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Triticeae Genomics and Genetics 2012, Vol.3, No.4, 38-43
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between the same genotype wheat and different corns
or in the crosses of different genotype wheat and the
same genotype corn. Furthermore, by means of the
treatments of low temperature, dark culture, different
concentrations of 2,4
-
D and processing times, as well
as corn pollen repeated pollination etc., the differences
of hybridization affinity also existed significantly (Cai
et al., 2006; 2007). These phenomena implied that the
Kr
gene should possess rich allele diversities and
complex expression and regulation.
39
Recently, Manickavelu obtained a 751 bp cDNA
sequence of
Kr
gene from the recombinant inbred
lines RIL (
Kr
/
kr
) derived from the cross of Chinese
Spring 5B monomer (
kr
) and Mara (
Kr
/
Kr
) by using
the Annealing Control Primer System, (ACP)
(GenBank accession No.: AB379558.1, due to the stop
codon existing in the sequence, therefore assigned as
pseudo-gene mRNA) (Manickavelu et al., 2009a). So
far, it was not yet to be reported about the molecular
structure of
Kr
gene in different wheat varieties or
germplasms.
For the above reasons, the six genotypes of common
wheat selected from 285 Chinese wheat core
germplasms were employed to isolate the
Kr
gene by
using homologous cloning approach with the
Kr
gene-specific primers, in order to predict the encoding
proteins of the
Kr
genes and their functions and to
provide a theoretical basis for explaining the
molecular mechanism of incompatibility of distant
hybridization in wheat and enhancing the distant
hybridization
efficiency,
promoting
genetic
improvement and germplasm innovation in wheat.
1 Results and Analysis
1.1 PCR amplification of the fragments of
Kr
gene
PCR amplification was carried out with specific
primers L4 under the same conditions. The results
showed that one expected PCR band appeared in three
genotypes of Mazhamai Xiaobaimang and Tuokexun-
yihao, and the sequencing results exhibited that the
length of three bands was 414 bp (Figure 1).
Figure 1 The fragment of
Kr
gene amplified by PCR with
primer L4
1.2 Alignment of the sequences of
Kr
gene
fragments
PCR products were recovered and purified as well as
TA cloned and sequenced. Alignment of the sequenced
target products were conducted by applying
DNAMAN Software. The results of sequence
homology comparison showed that the sequence
fragment homology of the Mazhamai and Xiaobaim-
ang shared 100% identity, while 99.8% with Tuoke-
xunyihao that only existed two SNP differenced at the
sites of 284 bp and 321bp (Figure 2), suggesting that
the sequences of this fragment in the
Kr
gene should
be highly conservative in different wheat genotypes.
1.3 Homologous sequences and their relationships
of
Kr
gene sequences in
Triticease
plants
Thirteen sequences of Triticeae plants with high
homology (E value <1e
-
20) were blasted at NCBI by
using the fragment sequence of
Kr
gene obtained in
this research as probe. The obtained sequences incl-
uded the S-locus receptor kinase gene (AY963808. 1),
high molecular weight glutenin gene (AY368673.1),
CRT/DRE binding factor (AY951949.1), EXPB1 gene
(AY533103.1). We applied these sequences to cons-
truct the cluster based on the Distance tree of results
program of NCBI website (Figure 3).
Comparing the obtained 414 bp fragment of
Kr
gene
with wheat S locus (S-locus) receptor kinase gene
(AY963808.1), the blast results showed that the
sequence homology reached 85% in the same regions
(Figure 4), indicated that the encoding proteins of the
obtained sequences might share a similar function
with S-locus receptor kinase in plant.