Assessing Ploidy Level Analysis and Single Pollen Genotyping of Diploid and Euploid Citrus Genotypes by Fluorescence-Activated Cell Sorting and Whole-Genome Amplification.

Miguel Garavello,Steven Dreissig,José Cuenca,Andreas Houben,Jörg Fuchs,Pablo Aleza

doi:10.3389/fpls.2019.01174

Abstract

Flow cytometry is widely used to determine genome size and ploidy level in plants. This technique, when coupled with fluorescence-activated cell sorting (FACS), whole genome amplification and genotyping (WGA), opens up new opportunities for genetic studies of individualized nuclei. This strategy was used to analyze the genetic composition of single pollen nuclei of different citrus species. The flow cytometry and microscope observations allowed us to differentiate the populations of pollen nuclei present in the diploid and euploid genotypes analyzed, showing that citrus has binuclear pollen. We have identified in the “CSO” tangor an additional nuclei population composed by the vegetative plus generative nuclei. Genotyping of this nuclei population revealed that vegetative and generative nuclei show the same genetic configuration. In addition, we have demonstrated the presence of unreduced gametes in the diploid genotype “Mexican lime.” Genomic amplification is a robust method for haploid nuclei genotyping with several molecular markers, whereas in diploid nuclei using heterozygous markers showed a bias towards one of the two alleles, limiting the use of this tool in this type of nuclei. We further discuss the importance and applications of single pollen genotyping in citrus genetic studies.

Highlights

Flow cytometry has become a widely used technique for genome size estimation and ploidy analysis in plant research because of its high throughput, accuracy and resolution as well as low operating cost per sample
We describe an effective methodology to determine the ploidy level in mature pollen grains of diploid, triploid, and tetraploid citrus genotypes, using fluorescence-activated cell sorting (FACS), followed by whole genome amplification and genotyping (WGA) and genotyping of individualized nuclei from pollen grains by Simple Sequence Repeats (SSR) and Single Nucleotide Polymorphism (SNP) molecular markers
To determine the necessary diameters of the pre- and bursting filters required for the pollen grain and nuclei isolation, respectively, we measured the diameters of pollen grains of 10 different citrus genotypes representing ancestral and secondary species

Summary

Introduction

Flow cytometry has become a widely used technique for genome size estimation and ploidy analysis in plant research because of its high throughput, accuracy and resolution as well as low operating cost per sample.

Methods

Results

Discussion

Conclusion