15 April 2008, Volume 20 Issue 1.1

    

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  Combining Ability and Heterotic Performance of Bt  Transgenic Lines in Cotton (Gossypium hirsutum L. )

  GUO Xiao-ping, ZHAO Yuan-ming, WU Jia-he, ZHANG Xian-long, NIE Yi-chun

  Cotton Science. 2008, 20(1.1): 1-5.

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  Five transgenic lines of cotton (Gossypium hirsutum L.) carrying the cry1Ac (Bt，Bacillus thuringiensis) gene were developed from a cultivar JH321 by independent transformation events. Fifteen F₁ hybrids were obtained from diallel crosses among the five transgenic lines and JH321. When compared with the transformation receptor cultivar JH321, significant variations in agronomic traits including improved yield or yield components were found among these transgenic lines. General combining abilities (GCA) among these transgenic lines varied greatly, and two of these lines with significant positive GCA effects were identified. Heterosis in F₁ hybrids also exhibited significant variations. It was found that the average heterosis of crosses with two transgenic lines was lower than that with only one transgenic line as parent in yield performance. The economic trait improvement for transgenic lines and the heterosis produced between transgenic line and receptor cultivar might be raised from different integration sites of Bt gene.
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  Heterosis Performance and Correlation Analysis on Economic Traits of Upland Cotton Hybrids in Different Ecological Environments

  XING Chao-zhu, YU Shu-xun, GUO Li-ping, MIAO Cheng-duo, FENG Wen-juan, WANG Hai-lin, ZHAO Yun-lei

  Cotton Science. 2008, 20(1.1): 6-10.

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  24 combinations by NCⅡmating design and their ten parents were used to evaluate their surpassing parental heterosis of yield and fiber quality traits in three different ecological environments. The results showed that surpassing parental heterosis of yield traits were widely existed in three ecological environments. Especially the lint and seed cotton yield performed the most prominent heterosis, followed by boll number. In the Yangtze River Valley experimental stations, the hybrids and their parents had higher yield, but heterosis was not significant, and in Sanya, Hainan and Anyang, Henan experimental station with relatively lower production levels, though the hybrids had relatively low yield, their parents were significantly lower than the hybrids in yield, so heterosis was significant. This indicated that hybrids exhibited relatively stable yield performances in different environments, but the parents were greatly affected by environments. Fiber quality traits varied with ecological environments, but the surpassing parental heterosis was not significant. Correlation analysis showed that the boll numbers had the closest relation with lint yield. Moreover, there were significantly positive correlations among boll number, boll weight and lint percentage, which indicated the three yield traits can be simultaneously improved through parents selection. Correlations were inconsistent among fiber quality traits in different environments, while the micronaire had significantly positive correlation with other fiber quality traits, hence it was very important to select materials with low micronaire value as parents in high yield and fine fiber quality hybrid breeding.
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  Regional Distribution of Cotton Fiber Quality in China

  TANG Shu-rong, YANG Wei-hua

  Cotton Science. 2008, 20(1.1): 11-16.

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  The fibre quality status is very important for super quality cotton production and  diverse requirements of textile industry in China.In this study,the quality of cotton fiber samples which are collected from 13 major cotton production provinces between 2001 and 2005 were analyzed. Eight quality traits including fiber length, uniformity index, specific strength, elongation, micronaire, reflectance degree, yellow degree, and spinning consistency index were tested. The data indicated that the cotton fiber length ranged around 28 to 29 mm, the fiber uniformity index ranged from 82% to 84%, fiber strength ranged from 27 to 29 cN·tex^-1, micronaire ranged from 4.0 to 5.0. These fibres could satisfy the requirements of middle and low grade spinning. The fibers from the Yangtze River Valley (Jiangxi, Hubei, and Hunan Province) were superer in length, strength, and uniformity index. However, the micronaire was a little higher, the color, character was not good enough, and,the grade was lower. The fiber qualities of the Northwest Inland Region (Xinjiang) were the best in terms of micronaire, color and grade. As for the Yellow River Valley Region, the index of its fibre qualities was between the above two regions.
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  Cloning and Characterization of a Novel Cotton Fiber  Expressed Protein (GhCFE) cDNA

  GUO Ying, GUO Wang-zhen, ZHANG Tian-zhen

  Cotton Science. 2008, 20(1.1): 17-23.

