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ThesisItem Open Access DNA Barcoding and Antioxidant profiling of macrophytes of Dal Lake(SKUAST Kashmir, 2022) Shafqat Mehraj; Masoodi, Khalid ZAquatic macrophytes are considered photosynthetic organisms of freshwater habitats, comprising vascular plants, aquatic bryophytes, and macroalgae growing permanently or temporally in aquatic environments. These play an essential role in the structure and functioning of freshwater ecosystems. The present study was carried out to validate the morphological identification of macrophytes of Dal Lake, Srinagar through DNA Barcoding and to determine the antioxidant potential of these macrophytes. Twenty macrophytes were collected from Dal Lake Srinagar. DNA was extracted using the CTAB method, and PCR products were sequenced. Twenty different macrophytes were annotated and identified based on the Mat-K sequence deposited in the GenBank. Bioinformatic analysis further revealed the association between these macrophytes at the molecular level. Phylogenetic relationships were identified which resulted in the classification of 20 nucleotide sequences. The following codon locations were included: 1st+, 2nd+, 3rd+ and Noncoding regions were depicted. The final dataset contained a total of 1571 locations. In addition to DNA barcoding, antioxidant potential of these macrophytes was also identified with respect to rutin which was taken as standard. DPPH radical scavenging activity was found significantly higher in leaves of Potamogeton praelongus followed by Polypogon monosplensis, Potamogeton natans, and Typha latifolia, with inhibition of 20.60±8.142, 20.06±14.13 and 18.71±8.43µg/ml respectively and IC50 values as 108.53389, 482.19697 and 271.53285 respectively. The data revealed that Potamogeton prolongeus was a better scavenger of DPPH free radical than rutin. These results revealed that the macrophytes having the highest antioxidant potential can be used as superfoods and sources of nutraceuticals for cattle feed. Further more Trapa bicornis, Potamogeton lucans, Phragmites pusillus, Lycopus uniflorous, Carex vestitata, Potamogeton praelongus are reported for the first time in Dal Lake.ThesisItem Open Access Biochemical Characterisation and DNA Barcoding of Kala zeera Germplasm Lines of Jammu and Kashmir(SKUAST Kashmir, 2022) Salima Fayaz; Zargar, Sajad MajeedKala zeera (Bunium persicum) is a high value and under-exploited spice crop that grows in mountainous regions of North western Himalayas. It has received very little attention in terms of standardization of production technology, production improvement, and plant protection management practices. The aim of this study was to characterise the Kala zeera germplasm of Jammu and Kashmir, India using morphological (seed traits), biochemical parameters and to establish DNA barcodes of desirables lines. Significant variation among Kala zeera genotypes for various seed traits was observed. Seed weight ranged from 0.05 to 0.35 (g). Seed length ranged from 2.21 to 2.28 (mm). We also observed significant variation in biochemical parameters. Antioxidant capacity was determined using the free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) method. Genotype RMZ12 showed highest flavonoid (4.15mg/g) and phenol content (22.56mg/g) content, whereas RMZ 12 also showed highest anti-oxidant property. Further, we established DNA barcode of desirable kala zeera genotypes using ITS markers. The sequences of same have been published in NCBI. This study helped us in identification of Kala zeera genotypes with high phenol, flavonoid and anti-oxidant content and their barcodes were also revealed. Best germplasm lines can be further evaluated and utilised in breeding program for development of promising kala zeera varieties.ThesisItem Open Access Studies on selenium (Se) induced phosphorus (P) stress tolerance in common bean (Phaseolus vulgaris L.)(SKUAST Kashmir, 2022) Madeeha Mansoor; Zargar, Sajad M.Mineral stress is one of the dominating abiotic stresses, which leads to decrease in crop production. Selenium (Se) seed priming is a recent approach to mitigate the plant’s mineral deficiency stress. Although not an essential element, Se has beneficial effects on the plants in terms of growth, quality, yield and plant defense system thus, enhancing plant tolerance to mineral deficiency. The present research was accomplished to find out the effect of Se priming on common bean plant (SFB-1 variety) under phosphorus (P) stress. The seeds were grown in vitro on four different MGRL media which are normal MGRL media as control with non-Se primed seeds (Se - P +), non -Se primed seeds grown on P deficient MGRL media (Se- P-), Se primed seeds grown on normal MGRL media (Se+P+) and