# Patent

## Title
Sorghum centromere sequences and minichromosomes

## Abstract

The invention is generally related to MCs containing sorghum centromere sequences. In addition, the invention provides for methods of generating plants transformed with these MCs. MCs with novel compositions and structures are used to transform plants cells which are in turn used to generate the plant. Methods for generating the plant include methods for delivering the MC into plant cell to transform the cell, methods for selecting the transformed cell, and methods for isolating plants transformed with the MC.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Phase of International Application No. PCT/US2010/043065, filed Jul. 23, 2010; which claims priority to U.S. Provisional Patent Application No. 61/228,015, filed Jul. 23, 2009, both of which are herein incorporated by reference in their entirety.


GOVERNMENT SUPPORT
Not applicable.
COMPACT DISC FOR SEQUENCE LISTINGS AND TABLES
Not applicable.
FIELD OF THE INVENTION
The present invention relates to sorghum centromere sequences that are useful, for example, in constructing artificial chromosomes comprising sorghum centromere sequences, and cells and organisms comprising such artificial chromosomes, including Sorghum bicolor and Sorghum sudanese. Methods the make and use the disclosed sorghum centromeres are also disclosed.
BACKGROUND OF THE INVENTION
Two general approaches are used for introduction of new heritable genetic information (“transformation”) into cells. One approach is to introduce the new genetic information as part of another DNA molecule, referred to as an “episomal vector,” or “minichromosome” (MC), which can be maintained as an independent unit (an episome) apart from the host chromosomal DNA molecule(s). Episomal vectors contain all the necessary DNA sequence elements required for DNA replication and maintenance of the vector within the cell. Many episomal vectors are available for use in bacterial cells (for example, see Maniatis et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982). However, only a few episomal vectors that function in higher eukaryotic cells have been developed. Higher eukaryotic episomal vectors were primarily based on naturally occurring viruses. In higher plant systems gemini viruses are double-stranded DNA viruses that replicate through a double-stranded intermediate upon which an episomal vector could be based, although the gemini virus is limited to an approximately 800 bp insert. Although an episomal plant vector based on the Cauliflower Mosaic Virus has been developed, its capacity to carry new genetic information also is limited (Brisson et al., Nature, 310:511, 1984).
The other general method of genetic transformation involves integration of introduced DNA sequences into the recipient cell's chromosomes, permitting the new information to be replicated and partitioned to the cell's progeny as a part of the natural chromosomes. The introduced DNA usually can be broken and joined together in various combinations before it is integrated at random sites into the cell's chromosome (see, for example Wigler et al., Cell, 11:223, 1977). Common problems with this procedure are the rearrangement of introduced DNA sequences and unpredictable levels of expression due to the location of the transgene integration site in the host genome or so called “position effect variegation” (Shingo et al., Mol. Cell. Biol., 6:1787, 1986). Further, unlike episomal DNA, integrated DNA cannot normally be precisely removed. A more refined form of integrative transformation can be achieved by exploiting naturally occurring viruses that integrate into the host's chromosomes as part of their life cycle, such as retroviruses (see Chepko et al., Cell, 37:1053, 1984).
One common genetic transformation method used in higher plants is based on the transfer of bacterial DNA into plant chromosomes that occurs during infection by the phytopathogenic soil bacterium Agrobacterium (see Nester et al., Ann. Rev. Plant Phys., 35:387-413, 1984). By substituting genes of interest for a portion of the naturally transferred bacterial sequences (called T-DNA), investigators have been able to introduce new DNA into plant cells. However, even this more “refined” integrative transformation system is limited in three major ways. First, DNA sequences introduced into plant cells using the Agrobacterium T-DNA system are frequently rearranged (see Jones et al., Mol. Gen. Genet., 207:478, 1987). Second, the expression of the introduced DNA sequences varies between individual transformants (see Jones et al., EMBO J., 4:2411-2418, 1985). This variability is presumably caused by rearranged sequences and the influence of surrounding sequences in the plant chromosome (i.e., position effects), as well as methylation of the transgene. Finally, insertion of extra elements into the genome can disrupt the genes, promoters or other genetic elements necessary for normal plant growth and function.
Another widely used technique to genetically transform plants involves the use of microprojectile bombardment to integrate DNA sequences into the genome. In this process, a nucleic acid containing the desired genetic elements to be introduced into the plant's native chromosome is deposited on or in small metallic particles, e.g., tungsten, platinum, or preferably gold, which are then delivered at a high velocity into the plant tissue or plant cells. However, similar problems arise as with Agrobacterium-mediated gene transfer, and as noted above expression of the inserted DNA can be unpredictable and insertion of extra elements into the genome can disrupt and adversely impact plant processes.
One attractive alternative to the commonly used methods of transformation is the use of an artificial chromosome. Artificial chromosomes are episomal nucleic acid molecules that exist autonomously from the native chromosomes of the host genome. They can be linear or circular DNA molecules that are comprised of cis-acting nucleic acid sequence elements that provide replication and partitioning activities (see Murray et al., Nature, 305:189-193, 1983). Desired elements include: (1) origin of replication, which are the sites for initiation of DNA replication, (2) centromeres (site of kinetochore assembly and responsible for proper distribution of replicated chromosomes into daughter cells at mitosis or meiosis), and (3) if the chromosome is linear, telomeres (specialized DNA structures at the ends of linear chromosomes that function to stabilize the ends and facilitate the complete replication of the extreme termini of the DNA molecule). An additional desired element is a chromatin organizing sequence. It is well documented that centromere function is crucial for stable chromosomal inheritance in almost all eukaryotic organisms (reviewed in Nicklas, J Cell Sci. 189:283-5, 1988). The centromere accomplishes this by attaching, via centromere binding proteins, to the spindle fibers during mitosis and meiosis, thus ensuring proper gene segregation during cell divisions.
Artificial chromosomes have been engineered using one of two approaches. The first approach identifies and assembles the desired chromosomal elements into an artificial construct. This is approach has been described as “bottom-up” and involves the use of a heterologous system (i.e. bacteria or fungal) to perform the various cloning steps necessary to assemble the artificial chromosome. Artificial chromosomes of this type will be referred to in this application as “minichromosomes or “MCs”. The second approach derives the artificial from existing chromosomes through chromosome fragmentation and, optionally, subsequent addition of desired elements including transgenes. For example, an existing chromosome can be induced to undergo breakage events that result in chromosomal fragments. Minimal fragments that possess the elements necessary for replication and segregation during cell division (i.e. centromere, origins of replication and telomeres) can be identified. These derived artificial chromosomes can then be used as targets for further manipulation including the addition of one or more transgenes. This approach has been described as “top-down” and does not require the use of a heterologous system (i.e. bacterial or fungal) since it doesn't require in vitro-based cloning steps. Artificial chromosomes of this type will be referred to in this application as “recombinant chromosomes.”
The essential chromosomal elements for construction of artificial chromosomes have been precisely characterized in lower eukaryotic species, and more recently in mouse and human Autonomous Replication Sequences (ARSs) have been isolated from unicellular fungi, including Saccharomyces cerevisiae (brewer's yeast) and Schizosaccharomyces pombe (see Stinchcomb et al., Nature 282:39-43, 1979 and Hsiao et al., Proc Natl Acad Sci USA 76:3829-33, 1979). An ARS behaves like an origin of replication allowing DNA molecules that contain the ARS to be replicated in concert with the rest of the genome after introduction into the cell nuclei of these fungi. DNA molecules containing these sequences replicate, but in the absence of a centromere they are not partitioned into daughter cells in a controlled fashion that ensures efficient chromosome inheritance.
Artificial chromosomes have been constructed in yeast using the three cloned essential chromosomal elements (see Murray et al., Nature, 305:189-193, 1983). None of the essential components identified in unicellular organisms, however, function in higher eukaryotic systems. For example, a yeast centromere sequence will not confer stable inheritance upon vectors transformed into higher eukaryotes.
In contrast to the detailed studies done in yeast, less is known about the molecular structure of functional centromeric DNA of higher eukaryotes. Ultrastructural studies indicate that higher eukaryotic kinetochores, which are specialized complexes of proteins that form on the centromere during late prophase, are large structures (mammalian kinetochore plates are approximately 0.3 μm in diameter) which possess multiple microtubule attachment sites (reviewed in Rieder, Int Rev Cytol; 79:1-58, 1982). It is therefore possible that the centromeric DNA regions of these organisms will be correspondingly large, although the minimal amount of DNA necessary for centromere function may be much smaller.
While the above studies have been useful in elucidating the structure and function of centromeres, it was not known whether information derived from lower eukaryotic or mammalian higher eukaryotic organisms would be applicable to sorghum. There exists a need for cloned centromeres from sorghum, which would represent a first step in the production of artificial chromosomes, or in the identification of recombinant chromosomes. There further exists a need for sorghum cells, plants, seeds and progeny containing functional, stable, and autonomous artificial or recombinant chromosomes capable of carrying a large number of different genes and genetic elements.
SUMMARY OF THE INVENTION
In one aspect, the present invention addresses sorghum MCs comprising a sorghum centromere having one or more repeated nucleotide sequences, described in further detail herein. In some embodiments, such MCs comprise a centromere comprising one or more selected repeated nucleotide sequences derived from sorghum, including those isolated from sorghum genomic DNA and synthetic arrays of repeat sequences. In other embodiments, the invention addresses sorghum recombinant chromosomes.
In another aspect, the invention provides modified or “adchromosomal” sorghum plants, containing functional, stable, autonomous MCs or recombinant chromosomes.
The invention provides for isolated sorghum MCs comprising a centromere, wherein the centromere comprises at least two copies of a repeated nucleotide sequences, and wherein the centromere confers the ability to segregate to daughter cells. The repeated nucleotide sequences may be short sorghum satellite sequences such as those sequences set out in SEQ ID NOs:23-176, or the consensus sorghum satellite sequence set out as SEQ ID NO:22. The repeated nucleotide sequences may be longer sequences such as the sorghum retrotransposon CRS sequence, set out as SEQ ID NO:21 or fragments thereof.
In exemplary embodiments, the invention provides for a sorghum plant cell comprising a sorghum MC comprising a sorghum centromere that comprises at least two repeat nucleotide sequences that have a sequence that hybridizes under conditions comprising hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C. to any one of the sorghum satellite sequence set out is SEQ ID NOs:23-176, or the consensus sorghum satellite sequence set out as SEQ ID NO:22, or the sorghum retrotransposon sequence of SEQ ID NO:21 or a fragment thereof, and wherein the centromere confers the ability to segregate to daughter cells. Alternatively, the hybridization conditions may comprise hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C.
In another exemplary embodiment, the invention provides for a sorghum plant cell comprising a sorghum MC comprising a sorghum centromere, wherein the centromere comprises at least two copies of a repeated nucleotide sequences that have a sequence that is at least 80% identical to any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or the consensus sorghum satellite sequence set out as SEQ ID NO:22, or the sorghum retrotransposon sequence of SEQ ID NO:21 or a fragment thereof, and wherein the centromere confers the ability to segregate to daughter cells. The invention also provides for a sorghum plant cell comprising a sorghum MCs wherein the repeated nucleotide sequence comprise a sequence that is at least 85% identical, or 90% identical or 95% identical or 98% identical to any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or the consensus sorghum satellite sequence set out as SEQ ID NO:22.
In another embodiment, the invention provides for a sorghum plant cell comprising a sorghum Applied MC comprising at least two copies of a repeated nucleotide sequence that is at least 80% identical to any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or the consensus sorghum satellite sequence set out as SEQ ID NO:22, or the sorghum retrotransposon sequence of SEQ ID NO:21 or a fragment thereof or hybridizes to the nucleotide sequence of any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or the consensus sorghum satellite sequence set out as SEQ ID NO:22, or the sorghum retrotransposon sequence of SEQ ID NO:21 or a fragment thereof under stringent conditions comprising hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C., and a Transgene Expression Cassette.
In a further embodiment, the invention provides for a sorghum plant cell comprising a sorghum MC comprising a sorghum centromere, wherein the centromere comprises (a) at least two copies of a sorghum satellite nucleotide sequence, and (b) at least two copies of the a sorghum CRS nucleotide sequence (SEQ ID NO:21) or fragments thereof, and wherein the centromere confers the ability to segregate to daughter sorghum cells. In another embodiment, the invention provides for a sorghum plant cell comprising a MC comprising a sorghum centromere, wherein the centromere comprises (a) at least one array of sorghum satellite nucleotide sequences, and (b) at least one array of sorghum CRS nucleotide sequence (SEQ ID NO:21) or fragments thereof, and wherein the centromere confers the ability to segregate to daughter sorghum cells. The sorghum satellite nucleotide sequence may be one of the sequences set out in SEQ ID NOs:23-176, or the consensus sorghum satellite sequence set out as SEQ ID NO:22, or a sequence that hybridizes under conditions comprising hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C. to any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or to the consensus sorghum satellite sequence set out as SEQ ID NO:22, or a sequence that is al least 70% identical to any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or to the consensus sorghum satellite sequence set out as SEQ ID NO:22.
In addition, the invention provides for a sorghum plant cell comprising a sorghum Applied MC comprising a sorghum centromere, wherein the sorghum centromere comprises (a) at least 5 copies of a repeated nucleotide sequence within 1 kb of nucleotide sequence, wherein the repeated nucleotide sequence is at least 80% identical to any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or to the consensus sorghum satellite sequence set out as SEQ ID NO:22, or hybridizes to the nucleotide sequence of any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or to the consensus sorghum satellite sequence set out as SEQ ID NO:22 under stringent conditions comprising hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C., and (b) at least 2 copies of a repeated nucleotide sequence that is at least 80% identical over its length to a fragment of the nucleotide sequence of SEQ ID NO:21 or hybridizes to a fragment of SEQ ID NO:21 under stringent conditions comprising hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C.
In another embodiment, the invention provides a sorghum plant cell comprising (a) a polynucleotide sequence that is transcribed as a first RNA, (b) a polynucleotide sequence that is transcribed as a second RNA, and (c) a polynucleotide sequence that is transcribed as a third RNA, wherein transcription of the polynucleotide sequences results in increased biomass of a sorghum plant.
In an additional embodiment, the invention provides for a sorghum plant cell comprising a transgene expression cassette not integrated into the plant cell genome, wherein the Transgene Expression Cassette comprises (a) a polynucleotide sequence that is transcribed as a first RNA, (b) a polynucleotide sequence that is transcribed as a second RNA, and (c) a polynucleotide sequence that is transcribed as a third RNA, wherein transcription of the polynucleotide sequences results in increased biomass of a sorghum plant.
The invention provides for a sorghum plant cell comprising a recombinant chromosome comprising at least two copies of a repeated nucleotide sequences, and wherein the centromere confers the ability to segregate to daughter cells. The repeated nucleotide sequences may be short sorghum satellite sequences such as those sequences set out in SEQ ID NOs:23-176, or the consensus sorghum satellite sequence set out as SEQ ID NO:22. The repeated nucleotide sequences may be longer sequences such as the sorghum retrotransposon sequence CRS, set out as SEQ ID NO:21.
In exemplary embodiments, the invention provides for a sorghum plant cell comprising a recombinant chromosome comprising a sorghum centromere that comprises at least two repeat nucleotide sequences that have a sequence that hybridizes under conditions comprising hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C. to any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or to the consensus sorghum satellite sequence set out as SEQ ID NO:22, or to the sorghum retrotransposon sequence of SEQ ID NO:21 or a fragment thereof, and wherein the centromere confers the ability to segregate to daughter cells. Alternatively, the hybridization conditions may comprise hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C.
In another exemplary embodiment, the invention provides for a sorghum plant cell comprising a recombinant chromosome comprising at least two copies of a repeated nucleotide sequences that have a sequence that is at least 80% identical to any one of the sorghum satellite sequence set out in the SEQ ID NOs:23-176, or to the consensus sorghum satellite sequence set out as SEQ ID NO:22, or to the sorghum retrotransposon sequence of SEQ ID NO:21 or a fragment thereof, and a transgene expression cassette comprising at least three exogenous nucleic acids. The invention also provides for sorghum recombinant chromosomes wherein the repeated nucleotide sequence comprise a sequence that is at least 85% identical, or 90% identical or 95% identical or 98% identical to any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or to the consensus sorghum satellite sequence set out as SEQ ID NO:22, or the sorghum retrotransposon sequence of SEQ ID NO:21 or a fragment thereof.
In a further embodiment, the invention provides for a sorghum plant cell comprising a sorghum recombinant chromosome comprising a sorghum centromere, wherein the centromere comprises (a) at least two copies of a sorghum satellite nucleotide sequence, and (b) at least two copies of the a sorghum CRS nucleotide sequence (SEQ ID NO:21) or a fragment thereof, and wherein the centromere confers the ability to segregate to daughter cells. In another embodiment, the invention provides for a sorghum recombinant chromosome comprising a sorghum centromere, wherein the centromere comprises (a) at least one array of sorghum satellite nucleotide sequences, and (b) at least one array of sorghum CRS nucleotide sequence (SEQ ID NO:21) or a fragment thereof, and wherein the centromere confers the ability to segregate to daughter cells. The sorghum satellite nucleotide sequence may be one of the sequences set out in SEQ ID NOs:23-176, or to the consensus sorghum satellite sequence set out as SEQ ID NO:22, or to a sequence that hybridizes under conditions comprising hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C. to a nucleotide sequence any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or to the consensus sorghum satellite sequence set out as SEQ ID NO:22, or to a sequence that is at least 80% identical to any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or to the consensus sorghum satellite sequence set out as SEQ ID NO:22.
Alternatively, the invention provides for sorghum plant cells comprising a recombinant chromosome that has not been maintained in a cell of a heterologous organism.
In another embodiment, the invention provides for a sorghum plant cell comprising (a) at least two copies of a repeated nucleotide sequence that is at least 80% identical to any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or to the consensus sorghum satellite sequence set out as SEQ ID NO:22, or to the sorghum retrotransposon sequence of SEQ ID NO:21 or a fragment thereof or hybridizes to any one of the sorghum satellite sequence set out in SEQ ID NOs:23-176, or to the consensus sorghum satellite sequence set out as SEQ ID NO:22, or to the sorghum retrotransposon sequence of SEQ ID NO:21 or a fragment thereof under stringent conditions comprising hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C., and (b) a Transgene Expression Cassette comprising at least three exogenous nucleic acids, wherein the nucleotide sequence and the Transgene Expression Cassette are not integrated into the genome of the sorghum plant cell.
The invention also provides for a sorghum plant cell comprising a sorghum MC comprising a sorghum centromere, wherein the centromere comprises at least two synthetic repeat sequences or a synthetic array of repeated nucleotide sequence, wherein the array comprises at least two copies of a repeated nucleotide sequence, and wherein the centromere confers the ability to segregate to daughter sorghum cells. These artificially synthesized repeated nucleotide sequences may be based on sequence information from natural sorghum centromere sequences, combinations or fragments of natural sorghum centromere sequences including a combination of repeats of different lengths, a combination of different sequences, a combination of both different repeat lengths and different sequences, a combination of different artificially synthesized sequences or a combination of natural sorghum centromere sequence(s) and artificially synthesized sorghum sequence(s). The polynucleotides comprising synthetic arrays of sorghum repeat sequences and synthetic arrays of sorghum repeat sequences may be generated using any technique known in the art including PCR from sorghum genomic DNA (or a clone thereof) or by custom oligonucleotide synthesis.
The invention provides for any of the preceding sorghum MCs or recombinant chromosomes having a centromere comprising an array of repeated nucleotide sequence that ranges from about 1 kb to about 200 kb in length, 1 kb to about 100 kb in length, about 1 kb to about 10 kb, about 2 kb to about 12 kb, about 5 kb to about 25 kb, about 10 kb to about 50 kb, about 25 kb to 100 kb.
The invention further contemplates any of the preceding sorghum MCs or recombinant chromosomes having centromeres comprising at least 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 750 bp, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 10.5 kb, 11 kb, 11.5 kb, 12 kb, 12.5 kb, 13 kb, 13.5 kb, 14 kb, 14.5 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 110 kb, 120 kb, 130 kb, 140 kb, 150 kb, 160 kb, 170 kb, 180 kb, 190 kb, 200 kb, 225 kb, 250 kb, 275 kb, 300 kb, 325 kb, 350 kb or 375 kb.
In another embodiment, any of the preceding sorghum MCs or recombinant chromosomes comprise centromeres having n copies of a repeated nucleotide sequence, wherein n is less than 2000, less than 1500, less than 1000, less than 500, less than 400, less than 300, less than 250, less than 200, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 25, less than 20, less than 15, less than 10, less than 9, less than 8, less than 7, less than 6 or less than 5. In exemplary embodiments, the centromeres of the sorghum MCs of the invention comprise n copies of a repeated nucleotide sequence, wherein n is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 1000. In additional exemplary embodiments, the centromeres of the sorghum MCs or recombinant chromosomes of the invention comprise n copies of a repeated nucleotide sequence where n ranges from 2 to 10, 2 to 20, 5 to 15, 5 to 25, 5 to 50, 5 to 100, 5 to 250, 5 to 500, 5 to 1000, 15 to 25, 15 to 50, 15 to 100, 15 to 250, 15 to 500, 15 to 1000, 25 to 50, 25 to 100, 25 to 250, 25 to 500, 25 to 1000, 50 to 100, 50 to 250, 50 to 500, 50 to 1000, 100 to 250, 100 to 500, 100 to 1000, 250 to 500, 250 to 1000, or 500 to 1000.
In an embodiment of the invention, any of the preceding sorghum MCs or recombinant chromosomes comprising a centromere having at least 5 consecutive repeated nucleotide sequences in “head to tail orientation.” In an embodiment of the invention, any of the preceding sorghum MCs or recombinant chromosomes comprising a centromere having at least 5 consecutive repeated nucleotide sequences in “tandem,” in which one repeat sequence is immediately adjacent to another repeat sequence in any orientation, e.g. head to tail, tail to tail, or head to head. The invention also provides for any of the preceding sorghum MCs or recombinant chromosomes comprising a centromere having at least 5 repeated nucleotide sequences that are consecutive. The term “consecutive” refers to the same or similar repeated nucleotide sequences (e.g., at least 80% identical) that follow one after another without being interrupted by other significant sequence elements. Consecutive repeated nucleotide sequences may be in any orientation, e.g. head to tail, tail to tail, or head to head, and need not be directly adjacent to each other (e.g., may be 1-50 bp apart).
The invention further provides for any of the preceding sorghum MCs or recombinant chromosomes comprising a centromere having at least 5 of the consecutive repeated nucleotide sequences separated by less than n number of nucleotides, wherein n ranges from 1 to 10, or 1 to 20, or 1 to 30, or 1 to 40, or 1 to 50 or wherein n is less than 10 bp or n is less than 20 bp or n is less than 30 bp or n is less that 40 bp or n is less than 50 bp.
The invention also provide for any of the preceding sorghum MCs or recombinant chromosomes comprising a centromere having at least two arrays of consecutive repeated nucleotide sequences, wherein the array comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000 or 2000 repeated nucleotide sequences. The repeats within an array may be in tandem in any orientation, e.g. head to tail, tail to tail, or head to head, or consecutive in any orientation, e.g. head to tail, tail to tail, or head to head. The arrays may be separated by less than n number of nucleotides, wherein n ranges from 1 to 10, or 1 to 20, or 1 to 30, or 1 to 40, or 1 to 50, or 1 to 60, or 1 to 70, or 1 to 80, or 1 to 90, or 1 to 100, or wherein n is less than 10 bp or n is less than 20 bp or n is less than 30 bp or n is less than 40 bp or n is less than 50 bp. The two arrays may comprise the same repeated nucleotide sequence or two different repeated nucleotide sequences (i.e. the first array can be comprised of repeat type 1 and the second array can be comprised of repeat type 2—here “type 1” and “type 2” are arbitrary designations).
In one embodiment, the sorghum MCs or recombinant chromosomes of the invention are 1000 kb or less in length, 900 kb or less in length, 800 kb or less in length or 700 kb or less in length. In exemplary embodiments, the sorghum MC is 600 kb or less in length, 500 kb or less in length, 250 kb or less in length, 100 kb or less in length, 50 kb or less in length, 10 kb or less in length, 5 kb or less in length, or 1 kb or less in length. For example, the sorghum MCs of the invention are 50 to 250 kb in length, 50 to 100 kb in length, 50 to 75 kb in length, 50 to 100 kb in length, 60 kb to 85 kb in length, 70 to 90 kb in length, 75 to 100 kb in length, 100 to 250 kb in length, 250 to 500 kb in length, 500 to 1000 kb in length. In an exemplary embodiment, the sorghum MC is 28 kb in length, 42 kb in length, 82 kb in length, 87 kb in length, 88 kb in length, 97 kb in length, 130 kb in length, 150 kb in length, 200 kb in length or ranges from 28-200 kb in length. The MC of the invention preferably has a segregation efficiency during mitotic division of at least 60%, at least 80%, at least 90% or at least 95% and/or a transmission efficiency during meiotic division of, e.g., at least 60%, at least 80%, at least 85%, at least 90% or at least 95%.
The sorghum MC or recombinant chromosomes of the invention preferably has a segregation efficiency during mitotic division of at least 60%, at least 80%, at least 90% or at least 95% and/or a transmission efficiency during meiotic division of, e.g., at least 60%, at least 80%, at least 85%, at least 90% or at least 95%.
In another embodiment, the sorghum MCs or recombinant chromosomes of the invention comprise a site for site-specific recombination.
The invention also provides for a sorghum MC, wherein the MC is derived from a donor clone or a centromere clone and has substitutions, deletions, insertions, duplications or arrangements of one or more nucleotides in the MC compared to the nucleotide sequence of the donor clone or centromere clone. In one embodiment, the sorghum MC is obtained by passage of the sorghum MC through one or more hosts. In another embodiment, the MC is obtained by passage of the MC through two or more different hosts. The host may be selected from the group consisting of viruses, bacteria, yeasts. In another embodiment, the sorghum MC is obtained from a donor clone by in vitro methods that introduce sequence variation during template-based replication of the donor clone, or its complementary sequence. In one embodiment this variation may be introduced by a DNA-dependent DNA polymerase. In a further embodiment a sorghum MC derived by an in vitro method may be further modified by passage of the MC through one or more hosts.
The invention also provides for a sorghum MC or recombinant chromosome, wherein the MC comprises at least one exogenous nucleic acid. In further exemplary embodiments, the sorghum MC or recombinant chromosome comprises at least two or more, at least three or more, at least four or more, at least five or more, at least ten or more, at least 20 or more, at least 30 or more, at least 40 or more, at least 50 or more exogenous nucleic acids.
In one embodiment, at least one exogenous nucleic acid of any of the preceding sorghum MCs or recombinant chromosome is operably linked to a heterologous regulatory sequence functional in plant cells, including but not limited to a plant regulatory sequence. The invention also provides for exogenous nucleic acids linked to a non-plant regulatory sequence, such as an arthropod, viral, bacterial, vertebrate or yeast regulatory sequence. The invention also provides for exogenous nucleic acids linked to a regulatory sequence from sorghum.
The invention also provides for a MC or recombinant chromosome comprising a gene or group of genes that act to improve the total recoverable sugar from sorghum. Such genes may act to increase the sugar concentration of the stem juice, increase the amount of juice, or increase the stem strength to improve yield, increase total bomass of the plant. Such genes may be derived from bacterial sequences such as a sucrose isomerase or from animal, plant fungal, or protist sequences. Such genes from plants may include genes involved in sugar metabolism or transport or genes of unknown function that have been shown to quantitatively increase total recoverable sugar. Such genes may also include genes that affect plant height, stem diameter, water metabolism or total biomass. Such genes may also include those that regulate the equilibrium between starch and sugar. Several genes have been shown to improve sugar accumulation. For example, expression of a bacterial sucrose isomerase can increase sugarcane sugar content by as much as two-fold (Birch, R. G., and Wu, L. (2007). Doubled sugar content in sugarcane plants modified to produce a sucrose isomer. Plant Biotechnology Journal 5: 109-117. The lignin-deficient “brown midrib” mutations improve sorghum sugar content via their effects on lignin; this phenotype is caused by mutations in cinnamyl alcohol dehydrogenase (CAD), and 14 CAD-like genes are present in the sorghum genome (Saballos, A et al. Genetics 181:783-95, 2009).
In another embodiment, the sorghum MC or recombinant chromosome comprises an exogenous nucleic acid comprises a QTL that confers a desirable trait. QTLs that affect total recoverable sugars have been mapped in sugarcane (Murray, S. C., et al. Crop Sci. 48:2165-2179, 2008).
In another embodiment, the sorghum MC or recombinant chromosome comprises an exogenous nucleic acid that confers herbicide resistance, insect resistance, disease resistance, or stress resistance on the sorghum plant. The invention provides for sorghum MCs or recombinant chromosomes comprising an exogenous nucleic acid that confers resistance to phosphinothricin or glyphosate herbicide. Nonlimiting examples include an exogenous nucleic acid that encodes a phosphinothricin acetyltransferase, glyphosate acetyltransferase, acetohydroxyadic synthase or a mutant enoylpyruvylshikimate phosphate (EPSP) synthase. Nonlimiting examples of exogenous nucleic acids that confer insect resistance include a Bacillus thuringiensis toxin gene or Bacillus cereus toxin gene. In related embodiments, the sorghum MC or recombinant chromosome comprises an exogenous nucleic acid conferring herbicide resistance, an exogenous nucleic acid conferring insect resistance, and at least one additional exogenous nucleic acid.
The invention further provides for sorghum MCs or recombinant chromosomes comprising additional copies of genes already found in the sorghum genome. The invention also provides for the additional copies of sorghum genes carried on the sorghum MC or recombinant chromosomes to be operably linked to either their native regulatory sequences or to heterologous regulatory sequences.
The invention further provides for sorghum MCs or recombinant chromosome comprising an exogenous nucleic acid that confers resistance to drought, heat, chilling, freezing, excessive moisture, ultraviolet light, ionizing radiation, toxins, pollution, mechanical stress or salt stress. The invention also provides for a sorghum MC that comprises an exogenous nucleic acid that confers resistance to a virus, bacteria, fungi or nematode.
The invention provides for sorghum MCs or recombinant chromosome comprising an exogenous nucleic acid selected from the group consisting of a nitrogen fixation gene, a plant stress-induced gene, a nutrient utilization gene, a gene that affects plant pigmentation, a gene that encodes an antisense or ribozyme molecule, a gene encoding a secretable antigen, a toxin gene, a receptor gene, a ligand gene, a seed storage gene, a hormone gene, an enzyme gene, an interleukin gene, a clotting factor gene, a cytokine gene, an antibody gene, a growth factor gene, a transcription factor gene, a transcriptional repressor gene, a DNA-binding protein gene, a recombination gene, a DNA replication gene, a programmed cell death gene, a kinase gene, a phosphatase gene, a G protein gene, a cyclin gene, a cell cycle control gene, a gene involved in transcription, a gene involved in translation, a gene involved in RNA processing, a gene involved in RNAi, an organellar gene, a intracellular trafficking gene, an integral membrane protein gene, a transporter gene, a membrane channel protein gene, a cell wall gene, a gene involved in protein processing, a gene involved in protein modification, a gene involved in protein degradation, a gene involved in metabolism, a gene involved in biosynthesis, a gene involved in assimilation of nitrogen or other elements or nutrients, a gene involved in controlling carbon flux, gene involved in respiration, a gene involved in photosynthesis, a gene involved in light sensing, a gene involved in organogenesis, a gene involved in embryogenesis, a gene involved in differentiation, a gene involved in meiotic drive, a gene involved in self incompatibility, a gene involved in development, a gene involved in nutrient, metabolite or mineral transport, a gene involved in nutrient, metabolite or mineral storage, a calcium-binding protein gene, or a lipid-binding protein gene.
The invention also provides for a sorghum MC or recombinant chromosome comprising an exogenous enzyme gene selected from the group consisting of a gene that encodes an enzyme involved in metabolizing biochemical wastes for use in bioremediation, a gene that encodes an enzyme for modifying pathways that produce secondary plant metabolites, a gene that encodes an enzyme that produces a pharmaceutical, a gene that encodes an enzyme that improves changes the nutritional content of a plant, a gene that encodes an enzyme involved in vitamin synthesis, a gene that encodes an enzyme involved in carbohydrate, polysaccharide or starch synthesis, a gene that encodes an enzyme involved in mineral accumulation or availability, a gene that encodes a phytase, a gene that encodes an enzyme involved in fatty acid, fat or oil synthesis, a gene that encodes an enzyme involved in synthesis of chemicals or plastics, a gene that encodes an enzyme involved in synthesis of a fuel and a gene that encodes an enzyme involved in synthesis of a fragrance, a gene that encodes an enzyme involved in synthesis of a flavor, a gene that encodes an enzyme involved in synthesis of a pigment or dye, a gene that encodes an enzyme involved in synthesis of a hydrocarbon, a gene that encodes an enzyme involved in synthesis of a structural or fibrous compound, a gene that encodes an enzyme involved in synthesis of a food additive, a gene that encodes an enzyme involved in synthesis of a chemical insecticide, a gene that encodes an enzyme involved in synthesis of an insect repellent, or a gene controlling carbon flux in a plant.
In another embodiment of the invention, any of the preceding sorghum MCs or recombinant chromosomes comprises a telomere.
The invention also provides embodiments wherein any of the preceding sorghum MCs or recombinant chromosomes is linear or circular.
In one embodiment, the invention provides for sorghum plants or plant cells comprising any of the preceding sorghum MCs or recombinant chromosomes. The invention also provides for sorghum plant tissue and sorghum seed obtained from the sorghum plants of the invention.
In another embodiment, the invention provides for sorghum plants comprising any of the preceding sorghum MCs or recombinant chromosomes, which may be referred to herein as “adchromosomal” sorghum plants. In addition, the invention provides for sorghum plant cells, tissues and seeds obtained from these modified plants.
In one embodiment, the invention provides for a sorghum plant cell comprising any of the preceding sorghum MCs or recombinant chromosomes that (i) is not integrated into the sorghum plant cell genome and (ii) confers an altered phenotype on the sorghum plant cell associated with at least one structural gene within the sorghum MC. The altered phenotype comprises increased expression of a native gene, decreased expression of a native gene, or expression of an exogenous gene. In a further embodiment, these sorghum plant cells also comprise one or more integrated exogenous structural gene(s).
Another embodiment of the invention is a part of any of the preceding sorghum plants. Exemplary sorghum plant parts of the invention include a pod, root, sett root, shoot root, root primordial, shoot, primary shoot, secondary shoot, tassle, panicle, arrow, midrib, blade, ligule, auricle, dewlap, blade joint, sheath, node, internode, bud furrow, leaf scar, cutting, tuber, stem, stalk, fruit, berry, nut, flower, leaf, bark, wood, epidermis, vascular tissue, organ, protoplast, crown, callus culture, petiole, petal, sepal, stamen, stigma, style, bud, meristem, cambium, cortex, pith, sheath, silk, ovule or embryo. Other exemplary sorghum plant parts are a meiocyte or gamete or ovule or pollen or endosperm of any of the preceding plants. Other exemplary plant parts are a seed, seed-piece, embryo, protoplast, cell culture, any group of plant cells organized into a structural and functional unit, ratoon, or propagule of any of the preceding sorghum plants.
An embodiment of the invention is a progeny of any of the preceding sorghum plants of the invention. These progeny of the invention may be the result of self-breeding, cross-breeding, apomyxis or clonal propagation. In exemplary embodiments, the invention also provides for progeny that comprise a sorghum MC or recombinant chromosome that is descended from a parental sorghum MC or recombinant chromosome that contained a centromere less than about 1000 kilobases in length, less than about 750 kilobases in length, less than about 600 kilobases in length, less than about 500 kilobases in length, less than about 400 kilobases in length, less than about 300 kilobases in length, less than about 250 kilobases in length, less than about 200 kilobases in length, less than about 150 kilobases, less than about 100 kilobases, less than about 90 kilobases in length, less than about 85 kilobases in length, less than about 80 kilobases in length, less than about 75 kilobases in length, less than about 70 kilobases in length, less than about 65 kilobases in length, less than about 60 kilobases in length, less than about 55 kilobases in length, less than about 50 kilobases in length, less than about 45 kilobases in length, less than about 40 kilobases in length, less than about 35 kilobases in length, less than about 30 kb in length, less than about 25 kilobases in length, less than about 20 kb in length, less than about 15 kilobases in length, less than about 12 kilobases in length, less than about 10 kb in length, less than about 7 kb in length, less than about 5 kb in length, or less than about 2 kb in length.
In another aspect, the invention provides for methods of making a sorghum MC for use in any of the preceding sorghum plants of the invention. These methods comprise identifying a centromere nucleotide sequence in a sorghum genomic DNA library using a multiplicity of diverse probes, and constructing a sorghum MC comprising the centromere nucleotide sequence. These methods may further comprise determining hybridization scores for hybridization of the multiplicity of diverse probes to genomic clones within the sorghum genomic nucleic acid library, determining a classification for genomic clones within the sorghum genomic nucleic acid library according to the hybridization scores for at least two of the diverse probes, and selecting one or more genomic clones within one or more classifications for constructing the sorghum MC.
The invention also contemplates methods of using any of the preceding sorghum plants to produce a recombinant protein, by growing a sorghum plant comprising a sorghum MC or recombinant chromosome that comprises an exogenous nucleic acid encoding the desired recombinant protein. Optionally the sorghum plant is harvested and the desired protein product is isolated from the plant. Exemplary protein products include industrial enzymes such as those useful for biofuel production.
The invention also contemplates methods of using any of the preceding sorghum plants to produce a chemical product, by growing a sorghum plant comprising a sorghum MC or recombinant chromosome that comprises an exogenous nucleic acid encoding and enzyme involved in the synthesis of the chemical product. Optionally the sorghum plant is harvested and the desired chemical product is isolated from the plant. Exemplary chemical products include sugars, lipids and carbohydrates useful in the production of biofuels.
Another aspect of the invention provides for methods of using any of the preceding sorghum plants comprising a sorghum MCs or recombinant chromosome for a food product, a pharmaceutical product or chemical product, according to which a suitable exogenous nucleic acid is expressed in sorghum plants or plant cells and the plant or plant cells are grown. The plant may secrete the product into its growth environment or the product may be contained within the plant, in which case the plant is harvested and desirable products are extracted.
Thus, the invention contemplates methods of using any of the preceding sorghum plants comprising a sorghum MC or recombinant chromosome to produce a modified food product, for example, by growing a plant that expresses a exogenous nucleic acid that alters the nutritional content of the plant, and harvesting or processing the sorghum plant.
The invention also provides for methods of constructing a synthetic array of repeated nucleotide sequence having sorghum centromere function comprising the steps of: (a) PCR amplifying a sorghum satellite sequence, (b) cloning the PCR amplified satellite sequence into a cloning vector, (c) sequencing the cloned satellite DNA, (d) use a restriction enzyme with an asymmetric recognition sequence to excise the cloned satellite sequence from the cloning vector, (e) ligate the satellite sequence to one another forming a synthetic tandem array, (f) ligate the synthetic array into a sorghum MC backbone vector. The invention also provides for an isolated sorghum MC comprising a synthetic array of repeated nucleotide sequence constructed according to the method of the invention, and sorghum plant cells and plants comprising these MCs.
In another embodiment, the invention provides for methods of contacting a sorghum cell with a sorghum MC comprising the steps of (a) delivering the MC to immature differentiated leaves of the apical region of the stem of a sorghum plant, wherein the MC comprises a selectable marker gene, and (b) selecting the sorghum cells expressing the marker gene, wherein expression of the marker gene indicates transformation with the MC. The leaves used in this method are immature but are fully differentiated, such as the inner immature leaves of the sorghum stem. In an exemplary embodiment, the MC may be delivered by bombarding the immature leaves with micro-particles comprising the sorghum MC.
The invention also provides for methods of regenerating a sorghum plant transformed with a sorghum MC comprising the steps of (a) obtaining a callus comprising a sorghum cell that is transformed by any of the methods of the invention, and (b) growing the callus in media that may comprise 1%-3% polyvinylpyrrolidone to form a plantlet, wherein the cells of the plantlet are transformed with the sorghum MC. In a further embodiment, the methods of culturing the callus comprise growing the cells in liquid media for a time period and subsequently culturing the cells in a solid culture media. In an exemplary embodiment, the sorghum MC comprises a growth regulating gene such as a gene in the auxin biosynthesis or perception pathways. Such genes may include iaaM (Trp mono-oxygenase), iaaH (Indole-3-acetamide hydrolase), and ipt (AMP iso-pentenyl transferase). When these three genes are expressed on a MC, IaaM converts Trp into indole-3-acetamide, which IaaH converts into auxin. Ipt converts AMP into a cytokinin. The expression of all three genes allows a cultured cell to grow in the absence of exogenously supplied hormones.
SEQUENCES OF THE INVENTION
The following list indicates the identity of the SEQ ID NOs in the sequence listing:
SEQ ID NOs:1-20—promoter sequences
SEQ ID NO:21—sorghum CRS sequence
SEQ ID NO:22—sorghum consensus satellite repeat sequence
SEQ ID NOs:23-177—sorghum satellite repeat sequences
SEQ ID NO:178—previously identified sorghum CRS sequence
SEQ ID NOs:179-180—forward and reverse primers for amplifying SEQ ID NO:21 of the invention
SEQ ID NOs:181-182—forward and reverse primers for making sorghum satellite repeat-specific probes for FISH analysis
SEQ ID NOs:183-275—sorghum centromere sequence contigs from BAC 42NM (identified as CRS-positive)
SEQ ID NOs:276-326—sorghum centromere sequence contigs from BAC 89F4 (identified as satellite-positive)


