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ThesisItem Open Access Cryopreservation of chethikoduveli (Plumbago rosea L.) and assessment of genetic fidelity of regenerated plantlets using molecular markers(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2014) Anand, Vishnu Prakash; KAUInvestigations on “Cryopreservation of Chethikoduveli (Plumbago rosea L.) and assessment of genetic fidelity of regenerated plantlets using molecular markers” were carried out at the Department of Plant Biotechnology, College of Agriculture, Vellayani during 2011-2013. Plumbago rosea var. Agni plants were collected from AMPRS, Odakkali, Ernakulam and maintained at the Department of Plant Biotechnology, College of Agriculture, Vellayani as source of explant during the course of the study. The objectives of the present study was to standardise cryopreservation protocol by encapsulation dehydration technique for long term conservation of P. rosea and genetic fidelity assessment of plantlets recovered and regenerated from cryostorage using molecular markers. The project was carried out in two phases viz., in vitro regeneration and in vitro conservation by cryopreservation of P. rosea. In vitro regeneration protocol was optimised for P. rosea var. Agni. Various steps of in vitro regeneration viz., surface sterilization, axillary shoot proliferation, in vitro rooting and acclimatization and planting out has been standardised. For surface sterilizing, single nodal explants (3-4 cm long) were subjected to fungicide treatment with 0.1 per cent carbendazim 50 per cent W. P. (for 30 min) followed by aseptic sterilisation dip with absolute alcohol. Further, the explants were surface sterilised with 0.2 per cent mercuric chloride (for 5 min) which gave 100 per cent survival without any contamination. Enhanced release of axillary buds from single nodal explants, with maximum shoot proliferation (5.28 shoots/culture) was obtained in the medium, MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. The best response (10.67 roots/culture) of in vitro rooting of plantlets was obtained in the medium, MS + NAA 1.0 mg l-1. In vitro rooted plants gave a maximum survival rate of 76 per cent and 72 per cent, when planted out in potting media consisting of red soil and coir pith (3:1) and red soil and coir pith (2:1) supplemented with VAM respectively at 25 per cent shade. In cryopreservation studies, preconditioning treatment (sucrose 0.5 M for 7 days) recorded maximum shoot proliferation (2.67 shoots/culture) when nodal segments with single axillary bud were cultured on MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1 medium. Among different encapsulation treatments, maximum shoot proliferation of (2.31 shoots/culture) was obtained in beads formed with sodium alginate 2.5 per cent and calcium chloride 100 mM, when cultured on the medium, MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. Pre-culture medium supplemented with sucrose 0.5 M for 3days gave maximum shoot proliferation (3.44 shoots/culture) when cultured on the medium, MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. A desiccation duration of 5 h at 18.13 per cent moisture level was found to be most effective giving 66.67 per cent survival and 62.50 per cent regeneration on thawing and culturing on the recovery medium MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. The beads when stored in liquid nitrogen for different duration and cultured on recovery medium did not show any significant variation with respect to survival per cent. RAPD markers were tried to study the genetic fidelity of the regenerated plantlets from encapsulated and cryopreserved axillary buds. Six primers were screened and RAPD banding patterns of the cryoregenerated plantlets and control plants were compared. Polymorphism was not found with any of the primers tested. RAPD profiles of cryoregenerated plantlets were identical to those of the control. The in vitro regeneration protocol optimized included surface sterilization of single node cuttings with 0.2 per cent HgCl2 for 5 min, axillary shoot proliferation in MS medium supplemented with BA 1.5 mg l-1 and IAA 1.0 mg l-1, in vitro rooting in MS medium supplemented with NAA 1.0 mg l-1 and planting out in potting medium, red soil and coir pith (3:1). The protocol for encapsulation dehydration technique of cryopreservation was standardised for the axillary buds of P. rosea with preconditioning in semi solid MS medium supplemented with sucrose 0.5 M for 7 days, encapsulation using sodium alginate 2.5 per cent and calcium chloride 100 mM followed by pre-culture in liquid MS supplemented with sucrose 0.5 M for 3 days and 5 h dehydration (MC 18.13 %), rapid freezing in LN for at least 2 h and recovery in the medium MS + BA 1.5 mg l-1 + IAA 1.0 mg l-1. The cryopreservation protocol using encapsulation-dehydration technique standardised could be utilised for long-term conservation of P. rosea.ThesisItem Open Access Characterisation of Pathogenesis related proteins for anthracnose resistance in vegetable cowpea, Vigna spp.