15 September 2014, Volume 26 Issue 5

    

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  Identification and Analysis of miRNAs in Cotton Leaves under Salt Stress

  Xu-Fei-Fei, PENG Zhen, GONG Wen-Fang, HE Shou-Pu, PANG Bao-Yin, DU Xiong-Ming

  Cotton Science. 2014, 26(5): 377-386.

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  microRNAs(miRNAs) are 21-24 nt non-protein-coding small RNAs (sRNAs) that regulate plant gene expression by directing target mRNA cleavage or translational inhibition. In this study, four miRNA libraries from Zaoshuchangrong-7 and Nandanbadidahua exposed to 200 mmol·L^-1 NaCl for 4 h were constructed and sequenced. Compared with the D5 genome (Gossypium raimondii L.) and the salt stress transcriptome sequences of upland cotton(Gossypium hirsutum L.) from our library, 360 miRNAs including 308 conserved miRNAs and 52 novel miRNAs were identified. A total of 94 differentially expressed miRNAs were detected in the two lines and they could be divided into two expression types. Of these, 20 candidate miRNAs were selected. Kyoto Encyclopedia of Genes and Genomes(KEGG) analysis indicated that the target genes of differentially expressed conserved miRNAs were significantly enriched in antigen processing and presentation pathway in salt-tolerant line, but in isoquinoline alkaloid biosynthesis in salt-sensitive line.
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  Association Analysis of Agronomic Traits in Upland Cotton Using SSR Markers

  LIANG Bing, FAN Shu-Li, SONG Mei-Zhen, PANG Chao-You, WEI Heng-Ling, YU Shu-Xun

  Cotton Science. 2014, 26(5): 387-395.

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  In this study, 68 samples of upland cotton sowed on two different dates were scanned using 160 pairs of SSR primers to perform association analysis of 17 agronomic traits, including those related to early maturity, yield and fiber quality, which are mostly quantitative traits. STRUCTURE 2.3.4 software was used to estimate the population structure of the 68 varieties, which could be divided into three subgroups. Cluster analysis based on Nei's genetic distance was almost identical to that the 68 samples could be divided into three subgroups. Using general linear model (GLM) and mixed linear model (MLM) to perform association analysis by TASSEL 4.0 software, six SSR loci were detected at the two sowing dates, of which CER0098-400 is extremely significantly associated with growth stage, DPL0375-250 and HAU2414-147 are extremely significantly associated with location of the first branch, three SSR loci, DPL0504-135, HAU0883-120 and NAU1298-524, were extremely significantly associated with lint percentage. This research provides the bases for marker-assisted selection (MAS) in upland cotton breeding.
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  Molecular Marker-assisted Selection and Pyramiding Effect of Major QTLs for Cotton Fiber Strength

  WANG Tian-Kang, SHI Yu-Zhen, SHANG Hai-Hong, LIU Ai-Ying, LI Jun-Wen, GONG Ju-Wu, WANG Tao, GONG Wan-Kui, CHEN Ting-Ting, LI Bo-Tao, YUAN You-Lu

  Cotton Science. 2014, 26(5): 396-403.

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  Two elite fiber quality materials, 0-153 and Xinluzao 24, and two commercial cotton cultivars (lines), Lumianyan 28 and Jimian 516, were used as parents to develop two double-cross combinations (Jimian 516 × 0-153)×(Jimian 516 × Xinluzao 24) (Pop1), (Lumianyan 28 × 0-153) × (Lumianyan 28 × Xinluzao 24) (Pop2). Four simple sequence repeat(SSR) markers, linked to three quantitative trait loci(QTL) of fiber strength and derived from 0-153 or Xinluzao 24, were used to study the efficiency of molecular marker-assisted selection and the pyramiding effect of fiber strength. The assisted selection of four markers was shown to have significant genetic effect. In the two groups of homozygous dominant/dominant genotype, the fiber strength of individuals varied from 31.21 to 32.62 cN·tex-1, while that of the heterozygous genotype varied from 30.77 to 32.50 cN·tex^-1. The selection effects of single marker were 0.80～1.51 cN·tex^-1; In the QTL-1×QTL-3 and QTL-2×QTL-3 combinations, the fiber strength of individuals involving the pyramiding of two QTLs varied from 33.40 to 34.08 cN·tex^-1. Selection effects were 2.73～3.56 cN·tex^-1 compared to individuals without the two QTLs, and 1.12～3.02 cN·tex^-1 compared to individuals with one of the two QTLs. Additionly, the pyramiding effect showed QTL-1 and QTL-2 might be the same QTL, and that QTL-2 and QTL-3 had obvious epistasis effect. The findings demonstrate the feasibility of using molecular marker to assist the pyramiding selection of fiber strength.
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  The Effect of DET2 Single Nucleotide Variation on Protein Structure and Fertility in Gossypium

