Meiotic behavior of small chromosomes in maize

The typical behavior of chromosomes in meiosis is that homologous pairs synapse, recombine, and then separate at anaphase I. At anaphase II, sister chromatids separate. However, studies of small chromosomes in maize derived from a variety of sources typically have failure of sister chromatid cohesion at anaphase I. This failure occurs whether there is pairing of two copies of a minichromosome or not. These characteristics have implications for managing the transmission of the first generation artificial chromosomes in plants. Procedures to address these issues of minichromosomes are discussed

A transgenic system for generation of transposon Ac/Ds-induced chromosome rearrangements in rice

The maize Activator (Ac)/Dissociation (Ds) transposable element system has been used in a variety of plants for insertional mutagenesis. Ac/Ds elements can also generate genome rearrangements via alternative transposition reactions which involve the termini of closely linked transposons. Here, we introduced a transgene containing reverse-oriented Ac/Ds termini together with an Ac transposase gene into rice (Oryza sativa ssp. japonica cv. Nipponbare). Among the transgenic progeny, we identified and characterized 25 independent genome rearrangements at three different chromosomal loci. The rearrangements include chromosomal deletions and inversions and one translocation. Most of the deletions occurred within the T-DNA region, but two cases showed the loss of 72 kilobase pairs (kb) and 79 kb of rice genomic DNA flanking the transgene. In addition to deletions, we obtained chromosomal inversions ranging in size from less than 10 kb (within the transgene DNA) to over 1 million base pairs (Mb). For 11 inversions, we cloned and sequenced both inversion breakpoints; in all 11 cases, the inversion junctions contained the typical 8 base pairs (bp) Ac/Ds target site duplications, confirming their origin as transposition products. Together, our results indicate that alternative Ac/Ds transposition can be an efficient tool for functional genomics and chromosomal manipulation in rice

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

Reactivation of an Inactive Centromere Reveals Epigenetic and Structural Components for Centromere Specification in Maize[W]

Stable maize (Zea mays) chromosomes were recovered from an unstable dicentric containing large and small versions of the B chromosome centromere. In the stable chromosome, the smaller centromere had become inactivated. This inactive centromere can be inherited from one generation to the next attached to the active version and loses all known cytological and molecular properties of active centromeres. When separated from the active centromere by intrachromosomal recombination, the inactive centromere can be reactivated. The reactivated centromere regains the molecular attributes of activity in anaphase I of meiosis. When two copies of the dicentric chromosome with one active and one inactive centromere are present, homologous chromosome pairing reduces the frequency of intrachromosomal recombination and thus decreases, but does not eliminate, the reactivation of inactive centromeres. These findings indicate an epigenetic component to centromere specification in that centromere inactivation can be directed by joining two centromeres in opposition. These findings also indicate a structural aspect to centromere specification revealed by the gain of activity at the site of the previously inactive sequences

Rapid and Repeatable Elimination of a Parental Genome-Specific DNA Repeat (pGc1R-1a) in Newly Synthesized Wheat Allopolyploids

Recent work in the Triticum-Aegilops complex demonstrates that allopolyploidization is associated with an array of changes in low-copy coding and noncoding sequences. Nevertheless, the behavior and fate of repetitive DNA elements that constitute the bulk of nuclear DNA of these plant species is less clear following allopolyploidy. To gain further insight into the genomic events that accompany allopolyploid formation, we investigated fluorescence in situ hybridization (FISH) patterns of a parental-specific, tandem DNA repeat (pGc1R-1) on three sets of newly synthesized amphiploids with different parental species. It was found that drastic physical elimination of pGc1R-1 copies occurred in all three amphiploids in early generations. DNA gel-blot analysis confirmed the FISH data and estimates indicated that ∼70–90% of the copies of the pGc1R-1 repeat family were eliminated from the amphiploids by the second to third selfed generations. Thus, allopolyploidy in Triticum-Aegilops can be accompanied by rapid and extensive elimination of parental-specific repetitive DNA sequences, which presumably play a role in the initial stabilization of the nascent amphiploid plants

Cytological Visualization of DNA Transposons and Their Transposition Pattern in Somatic Cells of Maize

Global genomic analysis of transposable element distributions of both natural (En/Spm, Ac–Ds, and MuDR/Mu) and modified (RescueMu) types was performed by fluorescence in situ hybridization (FISH) on somatic chromosomes coupled with karyotyping of each chromosome. In lines without an active transposable element, the locations of silent En/Spm, Ac–Ds, and MuDR/Mu elements were visualized, revealing variation in copy number and position among lines but no apparent locational bias. The ability to detect single elements was validated by using previously mapped active Ac elements. Somatic transpositions were documented in plants containing an engineered Mutator element, RescueMu, via use of the karyotyping system. By analyzing the RescueMu lines, we found that transposition of RescueMu in root-tip cells follows the cut-and-paste type of transposition. This work demonstrates the utility of FISH and karyotyping in the study of transposon activity and its consequences

The Behavior of the Maize B Chromosome and Centromere

The maize B chromosome is a non-essential chromosome with an accumulation mechanism. The dispensable nature of the B chromosome facilitates many types of genetic studies in maize. Maize lines with B chromosomes have been widely used in studies of centromere functions. Here, we discuss the maize B chromosome alongside the latest progress of B centromere activities, including centromere misdivision, inactivation, reactivation, and de novo centromere formation. The meiotic features of the B centromere, related to mini-chromosomes and the control of the size of the maize centromere, are also discussed

Minichromosome Analysis of Chromosome Pairing, Disjunction, and Sister Chromatid Cohesion in Maize[W]

With the advent of engineered minichromosome technology in plants, an understanding of the properties of small chromosomes is desirable. Twenty-two minichromosomes of related origin but varying in size are described that provide a unique resource to study such behavior. Fourteen minichromosomes from this set could pair with each other in meiotic prophase at frequencies between 25 and 100%, but for the smaller chromosomes, the sister chromatids precociously separated in anaphase I. The other eight minichromosomes did not pair with themselves, and the sister chromatids divided equationally at meiosis I. In plants containing one minichromosome, the sister chromatids also separated at meiosis I. In anaphase II, the minichromosomes progressed to one pole or the other. The maize (Zea mays) Shugoshin protein, which has been hypothesized to protect centromere cohesion in meiosis I, is still present at anaphase I on minichromosomes that divide equationally. Also, there were no differences in the level of phosphorylation of Ser-10 of histone H3, a correlate of cohesion, in the minichromosomes in which sister chromatids separated during anaphase I compared with the normal chromosomes. These analyses suggest that meiotic centromeric cohesion is compromised in minichromosomes depending on their size and cannot be maintained by the mechanisms used by normal-sized chromosomes