AFLP and MSAP analysis of early ripening bud mutant and its revertant materials of navel orange

Abstract:【Objective】Gannanzao is an early ripening bud sport material of Newhall, and its fruit maturity was over 30 days earlier than Newhall l. Early ripening revertant was the bud sport material of Gannanzao, and its fruit maturity was consistent with that of Newhall. The purpose of this study was to detect the differences of genome, methylation modification level and variation mode among the Newhall, Gannanzao and revertant. The relationship between these differences and early ripening character of navel orange was analyzed to further clarify the occurrence and development mechanism of fruit ripening character of the navel orange.【Methods】In this study, the Newhall, Gannanzao and revertant mutant were used as experimental materials. The amplified fragment length polymorphism (AFLP) and methylation sensitive amplified polymorphism analysis (MSAP) were employed to study the genome, methylation modification level and variation mode of the three different materials. The materials were collected from the orchard in Yudu county, Ganzhou city and the“citrus germplasm resource nursery”of Gannan Normal University. We used the improved CTAB method for the extraction of the genomic DNA of the samples. The quality and concentration of genomic DNA were detected by agarose gel electrophoresis combined with ultramicro nucleic acid protein analyzer (Thermo Scientific, USA). The genomic DNA meeting the requirements was diluted to 100 ng·μL^-1 and stored in the refrigerator at -20 ℃ for standby. Both AFLP and MSAP markers needed double enzyme digestion, ligation, pre-amplification and selective amplification. However, the endonuclease used in AFLP labeling was different from that used in MSAP labeling. EcoRⅠ-MseⅠ were used in AFLP, EcoRⅠ-HpaⅡ and EcoRⅠ-MspⅠ were used in MSAP. AFLP marker and MSAP marker selective amplification products were added with 20 μL mineral oil to prevent samples volatilization and put into Qsep 100TM type automatic nucleic acid protein analyzer (Bioptic, Inc. Taiwan). High Resolution Cartridge (S1) was used for electrophoresis analysis. The clearly identifiable strip was marked as“1”, and the vacancy was marked as“0”. The obtained dates were analyzed by Excel.【Results】Many polymorphic bands were found by AFLP and MSAP techniques. A total of 228 amplification sites and 660 bands were obtained from ten pairs of primer combinations labeled by AFLP. The total bands of Newhall, Gannanzao and revertant mutant were 218, 216 and 226, respectively. Using Newhall as the control variety, there were 12 differential bands in Gannanzao or revertant mutant, and the proportion of polymorphism was 5.504 6%. With Gannanzao as the control variety, there were 12 differential bands in revertant mutant, and the proportion of polymorphism was 5.555 6%. A total of 220 amplification sites were obtained from the ten primer combinations labeled with MSAP. The amplification sites of Newhall, Gannanzao and revertant mutant were 219, 218 and 220 respectively. A total of 1218 bands were obtained from the MSAP markers, including 190 bands of Newhall HpaⅡ (H), 218 bands of Newhall MspⅠ (M), 189 bands of Gannanzao H, 217 bands of Gannanzao M, 186 bands of revertant mutant H and 218 bands of revertant mutant M. Compared with Newhall l, there were three polymorphic bands and six polymorphic bands in Gannanzao and revertant mutating, respectively, and the proportions of polymorphisms were 1.369 9% and 2.752 3%, respectively. Compared with Gannanzao, there were seven polymorphic bands in revertant mutant, and the proportion of polymorphism was 3.211 0%. The proportion of polymorphism in AFLP (5.504 6%-5.555 6% ) was higher than those in MSAP (1.369 9%-3.211 0% ). It was preliminarily found that the eight polymorphic bands of the seven pairs of primers might be involved in the regulation of the navel orange ripening trait, including 115 bp of E1-M2 primer, 100 bp of E1-M10 primer, 255 bp of E2-M1 primer, 303 bp of E2-M3 primer, 112 bp and 282 bp of E3-M1 primer, 134 bp of Es1 Ms1 primer and 104 bp of Es8 Ms8 primer. Most of the sites of Gannanzao and revertant mutant maintained the original methylation state. The frequency of DNA methylation of test materials was 13.761 4%-16.818 2% , with an average of 14.911 6% . The full methylation was the main methylation mode. Among the 15 methylation variation patterns, only five and six were detected in Gannanzao and revertant mutant, respectively. The frequency of hyper-methylation was significantly higher than that of demethylation. The fruit ripening reverse of the revertant mutant might be related to the CHG of the demethylation.【Conclusion】There were genetic differences among Newhall, Gannanzao and revertant mutant, and the genetic difference between the revertant mutant and Gannanzao was greater than that between the revertant mutant and Newhall. We proved that the reverse mutation was the early ripening revertant mutant of Gannanzao at gene level. The hyper-methylation and demethylation of genomic DNA would occur simultaneously in the process of forming bud sport materials with different maturity period of the navel orange, but hyper-methylation was the main mode. This study would provide a clue for further elucidating the occurrence and development mechanism of early ripening character of the navel orange.