Starch characterisation and variability in GBSS loci of synthetic hexaploid wheats and their durum and <Emphasis Type="Italic">Aegilops tauschii</Emphasis> parents

Greater variability in starch properties is found in lower ploidy wheats than in commercial hexaploid wheats. This paper reports on the starch properties and variability in granule bound starch synthase (GBSS) loci of 17 diploid (Aegilops tauschii) and 12 tetraploid (durums) potential progenitors of wheat, compared with 29 synthetic hexaploid wheats produced from such progenitors. Starch properties examined were granule size distribution, swelling power, amylose content, gelatinisation and amylose-lipid dissociation properties. A PCR screening method was able to detect the presence or absence of each of the three GBSS genes. It also detected polymorphisms in eight diploids and nine hexaploids, all displaying the same 25 bases deletion in the D genome allele of GBSS. Two tetraploids and five hexaploids were null 4A for GBSS. There was little difference in the amylose contents and amylose-lipid dissociation peak temperatures of the synthetic hexaploids and the lower ploidy wheats. The synthetic hexaploids showed intermediate swelling power values with the durums giving the highest swelling powers. The durums also had higher B granule contents than the A. tauschii accessions, but not as high as the synthetics. However, the A. tauschii samples gave the highest gelatinisation peak temperatures. The presence of the null 4A mutation was positively correlated with swelling power, amylose content and DSC measurements. The new smaller D genome allele of GBSS was associated with slightly higher swelling power. These results confirm the value of wheat progenitor lines as sources of new starch properties for hexaploid wheat. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gene dosage effect of the wheat Wx alleles and their interaction on amylose synthesis in the endosperm

To find out gene dose effect of each of the three homoeologous Wx genes and their interaction on the production of granule-bound starch synthase (GBSS I) and amylose biosynthesis in the endosperm, Chinese Spring and its near-isogenic waxy types were crossed reciprocally and, obtained a plant population with varying doses of each Wx gene. The amount of GBSSI was increased linearly with increasing gene dose of either of Wxloci. In each of the three Wx loci, the change in amylose content was linear up to 3 doses, with a more potent capacity ofWx-B1a at any dose. Higher level of amylose production was observed in the reciprocal F₁ grains than the expected effect of dose/s of each gene or additive effect of different allelic combination by artificially blend starches which have amylose produced by equivalent number ofWx alleles to that of relevant F₁ cross. When Wx-B1a and Wx-A1a were combined, increase in amylase content was not in proportion to increase in gene dosage. The enhanced amylase synthesis was shown by 2-gene and 3-geneinteraction, indicating that not only type of the three Wx genes and its dose but the interaction among them have significant roles in determining the amylose content. This revised version was published online in July 2006 with corrections to the Cover Date.     

两个直链淀粉含量不同的小麦品种籽粒淀粉合成酶活性与淀粉积累特征的比较

以均含有3个Waxy蛋白亚基的普通小麦品种济麦20（低直链淀粉含量）和鲁麦21（高直链淀粉含量）为材料，对灌浆期籽粒淀粉合成相关酶活性的变化及淀粉积累特征进行了研究，并分析了两者之间的关系。结果表明，蔗糖合成酶(SS)、腺苷二磷酸葡萄糖焦磷酸化酶(AGPP)、束缚态淀粉合成酶(GBSS)、可溶性淀粉合成酶(SSS)和淀粉分支酶(SBE)活性均呈单峰曲线变化。鲁麦21的上述酶活性均高于济麦20。相关分析表明，支链淀粉积累速率与SS、AGPP、SSS和SBE呈显著或极显著正相关；直链淀粉积累速率与SS、AGPP和GBSS呈极显著正相关。Logistic方程拟合淀粉积累过程发现，支、直链淀粉最终积累量的高低取决于积累启动时间的早晚和积累速率的高低，而积累持续期的调节作用较小。直链淀粉的积累速率除受GBSS活性影响外，还受SS和AGPP活性的影响，其中，GBSS活性的变化与2品种籽粒直链淀粉积累量的变化情况基本吻合。籽粒灌浆后期的GBSS活性对直链淀粉最终积累量的调节作用大于灌浆前期，说明对同时具有3个Waxy蛋白亚基的不同品种，Waxy蛋白亚基表达量(GBSS活性)的差异可能是导致品种间籽粒直链淀粉含量较大差异的一个关键原因。     

The effect of the Waxy locus (Granule Bound Starch Synthase) on pasting curve characteristics in specialty rices (Oryza sativa L.)

