Shirish A. Ranade

Role of polyamines and ethylene as modulators of plant senescence

Under optimal conditions of growth, senescence, a terminal phase of development, sets in after a certain physiological age. It is a dynamic and closely regulated developmental process which involves an array of changes at both physiological and biochemical levels including gene expression. A large number of biotic and abiotic factors accelerate the process. Convincing evidence suggests the involvement of polyamines (PAs) and ethylene in this process. Although the biosynthetic pathways of both PAs and ethylene are interrelated, S-adenosylmethionine (SAM) being a common precursor, their physiological functions are distinct and at times antagonistic, particularly during leaf and flower senescence and also during fruit ripening. This provides an effective means for regulation of their biosynthesis and also to understand the mechanism by which the balance between the two can be established for manipulating the senescence process. The present article deals with current advances in the knowledge of the interrelationship between ethylene and PAs during senescence which may open up new vistas of investigation for the future.

RAPD profile based genetic characterization of chemotypic variants of Artemisia annua L.

The annual herbaceous plant, Artemisia annua L., belonging to family Asteraceae, is the natural source of the highly potent antimalarial compound, artemisinin, besides producing valuable essential oil. The plant is at present the sole commercial source for artemisinin production since all the chemical syntheses are non-viable. Therefore, economic and practical considerations dictate that plants with maximum content of artemisinin be found and/or ways to increase their artemisinin content be sought. The key to this selection and breeding is a comprehension of chemical and genetic variability and suitable selection(s) of elites from within the available population. In the present study, RAPD analyses of selected chemotypes from a decade old introduced population in India were carried out using arbitrary primers. The RAPD data clearly indicate the distinction amongst these plants. Further, the detection of highly polymorphic profiles (97 polymorphic markers out of a total of 101 markers) suggests the existence of very high levels of genetic variation in the Indian population despite geographical isolation and opens out a strong possibility of further genetic improvement for superior artemisinin content. UPGMA analyses of RAPD and phytochemical trait data indicate that the wide phytochemical diversity is included within the genetic diversity. These results further support the prospects for selection and breeding of superior artemisinin containing lines.

Molecular distinction amongst varieties of Mulberry using RAPD and DAMD profiles

BackgroundMulberry trees are the most important host for rearing mulberry silkworms in sericulture. Improved varieties of mulberry tree have been developed through traditional breeding procedures. Not much work, however, has been carried out on the molecular characterization of these varieties. Random Amplified Polymorphic DNA (RAPD) and Directed Amplification of Minisatellite DNA (DAMD) methods based on Polymerase Chain Reaction are important tools to analyze genetic diversity of mulberries. These have been used to determine variation amongst nine varieties of Morus spp. maintained at Banthra Research Station of National Botanical Research Institute, Lucknow.Results and DiscussionThe varieties were analyzed using 23 arbitrary sequence decamer primers for RAPD, and 3 minisatellite core sequence primers for DAMD reactions. The RAPD and DAMD band data, (a total of 200 bands), were used to determine the pair wise distances according to Jaccards algorithm. From these distance values Neighbour Joining (NJ) analyses were carried out separately for the RAPD and the DAMD data. The triploid varieties were found to be most similar to each other using RAPD analysis, while the varieties S13 and S34 were more similar using DAMD analysis. Nearly 85% of the RAPD bands and 91% of the DAMD bands were polymorphic across the nine varieties.ConclusionsThe mulberry varieties could be distinguished by their RAPD and DAMD profiles. As many as five RAPD primers and one DAMD primer generated profiles that can together differentiate all the nine varieties in terms of unique bands.

Genetic diversity in inter-simple sequence repeat profiles across natural populations of Indian pomegranate (Punica granatum L.).

