全 文 ：分子植物育种，2010年，第 8卷，第 1期，第 20-28页
Molecular Plant Breeding, 2010, Vol.8, No.1, 20-28
A Letter
研究报告
Molecular Characterization of α-gliadin Genes from Diploid Lophopyrum
elongatum and Elymus sibiricus
Wu Weidong 1,2* Chen Mengzhu 1* Chen Fanguo 1** Xia Guangmin 1
1 School of Life Sciences, Shandong University, Jinan, 250100; 2 School of Life Sciences, Qiannan Normal College for Nationalities, Duyun, 558000
* The authors who contribute equally
** Corresponding author, fanguo2002@sdu.edu.cn
DOI: 10.3969/mpb.008.000020
Abstract Sixty-three α-gliadin open reading frames (ORF) were characterized from diploid Lophopyrum elonga－
tum and tetroploid Elymus sibiricus. Sequence comparison revealed that about 90% of them contained an internal
stop codon or frameshift. Alignment between these ORFs and reported α-gliadin sequences reveal that they coded i-
dentical signal peptide domain, repetitive domain and C-terminal region, except polyglumatine domains. Analysis of
the first three codons in repetitive domains reveals two new types of motifs (GRV and RV). Novel types of motifs, in-
cluding (Q)3AR(Q)5, (Q)2AR(Q)5, (Q)3A , were characterized in the first polyglumatine region. Three known types of
celiac disease (CD) epitopes in Triticum were screened and compared in the repetitive and the C-terminal unique
domains. A potential real gene (EU016530) from E. sibiricus contained two types of CD epitode (glia-α2 and gli-
a-α), whereas, the real genes from diploid L. elongatum are found to contain no CD epitode, only with pseudogenes
EU018257 containing glia-α. Phylogenetic analysis showed that α-gliadins from diploid L. elongatum were closely
related to those from decaploid L. elongatum, while that from E. sibiricus were more homologous with those from
other Triticum species.
Keywords Lophopyrum elongatum, Elymus sibiricus, α-gliadin gene, Celiac disease (CD) epitope, Evolution
长穗偃麦草与老芒麦中 α-醇溶蛋白基因的分离与鉴定
吴卫东 1,2* 陈梦竹 1* 陈凡国 1** 夏光敏 1
1山东大学生命科学学院,济南, 250100; 2黔南民族师范学院生命科学系,都匀, 558000
*同等贡献作者
**通讯作者, fanguo2002@sdu.edu.cn
摘 要 本研究从长穗偃麦草与老芒麦中分离了 63个 α-醇溶蛋白基因的开放阅读框，并进行了序列分析，
结果表明 90%的基因中含有提前终止密码子或移码突变。与已经发表的 α-醇溶蛋白基因序列比对发现，这些
基因都编码相似的信号肽、重复区和 C-末端，但在两个多聚谷氨酰氨区差异明显。依据重复区中前三个氨基
酸序列分析，出现了 GRV和 RV两种新的亚基类型；在第一个多聚谷氨酰氨区出现了(Q)3AR(Q)5、(Q)2AR(Q)5、
(Q)3A新类型。乳糜泻抗原分析显示，来自老芒麦的一个真基因 EU016530中含有两种乳糜泻抗原(glia-α2和
glia-α)，而来自长穗偃麦草的真基因中没有发现乳糜泻抗原的存在，仅有假基因 EU018257含有乳糜泻抗原
glia-α。进化分析表明，二倍体长穗偃麦草 α-醇溶蛋白基因与十倍体的长穗偃麦草α-醇溶蛋白基因有较近
的亲缘关系；而老芒麦的 α-醇溶蛋白基因与其它小麦族的基因聚在一起。
关键词 长穗偃麦草,老芒麦, α-醇溶蛋白基因,乳糜泻抗原,进化
www.molplantbreed.org/doi/10.3969/mpb.008.000020
Program foundation: This work was supported by Natural Science Foundation of Shandong province (Y2007D48) and Doctoral Foundat-
ion of Shandong province (2008BS07012)
It is well established that the bread making poten-
tial of wheat flour depends largely on its gluten pro-
teins. Gluten proteins play a key role in determining the
unique baking quality of wheat by conferring water ab-
sorption capacity, cohesivity, viscosity and elasticity on
dough. They can be divided into two main fractions ac-
cording to their solubility in aqueous alcohols: the solu-
ble gliadins and the insoluble glutenins (Shewry et al.,
2003). The former presents as monomers and is initially
classified into four groups (α-, β-, γ-, ω-gliadin) on the
basis of the mobility at low pH in gel electrophoresis.
