Dosage effects of the three Wx genes on amylose synthesis in wheat endosperm

Amylose synthesis in wheat endosperm is mainly controlled by the granule-bound starch synthase of about 60 kDa, the so-called waxy (Wx) protein. The Wx proteins are the product of the Wx genes at a triplicate set of single-copy homoeoloci located on chromosomes 7A (Wx-A1), 4A (Wx-B1) and 7D (Wx-D1). Using Chinese Spring and its aneuploid lines, including nullisomic-tetrasomics, tetrasomics, ditelosomics and deletion stocks, together with single-chromosome substitution lines for these chromosomes, the effects of varying the dosage of whole chromosomes and chromosome arms, as well as the effects of null alleles, upon amylose synthesis were investigated. Nullisomic 4A and the deletion of chromosome segments carrying the Wx-B1 gene reduced the amylose content by more than 3%. A reasonable agreement was found in the substitution lines. This confirms that the absence of the Wx-B1 gene, or else substitution of this gene by its null allele, has the most striking effect on decreasing amylose synthesis. The removal of chromosomes carrying either the Wx-A1 or the Wx-D1 gene reduces the amylose content by less than 2%. A similar reduction was revealed by substitution of these two genes by the null alleles. Double dosages of chromosomes 7A, 4A and 7D did not increase amylose content, while the tetrasomic chromosomes produced more of the respective Wx proteins. This suggests that a certain level of Wx gene activity or of the Wx proteins led to the maximum amount of amylose.     

Origin and evolution of the waxy phenotype in Amaranthus hypochondriacus: evidence from the genetic diversity in the Waxy locus

The existence of polymorphism in the Waxy locus in a large gene pool of 53 strains with various waxy phenotypes from samples of Amaranthus hypochondriacus collected from different regions was investigated in an origin-and-evolution study. First, we screened all strains for a mutation point (G–A polymorphism in exon 6) by using PCR–RFLP and/or direct sequence analysis. The results showed that the nonsense mutation in the coding region (exon 6) of the Waxy gene was responsible for the change in perisperm starch, leading to a waxy phenotype in all strains. Second, phylogenetic analysis, which was based on the Waxy variation, indicated diverse waxy types occurring separately and independently in certain domesticated regions in Mexico. Finally, we designated nine molecular types by comparing obvious structural variations in the coding region of the Waxy gene. Among the molecular types, A. hypochondriacus contained Type III in three subtypes with the waxy phenotype, with evolutionary routes that could originate from Type II in accordance with G–A polymorphism. In addition, these types had the same mutation points by which the Waxy gene was converted into the waxy phenotype. Therefore, the present results showed that the nonsense mutation is a unique event in the evolution of waxy phenotypes in this crop. This study will provide useful information for understanding the evolutionary process of the waxy phenotype.     

The insertion/deletion polymorphisms in the waxy gene of barley genetic resources from East Asia

The length polymorphism in the waxy gene, which encodes a granule-bound ADP-glucose-glucosyl transferase [granule-bound starch synthase I (GBSS I), E.C. 2.4.1.11] in barley (Hordeum vulgare), was found. The 5′ leader sequence of the waxy gene of barley germplasm from Japan and Korea was analyzed by the polymerase chain reaction (PCR). The waxy gene of these genetic stocks had three types of length polymorphisms, suggesting that there are insertion/deletion mutations at the 5′ leader sequence of the waxy gene. DNA sequence analysis of the polymorphic PCR products showed that: (1) a 403-bp deletion mutation, which included a complete exon I, was found in the wax allele and a 193-bp insertion sequence was located in the intron I, and (2) the insertion sequence was also located in intron I of the Wax allele. The identity of the insertion sequence was completely conserved between the wax allele and the novel Wax allele. These finding s implying that the wax allele, which was found in indigenous waxy barley, originated in non-waxy barley with the novel Wax allele. Received: 12 January 2001 / Accepted: 17 April 2001     

Genetic control of amylose content in wheat endosperm starch and differential effects of three Wx genes

