| Since the first herbicide 2, 4-D being discovered in 1940, more and more herbicides have emerged constantly. Among those chemicals, glyphosate stands out because of its broad-spectrum, high-efficiency, low-toxicity and non-selection, and is widely used throughout the world. Therefore, it is necessary to develop glyphosate-resistant crops, and the development of transgenetic glyphosate-resistant crops indeed greatly facilitates weed control, but the safety of GR crops face the controversy. Particularly, in the process of cotton planting, almost all the problems will be resolved if we can excavate endogenous glyphosate-resistant genes from the existing cotton resources and serve for cotton breeding. In view of the existence of many excellent genes in cotton germplasm resources, we identified the natural resistance to glyphosate in wild cotton, G. hirsutum races and G. arboreum L, analysized the sequence of epsps and its expression in the glyphosate-resistant G. hirsutum races after the treatment by glyphosate, and tried to discover glyphosate-resistant gene resources with the method of association mapping in the natural population of G. hirsutum races. The results are as follows:1. Evaluation and comparative analysis of natural resistance to glyphosate in cotton germplasm resourcesIn this study, we identificated and evaluated the natural glyphosate-resistance of cotton resources. With the method of spraying solution to twigs in wild cotton, and tampons placed at specific positions on the leaves with different concentrations of glyphosate in G. hirsutum races and G. arboreum L, we rated them by the reaction of the treated position. Totally, the materials of wild cotton included 134 plants of 26 species, the G. hirsutum races included 403 accessions which belong to 7 races, and the G. arboreum L included 101 accessions which came from 5 different areas. Here, we obtained a method of identifying the natural glyphosate-resistance and a corresponding reference of grading standard. Moreover, it was demonstrated that 1 plant, 2 accessions and 5 germplasms exhibit high resistance to glyphosate from wild cotton, G. hirsutum races and G. arboreum L respectively, 16 and 1 showed highly susceptible in wild cotton and G. hirsutum races seperately, but no plant susceptible behavior in G. arboreum L. In addition, the lethal concentrations of CRI12 in the way of spraying in seedling, bud and flower-boll-forming stage are 0.1%, 0.15% and 0.25% respectively, we carried out in resistant and susceptible G. hirsutum races, finally 1 was authenticated resistant and 1 being sensitive materials. At the same time, we analyzed the diversity of glyphosate resistance and formed an order as: wild cotton > G. hirsutum races > G. arboreum L. Moreover, the order of general glyphosate-resistant level is G. arboreum L > G. hirsutum races > wild cotton. The evaluation of glyphosate-resistance contributed to understanding gylphosate-resistance of cotton comprehensively, and these materials provided a base for further in-depth study for the genetic mechanism, internal resistance genes mining and the resistance mechanism, and guided glyphosate-resistant cotton breeding.2. epsps gene sequencing and its transcription after glyphosate treatmentUsing sad1 as the reference gene, we detected the genetically modified ingredients of G. hirsutum races with the specific primers of Monsanto GR gene sequences in cotton, and proved the glyphosate-resistant genes of test materials did not come from genetically modified cotton. With the full-length primer of epsps in G. hirsutum(Coker 312), we obtained the amplified production of G. hirsutum races, sequenced, and translated into amino acid sequence. No specific amino acid sites were found by BLAST, and this suggested glyphosate-resistance in G. hirsutum races was not caused by the mutation of epsps gene. Using quantitative RT-PCR, epsps transcript abundance was measured relative to actin, a low-copy gene with known monogenic inheritance in cotton. Unfortunately, there is no significant changes with glyphosate treatment or not, and this suggested epsps gene expression was unaffected by glyphosate treatment in G. hirsutum races.3. Evaluating glyphosate-resistant gene resources in G. hirsutum races through association mappingIn this study, a broad range of variations were observed for shikimate content and relative dry weight in three natural environments, and these phenotypic variations were significantly affected by the environments, but genetic effects mainly contributed. Association mapping was applied to dissect the genetic basis of glyphosate-resistance in 202 accessions of G. hirsutum races using 182 simple sequence repeats(SSRs) markers covering all cotton chromosomes. Based on the model controlling for both population structure(Q) and relative kinship(K), 41 loci significantly associated with relative dry weight were detected, which could explain part of the phenotypic variance, ranging from 13% to 70%, whereas another 48 loci were associated with the accumulation content of shikimate, explaining the phenotypic variations from 10% to 29%. Gene sequences including the associated markers were searched from the public databases of G. arboreum L, G. raimondii and G. hirsutum. Among them, the ABC(ATP-binding cassette) transporter, which embodies the associated marker MON_CGR6680 and demonstrated in Ambrosia artemisiifolia through RT-PCR, was inferred to be concerned with the shikimate biosynthesis from Kyoto Encyclopedia of Genes and Genomes(KEGG). These results provide insights into the genetic basis of glyphosate resistance, meanwhile, the detected loci may be used as the candidate markers for the improvement of glyphosate-resistance in cotton breeding. |
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