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Gossypol and related sesquiterpene aldehydes in cotton function as defense compounds but are antinutritional in cottonseed products. By transcriptome comparison and coexpression analyses, we identified 146 candidates linked to gossypol biosynthesis. Analysis of metabolites accumulated in plants subjected to virusinduced gene silencing (VIGS) led to the identification of four enzymes and their supposed substrates. In vitro enzymatic assay and reconstitution in tobacco leaves elucidated a series of oxidative reactions of the gossypol biosynthesis pathway. The four functionally characterized enzymes, together with (+)-δ-cadinene synthase and the P450 involved in 7-hydroxy-(+)-δ-cadinene formation, convert farnesyl diphosphate (FPP) to hemigossypol, with two gaps left that each involves aromatization. Of six intermediates identified from the VIGS-treated leaves, 8-hydroxy-7-keto- δ-cadinene exerted a deleterious effect in dampening plant disease resistance if accumulated. Notably, CYP71BE79, the enzyme responsible for converting this phytotoxic intermediate, exhibited the highest catalytic activity among the five enzymes of the pathway assayed. In addition, despite their dispersed distribution in the cotton genome, all of the enzyme genes identified show a tight correlation of expression. Our data suggest that the enzymatic steps in the gossypol pathway are highly coordinated to ensure efficient substrate conversion. [ABSTRACT FROM AUTHOR]

A procedure for simultaneous identification and quantification of canrenone and its biotransformed product 11-a-hydroxycanrenone by high-performance liquid chromatography with ultraviolet detector (HPLC-UVD) and mass spectrometry (LC-MS) methods was proposed. The optimal determination variables on the HPLC-UVD or LC-MS coupled with a ZORBAX Eclipse XDBC18 column (150mm × 4.6 mm, 5 μm) were set as follows: detection wavelength of 280 nm, mobile phase of water and methanol gradient elution, temperature for the chromatographic column of 30°C, flow rate of mobile phase of 0.8mL/min, sample injection volume of 5 μL, and elution time of 40 min. The MS conditions were set as follows: the flow rate of sheath gas, aux gas, and sweep gas were kept at 35 arb, 5 arb, and 0 arb, respectively. The temperature of capillary was held at 300°C, and capillary voltage was set at 30.00 V. Tube lens were performed at 100.00 V. The proposed method was validated by linearity (r² ≥ 0.9910), average recovery (94.93%, RSD1.21%), precision (RSD ≥ 1.31%), limit of detection, and limit of quantification (LOD 0.1∼0.12 mg/L, LOQ 0.5²0.67 mg/L), which proved to be affordable for simultaneously determining canrenone and its bio-transformed product 11-a-hydroxy-canrenone. [ABSTRACT FROM AUTHOR]

nnexins constitute a family of multifunction and structurally related proteins. These proteins are ubiquitous in the plant kingdom, and are important calcium-dependent membrane-binding proteins that participate in the polar development of different plant regions such as rhizoids, root caps, and pollen tube tips. In this study, a novel cotton annexin gene (designated as GhFAnnx) was isolated from a fiber cDNA library of cotton ( Gossypium hirsutum). The full-length cDNA of GhFAnnx comprises an open reading frame of 945 bp that encodes a 314-amino acid protein with a calculated molecular mass of 35.7 kDa and an isoelectric point of 6.49. Genomic GhFAnnx sequences from different cotton species, TM-1, Hai7124 and two diploid progenitor cottons, G. herbaceum (A-genome) and G. raimondii (D-genome) showed that at least two copies of the GhFAnnx gene, each with six exons and five introns in the coding region, were identified in the allotetraploid cotton genome. The GhFAnnx gene cloned from the cDNA library in this study was mapped to the chromosome 10 of the A-subgenome of the tetraploid cotton. Sequence alignment revealed that GhFAnnx contained four repeats of 70 amino acids. Semi-quantitative reverse transcriptase-polymerase chain reaction revealed that GhFAnnx is preferentially expressed in different developmental fibers but its expression is low in roots, stems, and leaves. Subcellular localization of GhFAnnx in onion epidermal cells and cotton fibers suggests that this protein is ubiquitous in the epidermal cells of onion, but assembles at the edge and the inner side of the apex of the cotton fiber tips with brilliant spots. In summary, GhFAnnx influences fiber development and is associated with the polar expansion of the cotton fiber during elongation stages. [ABSTRACT FROM AUTHOR]

