S. R. Maloo

Assessment of genetic diversity in Isabgol ( Plantago ovata Forsk.) using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers for developing crop improvement strategies

Isabgol ( Plantago ovata F., family: Plantaginaceae) is one of the most important medicinal plants of South Asia. Two DNA based molecular marker techniques, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR), were used to study the genetic diversity among genotypes of Isabgol. A total of 38 polymorphic primers (22 random and 16 ISSR) were used. Amplification of genomic DNA of 24 genotypes, using RAPD analysis, yielded 208 fragments, of which 98 (47.12%) were polymorphic. The 16 ISSR primers produced 124 bands across 24 genotypes, of which 24 (19.35%) were polymorphic. RAPD markers appeared more informative than ISSR in determining the genetic diversity. The similarity coefficient ranged from 0.77 to 0.97, 0.81 to 1.00 and 0.84 to 0.98 with RAPD, ISSR and combined dendrogram, respectively. This indicates very low level of genetic diversity among genotypes. A poor mantel correlation ( r = 0.28) was found between both sets of genetic similarity data, suggesting that both sets of markers revealed unrelated estimates of genetic relationships. Therefore, the RAPD and ISSR markers show two genetic grouping of studied Isabgol genotypes. The genotypes RI-168, RI-167, RI-137, PB-62, RI-153, RI-148 and Gumary were spotted as genetically diverse in both sets of markers and could be efficiently utilized in crop improvement programmes. Keywords : Plantago ovata , molecular marker, RAPD, ISSR, genetic diversity, medicinal plant African Journal of Biotechnology Vol. 12(23), pp. 3622-3635

Assessment of the morphological and molecular diversity in Amaranthus spp.

Genetic diversity and relationships of 15 Amaranthus species were examined by using both morphological traits and random amplification of polymorphic DNA (RAPD) markers. Ten morphological observations were recorded out of which seven were subjected to analysis of variance. Mean squares due to genotypes were highly significant. Wide mean range performance was observed for number of effective tillers per plant (9 to 12.3), spike length (16.7 to 42.4 cm), spike mass (16.2 to 24.5 g), seed yield per plant (6.4 to 16.6 g), biological yield per plant (19.8 to 28.6 g), harvest index (32.9 to 57.1%) and seed protein content (15.75 to 16.49). RAPD analysis has been carried out using 12 arbitrary sequence decamer primers. Seventy four amplicons were obtained out of which 58 were polymorphic and the level of polymorphism was 78.3%. The average number of polymorphic bands per primer was 4.8. From the RAPD data, an unweighted pair-group method arithmetic (UPGMA) dendrogram illustrating the genetic relationship among fifteen genotypes were computed. The trends of genotypes relationship amongst the Amaranths spp. determined by RAPDs are consistent with their morphological traits.

Genetic Confirmation of Mungbean Genotypes (Vigna Radiata L. Wilczek) Using Molecular Markers

Molecular characterization is helpful in understanding the phylogenetic relationship among various germplasm to reveal the genetic diversity among the used parental genotypes. Among several efficient methods for revealing genetic variability within and among plant populations, one of the most widely applied method is marker analysis. RAPD and ISSR, markers are commonly used because they are quick, simple and environment non-sensitive enabling genetic diversity analysis in several types of plant material like natural populations, population in breeding programmes. Evaluation of genetic diversity would promote the efficient use of genetic variations, effective conservation and purity of the genotype to be determined as well as utilization of germplasm in crop improvement. The RAPD and ISSR data were evaluated to obtain a combined similarity matrix. The similarity coefficient values lay between 0.46–0.68. The RAPD and ISSR cluster tree analysis showed that the eight genotypes could be divided into 4 clusters. The genotype BM 4 was grouped in separate VI cluster. However, PDM 139 was grouped on cluster IIA. In the light of RAPD and ISSR study the parents of the cross BM 4 x PDM 139 were also noticed for their genetic diversity, having 53% dissimilarity and grouped into the separate clusters.

