25 Kb, 1.5 Kb, 1.75 Kb, 2 Kb, 2.25 Kb, 2.5 Kb, 2.75 Kb, 3 Kb, 3, 25 Kb, 3.5 Kb, 3.75 Kb, 4 Kb, 4.25 Kb, 4.5 Kb, 4.75 Kb, or 5 Kb of a maize B chromosome sequence selected from the group consisting of SEQ ID NO: 1-126 and 128-888; selecting a site-specific genome modification enzyme that specifically cleaves the target site; introducing the site-specific genome modification enzyme into the maize cell; introducing the DNA of interest into the corn cell; integrating the DNA of interest into the target site; and selecting a transgenic corn cell comprising at least one of the DNA of interest integrated into the B chromosome. In some embodiments, the DNA of interest is integrated into the double-strand break generated by one or more site-specific genome modification enzymes. In some embodiments, the DNA of interest encodes the site-specific genome modification enzyme. In some embodiments, the DNA of interest encodes one or more of an endonuclease, a recombinase, a transposase, a helicase, or any combination thereof. In some embodiments, the DNA of interest comprises one or more gene expression cassettes, wherein the gene expression cassettes are selected from the group comprising: an insecticide resistance gene expression cassette, herbicide tolerance gene expression cassette, nitrogen use efficiency gene expression cassette, a water use efficiency gene expression cassette, a nutritional quality gene expression cassette, a DNA binding gene expression cassette, a selectable marker gene expression cassette , an RNAi construct expression cassette, a site-specific genome modification enzyme gene expression cassette, or an expression cassette encoding one or more of a CRIPR-associated protein, a tracr RNA, and a guide RNA .

[0010] Various embodiments relate to a method of providing a site-specific genome modification enzyme to a maize cell comprising integrating at least one site-specific genome modification enzyme into a sequence having at least 90%, at least 91%, at least 92%, at least 93%, 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, sequence identity to at least 0.25 Kb, 0.5 Kb, 0.75 Kb, 1 Kb, 1.25 Kb, 1.5 Kb, 1.75 Kb, 2 Kb, 2.25 Kb, 2.5 Kb, 2, 75 Kb, 3 Kb, 3.25 Kb, 3.5 Kb, 3.75 Kb, 4 Kb, 4.25 Kb, 4.5 Kb, 4.75 Kb, or 5 Kb of a selected maize B chromosome sequence of the group consisting of SEQ ID NO: 1-126 and 128-888; and selecting a maize cell comprising site-specific genome modification enzyme integrated into the B chromosome. In some embodiments, the site-specific genome modification enzyme specifically cleaves a target site on one or more A chromosomes in which a DNA of interest is integrated. In some embodiments, the DNA of interest comprises one or more gene expression cassettes, wherein the gene expression cassettes are selected from the group comprising: an insecticide resistance gene expression cassette, herbicide tolerance gene expression cassette, nitrogen use efficiency gene expression cassette, a water use efficiency gene expression cassette, a nutritional quality gene expression cassette, a DNA binding gene expression cassette, a selectable marker gene expression cassette and an RNAi construct expression cassette. In some embodiments, progeny are selected that comprise the DNA of interest, but do not comprise the B chromosome comprising the site-specific genome modification enzyme. In some embodiments, the B chromosome comprises a negative selection marker.

[0011] Various embodiments relate to a recombinant nucleic acid comprising a nucleic acid sequence of at least 1 Kb having at least 90% sequence identity with a corn B chromosome sequence selected from the group consisting of SEQ ID NO: 1-126 and 128-888, and a DNA of interest integrated into the maize B chromosome sequence to produce the recombinant nucleic acid. In some embodiments, the recombinant nucleic acid comprises a DNA of interest that is integrated proximal to a target site for a site-specific genome modification enzyme. In some embodiments, the site-specific genome modification enzyme is an endonuclease. In some embodiments, the site-specific genome modification enzyme is a recombinase. In some embodiments, the site-specific genome modification enzyme is a transposase. In some embodiments, the site-specific genome modification enzyme is a helicase. In some embodiments, the site-specific genome modification enzyme is any combination of an endonuclease, a recombinase, a transposase, and a helicase. In some embodiments, the target site is specific to the maize B chromosome sequence. In another embodiment, the target site is specific to the maize B chromosome sequence is selected from one or more of the group comprising SEQ ID NO:1-126 and 128-888. In some embodiments, the genome modifying enzyme site-specific is a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a DNA-guided recombinase, a DNA-guided endonuclease, an RNA-guided recombinase, an RNA-guided endonuclease, a type I CRISPR-Cas system, type II CRISPR-Cas system, and a type III CRISPR-Cas system; In some embodiments, the endonuclease is selected from the group comprising Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 and Csf4 nuclease; RNA-guided recombinase. In some embodiments, the site-specific genome modification enzyme is a fusion protein comprising a recombinase and a CRISPR-associated protein. In some embodiments, the recombinase is a tyrosine recombinase linked to a DNA recognition motif, or a serine recombinase linked to a DNA recognition motif. In some embodiments, the recombinase is a Cre recombinase, a Flp recombinase and a Tnp1 recombinase, a PhiC31 integrase, an R4 integrase, or a TP-901 integrase. In some embodiments, the transposase is a DNA transposase linked to a DNA binding domain. In some embodiments, the DNA of interest does not encode a peptide. In some embodiments, the DNA of interest encodes a peptide. In some embodiments, the DNA of interest comprises one or more gene expression cassettes. In some embodiments, the gene expression cassette is selected from the group comprising an insecticide resistance gene expression cassette, a herbicide tolerance gene expression cassette, a nitrogen use efficiency gene expression cassette, a nitrogen use efficiency gene expression cassette, a water use efficiency gene expression cassette, a nutritional quality gene expression cassette, a DNA binding gene expression cassette, a selectable marker gene expression cassette, an RNAi construct expression cassette, an expression cassette site-specific genome modification enzyme gene, an expression cassette encoding a recombinant guide RNA of an RNA-guided endonuclease, or an expression cassette encoding a recombinant DNA guide. In some embodiments, the DNA of interest is integrated into the target site of the maize B chromosome via a homology-directed repair integration method. In some embodiments, the DNA of interest is integrated into the target site of the maize B chromosome via a non-homologous end joining integration method. In some embodiments, the DNA of interest and/or the maize B chromosome target site sequences are modified during integration of the DNA of interest into the target site of the maize B chromosome sequence. In some embodiments, the recombinant nucleic acid is present in a corn plant, in a corn plant part, in a corn seed or in a corn plant cell.

[0012] Various embodiments relate to a recombinant nucleic acid comprising a nucleic acid sequence of at least 1 Kb having at least 90% sequence identity with a corn B chromosome sequence selected from the group consisting of SEQ ID NO: 1-126 and 128-888, and a DNA of interest integrated into the maize B chromosome sequence to produce the recombinant nucleic acid. In some embodiments, the recombinant nucleic acid comprises a DNA of interest integrated between a pair of target sites for a site-specific genome modification enzyme. In some embodiments, the site-specific genome modification enzyme is an endonuclease. In some embodiments, the site-specific genome modification enzyme is a recombinase. In some embodiments, the site-specific genome modification enzyme is a transposase. In some embodiments, the site-specific genome modification enzyme is a helicase. In some embodiments, the site-specific genome modification enzyme is any combination of an endonuclease, a recombinase, a transposase, and a helicase. In some embodiments, the target site is specific to the maize B chromosome sequence. In another embodiment, the target site is specific to the maize B chromosome sequence is selected from one or more of the group comprising SEQ ID NO: 1-126 and 128888. In some embodiments, the maize B-chromosome specific genome modification enzyme site is a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a DNA-guided recombinase, a DNA-guided endonuclease, an RNA-guided recombinase, an RNA-guided endonuclease, a system CRISPR-Cas type I, CRISPR-Cas type II system, and a CRISPR-Cas type III system; In some embodiments, the endonuclease is selected from the group comprising Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 and Csf4 nuclease. In some embodiments, the site-specific genome modification enzyme is an RNA-guided recombinase. In some embodiments, the site-specific genome modification enzyme is a fusion protein comprising a recombinase and a CRISPR-associated protein. In some embodiments, the recombinase is a tyrosine recombinase linked to a DNA recognition motif, or a serine recombinase linked to a DNA recognition motif. In some embodiments, the recombinase is a Cre recombinase, a Flp recombinase and a Tnp1 recombinase, a PhiC31 integrase, an R4 integrase, or a TP-901 integrase. In some embodiments, the transposase is a DNA transposase linked to a DNA binding domain. In some embodiments, the DNA of interest does not encode a peptide. In some embodiments, the DNA of interest encodes a peptide. In some embodiments, the DNA of interest comprises one or more gene expression cassettes. In some embodiments, the gene expression cassette is selected from the group comprising an insecticide resistance gene expression cassette, a herbicide tolerance gene expression cassette, a nitrogen use efficiency gene expression cassette, a nitrogen use efficiency gene expression cassette, a water use efficiency gene expression cassette, a nutritional quality gene expression cassette, a DNA binding gene expression cassette, a selectable marker gene expression cassette, an RNAi construct expression cassette, an expression cassette site-specific genome modification enzyme gene, an expression cassette encoding a recombinant guide RNA of an RNA-guided endonuclease, or an expression cassette encoding a recombinant DNA guide. In some embodiments, the DNA of interest is integrated into the target site of the maize B chromosome via a homology-directed repair integration method. In some embodiments, the DNA of interest is integrated into the target site of the maize B chromosome via a non-homologous end joining integration method. In some embodiments, the DNA of interest and/or the maize B chromosome target site sequences are modified during integration of the DNA of interest into the maize B chromosome target site sequences. In some embodiments, the recombinant nucleic acid is present in a corn plant, a corn plant part, a corn seed, or a corn plant cell.

[0013] Various embodiments relate to a recombinant nucleic acid comprising a nucleic acid sequence of at least 1 Kb having at least 90% sequence identity with a corn B chromosome sequence selected from the group consisting of SEQ ID NO: 1-126 and 128-888, and a DNA of interest integrated into the maize B chromosome sequence to produce the recombinant nucleic acid. In some embodiments, the recombinant nucleic acid comprises one or more of the DNA of interest inserted near two or more target sites for a site-specific genome modification enzyme. In some embodiments, the site-specific genome modification enzyme is an endonuclease. In some embodiments, the site-specific genome modification enzyme is a recombinase. In some embodiments, the site-specific genome modification enzyme is a transposase. In some embodiments, the site-specific genome modification enzyme is a helicase. In some embodiments, the site-specific genome modification enzyme is any combination of an endonuclease, a recombinase, a transposase, and a helicase. In some embodiments, the target site is specific to the maize B chromosome sequence. In another embodiment, the target site is specific to the maize B chromosome sequence is selected from one or more of the group comprising SEQ ID NO: 1-126 and 128-888. In some embodiments, the genome modifying enzyme site-specific is a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a DNA-guided recombinase, a DNA-guided endonuclease, an RNA-guided recombinase, an RNA-guided endonuclease, a type I CRISPR-Cas system, type II CRISPR-Cas system, and a type III CRISPR-Cas system; In some embodiments, the endonuclease is selected from the group comprising Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 and Csf4 nuclease; RNA-guided recombinase. In some embodiments, the site-specific genome modification enzyme is a fusion protein comprising a recombinase and a CRISPR-associated protein. In some embodiments, the recombinase is a tyrosine recombinase linked to a DNA recognition motif, or a serine recombinase linked to a DNA recognition motif. In some embodiments, the recombinase is a Cre recombinase, a Flp recombinase and a Tnp1 recombinase, a PhiC31 integrase, an R4 integrase, or a TP-901 integrase. In some embodiments, the transposase is a DNA transposase linked to a DNA binding domain. In some embodiments, two or more of the DNA of interest are the same. In some embodiments, two or more of the DNA of interest are not the same. In some embodiments, the DNA of interest does not encode a peptide. In some embodiments, the DNA of interest encodes a peptide. In some embodiments, the DNA of interest comprises one or more gene expression cassettes. In some embodiments, the gene expression cassette is selected from the group comprising an insecticide resistance gene expression cassette, a herbicide tolerance gene expression cassette, a nitrogen use efficiency gene expression cassette, a nitrogen use efficiency gene expression cassette, a water use efficiency gene expression cassette, a nutritional quality gene expression cassette, a DNA binding gene expression cassette, a selectable marker gene expression cassette, an RNAi construct expression cassette, an expression cassette site-specific genome modification enzyme gene, an expression cassette encoding a recombinant guide RNA of an RNA-guided endonuclease, or an expression cassette encoding a recombinant DNA guide. In some embodiments, the DNA of interest is integrated into the target site of the maize B chromosome via a homology-directed repair integration method. In some embodiments, the DNA of interest is integrated into the target site of the maize B chromosome via a non-homologous end joining integration method. In some embodiments, two or more of the maize B chromosome target site sequences each comprise a DNA of interest integrated to produce two or more recombinant sequences. In some embodiments, the two or more recombinant sequences are located on the same B chromosome. In some embodiments, the two or more recombinant sequences are located on different B chromosomes. In some embodiments, the two or more recombinant sequences are located on different B chromosomes. which, during cell division, recombine to generate a new mg-focal on a new B chromosome. In some embodiments, the DNA of interest and/or maize B chromosome target site sequences are modified during integration of the maize B chromosome. interest in the target site of the corn B chromosome sequence. In some embodiments, the recombinant nucleic acid is present in a corn plant, in a corn plant part, in a corn seed, or in a corn plant cell.