Se primed seeds grown on P deficient MGRL media (Se+P -). The various morphological and biochemical parameters such as proline content, total sugar content, polyphenols and expression of proteins were analyzed under P stress. The results showed that Se priming has significantly (p≤0.05) affected the biochemical parameters under normal and P stress conditions. The morphological parameters-length, weight, number of nodes and leaves of Se+P+, Se+P- root and shoot tissue showed increase as compared to Se-P+, Se-P-. Similarly various biochemical parameters such as total chlorophyll content, proline and total sugar content of Se+P+, Se+P- increased significantly as compared to Se-P+, Se-P-. The differential protein expression in both Se+P+, Se+P- and Se-P+, Se-P- plants were determined using MALDI-TOF MS. The differentially expressed proteins in Se+P+, Se+P- plants were identified as caffeic acid-3-O-methyltransferase (COMT) and SecA protein (a subunit of Protein Translocan transporter) and are found responsible for lignin synthesis in root cell walls and ATP dependent movement of thylakoid proteins across the membranes in shoot respectively. The differential expression of proteins in plant tissues, validated morphological and biochemical responses such as maintaining membrane integrity, enhanced modifications in cellular metabolism, improved polyphenol activities and expression of defensive proteins against mineral deficiency. The study provided an understanding of Se application as a potential approach increasing tolerance and yield in crop plants against mineral deficiency.ThesisItem Open Access Genome-wide Association Mapping (GWAS) of essential amino acids in Buckwheat (Fagopyrum spp.)(SKUAST Kashmir, 2022) Nath, Adil RashidThe major aim of this study was to identify QTLs associated with essential amino acids (Lysine and Methionine) using 132 diverse genotypes of Buckwheat, that were collected from different regions of North-western Himalayas and also procured from NBPGR, New Delhi. Statistical model used for evaluation of germplasm for amino acids was complete Randomized Design (CRD) with three replications. The grain lysine content of the buckwheat germplasm ranged from 5.13 to 6.91g/16gN where as the grain methionine content ranged from 1.09 to 4.71 g/16gN. Further genotype by sequencing (GBS) method with Compressed Mixed Linear Model (CMLM) was followed for identification of QTLs contributing for grain lysine and methionine content. 15 SNPs were found significantly associated with buckwheat grain lysine content with 3 SNPs located on chromosome no.1, 2 SNPs on chromosome 4, 4 SNPs on chromosome 5, 3 SNPs on chromosome 7 and one each on chromosome 2 and 3. SNP (FT5:10662066) located on chromosome 5, has been found with highest R2 value (0.42). Where as 18 SNPs were found associated, with 6 SNPs located on chromosome 6, 4 SNPs on chromosome 4, 3 SNPs on chromosome 3, 2 SNPs each on chromosome 1 and 2 and 1 SNP each on chromosome 2, 5 and 7. SNP (FT6:33450014) located on chromosome 6 has been found with highest R2 value (0.211). Further analysis revealed identification of potential candidate genes present in 100 Kb regions of identified QTLs for these amino acids.ThesisItem Open Access Genetic Dissection of QTLs/Candidate Gene(s) Underlying Nutraceutical Traits in Buckwheat (Fagopyrum spp.)(SKUAST Kashmir, 2023) Madhiya Manzoor; Zargar, Sajad M.The pandemic COVID-19 has been very impactful in sensitising us to have healthy and nutritional food. Moreover, the importance of underutilised crops have been majorly focussed at various global forms due to which 2023 year has been declared as an International Year of Millets by UN. Among these underutilised crops, buckwheat has been of great significance owing to its nutritional and nutraceutical potential. In the present study, we have characterised 132 diverse set of buckwheat germplasm that was collected from North Western Himalayas and also procured from NBPGR, New Delhi for various nutritional and nutraceutical compounds. The evaluation of this germplasm for these nutri-nutraceutical compounds (phenol, flavonoids, antioxidants Fe, Zn, ascorbic acid and folate) helped us in establishing inventory of nutritional and nutraceutical of buckwheat. Further, we have adopted high throughput genotyping facility (GBS) which helped us in identification of 28 QTLs for these traits. These QTLs have been mapping on chromosomes and further potential candidate genes positioned in 100kB regions of QTLs have been identified. As such we could establish genetic as well as genomic resources from this study which have implications in development of nutritionally improved genetic stock in buckwheat. Furthermore, this study revealed that the defatted buckwheat flour and protein isolates can be used for preparation of bioactive peptides that can be an important component of functional food and could find usefulness in the food and pharmaceutical industries.ThesisItem Open Access Proteo-genomics based Molecular Characterization and Expression Studies of Flavonoid Biosynthesis Gene(s) in Buckwheat (Fagopyrum spp.)