De novo centromere formation on chromosome fragments with an inactive centromere in maize (Zea mays)

The B chromosome of maize undergoes nondisjunction at the second pollen mitosis as part of its accumulation mechanism. Previous work identified 9-Bic-1 (9-B inactivated centromere-1), which comprises an epigenetically silenced B chromosome centromere that was translocated to the short arm of chromosome 9(9S). This chromosome is stable in isolation, but when normal B chromosomes are added to the genotype, it will attempt to undergo nondisjunction during the second pollen mitosis and usually fractures the chromosome in 9S. These broken chromosomes allow a test of whether the inactive centromere is reactivated or whether a de novo centromere is formed elsewhere on the chromosome to allow recovery of fragments. Breakpoint determination on the B chromosome and chromosome 9 showed that mini chromosome B1104 has the same breakpoint as 9-Bic-1 in the B centromere region and includes a portion of 9S. CENH3 binding was found on the B centromere region and on 9S, suggesting both centromere reactivation and de novo centromere formation. Another mini chromosome, B496, showed evidence of rearrangement, but it also only showed evidence for a de novo centromere. Other mini chromosome fragments recovered were directly derived from the B chromosome with breakpoints concentrated near the centromeric knob region, which suggests that the B chromosome is broken at a low frequency due to the failure of the sister chromatids to separate at the second pollen mitosis. Our results indicate that both reactivation and de novo centromere formation could occur on fragments derived from the progenitor possessing an inactive centromere.

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.

The unusualdRempretrotransposon is abundant, highly mutagenic, and mobilized only in the second pollen mitosis of some maize lines

The frequent mutations recovered recently from the pollen of select maize lines resulted from the meiotic mobilization of specific low-copy number long-terminal repeat (LTR) retrotransposons, which differ among lines. Mutations that arise at male meiosis produce kernels with concordant mutant phenotypes in both endosperm and embryo because the two sperms that participate in double fertilization are genetically identical. Those are in a majority. However, a small minority of kernels with a mutant endosperm carry a nonconcordant normal embryo, pointing to a postmeiotic or microgametophytic origin. In this study, we have identified the basis for those nonconcordant mutations. We find that all are produced by transposition of a defective LTR retrotransposon that we have termeddRemp(defective retroelement mobile in pollen). This element has several unique properties. Unlike the mutagenic LTR retrotransposons identified previously,dRempis present in hundreds of copies in all sequenced lines. It seems to transpose only at the second pollen mitosis because alldRempinsertion mutants are nonconcordant yet recoverable in either the endosperm or the embryo. Although it does not move in most lines,dRempis highly mobile in the Corn Belt inbred M14, identified earlier by breeders as being highly unstable. Lastly, it can be recovered in an array of structures, ranging from solo LTRs to tandemdRemprepeats containing several internal LTRs, suggestive of extensive recombination during retrotransposition. These results shed further light on the spontaneous mutation process and on the possible basis for inbred instability in maize.

The Gametic Non-Lethal Gene Gal on Chromosome 5 Is Indispensable for the Transmission of the Co-Induced Semidwarfing Gene d60 in Rice

