Waxy Gene Research Articles

Trait‐Specific Improvement of a Cytoplasmic Male‐Sterile Line Using Molecular Marker‐Assisted Selection in Rice

ABSTRACTTo specifically improve preharvest sprouting (PHS) and amylose content (AC) in the cytoplasmic male‐sterile (CMS) line G46A, two simple sequence repeat (SSR) markers, RM3754 and RM447, that are closely linked to qPSR8, a major quantitative trait locus for PHS, were adopted to select for reduced PHS; SSR marker 484/485, which is tightly linked to the waxy gene, was used to select for AC. In the backcross progenies with G46B, the maintainer of G46A, as recurrent parent and ‘Lemont’ as donor parent, most plants with a homozygous Lemont allele at both RM3754 and RM447 showed good PHS resistance. Amylose content in the progeny of G46B/Lemont was significantly related to a difference in alleles at the 484/485 locus. Based on agronomic trait performance and evaluation for PHS and AC, 28 elite lines obtained from the BC3F5 generation were then used in further backcross schemes to breed improved CMS lines. Subsequently, two improved CMS lines showed traits similar to G46A but with improved PHS resistance and intermediate AC.

Phylogenetic relationships of Coryloideae based on waxy and atpB-rbcL sequences

Phylogenetic studies were conducted for 35 populations of the subfamily Coryloideae (Betulaceae) based on waxy gene of nuclear DNA and atpB-rbcL intergenic spacer region of chloroplast DNA. Waxy data analysis suggest that Coryloideae is monophyletic; Corylus is monophyletic and basally branching within the subfamily Coryloideae; Ostryopsis is sister to the Carpinus and Ostrya clade, and the Ostrya is monophyletic (BS=86, PP=99). AtpB-rbcL intergenic spacer region analysis shows that Ostryopsis appeared as the most basal clade within the Coryloideae; Corylus is monophyletic(BS=98, PP=100) and placed between Carpinus-Ostrya and Ostryopsis clade; Carpinus and Ostrya formed a clade with a high support value(BS=100, PP=100). Carpinus sect. Carpinus is monophyletic, whereas sect. Distegocarpus is paraphyletic in the waxy tree. Corylus formed two subclades, but discordance at the infrageneric classification based on morphological characters. In the atpB-rbcL tree, Carpinus and Corylus taxa form a polytomy within the each clade. Results from the two data sets differ mainly in the relative position of Ostryopsis, the monophyly of Ostrya, and the relationships within the Carpinus-Ostrya clade. Further studies are needed for clarify the taxonomic position and the generic limitation.

Hybridization, polyploidy and invasion: lessons from Spartina (Poaceae)

In this paper, we examine how the Spartina system has helped our understanding of the genomic aspects of allopolyploid speciation in the context of biological invasion. More specifically the respective roles of hybridization and genome duplication in the success of newly formed allopolyploid species are explored. Hybridization appears to have triggered genetic and epigenetic changes in the two recently formed European homoploid hybrids S. × towsendii and S. × neyrautii. Deviation from parental structural additivity is observed in both hybrids, with different patterns when considering transposable element insertions or AFLP and methylation alteration. No important changes are observed in the invasive allopolyploid Spartina anglica that inherited the identical genome to S. × townsendii. The repeated rRNA genes are not homogenized in the allopolyploid, and both parental repeats are expressed in the populations examined. Transcriptomic changes suggest possible gene silencing in both hybrids and allopolyploid. In the long-term of evolutionary time, older hexaploid Spartina species (Spartina alterniflora, Spartina maritima and Spartina foliosa) appear to have selectively retained differential homeologous copies of nuclear genes. Waxy gene genealogies suggest a hybrid (allopolyploid) origin of this hexaploid lineage of Spartina. Finally, nuclear and chloroplast DNA data indicate a reticulate origin (alloheptaploid) of the invasive Spartina densiflora. All together these studies stress hybridization as a primary stimulus in the invasive success of polyploid Spartina species.