[0014] Various embodiments relate to a recombinant nucleic acid comprising a nucleic acid sequence of at least 1 Kb having at least 90% sequence identity with a corn B chromosome sequence selected from the group consisting of SEQ ID NO: 1-126 and 128-888, and a DNA of interest integrated into the maize B chromosome sequence to produce the recombinant nucleic acid. In some embodiments, the recombinant nucleic acid comprises one or more of the DNA of interest inserted near two or more target sites for a site-specific genome modification enzyme. In some embodiments, the site-specific genome modification enzyme is an endonuclease. In some embodiments, the site-specific genome modification enzyme is a recombinase. In some embodiments, the site-specific genome modification enzyme is a transposase. In some embodiments, the site-specific genome modification enzyme is a helicase. In some embodiments, the site-specific genome modification enzyme is any combination of an endonuclease, a recombinase, a transposase, and a helicase. In some embodiments, the target site is specific to the maize B chromosome sequence. In another embodiment, the target site is specific to the maize B chromosome sequence is selected from one or more of the group comprising SEQ ID NO: 1-126 and 128-888. In some embodiments, the genome modifying enzyme site-specific is a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a DNA-guided recombinase, a DNA-guided endonuclease, an RNA-guided recombinase, an RNA-guided endonuclease, a type I CRISPR-Cas system, type II CRISPR-Cas system, and a type III CRISPR-Cas system; In some embodiments, the endonuclease is selected from the group comprising Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 and Csf4 nuclease; RNA-guided recombinase. In some embodiments, the site-specific genome modification enzyme is a fusion protein comprising a recombinase and a CRISPR-associated protein. In some embodiments, the recombinase is a tyrosine recombinase linked to a DNA recognition motif, or a serine recombinase linked to a DNA recognition motif. In some embodiments, the recombinase is a Cre recombinase, a Flp recombinase and a Tnp1 recombinase, a PhiC31 integrase, an R4 integrase, or a TP-901 integrase. In some embodiments, the transposase is a DNA transposase linked to a DNA binding domain. In some embodiments, two or more of the DNA of interest are the same. In some embodiments, two or more of the DNA of interest are not the same. In some embodiments, the DNA of interest does not encode a peptide. In some embodiments, the DNA of interest encodes a peptide. In some embodiments, the DNA of interest comprises one or more gene expression cassettes. In some embodiments, the gene expression cassette is selected from the group comprising an insecticide resistance gene expression cassette, a herbicide tolerance gene expression cassette, a nitrogen use efficiency gene expression cassette, a nitrogen use efficiency gene expression cassette, a water use efficiency gene expression cassette, a nutritional quality gene expression cassette, a DNA binding gene expression cassette, a selectable marker gene expression cassette, an RNAi construct expression cassette, an expression cassette site-specific genome modification enzyme gene, an expression cassette encoding a recombinant guide RNA of an RNA-guided endonuclease, or an expression cassette encoding a recombinant DNA guide. In some embodiments, the DNA of interest is integrated into the target site of the maize B chromosome via a homology-directed repair integration method. In some embodiments, the DNA of interest is integrated into the target site of the maize B chromosome via a non-homologous end joining integration method. In some embodiments, two or more of the maize B chromosome target site sequences each comprise a DNA of interest integrated to produce two or more recombinant sequences. In some embodiments, the two or more recombinant sequences are located on the same B chromosome. In some embodiments, the two or more recombinant sequences are located on different B chromosomes. In some embodiments, the two or more recombinant sequences are located on different B chromosomes. which, during cell division, recombine to generate a new mg-focal on a new B chromosome. In some embodiments, the DNA of interest and/or maize B chromosome target site sequences are modified during integration of the maize B chromosome. interest in the target site of the corn B chromosome sequence. In some embodiments, the recombinant nucleic acid is present in a corn plant, in a corn plant part, in a corn seed, or in a corn plant cell.

[0015] Various embodiments relate to a method for producing a corn plant cell comprising a recombinant nucleic acid with a DNA of interest integrated into a B chromosome, the method comprising: (a) selecting the target site at a genomic locus of the maize B chromosome having at least 90% sequence identity of the group consisting of SEQ ID NO: 1-126 and 128-888; (b) selecting a site-specific genome modification enzyme that specifically binds and cleaves the target site in the maize B chromosome genomic locus; (c) introducing the site-specific genome modification enzyme into a maize plant cell; (d) optionally, when the site-specific genome modification enzyme of step (c) is an RNA-guided endonuclease, introducing a guide RNA into a corn plant cell; or (e) optionally, when the site-specific genome modification enzyme of step (c) is a DNA-guided endonuclease, introducing a guide DNA into a maize plant cell; (e) introducing the DNA of interest into the corn plant cell; (f) integrate the DNA of interest proximal to the target site in the maize B chromosome genomic locus; and (g) selecting transgenic plant cells comprising the DNA of interest integrated into the target site of the maize B chromosome genomic locus. Various embodiments relate to a method for producing a corn plant cell comprising a recombinant nucleic acid with a DNA of interest integrated into a B chromosome, the method comprising: (a) selecting a pair of target sites at a genomic locus of the chromosome Corn B having at least 90% sequence identity to the group consisting of SEQ ID NO: 1126 and 128-888; (b) selecting one or more site-specific genome modification enzymes that specifically bind and cleave the pair of target sites in the maize B chromosome genomic locus; c) introducing site-specific genome modification enzymes into a maize plant cell; (d) optionally, when one of the site-specific genome modification enzymes of step (c) is an RNA-guided endonuclease, introducing a guide RNA into a corn plant cell; or (e) optionally, when one of the site-specific genome modification enzymes of step (c) is a DNA-guided endonuclease, introducing a guide DNA into a maize plant cell; (e) introducing the DNA of interest into the corn plant cell; (f) integrating the DNA of interest between the pair of target sites for site-specific genome modification enzymes at a maize B chromosome genomic locus; and (g) selecting transgenic plant cells comprising at least one of the DNA of interest integrated into at least one of the target sites of the maize B chromosome genomic locus. Various embodiments relate to a method for producing a corn plant cell comprising a recombinant nucleic acid with at least one DNA of interest integrated into a B chromosome, the method comprising: (a) selecting two or more target sites at a genomic locus of the maize B chromosome with at least 90% sequence identity of the group consisting of SEQ ID NO: 1-126 and 128-888; (b) selecting one or more site-specific genome modification enzymes that specifically bind and cleave the two or more target sites in the maize B chromosome genomic locus; (c) introducing the site-specific genome modification enzymes into a maize plant cell; (d) optionally, when one of the site-specific genome modification enzymes of step (c) is an RNA-guided endonuclease, introducing a guide RNA into a corn plant cell; or (e) optionally, when one of the site-specific genome modification enzymes of step (c) is a DNA-guided endonuclease, introducing a guide DNA into a maize plant cell; (e) introducing at least one DNA of interest into the corn plant cell; (f) integrating the at least one DNA of interest proximal to two or more target sites for site-specific genome modification enzymes at a genomic locus of the maize B chromosome; and (g) selecting transgenic plant cells comprising at least one of the DNA of interest integrated into at least one of the target sites of the maize B chromosome genomic locus. Various embodiments relate to a method for producing a corn plant cell comprising a recombinant nucleic acid with two or more DNA of interest integrated into a B chromosome, the method comprising: (a) selecting two or more target sites at a genomic locus of maize B chromosome having at least 90% sequence identity of the group consisting of SEQ ID NO:1-126 and 128-888; (b) selecting one or more site-specific genome modification enzymes that specifically bind and cleave the two or more target sites in the maize B chromosome genomic locus; (c) introducing the site-specific genome modification enzymes into a maize plant cell; (d) optionally, when one of the site-specific genome modification enzymes of step (c) is an RNA-guided endonuclease, introducing a guide RNA into a corn plant cell; or (e) optionally, when one of the site-specific genome modification enzymes of step (c) is a DNA-guided endonuclease, introducing a guide DNA into a maize plant cell; (e) introducing the two or more DNA of interest into the corn plant cell; (f) integrating the two or more DNA of interest proximal to two or more target sites for site-specific genome modification enzymes at a maize B chromosome genomic locus; and (g) selecting transgenic plant cells comprising at least one of the DNA of interest integrated into at least one of the target sites of the maize B chromosome genomic locus. In some embodiments, the site-specific genome modification enzyme is an endonuclease. In some embodiments, the site-specific genome modification enzyme is a recombinase. In some embodiments, the site-specific genome modification enzyme is a transposase. In some embodiments, the site-specific genome modification enzyme is a helicase. In some embodiments, the site-specific genome modification enzyme is any combination of an endonuclease, a recombinase, a transposase, and a helicase. In some embodiments, the target site is specific to the maize B chromosome sequence. In another embodiment, the target site is specific to the maize B chromosome sequence is selected from one or more of the group comprising SEQ ID NO: 1-126 and 128-888. In some embodiments, the genome modifying enzyme site-specific is a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a DNA-guided recombinase, a DNA-guided endonuclease, an RNA-guided recombinase, an RNA-guided endonuclease, a type I CRISPR-Cas system, type II CRISPR-Cas system, and a type III CRISPR-Cas system; In some embodiments, the endonuclease is selected from the group comprising Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 and Csf4 nuclease. In some embodiments, the site-specific genome modification enzyme is an RNA-guided recombinase. In some embodiments, the site-specific genome modification enzyme is a fusion protein comprising a recombinase and a CRISPR-associated protein. In some embodiments, the recombinase is a tyrosine recombinase linked to a DNA recognition motif, or a serine recombinase linked to a DNA recognition motif. In some embodiments, the recombinase is a Cre recombinase, a Flp recombinase and a Tnp1 recombinase, a PhiC31 integrase, an R4 integrase, or a TP-901 integrase. In some embodiments, the transposase is a DNA transposase linked to a DNA binding domain. In some embodiments, two or more of the DNA of interest are the same. In some embodiments, two or more of the DNA of interest are not the same. In some embodiments, the DNA of interest does not encode a peptide. In some embodiments, the DNA of interest encodes a peptide. In some embodiments, the DNA of interest comprises one or more gene expression cassettes. In some embodiments, the gene expression cassette is selected from the group comprising an insecticide resistance gene expression cassette, a herbicide tolerance gene expression cassette, a nitrogen use efficiency gene expression cassette, a nitrogen use efficiency gene expression cassette, a water use efficiency gene expression cassette, a nutritional quality gene expression cassette, a DNA binding gene expression cassette, a selectable marker gene expression cassette, an RNAi construct expression cassette, an expression cassette site-specific genome modification enzyme gene, an expression cassette encoding a recombinant guide RNA of an RNA-guided endonuclease, or an expression cassette encoding a recombinant DNA guide. In some embodiments, the site-specific genome modification enzyme is stably transformed in the corn plant cell. In some embodiments, the site-specific genome modification enzyme is transiently transformed in the corn plant cell. In some embodiments, the site-specific genome modification enzyme is constitutively expressed in the corn plant cell. In some embodiments, the site-specific genome modification enzyme is expressed in the corn plant cell under the control of a regulatable promoter. In some embodiments, the regulatable promoter is a heat shock promoter, a tissue-specific promoter, or a chemically inducible promoter. In some embodiments, the DNA of interest is integrated into the target site of the maize B chromosome via a homology-directed repair integration method. In some embodiments, the DNA of interest is integrated into the target site of the maize B chromosome via a non-homologous end joining integration method. In some embodiments, two or more of the maize B chromosome target site sequences each comprise a DNA of interest integrated to produce two or more recombinant sequences. In some embodiments, the two or more recombinant sequences are located on the same B chromosome. In some embodiments, the two or more recombinant sequences are located on different B chromosomes. In some embodiments, the two or more recombinant sequences are located on different B chromosomes. which, during cell division, recombine to generate a new mg-focal on a new B chromosome. In some embodiments, the DNA of interest and/or maize B chromosome target site sequences are modified during integration of the maize B chromosome. interest in the target site of the corn B chromosome sequence. In some embodiments, the recombinant nucleic acid is present in a corn plant, in a corn plant part, in a corn seed, or in a corn plant cell.

[0016] In some embodiments, specific probes are developed for the corn B chromosome sequence selected from the group consisting of SEQ ID NO: 1-126 and 128-888. In some embodiments, probes are generated by PCR amplification. In some embodiments, the PCR primers are selected from the group consisting of SEQ ID NO:889-894. In some embodiments, PCR primers are used to map maize Chromosome B/Chromosome A chromosomal translocations. In some embodiments, the corn B chromosome sequence is used to develop corn B chromosome markers. In some embodiments, the corn B chromosome sequence is used to identify the flanking sequence of transgenes integrated into a B chromosome.

[0017] Various embodiments relate to a method of identifying a unique sequence for the corn B chromosome including one or more of the steps: (1) identifying a corn plant with a high copy number of the B chromosome; (2) prepare DNA from tissue collected from the plant in step 1; (3) prepare a fosmid library with the DNA from step 2 and aliquot into pools; (3) sequence the assembled fosmid library; (4) use bioinformatics analysis to extract the unique sequence of the maize B chromosome. In some embodiments, the corn plant with a high B chromosome cell nucleus is selected from a plant with 1, 2, 3, 4, 5, 6, 7, 8, 8, 10, 11, 12, 13, 14 , 15 , 16, 17, 18, 19, 20 or more B chromosomes. In some embodiments, bioinformatics analysis includes: (1) extracting good quality, usable sequence from the assembled fosmid library; (2) assemble the sequence of reads from step 1 into individual assembly contigs; (3) analyze and mask the individual contig assembly from step 2 to (a) known maize A chromosome non-repeat sequence from a reference maize genome assembly, (b) to known maize mitochondrial DNA sequence from a reference corn genome, (c) for known corn chloroplast DNA sequence in a reference corn genome assembly; and (d) for E. coli and/or fosmid vector sequences; (4) analyzing the assembled sequence from step (3) against each other to identify the B chromosome repeat sequence or to filter out the maize A chromosome sequence that is not in the maize reference genome assembly; and (4) compare the resulting sequences to identify the longest representative sequence for each contig and assemble the largest contig from overlapping contigs. In other embodiments, the long-read sequence is generated from maize B chromosome DNA using single-molecule, real-time sequencing technology to generate a high-quality contiguous assembly of the genomic sequence. In other embodiments, the maize B chromosome contig assembly from fosmid library analysis and the long read sequence are combined to generate an assembly of a unique B chromosome sequence. DETAILED DESCRIPTION

[0018] Although maize B chromosomes have been studied for many decades, a comprehensive set of sequences unique to maize B chromosomes is not available. The present disclosure provides unique sequences of maize B chromosomes useful for the development of probes for mapping B chromosomes, the development of B chromosome genomic markers, and the identification of loci for site-specific integration of a DNA of interest into B chromosomes. .

[0019] Various embodiments described herein refer to ways to identify unique maize B chromosome sequences. These unique maize B chromosome sequences are useful for developing site-specific targeted integration of a DNA of interest into a single locus, integration of two or more DNAs of interest into separate loci, integration of two or more DNAs of interest at a single locus or integration of two or more DNA of interest at two or more loci. The integration of two or more DNA of interest at separate but linked loci is also known as "stacking".

