Wx Gene Research Articles

Evaluation of genetic effect nphysiochemical properties changes of Wx near isogenic lines of Y58S in rice

The Wx gene primarily influences the physiochemical properties changes of rice (Oryza sativa L.) To improve the grain qualities of the cv. Y58S with low amylose content (AC) value, the genetic effect of Wx gene was explored on quality traits. With the BC3F2 genetic population of Y58S line associated with different AC value as the materials, this study reported the genetic effect of three different Wx alleles (Wxa, Wxin, Wxb) under near-isogenic background. It was shown that Wx had major effects on the quality traits, and the genetic effect value was in the order Wxa > Wxin > Wxb. In addition, during the course of physiochemical properties changes of higher AC rice cultivars determined by Wxa or Wxin allele, accompanied by the increasing of AC, gel consistency and head milled rice recovery would be decreased in combination with the rising of chalky traits, all of which resulted in substantial fluctuations on quality traits (p < 0.01). However, the genetic law was not evident for Wxb allele. Therefore, except for the major gene Wx, the minor genes in the regulative network of starch-synthesis might be utilized for quality improvement.

Functional Characteristics of Bread Wheat (Triticum aestivum L.) Near‐Isogenic Lines Differing at the Waxy (Wx) Locus

The Waxy (Wx) gene in hexaploid wheat (Triticum aestivum L.) encodes granule‐bound starch synthase (GBSS1), which is involved in the synthesis of amylose, a mostly linear glucan polymer that makes up ∼25% of wheat starch. A null mutation of the Wx gene in each of the three genomes is associated with starch almost entirely consisting of the branched glucan polymer amylopectin (waxy starch), with corresponding changes in functionality. However, the rheological behavior of partially waxy starch remains unclear. The objective of this study was to characterize flour and baking quality in 16 near‐isogenic lines, null at the Wx locus on zero, one, two, or all three genomes, grown in four different environments. Across allelic groups, significant variations in amylose concentrations, flour paste viscosity, loaf structure and texture, dough stability, and proximate variables were observed. Because waxy wheat starch has greater water absorbance and resistance to retrogradation than normal starch, its inclusion in flour blends has been suggested as a means of improving the texture and appearance of bakery products and noodles. The results indicate that wheat encoding &lt;3 functional homeologs of GBSS1 produces starch that has potential in the production of certain food items, such as Asian noodles. However, further research is necessary to determine the optimal amylose‐to‐amylopectin ratio to improve baking quality.

QTL analysis for cooking traits of super rice with a high‐density SNP genetic map and fine mapping of a novel boiled grain length locus

AbstractHybrid rice has contributed substantially to the improvement of grain production worldwide, yet its poor cooking and tasting characteristics have long been recognized. In this study, 132 recombinant inbred lines derived from LYPJ were used to identify quantitative trait loci (QTLs) for 12 cooking traits with the high‐density SNP linkage map recently developed by our team. We identified 17 QTLs on chromosomes 1, 2, 4, 5, 6, 7, 8, 9 and 11, which accounted for 7.50% to 23.50% of the phenotypic variations. A novel major QTL qBGL7 for boiled grain length was further fine‐mapped to an interval of 440 Kb between the two markers RM21906 and gl3 using a BC3F2 population. Two near‐isogenic lines with extreme boiled grain length, GX5‐176 and GX5‐101, could be directly used in improving cooking quality. We also identified a QTL for soaked grain width expansion rate, qSGWE6, in the Wx gene region on chromosome 6. The Wx differential regulation coincided with sequential variation between the two parents. Our work offered a theoretical basis for molecular breeding of high‐quality hybrid rice.

Molecular characterization of several Wx alleles in durum wheat

The Wx gene, which encodes waxy proteins, is the sole gene responsible for amylose synthesis in the wheat seed endosperm. In this study, we characterized, at the molecular level, several Wx alleles in durum wheat (Triticum turgidum L. ssp. durum Desf.) that had previously been catalogued at the protein level. Our data show a misclassification of the alleles in both Wx genes: Wx-A1 alleles from Blanqueta (Wx-A1a) and Astrodur (Wx-A1b) have been reclassified as Wx-A1a and Wx-A1h, respectively. A sequence comparison of the Wx-B1e allele shows that there were up to five different alleles under this denomination, which confirms that the protein analysis by SDS-PAGE separation needs to be combined with the PCR amplification-sequencing analysis in order to obtain a correct classification of the alleles, which will facilitate their use in wheat quality improvement.

