15 September 2017, Volume 29 Issue 5

    

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  Development of EST-SNP Markers in Gossypium arboreum and Their Validation in G. hirsutum

  Xu Peng, Cai Jihong, Guo Qi, Zhang Xianggui, Xu Zhenzhen, Shen Xinlian

  Cotton Science. 2017, 29(5): 401-414. https://doi.org/10.11963/1002-7807.xpsxl.20170628

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  [Objective] With the development of the cotton genome sequence database and next-generation high-throughput sequencing techniques, the resources available for generating single nucleotide polymorphism (SNP) markers are gradually expanding. [Method] The Gossypium arboreum expressed sequence tags (ESTs) downloaded from the NCBI database were assembled into 7 187 contigs using the CAP3 program. Additionally, the QualitySNP program was used for SNP mining. [Result] A total of 2 690 SNPs were obtained from 807 contigs that consisted of more than four ESTs. We obtained 953 highly reliable candidate SNPs by screening for a minor loci frequency of more than 30%. Finally, a total of 149 candidate EST-SNPs that may be used in G. hirsutum were obtained through in silico screening. An allele-specific polymerase chain reaction and cleaved amplified polymorphic sequence molecular markers were used to validate the accuracy of the selected candidate SNPs in G. hirsutum. [Conclusion] The EST-SNP markers from G. arboreum may be used to analyze G. hirsutum. The obtained EST-SNP markers will be used to construct genetic maps, map important traits, and complete marker-assisted selection in G. hirsutum.
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  Genome-Wide Identification and Expression Analysis of the Gene Family Encoding Brassinazole Resistant Transcription Factors in Cotton

  Guo Xinlei, Lu Pu, Wang Yuanyuan, Cai Xiaoyan, Wang Xingxing, Zhou Zhongli, Wang Yuhong, Wang Chunying, Wang Kunbo, Liu Fang

  Cotton Science. 2017, 29(5): 415-427. https://doi.org/10.11963/1002-7807.gxllf.20170830

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  [Objective] Brassinazole resistant transcription factors (BZR) play important roles in Brassinosteroid (BR) signal transduction in plants. The BZR gene family is involved in plant developmental processes by regulating the expression of related genes. The aim of this study is to characterize the number, evolutionary characteristics, and biological function of BZR genes in cotton. [Method] We conducted genome-wide identification and expression analysis of the BZR gene family in Gossypium raimondii, G. arboreum, and G. hirsutum. [Result] In this study, seven, seven, and 14 BZR genes were identified from G. raimondii, G. arboreum, and G. hirsutum, respectively. These BZR genes were grouped into two groups, a and b, based on their amino acid sequences and phylogenetic analysis. Gene structure analysis showed that most BZR genes contain two exons. Additionally, BZR genes in "a" group had a longer intron than those in "b" group; within the same group, BZR genes had a similar gene structure.  Analysis of gene duplication revealed that segmental duplications played crucial roles in BZR gene family expansion, with cotton BZR genes experiencing purifying selection during evolution. Transcriptome data analysis showed that 14 GhBZRs had differing expression patterns in different organs; most GhBZRs were highly expressed in stem, leaf, petal, and pistil tissue, and were induced or repressed by abiotic stress. [Conclusion] These results suggest that GhBZR genes may be involved in stress resistance and fiber development in cotton. Furthermore, they provide information for further analysis aimed at uncovering the biological functions of BZR genes in cotton.
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  Genome-Wide Identification of PPO Gene Family Members and Their Response to Verticillium dahliae in Upland Cotton

  Zhang Hui, Tian Xinquan, Gao Wei, Cai Yingfan, Long Lu

  Cotton Science. 2017, 29(5): 428-436. https://doi.org/10.11963/1002-7807.zhll.20170727

