Shaik G. Mastan

Molecular characterization and identification of markers for toxic and non-toxic varieties of Jatropha curcas L. using RAPD, AFLP and SSR markers

Jatropha curcas L., a multipurpose shrub has acquired significant economic importance for its seed oil which can be converted to biodiesel, is emerging as an alternative to petro-diesel. The deoiled seed cake remains after oil extraction is toxic and cannot be used as a feed despite having best nutritional contents. No quantitative and qualitative differences were observed between toxic and non-toxic varieties of J. curcas except for phorbol esters content. Development of molecular marker will enable to differentiate non-toxic from toxic variety in a mixed population and also help in improvement of the species through marker assisted breeding programs. The present investigation was undertaken to characterize the toxic and non-toxic varieties at molecular level and to develop PCR based molecular markers for distinguishing non-toxic from toxic or vice versa. The polymorphic markers were successfully identified specific to non-toxic and toxic variety using RAPD and AFLP techniques. Totally 371 RAPD, 1,442 AFLP markers were analyzed and 56 (15.09%) RAPD, 238 (16.49%) AFLP markers were found specific to either of the varieties. Genetic similarity between non-toxic and toxic verity was found to be 0.92 by RAPD and 0.90 by AFLP fingerprinting. In the present study out of 12 microsatellite markers analyzed, seven markers were found polymorphic. Among these seven, jcms21 showed homozygous allele in the toxic variety. The study demonstrated that both RAPD and AFLP techniques were equally competitive in identifying polymorphic markers and differentiating both the varieties of J. curcas. Polymorphism of SSR markers prevailed between the varieties of J. curcas. These RAPD and AFLP identified markers will help in selective cultivation of specific variety and along with SSRs these markers can be exploited for further improvement of the species through breeding and Marker Assisted Selection (MAS).

Molecular characterization and genetic diversity analysis of Jatropha curcas L. in India using RAPD and AFLP analysis.

Jatropha curcas L. belongs to family Euphorbiaceae, native to South America and widely distributed in South and Central America, attained significant importance for its seed oil which can be converted to biodiesel, a renewable energy source alternative to conventional petro-diesel. Very few attempts were made to understand the extent of genetic diversity that exists in J. curcas. Therefore, the present investigation was undertaken to asses the genetic diversity among 28 diverse germplasm collected from distinct geographical areas in India. The overall percentage of polymorphism (PP) was found to be 50.70 and 60.95 by RAPD and AFLP, respectively. The mean PP was found to be 9.72 and 20.57 by RAPD and AFLP, respectively. The mean genetic similarity was observed to be 0.89 by RAPD and 0.88 by AFLP. Among the germplasm JCI20 found to be the most diverged one. The dendrogram analysis of RAPD and AFLP data showed good congruence, but better resolution and more polymorphism was observed with AFLP. When the dendrogram of RAPD was compared with AFLP dendrogram, the major clustering pattern was found to be similar; however, changes in minor grouping were observed. In both RAPD and AFLP analysis clustering of germplasm did not show any correlation with the geographical area of collection. Low genetic diversity observed in J. curcas and the clustering pattern indicates that the distribution of species might have happened through anthropogenic activity and warrants the need for widening the genetic base. The present study will provide pavement for further intra-population studies on narrow geographical areas, to understand the population genetic structure, phylogeography, molecular ecological studies. The marker information and the characterized germplasm help in further improvement of the species through marker assisted breeding programs.

Isolation of novel microsatellites using FIASCO by dual probe enrichment from Jatropha curcas L. and study on genetic equilibrium and diversity of Indian population revealed by isolated microsatellites.

Jatropha curcas L. belongs to family Euphorbiaceae, native to South America attained significant importance for its seed oil which can be converted to biodiesel, a renewable energy source alternative to conventional petrodiesel. Very few attempts were made to isolate novel microsatellite markers and assessment of the extent of genetic equilibrium and diversity that exists in J. curcas. Therefore, the present investigation was undertaken to isolate the novel microsatellites and access genetic equilibrium, diversity that exists among 44 diverse germplasm collected from distinct geographical areas in India using isolated microsatellites. The overall efficiency of the enrichment of microsatellite by dual probe in the present study found to be 54% and among the sequences obtained the percentage of sequences having suitable flanking regions for the primer designing was found to be 89.58%. The mean co-efficient of genetic similarity (CGS) was found to be 0.97. The overall diversity obtained by microsatellites was found to be low in comparison with the diversity reported by multilocus markers systems observed in earlier studies; however, the good allele polymorphism was observed. The overall dendrogram of microsatellite analysis resulted in random clustering of germplasm and not in accordance to geographical area of collection. The present study, diversity analysis using microsatellite markers concludes the low genetic diversity and genetic disequlibrium of J. curcas in India and will provide pavement for further intra-population studies on narrow geographical areas to understand the population genetic structure, phylogeography and molecular ecological studies. The germplasm characterized, and the microsatellite markers isolated and characterized in the present study can be employed efficiently in breeding programs for genetic improvement of the species through marker assisted selection and QTL analysis, for further genetic resource management and help in making the J. curcas as potential crop with superior agronomical traits.