[0020] Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one skilled in the art to which the present invention belongs. Where a term is provided in the singular, the inventors also contemplate aspects of the description described by the plural of that term. Where there are discrepancies in the terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given here. Other technical terms used have their common meaning in the technique in which they are used, as exemplified by several technique-specific dictionaries, e.g., "The American Heritage® Science Dictionary" (Publishers of American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), the "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or the "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to limit themselves to one mechanism or mode of action. Reference is provided for illustrative purposes only.

[0021] The practice of the present description employs, unless otherwise indicated, conventional techniques of biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and biotechnology, which are within the skill of the technique. See Green and Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, 4th edition (2012); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the METHODS IN ENZYMOLOGY series (Academic Press, Inc.):PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames, and G. R. Taylor eds. (1995)); Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL; ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)); RECOMBINANT PROTEIN PURIFICATION:PRINCIPLES AND METHODS, 18-1142-75, GE Healthcare Life Sciences; C. N. Stewart, A. Touraev, V. Citovsky, T. Tzfira eds. (2011) PLANT TRANSFORMATION TECHNOLOGIES (Wiley-Blackwell); and R. H. Smith (2013) PLANT TISSUE CULTURE. TECHNIQUES AND EXPERIMENTS (Academic Press, Inc.).

[0022] All references cited herein are incorporated by reference in their entirety.

[0023] As used herein, the singular form "a", "an" and "a/o" includes plural references unless the context clearly dictates otherwise. Thus, for example, reference to "plant", "the plant" or "a plant" also includes a plurality of plants; also, depending on the context, use of the term "plant" may also include genetically similar or identical progeny of that plant; use of the term "a nucleic acid" optionally includes, as a practical matter, many copies of that nucleic acid molecule; Similarly, the term "probe" optionally (and typically) encompasses many similar or identical probe molecules.

[0024] As used herein, "plant" refers to an entire plant or a cell or tissue culture derived from a plant, comprising any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells and/or progeny thereof. A progeny plant can be of any filial generation, for example, F1, F2, F3, F4, F5, F6, F7, etc. A plant cell is a biological cell of a plant, taken from a plant or derived through cultivation from a cell taken from a plant. Plant parts include harvestable parts and parts useful for propagating progeny plants. Plant parts useful for propagation include, for example and without limitation: seed; fruit; a cut; a seedling; a tuber; and a rootstock. A crop part of a plant may be any useful part of a plant, including, for example and without limitation: flower; pollen; changes; tuber; sheet; stem; fruit; seed; and root. Plant cells, as used herein, include protoplasts and protoplasts with a cell wall. A plant cell can be a protoplast, a gamete-producing cell, or a cell or collection of cells that can regenerate into an entire plant.

[0025] As used herein, the term "about" indicates that a value includes the inherent variation in error for the method being employed to determine the value, or the variation that exists among experiments.

[0026] As used herein, "corn" and "maize" interchangeably refer to Zea mays L and include all plant varieties that can be reproduced with corn, including wild corn species. As used here, "corn" and "corn" may be used interchangeably.

[0027] As used here, rye refers to Secale cereale.

[0028] In one aspect, a corn plant or seed provided in the present invention is Zea mays L. In another aspect, a corn plant or seed provided in the present invention is Zea mays ssp. mays. In yet another aspect, a corn plant or seed provided herein is a domesticated strain or variety.

[0029] As used herein, the term "B chromosome" refers to an extra or supernumerary chromosome. B chromosomes are found in addition to the normal diploid complement of chromosomes in a cell. In maize, the normal diploid complement of chromosomes is 20. Normal chromosomes may be called "A chromosomes". B chromosomes are dispensable and are not necessary for normal development. When two B chromosomes are present in a single plant, the two B chromosomes come together in meiotic prophase and recombination can occur. B chromosomes do not associate with A chromosomes.

[0030] In one aspect, the methods of the present invention incorporate DNAs of interest into a supernumerary chromosome. In some embodiments, the methods of the present invention incorporate DNAs of interest into a corn B chromosome sequence selected from the group consisting of SEQ ID NO: 1-126 and 128888.

[0031] One or more B chromosomes, in accordance with certain aspects of the present description, can be administered to a progeny without the rest of the genome (for example, through a haploid induction cross that retains the B chromosome), allowing conversion complete for a new variety in a single cross. In other aspects, a B chromosome can be transferred to other species, allowing testing of the DNA of interest or DNAs of interest in other cultures. For example, transmission of a B chromosome to oats has been demonstrated (Koo et al., Genome Research 21(6):908-914, 2011), as has transmission of a corn chromosome to wheat (Comeau et al ., Plant Science 81(1):117-125, 1992).

[0032] In certain cases, such as in corn and rye, B chromosomes have "accumulation mechanisms" that allow them to transmit at frequencies higher than Mendelian. For example, in maize, the sister chromatids of the B chromosome do not separate during the second pollen division (first generator). As a result, both sister chromatids are delivered to one of the pollen cells, while the other pollen cell receives neither. This effect, called nondisjunction, means that a plant with just a single B chromosome can provide two B chromosomes to the next generation when used as a male. Such an effect may be desirable during the process of trait introgression, as it allows individuals homozygous (as opposed to hemizygous) for a megalocus carried on a B chromosome to be recovered in a backcross, as long as the B chromosome is released from pollen.

[0033] The nondisjunction effect requires that specific portions of the B chromosome are present. A trans-active piece at the tip of the long arm and a cis-active piece near the centromere are required. Very small deletions at the tip of the long arm of the B chromosome are recoverable and the resulting B chromosomes do not exhibit any nondisjunction. In certain embodiments of the disclosure, such a B chromosome deletion variant may be desired, for example, for the purpose of providing a megalocus for commercial traits. In other embodiments, nondisjunction may be desired, for example, for the purpose of transiently providing one or more site-specific genome modification enzymes that modify one or more target sequences in the A genome to produce one or more gene edits or transgenic insertions and where B chromosomes comprising one or more site-specific genome modification enzymes are lost in subsequent generations.

[0034] Various modalities refer to the use of B chromosomes to quickly transfer the haploid induction effect to new lineages. Because the B chromosome can be retained at a low percentage, a line in which the haploid induction effect is caused by the B chromosome can allow the effect to be carried to additional lines in a single cross. This simplifies the creation of new haploid induction lines with desired agronomic or genetic properties. The genetic elements disclosed in US Patent Application 62/375. 618 (titled Compositions and Methods for Plant Haploid Induction, deposited on August 16, 2016) can be incorporated into the unique B chromosome sequences described here to produce plants containing the haploid-inducing B chromosome (HI-B chromosome). The description of US Patent Application 62/375. 618 is incorporated herein by reference in its entirety. Other haploid induction genes, for example, the CENH3-based transgenes (Kelliher, T et al., "Maternal Haploids Are Preferentially Induced by CENH3-tailswap Transgenic Complementation in Maize", Frontiers in Plant Science 7:414 (2016)) as well can be incorporated into unique B chromosome sequences, as described herein, to produce plants containing the HI-B chromosome. To move the haploid induction effect to new lines, a line containing the HI-B chromosome is crossed to the desired line and the progeny are screened for cases that are haploid and that have retained the HI-B chromosome.

[0035] In one aspect, a B chromosome provided here is a corn B chromosome. In one aspect, a corn B chromosome sequence or corn B chromosome genomic loci provided herein is selected from the group consisting of SEQ ID NO: 1-126 and 128-888. In one aspect, a B chromosome provided herein undergoes nondisjunction. In one aspect, a B chromosome provided herein undergoes nondisjunction in a pollen cell. In another aspect, a B chromosome provided here accumulates in a non-Mendelian manner. In one aspect, a B chromosome provided herein is truncated. In one aspect, a B chromosome provided herein may comprise one or more DNA of interest. In another aspect, a B chromosome provided herein can be used in any method provided herein. In one aspect, a B chromosome provided herein is heterogeneous. In one aspect, a B chromosome provided herein is univalent during meiosis. In one aspect, a B chromosome provided herein pairs with a second B chromosome during meiosis. In one aspect, a B chromosome provided herein pairs with a second B chromosome during meiosis, and the first and second B chromosomes recombine to create a new B chromosome. In one aspect, a B chromosome provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten target sites for site-specific genome modification enzymes. In one aspect, a B chromosome provided herein comprises a translocation between a B chromosome and an A chromosome, wherein the B chromosome comprises a centromere of the B chromosome. In one aspect, a first B chromosome provided herein has at least 50%, at least at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with a second B chromosome.

[0036] In one aspect, a plant cell provided herein may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more , ten or more, fifteen or more, twenty or more, or twenty-five or more B chromosomes.

[0037] The methods described herein can be applied to extract unique B chromosome sequences of sufficient length to provide gene targeting to multiple sites and to allow the development of B chromosome markers. site-directed genome modification, including site-specific integration of a DNA of interest. The methods described here can be applied to any genome with B chromosomes that utilizes an assembled bacterial artificial chromosome (BAC), a fosmid sequencing strategy, or a single-molecule real-time sequencing strategy.

[0038] The sequences described here can also be used to create gene editing tools to produce truncated B chromosomes with altered transmission properties, which would be useful in a 1-cross trait integration strategy. The sequences disclosed herein can also be used to identify polymorphisms between different sources of B chromosomes and such polymorphisms can be used to construct a genetic recombination map of the B chromosome. The sequences disclosed herein can also be used as FISH probes or in other mapping strategies. physical (B-A translocation mapping) to locate contig sequences relative to each other and other landmarks on the B chromosome (centromere, telomere, published B repeats, etc).

[0039] As used herein, "recombinant sequence" and "recombinant nucleic acid sequence" are used interchangeably.

[0040] As used herein, "inserted" and "integrated" are used interchangeably.

[0041] In some embodiments, a DNA of interest may be a transgene. In some embodiments, a DNA of interest may be a DNA molecule that is "cis-genic," which refers to a DNA sequence from the crop plant itself or from a sexually compatible donor plant. In some embodiments, a DNA of interest may comprise one or more "transgenes", where a transgene is a DNA sequence not found naturally on the maize B chromosome.

[0042] In one aspect, the present invention provides one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more , fifteen or more twenty or more, or twenty-five or more recombinant nucleic acid sequences comprising a nucleic acid sequence of at least 25 base pairs, at least 50 base pairs, at least 100 base pairs, at least 250 base pairs, at least 500 base pairs at least 1000 base pairs (1 Kb), at least 1500 base pairs (1.5 Kb), at least 2000 base pairs (2 Kb), at least 2500 base pairs bases (2.5 Kb), at least 3000 base pairs (3 Kb), at least 3500 base pairs (3.5 Kb), at least 4000 base pairs (4 Kb), at least 4500 base pairs ( 4.5 Kb), at least 5000 base pairs (5 Kb), at least 5500 base pairs (5.5 Kb), at least 6000 base pairs (6 Kb), at least 6500 base pairs (6, 5 Kb), at least 7000 base pairs (7 Kb), at least 7500 base pairs (7.5 Kb), at least 8000 base pairs (8 Kb), at least 8500 base pairs (8.5 Kb) ), at least 9000 base pairs (9 Kb), at least 9500 base pairs (9.5 Kb) or at least 10,000 base pairs (10 Kb) that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO:1-126 and 128-888, and one or more, two or more, three or more, four or more, five or more, six or more more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, or twenty-five or more DNAs of interest that can be integrated into a maize B chromosome nucleic acid sequence to produce the recombinant nucleic acid sequence. In another aspect, a recombinant nucleic acid sequence provided herein is integrated into a target site for a site-specific genome modification enzyme, wherein the target site is specific to the maize B chromosome DNA sequence. In yet another aspect, a recombinant nucleic acid sequence provided herein is integrated between two target sites for a site-specific genome modification enzyme, where the target site is specific to the maize B chromosome DNA sequence. In another aspect, one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, or twenty or more DNAs of interest are integrated into one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, or twenty or more target sites for a site-specific genome modification enzyme, wherein the target site is specific to the maize B chromosome DNA sequence. In one aspect, a recombinant nucleic acid provided herein comprises a DNA of interest that encodes a peptide. In another aspect, the recombinant nucleic acid provided herein comprises a DNA of interest that does not encode a peptide. In some embodiments, a recombinant nucleic acid provided herein comprises a DNA of interest that encodes one or more site-specific DNA modification enzymes. In some embodiments, a recombinant nucleic acid provided herein comprises a DNA of interest that encodes one or more guide RNAs and/or tracer RNAs. In some embodiments, a recombinant nucleic acid provided herein comprises a DNA of interest that encodes an siRNA. In some embodiments, a recombinant nucleic acid provided herein comprises a DNA of interest that encodes a microRNA.

[0043] In one aspect, the present invention provides one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more , fifteen or more twenty or more, or twenty-five or more recombinant nucleic acid sequences comprising one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more more, ten or more, fifteen or more, twenty or more, or twenty-five or more DNAs of interest integrated into a B chromosome. In another aspect, the present invention provides one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more twenty or more, or twenty-five or more recombinant nucleic acid sequences comprising one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more more, ten or more, fifteen or more, twenty or more, or twenty-five or more DNAs of interest integrated into two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more B chromosomes.

[0044] In one aspect, a DNA of interest provided herein is modified during integration into a B chromosome sequence. In another aspect, a DNA of interest provided herein is not modified during integration into a B chromosome. In one aspect, a B chromosome sequence provided herein is modified when a DNA of interest provided herein integrates into it. In another aspect, a B chromosome sequence provided herein is not modified when a DNA of interest provided herein integrates into it. In some embodiments, the DNA of interest comprises a nucleic acid sequence of at least 5 base pairs, at least 10 base pairs, at least 15 base pairs, at least 20 base pairs, at least 25 base pairs, at least 50 base pairs at least 100 base pairs at least 125 base pairs at least 150 base pairs at least 200 base pairs at least 250 base pairs at least 500 base pairs at least 1000 base pairs (1 Kb), at least 1500 base pairs (1.5Kb), at least 2000 base pairs (2 Kb), at least 2500 base pairs (2.5 Kb), at least 3000 base pairs (3 Kb), at least 3500 base pairs (3.5 Kb), 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% sequence identity with a sequence portion selected from the group consisting of SEQ ID NO: 1-126 and 128 -888 to promote homologous recombination between the B chromosome and the DNA of interest.

