Wx Locus Research Articles

Corrigendum to: Genetic mapping of commercially significant starch characteristics in wheat crosses

Starch properties were measured on the doubled haploid progeny of 2 crosses, one between Cranbrook and Halberd and the other between CD87 and Katepwa. Properties studied included starch peak and final viscosity measured by Rapid Visco Analyser, starch granule size distribution measured by laser light scattering, starch gelatinisation temperature by differential scanning calorimetry, and flour swelling volume. In the Cranbrook Halberd cross (samples from 2 environments), a highly significant quantitative trait locus (QTL) was located on chromosome 4A for both starch peak viscosity and starch/flour swelling volume, centred around the Wx-B1 locus. In previous studies, this locus has been found to be linked to Japanese noodle quality. The increases in starch peak viscosity and flour swelling volume are derived from the Halberd parent, consistent with the fact that Halberd is null for the Wx-B1 locus on chromosome 4A and is missing the respective granule-bound starch synthase protein, whereas Cranbrook is a wheat line carrying the normal 3 Wx loci. The final starch viscosity also showed an association with the Wx-B1 locus. In the CD87 Katepwa cross, the progeny showed an association between peak viscosity and a marker on chromosome 7A. This appeared to be near the Wx-A1 locus. In some experiments, flour viscosity showed a highly significant QTL on chromosome 7B, apparently at the same locus as the late maturity - amylase derived from the Cranbrook parent. Starch gelatinisation onset temperature indicated a significant QTL on chromosomes 2B and 7A (LOD = 2.58 and 3.66, respectively, in interval analyses). Starch gelatinisation peak temperatures indicated a QTL on chromosome 7A, which, although not in the significant (P = 0.05) class based on regression analyses, indicated a LOD score of 3.06 in interval analyses. Heat of gelatinisation (H) indicated a suggestive QTL (LRS = 14.5 with a threshold of 14.7 for P < 0.05, LOD = 2.65 for interval analysis), on chromosome 4A, at the Wx-B1 locus with an increased effect coming from the Halberd parent. The A:B granule ratio analysis indicated a significant QTL on chromosome 4B, but this was not observed in all environments and may be due to the fact that the QTL corresponded to the position of a major QTL controlling plant growth.

Development of near-isogenic lines of wheat carrying different null Wx alleles and their starch properties

The granule-bound starch synthase (GBSS I) encoded by the Wxgenes, is involved in amylose synthesis. For analyses of mechanisms of amylose synthesis and associated starch properties in hexaploid wheat, eight possible genotypes having different combinations of the three null alleles at the Wx loci with a common genetic background are a prerequisite. A near-isogenic population of doubled haploid (DH) lines was produced from Chinese Spring × waxy Chinese Spring F1 plants using the wheat × maize method. The Wx protein phenotypes of the DH progeny were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and found that the null alleles at each of the three Wx loci segregated in a Mendelian fashion. A field trial demonstrated no differences between the eight types for ear emergence time, plant height and grain yield traits. Amylose content in the endosperm starch was highest in the wild type while lowest in the waxy type having no Wx proteins. Comparison between single null types and double null types indicated that the amylose synthesis capacity of Wx-A1a allele is the lowest. Pasting properties of starch are the highest in the waxy type, followed by the double null types. Consequently, both peak viscosity and breakdown were negatively correlated with amylose content. The chain-length distribution analysis of amylopectin structure revealed no clear difference among the eight types,suggesting that the reduced GBSS I activity due to introgression of the null Wx alleles does not affect either the chain length or the degree of branching of amylopectin.