DETAILED DESCRIPTION OF THE INVENTION
While this invention is susceptible of embodiment in many different forms, and will be described herein in detail, specific embodiments thereof with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated.
The invention provides novel, isolated functional, stable, autonomous MCs and recombinant chromosomes comprising centromere comprising sorghum repeat sequences including synthetic sequences. The invention also provides for “adchromosomal sorghum plants,” described in further detail herein.
One aspect of the invention is related to plants containing functional, stable, autonomous MCs or recombinant chromosomes, preferably carrying one or more exogenous nucleic acids or carrying extra copies of a nucleic acid that already exists in the plant's genome. Such plants carrying MCs or recombinant chromosomes are contrasted to transgenic plants whose genome has been altered by integrating exogenous nucleic acid transgenes into the native plant chromosomes. Preferably, expression of the exogenous nucleic acid, either constitutively or in response to a signal (which may be induced by challenge or a stimulus), e.g. or tissue specific expression, or time specific expression, results in an altered phenotype of the plant.
The invention provides for MCs or recombinant chromosomes comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 250, 500, 1000 or more exogenous nucleic acids.
The invention contemplates that sorghum plants may be used to cary the autonomous MCs as described herein. A related aspect of the invention is a plant part or plant tissue, including a pod, root, sett root, shoot root, root primordial, shoot, primary shoot, secondary shoot, tassle, panicle, arrow, midrib, blade, ligule, auricle, dewlap, blade joint, sheath, node, internode, bud furrow, leaf scar, cutting, tuber, stem, stalk, fruit, berry, nut, flower, leaf, bark, wood, epidermis, vascular tissue, organ, protoplast, crown, callus culture, petiole, petal, sepal, stamen, stigma, style, bud, meristem, cambium, cortex, pith, sheath, silk, ovule or embryo. Other exemplary plant parts are a meiocyte or gamete or ovule or pollen or endosperm of any of the preceding plants. Other exemplary plant parts are a seed, seed-piece, embryo, protoplast, cell culture, any group of plant cells organized into a structural and functional unit, ratoon or propagule of any of the preceding plants.
In one preferred embodiment, the exogenous nucleic acid is primarily expressed in a specific location or tissue of a plant, for example, stem, epidermis, vascular tissue, meristem, cambium, cortex, pith, leaf, sheath, flower, root or seed. Tissue-specific expression can be accomplished with, for example, localized presence of the MC or recombinant chromosome, selective maintenance of the MC or recombinant chromosomes, or with promoters that drive tissue-specific expression.
Another related aspect of the invention is meiocytes, pollen, ovules, endosperm, seed, somatic embryos, apomyctic embryos, embryos derived from fertilization, vegetative propagules and progeny of the originally adchromosomal plant and of its filial generations that retain the functional, stable, autonomous MC or recombinant chromosome. Such progeny include clonally propagated plants, embryos and plant parts as well as filial progeny from self- and cross-breeding, and from apomyxis.
Preferably the MC or recombinant chromosome is transmitted to subsequent generations of viable daughter cells during mitotic cell division with a transmission efficiency of at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
During meiotic division, the MC or recombinant chromosome is preferably transmitted to viable gametes with a transmission efficiency of at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% when more than one copy of the MC recombinant chromosome is present in the gamete mother cells of the plant. Preferably, the MC or recombinant chromosome is transmitted to viable gametes during meiotic cell division with a transmission frequency of at least 1%, 10%, 20%, 30%, 40%, 45%, 46%, 47%, 48%, or 49% when one copy of the MC or recombinant chromosome is present in the gamete mother cells of the plant. For production of seeds via sexual reproduction or by apomyxis the MC or recombinant chromosome is preferably transferred into at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of viable embryos when cells of the plant contain more than one copy of the MC or recombinant chromosome. For production of seeds via sexual reproduction or by apomyxis from plants with one MC or recombinant chromosome per cell, the MC or recombinant chromosome is preferably transferred into at least 1%, 10%, 20%, 30%, 40%, 45%, 46%, 47%, 48%, or 49% of viable embryos.
Preferably, a MC or recombinant chromosome that comprises an exogenous selectable trait or exogenous selectable marker can be employed to increase the frequency in subsequent generations of adchromosomal cells, tissues, gametes, embryos, endosperm, seeds, plants or progeny that comprise the MC or recombinant chromosome. More preferably, the frequency of transmission of MCs or recombinant chromosomes into viable cells, tissues, gametes, embryos, endosperm, seeds, plants or progeny can be at least 95%, 96%, 97%, 98%, 99% or 99.5% after mitosis or meiosis by applying at least one selection that favors the survival of adchromosomal cells, tissues, gametes, embryos, endosperm, seeds, plants or progeny over such cells, tissues, gametes, embryos, endosperm, seeds, plants or progeny lacking the MC or recombinant chromosome.
Transmission efficiency may be measured as the percentage of progeny cells or plants that carry the MC or recombinant chromosome as measured by one of several assays taught herein including detection of reporter gene fluorescence, PCR detection of a sequence that is carried by the MC or recombinant chromosome, RT-PCR detection of a gene transcript for a gene carried on the MC or recombinant, Western analysis of a protein produced by a gene carried on the MC or recombinant chromosome, Southern analysis of the DNA (either in total or a portion thereof) carried by the MC or recombinant chromosome, fluorescence in situ hybridization (FISH) or in situ localization by repressor binding, to name a few. Any assay used to detect the presence of the MC (or a portion of the MC) or recombinant chromosome may be used to measure the efficiency that a parental cell or plant transmits the MC or recombinant chromosome to its progeny. Efficient transmission as measured by some benchmark percentage should indicate the degree to which the MC or recombinant chromosome is stable through the mitotic and meiotic cycles.
Plants of the invention may also contain chromosomally integrated exogenous nucleic acid in addition to the autonomous MCs or recombinant chromosome. The modified plants or plant parts, including plant tissues of the invention may include plants that have chromosomal integration of some portion of the MC (e.g. exogenous nucleic acid or centromere sequences) or recombinant chromosome in some or all cells the plant. In one aspect of the invention, the autonomous MC or recombinant chromosome can be isolated from integrated exogenous nucleic acid by crossing the modified plant containing the integrated exogenous nucleic acid with plants producing some gametes lacking the integrated exogenous nucleic acid and subsequently isolating offspring of the cross, or subsequent crosses, that are modified but lack the integrated exogenous nucleic acid. This independent segregation of the MC or recombinant chromosome is one measure of the autonomous nature of the MC.
Another aspect of the invention relates to methods for producing and isolating such modified plants containing functional, stable, autonomous MCs.
In one embodiment, the invention contemplates improved methods for isolating native centromere sequences. In another embodiment, the invention contemplates methods for generating variants of native or artificial centromere sequences by passage through other host cells such are bacterial or fungal hosts.
In a further embodiment, the invention contemplates methods for delivering the MC into plant cells or tissues to transform the cells or tissues, optionally detecting MC presence or assessing MC performance, and optionally generating a plant from such cells or tissues.
Exemplary assays for assessing MC performance include lineage-based inheritance assays, use of chromosome loss agents to demonstrate autonomy, exonuclease digestion, global mitotic MC inheritance assays (sectoring assays) with or without the use of agents inducing chromosomal loss, assays measuring expression levels of genes (including marker genes) carried by the MC over time and space in a plant, physical assays for separation of autonomous MCs or recombinant chromosomes from endogenous nuclear chromosomes of plants, molecular assays demonstrating conserved MC structure, such as PCR, Southern blots, MC rescue, cloning and characterization of MC sequences present in the plant, cytological assays detecting MC presence in the cell's genome (e.g. FISH) and meiotic MC inheritance assays, which measure the levels of MC inheritance into a subsequent generation of plants via meiosis and gametes, embryos, endosperm or seeds.
Another aspect of the invention relates to methods for using such plants containing a MC or recombinant chromosome for producing food products, pharmaceutical products, biofuels and chemical products by appropriate expression of exogenous nucleic acid(s) contained within the MC(s) or recombinant chromosome(s).
Yet another aspect of the invention provides novel autonomous MCs with novel compositions and structures which are used to transform plant cells which are in turn used to generate a plant (or multiple plants). Exemplary MCs of the invention are contemplated to be of a size 2000 kb or less in length. Other exemplary sizes of MCs include less than or equal to, e.g., 1500 kb, 1000 kb, 900 kb, 800 kb, 700 kb, 600 kb, 500 kb, 450 kb, 400 kb, 350 kb, 300 kb, 250 kb, 200 kb, 150 kb, 100 kb, 80 kb, 60 kb, 40 kb, 35 kb in length. In an exemplary embodiment, the MC is about 28 kb in length, 42 kb in length, 82 kb in length, 87 kb in length, 88 kb in length, 97 kb in length, 130 kb in length, 150 kb in length, 200 kb in length or ranges from 28 kb to 200 kb in length.
In a related aspect, novel centromere compositions as characterized by sequence content, size or other parameters are provided. Preferably, the minimal size of centromeric sequence is utilized in MC construction. Exemplary sizes include a centromeric nucleic acid segment derived from a portion of plant genomic DNA or a synthesized based on a plant satellite repeat sequence, that is less than or equal to 1000 kb, 900 kb, 800 kb, 700 kb, 600 kb, 500 kb, 400 kb, 300 kb, 200 kb, 190 kb, 150 kb, 100 kb, 95 kb, 90 kb, 85 kb, 80 kb, 75 kb, 70 kb, 65 kb, 60 kb, 55 kb, 50 kb, 45 kb, 40 kb, 35 kb, 30 kb, 28 kb, 25 kb, 20 kb, 17 kb, 15 kb, 12 kb, 10 kb, 7, kb, 6.4 kb, 5 kb, or 2 kb in length. Exemplary inserts may range in size 80 kb to 100 kb, 7 kb to 190 kb, 7 kb to 12 kb, 5 kb to 10 kb, 3 kb to 10 kb, 3 kb to 7 kb, 5 kb to 7 kb, 10 to 30 kb, 15 to 30 kb, and 15 to 28 kb. Another related aspect is the novel structure of the MC, particularly structures lacking bacterial sequences, e.g., required for bacterial propagation, referred to as backbone-free MCs.
In other exemplary embodiments, the invention contemplates MCs or other vectors comprising centromeric nucleotide sequence that when hybridized to 1, 2, 3, 4, 5, 6, 7, 8 or more of the probes described in the examples herein, under hybridization conditions described herein, e.g. low, medium or high stringency, provides relative hybridization scores. Exemplary stringent hybridization conditions comprise hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C. Additional exemplary stringent hybridization conditions comprise hybridization in 0.02 M to 0.15 M NaCl at temperatures of about 50° C. to 70° C. or 0.5×SSC 0.25% SDS at 65° for 15 minutes, followed by a wash at 65° C. for a half hour or hybridization at 65° C. for 14 hours followed by 3 washings with 0.5×SSC, 1% SDS at 65° C. Probe hybridization can be scored visually to determine a binary (positive versus negative) value, or more preferably the probes can be assigned a score based on the relative strength of their hybridization on a 10 point scale. For example, relative hybridization scores of 5 may be used to select clones that hybridize well to the probe. Alternatively, a hybridization signal greater than background for one or more of these probes can be used to select clones. Modified or adchromosomal plants or plant parts containing such MCs are contemplated.
The advantages of the present invention include: provision of an autonomous, independent genetic linkage group for accelerating breeding; lack of disruption of host genome; multiple gene “stacking” of large and potentially unlimited numbers of genes; uniform genetic composition exogenous DNA sequences in plant cells and plants containing autonomous MCs; defined genetic context for predictable gene expression; higher frequency occurrence and recovery of plant cells and plants containing stably maintained exogenous DNA due to elimination of inefficient integration step. In addition, MCs that increase total recoverable sugars, or enhance the utility of modified plants for use in biofuel production are specifically envisioned.
I. Composition of MCs and MC Construction
The MC vector of the present invention may contain a variety of elements, including (1) sequences that function as plant centromeres, (2) one or more exogenous nucleic acids, including, for example, plant-expressed genes, or genes for non-coding RNAs, (3) sequences that function as an origin of replication, which may be included in the region that functions as plant centromere, (4) optionally, a bacterial plasmid backbone for propagation of the plasmid in bacteria, (5) optionally, sequences that function as plant telomeres, (6) optionally, additional “stuffer DNA” sequences that serve to physically separate the various components on the MC from each other, (7) optionally “buffer” sequences such as MARs or SARs, (8) optionally marker sequences of any origin, including but not limited to plant and bacterial origin, (9) optionally, sequences that serve as recombination sites, and (10) “chromatin packaging sequences” such as cohesion and condensing binding sites.
The MCs of the present invention may be constructed to include various components which are novel, which include, but are not limited to, the centromere comprising novel repeating centromeric sequences, as described in further detail below.
Novel Centromere Compositions
The centromere in the MC of the present invention may comprise novel repeating centromeric sequences.
Vectors comprising one, two, three, four, five, six, seven, eight, nine, ten, 15 or 20 or more of the elements contained in any of the exemplary vectors described in the examples below are also contemplated.
The invention specifically contemplates the alternative use of fragments or variants (mutants) of any of the nucleic acids described herein that retain the desired activity, including nucleic acids that function as centromeres, nucleic acids that function as promoters or other regulatory control sequences, or exogenous nucleic acids. Variants may have one or more additions, substitutions or deletions of nucleotides within the original nucleotide sequence or consensus sequence. Variants include nucleic acid sequences that are at least 50%, 55%, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the original nucleic acid sequence. Variants also include nucleic acid sequences that hybridize under low, medium, high or very high stringency conditions to the original nucleic acid sequence. Similarly, the specification also contemplates the alternative use of fragments or variants of any of the polypeptides described herein.
The comparison of sequences and determination of percent identity between two nucleotide sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453 algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix. Preferably parameters are set so as to maximize the percent identity.
As used herein, the term “hybridizes under low stringency, medium stringency, and high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology (1989) John Wiley & Sons, N.Y., 6.3.1-6.3.6, which is incorporated by reference. Aqueous and non-aqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.5×SSC, 0.1% SDS, at least at 50° C.; 2) medium stringency hybridization conditions in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 55° C.; 3) high stringency hybridization conditions are hybridization at 65° C. for 12-18 hours and washing three times for 15-90 minutes with 0.25×SSC, 0.1% SDS at 65° C. Additional exemplary stringent hybridization conditions comprise 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. Other exemplary highly selective or stringent hybridization conditions comprise 0.02 M to 0.15 M NaCl at temperatures of about 50° C. to 70° C. or 0.5×SSC 0.25% SDS at 65° for 12-15 hours, followed by three washes at 65° C. for 15-90 minutes each.
MC Sequence Content and Structure
Plant-expressed genes from non-plant sources may be modified to accommodate plant codon usage, to insert preferred motifs near the translation initiation ATG codon, to remove sequences recognized in plants as 5′ or 3′ splice sites, or to better reflect plant GC/AT content. Plant genes typically have a GC content of more than 35%, and coding sequences which are rich in A and T nucleotides can be problematic. For example, ATTTA motifs may destabilize mRNA; plant polyadenylation signals such as AATAAA at inappropriate positions within the message may cause premature truncation of transcription; and monocotyledons, such as sorghum, may recognize AT-rich sequences as splice sites.
Each exogenous nucleic acid or plant-expressed gene may include a promoter, a coding region and a terminator sequence, which may be separated from each other by restriction endonuclease sites or recombination sites or both. Genes may also include introns, which may be present in any number and at any position within the transcribed portion of the gene, including the 5′ untranslated sequence, the coding region and the 3′ untranslated sequence. Introns may be natural plant introns derived from any plant, or artificial introns based on the splice site consensus that has been defined for plant species. Some intron sequences have been shown to enhance expression in plants. Optionally the exogenous nucleic acid may include a plant transcriptional terminator, non-translated leader sequences derived from viruses that enhance expression, a minimal promoter, or a signal sequence controlling the targeting of gene products to plant compartments or organelles.
The coding regions of the genes can encode any protein, including but not limited to visible marker genes (for example, fluorescent protein genes, other genes conferring a visible phenotype to the plant) or other screenable or selectable marker genes (for example, conferring resistance to antibiotics, herbicides or other toxic compounds or encoding a protein that confers a growth advantage to the cell expressing the protein) or genes which confer some commercial or agronomic value to the modified or adchromosomal plant. Multiple genes can be placed on the same MC vector. The genes may be separated from each other by restriction endonuclease sites, homing endonuclease sites, recombination sites or any combinations thereof. Alternatively, the cloning process can be executed in a manner that destroys the intervening restriction sites. Any number of genes can be present.
The MC vector may also contain a bacterial plasmid backbone for propagation of the plasmid in bacteria such as E. coli, A. tumefaciens, or A. rhizogenes. The plasmid backbone may be that of a low-copy vector or in other embodiments it may be desirable to use a mid to high level copy backbone. In one embodiment of the invention, this backbone contains the replicon of the F′ plasmid of E. coli. However, other plasmid replicons, such as the bacteriophage P1 replicon, or other low-copy plasmid systems such as the RK2 replication origin, may also be used. The backbone may include one or several antibiotic-resistance genes conferring resistance to a specific antibiotic to the bacterial cell in which the plasmid is present. Bacterial antibiotic-resistance genes include but are not limited to kanamycin-, ampicillin-, chloramphenicol-, streptomycin-, spectinomycin-, tetracycline- and gentamycin-resistance genes.
The MC vector may also contain plant telomeres. An exemplary telomere sequence is TTTAGGG or its complement. Telomeres are specialized DNA structures at the ends of linear chromosomes that function to stabilize the ends and facilitate the complete replication of the extreme termini of the DNA molecule (Richards et al., Cell, 53:127-36, 1988; Ausubel et al., Current Protocols in Molecular Biology, Wiley & Sons, 1997).
Additionally, the MC vector may contain “stuffer DNA” sequences that serve to separate the various components on the MC (centromere, genes, telomeres) from each other. The stuffer DNA may be of any origin, prokaryotic or eukaryotic, and from any genome or species, plant, animal, microbe or organelle, or may be of synthetic origin. The stuffer DNA can range from 100 bp to 10 Mb in length and can be repetitive in sequence, with unit repeats from 10 to 1,000,000 bp. Examples of repetitive sequences that can be used as stuffer DNAs include but are not limited to: rDNA, satellite repeats, retroelements, transposons, pseudogenes, transcribed genes, microsatellites, tDNA genes, short sequence repeats and combinations thereof. Alternatively, the stuffer DNA can consist of unique, non-repetitive DNA of any origin or sequence. The stuffer sequences may also include DNA with the ability to form boundary domains, such as but not limited to scaffold attachment regions (SARs) or matrix attachment regions (MARs). The stuffer DNA may be entirely synthetic, composed of random sequence. In this case, the stuffer DNA may have any base composition, or any A/T or G/C content. For example, the G/C content of the stuffer DNA could resemble that of the plant ( ̃30-40%), or could be much lower (0-30%) or much higher (40-100%). Alternatively, the stuffer sequences could be synthesized to contain an excess of any given nucleotide such as A, C, G or T. Different synthetic stuffers of different compositions may also be combined with each other. For example a fragment with low G/C content may be flanked or abutted by a fragment of medium or high G/C content, or vice versa.
In one embodiment of the invention, the MC has a circular structure without telomeres. In another embodiment, the MC has a circular structure with telomeres. In a third embodiment, the MC has a linear structure with telomeres, as would result if a “linear” structure were to be cut with a unique endonuclease, exposing the telomeres at the ends of a DNA molecule that contains all of the sequence contained in the original, closed construct with the exception of an antibiotic-resistance gene. In a fourth embodiment of the invention, the telomeres could be placed in such a manner that the bacterial replicon, backbone sequences, antibiotic-resistance genes and any other sequences of bacterial origin and present for the purposes of propagation of the MC in bacteria, can be removed from the plant-expressed genes, the centromere, telomeres, and other sequences by cutting the structure with, for example, an unique endonuclease. This results in a MC from which much of, or preferably all, bacterial sequences have been removed. In this embodiment, bacterial sequence present between or among the plant-expressed genes or other MC sequences would be excised prior to removal of the remaining bacterial sequences by cutting the MC with an endonuclease and re-ligating the structure such that the antibiotic-resistance gene has been lost. The unique endonuclease site may be the recognition sequence of any of a number of endonucleases including but not limited to restriction endonucleases, meganucleases, or homing endonuclease. Alternatively, the endonucleases and their sites can be replaced with any specific DNA cutting mechanism and its specific recognition site such as rare-cutting endonuclease or recombinase and its specific recognition site, as long as that site is present in the MCs only at the indicated positions.
Various structural configurations are possible by which MC elements can be oriented with respect to each other. A centromere can be placed on a MC either between genes or outside a cluster of genes next to one telomere or next to the other telomere. Stuffer DNAs can be combined with these configurations to place the stuffer sequences inside the telomeres, around the centromere between genes or any combination thereof. Thus, a large number of alternative MC structures are possible, depending on the relative placement of centromere DNA, genes, stuffer DNAs, bacterial sequences, telomeres, and other sequences. The sequence content of each of these variants is the same, but their structure may be different depending on how the sequences are placed. These variations in architecture are possible both for linear and for circular MCs.
Exemplary Centromere Components
Centromere components may be isolated or derived from native plant genome, for example, modified through recombinant techniques or through the cell-based techniques described below. Alternatively, wholly artificial centromere components may be constructed using as a general guide the sequence of native centromeres such as native sorghum satellite repeat sequences. Combinations of centromere components derived from natural sources and/or combinations of naturally derived and artificial components are also contemplated. As noted above, centromere sequences from one taxonomic plant species may be functional in another taxonomic plant species, genus and family.
In one embodiment, the centromere contains n copies of a repeated nucleotide sequence obtained by the methods disclosed herein; wherein n is at least 2. In another embodiment, the centromere contains n copies of interdigitated repeats. An interdigitated repeat is a DNA sequence that consists of two distinct repetitive elements that combine to create a unique permutation. Potentially any number of repeat copies capable of physically being placed on the recombinant construct could be included on the construct, including about 5, 10, 15, 20, 30, 50, 75, 100, 150, 200, 300, 400, 500, 750, 1,000, 1,500, 2,000, 3,000, 5,000, 7,500, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 and about 100,000, including all ranges in-between such copy numbers. Moreover, the copies, while largely identical, can vary from each other. Such repeat variation is commonly observed in naturally occurring centromeres. The length of the repeat may vary, but will preferably range from about 20 bp to about 360 bp, from about 20 bp to about 250 bp, from about 50 bp to about 225 bp, from 20 bp to 137 bp, from about 75 bp to about 210 bp, such as a 92 bp repeat, a 97 bp repeat and a 100 bp repeat, from about 100 bp to about 205 bp, from about 125 bp to about 200 bp, from about 150 bp to about 195 bp, from about 160 bp to about 190 and from about 170 bp to about 185 bp including about 180 bp. Larger repeats including those up to 3,465 bp or 3,500 bp or 3,600 bp or 3,700 bp are also anticipated by the current invention.
The invention contemplates that two or more of these repeated nucleotide sequences, or similar repeated nucleotide sequences, may be oriented head to tail within the centromere. The term “head to tail” refers to multiple consecutive copies of the same or similar repeated nucleotide sequence (e.g., at least 70% identical) that are in the same 5′-3′ orientation. The invention also contemplates that two or more of these repeated nucleotide sequences may be consecutive within the centromere. The term “consecutive” refers to the same or similar repeated nucleotide sequences (e.g., at least 70% identical) that follow one after another without being interrupted by other significant sequence elements. Such consecutive repeated nucleotide sequences may be in any orientation, e.g. head to tail, tail to tail, or head to head, and may be separated by n number of nucleotides, wherein n ranges from 1 to 10, or 1 to 20, or 1 to 30, or 1 to 40, or 1 to 50. Exemplary repeated nucleotide sequences derived from sorghum are set out in SEQ ID NOs:23-176, the consensus sorghum satellite sequence (SEQ ID NO:22, and the sorghum retrotransoposon sequence (SEQ ID NO:21) or a fragment thereof.
Modification of Centromeres Isolated from Native Plant Genome
Modification and changes may be made in the centromeric DNA segments of the current invention and still obtain a functional molecule with desirable characteristics. The following is a discussion based upon changing the nucleic acids of a centromere to create an equivalent, or even an improved, second generation molecule.
In particular embodiments of the invention, mutated centromeric sequences are contemplated to be useful for increasing the utility of the centromere. It is specifically contemplated that the function of the centromeres of the current invention may be based in part or in whole upon the secondary structure of the DNA sequences of the centromere, modification of the DNA with methyl groups or other adducts, and/or the proteins which interact with the centromere. By changing the DNA sequence of the centromere, one may alter the affinity of one or more centromere-associated protein(s) for the centromere and/or the secondary structure or modification of the centromeric sequences, thereby changing the activity of the centromere. Alternatively, changes may be made in the centromeres of the invention which do not affect the activity of the centromere. Changes in the centromeric sequences which reduce the size of the DNA segment needed to confer centromere activity are contemplated to be particularly useful in the current invention, as would changes which increased the fidelity with which the centromere was transmitted during mitosis or meiosis.
Modification of Centromeres by Passage Through Bacteria, Plant or Other Hosts or Processes
In the methods of the present invention, the resulting MC DNA sequence may also be a derivative of the parental clone or centromere clone having substitutions, deletions, insertions, duplications and/or rearrangements of one or more nucleotides in the nucleic acid sequence. Such nucleotide mutations may occur individually or consecutively in stretches of 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 4000, 8000, 10000, 50000, 100000, and about 200000, including all ranges in-between.
Variations of MCs may arise through passage of MCs through various hosts including virus, bacteria, yeast, plant or other prokaryotic or eukaryotic organism and may occur through passage of multiple hosts or individual host. Variations may also occur by replicating the MC in vitro.
Derivatives may be identified through sequence analysis, or variations in MC molecular weight through electrophoresis such as, but not limited to, CHEF gel analysis, column or gradient separation, or any other methods used in the field to determine and/or analyze DNA molecular weight or sequence content. Alternately, derivatives may be identified by the altered activity of a derivative in conferring centromere function to a MC.
Production or Synthesis of Synthetic Centromere Repeat Sequences
These artificially synthesized repeated nucleotide sequences of the invention may be derived from natural centromere sequences, combinations or fragments of natural centromere sequences including a combination of repeats of different lengths, a combination of different sequences, a combination of both different repeat lengths and different sequences, a combination of different artificially synthesized sequences or a combination of natural centromere sequence(s) and artificially synthesized sequence(s). The synthetic nucleotide sequences and arrays of these synthetic repeat sequences may be generated using any technique known in the art including PCR from genomic DNA, e.g. the methods described in Example 1, or by custom polynucleotide synthesis.
Polynucleotide synthesis is the non-biological, chemical synthesis of defined sequences of nucleic acids using automated synthesizers. Oligonucleotides may be chemically synthesized, purified and then these oligonucleotides are connected by specific annealing and standard ligation or polymerase reactions. Exemplary ligation methods include ligation of phosphorylated overlapping oligonucleotides (Gupta et al. PNAS USA, 60, 1338-1344, 1993, Fuhrmann et al. Plant J. 19:353-61, 1999), the Fokl method (Mandecki et al. Gene, 68, 101-107) and a modified form of ligase chain reaction for gene synthesis. In addition, PCR assembly approaches may be used which generally employ oligonucleotides of 40-50 nt long that overlap each other. These oligonucleotides are designed to cover most of the sequence of both strands, and the full-length molecule is generated progressively by overlap extension PCR (Stemmer et al. Gene, 164, 49-53), thermodynamically balanced inside-out PCR (Gao et al. Nucleic Acids Res. 15; 31(22):e143, 2003) or combined approaches (Young et al. Nucleic Acids Res. 15; 32(7):e59, 2004).
Exemplary Exogenous Nucleic Acids Including Plant-Expressed Genes
Of particular interest in the present invention are exogenous nucleic acids which when introduced into plants will alter the phenotype of the plant, a plant organ, plant tissue, or a portion of the plant. Exemplary exogenous nucleic acids encode polypeptides. Other exemplary exogenous nucleic acids alter expression of exogenous or endogenous genes, either increasing or decreasing expression, optionally in response to a specific signal or stimulus.
As used herein, the term “trait” can refer either to the altered phenotype of interest or the nucleic acid which causes the altered phenotype of interest.
One of the major purposes of transformation of crop plants is to add some commercially desirable, agronomically important traits to the plant. Such traits include, but are not limited to, enhanced production of total recoverable sugars; utility for production of biofuels; herbicide resistance or tolerance; insect (pest) resistance or tolerance; disease resistance or tolerance (viral, bacterial, fungal, nematode or other pathogens); stress tolerance and/or resistance, as exemplified by resistance or tolerance to drought, heat, chilling, freezing, excessive moisture, salt stress, mechanical stress, extreme acidity, alkalinity, toxins, UV light, ionizing radiation or oxidative stress; increased yields, increased biomass, whether in quantity or quality; enhanced or altered nutrient acquisition and enhanced or altered metabolic efficiency; enhanced or altered nutritional content and makeup of plant tissues used for food, feed, fiber or processing; physical appearance; male sterility; drydown; standability; prolificacy; starch quantity and quality; oil quantity and quality; protein quality and quantity; amino acid composition; modified chemical production; altered pharmaceutical or nutraceutical properties; altered bioremediation properties; increased biomass; altered growth rate; altered fitness; altered biodegradability; altered CO2 fixation; presence of bioindicator activity; altered digestibility by humans or animals; altered allergenicity; altered mating characteristics; altered pollen dispersal; improved environmental impact; altered nitrogen fixation capability; the production of a pharmaceutically active protein; the production of a small molecule with medicinal properties; the production of a chemical including those with industrial utility; the production of nutraceuticals, food additives, carbohydrates, RNAs, lipids, fuels, dyes, pigments, vitamins, scents, flavors, vaccines, antibodies, hormones, and the like; and alterations in plant architecture or development, including changes in developmental timing, photosynthesis, signal transduction, cell growth, reproduction, or differentiation. Additionally one could create a library of an entire genome from any organism or organelle including mammals, plants, microbes, fungi, or bacteria, represented on MCs.
In one embodiment, the sorghum plant comprising a sorghum MC or recombinant chromosome may exhibit increased or decreased expression or accumulation of a product of the plant, which may be a natural product of the plant or a new or altered product of the plant. Exemplary products include an enzyme, an RNA molecule, a nutritional protein, a structural protein, an amino acid, a lipid, a fatty acid, a polysaccharide, a sugar, an alcohol, an alkaloid, a carotenoid, a propanoid, a phenylpropanoid, or terpenoid, a steroid, a flavonoid, a phenolic compound, an anthocyanin, a pigment, a vitamin or a plant hormone. In another embodiment, the sorghum plant comprising a sorghum MC or recombinant chromosome has enhanced or diminished requirements for light, water, nitrogen, or trace elements. In another embodiment the sorghum plant comprising a sorghum MC or recombinant chromosome has an enhanced ability to capture or fix nitrogen from its environment. In yet another embodiment, the sorghum plant comprising a sorghum MC or recombinant chromosome is enriched for an essential amino acid as a proportion of a protein fraction of the plant. The protein fraction may be, for example, total seed protein, soluble protein, insoluble protein, water-extractable protein, and lipid-associated protein. The sorghum plant comprising a sorghum MC or recombinant chromosome may include genes that cause the overexpression, underexpression, antisense modulation, sense suppression, inducible expression, inducible repression, or inducible modulation of another gene.
A brief summary of exemplary improved properties and polypeptides of interest for either increased or decreased expression is provided below.
(i) Herbicide Resistance
A herbicide resistance (or tolerance) trait is a characteristic of a sorghum plant comprising a sorghum MC or recombinant chromosome that is resistant to dosages of an herbicide that is typically lethal to a wild type plant. Exemplary herbicides for which resistance is useful in a plant include glyphosate herbicides, phosphinothricin herbicides, oxynil herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and glufosinate herbicides. Other herbicides would be useful as would combinations of herbicide genes on the same MC or recombinant chromosome.
The genes encoding phosphinothricin acetyltransferase (bar), glyphosate tolerant EPSP synthase genes, glyphosate acetyltransferase, the glyphosate degradative enzyme gene gox encoding glyphosate oxidoreductase, deh (encoding a dehalogenase enzyme that inactivates dalapon), herbicide resistant (e.g., sulfonylurea and imidazolinone) acetolactate synthase, and bxn genes (encoding a nitrilase enzyme that degrades bromoxynil) are good examples of herbicide resistant genes for use in transformation. The bar gene codes for an enzyme, phosphinothricin acetyltransferase (PAT), which inactivates the herbicide phosphinothricin and prevents this compound from inhibiting glutamine synthetase enzymes. The enzyme 5 enolpyruvylshikimate 3 phosphate synthase (EPSP Synthase), is normally inhibited by the herbicide N (phosphonomethyl)glycine (glyphosate). However, genes are known that encode glyphosate resistant EPSP synthase enzymes. These genes are particularly contemplated for use in plant transformation. The deh gene encodes the enzyme dalapon dehalogenase and confers resistance to the herbicide dalapon. The bxn gene codes for a specific nitrilase enzyme that converts bromoxynil to a non herbicidal degradation product. The glyphosate acetyl transferase gene inactivates the herbicide glyphosate and prevents this compound from inhibiting EPSP synthase.
Polypeptides that may produce plants having tolerance to plant herbicides include polypeptides involved in the shikimate pathway, which are of interest for providing glyphosate tolerant plants. Such polypeptides include polypeptides involved in biosynthesis of chorismate, phenylalanine, tyrosine and tryptophan.
(ii) Insect Resistance
Potential insect resistance (or tolerance) genes that can be introduced include Bacillus thuringiensis toxin genes or Bt genes (Watrud et al., In: Engineered Organisms and the Environment, 1985). Bt genes may provide resistance to lepidopteran or coleopteran pests such as European Corn Borer (ECB). Preferred Bt toxin genes for use in such embodiments include the CrylA(b) and CrylA(c) genes. Endotoxin genes from other species of B. thuringiensis which affect insect growth or development also may be employed in this regard.
It is contemplated that preferred Bt genes for use in the MCs or recombinant chromosomes disclosed herein will be those in which the coding sequence has been modified to effect increased expression in plants, and for example, in monocot plants including sorghum. Means for preparing synthetic genes are well known in the art and are disclosed in, for example, U.S. Pat. No. 5,500,365 and U.S. Pat. No. 5,689,052, each of the disclosures of which are specifically incorporated herein by reference in their entirety. Examples of such modified Bt toxin genes include a synthetic Bt CrylA(b) gene (Perlak et al., PNAS USA, 88:3324-3328, 1991), and the synthetic CrylA(c) gene termed 1800b (PCT Application WO 95/06128). Some examples of other Bt toxin genes known to those of skill in the art are given in Table 1 below.