Assessment of Genetic Stability and Instability of Tissue Culture-Propagated Plantlets of Aloe vera L. by RAPD and ISSR Markers

Efficient plantlet regeneration with and without intermediate callus phase was achieved for a selected genotype of Aloe vera L. which is sweet in test and used as a vegetable and source of food. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) marker assays were employed to evaluate genetic stability of plantlets and validate the most reliable method for true-to-type propagation of sweet aloe, among two regeneration systems developed so far. Despite phenotypic similarities in plantlets produced through both regeneration systems, the differences in genomic constituents of plantlets produced through intermediate callus phase using soft base of inflorescence have been effectively distinguished by RAPD and ISSR markers. No polymorphism was observed in regenerants produced following direct regeneration of axillary buds, whereas 80% and 73.3% of polymorphism were observed in RAPD and ISSR, respectively, in the regenerants produced indirectly from base of the inflorescence axis via an intermediate callus phase. Overall, 86.6% of variations were observed in the plantlets produced via an intermediate callus phase. The occurrence of genetic polymorphism is associated with choice of explants and method used for plantlet regeneration. This confirms that clonal propagation of sweet aloe using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. On the other hand, a high degree of variations were observed in plantlets obtained through indirect regeneration and thus cannot be used for the mass multiplication of the genotype; however, it can be used for crop improvement through induction of somaclonal variations and genetic manipulations.

Cross species amplification ability of novel microsatellites isolated from Jatropha curcas and genetic relationship with sister taxa : Cross species amplification and genetic relationship of Jatropha using novel microsatellites ()

The present investigation was undertaken with an aim to check the ability of cross species amplification of microsatellite markers isolated from Jatropha curcas—a renewable source of biodiesel to deduce the generic relationship with its six sister taxa (J. glandulifera, J. gossypifolia, J. integerrima, J. multifida, J. podagrica, and J. tanjorensis). Out of the 49 markers checked 31 markers showed cross species amplification in all the species studied. JCDS-30, JCDS-69, JCDS-26, JCMS-13 and JCMS-21 amplified in J. curcas. However, these markers did not show any cross species amplification. Overall percentage of polymorphism (PP) among the species studied was 38% and the mean genetic similarity (GS) was found to be 0.86. The highest PP (24) and least GS (0.76) was found between J. curcas/J. podagrica and J. curcas/J. multifida and least PP (4.44) and highest GS (0.96) was found between J. integerrima/J. tanjorensis. Dendrogram analysis showed good congruence to RAPD and AFLP than nrDNA ITS data reported earlier. The characterized microsatellites will pave way for intraspecies molecular characterization which can be further utilized in species differentiation, molecular identification, characterization of interspecific hybrids, exploitation of genetic resource management and genetic improvement of the species through marker assisted breeding for economically important traits.

Genetic Improvement in Jatropha curcas Through Selection and Breeding

Jatropha curcas L. has been labeled globally as the most likely crop for future biofuels. However, it suffers from low seed yield and high plant growth. Therefore, systematic selection from the existing germplasm, their breeding for improved characteristics and later domestication are the prerequisites to achieve genetic improvement in this species. To initiate breeding programme in any species, it is essential to assess the extent of the existing variability before embarking upon selection of superior germplasm. Further knowledge is needed on the reproductive biology of Jatropha, including its phenology, and molecular diversity in order to improve its productivity, the ultimate objective being to mass multiply improved true-to-type germplasm. In this review, we made an attempt to discuss the currently available information on Jatropha particularly the genetic variability in the species and possibilities for exploitation of the genetic potential for its improvement.

