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Bangladesh decodes Jute Plant Genome

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  1. TopCat

    TopCat ELITE MEMBER

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    Bangladeshi researchers have successfully decoded the Jute Plant Genome.


    With the successful sequencing of jute genome, Bangladesh has become only the second country after Malaysia, among the developing nations, to achieve such a feat.

    Researchers from Dhaka University, Bangladesh Jute Research Institute and Software Company DataSoft in collaboration with Centre for Chemical Biology University of Science Malaysia and University of Hawaii, USA has decoded the genome.


    Prime Minister Sheikh Hasina made the announcement of Bangladesh’s scientific adherence in the Parliament on Wednesday.


    Dubbing it a ‘historic scientific advancement’, Sheikh Hasina said the discovery would rejuvenate the lost heritage of ‘golden fibre’ as gene mapping of jute would now help breeders develop jute varieties resistant to pests and climate adversities
     
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  2. Skies

    Skies SENIOR MEMBER

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    Plant Tissue Cult. & Biotech. 15(2): 145-156, 2005 (December)

    source: http://74.125.155.132/scholar?q=cac...equencing+of+Jute.&hl=en&as_sdt=2000&as_vis=1

    PTC&B
    Preliminary Progress in Jute (Corchorus species)
    Genome Analysis

    Ahmad S. Islam, Matthew Taliaferro, Christopher T. Lee, Craig
    Ingram, Rebecca J. Montalvo, Gerrit van der Ende, Shahabudin
    Alam1, Javed Siddiqui1
    and Kanagasabapathi Sathasivan*

    Molecular Cell and Developmental
    Biology, School of Biological Sciences, The University
    of Texas at Austin, USA

    Key words: Genomic DNA, Corchorus olitorius, C. capsularis, WU-BLAST, TAIR,
    18S rRNA, tRNA-Leu

    Abstract

    The paper summarizes the progress made in cloning and sequencing a limited
    number of genes from jute (Corchorus olitorius and C. capsularis) and discusses
    future applications of jute genome analysis. As of December 2005, slightly over
    200 DNA sequences have been deposited in GenBank. Although many of these
    sequences are partial and uncharacterized, this marks the beginning of a major
    step in unraveling of the hitherto unknown jute genome. We have constructed
    both cDNA and genomic DNA libraries for C. olitorius var. O4 and C. capsularis
    var. CVL-1 in the plasmid vectors pSMART and pBluescript, respectively.
    Random clones were isolated and sequenced. These DNA sequences have been
    deposited in GenBank and analyzed using TAIR (TAIR - Home Page) for
    similar sequences in Arabidopsis thaliana and other related plant species. The
    complete sequence for tRNA-Leu and partial sequence of DNA fragments
    encoding several proteins, such as RNA polymerase β subunit-1, 18S rRNA,
    mitochondrial DNA directed RNA polymerase and carboxytransferase β-subunit
    are reported in this paper. The analysis of DNA sequences of related taxa
    deposited in GenBank is also presented delineating the scope and applications of
    cloning genes of agronomic importance.

    Introduction

    Jute, the world’s second most cultivated fiber crop next to cotton, is extensively
    grown in India, Bangladesh, China, Thailand, Russia, Myanmar, Nepal,
    Uzbekistan, Chile and Brazil (FAOSTAT). India and Bangladesh
    rank as the top two countries in terms of jute production. In Bangladesh, jute is
    the principal cash crop and jute products contribute a significant portion to

    *Corresponding author (sata@mail.utexas.edu); 1DNA Technologies, Gaithersburg, Maryland. 146 Islam et al.
    country’s foreign exchange (bangladeshgov.org). Jute
    cultivation helps sustain millions of farmers in these two countries alone. The
    cultivated varieties of jute have been evolved from Corchorus olitorius L. and C.
    capsularis L. through conventional breeding and pure line selection (Ghosh 1983)
    based on their yield and agronomic performance. Synthetic fibers have been a
    major competition in the international market to the natural jute fibers. In recent
    years the situation has improved because natural fibers do not pollute the
    atmosphere as do synthetics and may help reduce the cutting of trees for making
    paper. In addition, jute is now being utilized to manufacture more value-added
    industrial products such as in the making of Geo-textiles (Dedicated Servers | Managed Dedicated Servers | Web Hosting | VPS | Liquid Web.
    jute.com/geojute.html) for protecting embankments against river erosion, fiber
    reinforced building materials, packaging materials and in the production of
    paper. However, there are problems in increasing the productivity and
    profitability of jute. Some of the major challenges include susceptibility of the
    jute crop to insect pests and fungal diseases, photoperiod sensitivity, poor fiber
    quality, and low yield under unfavorable growth conditions such as salinity,
    drought, flood or cold. Addressing these challenges through traditional plant
    breeding program has limitations due to lack of genetic diversity among
    cultivated jute varieties and sexual incompatibilities between the cultivars of the
    two jute species and between each of the two species and wild Corchorus species.

    In spite of developing successful hybrids between two species of jute namely, C.
    olitorius and C. capsularis (Islam and Rashid 1960), it was not possible to release
    any variety from the advanced progeny of the above interspecific crosses.
    Hence, in recent years molecular approaches to improve the agronomic traits of
    jute are being considered as alternatives.

    Recently, research in some universities in the Indian subcontinent has
    resorted to molecular approaches through systematic cloning and transgenic
    work. By means of a combined study of AFLP and RAPD, Hossain et al. (2002,
    2003) at Dhaka University have shown the importance of using molecular
    markers in distinguishing between cold-tolerant and cold sensitive jute varieties
    obtained from GenBank at Bangladesh Jute Research Institute (BJRI). Using
    simple sequence repeat (SSR) marker loci and AFLP assay, Basu et al. (2004) at
    the Indian Institute of Technology (IIT), Kharagpur, evaluated genetic diversity
    of 49 genotypes of the two jute species. More recently, P. K. Gupta at Charan
    Singh University, Meerat (CSUM), India and his associates as well as the Dhaka
    University team led by Haseena Khan developed genomic SSRs and deposited
    the sequences in GenBank. By developing more SSRs Gupta and his team at
    CSUM are planning to embark upon a program of gene tagging combined with
    the construction of a framework linkage map for QTL interval mapping. In
    addition to the research on markers, procedure for successful regeneration (Seraj Preliminary Progress in Jute (Corchorus spp.) Genome Analysis 147
    et al. 1992; Saha et al. 1999) and transformation of C. capsularis (Ghosh et al. 2002)
    have been developed.