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  [Objective] Polyphenol oxidases (PPOs) are ubiquitous among plants and have been shown to be involved in defense responses to biotic stresses, such as pathogens and insects. The aim of this study was to verify PPO genes and their expression patterns in upland cotton and determine the relationship between PPO and cotton resistance to Verticillium dahliae. [Method] We analyzed changes in PPO activity in allotetraploid cotton TM-1 roots after V991 (V. dahliae) inoculation, and isolated related genes from the TM-1 genome database. Bioinformatics and expression analysis were used to characterize the isolated genes. [Result] 13 putative PPO genes were isolated, all of which were intron-free and encoded proteins containing two copper ion binding sites and conserved PPO domains (tyrosinase, DWL and KFDV domain). Phylogenetic analysis revealed that there was greater genetic diversity in PPO genes within species than between species. GhPPO genes were duplicated, most of which were lowly expressed in all tissues examined, only a few showed tissue-specific expression in different organs. Quantitative real-time polymerase chain reaction showed that GhPPO6D was highly expressed in cotton roots and induced by V. dahliae inoculation. [Conclusion] These results indicated that GhPPO6D may participate in the interaction between cotton and V. dahliae and could be responsible for the up-regulation of PPO activity after V991 infection.
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  Analysis of the Correlation between Pigment Glands and (-)/(+)-Gossypol Contents in Upland Cotton

  Cotton Science. 2017, 29(5): 437-446. https://doi.org/10.11963/1002-7807.lhzsj.20170818

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   [Objective] The aim of this study was to examine the correlation between pigment glands and (-)/(+)-gossypol contents in different organs of different upland cotton genotypes. [Method] Using upland cotton materials CCRI 12, CCRI 12 (glandless), Coker 312, Coker 312 (glandless), IF11 and Jimian 14, we accurately measured the density and size of the pigment glands, tested (-)/(+)-gossypol contents by pre-column derivatization-high performance liquid chromatography (HPLC), and then analyzed the correlation between pigment glands and (-)/(+)-gossypol contents. [Result] The whole plants of low gossypol cotton lines were without pigment glands, but free gossypol could still be detected in different organs at mass fractions less than 0.02%. The organs of glanded cotton all had typical pigment glands, the gland density was highest in the seed and lowest in the bract. The gland size (diameter) was largest in the boll shell and smallest in the petal and seed. Across different organs, the total gossypol content was highest in the seed and lowest in the bract. The contents of (+)-gossypol in the seed and petal were higher than (-)- gossypol. Conversely, the (-)-gossypol contents of the calyx, boll shell, upper leaf, middle leaf, and lower leaf were higher than (+)-gossypol. There was a significant negative correlation between the density and size of glands in glanded cotton. There were significant positive correlations between total gossypol content and (-)/(+)-gossypol contents, and between (+)-gossypol content and (-)-gossypol content. Gland density was positively correlated with total gossypol content and (-)/(+)-gossypol contents. Gland size was negatively correlated with (+)-gossypol content, and showed no significant correlation with (-)-gossypol content or total gossypol content. [Conclusion] Overall, the strong positive correlation between total gossypol content in the seed and petal  suggests that we can use the petal gossypol content to infer the seed gossypol content.
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  Study on Light Spatial Distribution at Different Scales in the Cotton Canopy

  Liu Hui, Ji Lingyan, Zhao Tianlun, Chen Jinhong, Zhu ShuijinLiu Shuai, Li Yabing, Han Yingchun, Wang Guoping, Yang Beifang, Feng Lu, Lei Yaping, Fan Zhengyi, Du Wenli

  Cotton Science. 2017, 29(5): 447-455. https://doi.org/10.11963/1002-7807.lslyb.20170903