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  Through randomly sequencing the cotton fiber cDNA library from 7235 germplasm line with elite fiber quality in Gossypium hirsutum L., a cDNA clone encoding cotton fiber expressed protein was isolated, designated as GhCFE (GenBank accession number: DQ073045). The full length of this cDNA clone was 1274 bp, with the open reading frame encoded 332 amino acids. The putative protein of this gene had an isoelectric point of 6.14 and a calculated molecular weight of 37.7 kD. It had a signal peptide with 27 amino acid residues in N-terminal. RT-PCR analysis indicated this gene did not express in root or stem, only had a weak expression in leaf, while it could be detected in ovules and fiber cells of different developmental stages, especially in elongating fibers. Phylogenetic tree showed GhCFE had high homologies with other CFEs reported previously. We cloned GhCFE into the fission yeast (S. pombe) vector pREP5N and detected that this gene had no significant effect on elongating cells or thickening cell wall in the transformed yeast. Further, sense expression vector containing 35S promoter and both sense and antisense expression vectors containing E6 promoter using pBI121 plasmid were constructed, and the work of transferring these recombined vectors into Gossypium hirsutum L. by Agrobacterium tumefaciens is ongoing.
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  BAC Library Construction and Characterization of  Suyuan 7235, a Cotton Germplasm with High Fiber Strength

  WANG Xing-fen, MA Jun, MA Zhi-ying, ZHANG Gui-yin, ZHENG Yong-min

  Cotton Science. 2008, 20(1.1): 24-28.

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  The bacterial artificial chromosome (BAC) library of Suyuan 7235, a cotton germplasm with high fiber strength has been constructed following the partial digestion of genomic DNA with HindIII. The pIndigoBAC-5 (HindIII-cloning ready) cloning vector was used for the library. The BAC library included 30336 clones. Analysis of 96 recombinants showed that the insert DNA size ranged from 50 kb to 140 kb, averaged 120 kb with less than 2.1% of empty clones. As much as \{89.6%\} clones inserts are over 100 kb. As a genomic resource, the library has potential use in further research of fiber strength gene analysis and gene cloning, as well as SSR primer development.
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  Cloning and Characterization of an Organ Specific and Pathogen  Responsive Promoter from Cotton (Gossypium hirsutum L.)

  ZHANG Bao-long, YANG Yu-wen, NI Wan-chao, SHEN Xin-lian, SHE Jian-ming, He Xiao-lan, ZHANG Xiang-gui, XU Ying-jun, YAO Shu

  Cotton Science. 2008, 20(1.1): 29-35.

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  A promoter region was isolated from the 5′ flanking sequence of GHNBS in Gossypium hirsutum by Tail PCR. CAAT-box, TATA-box as well as several pathogen, SA, MeJA and ethylene responsive elements, such as W box, GT-1, MYB and MYBST1 motifs were identified by PLACE analysis while some root specific motifs were also identified in this region. Three enhancer elements including two as-1 and one EECCRCAH1 motifs that were responsible for the strong expression of GUS gene in transgenic plants were  located in the far upstream. Fusions of different 5′ promoter deletion derivatives with the coding region of the GUS gene were transformed into Arabidopsis. Histochemical localization showed strong staining in roots, phloem of the stem and leaf veins. PGN-1559 and PGN-1117 showed organ specific GUS staining patterns while PGN-476 did not. The fact that GHNBS promoter could enhance the expression level of the GUS gene in transgenic Arabidopsis when treated with SA, ABA, MeJA, ethylene, Fusarium oxyporum and DC3000 showed that there were some regulatory elements in the 5′ flanking sequence of GHNBS and which was most possible a pathogen responsive and organ specific promoter.
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  Molecular Markers of Verticillium Wilt Resistance in Upland Cotton  (Gossypium hirsutum L.) Cultivar and Their Effect on

  WANG Fu-rong, LIU Ren-zhong, WANG Liu-ming, ZHANG Chuan-yun, LIU Guo-dong, LIU Qin-hong, Ma Xiao-bo, ZHANG Jun

  Cotton Science. 2008, 20(1.1): 36-43.

