The role of DNA methylation in the maintenance of phenotypic variation induced by grafting chimerism in Brassica

Grafting facilitates the interaction between heterologous cells with different genomes,resulting in abundant phenotypic variation,which provides opportunities for crop improvement.However,how grafting-induced variation occurs and is transmitted to progeny remains elusive.A graft chimera,especially a periclinal chimera,which has genetically distinct cell layers throughout the plant,is an excellent model to probe the molecular mechanisms of grafting-induced variation maintenance.Here we regenerated a plant from the T-cell layer of a periclinal chimera,TCC(where the apical meristem was artificially divided into three cell layers–from outside to inside,L1,L2,and L3;T=Tuber mustard,C=red Cabbage),named rTTT0(r=regenerated).Compared with the control(rsTTT,s=self-grafted),rTTT0 had multiple phenotypic variations,especially leaf shape variation,which could be maintained in sexual progeny.Transcriptomes were analyzed and 58 phenotypic variation-associated genes were identified.Whole-genome bisulfite sequencing analyses revealed that the methylome of rTTT0 was changed,and the CG methylation level was significantly increased by 8.74%.In rTTT0,the coding gene bodies are hypermethylated in the CG context,while their promoter regions are hypomethylated in the non-CG context.DNA methylation changes in the leaf shape variation-associated coding genes,ARF10,IAA20,ROF1,and TPR2,were maintained for five generations of rTTT0.Interestingly,grafting chimerism also affected transcription of the microRNA gene(MIR),among which the DNA methylation levels of the promoters of three MIRs associated with leaf shape variation were changed in rTTT0,and the DNA methylation modification of MIR319 was maintained to the fifth generation of selfed progeny of rTTT0(rTTT5).These findings demonstrate that DNA methylation of coding and non-coding genes plays an important role in heterologous cell interaction-induced variation formation and its transgenerational inheritance.

Isolation and identification of symbiotic bacteria from the skin, mouth, and rectum of wild and captive tree shrews

Endosymbionts influence many aspects of their hosts’ health conditions, including physiology, development, immunity, metabolism, etc. Tree shrews(Tupaia belangeri chinensis) have attracted increasing attention in modeling human diseases and therapeutic responses due to their close relationship with primates. To clarify the situation of symbiotic bacteria from their body surface, oral cavity, and anus, 12 wild and 12 the third generation of captive tree shrews were examined. Based on morphological and cultural characteristics, physiological and biochemical tests, as well as the 16 S rDNA full sequence analysis, 12 bacteria strains were isolated and identified from the wild tree shrews: body surface: Bacillus subtilis(detection rate 42%), Pseudomonas aeruginosa(25%), Staphlococcus aureus(33%), S. Epidermidis(75%), Micrococcus luteus(25%), Kurthia gibsonii(17%); oral cavity: Neisseria mucosa(58%), Streptococcus pneumonia(17%); anus: Enterococcus faecalis(17%), Lactococus lactis(33%), Escherichia coli(92%), Salmonella typhosa(17%); whereas, four were indentified from the third generation captive tree shrews: body surface: S. epidermidis(75%); oral cavity: N.mucosa(67%); anus: L. lactis(33%), E. coli(100%). These results indicate that S. epidermidis, N. mucosa, L. lactis and E. coli were major bacteria in tree shrews, whereas, S. aureus, M. luteus, K. gibsonii, E. faecalis and S. typhosa were species-specific flora. This study facilitates the future use of tree shrews as a standard experimental animal and improves our understanding of the relationship between endosymbionts and their hosts.