Identification and characterization of microRNAs in Asiatic cotton (Gossypium arboreum L.)

To date, no miRNAs have been identified in the important diploid cotton species although there are several reports on miRNAs in upland cotton. In this study, we identified 73 miRNAs, belonging to 49 families, from Asiatic cotton using a well-developed comparative genome-based homologue search. Several of the predicted miRNAs were validated using quantitative real time PCR (qRT-PCR). The length of miRNAs varied from 18 to 22 nt with an average of 20 nt. The length of miRNA precursors also varied from 46 to 684 nt with an average of 138 ±120 nt. For a majority of Asiatic cotton miRNAs, there is only one member per family; however, multiple members were identified for miRNA 156, 414, 837, 838, 1044, 1533, 2902, 2868, 5021 and 5142 families. Nucleotides A and U were dominant, accounted for 62.95%, in the Asiatic cotton pre-miRNAs. The Asiatic cotton pre-miRNAs had high negative minimal folding free energy (MFE) and adjusted MFE (AMFE) and high MFE index (MFEI). Many miRNAs identified in Asiatic cotton suggest that miRNAs also play a similar regulatory mechanism in diploid cotton.     

Alternative approach for consistent yields of total genomic DNA from cotton (Gossypium hirsutum L.)

A persistent limitation to molecular biological research on cotton (Gossypium spp.) has been the difficulty in isolation of total genomic DNA from the plant tissue. This report describes a reliable strategy for isolation of genomic DNA from cotton. The mini-preparation procedure involves use of lyophilized, etiolated cotyledons and an anion exchange column kit. The isolated DNA had a molecular weight in excess of 50 kb with minimal degradation or shearing. Routine yields ranged from 5 to 7 μg DNA per etiolated cotyledon pair (corresponding to 100 ng/mg dry weight), in contrast to little or no DNA from equivalent amounts of either green cotyledons or mature leaf tissue. The decreased yields from the latter tissues appeared to be correlated with increased afmounts of flavonoid. The DNA was amenable to routine molecular applications as demonstrated by: digestibility with a number of restriction enzymes (Eco RI,HindIII,Sau 3A), and hybridization of a tomato genomic clone containing the gene for S-adenosylmethionine synthetase to a 13.3-kbEco RI fragment of cotton. Using DNA from an isoline immune to root-knot nematodes, we observed no impediment to genomic cloning.     

Characterization of GhRac1 GTPase expressed in developing cotton (Gossypium hirsutum L.) fibers

Cytoskeleton assembly plays an important role in determining cotton fiber cell length and morphology and is developmentally regulated. As in other plant cells, it is not clear how cytoskeletal assembly in fibers is regulated. Recently, several Rac/Rop GTPases in Arabidopsis were shown to regulate isotropic and polar cell growth of root hairs and pollen tubes by controlling assembly of the cytoskeleton. GhRac1, isolated from cottonseeds, is a member of the Rac/Rop GTPase family and is abundantly expressed in rapidly growing cotton tissues. GhRac1 shows the greatest sequence similarity to the group IV subfamily of Arabidopsis Rac/Rop genes. Overexpression of GhRac1 in E. coli led to the production of a functional GTPase as shown by in vitro enzyme activity assay. In contrast to other Rac/Rop GTPases found in cotton fiber, GhRac1 is highly expressed during the elongation stage of fiber development with expression decreasing dramatically when the rate of fiber elongation declines. The association of highest GhRac1 expression during stages of maximal cotton fiber elongation suggests that GhRac1 GTPase may be a potential regulator of fiber elongation by controlling cytoskeletal assembly.     

Initiation and characterization of a cotton (Gossypium hirsutum L.) photoautotrophic cell suspension culture