Evolution and phylogenetic utility of the PHOT gene duplicates in the Verbena complex (Verbenaceae): dramatic intron size variation and footprint of ancestral recombination

A well-resolved species level phylogeny is critically important in studying organismal evolution (e.g., hybridization, polyploidization, adaptive speciation). Lack of appropriate molecular markers that give sufficient resolution to gene trees is one of the major impediments to inferring species level phylogenies. In addition, sampling multiple independent loci is essential to overcome the lineage sorting problem. The availability of nuclear loci has often been a limiting factor in plant species-level phylogenetic studies. Here the two PHOT loci were developed as new sources of nuclear gene trees. The PHOT1 and PHOT2 gene trees of the Verbena complex (Verbenaceae) are well resolved and have good clade support. These gene trees are consistent with each other and previously generated chloroplast and nuclear waxy gene trees in most of the phylogenetic backbone as well as some terminal relationships, but are incongruent in some other relationships. Locus-specific primers were optimized for amplifying and sequencing these two loci in all Lamiales. Comparing intron size in the context of the gene trees shows dramatic variation within the Verbena complex, particularly at the PHOT1 locus. These variations are largely caused by invasions of short transposable elements and frequent long deletions and insertions of unknown causes. In addition, inspection of DNA sequences and phylogenetic analyses unmask a clear footprint of ancestral recombination in one species.

Variation in Granule Bound Starch Synthase I (GBSSI) loci amongst Australian wild cereal relatives (Poaceae)

A complex cascade of enzymes is responsible for the development of starch granules in grain endosperm. Granule Bound Starch Synthase I (GBSSI), encoded by the Waxy gene, is a key enzyme of starch synthesis and determines the accumulation of amylose in the starch granules. The complete genomic GBSSI sequence was ascertained for eight Australian cereal wild relatives (CWR) to determine diversity within the gene. A phylogeny derived from the coding sequence of the entire Waxy gene was compared to established phylogenetic relationships. Starch granule morphology observed in conjunction with this phylogeny suggests that small polygonal starch granules arranged as compound granules are the ancestral state, evolving subsequently to bimodal starch granules and to larger simple granules. Genomic sequence length varied within the species from 2800 to 3572 bp. Most variation occurred within the intron sequences, the largest insertion showing strong homology to a retrotransposon. One wild species was determined to have a deletion in the 3′-end of exon 1 resulting in a putatively non-functional allele. Alignment of the amino acid sequence showed strong homology throughout the central fragments of the gene but broad variation in the transit peptides. All putative functional alleles maintained the reported active sites for glycogen synthesis, though with variations in other highly conserved areas of the gene. These variations within the wild relatives of cultivated cereals may provide novel sources of genetic diversity for future cereal improvement programs.

Nucleotide polymorphisms in the waxy gene of NaN 3-induced waxy rice mutants

Spontaneous and induced waxy phenotype, associated with endosperm containing little or no amylose, has been recognized in rice ( Oryza sativa L.). Mutation of a dominant gene Wx into a recessive gene wx, which causes the inactivation or absence of granule bound starch synthase, is believed to be responsible for the change in endosperm starch leading to the waxy grain. In the present study, the nucleotide polymorphism in the Wx gene of rice genotype Tainung 67 (wild type) and its 35 NaN 3-induced wx mutants were examined. Iodine staining confirmed that all the mutants had waxy grain trait. The G-to-T single base substitution analysis indicated that the wild type genotype Tainung 67 and its waxy mutants carried Wx b allele. Moreover, 23-bp duplication in exon 2 was detected in all the waxy mutants. Microsatellite polymorphism (CT) n was also detectable on the Wx gene of the tested genotypes and mutants, with at least 5 classes of (CT) n microsatellites identified at the Wx locus. Electrophoretic analyses also confirmed the observed nucleotide polymorphsim. Thus, nucleotide polymorphsim exist among NaN 3-induced waxy mutants in rice. However, only the 23-bp duplication in exon 2 may be used as a molecular marker to characterize waxy grain trait in rice genotypes.

Waxy gene haplotypes: Associations with pasting properties in an international rice germplasm collection

Associations between RVA pasting properties and three single nucleotide polymorphism (SNP) sites in the Waxy gene intron 1, exon 6, and exon 10 were determined using rice genotypes of diverse geographic origin. A total of four SNP-haplotypes (combination of SNP alleles) were identified that explained high proportions of the variation in RVA pasting properties ( R 2 = 0.574–0.704). A haplotype containing DNA sequence variation in exon 10 (exon 10 cytosine nucleotide) was exclusively found in high-apparent amylose content (AAC) genotypes with a higher RVA viscosity profile compared to the high AAC genotypes with a different haplotype. The exon 10 SNP explained variances in coolpaste and setback (coolpaste–hotpaste) to 0.642 and 0.499, respectively. Across three haplotypes, which contained exon 10 adenine nucleotide, AAC was correlated with peak, hotpaste, breakdown and setback (coolpaste–hotpaste) at r = −0.85, −0.75, −0.79, and 0.49, respectively. Therefore, the exon 10 SNP differentiates high AAC types with a strong RVA profile and thus can be used by molecular breeding programs focused on quality improvement. Additionally, characterizing genotypes by their functional SNPs allowed us to better understand the relationship between the Waxy gene, its chemical product (i.e., AAC) and the functionality created by the product (i.e., pasting properties).

