15 July 2017, Volume 29 Issue 4

    

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  Integrated Structure of the Modified cry1Aa Gene in Cotton and Its Event-Specific Detection

  Wang Ye, Xie Jiajian, Huang Chunmeng, Peng Yufa

  Cotton Science. 2017, 29(4): 307-315. https://doi.org/10.11963/1002-7807.wyxjj.20170628

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  [Objective] We detected a modified cry1Aa gene in a Chinese cotton line in 2011. The objectives of this study were to establish a specific detection method applicable for commercial cotton varieties by integrating the results of structural analyses and to confirm the existence of the exogenous gene in cotton. [Method] The integrated structure of the modified cry1Aa gene (NN6) was determined by genome walking and a long-chain polymerase chain reaction (PCR). An event-specific qualitative PCR and a quantitative real-time PCR were established based on the linkage region between genomic DNA and the exogenous DNA of NN6. [Result] The NN6 structure mainly comprised the cry1Aa, aad, and nptII genes, which had been inserted into chromosome 7 at position 37 169 450, with a 91 bp deletion in the genome. The highly sensitive qualitative PCR was able to quickly identify the heterozygous and homozygous NN6. The limit of detection for the NN6-specific quantitative real-time PCR was 44 copies, which satisfied the requirements for analyzing Chinese and international cotton varieties. The seeds of three commercial cotton varieties carrying the cry1Aa gene were tested, with a resulting purity of 1.51%, 5.21%, and 21.09%. [Conclusion] We analyzed the integrated structure of NN6 and established a specific detection method that may be useful for ensuring transgenic cotton can be bred safely in China.
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  Methylation-sensitive Amplified Polymorphism Analysis of Epigenetic Changes during Fiber Development in Green Cotton (Gossypium hirsutum L.)

  Qian Senhe, Hong Liang, Wei Ming, Lin Yi, Cai Yongping

  Cotton Science. 2017, 29(4): 316-326. https://doi.org/10.11963/1002-7807.qshqsh.20170405

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  [Objective] The aim of this study was to analyze the DNA methylation during fiber development in green cotton. [Method] The products resulting from EcoRI/HpaII and EcoRI/MspI double-digestions were amplified by methylation-sensitive amplified polymorphism primers to analyze the methylation status of the 5'-CCGG-3' locus in the 'Lüxumian No. 1' genome. Meanwhile, the amplified fragments were sequenced to characterize their biological functions. [Result] A total of 4 112 fragments were amplified by 66 primer pairs, with an average of 822.4 fragments each. Additionally, each primer amplified an average of 12.46 fragments. The number of methylated fragments at 10, 15, 20, and 25 days post anthesis (DPA) was 11.45%, 13.86%, 20.10%, and 33.13% higher than the number of methylated fragments at 5 DPA, respectively. At 10, 15, 20, and 25 DPA, 3.15%, 3.43%, 3.65%, and 2.52% of the green cotton fiber genomic loci were methylated, respectively. In contrast, at the same time points, 12.87%, 14.72%, 13.31%, and 48.81% of the loci were demethylated, respectively. The sequencing results and BLAST searches revealed that 17 gene fragments were homologous to known functional genes, including those from the cotton mitochondrial genome as well as the genes encoding a serine protease and an esterase. These genes were methylated at 25 DPA. [Conclusion] Genes are methylated and demethylated during green cotton fiber development.
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  Polymorphism Analysis of Mitochondrial Genes Associated Cytoplasmic Male Sterility in Cotton (Gossypium harknessii Brandegee)

  Gong Yangcang, Zhang Xuelin, Wu Jianyong, Zhang Xingping, Peng Fanjia, Zhang Zhigang, He Yunxin, Mei Zhengding, Zhou Degui, Xing Chaozhu

  Cotton Science. 2017, 29(4): 327-335. https://doi.org/10.11963/1002-7807.gycxcz.20170630