Later studies on amino acid sequences, however, have
shown that the electrophoretic mobility does not always
reflect the protein relationship and that α-andβ-gliadins
fall into one group (α/β-type) (Wieser, 2007). Among all
gliadins or even whole gluten, α-gliadin is the most
abundant, comprising 15%~30% of the wheat seed pro-
teins. Therefore, it is one of the most consumed proteins
by human (van Herpen et al., 2006; Wang et al., 2007).
Genetically, α-gliadin is controlled by Gli-2 loci
(Gli-A2, Gli-B2, and Gli-D2), presenting on the short
arms of the homoeologous group 6 chromosomes
(Shewry et al., 2003; Payne, 1987). Estimated number of
α-gliadin ranges from 25 to 150 copies per haploid
genome of wheat and its ancestral species (Herberd et al.,
1985; Okita et al., 1985; Anderson et al., 1997). These
differences are probably caused by duplication and dele-
tion of chromosome fragments, such as a wheat line
lacking an entire cluster of α-gliadin genes (DOvidio et
al., 1991). However, the copy number of α-gliadin
genes is largely in excess of the total number of
α-gliadin proteins that can be separated by two-dimen
sional electrophoresis (Lafiandra et al., 1984). This can
be partially explained by the assumption that some pep-
tides encoded by the different gene family members are
pseudogenes (Gu et al., 2004). Anderson and Greene
found that half of the genomic sequences of α-gliadins
contained internal stop codons and were presumably
pseudogenes (Anderson et al., 1997; van Herpen et al.,
2006) further reported that 87% of α-gliadin genomic
sequences contained an internal stop codon (van Herp
en et al., 2006). Although a large number of α-giladin
genes were reported (van Herpen et al., 2006; Wang et
al., 2007; Sumner-Smith et al., 1985; Anderson et al.,
1997), the detailed constitution of the multi-gene locus
is absent.
The typical structure of the α-gliadin consists of a
short N-terminal signal peptide (S) followed by a repeti-
tive domain (R) and a longer non-repetitive domain (U1
and U2), separated by two polyglutamine repeats (Q1
and Q2) (Anderson et al., 1997; van Herpen et al., 2006).
The repetitive domain of α-gliadin contains two basic
repeat units PQPQPFP and PQQPY, which are modi-
fied by the substitution of single residues. With a few
exceptions, α-gliadin contains six conserved cystein
residues in the non-repetitive domains, which form
three homologous intrachain crosslinks (Shewry et al.,
2003). Both the number and distribution of cysteines in
α-gliadin could influence gluten quality. Changes in
the position of cysteine residues might affect the pattern
of disulphide bond formation, resulting in a failure of
two cysteine residues in a protein. Such two cysteine
residues would then be available for intermolecular
disulphide bond formation (Masci et al., 2002). Some
α-gliadins containing additional cysteine residues,
which allow the formation of interchain disulfide bonds,
have positive effect on pasta quality (Shewry et al., 1997;
Anderson et al., 2001).