Starch structure and functionality have a significant impact on the utilization of cereal grains as food and feed. Starch viscosity characteristics are used to characterize rice cooking, processing and eating quality. In order to examine the genetics of viscosity characteristics, we developed molecular markers for five of the major enzymes involved in starch synthesis in the endosperm: granule bound starch synthase, soluble starch synthase, rice branching enzymes 1 and 3 and starch debranching enzyme. These markers were polymorphic in a cross between specialty rice varieties of diverging amylose content and viscosity characteristics. Our results indicate that the Waxy locus, encoding the gene for granule bound starch synthase, has a significant effect on peak viscosity, hot paste viscosity, cool paste viscosity, breakdown and setback viscosity. We estimate that the tightly linked (5–10 cm)locus for starch synthase may have a lesser, additive effect. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of a single nucleotide polymorphism that controls the cooking quality of rice using a non-gel based assay

The waxy gene encoding granule-bound starch synthase (GBSS) is responsible for the synthesis of amylose in developing grain. Recent work has shown that a G-T polymorphism in the leader intron 5' splice site of GBSS plays a key role in determining the cooking and processing quality of rice. Cultivars with sequence AGGTATA at this location splice GBSS pre-mRNA efficiently and produce relatively large amounts of amylose. These varieties generally a have firm texture when cooked and the grains remain separate. In contrast, GBSS pre-mRNA splicing is temperature sensitive and generally less efficient in cultivars with the sequence AGTTATA. As a result, these cultivars generally have lower amylose content and produce soft and sticky cooked rice. We have used the READIT^TM assay, anon-gel based assay that uses the ability of DNA polymerase to perform pyrophosphoralysis, the reverse of DNA polymerization, to screen the critical G-T polymorphism in more than 750 samples from U.S. and Asian germplasm. We observed complete concordance between the results obtained using DNA sequencing or restriction enzyme digestion and the READIT^TM assay. It also gave accurate results with both heterozygous plants and with complex mixtures as might result when grain from advanced generation plants is pooled to obtain larger samples. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

A novel waxy allele in sorghum landraces in East Asia

A loss of granule‐bound starch synthase I (GBSS I) activity results in starch granules that contain mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles, wx^a, associated with no detectable GBSS I, and wx^b, associated with apparently inactive GBSS I in the endosperm, were reported in sorghum (Sorghum bicolor (L.) Moench). In this study, the waxy alleles in a sorghum core collection were investigated using DNA markers. Of the 337 sorghum accessions examined, 17 accessions that were confirmed to be waxy by a negative iodine staining result and 16 were found to be wx^a. A novel waxy allele, wx^c, was found in a Taiwanese landrace. This allele consists of a +1G to C mutation in the 5′ splice site at the intron 10–exon 11 boundary, a mutation that most likely resulted in the suppression of GBSS I gene expression. A DNA marker specific for wx^c was produced to distinguish the wx^c allele from other alleles, allowing the identification of heterozygous non‐waxy plants.     

小麦Wx基因近等基因系的创制及其对直链淀粉含量、面条感官品质的影响

为明确不同Wx基因对小麦直链淀粉含量的影响以及筛选面条品质优异的基因型,以糯小麦品系Caiwx (aabbdd)为3个Wx基因隐性突变供体亲本,以扬麦01-2 (AABBDD)为轮回亲本,利用连续回交结合花粉碘染、STS标记和同工酶标记检测方法,创制了8种Wx基因纯合基因型的近等基因系,其基因型分别为AABBDD、AABBdd、AAbbDD、AAbbdd、aaBBDD、aaBBdd、aabbDD和aabbdd。利用这些基因型探讨了不同Wx基因对直链淀粉含量及面条感官品质的影响。结果表明,各系的直链淀粉含量为0.9%~24.8%,系间差异显著;糯小麦型(aabbdd)直链淀粉含量最低,双缺失型和单缺失型其次,双缺失型中aaBBdd型含量最高,单缺失型中AAbbDD型含量最低,表明Wx-B1对直链淀粉的合成作用最大。糯小麦型面条的色泽、表观、软硬度、黏性、韧性得分以及总分显著低于其他类型及轮回亲本扬麦01-2;单缺失型面条的色泽、表观得分、总分显著高于轮回亲本扬麦01-2,其中aaBBDD型面条品质表现突出,与市售优质面条粉“雪花粉”制作的面条得分相当,而其他7种基因型及轮回亲本扬麦01-2的面条评分均显著低于雪花粉。说明可以通过遗传操作Wx基因培育优质面条小麦品种。     

Developing gene-tagged molecular markers for functional analysis of starch-synthesizing genes in rice (Oryza sativa L.)