Isolation and characterization of a rice mutant insensitive to cool temperatures on amylose synthesis

The Wx b gene, one of the alleles at the rice waxy(wx) locus, is activated at cool temperatures during seed development, andas a result, a large amount of amylose is accumulated causing a reductionin rice grain quality. We found that the seeds of a du mutant couldbe visibly distinguished depending on whether they matured at cool ornormal temperatures. Using these characteristics, we isolated a mutantcandidate insensitive to cool temperatures. While the amylose content inthe original line was about 2% at a normal temperature (28 °C)and 12% at a cool temperature (21 °C), in the mutant candidate(coi) the amylose content was not affected by temperatures, i.e. theamylose content was about 3% at both temperatures. This finding incombination with the results of an immunoblot analysis indicated that theabsence of an increase in the amylose content in this mutant was caused bya constant level of Wx gene expression at normal and cooltemperature. Genetic analysis revealed that this insensitivity to cooltemperatures was caused by a single recessive mutation. This mutantshould be useful in breeding programs designed to produce rice of desiredquality at cool temperatures and in understanding genetic and molecularmechanisms that respond to slight changes in temperature.

Genetic mapping of commercially significant starch characteristics in wheat crosses

Starch properties were measured on the doubled haploid progeny of 2 crosses, one between Cranbrook and Halberd and the other between CD87 and Katepwa. Properties studied included starch peak and final viscosity measured by Rapid Visco Analyser, starch granule size distribution measured by laser light scattering, starch gelatinisation temperature by differential scanning calorimetry, and flour swelling volume. In the Cranbrook Halberd cross (samples from 2 environments), a highly significant quantitative trait locus (QTL) was located on chromosome 4A for both starch peak viscosity and starch/flour swelling volume, centred around the Wx-B1 locus. In previous studies, this locus has been found to be linked to Japanese noodle quality. The increases in starch peak viscosity and flour swelling volume are derived from the Halberd parent, consistent with the fact that Halberd is null for the Wx-B1 locus on chromosome 4A and is missing the respective granule-bound starch synthase protein, whereas Cranbrook is a wheat line carrying the normal 3 Wx loci. The final starch viscosity also showed an association with the Wx-B1 locus. In the CD87 Katepwa cross, the progeny showed an association between peak viscosity and a marker on chromosome 7A. This appeared to be near the Wx-A1 locus. In some experiments, flour viscosity showed a highly significant QTL on chromosome 7B, apparently at the same locus as the late maturity - amylase derived from the Cranbrook parent. Starch gelatinisation onset temperature indicated a significant QTL on chromosomes 2B and 7A (LOD = 2.58 and 3.66, respectively, in interval analyses). Starch gelatinisation peak temperatures indicated a QTL on chromosome 7A, which, although not in the significant (P = 0.05) class based on regression analyses, indicated a LOD score of 3.06 in interval analyses. Heat of gelatinisation (H) indicated a suggestive QTL (LRS = 14.5 with a threshold of 14.7 for P < 0.05, LOD = 2.65 for interval analysis), on chromosome 4A, at the Wx-B1 locus with an increased effect coming from the Halberd parent. The A:B granule ratio analysis indicated a significant QTL on chromosome 4B, but this was not observed in all environments and may be due to the fact that the QTL corresponded to the position of a major QTL controlling plant growth.

Analysis of intragenic recombination at wx in rice: correlation between the molecular and genetic maps within the locus.

In plant genomes as well as other eukaryotic genomes, meiotic recombination does not occur uniformly. At the level of the gene, high recombination frequencies are often observed within genetic loci in maize, but this feature of intragenic recombination is not seen at the csr1 locus in Arabidopsis. These observations suggest that meiotic recombination in plant genomes varies considerably among species. In the present study we investigated meiotic recombination at the wx locus in rice. The mutation sites of wx mutants induced by ethyl methanesulfonate (EMS) treatment or gamma-ray irradiation and a spontaneous wx mutant were physically characterized, and the genetic distances between those wx mutation sites were estimated by pollen analysis. Based on these results, the recombination frequency at the wx locus in rice was estimated as 27.3 kb/cM, which was about 10 times higher than the average for the genome, suggesting that there was a radically different rate of meiotic recombination for intra- and intergenic regions in the rice genome.