Special gluten proteins of wheat cultivar can cause
celiac disease (CD) of the general population in 0.5%~1%
frequency. Among these proteins, the α-gliadin and
some glutenin contain several peptides in relation to the
disease (van Herpen et al., 2006; van de Wal et al.,
1998; Koning et al., 2003; Vader et al., 2003). There is
genetic diversity in the both contents of gluten proteins
with toxic epitopes. Thus, it is necessary to investigate
and select wheat and its relatives with less or no toxic
epitopes and use for the disease resistant breeding of
wheat cultivar.
There are many wild related species of wheat.
Most of them have been proved to contain gluten genes.
To date, a few of these genes have been characterized
(McIntosh et al., 1999, http://wheat.pw.usda.gov/gg-
pages/wgc). Diploid Lophopyrum elongatum and tetro-
ploid Elymus sibiricus own many excellent agronomi-
cal characters that have been widely applied in wheat
breeding for quality improvement, such as high content
of seed protein (Dong and Zhang, 2000). In this paper,
Molecular Characterization of α-gliadin Genes from Diploid Lophopyrum elongatum and Elymus sibiricus
长穗偃麦草与老芒麦中 α-醇溶蛋白基因的分离与鉴定 21
分子植物育种
Molecular Plant Breeding
we report the cloning and characterizing of the
α-gliadin genes from L. elongatum and E. sibiricus.
The purpose of this work is to characterize novel genes
in relation to their evolution, and to understand the di-
versity of CD epitopes in the α-gliadin gene family a-
mong wild relatives of the wheat cultivar.
1 Results
1.1 Is olating and sequencing of α-gliadin genes
from L. elongatum and E. sibiricus
Using genomic PCR amplification, DNA frag-
ments with the complete coding sequences were ob-
tained from L. elongatum PI531719 and E. sibiricus
PI598800, which are approximately 900 bp in size (Fig-
ure 1). Theywere thenpurified fromtheagarosegel, ligat-
ed into pMD18-T vector and transformed into E. coli
DH10B competent cells. The positive clones were
screened out and sequenced. As a result, we obtained 63
DNAsequences that are not found in the public databases,
which showed high similarity to other known α-gliadin
genes. Only 6 of these sequences contained non-in-
framed full-ORF α-gliadin gene (Table 1). The residual
57 sequences contained one or more internal stop
codons or a frameshift mutation, which were referred to
pseudogenes (Table 1). According to the deduced amino
acid sequences, the stop codons of pseudogenes were
almost located on the positions where the full-ORF
gene contains a glutamine residue codon. The predomi-
nant base transitions are from C to T, altering CAG or
CAA codons to TAG or TAA stop codons in glu-
tamines.
Number of synonymous (Ks) and non-synonymous
(Ka) substitutions per site was calculated from pair wise
comparisons among the obtained full-ORF genes and
the pseudogenes (Figure 2). The result indicated that ra-
tio of Ks/Ka were significantly higher in pseudogenes
than that in full-ORF genes.
1.2 Characteristic of derived amino acid sequences
of α-gliadin genes
The sixty-three deduced amino acid sequences share
an identity ofmore than 77%with one another, and an av-
erage of more than 85% with previously reported
α-gliadin gene sequences of wheat and its related grass
species. All of them possess a typical structure shared
by previously reported α-gliadins. These cloned gliadin
genes, therefore, were suggested to be new members of
α-gliadin family. Each subset consists of a signal pep-
tide with 19 or 20 amino acid residues, fol lowed by a
Figure 1PCRamplification ofα-gliadin gene fromdiploid L. elon－
gatumandE. sibiricus
Note: M: λDNA/EcoRⅠ+HindⅢ ; 1: Diploid L. elongatum; 2:
Tetroploid E. sibiricus
Table 1 Full-ORF and pseudogenes from diploid L. elongatum and E. sibiricus obtained in this study
Genome
Ee
StH
Total
Species, Accession
L. elongatum PI531719
E. sibiricus PI598800
Full-ORF
5 (EU018263-EU018267)
1 (EU016530)
6
Pseudogene
56 (EU018207-EU018262)
1 (EU016529)
57
Total
61
2
63
Figure 2 Relative numbers of Ka and Ks per site for pairwise
comparisons among full-ORF α-gliadins and pseudogene se-
quences
Note: The dotted line represents a Ka/Ks ratio of 1; Linear trend-
lines with the intercept set to zero are shown both for full-ORF
sequences and pseudogene sequences
www.molplantbreed.org
DOI: 10.3969/mpb.008.00002022
Molecular Characterization of α-gliadin Genes from Diploid Lophopyrum elongatum and Elymus sibiricus
长穗偃麦草与老芒麦中 α-醇溶蛋白基因的分离与鉴定
repetitive domain, a polyglutamine domain, a unique
region (except EU018215), a second polyglutamine do-
main, and finally a C-terminal unique domain (Figure 3).
