A Kinesin-14 Motor Activates Neocentromeres to Promote Meiotic Drive in Maize.

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.

Maize chromosomal knobs are located in gene-dense areas and suppress local recombination.

Knobs are conspicuous heterochromatic regions found on the chromosomes of maize and its relatives. The number, locations, and sizes of knobs vary dramatically, with most lines containing between four and eight knobs in mid-arm positions. Prior data suggest that some knobs may reduce recombination. However, comprehensive tests have not been carried out, primarily because most knobs have not been placed on the genetic map. We used fluorescent in situ hybridization and two recombinant inbred populations to map seven knobs and to accurately place three knobs from the B73 inbred on the genomic sequence assembly. The data show that knobs lie in gene-dense regions of the maize genome. Comparisons to 23 other recombinant inbred populations segregating for knobs at the same sites confirm that large knobs can locally reduce crossing over by as much as twofold on a cM/Mb scale. These effects do not extend beyond regions ~10 cM to either side of knobs and do not appear to affect linkage disequilibrium among genes within and near knob repeat regions of the B73 RefGen_v2 assembly.

Fitness Costs and Variation in Transmission Distortion Associated with the Abnormal Chromosome 10 Meiotic Drive System in Maize.

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, i.e., 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.

HtStuf: High-Throughput Sequencing to Locate Unknown DNA Junction Fragments.

Advances in high-throughput sequencing have led to many new technologies for assessing genomes and population diversity. In spite of this, inexpensive and technically simple methods for efficiently pinpointing the location of transgenes and other specific sequences in large genomes are lacking. Here we report the development of a modified TA cloning and Illumina sequencing method called high-throughput sequencing to locate unmapped DNA fragments (HtStuf). Transgenic insertion sites were identified and confirmed in nine out of 10 transgenic soybean [Glycine max (L.) Merr.] lines, and major rearrangements of the transgene were detected in these lines. Additionally this method was used to map insertions of the introduced DNA transposon, mPing, in four T6 lines derived from a single event. Fifteen of the mPing insertion sites were validated with polymerase chain reaction. Together, these data demonstrate the simplicity and effectiveness of this novel sequencing method.

Intragenomic conflict between the two major knob repeats of maize.

Examples of meiotic drive, the non-Mendelian segregation of a specific genomic region, have been identified in several eukaryotic species. Maize contains the abnormal chromosome 10 (Ab10) drive system that transforms typically inert heterochromatic knobs into centromere-like domains (neocentromeres) that move rapidly poleward along the spindle during meiosis. Knobs can be made of two different tandem repeat sequences (TR-1 and 180-bp repeat), and both repeats have become widespread in Zea species. Here we describe detailed studies of a large knob on chromosome 10 called K10L2. We show that the knob is composed entirely of the TR-1 repeat and is linked to a strong activator of TR-1 neocentromere activity. K10L2 shows weak meiotic drive when paired with N10 but significantly reduces the meiotic drive exhibited by Ab10 (types I or II) in Ab10/K10L2 heterozygotes. These and other data confirm that (1) there are two separate and independent neocentromere activities in maize, (2) that both the TR-1 and knob 180 repeats exhibit meiotic drive (in the presence of other drive genes), and (3) that the two repeats can operate in competition with each other. Our results support the general concept that tandem repeat arrays can engage in arms-race-like struggles and proliferate as an outcome.