Hybrid Liriodendron Chinense Rooting Genetic Variation And Genetic Screening

Liriodendron chinese×Liriodendron tulipifera as an important broadleaf tree for industrialcutstock and landscaping was been seriously obstructed the large-scale popularization because it is difficult in propagation particularly taking root in cutting. By the basis of crossing breeding in recent years, the main contents of this research included genetic variation in rooting ability of clones, screening clones of high rooting, variation of physiological and biochemical indicators, screening genes related to rooting formation, function verification. We hope to filer some high rooting clones and search for physiological and biochemical indicators used for early selection of Liriodendron chinese×Liriodendron tulipifera. Furthermore, we hope to preliminaryly explore the molecular mechanism of adventitious root formation. The main results were showed as following:(1) Genetic variation in clones and screening clones of high rooting. The test results showed that:clones of Liriodendron chinese×Liriodendron tulipifera have significantly different in rooting rates, and the coefficient of genetic variation reached 26.6%; the repeatability of rooting traits in clones was 0.34, which showed that the root traits are under genetic control nearly at the moderate level. Finally, combination with index in rooting rate and growth amount, three clones with high rooting rate and fast growing ability were obtained, which were labeled 11 (rooting rate is 77.8%),10 (rooting rate is 53.9%) and 17 (rooting rate is 51.9%). There clone 12 (rooting rate is 77.2%) and clone 20 (rooting rate is 77.3%), which have high rooting rates but lower growth amounts can be used as breeding materials.(2) Variation of GA,ZR,IAA,ABA and four kinds of enzymes involving POD, PPO, SOD and CAT. The results show that, the content of IAA, ZR and ABA basically had a rising trend before September, and to the end of August to the highest, then decreased slowly. But content of GA change smoothly which was stable condition basically. About of the regulation of changing of years, content of GA had a decreasing trend, especially after 6 years old. And the other three kinds of hormones irregular changed. Content of four hormones had a very significant difference in good growth and bad growth trees. Changing regulation of PPO in one year is basically on the contrary with growth rate of trees, but changing regulation of other enzymes of is not significant in one year and years. Furthermore, four enzymes in different growth trees are not significant difference, having no obvious relevance with height and diameter at breast height of Liriodendron hybrid. ZR and IAA can be used as preferred indicator of heterosis of hybrid Liriodendron, and GA as supplementary indicator.(3) Constructing a normalized uncut cDNA library. We constructed a high-quality Normalized uncut cDNA Library from mixture four stages of development of adventitious root of Liriodendron chinese×Liriodendron tulipifera without the use of restriction enzyme cloning technique which combines the performance of SuperScriptⅡReverse Transcriptase with the Gateway Technology. The titer of uncut cDNA library is 7.34×106 cfus/ml, and the recombinants accounted for more than 95%. Results of PCR using colonies directly as templates showed that the insert fragment length of the library ranged from 800bp-3500bp, and the average length was more than 1500bp. After normalizing, the titer of the library is 3.72×105cfu/ml, and the recombinants accounted for more than 95%. Results of PCR using colonies directly as templates showed that the insert fragment length of the library ranged from 850bp-3500bp, and the average length was more than 1300bp.By qPCR, copy number of housekeeping genes have reduced about 35-85 times after normalizing. The result suggested to some degree that the cDNA library was efficient normalized.(4) Analyzing ESTs and classifying genes of annotated function. From the library 5176 effective ESTs were sequenced, the average length of effective ESTs was 1001bp. The average GC content was 46.74%. The Phrap software was used for contig analysis of all the ESTs. From the 5176 effective ESTs of adventitious root of Liriodendron chinese×Liriodendron tulipifera, a total of 2921 different Unigenes which included 2055 contigs and 866 singlets were obtained.2921 unigenes were compared with the sequences in the non-redundant protein and nucleotide databases of NCBI, total 1796 contigs and 751 singlets which proportion 87.88% sharing significant similarity with sequences registered in these databases after being analyzed using BLASTX and BLASTN program. Aiming at annotate molecular biology functions of ESTs, furthermore, parts of these differential expressed genes were enumerated, such as genes possible related to auxin signal transport, genes possible related to degradation by ubiquitination, genes possible related to cell cycle,genes possible related to transcription factor SCR, SHR, NAC genes possible related to root hair formation, and so on. Undoubtedly, the outcome would contribute new ideas on the study of molecular mechanism for adventitious root development in Liriodendron chinese×Liriodendron tulipifera hardwood cuttings. Furthermore,2921 unique ESTs used to be developmented of EST-SSR markers.181 SSRs Containing 166 ESTs identified, including perfect di-, tri-, tetra-, penta- and hexanucleotide motifs. Among them, the dinucleotide and trinucletide repeats were the most abundant SSRs detected, accounting for 91.16%.(5) Cloning F-box gene. A complete-length 2177bp cDNAs encoding LHFB1 gene were isolated from cDNA library of adventitious rooting by RACE. The ORF is 1719bp, encoding 572 amino acids, and molecular weight of protein is 66.44KD. It is found that LHFB1protein is high homology with other F-box family proteins of other species by BLASTx. The analysis result of Realtime PCR showed the expression of LHFB1 gene is significant higher in adventitious rooting than leave and stem. And in four stages of developmental adventitious rooting expression of LHFB 1 presents wavy trend, which increase firstly, then drop but last increase again. That reflects the complex of expression pattern.(6) Spatial expression analysis of candidate ESTs. By Realtime PCR the spatial expression feature analysis of six candidate ESTs AUX1,ARF1,NAC1,RHD1,IRE,RHL1 in different tissues (root, stem, leave) and different developmental periods of adventitious rooting indicated that some genes were preferentially expressed in adventitious rooting, and also showed that different expressed patterns of different genes. The result showed expression levels of AUX1,IRE were significant higher in adventitious rooting than leave and stem, and amount of expression of NAC1,RHD1,ARF1 in different developmental stages of adventitious rooting was significant difference.