Molecular diversity and detection of Waxy genes in the Iranian wheat populations by multiplex PCR

Early and quick identification of wheat Waxy genes is important in different industries and in determining the quality of bread. A stable multiplex polymerase chain reaction (PCR) system was established to identify the three Waxy (Wx) genes in wheat. Three Waxy genes – A, B and D were amplified simultaneously by using two primer pairs that produce target sequences of 230/265 bp, 854 and 204 bp, respectively. By application of these two primer pairs Wx-A1, Wx-B1 and Wx-D1 genes can be detected in a single PCR, which can be helpful for breeders as a marker assisted selection in wheat quality breeding programmes. Also, by using two primer pairs, a maximum and minimum number of polymorphic bands were observed for Aegilops speltoites (B group) (100%) and Triticum durum (AB), and Triticum aestivum (ABD groups) (0.00%), respectively. The B group recorded the highest unbiased expected heterozygosity (0.282) and Shannon's index values (0.430), whereas the lowest values of unbiased expected heterozygosity (0.00) and Shannon's index (0.00) were recorded for the AB and ABD groups.

Effect of high temperature on the expressions of genes encoding starch synthesis enzymes in developing rice endosperms

High temperature is the major environmental factor affecting grain starch properties of cooking rice cultivars. In this study, two non-waxy indica rice genotypes, cv. 9311 and its mutant with extremely high amylose phenotype (9311eha) were used to study the differential expressions of genes in starch synthesis and their responses to high temperature (HT). Significant increase in apparent amylose content and hot-water-soluble starch content in mutant 9311eha were genetically caused by a substitution from AGTTATA to AGGTATA at the leader intron 5′ splice site in Wx gene. This mutation resulted in different mRNA transcript levels, mRNA splicing efficiencies and protein levels of Wx between the two rice genotypes, which also lead to the genotype-dependent alteration in the temporal pattern of Wx transcription and granule-bound starch synthase (GBSS) activity in response to HT. However, changes in the activities of other starch synthesizing enzymes and their expressions of distinct isoform genes were not significant with the Wx gene mutation, thus only minor difference in the particle size of starch granule, chain-length distribution and gelatinization enthalpy were found between the two genotypes. The temporal-specific expression of multiple isoform genes responsive to different temperature regiments indicated that the reduction of GBSS transcript expression under HT was generally accompanied by the decreased expressions of SSSIIa, SSSIIIa and SBEIIb. Consequently, high temperature-ripened grains in 9311eha showed high proportion of intermediate and long B chains and somewhat lower level of short A chain compared to the wildtype. The temperature-dependent alteration of amylose content was not only attributed to the reduced expression of GBSS, but also associated with the complimentary effect of SSSIIa and SBEIIb.

Characterization of Myanmar Paw San Hmwe Accessions Using Functional Genetic Markers

Paw San Hmwe (PSM) rice has been cultivated in many areas of Myanmar for a long time. Strong aroma, good taste and its elongation during cooking are the key characteristics of PSM rice. Thirty-one PSM accessions were genotypically characterized, and their physical grain and cooking quality traits were studied. We used specific gene markers associated with aroma, apparent amylose content (AAC) and alkali spreading value to determine the alleles carried by different PSM accessions. The results revealed that six PSM accessions (PSM10, PSM12, PSM13, PSM21, PSM22 and PSM30) had a 3-bp insertion in Os2AP gene. Gel consistency (GC) allele was predominant among the PSM accessions for gelatinization temperature (GT), however, the phenotype observed was between low and intermediate GT because of the combination of the GC allele with the presence of low GT allele at heterozygous state from the other loci of the SSIIa gene. Intermediate to high AAC was observed among the PSM accessions corresponding to the haplotype identified for the single nucleotide polymorphism G/T and the (CT)n repeat in the Wx gene. The characterization and grouping data of PSM accessions posted benefits to Myanmar seed banks, and our results will help in maintaining the integrity of PSM rice variety.