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  Genetic population of F₂ and its corresponding F_2：3 lines derived from a hybridization combination of a Verticillium wilt disease resistance cultivar, of pestresistant transgenic upland cotton (Gossypium hirsutum L.), crossed to a Verticillium wilt disease sensible upland cotton line (Luyuan 343), with high fiber quality genomic components introgressed from G. barbadense L., were used to evaluate the resistance of Verticillium wilt disease, and then dissected the resistance genes by SSR markers with the phenotype data of different developmental stage of cotton growth season. qVWR-16-1a, which was detected with the data of the vigorously developmental stage, is located between markers BNL2986 and NAU751 in the fragment of chromosome 16 with 5.73 cM to NAU751, and accounts for 16.53% of the phenotypic variation.The resistance is proved to be from the genotype of resistance parent. Meanwhile, with the data of the late stage of the cotton growth season, three QTLs were detected to be related to the Verticillium wilt resistance. qVWR-16-1b is located in the same interval to qVWR-16-1a with 1.73 cM to the marker NAU751 locus and accounts for 10.27% of the phenotypic variation, while another locus named qVWR-16-2b is located in another interval between BNL1604 and BNL1395, in the same chromosomal fragment with 1.39 cM to BNL1395, and accounts for 10.8% of the phenotype variation. Another QTL named qVWR-2-1b located in the interval between BNL3950 and BNL3971 with only 0.01 cM to BNL 3950 in the fragment of chromosome 2, accounts for 13.78% of the phenotypic variation. Evaluation on the disease resistance of some offspring (F₅) in the breeding population showed that pyramiding the resistant genotypes of marker NAU751 and BNL1395 can significantly improve the Verticillium wilt disease resistance. It was concluded molecular markers could be used to improve Verticillium wilt disease resistance in the process of breeding using the high fiber quality introgression line by assistant selection or pyramiding the resistant genotypes.
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  Effects of Calcium and Auxins on the Initiation  of Lateral Roots of Cotton Seedlings

  XIE Zhi-xia, ZHANG Yi, TIAN Xiao-li, LI Zhao-hu, HE Zhong-pei, ZHAI Zhi-xi, DUAN Liu-sheng

  Cotton Science. 2008, 20(1.1): 44-50.

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  The effects of Ca²⁺  and auxins on root growth of cotton (Gossypium hirsutum L.) were studied. Cotton seedlings were cultured in two layers of filter paper adhered on glass boards with the lower 2 cm dipped in water and grown in growth chamber at (25±1)^oC. Agar solutions containing test concentrations of Ca(NO₃)₂, indole butyric acid (IBA), Ca(NO₃)₂ + IBA, Ca₂+ antagonists or calmodulin (CaM) antagonists were pasted on the joint between seedling hypocotyl and radicle, respectively. Treatments with 50 mg·L^-1 Ca(NO₃)₂  and 5 mg·L^-1 IBA promoted lateral root initiation (LRI) by 14.8 and 29.6 %, but retarded primary root elongation (PRE) by 13.4 and 8.3%,respectively. The combination of 50 mg·L^-1 Ca²⁺ + 5 mg·L^-1 IBA showed synergistic effects as the LRI was 33% higher than the control, while the PRE was close to the control, but much higher than that treated with only Ca(NO₃)₂ or IBA. The PRE, LRI and free CaM content were inhibited by treatment with 3~10 mg·L^-1 LaCl₃ (Ca²⁺ inhibitor), 3~10 mg·L^-1  EGTA (calcium chelated agent) and 3~10 mg·L^-1 TFP (CaM antagonist). Treatment with 5 mg·L^-1  IBA enhanced indolacetic acid (IAA) in the basal radicle segment, repressed IAA and GAs in apical and middle segment of radicle, increased CTKs (cytokinin) content and decreased IAA/ CTKs in all segments of the radicle. Treatment with 3~10 mg·L^-1 TIBA reduced CTKs in the whole radicle, IAA and IAA/ CTKs in basal and middle segments, and increased IAA content as well as IAA/ CTKs in radicle apex, which might have contributed to its inhibition of LRI and enhancement of PRE.
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  Effects of the Salinity Priming on the NaCl Tolerance of  Transgenic Insect Resistant Cotton (Gossypium hirsutum L.)

  LIN Jun, SUN Yu-Jiang, Lü You-Jun, ZHU Shui-Jin

  Cotton Science. 2008, 20(1.1): 51-55.

