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Authors: Surender
Advisor: Kharb, Pushpa
Title: Cloning and characterisation of sex-specific DNA marker in jojoba (Simmondsia chinensis L.)
Publisher: CCSHAU
Language: en
Type: Thesis
Agrotags: Economic systems, Livestock, Animal husbandry, Dairy farms, Area, Productivity, Biological phenomena, Breeds (animals), Selection, Diseases
Abstract: Jojoba (Simmondsia chinensis L. Schneider) is a slow growing, dioecious shrub of arid zones and belongs to family Simmondsiaceace. It is commercially valued for multipurpose uses of its oil present in seeds. Sex of the young jojoba plant cannot be judged until the first flower buds appear which may take up to 3-4 years. Bhardwaj (2008) identified a male specific ISSR marker in jojoba, amplifying a unique allele of 1100 bp in male genotypes only. This study was undertaken to clone and characterize this sex-specific marker and to convert this marker into more specific SCAR marker. PCR amplification of DNA, from ten male and ten females plants of jojoba, was carried out using primer IS52. The amplified products were separated by agarose gel (1.5%) electrophoresis. A fragment of size 1100bp size was found to be present only in male plants. This specific band was eluted from the agarose gel using Qiagen gel extraction kit and cloned in to pJET1.2/blunt Cloning Vector using CloneJETTM PCR cloning kit supplied by Fermentas USA. The ligation mixture was transformed into freshly prepared competent cells of E.coli JM107 strain. The transformed colonies were allowed to grow on LB (Luria Bertani) plates containing ampicillin (100μg/ml).Only recombinant colonies appeared on the LB plates. The plasmid DNA from one colony was isolated using modified alkaline lysis method and then checked on 0.7% agarose gel. The male specific cloned DNA fragment was got sequenced using automated DNA sequencer ABI 3130XL Genetic Analyzer (Applied Biosystem, USA) located at Department of Animal Biotechnology, COVS, CCS HAU, Hisar. The sequence generated was compared with the sequences available in NCBI database and the maximum homology search was done using BLASTn. The sequence did not have any homology with any known nucleotide sequences in the available databases. Three open reading frames (108, 40 and 36 amino acids) were revealed in the sequences. Out of these, two ORF (40 and 36 amino acids) did not show any homology in available databases. One ORF of 108 amino acids showed 32% similarity with monothiol glutaredoxin-5 and mitochondrial precursor of Aspergillus terrus. SCAR scF (Simmondsia chinensis forward) and SCAR scR (Simmondsia chinensis reverse) primers were designed based on the sequence of male specific fragment. This primer pair amplified a unique allele of 1000 bp in male genotypes only (Patent filed).
Issue Date: 2010
Appears in Collections:M. Sc. Dissertations

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