    At the University of Texas at Austin, work on the construction of cDNA and
    genomic DNA library of the above two jute species has been initiated recently.
    As a first step towards achieving this goal, a rapid method for high quality RNA
    isolation from jute was developed in this lab (Khan et al. 2004). A cDNA and
    genomic DNA libraries have been constructed in pSMART and pBluescript
    vectors from C. olitorius and C. capsularis, respectively. We have isolated and
    sequenced random fragments with the objective of testing these DNA libraries
    and deposited some of the sequences in GenBank. This paper reports analysis of
    the DNA sequences that show homology with other plant genes including that of
    Arabidopsis thaliana. It summarizes the results from the analysis of selected DNA
    sequences currently available in GenBank. We present the challenges
    encountered in cloning genes from jute and suggest possible solutions to
    overcome such problems. In addition, this paper attempts to update jute
    researchers on the current status in jute molecular biology and to establish liaison
    with any other team(s) who may be interested to collaborate in the jute genome
    project.

    Material and Methods
    Plant materials and growth: Seeds of C. capsularis var. CVL-1 and of C. olitorius var.
    O4 were supplied by Bangladesh Jute Research Institute through the courtesy of
    Haseena Khan, Dhaka University. Seeds from C. olitorius were surface sterilized
    with 10 % bleach and incubated on a moist filter paper in Petri dishes inside an
    incubator at room temperature (23°C). Seven-day-old seedlings grown inside
    Petri dishes were collected and shipped to DNA technologies, Maryland for
    RNA isolation and cDNA library construction. The sample weight was roughly
    1g. Seeds of C. capsularis were surface sterilized in the same manner and planted
    in the greenhouse under normal lighting and allowed to grow to mature plants.
    The young leaves of mature plants were harvested, frozen in liquid nitrogen and
    shipped to DNA technologies, Maryland for genomic DNA isolation and library
    construction.

    Isolation of the total RNA: The frozen jute tissues were directly grinded in the
    RNA isolation solution (Trizol-Invitrogen) in liquid nitrogen. The lysates were
    centrifuged at 10,000 × g for 10 min to remove unlysed cells and contaminants.
    The supernatant was mixed with 0.2 volume of chloroform and incubated on ice
    for 15 min. The mixture was centrifuged at 10,000 × g for 10 min and the upper
    layer was collected. The upper layer was mixed with half volume of isopropanol
    and centrifuged for 30 min at 10,000 × g. The pellet was washed with 70% ethanol 148 Islam et al.
    and air dried. The RNA was dissolved in DEPC treated autoclaved water. An
    aliquot of RNA was checked on denaturing agarose gel.
    Isolation of the mRNA: Jute mRNA was isolated from total RNA using oligo
    dT cellulose. Briefly, the total RNA was mixed with 10 ml of binding buffer (10
    mM Tris pH 7.5, 500 mM NaCl) and heated at 70°C for 5 min. The samples were
    immediately chilled on ice for 5 min and mixed with oligo dT latex beads. The
    mixture was incubated at room temperature for 2 h to allow the complete
    binding of the mRNA with oligo dT. After incubation, the mixture was
    centrifuged at 5,000 ×g. The pellet was washed twice with the binding buffer.
    Now, the pellet was washed with excess of a low salt buffer (10 mM Tris pH 7.5,
    250 mM NaCl). This step was repeated several times to remove all unbound
    RNA molecules. The mRNA was finally eluted with 10 mM Tris pH 8.0. An
    aliquot of mRNA sample was checked on denaturing agarose gel.

    Construction of the cDNA library: Two µg (micrograms) of mRNA were mixed
    with oligo dT primer (18 mer) and heated at 65°C for 10 min. The mixture was
    cooled on ice and the following cDNA synthesis reagents were added: dNTP,
    RNase inhibitor, first strand cDNA synthesis buffer and superscript reverse
    transcriptase. The reaction mixture was incubated at 42°C for 2.5 h. The second
    strands were synthesized using dNTP mix, E. coli DNA polymerase and RNase H
    in second strand buffer at 16°C for 2h. After the second strand synthesis, the
    ends were polished using Pfu polymerase at 72°C for 30 min. The cDNA was
    extracted with phenol: chloroform: isoamyl alcohol.

    EcoRI adapter ligation, kinasing of cDNA ends and size fractionation of the cDNA:
    The end-polished cDNA was mixed with EcoRI adapter, T4 DNA ligase buffer
    and a high concentration of T4 DNA ligase. The mixture was incubated at 8°C
    for 48h. The mixture was heated at 50o
    C for 5 min. The ends of cDNA were
    phosphorylated (addition of 5’-phosphate) by T4 kinase. The reaction was
    continued at 37° C for 1 h. The kinased cDNA was precipitated with ethanol and
    3M sodium acetate. The adapter ligated cDNA was re-suspended in 10 mM TE
    buffer and electrophoresed on 1% low melting point agarose gel for 3 h. The
    cDNA in the size range of 0.5 kb and higher was recovered from low melting
    point agarose by extraction with phenol; phenol chloroform and chloroform. The
    cDNA was precipitated with ethanol.

