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2014 - 20156
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Subject: Biotechnology × Start date: 2010 × Thesis Type: Ph.D ×
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Now showing 1 - 6 of 6
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    ThesisItemOpen Access
    Genome Mapping and Identification of Molecular Markers for Various Agronomic and Fibre Quality Traits in Tetraploid Cotton
    (University of Agricultural Science, Dharwad, 2015-11) Ramesh; Katageri, I.S.
    Based on four testing trials, in three years, DHBR-25, DHBR-43, DHBR-81, DHBR-161, DHBR-128, DHBR-156, DHBR-20, DHBR-88, DHBR-58 and DHBR-153 recorded significantly higher seed cotton yield than best parent DS-28 (853.56 kg/ha) and commercial check, Sahana (1025.63 kg/ha). The genotype DHBR-20 (1313.78 kg/ha; 24.18 g/tex) recorded significantly higher seed cotton yield with significantly higher fibre strength and on par with commercial checks, Sahana (1025.63 kg/ha; 21.52 g/tex) and MCU-5 (659.49 kg/ha; 23.13 g/tex) on other traits like ginning out turn and boll weight. It indicates the possibility of simultaneous improvement for both seed cotton yield and fibre strength through interspecific hybridization. Using CottonSNP63K array BeadChip, 178 recombinant inbred lines were genotyped. Average success call rate was 70.17 per cent. Among them, 83.26 and 16.73 per cent alleles were in homozygous and heterozygous status respectively. The genetic linkage map constructed with 2188 SNP marker loci segregating in 1:1 ratio in mapping population, covered a genetic distance of 2162 cM with an average marker density of 0.98 cM on each chromosome. The largest chromosome was Chr.D19 followed by Chr.D24 which spans 517 cM with 518 marker loci and 288 cM with 289 loci respectively. The shortest were Chr.A04, Chr.D17 and Chr.D23 each covered 11 cM with 12 marker loci. The largest gap length was observed on Chr.A11 (41.1 cM) and lowest on Chr.D19 and Chr.D22 (3 gaps) with mean of 7.45. Using four season mean data, 295, 137 and 19 QTLs were identified by single marker analysis, interval mapping and composite interval mapping respectively. Their contribution to phenotypic variance range from 7.81 to 14.24 per cent in single marker analysis, 7.59 to 39.49 per cent in interval mapping and 9.29 to 24.82 per cent composite interval mapping. QTLs with higher contribution to phenotypic variation (>10 per cent) are useful in genetic enhancement of yield, yield contributing and fibre quality traits.
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    ThesisItemOpen Access
    Genetic Diversity and Transcriptome Analysis in Sugarcane in Response to Moisture Stress
    (University of Agricultural Science, Dharwad, 2015-09) Manel, Dapanage; Bhat, Sumangala
    Moisture stress causes significant yield loss in all crops and sugarcane is also not an exception. Studying the diversity among the commercial sugarcane varieties and molecular basis of stress tolerance is useful while developing moisture stress tolerant sugarcane varieties. Fifty-two commercial sugarcane varieties were subjected for moisture stress by withholding the irrigation and grouped into different tolerance categories based on the time taken for appearance of wilting symptoms. Two (Co 775 and Co 99010) out of 52 varieties showed wilting symptoms early (4 days after withholding the irrigation) were grouped as highly susceptible and three varieties namely Co 94008, CoT 8201 and ISH 100 showed very late wilting symptoms (12 days) were grouped as highly tolerant. Varieties showed significant variation in the traits such as proline content, EC, RWC and chlorophyll content and positive correlation were observed between RWC and chlorophyll content under moisture stress condition with time taken for appearance of wilting symptoms. Molecular diversity among 52 commercial varieties studied using 54 SSR markers revealed higher (58 per cent) similarity among the varieties. The transcriptome profile of a moisture stress tolerant sugarcane variety, Co 94008, under moisture stress were studied by total RNA sequencing in Illumina Solexa NGS platform and more than 17000 transcripts were identified. Among them, 1143 transcripts showed significant differential expression under moisture stress and expressions of 487 were found to be up-regulated and 656 were down-regulated. Of the top 20 up-regulated transcripts, nine showed more than 100 fold expression and expressions of sixteen genes were highly down regulated (> -5 folds) under moisture stress. Expression profiles of TFs generated using semi qRT-PCR followed by qRT-PCR revealed significant differential expression of nine TF genes among the susceptible and resistant varieties under different levels of moisture stress. The TF, bZIP was significantly up regulated in resistant variety under both moderate and sever stress conditions than the susceptible variety.