The endosperm starch of the wheat grain is composed of amylose and amylopectin. Genetic manipulation of the ratio of amylose to amylopectin or the amylose content could bring about improved texture and quality of wheat flour. The chromosomal locations of genes affecting amylose content were investigated using a monosomic series of Chinese Spring (CS) and a set of Cheyenne (CNN) chromosome substitution lines in the CS genetic background. Trials over three seasons revealed that a decrease in amylose content occurred in monosomic 4A and an increase in monosomic 7B. Allelic variation between CS and CNN was suggested for the genes on chromosomes 4A and 7B. To examine the effects of three Waxy (Wx) genes which encode a granule-bound starch synthase (Wx protein), the Wx proteins from CS monosomics of interest were analyzed using SDS-PAGE. The amount of the Wx protein coded by the Wx-B1 gene on chromosome arm 4AL was reduced in monosomic 4A, and thus accounted for its decreased amylose content. The amounts of two other Wx proteins coded by the Wx-A1 and Wx-D1 genes on chromosome arms 7AS and 7DS, respectively, showed low levels of protein in the monosomics but no effect on amylose content. The effect of chromosome 7B on the level of amylose suggested the presence of a regulator gene which suppresses the activities of the Wx genes.     

Deletion commonly found in Waxy gene of Japanese and Korean cultivars of Job’s tears (Coix lacryma-jobi L.)

We isolated the entire sequence of the coding region of Waxy gene of a non-waxy accession of Job??s tears (Coix lacryma-jobi) by PCR-based methods. We also compared the entire sequences of the gene between two non-waxy accessions and three waxy cultivars and found a 275-bp deletion in the coding region (exons 10?C11) of this gene specific to waxy cultivars. We showed by PCR genotyping that this deletion is commonly found in Japanese and Korean cultivars and confirmed that this deletion resulted in lack of Wx protein. We also confirmed that this polymorphism of the gene co-segregates with phenotypes in endosperm and pollen. These results suggest that this PCR-based marker will be useful in breeding of Job??s tears and that genetic information obtained in other grass species will be also useful in genetics and breeding of Job??s tears.     

Comparison of the waxy locus sequence from a non-waxy strain and two waxy mutants of spontaneous and artificial origins in barley

Molecular characterization of 3 alleles of the waxy gene from a non-waxy strain "Shikoku hadaka No. 84" (SH84), an indigenous waxy strain "Mochimugi D" (MMD), and an artificial waxy mutant strain "Shikoku hadaka No. 97" (SH97) of barley (Hordeum vulgare ssp. vulgare) was performed via a PCR direct sequencing strategy. The 3 haplotypes were analyzed in terms of single nucleotide polymorphisms, insertion/deletion mutations, and simple sequence repeat polymorphisms. In comparison with the barley non-waxy gene sequence deposited in the public DNA database, 110 polymorphic sites were found in the 5,190-bp sequenced region of the non-waxy strain SH84. A 418-bp deletion in the 5' non-coding sequence was identified in the indigenous waxy strain MMD. Except for the deletion in the promoter region, the spontaneous mutant wax allele and non-waxy allele were identical. Such highly conserved sequences provide evidence for the recent occurrence of a deletion event in the cultivated barley gene pool. Compared to the original variety SH84, induced waxy mutant SH97 had a base substitution of a C to T in the exon 5, which converting Gln-89 of the wild-type gene into a stop codon, suggesting the involvement of a nonsense-mediated mRNA decay. These results will be helpful for understanding the mechanism of the variable amylose content in waxy cultivars of cereal species.     

Excision of Ds produces waxy proteins with a range of enzymatic activities.

The waxy (wx) locus of maize encodes an enzyme responsible for the synthesis of amylose in endosperm tissue. The phenotype of the Dissociation (Ds) insertion mutant wx-m1 is characterized by endosperm sectors that contain different levels of amylose. We have cloned the Wx gene from this allele and from two germinal derivatives, S5 and S9, that produce intermediate levels of amylose. The Ds insertion in wx-m1 is in exon sequences, is 409 bp in length and represents an example of a class of Ds elements that are not deletion derivatives of the Activator (Ac) controlling element. The two germinal derivatives, S5 and S9, lack the Ds element but contain an additional 9 and 6 bp, respectively, at the site of Ds insertion. The level of Wx mRNA and Wx protein in S5 and S9 is essentially the same as in normal endosperm tissue but Wx enzymatic activity is reduced. Thus, the lesions in S5 and S9 lead to the addition of amino acids in the Wx protein, resulting in Wx enzymes with altered specific activities. This work supports the notion that the maize transposable elements may serve a function in natural populations to generate genetic diversity, in this case, proteins with new enzymatic properties.     

