Kim, Seong-Cheol
(Namhae Sub-Station, National Institute of Horticultural and Herbal Science, RDA)
,
Jung, Yong-Hwan
(Jeju Biodiversity Research Institute)
,
Seong, Ki-Cheol
(National Agricultural Research Center for Climate Change, NIHHS, RDA)
,
Chun, Seung-Jong
(National Agricultural Research Center for Climate Change, NIHHS, RDA)
,
Kim, Chun Hwan
(National Agricultural Research Center for Climate Change, NIHHS, RDA)
,
Lim, Chan Kyu
(National Agricultural Research Center for Climate Change, NIHHS, RDA)
,
Joa, Jae-Ho
(National Agricultural Research Center for Climate Change, NIHHS, RDA)
,
Lee, Dong-Sun
(Faculty of Biotechnology, Jeju National University)
A sex-linked random amplified polymorphic DNA (RAPD) marker was identified from Asparagus officinalis L. and was converted into a sequence-characterized amplified regions (SCAR) marker for the large-scale screening of male and female plants. A total of 100 arbitrary decamer oligonucleotide primers w...
A sex-linked random amplified polymorphic DNA (RAPD) marker was identified from Asparagus officinalis L. and was converted into a sequence-characterized amplified regions (SCAR) marker for the large-scale screening of male and female plants. A total of 100 arbitrary decamer oligonucleotide primers were used for the RAPD analysis. Among them, the primer UBC347 amplified one female-specific 400 base pair DNA. Subsequently, the amplified RAPD fragment was cloned and sequenced. The fragment was abundant in AT and shared sequence homology with retrotransposon elements. On the basis of the sequence obtained, a pair of SCAR primer was designed. The amplification product, named F400, was the same size as the respective RAPD fragment from which it was derived. The F400 SCAR marker resulted to be female-specific in the three asparagus varieties tested in this study. This SCAR marker can be used for an early and rapid identification of female and male plants during breeding programs of asparagus.
A sex-linked random amplified polymorphic DNA (RAPD) marker was identified from Asparagus officinalis L. and was converted into a sequence-characterized amplified regions (SCAR) marker for the large-scale screening of male and female plants. A total of 100 arbitrary decamer oligonucleotide primers were used for the RAPD analysis. Among them, the primer UBC347 amplified one female-specific 400 base pair DNA. Subsequently, the amplified RAPD fragment was cloned and sequenced. The fragment was abundant in AT and shared sequence homology with retrotransposon elements. On the basis of the sequence obtained, a pair of SCAR primer was designed. The amplification product, named F400, was the same size as the respective RAPD fragment from which it was derived. The F400 SCAR marker resulted to be female-specific in the three asparagus varieties tested in this study. This SCAR marker can be used for an early and rapid identification of female and male plants during breeding programs of asparagus.
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제안 방법
Since RAPD markers show poor reproducibility, they have to be converted into SCAR markers. For this purpose, we cloned and determined its nucleotide sequence and compared sequences of UBC400347 fragments from female plants (Fig. 3A). A 24-mer SCAR primer was designed on the basis of the UBC319 RAPD primer which amplified specific fragments only in male plants and a 22-mer SCAR primer was designed on the basis of the UBC347 RAPD primer which amplified specific bands only in female plants.
In this study, we report a SCAR marker to identify female and male plants in the dioecious asparagus.
5 × TBE buffer at 1500 V for 700 min. Sequences were aligned with CLUSTAL W (Thompson et al., 1994) and then adjusted manually to align several conserved regions. Sites with missing data or gaps were excluded from all analyses.
대상 데이터
A 24-mer SCAR primer was designed on the basis of the UBC319 RAPD primer which amplified specific fragments only in male plants and a 22-mer SCAR primer was designed on the basis of the UBC347 RAPD primer which amplified specific bands only in female plants. The primer named F400 (Table 1).
이론/모형
(B) Multiple sequence alignment of UBC400347 fragment and plant retrotransposon proteins belonging to UBN2 superfamily. The sequence alignment and conserved residues were displayed using the CLUSTALW and the BOXSHADE programs. Identical amino acid sequences are highlighted in black, and similar sequences in grey.
We collected three male and female plants from three varieties after flowering and immediately frozen in liquid nitrogen and stored at -70℃. Total DNA was purified from approximately 0.5 g of fresh leaves using the protein precipitation method of Dellaporta et al. (1983).
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