Waxy Alleles Research Articles

DNA Markers for Identifying waxy Mutations and Improving Noodle Quality in Wheat

Approximately 70% of wheat endosperm consists of starch, and variations in the quality and quantity of starch affect the processing characteristics of wheat flour. Amylose content in particular has a major effect on Asian noodle quality, and selection of wheat lines with slightly lower amylose levels is an important goal in Japanese wheat breeding programs. Accurately measuring amylose content by direct methods such as colorimetric assays was found to be problematic, suggesting there was a need for a more efficient and accurate method of screening for reduced amylose content. Therefore, we characterized mutations in the wheat waxy genes, which control amylose synthesis, and developed DNA markers for the identification of null waxy alleles. In this review, we describe the development of these markers and outline their utility for wheat breeding programs.

Waxy strains of three amaranth grains raised by different mutations in the coding region

Waxy strains raised by waxy mutation have been found for three amaranth grains. Three genes encoding waxy protein were isolated from Amaranthus caudatus (Wx-ca), A. cruentus (Wx-cr), and A. hypochondriacus (Wx-hy). Sequence analysis indicated that the Wx-ca, Wx-cr, and Wx-hy genes contained the same exon (13 exons) and intron (12 introns) structure. The lengths of the Wx-ca, Wx-cr, and Wx-hy genes were 3,236, 3,237, and 3,225 bp, respectively. The alignment of the coding sequence of the three Waxy genes showed 12 polymorphic sites, including 11 single nucleotide polymorphisms (SNPs) (in exons 10 and 12 and introns 1, 3, 4, 9, and 11) and 5 deletions or insertions (indels) (in introns 4, 9, and 11). In particular, major polymorphism was detected in 8- and 3-bp indels in intron 4. Moreover, the mutation in the waxy alleles (wx-ca, wx-cr, and wx-hy) of all three species was also isolated and characterized. Comparison of coding sequences of the three Waxy genes and their waxy alleles indicated one base insertion (wx-ca: insert of T base in exon 8) and a base substitution (wx-cr: a G-to-T base substitution in exon 10; wx-hy: a G-to-A base substitution in exon 6), which occurred as internal termination codon in the three Waxy genes, suggesting the involvement of a nonsense or frameshift mutation. Therefore, these different mutations in coding regions were considered to be the cause of the waxy (amylose-free) phenotype.

Identification and characterization of a novel Waxy allele from a Yunnan rice landrace

Low amylose content (AC) is a desirable trait for rice (Oryza sativa L.) cooking quality and is selected in soft rice breeding. To gain a better understanding of the molecular mechanism controlling AC formation, we screened 83 Yunnan rice landraces in China and identified a rice variety, Haopi, with low AC. Genetic analyses and transgenic experiments revealed that low AC in Haopi was controlled by a novel allele of the Wx locus, Wx(hp), encoding a granule-bound starch synthase (GBSSI). Sequence comparisons of Wx(hp) and Wx(b) alleles (from Nipponbare) showed several nucleotide changes in the upstream regulatory regions (including the promoter, 5'-untranslated region, and first intron 5' splicing junction site). Interestingly, these changes had no obvious effect on the expression level and splicing efficiency of Wx transcripts. In addition, an examination of the coding region revealed that the Wx(hp) allele carries an A-to-G change at nucleotide position +497 from the start codon, resulting in an Asp(165)/Gly(165) substitution. The amino acid substitution had no detectable effects on GBSSI activity in vitro; however, it notably reduced the binding of GBSSI to starch granules, resulting in a reduction of AC in rice seeds. Moreover, three other Yunnan landraces with low AC also carry a nucleotide substitution identical to Haopi at the +497 position of the Wx gene, suggesting common ancestry. Based on the single-nucleotide polymorphism, we have developed a new derived cleaved amplified polymorphic sequence marker for use in breeding practice to manipulate AC in rice endosperm.