Karyology and Genomics of Jatropha: Current Status and Future Prospects

Global warming, population, environmental degradation and food production are serious concerns to the well being of mankind. Development of sustainable energy resources is an essential component to many of these problems. Bioenergy holds great promise to contribute significantly to reduce petroleum consumption and emission of green house gases. Biodiesel derived from the oil of Jatropha curcas seed is emerging as an alternative to fossil fuel, since it has the desirable physiochemical characteristics and performance even superior to conventional petroleum diesel. In addition, the plant is able to grow on marginal lands, which eliminates the “food versus fuel” dilemma. However, large scale cultivation of J. curcas remains the single most important issue that will ultimately decide of its success. Despite the availability of a vast germplasm with wide variability, not much progress has been made in developing varieties or hybrids for higher oil yield, better agricultural and economic features. Additional information about the karyology, genetic diversity and genomics is necessary to generate mapping populations, marker assisted selection and to develop superior genotypes. In this review, we discuss the state of the art of genetic improvement research for J. curcas.

TDZ-Induced Plant Regeneration in Jatropha curcas : A Promising Biofuel Plant

In recent years, Jatropha curcas has pronounced attention due to its capacity of production of biodiesel. Uniform large-scale propagation of J. curcas is one of the significant keys that will eventually decide victory. Direct regeneration is one of the methods which help in the production of uniform and homogenous plant, and TDZ plays an important role in the production of plantlets by direct organogenesis in several number of plant species including J. curcas. Measuring the economical importance of J. curcas and the role of TDZ in shoot regeneration, the present book chapter briefly reviews the impact of TDZ on shoot bud induction from various explants of J. curcas.

Soil microbial diversity shift as affected by conversion of shallow and rocky wastelands to Jatropha curcas L. plantation

The cultivation in wastelands of Jatropha curcas as a biofuel crop avoids the alleged food vs. fuel dilemma. Converting wastelands into Jatropha plantation will produce changes in microbial composition which in turn may have a profound effect on biochemical activities and physico-chemical properties of soil. These changes may be permanent and the soil may attain a different equilibrium through this land use change. The objective of this study was to compare the microbial diversity of native undisturbed soil of barren rocky wasteland and soil drawn from 24 months old J. curcas plantation. Cultivation of Jatropha employing suitable agronomic practices improved soil organic carbon, available P and K. Total soil DNA extracts were used as template DNA to amplify 16S and 18S rDNA gene fragments that were further sequenced and taxonomically assigned by comparisons with gene bank resources. Diversity indices showed that the microbial diversity was higher and more evenly distributed in native soil than in Jatropha planted soil. A prominent diversity shift from Ascomycota in the native soil to Basidiomycota and Chytridiomycota in the Jatropha bulk soils was observed. Interestingly, the conversion from barren land to Jatropha cultivation with recommended agricultural practices also brought about a marked decrease in population of several fungal pathogens. There was a striking increase in members of Proteobacteria (1.7-fold) as well as of Bacteroidetes in Jatropha planted soil as compared to native soil. In contrast, there was a decrease in the Acidobacteria and Chloroflexi community in Jatropha planted soil. It would be useful to follow the microbial pattern over the long term and to study the evolution of the Jatropha soil ecosystem on wastelands.

Isolation and sequence characterization of DNA-A genome of a new begomovirus strain associated with severe leaf curling symptoms of Jatropha curcas L.

Begomoviruses belong to the family Geminiviridae are associated with several disease symptoms, such as mosaic and leaf curling in Jatropha curcas. The molecular characterization of these viral strains will help in developing management strategies to control the disease. In this study, J. curcas that was infected with begomovirus and showed acute leaf curling symptoms were identified. DNA-A segment from pathogenic viral strain was isolated and sequenced. The sequenced genome was assembled and characterized in detail. The full-length DNA-A sequence was covered by primer walking. The genome sequence showed the general organization of DNA-A from begomovirus by the distribution of ORFs in both viral and anti-viral strands. The genome size ranged from 2844 bp-2852 bp. Three strains with minor nucleotide variations were identified, and a phylogenetic analysis was performed by comparing the DNA-A segments from other reported begomovirus isolates. The maximum sequence similarity was observed with Euphorbia yellow mosaic virus (FN435995). In the phylogenetic tree, no clustering was observed with previously reported begomovirus strains isolated from J. curcas host. The strains isolated in this study belong to new begomoviral strain that elicits symptoms of leaf curling in J. curcas. The results indicate that the probable origin of the strains is from Jatropha mosaic virus infecting J. gassypifolia. The strains isolated in this study are referred as Jatropha curcas leaf curl India virus (JCLCIV) based on the major symptoms exhibited by host J. curcas.