The effect of DNA polymorphisms and natural variation on crossover hotspot activity in Arabidopsis hybrids.

In hybrid organisms, genetically divergent homologous chromosomes pair and recombine during meiosis; however, the effect of specific types of polymorphisms on crossover is poorly understood. Here, to analyze this in Arabidopsis, we develop the seed-typing method that enables the massively parallel fine-mapping of crossovers by sequencing. We show that structural variants, observed in one of the generated intervals, do not change crossover frequency unless they are located directly within crossover hotspots. Both natural and Cas9-induced deletions result in lower hotspot activity but are not compensated by increases in immediately adjacent hotspots. To examine the effect of single nucleotide polymorphisms on crossover formation, we analyze hotspot activity in mismatch detection-deficient msh2 mutants. Surprisingly, polymorphic hotspots show reduced activity in msh2. In lines where only the hotspot-containing interval is heterozygous, crossover numbers increase above those in the inbred (homozygous). We conclude that MSH2 shapes crossover distribution by stimulating hotspot activity at polymorphic regions.

Quantifying Meiotic Crossover Recombination in Arabidopsis Lines Expressing Fluorescent Reporters in Seeds Using SeedScoring Pipeline for CellProfiler.

The number of crossovers during meiosis is relatively low, so multiple meioses need to be analyzed to accurately measure crossover frequency. In Arabidopsis, systems based on the segregation of fluorescent T-DNA reporters that are expressed in seeds (fluorescent-tagged lines, FTLs) allow for an accurate measurement of crossover frequency in specific chromosome regions. A major advantage of FTL-based experiments is the ability to analyze thousands of seeds for each biological replicate, which requires the use of automatic seed scoring. Here, we describe a protocol to computationally count the proportion of seeds that experienced a crossover event within the tested FTL interval and so measure the recombination frequency within that interval. We describe SeedScoring, a CellProfiler pipeline where the total time needed to measure crossover frequency in a single FTL line is approximately 5 min using a series of three images taken under a fluorescent stereomicroscope (3 min) and passing these images through the SeedScoring pipeline described in this protocol (2 min).

MSH2 shapes the meiotic crossover landscape in relation to interhomolog polymorphism in Arabidopsis.

During meiosis, DNA double-strand breaks undergo interhomolog repair to yield crossovers between homologous chromosomes. To investigate how interhomolog sequence polymorphism affects crossovers, we sequenced multiple recombinant populations of the model plant Arabidopsis thaliana. Crossovers were elevated in the diverse pericentromeric regions, showing a local preference for polymorphic regions. We provide evidence that crossover association with elevated diversity is mediated via the Class I crossover formation pathway, although very high levels of diversity suppress crossovers. Interhomolog polymorphism causes mismatches in recombining molecules, which can be detected by MutS homolog (MSH) mismatch repair protein heterodimers. Therefore, we mapped crossovers in a msh2 mutant, defective in mismatch recognition, using multiple hybrid backgrounds. Although total crossover numbers were unchanged in msh2 mutants, recombination was remodelled from the diverse pericentromeres towards the less-polymorphic sub-telomeric regions. Juxtaposition of megabase heterozygous and homozygous regions causes crossover remodelling towards the heterozygous regions in wild type Arabidopsis, but not in msh2 mutants. Immunostaining showed that MSH2 protein accumulates on meiotic chromosomes during prophase I, consistent with MSH2 regulating meiotic recombination. Our results reveal a pro-crossover role for MSH2 in regions of higher sequence diversity in A. thaliana.

WOX14 promotes bioactive gibberellin synthesis and vascular cell differentiation in Arabidopsis.

Procambial and cambial stem cells provide the initial cells that allow the formation of vascular tissues. WOX4 and WOX14 have been shown to act redundantly to promote procambial cell proliferation and differentiation. Gibberellins (GAs), which have an important role in wood formation, also stimulate cambial cell division. Here we show that the loss of WOX14 function phenocopies some traits of GA-deficient mutants that can be complemented by exogenous GA application, whereas WOX14 overexpression stimulates the expression of GA3ox anabolism genes and represses GA2ox catabolism genes, promoting the accumulation of bioactive GA. More importantly, our data clearly indicate that WOX14 but not WOX4 promotes vascular cell differentiation and lignification in inflorescence stems of Arabidopsis.