Two distinct waxy alleles impact the granule-bound starch synthase in sorghum

The granule-bound starch synthase (GBSS) is the enzyme responsible for amylose synthesis in starch granules. Loss of GBSS activity results in starch granules containing mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles were identified in sorghum (Sorghum bicolor L. Moench) characterized by the absence (waxy a ; wx a ) or presence (waxy b ; wx b ) of the GBSS protein in the endosperm. To characterize these alleles, we examined endosperm architecture using scanning electron microscopy (SEM), assayed GBSS enzymatic activities, and identified DNA lesions associated with the mutations in the GBSS (Sb10g002140) gene. wx a , the allele present in B Tx630 and R Tx2907, contained a large insertion in the third exon, which was consistent with the absence of the GBSS protein previously observed. wx b , the allele present in B 9307 and B TxARG1, contained a missense mutation that resulted in conversion of glutamine 268 to histidine in a conserved domain in starch synthases. In wx b , GBSS activity was less than 25% that of the non-waxy line B Wheatland, and GBSS activity was not detected in wx a . SEM showed that endosperm architecture was very similar in both wx a and wx b alleles, but altered in comparison to non-waxy lines R Tx430 and B Wheatland. Both alleles may have a range of potential applications in grain sorghum because of low amylose content in their starch and the presence or absence of the GBSS protein. PCR based markers were developed for both the wx a and the wx b alleles to aid in molecular breeding of low amylose sorghum.

Variation for Glutenin and Waxy Alleles and Their Effect on Quality Properties in Sichuan Wheat Landraces

Variation for Glutenin and Waxy Alleles and Their Effect on Quality Properties in Sichuan Wheat Landraces

Amylose content and starch properties generated by five variant Wx alleles for granule-bound starch synthase in common wheat (Triticum aestivum L.)

In common and durum wheats (Triticum aestivum L. and T. durum Desf.), variant waxy (Wx) alleles have been reported for three Wx proteins (Wx-A1, -B1 and -D1), responsible for amylose synthesis in flour starch. Five variant alleles, Wx-A1c, -A1e, -B1c, -B1d and -D1c, were examined to elucidate their effects on amylose content in flour starch. Common wheat lines carrying a Wx protein produced by one variant (e.g., Wx-A1c) and one control (e.g., Wx-A1a) allele were bred and their starches were compared. Results showed that Wx-A1e did not produce amylose (waxy phenotype), whereas three alleles (Wx-A1c, -B1c and -B1d) reduced amylose, and -D1c might have increased it slightly. Most data on blue value, swelling power and starch paste clarity in water and dimethyl sulphoxide also suggested the variant Wx alleles either reduced or increased amylose content.

Molecular characterization of the waxy locus in sorghum

A comparison of approximately 4.5 kb of nucleotide sequence from the waxy locus (the granule-bound starch synthase I [GBSS I] locus) from a waxy line, BTxARG1, and a non-waxy line, QL39, revealed an extremely high level of sequence conservation. Among a total of 24 nucleotide differences and 9 indels, only 2 nucleotide changes resulted in altered amino acid residues. Protein folding prediction software suggested that one of the amino acid changes (Glu to His) may result in an altered protein structure, which may explain the apparently inactive GBSS I present in BTxARG1. This SNP was not found in the second waxy line, RTx2907, which does not produce GBSS I, and no other SNPs or indels were found in the approximately 4 kb of sequence obtained from RTx2907. Using one indel, the waxy locus was mapped to sorghum chromosome SBI-10, which is syntenous to maize chromosome 9; the waxy locus has been mapped to this maize chromosome. The distribution of indels in a diverse set of sorghum germplasm suggested that there are two broad types of non-waxy GBSS I alleles, each type comprising several alleles, and that the two waxy alleles in BTxARG1 and RTx2907 have evolved from one of the non-waxy allele types. The Glu/His polymorphism was found only in BTxARG1 and derived lines and has potential as a perfect marker for the BTxARG1 source of the waxy allele at the GBSS I locus. The indels correctly predicted the non-waxy phenotype in approximately 65% of diverse sorghum germplasm. The indels co-segregated perfectly with phenotype in two sorghum populations derived from crosses between a waxy and a non-waxy sorghum line, correctly identifying heterozygous lines. Thus, these indel markers or sequence-based SNP markers can be used to follow waxy alleles in sorghum breeding programs in selected pedigrees.

