High-resolution, genome-wide maps of meiotic DSBs in plants
SPO11 (SPORULATION11) is an evolutionary conserved transesterase enzyme that catalyses the formation of meiotic DSBs, as a subunit in hetero-hexameric topoisomerase VI like-complexes. During catalysis, each SPO11 protein covalently attaches to the 5’ end of the single strand DNA at the cut site. Downstream bidirectional activities of endo- and exonucleases of Mre11-Rad50-Xrs2/Nbs1-Sae2 complex release the SPO11 bound to about ~35 nucleotide DNA oligonucleotides molecules (SPO11-oligos). Yeast SPO11-oligos have been purified and sequenced deeply, which has demonstrated an important role of chromatin structure in meiotic DSB formation (Pan et al., 2011). We have developed a new protocol for genome-wide SPO11-1-oligo mapping in Arabidopsis by using a SPO11-1-myc transgene line which complements the spo11-1 fertility. Small oligonucleotides (~35-45 nt) associated with SPO11-1 protein were then purified, and deep-sequenced to profile meiotic DSB sites at the Arabidopsis genome with 32,000 transposons and complex epigenetic mechanisms.
Interconnections between Meiotic meiotic DSBs recombination and Chromatin: Because the SPO11-oligo map generates a single-nucleotide resolution of meiotic DSB landscape at genome, it is a sensitive genome-wide assay to investigate the mechanism how genetic, epigenetic and environmental changes affect chromatin structure and meiotic DSB formation. The meiotic DSB landscapes in Arabidopsis epigenetic mutants will be elucidated.
Meiotic DSBs and Temperature: Temperature alters CO rates along chromosomes in Arabidopsis and other plants. SPO11-oligo maps in Arabidopsis grown under different temperatures will determine whether temperature can affect the landscape of meiotic DSBs along whole chromosomes, or downstream events of DSB formation. The DSB maps will reveal the relationships between temperature, plant meiotic recombination initiation and chromatin structure.
Meiotic DSBs and Sex: Like most multicellular eukaryotes, Arabidopsis has a sex-specific CO pattern, with more crossovers in male than female at subtelomeric regions. However, it remains elusive whether plant meiotic DSBs occur more frequently in the male meiocyte than the female. It will determines if or not there is a differences in the pattern of meiotic DSBs along chromosomes occur in the male and female lineages. The results will provide insights into how sex specific recombination landscapes are controlled in other higher eukaryotes, as well as plants.
Meiotic DSB maps in Diverse Plants: The core mechanism of meiotic recombination including SPO11 is widely conserved among eukaryotes, but surprisingly the mechanisms that define meiotic recombination location and activity are varied between species [endif]--(Fowler et al., 2014; Pratto et al., 2014). It is important to study the variations of meiotic recombination control in diverse species, in order to investigate how meiotic recombination contributes to evolutionary changes of genomes via genetic and epigenetic information. To answer the question, we will apply the SPO11-oligo sequencing approach to diverse plant species and crops, including rice, tomato, cucumber and maize.![endif]--