The phytohormone ABA was known to play a vital role in modulating plant responses to drought stress, and AtMYC2 (a GhbHLH1 homolog) had been proved to act as transcriptional activator involved in the dehydration and ABA response pathway in Arabidopsis. Here, using AtMYC2 amino acid sequence as a querying probe to screen Gossypium hirsutum L. EST database and contigging the candidate ESTs, the putative full-length cDNA sequence of cotton bHLH transcription factor was assembled and confirmed by amplifying leaves cDNA of drought-treated upland cotton cv Jinmian 19. The full-length cDNA named as GhbHLH1 has an open-reading frame of 2,025 bp, encoding a protein of 674 amino acids with a calculated molecular mass of 73.6 kDa and an isoelectric point of 5.46. Sequence alignment shows that GhbHLH1 contains a 60 amino acid long bHLH domain and have high homology with bHLH domain proteins in Arabidopsis, especially with AtMYC2, which plays an important role in response to stress stimuli. Semi-quantitative RT-PCR reveals that GhbHLH1 is strongly expressed in 7-day- post anthesis fibers but weak in roots, stems, and leaves. Based on real-time quantitative RT-PCR, the expression of GhbHLH1 in leaves was transitorily induced by ABA and PEG treatments, although its transcripts were accumulated in various organs. However, its expression was not affected by salt and cold treatments. Our results may provide the basis for future research of bHLH domain gene’s roles as a regulator of ABA signaling in cotton. [ABSTRACT FROM AUTHOR]

Abstract  Two cotton genotypes, Simian 3 (SM 3) and WC, were co-transformed using a mixture of four Agrobacterium tumefaciens cultures of strain LBA4404, each carrying a plasmid harboring the following genes, Bt + sck (for Bacillus thuringenesis protein and modified Cowpea trypsin inhibitor), bar (for glufosinate), keratin, and fibroin. The frequency of callus induction, embryogenesis, and plant regeneration were notably different between the two genotypes. However, there were no differences between the two genotypes for number of plantlets carrying multiple gene copies of different gene combinations as well as transformation frequency for different gene combinations. PCR analysis indicated that more than 80% of plantlets carried the nptII gene for kanamycin resistance. Overall, the co-transformation frequency of two or more genes was about 35%. Southern blot analysis confirmed integration of target genes into the cotton genome, and the number of copies of the transgene(s) varied from one to four. Multiple transgene expression was confirmed by RT-PCR analysis in some transgenic lines. Further analysis of T1 plants demonstrated that multiple transgenes were inherited and expressed in progenies. [ABSTRACT FROM AUTHOR]

The open-bud mutant plant has a flower bud that is open at its tip due to the shortening of the corollas, resulting in exposure of the stigma and upper anthers. This mutant plant is potentially useful as a parent for producing hybrid cotton seeds. We have identified the open-bud trait to be inherited as recessive duplicate genes and finely mapped them in the homoelogous chromosome (chr) pair of chr. 18 (D13) and 13 (A13). Gossypium hirsutum substitution line 18, which has chr. 18 replaced by its corresponding homozygous chromosome in G. barbadense acc. 3-79 and the genotyped open-bud duplicate genes ob 1 ob 1 ob 2 ob 2, was crossed with the monomeric genotypic G. hirsutum acc. TM-1 and G. barbadense acc. 3-79 to produce two mapping populations. We then fine-mapped the duplicate open-bud gene ob 1 in the chr. 18 (D13) and ob 2 in the chr. 13 (A13) homoelogous chromosome pair in tetraploid cotton. Molecular markers closely linked with ob 1 and ob 2 will be useful tools in the development of open-bud lines by marker-assisted selection. [ABSTRACT FROM AUTHOR]

Agrobacterium- tumefaciens-mediated transformation of cotton embryogenic calli (EC) was enhanced by choosing appropriate EC and improving efficiency of coculture, selection cultivation, and plant regeneration. After 48-h cocultivation, the number of β-glucuronidase (GUS)-positive calli characterized by yellow, loose, and fine-grained EC was twofold greater than that of gray, brown, and coarse-granule EC. It indicated that efficiency of transient transformation was affected by EC morphology. And transient transformation efficiency was also improved by cocultivation on the medium adding 50 mg l−1 acetosyringone at 19°C for 48 h. Subculturing EC on the selection medium with low cell density was beneficial to production of more kanamycin-resistant (Km-R) calli lines. From an original 0.3-g EC, an average of 20 Km-R calli lines were obtained from a selection dish and the GUS-positive rate of Km-R clones was 81.97%. A large number of normal plants were rapidly regenerated on the differentiation medium with dehydration treatments and the GUS-positive rate of regeneration plants was about 72.60%. Polymerase chain reaction analysis of GUS-positive plantlets revealed a 100% positive detection rate for neomycin phosphotransferase II gene and uidA. Southern blot of transgenic plants regenerated from different Km-R calli lines demonstrated that the target gene, mostly with the low copy number, has been integrated into the cotton genome. [ABSTRACT FROM AUTHOR]

