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.

CRISPR/Cas9-mediated targeted T-DNA integration in rice.

KEY MESSAGE: Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome. Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard Agrobacterium binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (hptII) gene. One version of the vector had hptII expression driven by an OsAct2 promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless hptII gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the hptII gene would allow hptII expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50-80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that Agrobacterium-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions.

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.

Biolistic DNA Delivery in Maize Immature Embryos.

One of the key factors for ensuring a successful genetic transformation is to effectively introduce genetic materials, such as plasmid DNA, into plant cells. A biolistic gun is one of the two best established and most popular tools for delivery of DNA into maize cells. It is the method that generated the first fertile transgenic maize plant. In this chapter, we describe steps involved in introducing single or paired plasmid DNAs into immature embryos of maize Hi II hybrid genotype, using Biolistic® PDS-1000/He particle delivery system. While we focus on the biolistic delivery process in the protocol presented here, we also provide step-by-step information required for successful regeneration of transgenic maize plants.

Development of a Transformable Fast-Flowering Mini-Maize as a Tool for Maize Gene Editing.

Maize (Zea mays ssp. mays) is a popular genetic model due to its ease of crossing, well-established toolkits, and its status as a major global food crop. Recent technology developments for precise manipulation of the genome are further impacting both basic biological research and biotechnological application in agriculture. Crop gene editing often requires a process of genetic transformation in which the editing reagents are introduced into plant cells. In maize, this procedure is well-established for a limited number of public lines that are amenable for genetic transformation. Fast-Flowering Mini-Maize (FFMM) lines A and B were recently developed as an open-source tool for maize research by reducing the space requirements and the generation time. Neither line of FFMM were competent for genetic transformation using traditional protocols, a necessity to its status as a complete toolkit for public maize genetic research. Here we report the development of new lines of FFMM that have been bred for amenability to genetic transformation. By hybridizing a transformable maize genotype high Type-II callus parent A (Hi-II A) with line A of FFMM, we introgressed the ability to form embryogenic callus from Hi-II A into the FFMM-A genetic background. Through multiple generations of iterative self-hybridization or doubled-haploid method, we established maize lines that have a strong ability to produce embryogenic callus from immature embryos and maintain resemblance to FFMM-A in flowering time and stature. Using an Agrobacterium-mediated standard transformation method, we successfully introduced the CRISPR-Cas9 reagents into immature embryos and generated transgenic and mutant lines displaying the expected mutant phenotypes and genotypes. The transformation frequencies of the tested genotypes, defined as the numbers of transgenic event producing T1 seeds per 100 infected embryos, ranged from 0 to 17.1%. Approximately 80% of transgenic plants analyzed in this study showed various mutation patterns at the target site. The transformable FFMM line, FFMM-AT, can serve as a useful genetic and genomic resource for the maize community.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes.

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

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.

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.

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.

Fast-Flowering Mini-Maize: Seed to Seed in 60 Days.

Two lines of Zea mays were developed as a short-generation model for maize. The Fast-Flowering Mini-Maize (FFMM) lines A and B are robust inbred lines with a significantly shorter generation time, much smaller stature, and better greenhouse adaptation than traditional maize varieties. Five generations a year are typical. FFMM is the result of a modified double-cross hybrid between four fast-flowering lines: Neuffer's Early ACR (full color), Alexander's Early Early Synthetic, Tom Thumb Popcorn, and Gaspe Flint, followed by selection for early flowering and desirable morphology throughout an 11-generation selfing regime. Lines A and B were derived from different progeny of the initial hybrid, and crosses between Mini-Maize A and B exhibit heterosis. The ancestry of each genomic region of Mini-Maize A and B was inferred from the four founder populations using genotyping by sequencing. Other genetic and genomic tools for these lines include karyotypes for both lines A and B, kernel genetic markers y1 (white endosperm) and R1-scm2 (purple endosperm and embryo) introgressed into Mini-Maize A, and ∼24× whole-genome resequencing data for Mini-Maize A.

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.

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.

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.