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Background: Integrase strand transfer inhibitors (INSTIs) are first-line regimens for HIV treatment. We aimed to examine their impact on cognitive performance and depressive symptoms in women with HIV (WWH). Setting: Women's Interagency HIV Study, a multisite, prospective, cohort study. Methods: WWH who started or switched to INSTI-based antiretroviral therapy (ART) and completed neuropsychological testing and the Center for Epidemiological Studies-Depression (CES-D) scale before and after INSTI start/switch were included in the analyses. Primary outcomes were demographically corrected cognitive domain T-scores. Linear mixed-effects models adjusted for relevant covariates were used to examine effects of start/switch of any INSTI and individual INSTI drugs on cognition and CES-D scores. Results: Six hundred thirty-nine WWH, median age 49 (interquartile range 12) years, 66% Black non-Hispanic, had neuropsychological and CES-D scale data before and after INSTI start/switch. Although 14% started INSTI-based ART, the remainder switched to INSTI-based ART from another regimen. Overall, any INSTI use was associated with poorer learning post-INSTI. Specifically, use of dolutegravir and elvitegravir, but not raltegravir, was associated with poorer learning. In analyses restricted to INSTI switch, any INSTI use, and dolutegravir use, was associated with poorer learning. Among those switching from a PI-based regimen, INSTIs overall and dolutegravir remained associated with poorer learning; switching from a nonnucleoside reverse transcriptase inhibitor to dolutegravir was also associated with poorer learning. INSTI start/switch was not related to depressive symptom changes. Conclusions: INSTI use was associated with poorer learning among WWH. These changes were mainly observed in elvitegravir and dolutegravir users, indicating that the impact of INSTI on cognition in WWH may not be a class effect. [ABSTRACT FROM AUTHOR]

Properties of leaf and infusion colours, chemical components and volatile flavour compounds of oolong teas and their correlation with perceived quality score given by tea-tasting panel were analysed. The scores for appearance and infused leaf correlated strongly with concentrations of chlorophyll a (chl a), chlorophyll (chl b) and chlorophyll (chl) ( r = 0.355–0.433, P < 0.05) and the total quality score (TQS) positively correlated with concentrations of chl a, chl b and chl ( r = 0.517–0.533, P < 0.01). The perceived taste score and TQS positively correlated with the concentration of total free amino acid ( r = 0.514, 0.694, P < 0.01) and theanine ( r = 0.500, 0.684, P < 0.01). The volatile composition and their quantities varied widely among oolong tea samples. Nerolidol, indole, benzeneacetaldehyde, linalool, linalool oxide I, hexanal, benzyl nitrile, geraniol and 1-penten-3-ol were prevailing volatile compounds detected in most of oolong tea samples. Principal component analysis screened ten principal components with the first three (glutamic acid, total catechins and benzeneacetaldehyde) explaining 27.86%, 20.00% and 14.46% of the total variance, respectively. Regression analysis upon the ten principal components formulated a prediction model on the total quality score with 78.5% probability. The result showed that oolong teas could be partially classified by cluster analysis based on principal components. [ABSTRACT FROM AUTHOR]

Properties of leaf and infusion colours, chemical components and volatile flavour compounds of Longjing teas and their correlation with perceived quality score given by tea-tasting panel were analysed. The scores for appearance, infused leaves and for the Total Quality Score (TQS) negatively correlated with concentrations of chlorophyll-a (Chl-a), Chl-b and total chlorophyll ( r = −0.723 to −0.846, P < 0.01). The perceived taste score positively correlated with the concentration of total free amino acid ( r = 0.527, P < 0.05) and theanine ( r = 0.511, P < 0.05), whereas the TQS positively correlated with total free amino acid ( r = 0.533). The volatile composition and their quantities varied widely among Longjing tea samples. (E)-geraniol, linalool, hexanal, pentanal, heptanal, nonanal and jasmone were prevailing volatile compounds detected in all samples. Principal component analysis (PCA) screened twelve principal components with the first three explaining 18.82%, 15.31% and 11.83% of the total variance respectively. The concentrations of total free amino acids, pentanal and epigallocatechin gallate were with the highest component score coefficients for the first three principal components respectively. Regression analysis upon the twelve principal components formulated a prediction model on the TQS with 77.0% probability. [ABSTRACT FROM AUTHOR]