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    ThesisItemOpen Access
    CULTURE INDEPENDENT GENOMIC ANALYSIS OF ORGANIC AND INORGANIC FARM SOIL
    (University of Agricultural Sciences, Dharwad, 2015-06) PASHA, MALIK AHMED; BHAT, SUMANGALA
    Soil microorganisms play pivotal roles in various biogeochemical cycles and also influence above ground ecosystem by contributing to soil structure, fertility, plant health and nutrition. This study was conducted to estimate the soil bacterial richness, diversity, dynamics and their composition in organic and inorganic farming practices by culture independent analysis of 16S rDNA using DGGE and NGS. In this study, a protocol for isolation of good quality and quantity DNA from soil microbial community was optimized by combination of physical and chemical cell lysis methods followed by purification by chemical (CaCl2) flocculation. To study the effect of agronomic practices and crop stage specific root exudates, sample was taken at different growth stages of crop from experimental plot of organic and inorganic farming, institute of organic farming, UAS Dharwad. Biological indicator of soil health was analysed by whole metagenome sequencing. Finally, a soil metagenomic library was constructed to exploit the genomic resources of soil microbes. The difference in microbial diversity between organic and inorganic farming is subtle in cereal crop and significant in pulse crop ecto-rhizosphere soil. And the observed difference is because of inhibitory effects of synthetic fertilizers and pesticides. The dominant microbial species remains same, but their relative proportion is different in organic and inorganic farming. Inorganic farming practices reduced the population of naturally available plant beneficial microbes in soil. Selection of microbes by root exudates was also disturbed by chemical fertilizers and pesticides. Microbial activity was observed more in organic farming. However, basic function like nutrient cycling is maintained in both the soils, but by different processes recruited by different set of microbes. Seven year old organic farming did not showed any signs of disease suppressiveness, in turn it was not efficient for controlling plant pathogenic microbes. One positive clone for phosphate solubilisation was obtained in metagenomic library.
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    ThesisItemOpen Access
    CLONING AND EXPRESSION OF COLEOPTERAN SPECIFIC cry, vip GENES AND DEVELOPMENT OF A CONSTRUCT CONTAINING BOTH cry AND chi GENES
    (University of Agricultural Sciences, Dharwad, 2015-06) BHARTI, GUNJAN; KRISHNARAJ, P. U.
    The present work involved screening of B. thuringiensis isolates for different coleopteran-specific cry gene/s, cloning and expression of variants of cry and vip. The presence of different coleopteran specific cry genes (cry1I, cry3, cry7,8, cry14, cry18, cry23, cry26, cry28, cry34, cry35, cry36, cry37 and cry55) was determined in 993 native B. thuringiensis isolates. Strains carrying cry26 and cry3 were the most abundant representing 6.16 and 6.03 per cent of the isolates respectively. Many of the isolates screened (~60 per cent) did not amplify for any cry gene. The crude proteins extracted from the native isolates along with positive control were subjected to SDS-PAGE. The protein bands corresponding to approximately 130kDa were observed in seven potent isolates. Six of the potent isolates showed the presence of a protein band size of 45-50 kDa. None of the 90 native isolates tested, amplified for vip1A. Of the 90 isolates, only five of the isolates (DBT 141, DBT142, DBT642, DBT637, DBT658) were found to be positive for vip3A. DBT 730 was found to be positive for vip2A. The variant cry1I gene from CFE20(3) based on ARFLP patterns was cloned and expressed in E. coli M15(pREP4). The nucleotide sequence showed 99 per cent homology with the reference sequence (JN226100.1). The cry34 gene from CFE43(1) was cloned and expressed in E. coli M15(pREP4). The nucleotide sequence showed 95 per cent homology with the cry34Ba(AY552555.1). The vip3A and vip2A variants were cloned from DBT642 and DBT730 respectively and expressed in E.coli M15(pREP4). The nucleotide sequence of vip3A and vip2A showed 99 per cent homology with the reference sequence (AF500478.2) and reference sequence (EU940372.1) respectively. A construct with both cry1I and chi gene has been developed using the Gibson assembly master mix.
