Engineered minichromosomes in plants.

Artificial chromosome platforms are described in plants. Because the function of centromeres is largely epigenetic, attempts to produce artificial chromosomes with plant centromere DNA have failed. The removal of the centromeric sequences from the cell strips off the centromeric histone that is the apparent biochemical marker of centromere activity. Thus, engineered minichromosomes have been produced by telomere mediated chromosomal truncation. The introduction of telomere repeats will cleave the chromosome at the site of insertion and attach the accompanying transgenes in the process. Such truncation events have been documented in maize, Arabidopsis, barley, rice, Brassica and wheat. Truncation of the nonvital supernumerary B chromosome of maize is a favorite target but engineered minichromosomes derived from the normal A chromosomes have also been recovered. Transmission through mitosis of small chromosomes is apparently normal but there is loss during meiosis. Potential solutions to address this issue are discussed. With procedures now well established to produce the foundation for artificial chromosomes in plants, current efforts are directed at building them up to specification using gene stacking methods and editing techniques.

Site-specific recombinase genome engineering toolkit in maize.

Site-specific recombinase enzymes function in heterologous cellular environments to initiate strand-switching reactions between unique DNA sequences termed recombinase binding sites. Depending on binding site position and orientation, reactions result in integrations, excisions, or inversions of targeted DNA sequences in a precise and predictable manner. Here, we established five different stable recombinase expression lines in maize through Agrobacterium-mediated transformation of T-DNA molecules that contain coding sequences for Cre, R, FLPe, phiC31 Integrase, and phiC31 excisionase. Through the bombardment of recombinase activated DsRed transient expression constructs, we have determined that all five recombinases are functional in maize plants. These recombinase expression lines could be utilized for a variety of genetic engineering applications, including selectable marker removal, targeted transgene integration into predetermined locations, and gene stacking.

Meiotic Studies on Combinations of Chromosomes With Different Sized Centromeres in Maize.

Multiple centromere misdivision derivatives of a translocation between the supernumerary B chromosome and the short arm of chromosome 9 (TB-9Sb) permit investigation of how centromeres of different sizes behave in meiosis in opposition or in competition with each other. In the first analysis, heterozygotes were produced between the normal TB-9Sb and derivatives of it that resulted from centromere misdivision that reduced the amounts of centromeric DNA. These heterozygotes could test whether these drastic differences would result in meiotic drive of the larger chromosome in female meiosis. Cytological determinations of the segregation of large and small centromeres among thousands of progeny of four combinations were made. The recovery of the larger centromere was at a few percent higher frequency in two of four combinations. However, examination of phosphorylated histone H2A-Thr133, a characteristic of active centromeres, showed a lack of correlation with the size of the centromeric DNA, suggesting an expansion of the basal protein features of the kinetochore in two of the three cases despite the reduction in the size of the underlying DNA. In the second analysis, plants containing different sizes of the B chromosome centromere were crossed to plants with TB-9Sb with a foldback duplication of 9S (TB-9Sb-Dp9). In the progeny, plants containing large and small versions of the B chromosome centromere were selected by FISH. A meiotic "tug of war" occurred in hybrid combinations by recombination between the normal 9S and the foldback duplication in those cases in which pairing occurred. Such pairing and recombination produce anaphase I bridges but in some cases the large and small centromeres progressed to the same pole. In one combination, new dicentric chromosomes were found in the progeny. Collectively, the results indicate that the size of the underlying DNA of a centromere does not dramatically affect its segregation properties or its ability to progress to the poles in meiosis potentially because the biochemical features of centromeres adjust to the cellular conditions.

Plant minichromosomes.

Plant minichromosomes have the potential for stacking multiple traits on a separate entity from the remainder of the genome. Transgenes carried on an independent chromosome would facilitate conferring many new properties to plants and using minichromosomes as genetic tools. The favored method for producing plant minichromosomes is telomere-mediated chromosomal truncation because the epigenetic nature of centromere function prevents using centromere sequences to confer the ability to organize a kinetochore when reintroduced into plant cells. Because haploid induction procedures are not always complete in eliminating one parental genome, chromosomes from the inducer lines are often present in plants that are otherwise haploid. This fact suggests that minichromosomes could be combined with doubled haploid breeding to transfer stacked traits more easily to multiple lines and to use minichromosomes for massive scale genome editing.

Telomere-Mediated Chromosomal Truncation for Generating Engineered Minichromosomes in Maize.

Minichromosomes have been generated in maize using telomere-mediated truncation. Telomere DNA, because of its repetitive nature, can be difficult to manipulate. The protocols in this unit describe two methods for generating the telomere DNA required for the initiation of telomere-mediated truncation. The resulting DNA can then be used with truncation cassettes for introduction into maize via transformation. © 2016 by John Wiley & Sons, Inc.

Engineered Minichromosomes in Plants: Structure, Function, and Applications.

Engineered minichromosomes are small chromosomes that contain a transgene and selectable marker, as well as all of the necessary components required for maintenance in an organism separately from the standard chromosome set. The separation from endogenous chromosomes makes engineered minichromosomes useful in the production of transgenic plants. Introducing transgenes to minichromosomes does not have the risk of insertion within a native gene; additionally, transgenes on minichromosomes can be transferred between lines without the movement of linked genes. Of the two methods proposed for creating engineered minichromosomes, telomere-mediated truncation is more reliable in plant systems. Additionally, many plants contain a supernumerary, or B chromosome, which is an excellent starting material for minichromosome creation. The use of site-specific recombination systems in minichromosomes can increase their utility, allowing for the addition or subtraction of transgenes in vivo. The creation of minichromosomes with binary bacterial artificial chromosome vectors provides the ability to introduce many transgenes at one time. Furthermore, coupling minichromosomes with haploid induction systems can facilitate transfer between lines. Minichromosomes can be introduced to a haploid-inducing line and crossed to target lines. Haploids of the target line that then contain a minichromosome can then be doubled. These homozygous lines will contain the transgene without the need for repeated introgressions.