Some aspects of the intruder detection system (IDS) based on the leaky coaxial cable (LCX) sensor are still unknown due to its complex propagation characteristics. In order to study the field disturbance mechanism of human intruder and to reduce the phase error caused by the initial state of the detection signal, a novel method of improving the localization accuracy is proposed. At the frequencies of 40MHz, 100MHz and 200MHz, the IDS based on the LCX sensor is proposed by analyzing the scattering characteristics of human intruder and using IQ demodulation method. According to the characteristics of the IDS, the electric field distribution is obtained by irradiating the human intruder from three typical radiation directions, which are front, side and low-side direction in the mentioned frequency range. Combined with the method of pulse accumulation and synchronous subtraction, the intruder localization can be easily realized by using pulse delay positioning method. The results demonstrate the improvement in localization accuracy and the decrement in false positive rate, and the positioning accuracy is less than 3 meter. [ABSTRACT FROM AUTHOR]

The present study aimed to characterize the simple sequence repeat markers in cotton using the cotton expressed sequence tags. A total of 111 EST-SSR polymorphic molecular markers with trinucleotide motifs were used to evaluate the 79 accessions of Gossypium L., (G. darwinii, 59 and G. barbadense, 20) collected from the Galapagos Islands. The allele number ranged from one to seven, with an average value of 2.85 alleles per locus, while polymorphisminformation content values varied from 0.008 to 0.995, with an average of 0.520. The discrimination power ranks high for the majority of the SSRs, with an average value of 0.98. Among 111 pairs of EST-SSRs and gSSRs, a total of 49markers, comprising nine DPLs, one each of MonCGR, MUCS0064, and NAU1028, and 37 SWUs (D-genome), were found to be the best matched hits, similar to the 155 genes identified by BLASTx in the reference genome of G. barbadense, G. arboreum L., and G. raimondii Ulbr. Related genes GOBAR&lowbar;DD21902, GOBAR&lowbar;DD15579, GOBAR&lowbar;DD27526, and GOBAR&lowbar;AA04676 revealed highly significant expression 10, 15, 18, 21, and 28 days post-anthesis of fiber development. The identified EST-SSR and gSSR markers can be effectively used for mapping functional genes of segregating cotton populations, QTL identification, and marker-assisted selection in cotton breeding programs. [ABSTRACT FROM AUTHOR]

The extrusion of toxins and substances at a cellular level is a vital life process in plants under abiotic stress. The multidrug and toxic compound extrusion (MATE) gene family plays a large role in the exportation of toxins and other substrates. We carried out a genome-wide analysis of MATE gene families in Gossypium raimondii and Gossypium arboreum and assessed their expression levels under salt, cadmium and drought stresses. We identified 70 and 68 MATE genes in G. raimondii and G. arboreum, respectively. The majority of the genes were predicted to be localized within the plasma membrane, with some distributed in other cell parts. Based on phylogenetic analysis, the genes were subdivided into three subfamilies, designated as M1, M2 and M3. Closely related members shared similar gene structures, and thus were highly conserved in nature and have mainly evolved through purifying selection. The genes were distributed in all chromosomes. Twenty-nine gene duplication events were detected, with segmental being the dominant type. GO annotation revealed a link to salt, drought and cadmium stresses. The genes exhibited differential expression, with GrMATE18, GrMATE34, GaMATE41 and GaMATE51 significantly upregulated under drought, salt and cadmium stress, and these could possibly be the candidate genes. Our results provide the first data on the genome-wide and functional characterization of MATE genes in diploid cotton, and are important for breeders of more stress-tolerant cotton genotypes. [ABSTRACT FROM AUTHOR]

