Cloning and characterisation of sex-specific DNA marker in jojoba (Simmondsia chinensis L.)
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Date
2010
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CCSHAU
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).
Description
Keywords
Economic systems, Livestock, Animal husbandry, Dairy farms, Area, Productivity, Biological phenomena, Breeds (animals), Selection, Diseases