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15 January 2019, Volume 31 Issue 1

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The integration and insertion site of GbVe1 gene in the genome of transgenic cotton (Gossypium hirsutum)
Chen Tianzi, Ling Xitie, Yang Yuwen, Zhang Baolong
Cotton Science. 2019, 31 (1): 1-11.   DOI: 10.11963/1002-7807.ctzzbl.20190105
Abstract ( 5 PDF (3302KB) ( 0 )
[Objective] The aim of this study was to obtain the flanking sequences of T-DNA in the transgenic cotton containing a GbVe1 over-expression cassette. [Method] The T-DNA insertion copy number in the transgenic GbVe1 cotton was analyzed by southern blot. Flanking sequences of the transgenic lines with putative single T-DNA insertion copy were obtained using high-efficiency Thermal asymmetric interlaced polymerase chain reaction (hiTAIL-PCR). The T-DNA insertion sites were further confirmed by PCR with specific primers. [Result] RB-flanking sequences (119-1 018 bp) and LB-flanking sequences (243-516 bp) were obtained from three transgenic lines with low copy number of T-DNA insertion. The AT content was more than 63% in these flanking sequences. A same single insertion site in the intron of Gohir.D01G157600.1 was found in the two transgenic lines 7/100826-152 and 12/100826-393, while two separated insertion sites, one also in the intron of Gohir.-D01G157600.1 and the other in the intergenic region of A12 chromosome, were found in the transgenic line 1/w-ch14. A deletion of 21 bp was found in the insertion site in the intron of Gohir.D01G157600.1. The T-DNA insertion in the intron of Gohir.D01G157600.1 was further confirmed by the specific PCR. [Conclusion] The flanking sequences of T-DNA in the transgenic GbVe1 cotton were obtained and the specific transformation event in the intron of Gohir.D01G157600.1 was further confirmed by PCR.
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QTL Mapping of Boll Weight Trait Based on Recombinant Inbred Lines in Gossypium hirsutum L.
Qu Zhaoyang, Jia Xiaoyun, Ma Qifeng, Wang Hantao, Wei Hengling, Fan Shuli
Cotton Science. 2019, 31 (1): 12-22.   DOI: 10.11963/1002-7807.qzyfsl.20190115
Abstract ( 8 PDF (2836KB) ( 0 )
[Objective] The aim of this study was to explore the quantitative trait locus (QTL) related to the boll weight. [Method] A single seed descended population of 137 recombinant inbred lines (RILs) was developed from the cross of upland cotton (Gossypium hirsutum L.) CCRI 36 and G2005, an introgression inbred line introgressed from G. barbadense. Using restriction-site associated DNA sequencing (RAD-seq), a genetic linkage map composed of 6 434 makers, including 6 295 single nucleotide polymorphisms (SNP) and 139 simple sequence repeat (SSR) markers, was developed from the RILs population. [Result] This map spanned 4 071.98 cM with an average distance of 0.63 cM between adjacent markers. QTL mapping was performed by using boll weight data of five environments through WinQTLCart 2.5 software. Thirty-two QTL, with 4.46%-15.84% explained phenotypic variation related boll weight, were detected and found distributing on 15 chromosomes. qBW-A4-1, qBW-A4-2, qBW-A5-2, qBW-D9-1, and qBW-D9-2 were detected in two environments, which explained 5.07%-15.84% of the phenotypic variation. [Conclusion] Major QTLs detected in this study will provide an important reference for analysis of the genetic mechanism of boll weight.