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   [Objective] Cotton canopy photosynthetically active radiation (PAR) spatial distribution is a significant factor in the light energy coefficient of utilization. The aim of this study is to analyze spatial distribution characteristics of each component of PAR. [Method] PAR spatial distribution in the cotton canopy was presented intuitively using a geo-statistical method. [Result] (1) At different points, PAR spatial distribution was a "V" shape at the flowering stage. However, a short and shallow "V" shape was observed with high plant population density (PPD). (2) At different profiles, the PAR interceptions changed slightly in the vertical direction of the middle-lower canopy; and the extent of variation was small in the horizontal direction, 60 cm from the ground. (3) At different layers, PAR interceptions increased with PPD. In the horizontal direction, PAR interceptions between two adjacent cotton rows were lower than that between nearby cotton rows; in the vertical direction, PAR interceptions in the lower layers were above 0.83, and 0.27 less than in the upper layers. [Conclusion] PAR interception can be increased by increasing PPD suitably. Using geo-statistives to quantify PAR spatial distribution within the cotton canopy precisely and accurately is of great significance for reasonable plant density arrangement, plant architecture molding, and yield improvement variety selection.
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  Relation of Plant Type Traits with Fiber Yield and Quality in the Crossing Population of Mechanical-Harvested Cotton

  Qi Haikun, Yan Gentu, Wang Ning, Qiao Wenqing, Shi Jianbin, Xu Qinghua, Zhou Hong, Huang Qun

  Cotton Science. 2017, 29(5): 456-465. https://doi.org/10.11963/1002-7807.qhkhq.20170822

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  [Objective] This study aimed to determine the most important factors for mechanized production, make genetic improvements, and breed high yield and quality varieties suitable for mechanized production, so as to effectively improve the efficiency of cotton breeding. [Method] We used SAS V8 statistical software to analyze the variation, correlation, and variance of agronomic characteristics in the F_2:3 crossing population of upland cotton (Gossypium hirsutum L.) varieties Z571 and CCRI 49. [Result] There was a significant separation between the agronomic traits of F_2:3 population lines, and many transgressive individuals appeared in their descendants. When selecting in the field, we found it important to focus on the complex relationship among agronomic traits. Seed cotton yield per unit area was significantly positively correlated with plant height and number of fruit branches. The number of fruit branches was significantly negatively correlated with average fiber length and breaking tenacity, and significantly positively correlated with the micronaire value. Conversely, the number of vegetative branches was significantly positively correlated with the average fiber length and breaking tenacity, and significantly negatively correlated with the micronaire value. The boll number, one component factor of yield, was significantly positively correlated with the average fiber length and breaking tenacity, and was significantly negatively correlated with the micronaire value. Conversely, the boll weight, another component factor of yield, showed the opposite pattern. [Conclusion] Plant traits had significant or extreme sig nificant genetic correlations with yield and quality. High yield and high quality cotton had more vegetative branches. New varieties of cotton with high yield and quality should, therefore, be selected based on a great number of smaller bolls.
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  Study of the Tolerant Physiology of Cotton Seedlings Nursed with Simplified Techniques after Removal from the Seedling Bed

  Wang Guoping, Han Yingchun, Li Yabing, Fan Zhengyi, Tian Huaiqin, Feng Lu, Yang Beifang, Zhi Xiaoyu, Mao Shuchun

  Cotton Science. 2017, 29(5): 466-475. https://doi.org/10.11963/1002-7807.wgplyb.20170710

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  [Objective] The primary target of this experiment was to illustrate the tolerant physiological characteristics of cotton seedlings nursed with simplified techniques after removal from the seedling bed to show theoretical support for these techniques in terms of safe storage and protection during transportation. [Method] Plants of the cotton variety CCRI 50 were grown in nursing substrate with (W) and without (0) an added water-retaining agent. Physiological indexes of cotton leaves were tested, and the cellular structure of the leaves was observed with the scanning electron microscope. [Result] The results showed that the leaf relative water content (LRWR) decreased to 95% in 3 d under the two treatments but could return to 98% when the seedlings were rewatered and that the survival rate following transplanting was above 99%. Scaning electron microscopy indicated that the cellular structure of the leaves was quite integrated, with a continuous and smooth surface. Additionally, the cell junction was natural and stomata guard cells were relatively well-stacked. Five days after removal from the seedling bed was the demarcation line for the activity of cotton seedlings, with LRWR decreasing by 10 percentage points and only reaching 93% when rewatered. From 5 d to 7 d, when the seedlings were removed  from bed, the leaf gradually suffered increasing drought stress and water in the leaf was lost quickly. This lead to a decrease in the LRWR at a rate of up to 15 percentage points per day. By 11 d, the LRWR had dropped to 50%, the cells of the outer leaves had shrunk severely, the cytoderm was folded and overlapped, and the guard cells had collapsed, indicating that water loss dramatically weakened the recovery capability of these plants. The proline and malondialdehyde contents of the seedlings increased during their removal from the beds, and the activity of peroxidase increased. The activity of catalase and superoxide dismutase increased during the first 5 d before decreasing again. [Conclusion] The inclusion of a water-retaining agent in the nursing substrate will help to maintain water level in the plants that can help relieve drought stress associated with the bed-away-seedlings.
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  Effect of Indigenous Arbuscular Mycorrhizae  Fungi on the Root Morphological Characteristics of Cotton at the Seedling Stage