TABLE 1







Bacillus thuringiensis Endotoxin Genesa











New 
Old 
GenBank 



Nomenclature
Nomenclature
Accession







Cry1Aa
CryIA(a)
M11250



Cry1Ab
CryIA(b)
M13898



Cry1Ac
CryIA(c)
M11068



Cry1Ad
CryIA(d)
M73250



Cry1Ae
CryIA(e)
M65252



Cry1Ba
CryIB
X06711



Cry1Bb
ET5
L32020



Cry1Bc
PEG5
Z46442



Cry1Bd
CryE1
U70726



Cry1Ca
CryIC
X07518



Cry1Cb
CryIC(b)
M97880



Cry1Da
CryID
X54160



Cry1Db
PrtB
Z22511



Cry1Ea
CryIE
X53985



Cry1Eb
CryIE(b)
M73253



Cry1Fa
CryIF
M63897



Cry1Fb
PrtD
Z22512



Cry1Ga
PrtA
Z22510



Cry1Gb
CryH2
U70725



Cry1Ha
PrtC
Z22513



Cry1Hb

U35780



Cry1Ia
CryV
X62821



Cry1Ib
CryV
U07642



Cry1Ja
ET4
L32019



Cry1Jb
ET1
U31527



Cry1K

U28801



Cry2Aa
CryIIA
M31738



Cry2Ab
CryIIB
M23724



Cry2Ac
CryIIC
X57252



Cry3A
CryIIIA
M22472



Cry3Ba
CryIIIB
X17123



Cry3Bb
CryIIIB2
M89794



Cry3C
CryIIID
X59797



Cry4A
CryIVA
Y00423



Cry4B
CryIVB
X07423



Cry5Aa
CryVA(a)
L07025



Cry5Ab
CryVA(b)
L07026



Cry6A
CryVIA
L07022



Cry6B
CryVIB
L07024



Cry7Aa
CryIIIC
M64478



Cry7Ab
CryIIICb
U04367



Cry8A
CryIIIE
U04364



Cry8B
CryIIIG
U04365



Cry8C
CryIIIF
U04366



Cry9A
CryIG
X58120



Cry9B
CryIX
X75019



Cry9C
CryIH
Z37527



Cry10A
CryIVC
M12662



Cry11A
CryIVD
M31737



Cry11B
Jeg80
X86902



Cry12A
CryVB
L07027



Cry13A
CryVC
L07023



Cry14A
CryVD
U13955



Cry15A
34kDa
M76442



Cry16A
cbm71
X94146



Cry17A
cbm71
X99478



Cry18A
CryBP1
X99049



Cry19A
Jeg65
Y08920



Cyt1Aa
CytA
X03182



Cyt1Ab
CytM
X98793



Cyt2A
CytB
Z14147



Cyt2B
CytB
U52043







aAdapted from Crickmore, N, Zeigler, DR, Feitelson, J. et aL. Microbiol. Molec. Biol. Rev. 62:807-813, 1998.