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Evaluation and QTL Mapping of Tolerance to Salinity Using Interspecific Introgression Lines from Gossypium barbadense in G. hirsutum
Zhu Xiefei, Si Zhanfeng
Cotton Science. 2019, 31 (1): 23-30.   DOI: 10.11963/1002-7807.zxfzxf.20181226
Abstract ( 16 PDF (1495KB) ( 0 )
[Objective] Tolerance evaluation is the basis for cotton salinity-resistant breeding. [Method] Relative growth of cotton plants at seedling stage for 148 introgression lines was used to evaluate the tolerance to salinity. [Result] The results demonstrated that salt damage coefficients were significantly different from each other, with an average 61.22% ranging from 1.34% to 100%. Based on the salt damage coefficient, 148 introgression lines were divided into three groups; group 1 (23.65%) was more tolerant than the tolerant parent with the salt damage coefficient less than 29.35%; group 2 (39.19%) was more sensitive than the tolerant parent with the salt damage coefficient from 29.35%―71.70%; group 3 (37.16%) was more sensitive than the susceptible parent with the salt damage coefficient above 71.70%. [Condusion] Mapping of QTLs (Quantitative trait locus) for salt-tolerant traits indicated that 23 QTLs for tolerance-related traits were detected. Among them, 12 QTLs were aggregately distributed on chromosomes A05, A12, D05 and D11. Our results demonstrate that it is feasible for screening salt-tolerant introgression lines from Gossypium barbadense into G. hirsutum, especially by molecular marker-assisted selection for specific chromosomes with high efficiency.
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Functional Identification of Cotton U3 and U6 Promoters in the CRISPR/Cas9 Genome Editing System
Zang Xuyang, Dai Peihong, Li Jiyang, Pu Yan, Gu Aixing, Liu Xiaodong
Cotton Science. 2019, 31 (1): 31-39.   DOI: 10.11963/1002-7807.zxylxd.20181212
Abstract ( 3 PDF (3008KB) ( 0 )
[Objective] The function of U3 and U6 promoters that were cloned from sea-island cotton were identified in order to provide more available U3 and U6 promoters for the construction of cotton CRISPR/Cas9 multi-sites gene editing system. [Method] Two CRISPR/Cas9 gene editing vectors were constructed, in which sgRNA were driven by GbU6-7P and GbU3-2P, respectively, and GGB, a negative regulator in drought tolerance, was used as target gene. The function of the vector were identified in cotton leaf protoplast of Xinhai 16. The core fragment of CRISPR/Cas9 gene editing vector were enriched by Polymerase chain reaction method and were delivered into protoplast through PEG transient transformation. Then genomic DNA were extracted from protoplast. Gene mutations were analyzed using Enzyme digest/Polymerase chain reaction and sequenced. Finaly the mutation efficiencies of the CRISPR/Cas9 system were calculated and the frequency distribution of the mutation in target site were drawn in order to confirm the authenticity of the mutation. [Result] All type of mutation in target loci were base substitution. [Conclusion]Both CRISPR/Cas9 gene editing systems based on GbU3-2P and GbU6-7P promoters could successfully edit the sequence of cotton GGB gene and cause gene mutation.
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Physiological Mechanism of Different Varieties and Potassium Application Amounts on Cotton Resistance to Verticillium Wilt
Li Yan, Zhang Xihe, Yu Kai, Huo Yuyang, Wang Youhua, Chen Binglin
Cotton Science. 2019, 31 (1): 40-53.   DOI: 棉花|品种|施钾量|抗黄萎病性|生理机制
Abstract ( 1 PDF (6576KB) ( 0 )
[Objective] It has theoretical guiding significance for the comprehensive control technology of Verticillium wilt of cotton to explore the effect of potassium nutrition on mechanism of cotton Verticillium wilt resistance under the condition of pathogen stress of Verticillium dahliae. [Method] Three cotton cultivars Jimian 11 (JM-11 as susceptible cultivar), Zhongzhimian 2 (ZZM-2 as control cultivar), Nongda 601 (ND-601 as resistant cultivar) were adopted, and each cultivar had 0, 75, 150, 225, 300 kg· hm-2 potassium levels represented by K0, K75, K150, K225, K300. Two factors split plot experiment was designed under artificial nursery inoculated Verticillium dahlia, to observe disease index of Verticillium wilt, and researched resistance associated substances content and enzyme activity. [Result] (1) The lignin, total phenol contents and POD, PAL activity in leaves or roots of JM-11 were more greatly influenced by Verticillium wilt compared with ZZM-2, ND-601; the crucial disease resistance substances and enzymes were lignin, total phenol and PAL for JM-11, total phenol for ZZM-2 and ND-601. (2) The lignin, total phenol contents and POD, PAL activities of JM-11 were more sensitive to root potassium than ND-601, but weaker than that of ZZM-2. The sensitivity of POD, PAL activities in functional leaves of JM-11 to potassium were more sensitive than ND-601 and ZZM-2. (3) The best effect of potash fertilizer for relieving disease index for JM-11, ZZM-2, ND-601 were 32.5%, 26.4%, 16.5%, and corresponding potassium application amounts were 290.5 kg·hm-2, 253.5 kg·hm-2 and 229.5 kg·hm-2, respectively. [Conclusion] Application of potassium could increase the resistance to Verticillium wilt and reduce the occurrence of cotton Verticillium wilt.
