**7. Molecular characterization of artificial triploid loquats**

Seven artificial triploid loquats were obtained by sexual hybridization between tetraploid "Jiefangzhong" and diploid "Hunanzaoshu". Eleven ISSR primers amplified 1989 bands, to contrast with parents, the added, lost, agnate and maternal bands were 1, 4, 9 and 19 respectively, indicating that the genomes of hybrid progenies are more similar to maternal parent which provided more chromosomes, the added and lost bands different from both parents , suggesting substantial genome variations among the artificial triploid loquats.

Similar result was observed using AFLP analysis. A total of 3122 bands were amplified with 12 pairs of AFLP primers. The numbers of added, lost, agnate and maternal bands were 82, 58, 49 and 105 respectively. All these indicated that substantial degree of genome variation occurred druing the process of triploid formation (Table 9).

DNA methylation analysis of artificial triploid loquat individuals and their parents showed that, a total of 5302 bands were amplified with 12 pairs of primers, of which 605 bands were full methylated and 233 bands were hemimethylated. Total methylation ratios of triploidy F1 hybrids were between 13.2% and 17.8%, 15.4% on average, full methylation ratios were between 10.5% and 12.2%, 11.7% on average, hemimethylation ratios were between 2.2% and 5.5%, 3.9% on average. Relative to parental plants, transmutation tendency of toal methylation and full methylation ratios of the artificial triploids was not significant. However, the hemimethylation ratios in the artificial triploids all decreased. The frequencies of the five types of methylation, demethylation, hyper-methylation, hypomethylation and intermediate pattern were 24.2%, 28.8%, 38.5%, 6.6% and 1.9% respectively (Table 10). Hyper-methylation occurred mainly during the development of artificial triploids, then demethylation, accompanied hypo-methylation, and methylation maintain and intermediate pattern that methylation state maintained between parent plants.


Table 9. Statistics of polymorphic bands of artificial triploids by AFLP technique. a the ratios of one type of polymorphic bands and total polymorphic bands; b the ratios of polymorphic bands and total bands.


Table 10. Genomic DNA methylation of artificial triploid loquats and their parents. <sup>a</sup> including full methylated and hemimethylated sites; b Full methylation denotes 5′-C<sup>m</sup>CGG-3′in double strands; c Hemimethylation denotes 5′ <sup>m</sup>C CGG-3′in single strand.