Protease inhibitors also may provide insect resistance (Johnson et al., PNAS USA 86): 9871-9875, 1989), and will thus have utility in plant transformation. The use of a pinII gene in combination with a Bt toxin gene, the combined effect of which has been discovered to produce synergistic insecticidal activity is envisioned to be particularly useful. Other genes which encode inhibitors of the insect's digestive system, or those that encode enzymes or co factors that facilitate the production of inhibitors, also may be useful. This group may be exemplified by oryzacystatin and amylase inhibitors such as those from wheat and barley.
Amylase inhibitors are found in various plant species and are used to ward off insect predation via inhibition of the digestive amylases of attacking insects. Several amylase inhibitor genes have been isolated from plants and some have been introduced as exogenous nucleic acids, conferring an insect resistant phenotype that is potentially useful (Chrispeels, M J and D E Sadava, Plants, Genes, and Crop Biotechnology Jones and Bartlett Press, 2003).
Genes encoding lectins may confer additional or alternative insecticide properties. Lectins are multivalent carbohydrate binding proteins which have the ability to agglutinate red blood cells from a range of species. Lectins have been identified recently as insecticidal agents with activity against weevils, ECB and rootworm (Murdock et al., Phytochemistry, 29:85-89, 1990, Czapla & Lang, J. Econ. Entomol., 83:2480-2485, 1990). Lectin genes contemplated to be useful include, for example, barley and wheat germ agglutinin (WGA) and rice lectins (Gatehouse et al., J. Sci. Food. Agric., 35:373-380, 1984), with WGA being preferred.
Genes controlling the production of large or small polypeptides active against insects when introduced into the insect pests, such as, e.g., lytic peptides, peptide hormones and toxins and venoms, form another aspect of the invention. For example, it is contemplated that the expression of juvenile hormone esterase, directed towards specific insect pests, also may result in insecticidal activity, or perhaps cause cessation of metamorphosis (Hammock et al., Nature, 344:458-461, 1990).
Genes that encode enzymes that affect the integrity of the insect cuticle form yet another aspect of the invention. Such genes include those encoding, e.g., chitinase, proteases, lipases and also genes for the production of nikkomycin, a compound that inhibits chitin synthesis, the introduction of any of which is contemplated to produce insect resistant plants. Genes that code for activities that affect insect molting, such as those affecting the production of ecdysteroid UDP glucosyl transferase, also fall within the scope of the useful exogenous nucleic acids of the present invention.
Genes that code for enzymes that facilitate the production of compounds that reduce the nutritional quality of the host plant to insect pests also are encompassed by the present invention. It may be possible, for instance, to confer insecticidal activity on a plant by altering its sterol composition. Sterols are obtained by insects from their diet and are used for hormone synthesis and membrane stability. Therefore alterations in plant sterol composition by expression of novel genes, e.g., those that directly promote the production of undesirable sterols or those that convert desirable sterols into undesirable forms, could have a negative effect on insect growth and/or development and hence endow the plant with insecticidal activity. Lipoxygenases are naturally occurring plant enzymes that have been shown to exhibit anti nutritional effects on insects and to reduce the nutritional quality of their diet. Therefore, further embodiments of the invention concern modified plants with enhanced lipoxygenase activity which may be resistant to insect feeding.
Tripsacum dactyloides is a species of grass that is resistant to certain insects, including root worm. It is anticipated that genes encoding proteins that are toxic to insects or are involved in the biosynthesis of compounds toxic to insects will be isolated from Tripsacum and that these novel genes will be useful in conferring resistance to insects. It is known that the basis of insect resistance in Tripsacum is genetic, because said resistance has been transferred to Zea mays via sexual crosses (Branson and Guss, Proceedings North Central Branch Entomological Society of America, 27:91-95, 1972). It is further anticipated that other cereal, monocot or dicot plant species may have genes encoding proteins that are toxic to insects which would be useful for producing insect resistant plants.
Further genes encoding proteins characterized as having potential insecticidal activity also may be used as exogenous nucleic acids in accordance herewith. Such genes include, for example, the cowpea trypsin inhibitor (CpTI; Hilder et al., Nature, 330:160-163, 1987) which may be used as a rootworm deterrent; genes encoding avermectin (Avermectin and Abamectin; Campbell, W. C., Ed., 1989; Ikeda et al., J. Bacteriol., 169:5615-5621, 1987) which may prove particularly useful as a corn rootworm deterrent; ribosome inactivating protein genes; and even genes that regulate plant structures. Sorghum plant comprising a sorghum MC or recombinant chromosome comprising anti insect antibody genes and genes that code for enzymes that can convert a non toxic insecticide (pro insecticide) applied to the outside of the plant into an insecticide inside the plant also are contemplated.
Polypeptides that may improve plant tolerance to the effects of plant pests or pathogens include proteases, polypeptides involved in anthocyanin biosynthesis, polypeptides involved in cell wall metabolism, including cellulases, glucosidases, pectin methylesterase, pectinase, polygalacturonase, chitinase, chitosanase, and cellulose synthase, and polypeptides involved in biosynthesis of terpenoids or indole for production of bioactive metabolites to provide defense against herbivorous insects. It is also anticipated that combinations of different insect resistance genes on the same MC or recombinant chromosomes will be particularly useful.
Vegetative Insecticidal Proteins (VIP) are another class of proteins originally found to be produced in the vegetative growth phase of the bacterium, Bacillus cereus, but do have a spectrum of insect lethality similar to the insecticidal genes found in strains of Bacillus thuriengensis. Both the vip1a and vip3A genes have been isolated and have demonstrated insect toxicity. It is anticipated that such genes may be used in modified plants to confer insect resistance (“Plants, Genes, and Crop Biotechnology” by Maarten J. Chrispeels and David E. Sadava (2003) Jones and Bartlett Press).
(iii) Environment or Stress Resistance
Improvement of a plant's ability to tolerate various environmental stresses such as, but not limited to, drought, excess moisture, chilling, freezing, high temperature, salt, and oxidative stress, also can be affected through expression of novel genes. It is proposed that benefits may be realized in terms of increased resistance to freezing temperatures through the introduction of an “antifreeze” protein such as that of the Winter Flounder (Cutler et al., J. Plant Physiol., 135:351-354, 1989) or synthetic gene derivatives thereof. Improved chilling tolerance also may be conferred through increased expression of glycerol 3 phosphate acetyltransferase in chloroplasts (Wolter et al., EMBO J., 4685-4692, 1992). Resistance to oxidative stress (often exacerbated by conditions such as chilling temperatures in combination with high light intensities) can be conferred by expression of superoxide dismutase (Gupta et al., 1993), and may be improved by glutathione reductase (Bowler et al., Ann Rev. Plant Physiol., 43:83-116, 1992). Such strategies may allow for tolerance to freezing in newly emerged fields as well as extending later maturity higher yielding varieties to earlier relative maturity zones. Many sorghum genes are known to be modulated by various stresses including drought stress, salt stress and the application of stress response hormones (Buchanan C D, et al. Plant Mol. Biol. 58:699-720, 2005) and (Srinivas G, et al. Theor Appl Genet. 118:703-17, 2009).
It is contemplated that the expression of novel genes that favorably affect plant water content, total water potential, osmotic potential, or turgor will enhance the ability of the plant to tolerate drought. As used herein, the terms “drought resistance” and “drought tolerance” are used to refer to a plant's increased resistance or tolerance to stress induced by a reduction in water availability, as compared to normal circumstances, and the ability of the plant to function and survive in lower water environments. In this aspect of the invention it is proposed, for example, that the expression of genes encoding for the biosynthesis of osmotically active solutes, such as polyol compounds, may impart protection against drought. Within this class are genes encoding for mannitol L phosphate dehydrogenase (Lee and Saier, PNAS USA 78:7336-7340, 1981) and trehalose 6 phosphate synthase (Kaasen et al., J. Bacteriology, 174:889-898, 1992). Through the subsequent action of native phosphatases in the cell or by the introduction and coexpression of a specific phosphatase, these introduced genes will result in the accumulation of either mannitol or trehalose, respectively, both of which have been well documented as protective compounds able to mitigate the effects of stress. Mannitol accumulation in transgenic tobacco has been verified and preliminary results indicate that plants expressing high levels of this metabolite are able to tolerate an applied osmotic stress (Tarczynski et al., Science, 259:508-510, 1993, Tarczynski et al. PNAS USA, 89:1-5, 1993).
Similarly, the efficacy of other metabolites in protecting either enzyme function (e.g., alanopine or propionic acid) or membrane integrity (e.g., alanopine) has been documented (Loomis et al., J. Expt. Zoology 252:9-15, 1989), and therefore expression of genes encoding for the biosynthesis of these compounds might confer drought resistance in a manner similar to or complimentary to mannitol. Other examples of naturally occurring metabolites that are osmotically active and/or provide some direct protective effect during drought and/or desiccation include fructose, erythritol (Coxson et al., Biotropica 24:121-133, 1992), sorbitol, dulcitol (Karsten et al., Botanica Marina 35:11-19, 1992), glucosylglycerol (Reed et al., J. Gen. Microbiol. 130:1-4, 1984; Erdmann et al., J. Gen. Microbiol. 138:363-368, 1992), sucrose, stachyose (Koster and Leopold, Plant Physiol. 88:829-832, 1988; Blackman et al., Plant Physiol. 100:225-230, 1992), raffinose (Lugo and Leopold, Plant Physiol. 98:1207-1210, 1992), proline (Rensburg et al., J. Plant Physiol. 141:188-194, 1993), glycine betaine, ononitol and pinitol (Vernon and Bohnert, EMBO J. 11:2077-2085, 1992). Continued growth and increased reproductive fitness during times of stress may be augmented by introduction and expression of genes such as those controlling the osmotically active compounds discussed above and other such compounds. Currently preferred genes which promote the synthesis of an osmotically active polyol compound are genes which encode the enzymes mannitol 1 phosphate dehydrogenase, trehalose 6 phosphate synthase and myoinositol 0 methyltransferase.
It is contemplated that the expression of specific proteins also may increase drought tolerance. Three classes of Late Embryogenic Abundant (LEA) Proteins have been assigned based on structural similarities (see Dure et al., Plant Molec. Biol. 12:475-486, 1989). All three classes of LEAs have been demonstrated in maturing (e.g. desiccating) seeds. Within these 3 types of LEA proteins, the Type II (dehydrin type) have generally been implicated in drought and/or desiccation tolerance in vegetative plant parts (e.g. Mundy and Chua, EMBO J., 7:2279-2286, 1988; Piatkowski et al., Plant Physiol. 94:1682-1688, 1990; Yamaguchi Shinozaki et al., Plant Cell Physiol. 33:217-224, 1992). Expression of a Type III LEA (HVA 1) in tobacco was found to influence plant height, maturity and drought tolerance (Fitzpatrick, Gen. Engineering News 22:7, 1993). In rice, expression of the HVA 1 gene influenced tolerance to water deficit and salinity (Xu et al., Plant Physiol. 110:249-257, 1996). Expression of structural genes from any of the three LEA groups may therefore confer drought tolerance. Other types of proteins induced during water stress include thiol proteases, aldolases or transmembrane transporters (Guerrero et al., Plant Molecul. Biol. 15:11-26, 1990), which may confer various protective and/or repair type functions during drought stress. It also is contemplated that genes that effect lipid biosynthesis and hence membrane composition might also be useful in conferring drought resistance on the plant.
Many of these genes for improving drought resistance have complementary modes of action. Thus, it is envisaged that combinations of these genes might have additive and/or synergistic effects in improving drought resistance in plants. Many of these genes also improve freezing tolerance (or resistance); the physical stresses incurred during freezing and drought are similar in nature and may be mitigated in similar fashion. Benefits may be conferred via constitutive expression of these genes, but the preferred means of expressing these novel genes may be through the use of a turgor induced promoter (such as the promoters for the turgor induced genes described in Guerrero et al., Plant Molecul. Biol. 15:11-26, 1990 and Shagan et al., Plant Physiol. 101:1397-1398, 1993 which are incorporated herein by reference). Spatial and temporal expression patterns of these genes may enable plants to better withstand stress.
It is proposed that expression of genes that are involved with specific morphological traits that allow for increased water extractions from drying soil would be of benefit. For example, introduction and expression of genes that alter root characteristics may enhance water uptake. It also is contemplated that expression of genes that enhance reproductive fitness during times of stress would be of significant value. For example, expression of genes that improve the synchrony of pollen shed and receptiveness of the female flower parts, e.g., silks, would be of benefit. In addition it is proposed that expression of genes that minimize kernel abortion during times of stress would increase the amount of grain to be harvested and hence be of value.
Given the overall role of water in determining yield, it is contemplated that enabling plants to utilize water more efficiently, through the introduction and expression of novel genes, will improve overall performance even when soil water availability is not limiting. By introducing genes that improve the ability of plants to maximize water usage across a full range of stresses relating to water availability, yield stability or consistency of yield performance may be realized.
Polypeptides that may improve stress tolerance under a variety of stress conditions include polypeptides involved in gene regulation, such as serine/threonine-protein kinases, MAP kinases, MAP kinase kinases, and MAP kinase kinase kinases; polypeptides that act as receptors for signal transduction and regulation, such as receptor protein kinases; intracellular signaling proteins, such as protein phosphatases, GTP binding proteins, and phospholipid signaling proteins; polypeptides involved in arginine biosynthesis; polypeptides involved in ATP metabolism, including for example ATPase, adenylate transporters, and polypeptides involved in ATP synthesis and transport; polypeptides involved in glycine betaine, jasmonic acid, flavonoid or steroid biosynthesis; and hemoglobin. Enhanced or reduced activity of such polypeptides in modified plants will provide changes in the ability of a plant to respond to a variety of environmental stresses, such as chemical stress, drought stress and pest stress.
Other polypeptides that may improve plant tolerance to cold or freezing temperatures include polypeptides involved in biosynthesis of trehalose or raffinose, polypeptides encoded by cold induced genes, fatty acyl desaturases and other polypeptides involved in glycerolipid or membrane lipid biosynthesis, which find use in modification of membrane fatty acid composition, alternative oxidase, calcium-dependent protein kinases, LEA proteins or uncoupling protein.
Other polypeptides that may improve plant tolerance to heat include polypeptides involved in biosynthesis of trehalose, polypeptides involved in glycerolipid biosynthesis or membrane lipid metabolism (for altering membrane fatty acid composition), heat shock proteins or mitochondrial NDK.
Other polypeptides that may improve tolerance to extreme osmotic conditions include polypeptides involved in proline biosynthesis.
Other polypeptides that may improve plant tolerance to drought conditions include aquaporins, polypeptides involved in biosynthesis of trehalose or wax, LEA proteins or invertase.
(iv) Disease Resistance
It is proposed that increased resistance (or tolerance) to diseases may be realized through introduction of genes into plants, for example, into monocotyledonous plants such as sorghum. It is possible to produce resistance to diseases caused by viruses, viroids, bacteria, fungi and nematodes. It also is contemplated that control of mycotoxin producing organisms may be realized through expression of introduced genes. Resistance can be affected through suppression of endogenous factors that encourage disease-causing interactions, expression of exogenous factors that are toxic to or otherwise provide protection from pathogens, or expression of factors that enhance the plant's own defense responses.
Resistance to viruses may be produced through expression of novel genes. For example, it has been demonstrated that expression of a viral coat protein in a modified plant can impart resistance to infection of the plant by that virus and perhaps other closely related viruses (Hemenway et al., EMBO J. 7:1273-1280, 1988, Abel et al., Science 232:738-743, 1986). It is contemplated that expression of antisense genes targeted at essential viral functions may also impart resistance to viruses. For example, an antisense gene targeted at the gene responsible for replication of viral nucleic acid may inhibit replication and lead to resistance to the virus. It is believed that interference with other viral functions through the use of antisense genes also may increase resistance to viruses. Further, it is proposed that it may be possible to achieve resistance to viruses through other approaches, including, but not limited to the use of satellite viruses.
It is proposed that increased resistance to diseases caused by bacteria and fungi may be realized through introduction of novel genes. It is contemplated that genes encoding so called “peptide antibiotics,” pathogenesis related (PR) proteins, toxin resistance, or proteins affecting host pathogen interactions such as morphological characteristics will be useful. Peptide antibiotics are polypeptide sequences which are inhibitory to growth of bacteria and other microorganisms. For example, the classes of peptides referred to as cecropins and magainins inhibit growth of many species of bacteria and fungi. It is proposed that expression of PR proteins in plants, for example, monocots such as sorghum, may be useful in conferring resistance to bacterial disease. These genes are induced following pathogen attack on a host plant and have been divided into at least five classes of proteins (Bol et al. Annu. Rev. Pytopathol. 28:113-138, 1990). Included amongst the PR proteins are beta 1, 3 glucanases, chitinases, and osmotin and other proteins that are believed to function in plant resistance to disease organisms. Other genes have been identified that have antifungal properties, e.g., UDA (stinging nettle lectin), or hevein (Broakaert et al., PNAS USA 87:7633-7, 1989; Barkai Golan et al., Arch. Microbiol. 116:119-121, 1978). It is known that certain plant diseases are caused by the production of phytotoxins. It is proposed that resistance to these diseases would be achieved through expression of a novel gene that encodes an enzyme capable of degrading or otherwise inactivating the phytotoxin. It also is contemplated that expression of novel genes that alter the interactions between the host plant and pathogen may be useful in reducing the ability of the disease organism to invade the tissues of the host plant, e.g., an increase in the waxiness of the leaf cuticle or other morphological characteristics.
Polypeptides useful for imparting improved disease responses to plants include polypeptides encoded by cercosporin-induced genes, antifungal proteins and proteins encoded by R-genes or SAR genes.
Agronomically important diseases in sorghum include but are not limited to Exserohilum turcicum, Colletotrichum graminicola (Glomerella graminicola), Cercospora sorghi, Gloeocercospora soghi, Ascochyta sorghi, Pseudomonas syringae p.v. syringae, Xanthomonas campestris p.v. holcicola, Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Periconia circinata, FUSArium moniliforme, Alternaria alternate, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans), Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari Sporisorium relianum (Sphacelotheca reliana), Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis, Sclerospora graminicola, FUSArium graminearum, FUSArium Oxysporum, Pythium arrhenomanes, and Pythium graminicola. 
(v) Plant Agronomic Characteristics
Temperature also influences where crop plants can be grown. Within the areas where it is possible to grow a particular crop, there are varying limitations on the maximal time it is allowed to grow to maturity and be harvested. For example, a variety to be grown in a particular area is selected for its ability to mature within the required period of time with maximum possible yield. It is considered that genes that influence maturity can be identified and introduced into plant lines to create new varieties adapted to different growing locations or the same growing location, but having improved yield at harvest. Expression of genes that are involved in regulation of plant development may be especially useful.
It is contemplated that genes may be introduced into plants that would improve standability and other plant growth characteristics. Expression of novel genes in plants which confer stronger stalks, improved root systems, or prevent or reduce ear droppage or shattering would be of great value to the farmer. It is proposed that introduction and expression of genes that increase the total amount of photoassimilate available by, for example, increasing light distribution and/or interception would be advantageous. In addition, the expression of genes that increase the efficiency of photosynthesis and/or the leaf canopy would further increase gains in productivity. It is contemplated that expression of a phytochrome gene in crop plants may be advantageous. Expression of such a gene may reduce apical dominance, confer semidwarfism on a plant, or increase shade tolerance (U.S. Pat. No. 5,268,526). Such approaches would allow for increased plant populations in the field.
(vi) Nutrient Utilization
The ability to utilize available nutrients may be a limiting factor in growth of crop plants. It is proposed that it would be possible to alter nutrient uptake, tolerate pH extremes, mobilization through the plant, storage pools, and availability for metabolic activities by the introduction of novel genes. These modifications would allow a plant, for example, sorghum to more efficiently utilize available nutrients. It is contemplated that an increase in the activity of, for example, an enzyme that is normally present in the plant and involved in nutrient utilization would increase the availability of a nutrient or decrease the availability of an antinutritive factor. An example of such an enzyme would be phytase. It is further contemplated that enhanced nitrogen utilization by a plant is desirable. Expression of a glutamate dehydrogenase gene in plants, e.g., E. coli gdhA genes, may lead to increased fixation of nitrogen in organic compounds. Furthermore, expression of gdhA in plants may lead to enhanced resistance to the herbicide glufosinate by incorporation of excess ammonia into glutamate, thereby detoxifying the ammonia. It also is contemplated that expression of a novel gene may make a nutrient source available that was previously not accessible, e.g., an enzyme that releases a component of nutrient value from a more complex molecule, perhaps a macromolecule.
Polypeptides useful for improving nitrogen flow, sensing, uptake, storage and/or transport include those involved in aspartate, glutamine or glutamate biosynthesis, polypeptides involved in aspartate, glutamine or glutamate transport, polypeptides associated with the TOR (Target of Rapamycin) pathway, nitrate transporters, nitrate reductases, amino transferases, ammonium transporters, chlorate transporters or polypeptides involved in tetrapyrrole biosynthesis.
Polypeptides useful for increasing the rate of photosynthesis include phytochrome, ribulose bisphosphate carboxylase-oxygenase, Rubisco activase, photosystem I and II proteins, electron carriers, ATP synthase, NADH dehydrogenase or cytochrome oxidase.
Polypeptides useful for increasing phosphorus uptake, transport or utilization include phosphatases or phosphate transporters.
(vii) Male Sterility
Male sterility is useful in the production of hybrid varieties of sorghum. It is proposed that male sterility may be produced through expression of novel genes. For example, it has been shown that expression of genes that encode proteins, RNAs, or peptides that interfere with development of the male inflorescence and/or gametophyte result in male sterility. Chimeric ribonuclease genes that express in the anthers of transgenic tobacco and oilseed rape have been demonstrated to lead to male sterility (Mariani et al., Nature, 347:737-741, 1990).
A number of mutations were discovered in maize that confer cytoplasmic male sterility. One mutation in particular, referred to as T cytoplasm, also correlates with sensitivity to Southern corn leaf blight. A DNA sequence, designated TURF 13 (Levings, Science, 250:942-947, 1990), was identified that correlates with T cytoplasm. It is proposed that it would be possible through the introduction of TURF 13 via transformation, to separate male sterility from disease sensitivity. As it is necessary to be able to restore male fertility for breeding purposes and for grain production, it is proposed that genes encoding restoration of male fertility also may be introduced.
Male sterility systems have also been described in sorghum. These include cytoplasmic male sterility (Van Tang H, et al. Curr Genet. 29:265-74, 1996), gametophytic male sterility (Pring D R, et al. J. Hered. 90:386-93, 1999), and nuclear male sterility (J. F. Pedersen and J. J. Toy, Crop Science 41:607, 2001).
(viii) Altered Nutritional Content
Genes can be introduced into plants to improve or alter the nutrient quality or content. Introduction of genes that alter the nutrient composition of a crop can greatly enhance the feed, food or forage value. Limiting essential amino acids can include lysine, methionine, tryptophan, threonine, valine, arginine, and histidine. The levels of these essential amino acids can be elevated by mechanisms which include, but are not limited to, the introduction of genes to increase the biosynthesis of the amino acids, decrease the degradation of the amino acids, increase the storage of the amino acids in proteins, or increase transport of the amino acids to particular tissues.
Polypeptides useful for providing increased protein quantity and/or quality include polypeptides involved in the metabolism of amino acids in plants, particularly polypeptides involved in biosynthesis of methionine/cysteine and lysine, amino acid transporters, amino acid efflux carriers, seed storage proteins, proteases, or polypeptides involved in phytic acid metabolism.
The protein composition of a crop can be altered to improve the balance of amino acids in a variety of ways including elevating expression of native proteins, decreasing expression of those with poor composition, changing the composition of native proteins, or introducing genes encoding entirely new proteins possessing superior composition.
The introduction of genes that alter the oil content of a crop plant can also be of value. Increases in oil content can result in increases in metabolizable-energy-content. The introduced genes can encode enzymes that remove or reduce rate-limitations or regulated steps in fatty acid or lipid biosynthesis. Such genes can include, but are not limited to, those that encode acetyl-CoA carboxylase, ACP-acyltransferase, alpha-ketoacyl-ACP synthase, or other well known fatty acid biosynthetic activities. Other possibilities are genes that encode proteins that do not possess enzymatic activity such as acyl carrier protein. Genes can be introduced that alter the balance of fatty acids present in the oil providing a more healthful or nutritive feedstuff. The introduced DNA also can encode sequences that block expression of enzymes involved in fatty acid biosynthesis, altering the proportions of fatty acids present in crops.
Genes can be introduced that enhance the nutritive value of crops, or of foods derived from crops by increasing the level of naturally occurring phytosterols, or by encoding for proteins to enable the synthesis of phytosterols in crops. The phytosterols from these crops can be processed directly into foods, or extracted and used to manufacture food products.
Genes can be introduced that enhance the nutritive value or energy value of the starch component of crops, for example by altering increasing the degree of branching of starch molecules, resulting in improved utilization of the starch in biofuel feedstock applications. Additionally, other major constituents of a crop can be altered, including genes that affect a variety of other nutritive, processing, or other quality aspects. For example, pigmentation can be increased or decreased.
Carbohydrate metabolism can be altered, for example by increased sucrose production and/or transport. Polypeptides useful for affecting on carbohydrate metabolism include polypeptides involved in sucrose or starch metabolism, carbon assimilation or carbohydrate transport, including, for example sucrose transporters or glucose/hexose transporters, enzymes involved in glycolysis/gluconeogenesis, the pentose phosphate cycle, or raffinose biosynthesis, or polypeptides involved in glucose signaling, such as SNF1 complex proteins.
Feed or food crops can also possess sub-optimal quantities of vitamins, antioxidants or other nutraceuticals, requiring supplementation to provide adequate nutritive value and ideal health value. Introduction of genes that enhance vitamin biosynthesis can be envisioned including, for example, vitamins A, E, B12, choline, or the like. Mineral content can also be sub-optimal. Thus genes that affect the accumulation or availability of compounds containing phosphorus, sulfur, calcium, manganese, zinc, or iron among others would be valuable.
Numerous other examples of improvements of crops can be used with the invention. Introduction of DNA to accomplish this might include sequences that alter lignin production such as those that result in the “brown midrib” phenotype associated with superior feed value for cattle. Other genes can encode for enzymes that alter the structure of extracellular carbohydrates, or that facilitate the degradation of the carbohydrates so that it can be efficiently fermented into ethanol or other useful carbohydrates.
It can be desirable to modify the nutritional content of plants by reducing undesirable components such as fats, starches, etc. This can be done, for example, by the use of exogenous nucleic acids that encode enzymes which increase plant use or metabolism of such components so that they are present at lower quantities. Alternatively, it can be done by use of exogenous nucleic acids that reduce expression levels or activity of native plant enzymes that synthesize such components.
Likewise the elimination of certain undesirable traits can improve the food or feed value of the crop. Many undesirable traits must currently be eliminated by special post-harvest processing steps and the degree to which these can be engineered into the plant prior to harvest and processing would provide significant value. Examples of such traits are the elimination of anti-nutritionals such as phytates and phenolic compounds which are commonly found in many crop species. Also, the reduction of fats, carbohydrates and certain phytohormones can be valuable for the food and feed industries as they can allow a more efficient mechanism to meet specific dietary requirements.
In addition to direct improvements in feed or food value, genes also can be introduced which improve the processing of crops and improve the value of the products resulting from the processing. Novel genes that increase the efficiency and reduce the cost of such processing, for example by decreasing the time required at a particular step, can also find use. Improving the value of products derived from processed plants, such as sorghum, can include altering the quantity or quality of sugar, starch, oil, fiber, gluten, or the components. Elevation of sugar or starch can be achieved through the identification and elimination of rate limiting steps in sugar and starch and sugar biosynthesis by expressing increased amounts of enzymes involved in biosynthesis or by decreasing levels of the other components of crops resulting in proportional increases in sugar or starch. In addition, plants can be modified by introducing or expressing a gene or genes that produce novel products, such as secondary plant metabolites or pharmaceutical products, which can be purified during the processing step. Using MCs or recombinant chromosomes to both introduce genes for new products and optionally for improving processing steps could provide a cost effective option to produce these novel products.
Oil is another product of processing, the value of which can be improved by introduction and expression of genes. Oil properties can be altered to improve its performance in the production and use of cooking oil, shortenings, lubricants or other oil-derived products or improvement of its health attributes when used in the food-related applications. Novel fatty acids also can be synthesized which upon extraction can serve as starting materials for chemical syntheses. The changes in oil properties can be achieved by altering the type, level, or lipid arrangement of the fatty acids present in the oil. This in turn can be accomplished by the addition of genes that encode enzymes that catalyze the synthesis of novel fatty acids (e.g. fatty acid elongases, desaturases) and the lipids possessing them or by increasing levels of native fatty acids while possibly reducing levels of precursors or breakdown products. Alternatively, DNA sequences can be introduced which slow or block steps in fatty acid biosynthesis resulting in the increase in precursor fatty acid intermediates. Genes that might be added include desaturases, epoxidases, hydratases, dehydratases, or other enzymes that catalyze reactions involving fatty acid intermediates. Representative examples of catalytic steps that might be blocked include the desaturations from stearic to oleic acid or oleic to linolenic acid resulting in the respective accumulations of stearic and oleic acids. Another example is the blockage of elongation steps resulting in the accumulation of C8 to C12 saturated fatty acids.
Polypeptides useful for providing increased oil quantity and/or quality include polypeptides involved in fatty acid and glycerolipid biosynthesis, beta-oxidation enzymes, enzymes involved in biosynthesis of nutritional compounds, such as carotenoids and tocopherols.
Polypeptides involved in production of galactomannans or arabinogalactans are of interest for providing plants having increased and/or modified reserve polysaccharides for use in food, pharmaceutical, cosmetic, paper and paint industries.
Polypeptides involved in modification of flavonoid/isoflavonoid metabolism in plants include cinnamate-4-hydroxylase, chalcone synthase or flavones synthase. Enhanced or reduced activity of such polypeptides in modified plants will provide changes in the quantity and/or speed of flavonoid metabolism in plants and can improve disease resistance by enhancing synthesis of protective secondary metabolites or improving signaling pathways governing disease resistance.
Polypeptides involved in lignin biosynthesis are of interest for increasing plants' resistance to lodging and for increasing the usefulness of plant materials as biofuels.
(ix) Production or Assimilation of Chemicals or Biologicals
It may further be considered that a sorghum plant comprising a sorghum MC or recombinant chromosome prepared in accordance with the invention may be used for the production or manufacturing of useful biological compounds that were either not produced at all, or not produced at the same level, in the plant previously. Alternatively, plants produced in accordance with the invention may be made to metabolize or absorb and concentrate certain compounds, such as hazardous wastes, thereby allowing bioremediation of these compounds.
The novel plants producing these compounds are made possible by the introduction and expression of one or potentially many genes with the constructs provided by the invention. The vast array of possibilities include but are not limited to any biological compound which is presently produced by any organism such as proteins, nucleic acids, primary and intermediary metabolites, carbohydrate polymers, enzymes for uses in bioremediation, enzymes for modifying pathways that produce secondary plant metabolites such as falconoid or vitamins, enzymes that could produce pharmaceuticals, and for introducing enzymes that could produce compounds of interest to the manufacturing industry such as specialty chemicals and plastics. The compounds may be produced by the plant, extracted upon harvest and/or processing, and used for any presently recognized useful purpose such as pharmaceuticals, fragrances, and industrial enzymes to name a few.
(x) Other Characteristics
Cell cycle modification: Polypeptides encoding cell cycle enzymes and regulators of the cell cycle pathway are useful for manipulating growth rate in plants to provide early vigor and accelerated maturation. Improvements in quality traits, such as seed oil content, may also be obtained by expression of cell cycle enzymes and cell cycle regulators. Polypeptides of interest for modification of cell cycle pathway include cycling and EIF5α pathway proteins, polypeptides involved in polyamine metabolism, polypeptides which act as regulators of the cell cycle pathway, including cyclin-dependent kinases (CDKs), CDK-activating kinases, cell cycle-dependent phosphatases, CDK-inhibitors, Rb and Rb-binding proteins, or transcription factors that activate genes involved in cell proliferation and division, such as the E2F family of transcription factors, proteins involved in degradation of cyclins, such as cullins, and plant homologs of tumor suppressor polypeptides.
Plant growth regulators: Polypeptides involved in production of substances that regulate the growth of various plant tissues are of interest in the present invention and may be used to provide modified plants having altered morphologies and improved plant growth and development profiles leading to improvements in yield and stress response. Of particular interest are polypeptides involved in the biosynthesis, or degradation of plant growth hormones, such as gibberellins, brassinosteroids, cytokinins, auxins, ethylene or abscisic acid, and other proteins involved in the activity, uptake and/or transport of such polypeptides, including for example, cytokinin oxidase, cytokinin/purine permeases, F-box proteins, G-proteins or phytosulfokines.
Transcription factors in plants: Transcription factors play a key role in plant growth and development by controlling the expression of one or more genes in temporal, spatial and physiological specific patterns. Enhanced or reduced activity of such polypeptides in modified plants will provide significant changes in gene transcription patterns and provide a variety of beneficial effects in plant growth, development and response to environmental conditions. Transcription factors of interest include, but are not limited to myb transcription factors, including helix-turn-helix proteins, homeodomain transcription factors, leucine zipper transcription factors, MADS transcription factors, transcription factors having AP2 domains, zinc finger transcription factors, CCAAT binding transcription factors, ethylene responsive transcription factors, transcription initiation factors or UV damaged DNA binding proteins.
Homologous recombination: Increasing the rate of homologous recombination in plants is useful for accelerating the introgression of transgenes into breeding varieties by backcrossing, and to enhance the conventional breeding process by allowing rare recombinants between closely linked genes in phase repulsion to be identified more easily. Polypeptides useful for expression in plants to provide increased homologous recombination include polypeptides involved in mitosis and/or meiosis, DNA replication, nucleic acid metabolism, DNA repair pathways or homologous recombination pathways including for example, recombinases, nucleases, proteins binding to DNA double-strand breaks, single-strand DNA binding proteins, strand-exchange proteins, resolvases, ligases, helicases and polypeptide members of the RAD52 epistasis group.
Enhanced Biofuel Conversion
Biofuels can be produced from the conversion of biomass into liquid or gaseous fuels by converting the biomass into sugars, or by direct extract of sugars, that can be fermented or chemically converted to form a biofuel. Biofuels can also be generated by extracting oils from the biomass. Exemplary biofuels are ethanol, propanol, butanol, methanol, methane, 2,5-dimethylfurqan, dimethyl ether, biodiesel (short chain acid alkyl esters), biogasoline, syngas, parrafins (alkanes), other hydrocarbons or co-products of hydrogen.
The invention provides for MCs or recombinant chromosomes expressing at least one gene that enhance or increase sugar production or extractability, enhance or increase biomass, enhance the conversion of biomass to sugars or enhance sugar fermentation to biofuels. It may further be considered that a modified plant prepared in accordance with the invention may be used as biomass for the production of biofuels or the plant may facilitate conversion of biomass to sugars or facilitate fermentation of sugars to biofuels.
Enzymes that may be useful for biofuel production include those that break down glucans. In some embodiments, the enzymes are selected from the group consisting of: endo-β(1,4)-glucanase, cellobiohydrolase, β-glucosidase, α/β-glucosidase, mixed-linked glucanase, endo-β(1,3)-glucanase, exo-β(1,3)-glucanse and β-(1,6)-glucanase. In other embodiments the enzymes break down xyloglucans, xylans, mannans or lignins.
The enzyme genes may be controlled by inducible promoters that may be inactive until a desired time, such as at harvest or when the plant is added to the biofuels process (e.g. inactive at physiological conditions, then activated by heat or pH), or sequestered by subcellular localization. The enzymes may also be controlled by a tissue-specific promoter which may be active only in specific tissues (e.g seeds or leaves).
Non-Protein-Expressing Exogenous Nucleic Acids
Plants with decreased expression of a gene of interest can also be achieved, for example, by expression of antisense nucleic acids, dsRNA or RNAi, acatalytic RNA such as ribozymes, sense expression constructs that exhibit cosuppression effects, aptamers or zinc finger proteins.
Antisense RNA reduces production of the polypeptide product of the target messenger RNA, for example by blocking translation through formation of RNA:RNA duplexes or by inducing degradation of the target mRNA. Antisense approaches are a way of preventing or reducing gene function by targeting the genetic material as disclosed in U.S. Pat. Nos. 4,801,540, 5,107,065, 5,759,829, 5,910,444, 6,184,439, and 6,198,026, all of which are incorporated herein by reference. In one approach, an antisense gene sequence is introduced that is transcribed into antisense RNA that is complementary to the target mRNA. For example, part or all of the normal gene sequences are placed under a promoter in inverted orientation so that the complementary strand is transcribed into a non-protein expressing antisense RNA. The promoter used for the antisense gene may influence the level, timing, tissue, specificity, or inducibility of the antisense inhibition.
Autonomous MCs or recombinant chromosome may comprise exogenous DNA flanked by recombination sites, for example lox-P sites, that can be recognized by a recombinase, e.g. Cre, and removed from the MC or recombinant chromosome. In cases where there is a homologous recombination site or sites in the host genomic DNA, the exogenous DNA excised the MC or recombinant chromosome may be integrated into the genome at one of the specific recombination sites and the DNA flanked by the recombination sites will become integrated into the host DNA. The use of a MC or recombinant chromosome as a platform for DNA excision or for launching such DNA integration into the host genome may include in vivo induction of the expression of a recombinase encoded in the genomic DNA of a transgenic host, or in a MC or recombinant chromosome.
RNAi gene suppression in plants by transcription of a dsRNA is described in U.S. Pat. No. 6,506,559, US patent application Publication No. 2002/0168707, WO 98/53083, WO 99/53050 and WO 99/61631, all of which are incorporated herein by reference. The double-stranded RNA or RNAi constructs can trigger the sequence-specific degradation of the target messenger RNA. Suppression of a gene by RNAi can be achieved using a recombinant DNA construct having a promoter operably linked to a DNA element comprising a sense and anti-sense element of a segment of genomic DNA of the gene, e.g., a segment of at least about 23 nucleotides, more preferably about 50 to 200 nucleotides where the sense and anti-sense DNA components can be directly linked or joined by an intron or artificial DNA segment that can form a loop when the transcribed RNA hybridizes to form a hairpin structure.
Catalytic RNA molecules or ribozymes can also be used to inhibit expression of the target gene or genes or facilitate molecular reactions. Ribozymes are targeted to a given sequence by hybridization of sequences within the ribozyme to the target mRNA. Two stretches of homology are required for this targeting, and these stretches of homologous sequences flank the catalytic ribozyme structure. It is possible to design ribozymes that specifically pair with virtually any target mRNA and cleave the target mRNA at a specific location, thereby inactivating it. A number of classes of ribozymes have been identified. One class of ribozymes is derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants. The RNAs replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples include Tobacco Ringspot Virus (Prody et al., Science, 231:1577-1580, 1986), Avocado Sun blotch Viroid (Palukaitis et al., Virology, 99:145-151, 1979; Symons, Nucl. Acids Res., 9:6527-6537, 1981), and Lucerne Transient Streak Virus (Forster and Symons, Cell, 49:211-220, 1987), and the satellite RNAs from velvet tobacco mottle virus, Solanum nodiflorum mottle virus and subterranean clover mottle virus. The design and use of target RNA-specific ribozymes is described in Haseloff, et al., Nature 334:585-591 (1988). Several different ribozyme motifs have been described with RNA cleavage activity (Symons, Annu. Rev. Biochem., 61:641-671, 1992). Other suitable ribozymes include sequences from RNase P with RNA cleavage activity (Yuan et al., PNAS USA, 89:8006-8010, 1992; Yuan and Altman, Science, 263:1269-1273, 1994; U.S. Pat. Nos. 5,168,053 and 5,624,824), hairpin ribozyme structures (Berzal-Herranz et al., Genes and Devel., 6:129-134, 1992; Chowrira et al., J. Biol. Chem., 269:25856-25864, 1994) and Hepatitis Delta virus based ribozymes (U.S. Pat. No. 5,625,047). The general design and optimization of ribozyme directed RNA cleavage activity has been discussed in detail (Haseloff and Gerlach, Nature 334:585-91, 1988; Chowrira et al., J. Biol. Chem., 269:25856-25864, 1994).
Another method of reducing protein expression utilizes the phenomenon of cosuppression or gene silencing (for example, U.S. Pat. Nos. 6,063,947, 5,686,649, or 5,283,184; each of which is incorporated herein by reference). Cosuppression of an endogenous gene using a full-length cDNA sequence as well as a partial cDNA sequence are known (for example, Napoli et al., Plant Cell 2:279-289, 1990; van der Krol et al., Plant Cell 2:291-299, 1990; Smith et al., Mol. Gen. Genetics 224:477-481, 1990). The phenomenon of cosuppression has also been used to inhibit plant target genes in a tissue-specific manner.
In some embodiments, nucleic acids from one species of plant are expressed in another species of plant to effect cosuppression of a homologous gene. The introduced sequence generally will be substantially identical to the endogenous sequence intended to be repressed, for example, about 65%, 80%, 85%, 90%, or preferably 95% or greater identical. Higher identity may result in a more effective repression of expression of the endogenous sequence. A higher identity in a shorter than full length sequence compensates for a longer, less identical sequence. Furthermore, the introduced sequence need not have the same intron or exon pattern, and identity of non-coding segments will be equally effective. Generally, where inhibition of expression is desired, some transcription of the introduced sequence occurs. The effect may occur where the introduced sequence contains no coding sequence per se, but only intron or untranslated sequences homologous to sequences present in the primary transcript of the endogenous sequence.
Yet another method of reducing protein activity is by expressing nucleic acid ligands, so-called aptamers, which specifically bind to the protein. Aptamers may be obtained by the SELEX (Systematic Evolution of Ligands by EXponential Enrichment) method. See U.S. Pat. No. 5,270,163, incorporated herein by reference. In the SELEX method, a candidate mixture of single stranded nucleic acids having regions of randomized sequence is contacted with the protein and those nucleic acids having an increased affinity to the target are selected and amplified. After several iterations a nucleic acid with optimal affinity to the polypeptide is obtained and is used for expression in modified plants.
A zinc finger protein that binds a polypeptide-encoding sequence or its regulatory region is also used to alter expression of the nucleotide sequence. Transcription of the nucleotide sequence may be reduced or increased. Zinc finger proteins are, for example, described in Beerli et al. (1998) PNAS USA 95:14628-14633, or in WO 95/19431, WO 98/54311, or WO 96/06166, all incorporated herein by reference.
Other examples of non-protein expressing sequences specifically envisioned for use with the invention include: tRNA sequences, for example, to alter codon usage; rRNA variants, for example, which may confer resistance to various agents such as antibiotics.
It is contemplated that unexpressed DNA sequences, including novel synthetic sequences, could be introduced into cells as proprietary “labels” of those cells and plants and seeds thereof. It would not be necessary for a label DNA element to disrupt the function of a gene endogenous to the host organism, as the sole function of this DNA would be to identify the origin of the organism. For example, one could introduce a unique DNA sequence into a plant and this DNA element would identify all cells, plants, and progeny of these cells as having arisen from that labeled source. It is proposed that inclusion of label DNAs would enable one to distinguish proprietary germplasm or germplasm derived from such, from unlabelled germplasm.
Exemplary Plant Promoters, Regulatory Sequences and Targeting Sequences
Exemplary classes of plant promoters are described below.
Constitutive Expression promoters: Exemplary constitutive expression promoters include the ubiquitin promoter (e.g., sunflower—Binet et al. Plant Science 79: 87-94, 1991; maize—Christensen et al. Plant Molec. Biol. 12: 619-632, 1989; and Arabidopsis—Callis et al., J. Biol. Chem. 265: 12486-12493, 1990; and Norris et al., Plant Mol. Biol. 21: 895-906, 1993); the CaMV 35S promoter (U.S. Pat. Nos. 5,858,742 and 5,322,938); or the actin promoter (e.g., rice—U.S. Pat. No. 5,641,876; McElroy et al. Plant Cell 2: 163-171, 1990; McElroy et al. Mol. Gen. Genet. 231: 150-160, 1991, and Chibbar et al. Plant Cell Rep. 12: 506-509, 1993. Exemplary promoters for use in sorghum include the seed-specific SBEIIb promoter (Mutisya J, et al. J Plant Physiol. 163:770-80, 2005). Other promoters that may be useful in sorghum include the maize polyubiquitin 1 (Mubi-1) and the Sugarcane polyubiquitin 9 (SCubi9) promoters (Wang M L, et al. Transgenic Res. 14:167-78, 2005); and the Sugarcane polyubiquitin 4 (ubi4) promoter (Wei H, et al. J Plant Physiol. 160:1241-51, 2003).
Inducible Expression promoters: Exemplary inducible expression promoters include the chemically regulatable tobacco PR-1 promoter (e.g., tobacco—U.S. Pat. No. 5,614,395; Arabidopsis—Lebel et al., Plant J. 16: 223-233, 1998; maize—U.S. Pat. No. 6,429,362). Various chemical regulators may be employed to induce expression, including the benzothiadiazole, isonicotinic acid, and salicylic acid compounds disclosed in U.S. Pat. Nos. 5,523,311 and 5,614,395. Other promoters inducible by certain alcohols or ketones, such as ethanol, include, for example, the alcA gene promoter from Aspergillus nidulans (Caddick et al. Nat. Biotechnol 16:177-180, 1998). A glucocorticoid-mediated induction system is described in Aoyama and Chua (Plant Journal 11: 605-612, 1997) wherein gene expression is induced by application of a glucocorticoid, for example a dexamethasone. Another class of useful promoters is water-deficit-inducible promoters, e.g. promoters which are derived from the 5′ regulatory region of genes identified as a heat shock protein 17.5 gene (HSP 17.5), an HVA22 gene (HVA22), and a cinnamic acid 4-hydroxylase (CA4H) gene of Zea mays. Another water-deficit-inducible promoter is derived from the rab-17 promoter as disclosed by Vilardell et al. (Plant Molec. Biol, 17:985-993, 1990). See also U.S. Pat. No. 6,084,089 which discloses cold inducible promoters, U.S. Pat. No. 6,294,714 which discloses light inducible promoters, U.S. Pat. No. 6,140,078 which discloses salt inducible promoters, U.S. Pat. No. 6,252,138 which discloses pathogen inducible promoters, and U.S. Pat. No. 6,175,060 which discloses phosphorus deficiency inducible promoters.
As another example, numerous wound-inducible promoters have been described (e.g. Xu et al. Plant Molec. Biol. 22: 573-588, 1993; Logemann et al., Plant Cell 1: 151-158, 1989; Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792, 1993; Firek et al. Plant Molec. Biol. 22: 129-142, 1993; Warner et al. Plant J. 3: 191-201, 1993)). Logemann describe 5′ upstream sequences of the potato wunl gene. Xu et al. show that a wound-inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice. Rohrmeier & Lehle describe maize Wipl cDNA which is wound induced and which can be used to isolate the cognate promoter. Firek et al. and Warner et al. have described a wound-induced gene from the monocotyledon Asparagus officinalis, which is expressed at local wound and pathogen invasion sites.
Tissue-Specific Promoters: Exemplary promoters that express genes only in certain tissues are useful according to the present invention. For example root specific expression may be attained using the promoter of the maize metallothionein-like (MTL) gene described by de Framond (FEBS 290: 103-106, 1991) and also in U.S. Pat. No. 5,466,785, incorporated herein by reference. U.S. Pat. No. 5,837,848 discloses a root specific promoter. Another exemplary promoter confers pith-preferred expression (see WO 93/07278, herein incorporated by reference, which describes the maize trpA gene and promoter that is preferentially expressed in pith cells). Leaf-specific expression may be attained, for example, by using the promoter for a maize gene encoding phosphoenol carboxylase (PEPC) (see Hudspeth & Grula, Plant Molec Biol 12: 579-589 (1989)). Pollen-specific expression may be conferred by the promoter for the maize calcium-dependent protein kinase (CDPK) gene which is expressed in pollen cells (WO 93/07278). US Pat. Appl. Pub. No. 20040016025 describes tissue-specific promoters. Pollen-specific expression may be conferred by the tomato LAT52 pollen-specific promoter (Bate et al., Plant Mol. Biol. 37:859-69, 1998).
See also U.S. Pat. No. 6,437,217 which discloses a root-specific maize RS81 promoter, U.S. Pat. No. 6,426,446 which discloses a root specific maize RS324 promoter, U.S. Pat. No. 6,232,526 which discloses a constitutive maize A3 promoter, U.S. Pat. No. 6,177,611 which discloses constitutive maize promoters, U.S. Pat. No. 6,433,252 which discloses a maize L3 oleosin promoter that are aleurone and seed coat-specific promoters, U.S. Pat. No. 6,429,357 which discloses a constitutive rice actin 2 promoter and intron, US patent application Pub. No. 20040216189 which discloses an inducible constitutive leaf specific maize chloroplast aldolase promoter.
Optionally a plant transcriptional terminator can be used in place of the plant-expressed gene native transcriptional terminator. Exemplary transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons.
Various intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. For example, the introns of the maize Adhl gene have been found to significantly enhance expression. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al., Genes Develop. 1: 1183-1200, 1987). The intron from the maize bronze1 gene also enhances expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically within the non-translated leader. US Patent Application Publication 2002/0192813 discloses 5′, 3′ and intron elements useful in the design of effective plant expression vectors.
A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous cells. Specifically, leader sequences from Tobacco Mosaic Virus (TMV, the “omega-sequence”), Maize Chlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. Gallie et al. Nucl. Acids Res. 15: 8693-8711, 1987; Skuzeski et al. Plant Molec. Biol. 15: 65-79, 1990). Other leader sequences known in the art include but are not limited to: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, O, et al. PNAS USA 86:6126-6130, 1989); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al., Virology 154:9-20, 1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20); human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejak, D. G., and Sarnow, P., Nature 353: 90-94, 1991; untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., and Gehrke, L., Nature 325:622-625, 1987); tobacco mosaic virus leader (TMV), (Gallie et al., Molecular Biology of RNA, pages 237-256, 1989); or Maize Chlorotic Mottle Virus leader (MCMV) (Lommel et al., Virology 81:382-385, 1991). See also, Della-Cioppa et al., Plant Physiology 84:965-968 (1987).
A minimal promoter may also be incorporated. Such a promoter has low background activity in plants when there is no transactivator present or when enhancer or response element binding sites are absent. One exemplary minimal promoter is the Bz1 minimal promoter, which is obtained from the bronze1 gene of maize. Roth et al., Plant Cell 3: 317 (1991). A minimal promoter may also be created by use of a synthetic TATA element. The TATA element allows recognition of the promoter by RNA polymerase factors and confers a basal level of gene expression in the absence of activation (see generally, Mukumoto, Plant Mol Biol 23: 995-1003, 1993; Green, Trends Biochem Sci 25: 59-63, 2000).
Sequences controlling the targeting of gene products also may be included. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (e.g. Comai et al., J. Biol. Chem. 263: 15104-15109, 1988). These signal sequences can be fused to heterologous gene products to affect the import of heterologous products into the chloroplast (van den Broeck, et al. Nature 313: 358-363, 1985). DNA encoding for appropriate signal sequences can be isolated from the 5′ end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein or many other proteins which are known to be chloroplast localized. Other gene products are localized to other organelles such as the mitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol. 13: 411-418, 1989). Examples of sequences that target to such organelles are the nuclear-encoded ATPases or specific aspartate amino transferase isoforms for mitochondria. Targeting cellular protein bodies has been described by Rogers et al. (PNAS USA 82: 6512-6516, 1985). In addition, amino terminal and carboxy-terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783, 1990). Additionally, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368, 1990).
Another possible element which may be introduced is a matrix attachment region element (MAR), such as the chicken lysozyme A element (Stief et al. Nature 34:343-5, 1989), which can be positioned around an expressible gene of interest to effect an increase in overall expression of the gene and diminish position dependent effects upon incorporation into the plant genome (Stief et al., Nature, 341:343, 1989; Phi-Van et al., Mol. Cell. Biol., 10:2302-230, 1990).
Use of Non-Plant Promoter Regions Isolated from Drosophila melanogaster and Saccharomyces cerevisiae to Express Genes in Plants
The promoter in the sorghum MC or recombinant chromosome of the present invention can be derived from plant or non-plant species. In one embodiment, the nucleotide sequence of the promoter is derived from non-plant species for the expression of genes in plant cells, including but not limited to dicotyledon plant cells such as tobacco, tomato, potato, soybean, canola, sunflower, alfalfa, cotton and Arabidopsis, or monocotyledonous plant cell, such as wheat, maize, rye, rice, turf grass, oat, barley, sorghum, sugarcane and millet. In one embodiment, the non-plant promoters are constitutive or inducible promoters derived from insect, e.g., Drosophila melanogaster or yeast, e.g., Saccharomyces cerevisiae. Table 2 lists the promoters from Drosophila melanogaster and Saccharomyces cerevisiae that are used to derive the examples of non-plant promoters in the present invention. Promoters derived from any animal, protist, or fungi are also contemplated. SEQ ID NOs:1-20, or fragments, mutants, hybrid or tandem promoters thereof, are examples of promoter sequences derived from Drosophila melanogaster or Saccharomyces cerevisiae. These non-plant promoters can be operably linked to nucleic acid sequences encoding polypeptides or non-protein-expressing sequences including, but not limited to, antisense RNA and ribozymes, to form nucleic acid constructs, vectors, and host cells (prokaryotic or eukaryotic), comprising the promoters.







TABLE 2





Exemplary Promoters from D. melanogaster and S. cerevisiae







Drosophila melanogaster promoters (adapted from the Drosophila FlyBase, referenced in Grumbling, G.


and Strelets, Nucl. Acids Rsrch. 34:D484-8, 2006












SEQ ID


Standard promoter




NO:
Symbol
Flybase ID
gene name
Gene product
Chromosome





1
Pgd
FBgn0004654
Phosphogluconate
6-phosphogluconate
X





dehydrogenase
dehydrogenase



2
Grim
FBgn0015946
grim
grim-P138
3


3
Uro
FBgn0003961
Urate oxidase
Uro-P1
2


4
Sna
FBgn0003448
Snail
sna-P1
2


5
Rh3
FBgn0003249
Rhodopsin 3
Rh3
3


6
Lsp-1 γ
FBgn0002564
Larval serum protein 1
Lsp1γ-P1
3










Saccharomyces cerevisiae Promoters (adapted from information available from the Saccharomyces


Genome Database, referenced in Dwight SS et al. Brief Bioinform. 5:9-22, 2004).