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Morphogenesis of Pigment Glands and Metabolic Characteristics of Gossypol in Gossypium bickii
Hu Jiahui, Zhao Tianlun, Li Cheng, Yu Jingwen, Chen Jinhong, Zhu Shuijin
Cotton Science. 2019, 31 (1): 54-63.   DOI: 10.11963/1002-7807.hjhzsj.20181226
Abstract ( 3 PDF (1929KB) ( 0 )
[Objective]We studied the process of pigment gland formation and the dynamic change of gossypol synthesis, the dynamic relationship and molecular mechanism between the pigment glands and gossypol during the process of embryo formation and seed germination in G. bickii Prokh. [Method] We chose G. bickii as meterial and studied the tissue section observation, determination of gossypol contents and gene expression profile analysis of pigment gland formation and gossypol synthesis genes. [Result] The results showed that a large number of pigment protoglands formed on the cotyledons during embryo development process in G. bickii. The pigment protoglands formed to cavity structure at 12 hours after seed germination, and then converted into mature pigment glands. Analysis of gene expression profiles related to the pigment gland formation and gossypol synthesis during seed germination showing that GoPGF gene was highly expressed on the cotyledons of seed germination of 12 hours, which may be related to pigment protoglands forming to cavity structure of G.bickii. Farnesyl pyrophosphate synthase gene (FPS) was highly expressed on the cotyledons of seed germination of 24 hours showing it may be associated with the gossypol transported into the cotyledon pigment glands. CYP706B1 gene was highly expressed on the cotyledons of seed germination of 0 hours and HMG1 were highly expressed on the cotyledons of seed germination of 0 hours, 36 hours and 48 hours showing these genes may be associated with the formation of pigmented cavities and the storage and transport of gossypol; Cadinene synthase genes(CAD1-A) and HMG2 genes were not closely related to the pigment gland formation. [Conclusion] The 12 hours after seed germination is the key period of the morphogenesis of the pigment glands, and the morphogenesis of the pigment glands is synchrony with the time of the occurrence of gossypol in G.bickii. GoPGF, FPS, CYP706B1 and HMG1 are associated with the formation of pigmented cavities and the storage and transport of gossypol.
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Effect of Defoliants Application on Physiological Characters of Cotton Leaf without  Defoliants
Wang Xiaojing, Li Sijia, Liu Ruixian, Zhang Guowei, Yang Changqin, Ni Wanchao
Cotton Science. 2019, 31 (1): 64-71.   DOI: 10.11963/1002-7807.wxjlrx.20181228
Abstract ( 6 PDF (1768KB) ( 0 )
 [Objective]The aim of this study is to elucidate the physiological mechanism of cotton leaf without defoliant response to thidiazuron (TDZ). [Method] A pot experiment was conducted to study the effect of defoliants on the photosynthesis, chlorophyll fluorescence, antioxidant enzyme activity, and endogenous hormone contents of cotton leaf without defoliant at 1 d, 3 d, 6 d and 10 d after the application of TDZ. Cultivar CCRI-50 was used for this experiment. [Result] TDZ rapidly induced cotton leaf abscission, however, no abscission was detected to the cotton leaf without defoliants. The net photosynthetic rate (Pn) of cotton leaf without defoliants was reduced by 1 d, 3 d and 6 d after TDZ applied, but rise to the control treated with water level by 10 d. TDZ also caused a significant decline in the maximum photochemical efficiency of photosystem II (Fv/Fm), the quantum yield of electron transport (ΦPSII), the electron transport rate (Ret) and the photochemical quenching coefficient (qP), but an increase in the non-photochemical quenching coefficient (qNP). However, with the same changes as Pn, the chlorophyll fluorescence characters got to the control by 10 d. TDZ application significantly increased the superoxide dismutase activity,  did not affect the peroxidase activity, decease the malondialdehyde content. Abscisic acid contents in the cotton leaf without defoliant were lower than control by 3 d and 6 d after the applied of TDZ, but have the same level with control at 10 d, and the auxin had a little changes compared with control during the whole period of experimental time. [Conclusion] TDZ application significantly affect the photosynthesis, chlorophyll fluorescence, antioxidant enzyme activity, and endogenous hormone contents of cotton leaf without defoliant. When the leaf with defoliant drop off, the physiological characters of cotton leaf without of defoliants recovered to control level, and no abscission were detected.
