A molecular insight into papaya leaf curl-a severe viral disease.

Papaya leaf curl disease (PaLCuD) caused by papaya leaf curl virus (PaLCuV) not only affects yield but also plant growth and fruit size and quality of papaya and is one of the most damaging and economically important disease. Management of PaLCuV is a challenging task due to diversity of viral strains, the alternate hosts, and the genomic complexities of the viruses. Several management strategies currently used by plant virologists to broadly control or eliminate the viruses have been discussed. In the absence of such strategies in the case of PaLCuV at present, the few available options to control the disease include methods like removal of affected plants from the field, insecticide treatments against the insect vector (Bemisia tabaci), and gene-specific control through transgenic constructs. This review presents the current understanding of papaya leaf curl disease, genomic components including satellite DNA associated with the virus, wide host and vector range, and management of the disease and suggests possible generic resistance strategies.

Genetic diversity, population structure and association analysis in linseed (Linum usitatissimum L.).

The present investigation aimed to explore the level of genetic diversity, determine the population structure in a larger set of germplasm of linseed using microsatellite marker and identify linked markers through association mapping. A total of 168 accessions of linseed were evaluated for major agro-economic traits and SSRs markers deployed for diversity assessment. A total of 337 alleles were amplified by 50 SSRs ranging from 2 to 13 with an average of 6.74 ± 2.8 alleles per loci. The neighbor joining based clustering grouped all the accessions into three major clusters that were also confirmed by scatter plot of PCoA. While model based clustering determined four sub-populations (K = 4). Further, analysis of molecular variance analysis considering three population showed that maximum variation (79%) was within the population. We identified one putative SSR marker (Lu_3043) linked with days to 50% flowering through both GLM and MLM analysis of association mapping. The results of this preliminary study revealed genetic diversity, population structure in linseed and linked marker which could be utilized in future breeding program.

Diversity analysis in Indian genotypes of linseed (Linum usitatissimum L.) using AFLP markers.

AFLP fingerprinting of 45 Indian genotypes of linseed was carried out to determine the genetic relationship among them. Sixteen primer combinations produced 1142 fragments with 1129 as polymorphic and 13 as monomorphic fragments. Polymorphic fragments varied from 44 (E-ACA/M-CTA) to 94 (E-AGC/M-CAC) with an average of 70.6 fragments per primer combination. The frequency of polymorphism varied from 93.7% to 100% with an average of 98.8% across all the genotypes. The PIC value ranged from 0.19 to 0.31 with an average of 0.23 per primer combination. The primer pair E-AGC/M-CAC showed the maximum PIC value (0.31) followed by E-AGC/M-CAG (0.29), E-AAC/M-CAG (0.26) and E-AGC/M-CTA (0.25). Resolving power (RP) and marker index (MI) varied from 13.73 to 43.50 and 8.81 to 28.91 respectively. The Jaccard's similarity coefficient varied from 0.16 to 0.57 with an average of 0.26 ± 0.05. The maximum genetic similarities (57%) were detected between genotypes Him Alsi-1 and Him Alsi-2, followed by Him Alsi-1 and GS41 and GS41 and LC-54. The genotypes R-552, Himani, RKY-14, Meera, Indira Alsi-32 and Suyog were found to be more divergent genotypes. The NJ clustering grouped all the 45 genotypes into three major clusters. In general the genotypes of cluster III had high oil content and those of cluster I had low oil content. At the population level, within population variance was much higher than between populations variance.

Strategy for a generic resistance to geminiviruses infecting tomato and papaya through in silico siRNA search.

Use of siRNA is a powerful methodology to particularly knockdown the targeted genes in a sequence specific manner. The potential of siRNA can be harnessed for silencing specific geminiviral genes in papaya and tomato plant hosts, thus making them resistant to the respective viruses. The challenge is in designing exogenous siRNA which can trigger silencing of viral genes irrespective of the genetic variability in different viral isolates and at the same time the selected siRNA does not target any plant gene (off target silencing). In this study, we have designed siRNA from the most conserved regions of viral coat protein (AV1) and replicase (AC1) genes retrieved from different isolates of geminiviruses infecting papaya (PLCV), and tomato (TLCV & TLCV, Northern India), so as to give a broad spectrum resistance and efficient silencing as it is highly homology-dependent strategy. Software siRNA finder (Ambion) was used on the selected conserved sequences in order to select only those putative siRNA oligonucleotides which fulfill all the basic criteria required as per the algorithm. Finally, a cross search using BLAST was performed to confirm that the designed siRNAs do not have any homology to plant genome sequences. The putative siRNA sequences thus designed to target essential genes of geminiviruses and introduced into the plants may facilitate developing papaya and tomato crops with generic resistance to geminiviruses.

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 Nei's 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.

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.