[0045] In some embodiments, the DNA of interest may comprise a modified nucleic acid sequence. As used herein, a "modified" nucleic acid sequence comprises one or more nucleotide insertions, substitutions, deletions, duplications or inversions.

[0046] As used herein, a "nucleic acid of interest", "DNA of interest" or "donor" defined as a nucleic acid/DNA sequence that has been selected for targeted and site-directed insertion into the maize genome. A nucleic acid of interest may be of any length, for example between 2 and 50,000 nucleotides in length (or any integer therebetween or above), or between about 1,000 and 5,000 nucleotides in length (or any integer value in between). A DNA of interest may comprise one or more gene expression cassettes that encode actively transcribed and/or translated gene sequences. In some embodiments, the DNA of interest may comprise a polynucleotide sequence that does not comprise a functional gene expression cassette or a complete gene (e.g., it may simply comprise regulatory sequences such as a promoter) or may not contain any identifiable gene expression elements or any actively transcribed genetic sequence. The DNA of interest may optionally contain an analytical domain. After integration of the DNA of interest into the maize genome, the integrated sequences are referred to as the "integrated DNA of interest" or "inserted DNA of interest." Furthermore, the DNA of interest may be linear or circular and may be single-stranded or double-stranded. It may be delivered into the cell as naked nucleic acid, as a complex with one or more delivery agents (e.g., liposomes, poloxamers, protein-encapsulated T-strand, etc.), or contained in a bacterial or viral delivery vehicle, such as, for example, Agrobacterium tumefaciens or a geminivirus, respectively.

[0047] In one aspect, a DNA of interest provided herein may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or less ten genes. In one aspect, a DNA of interest provided herein does not contain genes. Without limitation, a gene provided herein may include an insecticide resistance gene, a herbicide tolerance gene, a nitrogen use efficiency gene, a water use efficiency gene, a nutritional quality gene, a DNA ligation, a selectable marker gene, an RNAi construct, a site-specific genome modification enzyme gene, a recombinant guide RNA from a CRISPR/Cas9 system, a geminivirus-based expression cassette, or a vector system of plant viral expression. In one aspect, a gene provided herein comprises a promoter nucleic acid sequence. In another aspect, a structural gene provided herein does not comprise a promoter nucleic acid sequence.

[0048] Examples of suitable genes of agronomic interest provided for by the present description include, but are not limited to, genes for tolerance to diseases, insects or pests; herbicide tolerance; genes for quality improvements such as yield, nutritional improvements, environmental or stress tolerances; or any desirable changes in plant physiology, growth, development, morphology or product(s), including starch production (US Patent 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295); modified oil production (US Patent 6,444,876; 6,426,447; 6,380,462); high oil production (US Patent 6,495,739; 5,608,149; 6,483,008; 6,476,295); modified fatty acid content (.Pate US 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,538; 6,589,767; 6,537,750; 6,461; 6,459,018); high protein production (US Patent 6,380,466); fruit ripening (US Patent 5,512,466); improved animal and human nutrition (US Patent 6,723,837; 6,653,530; 6,5412,59; 5,985,605; 6,171,640); or biopolymers (US Patent RE37,543; 6,228,623; 5,958,745 and Patent Publication US20030028917). Also resistance to environmental stress (US Patent 6,072,103); pharmaceutical peptides and secretable peptides (US Patent 6,812,379; 6,774,283; 6,140,075; 6,080,560); improved processing characteristics (US Patent 6,476,295); better digestibility (US Patent 6,531,648); low raffinose (US Patent 6,166,292); production of industrial enzymes (US Patent 5,543,576); improved taste (US Patent 6,011,199); nitrogen fixation (US Patent 5,229,114); hybrid seed production (US Patent 5,689,041); fiber production (US Patent 6,576,818; 6,271,443; 5,981,834; 5,869,720); biofuel production (US Patent 5,998,700). Any of these or other genetic elements, methods and transgenes may be used with the description as will be appreciated by those skilled in the art with a view to instantaneous description.

[0049] A DNA of interest provided herein may also include sequences that code for other sequences, such as a messenger RNA (mRNA). An mRNA produced from a nucleic acid molecule of the present disclosure may contain a 5' untranslated leader sequence (5'-UTR). This sequence can be derived from the promoter selected to express the gene and can be specifically modified to increase or decrease mRNA translation. A 5'-UTR can also be obtained from viral RNAs, suitable eukaryotic genes, or a synthetic gene sequence. Such "enhancer" sequences may be desirable to increase or alter the translational efficiency of the resulting mRNA. The present description is not limited to constructs in which the untranslated region is derived from the 5'-UTR accompanying the promoter sequence. Rather, the 5'-UTR sequence may be derived from unrelated promoters or genes (see, for example, US Patent 5,362,865). Examples of non-translation leader sequences include corn and petunia heat shock protein leaders (US Patent 5,362,865), plant virus coat protein leaders, rubisco company leaders, GmHsp (US Patent 5,659,122), PhDnaK (US Patent 5,362,865), AtAnt1, TEV (Carrington and Freed, Journal of Virology, (1990) 64:1590-1597), and AGRtu. nos (GenBank Accession V00087; Bevan et al., Nucleic Acids Research (1983) 11:369-385). Other genetic components that serve to increase expression or affect transcription or translation of a gene are also viewed as genetic components.

[0050] A DNA of interest provided herein may further comprise a 3' untranslated region (3'-UTR) of a gene. The 3'-UTRs provided may contain a transcription terminator, or an element having equivalent function, and a polyadenylation signal that functions in plants to cause the addition of polyadenylated nucleotides to the 3' end of an RNA molecule. DNA sequences are referred to herein as transcription termination regions. The regions are required for efficient mRNA polyadenylation. An RNA polymerase transcribes a DNA coding sequence through a site where polyadenylation occurs. Examples of suitable 3' regions are (1) the transcribed, untranslated 3' regions containing the polyadenylation signal of Agrobacterium Ti plasmid genes such as nopaline synthase (NOS; Fraley et al., Proceedings of the National Academy of Sciences , USA (1983) 80:4803-4807), and (2) plant genes, such as the soybean storage protein genes and the small subunit of the ribulose-1,5-bisphosphate carboxylase gene (ssRUBISCO). An example of a preferred 3' region is that of the pea ssRUBISCO E9 gene (European Patent Application 0385 962).

[0051] In a DNA of interest provided herein may encode one or more site-specific genome modification enzymes. In some embodiments, one or more site-specific genome modification enzymes are selected from an endonuclease, a recombinase, a transposase, a helicase, or any combination thereof. In a DNA of interest provided herein may encode one or more of a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a DNA-guided recombinase, a DNA-guided endonuclease, a guided RNA recombinase, an RNA-guided endonuclease, a type I CRISPR-Cas system, type II CRISPR-Cas system, and a type III CRISPR-Cas system. In a DNA of interest provided herein may encode one or more of Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3 , Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, C sx3 , Nuclease Csx1, Csx15, Csf1, Csf2, Csf3 and Csf4. In a DNA of interest provided herein may encode a dCas9-recombinase fusion protein. In a DNA of interest provided herein may encode one or more of a tyrosine recombinase, a serine recombinase, a Cre recombinase, a Flp recombinase, a Tnp1 recombinase, a PhiC31 integrase, an R4 integrase or a TP-901 integrase. In a DNA of interest provided herein may encode one or more of a tracer RNA and/or a guide RNA.

[0052] In one aspect, a DNA of interest provided herein may encode a selectable, traceable or demarcable marker gene. These genetic components are also referred to herein as functional genetic components, since they produce a product that serves a function in identifying a transformed plant, or a product of agronomic utility. DNA that serves as a selection or screening device can function in a regenerable plant tissue to produce a compound that would confer resistance to the plant tissue to an otherwise toxic compound. Various selectable or screenable marker genes are known in the art and may be used in the present disclosure. Genes of interest for use as a selectable, screenable or demarcable marker would include, but not limited to, β-glucuronidase (GUS), green fluorescent protein (GFP), luciferase (LUC), genes that confer tolerance to antibiotics such as kanamycin ( Dekeyser et al., Plant Physiology (1989) 90:217-223) or spectinomycin (e.g., spectinomycin aminoglycoside adenyltransferase (aadA); US Patent 5,217,902), genes encoding enzymes that provide tolerance to herbicides such as glyphosate (e.g. example, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS): Della-Cioppa et al., Bio/Technology (1987) 5:579-584); US Patent 5,627,061, US Patent 5,633,435, US Patent 6,040,497; US Patent 5,094,945; WO04074443 and WO04009761; glyphosate oxidoreductase (GOX; US Patent 5,463,175); glyphosate decarboxylase (WO05003362 and US Patent Application 20040177399; or glyphosate N-acetyltransferase (GAT): Castle et al., Science (2004) 304:1151-1154) US Patent Publication 20030083480), dalapon (e.g., dehI encoding to 2,2-dichloropropionic acid dehalogenase conferring tolerance to 2,2-dichloropropionic acid (Dalapon; WO9927116)), bromoxynil (haloarylnitrilase (Bxn) to confer tolerance to bromoxynil (WO8704181A1; US Patent 4,810,648; WO8900193A), sulfonyl herbicides ( for example, acetohydroxy acid synthase or acetolactate synthase conferring tolerance to acetolactate synthase inhibitors, such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide; 6,613,963; 5,013,659; 5,141,870; 5,378,824;5,605,011); US Patent 5,646,024, 5,561,236, 5,276,268; 5,637,489; 5,273,894; and EP 275,957), atrazine (encoding GST-III), dicamba (dicamba monooxygenase; US Patent Application Publications 20030115626, 20030135879), or sethoxydim (acetyl-coenzyme A carboxylase modified to confer tolerance to cyclohexanedione (sethoxydim) and aryloxyphenoxypropionate (haloxyfop) (US Patent 6,414,222), among others. Other selection procedures can also be implemented, including positive selection mechanisms (e.g., use of the Escherichia coli manA gene, allowing growth in the presence of mannose) , and dual selection (e.g., using simultaneously spectinomycin and glufosinate, or spectinomycin and dicamba) and would still fall within the scope of the present disclosure.

[0053] In one aspect, a selectable marker provided herein is a positive selection marker. A positive selection marker confers an advantage to a transformed cell. In one aspect, a DNA of interest provided herein comprises a selectable marker gene that confers antibiotic resistance or herbicide resistance. In another aspect, a selectable marker provided here is a negative selection marker. In another aspect, a selectable marker provided herein is both a positive selection marker and a negative selection marker. A negative selectable marker provided herein may be a lethal or non-lethal negative selectable marker. A non-lethal negative selectable marker gene can be one of any listed in US Publication 2004-0237142, such as GGPP synthases, GA 2-oxidase gene sequences, isopentenyltransferase (IPT), CKI1 (cytokinin-independent 1), ESR-2, ESR1-A, auxin-producing genes such as indole-3-acetic acid (IAA), iaaM, iaah, roLABC, genes that result in overexpression of ethylene biosynthetic enzymes, VP1 genes, AB13 genes, LEC1 genes, and Bas1 , for example. A non-lethal negative selectable marker gene can be included in any nucleic acid molecule provided herein. A non-lethal negative selectable marker gene provided herein is a gene that results in the overexpression of a class of enzymes that utilize substrates of the gibberellic acid (GA) biosynthetic pathway, but that do not result in the production of bioactive GA. In another aspect, a nucleic acid molecule provided herein comprises a non-lethal negative selectable marker gene, such as an Erwinia herbicola phytoene synthase gene (crtB).

[0054] In one aspect, a DNA of interest provided herein comprises a promoter. A promoter contains a sequence of nucleotide bases that signals RNA polymerase to associate with DNA and to initiate transcription into mRNA using one of the DNA strands as a template to form a corresponding complementary strand of RNA. In one aspect, a promoter provided herein is a constitutive promoter. In another aspect, a promoter provided herein is a regulatable promoter. In yet another aspect, a regulatable promoter provided herein is a heat shock promoter, a tissue-specific promoter, or a chemically inducible promoter.

[0055] A number of promoters that are active in plant cells have been described in the literature. Such promoters would include, but are not limited to, the nopaline synthase (NOS) and octopine synthetase (OCS) promoters that are carried on Ti plasmids of Agrobacterium tumefaciens, the kaulimovirus promoters, such as the 19S and 35S promoters of the cauliflower (CaMV) and the Figwort mosaic virus (FMV) 35S promoter and the improved CaMV35S promoter (e35S). A variety of other plant gene promoters that are regulated in response to environmental, hormonal, chemical and/or developmental signals can also be used for expression of heterologous genes in plant cells, including, for example, promoters regulated by ( 1) heat (Callis et al., Plant Physiology, (1988) 88:965-968), (2) light (e.g., pea RbcS-3A promoter, Kuhlemeier et al. Plant Cell, (1989) 1:471 -478; maize RbcS promoter, Schaffner et al., Plant Cell (1991) 3:997-1012); (3) hormones, such as abscisic acid (Marcotte et al., Plant Cell, (1989) 1:969-976), (4) wound (e.g., Siebertz et al., Plant Cell, (1989) 961-968 ); or other signals or chemicals. Tissue-specific promoters are also known.

[0056] In one aspect, a DNA of interest provided herein may comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten promoters. In one aspect, a DNA of interest provided herein does not comprise a promoter. In one aspect, a promoter provided herein may be part of a gene.

[0057] As described below, it is preferred that the particular promoter selected is capable of causing sufficient expression to result in the production of an effective amount of the gene product of interest. Examples describing such promoters include, without limitation, US Patent 6,437,217 (corn RS81 promoter), US Patent 5,641,876 (rice actin promoter), US Patent 6,426,446 (corn RS324 promoter), US Patent 6,429. 362 (PR-1 corn promoter), US Patent 6,232,526 (A3 corn promoter), US Patent 6,177,611 (corn constitutive promoters), US Patent 5,322,938, 5,352,605, 5,359,142 and 5,530. 196 (35S promoter), US Patent 6,433,252 (corn oleosin L3 promoter), US Patent 6,429,357 (rice actin 2 promoter as well as an intron of rice actin 2), US Patent 5,837,848 ( root-specific promoter), US Patent 6,294,714 (light-inducible promoters), US Patent 6,140,078 (salt-inducible promoters), US Patent 6,252,138 (pathogen inducible promoters), US Patent 6,175,060 (inducible promoters by phosphorus deficiency), US Patent 6,635,806 (gamma-coxin promoter), and US Patent Application 09/757,089 (maize chloroplast aldolase promoter). Additional promoters that may find use are a nopaline synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (which is carried on Agrobacterium tumefaciens tumor-inducing plasmids), the kaulimovirus promoters , such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Molecular Biology (1987) 9:315-324), the CaMV 35S promoter (Odell et al., Nature (1985) 313 :810812), the figwort mosaic virus 35S promoter (US Patent 6,051,753; 5,378,619), the sucrose synthetase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA (1990) 87: 4144-4148), the promoter of the R gene complex (Chandler et al., Plant Cell (1989) 1:1175-1183), and the promoter of the chlorophyll a/b binding protein gene, PC1SV US Patent 5,850. 019), and AGRtu promoters. nos (GenBank Accession V00087; Depicker et al. Journal of Molecular and Applied Genetics (1982) 1:561-573; Bevan et al. 1983).