    Vector digestion, purification and the ligation of cDNA: For making the library,
    pSMART vector from Lucigen was used. The sequences and the restriction map
    of the vector are available in Advanced Products for Molecular Biology - Lucigen Corporation. The vector was digested with
    EcoRI and treated with calf intestinal alkaline phosphatase (CIAP). The digested
    vector was purified on the agarose gel. An aliquot of digested vector was ligated
    and used to electroporate E. coli DH10B cells to test the efficiency of the
    digestion. After getting successful results, the cDNA was separately ligated into Preliminary Progress in Jute (Corchorus spp.) Genome Analysis 149
    the vector in the presence of T4 DNA ligase at various concentrations. The
    samples were incubated at 8°C for 48 h. Ligated cDNA was precipitated with
    ethanol and re-suspended in water. An aliquot of the ligated samples were
    electroporated in E. coli DH10B cells. Immediately after electroporation, the cells
    were mixed with SOC medium and allowed to recover for 1.5 h at 37°C. Libraries
    were centrifuged at 5000 rpm for 10 min. The medium was discarded and the
    pellet was re-suspended in SOC medium containing the appropriate antibiotic
    and 15% glycerol. The cells were plated on LB ampicillin plates and grown at
    37°C overnight. DNA from single colonies was isolated and screened for the
    presence of inserts.

    Genomic DNA library construction: Pre-chilled (– 80°C) sterile grinder and
    pestle were used to grind young jute (C. olitorius var. O-4) leaves until the leaf
    became powdery. Five ml of lysis buffer-I containing proteinase K were added to
    the powdered leaves and the mixture was incubated at 37°C in a shaker
    overnight with a low-speed agitation. The incubated material was spun at 10,000
    rpm for 15 min at 4°C and the supernatant was collected. After adding 5 ml
    phenol : chloroform : isoamyl alcohol (PCI) to the supernatant, it was vortexed
    and spun at 10,000 rpm for 5 min. The aqueous phase was saved and 3M sodium
    acetate (0.3M final) was added to it. Nearly 2.5 volumes of 100% ethanol (cold)
    were added to the aqueous phase; the material was well mixed and stored at
    –20°C for 30 min. The re-suspended pellet was spun down after 1 ml of TE was
    added to it. Genomic DNA was then isolated by Easy-DNA Isolation Kit
    (Invitrogen, Carlsbad, CA; (cf. manufacturer’s protocol). In short, 350 µl of
    solution A were added to the DNA and incubated at 65°C for 10 min. Thereafter,
    150 µl of solution B were added and the mixture was vortexed vigorously until
    the precipitation was found to move freely. After adding 500 µl chloroform to
    the previous mixture, it was vortexed until the viscosity of the material was
    found to decrease. The next step was centrifugation at 14,000 rpm for 20 min. To
    the upper transferred aqueous phase, 1 ml of cold 100% ethanol was added,
    mixed well and left at – 20°C for 30 min. The centrifugation at 14,000 rpm for 20
    min was repeated. To the transferred upper aqueous phase, 500 µl of cold 80%
    ethanol were added and spun at 14,000 rpm for 5 min. The pellet was re-
    suspended in 100 µl TE containing RNase. The last step was incubation at 37°C
    for 30 min and the incubated product was stored at 4°C. The quality of the
    isolated genomic DNA (gDNA) was checked by running the samples on a 0.7%
    agarose gel and taking OD readings at 260 nm.

    Restriction enzyme digestion of gDNA and size fractionation: Genomic DNA was
    partially digested with restriction enzyme Apo I and Sma I (Roche Diagnostics,
    Indianapolis, IN) in separate tubes for ligation with their specific vectors.
    Approximately, an amount of 10 µg of gDNA was taken for restriction enzyme 150 Islam et al.
    digestion for 90 min; about one-third of the partially digested gDNA was
    removed from the digestion reaction and mixed with 10 µl of 0.5 M EDTA every
    30 min, followed by their storage at 4°C. The three restriction enzyme-digested
    DNA fractions were combined. The next step was to purify the digested material
    using Qiagen MinElute Gel extraction Kit (cf. manufacturer’s protocol).

    Vector preparation: Seven µg of pBluescript SK (+/-) vector (www.Stratagene.
    com) was digested twice with EcoRI and SmaI (Roche Diagnostics, Indianapolis,
    IN) in separate tubes for the ligation with their specific inserts. Briefly, the DNA
    was digested for 2.5 h at the specific temperature; thereafter more enzymes were
    added and the digestion period was prolonged for another 2 h. The digested
    DNA was inactivated by heating at 70°C for 10 min and treated with Shrimp
    alkaline phosphatase, (SAP; Roche Diagnostics, Indianapolis, IN) after it had
    cooled down to room temperature, (cf. the manufacturer’s protocol). Restriction-
    enzyme digested and SAP-treated vector DNA was then purified with PCI
    followed by treatment with chloroform: isoamyl alcohol (CIA), and finally
    precipitated with ethanol. The DNA was then re-digested with the same set of
    enzymes and the same time frame as above followed by SAP treatment, purified
    with PCI followed by CIA and precipitated with ethanol. The vector DNA was
    re-suspended with an appropriate amount of sterile H2O/TE. The quality check
    (QC) of the isolated vectors was performed by running the samples on a 0.7%
    agarose gel.

    Ligation and electroporation: The ApoI digested gDNA was ligated with the
    pBluescript SK (+/-) vector which was earlier digested with EcoRI and the Sma I
    digested gDNA was ligated with the pBluescript SK (+/-) vector which was
    earlier digested with Sma I, respectively. The ligation reaction was made for 10 µl
    and incubated for two days at 4°C. Two µl of the ligated reaction mixture were
    added to 70 µl of Electro-10 Blue competent cells (thawed at 4°C) and
    electroporated in a 0.1 cm gap pre-chilled cuvette at 1700 volts. One ml of SOC
    was added immediately. Cells were transferred to a new 15 ml culture tube,
    incubated at 37oC for 1 h and 100 µl of cells were spread on LB-agar plates
    containing ampicillin. The plates were incubated at 37°C overnight. The number
    of colonies was counted.

