1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Identification of the donors for blast resistance from traditional rice varieties of Kerala using functional markers(Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, 2014) Henry Nickolas; Jayalekshmy, V GThe research project entitled “Identification of the donors for blast resistance from traditional rice varieties of Kerala using functional markers” was carried out in the Department of Plant Breeding and Genetics, College of Agriculture, Vellayani, Thiruvananthapuram and farmer’s field at Pattambi, Palakkad district during 2012 - 2014. The major objectives of the study were to identify the traditional rice varieties with blast resistance genes (Pi 1, Pi 2, Pi kh) using associated functional markers and the field scoring of the lines under disease stress condition. In the present study, thirty traditional rice varieties of Kerala were taken for resistance gene source identification and field level evaluation. The selected rice varieties were screened for the presence of the reported blast resistance genes Pi 1, Pi 2 and Pi kh using three SSR (Simple Sequence Repeat) markers RM224, RM527 and RM206 respectively. C101Lac (Pi 1), C101A51 (Pi 2) and Tetep (Pi kh) from DRR, Hyderabad were taken as the resistant gene source check. Functional marker analysis showed the presence of gene Pi 1 in nine varieties. Twenty varieties showed the presence of gene Pi 2 and six varieties showed the presence of gene Pi kh. Among these, three varieties were having a gene combination of Pi 1 and Pi 2. Three varieties were having genes Pi 2 and Pi kh and two varieties had Pi 1 and Pi kh gene combination. Five varieties did not show the presence of molecular marker linked to any of the genes under study. Field screening was done for scoring the varieties for blast disease resistance by growing in the disease prone farmer’s field at Pattambi. From screening, ten varieties were found to be moderately resistant and fifteen varieties displayed moderately susceptible response. Analysis of variance of the infection index calculated from disease score showed that, nine varieties were having low index and they were on par. All the remaining varieties showed susceptible response. None of the varieties were immune or resistant. Popular rice varieties Uma and Jyothi showed a high infection index. Comparing the disease score and the presence of genes, it was inferred that, genes Pi 1 and Pi kh in combination, imparted moderate resistance under Kerala condition. Varieties Parambuvattan and Kavunginpoothala having these two genes showed low infection index in the field screening. Considering single gene effect, gene Pi 1 imparted moderate resistance. Varieties Thekkan chitteni and Njavara from Kunnathoor are having this gene showed a low infection index. Pyramiding of genes Pi 1 and Pi kh can impart durable resistance to rice varieties of Kerala. Parambuvattan and Kavunginpoothala having these two genes in combination can be used as donors for the genes.Item Sex determination in nutmeg (Myristica fragrans Houtt.) through molecular and biochemical markers(Centre for Plant Biotechnology, College of Horticulture, Vellanikkara, 2010) Maddela Sudhamayee; Valsala, P ANutmeg (Myristica fragrans Houtt.) is an important tree spice of southern India, Malaysia and Indonesia yielding two products of commercial value, ‘nutmeg’ and ‘mace’. Nutmeg of commerce is the dried seed and mace is the dried aril. It is a spreading evergreen tree of the family Myristicaceae and is dioecious with long pre bearing period of 5 to 7 years. Presently, the dioecy in nutmeg is overcome by vegetative propagation or top working. Even though several vegetative methods have been reported, the large scale adoption of these methods is constrained due to insufficient number of orthotrops. So seedling continues to be the major propagating material. In the present study, an attempt was made to identify sex in nutmeg through molecular and biochemical markers so as to identify sex at seedling stage itself. Molecular marker used was RAPD and biochemical marker was isozymes of acid phosphatase and glutamate oxaloacetate transaminase (GOT). Based on bearing pattern and floral morphology, five typical male and female plants of age 10-15 years were selected for RAPD analysis. DNA isolation technique was standardised using CTAB method. Good quality DNA with UV absorbance ratio (A260/A280) 1.80- 1.84 was used for analysis. Sixty seven decamer primers were screened and four primers showing the polymorphism has been selected for further RAPD analysis. PCR amplification with selected primers viz. OPD 15, OPA 27, OPF 05 and OPK 01 was carried out with samples of bulk DNA from five male and female, DNA of individual male and female and negative control. Among them OPK 01 amplified reproducible female specific band (1.1 kb) in bulked and individual samples. The polymorphic female specific band amplified by OPK 01 primer was eluted and cloned in pDrive vector and transformed into E. coli JM 109 cells. Cloned cells were subjected to blue-white screening and transformed white clones were sequenced at Bioserve, Hyderabad. The sequence