Alignment between the deduced amino acid sequences
and the reported α-gliadin sequences reveals that high
identity exist in the signal peptide domain, repetitive do-
main and C-terminal region, except the two polygluma-
tine domains. Generally, differences of the amino acid
could be attributed to single nucleotide base changes,
sequence changes involving complete codons, or to the
frameshift variations.
The N-terminal repetitive domain following signal
peptide comprised of amino acid numbers varied from
75 (EU018236) to 102 (EU016530). This region consists
of the first three codon motifs for VRV, GRV and RV
and several repeat motifs. GRV (such as EU018215) and
RV (onlyEU018208)were two new types. Each of the lat-
ter, composed of 3 to 9 codons, was cleaved into two
parts: the conserved first three codons and the vari-
able-length glutamine-rich region. The repeat motifs
were encoded by a DNA pattern CCATA/TTCCA/G
CAR, mentioned by Anderson and Greece in 1997. CAR
presents a 0~6 glutamine-rich region.
The difference between α-gliadin and other glutens
is that α-gliadin possesses two polyglutamine regions.
Most amino acids in the two regions are glutamine
(Table 2). The residue numbers of these regions are
high-variable, which contribute to most of the differ-
ences in complete protein sizes of α-gliadin. Based on
these results, several new types of α-gliadin were char-
Figure 3 Classification of the deduced amino acid sequences
based on the distribution of cysteine
Note: S: Signal peptide; R: Repetitive domain; Q1: N-terminal
polyglutamine domain; U1: N-terminal unique domain; Q2: C-ter-
minal polyglumatine domain; U2: C-terminal unique domain
Table 2 Variation of two polyglumatine regions with fifty-two α-gliadin genes (excluding 11 frameshift pseudogenes)
Clone
EU018217, EU018229, EU018238, EU018251, EU018256
EU018221, EU018240, EU018245, EU018247-EU018250,
EU018252, EU018255, EU018258, EU018262
EU018232
EU018246
EU018220
EU018214, EU018228, EU018231, EU018237
EU018212
EU018222
EU018223, EU018233
EU016530
EU016529
EU018209, EU018263-EU018267, EU018218, EU018230,
EU018235, EU018241, EU018257, EU018261
EU018208, EU018210, EU018216, EU018227, EU018236,
EU018239, EU018253, EU018259
EU018226
EU018215
N-terminal polyglumatine
(Q)3A(Q)7H(Q)5
(Q)3A(Q)9ST(Q)6
(Q)3A(Q)9R(Q)5
(Q)3A(Q)9ST(Q)5
(Q)3A(Q)2R(Q)12
(Q)3A(Q)9ST(Q)6
(Q)3A(Q)10
(Q)3A(Q)9
(Q)3A(Q)5
(Q)11
(Q)7
(Q)3A(Q)8
(Q)3AR(Q)5
(Q)2AR(Q)5
(Q)3A
C-terminal polyglumatine
(Q)3ST(Q)2
(Q)6
(Q)22 (EU018250, EU018255)
(Q)6
(Q)6
(Q)6
(Q)6
(Q)9
(Q)9
(Q)19
(Q)8
(Q)4
(Q)6
(Q)4 (EU018257)
(Q)5 (EU018264)
(Q)23
(Q)22 (EU018259)
(Q)24
(Q)2R(Q)13
Note: ST presents stop codon
23
分子植物育种
Molecular Plant Breeding
Figure 4 Deduced amino acid sequence alignment of the six full-ORF α-gliadin genes
Note: 1: Presents glia-α2 and glia-α9 epitopes; 2: Presents glia-α20; 3: Presents glia-α; The underlined sequences represent the
primers region
acterized, such as (Q)3AR(Q)5, (Q)2AR(Q)5, and (Q)3A.