    Quality control (QC) of the library: Ten colonies were randomly picked up and
    grown in LB (+ antibiotic) overnight. QC was performed by restriction enzyme
    digestion (Fig. 1) or PCR methods. Once the pilot ligations were found effective
    (> 90% recombinants), large-scale ligations were set up to get at least 106 primary
    clones. The whole library was recovered in 10 ml of SOC at 37°C. One hundred
    µl of the library were plated on LB-plates (with ampicillin). The plates were
    incubated overnight. On the following day, the number of colonies was counted.
    The library was centrifuged and re-suspended in 2 ml of LB-antibiotic. Approxi- Preliminary Progress in Jute (Corchorus spp.) Genome Analysis 151
    mately, 40 - 50,000 colonies were spread on each 150 mm plate. The plates were
    incubated overnight. The last step consisted of aliquoting the libraries in 1 ml
    aliquot, labeling each container and storing them at – 70°C.

    Isolation of plasmid DNA: The plasmids containing either cDNA or gDNA
    cloned materials were isolated via alkaline lysis procedure through Qiagen spin
    columns as per manufacturer’s instructions. Once these plasmids were isolated,
    the quantity and quality of the samples were determined based on spectrophoto-
    metric (NanoDrop) analysis. In order to identify which plasmids possibly
    contained cDNA inserts, we digested each plasmid sample with EcoRV and
    EcoRI restriction enzymes and then separated the results of the digest on a 0.8%
    agarose gel stained with ethidium bromide, SyBr Green or SyBr Gold. The
    recombinant plasmids were identified and sent for sequencing at the core DNA
    sequencing facility at The University of Texas at Austin. Upon obtaining positive
    sequencing results, a BLAST nucleotide search was performed on each sample in
    order to determine to which genes the fragments were most closely related. In
    addition, we have confirmed the sequence alignment results with the WU-
    BLAST service in TAIR (TAIR - Home Page) and the results have been
    recorded.

    Results and Discussion
    The cDNA library constructed in pSMART cloning vector (Advanced Products for Molecular Biology - Lucigen Corporation)
    into the NotI/Blunt restriction site and stably maintained in the E. coli host cell
    DH10B-T1 contained 106 primary clones comprising about 90% recombinants.
    The average insert size as determined by PCR was 100 to 500 bp. The inserts
    were relatively small ranging from 100 to 500 bp. The short strands of cDNA
    could possibly be due to some inhibitory compound that might have co-purified











    Fig. 1. Results from gDNA library; ten randomly picked clones digested with
    Pvu II indicating the presence of DNA inserts in nine out of ten samples.
    with the jute RNA during the process of isolation. Special attention to prevent
    such contamination may help in the future cDNA synthesis and library
    MW 1 2 3 4 5 6 7 8 9 10
    3 kbp152 Islam et al.
    construction. Random cDNA library clones were cultured on LB kanamycin and
    the DNA isolated was sequenced.
    The total genomic DNA of C. capsularis was used in the genomic DNA
    library constructed in pBluescript KS (Genomics Master). The genomic
    library contained 106 to 107 primary clones and the average insert size was about
    100 to 1000 bp. Random clones were cultured in LB ampicillin and the plasmid
    DNA isolated was sent for sequencing. The cDNA and gDNA sequences were
    used to search for similar sequences through the TAIR (TAIR - Home Page)
    web site through WU-BLAST (Wisconsin University - BLAST). The sequence
    analysis from randomly selected clones is summarized in the Table 1.

    Table 1. Examples of the DNA sequences obtained from the jute genomic DNA and
    cDNA sequences. Repetitive gene names are mentioned here to illustrate the
    frequency of such clones in the library.

    Seq.
    No.
    TAIR result Score E value
    7
    AT1G68990. DNA-directed RNA polymerase,
    mitochondrial
    96
    2.00E-18
    13 AT3G41768. 18SrRNA 287 2.00E-76
    14 AT3G41768. 18SrRNA 344 4.00E-93
    5 ATCG00180. RNA polymerase β subunit-1 1068 0
    4 ATCG00180. RNA polymerase β subunit-1 337 2.00E-91
    9 ATCG00180. RNA polymerase β' subunit-1 1207 0
    11 ATCG00180. RNA polymerase β' subunit-1 371 1.00E-101
    15 ATCG00490. large subunit of RUBISCO. 492 1.00E-138
    3 ATCG00500. carboxytranserase β subunit 151 3.00E-35
    2 ATCG00860. hypothetical protein 101 3.00E-20
    8 ATCG00860. hypothetical protein 898 0
    10
    ATCG00905. chloroplast gene encoding ribosomal
    protein s12
    295
    8.00E-79
    12 ATCG01180. chloroplast-encoded 23S ribosomal RNA 315 9.00E-85
    6 ATCG01280. hypothetical protein 870 0
    1 tRNA-Leu 161 3.00E-38

    The information in the above Table was compiled from all sequences
    collected from June to November 2005. All sequences were compared against
    known sequences in the TAIR database (TAIR - BLAST) with
    introns and untranslated regions included (AGI Genes (+introns. +UTRs)
    (DNA)). Any entered sequence that gave homologous sequences with a
    maximum score of 50 bits or less was discarded. Thus, of 52 sequences, 37 were
    discarded. Those with maximum scores over 50 bits were analyzed further. The
    scores and error values for the homologous sequences were recorded. The
    highest score for each input sequence was noted. The probable location of the
    genes of interest, their placement in the chromosome, and the specific protein
    encoded by each individual gene were also taken note of. Analysis of the Preliminary Progress in Jute (Corchorus spp.) Genome Analysis 153
    sequences revealed the partial DNA sequence of three hypothetical proteins,
    RNA polymerase β subunit-1, 18S rRNA, mitochondrial DNA directed RNA
    polymerase, carboxytransferase β subunit, and tRNA-Leu. As mentioned above
    a known partial base sequence of putative phosphate transport ATP-binding
    protein gene, partial cds was determined in C. capsularis var. CVL-1.
    The early submission to GenBank (National Center for Biotechnology Information) of
    the DNA base sequences of Corchorus species was of C. olitorius from Harvard
    University Herbaria, MA, USA (Alverson et al. 1999). In the course of the next
    four years, this group (Whitlock et al. 2003) working at University of
    Massachusetts, Amherst published partial cDNA base sequences of NADH
    dehydrogenase (ndhF) gene of a number of wild Corchorus species namely, C.
    sidoides, C. bricchettii, C. argutus, C. siliquosus as well as fiber-yielding, species, C.
    olitorius and C. capsularis. On the basis of similarity of base sequence of ndhF
    gene, Whitlock et al. (2003) suggested that Oceanopapaver neocaledonicum be
    transferred to Corchorus.

