| Maize is one of the most important food and energy crops in the world. Although China is the second-largest maize producer in the world, it lacks of quality maize germplasm resources which are disease tolerance or resistance, high combining ability and wide adaptablity.. Transgenic breeding is one of important ways to improve maize germplasm resources. Maize embrogenic callus inducted from its immature embyro is one of the widely used corn receptor type in the filed of GMO(Genetically Modified Organism)IBut maize inbred lines can not be directly used as transgenic receptor material due to its low frequency of embryonic callus induction. Generally, the traditional process of transgenic maize breeding need to go through several critical steps including genetic transformation, self-crossing for homozygosis, backcross transformation and hybrid tissue culture, so it takes at least 3-6 years to obtain transgenic elite inbred lines. The difference of maize embryogenic callus induction rate is greatly due to the genotypes of different materials, so cloning major genes which control embryogenic callus induction rate, analysising mechanism of maize embryogenic callus induction and creating more maize inbred lines of high embryogenic callus induction ratio, are conducive to the rapid development of the maize transgenic breeding.In this study,a total of 301 maize inbred lines with representative embryogenic callus induction and rich diversity, and inbred lines 18-599R with the strong ability of embryonic culturing were used as test materials,combined with genome-wide association analysis and Solexa digital gene expression profiling method, screening genes related to embryogenic callus induction rate and differentially expressed genes. And the final results are as follows:1.A total of 301 maize inbred lines with rich diversity were used as test materials in 2014 and 2015, respectively. At 12 DAP (Days After Pollination), immature embryos with the size of 1.0-1.5mm were picked and cultured in induced culture media which contained 2,4-D to induce embryogenic callus.We counted the bud length and germination rate of each material in the 7th day, and investigated the embryogenic callus induction rate in the 15th,20th,25th day,respectively. As a result, there are three materials with the embryogenic callus induction rate of 40%-80%, another 3 of 30%-40%,12 of 20%-30%,25 of 10%-20%,69 of 0-10% and 188 of 0%2.Using 44243 high quality SNPs whose minor allele frequencies (MAF) were more than 5%, combined with population structure and genetic relationship, genome-wide association study (GWAS) was conducted. A total of 25 SNPs were detected significantly related to embryonic callus tissue in two environments (P <0.0001). Only 1 SNP detected in both enviroments distribute across maize chromosome bin 1.05, and 15 candidate genes were screened in 25 SNPs. Functional annotation indicated that these candidate genes may be involved in the physiological and biochemical process such as protein binding, ATP combination, mitochondria constitute, electron transfer, and protein kinase activity. In addition, we also conducted GWAS of bud length and germination rate. As a result, a total of 36 SNPs were detected significantly related to germination rate in these two environments, and 10 SNPs were detected in both environments.20 candidate genes were screened in 28 SNPs. Functional annotation showed that these candidate genes may be involved in transferase activity, protein kinase activity, energy transfer, isomerase activity. Meanwhile, a total of 53 SNPs were detected significantly related to bud length in two environments, and 6 SNPs were detected in both environments.31 candidate genes were screened in 31 SNPs. Functional annotation indicated these candidate genes may be associated with phosphate transfer reaction adjustment factor activity, helicase activity, methyltransferase activity, dehydrogenase activity and hydrolase activity.3.The inbred line 18-599R with the strong ability of embryonic culturing was used as test material. The immature embryo callus induction process was divided into embryo enlargement stage, primary callus formation stage, embryogenic callus formation stage. The DGE sequencing results show that there are 18951(14.55%)? 18359(56.42%),18140(15.83%),17870(15.5%) sequencing tags which are matched to the maize genome in CK?Stage ??Stage ??Stage ?, respectively; 4825,5119 and 5463 different expression genes(DEGs) were detected in the 3 training stages, respectively; 199,230,250 DEGs changed for at least 5 folds were up-regulated in the 3 training stages,respectively, and 52,94,63 DEGs changed for at least 5 folds were down-regulated in the 3 training stages,respectively. The results shows that a large number of genes was induced to express by the induction of maize immature embryos to embryogenic callus.4.GO functional annotation of DEGs indicated that both up-regulated genes and down-regulated genes could be classified into much categories, such as cellular composition, signal transduction, physiological metabolism. In addition, KEGG pathway analysis showed that significantly differentially expressed genes in the Stage ? enriched in fatty acid biosynthesis pathways, metabolism of xenobiotics by cytochrome P450 pathways, moreover, in the Stage ? pyruvate metabolism, biosynthesis of plant hormones, fatty acid biosynthesis were three of the most affected pathways, and in the Stage ? Pyruvate metabolism,glycolysis/gluconeogenesis, biosynthesis of phenylpropanoids were identified to be the three of the most enriched pathway. In addition, the hormone signaling pathway is a significant pathway in all Stages.5. In this study, we employed GWAS to analyze the rate of embryogenic callus of 301 maize inbred lines, and screened genes related to the induction of embryogenic callus, at the same time,, in order to find the differentially expressed genes during the dedifferentiation period of maize immature embryo, DGEs was conducted in the materials of stage ?(1-5 days), of the stage ?(6-10 days) and of the stage ? (11-15 days), while the 18-599R, which has high ability of embryonic induction, is used as a control. The same gene can be founded by compared with both of the results. This shows that the 2 experiment has some consistency, and these common genes will be the focus of attention in subsequent experiments.6.We have made a preliminary validation of the GA signaling pathway mediated by MYB138 in the plant hormone signaling pathway.The results showed that exogenous GA3 can promote the growth of adventitious buds from maize immature embryo and inhibit that of adventitious bud induction to embryogenic callus.The induction rate of ZMMYB138 gene of Arabidopsis thaliana mutant atmyb65 was decreased by about 20% compared with that of wild type COL embryogenic callus.This part of the tissue in the induction processwill produce abnormal buds, which lead to the failure of dedifferentiation. |
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