Cold stress is a potent abiotic stress resulting in cotton yield and fiber quality loss. We hypothesized that GhGTG1 could increase low temperature stress tolerance by improving the sensitivity to ABA. Genome-wide identification, biochemical traits and functional characterization of GhGTG1 , were investigated under different levels of ABA and cold stress. GhGTG1 had two highly conserved functional domains (ABA_GPCR, GPHR_N) in all cotton species. Under ABA treatment, over expression of GhGTG1 in Arabidopsis had a lower germination rate and root elongation. While under 4 ℃ stress, transgenic plants had a higher germination rate and root elongation. This indicate that GhGTG1 improved sensitivity to ABA under cold stress. In transgenic plants, CAT and SOD activity was increased, while H 2 O 2 and MDA content decreased. Less trypan blue and DAB staining area suggested that GhGTG1 enhances cold stress resistance. Under 4 ℃ treatment, silence cotton seedlings through VIGS method, resulted in a poor growth, lower relative leaf water content, chlorophyll content but a higher ion leakage and excised leaf water loss. The activity of CAT and SOD decreased, H 2 O 2 and MDA content was increased. Two marker genes (OST1 and ABCG40) are associated with the stomatal movement, but these genes were substantially suppressed. This suggesting that GhGTG1 can induce cold stress tolerance by improving ABA sensitivity Via., maintaining stomatal movement. These data will provide new insight of cold resistance mechanism in cotton and further will help the breeder to develop new cotton cold stress tolerance germplasm. [Display omitted] • GhGTG1 increased ABA sensitivity to improve cold stress tolerance in Arabidopsis. • GhGTG1 can improve cold tolerance by regulating stomata opening and closure through ABA pathway in cotton. • GhGTG1 can induce cold stress tolerance by improving ABA sensitivity to control the stomatal movement. [ABSTRACT FROM AUTHOR]

Background: Chromosome microdissection is one of the most important techniques in molecular cytogenetic research. Cotton (Gossypium Linnaeus, 1753) is the main natural fiber crop in the world. The resistance gene analog (RGA) cloning after its single chromosome microdissection can greatly promote cotton genome research and breeding. Results: Using the linker adaptor PCR (LA-PCR) with the primers of rice disease-resistance homologues, three nucleotide sequences PS016 (KU051681), PS054 (KU051682), and PS157 (KU051680) were obtained from the chromosome Ah01 of upland cotton (cv. TM-1). The Blast results showed that the three sequences are the nucleotide binding site-leucine rich repeat (NBS-LRR) type RGAs. Clustering results indicated that they are homologous to these published RGAs. Thus, the three RGAs can definitely be confirmed as NBS-LRR class of RGAs in upland cotton. Conclusions: Using single chromosome microdissection technique, DNA libraries containing cotton RGAs were obtained. This technique can promote cotton gene cloning, marker development and even the improvement of cotton genome research and breeding. [ABSTRACT FROM AUTHOR]

Plant height, which shows dynamic development and heterosis, is a major trait affecting plant architecture and has an indirect in uence on economic yield related to biological yield in cotton. In the present study, we carried out dynamic analysis for plant height and its heterosis by quantitative trait loci (QTL) mapping at multiple developmental stages using two recombinant inbred lines (RILs) and their backcross progeny. At the single-locus level, 47 QTL were identi ed at ve developmental stages in two hybrids. In backcross populations, QTL identi ed at an early stage mainly showed partial effects and QTL detected at a later stage mostly displayed overdominance effects. At the two-locus level, we found that main effect QTL played a more important role than epistatic QTL in the expression of heterosis in backcross populations. Therefore, this study implies that the genetic basis of plant height heterosis shows dynamic character and main effect QTL with dominance determines heterosis for plant height in Upland cotton. [ABSTRACT FROM AUTHOR]

Cotton fiber, a raw natural fiber material, is widely used in the textile industry. Understanding the genetic mechanism of fiber traits is helpful for fiber quality improvement. In the present study, the genetic basis of fiber quality traits was explored using two recombinant inbred lines (RILs) and corresponding backcross (BC) populations under multiple environments in Upland cotton based on marker analysis. In backcross populations, no significant correlation was observed between marker heterozygosity and fiber quality performance and it suggested that heterozygosity was not always necessarily advantageous for the high fiber quality. In two hybrids, 111 quantitative trait loci (QTL) for fiber quality were detected using composite interval mapping, in which 62 new stable QTL were simultaneously identified in more than one environment or population. QTL detected at the single-locus level mainly showed additive effect. In addition, a total of 286 digenic interactions (E-QTL) and their environmental interactions [QTL × environment interactions (QEs)] were detected for fiber quality traits by inclusive composite interval mapping. QE effects should be considered in molecular marker-assisted selection breeding. On average, the E-QTL explained a larger proportion of the phenotypic variation than the main-effect QTL did. It is concluded that the additive effect of single-locus and epistasis with few detectable main effects play an important role in controlling fiber quality traits in Upland cotton. [ABSTRACT FROM AUTHOR]