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  [Objective] Cytoplasmic male sterility (CMS) is closely associated with the mitochondrial genome. The aim of this study was to identify CMS-related mitochondrial genes in cotton. [Method] Ten mitochondrial gene probes (i.e., atpA, atp6, atp9, cob, coxI, coxII, coxIII, nad3, nad6, and nad9) were used to analyze restriction fragment length polymorphisms in a Gossypium harknessii Brandegee cytoplasmic male sterile line (S), maintainer line (F), and hybrid (H). [Result] Ten probe/enzyme combinations for four probes (i.e., atpA, coxIII, nad3, and nad6) revealed polymorphisms among lines S, F, and H. All enzyme digestions for two probes (i.e., atpA and nad6) displayed the polymorphisms in three lines, with the same patterns for lines S and H. For atpA, the EcoR I digestion revealed two fragments in the three lines. The 2.2 kb fragment was common to all three lines, while the second fragment was 3.2 kb in lines S and H, but 4.8 kb in line F. The atpA/PstI combination produced a 17.0 kb fragment in lines S and H, but a 10.2 kb fragment in line F. For nad6, one or two additional fragments of the same length were detected in lines S and H, while line F had more than two additional fragments. With coxIII as a probe, the EcoR I digestion resulted in a 2.5 kb fragment in three lines, as well as a 1.7 kb fragment in lines F and H, but not in line S. With the nad3 probe, the same patterns were observed for lines S and F, while the pattern for line H differed because of a lack of a 9.5 kb fragment. [Conclusion] Both atpA and nad6 are involved in regulating the development of CMS, while coxIII and nad3 may be regulated by nuclear genes to help restore fertility in cytoplasmic male sterile lines.
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  Optimization of EMS Mutagenesis Condition and Screening of Mutants in Gossypium arboretum L.

  Kong Depei, Qu Lingbo, Zhang Xueyan, Liu Ji, Wang Peng, Li Fuguang

  Cotton Science. 2017, 29(4): 336-344. https://doi.org/10.11963/1002-7807.kdplfg.20170703

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  [Objective] This research aimed to clarify the optimization of EMS concentration and treat duration on mutation of Gossypium arboretum L. and create excellent mutants library. [Method] Seeds of Gossypium arboreum line Shixiya1 were immersed in EMS solutions with volume fraction ranging from 0.4% to 1.5% by 4-8 h. Then, the seed germination index and seedling growth were analyzed and the morphological character of M₁, M₂, and M₃ generation were identified. [Result] The treatment with 0.6% EMS for 8 h was appropriate for Shixiya 1 mutagenesis. Under this treatment conditions, the seed germination index and the germination rate were 18.84 and 51%, respectively. To obtain abundant mutants, approximated 23 000 seeds of Shixiya 1were treated with 0.6% EMS for 8 h. 5559 mutants were screened out from M₁ generation, 825 and 57 from M₂ and M₃, respectively. The mutation frequency of Shixiya 1 was 48.37%. The mutation frequency of leaf type variants, plant type variants were the highest in M₂, which were 8.0% and 5.9%, respectively．The inheritable frequency of mutant characters from M₂ generation to M₃ generation was 31.30%, and the mutant frequency of M₃ generation was 18.26%. [Conclusion]This study have established the EMS mutagenesis system of Gossypium arboreum line Shixiya 1, and created 36 steadily inherited mutants. The mutant library we constructed will be a very useful genetic resource for functional genomics and genetic improvement in cotton．
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  Effect of Drip Irrigation Amount on the Agronomic Traits and Yield of Cotton Grown with a Chemical Topping in Northern Xinjiang, China

  Xu Shouzhen, Zuo Wenqing, Chen Minzhi, Sui Longlong, Dong Hengyi, Jiu Xingli, Zhang Wangfeng

  Cotton Science. 2017, 29(4): 345-355. https://doi.org/10.11963/1002-7807.xszzwf.20170605

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  [Objective] The aim of this study was to examine the effects of drip irrigation amount on the agronomic traits and yields of cotton plants grown using a chemical topping, which may have implications for the application of chemical topping technology during cotton production. [Method] Two-factor experiments with three drip irrigation treatments and two topping methods were conducted under field conditions during the cotton-growing period in 2016. We used manual and chemical topping methods. Flumetralin and DPC topping agents were selected for the chemical topping. Agronomic traits, defoliation state before machine harvesting, and changes in yield were assessed to clarify the effects of chemical toppings following different drip irrigation treatments. [Result] There was a significant interaction between the topping methods and drip irrigation amount, which influenced cotton plant height and branch length. For the chemically topped cotton plants, the average plant height and branch length under the middle drip irrigation treatment were 6% and 12% lower, respectively, than those under the high drip irrigation treatment, but were 13% and 14% higher, respectively, than those under the low drip irrigation treatment. There were no significant differences in the effects of the middle and high drip irrigation treatments on the yield of chemically topped cotton plants. In contrast, the cotton yield of chemically topped plants was 7%-8% higher under the middle drip irrigation treatment than that under the low drip irrigation treatment. There were no significant differences in the defoliation rate and amount of miscellaneous leaves between the chemical and manual topping methods. However, the low drip irrigation treatment may have increased the defoliation rate in the chemically topped cotton. Although the chemical topping method significantly decreased boll weight from the upper branches, it had no significant effect on the final yield and lint percentage. [Conclusion] Two rounds of a middle drip irrigation treatment (32 m³·667 m^-2) after spraying plants with a topping agent may affect plant growth and decrease the defoliation rate of chemically topped cotton plants. Furthermore, the middle drip irrigation treatment does not appear to decrease seed cotton yields. Therefore, spraying plants with a topping agent and applying the middle drip irrigation treatment may be beneficial for cotton production.
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  Effects of Different Nitrogen Applied on the Growth, Yield, and Fiber Quality of Cotton Genotypes with Different Potassium-Use Efficiency in Florescence