Comparison of amylopectin structure and activities of key starch synthesis enzymes in the grains of rice single-segment substitution lines with different Wx alleles

The Wx gene is the major gene controlling amylose synthesis in rice endosperm. So far, the Wx gene can be mainly classified into five common alleles, wx, Wx t, Wx g1, Wx g2, and Wx g3, according to their amylose phenotypes. Besides, the Wx allelic variation was also found to play an important role in regulating other physicochemical properties of rice starch. However, the specific physiological and starch structural mechanisms are not clear yet. With a set of single-segment substitution lines harboring five different Wx alleles, the genotype differences in amylopectin structure and dynamic changes of activities of key starch synthesis enzymes were investigated. The distinct Wx genotype difference in chain length distribution of amylopectin was confirmed by size exclusion chromatography. There were clear allelic differences in activities of starch synthesis enzymes during grain filling. The Wx allelic variation had the highest impact on the activities of starch branching enzyme, followed by pullulanase, and soluble starch synthase, while no influence on isoamylase activity. Differences in structural features of amylopectin and mean enzyme activities during grain filling period correlated well with most of the variations in starch property traits. These findings indicate that the Wx allelic variation may affect starch structure and physicochemical properties through selectively influencing activities of starch synthesis enzymes in developing rice grains. The present results will contribute to our understanding of the regulation of starch biosynthesis in rice endosperm.

Physicochemical and Thermal Characteristics of Starch Isolated from a Waxy Wheat Genotype Exhibiting Partial Expression of Wx Proteins

ABSTRACTA unique wheat genotype carrying waxy‐type allelic composition at the Wx loci, Gunji‐1, was developed, and its starch properties were evaluated in comparison to parental waxy and wild‐type wheat varieties. Gunji‐1 was null in all three of the Wx genes but exhibited a lower level of Wx proteins than the wild‐type. Starch amylose content and cold water retention capacity were 10.1 and 70.5% for Gunji‐1, 4.2 and 76.6% for waxy, and 27.9 and 65.0% for wild‐type, respectively. No significant differences were observed in microstructure, granule size distribution, and X‐ray diffractograms of the starch granules isolated from Gunji‐1 compared with those of waxy and wild‐type wheat varieties. Starch pasting peak, breakdown, and setback viscosities and peak temperature of Gunji‐1 were intermediate between waxy and wild‐type wheat. In starch gel hardness, Gunji‐1 (1.1 N) was more similar to waxy wheat (0.5 N) than to the wild‐type variety (17.6 N). Swelling power, swelling volume, paste transmittance during storage, and gelatinization enthalpy of Gunji‐1 were lower than those of waxy wheat but greater than those of wild‐type wheat. Retrogradation of starch stored for one week at 4°C expressed with DSC endothermic enthalpy was absent in the waxy wheat variety, whereas Gunji‐1 exhibited both retrogradation of amylopectin and amylose‐lipid complex melting similar to the wild‐type parent, even though enthalpies of Gunji‐1 were much smaller than the wild‐type parent.

Modulation of amylose content by structure-based modification of OsGBSS1 activity in rice (Oryza sativa L.).

The rice Waxy (Wx) gene encodes granule-bound starch synthase 1 (EC 2.4.1.242), OsGBSS1, which is responsible for amylose synthesis in rice seed endosperm. In this study, we determined the functional contribution of eight amino acids on the activity of OsGBSS1 by introducing site-directed mutated Wx gene constructs into the wx mutant glutinous rice. The eight amino acid residues are suspected to play roles in OsGBSS1 structure maintenance or function based on homologous enzyme sequence alignment and homology modelling. Both OsGBSS1 activity and amylose content were analysed in homozygous transgenic lines carrying the mutated OsGBSS1 (Wx) genes. Our results indicate that mutations at diverse sites in OsGBSS1 reduces its activity by affecting its starch-binding capacity, its ADP-glucose-binding capability or its protein stability. Our results shed new light on the structural basis of OsGBSS1 activity and the mechanisms of OsGBSS1 activity on amylose synthesis in vivo. This study also demonstrates that it is feasible to finely modulate amylose content in rice grains by modifying the OsGBSS1 activity.

