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

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.

Analysis of Crossover Events and Allele Segregation Distortion in Interspecific Citrus Hybrids by Single Pollen Genotyping

“…Then, dehiscent anthers were selected under magnifying glass, discarding those that were not fully open, and they were sealed with parafilm and stored at −20 • C until use. To isolate pollen nuclei we used the methodology described by Kron and Husband (2012) and validated in citrus by Garavello et al (2019). Briefly, 4-5 dehisced anthers were vortexed in a small tube with 300 µl of nuclei isolation buffer in order to recover all pollen grains.…”

“…This strategy also allows performing studies on the genetic structures of pollen grain populations as compared with those originated at the plant level, without interferences due to a potential partial crossincompatibility or gamete competition (Gu et al, 2013). In citrus, the strategy of ploidy level analysis, sorting, and genotyping of single pollen grains has been previously assessed for several diploid, triploid, and tetraploid genotypes (Garavello et al, 2019), with successful results when applied to haploid pollen grains.…”

Genotyping of polyploid plants using quantitative PCR: application in the breeding of white-fleshed triploid loquats (Eriobotrya japonica)

“…There are several methods for estimating polyploid allele dose, among which KASPar technology [ 39 , 43 ] and QF-PCR [ 44 , 45 ] are widely applicable. KASPar technology is similar to TaqMan technology, and this method requires a high-density SNP database and genome-wide association analysis [ 46 ].…”

SHORT COMMUNICATION: SIMPLIFIED APPROACH FOR EARLY IDENTIFICATION OF SPONTANEOUS OIL PALM HAPLOID (Elaeis guineensis)

“…Numerous studies have associated abnormal germinated seeds, or 'off types', with haploid formation (Garavello et al, 2019). The actual 'offtype' morphologies observed in germinated seedlings that may be linked to haploid plants have never been described clearly for oil palm.…”

“…FCM is fast and convenient (Dolezel et al, 2007;Sliwinska, 2018). It robustly determined the ploidy level of the putative haploid in numerous plant research involving induction of haploids and doubled haploids (Gu et al, 2013;Garavello et al, 2019). From the FCM result, the ploidy level was determined by comparing the known ploidy (represented as histogram peak) of a reference plant genome to the unknown species.…”