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    ThesisItemOpen Access
    STUDIES ON INTERACTION BETWEEN TRICHODERMA AND SOIL BORNE FUNGUS (Sclerotium rolfsii) AND ITS MOLECULAR BASIS
    (University of Agricultural Sciences Dharwad, 2015-04) CHIDANAND A. RABINAL; Dr. SUMANGALA BHAT
    Sclerotium rolfsii is a soil borne fungal pathogen, which infects more than 500 crop species. Trichoderma spp. are being used effectively for the control of S. rolfsii. In the present study, both in vitro and in vivo assays were carried out to identify the potent Trichoderma isolate to suppress the S. rolfsii growth. The ten Trichoderma isolates studied behaved differently for different assays. Of the ten isolates, T. virens IABT1010 showed highest linear growth rate (19.75 mm/day) followed by T. viride IABT1044 (18.87 mm/day). However, in direct confrontation assay T. virens IABT1002 showed 100% growth inhibition of S. rolfsii. Similarly, for volatile substance assay, the isolate T. harzianum IABT1041 significantly inhibited the S. rolfsii, whereas chitinolytic activity was highest in T. virens IABT1002 (84.16 pmol/µg/min) and gluconolytic activity was high in T. viride IABT1044 (596.9 pmol/µg/min). In the in vivo assay, least seed rot infestation (55.55%) was observed in T. koningii IABT1252 treated Groundnut seeds compared to 100% in untreated seeds sown in S. rolfsii inoculated soil. The isolate, which performed better under in vivo experiment (T. koningii IABT1252) was selected for gene expression profiling in two stages of interaction viz., prior and after contact with S. rolfsii. The differentially expressed transcripts in both the stages were encoding for hydrolytic enzymes, secondary metabolites biosynthesis, transcription factors, signalling proteins, transporter proteins and protein involved in establishing the initial interaction. Of the 58 clones sequenced six did not show homology with any of the genes deposited in NCBI database. The differentially expressed transcripts by SSH was validated by profiling the expression pattern of few differentially expressed genes by qRT PCR study. The present study identified T. koningii IABT1252 as most potent, which needs to be studied further for its field performance. The identified genes, with mycoparasitic activity can be used to genetically engineer the crop plants in order to improve disease tolerance ability.
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    ThesisItemOpen Access
    Agrobacterium tumefaciens MEDIATED TRANSFORMATION OF PIGEONPEA FOR INDEPENDENT EXPRESSION OF cry1Ac, cry2Aa, cry1F AND cry1Acm AGAINST Helicoverpa armigera AND MOLECULAR ANALYSES OF SELECTED EVENTS
    (University of Agricultural Sciences Dharwad, 2014-06) MAHALE BARKU MANOHAR; Dr. B. FAKRUDIN
    In ICPL87119 and BSMR736, MS medium supplemented with 2.0 mg/l BAP, 4.0 mg/l TDZ and 2.0 mg/l zeatin, separately, induced maximum shoot buds, 53.7, 46.1 and 40.9 respectively; any further increase in cytokinins levels resulted in reduced shoot buds. The MS basal with 0.5 mg/l IBA induced maximum and healthier roots (4.8±0.7). In planta transformation revealed 80.00, 85.00, 66.50% explant response, 53.75, 90.00, 90.98% explant survival and 3.0, 6.5, 12.0% transformation efficiency in Agrobacterium tumefaciens infection alone, A. tumefaciens culture with tobacco leaf extract and air evacuation, respectively. The 88 putative transformants carrying cry1Ac were developed, of which 48 showed 3:1 transgene segregation pattern in T2. Insect mortality ranged from 25.0 to 70.0% whereas, Cry1Ac protein level from 0.31 to 0.85 µg/g and cry1Ac transcript level from 15.6 to 165.1 ng/µl, validated through northern blotting in different tissues (leaf, flower and pod). In case of cry2Aa, 65 transformants developed, of which 16 showed 3:1 transgene segregation in T2. Insect mortality ranged from 5.25 to 65.75% whereas, Cry2Aa protein and transcripts ranged from 0.01 to 3.23 µg/g and 41.2 to 134.5 ng/µl, respectively. Southern and juncture analyses of selected three cry1Ac and five cry2Aa transformants confirmed T-DNA integration in plant genome. Fourteen transformants carrying cry1F were developed, of which seven showed 3:1 transgene segregation pattern in T2, wherein insect mortality ranged from 10.0 to 62.5%, Cry1F protein level from 0.113 to 1.032 µg/g and transcripts ranged from 45.2 to 105.3 ng/µl. Similarly, eleven cry1Acm transformants were developed, of which seven showed 3:1 transgene segregation in T2. Insect mortality ranged from 35.0 to 62.5% whereas, protein level and transcripts ranged from 0.19 to 0.91 µg/g and 41.2 to 134.5 ng/µl respectively, in tested tissues. Pigeonpea transformation procedures and generated events of present study could be prospected for their further use.