Directed Amplification Of Minisatellite DNA Research Articles

Molecular Identification of Genetic Diversity and Population Structure in Moroccan Male Date Palm (Phoenix dactylifera L.) Using Inter-Simple Sequence Repeat, Direct Amplification of Minisatellite DNA, and Simple Sequence Repeat Markers

Understanding genetic diversity and population structure plays a vital role in the efficient use of available material in plant-breeding programs and in germplasm conservation strategies. In the present study, we aim to evaluate the genetic variations and population structure of male date palms from Morocco. The genetic diversity of 100 date palm (Phoenix dactylifera L.) genotypes was investigated using the performance of three types of molecular markers: inter-simple sequence repeats (ISSRs), direct amplification of minisatellite DNA (DAMD), and simple sequence repeats (SSRs). On the basis of their polymorphic information content (PIC) (ISSRs = 0.38; DAMD = 0.4; SSRs = 0.33), effective multiplex ratio (EMR) (ISSRs = 27.34; DAMD = 52.31; SSRs = 22.20), Resolving power Rp (ISSR = 13.81; DAMD = 28.73; SSR = 14.6), and marker index (MI) (ISSRs = 9.22; DAMD = 20.23; SSRs = 7.54) values, all markers used in our study are considered informative markers. Among them, DAMD markers demonstrated slightly higher informativeness compared to ISSR and SSR markers. A total of 216, 438, and 248 bands were, respectively, detected using ISSRs, DAMD, and SSRs, with 95%, 98% and 94% of polymorphism, respectively. The AMOVA results revealed considerable diversity within date palms. The PCOa results showed that males of Tinghir and Errachidia were regrouped into the same cluster, while males of Goulmima were separated into another group. A cluster and structure analysis separated the studied genotypes into three groups. One group comprises genotypes of males from Zagora with some female varieties scattered in this group. The second group includes male genotypes from Goulmima along with accessions of female and male varieties. The third group contains males of Errachidia, Tata and Tinghir populations. The cluster and structure analysis separated the studied genotypes according to their origin.

Analysis of genetic diversity and population structure of Moroccan date palm (Phoenix dactylifera L.) using SSR and DAMD molecular markers

BackgroundDate palm, oasis pivot, plays a vital socio-economic part in the southern area of Morocco. However, with climate change and drought intensity and frequency increasing, the Moroccan palm grove is threatened with significant genetic degradation. Genetic characterization of this resource is key element for the development of effective conservation and management strategies in the current circumstances of climate change and various biotic and abiotic stresses. To evaluate the genetic diversity of date palm populations collected from different Moroccan oases, we used simple sequence repeats (SSR) and directed amplification of mini-satellite DNA (DAMD) markers. Our results showed that used markers could efficiently assess genetic diversity in Phoenix dactylifera L. ResultsA total of 249 and 471 bands were respectively scored for SSR and DAMD, of which 100% and 92.9% were polymorphic. The polymorphic information content (PIC = 0.95), generated by the SSR primer was nearly identical to that generated by the DAMD primer (PIC = 0.98). The resolving power (Rp) was higher in DAMD than SSR (29.46 and 19.51, respectively). Analysis of the molecular variance (AMOVA) based on the combined data sets for both markers revealed a higher variance within populations (75%) than among populations (25%). Principal coordinate analysis (PCoA) and the ascendant hierarchical classification showed that the population of Zagora and Goulmima regions were the closest populations. The STRUCTURE analysis clustering of the 283 tested samples into seven clusters based on their genetic composition. ConclusionThe results drawn from this study will orient genotypes selection strategies for a successful future breeding and conservation program, particularly under climate change context.