(SKUAST Kashmir, 2023) Ammarah Hami; Zargar, Sajad M.The present study entitled, Proteo-genomics based Molecular Characterization and Expression Studies of Flavonoid Biosynthesis Gene(s) in Buckwheat (Fagopyrum spp.)” revealed that the use of multiple DNA barcodes ITS, psbA and accD authenticates the speciation in case of buckwheat. Further proteomics technique was employed to standardize the protein extraction method from different tissues (Shoot, root and seeds) of buckwheat. Among various methods tested phosphate -TCA acetone method showed high recovery of proteins from buckwheat shoots and roots having highest protein concentration of 3.31±0.05μg/μl and 1.20±0.04 μg/μl respectively. Whereas, phenol-based protocol showed highest proteome recovery from seeds of buckwheat(3.61±0.07μg/μl). Further, proteomic analysis couple with MALDI-TOF MS, helped us in identification of species specific protein biomarkers, which have implications in authenticating the species of buckwheat and to understand the expression variations among species for a specific biological activity. Furthermore, PAL and FLS genes were targeted to have understanding about spatiotemporal expression of these genes among two different species. The PAL and FLS genes showed differential expression patterns at different growth stages of Fagopyrum species. The expression of both genes was observed higher in case of F. tataricum as compared to F. esculentum that was attributed to biosynthesis of high concentrations of metabolites in Tartary buckwheat as compared to common buckwheat. The expression of FLS gene was found highest in the inflorescence while in PAL gene the highest expression was found in seed, which correlated with the highest rutin in seed.ThesisItem Open Access Studies on Molecular and Biochemical characterization of Rice (Oryza sativa L.) germplasm of Kashmir Himalayas(SKUAST Kashmir, 2023) Deepika Pandita; Husaini, Amjad M.Rice (Oryza sativa L.) regarded as one of the most important cereal crops, is a major food grain contributor to the total world food grain basket. It belongs to the genus Oryza, family Gramineae (Poaceae) and tribe Oryzeae. Half of the world’s population is suffering from one or more vitamin and/or mineral deficiency. The developed countries are managing deficiency by adopting fortification programs, but same programs are not affordable to poor countries. Therefore, an alternative and less expensive strategy is to modify the nutritional quality of the major cereals consumed by the people. Simple Sequence Repeats (Microsatellite), being easy, reliable, robust and reproducible, were employed in carrying out fingerprinting of these landraces. These markers are multiallelic and usually resolve on agarose gel. In the present study 50 genotypes of rice was selected from the germplasm collection maintained at Khudwani.The study observed that the highest level of P concentration was found in genotype K-37 (4553.33ppm) and the lowest level was found in genotype K-44 (2133.33ppm). Highest level of Fe concentration was found in genotype K-13 (31.93ppm) and the lowest level was observed in genotype K-21 (2.27ppm). Highest levels of K concentration was found in genotype B-55 (1921.33ppm) while as lower level was found in genotype K-17 (921.33ppm). Mg concentration was highest in genotype K-35 (7625.33ppm) and lowest in genotype K-16 (4551.33ppm). Highest level of Ca was found in genotype K-13 (23799.33ppm) and lowest level in genotype K-19 (9618ppm). Highest level of Na was found in genotype K-29 (198.67ppm) and the lowest level in genotype HA-11 (38.67ppm) among all 50 rice genotypes. Genotype K-34 had the greatest Zn concentration (10.17ppm), while genotype K-43 had the lowest concentration (1.27ppm). Highest level of protein content was found in genotype B-54 (7.75%) while as, lowest level was found in genotype K-27 (4.06%). Maximum level of starch content was found in genotype K-37 (82.02%) and minimum level was found in genotype K-22 (75.55%) among all 50 genotypes. Highest level of amylose in genotype K-39 (25.46%) and the lowest level was found in genotype K-47 (19.01%). Genotype K-47 (80.99%) had the highest content of amylopectin, whereas genotype K-39 (74.54%) had the lowest content. Genotype K-36 (3.3%) demonstrated the highest crude fibre content, while as lower level was found in genotype K-45 (1.23%). In this study 40 primer pairs scored 114 alleles, with