Pomegranate (Punica granatum L.), in the monogeneric family Punicaceae, is found in Iran, Afghanistan, India and Mediterranean countries. Iran is considered to be its primary centre of origin. In India, pomegranate occurs naturally only in the Western Himalayan regions of Jammu and Kashmir, Himachal Pradesh and Uttarakhand States. However, there is no information about genetic variation in wild pomegranate at population level. In this paper, we describe genetic diversity across natural populations of Indian pomegranate based on inter-simple sequence repeat (ISSR) markers. Forty-nine accessions representing eight populations from two regions were analysed using ISSR. Seventeen ISSR primers resulted in 268 polymorphic bands, with 87.01% polymorphism throughout the accessions. Pair-wise population genetic distances ranged from 0.05 to 0.45, with a mean of 0.25 between populations. amova and Neis genetic diversity analyses revealed higher genetic variation within populations than among populations. A higher genetic differentiation (G(ST)) was observed between the spatially distant populations, indicating a low level of genetic exchange (Nm) among these populations. However, clustering of populations was not in accordance with their geographical affiliations in the tree. The results indicate that the ISSR method is sufficiently informative and powerful to assess genetic variability in pomegranate, and that patterns of genetic variability observed among populations of wild pomegranate from the Western Himalaya differ. Estimation of genetic variation reported here provides a significant insight for in situ conservation and exploitation of genetic resources for this economically important species as potential breeding material.

Single primer amplification reaction methods reveal exotic and indigenous mulberry varieties are similarly diverse.

Mulberry is the sole food source for mulberry silkworm and a number of indigenous and exotic varieties are used in sericulture. Studies on assessment of genetic diversity have been done amongst a few mulberry varieties using one or at the most two methods. However, no comprehensive study on a large number of varieties has been carried out. In present study, single primer amplification reaction (SPAR) methods have been used for determination of diversity in 27 mulberry varieties (exotic as well as indigenous), using four minisatellite core sequence primers for directed amplification of minisatellite DNA (DAMD), three simple sequence repeat (SSR) motifs as primers for inter simple sequence repeat (ISSR) and 20 arbitrary sequence decamer primers for random amplified polymorphic DNA (RAPD) reactions. The Jaccard coefficients were determined for the DAMD, ISSR and RAPD band data (total of 58, 39 and 235 bands respectively). All three methods revealed wide range of distances supporting a wide range of mulberry genetic diversity. A cumulative analysis of the data generated by three methods resulted in a neighbour-joining (NJ) tree that gave a better reflection of the relatedness and affinities of the varieties to each other. Comparison of the three methods by marker indices and the Mantel test of correlation indicated that though all methods were useful for the assessment of diversity in mulberry, the DAMD method was better. When considered as two groups (10 exotic and 17 indigenous varieties), the mulberry varieties in the exotic group were found to have slightly greater diversity than the indigenous ones. These results support the concept of naturalization of mulberry varieties at locales distant from their origins.

Variation in random amplified polymorphic DNA (RAPD) profiles specific to fluconazole-resistant and -sensitive strains of Candida albicans.

Random amplified polymorphic DNA analysis was used to detect genotype relatedness among clinical fluconazole-resistant and -sensitive strains of Candida albicans recovered from twenty HIV-infected patients having oropharyngeal candidiasis. Sensitive strains were obtained from a local hospital and were from patients that had not been treated with azole drugs while resistant strains were recovered from patients in different parts of Europe and their resistance was a consequence of drug-treatment given to the patients. On amplification with different arbitrary sequence decamer primers, the results demonstrated a homogeneous banding pattern for all sensitive strains that was distinct from that obtained in case of the resistant strains. The DNA profiles of strains were thus broadly clustered into two major groups of resistant and sensitive strains. The RAPD technique may be useful in differentiating fluconazole-resistant strains from the -sensitive ones for early identification of resistant isolates from AIDS patients.

SPAR profiles and genetic diversity amongst pomegranate (Punica granatum L.) genotypes.