Wx gene in diploid wheat: molecular characterization of five novel alleles from einkorn (Triticum monococcum L. ssp. monococcum) and T. urartu

The Wx gene encodes the granule-bound starch synthase I or waxy protein, which is the sole enzyme responsible for amylose synthesis in wheat seeds. Triticum urartu and einkorn (T. monococcum L. ssp. monococcum), which are related to the A genome of bread wheat, could be important sources of variation for this gene. This study evaluated the Wx gene variability in 52 accessions of these species and compared their nucleotide sequences with the Wx-A1a allele of bread wheat. The level of polymorphism found was high, although not distributed equally between the two species. Five different alleles were found in T. urartu, of which four were novel (Wx-A u 1b, -A u 1c, -A u 1d and -A u 1e). All einkorn accessions had the same allele, which was also novel and was named Wx-A m 1a. A comparison between the proteins deduced from the novel alleles and the Wx-A1a protein showed that there were up to 33 amino acid changes in both the transit peptide and the mature protein. These results showed that these species, especially T. urartu, are a potential source of novel waxy variants.

Bioinformatic analysis of maize granule-bound starch synthase gene

Local alignment of Wx gene and its homolog sequences has been conducted. Phylogenetic dendrogram displaying evolutionary relationship between Poaceae members has been built basing on results of the alignment. Ancient Wx gene transfer from Zea mays to Dimeria lawsonii has been assumed. Primers for the exons 8-10 polymorphic region have been designed. In silico PCR has been conducted.

Major QTLs reduce the deleterious effects of high temperature on rice amylose content by increasing splicing efficiency of Wx pre-mRNA

We discovered four QTLs that maintain proper rice amylose content at high temperature by increasing the splicing efficiency of Wx gene. Amylose content mainly controlled by Wx gene is a key physicochemical property for eating and cooking quality in rice. During the grain filling stage, high temperature can harm rice grain quality by significantly reducing the amylose content in many rice varieties. Here, we provide genetic evidences between Wx gene expression and rice amylose content at high temperature, and identified several quantitative trait loci (QTLs) in this pathway. We performed a genome-wide survey on a set of chromosome segment substitution lines (CSSLs) which carried chromosomal segments from the heat resistant indica 9311 in the heat-sensitive japonica Nipponbare background. Four QTLs, qHAC4, qHAC8a, qHAC8b and qHAC10, which can reduce the deleterious effects of amylose content at high temperature, were identified and mapped to chromosome 4, 8, 8 and 10, respectively. The major QTL qHAC8a, with the highest LOD score of 6.196, was physically mapped to a small chromosome segment (~300 kb). The CSSLs carrying the qHAC8a, qHAC8b and/or qHAC4 from 9311 have the high pre-mRNA splicing efficiency of Wx gene and likely lead to stable amylose content at high temperature. Thus, increasing pre-mRNA processing efficiency of Wx gene could be an important regulation mechanism for maintaining stable amylose content in rice seeds at high temperature. In addition, our results provide a theoretical basis for breeding heat-stable grain in rice.

UTILIZATION OF DNA MARKERS FOR EVALUATING AMYLOSE CONTENT OF GRAIN IN RICE

The sensory and functional properties of rice are predominantly associated with its amylose content. Granule-bound starch synthase (GBSS) encoded by the Waxy (Wx) gene determines the synthesis of amylose, while starch branching enzymes encoded by Sbe genes are involved in the formation of amylopectin. Some studies have demonstrated that Wx gene is the major controller of amylose content but there are one or more modifying genes affecting the amylose content. Three markers, microsatellite, Single – nucleotide – polymorphism (G/T SNP) in Wx gene and Single – nucleotide – polymorphism (T/C SNP) in Sbe1 gene, were tested for their association with amylose content using sixty-nine rice accessions from twenty countries. Of the three markers, two markers in Wx gene are significantly associated with amylose content. The combination of two markers in Wx gene (haplotypes) explained 83.8% of the variation in amylose content and discriminated the three market classes of glutinous, low, intermediate and high amylose content of rice from each other. And T/C SNP in Sbe1 locus was not a suitable marker for amylose content. Keywords: marker, amylose content, Waxy gene.