    Another two important submissions to GenBank were by Basu et al. (2003) at
    the Indian Institute of Technology, Kharagpur. Working with C. capsularis, they
    determined complete cDNA sequence of caffeoyl-CoA-O-methyltransferase and
    cinnamyl alcohol dehydrogenase which are two of the three genes involved in
    lignin biosynthesis. Recently, Liu et al. (2005) at the Chinese Medical University,
    Taiwan deposited at the GenBank, base sequence of 18S ribosomal RNA gene
    of C. olitorius and C. capsularis as well as that of C. aestuans var. brevicaulis, a wild
    Corchorus species from Taiwan. A phylogenetic tree was constructed comprising
    several species of Corchorus and Gossypium robinsonii. Nucleotide sequences of
    18s rRNA from the four plants were compared using a clustalw multiple
    alignment (Multiple Sequence Alignment - CLUSTALW) by uploading a Fasta file of the nucleotide
    sequences into the clustalw site. After the alignment a phylogenetic tree was
    constructed using the clustalw site from the alignment score page. The NJ tree
    format was used.






    Fig. 2. Phylogenetic tree of selected Corchorus species and Gossypium robinsonii
    based on 18S rRNA gene sequences.

    The phylogenetic tree shows that Gossypium and Corchorus diverged from a
    common ancestor and that all species of the Corchorus are more closely related to
    C._capsularis
    C._olitorius

    C._aestuans_var._brevicaulis

    Cossypium_robinsonii 154 Islam et al.
    each other than to Gossypium. Furthermore, the tree shows that C. capsularis and
    C. olitorius are more closely related than each of these two species to C. aestuans.
    Thus the molecular data presented in this paper are in agreement with the
    current taxonomic classification and the crossing relationships between different
    Corchorus species as reported by the first author (Islam 1958). This phylogenetic
    tree based on 18S rRNA sequences needs to be compared taking into
    consideration of similar molecular characters before a consensus tree is
    generated.

    The joint team led by P.K. Gupta at Charan Singh University, Meerat, India
    and Haseena Khan at Dhaka University, Bangladesh submitted to GenBank
    DNA base sequences of SSR markers in 195 accessions of C. olitorius. These
    markers were used to distinguish accessions that were cold-tolerant from
    susceptible ones. The SSR sequences submitted to Genbank
    (BLAST: Basic Local Alignment Search Tool) by Sharma et al (2005) were retrieved
    and entered in TAIR website (TAIR - Home Page) to check for homology to
    sequences in other plant species, especially the genome of Arabidopsis thaliana.
    The WU-BLAST search was set to include introns and UTRs (AGI Genes
    (+introns, +UTRs) (DNA)). Sequences that gave significant homology were
    recorded. In some instances, a sequence showed homology to multiple
    sequences in other plant taxa, in which case the sequence with the greatest score
    was selected and recorded. In the case of multiple sequences having the same
    score, the one with the lowest E value was selected and recorded. Any sequence
    that returned matches with a maximum score of 50 bits or less was recorded as
    “No Hits.”

    Table 2. Summary of the sequence analysis of the jute SSR sequences
    by Sharma et al. (2005) posted in the GenBank.

    Genes Frequency Score E value
    Nuclear 79 197 to 2074 0.79 to 2.7E-88
    Mitochondrial 6 308 to 2310 6.9E-12 to 2.6E-122
    Choloroplast 3 596 to 1550 3.1E-21 to 7.6E-65
    Unidentified 107

    Of the 195 sequences analyzed, 79 showed homology to nuclear DNA with
    scores ranging from 197 to 2074, six showed homology to mitochondrial DNA
    with scores ranging from 308 to 2310, three showed homology to chloroplast
    DNA with scores ranging from 596 to 1550, and 107 were unidentified.
    The DNA libraries were made using mRNA extracted from leaves of young
    seedlings of the two jute species. So far the results submitted to GenBank are
    partial cDNA sequences of putative phosphate transport ATP-binding protein
    gene of C. capsularis var. CVL-1 (Accession No. DQ151661) and partial genomic
    base sequences of 18S ribosomal RNA gene of C. olitorius var. O4 (Accession No.
    DQ151662). Study of the randomly selected genomic DNA sequences of inserts Preliminary Progress in Jute (Corchorus spp.) Genome Analysis 155
    indicated that they represent partial sequences of the following: RNA
    polymerase β subunit, chloroplast-encoded 23S ribosomal, chloroplast gene
    encoding ribosomal s-12, DNA-directed RNA polymerase, mitochondrial
    carboxytransferase β subunit and three hypothetical proteins. These sequences
    will soon be submitted to GenBank.

    There should be a note of caution in deciphering genomic base sequences of
    Corchorus. The record of nucleotide sequences in Corchrous in the GenBank show
    that the majority of authors have not given names of the cultivar associated with
    the two fiber-yielding species. It is an important omission because a large
    amount of differences exist between cultivars of the two species of Corchorus in
    both morphological and physiological traits that are bound to be reflected in
    their DNA profiles.