We are interested in studying the distribution and range of diversity amongst the pomegranates in India. Single Primer Amplification Reaction (SPAR) profiling using Random Amplified Polymorphic DNA (RAPD) and Directed Amplification of Minisatellite DNA (DAMD) methods enabled the determination of the genetic diversity amongst a total of 64 Indian pomegranate genotypes including 15 wild, 34 semi-wild and 14 cultivated types. SPAR profile data were scored for the computation of pairwise distances as well as a Neighbour Joining (NJ) tree of all the genotypes. Eight RAPD and four DAMD primers showed discrete polymorphic patterns amongst these genotypes. From the profiles obtained with all the 12 primers considered together, 259 bands were scored. The NJ tree generated after a 1000 bootstrap test using Jaccard coefficient showed separation of Lagerstroemia speciosa used as the out-group taxon, while the pomegranate genotypes were resolved into distinct genetic lineages such that all the cultivated (except CBd70), and wild genotypes (except W101) clearly separated from other genotypes in distinct sub clusters while the semi-wild genotypes were resolved into three sub-clusters. The greatest and least distances detected between genotypes were 0.94 and 0.12, 0.97 and 0.24 and 0.95 and 0.38, amongst the cultivated, semi-wild and the wild genotypes respectively. The results indicate the high levels of genetic diversity present amongst the genotypes. Significantly, the wild genotypes also have a reasonably good range of diversity. A good germplasm collection, especially including the wild genotypes will enable a better pomegranate improvement program. Both SPAR methods, RAPD and DAMD, are found to be useful for studying the genetic diversity of pomegranate.

Genetic diversity amongst landraces of a dioecious vegetatively propagated plant, betelvine (Piper betle L.) §

Betelvine (Piper betle L., family Piperaceae) is an important, traditional and widely cultivated crop of India. The cultivators and consumers recognize more than 100 cultivars (landraces) based on regional and organoleptic considerations, while in terms of phytochemical constituents only five groups have been identified for all the landraces. Since betelvine is an obligate vegetatively propagated species, genomic changes, if any, may have become ‘fixed’ in the landraces. We carried out random amplified polymorphic DNA (RAPD) analysis in several landraces considered in four groups, namely, ‘Kapoori’, ‘Bangla’, ‘Sanchi’ and ‘Others’ in order to ascertain their genetic diversity. On the basis of the data from eleven RAPD primers, we distinguished genetic variation within and among the four groups of landraces. The results indicate the’Kapoori’ group is the most diverse. The neighbour joining (NJ) tree after a bootstrap (500 replicate) test of robustness clearly shows the four groups to be well separated. Interestingly, all known male or female betelvine landraces have separated in the NJ tree indicating an apparent gender-based distinction among the betelvines.

Genome analysis of amaranths: Determination of inter- and intra-species variations

Amaranths are an important group of plants and include grain, vegetable and ornamental types. Despite the economic importance of the amaranths, there is very little information available about the extent and nature of genetic diversity present in the genusAmaranthusat molecular level. We now report the randomly amplified polymorphic DNA (RAPD) profiles of different species ofAmaranthusas well as different accessions of the species. These RAPD analyses have been carried out using 65 arbitrary sequence decamer primers. From the RAPD data, an UPGMA dendrogram illustrating the inter-as well as intra-species relationships has been computed. The putative hybrid origin of A.dubiousfromA. hybridusandA. spinosusis also ruled out by the RAPD data. The trends of species relationships amongst the amaranths determined by RAPDs is consistent with their cytogenetic and evolutionary relationships that have already been determined.

Molecular assessment of genetic diversity in mung bean germplasm

RAPD profiles were used to identify the extent of diversity among 54 accessions of mung bean that included both improved and local land races. Out of the 40 primers screened, seven primers generated 174 amplification products with an average of 24.85 bands per primer. The RAPD profiles were analysed for Jaccard’s similarity coefficients that was found to be in the range from 0 to 0.48, indicating the presence of wide range of genetic diversity at molecular level. Cluster analysis was carried out based on distances (1-similarity coefficient) using neighbour-joining method in Free Tree package. The dendrogram resolved all the accessions into two major clusters, I (with 11 accessions) and II (with 43 accessions). However, the cluster was further divided into four subclusters (II A with six, II B with nine, II C with 15 and II D with 13 accessions). The distribution of the accessions in different clusters and subclusters appears to be related to their performance in field conditions for 10 morphological traits that were scored. This study indicated that the RAPD profiles provide an easy and simple technique for preliminary genetic diversity assessment of mung bean accessions that may reflect morphological trait differences among them.