| Nicotine is the most important content in the tobacco leaf. In order to investigate the relationship between nicotine heterosis and related gene expression, and provide the basis for improvement and control of nicotine content in tobacco, this study selects five types of tobacco as material,including 13 varieties.This study used the NCII mating design genetic, equipping 40 hybrid combinations. In different development stages, the nicotine content in tobacco leaf of parents and hybrid F1 were determined.And discussed the content of nicotine differences and heterosis performance, analyzed the combining ability and heritability. In the study, the main change period of tobacco nicotine traits and dynamic change rule were cleared. In addition,this paper discusses the related gene differential expression in the different heterosis combination change trend, analyzed the relationship between the expression of nicotine synthesis relative key enzyme genes and heterosis by using real-time fluorescence quantitative(Real- time PCR)technique. The main results were as follows:1、Nicotine content in the different types tobacco leaf is different. In the four tobacco types:flue-cured tobacco, burley tobacco, air-cured tobacco and aromatic tobacco, the average nicotine content of burley tobacco types is highest, as 4.27%, and significantly higher than that the other types of tobacco; the average nicotine content of flue-cured tobacco types is the lowest, as1.49%.The nicotine content in different varieties of the same tobacco types is also different. The nicotine content of G70 is the highest in the seven flue-cured tobacco varieties, as 2.47%, and significantly higher than the other; the nicotine content of Nanjiang 3 is the lowest, only as 1.12%, and significantly is lower than the other. The nicotine content of Meitandamanyan is the highest in the four air-cured tobacco varieties, as 2.54%; the nicotine content of Qinggeng is the lowest, as0.86%. Results show that the genetic resources which can use to improve nicotine content are rich.2、Crossed flue-cured tobacco with different types of tobacco, the heterosis of hybrids of nicotine content is diversity. Crossed flue-cured tobacco with aromatic tobacco, the heterosis ofhybrids main show over high-patent, which easy to choose the hybrids combinations whose nicotine content is higher than it parents. Crossed flue-cured tobacco with flue-cured tobacco,burley tobacco and air-cured tobacco, the heterosis of hybrids main show mid-parent, which easy to choose the hybrid combinations whose nicotine content is between the parents. Cross flue-cured tobacco with air-cured tobacco and burley tobacco, can choose the hybrid combinations whose nicotine content is lower than the parents. In forty hybrid combinations, there have nine hybrid combinations which heterosis over high-parent, accounting for 22.5%, and the strongest heterosis rate combination is Honghuadajinyuan x Basima, as 63.39%. There have eigth hybrid combinations which heterosis less lower-parent., accounting for 20%, and the strongest heterosis rate combination is NC82 x Meitandamanyan, as 97.72%. There have twenty three hybrid combinations which heterosis show as the mid-parent, accounting for 57.5%. Results show that the hybrid combinations could be select whose nicotine content meet the requirements by heterosis utilization.3 、 The analysis of combining ability’s and heritability’s results showed that: in the five female parents, including flue-cured tobacco varieties, these general combining ability variance of nicotine content have reached significant level. And in the eight male parents, including four types of tobacco, theses general combining ability variance of the nicotine content reached extremely significant level. Theses indicated that the variation of combining ability of nicotine content in the different type tobaccos is bigger than in the same tobacco type. The general combining ability of flue-cured tobacco and air-cured tobacco are negative. The general combining ability of burley tobacco and aromatic tobacco are positive. The highest general combining ability of the male parent is Basima, as 26.36, the lowest is Qinggeng, as 24.59. The highest general combining ability of the female parent is Honghuadajinyuan, as 16.12, the lowest is K326, as 8.41. Moreover,the difference of the special combining ability of nicotine content in hybrid combinations is extremely significant. In the forty combinations, there have twenty combinations’ special combining ability show positive, and the highest is Maryland×K326,as 37.46. There have twenty combinations’ special combining ability show negative, the lowest is K326×Basima, as 28.59. So the hybrid combinations could be select whose nicotine content meet the requirements by selecting the different parents. Then the broad sense heritability of the nicotine content is up to79.18%, the narrow sense heritability is 51.16%, indicating the main effect to the nicotine contentis the additive effect, and selection in the early generation selection is better. And the varieties could be breeding which the nicotine content meet the requirements by restructuring breeding and cross breeding.4、The dynamic change regularity of nicotine content in different materials were basically identical: With delay of growth period, the nicotine content in tobacco leaves increased continuously, and reached the highest at the mature stage. Before topping, the accumulation speed of nicotine is relatively slow. After topping, the accumulation speed of nicotine increased rapidly,and reached the highest at the mature stage. At 56 days to 70 days after transplanting, the nicotine content of 53 materials have increased 0.16% on average, which TN90 increased the highest, as0.38%, G70 × GDH02 increase the lowest, only as 0.05%. At 70 days and 77 days after transplanting, the nicotine content of 53 materials have increased 0.50% on average, which Va116× Basima increased the highest, as 1.29%, Nanjiang 3 and Qinggeng increased the lowest, both as0.12%。At 77 days and 84 days after transplanting, the nicotine content of 53 materials increased0.48% on average, which TN90 increase the highest, as 1.43%, K326 x TN90 increased the lowest,as 0.10%. At 84 days and 91 days after transplanting, the nicotine content of 53 materials increased 0.35% on average, which TN90 increased the highest, as 1.13%, nanjiang 3 increased the lowest, only as 0.02%. As a result, the main period of nicotine content increase is the topping stage to mature stage. And topping is the turning point of tobacco nicotine synthesis strength, the increase speed of nicotine content may be associated with tobacco varieties after topping. In addition, the results of the correlation analysis of the nicotine content in different periods show that the nicotine content of mature period and the nicotine content of a week after topping was significantly correlated, so it is the best early period to choose the nicotine content of mature period.5、In order to analysis five key enzyme gene expression in different heterosis combination,this study made the average expression of five key enzyme in the parents as the control, the results show as follows: ODC, PMT, ADC, QPT, MPO these five enzyme genes expressed in different development stages of quantity is different, and topping has a promoting effect to five enzyme gene expression, five key enzyme genes have a lot of express after topping. ODC and PMT always have a lot express in a week to two weeks after topping; ADC, QPT and MPO always have a lot express in two weeks to three weeks after topping. At the same time, the expression of QPT in thecombinations, which nicotine content is over high-parent, is higher than the combination which nicotine content is less lower-parent and the combinations which nicotine content is as the mid-parents. Consequently, the expression of QPT may have the maximum impact on the formation of heterosis.6、Results of the correlation analysis among the five key enzyme gene expression of the nicotine biosynthesis pathway, the nicotine content and the heterosis show that: In 77 days after transplanting, the relative expression of QPT with nicotine content and heterosis rate are significantly positively related. In 84 days after transplanting, the relative expression of ADC,QPT, MPO with nicotine content and heterosis rate are very significantly positive related, as well as three relative expression of enzyme genes has significant positive correlation between each other. In 91 days after transplanting, the relative expression of PMT with nicotine content and heterosis rate are significantly positively related, and other periods have not reach significant level.Therefore, in different periods, nicotine content and heterosis rate associated with different key enzyme genes. When some enzyme genes is significantly positively related in a period, it indicated that, increasing expression of these enzyme genes can effect the growth of nicotine content and heterosis rate at the same time period, which can be used as the indirect indicators to select nicotine content and heterosis rate. |
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