[0058] Promoter hybrids can be constructed to improve transcriptional activity (US Patent 5,106,739), or to combine desired transcriptional activity, inducibility and tissue specificity or developmental specificity. Promoters that function in plants include, but are not limited to, inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, and spatially regulated promoters. Other promoters that are tissue-enhanced, tissue-specific, or developmentally regulated are also known in the art and considered useful in the practice of the present invention.

[0059] The promoters used in the provided nucleic acid molecules and transformation vectors of the present description can be modified, if desired, to affect their control characteristics. Promoters can be derived through ligation with operator regions, random or controlled mutagenesis, etc. Additionally, promoters can be altered to contain multiple "enhancer sequences" to help elevate gene expression.

[0060] The present nucleic acid molecules can be incorporated into any suitable plasmid or plant transformation vector containing a selectable or traceable marker and associated regulatory elements as described, together with one or more nucleic acids encoded by a structural gene.

1. Site-specific genome modification enzymes

[0061] As used herein, the term "double-strand break-inducing agent" refers to any agent that can induce a double-strand break (DSB) in a DNA molecule. In some embodiments, the double-strand break-inducing agent is a site-specific genome modification enzyme.

[0062] As used herein, the term site-specific genome modification enzyme refers to any enzyme that can modify a nucleotide sequence in a site-specific manner. In the present description, site-specific genome modification enzymes include endonucleases, recombinases, transposases, helicases and any combination thereof.

[0063] Various modalities relate to promoting recombination by providing a site-specific genome modification enzyme. As used herein, the term "site-specific enzyme" refers to any enzyme that can modify a nucleotide sequence in a sequence-specific manner. In some embodiments, recombination is promoted by providing a single-strand break-inducing agent. In some embodiments, recombination is promoted by providing a double-strand break-inducing agent. In some embodiments, recombination is promoted by providing a strand separation-inducing reagent. In one aspect, the site-specific genome modification enzyme is selected from an endonuclease, a recombinase, a transposase, a helicase, or any combination thereof. In some embodiments, recombination occurs between B chromosomes. In some embodiments, recombination occurs between a B chromosome and an A chromosome. In some embodiments, recombination occurs between a DNA of interest and a unique B chromosome sequence, as described herein. .

[0064] Various modalities refer to promoting the integration of one or more DNAs of interest, providing a site-specific genome modification enzyme. As used herein, the term "site-specific enzyme" refers to any enzyme that can modify a nucleotide sequence in a sequence-specific manner. In some embodiments, the integration of one or more DNAs of interest is promoted by providing a single-strand break-inducing agent. In some embodiments, the integration of one or more DNAs of interest is promoted by providing a double-strand break-inducing agent. In some embodiments, integration of one or more DNAs of interest is promoted by providing a strand separation-inducing reagent. In one aspect, the site-specific genome modification enzyme is selected from an endonuclease, a recombinase, a transposase, a helicase, or any combination thereof.

[0065] In one aspect, the endonuclease is selected from a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-type effector nuclease (TALEN), an Argonaute (non-limiting examples of Argonaute protein include Thermus thermophilus Argonaute ( TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium Gregoryi Argonaute (NgAgo), an RNA-guided nuclease such as CRISPR-associated nuclease (non-limiting examples of CRISPR-associated nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5 , Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3 , Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, homologs thereof, or modified versions thereof).

[0066] In some embodiments, the site-specific genome modification enzyme is a dCas9-Fok1 fusion protein. In another aspect, the site-specific genome modification enzyme is a dCas9-recombinase fusion protein. As used herein, a "dCas9" refers to a Cas9 endonuclease protein with one or more amino acid mutations that result in a Cas9 protein lacking endonuclease activity but retaining RNA-guided site-specific DNA binding. As used herein, a "dCas9-recombinase fusion protein" is a dCas9 with a protein fused to dCas9 in such a way that the recombinase is catalytically active on DNA.

[0067] In some embodiments, the site-specific genome modification enzyme is a recombinase. Non-limiting examples of recombinase include a tyrosine recombinase linked to a DNA recognition motif provided herein is selected from the group consisting of a Cre recombinase, a Gin recombinase, a Flp recombinase and a Tnp1 recombinase. In one aspect, a Cre recombinase or a Gin recombinase provided herein is tethered to a zinc finger DNA-binding domain, or a TALE DNA-binding domain, or a Cas9 nuclease. In another aspect, a serine recombinase tethered to A DNA recognition motif provided herein is selected from the group consisting of a PhiC31 integrase, an R4 integrase and a TP-901 integrase. In another aspect, a DNA transposase linked to a DNA binding domain provided herein is selected from the group consisting of a TALE-piggyBac and TALE-Mutator.

[0068] Site-specific genome modification enzymes such as meganucleases, ZFNs, TALENs, Argonaute Proteins (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium Gregoryi Argonaute (NgAgo), homologues or modified versions thereof), RNA-guided nucleases (non-limiting examples of RNA-guided nucleases include the CRISPR-associated nucleases such as Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2 , Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, homologues thereof, or modified versions thereof) and RNA-guided engineered nucleases (RGNs), induce a modification of the genome such as a double-stranded DNA break (DSB) or a single-stranded DNA break at the target site of a genomic sequence that is then repaired by the natural processes of homologous recombination (HR) or non-homologous end joining (NHEJ ). Sequence modifications then occur at the cleaved sites, which may include deletions or insertions that result in gene disruption in the case of NHEJ, or integration of exogenous sequences by homologous recombination.

[0069] In one aspect of the present disclosure, site-specific genome modification enzymes are selected to induce a genome modification in one, a few or many individual target sequences of the maize B chromosome sequences provided herein. After exposure to the site-specific genome modification enzyme, the resulting recombinant nucleic acid can be identified in a variety of ways including sequencing, PCR amplification, Southern analysis, or other molecular methods used to detect the recombinant nucleic acid sequence. Site-specific genome modification enzymes can be expressed in plants such that one or more genome modifications occur within a genomic locus and the resulting progeny are screened for molecular changes.

[0070] Any of the DNA of interest provided herein can be integrated into a target site of a B chromosome sequence by introducing the DNA of interest and the site-specific genome modification enzymes provided. Any method provided herein may utilize any site-specific genome modification enzyme provided herein.

2. ZFNs

[0071] Zinc finger nucleases (ZFNs) are synthetic proteins characterized by an engineered zinc finger DNA binding domain fused to the FokI restriction endonuclease cleavage domain. ZFNs can be designed to cleave almost any long stretch of double-stranded DNA for modification of the zinc finger DNA-binding domain. ZFNs form dimers of monomers composed of a FokI endonuclease-specific hand DNA cleavage domain fused to a zinc finger array engineered to bind to a target DNA sequence.

[0072] The DNA binding domain of a ZFN is typically composed of 3-4 arrays of zinc fingers. Amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger helix, which contribute to site-specific binding to target DNA, can be altered and customized to fit specific sequences of the target. The other amino acids form the consensus backbone to generate ZFNs with different sequence specificities. The rules for selecting target sequences for ZFNs are known in the art.

[0073] The FokI nuclease domain requires dimerization to cleave DNA and therefore two ZFNs with their C-terminal regions are required to bind to the DNA strands opposite the cleavage site (separated by 5-7 bp). ZFN monomer can create the target site if the binding sites of two ZFs are palindromic. The term ZFN, as used here, is broad and includes a monomeric ZFN that can cleave double-stranded DNA without the assistance of another ZFN. The term ZFN is also used to refer to one or both members of a pair of ZFNs that are engineered to work together to cleave DNA at the same site.

[0074] Since the DNA binding specificities of zinc finger domains can, in principle, be re-engineered using one of several methods, custom ZFNs can, theoretically, be constructed to target virtually any gene sequence. Publicly available methods for engineering zinc finger domains include Context-Dependent Assembly (CoDA), Oligomerized Assembly Engineering (OPEN), and Modular Assembly.

3. TALENs

[0075] Transcription activator-like effectors (TALEs) can be engineered to bind to virtually any DNA sequence. TALE proteins are DNA-binding domains derived from several bacterial plant pathogens of the genus Xanthomonas. X pathogens secrete TALEs into the host plant cell during infection. TALE moves to the nucleus, where it recognizes and binds to a specific DNA sequence in the promoter region of a specific DNA sequence in the promoter region of a specific gene in the host genome. TALE has a central DNA-binding domain composed of 13-28 repeating monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, with the exception of the hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called di-repeated variable residues (RVDs). The NI, NG, HD and NN amino acid pairs of RVDs preferentially recognize adenine, thymine, cytosine and guanine/adenine, respectively, and the modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition allowed the engineering of specific DNA-binding domains by selecting a combination of repeat segments containing the appropriate RVDs. The transcription activator-like effector (TALE) DNA-binding domain can be fused to a functional domain, such as a recombinase, a nuclease, a transposase, or a helicase, thereby imparting sequence specificity to the functional domain.

[0076] Transcription activator-type effector nucleases (TALENs) are artificial restriction enzymes generated by fusing the transcription activator-type effector (TALE) DNA-binding domain to a nuclease domain. The term TALEN, as used here, is broad and includes a monomeric TALEN that can cleave double-stranded DNA without the assistance of another TALEN. The term TALEN is also used to refer to one or both members of a pair of TALENs that work together to cleave DNA at the same site. In some embodiments, the nuclease is selected from a group consisting of PvuII, MutH, TevI, FokI, AlwI, MlyI, SbfI, SdaI, StsI, CleDORF, Clo051, and Pept071. When FokI is fused to a TALE domain that each member of the TALEN pair binds to DNA sites flanking a target site, FokI monomers dimerize and cause a DSB at the target site.

[0077] In addition to the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains to target sites on the genome with appropriate orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity. PvuII, MutH, and TevI cleavage domains are useful alternatives to FokI and FokI variants for use with TALEs. PvuII functions as a highly specific cleavage domain when coupled to a TALE (see Yank et al. 2013. PLoS One. 8:e82539). MutH is capable of introducing specific strand nicks into DNA (see Gabsalilow et al. 2013. Nucleic Acids Research. 41:e83). TevI introduces double-strand breaks into DNA at targeted sitessee Beurdeley et al. 2013. Nature Communications.4:1762).

[0078] The relationship between the amino acid sequence and DNA recognition of the TALE binding domain allows for assignable proteins. Software programs such as DNA Works can be used to design TALE constructs. Other methods of designing TALE constructs are known to those skilled in the art. Doyle et al. (2012) TAL Effector-Nucleotide Targeter (TALE-NT) 2.0: tools for TAL effector design and target prediction. Nucleic Acids Res. 40(W1):W117-W122; Cermak (2011). Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res 39(12):e82.

4. Meganucleases

[0079] Meganucleases, which are commonly identified in microbes, are unique enzymes with high activity and long recognition sequences (> 14 bp), resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases often have extended DNA recognition sequences (e.g., 14-40 bp).

[0080] Engineering meganucleases is more challenging than that of ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are interconnected in a single domain. Specialized mutagenesis and high-throughput screening methods have been used to create new meganuclease variants that recognize unique sequences and have improved nuclease activity.

5. Argonaute

[0081] The Argonaute family of proteins is a DNA-guided endonuclease. Argonaute isolated from Natronobacterium Gregoryi has been reported to be suitable for DNA-guided genome editing in human cells (Gao, et al. of Argonaute from other species (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium Gregoryi Argonaute (NgAgo), homologs thereof, or modified versions of these). associated with a sequence that encodes the DNA guide.

6. CRISPR

[0082] The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system is an alternative to synthetic proteins whose DNA-binding domains allow them to modify genomic DNA at specific sequences (e.g., ZFN and TALEN ). The specificity of the CRISPR/Cas system is based on a guide RNA that uses complementary base pairs to recognize target DNA sequences. In some embodiments, the site-specific genome modification enzyme is a CRISPR/Cas system. In one aspect, a site-specific genome modification enzyme provided herein may comprise any RNA-guided Cas nuclease (non-limiting examples of RNA-guided nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 , Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6 , Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, homologues thereof or modified versions thereof); and, optionally, the guide RNA necessary to target the respective nucleases.

[0083] CRISPR/Cas systems are part of the adaptive immune system of bacteria and archaea, protecting them against invasion by nucleic acids, such as viruses, by cleaving foreign DNA in a sequence-dependent manner. Immunity is acquired by the integration of small fragments of invading DNA known as spacers between two adjacent repeats at the proximal end of a CRISPR locus. CRISPR templates, including spacers, are transcribed during subsequent encounters with invasive DNA and are processed into CRISPR small interfering RNAs (crRNAs) approximately 40 nt in length, which combine with CRISPR trans-activating RNA (tracrRNA) to activate and guide the Cas9 nuclease. This cleaves homologous double-stranded DNA sequences known as protospacers in the invading DNA. A prerequisite for cleavage is the presence of a conserved protospacer adjacent motif (PAM) downstream of the target DNA, which usually has the sequence 5'-NGG-3', but less frequently NAG. Specificity is provided by the so-called "seed sequence", approximately 12 bases upstream of the PAM, which must match between the RNA and the target DNA. Cpf1 acts in a similar way to Cas9, but Cpf1 does not require a tracrRNA.