Polyploid and Hybrid Origins of Pacific Island Sandalwoods (Santalum, Santalaceae) Inferred from Low‐Copy Nuclear and Flow Cytometry Data

It has been argued that polyploids are better adapted than diploids for long‐distance dispersal to and establishment on oceanic islands. To address this issue in a molecular phylogenetic framework, the extensive history of auto‐ and allopolyploidization in Santalum (Santalaceae), the sandalwood genus, was studied by sequencing the low‐copy nuclear gene waxy and investigating the ploidy level of all 16 species. Ploidy level was estimated by measuring the C value (total amount of DNA per nucleus) using flow cytometry and calibrating it by known chromosome numbers and new root‐tip chromosome counts of several taxa. Results indicate four ploidy levels in Santalum: diploid (n=10), tetraploid (n=20), hexaploid (n=30), and octoploid (n=40). The waxy phylogeny suggests that at least six independent polyploid events occurred in the history of Santalum: two allopolyploid events between distantly related species and four putatively autopolyploid events. An additional hybrid event between two tetraploid Hawaiian clad...

Effects of the Barley amo1 and wax Genes on Starch Structure and Physicochemical Properties

AbstractStarch structural mutants showing abnormal endosperm characteristics have been used for investigating the effects of the mutation on structure and physicochemical properties of starches. Inbred lines of barley cultivars ‘Shikoku Hadaka 97’ and ‘Glacier AC38’ were used to investigate the impact of amo1 and waxy genes on starch properties. The amo1 type starch had high apparent amylose content and low starch content. The amo1+waxy type starch contained very little amylose. The content of long chains of amylopectin as detected with high‐performance size‐exclusion chromatography (HPSEC) was decreased, and that of amylopectin chains with the degree of polymerization (DP) of 12‐36 was increased in amo1 and amo1+waxy type starches. The amo1 and amo1+waxy type starches exhibited high gelatinization temperatures and low gelatinization enthalpies.

Molecular phylogeny and reticulate origins of the polyploid Bromus species from section Genea (Poaceae)

The origin of polyploid Bromus species of section Genea was investigated using molecular data. This group of annual species native from the Old-World is composed of three diploids, two tetraploids, one hexaploid, and one octoploid. Molecular cloning, sequencing, and phylogenetic analyses were performed on several accessions per species. We used the low copy nuclear gene Waxy, repeated rDNA spacers ITS1 and ITS2 and chloroplast spacers trnT-trnL and trnL-trnF. Our analyses revealed four different lineages involved in the parentage of the polyploids and confirmed their reticulate origin. Three of these lineages are closely related to the diploid species B. sterilis, B. tectorum, and B. fasciculatus. The fourth lineage could not be related to any diploid according to the available data. Our data gave insights on the origin of all the polyploids of section Genea, and chloroplast data allowed us to identify the maternal lineages. The Waxy gene was the most informative regarding origin of the polyploids. The Waxy copies duplicated by polyploidy appear selectively maintained in the polyploid species. No sequence heterogeneity was encountered in the ITS region, where concerted evolution seems to have occurred toward either maternal or paternal repeats. These results provide new information about the origin and molecular evolution of these polyploids and will allow a more accurate taxonomic treatment of the concerned species, based on their evolutionary history.