Differential effects of the null alleles at the three Wx loci on the starch-pasting properties of wheat

The amylose/amylopectin ratio and the pasting properties of wheat starch are important in producing marketable flour products, especially Japanese noodles. To determine if null mutations at the three Wx loci confer differences in starch-pasting viscosity, we analyzed the variation associated with the null mutations in three separate sets of recombinant substitution lines of chromosomes 7A, 4A and 7D produced from crosses between Chinese Spring and three single-chromosome substitution lines carrying the null Wx alleles. Differential effects of null alleles at the three Wx loci on starch-pasting properties were revealed. With respect to chromosome 4A, the effect of the Wx-B1b allele, giving a higher peak and breakdown viscosity, was unambiguous. In addition, a QTL of minor effect was identified near the centromere on the short arm. The presence or absence of the Wx-A1 protein gave some variation in peak and breakdown viscosity, but the effects of Wx-Alb were much smaller than those of the Wx-Blb allele. Associated effects of the Wx-D1 locus were detected for the breakdown viscosity as the null Wx-D1b allele produced a higher viscosity than the wild-type Wx-D1a. While negative correlations between amylose content and breakdown viscosity were common in the three populations, the null mutations at the Wx loci produced some variation independent of amylose content. The genetic variation detected for breakdown viscosity was more evident than that for peak viscosity in all three recombinant populations.

Gene-dosage effects of individual Wx loci on amylose content in wheat

Gene-dosage effects of individual Wx loci on amylose content in wheat

Biochemical Characterization of the Wheat Waxy A Protein and Its Effect on Starch Properties

ABSTRACTGranule bound starch synthase1 (GBSS1) is a key enzyme in amylose biosynthesis and is encoded by the A, B and D GBSS1 wx loci in wheat. Wheat lines with mutations at the three GBSS1 loci have been identified. We have characterized and compared the grain starch of CDCW6 wheat line (null B and D for GBSS1) with PI235238 (null A and B for GBSS1), waxy (null A, B and D for GBSS1), and AC Reed (wild type wheat) grain starches. The grain starch of waxy, CDCW6, PI235238, and AC Reed lines contained ≈0, 12, 23, and 25% amylose (w/w), respectively. Waxy, partially waxy, and wild wheat grain starches showed significant differences in onset and peak transition temperatures as determined by differential scanning calorimetric analysis. Grain starches extracted from waxy, CDCW6, and PI235238 also had higher enthalpy of gelatinization values than did wild wheat starch. X‐ray diffraction analysis revealed the highest crystallinity for starch extracted from waxy wheat, followed by CDCW6. The starch produced from the CDCW6 line may find special food and industrial applications because of its relatively low amylose concentration.

The three important traits for cooking and eating quality of rice grains are controlled by a single locus in an elite rice hybrid, Shanyou 63

The cooking and eating quality of the rice grain is one of the most serious problems in many rice-producing areas of the world. In this study, we conducted a molecular marker-based genetic analysis of three traits, amylose content (AC), gel consistency (GC) and gelatinization temperature (GT), that are the most important constituents of the cooking and eating quality of rice grains. The materials used in the analysis included F(2) seeds, an F(2:3) population, and an F(9) recombinant inbred-line population from a cross between the parents of 'Shanyou 63', the most widely grown hybrid in rice production in China. Segregation analyses of these three generations showed that each of the three traits was controlled by a single Mendelian locus. Molecular marker-based QTL (quantitative trait locus) analyses, both by one-way analysis of variance using single marker genotypes and by whole-genome scanning with MAPMAKER/QTL, revealed a single locus that controls the expression of all three traits. This locus coincided with the Wx region on the short arm of chromosome 6, indicating that all three traits were either controlled by the Wx locus or by a genomic region tightly linked to this locus. This finding has provided clues to resolving the molecular bases of GC and GT in future studies. The results also have direct implications for the quality improvement of rice varieties.

