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Theses

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2013 - 20153
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Subject: Biotechnology and Molecular Biology × Author: KAU × End date: 2015 × Start date: 2011 × Type: Thesis ×
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    ThesisItemOpen Access
    DNA barcoding of spider mites (Prostigmata: Tetranychidae) in vegetable crops
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2015) Shruti, Bennur; KAU; Abida, P S
    DNA barcoding is a novel system designed to provide rapid, accurate, and automatable species identification using short DNA sequences from a standardized region of the genome. It helps in precise identification of species that can be applicable as a diagnostic tool for quarantine and other pest management activities. DNA barcoding is based on the variation on the sequences identified in genomic regions, which can distinguish individuals of a species. Species identification through barcoding is usually achieved by the retrieval of a short DNA sequence i.e. the ‘barcode’ from a standard region of the genome, a specific gene region either from mitochondria or nuclear genome. The barcode sequence from the unknown specimen could be compared with a library of reference barcode sequences derived from individuals of known identity. The family Tetranychidae or spider mites include the most injurious plant-feeding mites. Some infest a wide range of host plant whereas some others are highly specific. The family includes about 12,000 species of phytophagous mites which can rapidly disperse to exploit new feeding sites, damage agricultural and horticultural crops causing severe economic losses. Precise identification up to species level is difficult since all species look similar. So molecular methods are increasingly applied in tetranychid mite taxonomy to establish species identity and DNA barcoding is the best option for this. The study entitled “DNA barcoding of spider mites (Prostigmata : Tetranychidae) in vegetables” was done at Centre for Plant Biotechnology and Molecular Biology and Department of Agricultural Entomology, College of Horticulture, Vellanikkara. The objective of this study was to generate DNA barcodes for different species of spider mites in vegetable crops and to study the intra and inter species genetic relationship. For this, spider mites were collected from different locations viz; Anthikad, Alathore, Elenad, KVK, Thrissur and Vellanikkara and from different vegetable crops viz; Amaranthus, brinjal, cowpea, cucumber, dolichos bean, okra and ridge gourd. After collection, spider mites were reared in the laboratory to get an isoline from which few males and females were used for microscopic slide preparation. Acarologists used slides to identify the spider mite species based on morphological taxonomic keys. Total genomic DNA isolated using modified CTAB method (Rogers and Benedict, 1994) was subjected to PCR assay using markers for two different loci ITS2 (second internal transcribed spacer) and COI (mitochondrial cytochrome c oxidase subunit I) which yielded bands of 620 and 868bp respectively. The obtained bands were eluted and subjected for sequencing. Nucleotide divergence among the sequences was calculated using Barcode of Life Data (BOLD) tool ‘Distance summary’. It is desirable for barcodes to show very low sequence divergence within species. Multiple sequence alignment using clustalW of MEGA6 software was performed for all the sequences and phylogenetic analysis has discriminated three different species of Tetranychidae family: Tetranychus truncatus, T.macferlanei and T.okinawanus. Barcoding gap, a position in the sequence at which unique nucleotide is present in all the members of a Tetranychus species was also assessed. Morphological and molecular analysis results were correlated with each other and results matched. Tetranychus okinawanus found on cucumber during the study is first time getting reported from India. The study had shown that both ITS2 (99.25%) and COI (98.45%) sequences efficiently classified the spider mite species. However, ITS2 was found to be an efficient tool which gave species level resolution in spider mites. This can be used as supporting marker for COI to barcode spider mites species. In future, these primers can be used to barcode other Tetranychus species also and sequence analysis can also be done using ITS1 locus and its efficiency can be measured for DNA barcoding of Tetranychus species.