Molecular evidence for post-domestication selection in the Waxy gene of Chinese waxy maize

Waxy maize was first reported in China in 1909 and is mainly used in food production in Asia. The evidence for strong domestication selection in the Waxy locus of rice and a selective sweep around its genomic region make us to wonder whether there has been similar selection in Waxy in glutinous maize. To address this issue, DNA sequences of Waxy, three flanking genes and an unlinked gene (Adh1) of 30 accessions sampled from Chinese waxy maize accessions, including representative landraces and inbred lines, were determined in this study. Sharp reduction of nucleotide diversity and significant neutrality tests (Tajima’s D and Fu and Li’s F*) were observed in the Waxy locus in Chinese waxy maize but not in nonglutinous maize; comparison with the unlinked gene confirmed that this pattern was different to Waxy. Sequence analysis across a 143 kb genomic segment centered on the Waxy locus revealed patterns consistent with a selective sweep in the upstream region of Waxy. The selective sweep detected based on current limited genomic sequences exceeded over 50 kb, indicating strong selection in this or a bigger region. However, No sweep effect was detected in the repetitive downstream region of Waxy. Phylogenetic analysis indicated that Chinese waxy maize was domesticated from the cultivated flint maize (Zea mays ssp. mays) that was introduced from the new world. At least two independent deletions in exon 7 (30 bp) and 10 (15 bp) were identified in the Chinese accessions respectively. These findings demonstrate a similar pattern of domestication selection in the Waxy genomic region in both glutinous maize and rice, suggesting that this pattern in the rise of glutinous phenotype is likely in other cereal crops.

<I>Wx</I> sequence analysis of a autotetraploid<I>indica</I>mutant rice and establishment of molecular marker for identifying

The amylose content of the mutant of autotetraploid indica rice D4063-1 is 5.23% anout, which was half of its origin diploid rice Minghui 63. The whole sequence of Waxy gene of D4063-1 was amplified and sequenced. A base was absent on the Wx of D4063-1 in exon sequence, which resulted in frameshift mutation and terminating codon occurred ahead in the 9 exon. The mutation of Wx also led to the change of some mutation in the 9 exon and terminating codon occurred early. The change of Wx also led to changes of the sites of common restriction endonuclease. The results showed that D4063-1 added two sph sites compared to indica and japonica rice; Compared to japonica rice, D4063-1decreased six Acc sites, and added 4 Xba, a Pst and a Sal restriction sites. Phylogenic analysis showed that the DNA sequence of Waxy gene of D4063-1 was closer to indica rice. We supposed that the Waxy gene of D4063-1 originated from genotype of Wxa. According to the differences of Wx in D4063-1, we deduced the absent base led to RNA splicing obstacle, which was the main cause of low amylose content and it might be related to the soft rice phenotype. Based on analysis of Wx of D4063-1, indica and japonica and according to the special sites of the three species, primers as markers-AUT4063-I were designed to distinguish D4063-1 from other rice. Combining with primer pair F5, dominant and codominant ways were established for discriminating them, and rapid and correct identification of D4063-1 from other rice could be done.

Utilization of a molecular marker for evaluation of hybridization success in spring barley genetics and breeding

The success in hybridization of two-rowed spring barley genotypes of various origins was verified using the codominant SSR (Simple Sequence Repeat) marker HVWAXY which is located in the coding sequence of the waxy gene. The primer pair F 5’ AAG ACG TGG TGT TCG TGT G 3’ and R 5’ ATG GTT CCA GGG GTA AGT TC 3’ generated the PCR product of approximately 200 bp in the varieties/breeding lines Maridol, Bojos, Malz, Xanadu, Isotta, Josefin, Native, Sebastian, Conrad, KM 2436, KM 2439, KM 2629, KM-H-1320, whereas the product of about 250 bp was amplified in the lines KM 2416 and Br 7571h33. The parental genotypes possessing different alleles of the marker were crossed and the segregating lines of the F<sub>3</sub> generation were characterized employing molecular methods (marker-assisted selection). This prescreening enabled to work with a lower number of individuals (selfed individuals were excluded) at the initial stages of the breeding process leading to the acceleration of selection as a particular phase of breeding.

Allelic variation of the Waxy gene in foxtail millet [Setaria italica (L.) P. Beauv.] by single nucleotide polymorphisms