  Cotton Science. 2017, 29(5): 476-486. https://doi.org/10.11963/1002-7807.gxlfg.20170706

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  [Objective] In this study, we explored the effects of the soil indigenous arbuscular mycorrhizae (AM) fungal community on cotton root morphology and architecture at the seedling stage. [Method] A pot test at a field site was conducted using the in-growth system and included static (AM fungal colonization allowed freely) and vibrating (patting the top core edge twice every day to break any extradical hyphae attempting to access core) treatments in slightly (Electrical conductivity σ 1.0 mS·cm^－1), highly (σ 2.1 mS·cm^－1), and extremely (σ 4.0 mS·cm^－1) saline soils. [Result] The results indicated that colonization of indigenous AM fungi was accompanied by an increase in cotton plant height, leaf number, bud number, and leaf area index, and improved salt resistance. Indigenous AM fungi symbiosis significantly increased total root length, root volume, root surface area, and the length of fine roots with a diameter (d) ≤0.5 mm compared with the control (vibrating treatment). The percentage of the root length with a d ≤0.5 mm  under the static treatment was significantly higher than under the vibrating treatment in slightly saline soil but not with highly and extremely saline soils. Correlation analysis results showed significant positive correlations between both total root length, root lengths with a d ≤0.5 mm and root colonization of AM fungi, leaf P/K content. A positive correlation was also observed between total biomass and leaf P/K content, while a significant negative correlation was observeds between both total root length, root lengths with a d ≤0.5 mm and leaf Na concentration. An indigenous AM fungal community was advantageous to cotton total root length and fine root growth in saline soils, improved P/K status, reduced Na/Cl salt absorption, and increased salt resistance. [Conclusion] These results suggest that increasing root length and the percentage of fine roots through the introduction of symbiotic indigenous AM fungi may improve P/K status and reduce Na/Cl acquisition in the cotton root system.
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  Development of a Multiplex Polymerase Chain Reaction Method for Simultaneous Detection of Six Events in Genetically Modified Cotton

  Li Yi, Yin Quan, Liu Yong

  Cotton Science. 2017, 29(5): 487-494. https://doi.org/10.11963/1002-7807.lyly.20170727

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  [Objective] The aim of this study was to establish a multiplex polymerase chain reaction (PCR) detection method for transgenic cotton. [Method] Six events in genetically modified cotton were selected for multiplex PCR. Specific primers were designed based on national standards or related literature. The ratio of primer concentration, annealing temperature, and sensitivity of the multiplex PCR reaction system were optimized. [Result] The optimal concentrations for primers MON1445, GHB614, MON15985, MON88913, LLCOTTON25 and MON531 were 0.25, 0.30, 0.25, 0.16, 0.30, and 0.20 μmol·L－1, respectively; the optimal annealing temperature of the multiplex PCR was 56 ℃; the detection sensitivity of the method was 66 copies of the cotton genome. The verification results of the system showed that the amplified bands of all the known samples were identical to those of the transgenic events. [Conclusion] The system established in this study can be used for the rapid detection of six events occurring in genetically modified cotton.