We found that only two gliadins from the tetroploid
genome, EU016529 and EU016530, possessed complete
glutamine residue composition in polyglutamine re-
gions. Non-glutamine codons among these regions could
mainly be accounted for single or two base changes in
glutamine (CAA to CAT, CAA to GCA, etc.).
Most α-gliadins contain six conserved cysteine
residues in the two unique regions that can form in-
tramolecular disulfide bonds (Figure 3). Four were in the
N-terminal unique domain, and two in the C-terminal
domain. Figure 3 shows examples of gliadins with odd
numbers of cysteine residues. Clone EU018246 has an
additional cysteine created by a tyrine-to-cysteine
residue change at position 215. Clones EU018207,
EU018229, EU018238 and EU018251 have the same
serine-to-cysteine substitutions at position 209 and 210.
Clones EU018212 and EU018222 lose a cysteine
residue through a cysteine-arginine change at position
188 and 187. Clone EU018215 does not contain the
complete N-termin al unique region, resulting in four
cysteine residues missing.
All the full-ORF α-gliadin genes in Figure 4 are so
similar that they can not be divided as unique proteins by
www.molplantbreed.org
DOI: 10.3969/mpb.008.00002024
physical or chemical procedures commonly used. Clones
EU018263, EU018265, and EU018266, only different
from each other in several single nucleotide polymor
phisms (SNPs) at the DNA level, own the same deduced
amino acid sequences. Clone EU018264 differs from
EU018263 in a single alanine/proline substitution in
position 88 and in an amino acid deletion in position
205. Clones EU018267 and EU018263 differ from each
other in two amino acids substitution in position 44 and
139. In comparison with the above five proteins, clone
EU016530 from tetroploid genome was different in a
single peptide QQQ addition and several amino acid
changes. Thus, it is likely that subunits encoded by such
genes are observed in the same band in PAGE analyses.
1.3 Analysis of CD-toxic epitopes
Four known CD epitopes (glia-α2, glia-α9, glia-
α20, and glia-α) have been checked in the obtained
full-ORF genes (Figure 4) and pseudogenes (results not
shown). In the sequences of diploid species, the above-
mentioned four epitopes were absent in all the full-ORF
genes (Figure 4) and pseudogenes except EU018257,
which contained glia-α. In the two genes from the
tetroploid, the full-ORF gene EU016530 contained two
types of epitode, glia-α2 and glia-α, while the pseudo-
gene EU016529 contained the epitope glia-α9 only.
Each epitope had its own position in the α-gliadin pro-
tein. Glia-α was usually present in the C-termial u-
nique domain (U2), whereas glia-α2, glia-α9 and glia-
α20 were all found in the repetitive domain (R). A fur-
ther look at these sequences revealed that a SNP, which
resulted in an amino acid change in a particular epi-
tope, was present in most genes obtained in this test.
Figure 4 shows that the glia-α epitope in all of the
full-ORF genes from dip loid specie were disrupted at
the fourth amino acid of the epitope by the presence of
V (valine) instead of F (phenylalanine).