  GUO Bao-Sheng, SHI Gong-Yao, WANG Kai-Hui, LIU Su-En, ZHAO Cun-Peng, GENG Jun-Yi, HUA Jin-Ping

  Cotton Science. 2014, 26(5): 404-410.

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  This research aimed to investigate the effects of single nucleotide variation(SNVs) of the steroid 5 alpha-reductase gene(DET2) in cotton plants(Gossypium) on the ligand-binding domain of the coded protein, and to develop single nucleotide polymorphism(SNP) markers. The DET2 coding regions in Gossypium germplasm were amplified and sequenced. Multiple sequence alignment, and secondary and tertiary structure prediction of encoded proteins were carried out using sequence analysis software such as Geneious Pro^TM and online bioinformatics analysis tools. Models of protein tertiary structures, including ligand-binding domain, were established to explore the correlation between SNVs and cotton fiber quality, and lant fertility. Among the 13 stocks of upland cotton tested, together with island cotton and G. raimondii, DET2 demonstrated a high sequence conservation of 98.7%. A total of 25 SNVs were detected, of which eight resulted in changes to the amino acid sequence. Predicted secondary structure and tertiary structure were similar, although the ligand-binding sites could be classified into eight different types following protein sequence changes.
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  Genome-wide Identification of Argonaute Gene Family in Gossypium raimondii Using Bioinformatic Analysis

  LIU Guo-Yuan, WU Man, YU Ji-Wen, PEI Wen-Feng, YU Shu-Xun

  Cotton Science. 2014, 26(5): 411-419.

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  Argonaute(AGO) proteins play a very important role in small RNA-regulated pathways related to growth and development, defense against virus, histogenesis, cell proliferation, and apoptosis. We identified 15 AGO family members in the Gossypium raimondii(G. raimondii) genome, and used analyses of phylogeneties, gene structure, domains, and motifs to show that this family is highly conserved. The AGO gene family members varied from 3335 to 9086 bp in length, and 11 genes were located in seven chromosomes. The family can be divided into three groups. Each gene has a specific expression pattern in different tissues, with one gene expressed in flower, three genes in ovule. This identification and bioinformatics analysis lays the foundations for the future study of AGO gene family in G. raimondii.
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  Genetic Diversity and Association Analysis of Upland Cotton Based on SSR Markers

  GUO Zhi-Jun, ZHAO Yun-Lei, CHEN Wei, LI Yun-Hai, WANG Hong-Mei, GONG Hai-Yan, SANG Xiao-Hui

  Cotton Science. 2014, 26(5): 420-430.

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  To provide a genetic basis for molecular-assisted breeding and to improve the efficiency of breeding, simple sequence repeat(SSR) markers associated with agronomic traits were screened and the genetic diversity of cultivated upland cotton was analyzed. In 172 upland cotton cultivars, 74 SSR markers were assayed for polymorphism, and cluster analysis was performed on the genetic similarity matrix using NTSYS-pc 2.20 software to ascertain the diversity. Based on the analysis of population structure, an association analysis between SSR markers and three agronomic traits were performed using the Tassel 2.1 GLM(general linear model) program. The 74 SSR markers included 246 alleles belong to 148 loci，with an average of 3.32 alleles per SSR marker(range, 2-7). The average polymorphic information content(PIC) for per marker was 0.2730(range, 0.0281-0.3733), and the mean genetic similarity was 0.5369(range, 0.2816-1). This revealed low levels of upland cotton genetic diversity in China, although some oversea varieties and some varieties of the northwest inland cotton showed abundant genetic variation. Cluster analysis showed that all the accessions could be divided into 12 subgroups, which was in agreement with the pedigree but not with the origin. Structure analysis indicated that the 172 lines were assigned to three substructures. Of the SSR loci, 30 were significantly associated with boll weight, lint percentage, and Verticilium wilt resistance under GLM program, and the ratio of explanation on the phenotype variation of related markers ranged from 2.24% to 5.27%.
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  Effects of Glyphosate on Soil Enzyme Activities in Cotton Fields

  JIANG Wei-Li, MA Xiao-Yan, PENG Jun, MA Ya-Jie, MA Yan

  Cotton Science. 2014, 26(5): 431-437.