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Agatha Shiny, A; KAU; Deepu MathewCowpea (Family: Fabaceae) is an important pulse cum vegetable crop of suitable for the tropical and sub-tropical regions of the world. The grain type cowpeas better tolerates the biotic and abiotic stresses against the vegetable types. Under humid conditions, vegetable types, especially the pole types are susceptible to many diseases and among them, anthracnose caused by Colletotrichum lindemuthianum (Sacc. & Magn.) Br. and Cav. is very severe. In Kerala, complete yield loss in vegetable cowpea is reported due to anthracnose during monsoons. The study entitled “Characterization of pathogenesis related proteins for anthracnose resistance in vegetable cowpea, Vigna spp.” was carried out with objective to develop the protein profiles of resistant and susceptible bush and pole genotypes through SDS-PAGE analysis at different time intervals of infection and to characterize the differentially expressed proteins by MALDI-TOF followed by in-silico analyses. Two bush type varieties Pusa Komal and Kanakamony, the former reported to be highly susceptible and the latter immune to anthracnose and two pole type varieties Lola and Arimbra Local, of which the former susceptible and the latter resistant were used in the study. Pure culture of the pathogenic fungus was developed and maintained on selective medium (Neopeptone-Glucose-Agar) at the Dept. of Plant Pathology. The identity of Colletotrichum lindemuthianum has been established from the spore characteristics observed under phase contrast microscope and the pathogenicity was confirmed through artificial inoculation under controlled conditions. The pot culture experiment was conducted with 50 pots per variety. Artificial inoculation of pathogenic fungus was done and the leaf samples were collected at 0, 6, 12, 18, 24, 48, 72, 96, 120, 144,168 and 192 hours after artificial inoculation. The total protein was extracted using Tris-HCl buffer (pH-7.5), quantified using spectrophotometer and analyzed by SDS-PAGE method. The defense enzymes like peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were assayed. By artificial inoculation, disease responses for anthracnose were confirmed to be highly susceptible in Pusa Komal and Lola; highly resistant in Arimbra Local and immune in Kanakamony. Protein expression was found to be higher from the initial hours in resistant varieties whereas in susceptible varieties, the expression was reduced immediately after infection then peaked at 18hr and gradually decreased later on. Two prominent and differentially expressed protein bands at 56 kD and 14 kD were sequenced in MALDI-TOF to obtain the peptide mass fingerprint. Through in-silico analyses using Mascot server software, they were identified to be the large and small subunits of the chloroplastic enzyme RuBisCo. Thus the capability of a variety to maintain high levels of RuBisCo was found to be the deciding factor for anthracnose disease resistance. Further, protein profiles developed after purification of proteins by dialysis have clearly identified the differentially expressed band at 29 kD in the resistant varieties which is in the size range of already reported PR proteins. PO and PAL activities were proportionate to the resistance behavior, with the peak values at 18 and 24 hr after inoculation. With the results of this study, these defense enzymes are recommended as biochemical markers for identifying the resistance in the accessions. Capability to maintain higher levels of RuBisCo, PO and PAL enzymes is the characteristic of anthracnose resistant vegetable cowpeas and the future breeding programmes could be oriented in this directionThesisItem Open Access DNA fingerprinting of selected black pepper (piper nigrum L.) varieties.(Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2011) Manjunath, Mogalayi; KAU; Nazeem, P ABlack pepper (Piper nigrum L.) famous as “Black Gold” and also known as “King of Spices” is one of the important agricultural commodities of commerce and trade in India since pre-historic period. The crop is the major source of income and employment for rural households in the predominantly pepper growing State of Kerala where more than 2.5 lakh farm families are involved in pepper cultivation. Karnataka, Tamil Nadu are the other major pepper producing States in the country. Kerala accounts for 80-90% of the total pepper production in the country. Idukki and Wynadu are the two major pepper producing districts in Kerala. Different cultivars/varieties are popular among the farmers and there phenotypic identity is not very distinct. The study entitled “DNA fingerprinting of selected black pepper (Piper nigrum L.) varieties” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture during the period 2009-2011. The objectives of the study were to characterize the released black pepper varieties of KAU using different molecular markers - RAPD, ISSR and SSR and to develop DNA fingerprint with which the variety could be identified and its fidelity detected. Seven black pepper varieties (Panniyur 1 to Panniyur 7) collected from Pepper Research Station, Panniyur and maintained at CPBMB, COH were used for the study. DNA extraction was done with CTAB (Rogers and Benedich, 1994) method with slight modification. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (A260/A280) 1.80 - 1.89 was used for further analysis. The PCR conditions were optimized for RAPD, ISSR and SSR assay. 30 RAPD, 34 ISSR and 29 SSR primers were screened with bulked DNA of black pepper varieties for amplification and those which gave reliable distinct banding pattern were selected for further amplification and fingerprinting. The PCR products obtained from RAPD, ISSR and SSR analysis were separated on 1.3 to 2 percent agarose gel and the amplification patterns recorded. The genomic DNA from each variety was amplified with 10 each of selected RAPD and ISSR primers and 8 SSR primer pairs. The amplification pattern was scored and depicted to develop fingerprint for each variety. The Resolving power (Rp) worked out for the different primers ranged between 7.42 to 9.42 in RAPD and 5.42 to 12.28 in ISSR analysis; indicating the capacity of the primers selected to distinguish the varieties. The Polymorphic Information Content (PIC) varied from 0.86 to 0.90 for RAPD analysis and it was between 0.80 and 0.89 in ISSR analysis indicating the variability among the genotypes. Distinct bands were used to develop DNA fingerprint of black pepper varieties Panniyur 1 to Panniyur 7 through RAPD, ISSR and SSR analysis. Sharing of amplicons developed for each primer with other varieties was also analyzed and demarcated with different colour codes in the fingerprints developed. Most of the amplicons were found shared among the varieties. However, the pattern of sharing was different and good enough to separate out the varieties. Combined DNA fingerprint of each variety with RAPD, ISSR and SSR data was also developed. The amplification pattern observed in RAPD, ISSR and SSR analysis was scored and analyzed for quantifying the variability among the varieties. The computer package NTSYS-Pc was used for cluster analysis. Maximum variability observed was 48 percent for the variety Panniyur 4. The varieties Panniyur 1 and Panniyur 3 having the same parentage indicated 76 percent similarity. The fingerprint developed was good enough to provide varietal identity and the analysis could reveal variability/relatedness among the seven varieties. Separate and combined fingerprints were developed for all the seven varieties through RAPD, ISSR and SSR analysis. The DNA fingerprints thus developed could be utilized for the variety registration and settling IPR issues.ThesisItem Open Access Genetic transformation of Amorphophallus paeoniifolius (Dennst) Nicolson(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2014) Leen Abraham, N; KAU; Makeshkumar, TA study on Agrobacterium-mediated genetic transformation of Amorphophallus paeoniifolius (Dennst.) Nicolson was conducted at the Central Tuber Crop Research Institute, Sreekariyam, Thiruvananthapuram during 2013- 2014. Calli were initiated using petiole and leaves of in vitro plantlets of elephant foot yam cv. Gajendra in callus induction media. After four weeks of incubation, actively dividing globular, hard and creamy white calli were developed. Subculture of developed calli was repeated periodically (20 days) in CIM with an approximate size of one cm2. 15 days old calli was found to be suitable for transformation study. Calli sufficient for the transformation study was obtained after 3 months of subculture. Experiments were conducted to evaluate the sensitivity of elephant foot yam calli to different doses of antibiotics viz. geneticin, hygromycin, ticarcillin. It was observed that complete death and discoloration of the calli obtained with 20 mgl-1 geneticin and 10 mgl-1 hygromycin from sixth week treatment. Statistical analysis of sensitivity response of calli indicated that LD100 was 20 mgl-1 and 5 mgl-1 with geneticin and hygromycin respectively. Sensitivity of the calli to ticarcillin was studied and the responses are analysed with ANOVA. The lowest lethal concentration of ticarcillin was found to be 650 mgl-1. So, concentration below 650 mgl-1 can be used for the successful elimination of Agrobacterium without affecting the regeneration potential of explant. 