The sexual reproduction of triploids induces chromosomal karyotype variations, which are significant for germplasm resource innovation. Most triploid plants are with low fertility. Therefore, triploid offspring karyotypes’ variation pattern and phenotypic response remain poorly understood. Here, we employed three diploids with diverse genetic distances as male parents to cross-pollinate the female fertile triploid loquat Q24 to construct three experimental populations. The chromosome numbers of 93.82% of hybrid plants were 34~46 in three hybrid populations. All 168 aneuploids with 160 karyotypes and a small percentage of euploids were detected among 178 hybrids by the improved molecular karyotype analysis method. Further analysis revealed that when being transmitted to offspring, chromosome 5 of Q24 as disomy had the highest frequency (>50%), while chromosome 12 had the lowest frequency (≤30%). The frequency of Q24’s chromosomes being transmitted to offspring as disomy was influenced by the gene function on the chromosomes and the number of interchromosome collinear gene links. Whole-genome resequencing showed that the Q24 alleles exhibited segregation distortions in the offspring aneuploid population. Transgenic experiments demonstrated that the EjRUN1 gene, which was on one segregation distortion region of Q24, promoted the seed viability of triploid Arabidopsis. Furthermore, chromosome number, dosage, and male parent genotype affected the aneuploid phenotype. These findings advance the understanding of genome genetic characteristics of triploid loquat, and provide a reference for germplasm innovation of loquat rapidly through triploid sexual reproduction.

Polyploidy plays a significant role in loquat breeding, particularly in triploid breeding for seedless fruit production. Currently, loquat polyploid breeding primarily relies on natural seedling selection and sexual hybridization approaches. In this study, unfertilized ovules from four loquat varieties were in vitro cultured. Gynogenesis and embryoid regeneration were achieved in ‘Xingning 1’ and ‘Huabai 1’, with ‘Xingning 1’ demonstrating the highest gynogenesis efficiency (21.63%). Flow cytometry and chromosome counting revealed that the obtained embryoid lines included haploid, diploid, tetraploid, hexaploid, and chimeric ploidy types. Further characterization of ‘Xingning 1’-derived embryoid lines through SSR markers and whole-genome resequencing confirmed that the haploid, diploid, tetraploid, and hexaploidy embryoid originated from haploid–somatic chimeras, diploid, doubled diploid and tripled diploid, respectively. Metabolic analysis showed a positive correlation between ploidy level and the content of both soluble sugars and organic acids. This study explored a novel platform for polyploid induction in loquat and may provide methodological insights for improvement of other perennial fruit trees.