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  The effects of glyphosate on the activities of catalase, urease, and phosphatase were determined indoor in soil samples collected from cotton fields in Anyang, Henan Province and Anqing, Anhui Province, China, in 2013. Catalase activity was found to be concentration-dependently inhibited by 50-5000 mg·kg^-1 glyphosate. In both soil samples, urease activity was inhibited then stimulated by lower concentrations of glyphosate (50, 250 mg·kg^-1, 500 mg·kg^-1). After being treated by the higher concentration of glyphosate (5000 mg·kg^-1), Anyang cotton field soil urease was continuously inhibited, while Anqing cotton field soil urease was first inhibited then stimulated. As for phosphatase activities, it was inhibited with 500 mg·kg^-1 glyphosate in Anyang sample, but stimulated in Anqing sample, while it was stimulated with 5000 mg·kg^-1 glyphosate in both samples.
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  Cloning and Functional Analysis of the Pectin Methylesterase Gene GhPME6 in Upland Cotton

  WANG Lin, SHANG Hai-Hong, LI Jun-Wen, WANG Shao-Gan, LIU Ai-Ying, SHI Yu-Zhen, GONG Ju-Wu, GONG Wan-Kui, CHEN Ting-Ting, YUAN You-Lu

  Cotton Science. 2014, 26(5): 438-444.

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  With the suppressive subtractive hybridization result of a cotton fiber cDNA library, an expressed sequence tag (EST) sequence, which is specifically expressed during the cotton fiber secondary wall thickening stage and encodes pectin methylesterase, was identified from the cotton EST database. The complete cDNA sequence was obtained by rapid amplification of cDNA ends(RACE) technology, and named GhPME6. Gene structural analysis showed that GhPME6 contained a complete open reading frame of 1,560 bp, including two exons and one intron, and the gene encodes 519 amino acids. Its amino acid sequence consisted of two conservative structural domains: a pectin methylesterase inhibitor region and a pectinesterase region. The PME6 sequence was found to have been highly conserved during the evolution of different Gossypium genomes. Quantitative real-time PCR analysis revealed that GhPME6 was highly expressed during the secondary cell wall thickening stage of fiber development, so is predicted to influence the cotton fiber strength. We built an Escherichia coli expression system of GhPME6, which resulted in production of a target protein of expected size, thus laying the foundations for the further study of GhPME6 function in cotton fiber strength.
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  Isolation, Identification, and Antifungal Activity of the Antagonistic Bacterial Strain TUBP1 against Verticillium dahliae Kleb.

  ZENG Hong, YANG Sheng-Qiang

  Cotton Science. 2014, 26(5): 445-451.

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  The bacterial strain TUBP1, capable of strongly inhibiting Verticillium dahliae ATCC 36211, was obtained by preliminary and secondary screening using the agar plate diffusion method. It was characterized by morphological and cultural feature observation, physiological and biochemical experiments, and 16S rDNA and gyrB sequence analysis, and was identified as Bacillus axarquiensis. The in vitro antifungal activity of strain TUBP1 fermentation broth against V. dahliae was analyzed by growth rate and the Oxford cup method, revealing an inhibition ratio of (78.62 ± 3.48)% and inhabiting V. dahliae growth diameter of (21 ± 3.8) mm. In a greenhouse study, the biocontrol efficacy of strain TUBP1 against cotton Verticillium wilt was 43.67%, and this was also similar in a field experiment, at 43.07%. This is the first known report of the role of B. axarquiensis in preventing and curing cotton Verticillium wilt.
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  Effects of Drought on the Sucrose Metabolism of Subtending Leaves of Cotton Bolls at Different Fruiting Branches and Boll Weight during Flowering and Bolling Stages

  YANG Chang-Qin, LIU Rui-Xian, YANG Fu-Qiang, ZHANG Guo-Wei, XU Li-Hua, ZHOU Zhi-Guo

  Cotton Science. 2014, 26(5): 452-458.