  Liu Aizhong, Zheng Cangsong, Li Pengcheng, Sun Miao, Liu Jingran, Zhao Xinhua, Dong Helin, Shi Shubing

  Cotton Science. 2017, 29(4): 356-364. https://doi.org/10.11963/1002-7807.lazdhl.20170602

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   [Objective] The purpose of this study was to characterize the changes to cotton growth, yield, and fiber quality induced by different nitrogen applied in florescence under different soil available potassium (K) concentrations. [Method] High K-use efficiency cotton genotypes (Jimian 958 and Liaomian 18) and a low K-use efficiency cotton genotype (NuCOTN99B) were grown in pots with low (38.01 mg·kg^-1) or high (152.24 mg·kg^-1) soil K concentrations. Ammonium sulfate (as ammonium nitrogen) or calcium nitrate (as nitrate nitrogen) was applied in florescence, respectively. [Result] The plants grown under low soil K conditions had lower first fruit node and fewer bolls than the plants exposed to high soil K contents. Additionally, the whole plant dry weight, total K accumulation, total K utilization index, and seed cotton yield were significantly higher in plants treated with a high soil K concentration. Furthermore, the NH₄⁺  treatment produced shorter stems, fewer fruit branches and bolls, and lower seed cotton yields and the whole plant dry weight than the NO₃^-  treatment. The NH₄⁺  treatment also resulted in a lower accumulation of total K and a lower total K utilization index. [Conclusion] The low K-use efficiency cotton genotype was sensitive to the adverse effects of NH4+  applied in florescence, which had more serious consequences for the later mature cotton cultivar.
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  Changes to the PAR Spatial Distribution, Biomass, and Fiber Quality in Response to Plant Densities

  Zhi Xiaoyu, Han Yingchun, Wang Guoping, Feng Lu, Yang Beifang, Fan Zhengyi, Du Wenli, Lei Yaping, Mao Shuchun, Li Yabing

  Cotton Science. 2017, 29(4): 365-373. https://doi.org/10.11963/1002-7807.zxylyb.20170407

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  The purpose of this study was to analyze how cotton (Gossypium hirsutum L.) growth periods, PAR distribution, LAI, and biomass are affected by different plant densities. [Method] A hybrid cultivar (CCRI 75) and conventional cotton cultivar (SCRC 28)) were used for field experiments in 2012 and 2013. Three plant densities were analyzed (i.e., 15 000, 51 000, and 87,000 plants·hm-2). [Result] The PAR distributions during various growth periods were significantly different among the three tested plant densities. The amount of transmitted light decreased as the plant density increased. Moreover, the canopy structure and plant type varied with plant densities, which influenced the specific distribution of PAR. Additionally, considerable changes occurred to the LAI over different growth periods. The LAI increased rapidly from 60 days after sowing (DAS) in 2012 and 2013. However, there was a sharp decrease in the LAI at 100 DAS in 2012 and 110 DAS in 2013. As the plant density increased, the maximal accumulated biomass decreased, while an increasing proportion of the biomass was derived from vegetative rather than reproductive organs. Plant density also significantly affected the Micronaire value, with the highest value obtained under high plant density. [Conclusion] These findings may be relevant for determining the optimal plant density for enhanced cotton cultivation.
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  Effects of Plastic Film Residues on Soil Nitrogen Content, Root Distribution, and Cotton Yield during the Long-Term Continuous Cropping of Cotton

  Zumilaiti Tuergan, Lin Tao, Wang Liang, Wang Jing, Li Jianwei, Tang Qiuxiang

  Cotton Science. 2017, 29(4): 374-384. https://doi.org/10.11963/1002-7807.zmlttegtqx.20170602