500 mgl-1 ticarcillin used in this study was observed sufficient for the successful elimination of Agrobacterium without affecting the regeneration potential of calli. For the optimization of parameters affecting transformation, experiments were conducted for the standardisation of optimum concentration of acetosyringone, time of co cultivation, temperature of co cultivation, and suitable Agrobacterium strain. In a study conducted for standardisation of optimum concentration of acetosyringone, increasing number of transformants was obtained with increase in acetosyringone. Significantly higher GUS staining of calli (21.5896) was achieved with the addition of 400μM acetosyringone in the co cultivation media. The effect of number of days of co cultivation on transformation was compared on the GUS expression of 14-day old selected calli. Two-three days of co-cultivation was determined to be the suitable for elephant foot yam because prolonged co-cultivation period (more than three days) was found to promote overgrowth of bacteria and subsequent death of the calli. Correspondingly the transformation percentage was found to decrease with the decrease (less than two days) of co-cultivation period. Investigation of the effect of temperature during co cultivation in elephant foot yam calli revealed that temperature plays an important role in transformation efficiency. Higher temperature, 28°C was found to be optimal to support the highest transient transformation frequency in elephant foot yam and dramatic transient expression reduction occurred when temperature decreased from 22 °C to 20°C. Transformation efficiency with respect to the different strain of Agrobacterium was investigated and the results showed that maximum percent of GUS stained tissue (24.5 percent) of transformants was obtained with the strain LBA4404 with pOYE153 vector followed by AGL0/pOYE153 (14 percent) and GV3103/pCAMBIA 1305.2 (6 percent). GUS assay of transformed callus showed blue colour and confirmation was done by PCR analysis with specific primers and southern blotting. PCR amplification of the DNA of the calli survived in selection medium yielded an expected band size of 280 bp for nptII primer, two bands of size 880bp and 700bp for GUS primer, 300 bp single band for hpt primer and GUSPlus primer. No amplification was obtained for untransformed calli DNA. Nucleic acid spot hybridisation of putative transformants of elephant foot yam further confirmation of the presence of transgene in the DNA. Hybridisation with nptII probe yield spots of varying intensity for all the transformants of AGL0/pOYE153 and LBA4404/pOYE153. Whereas only 5 out of the 8 transformants of GV3103/pCAMBIA1305.2 gave positive for hpt probe and the intensity of spot was low when compared to the spots obtained with nptII probe. Southern hybridisation with DIG labelled nptII probe yield a band for positive control (pOYE153 plasmid) whereas the bands in sample lane was not observed. It is possible that the concentration of DNA (10μl) used in the blot was too low for detection of T-DNA inserts. Hybridisation with hpt probe gave a single band corresponding to the putative transformants lane, which are visible after 30 min exposure indicated that successful hybridisation of the DIG-labelled hpt probe. But the absence of band for positive control was not expected.ThesisItem Open Access Development of biodegradable films from enzymatically modified cassava starch(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Edwin, K Wilson; KAU; Sajeev, M SCassava forms an important food crop in the tropical countries and are rich in starch (20-40%) having desirable physico-chemical and functional properties. Starch and starch derivatives form an important constituent in biodegradable film preparation due to its renewability, abundance availability, low cost, film forming properties, high oxygen barriers, odorless, tasteless, colourless, nontoxic, low solubility, biodegradability etc. But films from the native cassava starch often possess poor physico-mechanical and hydrophobic properties. Hence modified starches by chemical, physical and enzymatic methods offer better scope for the production of biodegradable films which has got wide applications in the food packaging industry. The objectives of the present study was to find out the film forming properties of enzymatically modified cassava starch viz., liquefaction by -amylase and debranching by pullulanase enzymes added with glycerol as plasticiser. Rheological properties were measured in terms of the dynamic mechanical spectra of the film forming solutions viz., storage modulus, loss modulus, phase angle and complex viscosity. Filmogenic solutions based on -amylase was prepared with starch 5%, glycerol 20%, amylase concentration: 100, 200 and 300 μl from the stock solution (0.l ml amylase