Molecular Characterization of the Waxy Gene in Einkorn Wheat
This study characterizes 15 waxy genes from 15 accessions of the einkorn wheats Triticum urartu, T. boeoticum and T. monococcum. The mature protein coding sequences of waxy genes were analyzed. Nucleotide sequence variations in these regions resulted from base substitution and/or indel mutations. This work identified 8 distinct haplotypes from the diploid wheat waxy gene sequences. A main haplotype was found in 7 gene samples from the Au genome and Am genome. The waxy gene sequences from the Au and Am genomes could be obviously clustered into two clades, but the sequences from the Am genome of T. boeoticum and T. monococcum could not be clearly distinguished. The phylogenetic analysis revealed that the waxy gene sequences from the Am genome had accumulated fewer variations and evolved at a slower rate than the sequences from the Au genome. These results would contribute to the understanding of functional aspects and efficient utilization of waxy genes.
Diploid wheats, including Triticum urartu (2n = 2x = 14, AuAu), T. boeoticum (2n = 2x = 14, AmAm) and T. monococcum (2n = 2x = 14, AmAm), were the A genome donors of polyploid wheats and they provided many characters or genes of agronomic interest (DEgidio and Nardi, 1993). Subsequent molecular studies identified T. urartu as the Au genome ancestor of emmer wheat, durum wheat and common wheat, whereas T. monococcum is the donor of the Am genome of Triticum zhukovskyi (Dvorak et al., 1993; Jiang and Gill, 1994; Feldman, 2001; Baum and Bailey, 2004), although, T. urartu and T. boeoticum are wild and T. monococcum is domesticated.
Granule-bound starch synthase (GBSSI, EC18.104.22.168), also called waxy
protein (Echt and Schwartz, 1981) is a nuclear-encoded enzyme of about 60 kDa,
which plays a crucial role in the amylose synthesis in the plastids of plants
(Vos-Scheperkeuter et al., 1986). The gene encoding the granule-bound
starch synthase (called waxy gene) has been cloned from maize (Shure
et al., 1983; Klosgen et al., 1986), potato (Visser et al.,
1989; Van-Der-Leij et al., 1991), barley (Rohde et al., 1988),
rice (Hirano and Sano, 1991; Okagaki, 1992), pea (Dry et al., 1992),
common wheat (Mason-Gamer et al., 1998; Murai et al., 1999) and
three diploid wheats only (Yan et al., 2000). In view of the importance
of the waxy genes to wheat quality attributes (Zhao et al., 1998),
it was of interest to analyze more genes from respective diploid progenitors
In this study, fifteen partial waxy genes from T. urartu (designated the wx-TuA gene), T. boeoticum (designated the wx-TbA gene) and T. monococcum (designated the wx-TmA gene) were cloned. The objectives of this study were to isolate and characterize the partial waxy genes from diploid wheats and to investigate the polymorphisms of these genes in the A genome, as well as to discuss the nucleotide variations.
MATERIALS AND METHODS
In total, 15 accessions of einkorn wheats, including 5 T. boeoticum,
5 T. monococcum and 5 T. urartu accessions, were used to
characterize waxy genes. The einkorn wheat accessions were kindly provided
by Dr. Harold Bockelman of American Germplasm Resources Information Network
(GRIN) of USDA. These diploid wheat accessions were collected from various countries,
including Turkey, Armenia, Iraq, Asia Minor, Iran and others (Table
Genomic DNAs were extracted from the young leaves of single plant. From
September 2007 to October 2008, two anchored primers (F: 5-TTGCTGCAGGTAGCCACAC-3;
R: 5-CTCAAGTGCTGCCTGGC AGAGAA-3) were selected for amplification
the target waxy gene (Yan et al., 2000), which include the extron
1 to extron 3 regions. Reactions were carried out in 50 μL containing 200
ng of genomic DNA, 1.5 mM MgCl2, 5 pmol of each primer, 200 μM
of each dNTP and 1.2 units of Taq polymerase (TaKaRa) with high fidelity
and 5 μL of 10xPCR buffer. The PCR amplification was run in Peltier Thermal
Cycler PTC-220 (MJ Research, USA) with the following program: an initial step
of 95°C for 2 min, followed by 39 cycles of 95°C for 1 min, 60°C
for 1 min and 72°C for 2 min, then 72°C for 10 min. The expected fragments
were recovered and cloned into pMD18-T vector (TaKaRa), then transformed
into the competent E. coli cells (DH5α) waxy gene is a single
copy gene in the diploid wheats (Gautier et al., 2000). Therefore, one
positive clone of each accession was sequenced by commercial company (TaKaRa)
in two directions.
materials used in this study
Multiple sequence alignment were conducted by DNAman (version 5.2.2; Lynnon
Biosoft), Clustal W (Higgins et al., 1994) and MEGA (version 3.1 Kumar
et al., 2004). Phylogenetic analysis was carried out by MEGA using neighbor-joining
method (Saitou and Nei, 1987).
