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Center, Agricultural Research Service (ARS) of the
United States Department of Agriculture (USDA), and
these materials had been studied preciously (Yan et al.,
2007; Agrama et al., 2009). In April 2010, the materials
were planted at the Xindu Testing Site of Sichuan
Academy of Agricultural Sciences. The experiment
was set up three replicates that three rows in each
replicate and seven plants in each row, with the
planting and rowing space of 16 cm×26 cm. In the
early June 2010, 5 plants in the midst of the midst row
in each replicate were sampled for their tillers. The
remaining data was collected from the data in 2006
(Yan et al., 2007).
3.2 Primers design and PCR amplification
The
OsSPL
primers were designed according to 19
sequences of
OsSPL
genes downloaded from http://
rice.plantbiology.msu.edu/ using Primer Premier 6.0
(Table 1).
The CTAB method was used to extract DNA from the
rice leaves using (Murray and Thompson, 1980) with
slight modifications. PCR amplification was performed
according to a previously described method in our lab
(Gao et al., 2008), and the annealing temperatures of
the primers were listed in Table 1.
3.3 Data processing
The means of phenotypic data of each accession were
calculated, and significance tests were performed
using DPS 7.0 software. The PCR amplification bands
were recorded by referring to the presence of a band
as “1” and the absence of a band as “0”. The data
analyses were performed using NTSYpc
-
2.0e software.
According to the Hardy-Weinberg equilibrium, the
gene frequency of a sample randomly selected from a
random population indicates no significant difference.
In other words, the mean of the phenotypic data of the
sample is not significantly different from that of the
population. Based on the results of cluster analysis
using PCR amplification, the phenotypic data mean of
each subgroup and the phenotypic data mean of the
171 accessions were calculated to test whether there
was any significant difference between them. Then
these results were used to judge if any traits were
linked to the
OsSPL
genes, namely, the gene-trait
association analyses.
3.4
OsSPL
genes DNA and amino acid sequences
alignments
A DNA sequence alignment was performed between
the
Keng
(
Japonica
)
OsSPL
genes and
Hsien
(
Indica
)
OsSPL
genes using http://www.gramene.org/Multi/
blastview/BLA_K8FkdkRLV. The amino acid alignment
was carried out using the amino acid sequences of all
of the
OsSPL
genes downloaded from http://pfam.
janelia.org/family/PF03110.7#tabview=tab2 using
DNAMAN 6.0 software.
Authors' contributions
Guangjun Ren and Juansheng Ren conceived of the project and
its components. Juansheng Ren, Yuchao Yu and Guangjun Ren
contributed to the original concept of the project. Fangyuan
Gao, Lihua Zeng, Xianjun Lu, Xianting Wu and Wengui Yan
collected samples, performed the phenotyping and corrected
the paper. Juansheng Ren, Yuchao Yu and Guangjun Ren
analyzed all of the data together and wrote the paper. All authors
have read and approved the final manuscript.
Acknowledgments
This research project was supported by grants from the “948”
project of Ministry of Agriculture of the People's Republic of
China (2006
-
G1), National Natural Science Foundation of
China (30900891), China Agricultural Research System and
National High-tech R&D Program of China (863 Program). We
also thanked two anonymous peer reviewers for their assessments
and advice.
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