Occurrence of the Waxy Alleles wxa and wxb in Waxy Sorghum Plant Introductions and Their Effect on Starch Thermal Properties

The existence of two waxy alleles, wxa associated with no detectable granule bound starch synthase (GBSS) and wxb associated with apparently inactive GBSS, was recently reported in sorghum [Sorghum bicolor (L.) Moench]. In this paper, the occurrence of the wxa and wxb alleles in the USDA‐ARS photoperiod‐insensitive sorghum collection was determined, and the effects of the wxa and wxb alleles on thermal properties of sorghum starch (gelatinization temperatures and energy requirements) measured by differential scanning calorimetry. Of the 51 purported waxy accessions examined, 14 tested positive for presence of amylose by iodine staining and were considered to be previously misclassified wild‐type lines. Nine accessions were mixed for presence or absence of amylose. Twenty‐four of the 28 accessions confirmed to be waxy by negative iodine staining for amylose had no detectable GBSS using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) (wxa), and four were show to contain GBSS (wxb). Mean gelatinization onset, peak, and end temperatures were significantly lower for wild‐type than either of the two waxy genotypes. Mean gelatinization onset temperature was slightly higher for waxy‐GBSS+ genotypes than waxy‐GBSS− genotypes. Mean gelatinization end temperature was slightly higher for waxy‐GBSS− genotypes than waxy‐GBSS+ genotypes. Significant genetic variation was observed within genotypic classes, suggesting influence of additional modifier genes affecting sorghum starch structure or micro‐environmental effects.

Variation for glutenin and waxy alleles in the US hard winter wheat germplasm

We conducted a survey of high- and low-molecular-weight glutenin subunits (HMW-GS and LMW-GS, respectively) and waxy ( Wx) allele composition in 111 winter wheat cultivars and advanced lines developed in the US Hard Winter Wheat Region since 1991. At the HMW-GS Glu-A1 locus, 76.1% of entries had allele b (encoding subunit 2*), 21.2% carried allele a (subunit 1), and 2.7% had allele c (null). Glu-B1 alleles were c (subunits 7+9; 47.7% of entries), b (subunits 7+8; 33.3%), e (subunits 20x+20y; 7.7%), i (subunits 17+18; 6.3%), and w (subunits 6*+8*; 5%). At Glu-D1, 80.6% of the entries carried allele d (subunits 5+10), 11.3% had allele a (subunits 2+12), 7.2% carried allele b (subunits 3+12), and 0.9% had allele e (subunits 2+10). Glu-B1 and Glu-D1 allele frequency differed significantly ( P<0.01) from a previous study of the US Hard Winter Wheat germplasm developed from 1970 to 1990. At LMW-GS loci, five Glu-A3 alleles, nine Glu-B3 alleles, and five Glu-D3 alleles were identified. Ten entries with the null Wx-B1b allele and seven with the null Wx-A1b allele were detected. This survey allows comparisons of HMW-GS and waxy allele compositions with the US Hard Winter Wheat germplasm from previous eras and provides baseline data on LMW-GS composition.