Production of waxy (amylose-free) wheats

The Waxy (Wx) protein has been identified as granule-bound starch synthase (GBSS; EC 24.1.21), which is involved in amylose synthesis in plants. Although common wheat (Triticum aestivum L.) has three Wx proteins, partial waxy mutants lacking one or two of the three proteins have been found. Using such partial waxy mutants, tetra- and hexaploid waxy mutants with endosperms that are stained red-brown by iodine were produced. Both mutants showed loss of Wx protein and amylose. This is the first demonstration of genetic modification of wheat starch.     

Diverse origins of waxy foxtail millet crops in East and Southeast Asia mediated by multiple transposable element insertions

The naturally occurring waxy and low-amylose variants of foxtail millet and other cereals, like rice and barley, originated in East and Southeast Asia under human selection for sticky foods. Mutations in the GBSS1 gene for granule-bound starch synthase 1 are known to be associated with these traits. We have analyzed the gene in foxtail millet, and found that, in this species, these traits were originated by multiple independent insertions of transposable elements and by subsequent secondary insertions into these elements or deletion of parts of the elements. The structural analysis of transposable elements inserted in the GBSS1 gene revealed that the non-waxy was converted to the low-amylose phenotype once, while shifts from non-waxy to waxy occurred three times, from low amylose to waxy once and from waxy to low amylose once. The present results, and the geographical distribution of different waxy molecular types, strongly suggest that these types originated independently and were dispersed into their current distribution areas. The patterns of GBSS1 variation revealed here suggest that foxtail millet may serve as a key to solving the mystery of the origin of waxy-type cereals in Asia. The GBSS1 gene in foxtail millet provides a new example of the evolution of a gene involved in the processes of domestication and its post-domestication fate under the influence of human selection. Electronic Supplementary Material Supplementary material is available for this article at     

Differential regulation of waxy gene expression in rice endosperm

Summary In order to examine the effects of different alleles on the gene expression at the waxy locus, the Wx gene product which controls the synthesis of amylose was isolated from endosperm starch of rice plants and analysed by electrophoretic techniques. The major protein bound to starch granules was absent in most of waxy strains and increased with the number of Wx alleles in triploid endosperms, suggesting that the major protein is the Wx gene product. In addition to wx alleles which result in the absence or drastic reduction of the Wx gene product and amylose, differentiation of Wx alleles seemed to have occurred among nonwaxy rice strains. At least two Wx alleles with different efficiencies in the production of the major protein as well as amylose were detected. These alleles are discussed in relation to regulation of the gene expression.     

Genetic analysis of waxy locus in rice (Oryza sativa L.)

Summary Inheritance of waxy locus was studied in crosses of a waxy variety with four non-waxy parents having high-, intermediate-, low- or very low-amylose content. The analysis for amylose content was done on a single grain basis in parents, F₁, F₂, B₁F₁, and B₂F₁ seeds. The waxy parent lacking synthesis of amylose content was found to differ from the ones having high-, intermediate-, low- or very low-amylose content by one gene with major effect. Dosage effects for amylose content were observed to have great influence on segregation pattern and efficiency of selection. Selection efficiency for amylose content can be enhanced by selecting for endosperm appearance in early segregating generations.     

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.     