Amylose synthesis capacity of the three Wx genes of wheat cv. Chinese Spring

The Wx locus controls amylose synthesis in the cereal endosperm. Hexaploid wheat (Triticum aestivum L.) has the three Wx loci on chromosomes 7A ( Wx-A1), 4A (Wx-B1) and 7D (Wx-D1). To verify the effects of null alleles on reducing amylose content and determine the amylose synthesis capacity of each Wx gene independently and accurately, we produced eight possible types of recombinant lines carrying different null alleles at the Wx loci under the ‘Chinese Spring’ genetic background. Amylose content varied from 0% of the waxy ‘Chinese Spring’ to 25% of the ‘Chinese Spring’ normal type. The reducing effect of the single null alleles was the largest in Wx-B1b, and there was no significant difference between Wx-A1b and Wx-D1b. More than 3% reductions in amylose content were detected in the double null types. The results of the double null lines further demonstrated that for the capacity of amylose synthesis, Wx-B1a predominates and produces 21–22% amylose, followed by Wx-D1a (20–21%) and Wx-A1a (15–18%). These significant differences were partly correlated with variation in the amounts of the Wx proteins produced by different Wx genes. However, comparisons of the double null lines with the single null or normal lines indicated that amylose content was not linearly proportional to the number of the Wx genes, suggesting that the Wx genes act in an epistatic manner.

Identification and Characterization of U.S. Wheats Carrying Null Alleles at the wx Loci

ABSTRACTGranule‐bound starch synthase (GBSS) is the primary enzyme responsible for the synthesis of amylose in amyloplasts of cereal endosperm cells. Bread wheats, due to their hexaploid genetic system, carry three genes (wx loci) encoding GBSS. Purification and separation of GBSS from more than 200 North American hexaploid wheats allowed the identification of genotypes that carry null alleles at either the wx‐A1 and wx‐B1 loci. In addition, the cultivar Ike carried both wx‐A1 and wx‐B1 null alleles. No wx‐D1 nulls were detected. Null alleles were found in 10% of the hard winter wheats tested, but in only 2% of the sampled soft winter wheats. Amylose contents of wheats carrying single null alleles at either the wx‐A1 or wx‐B1 loci often were lower than those of wild type wheats, but greater reduction in amylose content was observed in Ike. Monoclonal antibodies were used to quantify water‐extractable GBSS in both wild‐type and null genotypes. Gene dosage compensation was evident, although GBSS content, as measured by ELISA, was significantly lower in Ike than in all other wheats. The identification of null alleles in adapted genotypes suggests the development of wheats with a wide range of amylose contents will be possible by simple genetic crossing and selection.

Intraspecific differentiation and geographical distribution of Wx alleles for low amylose content in endosperm of foxtail millet, Setaria italica (L.) Beauv.

To clarify the genetic mechanism which controls the variation in amylose content among nonwaxy landraces of foxtail millet, the inheritance of different starch types in endosperm was examined by I2-KI staining. The level of starch granule bound protein in foxtail millet endosperm was also analyzed using SDS-PAGE. The segregation for starch types in F2 and F3 seeds determined by I2-KI staining showed that there are three different alleles at the waxy (wx) locus. A major protein bound to starch granules was detected in nonwaxy endosperm but it was absent in most of the waxy endosperm, suggesting that the protein is the Wx gene product which is responsible for the synthesis of amylose in endosperm. The level of Wx protein proportionally corresponded to the amylose content among nonwaxy landraces. This implies that two different Wx alleles regulate quantitative levels of Wx protein. Landraces with the allele for low amylose content are from Taiwan, the Philippines, Indonesia and Thailand.

Analysis of recombination sites within the maize waxy locus.