Single Kernel Near‐Infrared Analysis of Tetraploid (Durum) Wheat for Classification of the Waxy Condition

ABSTRACTPlant breeding programs are active worldwide in the development of waxy hexaploid (Triticum aestivum L.) and tetraploid (T. turgidum L. var. durum) wheats. Conventional breeding practices will produce waxy cultivars adapted to their intended geographical region that confer unique end use characteristics. Essential to waxy wheat development, a means to rapidly and, ideally, nondestructively identify the waxy condition is needed for point‐of‐sale use. The study described herein evaluated the effectiveness of near‐infrared (NIR) reflectance single‐kernel spectroscopy for classification of durum wheat into its four possible waxy alleles: wild type, waxy, and the two intermediate states in which a null allele occurs at either of the two homologous genes (Wx‐1A and Wx‐1B) that encodes for the production of the enzyme granule bound starch synthase (GBSS) that controls amylose synthesis. Two years of breeders' samples (2003 and 2004), corresponding to 47 unique lines subdivided about equally into the four GBSS genotypes, were scanned in reflectance (1,000–1,700 nm) on an individual kernel basis. Linear discriminant analysis models were developed using the best set of four wavelengths, best four wavelength differences, and best four principal components. Each model consistently demonstrated the high ability (typically &gt;95% of the time) to classify the fully waxy genotype. However, correct classification among the three other genotypes (wild type, wx‐A1 null, and wx‐B1 null) was generally not possible.

Characterization of waxy grain sorghum lines in relation to granule-bound starch synthase

The waxy phenotype, associated with endosperm containing little or no amylose, has been recognized in sorghum (Sorghum bicolor L. Moench) since 1933. Although variants of the waxy gene are well characterized in other cereals, the waxy trait has been assumed to be controlled by a single allele, wx, in sorghum. Recent improvements in technologies encourage re-examination of the waxy sorghums. The objectives of this research were therefore to identify and characterize sorghum lines with differing waxy alleles and to describe the actions of those alleles in crosses. Grain of eight waxy sorghum lines (BTxARG1, BTx630, Tx2907, B.9307, 94C274, 94C278, 94C289, 94C369), three wild-type checks (BWheatland, RTx430, BN122), and F2 families from crosses among a subset of these lines were evaluated for presence or absence of granule-bound starch synthase (GBSS), the gene product of the wx locus, and wild-type vs. waxy endosperm. The F2 segregation ratios were tested for fit to a 3:1 ratio using Chi-square analyses. Two distinctly different naturally occurring waxy alleles were identified: One with no GBSS (GBSS−), and one with apparently inactive GBSS present (GBSS+). We propose that the waxy allele with no GBSS be designated wx a , and that waxy allele with apparently inactive GBSS present be designated wx b . These two alleles are located in close proximity on the waxy locus. The wx b allele is dominant to the wx a allele in terms of GBSS production, and both are recessive to the wild-type Wx in terms of amylose content.

Variation of Wx microsatellite allele, waxy allele distribution and differentiation of chloroplast DNA in a collection of Thai rice (Oryza sativa L.)

Eighty-two varieties of rice from different regions in Thailand were selected to explore the Waxy(Wx)gene diversity and indica-japonica differentiation of chloroplast DNA. A comparison of the 5’ splice site in the first intron was made between glutinous and nonglutinous rice. It revealed that non-glutinous with low-amylose content and glutinous rice were characterized as the Wxb allele based on the G-to-T base substitution, whereas non-glutinous rice with intermediate and high amylose carried the Wxa allele. Four Wx microsatellite alleles, (CT)n repeat, (n = 16,17,18 and 19) were found in glutinous rice. In contrast, non-glutinous rice showed five Wx microsatellite alleles (n = 11, 16, 17, 18 and 19). The (CT)17 allele was prominent allele in Thai population, while the (CT)11 allele was found only in intermediate and high amylose rice varieties from southern Thailand. Almost all of upland rice grown by various ethnic groups in northern Thailand were characterized as japonica type based on their having the PstI-12 fragment in their cpDNA, whereas most of rainfed lowland varieties from other regions of Thailand were indica. This exploration of DNA-based genetic markers is important, as it enhances our ability to describe and manipulate sources of genetic variation for rice breeding programs.

Duplicated coding sequence in the waxy allele of tropical glutinous rice ( Oryza sativa L.)

