Mechanism underlying 2n male and female gamete formation in lemon via cytological and molecular marker analysis

Abstract

The unreduced (2n) gametes have long been used in triploid breeding of citrus. In lemon, the previously reported mechanisms for 2n megagametophyte formation were controversial, whereas that for 2n pollen production is still unknown. Herein, the frequency of and mechanism underlying 2n megagametophyte and 2n pollen formation in ‘Eureka’ lemon were investigated based on cytological observation and genotyping of the triploid hybrids between ‘Eureka’ lemon and ‘Early gold’ sweet orange. As a result, 4.79% of the viable pollens of ‘Eureka’ lemon were identified as the 2n pollen with a larger diameter (70.16 ± 3.92 µm). The 2n pollen might be resulted from the formation of parallel spindles at meiosis stage II. Among the 204 plantlets regenerated from embryo rescue following the sexual cross, 12 were triploids as identified by flow cytometry. According to the analysis of heterozygosity transmission using 13 pericentromeric single nucleotide polymorphism (SNP) markers and 20 randomly distributed simple sequence repeat (SSR) markers, 11 triploids were identified to be originated from the fertilization of 2n megagametophytes of ‘Eureka’ lemon, with a frequency of 5.39%. Among them, nine 2n megagametophytes were supposed to be arisen from the second division restitution (SDR), whereas the other two were from post-meiotic genome doubling (PMD). These results to understand the mechanism underlying 2n gamete formation in lemon are valuable for its efficient polyploid breeding.