Characterization of the Wx gene in diploid Aegilops species and its potential use in wheat breeding

Wx gene encodes for the granule-bound starch synthase I or waxy protein, which is the sole enzyme responsible for amylose synthesis in wheat seeds. The Aegilops species, which are related to wheat, could be important sources of variation in this gene. In addition to its role in starch quality, this gene has been used in phylogenetic studies of wheat. The current study evaluated the variability of Wx gene in seven diploid species of Aegilops genus and compared their nucleotide sequences with the wheat homeologous genes. Nineteen new Wx alleles were found in the seven species evaluated. The alleles detected in two species of the Sitopsis section, Ae. searsii and Ae. speltoides, were related to the Wx-B1 gene of wheat. Two more of the Sitopsis species did not appear to be associated with this genome, whereas the remaining species were related to the Wx-D1 gene of wheat. The results showed an important variation of the Wx gene present in the Aegilops genus, and the 19 new Wx alleles detected could enlarge the genetic pool of wheat.

Characterization of an Italian rice germplasm collection with genetic markers useful for breeding to improve eating and cooking quality

Gelatinization temperature and apparent amylose content are key parameters used to describe the eating and cooking qualities of rice. Sequence variants of SSIIa and Waxy genes are important determinants of gelatinization temperature and apparent amylose content, respectively. A collection of Italian non-glutinous japonica rice accessions was characterized for sequence polymorphisms in SSIIa and Waxy genes, in comparison with non-Italian japonica and indica genotypes. For SSIIa two markers, SNP3 and SNP4, were used. A PCR amplification of multiple specific alleles protocol was developed for the identification of G/T polymorphism in 5′ splice site of first intron and A/C polymorphism in exon 6 of the Waxy gene. Based on simple allele-specific PCR, it can be proposed as a user-friendly, cost-effective tool for marker-assisted selection of amylose content. The collection was characterized also for the (CT)n repeats in exon 1 of the Waxy gene. The results showed that while SSIIa haplotypes were rather similar between Italian and non-Italian japonica rice, the Waxy gene haplotype T/A/(CT)18 was largely predominant in Italian accessions, other haplotypes, well represented in non-Italian japonica [T/A/(CT)19] and indica [e.g. G/C/(CT)20] genotypes, were present at lower frequency. Grain starch quality traits as apparent amylose content and RVA profile were also analysed. The In1/Ex6 SNP haplotypes of Wx gene were found to explain 79 % of variation in apparent amylose content, and 36, 22 and 25 %, of variation in the RVA parameters peak viscosity, breakdown and setback, respectively. The additional use of (CT)n repeats marker further improved the association of haplotypes with RVA parameters.

Effect of wx genes on amylose content, physicochemical properties of wheat starch, and the suitability of waxy genotype for producing Chinese crisp sticks

The effect of wx genes on amylose content, physicochemical properties of wheat starches, and the quality of Chinese crisp stick were investigated using near-isogenic lines (NILs) with null wx alleles in Yangmai 17 and Yangmai 01-2 backgrounds. wx genes showed significant effects on amylose content and other traits. The triple-null genotype had the lowest amylose content among eight genotypes, followed by double-null, single-null, and wild-type genotypes. The triple-null also showed lower flour yield, higher percentage of type B-granules on a volume basis and higher crystallinity than non-waxy genotypes, and showed significant differences in all pasting and thermal transition parameters compared to non-waxy genotypes, except for degree of retrogradation at day 14. For the quality of Chinese crisp stick, the hardness, crispness, fracturability, and specific volume of waxy genotype were 3.91 kg, 11.0, 1.85 mm and 104.4 ml, whereas the corresponding ranges for non-waxy genotypes were 5.39–5.70 kg, 0.5–0.9, 0.69–0.86 mm and 49.5–57.6 ml, respectively, in Yangmai 17 background. This indicates that waxy genotypes showed significantly better crisp stick quality than non-waxy genotypes. A similar trend was also observed in Yangmai 01-2 background. This indicated the potential utilization of waxy wheat for producing traditional products.