Differential effects of Wx-A1, -B1 and -D1 protein deficiencies on apparent amylose content and starch pasting properties in common wheat

Waxy (Wx) protein is a granule-bound starch synthase (GBSS) responsible for amylose production in cereal endosperm. Eight isolines of wheat (Triticum aestivum L.) having different combinations of presence and absence of three Wx proteins, Wx-A1, -B1, and -D1, were produced in order to elucidate the effect of Wx protein deficiencies on the apparent amylose content and starch-pasting properties. An improved SDS gel electrophoresis showed that ’Bai Huo’ (a parental wheat) carried a variant Wx-B1 protein from an allele, Wx-B1e. Thus, wheat lines of types 1, 2, 4, and 6 examined in this study contained a variant Wx-B1 allele and not the standard allele, Wx-B1a. The results from 3 years of experiments using 176 lines derived from two cross-combinations showed that apparent amylose content increased the least in type 8 (waxy) having no Wx proteins and, in ascending order, increased in type 5 (only the Wx-A1 protein is present) <type 7 (Wx-D1) <type 6 (Wx-B1) <type 3 (Wx-A1 and -D1) <type 4 (Wx-A1 and -B1) <type 2 (Wx-B1 and -D1) <type 1 (three Wx proteins). However, Tukey’ s studentized range test did not detect significant differences in some cases. Densitometric analysis suggested that the amylose content was related to the amount of the Wx protein in the eight types. Parameters in the Rapid Visco-Analyzer test and swelling power were correlated to amylose content. Consequently, amylose content and pasting properties of starch were determined to be influenced the most by the lack of the Wx-B1 protein, followed by a lack of Wx-D1, and leastly by the Wx-A1 deficiency, which indicated the presence of differential effects of the three null alleles for the Wx protein. Received: 1 February 1999 / Accepted: 10 April 1999     

Allelic Variation in the Porcine MYF5 Gene Detected by PCR–SSCP

The MYF5 gene has been reported to be integral to muscle growth and development, and hence it has been considered as a candidate gene for meat selection programs in pig. To ascertain whether there was variation in the porcine MYF5 gene, we have developed a method of PCR–single-strand conformational polymorphism (PCR–SSCP) analysis. In this study, two coding regions of the MYF5 gene were investigated. Four unique SSCP patterns were detected in exon 1 and three patterns were identified in exon 3. Two SNPs detected in exon 1 led to a non-synonymous alanine/proline substitution. A nucleotide change in exon 3 did not affect the amino acid sequence. Five extended haplotypes were observed across the two regions. The variation detected in this study might underpin the development of gene markers for improved muscle growth in pig breeding.     

Identification of three Wx proteins in wheat (Triticum aestivum L.)

Nullisomic analysis of waxy (Wx) protein of hexaploid wheat (Triticum aestivum L.) cv. “Chinese Spring” using two-dimensional polyacrylamide gel electrophoresis revealed that threeWx loci,Wx-A1, Wx-B1, andWx-D1, located on chromosome arms 7AS, 4AL, and 7DS, produce three distinct Wx subunit groups, subunit group-A (SGA), SGB, and SGD, respectively. SGA has a higher molecular weight and a more basic isoelectric point (pI) than the other two. SGB and SGD have the same molecular weight but a slightly different pI range. Owing to the detection of these three subunit groups, we were able to identify the expression of three waxy genes in wheat endosperm and to find two types of mutants among Japanese wheat cultivars, one lacking SGA and the others SGB. These results suggest the possibility of breeding a waxy wheat.     

籼稻和粳稻中蜡质基因座位上微卫星标记的多态性及其与直链淀粉含量的关系

稻米直链淀粉是在由蜡质基因Wx编码的颗粒结合淀粉合成酶（GBSS）的催化下合成的。最近,在Wx基因的区段内发现了一段多态性微卫星序列（CT）n。对74个非糯籼稻和粳稻材料的（CT）n多态性进行了分析,并探讨了其与直链淀粉含量之间的关系。在74个品种（系）中共发现7种（CT）n片段（Wx等位基因）,即（CT）8,（CF）10,（CT）11,（CT）16,（CT）17,（CT）18,（Ch）19。在籼粳亚种间,不同（CT）n的分布存在差异较大：在籼稻中,以（CT）11和（CT）18为主,占92．6％,另有（CT）10和（CT）8各2份,（CT）17型1份;在粳稻中,以（CT）16、（CT）17为主,共占20份材料中的90．0％。在上述74个品种（系）中,以（CT）n表示的Wx基因型对稻米直链淀粉含量的决定系数R2达0．912,也即Wx基因型差异可解释这些材料直链淀粉含量变异的91．2％。还发现6份籼稻材料Wx座位上为杂合的（CT）18／（CT）11,其中2份为推广早籼优质品种浙9248和舟优903,并对其在遗传和育种研究中的意义作了探讨。     