A heterotrophic cotton (Gossypium hirsutum L. cv. Stoneville 825) cell suspension culture was adapted to grow photoautotrophically. After two years in continuous photoautotrophic culture at 5% CO₂ (balance air), the maximum growth rate of the photoautotrophic cell line was a 400% fresh weight increase in eight days. The Chl concentration was approximately 500 g per g fresh weight.Elevated CO₂ (1%–5%) was required for culture growth, while the ambient air of the culture room (600 to 700 ul CO₂ 1^–1) or darkness were lethal. The cell line had no net photosynthesis at 350 ul 1^–1 CO₂, 2% O₂, and dark respiration ranged from 29 to 44 mol CO₂ mg^–1 Chl h^–1. Photosynthesis was inhibited by O₂. The approximate 1:1 ratio of ribulose 1,5-bisphosphate carboxylase (RuBPcase) to phosphoenolpyruvate carboxylase (PEPcase) (normally about 6:1 in mature leaves of C₃ plants) was due to low RuBPcase activity relative to that of C₃ leaves, not to high PEPcase activity. The PEPcase activity per unit Chl in the cell line was identical to that of spinach leaves, while the RuBPcase activity was only 15% of the spinach leaf RuBPcase activity. RuBPcase activity in the photoautotrophic cells was not limited by a lack of activation in vivo, since the enzyme in a rapidly prepared cell extract was 73% activated. No evidence of enzyme inactivation by secondary compounds in the cells was found as can be found with cotton leaves. Low RuBPcase activity and high respiration rates are most likely important factors in the low photosynthetic efficiency of the cells at ambient CO₂.Abbreviations Chl chlorophyll - COT heterotrophic cotton cell line - COT-P photoautotrophic cotton cell line - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - RuBPcase RuBP carboxylase - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - MX Murashige and Skoog medium with 0.4 mg 1^–1 2,4-D - KT photomixotrophic medium with 1% sucrose - KT^o KT medium with no carbohydrate - KTP^o KT^o medium supplemented with 0.3 M Picloram - CER CO₂ exchange rate - PCER CO₂ exchange rate in the light     

Isolation and characterization of a sterile-dwarf mutant in Asian cotton （Gossypium arboreum L.）

Plant height is an important trait in cotton. To elucidate the molecular mechanisms of the dwarf phenotype, a sterile-dwarf mutant derived from Gossypium arboreum L. cv. Jinhuazhongmian was developed by ^60Co y-ray irradiation. The results demonstrated that the steriledwarf mutant phenotype was controlled by a pair of recessive gene, which was designated sd^a. Plants carrying the sd^a gene contained lower levels of indole-3-acetic acid （IAA） and abscisic acid （ABA） compared with wild-type （WT） plants. The chlorophyll content and net photosynthetic rate in mutant leaves were markedly decreased. However, it was possible that ABA biosynthesis or signaling was involved in governing the sd^a phenotype. Semi-quantitative RT-PCR analysis detected 13 differentially expressed ESTs, and the steriledwarf mutant exhibited decreased expression levels relative to the WT. The role of nine potential hormone biosynthetic genes in the synthesis of IAA, ABA, polyamines （PAs） and jasmonic acid （JA） were discussed.     

Mapping and genomic targeting of the major leaf shape gene (L) in Upland cotton (Gossypium hirsutum L.)

Key message

A major leaf shape locus (L) was mapped with molecular markers and genomically targeted to a small region in the D-genome of cotton. By using expression analysis and candidate gene mapping, two LMI1 -like genes are identified as possible candidates for leaf shape trait in cotton.

Abstract

Leaf shape in cotton is an important trait that influences yield, flowering rates, disease resistance, lint trash, and the efficacy of foliar chemical application. The leaves of okra leaf cotton display a significantly enhanced lobing pattern, as well as ectopic outgrowths along the lobe margins when compared with normal leaf cotton. These phenotypes are the hallmark characteristics of mutations in various known modifiers of leaf shape that culminate in the mis/over-expression of Class I KNOX genes. To better understand the molecular and genetic processes underlying leaf shape in cotton, a normal leaf accession (PI607650) was crossed to an okra leaf breeding line (NC05AZ21). An F₂ population of 236 individuals confirmed the incompletely dominant single gene nature of the okra leaf shape trait in Gossypium hirsutum L. Molecular mapping with simple sequence repeat markers localized the leaf shape gene to 5.4 cM interval in the distal region of the short arm of chromosome 15. Orthologous mapping of the closely linked markers with the sequenced diploid D-genome (Gossypium raimondii) tentatively resolved the leaf shape locus to a small genomic region. RT-PCR-based expression analysis and candidate gene mapping indicated that the okra leaf shape gene (L ^o ) in cotton might be an upstream regulator of Class I KNOX genes. The linked molecular markers and delineated genomic region in the sequenced diploid D-genome will assist in the future high-resolution mapping and map-based cloning of the leaf shape gene in cotton.     