an average of 2.85 per allele. Primer RM413 observed the highest PIC value (0.58), while the primer RM162 observed the lowest PIC value (0.30), with an average of 0.44. Predominant allele frequency of polymorphic markers ranged from 0.40 to 0.78, with an average of 0.59. Gene diversity ranged from 0.35 to 0.66, with an average value of 0.52. Heterozygosity ranged from 0.18 to 0.74, with an average of 0.39. Cluster analysis was performed to elucidate the relationship among the genotypes. All 50 genotypes were grouped into three major clusters generated through Neighbour-joining analysis comprising of 20, 15 and 15 genotypes in clusters I, II, and III, respectively. All basmati genotypes were grouped separately in Cluster I. STRUCTURE analysis was carried out to find out the number of populations from 50 genotypes using 40 SSR markers. For the first level of clustering at K=2, we got the admixture for all 50 genotypes. At K=3, the 50 accessions of Kashmir province got separated into three different populations. The sub-population A, B and C represents 26 per cent (13), 36 per cent (18) and 20 per cent (10) of genotypes used in structure analysis respectively. The remaining 18 per cent (9) of genotypes represents admixture. Fst values of 0.1145, 0.1062 and 0.1609 were found in the sub-populations (A, B and C), respectively, with an average value of 0.1272. The percentage of variation among populations and within populations was estimated using Analysis of variance (AMOVA) which shows that 74 per cent was attributed within individuals, 23 per cent was among individuals and 3 per cent was among populations With respect to mineral profiling, our results revealed that among cluster I, genotype K-44 and HA-8 has shown higher mineral uptake for four minerals (Fe, K, Mg and Na) and (Ca, Zn, Na, P) respectively, followed by genotype HA-4 which showed more mineral uptake for three minerals (P, K, Ca). Similarly, in cluster II, genotype K-16 and K-19 had higher mineral uptake for three minerals (P, Fe, K) and (Fe, K, Ca) respectively. In cluster III, genotype K-30 demonstrated higher mineral uptake for four minerals (Fe, Mg, Ca, Zn) followed by K-31 and K-34 which displayed higher mineral uptake for three minerals (P, Fe, Na) and (Fe, K, Ca) respectively. With respect to nutrient profiling, our findings revealed that among cluster I, genotype HA-4 showed higher nutrient content for three nutrients (Protein, Starch and Amylose) followed by six more genotypes that displayed nutrient content accumulation for different nutrients ciz. genotype K-47 and K-40 showed each for protein and amylose, genotype K-45 and K-41 displayed each for amylose and crude fibre, genotype HA-6 showed for protein and amylopectin and genotype K-42 demonstrated for amylopectin and crude fibre. In cluster II, genotype K-37 and K-23 displayed nutrient content accumulation for only two nutrients separately, ciz. (amylopectin and crude fibre) and (starch and amylose) respectively. Similarly, in cluster III, five genotypes displayed the higher/lower nutrient content accumulation for only two nutrients separately, ciz. genotypes K-27 and HA-10 showed for starch and crude fibre, genotypes K-13 and K-30 displayed for protein and amylopectin and genotype K-24 showed for starch and amylose.ThesisItem Open Access Genome-Wide Association Mapping (GWAS) for Morphological and Seed Quality Traits in Buckwheat (Fagopyrum spp.)(SKUAST Kashmir, 2023) Naik, Samiullah; Zargar, Sajad M.Knowledge about genetic variability parameters is imperative for improving crop plants for traits of economic importance. This study reports the characterization of a large germplasm set of an underutilized pseudo-cereal buckwheat endemic to North Western Himalayas in order to identify and to elucidate the nature of variability, identify trait specific genotypes for use in buckwheat breeding programmes and to identify QTLs associated with different morphological and seed quality traits using Genome Wide Association Mapping (GWAS). The major aim was to estimate the genetic variability and to identify QTLs associated with morphological and seed quality traits in 162 diverse germplasm lines of buckwheat collected from different regions of North Western Himalayas. The material was evaluated in Randomized Completely Block Design (RCBD) with five replications at two different locations (SKUAST-Kashmir, Shuhama and Mountain Agricultural Research and Extension Station (MAR & ES), SKUAST-Kashmir, Izmarg, Gurez) during Year 2020 and 2021. The analysis of results indicated significant variability in the buckwheat germplasm for all morphological and seed quality traits across locations. The phenotypic coefficient of variance (PCV), genotypic coefficient of variance (GCV), broad