Rice starch consists of amylopectin and amylose, the latter component being controlled by the Waxy ( Wx) gene. Two alleles Wx a and Wx b were found to regulate the quantitative level of the Wx protein as well as the amylose content. A comparison of the genomic sequences of rice Wx genes responsible for the synthesis of grain amylose was made between two pairs of glutinous and non-glutinous reverse mutants. By comparing their 5′ splice codons in the first intron, the low-amylose and glutinous rices were characterized as the Wx b allele based on the G-to-T base substitution, which caused an aberrant splicing. When the entire genomic sequences of Wx b were compared, a unique duplication of 23 bp was found to be present only in the glutinous mutants. This 23 bp insertion was truly unique for tropical glutinous rice because of its similarity between the 800 bp sequences around the duplicated sequences of 24 glutinous varieties and two annual diploid wild rice accessions. This in-frame duplication created a premature translation stop codon at 78 bp downstream. The insertion was unique and none of the structure was identical to any transposon or retrotransposon ever reported in rice before. This unique duplicated sequence opens up opportunities to develop PCR-based markers useful for classifying grain amylose in rice.

The Effects of Hardness Allele Pinb-D1b on the Flour Quality of Wheat for Japanese White Salty Noodles

We studied the effects of hardness allele Pinb-D1b on the flour quality characters for Japanese white salty noodles. With 110 doubled haploid lines derived from ‘Chugoku 140’ (hard,Pinb-D1b )בChikugoizumi’ (soft, Pinb-D1a), Pinb-D1b hard lines were found to have larger L* and a* of 60% flour colour, and smaller RVA breakdown than those of Pinb-D1a soft lines in addition to the higher flour yield, and larger average flour particle size. The Pinb-D1b allele is thus regarded to effectively improve milling efficiency and flour brightness. The inferior flour viscosity of Pinb-D1b lines is compensated for by introducing waxy allele(s) to increase viscosity by lowering amylose content.

Structural and Physicochemical Properties of Endosperm Starches Possessing Different Alleles at the Amylose-extender and Waxy Locus in Maize (Zea mays L.)

The structure and some physicochemical properties of endosperm starches from seven amylose-extender (ae) and two waxy (wx) alleles of maize (Zea mays L.) were studied. Starches prepared from mature kernels of six ae mutants, Oh43 inbred line ae (standard ae), ae-RWB-2 and ae-RWB-3, and W23 x L317 hybrid line ae-PP, ae-Bol 561 and ae-emll, were uniquely ae type, as was concluded from B type X-ray diffractograms; high gelatinization temperatures determined by differential scanning calorimetry (DSC); with poor starch-granule digestibility to amylase; high amylose (37-45 %) and high intermediate fraction (13-18%) contents; and low ratios (1.0-1.2) of long α-1,4-chains to short α-1,4-chains of amylopectin determined by gel permeation chromatography (GPC) of isoamylase-debranched starches. The results also indicated that different ae alleles had different effects on the amylose content of endosperm starches. Mature kernels of Oh43 ae-RWB-1 mutant showed tarnished and translucent phenotype characteristics of the ae genotype but contained endosperm starch with 21-22% of amylose which was lower than that of the Oh43 normal counterpart. Both wx-B and wx-C (standard wx) genes have similar effect on structure and physicochemical properties of waxy starches of A632, B37, C105, Oh43, and W64A inbred lines.

Structure and Physicochemical Properties of Endosperm Starches of a Waxy Allelic Series and Their Respective Normal Counterparts in the Inbred Oh43 Maize Background