Relationship between Polymorphism of &lt;I&gt;Wx&lt;/I&gt; Gene and Amylose Content in Hulless Barley

Hulless barley ( Hordeum vulgare L. var. nudum Hook. f.) is a typical crop in Tibetan Plateau in China. Starch, including amylopectin and amylose, is the main storage component in hulless barley, of which amylose is in low content or even absent in the endosperm of waxy barley. The Waxy gene ( Wx ) located on the short arm of chromosome 7H is a key gene responsible for amylose synthesis, and also acts as a favorable gene in breeding of hulless barley. However, it is difficult to select hulless barley germplasm with Wx due to lack of effective molecular markers. This study aimed to find out molecular markers applicable in the identification of Wx gene. A total of 150 accessions of hulless barley were primarily tested by staining endosperm with I 2 -KI, and four accessions (IG107028, Puebla, Huzhushuangcaoren, and APM-HC1905) were found as waxy genotypes. The amylose contents (AC) of the 150 accessions measured using dual-wave length spectrophotometry method varied from 12.4% to 38.5% with an average of 26.0%. The amylose contents of the four waxy genotypes namely IG107028, Brachvtic, Kunlun 2, and Huzhushuangcaoren，were 12.4–18.6% with an average of 16.7%. No genotype was found without amylose. We selected 51 accessions with obvious difference in AC to validate the polymorphism of four pairs of SSR markers. Primers P1 and P3 showed no polymorphism among the 51 accessions, and primer P4 exhibited four types of amplified product, and the fragment size positively correlated with amylose content. The P4 primer was designed according to the sequence of Wx gene on chromosome 7H of barley. Primer P4 can be used as an effective marker in the selection of waxy hulless barley cultivars.

Marker-assisted selection for pyramiding the waxy and opaque-16 genes in maize using cross and backcross schemes

The waxy (wx) gene in maize is associated with higher content of amylopectin in the endosperm and better flavor. The opaque-16 (o16) gene is associated with higher lysine content in the endosperm and better nutritional value. To pyramid the wx and o16 genes, cross and backcross populations were constructed using the o16 line QCL3024 and the two waxy lines, QCL5019 and QCL5008, as parents. The linkage marker umc1141 for the o16 gene and the internal marker phi027 for the wx gene were used to select the target genes. Simple sequence repeat markers covering the whole genome were used for background selection in individual progenies of the backcross population. The grain lysine content was determined using the Acid Orange-12 Dye Binding Lysine method. Qualitative and quantitative analyses of the grain content of amylopectin were performed using the I2-KI procedure and double-wavelength spectrophotometry, respectively. Four lines of the double recessive genotype wxwxo16o16 were obtained from the F4 generation of the cross popula- tion and three lines of the same genotype were obtained from the BC2F4 generation of the backcross population. The lysine content of the pyramid lines was 16-27 and 18-28 % higher than the waxy parents QCL5019 and QCL5008, respectively. The pyramid lines had 61-63 % more amylopectin than the high- lysine parent QCL3024. The three pyramid lines from the backcross population had similar genetic back- ground to the waxy parent QCL5008. Our results are of significance for the improvement of maize quality.

Genetic Diversity of Waxy Gene in Chinese Glutinous Maize

Waxy (wx) gene is a key gene for glutinous trait in many waxy crops including waxy maize. wx diversity investigation in maize population has important significance for quality breeding and germplasm utility of Chinese waxy maize. In this study, 325 waxy maize germplasm accessions (309 from China, 11 from Thailand and five from South Korea) were used to investigate their wx genotypes and determine their apparent amylose content (AAC). the results demonstrated that genetic diversity of wx gene in Chinese waxy maize was low, and two mutation types, wx-D7 and wx-D10, accounted for 96.9% of the 325 accessions. Among these accessions, those from North of China and South Korea were the type of wx-D7 while others from South of China and Thailand were the type of wx-D10; most of landraces were the type of wx-D10 (78.9%) while most commercial hybrids and inbred lines were the type of wx-D7 (88.7% and 86.6%, respectively). AAC estimations indicated that mean values of AAC in commercial hybrids and inbred lines in China were lower than 2.2% while that in landraces was relatively high. Comparing with wx-D7, wx-D10 waxy maize had higher AAC with larger changing range. Geographical distribution and origins of two main wx genotypes and other wx genotypes were discussed also.