Analysis of diversity amongst widely distributed and endemic Atalantia (family Rutaceae) species from Western Ghats of India.

The genus Atalantia Correa is represented in India with four species and two varieties. The infra - generic classification and the species limits in Atalantia are, however, not well defined, due to the occurrence of intermediate forms. Two species, A. racemosa Wight and A. monophylla (L.) DC., are widely distributed, while the third species, A. wightii Tanaka is endemic in the Western Ghats, a well-known biodiversity hotspot. PCR-based methods have been commonly used for the assessment of genetic diversity in plants. We report for the first time the genetic diversity within and between populations of the above three species using two Single Primer Amplification Reaction (SPAR) methods. AMOVA analysis clearly indicates the lesser diversity among the species than within them. The UPGMA tree included all of the doubtful species in one single sub-cluster within the major cluster of A. racemosa and A. wightii, suggesting that these are probably hybrids derived from these two species. At the population level, all A. monophylla populations grouped together in a cluster that was clearly separated from all other species and populations.

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.

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.

Molecular distinction amongst varieties of mulberry using RAPD and DAMD profiles.

BACKGROUND: Mulberry 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 DISCUSSION: The 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 Jaccard's 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. CONCLUSIONS: The 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.

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.

Aspergillus fumigatus strains recovered from immunocompromised patients (ICP): subtyping of strains by RAPD analysis.

Invasive pulmonary aspergillosis in immunocompromised patients (ICP) is the second most frequent opportunistic fungal infection. The causative organism includes 16 species of Aspergillus, of which A. fumigatus dominates the ubiquitous incidence of invasive or allergic broncho-pulmonary aspergillosis (ABPA). The definitive diagnosis of invasive aspergillosis is difficult. We have analyzed 24 strains of A. fumigatus recovered from ICP using the RAPD technique. The profiles generated with the 20 primers tested were mostly unique. These results may have a profound impact on the management of aspergillosis, especially in the ICP.

RAPD profile variation amongst provenances of neem.

Neem, described as a tree for solving global problems, is an evergreen, long-lived, multipurpose tree of the tropics with a wide distribution range in India. It is believed to be highly cross-pollinated. Inter-provenance variations have been reported in neem in case of morphological and physiological characters. Yet no reports about the genetic determinism for these variations are available to our knowledge. In order to have an idea about the extent and/or nature of genetic (DNA) variation in neem, the powerful RAPD technique has been employed. RAPD profiles of 34 accessions/provenances of neem were generated with 200 decamer random primers, of which the data from the 49 primers, that resulted in reproducible amplification products, were considered for analysis. Based on the presence/absence of bands, a similarity matrix was computed. Dendrogram was constructed by UPGMA method based on the pairwise similarities amongst the RAPD profiles. The similarities in RAPD profiles amongst the different DNAs was more than that expected due to the cross-pollinated nature of the tree and furthermore, these more-than-expected similarities were not due to random chance. These results suggest that neem may have a narrow genetic base.

RAPD analysis of Candida albicans strains recovered from different immunocompromised patients (ICP) reveals an apparently non-random infectivity of the strains.

The opportunistic imperfect fungus Candida albicans causing life-threatening infections in immunocompromised patients (ICP), especially in HIV-positive cases, is recognized to be one of the most important nosocomial pathogens in the recent decades. The extent of strain-to-strain variation within a species and its relationship to the ability of the organism to colonize or invade a specific group of patients or even a body site is, however, not well known. We have analysed 19 strains of C. albicans recovered from ICP at different locales and times, employing the RAPD technique. No two strains generated identical RAPD profiles with any of the 21 primers tested. Further, the UPGMA clustering of the strains seemingly reflected a certain relationship or nonrandomness in the infection of the patients with the strain of C. albicans vis-a-vis the immunocompromised status due to underlying disease such as diabetes, cancer, asthma and meningitis. These results may have a profound impact on the management of candidiasis, especially in the ICP.

Identification of a dispersed MboI repeat family in five higher plant genomes.

Digestion of nuclear DNAs of five plants, namely Cucurbita maxima (red gourd), Trichosanthes anguina (snake gourd), Cucumis sativus (cucumber), Cajanus cajan (pigeon pea) and Phaseolus vulgaris (french bean) with the restriction endonuclease MboI yielded discrete size classes with molecular weights in the range of 0.5 to 5 kbp. The MboI digestion pattern of Cot 0.1 DNA in french bean is comparable with that of total DNA, indicating that these bands represented highly repeated DNA sequences. Cleavage of the DNAs with varying amounts of MboI indicated the dispersed nature of the repeat families. Southern hybridization studies using french bean highly repetitive DNA as a probe indicated more homology with repeats of pigeon pea and less homology with red gourd, snake gourd and cucumber repeats.