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.

Generation of a Maize B Centromere Minimal Map Containing the Central Core Domain.

The maize B centromere has been used as a model for centromere epigenetics and as the basis for building artificial chromosomes. However, there are no sequence resources for this important centromere. Here we used transposon display for the centromere-specific retroelement CRM2 to identify a collection of 40 sequence tags that flank CRM2 insertion points on the B chromosome. These were confirmed to lie within the centromere by assaying deletion breakpoints from centromere misdivision derivatives (intracentromere breakages caused by centromere fission). Markers were grouped together on the basis of their association with other markers in the misdivision series and assembled into a pseudocontig containing 10.1 kb of sequence. To identify sequences that interact directly with centromere proteins, we carried out chromatin immunoprecipitation using antibodies to centromeric histone H3 (CENH3), a defining feature of functional centromeric sequences. The CENH3 chromatin immunoprecipitation map was interpreted relative to the known transmission rates of centromere misdivision derivatives to identify a centromere core domain spanning 33 markers. A subset of seven markers was mapped in additional B centromere misdivision derivatives with the use of unique primer pairs. A derivative previously shown to have no canonical centromere sequences (Telo3-3) lacks these core markers. Our results provide a molecular map of the B chromosome centromere and identify key sequences within the map that interact directly with centromeric histone H3.

Punctate vascular expression1 is a novel maize gene required for leaf pattern formation that functions downstream of the trans-acting small interfering RNA pathway.

The maize (Zea mays) gene RAGGED SEEDLING2-R (RGD2-R) encodes an ARGONAUTE7-like protein required for the biogenesis of trans-acting small interfering RNA, which regulates the accumulation of AUXIN RESPONSE FACTOR3A transcripts in shoots. Although dorsiventral polarity is established in the narrow and cylindrical leaves of rgd2-R mutant plants, swapping of adaxial/abaxial epidermal identity occurs and suggests a model wherein RGD2 is required to coordinate dorsiventral and mediolateral patterning in maize leaves. Laser microdissection-microarray analyses of the rgd2-R mutant shoot apical meristem identified a novel gene, PUNCTATE VASCULAR EXPRESSION1 (PVE1), that is down-regulated in rgd2-R mutant apices. Transcripts of PVE1 provide an early molecular marker for vascular morphogenesis. Reverse genetic analyses suggest that PVE1 functions during vascular development and in mediolateral and dorsiventral patterning of maize leaves. Molecular genetic analyses of PVE1 and of rgd2-R;pve1-M2 double mutants suggest a model wherein PVE1 functions downstream of RGD2 in a pathway that intersects and interacts with the trans-acting small interfering RNA pathway.

ragged seedling2 Encodes an ARGONAUTE7-like protein required for mediolateral expansion, but not dorsiventrality, of maize leaves.

Leaves arise from the flank of the shoot apical meristem and are asymmetrical along the adaxial/abaxial plane from inception. Mutations perturbing dorsiventral cell fate acquisition in a variety of species can result in unifacial (radially symmetrical) leaves lacking adaxial/abaxial polarity. However, mutations in maize (Zea mays) ragged seedling2 (rgd2) condition cylindrical leaves that maintain dorsiventral polarity. Positional cloning reveals that rgd2 encodes an ARGONAUTE7 (AGO7)-like protein required to produce ta-siARF, a trans-acting small interfering RNA that targets abaxially located auxin response factor3a (arf3a) transcripts for degradation. Previous studies implicated ta-siARF in dorsiventral patterning of monocot leaves. Here, we show that arf3a transcripts hyperaccumulate but remain abaxialized in rgd2 mutant apices, revealing that ta-siARF function is not required for arf3a polarization. RGD2 also regulates miR390 accumulation and localization in maize shoot apices. Similar to the abaxialized maize mutant leafbladeless1 (lbl1), rgd2 mutants exhibit ectopic accumulation of the abaxial identity factor miR166 in adaxial domains. Thus, hyperaccumulation of arf3a and ectopic accumulation of miR166 are insufficient to condition abaxialized leaf phenotypes in maize. Finally, transcripts of a maize ago1 paralog overaccumulate in lbl1 but not in rgd2 mutants, suggesting that upregulation of ago1 combined with ectopic accumulation of miR166 contribute to abaxialized leaf formation in lbl1. We present a revised model for the role of small RNAs in dorsiventral patterning of maize leaves.