Assessment of genetic diversity among 16 promising cultivars of ginger using cytological and molecular markers

Ginger (Zingiber officinale Roscoe) is an economically important plant, valued all over the world. The existing variation among 16 promising cultivars as observed through differential rhizome yield (181.9 to 477.3 g) was proved to have a genetic basis using different genetic markers such as karyotype, 4C nuclear DNA content and random amplified polymorphic DNA (RAPD). The karyotypic analysis revealed a differential distribution of A, B, C, D and E type of chromosomes among different cultivars as represented by different karyotype formulas. A significant variation of 4C DNA content was recorded in ginger at an intraspecific level with values ranging from 17.1 to 24.3 pg. RAPD analysis revealed a differential polymorphism of DNA showing a number of polymorphic bands ranging from 26 to 70 among 16 cultivars. The RAPD primers OPC02, OPA02, OPD20 and OPN06 showing strong resolving power were able to distinguish all 16 cultivars. The extent of genetic diversity among these cultivars was computed through parameters of gene diversity, sum of allele numbers per locus and Shannon's information indices. Cluster analysis, Nei's genetic similarity and genetic distances, distribution of cultivars into special distance classes and principal coordinate analysis and the analysis of molecular variance suggested a conspicuous genetic diversity among different cultivars studied. The genetic variation thus detected among promising cultivars of ginger has significance for ginger improvement programs. Key words: Ginger, Karyotype, RAPD Introduction Zingiber officinale is rich in secondary metabolites such as oleoresin. It possesses an unique combination of properties like anti-inflammatory, aphrodisiac, antioxidant and antibacterial activity S. Nayak et al. · Genetic Diversity among Ginger Cultivars In the past decade, DNA polymorphism has become the marker of choice for the identification and characterization of plants. It is a relatively reliable, generally applicable method to obtain large samples of markers from any species of plant. However, each marker system samples a different fraction of the genomes and therefore has a different resolving power, range of applicability and probability of homology. The random amplified polymorphic DNA (RAPD) technique has been widely used in cultivar identification programs The objectives of this study were to (1) fingerprint ginger cultivars for identification and (2) detect the genetic diversity and relatedness of 16 cultivars sampled from different geographical regions using karyotypic analysis, 4C DNA content and RAPD analysis. In this study, many analytical procedures such as a n-j method, bootstrapping, spatial genetic structure analysis (SGS), and analysis of molecular variance (AMOVA) have been widely used to derive genetic distances among cultivars and to assess the structure of genetic data in a reduced dimensional space. Materials and Methods Plant materials Sixteen promising cultivars of ginger (Zingiber officinale) were included in the present study which were collected from the turmeric germplasm collection of the Orissa University of Agriculture and Techology (OUAT), Bhubaneswar, Orissa, India. These cultivars were initially collected from different parts of India Karyotype analysis Young and healthy root-tips of different cultivars of ginger were pre-treated in a (0.02 m) hydroxyquinoline mixture (1:1) for 