in 100 ml distilled water), temperature: 80, 85 and 90°C and time: 20, 30 and 40 min for gelatinization. For developing the pullulanase modified starch based films, the starch (5%) was incubated with pullulanase at 2, 3 and 4 units concentrations at 45, 50, 55 0C for 8, 16 and 24 h and the filmogenic solutions added with 20% glycerol were gelatinised at 90 0C for 20 min. Both the experiments were designed using response surface methodology using Box- Behnken design. The physico-mechanical, functional hygroscopic, biodegradation and storage studies of the films were carried out. The dextrose equivalent of the filmogenic solutions varied between 1.6-8.4 with the amylase and 2.2-16.0 with the pullulanase treated starch. The higher values of storage modulus, complex viscosity and low phase angle of the solution containing 153pullulanase treated starch compared to -amylase treated solutions showed that the gel formed during gelatinization of the solution is having more solid nature in their visco-elastic character. The films containing -amylase treated cassava starch showed better whiteness properties. Thickness, moisture content, tensile force, elongation at break and swelling capacity of the films containing pullulanase treated starch was higher than that of the films with -amylase treated starch. The higher solubility of the -amylase based starch films helps easy degradation of the films in the soil whereas offers poor packing ability. Pullulanase film‘s packing ability is better owing to low permeability and solubility. Though the films with both the modified starch is easily biodegradable, pullulalanase took 4 weeks for completely degrade into the soil as evidenced from the soil burial test. The microbial analysis studies showed that the soil in which -amylase treated film buried for degradation had highest bacterial, fungal and actinomycetes population than that of the soils with pululanase treated films. Considering various physical, mechanical and functional properties, the pullulanase modified starch offers better scope for the production of biodegradable packing materials.ThesisItem Open Access Development of molecular markers for anthracnose resistance in vegetable cowpea [vigna unguiculata (L.) walp](Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2015) Dolagobinda, Pradhan; KAU; Deepu, MathewCowpea is one of the most important food legume crops in the semi-arid tropics, covering Asia, Africa, Southern Europe and Central and South America. Nigeria is the centre of origin for cowpea and in India, it is exclusively known as a kharif season pulse but the demand of cowpea as vegetable is increasing. In India, cowpea is grown in about 0.5 million ha but productivity is as low as 600-750 kg grains ha-1. All the pole type vegetable cultivars are highly susceptible to anthracnose caused by Colletotrichum lindemuthianum, which is the most destructive disease, not only for cowpea but also in other legumes. Since no effective measures are capable to manage this disease, the losses can be up to 74-100 per cent and hence breeding the resistant cultivars is suggested as the only promising strategy. Molecular marker assisted selection offers precision in the breeding process but so far no markers are identified in this crop for the anthracnose resistance gene. With this background, the study on “Development of molecular markers for anthracnose resistance in vegetable cowpea [Vignaunguiculata (L.) Walp.]” was carried out at Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, during August 2013 to June 2015. The objective of the study was to identify an ISSR or RAPD molecular marker linked with anthracnose disease resistance in vegetable cowpea, using bulked segregant analysis to enable marker assisted selection. The dual purpose semi-trailing cultivar Kanakamony, which is reported as immune to anthracnose and the high yielding trailing vegetable type cultivar Sharika, which is susceptible, were used for the development of the mapping population for BSA to identify the marker. The parental populations were grown in separate fields in the month of October 2014 as per the package and practices recommended from KAU. Controlled crosses were made to develop F1 seeds during November and December months. Thirty F1 plants were grown during January - March 2015 and the F2 seeds were developed through selfing. The F2 segregating progenies were raised during May-June, in the pots and maintained in greenhouse. Evaluation of parents with F1 of Sharika × Kanakamony and reciprocal cross for morphological characters revealed that pole type and black seed colour characters of Sharika were dominant over semi-trialing and red colour seed of Kanakamony. To isolate the fungal spores for artificial inoculation on the F2 plants, infected plant parts were collected and initially cultured