Background: Dissecting genome organization is indispensable for further functional and applied studies. As genome sequences data shown, cotton genomes contain more than 60% repetitive sequences, so study on repetitive sequences composition, structure, and distribution is the key step to dissect cotton genome. Results: In this study, a bacterial artificial chromosome (BAC) clone enriched in repetitive sequences, was discovered initiatively by fluorescence in situ hybridization (FISH). FISHing with allotetraploidy cotton as target DNA, dispersed signals on most regions of all A sub-genome chromosomes, and only middle regions of all D sub-genome chromosomes were detected. Further FISHing with other cotton species bearing A or D genome as target DNA, specific signals were viewed. After BAC sequencing and bioinformational analysis, 129 repeat elements, size about 57,172 bp were found, accounting for more than 62% of the BAC sequence (91,238 bp). Among them, a type of long terminal repeat-retrotransposon (LTR-RT), LTR/Gypsy was the key element causing the specific FISH results. Using the fragments of BAC matching with the identified Gypsy-like LTR as probes, the BAC-57I23-like FISH signals were reappeared. Running BLASTN, the fragments had good match with all chromosomes of G. arboreum (A2) genome and A sub-genome of G. hirsutum (AD1), and had relatively inferior match with all chromosomes of D sub-genome of AD1, but had little match with the chromosomes of G. raimondii (D5) genome, which was consistent with the FISH results. Conclusion: A repeats-enriched cytogenetic marker to identify A and D sub-genomes of Gossypium was discovered by FISH. Combined sequences analysis with FISH verification, the assembly quality of repetitive sequences in the allotetraploidy cotton draft genome was assessed, and better chromosome belonging was verified. We also found the genomic distribution of the identified Gypsy-LTR-RT was similar to the distribution of heterochromatin. The expansion of this type of Gypsy-LTR-RT in heterochromatic regions may be one of the major reasons for the size gap between A and D genome. The findings showed here will help to understand the composition, structure, and evolution of cotton genome, and contribute to the further perfection of the draft genomes of cotton. [ABSTRACT FROM AUTHOR]

Two bacterial artificial chromosome (BAC) clones (350B21 and 299N22) of Pima 90-53 cotton [Gossypium barbadense Linnaeus, 1753 (2n=4x=52)] were screened from a BAC library using SSR markers. Strong hybridization signals were detected at terminal regions of all A genome (sub-genome) chromosomes, but were almost absent in D genome (sub-genome) chromosomes with BAC clone 350B21 as the probe. The results indicate that specific sequences, which only exist at the terminal parts of A genome (sub-genome) chromosomes with a huge repeat number, may be contained in BAC clone 350B21. When utilizing FISH with the BAC clone 299N22 as probe, a pair of obvious signals was detected on chromosome 13 of D genome (sub-genome), while strong dispersed signals were detected on all A genome (sub-genome) chromosomes. The results showed that peculiar repetitive sequence, which was distributed throughout all A genome (subgenome) chromosomes, may exist in BAC clone 299N22. The absence of the repetitive sequences, which exist in the two BAC clones, in D genome may account for the genome-size variation between A and D genomes. In addition, the microcolinearity analysis of the clone 299N22 and its homologous region on G. raimondii Ulbrich, 1932 chromosome 13 (D513) indicated that the clone 299N22 might come from A sub-genome of sea island cotton (G. barbadense), and a huge number of small deletions, illegitimate recombination, translocation and rearrangements may have occurred during the genus evolution. The two BAC clones studied here can be used as cytological markers but will be also be helpful to research in cotton genome evolution and comparative genomics. [ABSTRACT FROM AUTHOR]

Tea (Camellia sinensis L.) is a perennial woody plant that is widely cultivated to produce a popular non-alcoholic beverage; this beverage has received much attention due to its pleasant flavor and bioactive ingredients, particularly several important secondary metabolites. Due to the significant changes in the metabolite contents of the buds and the young expanding leaves of tea plants, high-performance liquid chromatography (HPLC) analysis and isobaric tags for relative and absolute quantitation (iTRAQ) analysis were performed. A total of 233 differentially expressed proteins were identified. Among these, 116 proteins were up-regulated and 117 proteins were down-regulated in the young expanding leaves compared with the buds. A large array of diverse functions was revealed, including roles in energy and carbohydrate metabolism, secondary metabolite metabolism, nucleic acid and protein metabolism, and photosynthesis- and defense-related processes. These results suggest that polyphenol biosynthesis- and photosynthesis-related proteins regulate the secondary metabolite content of tea plants. The energy and antioxidant metabolism-related proteins may promote tea leaf development. However, reverse transcription quantitative real-time PCR (RT-qPCR) showed that the protein expression levels were not well correlated with the gene expression levels. These findings improve our understanding of the molecular mechanism of the changes in the metabolite content of the buds and the young expanding leaves of tea plants. [ABSTRACT FROM AUTHOR]

