Maize abnormal chromosome 10 (Ab10) encodes a classic example of true meiotic drive that converts heterochromatic regions called knobs into motile neocentromeres that are preferentially transmitted to egg cells. Here, we identify a cluster of eight genes on Ab10, called the Kinesin driver (Kindr) complex, that are required for both neocentromere motility and preferential transmission. Two meiotic drive mutants that lack neocentromere activity proved to be kindr epimutants with increased DNA methylation across the entire gene cluster. RNAi of Kindr induced a third epimutant and corresponding loss of meiotic drive. Kinesin gliding assays and immunolocalization revealed that KINDR is a functional minus-end-directed kinesin that localizes specifically to knobs containing 180 bp repeats. Sequence comparisons suggest that Kindr diverged from a Kinesin-14A ancestor ∼12 mya and has driven the accumulation of > 500 Mb of knob repeats and affected the segregation of thousands of genes linked to knobs on all 10 chromosomes.

Meiotic drive describes a process whereby selfish genetic elements are transmitted at levels greater than Mendelian expectations. Maize abnormal chromosome 10 (Ab10) encodes a meiotic drive system that exhibits strong preferential segregation through female gametes. We performed transmission assays on nine Ab10 chromosomes from landraces and teosinte lines and found a transmission advantage of 62-79% in heterozygotes. Despite this transmission advantage, Ab10 is present at low frequencies in natural populations, suggesting that it carries large negative fitness consequences. We measured pollen transmission, the percentage of live pollen, seed production, and seed size to estimate several of the possible fitness effects of Ab10. We found no evidence that Ab10 affects pollen transmission,, Ab10 and N10 pollen are transmitted equally from heterozygous fathers. However, at the diploid (sporophyte) level, both heterozygous and homozygous Ab10-I-MMR individuals show decreased pollen viability, decreased seed set, and decreased seed weight. The observed fitness costs can nearly but not entirely account for the observed frequencies of Ab10. Sequence analysis shows a surprising amount of molecular variation among Ab10 haplotypes, suggesting that there may be other phenotypic variables that contribute to the low but stable equilibrium frequencies.

1) CHAPTER 7(a) Recent experiments have shown that the Percoll gradient step is not necessary to get nuclear preparations of sufficient quality. Thus this step has been deleted. (b) EGTA and L-lysine have been made standard components of MEB and MPDB solutions. These compounds greatly reduce DNA damage caused by endogenous nucleases. 2) CHAPTERS 7, 9, and 10 -PVP is no longer added to the lysis buffer or wash buffers (WB-A, WB-B, and WB-C). PVP often precipitates out of solution to form a brown gel in which the agarose/nuclei plugs may get stuck. Lysis buffer is now made 6 mM EGTA and 200 mM L-lysine. 3) CHAPTER 13 -We no longer recommend the use of GELase to isolate BAC insert DNA from plugs. Our experience suggests that the DNA may be damaged by GELase. Electroelution has proven the most effective means of obtaining clonable DNA from agarose plugs. 4) CHAPTER 9 -Mathematical errors in Table 9.1 have been corrected. ABSTRACTBacterial artificial chromosome (BAC) libraries have become invaluable tools in plant genetic research. However, it is difficult for new practitioners to create plant BAC libraries de novo because published protocols are not particularly detailed, and plant cells possess features that make isolation of clean, high molecular weight DNA troublesome. In this document we present an illustrated, step-by-step protocol for constructing plant BAC libraries. This protocol is sufficiently detailed to be of use to both new and experienced investigators. We hope that by reducing the obstacles to BAC cloning in plants, we will foster new and accelerated progress in plant genomics.

A maize chromosome variant called abnormal chromosome 10 (Ab10) converts knobs on chromosome arms into neocentromeres, causing their preferential segregation to egg cells in a process known as meiotic drive. We previously demonstrated that the gene Kinesin driver (Kindr) on Ab10 encodes a kinesin-14 required to mobilize neocentromeres made up of the major tandem repeat knob180. Here we describe a second kinesin-14 gene, TR-1 kinesin (Trkin), that is required to mobilize neocentromeres made up of the minor tandem repeat TR-1. Trkin lies in a 4-Mb region of Ab10 that is not syntenic with any other region of the maize genome and shows extraordinary sequence divergence from Kindr and other kinesins in plants. Despite its unusual structure, Trkin encodes a functional minus end-directed kinesin that specifically colocalizes with TR-1 in meiosis, forming long drawn out neocentromeres. TRKIN contains a nuclear localization signal and localizes to knobs earlier in prophase than KINDR. The fact that TR-1 repeats often co-occur with knob180 repeats suggests that the current role of the TRKIN/TR-1 system is to facilitate the meiotic drive of the KINDR/knob180 system.

Birds are considered dinosaurs that passed the 65 million years ago bottleneck. Songbirds (Passeriformes) include about half extant bird species (about 5000) and are generally the most air-thriving bird species, concordantly with their small size. Mayor Histocompatibility complex (MHC) molecules stimulate immune responses against microbes and its class I molecules have seven conserved residues in all vertebrates from jawed-fishes, 300 million years ago, to humans, including chickens.All wild songbird species tested by us (n=18) and others (n= 2) differ in α1 domain residue 10 and α2 residue 96 from all other vertebrates. Amplification, cloning and sequencing were performed by standard methods. Sequences alignment were done by using PAUP and MEGA programs software. Crystallographic studies were performed by using mammal and bird MHC molecules from MPID database and other sources and showed that these changes did not significantly vary the MHC class I molecule stability in songbirds.Further α1 and α2 domain comparisons by simple Composition Distances and Bayesian Inference showed that songbirds overall MHC class I molecules are phylogenetically more separated from mammal than other birds molecules. In addition MHC class I introns from Passeriformes (songbirds) were found to be longer than humans, chicken introns being the shortest ones.These small mainly air-borne dinosaurs (Passeriformes) have undergone a different evolutive pathway, regarding to MHC, than all other tested vertebrates and more terrestrial birds. This may have been originated by an altogether different dinosaurs linage origin or to adaptation to more aerial than terrestrial environment or other unknown cause. In any case, the specific changes observed in this work for class I molecules in songbirds have reached a entropic, stable solution similar to that reached by other vertebrates.