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  This study aimed to illustrate the mechanism of sucrose metabolism in subtending leaves of cotton bolls and its relationship with boll weight under drought stress during flowering and bolling stages. The 2-year-long experiments were conducted using pools of upland cotton(Gossypium hirsutum L.) plants, NuCOTN 33B, subjected to drought stress (relative water content reduced naturally until the boll opening stage) on the day of anthesis of the middle fruiting branches (MFB), using well-watered conditions as the control. The net photosynthetic rate declined under drought stress in the boll's subtending leaf, respectively, and the decline range was MFB>upper fruiting branch (UFB)>lower fruiting branch (LFB). Drought stress increased the soluble sugar contents of subtending leaves at LFB and UFB but decreased them at MFB. Sucrose content was increased and starch content decreased concomitantly, indicating that drought facilitated the partitioning of photosynthate into sucrose. Drought stress also depressed the activities of sucrose phosphate synthase and sucrose synthase, while acid invertase activity was initially decreased and then increased. The changes of MFB and UFB exceeded those of LFB, indicating that sucrose synthesis was inhibited, the degradation was first inhibited then promoted, causing a reduction in the amount of sucrose available for export. As a result, the boll weight declined during drought, and the decline range was MFB>UFB>LFB. In conclusion, the decreased sucrose synthesis and the impaired sucrose efflux of subtending leaves of bolls were the main causes for decreased boll weight under drought conditions during flowering and bolling stages of cotton.
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  Effects of Plant Pruning and Plant Population Density on Yield and Fiber Quality of Cotton in a Garlic-cotton Intercropping System

  XIE Zhi-Hua, LI Wei-Jiang, SU Min, MIAO Lei, CHENG Li-Juan, DONG He-Zhong

  Cotton Science. 2014, 26(5): 459-465.

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  Field experiments were conducted in three sites to study the effects of plant pruning mode (with or without vegetative branches) and plant density (2.7×10⁴, 3.3×10⁴, 3.9×10⁴, 4.5×10⁴ plants·hm^-2) on yield and fiber quality of cotton in a garlic-cotton intercropping system in southwestern Shandong province. The results showed that pruning mode, planting density or their interaction had no significant effect on cotton fiber quality, but there existed a significant interaction effect of pruning mode and planting density on cotton yield. Three combinations, 3.3×10⁴ plants·hm-2 with vegetative branches(VB), 2.7×10⁴ plants·hm^-2 with VB, and 3.3×10⁴ plants·hm^-2 without VB, improved lint yield by 20.4%, 9.2% and 10%, respectively, compared with the traditional cultivation method(2.7×10⁴ plants·hm^-2 without VB). They were also significantly higher than other treatment combinations in lint yield. Increased plant density, maitaining VB or their combination significantly increased biomass, but decreased the harvest index. Compared with traditional cultivation method, 3.3×10⁴ plants·hm^-2 with VB,  2.7×10⁴ plants·hm^-2 with VB and 3.3×10⁴ plants·hm^-2 without VB increased biomass by 13.7%, 27.8% and 11.6%, decreased harvest index by 5.3%, 11.5% and 4.5%, respectively. Significant improvement on lint yield by the three combinations was attributed to the increased biomass and total number of bolls per unit area on the basis of maintaining relatively higher harvest index and boll weight. It is concluded that appropriate increase in plant density combined with maintaining VB is an important practice to improve cotton yield and benefit in the garlic-cotton intercropping system.
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  Effects of Cadmium Stress on Growth and Cadmium Accumulation in Cotton (Gossypium hirsutum L.) Seedlings

  LIU Lian-Tao, CHEN Jing, SUN Hong-Chun, ZHANG Yong-Jiang, LI Cun-Dong

  Cotton Science. 2014, 26(5): 466-470.

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  Using transgenic cotton varieties Nongda 9 and Guoxin 3 as study materials, four different cadmium(Cd) concentrations were designed to investigate the effects of Cd on growth effect and Cd accumulation in cotton seedlings. With increasing Cd concentrations, the dry matter accumulation, total root length, total surface area, and total volume of cotton seedlings decreased significantly, while the root/shoot ratio and root diameter increased. Cd accumulation showed that: (blade + petiole) > root > stem. Moreover, translocation factors in 50 μmol·L^-1 Cd treatment peaked at 2.2 times the control level, and declined at 100 μmol·L^-1 Cd. These experiments showed that Cd stress increased Cd accumulation, inhibited root growth, and hindered above-ground plant growth, resulting in a toxic effect on cotton seedlings.