Single-pollen genotyping to estimate mode of unreduced pollen formation in Citrus tamurana cv. Nishiuchi Konatsu

Abstract

2n pollen formed by FDR in citrus. The Japanese local citrus cultivar, Nishiuchi Konatsu (Citrus tamurana hort. ex Tanaka; NK hereafter), has the ability to produce unreduced 2n pollen grains, allowing generation of polyploid progenies via sexual polyploidization. In this study, we developed a method of single-pollen genotyping for citrus and applied it to the analysis of transmission of heterozygosity in NK 2n pollen grains. Heterozygosity transmission was expressed as the percentage inheritance of a set of heterozygous alleles from the parent to the 2n gamete. The pathway of 2n pollen development was investigated by applying the observed heterozygosity transmission and genetic distance to two different map functions, for first division restitution (FDR) and second division restitution (SDR). The fit of the values observed for both functions was calculated, while virtually moving the centromere position. We screened for six heterozygous SSR (codominant microsatellite marker loci) in NK, all of which were expected to lie within the same linkage group. Pollen germination prior to DNA extraction was essential for this work, and 6-h incubation proved to be optimal for subsequent PCR amplification. Single-pollen genotyping unreduced NK 2n pollen grains revealed that heterozygosity transmission exceeded 50 % in all six alleles, and fitness tests indicated that the FDR map function better fitted the heterozygosity transmission observed rather than the SDR function. Our data thus strongly indicate that 2n pollen in NK is a result of first division restitution.

High-quality genetic mapping with ddRADseq in the non-model tree Quercus rubra

“…In conifers, genetic maps can be constructed using the haploid megagametophyte seed storage tissue from the seeds of a single tree [75–77], as was recently done with ddRADseq for the white cypress pine, Callitris glaucophylla [78]. The technology for genotyping single pollen grains, the only easily accessible haploid tissue in angiosperms, exists [79] and was recently demonstrated in hyūganatsu ( Citrus tamurana ) [80], but the technical challenges are considerable. For undomesticated angiosperm forest trees, especially the ecologically dominant, economically valuable and speciose oaks, the only feasible method at present is to use the progeny of known parents.…”

Flowering and Fruiting Haploid and Doubled Haploid Pummelos

“…By using single pollen genotyping, Honsho et al [46] demonstrated that unreduced 2n pollen grains of 'Nishiuchi Konatsu' hyuganatsu (Citrus tamurana hort. ex Tanaka) had heterozygosity transmission exceeding 50% in all six alleles, and fitness tests indicated that the FDR map function better fitted the heterozygosity transmission observed rather than the second division restitution (SDR) function.…”

Genetic Structure of Diploid Gametes for the Production of Triploid Citrus Hybrids

“…However, we also found that 18.8% of 2n `CSO´ pollen arisen from SDR and 7% of 2n megagametophytes of `Eureka frost´ and `Fino´ lemons arisen from (FDR) or (PRD). Honsho et al (2016) also concluded for the occurrence of FDR unreduced pollen from a molecular marker study performed at individual pollen grain level. However no plant resulting from such FDR pollen where described prior our work.…”

“…Later, Chen et al (2008) recognized triploid hybrids resulting from 2n-pollen of P. trifoliata in hybridizations with sweet orange. Recently, Honsho et al (2016), identified giant pollen grains in `Nishiuchi Konatsu´ mandarin (C. tamurana Hort. ex Tanaka) and based in single-pollen genotyping, revealed that FDR was the mechanism for the 2n pollen gamete formation, although no plants were recovered.…”

“…analyzed 543 triploid hybrids obtained from nineteen different genotypes of mandarins and only three triploid plants were obtained by FDR-2n female gametes and no 2n pollen gametes contributed to the production of triploid hybrids. In a `Nishiuchi Konatsu´, a Japanese mandarin (C. tamurana), Honsho et al (2016) reported the production of pollen grains with different sizes. They demonstrated that the biggest pollen grains were diploid resulting from FDR, although no progenies were recovered.…”