3.5 h at 14 ∞C followed by overnight fixation in propionic ethanol. Chromosome staining was made in 2% lacto propionic orcin after cold hydrolysis in 5 n HCl for 7 min. Root-tips were squashed in 45% propionic acid. Ten well scattered metaphase plates were selected for karyotype analysis of each species. The chromosome morphology was determined following the method of 4C DNA content For Feulgen cytophotometric estimation of 4C DNA, ten fixed root-tips from each cultivar (2n = 22 chromosomes) were hydrolysed in 1 n HCl for 12 min at 60 ∞C, washed in distilled water and stained in Schiff's reagent for 2 h at 14 ∞C; each root-tip squash was prepared in 75% acetic acid. Ten scorings were made from each slide and the 4C DNA content was estimated from metaphase chromosomes using a NIKON Optiphot microscope with a microspectrophotometer following the method of Sharma and Sharma (1980) with monochromatic light at 550 nm. In situ DNA values were obtained on the basis of optical density measurements which were converted to picograms (pg) using Vant Hoff's 4C nuclear DNA value (67.1 pg) for Allium cepa as standard Isolation of DNA Total plant DNA was isolated from fresh and young leaves. The leaves were harvested freshly and washed thoroughly with cold autoclaved distilled water and then blotted to dry. About 2 g leaf was excised from the upper tip portion of the buds. DNA extraction was done on the day of collection. The genomic DNA was isolated following the protocol of Doyle and Doyle (1990) with a little modification. Insoluble poly(vinylpyrrolidone) was added to the leaf tissue prior to grinding. The crude DNA was purified with RNase A (@ 60 µg ml Ð1 of DNA solution) followed by washing with S. Nayak et al. · Genetic Diversity among Ginger Cultivars 487 purified chloroform/isoamylalcohol (24:1). To test the quality and quantity of the purified DNA, the samples were electrophoresed in a 0.8% agarose gel along with a known amount of uncut lambda DNA (Bangalore Genei Pvt. Ltd, Bangalore, India) as standard. The sample DNA was diluted as 25 ng µl Ð1 for RAPD-PCR analysis. RAPD amplification Twenty random decamer primers (Operon Tech., USA) from A, C, D and N series (OPA02, 03, 04, 08, 16; OPAF14; OPC02, 05; OPD03, 07, 08, 18, 20; and OPN02, 03, 04, 06, 07, 10, 12) were used for RAPD analysis. RAPD assays were performed in a final volume of 25 µl containing 10 mm Tris-HCl [tris(hydroxymethyl)aminomethane], pH 9.0, 1.5 mm MgCl 2 , 50 mm KCl and 0.01% gelatin, 200 µm of each dNTPs, 0.4 µm primer, 25 ng template DNA and 0.5 unit of Taq DNA polymerase (Bangalore Genei, Bangalore, India). The RAPD analysis was performed as per the methodology described by The amplification products were electrophoresed in 1.5% agarose gel containing ethidium bromide (@ 0.5 µg ml Ð1 ) in TAE buffer (40 mm Tris base, 20 mm sodium acetate, 20 mm EDTA, glacial acetic acid to pH 7.2) for 3 h at 60 V. A total of 2.5 µl loading buffer (1.0 X TAE, 50% glycerol, 0.25% bromophenol blue and 0.25% xylene cyanol) was added to each reaction before electrophoresis. After electrophoresis, the gels were observed under an UV-transilluminator, documented in Gel-Doc 2000 (Bio-Rad) and photographed. Resolving power According to Prevost and Wilkinson (1999) the resolving power (Rp) of a primer is: Rp = Σ IB, where IB (band informativeness) takes the value of: 1-[2 ¥ (0.5 Ð p)], p being the proportion of the 16 genotypes (ginger cultivars analyzed) containing the band. Data collection and analysi

