Molecular Plant Breeding 2016, Vol.7, No.27, 1
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9
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3.2.1 Total DNA extraction
SDS method was used to extract the total DNA of the experimental materials, the concentration of DNA was
detected by 1% agarose gel electrophoresis (Figure 1).
3.2.2 Amplification of chloroplast
trnH-psbA
gene
According to reference documentation (
Fazekas
et al., 2008), we selected universal primers for the amplification of
chloroplast
trnH-psbA
gene.
The
upstream
and
downstream
primer
sequences
are
F:5’-CGCGCATGGTGGATTCACAA-3’, R: 5’-GTTATGCATGAACGTAATGCTC-3’. PCR amplification
system was as follows: 10×Buffer 2.5 μL, dNTPs 2.5 μL, upstream and downstream primers were 0.5 μL, LA
Taq
enzyme0.25 μL, template DNA 5 μL, ddH
2
O 14.25 μL, and total volume 25 μL. PCR reaction conditions were as
follows: 94
℃
pre denaturation 5 min, 94
℃
denaturation 30s, 56
℃
annealing 30s, 72
℃
extending 30s, 35
circulating, and finally 72
℃
extending reaction 10 min, 4
℃
conservation.
3.2.3 Cloning and sequences analyzing of chloroplast
trnH-psbA
gene
After purified the above-mentioned PCR product, the product connected with the carrier pMD19-T, and then
connected to the cells of the feeling state top 10. Selected white spots to culture, then PCR detected bacterial
colony, eventually sent purified product to Beijing Liuhe Huada gene polytron technologies Inc. for sequencing
analysis.
3.2.4 Sequence analysis
The chloroplast trnH-psbA gene sequences of tested 67 samples were analyzed in this research. After removing
the primer sequence in the sequencing results, Clustalx software was used for sorting, some loci should be revised
manually, DNAMAN software was applied to analyze homologous analysis and Phylip3.68 software was used for
phylogenetic analysis. The phylogenetic tree were builded by parsimony approach. The calculation of the support
rate by bootstrap for each branch was set to 1000 times repeated sampling.
Authors’ contributions
CJT did the data analysis, discussion of the results and drafted the manuscript. YXZ performed the experiments of the research; ZKZ
conceived the project, guided the design of the experiments, revised the manuscript and is responsible for the project; JYH did the
collection and the preparation of part of the experimental materials. All authors read and approved the final version of manuscript.
CJT and YXZ are the experimental designer and executive of this research; CJT and ZKZ did the data analysis and the draft; JYH
involved in the experiment design and the test results analysis; ZKZ conceived the project, guided the design of experiments,
analyzed the data, drafted and revised the manuscript. All authors read and approved the final version of manuscript.
Acknowledgement
The work was financially supported by the Beijing Municipal Education Commission(KM201510020011
，
CEFF-PXM
2016_014207_000038), Project of Construction of Innovative Teams and Teacher Career Development for Universities and Colleges
Under Beijing Municipality (IDHT20150503), Beijing Municipal Science and Technology Promotion Program
(TJSHG201310020020), Beijing laboratory of Urban and rural ecological environment Project (PXM2016-014207-000003).
References
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