Background: Cytogenetic map can provide not only information of the genome structure, but also can build a solid foundation for genetic research. With the developments of molecular and cytogenetic studies in cotton (Gossypium), the construction of cytogenetic map is becoming more and more imperative. Results: A cytogenetic map of chromosome 1 (A101) of Gossypium herbaceum (A1) which includes 10 bacterial artificial chromosome (BAC) clones was constructed by using fluorescent in situ hybridization (FISH). Meanwhile, comparison and analysis were made for the cytogenetic map of chromosome 1 (A101) of G. herbaceum with four genetic linkage maps of chromosome 1 (Ah01) of G. hirsutum ((AD)1) and one genetic linkage map of chromosome 1 of (A101) G. arboreum (A2). The 10 BAC clones were also used to be localized on G. raimondii (D5) chromosome 1 (D501), and 2 of them showed clear unique hybridized signals. Furthermore, these 2 BAC clones were also shown localized on chromosome 1 of both A sub-genome and D sub-genome of G. hirsutum. Conclusion: The comparison of the cytogenetic map with genetic linkage maps showed that most of the identified marker-tagged BAC clones appearing same orders in different maps except three markers showing different positions, which might indicate chromosomal segmental rearrangements. The positions of the 2 BAC clones which were localized on Ah01 and Dh01 chromosomes were almost the same as that on A101 and D501 chromosomes. The corresponding anchored SSR markers of these 2 BAC clones were firstly found to be localized on chromosome D501 (Dh01) as they were not seen mapped like this in any genetic map reported. [ABSTRACT FROM AUTHOR]

Roots are involved in acquisition of water and nutrients, as well as in providing structural support to plant. The root system provides a dynamic model for developmental analysis. Here, we investigated quantitative trait loci (QTL), dynamic conditional QTL and epistatic interactions for seedling root traits using an upland cotton F2 population and a constructed genetic map. Totally, 37 QTLs for root traits, 35 dynamic conditional QTLs based on the net increased amount of root traits (root tips, forks, length, surface area and volume) (i) after transplanting 10 days compared to 5 days, and (ii) after transplanting 15 days to 10 days were detected. Obvious dynamic characteristic of QTL and dynamic conditional QTL existed at different developmental stages of root because QTL and dynamic conditional QTL had not been detected simultaneously. We further confirmed that additive and dominance effects of QTL qRSA-chr1-1 in interval time 5 to 10 DAT (days after transplant) offset the effects in 10 to 15 DAT. Lots of two-locus interactions for root traits were identified unconditionally or dynamically, and a few epistatic interactions were only detected simultaneously in interval time of 5-10 DAT and 10-15 DAT, suggesting different interactive genetic mechanisms on root development at different stages. Dynamic conditional QTL and epistasis effects provide new attempts to understand the dynamics of roots and provide clues for root architecture selection in upland cotton. [ABSTRACT FROM AUTHOR]

Construction of single chromosomal DNA libraries by chromosome micromanipulation is a useful tool for pursuing genomic studies. Thus far, micromanipulation in cotton has not been reported yet, which may be due to difficulty in preparing chromosomes of similar sizes. In this study, single chromosome micromanipulation was successfully achieved in cotton. A single chromosome 5 of Gossypium arboreum (cultivar Shixiya-1) carrying a large satellite at mitotic metaphase was isolated by microdissection using the Cell Cut Plus Laser micromanipulation system. The chromosomal DNA was digested by Sau 3A and ligated to Sau 3A linker adaptors. After two rounds of linker adaptor PCR (LA-PCR) amplification, DNA fragments ranging from 300 to 2,500 bp were acquired. Southern hybridization revealed that the PCR products had homology with genomic DNA of the cultivar Shixiya-1, indicating that DNA of chromosome 5 has been successfully amplified. The second round LA-PCR products and 45S rDNA and chromosome-specific BAC clones were used as probes for fluorescence in situ hybridization analysis on metaphase chromosome. The results confirmed that the LA-PCR products were derived from the isolated target chromosome. Hybridization signals of the second round LA-PCR products were mainly detected along the entire chromosome 5; in addition, weak signals were also observed on other chromosomes, indicating that there were some homologous nucleotide sequences in other chromosomes. The second round LA-PCR products were cloned to generate a chromosome-specific DNA library which contains approximately 173,000 clones. Evaluation based on 136 randomly selected clones showed that the size of the inserts varied from 500 to 1,800 bp with an average of 750 bp. The no-load rate was less than 1 %, the titer of the library was 1.2 × 10 pfu mL, and the rate of the single and low copy sequences was over 47 %. This library will facilitate specific probe screening, molecular mapping, gene cloning, and DNA sequencing for this chromosome. [ABSTRACT FROM AUTHOR]

