Assessment of genetic diversity and population structure in wild Ziziphus species from northwest India using SSR marker technique.

BACKGROUND: Ziziphus species particularly Ziziphus mauritiana and Ziziphus nummularia constitute an important part of genetic resources in India. They contribute economically as a fruit crop with lots of morphological and pomological variability. In current study, 48 accessions belonging to two wild Ziziphus species, i.e., Z. mauritiana and Z. nummularia, were characterized using SSR markers. In addition, external features were also examined using stereomicroscope. RESULTS: Present investigation was done to explore the genetic structure of North Indian jujube. In total, 23 SSR markers detected 57 SSR alleles with an average of 2.47 alleles. Highest number of alleles (4) were detected by three primers, namely BFU1178, BFU479, and ZCMS14, while lowest number of alleles (2) were detected by fifteen primers. Highest Polymorphism Information Content (PIC) was 0.500 and shown by two primers, namely BFU528 and BFU1248, while lowest PIC (0.041) was observed in primers BFU286 with mean value of 0.443. Similarly, highest value of marker index (MI) was detected by primer BFU1178 i.e. 1.969, and lowest value of marker index was observed in primer BFU286 i.e. 0.021. Dendrogram generated using SSR markers data and principal component analysis showed two major groups of the analyzed germplasm with intermixing. STRUCTURE analysis also clustered all the accessions into two groups. We did not found correlation between geographic and genetic distances. CONCLUSIONS: The preliminary results suggest that there is high level of gene pool mixing in these species which can be attributed to their cross-pollination habit. However, more such studies with large numbers of samples are required in future to gain concrete insights of the genetic structure in these species.

Analysis of genetic diversity and population structure in Asparagus species using SSR markers.

BACKGROUND: Various Asparagus species constitute the significant vegetable and medicinal genetic resource throughout the world. Asparagus species serve as important commodity of food and pharmaceutical industries in India. A diverse collection of Asparagus species from different localities of Northwest India was investigated for its genetic diversity using simple sequence repeat (SSR) markers. RESULTS: Polymorphic SSR markers revealed high genetic diversity. Primer SSR-15 amplified maximum of 8 fragments while 3 primers, namely, SSR-43, SSR-63, and AGA1 amplified minimum of 3 fragments. Collectively, 122 alleles were amplified in a range between 3 and 8 with an average of 5 alleles per marker. The size of the amplified alleles ranged between 90 and 680 base pairs. Polymorphism information content (PIC) value varied from a highest value of 0.499 in primer AGA1 to a lowest value of 0.231 in primer SSR-63 with a mean value of 0.376 showing considerable SSR polymorphism. Dendrogram developed on the basis of Jaccard's similarity coefficient and neighbor-joining tree segregated all the studied Asparagus species into two discrete groups. Structure analysis based on Bayesian clustering allocated different accessions to two independent clusters and exhibited low level of individual admixture. CONCLUSIONS: The genetic diversity analysis showed a conservative genetic background for maximum species of asparagus. Only Accessions of Asparagus adscendens were split into two diverse clusters suggesting a wide genetic base of this species as compared to other species. Overall genetic diversity was high, and this germplasm of Asparagus can be used in future improvement programs. The findings of current research on Asparagus germplasm can make a momentous contribution to initiatives of interbreeding, conservation, and improvement of Asparagus in future.

Quantification of withaferin-A and withanolide-A in diploid (n = 12) and tetraploid cytotypes (n = 24) of "Rassbhary", Physalis angulata L.

During the present study an analytical method based on reverse-phase high performance liquid chromatography photodiode array detection method was developed for simultaneous determination of withaferin-A and withanolide-A in plant parts of two cytotypes (diploid n = 12 & tetraploid n = 24) of Physalis angulata. All the plant parts were extracted in different solvent solutions i.e., acidic [HCl] methanol (i.e., methanol containing 0.3% of HCl), methanol, n-hexane, chloroform. Both the compounds were comparatively analysed. The results revealed that tetraploid cytotype (n = 24) showed the higher composition of both the reference compounds. The method is simple, rapid and provides better resolution can be easily applied to the quantitative analyses of withanolides in plant matrices.

HPTLC Analysis of Solanum xanthocarpum Schrad. and Wendl., a Siddha Medicinal Herb.

In the present study, HPTLC is used to detect the presence and amount of triterpenoids and phytosterols in different plant parts (fruit, stem, leaf, and root) of Solanum xanthocarpum Schrad. and Wendl.; such analysis is done for the first time. Each plant part has its own medicinal value and is used as Siddha medicinal herb. The employed statistical analysis ensures that the developed method is reproducible and selective. The results show that the fruit samples contain highest amount of tested phytochemicals. This method can be used as an important tool to ensure the therapeutic dose in herbal formulations, standardization, and quality control of bulk drugs.

