Sequence of the supernumerary B chromosome of maize provides insight into its drive mechanism and evolution.

B chromosomes are enigmatic elements in thousands of plant and animal genomes that persist in populations despite being nonessential. They circumvent the laws of Mendelian inheritance but the molecular mechanisms underlying this behavior remain unknown. Here we present the sequence, annotation, and analysis of the maize B chromosome providing insight into its drive mechanism. The sequence assembly reveals detailed locations of the elements involved with the cis and trans functions of its drive mechanism, consisting of nondisjunction at the second pollen mitosis and preferential fertilization of the egg by the B-containing sperm. We identified 758 protein-coding genes in 125.9 Mb of B chromosome sequence, of which at least 88 are expressed. Our results demonstrate that transposable elements in the B chromosome are shared with the standard A chromosome set but multiple lines of evidence fail to detect a syntenic genic region in the A chromosomes, suggesting a distant origin. The current gene content is a result of continuous transfer from the A chromosomal complement over an extended evolutionary time with subsequent degradation but with selection for maintenance of this nonvital chromosome.

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.

Production of Engineered Minichromosome Vectors via the Introduction of Telomere Sequences.

Artificial minichromosomes are non-integrating vectors capable of stably maintaining transgenes outside of the main chromosome set. The production of minichromosomes relies on telomere-mediated chromosomal truncation, which involves introducing transgenes and telomere sequences concurrently to the cell to truncate an endogenous chromosomal target. Two methods can be utilized; either the telomere sequences can be incorporated into a binary vector for transformation with Agrobacterium tumefaciens, or the telomere sequences can be co-introduced with transgenes during particle bombardment. In this protocol, the methods required to isolate and introduce telomere sequences are presented. Following the methods presented, standard transformation procedures can be followed to produce minichromosome containing plants.

Preparation of Chromosomes from Zea mays.

High-quality preparations of chromosomes are useful for many purposes. To prepare metaphase chromosome spreads in maize, root tips are harvested and treated with nitrous oxide to stop cell division at metaphase before being fixed in acetic acid. This process allows a high number of condensed chromosome spreads to be obtained at the end of the procedure. To prepare chromosome spreads from various stages of meiosis, anthers are first fixed before being examined for developmental stage. Cells are digested with a mixture of enzymes before the chromosomes are dropped onto glass sides and fixed under UV light. © 2016 by John Wiley & Sons, Inc.

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.

Fluorescence In Situ Hybridization to Maize (Zea mays) Chromosomes.

Fluorescence In Situ Hybridization (FISH) is the annealing of fluorescent DNA probes to their complementary sequences on prepared chromosomes and subsequent visualization with a fluorescent microscope. In maize, FISH is useful for distinguishing each of the ten chromosomes in different accessions (karyotyping), roughly mapping single genes, transposable elements, transgene insertions, and identifying various chromosomal alterations. FISH can also be used to distinguish chromosomes between different Zea species in interspecific hybrids by use of retroelement painting. © 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.

In vivo modification of a maize engineered minichromosome.

Engineered minichromosomes provide efficient platforms for stacking transgenes in crop plants. Methods for modifying these chromosomes in vivo are essential for the development of customizable systems for the removal of selection genes or other sequences and for the addition of new genes. Previous studies have demonstrated that Cre, a site-specific recombinase, could be used to modify lox sites on transgenes on maize minichromosomes; however, these studies demonstrated somatic recombination only, and modified minichromosomes could not be recovered. We describe the recovery of an engineered chromosome composed of little more than a centromere plus transgene that was derived by telomere-mediated truncation. We used the fiber fluorescence in situ hybridization technique and detected a transgene on the minichromosome inserted among stretches of CentC centromere repeats, and this insertion was large enough to suggest a tandem insertion. By crossing the minichromosome to a plant expressing Cre-recombinase, the Bar selection gene was removed, leaving behind a single loxP site. This study demonstrates that engineered chromosomes can be modified in vivo using site-specific recombinases, a demonstration essential to the development of amendable chromosome platforms in plants.

Synthetic chromosome platforms in plants.

Synthetic chromosomes provide the means to stack transgenes independently of the remainder of the genome. Combining them with haploid breeding could provide the means to transfer many transgenes more easily among varieties of the same species. The epigenetic nature of centromere formation complicates the production of synthetic chromosomes. However, telomere-mediated truncation coupled with the introduction of site-specific recombination cassettes has been used to produce minichromosomes consisting of little more than a centromere. Methods that have been developed to modify genes in vivo could be applied to minichromosomes to improve their utility and to continue to increase their length and genic content. Synthetic chromosomes establish the means to add or subtract multiple transgenes, multigene complexes, or whole biochemical pathways to plants to change their properties for agricultural applications or to use plants as factories for the production of foreign proteins or metabolites.

Recovery of a telomere-truncated chromosome via a compensating translocation in maize.

Maize-engineered minichromosomes are easily recovered from telomere-truncated B chromosomes but are rarely recovered from A chromosomes. B chromosomes lack known genes, and their truncation products are tolerated and transmitted during meiosis. In contrast, deficiency gametes resulting from truncated A chromosomes prevent their transmission. We report here a de novo compensating translocation that permitted recovery of a large truncation of chromosome 1 in maize. The truncation (trunc-1) and translocation with chromosome 6 (super-6) occurred during telomere-mediated truncation experiments and were characterized using single-gene fluorescent in situ hybridization (FISH) probes. The truncation contained a transgene signal near the end of the broken chromosome and transmitted together with the compensating translocation as a heterozygote to approximately 41%-55% of progeny. Transmission as an addition chromosome occurred in ~15% of progeny. Neither chromosome transmitted through pollen. Transgene expression (Bar) cosegregated with trunc-1 transcriptionally and phenotypically. Meiosis in T1 plants revealed eight bivalents and one tetravalent chain composed of chromosome 1, trunc-1, chromosome 6, and super-6 in diplotene and diakinesis. Our data suggest that de novo compensating translocations allow recovery of truncated A chromosomes by compensating deficiency in female gametes and by affecting chromosome pairing and segregation. The truncated chromosome can be maintained as an extra chromosome or together with the super-6 as a heterozygote.