SEQ ID


Standard promoter




NO:
Symbol
Systematic Name
gene name
Gene product
Chromosome





7
Tef-2
YBR118W
TEF2 (Translation
Translation elongation
2





elongation factor
factor EF-1 alpha






promtoer)




8
Leu-1
YGL009C
LEU1 (LEUcine
isopropylmalate
7





biosynthesis)
isomerase



9
Met16
YPR167C
METhionine requiring
3′phosphoadenylyl-
16






sulfate reductase



10
Leu-2
YCL018W
LEU2 (leucine
beta-IPM
3





biosynthesis)
(isopropylmalate)







dehydrogenase



11
His-4
YCL030C
HIS4 (HIStidine requiring)
histidinol
3






dehydrogenase



12
Met-2
YNL277W
MET2 (methionine
L-homoserine-O-
14





requiring)
acetyltransferase



13
Ste-3
YKL178C
STE3 (alias DAF2 Sterile)
a-factor receptor 
11


14
Arg-1
YOL058W
ARG1(alias ARG10
arginosuccinate
15





ARGinine requiring)
synthetase



15
Pgk-1
YCR012W
PGK1 (phosphoglycerate
phosphoglycerate
3





kinase )
kinase



16
GPD-1
YDL022W
GPD1 (alias
glycerol-3-phosphate
4





DAR1/HOR1/OSG1/OSR
dehydrogenase






5:glycerol-3-phosphate







dehydrogenase activity




17
ADH1
YOL086C
ADH1 (alias ADC1)
alcohol
15






dehydrogenase



18
GPD-2
YOL059W
GPD2 (alias GPD3:
glycerol-3-phosphate
15





glycerol-3-phosphate
dehydrogenase






dehydrogenase activity




19
Arg-4
YHR018C
ARGinine requiring
argininosuccinate
8






lyase



20
Yat-1
YAR035W
YAT-1(carnitine
carnitine
1





acetyltransferase)
acetyltransferase









In the MCs or recombinant chromosome of the present invention, the promoter may be a mutant of the promoters having a substitution, deletion, and/or insertion of one or more nucleotides in the nucleic acid sequence of SEQ ID NOs:1 to 20, hybrid or tandem promoters.
The techniques used to isolate or clone a nucleic acid sequence comprising a promoter of interest are known in the art and include isolation from genomic DNA. The cloning procedures may involve excision or amplification, for example by polymerase chain reaction, and isolation of a desired nucleic acid fragment comprising the nucleic acid sequence encoding the promoter, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into the plant cell.
DEFINITIONS
The term “adchromosomal” plant or plant part means a plant or plant part that contains functional, stable and autonomous MCs. Adchromosomal plants or plant parts may be chimeric or not chimeric (chimeric meaning that MCs are only in certain portions of the plant, and are not uniformly distributed throughout the plant). An adchromosomal plant cell contains at least one functional, stable and autonomous MC.
The term “autonomous” means that when delivered to plant cells, at least some MCs are transmitted through mitotic division to daughter cells and are episomal in the daughter plant cells, i.e. are not chromosomally integrated in the daughter plant cells. Daughter plant cells that contain autonomous MCs can be selected for further replication using, for example, selectable or screenable markers. During the introduction into a cell of a MC, or during subsequent stages of the cell cycle, there may be chromosomal integration of some portion or all of the DNA derived from a MC in some cells. The MC is still characterized as autonomous despite the occurrence of such events if a plant may be regenerated that contains episomal descendants of the MC distributed throughout its parts, or if gametes or progeny can be derived from the plant that contain episomal descendants of the MC distributed through its parts.
A “centromere” is any DNA sequence that confers an ability to segregate to daughter cells through cell division. In one context, this sequence may produce a transmission efficiency to daughter cells ranging from about 1% to about 100%, including to about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or about 95% of daughter cells. Variations in transmission efficiency may find important applications within the scope of the invention; for example, MCs carrying centromeres that confer 100% stability could be maintained in all daughter cells without selection, while those that confer 1% stability could be temporarily introduced into a transgenic organism, but be eliminated when desired. In particular embodiments of the invention, the centromere may confer stable transmission to daughter cells of a nucleic acid sequence, including a recombinant construct comprising the centromere, through mitotic or meiotic divisions, including through both meiotic and meiotic divisions. A plant centromere is not necessarily derived from plants, but has the ability to promote DNA transmission to daughter plant cells.
The term “circular permutations” refer to variants of a sequence that begin at base n within the sequence, proceed to the end of the sequence, resume with base number one of the sequence, and proceed to base n−1. For this analysis, n may be any number less than or equal to the length of the sequence. For example, circular permutations of the sequence ABCD are: ABCD, BCDA, CDAB, and DABC.
The term “co-delivery” refers to the delivery of two nucleic acid segments to a cell. In co-delivery of plant growth inducing genes and MCs, the two nucleic acid segments are delivered simultaneously using the same delivery method. Alternatively, the nucleic acid segment containing the growth inducing gene, optionally as part of an episomal vector, such as a viral vector or a plasmid vector, may be delivered to the plant cells before or after delivery of the MC, and the MC may carry an exogenous nucleic acid that induces expression of the earlier-delivered growth inducing gene. In this embodiment, the two nucleic acid segments may be delivered separately at different times provided the encoded growth inducing factors are functional during the appropriate time period.
The term “coding sequence” is defined herein as a nucleic acid sequence that is transcribed into mRNA which is translated into a polypeptide when placed under the control of promoter sequences. The boundaries of the coding sequence are generally determined by the ATG start codon located at the start of the open reading frame, near the 5′ end of the mRNA, and TAG, TGA or TAA stop codons at the end of the coding sequence, near the 3′ end of the mRNA, and in some cases, a transcription terminator sequence located just downstream of the open reading frame at the 3′ end of the mRNA. A coding sequence can include, but is not limited to, genomic DNA, cDNA, semisynthetic, synthetic, or recombinant nucleic acid sequences.
The term “consensus” refers to a nucleic acid sequence derived by comparing two or more related sequences. A consensus sequence defines both the conserved and variable sites between the sequences being compared. Any one of the sequences used to derive the consensus or any permutation defined by the consensus may be useful in construction of MCs.
The term “exogenous” when used in reference to a nucleic acid, for example, is intended to refer to any nucleic acid that has been introduced into a recipient cell, regardless of whether the same or similar nucleic acid is already present in such a cell. Thus, as an example, “exogenous DNA” can include an additional copy of DNA that is already present in the plant cell, DNA from another plant, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene. An “exogenous gene” can be a gene not normally found in the host genome in an identical context, or an extra copy of a host gene. The gene may be isolated from a different species than that of the host genome, or alternatively, isolated from the host genome but operably linked to one or more regulatory regions which differ from those found in the unaltered, native gene.
The term “functional” to describe a MC means that when an exogenous nucleic acid is present within the MC the exogenous nucleic acid can function in a detectable manner when the MC is within a plant cell; exemplary functions of the exogenous nucleic acid include transcription of the exogenous nucleic acid, expression of the exogenous nucleic acid, regulatory control of expression of other exogenous nucleic acids, recognition by a restriction enzyme or other endonuclease, ribozyme or recombinase; providing a substrate for DNA methylation, DNA glycolation or other DNA chemical modification; binding to proteins such as histones, helix-loop-helix proteins, zinc binding proteins, leucine zipper proteins, MADS box proteins, topoisomerases, helicases, transposases, TATA box binding proteins, viral protein, reverse transcriptases, or cohesins; providing an integration site for homologous recombination; providing an integration site for a transposon, T-DNA or retrovirus; providing a substrate for RNAi synthesis; priming of DNA replication; aptamer binding; or kinetochore binding. If multiple exogenous nucleic acids are present within the MC, the function of one or preferably more of the exogenous nucleic acids can be detected under suitable conditions permitting function thereof.
“Library” is a pool of cloned DNA fragments that represents some or all DNA sequences collected, prepared or purified from a specific source. Each library may contain the DNA of a given organism inserted as discrete restriction enzyme generated fragments or as randomly sheared fragments into many thoUSAnds of plasmid vectors. For purposes of the present invention, E. coli, yeast, and Salmonella plasmids are particularly useful for propagating the genome inserts from other organisms. In principle, any gene or sequence present in the starting DNA preparation can be isolated by screening the library with a specific hybridization probe (see, for example, Young et al., In: Eukaryotic Genetic Systems ICN-UCLA Symposia on Molecular and Cellular Biology, VII, 315-331, 1977).
The term “linker” refers to a DNA molecule, generally up to 50 or 60 nucleotides long and composed of two or more complementary oligonucleotides that have been synthesized chemically, or excised or amplified from existing plasmids or vectors. In a preferred embodiment, this fragment contains one, or preferably more than one, restriction enzyme site for a blunt cutting enzyme and/or a staggered cutting enzyme, such as BamHI. One end of the linker is designed to be ligatable to one end of a linear DNA molecule and the other end is designed to be ligatable to the other end of the linear molecule, or both ends may be designed to be ligatable to both ends of the linear DNA molecule.
A “MC” is a recombinant DNA construct including a centromere that is capable of transmission to daughter cells. A MC may remain separate from the host genome (as episomes) or may integrate into host chromosomes. The stability of this construct through cell division could range between from about 1% to about 100%, including about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and about 95%. The MC construct may be a circular or linear molecule. It may include elements such as one or more telomeres, origin of replication sequences, stuffer sequences, buffer sequences, chromatin packaging sequences, linkers and genes. The number of such sequences included is only limited by the physical size limitations of the construct itself. It could contain DNA derived from a natural centromere, although it may be preferable to limit the amount of DNA to the minimal amount required to obtain a transmission efficiency in the range of 1-100%. The MC could also contain a synthetic centromere composed of tandem arrays of repeats of any sequence, either derived from a natural centromere, or of synthetic DNA. The MC could also contain DNA derived from multiple natural centromeres. The MC may be inherited through mitosis or meiosis, or through both meiosis and mitosis, the term MC specifically encompasses and includes the terms “plant artificial chromosome” or “PLAC,” or engineered chromosomes or microchromosomes and all teachings relevant to a PLAC or plant artificial chromosome specifically apply to constructs within the meaning of the term MC.
The term “non-protein expressing sequence” or “non-protein coding sequence” is defined herein as a nucleic acid sequence that is not eventually translated into protein. The nucleic acid may or may not be transcribed into RNA. Exemplary sequences include ribozymes or antisense RNA.
The term “operably linked” is defined herein as a configuration in which a control sequence, e.g., a promoter sequence, directs transcription or translation of another sequence, for example a coding sequence. For example, a promoter sequence could be appropriately placed at a position relative to a coding sequence such that the control sequence directs the production of a polypeptide encoded by the coding sequence.
“Phenotype” or “phenotypic trait(s)”, refers to an observable property or set of properties resulting from the expression of a gene. The set of properties may be observed visually or after biological or biochemical testing, and may be constantly present or may only manifest upon challenge with the appropriate stimulus or activation with the appropriate signal.
The term “plant” refers to any type of plant. Modified plants of the invention include, for example, dicots, gymnosperm, monocots, mosses, ferns, horsetails, club mosses, liver worts, hornworts, red algae, brown algae, gametophytes and sporophytes of pteridophytes, and green algae.
One modified crop plant of particular interest in the present invention is Sorghum, including but not limited to Sorghum bicolor (primary cultivated species), Sorghum almum, Sorghum amplum, Sorghum angustum, Sorghum rundinaceum, Sorghum brachypodum, Sorghum bulbosum, Sorghum burmahicum, Sorghum controversum, Sorghum drummondii, Sorghum carinatum, Sorghum exstans, Sorghum grande, Sorghum halepense, Sorghum interjectum, Sorghum intrans, Sorghum laxiflorum, Sorghum leiocladum, Sorghum macrospermum, Sorghum matarankense, Sorghum miliaceum, Sorghum nigrum, Sorghum nitidum, Sorghum plumosum, Sorghum propinquum, Sorghum purpureosericeum, Sorghum stipoideum, Sorghum timorense, Sorghum trichodadum, Sorghum versicolor, Sorghum virgatum, and Sorghum vulgare (including but not limited to the variety Sorghum vulgare var. sudanens also known as sudangrass). Hybrids of these species are also of interest in the present invention as are hybrids with other members of the Family Poaceae.
The term “plant part” includes a pod, root, sett root, shoot root, root primordial, shoot, primary shoot, secondary shoot, tassle, panicle, arrow, midrib, blade, ligule, auricle, dewlap, blade joint, sheath, node, internode, bud furrow, leaf scar, cutting, tuber, stem, stalk, fruit, berry, nut, flower, leaf, bark, wood, epidermis, vascular tissue, organ, protoplast, crown, callus culture, petiole, petal, sepal, stamen, stigma, style, bud, meristem, cambium, cortex, pith, sheath, silk, ovule or embryo. Other exemplary sorghum plant parts are a meiocyte or gamete or ovule or pollen or endosperm of any of the preceding plants. Other exemplary plant parts are a seed, seed-piece, embryo, protoplast, cell culture, any group of plant cells organized into a structural and functional unit, ratoon or propagule.
The term “promoter” is a DNA sequence that allows the binding of RNA polymerase (including but not limited to RNA polymerase I, RNA polymerase II and RNA polymerase III from eukaryotes) and directs the polymerase to a downstream transcriptional start site of a nucleic acid sequence encoding a polypeptide to initiate transcription. RNA polymerase effectively catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of the coding region.
A “promoter operably linked to a heterologous gene” is a promoter that is operably linked to a gene that is different from the gene to which the promoter is normally operably linked in its native state. Similarly, an “exogenous nucleic acid operably linked to a heterologous regulatory sequence” is a nucleic acid that is operably linked to a regulatory control sequence to which it is not normally linked in its native state.
The term “recombinant chromosome” refers to an engineered or artificial chromosome that has been constructed by fragmenting a natural chromosome and identifying fragmentation products that are capable of segregation through mitotic and/or meiotic cell divisions. Recombinant chromosomes are distinct from MCs in that they are not constructed in vitro from constituent parts and have not been passaged through an heterologous cell such as a bacteria or fungus (as is commonly used in standard cloning techniques). Recombinant chromosomes may the used as targets for addition of transgene expression cassettes.
The term “Basic MC” is defined as a recombinant DNA construct that when present within a cell is capable of mitotic and/or meiotic transmission to daughter cells under appropriate conditions and comprises a Assembled Centromere and, optionally, one or more of the following: (a) one or more telomeres; (b) one or more sequences for regulating, maintaining, or imparting topological or chromatin structure, molecular integrity, or stability of gene expression or inheritance in a cell; (c) the required vector DNA that allows for propagation of MC in and DNA that facilitates the selective removal of unwanted portions of MC prior to or after transformation; or (d) a Transgene Expression Cassette, wherein the Transgene Expression Cassette serves only to regulate, maintain, or impart function or stability to a MC in a cell.
A “Basic MC” does not include a Transgene Expression Cassette that imparts one or more functions other than those expressly set forth in subsection (d), above.
An “Assembled Centromere” means a polynucleotide sequence having the properties of a Centromere that is assembled from one or more fragments of native Centromere(s) and/or other polynucleotide sequence, which are (i) isolated from a plant cell, and/or based on plant Centromere sequence motifs, (ii) inserted into a plasmid vector that is propagated and maintained in a cell of a heterologous organism, and (iii) delivered back into a plant cell as part of a Basic or Applied MC. An Assembled Centromere may possibly be modified by an endogenous in vivo process after it is delivered into a plant cell such that its sequence now differs from that contained in the parental Basic or Applied MC as propagated in a cell of a heterologous organism. For the avoidance of doubt an Assembled Centromere does not include derivatives or deletions of native Centromeres that are constructed within the plant cell, and are never maintained in their entirety in a cell of a heterologous organism.
An “Applied MC” means a genetic construct formed by integrating one or more Transgene Expression Cassettes into a Basic MC, wherein said Transgene Expression Cassettes impart one or more functions other than to regulate, maintain, or impart function or stability to a MC.
The term “hybrid promoter” is defined herein as parts of two or more promoters that are fused together to generate a sequence that is a fusion of the two or more promoters, which is operably linked to a coding sequence and mediates the transcription of the coding sequence into mRNA.
The term “tandem promoter” is defined herein as two or more promoter sequences each of which is operably linked to a coding sequence and mediates the transcription of the coding sequence into mRNA.
The term “constitutive active promoter” is defined herein as a promoter that allows permanent stable expression of the gene of interest.
The term “Inducible promoter” is defined herein as a promoter induced by the presence or absence of biotic or an abiotic factor.
The term “polypeptide” does not refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. The term “exogenous polypeptide” is defined as a polypeptide which is not native to the plant cell, a native polypeptide in which modifications have been made to alter the native sequence, or a native polypeptide whose expression is quantitatively altered as a result of a manipulation of the plant cell by recombinant DNA techniques.
The term “pseudogene” refers to a non-functional copy of a protein-coding gene; pseudogenes found in the genomes of eukaryotic organisms are often inactivated by mutations and are thus presumed to be non-essential to that organism; pseudogenes of reverse transcriptase and other open reading frames found in retroelements are abundant in the centromeric regions of Arabidopsis and other organisms and are often present in complex clusters of related sequences.
The term “regulatory sequence” refers to any DNA sequence that influences the efficiency of transcription or translation of any gene. The term includes, but is not limited to, sequences comprising promoters, enhancers and terminators.
The term “repeated nucleotide sequence” refers to any nucleic acid sequence of at least 25 bp present in a genome or a recombinant molecule, other than a telomere repeat, that occurs at least two or more times and that are preferably at least 80% identical either in head to tail or head to head orientation either with or without intervening sequence between repeat units.
The term “retroelement” or “retrotransposon” refers to a genetic element related to retroviruses that disperse through an RNA stage; the abundant retroelements present in plant genomes contain long terminal repeats (LTR retrotransposons) and encode a polyprotein gene that is processed into several proteins including a reverse transcriptase. Specific retroelements (complete or partial sequences) can be found in and around plant centromeres and can be present as dispersed copies or complex repeat clusters. Individual copies of retroelements may be truncated or contain mutations; intact retroelements are rarely encountered.
The term “satellite DNA” refers to short DNA sequences (typically <1000 bp) present in a genome as multiple repeats, mostly arranged in a tandemly repeated fashion, as opposed to a dispersed fashion. Repetitive arrays of specific satellite repeats are abundant in the centromeres of many higher eukaryotic organisms.
A “screenable marker” is a gene whose presence results in an identifiable phenotype. This phenotype may be observable under standard conditions, altered conditions such as elevated temperature, or in the presence of certain chemicals used to detect the phenotype. The use of a screenable marker allows for the use of lower, sub-killing antibiotic concentrations and the use of a visible marker gene to identify clusters of transformed cells, and then manipulation of these cells to homogeneity. Preferred screenable markers of the present include genes that encode fluorescent proteins that are detectable by a visual microscope such as the fluorescent reporter genes DsRed, ZsGreen, ZsYellow, AmCyan, Green Fluorescent Protein (GFP) and modifications of these reporter genes to excite or emit at altered wavelengths. An additional preferred screenable marker gene is lac.
Alternative methods of screening for modified plant cells may involve use of relatively low, sub-killing concentrations of a selection agent (e.g. sub-killing antibiotic concentrations), and also involve use of a screenable marker (e.g., a visible marker gene) to identify clusters of modified cells carrying the screenable marker, after which these screenable cells are manipulated to homogeneity, a “selectable marker” is a gene whose presence results in a clear phenotype, and most often a growth advantage for cells that contain the marker. This growth advantage may be present under standard conditions, altered conditions such as elevated temperature, specialized media compositions, or in the presence of certain chemicals such as herbicides or antibiotics. Use of selectable markers is described, for example, in Broach et al. (Gene, 8:121-133, 1979). Examples of selectable markers include the thymidine kinase gene, the cellular adenine phosphoribosyltransferase gene and the dihydrylfolate reductase gene, hygromycin phosphotransferase genes, the bar gene, neomycin phosphotransferase genes and phosphomannose isomerase, among others. Preferred selectable markers in the present invention include genes whose expression confer antibiotic or herbicide resistance to the host cell, or proteins allowing utilization of a carbon source not normally utilized by plant cells. Expression of one of these markers should be sufficient to enable the survival of those cells that comprise a vector within the host cell, and facilitate the manipulation of the plasmid into new host cells. Of particular interest in the present invention are proteins conferring cellular resistance to kanamycin, G418, paramomycin, hygromycin, bialaphos, and glyphosate for example, or proteins allowing utilization of a carbon source, such as mannose, not normally utilized by plant cells.
The term “stable” means that the MC can be transmitted to daughter cells over at least 8 mitotic generations. Some embodiments of MCs may be transmitted as functional, autonomous units for less than 8 mitotic generations, e.g. 1, 2, 3, 4, 5, 6, or 7. Preferred MCs can be transmitted over at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 generations, for example, through the regeneration or differentiation of an entire plant, and preferably are transmitted through meiotic division to gametes. Other preferred MCs can be further maintained in the zygote derived from such a gamete or in an embryo or endosperm derived from one or more such gametes. A “functional and stable” MC is one in which functional MCs can be detected after transmission of the MCs over at least 8 mitotic generations, or after inheritance through a meiotic division. During mitotic division, as occurs occasionally with native chromosomes, there may be some non-transmission of MCs; the MC may still be characterized as stable despite the occurrence of such events if an adchromosomal plant that contains descendants of the MC distributed throughout its parts may be regenerated from cells, cuttings, propagules, or cell cultures containing the MC, or if an adchromosomal plant can be identified in progeny of the plant containing the MC.
A “structural gene” is a sequence which codes for a polypeptide or RNA and includes 5′ and 3′ ends. The structural gene may be from the host into which the structural gene is transformed or from another species. A structural gene will preferably but not necessarily include one or more regulatory sequences which modulate the expression of the structural gene, such as a promoter, terminator or enhancer. A structural gene will preferably but not necessarily confer some useful phenotype upon an organism comprising the structural gene, for example, herbicide resistance. In one embodiment of the invention, a structural gene may encode an RNA sequence which is not translated into a protein, for example a tRNA or rRNA gene.
The term “telomere” or “telomere DNA” refers to a sequence capable of capping the ends of a chromosome, thereby preventing degradation of the chromosome end, ensuring replication and preventing fusion to other chromosome sequences. Telomeres can include naturally occurring telomere sequences or synthetic sequences. Telomeres from one species may confer telomere activity in another species. An exemplary telomere DNA is a heptanucleotide telomere repeat TTTAGGG (and its complement) found in the majority of plants.
“Transformed,” “transgenic,” “modified,” and “recombinant” refer to a host organism such as a plant into which an exogenous or heterologous nucleic acid molecule has been introduced, and includes meiocytes, seeds, zygotes, embryos, endosperm, or progeny of such plant that retain the exogenous or heterologous nucleic acid molecule but which have not themselves been subjected to the transformation process.
When the phrase “transmission efficiency” of a certain percent is used, transmission percent efficiency is calculated by measuring MC presence through one or more mitotic or meiotic generations. It is directly measured as the ratio (expressed as a percentage) of the daughter cells or plants demonstrating presence of the MC to parental cells or plants demonstrating presence of the MC. Presence of the MC in parental and daughter cells is demonstrated with assays that detect the presence of an exogenous nucleic acid carried on the MC. Exemplary assays can be the detection of a screenable marker (e.g. presence of a fluorescent protein or any gene whose expression results in an observable phenotype), a selectable marker, or PCR amplification of any exogenous nucleic acid carried on the MC.
I. Constructing MCs by Site-Specific Recombination
Plant MCs may be constructed using site-specific recombination sequences (for example those recognized by the bacteriophage P1 Cre recombinase, or the bacteriophage lambda integrase, or similar recombination enzymes). A compatible recombination site, or a pair of such sites, is present on both the centromere containing DNA clones and the donor DNA clones. Incubation of the donor clone and the centromere clone in the presence of the recombinase enzyme causes strand exchange to occur between the recombination sites in the two plasmids; the resulting MCs contain centromere sequences as well as MC vector sequences. The DNA molecules formed in such recombination reactions is introduced into E. coli, other bacteria, yeast or plant cells by common methods in the field including, but not limited to, heat shock, chemical transformation, electroporation, particle bombardment, whiskers, or other transformation methods followed by selection for marker genes including chemical, enzymatic, color, or other marker present on either parental plasmid, allowing for the selection of transformants harboring MCs.
II. Methods of Detecting and Characterizing MCs in Plant Cells or of Scoring MC Performance in Plant Cells
Identification of Candidate Centromere Fragments by Probing BAC Libraries
Centromere clones are identified from a large genomic insert library such as a Bacterial Artificial Chromosome library. Probes are labeled using nick-translation in the presence of radioactively labeled dCTP, dATP, dGTP or dTTP as in, for example, the commercially available REDIPRIMETM kit (GE Healthcare; Piscataway, N.J.; USA) as per the manufacturer's instructions. Other labeling methods familiar to those skilled in the art could be substituted. The libraries are screened and deconvoluted. Genomic clones are screened by probing with small centromere-specific clones. Other embodiments of this procedure would involve hybridizing a library with other centromere sequences. Of the BAC clones identified using this procedure, a representative set are identified as having high hybridization signals to some probes, and optionally low hybridization signals to other probes. These are selected, the bacterial clones grown up in cultures and DNA prepared by methods familiar to those skilled in the art such as alkaline lysis. The DNA composition of purified clones is surveyed using for example fingerprinting by digesting with restriction enzymes such as, but not limited to, HinfI or HindIII. In a preferred embodiment the restriction enzyme cuts within the tandem centromere satellite repeat (see below). A variety of clones showing different fingerprints are selected for conversion into MCs and inheritance testing. It can also be informative to use multiple restriction enzymes for fingerprinting or other enzymes which can cleave DNA.
Fingerprinting Analysis of BACs and MCs
Centromere function may be associated with large tandem arrays of satellite repeats. To assess the composition and architecture of the centromere BACs, the candidate BACs are digested with a restriction enzyme, such as HindIII, which cuts with known frequency within the consensus sequence of the unit repeat of the tandemly repeated centromere satellite. Digestion products are then separated by agarose gel electrophoresis. Large insert clones containing a large array of tandem repeats will produce a strong band of the unit repeat size, as well as less intense bands at 2× and 3× the unit repeat size, and further multiples of the repeat size. These methods are well-known and there are many possible variations known to those skilled in the art.
Determining Sequence Composition of MCs by Shotgun Cloning/Sequencing, Sequence Analysis
To determine the sequence composition of the MC, the centromeric region of the MC is sequenced. To generate DNA suitable for sequencing MCs are fragmented, for example by using a random shearing method (such as sonication, nebulization, etc). Other fragmentation techniques may also be used such as enzymatic digestion. These fragments are then cloned into a plasmid vector and sequenced. The resulting DNA sequence is trimmed of poor-quality sequence and of sequence corresponding to the plasmid vector. The sequence is then compared to known DNA sequences using an algorithm such as BLAST to search a sequence database such as GenBank.
To determine the consensus of the satellite repeat in the MC, the sequences containing satellite repeat are aligned using a DNA sequence alignment program such as CONTIGEXPRESS® from Vector NTI®. The sequences may also be aligned to previously determined repeats for that species. The sequences are trimmed to unit repeat length using the consensus as a template. Sequences trimmed from the ends of the alignment are realigned with the consensus and further trimmed until all sequences are at or below the consensus length. The sequences are then aligned with each other. The consensus is determined by the frequency of a specific nucleotide at each position; if the most frequent base is three times more frequent than the next most frequent base, it was considered the consensus.
Methods for determining consensus sequence are well known in the art, see, e.g., US Pat. App. Pub. No. 20030124561; Hall et al. Plant Physiol. 129:1439-1447, 2002. These methods, including DNA sequencing, assembly, and analysis, are well-known and there are many possible variations known to those skilled in the art. Other alignment parameters may also be useful such as using more or less stringent definitions of consensus.
Non-Selective MC Mitotic Inheritance Assays
The following list of assays and potential outcomes illustrates how various assays can be used to distinguish autonomous events from integrated events.
Assay #1: Transient Assay
MCs are tested for their ability to become established as chromosomes and their ability to be inherited in mitotic cell divisions. In this assay, MCs are delivered to plant cells, for example suspension cells in liquid culture. The cells used can be at various stages of growth. In this example, a population in which some cells were undergoing division was used. The MC is then assessed over the course of several cell divisions, by tracking the presence of a screenable marker, e.g. a visible marker gene such as a fluorescent protein. MCs that are established and inherited well may show an initial delivery into many single cells; after several cell divisions, these single cells divide to form clusters of MC-containing cells. Other exemplary embodiments of this method include delivering MCs to other mitotic cell types, including roots and shoot meristems.
Assay #2: Non-Lineage Based Inheritance Assays on Modified Transformed Cells and Plants
MC inheritance is assessed on modified cell lines and plants by following the presence of the MC over the course of multiple cell divisions. An initial population of MC containing cells is assayed for the presence of the MC, by the presence of a marker gene, including but not limited to a fluorescent protein, a colored protein, a protein assayable by histochemical assay, and a gene affecting cell morphology. In the use of a DNA-specific dye, all nuclei are stained with a dye including but not limited to DAPI, Hoechst 33258, OliGreen, Giemsa YOYO, or TOTO, allowing a determination of the number of cells that do not contain the MC. After the initial determination of the percent of cells carrying the MC, the cells are allowed to divide over the course of several cell divisions. The number of cell divisions, n, is determined by a method including but not limited to monitoring the change in total weight of cells, and monitoring the change in volume of the cells or by directly counting cells in an aliquot of the culture. After a number of cell divisions, the population of cells is again assayed for the presence of the MC. The loss rate per generation is calculated by the equation (1):

Loss rate per generation=1−(F/I)1/n  (1)

