Genetic diversity and gene flow estimation in Prosopis cineraria (L.) Druce: A key stone tree species of Indian Thar Desert

Selected amplicon data obtained through our earlier study using ISSR and DAMD markers were utilized for determination of diversity within and among the populations of Prosopis cineraria (L.) accessions collected from different districts of Rajasthan (India). A total of 83 bands were generated from eight ISSR and five DAMD primers of which 79 were found to be polymorphic (95.18%). Nei’s gene diversity (h) ranged between 0.185 and 0.301 with overall diversity of 0.316 while Shannon’s information index (I) values recorded between 0.253 and 0.438 with an average value of 0.243. The gene flow value (1.713) and the diversity among populations (0.226) demonstrated higher genetic variation within the population. It is concluded that P. cineraria is accompanied by high genetic diversity within the population and elevated gene flow showing indications of adaptation to callous and fragile dry conditions of arid environment.     

Carnivorous Plants as a Source of Potent Bioactive Compound: Naphthoquinones

Carnivorous plants are among the curiosities of nature being different from the normal plants in their mode of nutrition. These plants have fascinated several researchers for centuries. They are also characterized by synthesis of bioactive compounds which are used as a mechanism for self defense. These compounds possess a broad spectrum of biological activities such as antiparasitic, antibacterial, insecticidal, fungicidal, anti-inflammatory, antipyretic, antiproliferative activities. Although, several antimicrobial drugs have been introduced during the recent decades, the problems of microbial infections resistant to synthetic pesticides still exist which necessitate the introduction of novel antimicrobial agents with additional modes of actions than the currently available therapeutic agents. Naphthoquinones are one of the most studied bioactive compounds which have been reported to inhibit the growth of proliferative cells and microbes. Efforts have been made to induce the biosynthesis of naphthoquinone in different species of carnivorous plants. It has been demonstrated that the accumulation of naphthoquinones in carnivorous plants was increased by injecting chitin into the plant tissues. Also, their biosynthesis could be enhanced by the incorporation of elicitors in in vitro cultures of plants. In the present review, we discuss the applications of naphthoquinones and its biosynthesis in carnivorous plants.     

Applications of CRISPR/Cas9 technology for modification of the plant genome

Genetica - The CRISPR/Cas (Clustered regularly interspaced short palindromic repeats/ CRISPR associated protein 9) system was discovered in bacteria and archea as an acquired immune response to...     

Assessment of genetic variation and identification of species-specific ISSR markers in five species of Cymbidium (Orchidaceae)

Thirty-five inter-simple sequence repeat (ISSR) markers were used to analyze the genetic variation in Cymbidium spp. High number of polymorphic bands (217) with overall 90 % of polymorphism at inter-specific level was observed. Cumulative genetic similarity ranged from 0.40–0.93 with an average value of 66 % among the species. At intra-specific level, average polymorphism detected, ranged from 29.8 to 69.9 % within the five species of Cymbidium. All the species were apparently endowed with low genetic variation at intra-specific level compared to inter-specific level. UPGMA clustering evidently distinguished the representatives of C. aloifolium and C. tigrinum which may be linked to entirely different climatic conditions in which they grow, besides their discrete morphological characteristics. Nine ISSR primers revealed 11 unique species-specific banding patterns belonging to three Cymbidiums, which can further developed as SCAR markers. Thus, present investigation provides valuable baseline data of genetic variation in five species of Cymbidium and addresses the conservation concerns of this horticulturally important orchid.     

Single primer amplification reaction (SPAR) reveals inter- and intra-specific natural genetic variation in five species of Cymbidium (Orchidaceae)

A total of 53 primers belonging to three SPAR methods, viz. RAPD, ISSR and DAMD, collectively produced 456 polymorphic amplicons with 96.6% polymorphism at inter-specific level in five species of Cymbidium, viz. C. aloifolium, C. mastersii, C. elegans, C. eburneum and C. tigrinum, whereas at intra-specific level, the observed polymorphism ranged from 51.2% to 77.1% among them. Three SPARs collectively revealed 25 unique species-specific amplicons; most of them were amplified with RAPD and DAMD primers besides few bands which were either missed (absent) or lost (heterozygosity). UPGMA clustering evidently distinguished the representatives of C. aloifolium and C. tigrinum, with distinct genetic distance, which may be due to their entirely different habitats as well as discrete morphological characteristics. Upon analysis of the data generated, all the three SPAR methods, either independently and/or in combination, revealed wide range of genetic variation between and within five species of Cymbidium. Comparison of matrix of individual SPAR method revealed that analysis of natural genetic variation using combination of SPAR methods, rather than an isolated approach, is highly effective. The critical analyses of the amplicon data are indicative of DAMD as the most powerful SPAR method by showing highest resolving power (Rp) followed by ISSR and RAPD. Alternatively, the total polymorphic information content was highest in case of RAPD followed by other two SPAR methods. Thus, the present investigation for the first time provides a valuable baseline data for genetic variation at inter- and intra-specific levels in horticultural Cymbidiums and also addresses conservation concerns.     