Development of low cost micropropagation protocol for Nardostachys jatamansi: A critically endangered medicinal herb of Himalayas

The study reports a low-cost micropropagation protocol for Nardostachys jatamansi, a high value, critically endangered Himalayan herb. Shoot-bud bearing rhizome segments of the plant were made to sprout and elongate on 0.8% agar-gelled Murashige and Skoog's medium containing 3% sucrose. The sprouted shoots were then multiplied on agar-gelled as well as liquid MS medium containing various concentrations and combinations of kinetin (Kn) and 1-phenyl-3- (1,2,3-thiadiazol-5-yl) urea (thidiazuron or TDZ). Maximum number of shoots developed per explant after 30 days on 100 ml agar gelled (6.7 shoots) and 30 ml liquid (6 shoots) MS medium containing 0.5 mg l−1 Kn and 0.75 mg l−1 TDZ. Although rooting of in vitro shoots was attempted on full as well as half-strength MS medium containing different auxins, rooting was achieved in 100% shoots (3.07 roots/ shoot) after 30 days on half-strength MS medium supplemented with 1.0 mg l−1 indole-3- acetic acid (IAA). Different potting mixtures were used to acclimatize the micropropagated plantlets under polyhouse condition, but a maximum of 83% survival was recorded in sand and sand plus soil. The genetic fidelity of the in vitro plantlets was also assessed using Start Codon Targeted Polymorphism (SCoT) and Directed Amplification of Minisatellite DNA (DAMD) markers, and a total of 8.03% polymorphism was recorded. Eventually, the cost of micropropagation was reduced by 6.4 times during shoot multiplication and further by half during rooting. The present micropropagation method developed for N. jatamansi can have potential applications in medicinal plant-based industry.

SPAR methods reveal high genetic diversity within populations and moderate gene flow of pointed gourd (Trichosanthes dioica Roxb.) germplasm

Abstract Pointed gourd (Trichosanthes dioica Roxb.) is one of the most nutritive cucurbitaceous vegetable crops cultivated in tropical and subtropical regions around the world. Poor characterization of the available germplasm, vegetative means of propagation and dioecy are the main constraints in developing a systematic breeding programme in pointed gourd. In this investigation, genetic variability of pointed gourd accessions belonging to four agro-climatic zones of Bengal region of India was evaluated by employing polymerase chain reaction (PCR) based single primer amplification reaction (SPAR) methods, viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR) and directed amplification of minisatellite DNA (DAMD). Percentage of polymorphic loci, Nei’s gene diversity index (h) and Shannon’s Information index (I) indicated a moderate level of genetic heterogeneity in studied germplasm. The mean coefficient of genetic differentiation (Gst) between populations for RAPD, ISSR and DAMD were 0.419, 0.305 and 0.315 respectively, indicating that 58.1%, 69.5% and 68.5% of genetic variability prevailed within the population. Similarly, analysis of molecular variance (AMOVA) revealed that majority of the genetic variation were partitioned among the individuals within a population. Gene flow estimate (Nm) calculated for RAPD, ISSR and DAMD marker were 0.692, 1.139 and 1.086 respectively indicating a moderate gene flow among the populations. The clustering pattern based on the combined marker systems was primarily related to the geographical distribution of the accessions. This study clearly demonstrates the efficiency of single primer based amplification reactions (SPARs) in duplicate identification, core collection and improvement of pointed gourd germplasm.

Evaluation of subculture ages on organogenic response from root callus and SPAR based genetic fidelity assessment in the regenerants of Hibiscus sabdariffa L.