[0084] As used herein, the term "target site" broadly refers to a genomic sequence selected for integration into a DNA of interest. In some aspects, the target site is in a genic region. In other aspects, the target site is in an intergenic region. In yet another aspect, the target site may include both a genic region and an intergenic region. In one aspect, a target site provided herein is recognized and cleaved by a double-strand break-inducing agent, such as a site-specific genome modification enzyme, a site-specific recombinase, or a site-specific transposase. In some embodiments, a target site comprises a nucleic acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, at least 99%, or 100%, sequence identity with a portion of a maize B chromosome sequence selected from the group consisting of SEQ ID NO: 1-126 and 128-888.

[0085] As used herein, the term "recombination" refers to the exchange of nucleotide sequences between two DNA molecules.

[0086] As used herein, the term "homologous recombination" refers to the exchange of nucleotide sequences in a conserved region shared by two genomic loci. Homologous recombination includes symmetric homologous recombination and asymmetric homologous recombination. Asymmetric homologous recombination can also be referred to as unequal recombination.

[0087] Methods for detecting recombination include, but are not limited to: 1) phenotypic screening, 2) molecular marker technologies such as single nucleotide polymorphism (SNP) analysis by TaqMan® or Illumina/Infinium PCR technology, 3 ) Southern blot analysis and 4) sequencing (e.g. Sanger, Illumina, 454, Pac-Bio, Ion Torrent). An example of a method for identifying the integration of a DNA of interest into a chromosome is inverse PCR (iPCR).

[0088] In one aspect, integration of a DNA of interest provided herein occurs through homologous recombination (HR). In another aspect, integration of a DNA of interest provided herein occurs via non-homologous end joining (NHEJ).

[0089] As used herein, "transgenic" means a plant or seed whose genome has been altered by the stable integration of recombinant DNA. A transgenic line includes a plant regenerated from an originally transformed plant cell and transgenic plants from progeny of later generations or crosses from a transformed plant. As used herein, "exogenous" is intended to refer to genes that are not normally present in the cell being transformed, or are not present in the form, structure, etc., as found in the DNA segment or gene being transformed. Thus, the term "exogenous" gene or DNA is intended to refer to any gene or segment of DNA that is introduced into a recipient cell, regardless of whether a similar gene is already present in such a cell. The type of DNA included in exogenous DNA may include DNA that is already present in the plant cell, DNA from another plant, DNA from a different organism, or an externally generated DNA such as a DNA sequence containing an antisense message from a gene or a DNA sequence that encodes a synthetic or modified version of a gene.

[0090] Plant cell transformation methods are well known to those skilled in the art. For example, specific instructions for transforming plant cells by bombarding microprojectiles with particles coated with recombinant DNA are found in US Patents 5,015,580 (soy); 5,550,318 (corn); 5,538,880 (corn); 5,914,451 (soy); 6,160,208 (corn); 6,399,861 (corn) and 6,153,812 (wheat); 6,002,070 (rice); 7,122,722 (cotton); 6,051,756 (Brassica); 6,297,056 (Brassica); US Patent Publication 20040123342 (sugar cane) and Agrobacterium-mediated transformation is described in US Patents 5,159,135 (cotton); 5,824,877 (soy); 5,591,616 (corn); 6,384,301 (soy); 5,750,871 (Brassica); 5,463,174 (Brassica), 5,188,958 (Brassica), all of which are incorporated herein by reference. Methods for transforming other plants can be found, for example, in Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Any appropriate method known to those skilled in the art can be used to transform a plant cell with any of the provided nucleic acid molecules.

[0091] As used herein, "stably transformed" is defined as a transfer of DNA into the genomic DNA of a target cell that allows the target cell to regenerate into a complete organism and pass the transferred DNA to the next generation of the transformed organism . Stable transformation requires integration of the transferred DNA within the reproductive cell(s) of the transformed organism. As used herein, "transiently transformed" is defined as a transfer of DNA to a cell that is not transferred to the next generation of the transformed organism. Transient transformations are often leaf or root (e.g., non-reproductive) transformations in plants. Transformed DNA does not normally integrate into the transformed cell's genomic DNA during a transient transformation. In one aspect, a method provided herein stably transforms a plant cell. In another aspect, a method provided herein transiently transforms a plant cell.

[0092] In one aspect, the present invention provides methods for transforming a plant cell with a nucleic acid sequence encoding a site-specific genome modification enzyme. In one aspect, a method provided herein comprises stable transformation of a nucleic acid sequence encoding a site-specific genome modification enzyme. In another aspect, a method provided herein comprises transient transformation of a nucleic acid sequence encoding a site-specific genome modification enzyme. In one aspect, a method provided herein comprises a constitutively expressed nucleic acid encoding a site-specific genome modification enzyme. In another aspect, a method provided herein comprises a nucleic acid sequence encoding a site-specific genome modification enzyme under the control of a regulatable promoter.

[0093] In one aspect of the present disclosure, a recombinant nucleic acid sequence provided herein is capable of being fully integrated into a chromosome.

[0094] In one aspect, transformation of a plant cell is carried out by an Agrobacterium-mediated method, and the nucleic acid molecules of interest are present in one or more DNA sequences (US Patent 6,265,638, 5,731. 179; Patent Application Publications US2005/0183170; 2003110532) or other nucleic acid sequence (e.g., vector backbone) that is transferred into a plant cell. Sequences that can be transferred to a plant cell can be present in a transformation vector in a bacterial strain to be used for transformation. In another aspect, the sequences may be present in separate transformation vectors in the bacterial strain. In yet another aspect, the sequences may be found in separate bacterial cells or strains used together for transformation.

[0095] A "transformation vector", as used herein, is plasmid DNA that is capable of transforming a plant cell. In one aspect, a transformation vector provided herein may comprise any DNA of interest provided herein. In another aspect, a transformation vector provided herein may comprise any nucleic acid molecule provided herein.

[0096] The DNA constructs used for transformation in the ways of the present description generally also contain the plasmid backbone DNA segments that provide replication and antibiotic selection function in bacterial cells, for example, an Escherichia coli origin of replication, such as ori322, an Agrobacterium origin of replication such as oriV or oriRi, and a coding region for a selectable marker such as Spec/Strp encoding the Tn7 aminoglycoside adenyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or a selectable marker gene of gentamicin (Gm, Gent). For plant transformation, the host bacterial strain is often Agrobacterium tumefaciens ABI, C58, LBA4404, AGLO, AGL1, EHA101 or EHA105 containing a plasmid with a transfer function for the expression unit. Other strains known to those skilled in the art of plant transformation may function in the present disclosure.

[0097] To confirm the presence of exogenous DNA or a "transgene(s)" in a transgenic cell, a variety of assays can be performed. These assays include, for example, "molecular biological" assays such as Southern and northern blotting and PCR, "biochemical" assays such as detection of the presence of a protein product, for example by immunological means (ELISAs and western blots) or by enzyme function (e.g., GUS assay); pollen histochemistry; plant part testing, such as leaf or root testing; and also, analyzing the phenotype of the entire regenerated plant.

[0098] The present invention provides a method for producing a transgenic plant cell comprising a DNA of interest, wherein the method comprises selecting a target B chromosome genomic locus having at least 70%, at least 75%, at least 80% , at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , at least 99%, at least 99% or 100% sequence identity to any selected sequence of SEQ ID NO:1-126 and 128-888, select a site-specific genome modification enzyme that specifically binds and cleaves the locus genomic target B chromosome; introducing the site-specific genome modification enzyme into a plant cell; introduce a DNA of interest into a plant cell; and inserting the DNA of interest into the genomic locus of the target B chromosome; and selecting plant cells comprising the DNA of interest integrated into the target site of the B chromosome locus. In one aspect, a method provided herein utilizes homology-directed repair integration to integrate a DNA of interest into a B chromosome genomic locus. Another aspect, a method provided herein utilizes non-homologous end joining integration to integrate a DNA of interest into a B chromosome genomic locus.

[0099] In one aspect, a method provided herein integrates one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, or twenty-five or more DNAs of interest in one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, or twenty-five or more B chromosome genomic loci located on a B chromosome. In another aspect, a method provided herein integrates one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, or twenty-five or more DNAs from interest in one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, or twenty-five or more B chromosome genomic loci located on two or more, three or more, four or more, five or more, six or more, seven or more eight or more, nine or more, or ten or more B chromosomes .

[00100] In one aspect, a method of manufacturing a transgenic plant cell comprises a DNA of interest that is modified during integration into a B chromosome sequence. In another aspect, a method of producing a transgenic plant cell, provided herein, comprises a B chromosome sequence that is modified when a DNA of interest provided herein integrates into it.

[00101] In one aspect, the present invention provides a method of producing a transgenic plant cell wherein two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more or twenty five or more DNAs of interest are integrated into two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, or twenty-five or more target B chromosome genomic loci on separate B chromosomes; and where the B chromosomes recombine so that the two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more , twenty or more, or twenty-five or more target B chromosome genomic loci generate a megalocus comprising the two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, or twenty-five or more DNAs of interest.

[00102] As used herein, a "megalocus" refers to a block of genetically linked transgenic traits that are normally inherited as a single unit. A megalocus as described may provide a plant with one or more desired traits, which may include, but are not limited to, improved growth, drought tolerance, salt tolerance, herbicide tolerance, insect resistance, pest resistance, disease resistance and the like. In specific embodiments, a megalocus comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13 or 15 transgenic loci (events) that are physically separate but genetically linked in such a way that which can be inherited as a single unit. Each transgenic locus in the megalocus can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2 , 2.5, 3, 5, 10, 15 or 20 cM apart from each other. In some embodiments, a megalocus comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13, or 15 transgenic loci (events) that are not genetically linked, but are located in the same B chromosome.

[00103] As used herein, a centimorgan ("cM") is a unit of measurement of recombination frequency and genetic distance between two loci. A cM is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.

[00104] As used herein, "closely linked" means that the marker or locus is about 20 cM, 15 cM, 10 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0, 5 cM, or less than 0.5 cM from another marker or locus. For example, 20 cM means recombination occurs between the marker and the locus with a frequency equal to or less than about 20%.

[00105] As used herein, "proximal" refers to the relative location of two nucleic acid sequences. In one aspect, a first nucleic acid sequence is proximate to a second nucleic acid sequence if the two nucleic acid sequences are separated by less than 50,000 base pairs, less than 25,000 base pairs, less than 15 .000 base pairs, less than 10,000 base pairs, less than 7500 base pairs, less than 5000 base pairs, less than 4000 base pairs, less than 3000 base pairs, less than 2500 base pairs, less than 2000 base pairs, less than 1500 base pairs, less than 1000 base pairs, less than 750 base pairs, less than 500 base pairs, less than 250 base pairs, less than 100 base pairs, less of 75 base pairs, less than 50 base pairs, less than 40 base pairs, less than 30 base pairs, less than 20 base pairs, less than 10 base pairs, less than 5 base pairs, less than 3 base pairs or 1 base pair.

[00106] In one aspect, the present invention provides plant cells that are not reproductive materials and do not mediate natural plant reproduction. In another aspect, the present invention also provides corn plant cells that are reproductive material and mediate the natural reproduction of the plant. In another aspect, the present invention provides corn plant cells that cannot maintain themselves through photosynthesis. In another aspect, the present invention provides somatic plant cells. Somatic cells, unlike germ cells, do not mediate plant reproduction.

[00107] The plant cells or plant parts provided may be seed, fruit, leaf, cotyledon, hypocotyl, meristem, embryo, endosperm, root, shoot, stem, pod, flower, inflorescence, stalk, pedicel, style, stigma , receptacle, petal, sepal, pollen, anther, filament, ovary, ovule, pericarp, phloem bud and vascular tissue. In another aspect, the present invention provides a plant chloroplast. In a further aspect, the present invention provides an epidermal cell, a stomatal cell, a trichome cell, a root hair cell, a storage root cell or a tuber cell. In another aspect, the present invention provides a protoplast cell. In another aspect, the present invention provides a plant callus cell. In one aspect, any plant, plant part or plant cell provided herein may comprise any recombinant sequence provided herein.

[00108] The nucleic acid molecules provided herein include deoxynucleic acids (DNA) and ribonucleic acids (RNA) and functional analogues thereof, such as complementary DNA (cDNA). The nucleic acid molecules provided herein can be single-stranded or double-stranded. Nucleic acid molecules comprise the nucleotide bases adenine (A), guanine (G), thymine (T), cytosine (C). Uracil (U) replaces thymine in RNA molecules. The symbol "N" can be used to represent any nucleotide base (e.g., A, G, C, T, or U). As used herein, "complementary" in reference to a nucleic acid molecule or nucleotide bases refers to A being complementary to T (or U), and G being complementary to C. Two complementary nucleic acid molecules are capable of hybridizing to each other. yes. As an example, the two strands of double-stranded DNA are complementary to each other. In one aspect of the present disclosure, two nucleic acid sequences are homologous, or essentially homologous, if they are 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to each other. A polynucleotide molecule of the present description comprises at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 250, at least 500, at least 1000 , at least 1500, at least 2000, at least 2500, or at least 3000 nucleotide bases. As used herein, "coding" refers to a polynucleotide that codes for the amino acids of a polypeptide. A series of three nucleotide bases encodes an amino acid. As used herein, "expressed", "expression" or "expressing" refers to the transcription of RNA from a DNA molecule. In one aspect, expression of a DNA of interest provided herein requires a promoter. In another aspect, expression of a DNA of interest provided herein does not require a promoter. In one aspect, an express transcript provided here is amended. In another respect, an express transcript provided here is not amended.

[00109] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally occurring amino acids.

[00110] In one aspect of the present disclosure, a nucleic acid sequence may be physically linked to another nucleic acid sequence, operatively linked to another nucleic acid sequence, or both physically and operatively linked to another nucleic acid sequence. As used herein, physically linked means that physically linked nucleic acid sequences are located on the same nucleic acid molecule. A physical connection can be adjacent or proximal. A nucleic acid sequence provided herein may be adjacent to another nucleic acid sequence. As used herein, operably linked means that the operably linked nucleic acid sequences exhibit their desired function. For example, in one aspect of the present invention, a provided DNA promoter sequence can initiate transcription of an operably linked DNA sequence into RNA. A nucleic acid sequence provided herein may be upstream or downstream of a physically or operably linked nucleic acid sequence. As used herein, upstream means the nucleic acid sequence positioned before the 5' end of a linked nucleic acid sequence. As used herein, downstream means that the nucleic acid sequence is positioned after the 3' end of a linked nucleic acid sequence. As used herein, 5' means the beginning of a coding DNA sequence or the beginning of an RNA molecule. As used herein, 3' means the end of a DNA coding sequence or the end of an RNA molecule. As used herein, "opposite side" refers to the 5' or 3' side of a nucleic acid molecule. For example, a nucleic acid molecule sequence on the 5' side of a second nucleic acid molecule is on the opposite side of a third nucleic acid molecule sequence on the 3' end of the second nucleic acid molecule.