The gametic lethal gene gal in combination with the semidwarfing gene d60 causes complementary lethality in rice. Here, we attempted to ascertain the existence of gal and clarify male gamete abortion caused by d60 and gal. Through the F2 to F4 generations derived from the cross between D60gal-homozygous and d60Gal-homozygous, progenies of the partial sterile plants (D60d60Galgal) were segregated in a ratio of 1 semidwarf (1 d60d60GalGal):2 tall and quarter sterile (2 D60d60Galgal):6 tall (2 D60d60GalGal:1 D60D60GalGal:2 D60D60Galgal:1 D60D60galgal), which is skewed from the Mendelian ratio of 1 semidwarf:3 tall. However, the F4 generation was derived from fertile and tall heterozygous F2 plants (D60d60GalGal), which were segregated in the Mendelian ratio of 1[semidwarf (d60d60GalGal)]:2[1 semidwarf:3 tall (D60d60GalGal)]:1[tall (D60D60GalGal)]. The backcrossing of D60Gal-homozygous tall F4 plants with Hokuriku 100 resulted in fertile BCF1 and BCF2 segregated in a ratio of 1 semidwarf:3 tall, proving that d60 is inherited as a single recessive gene in the D60d60GalGal genetic background (i.e., in the absence of gal). Further, gal was localized on chromosome 5, which is evident from the deviated segregation of d1 as 1:8 and linkage with simple sequence repeat (SSR) markers. Next-generation sequencing identified the candidate SNP responsible for Gal. In F1 and sterile F2, at the binucleate stage, partial pollen discontinued development. Degraded pollen lost vegetative nuclei, but second pollen mitosis raising two generative nuclei was observed. Thus, our study describes a novel genetic model for a reproductive barrier. This is the first report on such a complementary lethal gene, whose mutation allows the transmission of a co-induced valuable semidwarfing gene d60.

Locating a site on the maize B chromosome that controls preferential fertilization

In maize, the B chromosome can undergo nondisjunction at the second pollen mitosis, producing sperm with two B chromosomes and sperm with zero B chromosomes. Preferential fertilization is the ability of the sperm carrying two B chromosomes to transmit more frequently to the embryo of a kernel than the sperm lacking the B chromosome. A translocation involving the B chromosome and chromosome 9, TB-9Sb, has been used to study preferential fertilization. The B-9 chromosome has the same properties of nondisjunction and preferential fertilization as the standard B chromosome. Deletion derivatives of B-9, which lack the centric heterochromatin and possibly some adjacent euchromatin, were tested for their ability to induce preferential fertilization. They were found to lack the capacity for preferential fertilization.

Unstable inheritance of maize B-type chromosomes that lack centric heterochromatin

The B chromosome of maize undergoes frequent non-disjunction at the second pollen mitosis. In B–A translocations, the B–A chromosome retains the capacity for non-disjunction. We have collected deletion-derivative TB-9Sb stocks. One derivative, the "type 1 telocentric", has a B–9 chromosome that lacks centric heterochromatin. It produces few recessive (non-disjunctional) phenotypes in pollen parent testcrosses of the translocation heterozygote, 9 9–B telo B–9. The finding helped demonstrate the role of centric heterochromatin in non-disjunction. An isochromo some derivative of the type 1 telocentric was also recovered. It was tested in the 9–B 9–B iso B–9 constitution. This is equivalent to 9 9–B telo B–9 in terms of chromosome 9 dosage. Surprisingly, crosses with the isochromosome gave significant levels of recessive phenotypes. In addition, high levels of variegated phenotypes were found. Recently, a circumstance was found that makes inheritance of the type 1 telocentric chromosome somewhat similar to that of the isochromosome. Crosses with hypoploid 9–B 9–B telo B–9 plants showed significant levels of recessive and variegated phenotypes. These crosses were investigated to help explain the source(s) of the phenotypes. Cytological and genetic studies were performed. Centric misdivision was found to account for the variegated phenotypes. A mixture of conventional B non-disjunction and centric misdivision produced the recessive phenotypes. The significance of conventional non-disjunction in the absence of centric heterochromatin is discussed.Key words: cytogenetics, B chromosome, centromere, maize.