    The ultimate objective of the present study was to construct a complete
    cDNA and genomic DNA libraries of both C. olitorius and C. capsularis so that the
    genes of interest can be cloned and used to transform jute cultivars for obtaining
    value-added industrial products. Since this enormous task cannot be undertaken
    without adequate funding and collaboration with institutions willing to work on
    a tropical crop, we have been studying the partial cDNA and genomic DNA
    libraries on a limited scale. So far the genes we have isolated are either
    chloroplast-, ribosomal- or mitochondrial genes or the enzymes that reside in
    these structures. The main reason for obtaining genes from the above organelles
    alone is that DNA has so far been isolated from young leaves that contain
    numerous chloroplasts and mitochondria, and such genes are more abundant
    than others. No efforts were made so far to select unique DNA fragments. Future
    analysis will involve screening of the current library with probes of these
    abundantly expressed genes to avoid repeated sequencing. Also, many of the
    sequences are partial and full length sequences need to be isolated by cloning the
    cDNA ends. Further study aims at collecting suitable material from other parts of
    the plant such as young stem, bark tissue and roots to clone genes controlling
    many important traits of agronomic importance including those that confer
    resistance to insects, fungal and viral pathogens, and those for regulating lignin
    biosynthesis. Both random sequencing and selective cloning strategies will be
    employed. In addition, tissue specific promoters will be also be cloned for
    potential genetic improvement. Since the entire jute genome analysis is a major
    effort, an international collaboration among the scientists from India,
    Bangladesh, USA and other countries is essential to help accomplish this task.
    The clones and sequences should be made available for the genetic improvement
    of jute to ultimately benefit the millions of jute farmers in the long run.

    Acknowledgment

    The authors thank Professor Haseena Khan and Samuil Haque of Dhaka
    University, Bangladesh for providing jute seeds with proper phytosanitary
    certificate. They thank Ray Neubauer, Thomas Garrad, Mohamad Wazni,
    Lindsay Stone and Nabila Anwar who helped them with the plasmid DNA 156 Islam et al.
    isolation and laboratory work. Sincere thanks to Dena Sutton and her associates
    for helping with the growing of jute plants in the greenhouse of The University
    of Texas at Austin. Finally they express their gratitude to the University CoOp
    and the University of Texas at Austin for supporting the undergraduate research
    fellowship program without which the present research could not have been
    undertaken.

    References


    Alverson WS, Whitlock BA, Nyffeler R, Bayer C and Baum DA (1999) Phylogeny of the
    core Malvales: evidence from ndhF sequence data. Am. J. Bot. 86(10), 1459-1471 (1999)
    Basu A, Maiti MK and Sen SK (2003) Cloning and sequencing of mRNA for caffeoyl-
    CoA-o-methyltransferase from Corchorus capsularis. Direct submission to GenBank.
    [Accession No. AY500159]
    Basu A, Maiti MK and Sen SK (2003) Nucleotide sequence of cinnamyl alcohol cDNA
    isolated from Corchorus capsularis. Direct submission to GenBank [Accesssion No.
    AY504425]
    Basu A, Ghosh M, Meyer R, Powell W, Basak SL and Sen SK (2004) Analysis of Genetic
    Diversity in Cultivated Jute Determined by Means of SSR Markers and AFLP
    Profiling. Crop Sci. 44: 678-685.
    Ghosh T (1983) Handbok on jute. FAO, Rome.
    Ghosh M, Saha T, Nayak P and Sen SK (2002) Genetic transformation by particle gun
    bombardment of cultivated jute, Corchorus capsularis L. Plant cell reports. 20: 936-942.
    Hossain MB, Haque S and Khan H (2002). DNA Fingerprinting of Jute Germplasm by
    RAPD. J. Biochem. and Mol. Biol. 35(4): 414-419. (BSRK and Springer-Verlag)
    Hossain MB, Awal A, Rahman MA, Haque S and Khan H (2003) Distinction between
    cold sensitive and cold tolerant jute by a combined RAPD and AFLP study. J.
    Biochem. and Mol. Biol. 36(5): 427-432 (BSRK and Springer-Verlag).
    Islam AS (1958) Crossing relationship in the genus Corchorus. Proc. Intl. Botanic Congress
    held in Montreal, Canada.
    Islam AS and Rashid A (1960) First successful hybridization between the two jute
    yielding species. Nature 185: 258-259.
    Khan F, Islam AI and Sathasivan K (2004) A rapid method for high quality RNA
    isolation from Jute: Corchorus capsularis and C. olitorius L. Plant Tissue Cult. 14: 63-68.
    Liu SL, Chang YS and Hsieh CC (2005) Identification of Corchorus species based on ITS
    sequence of nuclear ribosomal DNA in Taiwan. GenBank Submission. 08-NOV-2005.
    Graduate Institute of Chinese Pharmaceutical, China Medical University, Taichung,
    Taiwan
    Saha T, Ghosh M and Sen SK (1999) Plant regeneration from cotyledonary explants of
    jute. Corchorus capsularis L. Plant Cell Rep. 18: 544-548.
    Seraj ZI, Sarker AB and Islam, AI (1992) Plant regeneration in a jute species (C. capsularis)
    and its possible relationship with glyoxylase-I. Plant Cell Rep. 12: 29-33.
    Sharma S, Singh R, Rustgi S, Goyal A, Gau A, Tyagi AK, Khan H, Sinha MK, Balyan
    HS and Gupta PK (2005) Development and use of simple sequence repeat (SSR)
    markers for the study of DNA polymorphism, transferability and genetic diversity in
    jute. (Personal communication and sequences posted in GenBank as of Dec 2005)
    Whitlock BA, Karol KG and Alverson WS (2003) Chloroplast DNA sequences confirm
    the placement of the enigmatic Oceanopapaver within Corchorus (Grewioideae:
    Malvaceae sensu lato., formerly Tiliaceae). Intl. J. Plant Sci. 164(1): 35-41. Preliminary Progress in Jute (Corchorus spp.) Genome Analysis 157


    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

    Preliminary Progress in Jute (Corchorus spp.) Genome Analysis

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    Last edited: Jun 27, 2010
  3. Skies

    Skies SENIOR MEMBER

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    Jute Genome Sequencing:

    How it all began

    It all began in February 2008 when Dr. Maqsudul Alam approached Professor Ahmed Shamsul Islam, Coordinator of GNOBB (Global Network of Bangladeshi Biotechnologists) regarding the possibility of sequencing the jute genome. Bangladeshi scientist community already looking into the possibility of getting the jute genome sequenced jumped up at this offer and this set the ball rolling. The whole process kicked off with many long conference calls between Dr. Alam and plant molecular biologists, Professors Haseena Khan and Zeba Seraj of the Department of Biochemistry and Molecular Biology, University of Dhaka.