in 3 different media viz. PDA, NGA and CDA. NGA and PDA medium were selected for artificial inoculation based on the characteristics of fungal spores. Artificial inoculation was initially done on 15 days old seedlings and was repeated after 15 days with an approximate concentration of 106 spores/ml and the highly resistant and susceptible F2 plants were identified for BSA. Good quality DNA was isolated from the parents and 47 RAPD and 43 ISSR primers were screened for their capability to generate polymorphic bands. Subsequently, based on the initial screening, 12 RAPD and 10 ISSR primers were selected for bulk segregant analysis. The bulked segregant analysis was done using the DNA from the resistant parent, susceptible parent, resistant F2 bulk and susceptible F2 bulk. BSA with the RAPD primer OPA 02 revealed a polymorphic band at 850 bp which was present in the susceptible parent and susceptible bulk and absent in the resistant parent and bulk. BSA with ISSR primers UBC 810 and UBC 811 produced polymorphic bands at 1.4 kb and 1.5 kb, respectively, which were present in resistant parent and resistant bulks. The co-segregation analysis has to be performed, the marker has to be characterized and validated for the use in breeding programmes.ThesisItem Open Access Genetic diversity analysis in taro (Colocasia esculenta (L.) Schott) of north east India(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2014) Vinutha, K B; KAU; Asha, Devi AThe study entitled “Genetic diversity analysis in taro [Colocasia esculenta (L.) Schott] of North East India” was carried out at the Division of Crop Improvement, Central Tuber Crops Research Institute, Sreekariyam, Thiruvananthapuram during 2013-2014. The objective of the study was to assess the genetic diversity among taro accessions from North East India, which is considered to be one of the centers of origin of taro, using morphological descriptors and SSR markers. The knowledge can be exploited in the heterotic breeding of taro to develop improved varieties suiting various needs. National and international germplasm repositories conserving root and tuber crops can also use the data to maintain taro germplasm efficiently. Twenty five accessions of taro collected from the various North Eastern states of India and maintained at CTCRI were selected for the study. Morphological characterization was performed at the maximum vegetative stage and tuber characters at harvest using ten quantitative and 28 qualitative traits. A combination of NBPGR and IPGRI descriptors were used to explain the wide range of morphological variability. The data was statistically interpreted in terms of diversity indices, PCA, ANOVA and cluster analysis using R statistical package and SAS program. The diversity indices (H’=0.87; D=1.00) revealed a high level of morphological diversity among the taro accessions. The first four components explained 76.59 per cent of the total variation with leaf margin colour, petiole colour (top 1/3rd, middle and base), leaf colour lower, sheath colour and sinus colour contributing maximum to the variability. ANOVA showed significant (P<0.01) variation for 7 out of the 10 quantitative traits studied. Duncan’s multiple range test gave a grouping based on the mean values of quantitative traits. Five major groups were revealed after hierarchical cluster analysis based on Euclidean distance, which did not bear any relation to the geographical origins of the accessions. A protocol was developed for the isolation of good quality DNA overcoming the high levels of secondary metabolites in taro. PCR conditions for SSR detection in taro were also optimized successfully. Ten out of 18 SSR primers were selected for the study after screening. Denaturing PAGE followed by silver staining was performed to analyze the variability among accessions at the molecular level. The average number of alleles and Shannon’s diversity index ranged from 6.0-12.57 and 1.59-2.37, respectively. The polymorphic marker ratio was found to be high for all the primers (0.76-1.0); however, Ce1 A06, Ce1 B03, Ce1 C06, Ce1 F04 and uq201-302 gave the maximum ratio of one. Cluster analysis based on Jaccard’s distance revealed five broad clusters which could not be correlated to the geographical similarities among the accessions. The parameters estimated from molecular and morphological characterization data established a high level of genetic diversity prevalent in the center of origin. The study revealed the absence of congruence between the clustering pattern and geographical origin suggesting that geographically diverse regions share ecologically similar characteristics and vice versa. Differences in morphological and molecular clustering patterns indicate the wide range of adaptations of the crop to the diverse environments inhabited. Though the Mantel’s test