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  [Objective] The objective of this field experiment was to study the effects of plastic film residues on soil nitrogen content, root distribution, and cotton yield in field for long-term continuous cropping of cotton (Gossypium hirsutum L.). [Method] The cotton variety 'Xinluzhong 47' was used in a field experiment conducted during the cotton-growing season in 2013-2014. The experiment involved the following five plastic film residue treatments: 0 (control), 225, 450, 675, and 900 kg·hm^-2. We analyzed changes in soil nitrogen content, root morphology, and cotton yields. [Result] Seed cotton yields decreased with increasing amounts of plastic film residue, and significantly affected the number of bolls per plant and boll weight, but not by lint percentage. The treatments with 450, 675, and 900 kg·hm^-2 plastic film residues had significant effects on seed cotton yields (P < 0.05). The seed cotton yield following the 900 kg·hm^-2 treatment was 22.2% lower than the control yield. Additionally, exposure to the plastic film residues decreased root growth, root length, root diameter, root surface area, root volume, and the number of root tips. There were significant differences in these root-growth indices (P < 0.05) between the control and plastic film residue treatments. The 900 kg·hm^-2 treatment decreased the root length by 33.7%, root diameter by 24.3%, root surface area by 19.72%, root volume by 66.4%, and the number of root tips by 35.3%. These results confirmed that the ammonium and nitrate levels increased with increasing concentrations of residual film. At the boll stage, the ammonium and nitrate levels following the 900 kg·hm^-2 treatment were 36.6% and 40.1% higher than the control levels, respectively. [Conclusion] Exposure to plastic film residues inhibits cotton root growth and decreases nitrogen use, leading to decreased cotton yields.
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  Synchronized Somatic Embryogenesis of 'Xinhai 16' Induced by Different Treatments

  Guo Jiayan\, Zhang Xia, Ding Xilian, Li Juan, Deng Li, Chen Quanjia, Sun Guoqing, Qu Yanying

  Cotton Science. 2017, 29(4): 385-392. https://doi.org/10.11963/1002-7807.gjyqyy.20170531

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  [Objective] The aim of this study was to develop an efficient method for synchronizing the somatic embryogenesis of Gossypium barbadense L. [Method] We attempted to synchronize the somatic embryogenesis of G. barbadense L. cv. 'Xinhai 16' embryonic calli. The somatic embryos induced by low temperature, starvation, and osmotic pressure were counted and analyzed. [Result] In the low-temperature treatment group, the number of somatic embryos increased significantly at 4 ℃. While, there were no significant differences in the number of somatic embryos between treatment times at 10 ℃. Nevertheless, more somatic embryos were produced at 10 ℃ than at 28 ℃ (control temperature). Somatic embryogenesis was inhibited by the nutrient starvation treatment (i.e., phosphate, nitrogen, or inositol deficiency). In the osmotic stress group, the best results were observed following a 7-day culture in medium containing 40 g·L^-1 glucose. [Conclusion] 'Xinhai 16' somatic embryogenesis can be synchronized by incubating calli at 4 ℃ for 3 days, simulating phosphate or nitrogen starvation conditions for 5 days, or maintaining calli in a medium containing 40 g·L^-1 glucose for 7 days. The low-temperature treatment may provide optimal synchronization conditions.
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  Cloning and Characterization of the GbWRKY40 Transcription Factor Gene from Gossypium barbadense L.

  Ni Zhiyong, Jiadela Tuliuhan, Qiu Yingfeng, Qu Yanying, Chen Quanjia

  Cotton Science. 2017, 29(4): 393-400. https://doi.org/10.11963/1002-7807.nzycqj.20170601

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  [Objective] The WRKY transcription factors modulate various biological processes, including plant growth, development, and responses to various environmental stresses. This study aimed to clarify the role of WRKY transcription factors related to cotton fiber development. [Method] The GbWRKY40 gene, which encodes a WRKY transcription factor, was isolated from cotton (Gossypium barbadense L.). The obtained sequence was analyzed for homology with genes from other cotton species using the DNAMAN7 program. The sequences of the homologous genes were aligned with Clustalx1.83. Additionally, the GbWRKY40 expression pattern was analyzed by a quantitative real-time polymerase chain reaction. Furthermore, a yeast expression vector was constructed and inserted into yeast strain AH109 cells to investigate the transcriptional activity of GbWRKY40. [Result] Full-length GbWRKY40 cDNA contains 1713 nucleotides, including a 942 bp open reading frame, a 261 bp 5'-untranslated region, and a 510 bp 3'-untranslated region. The open reading frame was predicted to encode a 313 amino acid protein with a relative molecular mass of 34.138 × 103 and an isoelectric point of 8.46. The genomic GbWRKY40 sequence was observed to comprise five exons and four introns. The predicted GbWRKY40 protein includes a WRKY domain consisting of approximately 60 amino acids, including the conserved WRKYGQK sequence and a zinc-finger motif (C-X₅-C-X₂₃-H-X₁-H). These characteristics indicated that GbWRKY40 belongs to group IIa of the WRKY family. Moreover, GbWRKY40 contains three putative nuclear localization signals. GbWRKY40 is highly homologous to GhWRKY40. Expression analyses involving a quantitative real-time polymerase chain reaction revealed that GbWRKY40 is highly expressed in cotton roots and fibers at 25 days post anthesis. Transcriptional activation assay results suggested that the function of the GbWRKY40 transcription factor is unrelated to the activation of transcription. [Conclusion] These results imply that GbWRKY40 may be involved in regulating secondary cell wal development in cotton fibers.