Assessment of Genetic Diversity Using ISSR Markers in Green Gram [Vigna radiata (L.) Wilczek]

Introduction Pulses offer one of the viable options for diversification of contemporary agriculture and management of natural resources. India is the largest producer and consumer of pulses in the world accounting 33 per cent of the area and 25 per cent of the global out-put. Green gram [Vigna radiata (L.) Wilczek) is the most important legume crop in India after chickpea and pigeonpea. It belongs to family Leguminaceae, subfamily Papillionaceae and its chromosome number is 2n = 2 x = 22. India is the primary green gram producer and contributes to about 75 per cent of the world pulses production. It contributes to about 14% of total pulses cultivation area and 7% of total pulses production in India. Green gram is extensively grown in India under varying soil types and climatic conditions and it improves soil fertility by fixing atmospheric nitrogen. It is a small herbaceous annual drought tolerant crop and suitable for dry land farming and predominantly used as intercrop with other crops. Being a short duration (60-65 days) crop with wide adaptability green gram grown International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 5 (2017) pp. 1150-1158 Journal homepage: http://www.ijcmas.com Molecular characterization is helpful in understanding the phylogenetic relationship among various germplasm to reveal the genetic diversity among the used parental genotypes. Among several efficient methods for revealing genetic variability within and among plant populations, one of the most widely applied methods is ISSR marker analysis. ISSR, markers are commonly used because they are quick, simple and environment non-sensitive enabling genetic diversity analysis in several types of plant material like natural populations, population in breeding programmes. Evaluation of genetic diversity would promote the efficient use of genetic variations, effective conservation and purity of the genotype to be determined as well as utilization of germplasm in crop improvement. ISSR marker analysis was performed to detect relatedness and diversity among eight parental genotypes. ISSR markers are useful in detecting polymorphism among accessions by generating a large number of markers that target multiple microsatellite loci distributed across the genome. Out of 109 scorable bands, 88 bands were polymorphic and the level of polymorphism was 81 per cent. Twenty five ISSR primers were used, out of which eighteen primers showed amplification in all genotypes. The average number of bands per primer was found to be 6.22 and average numbers of polymorphic bands per primer were 4.89. ISSR-01 proved to be best primer in the present investigation with total 29 fragments and eight highest scorable bands as well as 100 per cent polymorphism. K e y w o r d s Mungbean, ISSR Markers, Yield and yield components. Accepted: 12 April 2017 Available Online: 10 May 2017 Article Info Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 1150-1158 1151 all over the world as a sole crop and as an inter crop or mixed crop with cereals. Besides being a rich source of protein, green gram enriches soil fertility through atmospheric nitrogen fixation with the help of Rhizobium bacteria in nodules and humus thus, plays a crucial role in furthering sustainable agriculture. For any successful breeding programme to improve grain yield and component characters, it is essential to know precisely the genetic architecture of these characters under prevailing conditions. Application of molecular markers to plant breeding has established the need for information on variation in DNA sequence even in those crops in which little classical genetic and cytogenetic information is available. Currently, the genetic diversity of plants has been assessed more efficiently after the introduction of the methods that reveal polymorphism directly at the DNA levels. Materials and Methods Final experimental trial comprising 8 parents along with 28 F1s was evaluated during kharif, 2014 in randomized block design with three replications at RCA college farm, MPUAT, UDAIPUR. Eight diverse and well adapted genotypes of green gram were selected as parents for crossing programme, namely IPM-99-125, BM-4, ML-131, IPM 02-03, PDM-139, RMG-1035, RMG-344 and RMG-1045 (Table 1). All recommended cultural practices and plant protection measures were adopted to raise a good crop. Molecular analysis using ISSR markers was done exclusively for the parental material only. D Molecular marker analysis was done for the parental material to see the diversity present among the parental material. NA extracted from different green gram cultivars were compared using ISSR methodology. The leaves were harvested after 21 days and DNA was isolated with the help of Doyle and Doyle, 1987 protocol. DNA was extracted from young leaves (3–4 weeks old) using CTAB method and was amplified by using decamer random oligonucleotide primer in a DNA thermo cycler (Biometra). For the ISSR reactions, 25 primer pairs were used. The DNA content in 20 μl of the reaction mixture was 50 ng. The sequences of these primers were purchased from Bangalore Genei Pvt. Ltd. The details of operon code sequence of the primer and G:C contents are given on table 3. The reaction contained 10X reaction buffer, 200 μM each of dNTPs (“Bangalore Genei”), 0.5 μM of each primer and 1 unit of Taq DNA polymerase (Table 2 and Fig. 1). Submerged gel electrophoresis unit was used for fractionating amplified PCR products on 1.2% agarose gel. The gel was prepared in 1X TAE buffer containing 0.5 μg/ml of ethidium bromides. The samples and loading dye were mixed in 1:1 ratio and loaded with micropipette. In order to score and preserve banding patterns, photographs of the gel were taken by a Gel Documentation System, under UV transilluminator. ISSR bands were designated on the basis of their molecular size ranging between 1001000 bp. Electrophoresis was carried out at 100 V for 3 hr in 1X TAE electrophoresis buffer. Gel was viewed under UV transilluminator and photographed by gel documentation system. Presence of amplified products was scored as 1 and its absence as 0 for all genotypes and primer combinations. These data matrices were then entered into NTSYSPC developed by Rohlf (1993). The genetic distances obtained from cluster analysis through UPGMA were used to construct the dendrogram, depicting the relationships of the genotypes using computer program NTSYSpc version 2.02. Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 1150-1158 1152 Results and Discussion Total genomic DNA was isolated with CTAB method Doyle and Doyle (1987). The powdered plant tissues extracted with extraction buffer containing chelating agent (EDTA) which helped to inactivate nucleases released from the plant cells which could cause serious degradation of the genomic DNA. The amount of DNA isolated from various genotypes of V. radiata L. ranged from 757 to 1518 ng/μl. The genotype IPM 02-03 yielded the highest amount of DNA (1518 ng/μl). Whereas the lowest amount of DNA (757 ng/μl) was obtained from genotype RMG-344. The ratio of absorbance (A260/A280) ranged from 1.70 to 1.89 revealing that the DNA obtained was free from contaminants like polysaccharides, protein and RNA. The quality of DNA as also checked by gel electrophoresis revealed a single discrete band in all genotypes showing that genomic DNA was intact and had high molecular weight, free from any mechanical or enzymatic degradation, free from RNA contamination and was of high quality (Table 5). Twenty five ISSR primers were used for the present investigation out of which eighteen primers showed amplification in all genotypes (Fig. 2). A total of 112 amplified bands were obtained from the 18 primers, out of which 88 were polymorphic. The total number of amplified bands varied between 5 and 8 (Table 3). The average number of bands per primer was found to be 6.22 and average numbers of polymorphic bands per primer were 4.89. The polymorphism percentage ranged from 43 % (UBC-845) to 100% for five primers (ISSR-01, UBC-817, UBC-818, UBC-820 and UBC-854) used. Average polymorphism across all the genotypes of V. radiata L. was found to be 79%. Overall size of PCR amplified products ranged between 100 bp to 2000 bp. The PCR amplification using ISSR primers gave rise to reproducible amplification products. The number of potential ISSR markers depends on the variety and frequency of microsatellites, which tends to change with species (Table 6). Similar results were shown by Das et al., (2014), Singh et al., (2011) and Tantasawat et al., (2010). Genetic relationship and cluster tree analysis The data obtained by using ISSR primers were used to construct similarity matrix of eight V. radiata L. genotypes using „Simqual‟ sub-programme of software NTSYS-pc. Dendrograms were constructed using similarity matrix values as determined from ISSR data for V. radiata L. genotypes using unweighted pair group method with arithmetic average (UPGMA) sub-programme of NTSYS-pc software.