AbstractResearch over the past three decades has greatly increased our understanding of the biochemical genetics of various waxy (wx) alleles of maize, but our knowledge about the structure and physicochemical properties of endosperm starches obtained from the wx alleles is still incomplete. We further investigated the structure and physicochemical properties of endosperm starches from a wx allelic series and their normal counterparts in the Oh43 inbred background.Starch granules were prepared from mature kernels of wx mutant alleles; wx‐C31, wx‐R, wx‐90, wx‐a, wx‐B3, wx‐m‐1, wx‐m‐8, and wx‐S5, and their respective normal counterparts in the inbred Oh43 maize background. Measurements of absorption spectra of starch‐iodine complexes and by gel permeation chromatography of Pseudomonasisoamylase‐debranched starches showed that all of the starches from the wx allelic series were uniquely waxy type in characteristics, and their normal counterparts were characteristically normal type. Pasting characteristics of starch granules and retrograded starches of the wx allelic series and their respective normal counterparts were investigated by differential scanning calorimetry (DSC). The peak temperature (Tp) and conclusion temperature (Tc) values from starches of the wx allelic series were slightly higher than those of their respective normal counterparts and the ΔHs of starches of the wx allelic series were greater than those of their respective normal counterparts. The onset temperature (To), Tp, and Tc of all retrograded starches were very similar, however, the ΔHs of the retrograded starches of the wx allelic series were greate than those of their respective normal counterpart. Starch granules of the wx allelic series were hydrolyzed more rapidly than those of their respective normal counterparts.

Thermal Properties of Sorghum Starch

AbstractThe differential scanning calorimeter (DSC) was used to measure thermal properties of sorghum starch and flour. DSC gelatinization temperatures of 24 nonwaxy sorghum varieties were: To (onset temperature): 71.0 ± 1.0°C; Tp (peak temperature): 75.6 ± 0.9°C and Te (end temperature): 81.1 ± 1.1°C, respectively. The gelatinization energies ranged from 2.51 to 3.96 cal/g. There were no consistent relationships between DSC gelatinization temperatures or gelatinization energies and grain characteristics or physicochemical starch properties. DSC gelatinization temperature for nonwaxy, heterowaxy and waxy sorghum tended to increase as the number of waxy alleles (wx) increased. However, it appeared that at least a difference of two waxy alleles was required before differences in thermal properties of the samples were significant.

The DNA sequnce of the transposable element Ac of Zea mays L.

The sequence of the Ac element isolated from the wx-m7 allele has been determined. The Ac element is 4563 bp long. A central portion of roughly 3.1 kb is occupied by three open reading frames, two of which point in one direction and the third in the opposite direction. One of the reading frames potentially encodes a protein with a ten-fold repeat of pro gluN and pro glu dipeptides near its N-terminus. The sequences outside the open reading frames are characterized by the presence of a number of direct and inverted repeats. The Ac element may thus have evolved from a simpler progenitor structure. The sequence we have determined for the Ac from the wx-m7 allele differs in a few key positions from that reported for the Ac element from the wx-m9 allele (Pohlman et al. 1984). We have resequenced these positions in both Ac elements and find them to be identical. We conclude that the phenotypic differences between the two waxy alleles are not caused by structural differences in the Ac elements but rather may be attributable to the differences in their insertion sites.

Difference Between the Size of Waxy and Non-waxy Kernel in the F&lt;SUB&gt;1&lt;/SUB&gt; Rice Plant. : Unbalanced Growth in Floral Glumes and Caryopsis in Rice. V

The substantial difference between waxy and non-waxy endosperm character lies in the amylose content of the endosperm starch. In the rice plant, waxy starch consists of only arnylopectine, and non-waxy starch contains seven to 33 percent amylose (WEBB et al. 1968 and JULIANO 1970). Endosperm character of the kernels of the F1 plants between waxy and non-waxy varieties segregates into waxy and non-waxy, because of the xenia. In such F1 plants, the dimentional capacity of floral glumes and the photosynthate supply to the developing kernel of waxy grain is considered be equal to those of non-waxy grain, therefore any dimentional difference between waxy and non-waxy kernel should be attributable to the waxyness itself and or the interaction between the waxy allele and the genetic background.

Gene dosage effect of waxy alleles on amylose content in endosperm starch of rice.

Gene dosage effect of waxy alleles on amylose content in endosperm starch of rice.