[00111] In one aspect, a nucleic acid molecule provided herein is a transformation vector. In one aspect, a transformation vector is a bacterial plasmid. As used here, a "plasmid" is a DNA molecule that is physically separate from chromosomal DNA and has the ability to replicate. In another aspect, the bacterial plasmid is an Agrobacterium (Ti) tumor-inducing plasmid. In another aspect, a transformation vector is a synthetic plasmid. As used herein, a "synthetic plasmid" is an artificially created plasmid that is capable of the same functions (e.g., replication) as a natural plasmid (e.g., Ti plasmid). Without being limited, one skilled in the art can create a synthetic plasmid de novo by synthesizing a plasmid by individual nucleotides, or by splicing nucleic acid molecules from different pre-existing plasmids.

EXAMPLES Example 1: Obtaining a unique sequence from the maize B chromosome

[00112] To identify sequences unique to the corn B chromosome, the following approach was carried out. A line backcrossed several times to B73 corn containing multiple B chromosomes (referred to as B73 plus) was obtained at the University of Missouri and grown from seed in a greenhouse using standard stuffing and growing conditions. Due to non-Mendelian segregation of B chromosomes, root tip tissue was harvested from each individual plant grown from the B73 plus seed. The root tip tissue sample was used to create a chromosome smear for karyotyping, essentially as described by Kato et al (2011) Chromosome Painting for Plant Biotechnology, Plant Chromosome Engineering in Methods in Molecular Biology, ed. J. A. Birchler 701, 67-96. These chromosomal smears, stained with the chromosomal stain 4',6-diamidino-2-phenylindole (DAPI), were visually marked to determine the number of B chromosomes per individual nucleus. See, for example, Figure 1. From these data, at least one plant was identified as containing 20 individual B chromosomes per nucleus.

[00113] Leaf tissue was also collected from each plant and genomic DNA extracted. Genomic DNA was used in a Taqman® PCR copy number assay to confirm the number of B chromosomes in each individual plant. For this assay, a B-chromosome-specific marker and an A-chromosome-specific gene (alcohol dehydrogenase (ADH)) were amplified in the same PCR reaction. Controls included a B73 plant without B chromosomes and 4 plants from a non-B73 germplasm with 1, 2, 3, or 4 copies of the B chromosome. The ratio of the signal from the B chromosome-specific probe to the A chromosome-specific ADH probe showed an almost linear result when represented graphically. The individual plant identified by karyotyping as having 20 copies of the B chromosome was consistent with the Taqman PCR copy number assay to have 20 copies of the B chromosome. See Table 1.

[00114] Taking advantage of the 20X representation of the B chromosome for a chromosome in DNA extracted from the plant identified with 20 copies of B chromosomes, it was recognized that a fosmid sequencing strategy could be used to identify unique B chromosome sequences by fragment sequencing. of maize chromosomal DNA and extract high-confidence non-repetitive unique B sequences from the data. TABLE 1: Correlation of B chromosome copy number as determined by karyotyping to relative B marker value determined by Taqman PCR assay

[00115] Leaf tissue was collected from the individual plant with 20 copies of the B chromosome and genomic DNA was extracted using a modified CTAB method where 0.8 g of plant tissue was crushed by mechanical grinding, the powder was transferred to a tube of 50 ml with 20 ml of extraction buffer (1.5% cetyltrimethylammonium bromide (CTAB) (Sigma-Aldrich, Saint Louis, MO); 75 mM Tris-HCl (pH 8.0); 15 mM EDTA (pH 8.0) and 1 M NaCl) and the mixture was incubated in a water bath at 56°C for 20 minutes. After incubation, the tube was centrifuged and the top layer containing DNA was transferred to a new tube. Extraction was performed by adding 1/10 volume of 10% CTAB (10% cetyltrimethylammonium bromide (CTAB) and 0.7 M NaCl) and an equal volume of chloroform/iso-amyl alcohol (24:1), and this was mixed by rotating and inverting for 20 minutes, followed by centrifugation. The top layer was transferred to a new tube and an equal volume of isopropyl alcohol was added. This was centrifuged to sediment the DNA and the supernatant was discarded. The DNA pellet was resuspended in 5 ml of 1 M NaCl and 5 uL of 10 mg/ml RNase, and then the DNA was reprecipitated by adding 2 volumes of ethanol. The DNA pellet was washed twice with 5 ml per 70% ethanol wash and the final DNA pellet was resuspended in 1 mM Tris-HCl + EDTA + 0.1 mM (pH 8.0).

[00116] Genomic DNA isolated from the plant with 20 copies of the B chromosome was then used to prepare a fosmid library using the CopyRight® v2 Fosmid Cloning Kit. 0 (Lucigen Corporation, Middleton, WI). The fosmid constructs contained insertions of up to 40 kb of maize genomic sequence per construct. The fosmid constructs were pooled in 192 pools, each with approximately 1000 fosmid clones per pool. Fosmid DNA was extracted from each pool and subjected to Illumnia® sequencing following the manufacturer's protocol. Based on this sequencing, 162 of the 192 sets contained usable sequence or "reads". Reads from each of the 162 assemblies were assembled separately using PCAP assembly software tools ("Application of a superword array in genome assembly" Xiaoqiu Huang et. al Nucleic Acids Research, 2006, Vol. 34, No. 1 201-205 ) and CLCbio assembly cell (www. clcbio. com/products/clc-assembly-cell; Qiagen, Waltham, MA), and the sequence repeats for each well were masked using A chromosome repeats from a known genomic repeat library of corn (corn repeat library available at Smit, AFA, Hubley, R & Green, P RepeatMasker Open-4. 0. 2013-2015, www. repeatmasker. org). Next, the assembled sequence from each assembly was used to filter out non-repeated sequences specific to a chromosome by comparing the fosmid assembly sequence assemblies to the maize B73 reference genome (Schnable et al., (2009) The B73 Maize Genome:Complexity, Diversity, and Dynamics. Science 326, 1112-5) (representing the maize A chromosome sequence) using the Cross_Match alignment software (with parameters -minmatch 17 -minscore 200 -tags -masklevel 0 -penalty - 3 ), Phil Green, www. phrap. org/phredphrapconsed. html and using Monsanto's internal scripts to parse the alignment outputs and trim and filter the corresponding sequences from the A chromosome. Additionally, mitochondrial B73 (NCBI Reference Sequence:NC_007982. 1) and chloroplast genomic DNA (Reference Sequence reference NCBI:NC_001666. 2) was filtered in this process. The next step was to align the sequence of each meeting with the other 161 meetings, so that all meetings were individually compared to each other. This analysis was done to filter out "B chromosome repeats" that were identified by a sequence that occurred in 130 or more wells, or to filter out "A chromosome sequence that is not in the reference data set" that was identified by a sequence that occurred only one to three times in all meetings. Finally, the remaining sequences were compared to find the longest representative sequence for each of the contig groups with multiple matches between contigs in the group. Contig sequences that were greater than 2 kb are identified as unique sequences (SEQ ID NO: 1-126, 128-791) on the maize B chromosome.

Example 2: Maize B chromosome reference assembly

[00117] To generate a B chromosome reference assembly, genomic DNA from the B73 plant containing 20 copies of the B chromosome was prepared for PacBio® sequencing following the manufacturer's protocols. PacBio sequencing generates a long-read sequence to generate a high-quality contiguous assembly of the genome. The long-read B chromosome sequence data was combined with the unique B chromosome sequence generated from fosmid library sequencing and analysis, as described in Example 1. The combined data sets were used to generate the assembly sequences. of reference chromosome B 792-889. This B reference chromosome can be used to map small RNA and DNA methylation patterns, to identify new maize genes and their respective promoters, introns, terminators and insulators, to develop new B chromosome markers, to physically map the new markers of B chromosome, to enable experiments to measure the recombination that can occur between B chromosomes, and to design gene editing tools such as TALENs, CRISPRs, Cpf1, zinc fingers, or meganucleases.

Example 3: Site-Directed Genomic Modification of the Maize B Chromosome

[00118] The maize B chromosome sequences identified here are used for site-directed genome modification. A meganuclease is modified to target one of the unique maize B chromosome sites selected from a sequence in any of SEQ ID NOs: 1-126 and 128-888. A maize cell is contacted with (1) meganulcease under conditions to allow double-strand breakage at the selected site and with (2) a donor DNA molecule comprising a sequence of interest to be integrated at the selected site. The donor DNA molecule can integrate by non-homologous end joining (NHEJ) or the donor DNA molecule can integrate by homologous recombination.

[00119] Alternatively, a TALEN is engineered to target one of the unique maize B chromosome sites selected from a sequence in any of SEQ ID NOs: 1-126 and 128-888. A maize cell is contacted with (1) the TALEN under conditions to allow double-strand breakage at the selected site and with (2) a donor DNA molecule comprising a sequence of interest to be integrated at the selected site. The donor DNA molecule can integrate by non-homologous end joining (NHEJ) or the donor DNA molecule can integrate by homologous recombination. TALEN can be a single molecule or can function as a pair of molecules.

[00120] Alternatively, an RNA-guided DNA nuclease, such as CRISPR-Cas9 or Cpf1 endonuclease, is used to target one of the unique maize B chromosome sites selected from a sequence in any of SEQ ID NO: 1-126 and 128-888. A maize cell is contacted with (1) the CRISPR and Cas9 endonuclease, or Cpf1 endonuclease under conditions to allow double-strand breakage at the selected site and with (2) a donor DNA molecule comprising a sequence of interest to be integrated into the selected site. The donor DNA molecule can integrate by non-homologous end joining (NHEJ) or the donor DNA molecule can integrate by homologous recombination.

[00121] The donor DNA may comprise one or more gene expression cassettes, wherein the gene expression cassettes have one or more expression elements (e.g., promoter, intron, target peptide, 3' untranslated region/terminal signal ) to allow expression of a protein and/or RNA of interest in a plant cell. The protein of interest may be encoded by an insecticide resistance gene, a nitrogen use efficiency gene, a water use efficiency gene, a nutritional quality gene, a DNA binding gene, a selectable marker gene , a site-specific endonuclease gene, a meganuclease gene, a DNA-directed recombinase gene, or a gene encoding an RNA-guided endonuclease, such as Cas9 or Cpf1. In some cases, the donor DNA may encode one or more of an RNAi construct, a guide sequence capable of hybridizing to a target sequence, a tracr mate sequence, and a tracr sequence.

[00122] The maize B chromosome sequences identified here are used for genome editing. One or more sites are selected from a sequence in any of SEQ ID NO: 1-126 and 128-888. The one or more sites are targeted with a site-specific engineered meganuclease, endonuclease, or DNA-directed recombinase. Next, a maize cell is placed in contact with (1) the site-specific engineered meganuclease, endonuclease, or DNA-directed recombinase under specific conditions to allow double-strand breakage at the selected genomic site, and the DNA is deleted or inserted (indels) or a DNA base is changed.

Example 4: Maize B Chromosome Probe Development

[00123] The corn B chromosome sequences identified here were used to develop sets of PCR primers specific to the corn B chromosome. For example, Table 2 details three sets of PCR primers that amplify a region of the B chromosome-specific sequence of SEQ ID NO:175, 177, and 183. Specifically, the pair of PCR primers SEQ ID NO:889 and SEQ ID NO:890 amplify a region at coordinates 2626 - 2825 of SEQ ID NO: 175; the pair of PCR primers SEQ ID NO:891 and SEQ ID NO:892 amplify a region at coordinates 1170-1368 of SEQ ID NO:177; and the pair of PCR primers SEQ ID NO:893 and SEQ ID NO:894 amplify a region at coordinates 1118-1319 of SEQ ID NO:183. A control set of PCR primers SEQ ID NO:895 and SEQ ID NO:896 for a proximal region of the B chromosome, marked BCHR0004, was identified from the sequence. DU978594. 1 pCL7T-2 CL-repeat reported by Cheng et al.Chromosome Research (2010) 18:605–619. A control set of PCR primers SEQ ID NO:897 and SEQ ID NO:898 for a proximal euchromatin region 2/distal heterochromatin region 1, marked with BCHR0006, was identified from the sequence DU820494 pCL38T-2 CL-repeat DH1 reported by Cheng et al. Chromosome Research (2010) 18:605–619. And a control set of PCR primers SEQ ID NO:899 and SEQ ID NO:900 for chromosome B region of distal heterochromatin regions 3-4, marked BCHR0007, was identified from the StarkB repeat sequence reported by Lamb et al . Chromosome Research (2007) 15:383-398. TABLE 2. PCR primers specific for the B chromosome

[00124] PCR amplicons were mapped to the B chromosome using previously characterized B-A translocation strains. Corn with B-A translocation events were purchased from the Maize Coop Genetics Stock Center (www. maizegdb. org/stock_catalog). The samples used to test the B-specific control primers and probes were tertiary trisomic strains containing the B-A translocation (in addition to two normal A chromosomes) but not the reciprocal A-B translocation. Due to transmission issues, the Maize Genetics Coop stock center maintains these B-A translocations as heterozygotes. Thus, the distributed seed packet does not always contain tertiary trisomic material. If this is the case, then the translocation heterozygote (verified by FISH screening of the package received from the coop) was crossed to a tester germplasm with a test line, and color markers were used to identify likely tertiary trisomics in the F1 generation, which were then cultured and sampled.