Differential content of secondary metabolites in diploid and tetraploid cytotypes of Siegesbeckia orientalis L.

Siegesbeckia orientalis L. is an annual herb widely distributed throughout the world and has many medicinal properties. In Chinese traditional system, it is popularly known as Xi-Xian and used for its anti-inflammatory properties. In the present study, two cytotypes (diploid and tetraploid) have been investigated for their secondary metabolites. The different plant parts have been explored in terms of total phenolics, total flavonoids, DPPH radical scavenging acitivity and total antioxidant capacity. Out of different plant parts, leaves have the maximum amount of secondary metabolites and antioxidant potential. HPTLC technique has been applied to quantify six marker compounds in the two cytotypes. Tetraploid cytotype has been compared with diploid cytotype, which shows that tetraploid has the maximum amount of studied secondary metabolites with high antioxidant potential.

Development and characterization of novel microsatellite markers in Trillium govanianum: a threatened plant species from North-Western Himalaya.

Trillium govanianum is a temperate forest understory plant species of high value belonging to the family Melanthiaceae. It is endemic to Himalayan region and facing a bottleneck situation due to reckless extractions from its natural strands. In the present study, 21 microsatellite markers were developed and characterized in 20 accessions of T. govanianum. Collectively, the polymorphic markers amplified 31 alleles in a range of 2-4 with an average of 2.6 alleles per marker. The mean observed heterozygosity (H o), expected heterozygosity (H e), and Shannon information index (I) were 0.46, 0.48, and 0.73, respectively. Average polymorphism information content (PIC) was 0.385. The cross-transferability in a related species, namely, Polygonatum verticillatum, showed amplification of ten markers. The newly developed microsatellite markers efficiently distinguished the different accessions on the basis of their geographic origin. Thus, these microsatellites can be useful in exploring genetic diversity in various existing populations of T. govanianum in north-western Himalaya, which may be useful for their conservation, management, and improvement in future.

Anticonvulsant activity and acute neurotoxic profile of Achyranthes aspera Linn.

ETHNOPHARMACOLOGICAL RELEVANCE: Root powder of Achyranthes aspera Linn. (A. aspera) belongs to family Amaranthaceae is used in Indian traditional medicine for the management of epilepsy and its efficacy is widely acclaimed among the different rural communities. AIM OF THE STUDY: The present study was aimed to establish the possible anticonvulsant effect of A. aspera methanolic root extract using acute anticonvulsant models and to evaluate the acute toxicity and neurotoxic potential A. aspera extract. MATERIAL AND METHODS: A. aspera methanolic extract was standardized with respect to betaine using HPTLC. The maximal electroshock (MES), pentylenetetrazol (PTZ), picrotoxin and bicuculline induced seizure models were used to evaluate the anticonvulsant potential of standardized A. aspera root extract. The GABA content in cortex and hippocampus of extract treated mice was evaluated using HPLC. Moreover, the animals were also evaluated for acute toxicity study and neurotoxicity test. RESULTS: A significant enhancement in the seizure threshold was observed by A. aspera extract (5 and 10mg/kg) treated mice in PTZ, picrotoxin and bicuculline models as compared to saline treated mice respectively, whereas the extract failed to show protection in MES induced seizures. Moreover, A. aspera treatment (5 and 10mg/kg) significantly enhances the GABA levels in hippocampus and cortex as compared to saline treated group. A. aspera root extract was devoid of any sign of acute toxicity as well as neurotoxicity. CONCLUSIONS: A. aspera root extract exhibits significant anticonvulsant effect by facilitation of GABAergic neurotransmission in the brain.

New chromosome reports in Lamiaceae of Kashmir (Northwest Himalaya), India.

Meiotic studies and chromosome data are imperative in order to have an overall germplasm evaluation of a taxon. In the present effort, the meiotic study is carried out in 48 populations belonging to 26 species of Lamiaceae collected from their natural habitats in Kashmir Himalaya, which forms an important part of Northwest Himalaya. Chromosome counts in the five species viz. Dracocephalum nutans (2n = 10), Lycopus europaeus (2n = 22), Marrubium vulgare (2n = 54), Nepeta nervosa (2n = 18) and Salvia sclarea (2n = 22) are first time reported from India. Besides, 17 species are cytologically evaluated for the first time from the study area-Kashmir Himalaya. In Marrubium vulgare, hexaploid cytotype (2n = 6 × =54) is reported for the first time. Also, diploid and tetraploid cytomorphovariants are observed in Calamintha vulgaris (2n = 20, 40), Elsholtzia ciliata (2n = 16, 32) and Mentha longifolia (2n = 20, 40). Various meiotic abnormalities like chromatin stickiness, cytomixis, nonsynchronous disjunction, laggards, chromatin bridges, etc. leading to pollen abnormalities have been documented for the first time in some species. The worldwide status of chromosome number data in each genus is presented.