DNA Sequence Variation
Fifteen sequences were obtained and analyzed using BLASTn in NCBI. It was
found that these sequences had the closest homology with waxy genes,
confirming the new sequences as waxy. The 15 gene sequences share a high
sequence identity (97.06%), among which the identity values of the coding and
the noncoding sequences are 99.31 and 89.57%, respectively. Among the 691 nucleotides
of 5 gene sequences from the Au genome, there were 663 conserved
positions, 28 variable positions including 7 polymorphic sites (single nucleotide
polymorphisms, SNPs) and 21 singleton positions (mutation in only one sequence).
The frequency of SNPs in the waxy genes from the Au genome
was 1 in 98 bp. In the 10 Am genome-coding genes, which from the
Am genome were more conserved than those genes of the Au
genome. Among the 691 nucleotides, there were 682 conserved positions, 9 variable
positions, 4 polymorphism sites (SNPs) and 5 singleton positions. The frequency
of SNPs in the α-amylase inhibitor genes from the Am genome
The SNPs were classified into transitions (C-T and A-G) and transversions (A-C, A-T, C-G and G-T), according to their substitution types; the transitions were more common than the transversions (Cooper and Krawczak, 1990). The distribution and proportion of transitions and transversions in the SNPs of waxy genes from diploid wheats were investigated. As expected, most of the SNPs (81.25%, 8/9 in T. monococcum and 5/7 in T. urartu) were transitions and the rest (18.75%) were transversions.
A total of 8 haplotypes were defined on the basis of SNP and indel analysis,
among which 6 haplotypes only had one gene sample (Table 2).
Haplotype 4 was found in two out of 15 gene sequences. Haplotype 1 appeared
most frequently, which was involved in 7 gene samples. One and 4 haplotypes
were identified in T. urartu, T. boeoticum and T.
monococcum, respectively. Meanwhile, 2, 3, 5, 6, 7 and 8 haplotypes were
only found in T. urartu, T. boeoticum and T. monococcum,
patterns of SNPs and indels of 8 haplotypes in the 15 waxy genes
from einkorn wheat
GGTACA, II: TG, -: Deletion
Haplotypes 2, 3, 5, 6, 7 and 8 were observed in one species or subspecies.
Haplotype 1 was shared by the two species or subspecies (5 gene samples form
T. boeoticum; 2 gene samples from T. monococcum). Haplotypes 4
was shared by both T. monococcum and T. urartu. It can be concluded
that waxy genes from T. urartu were more diverse. Compared the
waxy alleles of einkorn wheat with that of bread wheat (Murai et al.,
1999), new alleles were frequently observed in einkorn wheat. Therefore, einkorn
wheat could be potentially valuable sources of novel waxy alleles to
improve the amylose synthesis in bread wheat.
Deduced Amino Acid Polymorphism
Analysis of the deduced amino acid sequences of the waxy protein
found that the sequences of all the haplotypes from T. urartu, T. boeoticum
and T. monococcum could encode proteins. Changes in the amino acid residues
at 6 positions could be attributed to the polymorphic sites in the nucleotide
sequences (Table 3). There were 6 different putative waxy
protein amino acid residues in einkorn wheats.
Phylogenetic distances were calculated and used to construct the neighbor-joining
trees showing phylogenetic relationships among the waxy genes in diploid
wheats. In addition to the NJ method, the same data set was analyzed by Minimum
Evolution (ME) and Maximum Parsimony (MP) methods. As the results of the 3 methods
were similar, only the NJ tree is presented. It was found that the waxy
genes were obviously divided into two groups. Four genes from the Au
genome of T. urartu clustered into one group, the other genes
were included in the other group (Fig. 1). Furthermore, the
genes, which are from T. urartu (Accession number was PI538736) and T.
monococcum (Accession number was PI428002), could be separated with the
other genes from T. boeoticum and T. momococcum in the subgroup
by phylogenetic analysis. Neighbor-joining trees were also constructed for haplotypes
of waxy genes from T. boeoticum, T. monococcum and
of amino acids caused by the nucleotide changes in genes
tree of waxy genes
Both the phylogenetic trees and haplotypes show that the waxy genes
from the Au genome were more divergent than those from the Am
genome. Moreover, the species T. urartu contained more haplotypes
than the species T. monococcum and T. boeoticum.