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  The effect of seed priming with salts on germination and emergence under NaCl tolerance was studied with two commercial transgenic insect resistant cotton cultivars, CCRI 41 and Z-905, as the materials, and their genetic background cultivar, CCRI 23, as check. The results indicated that it was inhibitive for seed germination and field emergence of the upland cotton when treated by 0.5% NaCl, especially for the transgenic insect resistant cotton cultivars which the germination percentage and field emergence stressed by 0.5% NaCl was much lower than that of their genetic background check. However, priming treatment with a certain concentration salts could alleviate the harmful of the NaCl stress significantly. After the treatment of seed priming, the germination percentage and seedling emergence on the condition of 0.5% NaCl stress increased significantly, and the seed tolerance to the salt stress enhanced greatly. There were some differences in the effect of the seed priming treatments on the seed germination and field emergence, stressed by NaCl, between the transgenic insect resistant cotton and their genetic background one, and the formers, with weak tolerance against NaCl stress, had a better effect than that of their genetic background one without exogenous insect resistant genes. There were some differences among the priming agents, of which Na₂ SO₄ was the best. In addition, there existed interactions between the concentration of the priming agents and the process time. The process time should be longer for the priming agents with lower concentration than that with higher concentration.
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  Drought-tolerance Evaluation of Cotton with PEG Water-stress Method

  ZHANG Xue-yan, LIU Chuan-liang, WANG Jun-juan, LI Fu-guang, YE Wu-wei

  Cotton Science. 2008, 20(1.1): 56-61.

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  Eight upland cotton cultivars, two sea island cotton ones and three Asian cotton ones with varied levels of drought tolerance were used. Cotton samples at germination, bud-stage, cotyledon-stage and real-leaf stage were exposed to PEG6000 stress for 12 hours. After 12-hour osmotic treatment，the survival rates were calculated, then by statistical method, curves of the mutative level of drought tolerance were obtained, proving that the 3～6-leaf stage is the key period related to cotton drought tolerance. With different PEG6000 concentrations, the rates which 3～6-leaf seedlings revived were calculated to compare with the revival rate in real drought situation. And the results revealed that: With 17% PEG6000 treatment, the rate which seedlings revived corresponded with the results in drought-shed. The results suggested that osmotic adjustment of cotton could be used to evaluate simply the drought tolerance of cotton, though the cotton after PEG-treated differed slightly in physiology with cotton after drought-treated. The method with osmotic adjustment of PEG is simple, fast and easily operated, could be used to evaluate the drought tolerance of cotton in principle, and will establish foundations for study on cotton drought-tolerance molecular biology.
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  Generation of Antibiosis-free Mutants of Bacillus subtilis NCD-2  with Transposon Tn917 and the Role of Antibiosis of NCD-2 in Controlling Cotton Sore Shin

  SUN Hui-gang, JIANG Ji-zhi, LI She-zeng, LU Xiu-yun, MA Ping

  Cotton Science. 2008, 20(1.1): 62-65.

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  The Bacillus subtilis NCD-2, isolated from the rhizosphere of cotton, could control cotton Verticillium wilt effectively in field. This bacterial strain inhibited the phytopathogen Verticillium dahliae and Rhizoctonia solani, which caused cotton Verticillium wilt and cotton sore shin, respectively, by secreting antipeptides.  To determine the role of the antipeptide in controlling cotton disease, the transposon Tn917 insertional mutagenesis was applied to NCD-2 strain.  In this study, B.subtilis NCD-2 was transformed with a plasmid pTV1 carrying Tn917 by protoplast methods.  1500 mutants were screened against V. dahliae and two antibiosis-free mutants were obtained with resistance to erythromycin and lincomycin but susceptible to chloromycetin.  The greenhouse experiment on cotton sore shin control with NCD-2 wild stain and antibiosis-free mutants showed that antibiosis of NCD-2 played an important role in controlling this disease.  The result suggested that the functional gene of B.subtilis NCD-2 encoding the antipeptide against V. dahliae had a significant function in controlling of cotton sore shin disease.
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  A New Detection Method of Wilting Virulence Induced by Phytotoxin from Verticillium dahliae on Cotton through Leaf Pricking and Spreading

  QI Jun-sheng, LI Huai-fang

  Cotton Science. 2008, 20(1.1): 66-72.