obtained after VecScreen (Nut seq 816 bp) was analysed with various bioinformatic tools like Blastn, Blastp, GenScan, ORF Finder, Transeq, GOR and Protparam. Three open reading frames identified in the sequence Nut seq816 sing NCBI tool “ORF Finder”. The +2 ORF strand encodes two domains for amino asparate transaminase (GOT) and cystathionine beta- lyases with E value 3.6. The other ORFs didn’t encode any domain. GenScan predicted no gene in the Nut seq816. Based on sequence information two pairs (SP1 and SP2) of SCAR primers (24 bp) were designed using primer3 programme. The efficiency of SCAR primer to distinguish male and female plants was tested by PCR amplification of DNA from five male and females. The SCAR primers amplified around 300 bp single band in five females. SCAR primer was again checked with four occasional fruiting males and it amplified 300 bp band in one occasional fruiting male. Expression of SCAR primers was tested in ten seedlings and got amplified in four samples. Leaf samples from identified male and female trees were used for the biochemical marker analysis. Polyacrylamide gel electrophoresis (PAGE) for isozyme assay was standardized with standard protein and nutmeg leaf protein. Acid phosphatase assay recorded four zones of activity and all were monomorphic. Glutamate oxaloacetate transaminase showed polymorphism and need protocol refinement. Accuracy of SCAR primer SP1 to distinguish male, female and occasional fruiting male has to be done with more samples. Commercialization of the technique can be explored based on the accuracy and cost benefit analysis.Item DNA barcoding in momordica spp.(Centre for plant biotechnology and molecular biology, College of horticulture, Vellanikkara, 2014) Girme Aoudumbar Ramesh; Deepu MathewThe genus Momordica comprises of 59 species, among which 7 are of Indian origin. Though the vegetables belonging to this genus are nutritionally rich with medicinal properties, their taxonomy remains confusive. The botanical names are often used incorrectly and interchangeably. Different taxonomic classification approaches have resulted in controversies about the number of species that exist and the phylogenetic relationships among these species. This situation necessitates an accurate, sensitive and simple alternate for the traditional taxonomy. DNA barcoding is a novel system designed to provide rapid, accurate, and automatable species identification using short, standardized genomic regions as internal species tags. DNA barcoding is based on the variation on the sequences identified genomic regions, which can distinguish individuals of a species because genetic variation between species exceeds that within species. Species identification through barcoding is usually achieved by the retrieval of a short DNA sequence i.e. the „barcode‟ from a standard part of the genome (i.e. a specific gene region either from chloroplast, mitochondria or nuclear genome). The barcode sequence from each unknown specimen is then compared with a library of reference barcode sequences derived from individuals of known identity. The study entitled “DNA barcoding in Momordica spp.” was done at the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara. The objective of this study was to develop the barcodes for the 7 Momordica spp. which were found in India. For this study, 25 accessions of seven Momordica species M. charantia, M. balsamina, M. dioica, M. sahyadrica, M. cochinchinensis, M. subangulata subsp. renigera and M. cymbalaria and two accession of Luffa has been taken. Total genomic DNA isolated using CTAB method (Rogers and Bendich, 1994) was subjected to PCR assay using various combinations of universal barcode primers for three loci matK, ITS2 and trnH-psbA. As ITS2 and trnH-psbA gave the bands of 600 and 300bp, respectively, and which are not sufficient to develop the complete barcodes, these loci were not used in the study. Thus the bands generated using the matK primers from all the 25 Momordica and 2 Luffa were used for sequencing. Phylogenetic analysis using ClustalW has discriminated the various species under Momordica, except dioica and sahyadrica. Kimura 2 Parameter (K2P) model using MEGA software. A wide level of molecular diversity detected with both the method which shows the high level of genetic variation among the species of 25 accessions of Momordica of Indian origin and Momordica and Luffa. Barcoding gap, a position in the sequence at which unique nucleotide is present in all the members of a particular species, was assessed for all the Momordica species and the Luffa, and these gaps were used to generate the barcodes for that species. From this study, barcodes were successfully generated for M. charantia, M. subangulata, M. cochinchinensis, M. cymbalaria, M. balsamina and Luffa acutangula. The BLAST analysis had shown that matK is 95 per cent efficient for species discrimination in Momordica. From the base sequence of matK generated from this study, barcoding primers were designed for Momordica and were successful in all the 25 accessions. The matK barcodes developed in this study could be successfully used to solve the taxonomic confusion in Momordica.