Background: Cotton (Gossypium spp.) is a model system for the analysis of polyploidization. Although ascertaining the donor species of allotetraploid cotton has been intensively studied, sequence comparison of Gossypium chloroplast genomes is still of interest to understand the mechanisms underlining the evolution of Gossypium allotetraploids, while it is generally accepted that the parents were A- and D-genome containing species. Here we performed a comparative analysis of 13 Gossypium chloroplast genomes, twelve of which are presented here for the first time. Methodology/Principal Findings: The size of 12 chloroplast genomes under study varied from 159,959 bp to 160,433 bp. The chromosomes were highly similar having .98% sequence identity. They encoded the same set of 112 unique genes which occurred in a uniform order with only slightly different boundary junctions. Divergence due to indels as well as substitutions was examined separately for genome, coding and noncoding sequences. The genome divergence was estimated as 0.374% to 0.583% between allotetraploid species and A-genome, and 0.159% to 0.454% within allotetraploids. Forty protein-coding genes were completely identical at the protein level, and 20 intergenic sequences were completely conserved. The 9 allotetraploids shared 5 insertions and 9 deletions in whole genome, and 7-bp substitutions in protein-coding genes. The phylogenetic tree confirmed a close relationship between allotetraploids and the ancestor of A-genome, and the allotetraploids were divided into four separate groups. Progenitor allotetraploid cotton originated 0.43-0.68 million years ago (MYA). Conclusion: Despite high degree of conservation between the Gossypium chloroplast genomes, sequence variations among species could still be detected. Gossypium chloroplast genomes preferred for 5-bp indels and 1-3-bp indels are mainly attributed to the SSR polymorphisms. This study supports that the common ancestor of diploid A- genome species in Gossypium is the maternal source of extant allotetraploid species and allotetraploids have a monophyletic origin. G. hirsutum AD1 lineages have experienced more sequence variations than other allotetraploids in intergenic regions. The available complete nucleotide sequences of 12 Gossypium chloroplast genomes should facilitate studies to uncover the molecular mechanisms of compartmental co-evolution and speciation of Gossypium allotetraploids. [ABSTRACT FROM AUTHOR]

Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established. [ABSTRACT FROM AUTHOR]

A transgenic male sterile line of upland cotton was generated by the ectopic expression of the monooxygenase ( MNX) gene from Arabidopsis thaliana via Agrobacterium-mediated transformation. The bacterium harbored a plasmid pBinplus carrying a 1.25-kb MNX coding sequence together with a GUS reporter gene; the former was driven by the MS2 promoter of a male sterility gene in Arabidopsis, and the latter was under the control of CaMV 35S promoter. Twenty-seven putative transgenic plants (T0) were obtained, all of which showed GUS activity and positive signals of NPTII and MNX genes by PCR analysis, and also showed male sterility to some extent. It was further confirmed by Southern blotting that one copy of the NPTII and MNX gene was integrated in the genome of the plants which expressed male sterility to a higher degree. Northern blotting assay also demonstrated that the transgenes stably transcribed in the genome of the transgenic plants in F4 generation. The male sterile plants usually display lower plant height, shortened internodes, shrunken anthers without pollen grains or with some abortive pollen grains, and unusual leaves with deeper multi-lobes. Microscope observations on the meiosis processes of pollen mother cells (PMCs) showed that the abortion of pollen grains mainly resulted from abnormalities of meiosis such as direct degeneration of PMCs, degenerations of dyad and tetrads, amitosis, lagging chromosomes, and the multi-polar segregations of chromosomes and so on. This study indicates a method of developing novel cotton male sterile materials for potential application in agriculture and for engineering of male sterility in other important crops. [ABSTRACT FROM AUTHOR]