Single primer amplification reaction (SPAR) methods reveal subsequent increase in genetic variations in micropropagated plants of Nepenthes khasiana Hook. f. maintained for three consecutive regenerations

The genetic fidelity of in vitro-raised plants of three successive regenerations of Nepenthes khasiana Hook. f. was assessed using three different single primer amplification reaction (SPAR) methods, viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of minisatellite DNA region (DAMD) markers. Out of 80 RAPD primers screened, 14 primers reflected a genetic variation of 4.1% in the first regeneration which was increased to 9.4% in the third regeneration. In the case of ISSR, out of 36 primers screened for assessment of genetic homogeneity of the regenerated plantlets, 12 primers showed an increase of genetic variation from 4.3% to 10% from the first to the third regenerations. In DAMD profiling, 15 primers were used for the evaluation of genetic fidelity where 8.47% of polymorphism was observed in the first regeneration which was increased to 13.33% in the third regeneration. The cumulative analysis reflected a genetic variation of 5.65% in the first regeneration which increased subsequently to 7.77% in the second regeneration and 10.87% in the third regeneration. The present study demonstrates SPAR technique to be an efficient tool for the assessment of clonal fidelity of in vitro-raised plants.     

Single primer amplification reaction (SPAR) reveals intra-specific natural variation in Prosopis cineraria (L.) Druce

Prosopis cineraria, an important multipurpose tree and vital component of the otherwise fragile ecosystem of arid and semiarid regions of India. It is highly drought tolerant and sprouts profusely during the extreme dry summer months when most other trees are leafless. P. cineraria is known to exhibit comparable genetic variations at intra-specific and inter-population levels reflected through morphological and cytogenetical diversity in regions, where this plant grows naturally. In the present study, single primer amplification reaction (SPAR) methods have been used for determination of diversity at DNA level in 30 accessions of P. cineraria collected from different districts of Rajasthan. The analyses include the use of six minisatellite core sequence primers for direct amplification of minisatellite DNA (DAMD), eight inter simple sequence repeats (ISSR) and 20 arbitrary primed decamer sequences for random amplification (RAPD) reactions. Upon analysis of the data generated, all the three SPAR methods, either independently and/or in combination, revealed wide range of genetic variation among accessions. Comparison of matrix of individual SPAR method using MxComp component of NTSYS-pc 2.02 K software proving that analysis of natural genetic variation using combination of SPAR methods particularly ISSR and DAMD, rather than an isolated approach, is very effective. Such an approach also yields better information and reflection of the relatedness and affinities at intra-species and inter-population levels. Therefore, it is opined that in order to reveal the intrinsic intra-specific variation, SPAR approaches involving more than one DNA marker may reveal more authentic genetic variation in tropical tree species like P. cineraria.     

Species-genomic relationships among the tribasic diploid and polyploid Carthamus taxa based on physical mapping of active and inactive 18S–5.8S–26S and 5S ribosomal RNA gene families,and the two tandemly repeated DNA sequences

In the genus Carthamus (2n = 20, 22, 24, 44, 64; x = 10, 11, 12), most of the homologues within and between the chromosome complements are difficult to be identified. In the present work, we used fluorescent in situ hybridisation (FISH) to determine the chromosome distribution of the two rRNA gene families, and the two isolated repeated DNA sequences in the 14 Carthamus taxa. The distinctive variability in the distribution, number and signal intensity of hybridisation sites for 18S–26S and 5S rDNA loci could generally distinguish the 14 Carthamus taxa. Active 18S–26S rDNA sites were generally associated with NOR loci on the nucleolar chromosomes. The two A genome taxa, C. glaucus ssp. anatolicus and C. boissieri with 2n = 20, and the two botanical varieties of B genome C. tinctorius (2n = 24) had diagnostic FISH patterns. The present results support the origin of C. tinctorius from C. palaestinus. FISH patterns of C. arborescens vis-à-vis the other taxa indicate a clear division of Carthamus taxa into two distinct lineages. Comparative distribution and intensity pattern of 18S–26S rDNA sites could distinguish each of the tetraploid and hexaploid taxa. The present results indicate that C. boissieri (2n = 20) is one of the genome donors for C. lanatus and C. lanatus ssp. lanatus (2n = 44), and C. lanatus is one of the progenitors for the hexaploid (2n = 64) taxa. The association of pCtKpnI-2 repeated sequence with rRNA gene cluster (orphon) in 2–10 nucleolar and non-nucleolar chromosomes and the consistent occurrence of pCtKpnI-1 repeated sequence at the subtelomeric region in all the taxa analysed indicate some functional role of these sequences.     

In vitro propagation and assessment of clonal fidelity of Nepenthes khasiana Hook. f.: a medicinal insectivorous plant of India

An efficient in vitro protocol for large-scale multiplication of Nepenthes khasiana, a threatened insectivorous plant of India, has been developed from nodal stem segments. The highest shoot proliferation of 19.16 ± 0.23 shoots/explant was recorded in half-strength Murashige and Skoog (MS) medium supplemented with 2.5 mg/l kinetin, 2.0 mg/l 6-benzyl aminopurine, 3 % sucrose and 0.8 % agar. The best rooting was achieved in half-strength MS medium supplemented with 2.0 mg/l α-naphthalene acetic acid with an average of 9.04 ± 0.46 roots/shoot. The plantlets were successfully transferred to the greenhouse with survival rate of 92 %, exhibiting normal development. Cytological and random amplified polymorphic DNA (RAPD) analyses were carried out to assess the genetic integrity of the regenerated plantlets. Cytological analysis revealed no change in chromosome number with cells studied showing 2n = 80. Of the 80 primers screened for RAPD analysis, 14 primers resulted in clear and scorable bands. A total of 72 amplification products were obtained out of which only 4.1 % bands were polymorphic. Cluster analysis of the RAPD profile revealed an average similarity coefficient ranging from 0.98 to 1.0, thus suggesting genetic stability in the micropropagated plants of N. khasiana.