sense heritability and genetic advance values varied between locations. Pooled analysis across locations shows that highest PCV values were recorded for number of primary branches. Heritability ranged from low for number of secondary branches to medium for days to 50% flowering. GWAS analysis shows that a total of seventy four SNPs were significantly associated with different morphological and seed quality traits across both locations. Among seventy four SNPs, thirteen QTLs were common/stable QTLs between two locations. The result of the present study provided valuable insights about the variability and QTLs identification in buckwheat germplasm under North Western Himalayan conditions. The information on variability parameters can be harnessed to develop an effective breeding strategy for improving buckwheat for traits of economic interest. The results of this research reveal the marker-trait associations and environmental interactions for morphological and seed quality traits in buckwheat. This study supports the use of GBS technology which allows the identification of a large number of SNP markers with many advantages for further association mapping and QTL studies for the detection of candidate genes related to morphological and seed quality traits for breeding programmes.ThesisItem Open Access Studies on DNA-barcoding and Molecular Diversity in Fusarium spp. Causing Wilt in Solanaceous Crops(SKUAST Kashmir, 2022) Parihar, Tasmeen Javeed; Masoodi, Khalid Z.Solanaceous crops (tomato, brinjal, chili and bell pepper) are grown globally and contribute incalculable values to wellness of human beings but are prone to many pathogen invasions. The current research “Studies on DNA- barcoding and Molecular Diversity in Fusarium spp. Causing Wilt in Solanaceous Crops” was carried out to identify the evolving species of Fusarium wilt infecting solanaceous crops. Wilt being one of the most common and destructive disease in terms of crop yield and growth in Solanaceous crops, a survey of solanaceous crops infected with Fusarium wilt was carried out in Kashmir valley. Samples were collected from different areas of Anantnag, Pulwama, Srinagar, Baramulla and Bandipura districts. A total of 75 isolates of Fusarium spp. were collected from tomato, brinjal, chili, and capsicum from three belts of Kashmir valley (North, South, and Central). Pathogens were was isolated from root portion and pure single spore cultures were maintained on PDA medium. Pathogenicity was carried out using rhizosphere inoculation technique. Initially plants showed light green to yellowish discoloration of leaves followed by their drooping, shriveling and finally death of whole plant after sixth weeks of inoculation. To identify the associated pathogens, pure cultures were established on PDA medium, and studied based on their morphological characterization viz., shape, size, septation of microconidia, macroconidia, mycelium, besides colony characters. DNA extraction and PCR amplification was performed using methods strandarized in Transcriptomics laboratory. Internal Transcribed Spacer (ITS) along with Transcription elongation factor (TEF) gene regions were used as DNA barcoding markers for sequencing. ITS prefers high degree of inter-specific variability and conserved primer sites but in some cases ITS is not suitable for closely related species, therefore TEF gene amplification was also persuaded and Fusarium avenaceum, Fusarium flocciferum, Fusarium oxysporum, Fusarium equisite, Fusarium incarnatum, Fusarium chlamydosporum, Fusarium latritium, Fusarium accuminatum and Fusarium tricinctum were identified, out of which Fusarium avenaceum, Fusarium flocciferum and Fusarium incarnatum are the new reports from solanaceous crops; depicting the diversifying and evolving nature of Fusarium species with respect to hosts within solanaceous crops and also from other host crops. The diversification of pathogens is also due to more selection pressure exerted by differential resistant pathogens of hosts which reduce change in genetic structure of the pathogen. In current study, seven microsatellite markers were used to predict evolutionary relationships, genetic variation, and population structure of different Fusarium species infecting tomato, brinjal, chili, and capsicum. Analysis of molecular variance (AMOVA), revealed high genetic variation within the population and low genetic variation between the populations. Structure analysis of the population resulted in grouping of 75 isolates into two clusters with quite low admixture in populations and same results were revealed from radial clustering using Darwin Neighbor Joining method. Based on dendrogram isolates were grouped into different clusters irrespective of their species and host
