The Application of DNA Molecular Marker Techniques in Hevea Brasiliensis
DNA was extracted from several Hevea sources; namely, various Hevea species, several cultivars from within the Hevea brasiliensis species such as clones and in vitro cultured H. brasiliensis. Four DNA molecular marker techniques were used to analyze the DNA. These techniques included a hybridiza...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2001
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/8452/1/FSMB_2001_28_A.pdf |
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| Summary: | DNA was extracted from several Hevea sources; namely, various Hevea
species, several cultivars from within the Hevea brasiliensis species such as
clones and in vitro cultured H. brasiliensis. Four DNA molecular marker
techniques were used to analyze the DNA. These techniques included a
hybridization-based marker technique called restriction fragment length
polymorphism (RFLP) and three polymerase chain reaction (PCR)-based
techniques viz. random amplified polymorphic DNA (RAPD), DNA amplified
fingerprinting (DAF) and sequence-tagged microsatellite sites (STMS). In the
RFLP study, a wheat ribosomal DNA, pTa71 (rDNA) probe was able to detect a
reduction in rDNA loci number in DNA from in vitro cultured plants compared to
DNA from control plants. Hybridization with M13 DNA fragments revealed interand
intraspecific variations among the DNA samples. Neither of thesehybridization probes could detect somac1onal variation within a sample of in vitro
cultured plants. On the other hand, RAPD and DAF were able to detect
somaclonal variation within the in vitro cultured plants. The polymorphic patterns
produced by RAPD could be neither correlated with any particular morphological
trait nor the source of calli i.e. anther or ovule. Meanwhile, DAF proved to be
more sensitive as it was able to detect a high degree of variation in the DNA
extracted from anther derived calli. STMS could not detect any variation nor
insertion/deletion mutation at the HMGR-l gene within the in vitro culture DNA.
RAPD and DAF molecular markers were found to be dominant while RFLP and
STMS markers were co-dominant in all of the H brasiliensis crosses tested in this
study. No change in the methylation sites for both in vitro culture and control
plants were detected when the DNAs were digested with both isoschizomeric
restriction enzymes Hpall and MspI. A micro satellite enriched library was
constructed and was found to be enriched with (GA)n repeats (39%).
Hybridization with one of these clones revealed inter- and intraspecific variations
with DpnII -restricted DNAs. This clone was subsequently sequenced and found to
be an imperfect repeat. |
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