Modification Of DNA Methylation By Grafting To Impact On Phenotype Of Walnut

Graftingmaintains the relative consistency of characters in varieties, while the phenotypesof the tree are significantly changed through different rootstocks. However, how the rootstockaffects phenotypic traits of scions, and its molecular mechanism remains unclear. In thisresearch, the walnut variety ‘SHS’ were respectively grafted on the rootstocks ‘ZNQ’ and ‘NY’,and the results showed that the phenotypic traits and physiological characteristics of walnutgrafts (such as growth, photosynthetic characteristics, etc.) changed obviously. Also, thegenomic DNA methylation level and patterns of walnut grafts (SHS/ZNQ and SHS/NY) werechanged, and some sequences in variated sites showing methylation pattern were closelyrelated to photosynthesis and metabolism. The research confirmed that walnut scion growthwas affected by the rootstocks influencing the genomic DNA methylation levels and patterns,which would enrich the understanding of tree growth potential affected by rootstocks.The main results are as follows:1. The optimized experiment system of walnut genomic DNA methylation was established.The walnut cotyledons were adopted as materials to compare three methods for extraction ofgonomic DNA, and the DNA isolated by modified high-salt CTAB protocol showed bestquality. After that, forty-three pairs of primer combinations, with clear and reproducible bands,were screened out in ninty pairs of primer combinations.The modifed MSAP experimentalsystem was established for walnut genomic DNA methylation analyses. Using modified MSAPexperimental system, the methylation level of genomic DNA in walnut leaves were analyzedand1060clear and reproducible bands were amplified. What is more, fourteen methylated siteswere recycled, cloned and sequenced, and BLASTn analysis showed that many types of DNAsequences, including transcription factors, presented methylation phenomenon in walnutleaves. 2. Genomic DNA methylation was analyzed using MSAP technology in various tissues ofthe same walnut variety (mature leaf, stem, root, pollen, cotyledon and husk), and972clearand reproducible bands were amplified. The methyltion levels of genomic DNA variedbetween different tissues of the same variety of walnut with nonsignificance, and themethylation patterns of variation ranged from0.199%~2.493%, with the highest CG-hyperrate (2.493%) in cotyledon tissue compaired with pollen tissue, and the lowest CG-hyper rate(0.199%) in leaf tissue compaired with steem tissue. Which demonstrated that the genomicDNA methylation levels kept relatively stable between different tissues in the same walnutvariety.3. Variances of growth vigour in walnut were assessed by measuring tree height, diameterat breast height, crown diameter, shoot length, shoot diameter and developing branch length of3-year-old SHS scions grafted on ZNQ and NY. The average height of SHS/ZNQ was5.33m,which was significantly higher than SHS/NY (4.18m). Other phenotypic traits of growthpotential (diameter at breast height, crown diameter, shoot length, shoot diameter anddeveloping branch length) of SHS/ZNQ were as well significantly higher than that of SHS/NY(P<0.01). The result showed that rootstocks had a significant impact on growth anddevelopment of the scions grafted on them.4. The photosynthetic characteristics were measured by Li-6400portable gas exchangesystem. At the same site conditions and the environment, the variations of photosyntheticcharacteristics between two scion combinations were different significantly. The light-saturatedrate of photosynthesis of SHS/ZNQ (21.52μmol m-2s-1) was significantly higher than that ofSHS/NY(19.96μmol m-2s-1). However, the photosynthetic parameters of LSP (B), LCP (C),and Rd(D) of SHS/ZNQ were significantly lower than that of SHS/NY(P<0.05).5. Genomic DNA methylation was analyzed using MSAP technology in phloem tissues ofSHS/ZNQ and SHS/NY, and1607clear and reproducible bands were amplified. The relativetotal methylation levels, CG and CHG on SHS/ZNQ (25.39%,17.67%, and7.72%respectively) were slightly lower than that on SHS/NY (27.57%,18.73%, and8.84% respectively. Nevertheless, the difference detected between SHS/ZNQ and SHS/NY was notsignificant (Utotal=0.72,<1.96), and the variation of methylation patterns ranged from0.85%to1.95%. Hence, it would be an important and substantive research for identifing specific sites ofmethylation changes and understanding its biological functions.6. In order to explore possible functional relevance of the loci with DNA methylationalterations, we isolated and sequenced a subset of variant MSAP bands from SHS walnutscions grafted on ZNQ and NY rootstocks. Based on BlastX analysis, we found that of the73variant MSAP bands that gave quality sequencing,49.32%showed significant homology toprotein-coding genes with known functions,12.33%fragments were homologous tophotosynthesis related genes and6.85%fragments were homologous to plastid sequences.7. This research demonstrated that the grafting can change the physiology and metabolismof the tree and affect tree growth. Also, grafting can cause genomic DNA methylation changesin specific sites, which were closely related to the growth and physiological metabolism.Therefore, the change of genomic DNA methylation patterns induced by rootstocks may be animportant reason to change its physiological and metabolic characteristics, which affected thegrowth and development of the grafts.