Studies On Mechanism Of Hybrid Vigor In Better-parent Hybrids Of Populus Deltoides Marsh

The poplar plantation area in China is 12 million mu（8 millon Ha）, which is four times than that of the total area of other countries. But Chinese poplar production at the international middle and lower level. Thus, in order to improve poplar production, varieties of poplar with fast growing and high quality characters were cultured by different approachs. Among these approaches, heterosis is a effective solution to increasing poplar’s production. Therefore, to explore mechanism of heterosis possesses important significance of theroretical direction in improving poplar production. In this study, Populus deltoides Marsh parental clones and their intraspecific F₁ hybrid clones with different growth vigor were used as materials to explore mechanism of heterosis.Based on the the analysis differences of water use efficient, nitrogen use efficient and photosynthesis use efficiency, physiological mechanism of different growth vigor of P. deltoides Marsh was comprehensive analysed. Meanwhile, to explore the molecular basis of heterosis at the epigenetic level, methylated DNA immunoprecipitation, combined with a high-throughput sequencing（MeDIP-seq） approach were applied to study the divergence of genome-wide DNA methylation landscapes in P. deltoides Marsh and their F₁ hybrids, and the contribution of DNA methylation to heterosis was analyzed. This study has great significance for enriching forest cross breeding theory, exploiting heterosis of poplar’s intraspecific hybridization and promoting improved variety system of forest production. The main results are described as following:（1） Evaluation of P. deltoides heterosis. After intraspecific hybridization of P. deltoides, the values of tree height and DBH of the H1, H2, and H3 clones at 5-year ages were greater than those of the parents and showed positive heterosis over better parent, while values of the L1 and L2 and L3 clones at 5-year ages were lower than those of the parent and showed negative heterosis. F₁ hybrids of P. deltoides with opposite growth vigor could obtained by intraspecific hybridization and this divergence was stable existed. The fastest growing points of tree height and DBH of H1、H2、H3 were at 3 years and 2 year, respectively, while those of L1、L2、L3 were both 2 year.（2） Evaluation of water use efficient and nitrogen use efficient of different growth vigor F₁ Hybrids clones. The δ¹³C value, nitrogen content and carbon content of better-parent F₁ hybrids（H1, H2, H3） were higher than those of maternal parent, 1.63%, 4.27%, 6.07%, respectively, and were higher than those of paternal parent, 1.31%, 7.84%, 4.72%, respectively. While those of lower-parent F₁ hybrids（L1, L2, L3） were significant lower than those of parent, which explained the better-parent F₁ hybrids had greater water use efficient（WUE） than lower-parent F₁ hybrids. The ratio of C and N of better-parent F₁ hybrids（mean value of H1、H2、H3 was 36.90） was significant lower than that of lower-parent F₁ hybrids（mean value of L1、L2、L3 was 38.89）, which meaned the the nitrogen use efficient（NUE） of better-parent F₁ hybrids was lower that of lower-parent F₁ hybrids. The significant negative correlation between WUE and NUE（R2=-0.652） indicated different growth vigor F₁ Hybrids of P. deltoides had different utilization strategy of energy, better-parent F₁ hybrids had the largest WUE, but due to their N utilization of speed was much greater than the speed of C assimilation, eventually leading to lower C/N, and thus the lowest nitrogen use efficiency, while lower-parent F₁ hybrids had heigher NUE. P. deltoides can not be optimal water and nitrogen use efficiency at the same time. WUE and NUE had certain constraints with each other. The δ¹³C value, nitrogen content and carbon content had divergency of spatial distribution. Those of upper canopy were higher than those of middle canopy, and those of the lower canopy were the lowest.（3） Evaluation of photosynthesis use efficiency of different growth vigor F₁ Hybrids clones. Compared with parents and lowe-parents F₁ hybrids, the better-parent F₁ hybrids possessed the larger photosynthetic area（including single leaf area and leaf area per plant）, the highest photosynthetic productivity（the mean value was 34.20% higher than maternal parent and that was 53.18% higher than paternal parent）, chlorophyll content（the mean value was 7.02% higher than maternal parent and that was 11.64% higher than paternal parent）, RuBPcase activity（the mean value was 9.00% higher than maternal parent and that was 47.65% higher than paternal parent）, the ratio of stomata number of upper epidermis and lower epidermis（the mean value was 5.83% higher than maternal parent and that was 28.28% higher than paternal parent） and stomatal frequency（the mean value was 1.93% higher than maternal parent and that was 16.43% higher than paternal parent） as well as light saturation point（the mean value was 1.93% higher than maternal parent and that was 16.43% higher than paternal parent）. Meanwhile the light compensation point, apparent quantum yield, dark respiration rate of better-parent F₁ hybrids were lower than those of parents and lower-parent F₁ hybrids. These characteristics of better-parents F₁ hybrids might contribute to broaden light intensity adaptability, accelerate rate of energy absorption conversion and carbon fixation, enhance the ability of dry matter accumulation and organ morphogenesis and improve the growth rate. while lower-parent had the opposite trend. They had lower nitrogen content and smaller photosynthetic area, resulting chlorophyll content and RuBP activity decreased, photosynthesis use efficiency was reduced, so the growth was slowly. Leaf area per plant, photosynthetic productivity and chlorophyll content were found to be used as indicators for evaluating of growth.（4） Analysis of the leaf methylomes of parents and their hybrids. Using methylated DNA immunoprecipitation and high-throughput sequencing, a total of 670 million reads were produced from the parents and their hybrids. 486.27 million of which were mapped to the reference genome of Populus trichocarpa, with an average mapped rates of 72.31% and average unique mapping rate of 57.8%. The P. deltoides intraspecific hybridization parents with similar genetic background had distinct DNA methylation levels. F₁ hybrids with growth vigor possessed elevated DNA methylation levels relative to mid-parent values, suggesting a nonadditive inheritance of DNA methylation in Populus, which showed that the methylation patterns of the parents were partially and dynamically inherited by their hybrids and was remodeled. The DNA methylation levels in promoter and CpG island regions which had great roles in regulating gene express and in repetitive sequences and transposable element which played an important roles in the maintenance of genomic stability of better-parent F₁ hybrids and parents and lower-parent F₁ hybrids were significant different. Compared with the maternal parent, better-parent F₁ hybrids had fewer hypermethylated genes and more hypomethylated ones. Compared with the paternal parent and lower-parent L1, better-parent F₁ hybrids had more hypermethylated genes and fewer hypomethylated ones. The differentially methylated genes between better-parent F₁ hybrids, the parents and lower-parent F₁ hybrids were enriched in the categories metabolic processes, response to stress, binding, and catalytic activity, development, and involved in hormone biosynthesis, signaling pathway. A multidimensional process was involved in the formation of heterosis.