Biochemical characteristics and microbial association of Isabgol (Plantago ovata Forks.) growing soils in Western Arid region of India

Isabgol (Plantago ovata Forks.) is one of the important cash crops in arid and semi-arid regions of India. The husk of Isabgol seeds are primarily used as laxative in medicinal preparations. The cultivation of Isabgol crop is very much dependent on soils and weather conditions as this crop is highly susceptible to many biotic and abiotic stress parameters. Soil microbial population is involved in many direct and indirect interactions with crop plants. The type of microbes present in the soils also affects the plant health. Soil samples from the rhizospheric zone of the plants were collected from Barmer, Jalore and Ajmer Districts of Rajasthan State in western arid region of India. The soil EC and pH (1:2.5) recorded were in the range of 0.12 to 0.46 dS/m and 7.4 to 8.9, respectively, depicting neutral to alkaline soils, the macro nutrient viz; N, P, K were found to be in the range of 128.0 to 192.54, 19.4 to 80.4 and 149.8 to 338.8 kg/ha, respectively. The DTPA extractable micronutrients Cu, Zn, Mn and Fe were in the range of 0.29 to 3.50, 0.26 to 1.5, 0.51 to 4.51 and 1.0 to 5.38 ppm, respectively, in the soil samples of Isabgol growing regions of Rajasthan. Total viable count (TVC) gives a quantitative idea about the presence of microorganism such as bacteria and fungi in samples. The total bacterial count of soil microorganisms varied from 0.8x107 to 1.96x107 cfu/g whereas, total fungal count varied from 1.52x106 to 2.85x106 cfu/g. Beneficial microorganism population in terms of total Azotobacter count (0.1x105 to 1.0 x105 ) psuedomonads counts (3.33x105 to 5.8x105 cfu/g) and phosphate solubilizing bacterial count (0.1x103 to 1.0x103 cfu/g) varied highly in different soils. Key words: Isabgol (Plantago ovata Forks.), bacterial count, fungal count, N, P, K, macronutrients, micronutrients.