established no correlation (r = 0.1432; p = 0.0648) between the molecular and morphological distance measures, the study could identify two groups of accessions that clustered together in both the methods.ThesisItem Open Access Identification and characterization of viruses infecting lesser yam (Dioscorea esculenta (Lour.) Burkill)(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Sudheer, K S; KAU; Jeeva, M LThe study entitled “Identification and characterization of viruses infecting lesser yam (Dioscorea esculenta ( Lour. ) Burkill )” was carried out at the Division of Crop Protection, ICAR-Central Tuber Crops Research Institute (CTCRI), Sreekariyam, Thiruvananthapuram during 2014-2015 with an aim to identify and characterize the viruses of lesser yam at molecular level and to design virus specific primers for detection. Lesser yam is a vegetatively propagated crop, grown for its high calorific value tubers. Yam viruses causing significant reduction in tuber yield and quality are poorly characterized which is restrict the international exchange of germplasm. Lesser yam leaf and tuber sample with different virus symptoms were collected from the germplasm repository and experimental fields of ICAR-CTCRI. The serological and nucleic acid based methods were employed for the detection of Yam Mild Mosaic Virus (YMMV), Yam Macluravirus and Yam Badnavirus which were reported in other yams from India. The leaf and tuber samples of lesser yam were indexed for YMMV, Yam Macluravirus by double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) and reverse transcription (RT-PCR) whereas Yam Badnavirus was by triple antibody sandwich enzyme linked immunosorbent assay (TAS-ELISA) and polymerase chain reaction (PCR). Yam Mild Mosaic Virus is the most prevalent virus detected in 74.28% of the samples followed by Yam Badnavirus (42.85%) and Yam Macluravirus (40%). Mixed infections of YMMV-Macluravirus (34.28%), YMMV-Badnavirus (31.42%), Macluravirus-Badnavirus (14.2%) and combination of these three viruses (14.2%) were also observed. Two pairs of novel species specific primers were developed to amplify the partial coat protein gene of YMMV and Yam Macluravirus. After identification, one sample each for YMMV, Yam Macluravirus and Yam Badnavirus were cloned and sequenced. The sequence data was analyzed through BLAST and111 sequence similarity was studied. YMMV has maximum similarity of 86% to Yam Mild Mosaic Virus isolate CN20, complete genome, whereas Yam Macluravirus, 95% to YMCTCRI-01 polyprotein gene and Yam Badnavirus, 99% to Dioscorea bacilliform virus 1 gene for polyprotein, isolate FJ65c De. The result obtained from this study will be useful for indexing the planting materials which helps in production of healthy planting material and germplasm there by helping the farming communities to get potential yield.ThesisItem Open Access Molecular characterization of cassava mosaic disease (CMD) resistant varieties and wild relatives of cassava (Manihot esculenta Crantz) using SSR and SNP markers(Department of Plant Biotechnology, College of Agriculture, Vellayani, 2015) Dhanya, O G; KAU; Mohan, CIn the present study an attempt was made to estimate the extent of genetic diversity between 25 CMD resistant, 16 susceptible and seven wild relatives of cassava using 14 SSR and two SNP primers. Out of the 16 primers, nine primers was analysed using PAGE and remaining seven using capillary electrophoresis on genetic analyzer. The primers produced a total of 53 alleles across the 48 cassava accessions. NS198 was found to be highly polymorphic with 6 alleles followed by NS169, SSR36 and SSR39 (5allelles). The two SNP marker analysis on genetic analyzer namely SNPAPX3 and SNP ERF revealed that out of the two peaks generated, one of the peak at a range of 650-690 and 500-530bp respectively was common to all the 48 accessions and the other peak was variable between samples. The dendrogram constructed with 16 primers using UPGMA had four major clusters which clearly distinguished the resistant, susceptible and wild collections of cassava. The close observation made on one of the sub cluster within major resistant cluster revealed the resistant cultivars TME3 and TME4 were closely related with a similarity coefficient of 0.98. Clustering analysis was well supported by PCA, made representation of distinct location of CMD resistant, susceptible and wild relatives of cassava on 2D and 3D dimensions. Comparison of PIC value for all the 16 primers found out the PIC value for individual SSR markers is higher than the individual SNP PIC value at a range of 0.19 - 0.24. SSR PIC values ranged from 0.347 in SSR32 with observed heterozygosity (He) 0.447 to 0.72 in NS198 with He 0.76. Based on the PIC ranges NS198, NS169, SSRY39, RME-1, SSR106 and NS158 were selected as highly polymorphic markers.
