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

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

doi:10.3389/fpls.2020.00615

Abstract

In citrus, a classical method of studying crossovers and segregation distortion (SD) is the genetic analysis of progenies. A new strategy combining fluorescence-activated cell sorting and whole genome amplification of haploid pollen nuclei with a large set of molecular markers, offers the opportunity to efficiently determine the frequency of crossovers and the identification of SD without the need to generate segregating populations. Here we have analyzed meiotic crossover events in a pollen nuclei population from “Eureka” lemon and the allelic SD was evaluated in a pollen nuclei population from a clementine × sweet orange hybrid (“CSO”). Data obtained from the “CSO” pollen nuclei population were compared to those obtained from genotyping of a segregating population (“RTSO”) arising from a hand-made sexual hybridization between diploid non apomictic selected tangor (mandarin × sweet orange; “RTO” tangor) as female parent pollinated with “CSO” tangor as male parent. The analysis of crossovers rates on chromosome 1 revealed the presence of up to five crossovers events on one arm and four on the corresponding other arm, with an average of 1.97 crossovers per chromosome while no crossover events were observed in five “Eureka” lemon pollen nuclei. The rate of SD observed in “CSO” pollen nuclei (13.8%) was slightly lower than that recovered in the “RTSO” population (20.7%). In the pollen nuclei population, SD was found on linkage group (LG) 2, while the “RTSO” population showed SD on LGs 2 and 7. Potential male gametic selection mechanisms were distinguished in pollen grains, while in the population, mechanisms of gametophytic selection and/or zygotic selection were observed. This methodology is a very useful tool to facilitate research focused on the reproductive biology of citrus and study the mechanisms that affect crossovers and SD.

Highlights

Most plant species that reproduce sexually alternate the life cycle between a diploid sporophytic phase and a reduced gametophytic haploid phase (Xu et al, 2013)
We have demonstrated that fluorescence activated cell sorting (FACS) technique coupled with whole genome amplification (WGA) is an adequate methodology for multilocus simple sequence repeats (SSR) and single nucleotide polymorphism (SNP) genotyping of citrus haploid pollen nuclei (Garavello et al, 2019)
We display for the first time a multi-locus genotyping study of the “clementine × sweet orange hybrid (CSO)” haploid pollen gametes with and without the interference of the female parent. This approach allows us to identify mechanisms that could be related with specific genomic regions associated to non-random segregation of gametes during meiosis or with male and female gametic interactions, or zygotic mechanisms (LG 7)

Summary

Introduction

Most plant species that reproduce sexually alternate the life cycle between a diploid sporophytic phase and a reduced gametophytic haploid phase (Xu et al, 2013). The formation of crossovers between homologous parental chromosomes occurs in the prophase of meiosis I (Hunter, 2015; Lambing et al, 2017; Li et al, 2017), and consists in the exchange of genetic information between two non-sister homologous chromatids (Zamariola et al, 2014; Lambing et al, 2017). Many studies have demonstrated that the distribution of meiotic crossover events along chromosomes is non-random in plants, varying locally and displaying several hotspots (Mézard, 2006; Choi et al, 2008; Lambing et al, 2017). SD may result mainly from non-random segregation of gametes during meiosis (due to several causes such as the presence of deleterious alleles or gamete competition), post-meiotic gamete dysfunction or differential gamete success (such as differential pollen tube growth, pollen–pistil interactions, or partial incompatibility) and differential zygotic fitness (Li et al, 2014; Bodénès et al, 2016; Fishman and McIntosh, 2019; Seymour et al, 2019)

Methods

Results

Conclusion