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    ThesisItemOpen Access
    DNA fingerprinting of selected chilli (Capsicum spp.) varieties
    (Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara, 2013) Manibala Kumari; KAU; Kesavachandran, R
    Chilli (Capsicum spp.) is considered as one of the most important commercial spice crops and is a widely used universal spice, named as the wonder spice. It is raised over an area of 18 lakh ha. in the world, with a production of 29 lakh t. India is not only the largest producer but also the largest consumer of chilli in the world. The study entitled “DNA fingerprinting of selected chilli (Capsicum spp.) varieties” was carried out at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture during the period 2012-2013. The objectives of the study were to characterize the released chilli 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. Six chilli varieties, namely Ujwala, Anugraha, Jwalamukhi, Jwalasakhi, Vellayani Athulya and Vellayani Samrudhi collected from CoH, Vellanikkara and CoA, Vellayani and maintained at CPBMB, CoH were used for the study. Morphological parameters of six chilli varieties were taken such as stem colour, branching habit, leaf size, leaf colour, fruit colour, fruit shape and fruit surface. DNA extraction was done by CTAB (Rogers and Bendich, 1994) method. The RNA contamination was completely removed through RNase treatment. Good quality DNA with UV absorbance ratio (A 260 /A ) 1.80 - 1.91 was used for further analysis. The PCR conditions were optimized for RAPD, ISSR and SSR assays. Thirty RAPD, 30 ISSR and 30 SSR primers were screened with bulked DNA of Ujwala, Anugraha and Jwalamukhi variety for amplification and those which gave reliable distinct banding patterns were selected for further amplification and fingerprinting. The PCR products obtained from RAPD, ISSR and SSR analyses were separated on two per cent agarose gel and the amplification patterns were recorded. Genomic DNA from each variety was amplified with ten selected primers of RAPD, ISSR and SSR primer pairs. The amplification patterns were scored and depicted to develop DNA fingerprint for each variety. The Resolving power (Rp) worked out for the different primers ranged between 8.33 (S 12) to 12.9 (OPAH 06) for RAPD primers and 8.66 (SPS 03) to 14.33 (ISSR 07) for ISSR primers, indicating the capacity of the primers selected to distinguish the varieties. The Polymorphic Information Content (PIC) varied from 0.80 (S 12) to 0.86 (OPAH 06) for RAPD primers and it was 0.82 (SPS 03) to 0.88 (UBC 840) for ISSR primers. Distinct bands were used to develop DNA fingerprint of chilli varieties (Ujwala, Anugraha, Jwalamukhi, Jwalasakhi, Vellayani Athulya and Vellayani Samrudhi) through RAPD, ISSR and SSR analyses. 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 for each variety with RAPD, ISSR and SSR data was also developed. The amplification patterns observed in RAPD, ISSR and SSR analyses were scored and analyzed for quantifying the variability among the varieties. The computer software NTSYS-Pc was used for cluster analysis (Rohlf, 2005). Maximum variability observed was 41 per cent for the variety Vellayani Samrudhi. The varieties Ujwala and Anugraha indicated 91 per cent similarity. The fingerprint developed was sufficient to provide varietal identity and the analysis could reveal variability/ relatedness among the six varieties.
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    ThesisItemOpen Access
    Identification of molecular marker for self-incompatibility in selected germplasm accessions of cocoa (Theobroma cacao L.)
    (College of Horticulture, Vellanikkara, 2015) Goradevaishali, Shivaji; KAU; Abida, P S
    Cocoa (Theobroma cacao L.) is a diploid species (2n = 2X = 20) with a small genome size of 380Mbp. It belongs to the family of Malvaceae (alternatively Sterculianceae). Theobroma cacao L. is a highly cross pollinated crop due to its peculiar flower structure and existence of self-incompatibility. Self-incompatibility is exploited in hybridization process of breeding programmes and also in selecting clones for polyclonal garden. Traditional method to access self- incompatibility is by hand pollinating 100 flowers per tree. This is very tedious and time consuming method. This necessitated the identification of a marker associated with self-incompatibility. The cocoa germplasm accessions are maintained at Cocoa Research Centre, KAU, Vellanikkara. The centre has identified and maintained Selfincompatible and self-compatible cocoa accessions. Self-incompatibility was manually assessed by selfing 100 flowers per accessions. The cocoa accession setting fruits on selfing classified as self-compatible and which do not set fruit as self-incompatible. Five self-incompatible [IMC 20, PNG 299, IMC 105, ICS 5 and R 10 (MEX)] and five self-compatible cocoa accessions (NA 149, EET 397, SIC 5, POUND 18, GV 13.5) were selected for the present study. Isolation of good genomic DNA from all self-incompatible and self-compatible accessions of cocoa was carried out from the young leaves; using Doyle and Doyle (1987) method was used for analysis of RAPD, ISSR and SSR markers. Random Amplified Polymorphic DNA (RAPD) fragments were generated in the bulked DNA samples in order to identify markers that were polymorphic between self-incompatible and self-compatible plants. Among the 76 RAPD primers screened, 15 primers were selected based on amplification. A 550 basepairs (bp) DNA fragments were generated with RN-6 primers in three selfcompatible specific genotypes. The polymorphic band was cloned and sequenced at SciGenom Pvt. Ltd., Cochin. The sequence analysis gave no information to develop SCAR marker. In Inter Simple Sequence Repeats (ISSR) assay, among the 42 primers screened initially, 20 primers were selected for the study. There was no polymorphism between self-compatible and self-incompatible genotypes. Additionally Simple Sequence Repeats (SSR) assay was also performed with genome specific primers. Total genomic DNA of the self-incompatible and self-compatible accessions was amplified with 11 SSR primers and out of them seven primers were selected. The primer mTcCIR 33 yielded distinctly polymorphic band of 350-400bp size in self-incompatible genotypes. mTcCIR 33 SSR primer also produced polymorphic band obtained on PAGE. The PCR product was directly sequenced at SciGenom Pvt. Ltd., Cochin. The sequence analysis showed 86 per cent identity to Theobromo cacao. More number of genus specific primers has to be screened to develop better markers to distinguish self-comptability and self-incompatability in cocoa or an attempt to biochemical markers can also be resorted.