The population of MC-containing cells may include suspension cells, callus, roots, leaves, meristems, flowers, or any other tissue of modified plants, or any other cell type containing a MC.
These methods are well-known and there are many possible variations known to those skilled in the art; they have been used before with human cells and yeast cells.
Assay #3: Lineage Based Inheritance Assays on Modified Cells and Plants
MC inheritance is assessed on cell lines and plants comprising the MCs by following the presence of the MC over the course of multiple cell divisions. In cell types that allow for tracking of cell lineage, including but not limited to root or leaf cell files, trichomes, and leaf stomata guard cells, MC loss per generation does not need to be determined statistically over a population, it can be discerned directly through successive cell divisions. In other manifestations of this method, cell lineage can be discerned from cell position, or methods including but not limited to the use of histological lineage tracing dyes, and the induction of genetic mosaics in dividing cells.
In one simple example, the two guard cells of the stomata are daughters of a single precursor cell. To assay MC inheritance in this cell type, the epidermis of the leaf of a plant containing a MC is examined for the presence of the MC by the presence of a marker gene, including but not limited to a fluorescent protein, a colored protein, a protein assayable by histochemical assay, and a gene affecting cell morphology. The number of loss events in which one guard cell contains the MC (L) and the number of cell divisions in which both guard cells contain the MC (B) are counted. The loss rate per cell division is determined as L/(L+B). Other lineage-based cell types are assayed in similar fashion. These methods are well-known and there are many possible variations known to those skilled in the art; they have been used before with yeast cells (though, instead of observing the marker in stomates, a color marker was observed in yeast colonies).
Linear MC inheritance may also be assessed by examining leaf or root files or clustered cells in callus over time. Changes in the percent of cells carrying the MC will indicate the mitotic inheritance.
Assay #4: Inheritance Assays on Modified Cells and Plants in the Presence of Chromosome Loss Agents
Any of the above three assays can be done in the presence of chromosome loss agents (including but not limited to colchicine, colcemid, caffeine, etopocide, nocodazole, oryzalin, trifluran). It is likely that an autonomous MC will prove more susceptible to loss induced by chromosome loss agents; therefore, autonomous MCs should show a lower rate of inheritance in the presence of chromosome loss agents. These methods have been used to study chromosome loss in fruit flies and yeast; there are many possible variations known to those skilled in the art.
III. Transformation of Plant Cells and Plant Regeneration
Various methods may be used to deliver DNA into plant cells. These include biological methods, such as Agrobacterium, E. coli, and viruses, physical methods such as biolistic particle bombardment, nanocopoea device, the Stein beam gun, silicon carbide whiskers and microinjection, electrical methods such as electroporation, and chemical methods such as the use of poly-ethylene glycol and other compounds known to stimulate DNA uptake into cells. Examples of these techniques have been described (Paszkowski et al., EMBO J. 3:2717-2722, 1984); Potrykus et al., Mol. Gen. Genet. 199:169-177. 1985; Reich et al., Biotechnol. 4:1001-1004; 1986; and Klein et al., Nature 327:70-73, 1987). Transformation using silicon carbide whiskers, e.g. in maize, is described in Brisibe (J. Exp. Bot. 51:187-196, 2000) and Dunwell (Methods Mol. Biol. 111:375-82, 1999) and U.S. Pat. No. 5,464,765.
Agrobacterium-Mediated Delivery
Agrobacterium-mediated transformation is one method for introducing a desired genetic element into a plant. Several Agrobacterium species mediate the transfer of a specific DNA known as “T-DNA” that can be genetically engineered to carry a desired piece of DNA into many plant species. Plasmids used for delivery contain the T-DNA flanking the nucleic acid to be inserted into the plant. The major events marking the process of T-DNA mediated pathogenesis are induction of virulence genes, processing and transfer of T-DNA.
There are three common methods to transform plant cells with Agrobacterium. The first method is co-cultivation of Agrobacterium with cultured isolated protoplasts. This method requires an established culture system that allows culturing protoplasts and plant regeneration from cultured protoplasts. The second method is transformation of cells or tissues with Agrobacterium. This method requires (a) that the plant cells or tissues can be modified by Agrobacterium and (b) that the modified cells or tissues can be induced to regenerate into whole plants. The third method is transformation of seeds, immature or mature embryos, apices or meristems with Agrobacterium. This method requires exposure of the meristematic cells of these tissues to Agrobacterium and micropropagation of the shoots or plan organs arising from these meristematic cells.
Those of skill in the art are familiar with procedures for growth and suitable culture conditions for Agrobacterium as well as subsequent inoculation procedures. Liquid, solid or semi-solid culture media can be used. The density of the Agrobacterium culture used for inoculation and the ratio of Agrobacterium cells to explant can vary from one system to the next, as can media, growth procedures, timing and lighting conditions.
Transformation of dicotyledons using Agrobacterium has long been known in the art, and transformation of monocotyledons using Agrobacterium has also been described. See, WO 94/00977 and U.S. Pat. No. 5,591,616, both of which are incorporated herein by reference. See also, Negrotto et al. (Plant Cell Rep. 19:798-803, 2000, incorporated herein by reference).
A number of wild-type and disarmed strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes harboring Ti or Ri plasmids can be used for gene transfer into plants. Preferably, the Agrobacterium hosts contain disarmed Ti and Ri plasmids that do not contain the oncogenes that cause tumorigenesis or rhizogenesis. Exemplary strains include Agrobacterium tumefaciens strain C58, a nopaline-type strain that is used to mediate the transfer of DNA into a plant cell, octopine-type strains such as LBA4404 or succinamopine-type strains, e.g., EHA101 or EHA105. The use of these strains for plant transformation has been reported and the methods are familiar to those of skill in the art.
US Application No. 20040244075 published Dec. 2, 2004 describes improved methods of Agrobacterium-mediated transformation. The efficiency of transformation by Agrobacterium may be enhanced by using a number of methods known in the art. For example, the inclusion of a natural wound response molecule such as acetosyringone (AS) to the Agrobacterium culture has been shown to enhance transformation efficiency with Agrobacterium tumefaciens (Shahla et al., (1987) Plant Molec. Biol. 8:291-298). Alternatively, transformation efficiency may be enhanced by wounding the target tissue to be modified or transformed. Wounding of plant tissue may be achieved, for example, by punching, maceration, bombardment with microprojectiles, etc. (See e.g., Bidney et al., Plant Molec. Biol. 18:301-313, 1992).
In addition, another recent method described by Broothaerts, et al. (Nature 433:629-633, 2005) expands the bacterial genera that can be used to transfer genes into plants. This work involved the transfer of a disarmed Ti plasmid without T-DNA and another vector with T-DNA containing the marker enzyme beta-glucuronidase, into three different bacteria. Gene transfer was successful and this method significantly expands the tools available for gene delivery into plants.
Microprojectile Bombardment Delivery
Another widely used technique to genetically transform plants involves the use of microprojectile bombardment. In this process, a nucleic acid containing the desired genetic elements to be introduced into the plant is deposited on or in small dense particles, e.g., tungsten, platinum, or preferably 0.5 to 1.0 micron gold particles, which are then delivered at a high velocity into the plant tissue or plant cells using a specialized biolistics device. Many such devices have been designed and constructed; one in particular, the PDS1000/He sold by Bio-Rad Laboratories (Hercules, Calif.; USA), is the instrument most commonly used for biolistics of plant cells. The advantage of this method is that no specialized sequences need to be present on the nucleic acid molecule to be delivered into plant cells; delivery of any nucleic acid sequence is theoretically possible.
For the bombardment, cells in suspension are concentrated on filters, petri dishes or solid culture medium. Alternatively, immature embryos, seedling explants, or any plant tissue or target cells may be arranged on filters, petri dishes or solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the microprojectile stopping plate.
Various biolistics protocols have been described that differ in the type of particle or the manner in which DNA is coated onto the particle. Any technique for coating microprojectiles that allows for delivery of transforming DNA to the target cells may be used. For example, particles may be prepared by functionalizing the surface of a gold particle by providing free amine groups. DNA, having a strong negative charge, will then bind to the functionalized particles.
Parameters such as the concentration of DNA used to coat microprojectiles may influence the recovery of transformants containing a single copy of the transgene. For example, a lower concentration of DNA may not necessarily change the efficiency of the transformation but may instead increase the proportion of single copy insertion events. In this regard, ranges of approximately 1 ng to approximately 10 μg (10,000 ng), approximately 5 ng to 8 μg or approximately 20 ng, 50 ng, 100 ng, 200 ng, 500 ng, 1 μg, 2 μg, 5 μg, or 7 μg of transforming DNA may be used per each 1.0-2.0 mg of starting gold particles (in the 0.5 to 1.0 micron range).
Other physical and biological parameters may be varied, such as manipulation of the DNA/microprojectile precipitate, factors that affect the flight and velocity of the projectiles, manipulation of the cells before and immediately after bombardment (including osmotic state, tissue hydration and the subculture stage or cell cycle of the recipient cells), the orientation of an immature embryo or other target tissue relative to the particle trajectory, and also the nature of the transforming DNA, such as linearized DNA or intact supercoiled plasmids. One may also want to use agents to protect the DNA during delivery. One may particularly wish to adjust physical parameters such as DNA concentration, gap distance, flight distance, tissue distance, and helium pressure.
The particles delivered via biolistics can be “dry” or “wet.” In the “dry” method, the MC DNA-coated particles such as gold are applied onto a macrocarrier (such as a metal plate, or a carrier sheet made of a fragile material such as mylar) and dried. The gas discharge then accelerates the macrocarrier into a stopping screen, which halts the macrocarrier but allows the particles to pass through; the particles then continue their trajectory until they impact the tissue being bombarded. For the “wet” method, the droplet containing the MC DNA-coated particles is applied to the bottom part of a filter holder, which is attached to a base which is itself attached to a rupture disk holder used to hold the rupture disk to the helium egress tube for bombardment. The gas discharge directly displaces the DNA/gold droplet from the filter holder and accelerates the particles and their DNA cargo into the tissue being bombarded. The wet biolistics method has been described in detail elsewhere but has not previously been applied in the context of plants (Mialhe et al., Mol Mar Biol Biotechnol. 4(4):275-83, 1995). The concentrations of the various components for coating particles and the physical parameters for delivery can be optimized using procedures known in the art.
A variety of plant cells/tissues are suitable for transformation, including immature embryos, scutellar tissue, suspension cell cultures, immature inflorescence, shoot meristem, epithelial peels, nodal explants, callus tissue, hypocotyl tissue, cotyledons, roots, leaves, meristem cells, and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspore-derived embryos, roots, hypocotyls, cotyledons and the like. Those cells which are capable of proliferating as callus also are recipient cells for genetic transformation.
Any suitable plant culture medium can be used. Examples of suitable media would include but are not limited to MS-based media (Murashige and Skoog, Physiol. Plant, 15:473-497, 1962) or N6-based media (Chu et al., Scientia Sinica 18:659, 1975) supplemented with additional plant growth regulators including but not limited to auxins such as picloram (4-amino-3,5,6-trichloropicolinic acid), 2,4-D (2,4-dichlorophenoxyacetic acid), naphalene-acetic acid (NAA) and dicamba (3,6-dichloroanisic acid), cytokinins such as BAP (6-benzylaminopurine) and kinetin, and gibberellins. Other media additives can include but are not limited to amino acids, macroelements, iron, microelements, vitamins and organics, carbohydrates, undefined media components such as casein hydrolysates, an appropriate gelling agent such as a form of agar, a low melting point agarose or Gelrite if desired. Those of skill in the art are familiar with the variety of tissue culture media, which when supplemented appropriately, support plant tissue growth and development and are suitable for plant transformation and regeneration. These tissue culture media can either be purchased as a commercial preparation, or custom prepared and modified. Examples of such media would include but are not limited to Murashige and Skoog, N6, Linsmaier and Skoog (Physio. Plant, 18:100, 1965), Uchimiya and Murashige (Plant Physiol. 15:473, 1962), Gamborg's B5 media (Exp. Cell Res., 50:151, 1968), D medium (Duncan et al., Planta, 165:322-332, 1985), Mc-Cown's Woody plant media (McCown and Lloyd, HortScience 6:453, 1981), Nitsch and Nitsch (Science 163:85-87, 1969), and Schenk and Hildebrandt (Can. J. Bot. 50:199-204, 1972) or derivations of these media supplemented accordingly. Those of skill in the art are aware that media and media supplements such as nutrients and growth regulators for use in transformation and regeneration and other culture conditions such as light intensity during incubation, pH, and incubation temperatures can be varied.
Those of skill in the art are aware of the numerous modifications in selective regimes, media, and growth conditions that can be varied depending on the plant system and the selective agent. Typical selective agents include but are not limited to antibiotics such as geneticin (G418), kanamycin, paromomycin or other chemicals such as glyphosate or other herbicides. Consequently, such media and culture conditions disclosed in the present invention can be modified or substituted with nutritionally equivalent components, or similar processes for selection and recovery of transgenic events, and still fall within the scope of the present invention.
MC Delivery without Selection
The MC is delivered to plant cells or tissues, e.g., plant cells in suspension to obtain stably modified callus clones for inheritance assay. Suspension cells are maintained in a growth media, for example MS liquid medium containing an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D). Cells are bombarded using a particle bombardment process, such as the helium-driven PDS-1000/He system, and propagated in the same liquid medium to permit the growth of modified and non-modified cells. Portions of each bombardment are monitored for formation of fluorescent clusters, which are isolated by micromanipulation and cultured on solid medium. Clones modified with the MC are expanded and homogenous clones are used in inheritance assays, or assays measuring MC structure or autonomy.
MC Transformation with Selectable Marker Gene
Isolation of MC-modified cells in bombarded calluses or explants can be facilitated by the use of a selectable marker gene. The bombarded tissues are transferred to a medium containing an appropriate selective agent for a particular selectable marker gene. Such a transfer usually occurs between 0 and about 7 days after bombardment. The transfer could also take place any number of days after bombardment. The amount of selective agent and timing of incorporation of such an agent in selection medium can be optimized by using procedures known in the art. Selection inhibits the growth of non-modified cells, thus providing an advantage to the growth of modified cells, which can be further monitored by tracking the presence of a fluorescent marker gene or by the appearance of modified explants (modified cells or explants may be green under light in selection medium, while surrounding non-modified cells are weakly pigmented). In plants that develop through shoot organogenesis, the modified cells can form shoots directly, or alternatively, can be isolated and expanded for regeneration of multiple shoots transgenic for the MC. In plants that develop through embryogenesis, additional culturing steps may be necessary. Sorghum can be regenerated through embryogenesis (Wernicke & Brettell, Nature 287:138-139, 1990; and Bhaskaran and Smith, In Vitro Cell. Devel. Biol. Plant 24:65-70, 1987) and can also be regenerated by shoot organogenesis (Nirwan and Kothari, J. Plant Biochem. Biotech., 13:149-152, 2004).
Useful selectable marker genes are well known in the art and include, for example, herbicide and antibiotic resistance genes including but not limited to neomycin phosphotransferase II (conferring resistance to kanamycin, paramomycin and G418), hygromycin phosphotransferase (conferring resistance to hygromycin), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, conferring resistance to glyphosate), phosphinothricin acetyltransferase (conferring resistance to phosphinothricin/bialophos), MerA (conferring resistance to mercuric ions). Selectable marker genes may be transformed using standard methods in the art.
The first step in the production of plants containing novel genes involves delivery of DNA into a suitable plant tissue (described in the previous section) and selection of the tissue under conditions that allow preferential growth of any cells containing the novel genes. Selection is typically achieved with a selectable marker gene present in the delivered DNA, which may be a gene conferring resistance to an antibiotic, herbicide or other killing agent, or a gene allowing utilization of a carbon source not normally metabolized by plant cells. For selection to be effective, the plant cells or tissue need to be grown on selective medium containing the appropriate concentration of antibiotic or killing agent, and the cells need to be plated at a defined and constant density. The concentration of selective agent and cell density are generally chosen to cause complete growth inhibition of wild type plant tissue that does not express the selectable marker gene; but allowing cells containing the introduced DNA to grow and expand into adchromosomal clones. This critical concentration of selective agent typically is the lowest concentration at which there is complete growth inhibition of wild type cells, at the cell density used in the experiments. However, in some cases, sub-killing concentrations of the selective agent may be equally or more effective for the isolation of plant cells containing MC DNA, especially in cases where the identification of such cells is assisted by a visible marker gene (e.g., fluorescent protein gene) present on the MC. Such sub-killing concentrations of the selective agent may be administered during part or all of the selection timing.
In some species (e.g., tobacco or tomato), a homogenous clone of modified cells can also arise spontaneously when bombarded cells are placed under the appropriate selection. An exemplary selective agent is the neomycin phosphotransferase II (nptII) marker gene, which is commonly used in plant biotechnology and confers resistance to the antibiotics kanamycin, G418 (geneticin) and paramomycin. In other species, or in certain plant tissues or when using particular selectable markers, homogeneous clones may not arise spontaneously under selection; in this case the clusters of modified cells can be manipulated to homogeneity using the visible marker genes present on the MCs as an indication of which cells contain MC DNA.
Regeneration of Modified Plants from Explants to Mature, Rooted Plants
In instances where shoot organogenesis is less efficient or for other reasons undesirable, an embryogenic step is necessary for regeneration. In these cases explant tissue is cultured on an appropriate media for embryogenesis, and the embryo is cultured until shoots form. The regenerated shoots are cultured in a rooting medium to obtain intact whole plants with a fully developed root system. These plants are potted in soil and grown to maturity in a greenhouse.
Generally, regeneration and tissue culture of sorghum plant parts and whole plants is challenging as sorghum produces phenolic compounds while in culture. The present invention provides for methods of culturing sorghum cells and tissues in media containing polyvinylpyrrolidone (PVP), see Examples, below. The PVP acts as a sink for the phenolic compounds produced by sorghum and enhances callus growth during selection as well as facilitating callus and plantlet regeneration. Furthermore, generation of sorghum callus can be facilitated by delivering to the plant cells and/or tissues MCs of the invention that contain auxin genes. The presence of the auxin genes will facilitate callus induction of the transformed tissue. The invention also provides for tissue culture methods which cycle between the liquid culture media and solid culture media in order to promote the frequency and the morphogenic competence of the regenerable sorghum callus.
For plants that develop through shoot organogenesis, regeneration of a whole plant involves culturing of regenerable explant tissues taken from sterile organogenic callus tissue, seedlings or mature plants on a shoot regeneration medium for shoot organogenesis, and rooting of the regenerated shoots in a rooting medium to obtain intact whole plants with a fully developed root system. These plants are potted in soil and grown to maturity in a greenhouse.
Explants are obtained from any tissues of a plant suitable for regeneration. Exemplary tissues include hypocotyls, internodes, roots, cotyledons, petioles, cotyledonary petioles, leaves and peduncles, prepared from sterile seedlings or mature plants.
Explants are wounded (for example with a scalpel or razor blade) and cultured on a shoot regeneration medium (SRM) containing Murashige and Skoog (MS) medium as well as a cytokinin, e.g., 6-benzylaminopurine (BA), and an auxin, e.g., α-naphthaleneacetic acid (NAA), and an anti-ethylene agent, e.g., silver nitrate (AgNO3). For example, 2 mg/L of BA, 0.05 mg/L of NAA, and 2 mg/L of AgNO3 can be added to MS medium for shoot organogenesis. The most efficient shoot regeneration is obtained from longitudinal sections of internode explants.
Shoots regenerated via organogenesis are rooted in a MS medium. Plants are potted and grown in a greenhouse to sexual maturity for seed harvest.
To regenerate a whole plant with a MC, explants are pre-incubated for 1 to 7 days (or longer) on the shoot regeneration medium prior to bombardment with MC (see below). Following bombardment, explants are incubated on the same shoot regeneration medium for a recovery period up to 7 days (or longer), followed by selection for transformed shoots or clusters on the same medium but with a selective agent appropriate for a particular selectable marker gene (see below).
Method of Co-Delivering Growth Inducing Genes to Facilitate Isolation of Modified Plant Cell Clones
Another method used in the generation of cell clones containing MCs involves the co-delivery of DNA containing genes that are capable of activating growth of plant cells, or that promote the formation of a specific organ, embryo or plant structure that is capable of self-sustaining growth. In one embodiment, the recipient cell receives simultaneously the MC, and a separate DNA molecule encoding one or more growth promoting, organogenesis-promoting, embryogenesis-promoting or regeneration-promoting genes. Following DNA delivery, expression of the plant growth regulator genes stimulates the plant cells to divide, or to initiate differentiation into a specific organ, embryo, or other cell types or tissues capable of regeneration. Multiple plant growth regulator genes can be combined on the same molecule, or co-bombarded on separate molecules. Use of these genes can also be combined with application of plant growth regulator molecules into the medium used to culture the plant cells, or of precursors to such molecules that are converted to functional plant growth regulators by the plant cell's biosynthetic machinery, or by the genes delivered into the plant cell.
The co-bombardment strategy of MCs with separate DNA molecules encoding plant growth regulators transiently supplies the plant growth regulator genes for several generations of plant cells following DNA delivery. During this time, the MC may be stabilized by virtue of its centromere, but the DNA molecules encoding plant growth regulator genes, or organogenesis-promoting, embryogenesis-promoting or regeneration-promoting genes will tend to be lost. The transient expression of these genes, prior to their loss, may give the cells containing MC DNA a sufficient growth advantage, or sufficient tendency to develop into plant organs, embryos or a regenerable cell cluster, to outgrow the non-modified cells in their vicinity, or to form a readily identifiable structure that is not formed by non-modified cells. Loss of the DNA molecule encoding these genes will prevent phenotypes from manifesting themselves that may be caused by these genes if present through the remainder of plant regeneration. In rare cases, the DNA molecules encoding plant growth regulator genes will integrate into the host plant's genome or into the MC.
Alternatively the genes promoting plant cell growth may be genes promoting shoot formation or embryogenesis, or giving rise to any identifiable organ, tissue or structure that can be regenerated into a plant. In this case, it may be possible to obtain embryos or shoots harboring MCs directly after DNA delivery, without the need to induce shoot formation with growth activators supplied into the medium, or lowering the growth activator treatment necessary to regenerate plants. The advantages of this method are more rapid regeneration, higher transformation efficiency, lower background growth of non-modified tissue, and lower rates of morphologic abnormalities in the regenerated plants (due to shorter and less intense treatments of the tissue with chemical plant growth activators added to the growth medium).
Determination of MC Structure and Autonomy in Adchromosomal Plants and Tissues
The structure and autonomy of the MC in adchromosomal plants and tissues can be determined by methods including but not limited to: conventional and pulsed-field Southern blot hybridization to genomic DNA from modified tissue subjected or not subjected to restriction endonuclease digestion, dot blot hybridization of genomic DNA from modified tissue hybridized with different MC specific sequences, MC rescue, exonucleas activity, PCR on DNA from modified tissues with probes specific to the MC, or Fluorescence Hybridization (FISH) to nuclei of modified cells. Table 3 below summarizes these methods.







TABLE 3







Examples of methods to determin MC structure and autonomy










Assay
Assay details
Potential outcome
Interpretation





Southern blot
Restriction digest of
1. Native sizes and pattern 
1. Autonomous or integrated via



genomic DNA* compared
of bands
CEN fragment



to purified MC
2. Altered sizes or pattern 
2. Integrated or rearranged




of bands



CHEF gel
Restriction digest of
1. Native sizes and pattern 
1. Autonomous or integrated via


Southern blot
genomic DNA compared
of bands
CEN fragment



to purified MC
2. Altered sizes or pattern 
2. Integrated or rearranged




of bands




Native genomic DNA (no
1. MC band migrating
1. Autonomous circles or linears



digest)
ahead of genomic DNA
present in plant




2. MC band co-migrating 
2. Integrated




with genomic DNA





3. >1 MC bands observed
3. Various possibilities


Exonuclease
Exonuclease digestion of
1. Signal strength close to
1. Autonomous circles present


assay
genomic DNA followed
that w/o exonuclease




by detection of circular
2. No signal or signal
2. Integrated



MC by PCR, dot blot, or
strength lower that w/o




restriction digest
exonuclease




(optional),





electrophoresis and





southern blot (useful for





circular MCs)




MC
Transformation of plant
1. Colonies isolated only 
1. Autonomous circles present,


rescue
genomic DNA into E. coli
from MC plants with MCs, not
native MC structure



followed by selection for
from controls; MC structure




antibiotic resistance
matches that of the parental




genes on MC
MC





2. Colonies isolated only from
2. Autonomous circles present,




MC plants with MCs, not from
rearranged MC structure OR MCs




controls; MC structure
integrated via centromere




different from parental MC
fragment




3. Colonies observed both in
3. Various possibilities




MC-modified plants and in





controls



PCR
PCR amplification of
1. All MC parts detected by
1. Complete MC sequences



various parts of the MC
PCR
present in plant




2. Subset of MC parts
2. Partial MC sequences present in




detected by PCR
plant


FISH
Detection of MC
1. MC sequences detected,
1. Autonomous



sequences in mitotic or
free of genome




meiotic nuclei by
2. MC sequences detected,
2. Integrated



fluorescence in situ
associated with genome




hybridization
3. MC sequences detected,
3. Both autonomous and




both free and associated
integrated MC sequences present




with genome





4. No MC sequences detected
4. MC DNA not visible by FISH





*Genomic DNA refers to total DNA extracted from plants containing a MC






Furthermore, MC structure can be examined by characterizing MCs ‘rescued’ from adchromosomal cells. Circular MCs that contain bacterial sequences for their selection and propagation in bacteria can be rescued from an adchromosomal plant or plant cell and re-introduced into bacteria. If no loss of sequences has occurred during replication of the MC in plant cells, the MC is able to replicate in bacteria and confer antibiotic resistance. Total genomic DNA is isolated from the adchromosomal plant cells by any method for DNA isolation known to those skilled in the art, including but not limited to a standard cetyltrimethylammonium bromide (CTAB) based method (Current Protocols in Molecular Biology. John Wiley & Sons, NY, 1994 et seq.) The purified genomic DNA is introduced into bacteria (e.g., E. coli) using methods familiar to one skilled in the art (for example heat shock or electroporation). The transformed bacteria are plated on solid medium containing antibiotics to select bacterial clones modified with MC DNA. Modified bacterial clones are grown up, the plasmid DNA purified (by alkaline lysis for example), and DNA analyzed by restriction enzyme digestion and gel electrophoresis or by sequencing. Because plant-methylated DNA containing methylcytosine residues will be degraded by wild-type strains of E. coli, bacterial strains (e.g. DH10B) deficient in the genes encoding methylation restriction nucleases (e.g. the mcr and mrr gene loci in E. coli) are best suited for this type of analysis. MC rescue can be performed on any plant tissue or clone of plant cells comprising a MC.
MC Autonomy Demonstration by In Situ Hybridization (ISH)
To assess whether the MC is autonomous from the native plant chromosomes, or has integrated into the plant genome, In Situ Hybridization is carried out (Fluorescent In Situ Hybridization or FISH is particularly well suited to this purpose). In this assay, mitotic or meiotic tissue, such as root tips or meiocytes from the anther, possibly treated with metaphase arrest agents such as colchicines or nitrous oxide is obtained, and standard FISH methods are used to label both the centromere and sequences specific to the MC. For example, a sorghum centromere is labeled using a probe from a sequence that labels all sorghum centromeres, attached to one fluorescent tag (Molecular Probes Alexafluor 568, for example), and sequences specific to the MC are labeled with another fluorescent tag (Alexafluor 488, for example). All centromere sequences are detected with the first tag; only MCs are detected with both the first and second tag. Chromosomes are stained with a DNA-specific dye including but not limited to DAPI, Hoechst 33258, OliGreen, Giemsa YOYO, and TOTO. An autonomous MC is visualized as a body that shows hybridization signal with both centromere probes and MC specific probes and is separate from the native chromosomes.
Determination of Gene Expression Levels
The expression level of any gene present on the MC can be determined by methods including but not limited to one of the following. The mRNA level of the gene can be determined by Northern Blot hybridization, Reverse Transcriptase-Polymerase Chain Reaction, binding levels of a specific RNA-binding protein, in situ hybridization, or dot blot hybridization.
The protein level of the gene product can be determined by Western blot hybridization, Enzyme-Linked Immunosorbant Assay (ELISA), fluorescent quantitation of a fluorescent gene product, enzymatic quantitation of an enzymatic gene product, immunohistochemical quantitation, or spectroscopic quantitation of a gene product that absorbs a specific wavelength of light.
Use of Exonuclease to Isolate Circular MC DNA from Genomic DNA
Exonucleases may be used to obtain pure MC DNA, suitable for isolation of MCs from E. coli or from plant cells. The method assumes a circular structure of the MC. A DNA preparation containing MC DNA and genomic DNA from the source organism is treated with exonuclease, for example lambda exonuclease combined with E. coli exonuclease I, or the ATP-dependent exonuclease (Qiagen Inc.; Valencia, Calif.; USA). Because the exonuclease is only active on DNA ends, it will specifically degrade the linear genomic DNA fragments, but will not affect the circular MC DNA. The result is MC DNA in pure form. The resultant MC DNA can be detected by a number of methods for DNA detection known to those skilled in the art, including but not limited to PCR, dot blot followed by hybridization analysis, and southern blot followed by hybridization analysis. Exonuclease treatment followed by detection of resultant circular MC may be used as a method to determine MC autonomy.
Structural Analysis of MCs by BAC-End Sequencing
BAC-end sequencing procedures, known to those skilled in the art, can be applied to characterize MC clones for a variety of purposes, such as structural characterization, determination of sequence content, and determination of the precise sequence at a unique site on the chromosome (for example the specific sequence signature found at the junction between a centromere fragment and the vector sequences). In particular, this method is useful to prove the relationship between a parental MC and the MCs descended from it and isolated from plant cells by MC rescue, described above. This method also fosters identification of specific sorghum MCs if more than one unique sorghum MC is present in a plant cell simultaneously.
Methods for Scoring Meiotic MC Inheritance
A variety of methods can be used to assess the efficiency of meiotic MC transmission. In one embodiment of the method, gene expression of genes encoded by the MC (marker genes or non-marker genes) can be scored by any method for detection of gene expression known to those skilled in the art, including but not limited to visible methods (e.g. fluorescence of fluorescent protein markers, scoring of visible phenotypes of the plant), scoring resistance of the plant or plant tissues to antibiotics, herbicides or other selective agents, by measuring enzyme activity of proteins encoded by the MC, or measuring non-visible plant phenotypes, or directly measuring the RNA and protein products of gene expression using microarray, northern blots, in situ hybridization, dot blot hybridization, RT-PCR, western blots, immunoprecipitation, Enzyme-Linked Immunosorbant Assay (ELISA), immunofluorescence and radio-immunoassays (RIA). Gene expression can be scored in the post-meiotic stages of microspore, pollen, pollen tube or female gametophyte, or the post-zygotic stages such as embryo, seed, or progeny seedlings and plants. In another embodiment of the method, the MC can de directly detected or visualized in post-meiotic, zygotic, embryonal or other cells in by a number of methods for DNA detection known to those skilled in the art, including but not limited to fluorescence in situ hybridization, in situ PCR, PCR, southern blot, or by MC rescue described above.
FISH Analysis of MC Copy Number in Meiocytes, Roots or Other Tissues of Adchromosomal Plants
The copy number of the MC can be assessed in any cell or plant tissue by FISH is particularly well suited to this purpose). In an exemplary assay, standard FISH methods are used to label the centromere, using a probe which labels all chromosomes with one fluorescent tag (e.g., ALEXA FLUOR® 568; Invitrogen Corp.; Carlsbad, Calif.; USA), and to label sequences specific to the MC with another fluorescent tag (ALEXA FLUOR® 488, for example). All centromere sequences are detected with the first tag; only MCs are detected with both the first and second tag. Nuclei are stained with a DNA-specific dye including but not limited to DAPI, Hoechst 33258, OliGreen, Giemsa YOYO, and TOTO. MC copy number is determined by counting the number of fluorescent foci per cell that label with both tags.
Induction of Callus and Roots from Adchromosomal Plants Tissues for Inheritance Assays
MC inheritance is assessed using callus and roots induced from transformed plants. To induce roots and callus, tissues such as leaf pieces are prepared from adchromosomal plants and cultured on a MS or N6 medium that may contain a cytokinin, e.g., 6-benzylaminopurine (BA), and an auxin, e.g., α-naphthaleneacetic acid (NAA). Any tissue of an adchromosomal plant can be used for callus and root induction, and the medium recipe for tissue culture can be optimized using procedures known in the art.
Clonal Propagation of Adchromosomal Plants
To produce multiple clones of plants from a MC-transformed plant, any tissue of the plant can be tissue-cultured for shoot organogenesis using regeneration procedures described under the section regeneration of plants from explants to mature, rooted plants (see above). Alternatively, multiple auxiliary buds can induced from a MC-modified plant by excising the shoot tip, which can be rooted and subsequently be grown into a whole plant; each auxiliary bud can be rooted and produce a whole plant. Additionally, multiple shoots that result from one plant can be subdivided in culture to produce multiple individual plants.
Scoring of Antibiotic- or Herbicide Resistance in Seedlings and Plants (Progeny of Self- and Out-Crossed Transformants)
Progeny seeds harvested from MC-modified plants can be scored for antibiotic- or herbicide resistance by seed germination under sterile conditions on a growth media (for example MS medium) containing an appropriate selective agent for a particular selectable marker gene. Only seeds containing the MC can germinate on the medium and further grow and develop into whole plants. Alternatively, seeds can be germinated in soil, and the germinating seedlings can then be sprayed with a selective agent appropriate for a selectable marker gene. Seedlings that do not contain MC do not survive; only seedlings containing MC can survive and develop into mature plants.
Genetic Methods for Analyzing MC Performance
Though sorghum is typically propagated vegitatively, it is possible to use sexual propagation techniques as well. In addition to direct transformation of a plant with a MC, plants containing a MChromsome can be prepared by crossing a first plant containing the functional, stable, autonomous MC with a second plant lacking the MC.
Fertile plants modified with MCs can be crossed to other plant lines to study MC performance and inheritance. In the first embodiment of this method, pollen from an adchromosomal plant can be used to fertilize the stigma of a non-adchromosomal plant. MC presence is scored in the progeny of this cross using the methods outlined in the preceding section. In the second embodiment, the reciprocal cross is performed by using pollen from a non-adchromosomal plant to fertilize the flowers of an adchromosomal plant. The rate of MC inheritance in both crosses can be used to establish the frequencies of meiotic inheritance in male and female meiosis. In a third embodiment of this method, pollen for an adchromosomal plant is used to fertilize another or the same adchromosomal plant (e.g. self or sibling pollination). In the fourth embodiment of this method, the progeny of one of the crosses just described are back-crossed to a non-adchromosomal parental line, and the progeny of this second cross are scored for the presence of genetic markers in the plant's natural chromosomes as well as the MC. Scoring of a sufficient marker set against a sufficiently large set of progeny allows the determination of linkage or co-segregation of the MC to specific chromosomes or chromosomal loci in the plant's genome. Genetic crosses performed for testing genetic linkage can be done with a variety of combinations of parental lines; such variations of the methods described are known to those skilled in the art.
It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.
EXAMPLES
Example 1
Sorghum Centromere Discovery
Identification of Sorghum Satellite Repeat Sequences
The investigators compiled Sorghum repetitive genomic DNA as candidate probes for hybridization with the BAC libraries. Sorghum sequence was extracted from GenBank and analyzed the sequence by homology to a known Sorghum satellite sequence (Zwick, M S, et al. Am J Bot 87: 1757-1764, 2000), set out in SEQ ID NO:177:








tacgtaagct tcgtttcgtc tgtttggaca tagtgctaat ctttatgcaa gatagatgca   60



 


cggtttacgt ggaacatatg atatgctcag aagcaattta ggacgcacct aatataactc  120


 


cttgatgatg tgtgtcacat ggaatcttgc ttcggtttct ttagagacag tgttagtttt  180


 


ggtagaagat atgtgcacag tgtacgccta atgcaccata ggctaaagaa accattttag  240


 


acgcacccga tggtactcgt agttgaagag gctcaactgg aggctcgatt tggtctgttc  300


 


ggatatagtg ctaatcttga tgcaagatag ttgcacaatt tgcaggcaac gtaccatatg  360


 


ttaagaaatc aatttggacg cacccaatgg aactcctaga tgacgtgtgt catatggaac  420


 


tcgcttcggt ctgtttggtg accatattag tttcactgca ggaaaggtgc atagtttgtg  480


 


cctaatgcac catagtctaa gaaaaccatt tttgatgcac ctgtttgtac ttctatgaag  540


 


aggctcaagt ggaagctcgg ttcggtctgt ttggagatag tgctaatctt gatgcaagat  600


 


aggtgtacgg tttgtatgga acataccata tgcttggaaa tcaatttgga tgcacccgtt  660


 


ggaactcctt gagaagtgtg tcttatgtac cctcgctttg gtctgtttag aaatagtgtt  720


 


agtttcagtg caagatatga gcatggtttg cgcctaacgc accatagtct aagaaaccat  780


 


tttggaagca cctgttggta cttcggtgaa gaagctcaag tggaagctcg gtttgacctg  840


 


tttggagata gtgctaatct tgatgcaaga tagtgcatga tttgcaagga acataccata  900


 


tgcttagaaa tcaacttgga cgcacgcccc gcaactccta catcacgtgt gtcatatgga  960


 


atcttacttc ggtccatttg taacattgta agttttagtg caag                  1004






The investigators identified the sequences listed in Table 4 (SEQ ID NOs:23-176). Each sequence represents a different repetitive DNA sequence from S. bicolor.