Isolation and characterization of resistance and defense gene analogs in cotton (Gossypium barbadense L.)

Plant disease resistance gene (R gene) and defense response gene encode some conserved motifs. In the present work, a PCR strategy was used to clone resistance gene analogs (RGAs) and defense gene analogs (DGAs) from Sea-island cotton variety Hai7124 using oligonucleotide primers based on the nucleotide-binding site (NBS) and serine/threonine kinase (STK) in the R-gene and pathogenesis-related proteins of class 2 (PR2) of defense response gene. 79 NBS sequences, 21 STK sequences and 11 DGAs were cloned from disease-resistance cotton. Phylogenic analysis of 79 NBS-RGAs and NBS-RGAs nucleotide sequences of cotton already deposited in GenBank identified one new sub-cluster. The deduced amino acid sequences of NBS-RGAs and STK-RGAs were divided into two distinct groups respectively: Toll/Interleukin-1 receptor (TIR) group and non-TIR group, A group and B group. The expression of RGAs and DGAs having consecutive open reading frame (ORF) was also investigated and it was found that 6 NBS-RGAs and 1 STK-RGA were induced, and 1 DGA was up-regulated by infection of Verticillium dahliae strain VD8. 4 TIR-NBS-RGAs and 4 non-TIR-NBS-RGAs were arbitrarily used as probes for Southern-blotting. There existed 2–10 blotted bands. In addition, since three non-TIR-NBS-RGAs have the same hybridization pattern, we conjecture that these three RGAs form a cluster distribution in the genome.     

Isolation and characterization of resistance and defense gene analogs in cotton (Gossypium barbadense L.)

Plant disease resistance gene (R gene) and defense response gene encode some con-served motifs. In the present work,a PCR strategy was used to clone resistance gene analogs (RGAs) and defense gene analogs (DGAs) from Sea-island cotton variety Hai7124 using oligonucleotide primers based on the nucleotide-binding site (NBS) and serine/threonine kinase (STK) in the R-gene and pathogenesis-related proteins of class 2 (PR2) of defense response gene. 79 NBS sequences,21 STK sequences and 11 DGAs were cloned from disease-resistance cotton. Phylogenic analysis of 79 NBS-RGAs and NBS-RGAs nucleotide sequences of cotton already deposited in GenBank identified one new sub-cluster. The deduced amino acid sequences of NBS-RGAs and STK-RGAs were divided into two distinct groups respectively: Toll/Interleukin-1 receptor (TIR) group and non-TIR group,A group and B group. The expression of RGAs and DGAs having consecutive open reading frame (ORF) was also investigated and it was found that 6 NBS-RGAs and 1 STK-RGA were induced,and 1 DGA was up-regulated by infection of Verticillium dahliae strain VD8. 4 TIR-NBS-RGAs and 4 non-TIR-NBS-RGAs were arbitrarily used as probes for Southern-blotting. There existed 2-10 blotted bands. In addition,since three non-TIR-NBS-RGAs have the same hybridization pattern,we conjecture that these three RGAs form a cluster distribution in the genome.     

Genetics of allozyme variation in Gossypium arboreum L. and Gossypium herbaceum L. (Malvaceae)

Summary Seed protein extracts from 90 accessions of Gossypium arboreum and 70 accessions of Gossypium herbaceum were electrophoretically analyzed for isozyme variation. Eighteen enzyme systems were resolved, ten of which were polymorphic among accessions. No within accession isozyme variation was observed within these highly inbred lines. A minimum of 24 genes encode the isozymes resolved and data is presented for codominant inheritance at 13 loci. Tests for non-random joint segregation in 63 of the 78 possible two-locus combinations from the 13 characterized loci give evidence for four pairs of linked genes (Lap2/Me1 [r=0.160+/-0.027], Lap2/Pgi1 [r= 0.285+/-0.055], Mdh6/Tpi1 [r= 0.197+/-0.028], and 6Pgd2/6Pgd3[r 0.000]. Numerous presumptive duplicate isozyme loci were observed and these were usually expressed as patterns of nonsegregating heteromultimers within accessions. Single gene expression was also observed at several loci. The observed results are in agreement with those of previous cytological investigations which have proposed the polyploid origin of the diploid Old World Gossypiums.     