    Dr. Maqsudul Alam and his Visions:

    In an email titled Jute Genome, Initial Planning by Dr. Alam written on February 13, 2008 to Dr. Ahmed Shamsul Islam, Dr. Haseena Khan and Dr. Zeba Seraj on how to undertake such a mammoth he wrote: “I am not that concerned at this moment about the raw data generation part (it should be technically feasible for a ~1,200Mbp project), my thinking is post raw data generation scenario.”


    The following is the content of another mail of the same day titled: Jute Genome Initial Planning II.


    “We are talking about minimum 12 months (will be a miracle) to 36 months to have a 85-95% draft genome with minimum 4-5x coverage done (for publication). Raw data generation, based on the technology of choice can be reduced dramatically, but assembly and annotation will take time. We want to create a new generation of researchers in Bangladesh who can continue moving the frontier during and after the Jute genome and unfortunately there is no shortcut. We need to create "out-of-box" creative infrastructure that takes potential/ talents of local graduate students and post-doctoral researchers both in biological, agricultural and computer science from public & private academic/research institutions including existing young research staffs at Jute related research institutes”.

    Involvement of Department of Biochemistry and Molecular Biology, University of Dhaka and the talents of its graduates:

    In the backdrop of such exchanges Dr. Alam visited Bangladesh in March 2008. During his visit he was invited by the Bangladesh Society for Biochemistry and Molecular Biology to give a talk on his work on blood supplement and this set the stage for a number of talks and presentation including one presentation, which ran for several hours at the Department of Biochemistry and Molecular Biology on the ‘nuts and bolts’ of genome sequencing. The audience included many students and faculty from many departments of the biological science and also personnel from renowned computer software company DataSoft.


    This captivating audience helped to form the first working body more than two years before the actual project kicked off. This team was called the “Swapna Jaatra”, or the Dream Journey, a name suggested by a graduate student from Dhaka University participating in Dr. Alam’s presentation. Also in this meeting the core team was formed with two major components, biology and computing. Dr. Haseena Khan from the Department of Biochemistry and Molecular Biology, DU and Mr. Mahboob Zaman of DataSoft were made the two team leaders of the two components respectively.


    Through this brief interaction with the biology graduates from the Department of Biochemistry and Molecular Biology and also the Department of Genetic Engineering and Biotechnology of University of Dhaka Dr. Maqsudul Alam got his first very positive impression of the intellectual capability of the young individuals. This impression was later translated in the excellent handling of the raw data and this would make him to comment later that he has never come across such talented students.

    Abed Chowdhury and the Creation of Public Opinion for Jute Genome Sequencing:

    Abed Chowdhury realized the importance of sequencing the jute genome from a nationalistic point of view and also from the point of Intellectual Property Rights issue. He therefore took up the issue in local dailies and wrote articles on the need of the jute genome sequencing and how this could help to bring back the lost glory of jute. He met with the then Chief of the Caretaker Government, Dr. Fakruddin Ahmed and discussed the need for sequencing the jute genome.

    His participation in the initial planning through emails between Dr. Alam and Bangladeshi researchers of the genome project helped the project to have a firm foundation. Due to this it was easy to jump start when funds were ultimately obtained for the project.

    Involvement of BAS:

    During this visit to Bangladesh Dr. Alam with Drs. Haseena Khan and Zeba Seraj from University of Dhaka and Dr. Abed Choudhury met the General Secretary of the Bangladesh Academy of Sciences (BAS) to discuss funding opportunities by the BAS. BAS appeared positive. This first positive response led to many eventually long distance conference calls between the President and General Secretary of BAS and research team of CCB, Universiti Sains Malaysia, Penang, Malaysia. However a firm commitment from BAS was never obtained.

    Dark days of the genome project:

    After Dr. Alam’s return to the USA discussion started through telephone and email on which jute species should be sequenced. Discussions also ensued on how to prepare the DNA from a single plant for sequencing. Once the jute variety to be sequenced was decided work started with sowing of a single plant of the desired jute variety. The idea was that as soon as funds for sequencing was obtained there should not be any delay in getting quality DNA for the project. It was realized that there was tremendous competition from other jute producing countries to get the jute genome sequenced and we should in no way lose to others.

    Dr. Alam prepared a draft proposal. This proposal of ~6 million USD was used as a basis for several project proposals prepared for several funding agencies during the last two years. The two team leaders from Bangladesh would eventually meet many individuals for funding possibilities. There were many assurances but no commitment. There was a growing frustration among the Swapna Jaatra team and many members of the team left for higher studies or jobs in other countries.

    Vice Chancellors of University of Dhaka step in:

    During the two years of gestation period of this national project two separate VCs of Dhaka University took many positive steps for obtaining funds for this important project. Professor SMA Faiz, the then VC of University of Dhaka met the then chief of the Caretaker Government and discussed the possibility of funding this project. He even arranged a meeting with the Advisor for Education, CTG government and quickly endorsed a proposal made on behalf of Dhaka University on jute genome sequencing. However before any commitment from them was obtained the tenure of CTG was over.