1.4 Evolutionary relationship of the gliadins
Five α-gliadin genes from decaploid L. elongatum
(unpublished data by author) and eight from other plants
of Triticum were retrieved from the NCBI in order to get
more informati on a bout the relationship be tween the
α-gliadin genes cloned in this work, Phylogenetic trees
were drawn from the deduced amino acid sequences of
the α-gliadin genes with or without the two polyglum-
atine regions (Figure 5A; Figure 5B ). Giadins from the
two species are clearly clustered into different groups
(Figure 5). Most of the gliadins from diploid L. elon－
gatum are closely related to those from decaploid L. e－
longatum. And gliadins from E. sibiricus seem to be
more homologous with those from other Triticum
species. The two phylogenetic trees drawn are only
slightly different from each other (Figure 5).
The sequences compared were from the start
codon to the stop codon, containing (A) or missing (B)
the two polyglumatine regions. EU018331-EU018335
from decaploid L. elongatum were cloned (unpublished);
DQ14035 and DQ296195 are from T. turgidum subsp.
Durum (durum wheat); M16496 is from Triticum urartu;
EU016529 and EU016530 are from E. sibiricus; EU01-
8208, EU018209, EU018215, EU018217; EU018220-
EU018223, EU018226, EU018232, EU018263-EU01-
8267 are from diploid L. elongatum. Sequences are se-
lected according to the differences of two polygluma-
tine regions; DQ246447, K03074, M01192, X01130,
X17361 are from common wheat (T. aestivum).
2 Discussion
2.1 α-gliadin gene composition, copy number and
complexity
Former researches indicated that each genome of
wheat cultivar and its related wild grasses all contained
25~150 α-gliadin gene copies (Anderson et al., 1997).
Here we have obtained sixty-one sequences from L. e－
longatum and two from E. sibiricus. The reason why we
having not gotten more α-gliadin genes from E. sibiri－
cus on one hand may attribute to the primers used,
which were designed according to the conserved region
of α-gliadin gene from the common wheat and its close
relatives, And E. sibiricus are not direct ancestors nor
close relatives of common wheat so that it may possible
to get the unexpected PCR results. On the other hand,
the genes were less polymorphic/heterozygous in E.
sibiricus than in L. elongatum. There should be clearly
different in α-gliadin gene composition between the
two species if the former suggestion is correct. So, new
strategies must be used to understand α-gliadin gene
Molecular Characterization of α-gliadin Genes from Diploid Lophopyrum elongatum and Elymus sibiricus
长穗偃麦草与老芒麦中 α-醇溶蛋白基因的分离与鉴定 25
分子植物育种
Molecular Plant Breeding
composition of E. sibiricus.
The major part of α-gliadin genes (about 90% )
from diploid L. elongatum possesses in-frame stop
codon or frame shift mutation that resulted in their in-
ability to express normal proteins in seeds. The per-
centage of pseudogenes appeared to be higher than that
has been reported previously by Anderson et al (1997),
but similar to van Herpen et al. (2006). Analysis of the
synonymous and non-synonymous substitutions in the
obtained gene sequences indicated that the pseudogenes
contained about 4 times of non-synonymous substitu-
tions than the full-ORF genes. This is consistent with
the reduced selection pressure on the pseudogenes.
2.2 Evolution relationship with decaploid L. elon－
gatum
Decaploid L. elongatum is an allopoplyploid. The
genomic type of de caploid L. elongatum was StStSt-
StEeEeEbEbExEx (Zhang et al., 1996). Sequence align
ment and phylogenetic analysis of α-gliadin genes
from diploid, decaploid L. elongatum, E. sibiricus and
Triticum indicate that genes from diploid L. elongatum
have very high similarity with those from decaploid,
whereas the two E. sibiricus genes are more homolo-
gous with others from Triticum species. This further
confirmed that the diploid L. elongatum was the donor
of the decaploid L. elongatum.
Anderson and Greece (1997) reported that two po-
lyglumatine regions were the most variable in α-gliadin
genes. They suggested that the two polyglumation re-
gions should be neglected in constructing phylogenetic
tree so as to avoid the influence of random mutation in
long glumatine repeat. Otherwise, the average number of
glutamne residues in the two polyglutamine regions
were special to different genomes (van Herpen et al.,
2006). Our results showed that there is only slight differ-
ence in constructing phylogenetic tree with or without
the two polyglumatine regions (Figure 5A; Figure 5B).