AbstractCotton (Gossypiumspp.) is an important source of edible oil and protein meals. Complex genetics and strong environmental effects hinder progress in seed quality trait breeding in this species. The use of molecular markers can improve an understanding of the genetic factors conditioning seed quality traits, and is expected to assist in selection of superior genotypes. This study was conducted to identify quantitative trail loci (QTL) associated with seed physical and nutrient traits in cotton. To achieve this objective, a population of 140 BC1S1lines developed from a cross between ?TM-1? and ?Hai7124? was evaluated in 2003 and 2004. A linkage map consisting of 918 markers from this population was used to identify QTL using QTLNetwork-2.0 software. Eleven single QTL were identified for kernel percentage, kernel oil percentage, kernel protein percentage and seven amino acids (Asp, Ser, Gly, Ile, Leu, Phe and Arg). Phenotypic variation explained by each individual QTL ranged from 10.89 to 46.28%. Two epistatic QTL for Cys and Leu were detected, explaining 9.55 and 4.43% of the phenotypic variation. These QTL detected for seed quality traits in cotton are expected to be useful for further breeding programmes targeting development of cotton with improved nutrient quality. [ABSTRACT FROM AUTHOR]

β-1,4-glucosidase (BG, EC3.2.1.21), one of three cellulases, is a widespread family of enzymes involved in the metabolism of cell wall polysaccharides in both prokaryocytes and eukaryotes. Here, we report the isolation of a full-length cDNA encoding β-1,4-glucosidase protein (designated as GhBG) and its putative function in the process of fiber development and in yeast. Through random sequencing of the cotton fiber cDNA library from 7235 germplasm line, with elite fiber quality in Gossypium hirsutum L. and utilizing the 5' rapid amplification of cDNA ends (RACE) technique, a 2133 bp cDNA clone encoding a cotton fiber specifically expressed protein (accession number: DQ103699) was isolated. GhBG was composed of a 1884 bp open reading frame (ORF) encoding 627 amino acid residues. This putative protein had an isoelectric point of 8.17, a calculated molecular weight of 68.78 KD and a signal peptide with 23 amino acid residues at the N-terminal. RT-PCR analysis indicated GhBG was specifically expressed in fiber cells and was highly abundant in 5-17 day post anthesis (DPA). It was not, however, expressed in root, hypocotyls or leaves. Southern blotting analysis showed there were two copies of GhBG in the upland cotton genome; most likely contained in sub-genome A and sub-genome D. GhBG was then integrated into a yeast expression vector, pREP-5N and electro-transformed into fission yeast Schizosaccharomyces pombe Q-01. The results demonstrated that GhBG led to a significant increase in cell length and width and a remarkable decrease of the length/width ratio. Compared to vector control transformants, cells were significantly larger and rounder and their growth velocity was also reduced. [ABSTRACT FROM AUTHOR]

Asiatic cotton ( Gossypium arboreum L.) is an “Old World” cultivated cotton species, the sinense race of which is planted extensively in China. This species is still used in the current tetraploid cotton breeding program as an elite germplasm line, and is also used as a model for genomic research in Gossypium. In the present study, 60 cotton microsatellite markers, averaging 4.6 markers for each A-genome chromosome, were chosen to assess the genetic diversity of 109 accessions. These included 106 G. arboreum landraces, collected from 18 provinces throughout four Asiatic cotton-growing regions in China. A total of 128 alleles were detected, with an average of 2.13 alleles per locus. The largest number of alleles, as well as the maximum number of polymorphic loci, was detected in the A03 linkage group. No polymorphic alleles were detected on chromosome 10. The polymorphism information content for the 22 polymorphic microsatellite loci varied from 0.52 to 0.98, with an average of 0.89. Genetic diversity analysis revealed that the landraces in the Southern region had more genetic variability than those from the other two regions, and no significant difference was detected between landraces in the Yangtze and the Yellow River Valley regions. These findings are consistent with the history of sinense introduction, with the Southern region being the presumed center of origin for Chinese Asiatic cotton, and with subsequent northeastward extension to the Yangtze and Yellow River Valleys. Cluster analysis, based on simple sequence repeat data for 60 microsatellite loci, clearly differentiated Vietnamese and G. herbaceum landraces from the sinense landrace. No relationship between inter-variety similarity and geographical ecological region was observed. The present findings indicate that the Southern region landraces may have been directly introduced into the provinces in the middle and lower Yangtze River Valley, where Asiatic cotton was most extensively grown, and further race sinense crops were subsequently produced. (Managing editor: Ya-Qin Han) [ABSTRACT FROM AUTHOR]

