Single pollen nuclei sequencing provides better profiling of meiotic recombination

When plant breeders want to develop new varieties with new combinations of traits, they need to rely on the recombination of genes between mother- and father plants that occurs during meiosis. This recombination is possible thanks to ‘crossing over’ events that happen between homologous chromosomes coming from the mother and father during meiotic cell division upon fertilization. This process occurs during the meiosis step in the formation of gametes that eventually give rise to an offspring population.

To optimize crop improvement, breeders want to gain insight into the frequency and preferred locations in the chromosomes where cross over events take place in order to generate novel allelic combinations of genes involved in important crop traits. Currently, DNA of F2 progeny plants from crosses between parents of interest are evaluated for parental SNP inheritance to generate recombination landscapes. Because genotyping F2 plants requires the growth to maturity of the F1 and the growth of F2 plants this is a very costly, slow and labor-intensive procedure.

Fast and cost-effective

Studying crossing overs in gametes, so shortly after meiosis, holds the promise of a fast and cost-effective approach. For an approach like that, one would need a way to isolate nuclei of individual gametes, an effective platform to sequence the DNA of individual nuclei, and smart bioinformatics to analyze the results and draw conclusions.

Combination of strengths

KeyGene is well known for its innovative protocols to isolate high quality high molecular weight DNA from plant tissue. KeyGene also developed proprietary protocols for isolating intact individual nuclei from pollen in a high throughput way.

For research performed together with Ultima Genomics, hundreds of pollen nuclei were isolated by KeyGene. A sub-sample of each nucleus was sequenced with KeyGene’s in-house DNBSEQ-G400 platform from MGI. An identical sub-sample of each nucleus was sequenced by Ultima Genomics, using Ultima’s new UG 100^tm platform. The sequencing results of millions of single nucleotide polymorphisms (SNPS) on both platforms were then analyzed using KeyGene’s data science capabilities.

Results

Meiotic recombination profiling analysis delivered evidence-based maps of recombination in individual gametes at extremely high resolution of approximately 5 Kbp. It also delivered valuable conclusions about recombination frequencies, the distribution of recombination along the chromosomes and even evidence of double crossover events (2 crossovers nearby each other on the same homologous chromosome). Such double crossovers are often overlooked or dubbed as ‘false positives’ by the more traditional F2 progeny plant DNA approach.

The Ultima Genomics platform produced slightly better results, probably thanks to the length of the reads, which were approximately twice as long as the reads on MGI. The Ultima Genomics platform produces single-end 300 bp reads, and MGI generates paired-end reads of 150 bp.
On top of that, the costs of the use of the UG 100^tm Ultima Genomics platform were substantially lower than the costs of sequencing on the MGI platform.

Perspectives outside breeding

KeyGene experts foresee that the substantial cost reduction of the Ultima Genomics sequencing platform will make single cell sequencing applications accessible for other research objectives too.

Read more

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