Structural variation in the Waxy gene and differentiation in foxtail millet [Setaria italica (L.) P. Beauv.]: implications for multiple origins of the waxy phenotype

The origin and evolution of the waxy type of foxtail millet [Setaria italica (L.) P. Beauv] were studied by analyzing structural variation in the Waxy gene. Initially, the Waxy gene was amplified by RT-PCR, RACE and genomic PCR from a non-waxy strain to determine the structure of the wild-type gene. Secondly, we screened by PCR for polymorphisms at the Waxy locus in 79 strains with various waxy phenotypes. We then carried out genomic Southern analysis on 67 strains and identified seven RFLP classes which were designated as types I-VII. RFLP type was correlated with phenotype, such that types I and II corresponded to non-waxy, types III and VI to low-amylose, and types IV, V and VII to waxy phenotypes. The differences between RFLP types could be attributed to insertions in the Waxy gene. Types II and VI were caused by the insertion of a Tourist element into intron 1 and a SINE-like sequence into intron 12, respectively. Types III, IV, V and VII were characterized by the insertion of large sequences into the Waxy gene that may alter the expression of the gene. Thus, multiple, independent insertions in the Waxy gene appear to have caused the loss-of-function waxy phenotypes. Furthermore, the geographical distributions of the three RFLP types associated with the waxy phenotype (types IV, V and VII) were distinct, with type IV being found mainly in Taiwan and Japan, type V in Korea, and type VII in Myanmar. These results indicate a polyphyletic origin for the waxy phenotype in landraces of foxtail millet.     

Classification of cultivated rices into indica and japonica types by the isozyme,RFLP and two milled-rice methods

Four methods for classifying cultivated rices (Oryza sativa L.) (including IR varieties) into indica and japonica types — waxy gene product in endosperm starch, glutelin ₃ molecular weight in milled rice, RFLP polymorphism at the Wx locus and Glaszmann's isozyme method — were compared. On the basis of the two endosperm traits and the RFLP method Glaszmann's group 1 (indica) was classified as mainly indica and intermediate groups 2, 3 and 4 as exclusively indica. However, the endosperm traits classified Glaszmann's group 5 as mainly indica, while the RFLP method classified it as japonica. The RFLP waxy gene probe was closest to the isozyme method in classifying group 6 as japonicas; the waxy gene product gave mainly indica reaction even in group 6, and the glutelin ₃ method was intermediate. All IR rices were classified as being indica on the basis of Wx gene product and by Glaszmann's method, but a few were classified as japonica by the glutelin ₃ method and by the RFLP waxy gene probe.     

Developmental Changes in the Amylose Content of Endosperm Starch of Barley (Hordeum vulgare L.) during the Grain Filling Period after Anthesis

Large and small starch granules were isolated and characterized from kernels of non-waxy (Bozu) and waxy (Yatomi mochi) barleys at their developmental stages of 8, 16, 28 and 40 days after flowering. The amylose content of the large and small granules of the non-waxy barley starch, as determined by the blue value and enzyme-chromatography, increased with the increasing age of the endosperm. Large granules of the non-waxy barley at any given developmental stage contained more amylose than small granules at the same stage, as in the case of mature non-waxy barley starches. Large granules of either the non-waxy or waxy barleys at any given developmental stage had a lower fraction III: fraction II ratio, one of the structural characteristics of amylopectin, than did small granules of the same cultivar at the same developmental stage. The amylose content in large granules of the waxy barley appeared to increase with the increasing age of the endosperm. The amylose content in small granules of the waxy barley at 8 days after flowering was 10%, although that at 16 and 28 days after flowering and at maturity was only 0~1%.