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; José Cuenca; Steven Dreissig; Jörg Fuchs; Andreas Houben; Pablo Aleza

doi:10.3389/fpls.2019.01174

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

Front Plant Sci. 2019 Sep 24:10:1174. doi: 10.3389/fpls.2019.01174. eCollection 2019.

Authors

Miguel Garavello^{1

2}, José Cuenca¹, Steven Dreissig³, Jörg Fuchs³, Andreas Houben³, Pablo Aleza¹

Affiliations

¹ Centro de Citricultura y Producción Vegetal, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, Valencia, Spain.
² INTA, Concordia Agricultural Experiment Station, Concordia, Argentina.
³ Department of Breeding Research, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Seeland, Germany.

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.

Keywords: SSR and SNP markers; flow cytometry; fluorescence-activated cell sorting; tetraploid; triploid; unreduced gametes; whole genome amplification.