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Analysis and Assessment on Heavy Metal Contamination in Cotton Seeds of Hunan Province
Xu Shuangjiao, Fang Dan, Tian Xinquan, Shi Meng, Ma Lei
Cotton Science. 2019, 31 (1): 72-78.   DOI: 10.11963/1002-7807.xsjml.20190122
Abstract ( 0 PDF (703KB) ( 0 )
[Objective]  In order to guarantee the quality and safety of cotton products and to explore the feasibility of alternative planting, the contamination condition of the heavy metals in cotton seeds in Hunan province was investigated and evaluated. [Method] Fifty-six cotton seed samples, collected in 6 counties of Hunan province, were collected to determine 6 indicators including cadmium, lead, copper, chromium, mercury and inorganic arsenic contents. This was followed by pollution index calculation, correlation analysis, and cluster analysis. [Results] The range of comprehensive pollution indexes of Junshan district, Anxiang county, Nanxian county, Wangcheng and Dingcheng districts varied between 0.366 5–0.577 0, which belonged to a secure level. However, the single pollution indexes of Hengyang county are relatively high. The comprehensive pollution index of Hengyang county was 0.801 6, meaning a mild pollution level. [Conclusion] The pollution status of cadmium, lead, copper, chromium, mercury and arsenic in Hunan cottonseed samples are generally acceptable. Cotton could be a substitute for rice on contaminated soil.
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Effects of Pruning Methods and Canopy Patterns on the Temporal-Spatial Distribution of Cotton Bolls and Yield
Zhu Xiaowei, Liu Liantao, Wan Hualong, Zhang Yongjiang, Sun Hongchun, Li Cundong
Cotton Science. 2019, 31 (1): 79-88.   DOI: 10.11963/1002-7807.zxwlcd.20190103
Abstract ( 4 PDF (1043KB) ( 0 )
[Objective] The purpose of this study was to shape the good canopy structure and achieve efficiency production of cotton. [Method] With Jimian 958 as test material, a split-plot field experiment was conducted over two years (2016—2017) to determine the effect of different pruning methods (removal and retention of vegetative branches) and canopy height treatments (high and low staggered canopy, low canopy and high canopy) on the temporal-spatial distribution of cotton bolls, yield and yield components in Baoding. [Result] Pruning method and canopy pattern had significant effects on the temporal-spatial distribution of cotton bolls. Regarding temporal distribution, retention of vegetative branch treatment (P2) significantly increased the number of total bolls (pre-summer, summer and autumn bolls) compared with removal of vegetative branch treatment (P1). Staggered canopy (C1) increased the number of pre-summer bolls but resulted in relatively fewer autumn bolls to that of the high canopy (C3) pattern. Spatial distribution of bolls varied by pruning method, with P2 treatment producing a greater number of fruit bolls concentrated in the middle part of the plant. P1 treatment produced a greater number of fruit bolls concentrated in the lower canopy. The combined effects of pruning methods and canopy pattern on seed cotton yield were significant, with the largest yield observed in P1C1 (vegetative branch removal×staggered canopy). The P1C1 group did not make significant difference in lint yield to the P2C1 (vegetative branch retention×staggered canopy) group. [Conclusion] The P2C1 canopy structure can produce a greater number of fruit bolls concentrated in the middle part of the cotton, enhance the number and proportion of summer bolls, increase the number of bolls per unit area, and produce higher yields while reducing labour usage, material resources and costs. This simplified cultivation measure offers an important opportunity to efficiently achieve high yields of cotton in the Yellow River Region.
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