Using 219 F2 individuals developed by crossing the genetic standard line TM-1 and the multiple dominant marker line T586 in Gossypium hirsutum L., a genetic linkage map with 19 linkage groups was constructed based on simple sequence repeat (SSR) markers. Compared with our tetraploid backboned molecular genetic map from a(TM-1 × Hai 7124) × TM-1 BC1 population, 17 of the 19 linkage groups were combined and anchored to 12 chromosomes (sub-genomes). Of these groups, four morphological marker genes in T586 had been mapped into the molecular linkage map. Meanwhile, three quantitative trait loci for lint percentage were tagged and mapped separately on the A03 linkage group and chromosome 6. (Managing editor: Li-Hui Zhao) [ABSTRACT FROM AUTHOR]

A bacterial artificial chromosome (BAC) library containing a large genomic DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA inserts is needed. We have developed a BAC library of the restoring line 0-613-2R for isolating the fertility restorer (Rf1) gene and genomic research in cotton (Gossypium hirsutum L.). The BAC library contains 97 825 clones stored in 255 pieces of a 384-well microtiter plate. Random samples of BACs digested with the NotI enzyme indicated that the average insert size is approximately 130 kb, with a range of 80–275 kb, and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 × haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with NotI and HindIII enzymes. Thus, the stability of a single BAC clone can be sustained at least for 100 generations. Eight simple sequence repeat (SSR) markers flanking the Rf1 gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker. (Managing editor: Li-Hui Zhao) [ABSTRACT FROM AUTHOR]

To determine the level of microsatellite sequence differences and to use the information to construct a phylogenetic relationship for cultivated tetraploid cotton ( Gossypium spp.) species and their putative diploid ancestors, 10 genome-derived microsatellite primer pairs were used to amplify eight species, including two tetraploid and six diploid species, in Gossypium. A total of 92 unique amplicons were resolved using polyacrylamide gel electrophoresis. Each amplicon was cloned, sequenced, and analyzed using standard phylogenetic software. Allelic diversities were caused mostly by changes in the number of simple sequence repeat (SSR) motif repeats and only a small proportion resulted from interruption of the SSR motif within the locus for the same genome. The frequency of base substitutions was 0.5%-1.0% in different genomes, with only few indels found. Based on the combined 10 SSR flanking sequence data, the homology of A-genome diploid species averaged 98.9%, even though most of the amplicons were of the same size, and the sequence homology between G. gossypioides (Ulbr.) Standl. and three other D-genome species (G. raimondii Ulbr., G. davidsonii Kell., and G. thurberi Tod.) was 98.5%, 98.6%, and 98.5%, respectively. Phylogenetic trees of the two allotetraploid species and their putative diploid progenitors showed that homoelogous sequences from the A- and D-subgenome were still present in the polyploid subgenomes and they evolved independently. Meanwhile, homoelogous sequence interaction that duplicated loci in the polyploid subgenomes became phylogenetic sisters was also found in the evolutionary history of tetraploid cotton species. The results of the present study suggest that evaluation of SSR variation at the sequence level can be effective in exploring the evolutionary relationships among Gossypuim species. (Managing editor: Wei WANG) [ABSTRACT FROM AUTHOR]

Genetic mapping provides a powerful tool for quantitative trait loci (QTL) analysis at the molecular level. A simple sequence repeat (SSR) genetic map containing 590 markers and a BC1 population from two cultivated tetraploid cotton ( Gossypium hirsutum L.) cultivars, namely TM-1 and Hai 7124 ( G. barbadense L.), were used to map and analyze QTL using the composite interval mapping (CIM) method. Thirty one QTLs, 10 for lobe length, 13 for lobe width, six for lobe angle, and two for leaf chlorophyll content, were detected on 15 chromosomes or linkage groups at logarithm of odds (LOD)> 2.0, of which 15 were found for leaf morphology at LOD > 3.0. The genetic effects of the QTL were estimated. These results are fundamental for marker-assisted selection (MAS) of these traits in tetraploid cotton breeding. (Managing editor: Li-Hui ZHAO) [ABSTRACT FROM AUTHOR]

The article presents case studies on patients who undergo endoscopic tracheal tumor resection using tracheotomy incision. One of the patients was intensively monitored by anaesthetic and electrocardiac instrument and a chest surgeon after a computed tomography (CT) scan showed a round neoplasm in the trachea under the superior aperture of thorax. It discusses the incidence of primary tracheal tumor as well as the causes symptoms of gradually aggravating airway obstruction.