The efficacy of an organogenesis based regeneration protocol and the occurrence of somaclonal variation has been studied up-to eighth subculture of Hibiscus sabdariffa L. var. HS4288. Initially, the induction of organogenic calli from 83.33% root explants was standardized in 2.26 μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65 μM kinetin (KIN) supplemented Driver-Kuniyuki Walnut (DKW) basal medium. The calli were maintained up to the eighth subculture (each with 6-weeks duration) in the same basal medium along with 1.13 μM 2, 4-D and 4.65 μM KIN. The highest regenerative efficiency of the root-derived calli was found in 1.08 μM α-Naphthaleneacetic acid (NAA) and 8.88 μM 6-Bezyladenine (BA) containing DKW medium. This particular medium produced 14.30 ± 0.47 numbers of visible shoots (>10 mm in length) at the first subculture from 425 ± 35 mg of callus through the process of organogenesis. The morpho-histological study confirmed the organogenic mode of regeneration. With the gradual increase of culture passage, the frequency of shoots was steadily declined. The genetic polymorphism of the regenerated plants from eight successive sub-cultures was assessed using three different single primer amplification reaction (SPAR) methods; viz. Random amplified polymorphic DNA (RAPD), Direct amplification of minisatellite DNA (DAMD) and Inter simple sequence repeat (ISSR) markers. The SPAR analysis revealed that up to fifth subculture all the three types of primers produced the monomorphic banding pattern among the regenerated plants with that of their mother plant. From the sixth subculture onwards there were some alterations in banding profiles both in RAPD and ISSR markers but DAMD marker did not show any polymorphism up to eighth subculture. In the present work, the highest polymorphism percentage was recorded among the plants of eighth subculture by using both RAPD (41.61%) and ISSR (31.65%) markers. The SPAR method ensured that the present protocol may be used for true to type plant production up-to five consecutive subcultures.

Molecular analysis of genetic diversity and population genetic structure in Ephedra foliata: an endemic and threatened plant species of arid and semi-arid regions of India.

Studies on the genetic diversity and structure in endangered and threatened species are of utmost importance to design and promote effective conservation and management programs. Ephedra foliata, an endemic and threatened species growing naturally in arid and semi-arid regions of north western India, was investigated to estimate genetic variability and population structure using inter-simple sequence repeats (ISSR) and directed amplification of mini-satellite DNA (DAMD) markers. Twenty-five (ISSR 15; DAMD 10) markers produced 449 fragments, of which 382 were polymorphic in nature, revealing 84.59% polymorphism. ISSR markers revealed higher levels of polymorphism, polymorphic information content, marker index, diversity index and effective multiplex ratio than DAMD markers. Higher values of polymorphism, genetic diversity and Shannon information index at species level than at population level revealed that E. foliata possess high genetic diversity. AMOVA revealed much higher variance within populations than among the populations. The three clustering approaches viz., UPGMA, PCoA, and STRUCTURE, grouped the eleven investigated populations into two clusters revealing two genetic populations and the patterns of clustering of populations was in accordance with their geographic distribution, suggesting that these populations have evolved in response to their local environments. The high level of genetic differentiation (G ST = 0.31) and moderate gene flow (N m = 1.11) among populations could be due to geographic isolation, regional climatic conditions, over-exploitation and improper seed setting. To the best of our knowledge, this study is the first endeavour to estimate genetic diversity and population structure of E. foliata using molecular markers.

Evaluation of genetic diversity of carnation cultivars using CDDP and DAMD markers and morphological traits

This study was aimed to evaluate genetic diversity using molecular markers and morphological traits in carnation, Dianthus caryophyllus (fam: Cryophyllaceae) widely grown as an ornamental. Fifteen cultivars were examined employing seven Conserved DNA-derived Polymorphism (CDDP) and seven Directed Amplification of Minisatellite DNA (DAMD) markers. These varieties were planted in a randomized complete block design in three replicates at the experimental farm of the National Institute of Ornamental Plants, Mahallat, Iran. Analysis of variance for morphological traits showed significant differences among the examined cultivars. Morphological traits such as flower longevity, flower diameter and number of branches were statistically significant at 1 percent probability level. CDDP and DAMD markers produced an average polymorphic index content of 0.38 and 0.29 for CDDP and DAMD markers, respectively. Genetic distance matrix ranged from 0.02 to 0.84 in CDDP and from 0.09 to 0.78 in DAMD marker analysis. Cluster analysis for the two markers revealed that cultivars were grouped in three clusters. Results of molecular evaluation showed that CDDP and DAMD markers have high ability to differentiate carnation cultivars through assessment of the genetic diversity.