Assessment of genetic diversity in a highly valuable medicinal plant Catharanthus roseus using molecular markers

Genetic diversity was evaluated among 14 cultivars of Catharanthus roseus using RAPD and ISSR markers.The RAPD primers resulted in the amplification of 56 bands, among which 46 (82%) bands were polymorphic Four ISSRprimers amplified 31 loci out of which 17 were polymorphic and 14 are monomorphic. The Jaccard's similarity derived fromthe combined marker system showed that the varieties First Kiss Coral and Cooler Orchid were the most closely relatedcultivars, with 98% similarity. In the dendrogram constructed on the basis of both RAPD and ISSR data two clear clusterswere obtained. The smaller cluster included C. roseus Cv Blue Pearl and C. roseus Cv. Patricia White and the larger clusterwas subdivided into two sub clusters with C. roseus Cv. First Kiss Polka Dot isolated from the rest of the cultivars. This maybe useful for breeding for improved quality

Genetic Stability of Micropropagated Ginger Derived from Axillary Bud through Cytophotometric and RAPD Analysis

A protocol was developed for the in vitro propagation of ginger (Zingiber officinale) cv. Suprava using dormant axillary buds from unsprouted rhizomes. The dormant axillary buds embedded in the rhizome nodes were induced to sprout when cultured on MS medium supplemented with 6-benzyladenine (BA) alone (1Ð6 mg/l) or with a combination of BA (1Ð 6 mg/l) and indole-3-acetic acid (IAA) (0.5, 1 mg/l). In vitro sprouted buds were transferred to the multiplication medium containing various combinations of auxins and cytokinins. MS basal medium supplemented with BA (1 mg/l), IAA (1 mg/l) and adenine sulfate (100 mg/l) was found optimum for the in vitro multiplication of shoots producing (8.2 ð 0.2) shoots from a single explant within 30 days of culture. The multiplication rate remained unchanged in subsequent subcultures. Rooting of shoots occurred in the same multiplication media. Upon transfer of the in vitro culture to ex vitro in pots, 96% of plants survived and established successfully under natural conditions. Tissue culture-raised plantlets of ginger could be conserved in vitro through subculturing at an interval of 4 months. The genetic stability of micropropagated clones was evaluated at regular intervals of 6 months up to 24 months in culture using cytophotometric estimation of 4C nuclear DNA content and random amplified polymorphic DNA (RAPD) analysis. Cytophotometric analysis revealed a unimodal distribution of the DNA content with a peak corresponding to the 4C value (23.1 pg), and RAPD analysis revealed monomorphic bands showing the absence of polymorphism in all fifty regenerants analyzed, thus confirming the genetic uniformity among in vitro grown somaclones of Z. officinale. This study is of commercial significance as axillary bud explants are available throughout the year for initiating a fresh culture of the elite ginger cv. Suprava to be used as a source of true-to-type disease-free planting material thereby minimizing the adverse effect of repeated subculturing from the same explant source

In Silico Biology of H1N1: Molecular Modelling of Novel Receptors and Docking Studies of Inhibitors to Reveal New Insight in Flu Treatment

Influenza is an infectious disease caused by RNA viruses of the family Orthomyxoviridae. The new influenza H1N1 viral stain has emerged by the genetic combination of genes from human, pig, and bird’s H1N1 virus. The influenza virus is roughly spherical and is enveloped by a lipid membrane. There are two glycoproteins in this lipid membrane; namely, hemagglutinin (HA) which helps in attachment of the viral strain on the host cell surface and neuraminidase (NA) that is responsible for initiation of viral infection. We have developed homology models of both Hemagglutinin and Neuraminidase receptors from H1N1 strains in eastern India. The docking studies of B-Sialic acid and O-Sialic acid in the optimized and energy-minimized homology models show important H-bonding interactions with ALA142, ASP230, GLN231, GLU232, and THR141. This information can be used for structure-based and pharmacophore-based new drug design. We have also calculated ADME properties (Human Oral Absorption (HOA) and % HOA) for Oseltamivir which have been subject of debate for long

Studies on genetic diversity in elite varieties of <i>Chrysanthemum</i> using RAPD and ISSR markers

161-169Genetic diversity in 40 elite varieties of Chrysanthemum was evaluated through two types of PCR based markers, random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR). These elite varieties resulted in the amplification of 214 bands with 16 RAPD and 4 ISSR primers. Of these amplicons, 6 were found to be monomorphic and rests were polymorphic, indicating presence of high level of genetic diversity in the studied varieties. All the varieties were related with each other with an average correlation of 0.42 Jaccard’s coefficient of similarity. The varieties ‘Robinhood’ and ‘Classic Red’ were most closely related (68% similarity level), while ‘Thyokangha’ and ‘Phil Houghton’ were widely apart from the variety ‘Maharaja’ (23% similarity level). The cluster analysis among the variant shows two major clusters containing the spray types and standard types. The above results illustrate that although the genetics of Chrysanthemum is very complex, RAPD and ISSR are powerful tools to detect the genetic diversity in its varieties. </span

Separation of the genera in the subtribe Cassiinae (Leguminosae: Caesalpinioidae) using molecular markers

Random amplified polymorphic DNA (RAPD), Inter simple sequence repeat (ISSR) and Amplified fragment length polymorphism (AFLP) markers were used to verify the segregation of the genus Cassia L. senso lato into three distinct genera namely Chamaecrista Moench., Senna P. Mill. and Cassia L. sensostricto Eighteen representatives of the three taxa were characterized using the molecular markers. 25 RAPD, six ISSR primers and six AFLP primer combinations resulted in the amplification of 612, 115 and 622 bands (loci) respectively. Most of the loci are found to be polymorphic, showing high degrees of genetic diversity among the different taxa studied. The dendrogram constructed on the basis of the RAPD, ISSR and AFLP data using SHAN clustering, divided Cassia L. senso lato. into three different clusters as Chamaecrista Moench. Senna P. Mill. and Cassia L. senso stricto High bootstrap value revealed that all the clusters were stable and robust. It was observed from the present investigation that these genera have their identity at molecular level, which supports the elevation of the genus Cassia L. senso lato to the level of subtribe Cassiinae and segregation into three distinct genera instead of intrageneric categories