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  A new method to measure the virulence of phytotoxin wilting caused by Verticillium dahliae through pricking on leaf surface and spreading phytotoxin on pricked area has been developed. First, phytotoxin of V. dahliae（V. d.-toxin）of V₉₉₁ strain，a severe defoliating virulence strain of V. dahliae to cotton， was partially purified with Sephacryl S-200 HR; next, 20~50 pits were pricked with a small needle in 0.5 cm² on the surface of cotyledon or euphylla of cotton seedling; then, certain volume of V. d.-toxin solution was spread on the 0.5 cm² of the leaves; and in 24 h after the treatment, wilting spots were appeared and recorded it. The water and BSA treatments were employed as controls. The virulence of wilting could be divided into five classes from 0 to Ⅳ.  Apart from the purified V.d.-toxin, the filtrate (liquid culture medium filtered after culturing V.d. over 7 days) can be also used for this method. To identify its general use of this method, eight V.d.strains were inoculated on leaves of 3 cotton cultivars. These V.d.strains included: severe wilting virulence (V₉₉₁, T₉, V₅₆ and V₁₄₆),  weak wilting virulence (V₁₁₀, V₂₄₆, V₂₅₀) and medium wilting virulence (V_3-10-1). By pricking small pits on cotton leaf surface and spreading V.d.-toxin onto the pricked area, it was easily to observe the wilting symptom and to evaluate their wilting virulence. With this method, the 8 tested strains were classified into corresponded groups, for instance, V₁₂₀, V₉₉₁ and T₉ were divided into the severe type of wilting virulence. This grouping result was as same as that formerly conducted through traditional methods (such as soaking seedling method). To determine the optimum cotton growing stage and part of the leaf on cotton plant, both cotyledon and euphylla in seedling and euphylla in adult plant were tested. As the result, this method could be utilized as early as in seedling stage, and cotyledon was better than euphylla since it was easy to prick but not easy to get through. However the extent of wilting varied with the temperature of environment, cotyledon or euphylla, and the concentration of V.d.-toxin used. To determine the differential reaction of cotton cultivars to V.d.-toxin with this method, four upland cotton cultivars (Ejing-3, B99261, CCRI 12 and Xingluzao-7) were used, and the coherence between V.d.-toxin soaking method and our new method was compared. The result proved that both methods got very similar by multiply tests.     From the experiments, this method revealed some advantages, such as swiftness, simplicity, high reliability, minute requirement of V.d.-toxin (less than 8 μg), and quickness in symptom development (in 24 h ) after treatment etc.. This new method can be used to identify both the resistance of cotton cultivars to Verticillium dahliae and the wilting virulence of pathovars of Verticillium dahliae.
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  Indirect ELISA Method for Detection of Verticillium  dahliae in Cottonseeds

  LIN Ling, CHEN Zhi-shi, GONG Wei-rong, ZHANG Ai-xiang, GU Gan-yu, GU Ben-kang

  Cotton Science. 2008, 20(1.1): 73-78.

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  Apart from condign climate and lack of resistant varieties，one of the important reasons causing Verticillium wilt more and more serious is seed-borne pathogen. To establish an effective method to detect Verticillium dahliae in cottonseeds, we have developed an indirect ELISA method．The toxin of Verticillium dahliae strain Bp₂ was used as antigen to prepare polyclonal antiserum．The indirect ELISA method was developed by using alkaline phosphatase-conjugated goat antimouse antibody as secondary antibody according to standard ELISA procedures．If the value of P/N (ratio of OD₄₀₅ of a sample to that of negative control)≥2，the sample was positive． The titer of the antiserum was 1: 204800 and the toxin could be detected as 1ow as 7.81 μg·L^-1．A11 of Verticillium dahliae strains, collected from different areas in Jiangsu Province, performed positive reaction. No cross-reactivity was observed among the isolates representing other species of fungi such as Rhizoctonia solani, Fusarium moniliforme, Fusarium graminearum, Sclerotinia sclerotiorum, Magnaporthe grisea. After the [WTBX]Verticillium dahliae[WT] strain Bp₂ or T₉ was cultured for one day， or the toxin could be detected in the culture solution．Two of strains were cultured after three days，the value of P/N was 9.05 and 8.82, respectively，and these high values could remain for 11 days．So Verticillium dahliae in cottonseeds could be deduced by detecting the toxin with this developed indirect ELISA．81 cottonseed samples, randomly chosen from different seed companies, were detected if there was Verticillium dahliae with them. Firstly, 100 g cottonseeds from each sample were washed by glide．Coated cotton-seeds were washed till the chemical coating was moved clearly．Then, the cottonseeds were dried at room temperature and separated to five shares each．Each share was put in 150 mL Czapek＇s medium and then cultured for three days．The culture medium was centrifuged for 30 min at 5000 r·min^-1, and the supernatant was tested by using indirect ELISA. 12 out of 81 cottonseed samples performed positive reaction．Cotton seedlings were infected by Verticillium wilt inoculated with the culture solution of the cottonseed samples that performed positive reaction．These results demonstrated that the method of indirect ELISA could be used for quickly detection of Verticillium dahliae in cottonseeds．