Thidiazuron induced in vitro morphogenesis for sustainable supply of genetically true quality plantlets of Brahmi

An effective and rapid method for propagating genetically true plantlets of a pharmaceutically important plant Bacopa monnieri L. was developed from thidiazuron (TDZ) pulse treated nodal segment explants. Explants treated for 24 h with different concentration of 0.0, 5.0, 10, 20 or 40 μM thidiazuron (TDZ) prepared in liquid Murashige and Skoog (MS) medium induces multiple shoot on MS basal medium without growth regulators. The highest frequency 95.3 ± 2.3% of regeneration and maximum number 43.7 ± 2.0 of shoots per explants with an average shoot height of 5.4 ± 0.29 cm were achieved after 08 weeks of culture from the explants treated with 20 μM TDZ. Simultaneously, the regenerated microshoots were rooted on the same growth regulator free media. Elongated shoots with roots were successfully acclimated to ex vitro condition with a 97% survival rate. Genomic stability of in vitro developed plants of B. monnieri was first time determined using flow cytometry and single primer amplification reaction (SPAR) methods viz., random amplified polymorphic DNA (RAPD), intersimple sequence repeat polymorphic DNA (ISSR) and directed amplification of minisatellite DNA (DAMD) and compared with donor plant. Nuclear DNA (nDNA) content of in vitro propagated B. monnieri plants has been estimated as 2.08 pg/2C based on the flow cytometric investigation of nuclei isolated from leaf tissues. No significant variations in nDNA content, plant genome size and ploidy level were observed between micropropagated plants and field grown donor plants. Likewise, the SPAR markers also revealed monomorphic banding profiles in micropropagated plantlets of B. monnieri and similar with to that of the donor plants verifying their genomic stability.

Characterization of Gladiolus Germplasm Using Morphological, Physiological, and Molecular Markers.

Estimation of variability and genetic relationships among breeding materials is one of the important strategies in crop improvement programs. Morphological (plant height, spike length, a number of florets/spike), physiological (chlorophyll content, chlorophyll fluorescence, and rapid light curve parameters) and Directed amplification of minisatellite DNA (DAMD) markers were used to investigate the relationships among 50 Gladiolus cultivars. Cluster analysis based on morphological data, physiological characteristics, molecular markers, and cumulative data discriminated all cultivars into seven, five, seven, and six clusters in the unweighted pair-group method using arithmetic mean (UPGMA) dendrogram, respectively. The results of the principal coordinate analysis (PCoA) also supported UPGMA clustering. Variations among the Gladiolus cultivars at phenotypic level could be due to the changes in physiology, environmental conditions, and genetic variability. DAMD analysis using 10 primers produced 120 polymorphic bands with 80% polymorphism showing polymorphic information content (PIC=0.28), Marker index (MI=3.37), Nei's gene diversity (h=0.267), and Shannon's information index (I=0.407). Plant height showed a positive significant correlation with Spike length and Number of florets/spike (r=0.729, p<0.001 and r=0.448, p=0.001 respectively). Whereas, Spike length showed positive significant correlation with Number of florets/spike (r=0.688, p<0.001) and Chlorophyll content showed positive significant correlation with Electron transport rate (r=0.863, p<0.001). Based on significant morphological variations, high physiological performance, high genetic variability, and genetic distances between cultivars, we have been able to identify diverse cultivars of Gladiolus that could be the potential source as breeding material for further genetic improvement in this ornamental crop.

Assessment of genetic diversity and population structure of Bergenia stracheyi (Saxifragaceae) in the Western Himalaya (India)