TABLE 4







Sorghum satellite sequences








SEQ ID



NO:
Nucleic acid sequence












23
aaactgagct tccacttgag cccctttacc caggagtatc atcgggtgca tccaaaatgg
60



tttcttagcc aatgatgcat taggcacaaa ttgtgtacct atcttgtacc aaaactaact
120



ctgtctccaa aca
133


 


24
aaactgagct tccacttgag cccctttaca caggagtatc atcgggtgca tccaaaatgg
60



tttcttagcc aatgatgcat taggcgcaaa ttgtgtacct atcttgtacc aaaactaact
120



ctgtctccaa aca
133


 


25
caaactgagc ttccacttga gcccctttac ctaggagtat gatcaggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgtac caaaactaac
120



tctgtctcca aac
133


 


26
caaactgagc ttccacttga gcccctttac ccaggagtat cttcaggtgc atccaaaatt
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtatc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


27
caaactgagc ttccacttga gcacctttac ccacgagtat catcgggctc atccaaaatg
60



gtttcttagc ccatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


28
caaactgagc ttccacttga gcccctgtac ccaggagtat catcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac taaaactacc
120



tttgtctcca aac
133


 


29
gaaccgacct tccacttgag cctctccacc taggattatc atcgggtgct tgcataatgg
60



tttctgagcc tatggtgcat tatgcgcaaa ccatgcacca atcctgcacc taaactaaca
120



ctgtctccaa aca
133


 


30
caaactgagc ttccacatga gcccctttac ccaggagtat attcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa agtatgtacc gatcttgcac caaaactaac
120



tctgtctcca aac
133


 


31
caaactgaga ttccacttga gcccctttac cggggagtat catcgtgtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actatgtacc tatcttacac caaaactaac
120



tctgtctcca aac
133


 


32
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggtgc atccaaaatg
60



gtttcttagc ctgtgatgca ttaggagaaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


33
aaactgagct tccacttgag cccctttacc caggagtgtt atcgagtgca tccaaaatag
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtacct atcttgcacc aaaactatct
120



ctgtctccaa aca
133


 


34
aaactgagct tccacttgag cccctttact caggagtatc atcgggttca tccaaaatgg
60



tttcttagcc aatgatgcat ttggcgcaaa ctgtgtacct atctcgcacc aaaactaact
120



ttgtctccaa aca
133


 


35
caaactgagc ttccacttga gcccctttac ccaggagtat catcgagtgc atccaaaata
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactgcc
120



tctgtctcca aac
133


 


36
caaactgagc ttccacttga gcccctttac ccaggagtat catcaggttc atccaaaatt
60



gtttcttagc ctatgatgca ttaggcgtaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


37
caaactgagc ttccacttga gcccctttgc ccaggagtat catcaggttc atccaaaatt
60



gtttcttagc ctttgatgca ttaggcgtaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


38
aaactgagct tccacttgag cccctttacc caggagtatc gtcgggtgca tccaaaatgg
60



tttcttaccc tatgatgcat taggcgcaaa atgtgtacct atcttgaacc aaaactaact
120



ctgtctccaa aca
133


 


39
caaactgagc ttccacttga gcccctttac ccaggagtat gatcgggtgc atctaaaatg
60



gtttcttagc ctatgatgca ttaggcgaaa actgtgtacc tattttgcac caaaactaac
120



tctgtctcca aac
133


 


40
caaactgagc ttccacttga gcccctttac ccaggagtat catcgggtgc atccaaaatg
60



gtttcttagg ctatgatgca ttaggcgcaa actgtgcacc tatcctgtac ctaaactaac
120



actgtctcca aac
133


 


41
caaactgagc ttccacttga gccccattac ctaggagtat gttcgggtgg attcaaaatg
60



gtttcttagc ctatgatgca ttatgcgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


42
aaactgagct tccacttgag cccctatacc tagtagtatc atcgggtgca tccaaaatga
60



tttcttatcc tatgatgcat taggcgcaaa ctgtgtacct atcttgcacc aaaactaact
120



ctgtctccaa aca
133


 


43
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcacag actatgtacc tatcttgcac caaaactaac
120



tccgtctcca aac
133


 


44
caaaccgacc ttccacttca gcctctttac ctaggattat catcgggtgc ttccataatg
60



gtttttgagc ctatggtgca tattgcgcaa accatgcacc aatcttgcat ctaaactaac
120



actgtctcca aac
133


 


45
aaactgagct tccaattgag cccctttacc caggagtatc atcgggtgca aacaaaatgg
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtacct atcttgcacc aaaaataact
120



ctgtctccaa aca
133


 


46
caaactgagc ttccacttga gcccctttac ctaggagtgt catcgggtgc attcaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tttgtctcta aac
133


 


47
caaactgagc ttccatttga gcccctttac ccacgagtat aattgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actgtatacc tatattgcac caaaactacc
120



tctgtctcca aac
133


 


48
caaactgagc ttccacttga gcccctatac cgaggagtat catcgggtgc attcaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac tgaaactaac
120



tctgtctcca aac
133


 


49
caaactgagc tttcacttga gcccctttac ctaggagtat gaacgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcta aac
133


 


50
aaactgagct tccacttgag cccctttaca caggagtatc atcgggtgca tccaaaatgg
60



tttcttagcc tatgatacat aaggcgcaaa ctgtgtatgt atcttgcacc aaatctaact
120



ctatctccaa aca
133


 


51
caaactgagc ttccacttga gcccttttac ccaggagtat catcgagtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactacc
120



tccgtctcca aac
133


 


52
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggtgc atccaaaatg
60



gtttcttcgc ctatgatgca ttaggcgcaa actatgtacc tatcttgcac caaaactaac
120



tttgtctcca aac
133


 


53
aaactgagct tccacttgag cccctttacc caggtgtatc atcgggtgca tccaaactgg
60



tttcttagcc tatgacgcat taggcgcaaa ctgtgtacct atcttgcacc aaaactacct
120



ctgtctccaa aca
133


 


54
caaactgagc ttccacttga gcccctttac ccaggagtat attcgggtgc atccaaaatg
60



gtttcttagc ctatgatgcc ttaggcgcaa agtgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


55
caaactgagc ctccacttga gcccctttac ccaggagtat catcaggtgc atccaaaatg
60



gtttcttagc atatgatgca ttaggcgtaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


56
caaactgagc ttccacttga gcccctttac ccaggagaat caacagatgc atccaaaata
60



gtttcttagc ctttgatgca ttaggtgcaa actgtgtagc tatcttgcac caatactaac
120



tctgtctcca aac
133


 


57
gaactgacct tccacttgag cctctttacc taggattatc atcgggtgct tccataatgg
60



tttctgagcc tatggtgcat tatgcgcaaa ccatgcacca atcttgcacc taaactaaca
120



ctgtctccaa aca
133


 


58
aaactgagct tccacttgag cccttttacc caggagtatc atcgagtgca tccaaaatga
60



tttcttaccc tatgatgcat taggcgcaaa ctgtgaacct atcttgcacc aaaactacct
120



ctgtctccaa aca
133


 


59
caaactgagt ttccacttga gcccctttac ccaggagtat catcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttcggcgcaa attgtgtacc taacttgcac caaaactaac
120



tctgtctcca aac
133


 


60
aaactgagct tccacttgag cccctttacc taggattatc atcgggtgca tccaaaatgg
60



tttcttagcc tattatgcat taggcgtaaa ctgtgtacca atcttgcacc aaaactaact
120



ctctctccaa ac
132


 


61
aaactgagct tccatttgag cccctttacc caggattatc atcgggtgcg tccaaaatgg
60



tttctgagcc tatgatgcat taggtggaaa ctgtgtacct attttgcacc aaaactaact
120



ctgtctccaa aca
133


 


62
accaaactgt gcttccactt aagcctcttc acctaggatt accatcaagt gcatccaaaa
60



tggtttctta gactatggtg aattaggcaa aaactgtgca cctatcttgc accaaaacta
120



acactatgtc caa
133


 


63
caaactgagc ttctgcttga gcccctttac ctaggagtat catcgggtgc atccaaaatg
60



gtttctcagc ctttgatgca ttaggcgtaa actgtgtacc tatgttgcac caaaactaac
120



tctatctcca aac
133


 


64
gatcaaacca agcttccact tgagcccctt ttcctaggag taccattagg tgtgtccaaa
60



aaggttctta gcctatggtg cattaggcgc aaaccattca cctatcttgc acagaaacta
120



atactgtctc aaa
133


 


65
cgaaccgacc ttccacatga gactcttcac ctaggattat catcgggtgc ttccataatg
60



gtttctgtgc ctatggtgca ttatgcgcaa accatgcacc aatcttgcac ctaaactaac
120



actctctcca aac
133


 


66
caaactgagc tttcccttga gccccttgag ccaggagtat catcgggtgc atccaaaatg
60



gtttcttagc tgtatgaagc attaggcgca aactgtgtac ttatcttgca ccaaaactaa
120



ctctgtctcc aaa
133


 


67
caaactgagc ttccacttga gcccctttac cttggagtat caacgggtgc atccaaaatg
60



ttttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tttgtctcca aac
133


 


68
aaactgagct tccacttgag cccctttacc caggagtatc atcgggtgca tccaaaatgg
60



attcttagcc tatgatgcat taggcgtaaa ctgtgtacct ttcttgtacc aaaactaact
120



ctgtctccaa aca
133


 


69
caaactgagc ttccacttga gcccctttac ctaggagtat catcggctcc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcct aac
133


 


70
caaactgagc ttccgcttga gcccctttac ctaggagtat catcgggtgc atccaaaatg
60



gtttctcagc ctatgatgcc ttaggagcaa actgtgtacc tatcttgcac caaaactaag
120



tctgtctcca aac
133


 


71
aaactgagct tccacttgag cccctttgcc caggagtatc atcaggttca tccaaaatgg
60



tttcttagcc tttgatgcat taggcgtagc ctgtgtacct atcttgcacc ataactaact
120



ctgtctccaa aca
133


 


72
accaaactgt gcttccactt gagcctcttc atctaggatt accatcaagt gcatccaaaa
60



tggtttctta gactacggtg aattaggcta aaattgtgca cctatcttgc accaaaacta
120



acactatgtc caa
133


 


73
caaactgagc ttccacttga gccccgttac ctaggagtat cttcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcacaa actatgtacc tatcttacac taaaactaac
120



tctgtctcca aac
133


 


74
aaactgagct tccacctgag cccctttacc caggagtatc atcgggtgca tccaaaatgg
60



tttcttagcc tatgatgctt taggcgcaaa ctgtgtacct atctagcacc aaaactagct
120



ctgtctccaa aca
133


 


75
cgaaccgacc tttcaattga gcctcttcac ctaggattat catcgggtgt ttccataatg
60



gtttctgagc ctatggtgca ttatgcgcaa accatgcacc aatcttgcac ctaaactaac
120



actgtctcca aac
133


 


76
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggagaaa actgtgtccc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


77
aaactgagct tccacttgag cccctttacc caggagtatc attgggtgca tccaaactgg
60



tttcttagcc tatgatgcat ttggcgcaaa ctgtgtacct atcttgcacc aaaactgact
120



ctgtctccaa aca
133


 


78
aaactgagat tccacttgag cccctttacc caacagtata atcgggtgca tccaaaatgg
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtaccg atcttgcacc aaaactaact
120



ctgtctccaa aca
133


 


79
caaactgagc ttccacttgg gcccctttac ccaggagtat caacagatgc atccaaaata
60



gtttcttagc ctttgatgca ttaggtgcaa actgtgtagc tatcttgcac caatactaac
120



tctgtctcca aac
133


 


80
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggtgc atacaaaatg
60



gttccttagc ctatgatgca ttaggcgcaa actgtgtact tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


81
aaactgagct tccacttgag cccctttacc taggagtatc atcgggtgca tccaaaatgg
60



tttcttagcc tatgatgcat ttggcacaaa ctgtgtacct atcctgcacc aaaactaact
120



ctgtctccaa aca
133


 


82
aaactgagct tccagttgag cccctttacc gaggagtatc atcaggtgca tccaaaatgg
60



tttcttagcc tatgatgcat taggcgcaac ctgtgtacct atcttgcacc aaaactacct
120



ctgtatccaa aca
133


 


83
caaactgagc ttccacttga gcccctttac ccaggagtat caacagattc atccaaaata
60



gtttcttagc ctttgatgca ttaggtgcaa actgtgtagc tatcttgcac caatactaac
120



tctgtctcca aac
133


 


84
aaacctacct tccacttgag cctctccacc taggagtatt atcgggtgct tccataatgg
60



tttccgagcc tatggtgcat tatgcgcaaa ccatgcacca atcttgcacc taaactaaca
120



ctgtctccaa aca
133


 


85
caaactgacc ttccacttga cactcttcac ctaggagtat tatccggtgc ttccataatg
60



gtttctgagc ctatggtgca ttatgcgcaa accatgcacc aatcttgcac ctaaactaac
120



actatctcca aac
133


 


86
caaactgagc ttccacttga gcccctttac cctggagtat cttcaggtgc atccaaaatt
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


87
caaactgagc ttccacttga gcccctttac ccaggagtat catcaggtgc atcagaaatg
60



gtttcttagc ctatgatgca ttaggtgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


88
caaactgagc ttccacttga gcccctttac ccaagagtat catcgggtgc atccaaaatg
60



gtttcttagc ctatgacgca ttaggcacaa actgtgtacc tatgttgcac caaaactaac
120



tctgtctcca aac
133


 


89
caaactgagc ttcctcttga gcccctttac ctaggagtat catcggttgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcgtaa actgtgtacc tatcttgcac caaaacttac
120



tctgtctcca aac
133


 


90
caaactgagc ttccacttga gcccctttac ccaggagtat catcgggtgc atacaaaatg
60



gattcttagc ctatgacgca ttaggcgcaa actatgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


91
aaactgagtt tccacatgag cacctttacc ctggagtatc atcaggtgca tccaaaatgg
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtacct atattgcacc aaaactaact
120



ctttctccaa aca
133


 


92
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggcgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcacaa actatgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


93
aaactgagct tccagttgag cccctttacc cagcagtatc atcgggtgga tccaaaatgg
60



tttcttcacc tatgatgcat taggcgcaaa ctgtgtacct atcttgcacc aaaactaact
120



ctatctcgaa aca
133


 


94
aactgagctt ccacttgagc ccctttagcc aggagtatca tcgggtgcat ccaaaatggt
60



ttcttagcct atgaaatcat taggcgcaaa ctgtgtacct atcttgcacc aaaactaact
120



ctgtctctaa aca
133


 


95
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggagc atccaaaatg
60



gtttcttagc ctatgatgca taaggagcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


96
caaactgagc ttccacttaa gcccctttac ctaggagtat catcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttacgcgcaa actgtgtacc tatcttgcac caaaactaac
120



tttgtctcca aac
133


 


97
caaactgagc ttccacttga gcccctttac ctaggagtat aatcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttagacgtaa actatgtacc tatcttgcac caaaactaac
120



tctgtctccg aac
133


 


98
aaactgagct tccacttgag cccctttacc taggagtatc atcgggtgta tccaaaattg
60



tttcttagcc tacgatgcat taggcgcaaa ctgtgtacct atcttgcacc aaaactaact
120



ctgtctccaa aca
133


 


99
caaactgagc ttccacttga gcccctttac ctaggagtat cattgggtgc atccaaaatg
60



ctttcttagc ctatgatgca ttaggtgcaa actgtgtagc tatcttgcac caaaactatc
120



tctatctcca aac
133


 


100
aaactgagct tccacttgag cccgtttacc gaggagtatc atcgagtgca tctaaaatga
60



tttcttagcc tatgatgcat taggcacaaa ctgtgtacct atctagcacc aaaactaact
120



ttctctccaa aca
133


 


101
accgaccttc cacttgagac tcttcaccta ggattatcat cgggtgcttc cataatggtt
60



tctgagccta tggtgcatta tgcacaaacc atgcaccaat attgcaccga aactaacact
120



gtctccaaac a
131


 


102
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggtgc atccaaaatg
60



gtttcttagc caatgatgca ttaggagaaa actgtgtacc aatcttgcac caaaactaac
120



tctgtctcca aac
133


 


103
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggtgc atccaaaaag
60



gtctcttagc ctatgatgcc ttaggagaaa actatgtacc tgtcttgcac cataactaac
120



tctgtctcca aac
133


 


104
aaactgtgct tgcacttgag cccctttacc caggagtatc atcgggtgca tccaaaatgg
60



tttcttagcc tatgatgcat taggcgcata ctgtgtacct atcttgcagt aaaactaact
120



ctgtctccaa aca
133


 


105
aaactgagct tccacttgag gccctttatc taggagtatc atcgggtgca tccaaaatgg
60



tttcttagcc tatgatgcgt taggcgcaaa ctatgtacct atcttgcacc aaaactaact
120



ctgtctccaa aca
133


 


106
aaactgagct tccacttgag cccctttacc taggagtatc ttcgggtgca tcagaaatgg
60



tttcttagcc tatcatgcat taggcacaaa ctgtgcacct atcttacatc aaaattaact
120



ctgtctccaa aca
133


 


107
caaactgagc ttccacttga gcccctttac ccaggagtat attcgggtgc atccaaaatg
60



gtttcttagc ctatgatgcg ttaggcgcaa actgtgtacc tatcttgcac cacaactaaa
120



tctgtctcca aac
133


 


108
caaactgagc ttccacttga gcccctttac ccaggagtat caacagatgc atccaaaata
60



gtttcttagc ctttgatgca ttaggtgcaa actgtgtagc tatcttgcac caatactaac
120



tctgtctgca aac
133


 


109
caaactgagg ttccgcttga gcccctttac ctaggagtat catcgggttc atccaaaatg
60



gtttctcagc ctatgatgcc ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


110
aaactgagct tccacttgag cccctttacc caggagtatc atcgggtgca tccaaaatgg
60



attcttcgcc tatgatgcat taggcgcaaa ctgtgtacct atcttgcacg aaaactaact
120



ctatctccaa aca
133


 


111
cgaaccgacc ttccacttga gccccttcac ctaggattat catcgggtgc ttccataatg
60



gtttttgagc ctatggtgca ttatgcacaa accatgcacc aatcttgcac ctaaactaac
120



actgtctcca aac
133


 


112
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggtgc atccaaaatg
60



gttaattagc ctatgatgca ttaggcgcta actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


113
caaactgagc ttccacttaa gcccctttac ccaggagtat cttcaggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcacaa actatgtacc tatcttacac caaaactaac
120



tctgtctcca aac
133


 


114
aaccgagacc tccacttgag gcctcttcac ctaggagata ccatcggatg cgtctaagat
60



ggtttcttat cctatggtgc attatgcgta acccgtgcac atatcttgct ccaaaactaa
120



tgctgtctct aaa
133


 


115
caaactgagc ttccacttga gcccctttac ccagtagtat catcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttatgcgaaa attgtgtacc tatattgcac caaaactaac
120



tctgtctcca aac
133


 


116
caaactgagc atccacttga gcccctttac ctaggagtat catcgggtgc atacaaaatg
60



gtttcttaac ctatgatgca ttagacgcaa actgtgtacc tatattgcac caaaactaac
120



tctgtctcca aac
133


 


117
caaactgagc ttccacttga gcccctttac cttggagtat catcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcacaa actgtgtacc tatcttgaac caaaactaac
120



tctgtctcca aac
133


 


118
aaactgagct tccacttgag cccctttacc caggagtatc ttcaggtgca tccaaaattg
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtacct atctttcacc aaaactaaca
120



ctgtctccaa aca
133


 


119
caaactgagc ttccacttga gcccctttac ctagaagtat catcgggtgc atccaaaagg
60



gtttcttagc ctatgatgta ctaggcgtaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


120
caaactgagc taccacttga gcccctttac ctaggagtat catcaggttc atccaaaatt
60



gtttcttagc ctatgatgcg ttaggcgtaa actgtttacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


121
caaactgagc ttccatttga gcccctttgc ctaggagtat catcgggtgc atccaaaatg
60



gttccttagc ctatgatgca ttaggtgcaa actgtgtacc tatcttgcac caaaactaac
120



tttgtctcca aac
133


 


122
caaactgagc ttccacctga gccactttaa ccaggagtat catcgggtgc atccaaaatg
60



ttttcttagc ctatgatgct ttaggcgcaa attgtgtacc tatcttgcac caaaactaac
120



tctgcctcca aac
133


 


123
caaactgagc ttccacttga gcatctttac ccaggagtat catcaggtgc atccaaaata
60



gtttcttagc ctatgatgca ttaggcacaa actgtgtacc tatcttgcaa caaaactaac
120



tctgtctcca tac
133


 


124
caaactgagc ttccacttga gcccctttac ctaggggtaa catcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcacaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


125
caaactgagc ttccacctga gcccctttac ctaggagtat catcgtgtgc atctaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


126
caaactgagc ttccacttga gcccctttac ctaggagtat catcgtgtgc atcaaaaatg
60



gtttcttagc ctatgaagca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


127
caaactgagc ttccacatga gcccctttac ctaggagtat catcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcacaa actgtgtacc tatcttgcac caaaactaac
120



tctgtatcca aac
133


 


128
caaactgagc ttccacttga gcccctttac ccaggagtat catcgagtgc atctaaaaag
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaagctacc
120



tctgtctcca aac
133


 


129
caaactgagc ttccacttga gcccctttac ccaggagtat caacagatgc atccaaaata
60



gtttcttagc ctttcatgca ttaggtgcaa actgtgtagc tatcttgcac caatactaac
120



tctgtctcca aac
133


 


130
aaactgagct tccacttgag cccctttacc ctggaatatc atcgggtgca tcccaaatgg
60



tttcttagcc tatgatgcat taggcgcaaa gtgtgtacct atcttgcacc aaaactaact
120



ttgtctccaa aca
133


 


131
aaactgagct tcaacttgag cccctttacc taggagtatc atcgggtgca tccaaaatgg
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtacct gtcttgcacc aaaactaacc
120



ctgtctccaa aca
133


 


132
caaactgagc ttccacttga gcccctttac ccaggagtat caacagatgc ttccaaaata
60



gtttcttagc ctttgatgca ttaggtgcaa actgtgtagc tatcttgcac caatactaac
120



tctgtctcca aac
133


 


133
caaactgagc ttccacttga gcccctttac ccaggagtat caacagatgc atccaaaata
60



gtttcttagt ctttgatgca ttaggtgcaa actgtgtagc tatcttgccc caatactaac
120



tctgtctcca aac
133


 


134
aaactgagct tccacgtgag cccctttacc caggagtata atcgggtgca tccaaaatgg
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtacct atcttgcacc aaagctatct
120



ctgtctccaa aca
133


 


135
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggtgc attcaaaatg
60



gtttcatagc ctatgatgca ttaggcgcaa actatgtacc tatcttgcac caaaactacc
120



tccgtctcca aac
133


 


136
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggtgt atccaaaatg
60



gtttcttagc ctatgatgca ttaggcatag actgtgtacc tatattgcac caaaactaac
120



tccgtctcca aac
133


 


137
caaactgagc ttccacttga gcccctttac ccaggagtat catcgggtgc atccaaaatt
60



gtttcttagc ttatgatgca ttaggtgtaa actgtgtacc tatcttgcat caaaactcac
120



tctgtctcca aac
133


 


138
caaactgagc ttccacttga gcccctttac ccaggagtat cttcaggtgc atccaaaatt
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tttcttgcac caaaactaac
120



tctgtctcca aac
133


 


139
aaactgagct tccacttgag cccctttacc caagagtacc atcgggtgca tccaaaatgg
60



tttcttagcc aatgatgcat taggcgcaaa ttgtgtacct atcttgtacc aaaactaact
120



ttgtctccaa aca
133


 


140
caaactgagc ttccacttga gcccctttac ctagcagtat aatcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcacaa actatgtact tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


141
aaactgagct tccacttgag cccctttacc gaggagtatc atcgggtgca ttcaaaatgg
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtacct atcttgcaca aaaactagct
120



ctgtctccaa aca
133


 


142
caaactgagc ttccacttga gcccctttac ccaggagtat catcaggttc atccaaaatt
60



gtttcttagc ctttgatgca ttaggcgtaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


143
aaactgagtt tccacatgag cacctttacc caggagtatc atcaggtgca tccaaaatgg
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtacct atcttgcacc aaaactaact
120



ctgtctccaa aca
133


 


144
aaactgagct tccacttgag ccccttttct caggagtatc attgggttca tccaaaatgg
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtacct atcttgtacc aaaactaact
120



ctgtctccaa aca
133


 


145
caaactgagc ttccacttga gcccctttac ccaggagtat catcgggtgc atccaaaatg
60



gtttctttgc ctatgatgca ttaggcggaa actgtgtacc tgttttgcac caaaactaac
120



tctatctcca aac
133


 


146
aaactgtgct tccacttgag cccctttacc taggagtatc atcagggtgc atccacaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


147
caaactgagc ttccacttgg gcccctttac ccaggagtat cttcaggtgc atccaaaatt
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


148
aaactgagct tccgcttgtg cccctttacc caagagtatc gacgggtgca tccaaaatgg
60



tttcttagcc tacgatgcat taggcgcaaa cagtgtagct atcttgcacc aaaactaact
120



ttgtctccaa aca
133


 


149
caaactgagc ttccacttga gcccctttac ctaggagtat catcaggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggagaaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


150
caaactgagc ttccacttga gcccctttac ctaggagtat catcgtgtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcacaa actgtgtacc tatcttgcac caaaagtaac
120



tctgtctcca aac
133


 


151
caaactgagc ttccacttga gcccctttac caaggagtat catcgggtgc atccaaaatg
60



gtttcttagc ctctggtgca ttaggcacaa gctaggtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


152
aaactgagct tccacttgag cccctttact caggagtatc atcgtgtgcc tccaaaatgg
60



tttcttagcc tatgatgcat taggcgcata ctgtgtacct atcttgcacc aaaactacct
120



ctatctccaa aca
133


 


153
aaactgagct tccacttgag cccctttaca cacgagtatc atcgggtgca tccaaaatgg
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtacct atcttgtacc aaaactaact
120



ctggctctaa aca
133


 


154
caaactgagc ttccacttga gtccctttac ccaggagtat catagggtgc atccaaaatg
60



ttttcttagc ctatgatgca ttaggcgtaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


155
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggtgc atccaagatg
60



gtttcttagc ctatgatgca ttagacgcaa actgtgtacc tatcttgcac caaaactaac
120



tttgtctcca aac
133


 


156
aaactgagct tccacttgag cccctttaca caggagtatc atcgggtgca tccaaaatgg
60



tttcttagca tatgatgcat tagtcgcaaa ctgtgtacct atcttgtacc aaaactaact
120



ctgtctccaa aca
133


 


157
caaacggagc ttccgcttga gcccctttac ctaagagtat catcgggtgc atccaaaatg
60



gtttgtcagc ctatgatgca ttaggtgcaa actgtgtacc tatcttgccc caaaactaac
120



tctgtctcca aac
133


 


158
caaactgagc ttccacttga gcccctttac ctaggagtat catcgggtgc atccaaaaag
60



gtttcttagc ctatgatgct ctaggagaaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcta aac
133


 


159
caaactgagc ttccacttga gcccctttac ccaggagtat catcgggtgc atctaaagtg
60



gtttcttagc ctacgatgca gtaggcgcaa actgtgtaca tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


160
tgaaacggag ctttcacttg agccccttga cctaggagta ccatcgggtg catccaaaat
60



ggtttcttat cctatggtgc attaggtgta aaccgtgcac ctatcttgca ccgaaactaa
120



cgttgtctct aaa
133


 


161
caaactgagc ttccacttga gcccctttac ccgggagtat catcgggtgc atccaaaatg
60



gtttcttatc caatgatgcg ttaggcgcaa actatgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


162
caaactgagc ttccagttga gcctctttac ccaggagtat catcgggtgg atccaaaatg
60



gtttgttagc ctatgatgca ttaggagcaa actatgtacc tatcttgcac caaaactaat
120



tctgtctcca aac
133


 


163
caaactgagc ttccacttga gcccctttac ccaggaggat cttcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tttcatgcac caaaactaac
120



tctgtctcca aac
133


 


164
caaaccgagc tttcacttta gccccttgac ctaggagtac catcgggtgc gttcaaaacg
60



gtttcttatc ctatggtgca ttaggtgcaa accgtgcacc tatcttgcac tgaaactaac
120



actgtctcta aac
133


 


165
aaactgagct tcgacttgag cccctttacc caggagtatc atcgggtgca tccaaaaggg
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtacgt atcttgcacc aaaactacct
120



ctgtctctaa aca
133


 


166
aaactgagct tccacttgag cccctttacc caggagtatc atcgggtgca tccaaaagag
60



tttcttagcc tatgatgcat taggcgcaaa ctgtgtacgt atcttgcacc aaaactacct
120



ctgtctccaa aca
133


 


167
caaactgagc ttccacttca gcccctttaa tcaggaatat catcgggtgc atccaaagta
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


168
caaactgagc ttccacttga gcccctttac ccaggagtat catcgggtgc atccaaaata
60



gtttcttagc ctacgatgca gtaagcgcaa actgtgtacc tatcttgcac caaaactaac
120



tcggtctcca aac
133


 


169
caaactgagc ttccacttga gcccctttac ctaggagtat gatcaggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcacaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


170
aaactgagct tccacttgag cacctttacc caggagtatc atcaggtgca tccaaaatgg
60



gttcttagcc tatgatgcat taggcgcaaa ctgtgtacct atcttgaacc aaaactaact
120



ctatctccaa aca
133


 


171
caaactgagc ttccacttga gcccctttac ccaggagtat cttcaggtgc atccaaaatt
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tttgtctcca aac
133


 


172
caaactgagc ttccacttga gcccctttac ctaggagtat aatcgggtgc atccaaaatg
60



gtttcttagc ctatgatgca ttaggtgcaa actatgtacc tatcttgcac caaaactaac
120



tttgtctccg aac
133


 


173
caaacagagc ttccaattga gaccctttac tcaggagtat catcgggttc atccaaaatg
60



gtttcttagc ctatgatgca ttaggcgcaa actgtgtacc tattttgcac caaaactaac
120



tctgtctcca aac
133


 


174
caaactgagc ttccacttga gcccctttac ccacgagtat catcgggctc atccaaaatg
60



atttcttagc ctatgatgca ttaggcgcaa actgtgtacc tatcttgcac caaaactaac
120



tctgtctcca aac
133


 


175
actgcgcttc cacttgagcc ccattaccca ggagtatcat cgggtgcatc caaaatagtt
60



tcatagccga tgatgcatta ggtgtaaact gtgtacctat cttgcaccaa aactaactct
120



gtctccaaac a
131


 


176
aactgagctt gcacttgagc ccctttaccc aggagtatca tcgagtgcat ccaaaattgt
60



ttcttagcct gtgatgcatt aggcgcaaac tgtgtacctg tcttgcacca aaactaactc
120



tgtctccaaa c
131









To identify the consensus sorghum satellite sequence from the sequences of SEQ ID NOs:23-176, these sequences were aligned using ALIGN (publicly available software; Altschul, S F, et al., J Mol. Biol. 215:403-10, 1990). The sequences were trimmed to unit repeat length using the consensus as a template. Sequences trimmed from the ends of the alignment were realigned with the consensus and further trimmed until all sequences were at or below the consensus length. The consensus was determined by the frequency of a specific nucleotide at each position; if the most frequent base was three times more frequent than the next most frequent base, it was considered the consensus. An exemplary consensus sorghum satellite sequence is set out as SEQ ID NO:22:








aaactgagct tccacttgag cccctttacc aggagtatca tcgggtgcat ccaaaatggt    60



 


ttcttagcct atgatgcatt aggcgcaaac tgtgtaccta tcttgcacca aaactaactc   120


 


tgtctccaaa cc                                                       132






The sorghum centromere specific retrotransposon sequence (CRS) was amplified using PCR and sequenced using primers designed from published sequence (set forth in SEQ ID NO:178; Presting, G G, et al., Plant J 16: 721-728, 1998 and Miller, J T, et al. Genetics 150: 1615-1623, 1998) and are set forth in SEQ ID NOs:179 and 180. The sequence for sorghum CRS is set out as SEQ ID NO:21:








tggattcgga ctggaaaata actctaactt gtatggatca ccacgacgtc atatggactc   60



 


caactgggac gttcctatac ttgttggaaa gctcatgaag tctactttcc aatgggtcca  120


 


accacatatc tatgcggctt atgagtcggg cgcagtcctt gttttcgtgc cgacaccttt  180


 


ttctgttttg gtgctgcgtc actctatttt ggaccaatgg cccatgtatc aagttgagtc  240


 


cattagggac gcatcctagg gttggaggac gactctagca cccctttggt cgtcctcccc  300


 


tctatttatt tacatctaga gccgccatga acaactggat tttgtttaga tcaagtttag  360


 


ccttcgctac ttgcttgtag gcgcgcgtgc aggatcagcc gcccgcctcc ttgtcttcgg  420


 


aaccccattg ttgattaaga ttcagtttaa aaccttcaat tcatcttgca aattcagtgc  480


 


ttgtttcctc gttcttgcta gttcttcgat tgcttgcagg acgggagccc taggggctgg  540


 


ttgtcgcgct ccacaagatc gtgacggttg ttggacgtgg tgtatcggtt gctaaggcgc  600


 


ggtcttgagg gctgtagtcg ggccgtgaac gtcatctcca tccactaatc gagttatcca  660


 


gcgcctctca tcgaaagatc aggccaaaaa ccctagcggg ctcgcatcag ttggtaatca  720


 


gagcaaggtt cttcggtgag agacttctaa tcctttgctg tttttaatta atttcctata  780


 


gtccagaaaa gccaaaaaaa tatagtagat tagtttttcc ataatcctat taaacctttg  840


 


tgccttggct agtaccgttt tagttagggc ttgttgaatt tgcgttgctt cggtttgtgt  900


 


cgagttgctg gtcttagtgt ctagtccttt agagtttcga gttcttgtca ccatctatac  960


 


acagccgagt attaccatat cttctctctg tcgaatctgt tgcgaagtct gaattgaact 1020


 


gtggtcatgg tccggatcga gtagagttcc aatctgagtt caaaaagaaa gataactcta 1080


 


cttgttcggc cttactctag agagagagag agagtgtgga gcgaaaaaag tgtgtggagc 1140


 


gaattgctct tttgtattct tttgttcata taatcagttt tggaggttgc ccacaaaaaa 1200


 


agaaaaaaaa gaaaagagaa aagattcaaa aaaaagaggc tgtttttcat attgatttta 1260


 


ggtttgtccc accttgtttt cgggggtgtg ctgtggtttt cctttgtgtc caggctcgcg 1320


 


tctctagcac ggtctagcct aggaccagca cagtaccatc gtcgaacgct tattcagctc 1380


 


gcttttataa ctaacgtggt gctagttcgt tccttgtttc agcccaccta tagctccaca 1440


 


tactctacag cttgacaggt cttgtgctgc agcaccgata cacttcgtcc attgctatac 1500


 


acttgttggc agacgacccc tcctgtcaag caagataaga attggtaaga acttgtgtta 1560


 


caggttgagt gtgagcgact tgctatagct acatcctagt agttgtaggg attttatttc 1620


 


ttcacttgct ttttgttgtc tttgtctttg aaccatgcca ggggcagatg atggtaacga 1680


 


aacaccactt acacctcgca ctatgggcat catacaatat tttgaaagga aagtgaagct 1740


 


gcacacagag ggacttgata acgacttgca ggtgacgaat gaaaagctgg ggcagttgga 1800


 


ggctacgcag attgccacaa acaacaagct cacaagtttg gaggaatccg ttgctagtgt 1860


 


ggacaaaagc cttgctgctc tcctaaggcg atttgatgct ttccacaccg aagataaaga 1920


 


gaagcataaa gaagaaaagg agggagatcg agagcacggt agtcatgaag atgactacac 1980


 


tggtgatact gaacatgatg atcaagacac tcgtgatcga cgtcgccttc gtcacaaccg 2040


 


tagaggtatg ggtggcaacc gccgacgcga ggtacacaat aatgatgatg ctttcagtaa 2100


 


gattaaattt aagatacccc tttttgatgg taaatatgac cctgatgctt acatcacttg 2160


 