Development of novel SSR markers for evaluation of genetic diversity and population structure in Tribulus terrestris L. (Zygophyllaceae).

Tribulus terrestris L., commonly called puncture vine and gokhru, is an important member of Zygophyllaceae. The species is highly important in context to therapeutic uses and provides important active principles responsible for treatment of various diseases and also used as tonic. It is widely distributed in tropical regions of India and the world. However, status of its genetic diversity remained concealed due to lack of research work in this species. In present study, genetic diversity and structure of different populations of T. terrestris from north India was examined at molecular level using newly developed Simple Sequence Repeat (SSR) markers. In total, 20 primers produced 48 alleles in a size range of 100-500 bp with maximum (4) fragments amplified by TTMS-1, TTMS-25 and TTMS-33. Mean Polymorphism Information Content (PIC) and Marker Index (MI) were 0.368 and 1.01, respectively. Dendrogram showed three groups, one of which was purely containing accessions from Rajasthan while other two groups corresponded to Punjab and Haryana regions with intermixing of few other accessions. Analysis of molecular variance partitioned 76 % genetic variance within populations and 24 % among populations. Bayesian model based STRUCTURE analysis detected two genetic stocks for analyzed germplasm and also detected some admixed individuals. Different geographical populations of this species showed high level of genetic diversity. Results of present study can be useful in identifying diverse accessions and management of this plant resource. Moreover, the novel SSR markers developed can be utilized for various genetic analyses in this species in future.

Cytomictic anomalous male meiosis and 2n pollen grain formation in Mertensia echioides Benth. (Boraginaceae) from Kashmir Himalaya.

Presently Mertensia echioides Benth. (Boraginaceae) collected from Kashmir Himalaya, India, is cytologically analyzed for the first time revealing 2n = 2x = 24 (diploid). Interestingly we found 4.3-6.2% syncytic meiocytes/PMCs with 2n = 4x = 48 (tetraploid) in addition to normal meiocytes (2n = 24) during male meiosis. These comparatively larger PMCs (pollen mother cells) lead to the formation of fertile giant 2n pollen grains. A frequency of 6.4-13.3% PMCs shows transfer of chromatin material at prophase-I and, therefore, results in aneuploid meiocytes. Whole chromatin transfer by the process of cytomixis could also have led to the formation of tetraploid cells. Translocation heterozygosity is also evident in the form of multivalents in 12-17% diploid (2x) meiocytes at diakinesis and metaphase-I and is reported for the first time in this species. The syncytes formed depict open chain hexavalent and quadrivalent formation in the three populations with different frequencies. Moreover chromatin stickiness at metaphase-I is observed in 45% of PMCs in population-1 (P-1). Syncyte or unreduced PMC formation leading to unreduced fertile gametes is here speculated to act as a possible way out for infraspecific polyploidization in the species.

Meiotic studies in some species of tribe Cichorieae (Asteraceae) from Western Himalayas.

The present paper deals with meiotic studies in 15 species belonging to 6 genera of the tribe Cichorieae from various localities of Western Himalayas. The chromosome number has been reported for the first time in Hieracium crocatum (2n = 10) and Lactuca lessertiana (2n = 2x = 16). Further, intraspecific variability has been reported for the first time in H. umbellatum (2n = 2x = 10 and 2n = 6x = 54), Tragopogon dubius (2n = 2x = 14 and 2n = 4x = 28), and T. gracilis (2n = 2x = 14). The chromosome report of 2n = 2x = 10 in Youngia tenuifolia is made for the first time in India. Maximum numbers of the populations show laggards, chromosome stickiness, and cytomixis from early prophase to telophase-II, leading to the formation of aneuploid cells or meiocytes with double chromosome number. Such meiotic abnormalities produce unreduced pollen grains and the reduced pollen viability.

Cytology of five species of subfamily Papaveroideae from the Western Himalayas.

During the present course, population-based meiotic studies were carried out on five species of subfamily Papaveroideae from selected localities of Kashmir and Himachal Pradesh in the Western Himalayas (India). Varied intraspecific chromosome counts were reported for the first time in Argemone mexicana and Meconopsis latifolia, both existing on 2n = 2x = 14. The x = 7, confirmed for the first time from the newly found diploid cytotype, is suggested to be the primary chromosomal basic number for the Meconopsis. Furthermore, meiotic course was noted to be normal in Argemone ochroleuca, it varied from normal to abnormal in the populations of A. mexicana and Papaver dubium whereas it was invariably found to be abnormal in all the populations of Meconopsis aculeata and M. latifolia. These anomalous taxa were marked with meiotic abnormalities in the form of cytomixis, chromosomal stickiness, unoriented bivalents, formation of laggards and bridges resulting in abnormal microsporogenesis, and production of heterogeneous-sized fertile pollen grains along with reduced pollen fertility.