Genetic diversity and population structure in Bergenia stracheyi, a threatened medicinal herb in the Western Himalaya of India was analysed using directed amplification of minisatellite DNA (DAMD) and inter simple sequence repeats (ISSR) markers. A total of 41 accessions of B. stracheyi representing three populations (Khillenmarg –KLM, Jalori Pass-JLP and Rohtang-RTG) were considered in the present study. The cumulative data analysis for 26 (10 DAMD + 16 ISSR) markers revealed 87.1% polymorphism. The maximum inter-population genetic distance was found between KLM and JLP, whereas the minimum genetic distance was found between RTG and JLP populations. The analysis of molecular variance (AMOVA) revealed maximum percentage of variation among individuals within populations (75%) than among the populations (25%). Clustering pattern of the three sample populations in STRUCTURE and PCoA analyses showed high genetic variation at population level. The present study revealed that distribution patterns, high altitudinal ranges, high habitat specificity, relatively high gene flow, small and isolated population size have shaped the current population structure of B. stracheyi in the Western Himalayan region. DAMD and ISSR markers have provided significant insights into characterization of B. stracheyi populations, and facilitate selection of appropriate accessions for further utilization in conservation and bioprospecting programmes.

Estimation of genetic variability and population structure in Ephedra gerardiana Wall. ex Stapf (Ephedraceae): An endangered and endemic high altitude medicinal plant

Ephedra gerardiana is commonly known as ‘Gerard's Joint fir’, used in the indigenous systems as well as in modern systems of medicine. Almost every part of the plant possesses innumerable medicinal properties. Here we examined the genetic diversity and population structure of E. gerardiana employing Direct Amplification of Minisatellite DNA (DAMD) and Inter Simple Sequence Repeats (ISSR) methods. The cumulative (DAMD and ISSR) marker data revealed a high genetic diversity (P=90.84%, H=0.30, I=0.45) at species level than at population levels (P=62.43%, H=0.22, I=0.33), and moderate genetic differentiation (GST=0.27) among populations. Unweighted pair group method for arithmetic mean (UPGMA), Principle coordinate analysis (PCoA) and Bayesian clustering analysis revealed a significant population structure, and are in congruence with geographic distribution of the accessions. Mantel test revealed a significant correlation between geographic and genetic distances (r=0.86, P=0.007). Comparatively, high genetic diversity at species level than the population levels showed that these populations have evolved in response to their respective environments, and geographical isolation have played an important role in genetic differentiation and population structuring. However, increasing anthropogenic pressure on natural habitats of E. gerardiana, warrants immediate in-situ and ex-situ conservation measures to preserve diverse genotypes available in western Himalayan region for long term sustainability of E. gerardiana. To the best of our knowledge, present study seems to be the first endeavour to analyse genetic diversity and population structure of E. gerardiana using molecular markers.

Phenetic and genetic diversity in Indian Luffa (Cucurbitaceae) inferred from morphometric, ISSR and DAMD markers

Genetic variations and relationships among cultivated and wild genotypes of five taxa of Indian Luffa were examined using inter simple sequence repeats (ISSR), directed amplification of minisatellite DNA (DAMD) markers and morphological characterization. Morphometric evaluation of 21 discrete characters in 51 representative accessions segregated the five taxa of Luffa in three main clusters: the two wild species (L. echinata, L. graveolens) in the first, and the cultivated L. aegyptiaca (smooth gourd) and L. acutangula (ridged gourd)/L. hermaphrodita (Hermaphrodite luffa) in the second and third clusters, respectively. Cumulative data analysis of 15 ISSR and seven DAMD markers revealed high percentage polymorphism (97.67 %), moderate genetic distance (0.06–0.72, avg. 0.51), and low heterozygosity and Shannon index values (H = 0.15; I = 0.22) across all the 76 genotypes assayed. A UPGMA dendrogram, based on the combined marker data, resolved the five taxa in two main clusters with high bootstrap support. The morphological and molecular trees showed incongruence in the number of main clusters resolved and in the disposition of the wild and cultivated taxa in different sub-clusters. The cluster analyses and PCoA plots revealed a nested grouping of the hermaphrodite luffa within the ridge gourd group. The Bayesian STRUCTURE analysis identified three genetic clusters for the five assumed taxa. Outcrossing test revealed a mixed mating system in Indian Luffa. This is the first ever report on the mating system in Luffa using molecular markers. The study also demonstrates the utility of using more than one DNA marker in the assessment of molecular diversity in a widely cultivated crop genus like Luffa with a narrow genetic base.