ggagattgct gttgatcaaa agtttgcatg tcatgaattt cctgagacta cacgtgttag 2220


 


ggctgctact agtgagttta cagattttgc ttctgtttgg tggatagaat atggaaagaa 2280


 


aaatcataat aacttaccta gaacttggga tgcgctgaaa agggccatga gagctagatt 2340


 


tgttccatct tactatgcgc gtgatatgat aaataagttg cagcagttaa gacaaggtgc 2400


 


taaaagtgta gaagaatatt atcaggaatt acaaacgggt atgttgcgtt gtaacctaga 2460


 


ggaggatgag gaaccggcta tggctagatt tttgggtggg ttaaatcggg aaattcagga 2520


 


catcctcgct tacaaagaat acaataatgt aacccgtttg tttcatcttg cttgtaaagc 2580


 


tgaaagggaa gtgcagagac gacgtgctag cacaaggagt aatatttctg cagggaaggc 2640


 


taattcatgg cagcaacgcg tggcttcaac tccatctaca cgtatttcta ctccatcatc 2700


 


tagtgacaag actcgagctg cccccaccaa ttcagttgcg aagacgatgc aaaagcctgc 2760


 


tgcgagtact tcatccgtgg catcgacggg tagaacaagc aacatacaat gtcaccggtg 2820


 


caagggatat gggcacatga tgcgtgactg tccaaacaag cgagttatga ttgtcaggga 2880


 


tgatggtgag tactcatctg ctagtgattt tgatgaggat acacttgcac tgcttgcgac 2940


 


tgaccatgca ggtaatgaag atcaaataga agaatatatt aatgcaggtg aagcggacca 3000


 


ctatgagagc ttgatcgtgc agcgagtgct tagtgcacaa atggagatgg cggaacaaaa 3060


 


tcagcgacac attttattcc aaacaaagtg tgtcatcaaa gagcgttctt gtcgcatgat 3120


 


cattgatgga ggtagctgca acaacttggc aagcagcgat atggtgcaga agcttgccct 3180


 


caacaccaaa ccacacccgc atccctacta catccaatgg ctgaacaaca gtggtaaggc 3240


 


aaaggtaact agacttgtga gaattaattt ttccatcgga tcctacaaag atattgttga 3300


 


atgtgatgtt gtgcctatgc aagcttgtaa cattctgcta ggtagacctt ggcaatttga 3360


 


tagagattct atgcatcatg gtagatcaaa tcagtattct tttctatacc atgatcgcaa 3420


 


aattgtgttg catcctatat cccctgaaac tattatgcaa actgatgttg ctagggctac 3480


 


taaagcaaag agcaagagca ataaaaatga taaatctgta attggtaaca aagatgagat 3540


 


aaaactgaaa ggacattgta tgatagctac caaatcagat attaatgagt tcaatgcatc 3600


 


cacttctgtt gcttatgctt tgatatgcaa ggatgctttg atttcagttg aggatatgca 3660


 


atgttctttg ccccctgctg ttgctaacgt tttgcaggag tattctgatg tgtttccaag 3720


 


tgatgtacca gcggggctgc ctccactacg cgggattgag caccaaattg atcttattcc 3780


 


tggatcagtt ttgccaaatc gtgcaccata caggacaaac ccggaggaaa caaaggaaat 3840


 


tcagcgacaa gtgcaagaac tactagacaa aggttatgtc cgagaatctc ttagtccttg 3900


 


tgctgttcca gtaattttag tgcctaagaa agatggaaca tggcgtatgt gtgttgattg 3960


 


tagagctatt aataatatca ccattcgata ttgacaccct attccacgat tagatgatat 4020


 


gctagatgaa ctgagtggtg ctgttgtgtt ttcaaaagtt gatttacgta gtgggtacca 4080


 


ccagattcgt atgaaattgg gagatgaatg gaaaactgct ttcaaaacta agttcggttt 4140


 


gtatgagtgg ttagtcatgc cttttgggtt aactaatgca cctagtactt tcatgagatt 4200


 


aatgaacgag gtcttgcgtg ctttcattgg gaaatttgtt gtcgtatatt ttgatgacat 4260


 


attgatttac agcaaatcat tggatgaaca tcttgatcat ttacgtgctg tttttaatgc 4320


 


actacgcgag gcacgtttat ttggtaacct tgagaagtgc accttttgca ccgatcgagt 4380


 


gtcttttctt ggttatgttg tgactccaca gggaattgag gttgatcaag ccaaggtgga 4440


 


agctatacag ggatggcctg tcccaaatac tatcacccag gtgcggagtt tcctaggact 4500


 


tgctagattc tatcgccgtt ttgtgaagga tttcagcacc attgctgcac cattgaatga 4560


 


gcttacaaag aagggggtgc cttttgattg gggcaaagca caagagaatt cattcaacat 4620


 


gttgaaagat aagttaactc atgcacctct cctacaactt cctgatttta ataagacttt 4680


 


tgagcttgaa tgtgatgcta gtggaattgg tttgggaggt gttttattac aagagggaaa 4740


 


acctattgca tattttagtg agaaattgag tgggcctgtt ctcaaattca acttatgata 4800


 


aagaactcta tgctcttgtt agaacattag agacatggca gcattatttg tggcccaaag 4860


 


agtttattat ccattttgat catgaatctt tgaaacatat tcgtagtcaa ggaaaactga 4920


 


atcgtaggca tgcaaagttg gttgaattta ttgaatcttt tccttatatt attaagcaca 4980


 


agaaagggaa ggaaaatatt attgctgatg ctttatcacg gagatatact ttgctgaatc 5040


 


aacttgatta caagatattt gggttagaaa caattaaaga ccaatatgtt catgatgctg 5100


 


attttagaga cgtgttgctg cattgtaaag atggaaaagg gtggaataaa ttcatcgtta 5160


 


gtgatgggtt tgtgtttaga gctaacaagc tatgcattcc agctagctct gttcgtttgt 5220


 


tgttgttgca ggaagcgcat ggaggtggct tgatgggaca ttttggagca aagaagaccg 5280


 


aggacatact tgctggtcat ttcttttggc ccaggatgaa gagagatgtg gagaggtttg 5340


 


ttgctcgttg cacaacatgt caaaaggcaa agtcacggtt aaatccccac ggtttgtatt 5400


 


tacctcttcc tgttcctaat gctccttggg aggatatatc tatggatttt gtgttgggac 5460


 


taccaaggac taggagggga cgtgatagtg tgtttgtggt tgttgataga ttttctaaga 5520


 


tggcacattt cataccatgt cataaaactg atgatgctac aaatattgct gatttgtttt 5580


 


ttcgagaaat tgttcgctta catggtgtgc ccaacacaat tgtttctgat cgtgatgcta 5640


 


aatttcttag tcatttttgg aagactttgt ggttcaaatt ggggactaag cttttatttt 5700


 


ccaccacctg tcatccccaa actgatggtc aaactgaagt tgttaataga actttatcca 5760


 


ctatgttaag ggctgtttta aagaagaata ttaagatgtg ggaagaatgt ttgcctcatg 5820


 


ttgagttcgc ctataatcgt tcattgcatt ctactacaaa aatgtgtcct tttgagattg 5880


 


tctatggctt cttgccacgt gctcctattg atttaatgcc tttgccaagt tctgaaaaaa 5940


 


taaattttga tgctaagcaa catgctgaat tgatgttaaa attgcatgaa gccactaaac 6000


 


aaaacataga gcgcatgaat gctaagtaca aatgcactgg agataaaggt agaaagcaat 6060


 


tgattctgga acctggggat ttggtttggt tgcatttgcg aaaagataga tttccagaac 6120


 


tgataaaatc caaattgatg cctagagctg atggtccttt taaagtgctg caacgaatta 6180


 


atgagaatgc atataagctt gatcttcctg cagattttgg ggttagtccc acatttaaca 6240


 


ttgcagattt gaagccttat ttgggtgagg aagatgagct tgagtcgagg acgactcaaa 6300


 


tgcaagaaag ggaggatgat gaggacatca acac                             6334


 


SEQ ID NO: 179 (forward primer): gggaagtaca gggacgaaga gc           22


 


SEQ ID NO: 180 (reverse primer): tgcaaccaaa ccaaatcacc ag           22






BAC Library Construction
A Bacterial Artificial Chromosome (BAC) library was constructed from sorghum genomic DNA. The sorghum genomic DNA was isolated from Sorghum bicolor, and digested with a restriction enzyme that was methylation insensitive to enrich BAC libraries for centromere DNA sequences.
Probe Identification and Selection
Groups of sorghum repetitive genomic DNA, including specific centromere-localized sequences, were initially compiled as candidate probes for hybridization with the BAC libraries. The satellite sequences set out in SEQ ID NOs:22-176 were used as probes for interrogating BAC libraries. These probes were prepared and labeled with standard molecular methods.
Library Interrogation and Data Analysis
The BAC clones from the libraries were spotted onto filters for further analysis. The filters were hybridized with the probes to identify specific BAC clones that contained DNA from the group of sequences represented by probes to identify those BAC clones that were positive for satellite sequence (the probe being amplified from sorghum genomic DNA using for a forward primer gtcacccagc agttccatcg ggtgc (SEQ ID NO:181) and for the reverse primer, actgctgggt gacgtggctc aagt (SEQ ID NO:182). Hybridization was at 65° C. for 12-15 hours and washing three times for 15-90 minutes with 0.25×SSC, 0.1% SDS at 65° C. Other exemplary stringent hybridization conditions comprise 0.5×SSC 0.25% SDS at 65° C. for 15 minutes, followed by a wash at 65° C. for a half hour. Unique clones that hybridized with one or more of the probes were isolated. Probe hybridization was scored visually to determine a binary (positive versus negative) value, and the signal was assigned a score based on the relative strength of hybridization on a 10 point scale.
From this experiment, 211 BACs were CRS-positive and 624 BACs were 137 bp satellite positive, providing at least 624 BACs as centromere candidates for MC construction. Exemplary BACs are shown in Table 5.







TABLE 5







Sorghum centromere sequence-containing BACs










Satellite-positive BACs
CRS-positive BACs







81J23
41A11



82O22
41D7



84B23
42D9



85K22
42N11



89A3
43C3



89E2
43D8



89F4
43E5



89H10
44B3



89H10
44K3



89H8
44N12



89I6
45C3



89J10
45M8



89J9
45P2



89N4
46F1



89N6
46F4



89P4
46P12



90C2
47F1



90I3
47H1



90L2
48D6



90L3
48F2










Of the BACs shown in Table, 42N11 and 89F4 were sequenced. BAC 42N11, identified for containing the CRS sequence, assembled into 90 contigs (set forth in SEQ ID NOs:183-275), yielding about 65 kb, of which 104 bp aligned to the consensus satellite sequence (SEQ ID NO:22), and 3325 bp aligned with the CRS sequence (SEQ ID NO:21). Further analysis showed that this BAC also aligned 2852 bp with CRM2, a corn centromere retroelement sequence related to CRS. BAC 89F4, identified for containing the CRS sequence, assembled into 50 contigs (set forth in SEQ ID NOs:276-326), yielding a total of about 47 kb, of which 857 bp aligned with the CRS sequence (SEQ ID NO:21), another 6314 aligned with CRM2, and 1632 aligned with the consensus satellite sequence of SEQ ID NO:22. These results also demonstrate that BAC clones containing centromere sequence as identified by SEQ ID NO:21 or SEQ ID NO:22 contain both sequences.
Example 2
Construction of Sorghum MCs Containing Genomic DNA (Prophetic)
A subset of BAC clones, identified as described in Example 1, are grown up and DNA is extracted for MC construction using NUCLOBONDTM Purification Kit (Clontech). To determine the molecular weight of centromere fragments in the BAC libraries, a frozen sample of bacteria harboring a BAC clone is grown in selective liquid media, and the BAC DNA harvested using standard alkaline lysis. The recovered BAC DNA is restriction digested and resolved on an agarose gel. Centromere fragment size is determined by comparing to a molecular weight standard.
The components of a exemplary sorghum MCs are described in Table 6. The UBQ10 promoter is used to express DsRed in MCs constructed with the backbone vector CHROM-SB.







TABLE 6







Donor components of CHROM-SB











Size




Element
(bp)
Location (bp)
Details













YAT1 yeast 
2000
7110-9109
PCR amplified YAT1 promoter 


Promoter


from chromosome I of S. 





cerevisiae for expression of 





NptII in sorghum


A. UBQ10 
360
9123-9482
PCR amplified A. thaliana


Intron


intron from UBQ10 gene 





(At4g05320) for stabilization 





of NptII gene transcript and 





increase protein expression level


NPTII
795
9510-10304
Neomycin phosphotransferase 





II plant selectable marker


Rps16A 
489
10368-10856
Amplified from A. thaliana 40S 


terminator


ribosomal protein S16 





(At2g09990) for termination 





of NptII gene


Bacterial 
817
11039-11855
Bacterial kanamycin selectable 


Kanamycin


marker


Terminator 6
332
12000-12331
Terminator 6


DsRed2 + NLS
780
12466-13245
Nuclear localized red fluorescent 





protein from Discosoma sp.


UBQ10 
2038
13282-15319
PCR amplified A. thaliana


Promoter


promoter from UBQ10 gene 





(At4g05320) for stabilization 





of DsRedI gene transcript and 





increase protein expression level


LoxP
34
7057-7090
Recombination site for Cre 




and 
mediated recombination




15335-15368









The MCs are constructed by following a two-step procedure: Step 1: Preparation of donor DNA for retrofitting with BAC centromere vectors and Step 2: Cre-Lox Recombination-BAC and Donor DNA to generate the MC. The resulting MChromsomes are subsequently tested in several different sorghum cell line.
Preparation of Donor DNA for Retrofitting
Cre recombinase-mediated exchange is used to construct MCs by combining the centromere fragments cloned in pBeloBAC11 with a donor plasmid (i.e. CHROM-SB Table 5). The recipient BAC vector carrying the sorghum centromere fragment contained a loxP recombination site; the donor plasmid contained two such sites, flanking the sequences to be inserted into the recipient BAC.
Sorghum MCs are constructed using a two-step method. First, the donor plasmid is linearized to allow free contact between the two loxP sites; eliminating the backbone of the donor plasmid. Second, the donor molecules are combined with sorghum centromere BACs and treated with Cre recombinase, generating circular sorghum MCs with all the components of the donor and recipient DNA. The MCs are delivered into E. coli and selected on medium containing kanamycin and chloramphenicol. Only vectors that successfully cre-recombined and contain both selectable markers survive in the medium. The MCs are extracted and restriction digested to verify DNA composition and calculate sorghum centromere insert size.
To determine the molecular weight of the sorghum centromere fragments in the sorghum MCs, three bacterial colonies from each transformation event are independently grown in selective liquid media, and the MC DNA harvested using alkaline lysis methods. The recovered MC is restriction digested and resolved on an agarose gel. Sorghum centromere fragment size is determined by comparing to a molecular weight standard. If variation in sorghum centromere size is noted, the MC with the largest sorghum centromere insert is used for further experimentation.
Functional Testing of Sorghum MCs Using Transient Assays
The MCs are tested, for example, in several sorghum cell lines, and the procedure optimized for antibiotic selection, cell pre-treatments, and bombardment conditions. All assays are transient and fluorescent cells are counted at several time points.
Example 3
Construction of Sorghum MCs Containing Synthetic Arrays of Repeat Sequence (Prophetic)
A synthetic array of the sorghum satellite repeat sequences is generated using PCR and directional cloning. A block of sorghum satellite repeats are PCR amplified and sequenced, and this sequence used as the basis for building the synthetic array.
For example, MCs containing synthetic arrays of sorghum satellite repeats may also contained either 5 or 8 stacked exogenous genes. The five-gene stack may include the genes NptII, DsRed, Anthocyanin, ZsGreen, and ZsYellow. The eight-gene stack may include those of the five-gene stack plus three additional genes from the A. tumefaciens tumor-inducing (Ti) pathway. These include iaaM (Trp mono-oxygenase), iaaH (Indole-3-acetamide hydrolase), and ipt (AMP iso-pentenyl transferase).
In order to investigate whether the MCs can carry a large number of genes, MCs containing a gene stack, a synthetic array of sorghum repeat nucleotide sequence and about 20 kb of peace lily (Spathiphyllum spp.) DNA is constructed. The total size of these MCs ranges between 82 kb and 87 kb.
In addition, MCs with a gene stack with two genes in addition to a synthetic sorghum centromere repeat array and an approximately 50 kb insertion of peace lily DNA may be constructed using the methods described above. The functionality of this MC demonstrates that the MCs of the invention can accommodate a large payload of genes, as 50 kb of the peace lily DNA includes a wide variety of genes.
Example 4
MC Delivery into Sorghum Cells and Regeneration (Prophetic)
To enhance the efficiency with which sorghum cells transformed with MCs can be regenerated into sorghum plants, where the MCs contain the auxin gene pathway and are delivered into fully differentiated leaf rolls rather than undifferentiated tissue, e.g. embryos. In addition, growth conditions are modified to enable development and propagation of transformed sorghum callus.
Sorghum is grown in the greenhouse for up to 6 months without floral initiation due to the growth time as well as the daylength settings on greenhouse supplemental lighting for those varieties that are day-length-sensitive. Stalks from several (clonal) plants are used to generate leaf rolls that do not include any developing meristematic tissue.
The MCs with a synthetic sorghum centromere are delivered to leaf rolls. For example, the MC may contain an eight-gene stack (“eight-gene MC”), or the MC may contain a five-gene stack (“five-gene MC”). In addition, a control plasmid (lacking a centromere) containing eight genes is also delivered, in which the eight-gene stack is identical to that delivered on the eight-gene MC.
The eight-gene MC includes A. tumefaciens tumor inducing (Ti) pathway genes (iaaM, iaaH, and ipt). The inclusion of these genes minimizes the time the transformed cells are in culture. IaaM converts Trp into indole-3-acetamide, which IaaH converts into auxin. Isopentenyl transferase (Ipt) converts 3′,5′-adenosine monophosphate (AMP) into a cytokinin. Auxin is used in cell culture to stimulate plant cells to form callus. Media with auxin promotes callus growth from plant cells whereas plant cells cultured on media lacking auxin either germinate (for embryogenic material) or are unable to grow (non-embryogenic or meristematic tissue such as leaf tissue). Thus, the eight-gene MC induces callus formation without supplementing the media with auxin.
A biolistic delivery method using dry gold particles is used to deliver MCs to the sorghum leaf rolls. MC DNA (in 1×TE) is precipitated onto 2.1 mg of sterilized and washed 0.6μ gold particles. The DNA-containing gold particles are resuspended in 2.5 M CaCl2 solution. 0.1 M spermidine (free base) are added to the mixture. The mixture is incubated on ice for 1.5 hours, with gentle finger vortexing (3×) for 45 minutes. The precipitated DNA is then washed with 100% ethanol, resuspended in 100% ethanol, and then the ethanol evaporated prior to bombardment.
The apical region of the sorghum stem is collected (20-30 cm long), after removing the outermost mature leaves, and the remaining leaves are sterilized by submersion in a solution of 50 ml bleach in 1 liter of water for 10 minutes. The remaining mature leaves are aseptically removed, and the young inner immature leaves are sliced into sections/discs approximately 2-3 mm thick. The leaf rolls are placed in Sorghum Osmotic Medium (SCOM; 4.3 g/l MS salts and vitamins, supplemented with 20 g/l sucrose, 0.5 g/l casein, 3 mg/l 2,4-D, 0.2 M mannitol, 0.2 M sorbitol pH to 5.8 and solidify with 2 g/L Gelrite) at 28° C. for 4-5 hours before the bombardment.
The three constructs are each initially tested by delivery into the leaf rolls. For delivery, the leaf rolls are bombarded with the MC DNA using the BioRad PDS-1000/He with a rupture disk rating of 900-1800 psi (1350 psi is preferred with one shot per plate). The gap distance (distance from rupture disk to macrocarrier) is 6 mm. Target shelf for tissue is L2-L4; L2 or L3 is preferred. The vacuum pressure of 25-29 in Hg; 27.5 in Hg is preferred. The bombarded leaf rolls are stored at 28° C. (dark) for an additional 16-18 hours on SCOM.
Subsequently, the bombarded leaf rolls are transferred to MSO (4.3 g/l MS salts and vitamins, supplemented with 20 g/l sucrose, 0.5 g/l casein, with NO 2,4-D. pH to 5.8 and solidify with 2 g/L Gelrite) and stored at 28° C., in the dark, for 2 weeks. The leaf rolls are visually assessed for callus production two and four weeks after bombardment.
Callus arising from the bombarded tissue is phenotypically evaluated for DsRed expression using a fluorescent dissecting microscope. If DsRed is observed in the tissue, then the callus is transferred to Regeneration Sorghum Medium (RSCM; 4.3 g/l MS salts and vitamins supplemented with 20 g/l sucrose, 0.5 g/l casein, 0.5 mg/l kinetin. pH to 5.8 and solidify with 2 g/L Gelrite) in low light (e.g., 16 hour day length, 26° C.) to initiate regeneration. This media does not contain auxin. If after a couple of additional weeks of culture, callus has also started to differentiate into root (primarily) and shoot material, which is not expected in the presence of auxin, then this result suggests either silencing or loss of the 3 genes from the A. tumefaciens tumor-inducing (Ti) pathway. After 2 additional weeks on media, PCR evaluation of this material or presence of the DsRed gene is carried out. If PCR results are negative, further suggesting loss of the entire MC and verifies that the genes are not silenced.
The advantages of including the genes of the Ti pathway on a MC are that the non-meristematic tissues are transformed and the need for callus initiation prior to DNA delivery is eliminated. In addition, the time in culture is reduced and as a result somaclonal variation, endogenous chromosome number changes and the like are also reduced. Furthermore, the inclusion of the Ti pathway genes eliminated the need for selectable marker genes.
In a separate experiment, five-gene (NptII, DsRed, Anthocyanin, ZsGreen, and ZsYellow) MCs are delivered into sorghum callus. These MCs are delivered to the callus cells using the wet biolistic method as described above.
Following delivery, Callus from the tissue is phenotypically evaluated for DsRed expression using a fluorescent dissecting microscope. If DsRed is observed in the tissue, the calluses are transferred to Selection Sorghum Medium MS3-50 (4.3 g/l MS salts and vitamins supplemented with 20 g/l sucrose, 0.5 g/l casein, 3.0 mg/l 2,4-D, 0.5 g/l polyvinylpyrrolidone (PVP). pH to 5.8 with 2 g/l Gelrite; further supplemented with 50 mg/l G418) for initial selection for 2 weeks. All calluses are subsequently transferred to additional selection on Selection Sorghum Medium MS3-75 (4.3 g/l MS salts and vitamins supplemented with 20 g/l sucrose, 0.5 g/l casein, 3.0 mg/l 2,4-D, 0.5 g/l polyvinylpyrrolidone (PVP) pH to 5.8 with 2 g/l Gelrite; further supplemented with 75 mg/l G418) for 4 additional weeks. Tissue is then visually assessed for sorghum callus tissue that is able to grow. Those identified events are transferred to Regeneration Sorghum Medium RSCM-25 (4.3 g/l MS salts and vitamins supplemented with 20 g/l sucrose, 0.5 g/l casein, 0.5 mg/l kinetin. pH to 5.8 and solidify with 2 g/L Gelrite; further supplemented with 25 mg/l G418 after autoclaving) in low light (16 hour day length, 26° C.) to initiate regeneration. Simultaneous with initiating regeneration, this callus tissue is evaluated by PCR for presence of the genes on the MC.
After an additional 4-6 weeks on regeneration, plantlets (with and without initial root initiation) are transferred to Rooting Medium RtSC-25 (2.15 g MS salts and vitamins supplemented with 20 g/l sucrose, 0.5 g/l casein, 0.5 mg/l kinetin. pH to 5.8 and solidify with 2 g/L Gelrite; further supplemented with 25 mg/l G418 after autoclaving). Rooting occurs in 2 to 6 additional weeks of culture with 16 hour day length at 26° C. Rooting occurs in sundae cups (Solo Cup Co.; Joliet, Ill.; USA) for additional plantlet growth and root development. Plantlets with well established root systems are transferred into pre-moistened soil-less mix (LC1, BFG Supply Co.; Burton, Ohio; USA) under a humidome in an 18 well flat in a growth chamber (28° C., 16 hour day length). The dome is opened slightly 3-4 days after transplanting to slowly reduce humidity. The dome is removed completely 2 days later and the plantlets are then transferred to a greenhouse (28° C., 16 hour day length). The plants are watered from trays beneath the pots when soil began to dry. The plants are subsequently transplanted and grown to maturity in 1.6 gallon pots with Soil:Peat:Perlite (1:1:1) supplemented with Osmocote fertilizer (Scotts Co.; Marysville, Ohio; USA).
Sorghum callus and tissues produce phenolic compounds while in tissue culture, and these phenolic compounds reduce or inhibit callus growth and plantlet regeneration. In order to promote sorghum plantlet regeneration in culture, the media described above (MS0, MS3 and variants thereof, and RSCM) are supplemented with PVP at a concentrations of 1% to 3% w/v according to the intensity of the exudation of the phenolic compounds. The PVP acts as a sink for phenolic compounds and enhances subsequent callus growth and plantlet regeneration.
In order to promote the frequency and the morphogenetic competence of regenerable sorghum callus, the cells are cycled from a liquid culture to a solid culture. The apical region of the sorghum stem (20-30 cm long) is collected and the mature leaves are removed. The stem is surface sterilize by submerging the tissue in a solution of 50 ml bleach in 1 liter of water for 10 minutes. The remaining outermost mature leaves are aseptically removed and the young inner immature leaves are sliced into sections/discs approximately 2-3 mm thick.
The resulting leaf roll discs are placed on sorghum MS3 Medium (MS3; 3 g/l MS salts and vitamins with 20 g/l sucrose, 0.5 g/l casein, 3 mg/l 2,4-D. pH to 5.8 and solidified with 2 g/L Gelrite) at 28° C. for 2 weeks in the dark. The resulting regenerable sorghum callus (white nodular embryogenic pieces) is then removed and placed into liquid sorghum MS1 Medium (MS1; 4.3 g/l MS salts and vitamins with 20 g/l sucrose, 0.5 g/l casein, 1 mg/l 2,4-D. pH to 5.8) at 28° C. for 2 weeks on a rotating orbital shaker (100-150 rpm) in the dark. After the 2 weeks, the regenerable sorghum callus (white nodular embryogenic pieces) is removed and sub-cultured back onto sorghum MS3 Medium (MS3) at 28° C. for 2 additional weeks in the dark. Sorghum callus can be subcultured in 2-week intervals between solid MS medium containing 3 mg/l 2,4-D and liquid MS medium containing 1 mg/l 2,4-D to maintain embryogenic callus.
Example 5
Evaluation of Autonomous MCs (Prophetic)
To evaluate whether the candidate MCs are maintained autonomously, FISH can be performed on mitotic metaphase chromosome spreads from root tips. FISH can be performed essentially as described in Kato et al. (PNAS USA. 101: 13554-13559, 2004).
For FISH, root tips can be collected approximately 10 days after transplanting regenerated T0 plants to soil or after germination (T1-T4 plants). Sampled roots (3-6 per plant) are moistened and exposed to nitrous oxide at 150 psi for 3 hours to arrest chromosomes in metaphase as described in Kato (Biotech. Histochem 74: 160-166, 1999). Roots are fixed in 90% acetic acid, and spread onto poly-lysine coated glass slides by squashing thin cross sections. Following hybridization with ALEXA FLUOR® 488 and ALEXA FLUOR® 568-labeled probes, slides are counter-stained with DAPI (0.04 mg/ml) and ≧15 metaphase cells are evaluated per plant using a Zeiss Axio-Imager equipped with rhodamine, FITC, and DAPI filter sets (excitation BP 550/24, emission BP 605/70; excitation BP 470/40, emission: BP 525/50; and excitation G 365, emission BP 445/50, respectively).
Extra-chromosomal signals are only considered to indicate autonomous MCs if ≧70% of the images (n≧15 cells analyzed) show co-localization of the ALEXA FLUOR® 488 and ALEXA FLUOR® 568 signals within 1 nuclear diameter of the endogenous metaphase maize chromosomes. Gray-scale images can be captured in each panel, merged and adjusted with pseudo-color using Zeiss AxioVision software (Zeiss; Thornwood, N.Y.; USA); fluorescent signals from doubly labeled MCs can be detected in both the red and green channels.
Integrated constructs result in two FISH signals, each on a replicated metaphase chromatid. The MCs can be considered autonomous when (i)≧70% of the cells examined (n≧15) contained signals that are clearly distinct from the DAPI-stained host chromosomes, (ii) integrated signals are not detected, and (iii) the fluorescent probe corresponding to the MC-encoded genes co-localize with the probe to repetitive centromeric DNA, suggesting an intact construct and making it unlikely that the signal is due to noise.

## Claims

1. A sorghum plant cell comprising a sorghum mini-chromosome comprising at least one exogenous nucleic acid and a sorghum centromere, wherein the sorghum centromere comprises at least five copies of a first repeated nucleotide sequence that (i) is at least 80% identical to the nucleotide sequence of any one of SEQ ID NOs: 22-176, or (ii) hybridizes to a nucleotide sequence of any one of SEQ ID NOs: 22-176 under stringent conditions comprising hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C., wherein the centromere confers the ability to segregate to daughter cells.

2. The sorghum plant cell of claim 1, further comprising a transgene expression cassette comprises at least one exogenous nucleic acid.

3. The sorghum plant cell of claim 1, further comprising at least two copies of a second repeated nucleotide sequence that is at least 80% identical over its length to a fragment of a sorghum retrotransposon sequence of SEQ ID NO: 21 or hybridizes to a fragment of the sorghum retrotransposon sequence of SEQ ID NO: 21 under stringent conditions comprising hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C.

4. A sorghum plant cell of claim 3, wherein the sorghum centromere comprises:
(a) at least 5 copies of the first repeat within 1 kb of nucleotide sequence and
(b) a transgene expression cassette comprising at least one exogenous nucleic acid.

5. The sorghum plant cell of claim 1, wherein the sorghum mini-chromosome exhibits a mitotic segregation efficiency in sorghum cells of at least 90%.

6. The sorghum plant cell of claim 2, wherein at least one of the exogenous nucleic acids is operably linked to a heterologous regulatory sequence functional in sorghum cells.

7. The sorghum plant cell according to claim 6, wherein the exogenous nucleic acid is selected from the group consisting of a herbicide resistance gene, a nitrogen fixation gene, an insect resistance gene, a disease resistance gene, a plant stress-induced gene, a nutrient utilization gene, a gene that affects plant pigmentation, a gene that encodes an antisense or ribozyme molecule, a gene encoding a secretable antigen, a toxin gene, a receptor gene, a ligand gene, a seed storage gene, a hormone gene, an enzyme gene, an antibody gene, a growth factor gene, a drought resistance gene, a heat resistance gene, a chilling resistance gene, a freezing resistance gene, an excessive moisture resistance gene, or a salt stress resistance gene or a biofuel gene.

8. The sorghum plant cell according to claim 1 wherein:
(a) a polynucleotide sequence is transcribed as a first RNA;
(b) a polynucleotide sequence is transcribed as a second RNA; and
(c) a polynucleotide sequence is transcribed as a third RNA, and wherein transcription of the polynucleotide sequences results in increased biomass of a sorghum plant compared to the biomass of a wild type sorghum plant.

9. A sorghum plant cell comprising a transgene expression cassette comprising at least one exogenous nucleic acid not integrated into the plant cell genome, wherein the transgene expression cassette comprises
(a) a polynucleotide sequence that is transcribed as a first RNA;
(b) a polynucleotide sequence that is transcribed as a second RNA; and
(c) a polynucleotide sequence that is transcribed as a third RNA; wherein transcription of the polynucleotide sequences results in increased biomass of a sorghum plant compared to the biomass of a wild type sorghum plant.

10. The sorghum plant cell of claim 1, wherein the recombinant chromosome has not been maintained in a cell of a heterologous organism.

11. The sorghum plant cell of claim 1, wherein the transgene expression cassette comprises at least three exogenous nucleic acids, and
wherein the first repeated nucleotide sequence and the transgene expression cassette are not integrated into the genome of the sorghum plant cell.

12. The sorghum plant cell of claim 2 that exhibits an altered phenotype associated with at least one exogenous nucleic acid within the sorghum mini-chromosome.

13. The sorghum plant cell of claim 12, wherein the altered phenotype comprises altered expression of a native active gene or the expression of an exogenous gene.

14. A sorghum plant, plant tissue or sorghum plant part comprising the plant cell of claim 1.

15. A sorghum seed obtained from the plant of claim 14, wherein the seed comprises a sorghum mini-chromosome comprising at least one exogenous nucleic acid and a sorghum centromere, wherein the sorghum centromere comprises at least five copies of a first repeated nucleotide sequence that (i) is at least 80% identical to a nucleotide sequence of any one of SEQ ID NOs: 22-176, or (ii) hybridizes to a nucleotide sequence of any one of SEQ ID NOs: 22-176 under stringent conditions comprising hybridization at 65° C. and washing three times for 15 minutes with 0.25×SSC, 0.1% SDS at 65° C. and wherein the centromere confers the ability to segregation to daughter cells.

16. A sorghum plant progeny comprising a sorghum mini-chromosome, wherein the plant progeny is the result of breeding a plant of claim 14.

17. A method of using a sorghum plant of claim 14, the method comprising growing the plant to produce a recombinant protein encoded by an exogenous nucleic acid of the mini-chromosome, and alternatively, further comprising a step of harvesting or processing the sorghum plant.

18. The sorghum plant cell of claim 4, wherein at least one exogenous nucleic acid is operably linked to a heterologous regulatory sequence functional in sorghum cells.

19. A sorghum plant of claim 4 that exhibits an altered phenotype associated with at least one exogenous nucleic acid within the sorghum mini-chromosome.