Single Cell Genomic Sequencing in Wheat: Application in Monitoring Recombination Frequency

Transfer of high value target loci from genetically diverse wild relatives to adapted elite varieties is seen as a key to the future of wheat breeding. Efforts to modulate homoeologous recombination between the chromosomes of alien donor and those of cultivated wheat via mutating the Ph1 (Pairing homoeologous 1) locus, chemical treatment or changes in environmental conditions are currently underway. However, the fast progress of such projects is hindered by the lack of a rapid screening method for monitoring the impact of modulation of homoeologous recombination in wheat. We have devised a microspore-based single cell genomic sequencing and genotyping strategy for monitoring the frequency of homoeologous recombination in wheat. Our method involves DNA or RNA isolation from single microspores derived from F1 progenies using either the Fluidigm C1 single cell platform or FACS (Fluorescence Assisted Cell Sorting) based cell microspore sorting combined with ultrasonic lysis. Subsequent sequencing of DNA or RNA and genotyping of multiple segregating microspores facilitates assessment of the frequency of homoeologous recombination. Based on our results, the FACS based cell sorting system seems to be the most appropriate  method for sorting wheat microspores as their large size (40-60 µM) exceeds the maximum limit imposed by the Fluidigm system (17-25 µM). These results along with the broad applications and challenges of single cell genomics in plants will be presented.