Interactive Effects of Growth Regulators, Carbon Sources, pH on Plant Regeneration and Assessment of Genetic Fidelity Using Single Primer Amplification Reaction (SPARS) Techniques in Withania somnifera L.

An improved and methodical in vitro shoot morphogenic approach through axillary bud multiplication was established in a drug yielding plant, Withania somnifera L. Effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyladenine (2iP), and thidiazuron (TDZ)] either singly or in combination with α-napthalene acetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium were tested. The highest regeneration frequency (90 %) with optimum number of shoots (32 ± 0.00)/explant were obtained on MS medium fortified with 2.5 μM 6-benzyladenine (BA) and 0.5 μM NAA and 30 g/l sucrose at pH 5.8. Among the tried TDZ concentrations, 0.5 μM resulted in maximum number of shoots (20.4 ± 0.40)/explant after 4 weeks of exposure. The proliferating shoot cultures established by repeated subculturing of the mother explants on the hormone-free medium produced the highest shoot number (29.4 ± 0.40) with shoot length (6.80 ± 0.12 cm)/explant at fourth subculture passage, which a decline in shoot proliferation was recorded. Different concentrations of NAA were tested for ex vitro rooting of microshoots. The maximum percentage of rooting 100 % with maximum roots (18.3 ± 0.1) was achieved in soilrite when basal portion of the microshoots were treated with 200 μM (NAA) for 15 min per shoot. The plantlets went through hardening phase in a growth chamber, prior to ex vitro transfer. The PCR-based single primer amplification reaction (SPAR) methods which include random amplified polymorphic DNA (RAPD) and direct amplification of minisatellite DNA (DAMD) markers has been used for assessment of genetic stability of micropropagated plantlets. No variation was observed in DNA fingerprinting patterns among the micropropagated and the donor plants illustrating their genetic uniformity.

Genetic variability and population structure of Bergenia ciliata (Saxifragaceae) in the Western Himalaya inferred from DAMD and ISSR markers

Genetic variability and population structure of Bergenia ciliata (Haw.) Sternb., commonly known as “Pashanbheda” (Stone-breaker), collected from the Western Himalayan region of India were estimated using two DNA fingerprinting methods viz., directed amplification of minisatellite DNA (DAMD) and inter simple sequence repeats (ISSR). The cumulative data analysis of DAMD and ISSR markers for 74 accessions from eight populations showed 86.1% polymorphism. Analysis of molecular variance (AMOVA) showed highest percentage of variation within individuals of populations (73.6%) and 21.7% among populations. STRUCTURE and PCoA analyses on the hierarchical partitioning of genetic diversity showed strong admixture of individuals among the eight assumed geographical populations of B. ciliata. The data suggests that high genetic flow is one of the major factors responsible for low genetic differentiation. Preservation of genetic diversity of B. ciliata is important, both to promote adaptability of the populations to changing environment as well as to preserve a large gene pool for future prospection. The present study using DAMD and ISSR markers, therefore, provide the means of rapid characterization of accessions within the populations, and thus enable the selection of appropriate accessions for further utilization in conservation and prospection programmes.

Assessment of genetic diversity in indigenous turmeric (Curcuma longa) germplasm from India using molecular markers.

Curcuma longa L., commonly known as turmeric, is one of the economically and medicinally important plant species. It is predominantly cultivated in the tropical and sub tropical countries. India is the largest producer, and exporter of turmeric in the world, followed by China, Indonesia, Bangladesh and Thailand. In the present study, Directed Amplification of Minisatellite DNA (DAMD) and Inter Simple Sequence Repeats (ISSR), methods were used to estimate the genetic variability in indigenous turmeric germplasm. Cumulative data analysis for DAMD (15) and ISSR (13) markers resulted into 478 fragments, out of which 392 fragments were polymorphic, revealing 82% polymorphism across the turmeric genotypes. Wide range of pairwise genetic distances (0.03-0.59) across the genotypes revealed that these genotypes are genetically quite diverse. The UPGMA dendrogram generated using cumulative data showed significant relationships amongst the genotypes. All 29 genotypes studied grouped into two clusters irrespective of their geographical affiliations with 100% bootstrap value except few genotypes, suggesting considerable diversity amongst the genotypes. These results suggested that the current collection of turmeric genotypes preserve the vast majority of natural variations. The results further demonstrate the efficiency and reliability of DAMD and ISSR markers in determining the genetic diversity and relationships among the indigenous turmeric germplasm. DAMD and ISSR profiling have identified diverse turmeric genotypes, which could be further utilized in various genetic improvement programmes including conventional as well as marker assisted breeding towards development of new and desirable turmeric genotypes.

