Research On The Features Of DNA Methylation In Bamboo During Aging And Rejuvenation

In order to research the epigenetic changes during aging and rejuvenation of the bamboo,methylation-sensitive amplification polymorphism was used to detect the DNA methylationstatus in leaves of different chronological ages of Phyllostachys heterocycla var. pubescens,different explants and callus of Phyllostachys heterocycla var. pubescens, and different organsof Phyllostachys bamusoides Sieb. et Zucc during flowering and asexual rejuvenation, theresults show that:1. DNA methylation ratio of Phyllostachys heterocycla var. pubescens increased withchronological ages.Samples taken from a stand containing plants of five ages (2,7,34,44, and>60yearsafter seed germination) were subjected to MSAP using six primer pairs that have previouslybeen shown to yield methylation rates that reflect the age of Moso bamboo. Genomic DNAmethylation level in Moso bamboo increased with age, from19.1%to36.29%. The results alsoshowed that the total genomic DNA methylation rates in Moso bamboo at differentchronological ages were significantly different, and the greater the ages difference in Mosobamboo, the more significant the variations in genomic DNA methylation rates.2.We can always build a superior fitting effect quadratic curve based on the distribution ofthe DNA methylation levels in Moso bamboo at different chronological ages, and this curvecan provide a method to detemine the chronological age of bamboo.we adopted principal component analysis to identify an integrated factor for clarifying theoverall information expressed by the six primer pairs in each age group. The equation of theintegrated factor is, Y=0.181x1+0.181x2+0.170x3+0.182x4+0.184x5+0.160x6.Put the totalDNA methylation rate obtained from the integrated factor equation into the quadratic curve thatwe built before, the result show that they can not coincide perfectly. However, Curve-fittingalso resulted in a quadratic curve exhibiting a superior fitting effect based on the distribution of the DNA methylation levels in Moso bamboo at this five different chronological ages, theequation for the quadratic curve is, Z=0.071y2-0.278y-19.182, R2=0.997,P＜0.01, and theoverall increasing trend of the total methylation rate is consistent with the quadratic curve thatwe built before.Methylation-sensitive amplification polymorphism (MSAP) analysis can be performed onthe genomic DNA of some bamboos whose chronological ages are already known, as well asthe bamboo whose chronological age is unknown. We can build a superior fitting effectquadratic curve based on distribution of the DNA methylation levels in bamboos whose agesare known, and then estimate the chronological age of the bamoo whose age is unknown by itsDNA methylation level.3. Methylation level in Phyllostachys heterocycla var. pubescens decreased duringexplants dedifferentiation, and methylation level in embryogenic callus was lower than that innon-embryonic callus.MSAP was used to detect the DNA methylation status of Phyllostachys heterocycla var.pubescens during explants dedifferentiation. Methylation level in callus (10.53%) was lowerthan that in explants (17.34%). Methylation and demethylation both occurred simultaneously indedifferentiation, with the higher probability of demethylation (48.86%) than of methylation(12.5%) eventually causing the decrease in methylation levels in its genomic DNA. We alsofind that methylation level in embryogenic callus was lower than that in non-embryonic callus,and73.44%of sites were significantly different in methylation status.4.Methylation level in P. bambusoides blades decreased during the flowering stage andthen increased gradually after flowering when new bamboo stands were produced via asexualrejuvenation.Before and after P. bambusoides flowering and during asexual regeneration,morphological observations and DNA methylation levels were detected for blades during fourdifferent representative growth stages. Results demonstrated that blade morphology during thefour periods exhibited significant differences. In addition, the methylation level of genomic DNA decreased during flowering but gradually increased during rejuvenation, indicating thatthe blades of P. bambusoides, regardless of morphology or methylation level, varied duringflowering but returned to normal during self-regeneration and self-rejuvenation after flowering.Compared with non-flowering bamboo stands,29.09%of methylation sites were mutatedduring P. bambusoides flowering, with17.88%of sites completely demethylated in floweringplants, which was much higher than that of the methylation sites (3.61%). The number andratio of polymorphic sites (23.64%) where changes in methylation status occurred inrejuvenated and non-flowering bamboo stands, especially demethylated sites (14.53%), werelower than those in flowering bamboo stands.5. Methylation in floral organs was lower than that in blades in flowering P. bambusoides.DNA methylation levels were detected for floral organs and blades during flowering ofPhyllostachys bamusoides Sieb. et Zucc. Results demonstrated that methylation in floral organs(27.44%) was lower than that in blades (29.96%), and28.85%of sites exhibited changes inmethylation status, mainly in the form of demethylation.