Figure 5 Phylogenetic trees of α-gliadin DNA coding sequences
Note: A: With the two polyglumatine regions; B: Without the two polyglumatine regions
www.molplantbreed.org
DOI: 10.3969/mpb.008.00002026
2.3 α-gliadin in relation to wheat breeding
Most α-gliadins conta in six conserved cysteine
residues in the two unique regions that can form in-
tramolecular disulfide bonds (Shewry et al., 2003; An-
derson et al., 1997). It is more conserved in full-ORF
genes than in pseudogenes obtained in this paper. Sev-
eral gliadins with odd number of cystenine residue were
found, but none of them were full-ORF genes. Thus we
suggest that conserved region evolve more quickly in
pseudogenes than in full-ORF genes.
Wheat gluten proteins cause celiac disease (CD) in
0.5% to 1% of the general population (van Herpen et al.,
2006). Among them, the α-gliadin contains several
peptides that are linked to the disease. Here we firstly
report CD epitopes in full-ORF genes and pseudogenes
from the two wild species related to wheat (van Herpen
et al., 2006). It was reported that one or more types of
epitope is related to special genome. Our results
showed that α-gliadin of E. sibiricus contained two
types of CD epitode in potential real gene, whereas,
those of diploid L. elongatum has only one type in pseu-
dogenes, with none in potential real genes.
3 Materials and methods
3.1 Plant materials
Seeds of Lophopyrum elongatum (EeEe, 2n=14)
and Elymus sibiricus (StStHH, 2n=28) were originally
collected from France. Their accession number is
PI531719 and PI598800, respectively.
3.2 Cloning and sequencing of α-gliadin gene ORFs
from diploid L. elongatum and tetroploid E. sibiricus
The seedlings of L. elongatum and E. sibiricus
were grown in darkness for 14 days under 23℃ . Ge-
nomic DNA was extracted from these seedlings by the
CTAB method according to Murray and Thompson
(1980). A pair of primers (P1: 5-ATGAAGACCTTTC
TCATCCT-3 and P2: 5-TCAGTTAGTACCGAAGA
TGCC-3) was designed according to the coding region
of α-gliadin genes in public database. A high fidelity
polymerase LA Taq with GC buffer (TaKaRa) was used
in genomic PCR. PCR started with a denaturation step
at 94℃ for 3 min, followed by 30 cycles each with de-
naturation at 94℃ for 40 s, anneal at 55℃ for 1 min,
and elongation at 72℃ for 2 min, with a final elonga-
tion step at 72℃ for 10 min. The purified PCR prod-
ucts were cloned into the vector pMD 18-T (TaKaRa)
and introduced into E. coli DH10B competent cells by
standard methods (Sambrook et al., 1989). Then PCR
am plification was performed to identify the positive
clones. The selected clones were sequenced by a com-
mercial company (Yingjun, Shanghai, China). At least
two independent sequences of each clone were checked.
3.3 Sequence analysis
Sequence analysis was performed using MEGA
version 3.1 (Kumar et al., 2004) and programs deposit-
ed in NCBI and EBI networks.
3.4 Analysis on synonymous and non-synonymous
substitution
The obtained n ucleotide sequences were aligned
codon-by-codon using Clustal W. The general selection
patterns were analyzed at the molecular level using
DnaSp 4.00 (Rozas et al., 2003). Insertions or deletions
that cause a frame-shift were treated as non-synony-
mous substitutions. The number of synonymous (Ks)
and non-synonymous substitutions (Ka) per site was cal-
culated from pair wise comparisons with incorporation
of the Jukes-Cantor correction, as described by Nei and
Gojobori in 1986 (van Herpen et al., 2006).
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www.molplantbreed.org
DOI: 10.3969/mpb.008.00002028