ISSR and DAMD markers revealed high genetic variability within Flavoparmelia caperata in Western Himalaya (India).

Flavoparmelia caperata (L.) Hale is medicinally very important and possesses antifungal and antibacterial activities. F. caperata is the only species found in India. Inter simple sequence repeat (ISSR) and Directed amplification of minisatellite DNA (DAMD) methods were used to analyze the genetic variability within F. caperata from the Western Himalayan region of India. Eleven ISSR and 10 DAMD primers produced 139 and 117 polymorphic bands, and detected 91.44 and 82.34% polymorphisms, respectively. Cumulative band data generated for ISSR and DAMD markers resulted in 86.86% polymorphism across all the accessions of F. caperata. The average Polymorphic information content (PIC) value obtained with ISSR, DAMD, and cumulative band data were 0.28, 0.27, and 0.27, respectively. The clustering of the F. caperata accessions in the UPGMA dendrogram showed that these accessions are intermingled with each other in different subclusters irrespective of their geographical affiliations. The pattern of genetic variations within F. caperata accessions could be due to free exchange of spores that might have taken place among these accessions in the wild. ISSR and DAMD markers efficiently and reliably resulted in discrete banding patterns and polymorphic profiles. These markers despite targeting different regions of genome, revealed almost similar levels of polymorphism across all the accessions. The wide range of genetic distance and high level of polymorphism detected by ISSR and DAMD reflected a high genetic variability among the different accessions of F. caperata.

Genetic variation and phylogenetic relationships among Indian Citrus taxa revealed by DAMD-PCR markers

Genetic variation and phylogenetic relationships among 50 wild and cultivated accessions of 19 Indian Citrus genotypes were examined through comparison of Directed Amplification of Minisatellite DNA (DAMD) markers and morphological characters. DAMD-PCR analysis with four primers resulted in amplification of a total of 45 bands, of which 35 (78 %) were polymorphic. Morphometric evaluation using 76 morphological characters showed high level of variability ranging from 0.18 to 1.00 (avg 0.39), whereas the Jaccard’s coefficient values of genetic similarity calculated from DAMD data ranged from 0.41 to 1.00 (avg 0.68), indicating moderate genetic divergence among the accessions studied. UPGMA dendrograms generated separately from morphometric and DAMD data segregated all the accessions of Citrus into four main clusters, each containing a true basic species and their probable hybrids. The grouping of individual accessions/genotypes under respective species or cultivars in DAMD dendrogram was based purely on their genetic relationships rather than geographical origin. There was no absolute congruence between the data and dendrograms generated from morphometric and DAMD analyses. The study demonstrates the resolving power of DAMD markers for discrimination of individual genotypes of Citrus under its respective species, hybrid or cultivar groups and inferring their genetic and phylogenetic relationships as well. This is the first report on application of DAMD markers in Citrus.

