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Molecular Plant Breeding 2011, Vol.2, No.07, 41
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47
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46
ground fresh leave tissue was suspended in a 1.5 ml
centrifugal tube with preheated 750 µl CTAB. The
suspension was shaken up, kept at 65
℃
for 45 min,
and then centrifuged with 12 000 rpm for 10 min. The
supernatant was abandoned later. Extracted by
chloroform isoamylalcohol (24:1) twice and
precipitation with two-thirds volume of isopropanol
under
-
4
℃
, and then centrifuged at 12 000 rpm for 10
min. The pellet was washed with 70% ethanol twice.
Then the pellets were air dried. DNA was dissolved in
50 µl deionized water. The DNA quality was
inspected by 1% agarose gel electrophoresis and
stored at
-
20
℃
.
3.3 AFLP analysis
AFLP was performed on the basis of the protocol
introduced by Vos et al. (1995) with a minor
modifications (Shi, 2006). Double digested the genomic
DNA with
Eco
R I/
Mse
I. The enzymed DNA was
ligated to the adaptors which had the
Eco
R I/
Mse
I
restriction sites at both sides. The preselective
amplification was performed with non-selective
Eco
R
I/
Mse
I primer combination. The PCR products were
diluted 10
-
fold with TE and used to be template for
selective amplification. The selective amplification
primer combination was
Eco
R+3/
Mse
l+3 (Table 2).
The selective amplification PCR products were added
with the same volume of loading buffer. Set in 95
℃
for 3 min for denature. Put on the ice immediately.
Draw 5.5 µl to% (w/v) polyacrylamide gel containing
which was preheated for 45 min at 55W. The gels
were silver stained (Lu et al., 2001) and placed in the
air to dry.
3.4 Data analysis
All distinct bands on AFLP gel were identified and
wrote down according to their position independently
for each cultivar and primer combination. A matrix
was constructed with all these data. The polymorphic
rate (P) and Polymorphism information content (PIC)
values were calculated as described by Botstein et al
(1980). GenAlEx 6.4 software (Rod Peakall.
http://www.anu.edu.au/BoZo/GenAlEx/new_version.p
hp) was used to calculate the amount of index of
genetic diversity including GS (Genetic similarity),
GD (Genetic distance) and AMOVA. Cluster analysis
was conducted based on the UPGMA (Unweighted
Pair Group Mean Average) method with MEGA4
(http://www.megasoftware.net/).
Authors’ contributions
Weidong Shi fulfilled major part of this paper, including varieties collection
and lines selection and cultivation, experiment design and accomplishment,
data analysis and manuscript preparation. Ruikui Huang participated in
germplasm collection. Shengmao Zhou participated in germplasm collection
and experiment design. Faqian Xiong gave great assistance to experimental
accomplishment. All authors read and approved the final manuscript.
Acknowledgements
This study was jointly supported by Agricultural Science Institute of
Guangxi Basic Research Project (200805Z) and (0815011
-
6
-
1) and Natural
Science Foundation of Guangxi Zhuang Autonomous Region
(2010GXNSFA013084). Mention of trade names or commercial products in
this paper is solely for the purpose of providing specific information and
does not imply recommendation or endorsement by authors or institutes or
university involved in this study.
References
Blears M.J., de Grandis S.A., Lee H., and Trevors J.T., 1998, Amplified
fragment length polymorphism (AFLP): a review of the procedure and
its applications, J. Indu. Microb. & Biotec., 21: 99-114
Botstein D., White R.L., Skolmick M., and Davis R.W., 1980, Construction
of a genetic linkage map in man using restriction fragment length
polymorphism, Amer. J. of Hum. Genet., 32: 314-332
Chu L.T., 2002, Studies on genetic polymorphisms with AFLP and
taxonomy in Brassica campestris (2n=20), thesis for M.S., Zhejiang
University, Supervisor: Cao J.S.
Guo J.X., Zhou N.Y., Ma R., and Cao M.Q., 2002, Genetic diversity in
Brassica
rapa
revealed by AFLP molecular mark, Journal of
Agricultural Biotechnology, 10(2): 138-143
Han J.M., Hou X.L., Xu H.M., and Wang J.J., 2008, Rapd analysis of
genetic diversity of non-heading chinese cabbage (
Brassica campestris.
ssp.
chinensis
makino) germplasm, Journal of Nanjing Agricultural
University, 31(3): 31-36
He Y.T., Tu J.X., Fu T.D., Li D.Y., and Chen B.Y., 2002, Genetic diversity of
germplasm resources of
Brassica campestris
. L in china by RAPD
markers, Acta Agronomica Sinica, (28): 697-703
Lu G.Y., Yang G.S., and Fu T.D., 2001, Silver-Stained AFLP Novel Assay
for DNA Fingerprinting in
Brassica napus
, Jourtud of Huazhong
Agricultural University, 20(5): 413-415
Ma J.J., 2008, Preliminary study of germplasm genetic diversity in
non-heading chinese cabbage with molecular marker, Thesis for M.S.,
Yangzhou University, Supervisor: Bo T.Y., and Chen X.H.
Ma S.M., and Wang Y.F., 2006, Progress on breeding of flowering Chinese
cabbage, Northern Horticlture, (3): 40-41
Shi W.D., 2006, Analysis of oilseed rape flowering time variation and
cloning of Arabidopsis late flowering time gene, Dissertation for Ph.D.,
Chinese Academy of Agriculture Sciences, Supervisor: Liu S.Y.
Sun D.L., Zhao Q.C., Song W.Q., and Chen R.Y., 2001, Relationships
analysis of Chinese cabbage species by AFLP, Acta Horticulture Sinica,
28(4): 331-335
Sun X.M., Qiao A.M., Sun M., Gui T.Q., and Yin C.X., 2010, ISSR anylysis
of genetic diversity of 27 flowering Chinese cabbage, Journal of
Southerwest China Normal University (natural science edition), 35(1):
119-123
Tan X., Zhang J.D., Qiao A.M., Li L.F., and Zhen S.W., 2009, RAPD
analysis of genetic diversity of flowering Chinese cabbage, Guangdong
Agricultural Sciences, 10: 154-157
Vos P., Hogers R., Bleeker M., Reijans M., van de Lee T., Hornes M.,