Time Course Study of the Chromosome-Type Breakage-Fusion-Bridge Cycle in Maize

The B chromosome of maize has been used in a study of dicentric chromosomes. TB-9Sb is a translocation between the B and chromosome 9. The B-9 of TB-9Sb carries 60% of the short arm of 9. For construction of dicentrics, a modified B-9 chromosome was used, B-9-Dp9. It consists of the B-9 chromosome plus a duplicated 9S region attached to the distal end. In meiosis, fold-back pairing and crossing over in the duplicated region gives a chromatid-type dicentric B-9 that subsequently initiates a chromatid-type breakage-fusion-bridge cycle. In the male, it forms a single bridge in anaphase II of meiosis and at the first pollen mitosis. However, the cycle is interrupted by nondisjunction of the B centromere at the second pollen mitosis, which sends the B-9 dicentric to one pole and converts it from a chromatid dicentric to a chromosome dicentric. As expected, the new dicentric undergoes the chromosome-type breakage-fusion-bridge cycle and produces double bridges. A large number of plants with chromosome dicentrics were produced in this way. The presence of double bridges in the root cells of plants with a chromosome dicentric was studied during the first 10 wk of development. It was found that the number of plants and cells showing double bridges declined steadily over the 10-wk period. Several lines of evidence indicate that there was no specific developmental time for dicentric loss. “Healing” of broken chromosomes produced by dicentric breakage accounted for much of the dicentric loss. Healing produced a wide range of derived B-9 chromosomes, some large and some small. A group of minichromosomes found in these experiments probably represents the small end of the scale for B-9 derivatives.

Maize microsporogenesis

The use of maize (Zea mays L.) pollen for basic scientific research has been well documented, but the progression of clear cytological features of maize microsporogenesis has not been fully documented. This study was undertaken to identify cytologically the different developmental stages of maize pollen and to correlate them with morphological features of the developing maize tassel. Morphological changes in the length of the tassel, floret, and anther were recorded and correlated with six cytologically defined stages of microsporogenesis: premeiosis, meiosis, uninucleate stage, first pollen mitosis, second pollen mitosis, and mature pollen.Key words: cytogenetics, gametophyte, maize, microsporogenesis, pollen.

A new method for producing homozygous duplications in maize

Translocations between the B chromosomes of maize and standard (A) chromosomes have been widely used to manipulate the dosage of A chromosome segments. The B chromosome frequently undergoes nondisjunction at the second pollen mitosis as part of its normal pattern of inheritance. BA chromosomes also undergo nondisjunction and, therefore, are used to produce duplications and deficiencies of A-chromatin. Duplications are useful in gene dosage studies and some may have agronomic value if they are associated with useful phenotypes. However, duplications produced by BA nondisjunction cannot be easily maintained during propagation. The BA undergoes nondisjunction each generation, destabilizing its inheritance. A new method is presented here for systematically duplicating segments of the maize genome using B–A translocations. The method uses meiotic segregation rather than nondisjunction to produce duplications. It employs AB chromosomes instead of BA chromosomes. As a test of the method, homozygous duplications (segmental tetrasomics) were constructed for a region on chromosome 3 and for another region on chromosome 9.Key words: maize, B chromosome, duplication.

Extrême précocité et conditions thermiques du développement apical et floral chez Claytonia caroliniana var. caroliniana

The apical and floral development of Claytonia caroliniana var. caroliniana has been studied concurrently with soil temperature, in a sugar maple forest of the Stoneham mountain, Québec. Apical cellular activity begins early in May, while the flowering stems of the year are present. At the beginning of July, external apical development becomes visible. In the first days of August, 9 months before flowering, the foliar and floral structures of the next year are already present in the soil. Meiosis takes place at the beginning of October and first pollen mitosis follows shortly after, in the middle of the same month. From that time, well developed individuals, without chlorophyll, are present just under the litter. They can occasionally turn green and reach the upper surface of the litter in November or December, where they will spend wintertime under the snow, at a temperature oscillating between 0 and −4 °C. This behaviour is quite close to the survival strategy of hemicryptophytes. The active epigeous growth period begins in the middle of April, with the melting of snow. Second pollen mitosis and flowering take place at this time, rapidly followed by seed setting, dissemination, and destruction of the aerial portion of the plant. Cytoecological investigations to study possible influence of environmental factors on chromosomal anomalies in primordia should thus be conducted during the year preceding the flowering of Claytonia.