Estimation of genetic variability and population structure in Sapindus trifoliatus L., using DNA fingerprinting methods

Genetic variability and population structure of Sapindus trifoliatus L. (Sapindaceae), collected from Gujarat, Karnataka and Uttar Pradesh states were estimated using three DNA fingerprinting methods viz., random amplified polymorphic DNA (RAPD), directed amplification of minisatellite DNA (DAMD) and inter-simple sequence repeats (ISSR). The cumulative data analysis carried out for all three markers showed 69.42 % polymorphism. The intra-population genetic diversity analysis revealed the highest values of Nei’s genetic diversity (0.16), Shannon information index (0.24) and polymorphic loci (43.99 %) among Bhavnagar (BH) population, whereas lowest values were found in Junagarh (JU) population. The maximum inter-population average genetic distance (0.20) was between Allahabad (AL) and JU populations. Analysis of molecular variance (AMOVA) showed highest percentage of variation among individuals of populations (56 %) followed by 25 % among populations and 19 % among regions. Principal coordinate analysis and UPGMA dendrogram revealed that genetic diversity was in congruence with the geographical diversity. The data strongly suggest that low genetic flow, geographic isolation and to some extent genetic drift are the major factors responsible for high genetic differentiation. Preservation of genetic diversity of S. trifoliatus is important, both to promote adaptability of the populations to changing environment as well as to preserve a large gene pool for future genetic improvement. The present study using RAPD, DAMD and ISSR profiles of S. trifoliatus provide the means of rapid characterization of accessions within the populations, and thus enable the selection of appropriate accessions for further utilization in conservation and prospection programs of this important plant genetic resource.

Genetic Relationships Among Wild and Cultivated Accessions of Curry Leaf Plant (Murraya koenigii (L.) Spreng.), as Revealed by DNA Fingerprinting Methods

Murraya koenigii (L.) Spreng. (Rutaceae), is an aromatic plant and much valued for its flavor, nutritive and medicinal properties. In this study, three DNA fingerprinting methods viz., random amplification of polymorphic DNA (RAPD), directed amplification of minisatellite DNA (DAMD), and inter-simple sequence repeat (ISSR), were used to unravel the genetic variability and relationships across 92 wild and cultivated M. koenigii accessions. A total of 310, 102, and 184, DNA fragments were amplified using 20 RAPD, 5 DAMD, and 13 ISSR primers, revealing 95.80, 96.07, and 96.73% polymorphism, respectively, across all accessions. The average polymorphic information content value obtained with RAPD, DAMD, and ISSR markers was 0.244, 0.250, and 0.281, respectively. The UPGMA tree, based on Jaccard's similarity coefficient generated from the cumulative (RAPD, DAMD, and ISSR) band data showed two distinct clusters, clearly separating wild and cultivated accessions in the dendrogram. Percentage polymorphism, gene diversity (H), and Shannon information index (I) estimates were higher in cultivated accessions compared to wild accessions. The overall high level of polymorphism and varied range of genetic distances revealed a wide genetic base in M. koenigii accessions. The study suggests that RAPD, DAMD, and ISSR markers are highly useful to unravel the genetic variability in wild and cultivated accessions of M. koenigii.

Characterization of Prosopis cineraria (L.) Druce germplasm with suitable horticultural traits from the hot arid region of Rajasthan, India

Sixteen germplasm accessions of Prosopis cineraria with suitable horticultural traits were identified from north-western Rajasthan, India, propagated clonally by budding on seedling rootstock and maintained in the field gene bank. Morphological characterization of seven-year-old trees of these accessions by 21 traits indicated a lot of variation among the accessions tested. Higher number of flowers per raceme was found in accession CIAH/K2, higher width of ripened pod in CIAH/K5, higher number of seeds per pod in CIAH/K12 and a higher weight of seed per pod in CIAH/K6. Overall, CIAH/K16 was found to be a superior genotype for most of the useful traits. High significant positive correlation was obtained with traits useful for horticultural values. Out of 62 random decamer primers for random amplification (RAPD) reaction, and four minisatellite core sequence for direct amplification of minisatellite DNA (DAMD) screened with these accessions, 12 RAPD and 2 DAMD primers were found polymorphic. Average polymorphism resolved by these markers among the accessions was 93.2%. Genetic diversity revealed by Jaccard’s co-efficient was between 0.11 and 0.77, and four major clusters were identified among these accessions by phylogenetic analysis using NTSYSpc-2.02e software. This study shows the existence of high genetic diversity within these accessions.