The Waxy (Wx) gene product controls the formation of a straight chain polymer of amylose in the starch pathway. Dominance/recessiveness of the Wx allele is associated with amylose content, leading to non-waxy/waxy phenotypes. For a total of 113 foxtail millet accessions, agronomic traits and the molecular differences of the Wx gene were surveyed to evaluate genetic diversities. Molecular types were associated with phenotypes determined by four specific primer sets (non-waxy, Type I; low amylose, Type VI; waxy, Type IV or V). Additionally, the insertion of transposable element in waxy was confirmed by ex1/TSI2R, TSI2F/ex2, ex2int2/TSI7R and TSI7F/ex4r. Seventeen single nucleotide polymorphims (SNPs) were observed from non-coding regions, while three SNPs from coding regions were non-synonymous. Interestingly, the phenotype of No. 88 was still non-waxy, although seven nucleotides (AATTGGT) insertion at 2,993 bp led to 78 amino acids shorter. The rapid decline of r (2) in the sequenced region (exon 1-intron 1-exon 2) suggested a low level of linkage disequilibrium and limited haplotype structure. K (s) values and estimation of evolutionary events indicate early divergence of S. italica among cereal crops. This study suggested the Wx gene was one of the targets in the selection process during domestication.

Phylogeny of Nolana (Nolaneae, Solanoideae, Solanaceae) as inferred from granule‐bound starch synthase I (GBSSI) sequences

The phylogenetic relationships of Nolana (Nolaneae, Solanaceae) were constructed using partial sequences (ca. 891 bp) of the granule‐bound starch synthase I (GBSSI) or the waxy gene. Nolana, with 89 species, is primarily distributed in coastal Chile (49 spp.) and Peru (43 spp.), and of these, four species are recorded in Peru and Chile, and another from the Galápagos Islands, Ecuador. Our phylogenetic analysis, utilizing a sampling of 63 of the 89 species, supports the monophyly of Nolana and recovered three clades with 95%–100% bootstrap support. Nolana sessiliflora is the sister taxon to the remainder of the genus. Two large, highly supported clades are evident; one containing taxa from Chile, Peru and the Galápagos Islands, and another containing taxa from Chile and Peru. Nolana galapagensis, an endemic to the Galápagos Islands, is suggested to be sister to N. arenicola in a clade that also includes N. adansonii from southern Peru and northern Chile. These two species differ substantially in habitat preference, habit, leaf shape, and mericarp morphology. The monophyly is confirmed for a morphologically cohesive group composed of N. acuminata, N. baccata, N. elegans, N. reichei, N. parviflora, N. pterocarpa, and N. paradoxa, a clade of essentially Chilean species.

Relationship Between the First Base of the Donor Splice Site of Waxy Gene Intron 1 and Amylose Content in Yunnan Indigenous Rice Varieties

There exists a single nucleotide polymorphism, G or T, at the first base of the donor splice site of waxy gene intron 1 in rice. In order to study the relationship between the first base of the donor splice site of waxy gene intron 1 and amylose content in rice, the one-step PCR method was used to determine whether it is G or T in 220 Yunnan indigenous rice varieties from 14 districts, 55 towns/counties of Yunnan Province, and 101 varieties of which were validated by the PCR- Acc I method. According to the G/T polymorphism, 164 rice varieties showed GG-genotype, while the other 56 fell into TT-genotype, accounting for 74.5% and 25.5% of all the test varieties, respectively. When all the rice varieties were divided into indica and japonica subspecies, it was found that 80.5% of indica rice and 67.0% of japonica rice belonged to GG-genotype. The rice varieties with GG-genotype had significantly higher amylose content (18.95% on average) than those with TT-genotype (all below 16%), but 33 rice varieties with GG-genotype still had low amylose content ranging from 3.91% to 15.93%, and most of them came from the Dai minority area in the Southwest of Yunnan Province. However, there was no significant difference in the mean amylose content of the same GG or TT genotypes between indica and japonica rice, suggesting that different genetic backgrounds, indica or japonic a, had no effect on amylose content. The coefficient of correlation between the genotype and amylose content was 0.733 ( P<0.01).

Molecular and Biochemical Analysis of the Plastidic ADP-glucose Transporter (ZmBT1) from Zea mays

Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous synthesis of ZmBT1 in Escherichia coli cells leads to the functional integration of ZmBT1 into the bacterial cytoplasmic membrane. ZmBT1 transports ADP-Glc in counterexchange with ADP with apparent affinities of about 850 and 465 mum, respectively. Recently, a complete ferredoxin/thioredoxin system has been identified in cereal amyloplasts and BT1 has been proposed as a potential Trx target protein (Balmer, Y., Vensel, W. H., Cai, N., Manieri, W., Schurmann, P., Hurkman, W. J., and Buchanan, B. B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 2988-2993). Interestingly, we revealed that the transport activity of ZmBT1 is reversibly regulated by redox reagents such as diamide and dithiothreitol. The expression of ZmBT1 is restricted to endosperm tissues during starch synthesis, whereas a recently identified BT1 maize homologue, the ZmBT1-2, exhibits a ubiquitous expression pattern in hetero- and autotrophic tissues indicating different physiological roles for both maize BT1 isoforms. BT1 homologues are present in both mono- and dicotyledonous plants. Phylogenetic analyses classify the BT1 family into two phylogenetically and biochemically distinct groups. The first group comprises BT1 orthologues restricted to cereals where they mediate the ADP-Glc transport into cereal endosperm storage plastids during starch synthesis. The second group occurs in mono- and dicotyledonous plants and is most probably involved in the export of adenine nucleotides synthesized inside plastids.

Identification of Variant Transcripts of Waxy Gene in Non-glutinous Rice (O. sativa L.) With Different Amylose Content

The identification of a number of cDNA sequences at the splice sites of Waxy (Wx) gene in rice endosperm point to the variation of gene expression associated with the G to T mutation at the 5' splice site of intron 1 and the alternative splicing produces variants transcripts by shortening. As consequence, variation of amylose content in this tissue. To determine whether the splicing type of Wx genes are also found in rice endosperm of Thai rice cultivars, cDNA of the gene have been isolated by using RT-PCR and sequenced. Analysis of the twenty-five cDNA clones led to the identification of the splicing pattern of the gene, four cryptic splice sites was found in cDNA sequences of low-amylose cultivars (<20%), while two cryptic splice sites of intermediate (20-25%) and high (>25%) amylose cultivars were characterized. The alternatively spliced transcripts were observed in much higher proportion in low amylose cultivars than that in intermediate and high amylose cultivars. These findings raise a possibility explanation that the degree of amylose content in non-glutinous rice cultivars may reflect to the amount of efficiently spliced transcripts.

Waxy gene haplotypes: Associations with apparent amylose content and the effect by the environment in an international rice germplasm collection

Apparent amylose content (AAC), the key determinant of rice end-use quality attributes, is primarily controlled by the Waxy gene which codes for granule bound starch synthase (GBSS). We examined the combination of sequence variation in the Waxy gene and environmental effects, and their associations with AAC using 171 rice accessions originating from 43 countries. The combination of two single-nucleotide-polymorphism (SNP) markers in the Waxy gene allows for the identification of three marker haplotypes in this gene. The first SNP is at the leader intron splice site (In1 SNP), and the second polymorphism is in exon 6. The haplotypes explained 86.7% of the variation in AAC and discriminated the three market classes of low, intermediate and high AAC rice from each other. The environment affected the AAC of all haplotypes. Higher air temperature during grain development associated with a decrease in AAC of low and intermediate AAC-types, but with an increase in AAC of high AAC-type. The association of AAC with several Waxy RM190 microsatellite-(CT n ) alleles in combination with the In1 SNP was also examined. In conclusion, the Waxy haplotypes studied appear to be useful markers for selecting the AAC of breeding lines developed from the world's rice germplasm.

Mutation loci and intragenic selection marker of the granule-bound starch synthase gene in waxy maize

Four pairs of specific PCR primers have been designed on the basis of the sequence of the granule-bound starch synthase gene (GBSS; dominant non-waxy gene Wx) and used to amplify its homologous sequence from thirteen waxy and two non-waxy inbred lines. Results from electrophoresis indicated that the recessive waxy gene was wx, derived from the dominant non-waxy gene Wx by mutation at its 3′ end. The sequence of the mutated 3′ end was amplified by the TAIL-PCR technique. Sequence alignment showed that the mutation of the wx gene was caused by transposition of the aldehyde dehydrogenase gene rf2. Two pairs of specific primers were designed on the basis of the sequence difference between the dominant gene Wx and its mutated recessive allele wx and used as intragenic selection markers to identify individual plants of genotypes WxWx, Wxwx, and wxwx by PCR amplification from the segregating population of the F2 generation crossed between waxy and non-waxy inbred lines. Iodine solution staining and starch component assay showed that all the 35 F2 plants identified as genotype WxWx produced non-waxy kernels of the F3 generation and that all 33 F2 plants identified as genotype wxwx produced waxy kernels of the F3 generation. This result can be used to improve the selection efficiency of waxy maize breeding and for selection of other single genes and major polygenes.