The information in this study describes a set of homoeologous genes encoding granule-bound starch synthase (waxy gene), from T. urartu, T. boeoticum and T. monococcum (closely related to the A genome donors of hexaploid wheat). The wx-TuA, -TbA and -TmA genes possessed high homology in exons, compared with other known waxy genes (Ainsworth et al., 1993). The alignment of the fifteen genes showed only one gap in exon1 and the observed variation among the wx-A genes provides a basis for primer design and PCR-based assays for assaying the mutants of the waxy genes in common wheat.
It has been found that the A genome of polyploid wheat is complex; obviously, more than one diploid wheat was involved in its formation (Metakovsky and Baboev, 1992). Therefore, a comparative intraspecific study of polymorphism is needed for a better understanding of the phylogenetic relationships between wheat species. An obvious feature of the waxy genes from diploid wheats with Am and Au genomes is the single copies gene. Isoelectric focusing, two-dimensional gel electrophoresis and both direct and clone sequencing also revealed single copies of the waxy genes in polyploid wheat (Zhao and Sharp, 1996; Murai et al., 1999; Yan et al., 2000). In our study, 15 waxy gene sequences were obtained from 15 diploid wheat accessions, indicating that there were waxy genes in the diploid wheats and all of them were single copy.
The gene sequences of the waxy gene from the diploid wheats with Am and Au genomes were obviously clustered in two groups with high bootstrap values and the genes from T. boeoticum and T. monococcum could not be distinguished by phylogenetic analysis. Several studies indicate a high level of similarity between the chromosomes of T. monococcum and T. aestivum (Dubcovsky et al., 1995; Luo et al., 2000), suggesting that Au and Am genomes, notwithstanding their high sterility when crossed, have not diverged very significantly since their separation from a common progenitor. In previous investigations, it was possible to distinguish T. urartu from T. boeoticum and T. monococcum by microsatellite and RFLP markers (Corre and Bernard, 1995; Hammer et al., 2000). In recent studies, the A genome bearing T. urartu clearly differs from the A bearing T. boeoticum and T. monococcum in that it lacks the short A1 5S DNA gene unit class (Baum and Bailey, 2004). Earlier studies have shown that Am and Au are related but distinct genomes because the hybrids between T. urartu and T. monococcum are not fertile (Johnson and Dhaliwal, 1976). The AFLP markers were exploited to assess the differences among A genomes of diploid Am and Au wheats and their polyploid relatives (Brandolini et al., 2006). This indicated that all Am genome samples (T. monococcum) clustered together and were separated from the Au diploids (T. urartu). Isoenzyme analysis also showed a very low level of genetic variation within populations of T. monococcum or T. boeoticum (Smith-Huerta et al., 1989; Moghaddam et al., 2000). The RFLP analysis, however, showed high levels of polymorphism (Castagna et al., 1994; Corre and Bernard, 1995). Thus, T. monococcum and T. boeoticum could be regarded as two subspecies of one species with the same Am genome.
The haplotype analysis revealed 8 haplotypes in 15 diploid wheat waxy gene samples. Haplotype 1 was found to be the main haplotype, occurring in 7 samples from T. urartu and T. monococcum. Out of the 8 haplotypes, 6 were found in only one single gene sample; haplotype 2 only had 2 samples, indicating that the waxy genes in einkorn wheats had less diversity.
This study was supported by the National High Technology Research and Development Program of China (863 program 2006AA10Z179 and 2006AA10Z1F8), the Key Technologies R&D Program (2006BAD01A02-23) and the FANEDD project (200357 and 200458) from Ministry of Education, China. Yu-Ming Wei was supported by the Program for New Century Excellent Talents in Universities of China (NCET-05-814). You-Liang Zheng was supported by the Program for Changjiang Scholars and Innovative Research Teams in Universities of China (IRT0453).
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