[00125] DNA was extracted from tissue collected from tertiary trisomic plants containing one of the translocation chromosomes (specifically, TB-10L7, TB-10L37, TB-10L3, TB-10L30, TB-10L19, TB-10L26 and TB-10L36 ); or a sample containing a full-length B chromosome (positive control for all probes); or a sample lacking a B chromosome (negative control); or a sample from a line with a spontaneously truncated B chromosome. DNA was used for PCR amplification using the BCHR004 PCR amplification regions for the proximal centromere region; BCHR006 for a euchromation region; and BCHR007 for a distal heterochromatin region; and the PCR amplification primer pairs identified from the B chromosome sequence (SEQ ID NO:889 +890; SEQ ID NO:891 +892; and SEQ ID NO:893 +894). The results for the presence or absence of a PCR amplicon are presented in Table 3. These results are illustrated in Figure 2, where the regions of the B chromosome mapped by the probes are indicated by parentheses. For example, probe BCHR0004 was mapped to the proximal/centromere region of the B chromosome; probe BCHR0006 was mapped to the proximal euchromatin region 2 (PE2) and DH1; the BCHR0007 probe was mapped in the distal region (DH3 and DH4); the amplification product of the PCR primer pair SEQ ID NO:889 +890 mapped to the proximal euchromatin 2 (PE2) region; and both amplicons of the PCR primer pair SEQ ID NO:891 +892 and SEQ ID NO:894 +894 mapped to the proximal euchromatin 2 region (PE2) and DH1. These results demonstrate that the unique B chromosome sequences presented as SEQ ID NO: 1-126 and SEQ ID NO: 128-888 are useful for the development of PCR primer amplicons useful for B chromosome mapping and single probe development. B chromosome. PCR amplicons and probes unique to the B chromosome have utility in Southern analysis and Northern analysis applications, as well as other protocols related to molecular biology and sequence. Table 3. BA Translocation mapping result. (+) indicates PCR amplicon present, (-) indicates no PCR amplicon

Example 5: Insertion of the B chromosome transgene

[00126] Transformation of transgenes into corn germplasm containing B chromosomes was carried out to evaluate methods for detecting transgene insertion into the corn B chromosome. Two transgenic transformation vectors (transformation vector A and transformation vector B) conferring tolerance to glyphosate were transformed into immature corn embryos using Agrobacterium tumefaciens to produce transgenic seedlings according to methods known to those skilled in the art. The immature F1 embryos used with transformation vector A were derived from a cross of B73 + B chromosomes (ranging from 6 to 15 B chromosomes) (females) X LH244 (males) parental germplasm. The immature F1 embryos used with the B transformation vector were derived from a cross of LH244 (females) X B73+B (ranging from 4 to 16 B chromosomes) parental germplasm (males). After transformation, glyphosate was used to select progeny containing the transgenes. In the stage of transferring the seedlings to the soil, root tip samples were collected for FISH analysis. FISH probes were designed for transgenes randomly integrated into the maize genome during transformation, and FISH was done essentially as described by Kato et al. (2011), including a DAPI contrast. From this analysis, no B chromosome insertions were identified with the A transformation vector. FISH analysis of root tip tissue from seedlings generated with the B transformation vector identified 10 random transgene insertion events into the B chromosome. Leaf samples were taken from plants identified by FISH analysis as having confirmed B chromosome transgene insertion and DNA was prepared. The DNA was used with inverse PCR (Hui, et al. (1998) Cell Mol Life Sci. 54:1403-1422; Tonooka and Fujishima (2009) Appl Microbiol Biotechnol 85:37-43.) to identify sequences flanking the insertion of the transgene. Of the 10 events, 5 had a single copy of the transgene inserted into the B chromosome, and the left and right flank sequence was obtained (Table 4). The flanking sequence was used to map the transgene insertion site to the B chromosome sequence, thus confirming the FISH analysis.

[00127] The unique B chromosome sequences presented as SEQ ID NO:1-126 and SEQ ID NO:128-888 are used to select specific target sites for genome modification, to select specific target sites for transgene integration and to facilitate the identification of the flank of the B chromosome transgene. Additionally, the inclusion of the unique B chromosome sequences presented as SEQ ID NO:1-126 and SEQ ID NO:128-888 in the genome sequence analysis is used to improve specificity ( and thus reduce off-target effects) of target site selection of both chromosome A and chromosome B genome modification. TABLE 4. Flank of chromosome B transgene SEQ ID NO

Example 6: Development of the B chromosome marker

[00128] The B chromosome sequences identified here are used for the development of the B chromosome marker, essentially as described in US20060141495, which is incorporated by reference in its entirety. Identification of SNP and Indel polymorphisms is performed by comparing sequence alignments of contigs and singletons from at least two separate maize lines. Genomic libraries from multiple corn lines containing B chromosomes are made by isolating genomic DNA from different corn lines by standard methods known in the art. For genomic libraries, genomic DNA is digested with a restriction endonuclease enzyme (e.g., PstI), the digested DNA is size fractionated on a 1% agarose gel, and recovered DNA fragments are ligated into a plasmid vector for sequencing. by standard molecular biology techniques as described in Green and Sambrook (2012). All sequences are assembled to identify non-redundant sequences as described in Example 1 and Example 2. Sequence differences from multiple clones in assembled contigs are identified as single or multiple nucleotide polymorphisms. The B chromosome sequence of multiple maize lines is assembled at sites with one or more polymorphisms, i.e., SNPs and/or Indels. Candidate polymorphisms are qualified by the following parameters: (a) The minimum length of a contig or singleton for a consensus alignment is 200 bases. (b) The percentage of base identity observed in a 15-base region on either side of a candidate SNP is at least 75%. (c) The minimum sequence of reads in a given contig is 4.

[00129] Once regions of polymorphism have been identified, Taqman® probes are designed to detect the specific genotype in a corn DNA sample. These probes can be designed and supplied by Applied Biosystems for their proprietary Taqman (trademark) assay (Applied Biosystems, Foster City, California). To confirm that an assay produces accurate results, each new assay is performed on multiple replicates of samples of known genotypic identity representing each of the three possible genotypes, for example, two homozygous alleles and one heterozygous sample. To be a valid and useful test, clusters of clearly separable data points such as that of the three genotypes can be assigned to at least 90% of the data points, and the assignment has been observed to be correct in at least 98% of the data points. data points. Subsequent to this validation step, the assay is applied to the progeny of a cross between two highly inbred individuals to obtain segregation data, which is then used to calculate a genetic map position for the polymorphic locus. SNP analysis is also used to aid in the selection of B chromosome target sites for site-directed genome modification, as detailed in Example 3.

Claims (8)

1. Recombinant nucleic acid, characterized in that it comprises: an acid sequence of a portion of a corn B chromosome sequence selected from the group consisting of SEQ ID NOs: 10-13, 16, 19, 21, 22, 24, 25, 32, 34, 38, 4045, 17-49, 55, 56, 60, 62, 64, 65, 68, 70, 72, 73, 75-79, 81, 83-86, 8892, 94, 95, 98, 100, 102, 103, 107, 108, 110-112, 114, 116-118, 121, 123, 124, 126, 129-133, 135, 137, 138, 140-150, 154-159, 162-166, 168, 171-180, 183-197, 200-212, 214, 215, 217, 218, 220, 222-225, 228-232, 234-236, 238-241, 243, 244, 247, 249, 251, 253, 254, 256-258, 260-266, 269, 270, 273, 275-279, 281-295, 299, 301, 302, 304-316, 318-320, 322, 323, 325, 327-329, 331-335, 338-344, 346, 347, 349-354, 357-360, 363, 365, 366-373, 375-379, 381, 383, 384, 386-388, 391, 392, 395-398, 400404, 406-412, 415, 416, 418-420, 422, 423, 428-433, 435-441, 443-446, 448-450, 452, 456-459, 461-464, 466- 475, 478, 479, 481-484, 486, 487, 490-495, 497-499, 501-503, 505-508, 510-513, 517-520, 524, 525, 527529, 531-543, 545- 547, 549-554, 556-560, 563-577, 579-584, 586-593, 595, 596, 598-609, 611-614, 616-619, 621-624, 626, 627, 629-636, 638640, 643-652, 655, 657-661, 663, 664, 666-668, 670-674, 676-680, 683688, 690-701, 703, 705-708, 711-717, 719-729, 731- 734, 736-738, 740, 741, 743-748, 750, 752-764, 766, 767, 770, 771, 773, 774, 776-785, 787-791, and 793-888 and at least one DNA from interest, with at least one of the DNA of interest being integrated into the maize B chromosome sequence to produce the recombinant nucleic acid. 2. Recombinant nucleic acid, according to claim 1, characterized by the fact that the DNA of interest is integrated into the double-strand break generated by one or more site-specific genome modification enzymes. 3. Recombinant nucleic acid according to claim 2, characterized by the fact that the one or more site-specific genome modification enzymes are: (a) an endonuclease selected from a meganuclease, a zinc finger nuclease, a transcription activator-type effector nuclease (TALEN), an Argonaute, a DNA-guided recombinase, a DNA-guided endonuclease, an RNA-guided recombinase, an RNA-guided endonuclease, a type I CRISPR-Cas system, a CRISPR- Type II Cas and a type III CRISPR-Cas system; (b) an endonuclease selected from the group consisting of Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, x1, Csx15, Csf1, Csf2, Csf3 and Csf4 nuclease; (c) a dCas9-recombinase fusion protein; or (d) a tyrosine recombinase, a serine recombinase, a Cre recombinase, a Flp recombinase, a Tnp1 recombinase, a PhiC31 integrase, an R4 integrase or a TP-901 integrase. 4. Method for producing a transgenic maize cell comprising at least one DNA of interest integrated into a B chromosome: (a) selecting at least one target site in a portion of a selected maize B chromosome sequence of the group consisting of SEQ ID NOs: 10-13, 16, 19, 21, 22, 24, 25, 32, 34, 38, 4045, 17-49, 55, 56, 60, 62, 64, 65, 68 , 70, 72, 73, 75-79, 81, 83-86, 8892, 94, 95, 98, 100, 102, 103, 107, 108, 110-112, 114, 116-118, 121, 123, 124 , 126, 129-133, 135, 137, 138, 140-150, 154-159, 162-166, 168, 171-180, 183-197, 200-212, 214, 215, 217, 218, 220, 222 -225, 228-232, 234-236, 238-241, 243, 244, 247, 249, 251, 253, 254, 256-258, 260-266, 269, 270, 273, 275-279, 281-295 , 299, 301, 302, 304-316, 318-320, 322, 323, 325, 327-329, 331-335, 338-344, 346, 347, 349-354, 357-360, 363, 365, 366 -373, 375-379, 381, 383, 384, 386-388, 391, 392, 395-398, 400-404, 406-412, 415, 416, 418-420, 422, 423, 428-433, 435 -441, 443-446, 448-450, 452, 456-459, 461-464, 466-475, 478, 479, 481-484, 486, 487, 490-495, 497-499, 501-503, 505 -508, 510-513, 517-520, 524, 525, 527-529, 531-543, 545-547, 549-554, 556-560, 563-577, 579-584, 586-593, 595, 596 ,598-609, 611-614, 616-619, 621-624, 626, 627, 629-636, 638-640, 643-652, 655, 657-661, 663, 664, 666-668, 670-674 , 676-680, 683-688, 690-701, 703, 705-708, 711-717, 719-729, 731-734, 736-738, 740, 741, 743-748, 750, 752-764, 766 , 767, 770, 771, 773, 774, 776-785, 787-791, and 793-888; (b) selecting a site-specific genome modification enzyme that specifically cleaves the target site; (c) introducing the site-specific genome modification enzyme into the maize cell; (d) introducing the DNA of interest into the maize cell; (e) integrate the DNA of interest into the target site; and (f) selecting a transgenic maize cell comprising at least one of the DNA of interest integrated into the B chromosome. 5. Method according to claim 4, characterized by the fact that the at least one DNA of interest is integrated into the B chromosome by homology-directed repair or non-homologous end joining. 6. Method, according to claim 4 or 5, characterized by the fact that: (a) two or more DNAs of interest are integrated into two or more loci that are genetically linked, but physically separated; or (b) two or more DNAs of interest are integrated into different B chromosomes. 7. Method according to claim 6, characterized in that the two or more loci present a portion of a maize B chromosome sequence selected from the group consisting of SEQ ID NOs: 10-13, 16, 19, 21, 22, 24, 25, 32, 34, 38, 4045, 17-49, 55, 56, 60, 62, 64, 65, 68, 70, 72, 73, 75-79, 81, 83-86, 8892, 94, 95, 98, 100, 102, 103, 107, 108, 110-112, 114, 116-118, 121, 123, 124, 126, 129-133, 135, 137, 138, 140-150, 154-159, 162-166, 168, 171-180, 183-197, 200-212, 214, 215, 217, 218, 220, 222-225, 228-232, 234-236, 238-241, 243, 244, 247, 249, 251, 253, 254, 256-258, 260-266, 269, 270, 273, 275-279, 281-295, 299, 301, 302, 304-316, 318-320, 322, 323, 325, 327-329, 331-335, 338-344, 346, 347, 349-354, 357-360, 363, 365, 366-373, 375-379, 381, 383, 384, 386-388, 391, 392, 395-398, 400404, 406-412, 415, 416, 418-420, 422, 423, 428-433, 435-441, 443-446, 448-450, 452, 456-459, 461- 464, 466-475, 478, 479, 481-484, 486, 487, 490-495, 497-499, 501-503, 505-508, 510-513, 517-520, 524, 525, 527529, 531- 543, 545-547, 549-554, 556-560, 563-577, 579-584, 586-593, 595, 596, 598-609, 611-614, 616-619, 621-624, 626, 627, 629-636, 638640, 643-652, 655, 657-661, 663, 664, 666-668, 670-674, 676-680, 683688, 690-701, 703, 705-708, 711-717, 719- 729, 731-734, 736-738, 740, 741, 743-748, 750, 752-764, 766, 767, 770, 771, 773, 774, 776-785, 787-791, and 793-888. 8. Method according to any one of claims 4 to 7, characterized by the fact that the site-specific genome modification enzyme is: (a) an endonuclease selected from a meganuclease, a zinc finger nuclease, a nuclease transcription activator-like effector (TALEN), an Argonaute, a DNA-guided recombinase, a DNA-guided endonuclease, an RNA-guided recombinase, an RNA-guided endonuclease, a type I CRISPR-Cas system, a CRISPR-Cas system type II and a type III CRISPR-Cas system; (b) an endonuclease selected from the group comprising Cpf1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, x1, Csx15, Csf1, Csf2, Csf3 and Csf4 nuclease; (c) a dCas9-recombinase fusion protein; or (d) a tyrosine recombinase, a serine recombinase, a Cre recombinase, a Flp recombinase, a Tnp1 recombinase, a PhiC31 integrase, an R4 integrase or a TP-901 integrase.