Genetic fine structure analysis of the maize wx locus has determined that the ratio of genetic to physical distance within wx was one to two orders of magnitude higher than the average for the maize genome. Similar results have been found at other maize loci. In this study, we examined several mechanisms that could account for this pattern. First, crossovers in two other maize genes resolve preferentially at specific sites. By mapping exchanges between wx-B1 and wx-I relative to a polymorphic SstI site, we found no evidence for such a hotspot at wx. Second, deletion of promoter sequences from wx alleles had little effect on recombination frequencies, in contrast to results in yeast where promoter sequences are important for initiating recombination in some genes. Third, high levels of insertion polymorphism may suppress intergenic recombination. However, the presence of a 2-kb Ds element 470 bp upstream of the wx transcription start site did not further suppress recombination between Ds insertions in nearby wx sequences. Thus, none of these mechanisms is sufficient to explain the difference between intergenic and intragenic recombination rates at wx.

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 α3 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 α3 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 α3 method and by the RFLP waxy gene probe.

Gamete Eliminator Adjacent to the wx Locus as Recealed by Pollen Analysis in Rice

Gamete Eliminator Adjacent to the wx Locus as Recealed by Pollen Analysis in Rice

Retrotransposition of a plant SINE into the wx locus during evolution of rice

A new type of plant retroposon, p-SINE1, has been found in the wx locus of rice (Oryza sativa). It has some structural characteristics similar to those of mammalian SINEs, such as members of the Alu or B1 family. In order to estimate the time at which the integration of p-SINE1 into a single locus occurred during rice evolution, we examined the distribution of two members of p-SINE1 in several species of the Oryza genus by the polymerase chain reaction (PCR). We found that one member of p-SINE1 (p-SINE1-r2) in the ninth intron of the wx+ gene was present only in two closely related species, O. sativa and O. rufipogon, and was not present in the other species carrying the AA genome within the Oryza genus. This result indicates that p-SINE1-r2 was integrated into the wx locus after O. sativa and O. rufipogon had diverged from other species with the AA genome. In contrast to p-SINE1-r2, another member (p-SINE1-r1) located in the untranslated 5'-region of the wx+ gene was present not only in all species with the AA genome but also in species with a different genome (CCDD). This result suggests that p-SINE1-r1 was integrated into that position prior to the genomic divergence. Thus, it appears that each member of p-SINE1 was retroposed at a specific site at a different time during rice evolution.

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 three Wx loci, Wx-A1, Wx-B1, and Wx-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.

イネにおけるアミロースの合成とその遺伝的制御

The wx locus controls amylose synthesis in the endosperm of rice . Since the amylose contents in the endosperm greatly affect the quality of rice, it is important for agricultural application to regulate the extent of amylose synthesis . In addition, the wx locus of rice is quite suitable for the molecular studies of gene regulation because the locus has some particular advantages for genetic research and it provides us various regulation mechanisms to be eluci dated at the molecular level. In this article, we review the genetic and molecular biological research, which have been carried out in our laboratory, about the regulation of gene expression at the wx locus and amylose synthesis in rice.

イネの<I>wx</I>遺伝子座の機能と米の品質

イネの<I>wx</I>遺伝子座の機能と米の品質

Molecular Characterization of the waxy Locus of Rice (Oryza sativa)

The gene at the waxy (wx) locus in rice was cloned and the nucleotide sequence of the coding region was determined. The wx+ gene from rice was composed of a number of exons and in-trons. The deduced amino acid sequence indicated that the predicted polypeptide is composed of a putative transit peptide of 77 amino acids and a mature protein region of 532 amino acids. The putative transit peptide, which may be essential for transporting the product of the gene at the wx locus (wx+ protein) into the amyloplast, has some characteristic features similar to those of the transit peptide associated with proteins to be transported into the chloroplast. An antiserum against the rice wx+ protein (anti-WXP serum) was prepared and the expression of the wx locus was examined by the Northern blotting analysis using DNA cloned from the rice wx locus and by the immunoblotting analysis using the anti-WXP serum. The expression of the wx+ gene was regulated in a tissue-specific manner, namely, it was expressed only in the endosperm and pollen. The wx+ gene was active at the early and middle stages and was almost inactive at the late stage of seed development. During active transcription of the gene, the wx+ protein appears to accumulate linearly.