Transformation of cotton (Gossypium hirsutum L.) via particle bombardment

Embryogenic suspension cultures of cotton (Gossypium hirsutum L.) were subjected to particle bombardment, where high density particles carrying plasmid DNA were accelerated towards the embryogenic plant cells. The plasmid DNA coating the particles encoded hygromycin resistance. One to two weeks following bombardment, embryogenic cotton cells were placed in proliferation medium containing 100 g/ml hygromycin. Clumps of tissue which grew in the presence of hygromycin were subcultured at low density into fresh hygromycin-containing proliferation medium. Following sequential transfer of embryogenic tissue to development and then germination media, plants were recovered from transgenic embryogenic tissue. Southern hybridization confirmed the presence of the hygromycin resistance gene in embryogenic suspension culture tissue and regenerated plants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Aph IV aminoglycoside phosphotransferase type IV Salaries and research support were provided by State and Federal funds appropriated to OSU/OARDC and USDA-ARS. Mention of trademark or proprietary products does not constitute a guarantee or warranty of the product by OSU/OARDC or USDA, and also does not imply approval to the exclusion of other products that may also be suitable. Journal Article No. 354-89     

Bin mapping of genomic and EST-derived SSRs in melon (Cucumis melo L.)

We report the development of 158 primer pairs flanking SSR motifs in genomic (gSSR) and EST (EST-SSR) melon sequences, all yielding polymorphic bands in melon germplasm, except one that was polymorphic only in Cucurbita species. A similar polymorphism level was found among EST-SSRs and gSSRs, between dimeric and trimeric EST-SSRs, and between EST-SSRs placed in the open reading frame or any of the 5′- or 3′-untranslated regions. Correlation between SSR length and polymorphism was only found for dinucleotide EST-SSRs located within the untranslated regions, but not for trinucleotide EST-SSRs. Transferability of EST-SSRs to Cucurbita species was assayed and 12.7% of the primer pairs amplified at least in one species, although only 5.4% were polymorphic. A set of 14 double haploid lines from the cross between the cultivar “Piel de Sapo” and the accession PI161375 were selected for the bin mapping approach in melon. One hundred and twenty-one SSR markers were newly mapped. The position of 46 SSR loci was also verified by genotyping the complete population. A final bin-map was constructed including 80 RFLPs, 212 SSRs, 3 SNPs and the Nsv locus, distributed in 122 bins with an average bin length of 10.2 cM and a maximum bin length of 33 cM. Map density was 4.2 cM/marker or 5.9 cM/SSR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rapid in-vitro plant regeneration of cotton (Gossypium hirsutum L.)

A rapid, clonal propagation procedure has been developed to regenerate mature cotton (Gossypium hirsutum L.) plants from pre-existing meristems that were excised from in-vitro-grown tissues. This plant regeneration procedure was applicable to diverse cotton germplasms and required specific concentrations of 6-benzylaminopurine (BA) depending on the origin of the meristems. All shoots regenerated directly without a callus phase. Screening BA concentrations (0.0–10.0 μm) demonstrated that shoot meristems (apices), secondary leaf nodes, primary leaf nodes, and cotyledonary nodes derived from in-vitro-grown 28-day-old seedlings (Paymaster HS26) varied in their ability to form elongated shoots depending on the level of BA. Indicative of a germplasm-independent procedure, a BA concentration screen (0.0, 0.3, 1.0 μm) demonstrated that explants with pre-existing meristems, excised from diverse germlines, were also able to form elongated shoots at 0.3 μm BA. In most cases, elongated shoots derived from this procedure were rooted by a two-step process: an in-vitro maturation step (Murashige and Skoog medium-activated charcoal) followed by planting into soil after basal application of Rootone. This BA plant regeneration procedure was rapid, reproducible, and highly efficient for Stoneville 7A, Paymaster HS26, and other high-fiber-yielding germlines. Regenerated plants were phenotypically normal and all of the mature plants regenerated to date have initiated flowers and set viable R₁ seeds. Received: 15 March 1997 / Revision received: 28 August 1997 / Accepted: 5 September 1997     

Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.)

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.     

Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.)

Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.Abbreviations NAA 1 naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog - BA 6 benzylamino purine - 2i P N⁶-(²-isopentenyladenine