    Begum Motia Chowdhury and the Genome Project:

    In November, 2009 Professor Arefin Siddique, VC, DU very kindly invited Begum Motia Chowdhury, Minister of Agriculture, to come to University of Dhaka and appreciate the research being carried out DU faculty at the Department of Biochemistry and Molecular Biology on jute and rice biotechnology. During her visit to DU she was informed about Dr. Maqsudul Alam and his interest to get the jute genome sequenced. Begum Motia Chowdhury enthusiastically asked for Dr. Alam’s telephone number so that she could get in touch with him. This coincided with Munir Hasan’s article in Prothom Alo on Dr. Alam and the need to get the jute genome sequenced, which appeared two days later after Begum Motia Chowdhury’s visit to DU. This article further prompted Begum Motia Chowdhury to act immediately. She called Dr. Alam in Malaysia and invited her to visit Bangladesh some time soon.


    When Dr. Alam came in December 2009 several meetings with the Agriculture Minister were arranged at different venues which included the Department of Biochemistry and Molecular Biology, DU, BARC and also the Ministry of Agriculture. During these meetings the need for urgent approval of the genome project and also the immediate release of funds was emphasized. It did not take the minister long to appreciate the importance of the project and the necessity to embark on the project as soon as possible. She understood the urgency of the limited time because of tight competition from other countries. During this short stay Begum Matia Chowdhury, Dr Maqsudul Alam, Project Manager of Centre of Chmical Biology, Malaysia Dr. Jennifer Saito met with Prime Minister. She was enthusiastic about the project to jump start. She, her Secretary and many senior staff of her ministry made all out efforts in approving the project and releasing the money in record time.

    DataSoft and the Talented Team:

    As the release of funds was being processed DataSoft with its own funds immediately set up a state-of-the-art bioinformatics laboratory with all the necessary computing facilities under very tight security surveillance. They created an excellent congenial atmosphere so that two very diverse groups, from biology and computer science could work and learn from each others strength and complete the project in record time in order to stick to the time line given to the Agriculture Minister. They very efficiently handled all the problems that came with handling huge genome data, unprecedented power outage and computer breakdowns etc. Without all these support completion of the project within the time frame would not have been possible.

    The Very Supportive Director General and talented Researchers of the Bangladesh Jute Research Institute:

    Dr. Kamaluddin, DG, BJRI provided an excellent bridge between the academia (University of Dhaka) and the private enterprise (DataSoft). This helped to form a smooth transition from analyses of raw data to the functional genomics of jute. His enthusiasm and efficient handling of delicate bureaucratic issues was the main force in a successful completion of the first stage of genome sequencing. His researchers were extremely eager to learn the newer issues of genomics and to learn how to turn the raw sequence data into a wealth of functional information.

    All these players formed a perfect winning team between public, private and semi-autonomous institutions of Bangladesh. Something that was unheard of before was turned into a reality by this winning team.

    ASGPB (USA), CCB@USM (Malaysia)

    Advance Studies in Genomics, Proteomics, Bioinformatics (ASGPB) at the University of Hawaii, USA and Centre for Chemical Biology (CCB@USM), Malaysia since the incipation all along been a solid Collaborator with their infrastructure from Raw data generation to High Performance Computer Usage.

    Future Prospects of Jute Genome Project

    Science and technology with its proper, sustainable and well-timed implementation is one of the mainstays for the development of any country. Being a developing country, Bangladesh is moving forward for her sustainable developments with diminutive pace and one of the main reasons of this is the lack of improvement in the science and technology sector. It is noteworthy that, the focus of research and development in science and technology must have to go along with the trends of the world of the present time. The 21st century is the century of biology which is mainly focused by genome research. The developed countries have already captured this drift of this research and many developing countries are underway to follow them. Being a developing country, it’s high time for Bangladesh to start actively thinking about this.



    Research and development of any country must have to congregate the country’s need, benefit her population, economy and socio-economic upgrading. Bangladesh has an agro-based economy where agriculture accounts for nearly 30% of country’s GDP and employs 65% of her labor force. So in this arena of genome research, Bangladesh should select her appropriate indigenous species which not only facilitate the improvement of her socio-economic and economic growth but also bring pride to the nation. As a country Bangladesh may be symbolized to the rest of the world by jute. Bangladesh has an old tradition of jute cultivation. Jute industry is the second largest industrial employer in the country which provides livelihoods to more than 35 million people including farmers, businessmen, workers, laborers and self employed artisans and weavers of the country. It is still the third largest source of foreign exchange. Therefore, the importance of jute research to the national welfare of Bangladesh cannot be underestimated and the focus must be narrowed down to the jute genome research to improve its quality and production. Though jute is our heritage, currently our farmers have to import them at high price. Through this project we can deliver high quality seeds having improved jute fibre and disease resistance in very low cost to the farmers. At present we only export raw jute, our neighboring countries make processed products out of this and earn five to ten times more foreign currency than us. After successful accomplishment of jute genome sequencing and its field application, we can produce export quality jute products and multiply our jute business in global market up to several folds. The government of the people’s republic of Bangladesh has realized this need and had taken the initiatives of sequencing the jute genome. The fruition of this initiative is now coming to broad day lights.



    To successfully complete this project the government in collaboration with the University of Dhaka and Data Soft, a non-government organization, has developed the work force and knowledge-base by incorporating the young graduate students from the University and the enthusiastic computer programmers from Data Soft. The whole work was co-ordinated by Bangladesh Jute Research Institute (BJRI). This step of including the young graduates is a positive sign of developing the youths of this country who in future will enlighten this nation with their knowledge and achieved experiences from this project. None the less, this type of effort will stop the threatening brain drain from this country. From the very beginning of this project, state-of-art technologies were used along with rigorous quality control in each and every step to ensure the standard of the work. These technologies established in Bangladesh will definitely shape up the research of this country to the right course and will encourage the scientists to join the technological super highway to keep pace with the contemporary researches.



    This project was an ultimate challenge for Bangladesh and the challenge has been overcome successfully. It was an eventual result of dreams and promises to do something for this country, days of hard works of the contributors and workers with the expenditure of money of the people of this country. Bangladesh with this project can now proclaim her existence with pride and honor to the rest of the world.