MOLECULAR CHARACTERIZATION OF THE NEW CYTOPLASM DERIVED FROM Helianthus argophyllus USING SOUTHERN ANALYSIS AND rep-PCR
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Date
2006
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ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY RAJENDRANAGAR, HYDERABAD
Abstract
Cytoplasmic male sterility (CMS) in higher plants is encoded by the
mitochondrial genome. Cytoplasmic male sterility is the most viable system for
hybrid seed production in sunflower. Cultivated hybrid sunflower (Helianthus
annuus) production depends on a single male- sterile cytoplasm PET1, derived from
H. petiolaris Nutt. And few restoration genes. The development of commercial
sunflower hybrids based on new CMS sources is of special interest for reducing the
potential risk of vulnerability to pathogens and for increasing the genetic diversity.
In this regard, the present investigation was carried out with the aim of molecular
characterization of the new ARG for hybrid seed production.
This study was undertaken to find the genetic relatedness of the new ARG
cytoplasm with conventional PET1 and other cytoplasms derived from H.
argophyllus ARG 2, ARG 3 using Southern analysis and rep-PCR.
For characterization of the new ARG cytoplasm, we have used DOR
developed ARG and CMS lines, PET 1 was used for comparison. Here the
comparison was done by rep-PCR analysis using the Box primer (BoxA1R). The
primer yielded 3 to 7 amplicons of 1.9 kb with all the mitochondrial DNA samples.
The banding pattern was repeatable and consistent. The new ARG cytoplasm and
2023A showed a similar banding pattern, whereas the ARG wild species had an
additional unique band. Hence, rep-PCR with Box primer was able to differentiate
among the new ARG cytoplasm, PET1, ARG 2 and ARG 3 cytoplasms. Rep-PCR
with all the three sets of primers i.e., REP, BOX and ERIC was done for
characterization of different Helianthus wild species. Though REP and ERIC
showed non-polymorphic profiles, BOX was able to distinguish among few species.
H. resinosus could be characterized by the absence of a prominent band where as H.
strumosus showed an additional band of lower size.
For hybrid sunflower development, several introgression lines have been
developed and they need to be characterized at the molecular level i.e., with respect
of the organelle genomes they carry. This confirmation is to be done with Southern
analysis using organelle genome specific probes. In present study, Southern blot
analysis was performed using genomic DNA digested with Bgl II restriction
endonuclease enzyme and atp 9 mitochondrial probe. The results show that the atp 9
probe hybridized to DNA fragments of different sizes which indicates the
differences in the cytoplasms of DOR developed lines with H. petiolaris though
some are identical. Helianthus anuus was found to be unique and clearly different
from other cytoplasms. Also ARG2, ARG3 and PET 1 cytoplasms showed clear and
unique differences from other samples. ARG2 and 4011A showed a similar pattern
but 2023A had a different pattern. There were no bands with respect to the new
ARG cytoplasm.
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Keywords
MOLECULAR, CHARACTERIZATION, CYTOPLASM, DERIVED, USING, SOUTHERN, ANALYSIS, rep-PCR