Abstract :
[en] Leaf senescence plays a vital role in the overall development of plant. And it is also an important agronomic trait of crop that affects the yield and quality of crop, such as grain size and weight. Tobacco (Nicotiana tabacum L.) is an important economic crop widely cultivated in the world and an important model plant in plant genetic study. In tobacco production, mature yellow leaves are harvesting targets. Leaf senescence is critical to the yield and quality of tobacco products. Investigation of tobacco leaf senescence regulating genes and analysis of their functions can enhance the understanding of the regulating mechanisms of tobacco leaf senescence, and provide new genetic resources for the breeding of new tobacco varieties.
In this study, two tobacco premature leaf senescence mutants yellow leaf 1 (yl1) and yellow leaf 2 (yl2) obtained by ethyl methane sulfonate (EMS) mutagenesis were studied. Leaf senescence of mutants were initiated earlier than wild type. The mutants yl1 and yl2 were studied for phenotypic characterization, genetic analysis and preliminary mapping of target genes. This study laid the foundation for further cloning of tobacco leaf senescence regulating gene and in-depth analysis of tobacco leaf senescence regulating mechanisms.
The main contents and results are as follows:
(1) yl1 mutant
The tobacco leaf senescence mutant yl1 was selected from the EMS mutagenesis mutant library of tetraploid common tobacco (Nicotiana tabacum L.) variety Honghuadajinyuan (HD). Phenotypic identification showed that yl1 had premature leaf senescence characteristics. The leaf senescence of yl1 was initiated earlier in the early developmental stage. The lower leaves of yl1 began to turn yellow at 50 days after transplanting (DAT). The chlorophyll in yl1 leaves degraded earlier than that in wild type. In leaves of the same developmental stage and at the same leaf position, the photosynthesis efficiency, protein content, and the expression level of photosynthesis related gene of yl1 were lower than that of wild type, while the expression level of senescence related gene was higher than that of wild type. In the late developmental stage, the chlorophyll content of yl1 decreased more rapidly, the senescence rate of yl1 was faster than that of wild type HD. The wild type tobacco varieties HD and Gexin3 (GX3) were crossed with yl1, respectively. All the F1 plants showed a normal leaf senescence phenotype similar to the wild type. In F2 populations, mutant type plants and wild type plants showed a separation ratio of 1:3. In BC1F1 population, mutant type plants and wild type plants showed a separation ratio of 1:1. The phenotypic separation ratio of F2 and BC1F1 population indicated that the phenotype of yl1 was controlled by a single recessive gene. The BC1F1 population developed between parent GX3 and yl1 was used to map the target gene. The target gene was mapped using the published SSR molecular markers, the result indicated that the target gene was located on the tobacco linkage group (LG) 11 between the markers PT60305 and PT53066, and the genetic distance was 3.51 and 1.08 cM, respectively. The treatment of exogenous plant hormones on detached leaves showed that yl1 was more sensitive to the ethylene and jasmonic acid than wild type.
(2) yl2 mutant
The tobacco leaf senescence mutant yl2 was selected from the EMS mutagenesis mutant library of tetraploid common tobacco (Nicotiana tabacum L.) variety Zhongyan100 (ZY100). The yl2 plants displayed early leaf yellowing. The lower leaves of yl2 began to turn yellow at 65 DAT. Physiological parameters and marker genes expression indicated that the yl2 phenotype was caused by premature leaf senescence. The chlorophyll content and protein content of yl2 were lower than wild type ZY100 throughout the developmental stages. The chlorophyll content was rapidly decreased in the later developmental stage. The wild type tobacco varieties Cuibi1 (CB1) and Gexin3 (GX3) were crossed with yl2, respectively. All the F1 plants showed a normal leaf senescence phenotype similar to the wild type. The phenotypic separation ratio of F2 population indicated that the phenotype of yl2 was controlled by a single recessive gene. Using the whole genome sequencing (WGS), 18 candidate SNPs associated with yl2 mutant phenotype were obtained. The possibility of 18 SNPs leading to the mutant phenotype was ruled out by PCR validation. The F2 population developed between CB1, GX3 and yl2 was used to map the target gene. The target gene was mapped using the published SSR molecular markers, the result indicated that the target gene was located on one end of tobacco linkage group (LG) 24. The genetic distance between target gene to the nearest polymorphic SSR marker PT60487 was 14.2 cM.
