| The tobacco leaves are harvested,and the tobacco stem in the leaves is difficult to use.Reducing the stem content of tobacco leaves has become an important goal to reduce costs and improve the benefits of cigarettes.At present,the study on the stem content of tobacco leaves is mainly based on the difference analysis of environment,cultivation and different varieties,while the study on the influence of genetic factors on the stem content of tobacco leaves is less.In order to explore the relationship between stem content of tobacco leaves and their heterosis performance and gene differential expression,and to provide a basis for the improvement and regulation of stem content of tobacco leaves,13 tobacco materials with large differences in stem content were selected as parents in this study,and 40 hybrid combinations were matched by NCII genetic mating design.The stem content of middle leaves was measured before and after topping,and the differences in stem content of tobacco leaves were explored.The variation trend and heterosis performance were analyzed for their combining ability and heritability.Hybrid combinations with large differences in heterosis performance were screened out.Real-time fluorescence quantitative PCR was used to analyze the expression of genes that may affect the stem content and Heterosis of tobacco at the gene level,and the related genes regulating the stem content and Heterosis of tobacco were preliminarily identified.The main findings are as follows:1.There are significant or even extremely significant differences in the stem content of tobacco leaves among parents and hybrid combinations.The trend of stem content of tobacco leaves before and after topping is the same for all materials.The higher stem content rates are K326(on the day of topping: 20.12%,one week after topping: 19.53%),TN90(on the day of topping: 22.82%,one week after topping: 21.64%),Basima(on the day of topping: 22.06%,one week after topping: 23.06%).)and Jiucaiping 2(the day of topping: 22.58%,the week after topping: 19.46%),the lower were Qinggeng(the day of topping: 11.67%,the week after topping:13.04%),Bina 1(the day of topping: 15.35%,the week after topping: 16.68%),GDH94(the day of topping: 14.02%,the week after topping: 15.45%)and Meidanmanyan(the day of topping:10.62%,the week after topping: 11.08%).The stem content of tobacco showed an upward trend after topping.There were obvious differences in the stem content of tobacco among varieties and materials,which showed genetic diversity,indicating that the genetic resources for improving the stem content of tobacco were abundant.2.Analysis of the influence of moisture content on stem percentage: the stem percentage of tobacco leaves in each period of moisture absorption was measured after the sample was killed and dried.The results showed that the change trend of stem percentage was gentle in the process of gradually increasing moisture content of tobacco leaves,and there was no obvious lawwith the change of moisture content.Taking K326 as an example,the moisture content of tobacco leaves in each period of moisture absorption was significantly different,but the difference of stem percentage of tobacco leaves in each period of moisture absorption was not significant So is his material.The results showed that the moisture content of the treated tobacco had no effect on the stem content,so it could be considered to shorten the moisture absorption time of the stem content to improve the research efficiency.3.The results of combining ability and genetic analysis showed that the broad heritability of tobacco stem content was 96.11%,indicating that the trait was selected by phenotype;the narrow heritability of tobacco stem content(72.32%)was higher,and the variance of GCA(75.25%)was greater than that of SCA(24.75%),indicating that tobacco stem content was mainly controlled by the additive effect of genes,but also had the effect of non-additive effect.The GCA relative effect values of tobacco stem content among different parents showed significant or extremely significant differences.Among them,Nanjiang 3(17.23)and Basima(12.82)had the greater positive value of GCA effect,while Meitandamanyan(-21.15),Qinggeng(-15),GHD88(-7.34)and NC82(-5.29)had the greater negative value of GCA effect,and these four materials had the stronger genetic transmissibility of low stem content.The negative effect values of SCA were higher than those of GDH94 TN90(-11.63),G70 Basima(-10.88),G70 Qinggeng(-9.19),NC82 Nanjiang 3(-8.93)and VA116 Jiucaiping 2(-8.85),and these combinations had potential for direct utilization in tobacco production.4.The results showed that there were significant differences in the Heterosis of middle parents among the hybrid combinations.After comparing the values of the Heterosis of middle parents before and after topping,the combination with the same trend of the Heterosis of strong and weak middle parents in the two periods was selected for gene differential expression analysis.GDH94 × Meitandamanyan(5.11%)and GDH94 × Bina 1(10.86%)had the highest heterosis;va116 × Nanjiang 3(0.66%)and nc82 × Jiucaiping 2(0.72%)had the weakest heterosis;K326× TN90(-14.64%)and va116 × Meitandamanyan(-19.16%)had the lowest heterosis.The above materials were used to analyze the differential expression of related genes.5.The relative expression of seven related genes in different cross combinations was analyzed one by one.The relative expression of the same gene in different cross combinations was different.The relative expression of Cp SRP43 gene in GDH94 × Bina 1 was the highest,22.14 times of the average expression of parents,and the lowest in va116 × Nanjiang 3,0.15 times of the average expression of parents.The relative expression of AGD 3,VOC1,GRF1,LNG1 and Cp SRP43 genes in the negative mesophilic dominant combination was very low,and the low expression of these genes may be related to the formation of negative mesophilic dominance.The results showed that there was a significant positive correlation between the relative expression of LNG1 gene and GRXC9 gene,Cp SRP43 gene and LNG1 gene,Cp SRP43 gene and GRXC9 gene There was a significant positive correlation between the relative expression of genes,which suggested that there might be interaction between the three genes.There was a positive correlation between the relative expression of seven genes and the Heterosis of stemmed rate,among which the relative expression of VOC1 gene was significantly positive correlated with the Heterosis of stemmed rate,and the relative expression of Cp SRP43 gene was significantly positive correlated with the Heterosis of stemmed rate.The results showed that the two genes regulated the formation of Heterosis of stem percentage. |
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