The granule-bound starch synthase(GBSS), starch branching enzymes 1 (SBE1)and 3 (SBE3) are major enzymes involved in starch biosynthesis in rice endosperm. Available sequences of Sbe1 and Sbe3 genes encoding corresponding SBE1 and SBE3 have been used to identify homologous regions from genome databases of both the indica rice 93-11 and the japonica rice Nipponbare. Sequence diversities were exploited to develop gene-tagged markers to distinguish an indica allele from a japonica allele for both Sbe1 and Sbe3 loci. With these newly developed gene-tagged markers and available Wx gene markers, the roles of these starch-synthesizing genes (Sbe1, Sbe3, and Wx) in determining amylose content (AC) in the rice endosperm were evaluated using a double-haploid (DH)population derived from a cross between the indica rice cv. Nanjing11 and the japonica rice cv. Balilla. Only the Wx and Sbe3 loci had significant effects on the AC variation. On average, indica Wx ^a genotypes showed ∼9.1% higher AC than japonica Wx ^b genotypes, while indica Sbe3 ^a genotypes showed ∼1.0% lower AC than japonica Sbe3 ^b genotypes. A significant interaction was also observed between Wx and Sbe3 loci whereby the amylose content was 0.3% higher in Sbe3 ^a than Sbe3 ^b genotypes in the presence of the Wx ^a allele, but it was lower by 2.3% in the presence of the Wx ^b allele. Overall, polymorphisms at the Wx and Sbe3 loci together could account for 79.1% of the observed AC variation in the DH population. The use of gene-tagged markers in marker-assisted selection and gene functional analysis was also discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

灌浆期高温对小麦籽粒淀粉的积累、粒度分布及相关酶活性的影响

以不同耐热性品种济麦20和鲁麦21为材料, 于花后5~9 d进行高温处理, 研究了小麦灌浆期高温对籽粒淀粉的积累、粒度分布及合成相关酶活性的影响。结果表明, 灌浆期高温显著降低籽粒淀粉积累量, 显著降低籽粒淀粉、支链淀粉含量, 提高直链淀粉含量和直/支链淀粉比例。高温对济麦20籽粒淀粉积累的影响程度较鲁麦21大。灌浆期高温使小麦籽粒A型淀粉粒的体积、数量和表面积百分比显著增加, B型淀粉粒这3指标则显著降低。高温处理后, 济麦20籽粒蔗糖合酶(SS)、ADPG焦磷酸化酶(AGPP)、可溶性淀粉合酶(SSS)、束缚态淀粉合酶(GBSS)活性与对照无显著差异, 而鲁麦21上述酶活性则高于对照。济麦20、鲁麦21籽粒上述酶活性分别于花后15 d和20 d开始低于对照。与其他淀粉合成相关酶相比, 高温对籽粒GBSS活性的影响程度较小。两品种处理间籽粒蔗糖含量及SS、AGPP、SSS和GBSS活性的变化趋势, 与其籽粒淀粉积累量的变化趋势基本一致。灌浆期高温使籽粒淀粉积累量降低, 主要因高温抑制了籽粒灌浆中后期的淀粉合成, 这是由籽粒蔗糖供应不足和籽粒淀粉合成相关酶活性下降所造成的。     

Genetic variation for waxy proteins and starch properties in Italian wheat germplasm

Granule-bound starch synthase (GBSS) is the primary enzymeresponsible for the synthesis of amylose in amyloplasts of cereal endospermcells. In bread wheat there are three structural genes (Wx-A1, Wx-B1,and Wx-D1) encoding for isoforms of GBSS. The loss of one or moreGBSS isoforms results in the reduction (partial-waxy) or absence (waxy) of amylose in the starch. Waxy wheats may find application inthe production of modified food starch and their flour may be used toextend the shelf life of baked products. In order to breed high qualitywheats able to produce bread with delayed staling, the genetic variabilityfor the waxy trait in our germplasm has been investigated. Weanalysed 288 cultivars of bread wheat, 139 cultivars of durum wheat andabout 200 accessions from other Triticum species. Gel electrophoresisshowed 63 bread wheats deficient in the Wx-B1, one in the Wx-A1 and one in the Wx-D1 protein isoforms, as well as one Triticum dicoccum lacking the Wx-A1 isoform. None of the analysedTriticum monococcum, Triticum durum, Triticum speltaand Triticum timopheevi accessions showed mutations at the Wxloci. The wheat accessions with Wx mutations were evaluated with aRapid Visco Analyser (RVA) to investigate starch properties. All theanalysed cultivars showed Peak Viscosity and Final Viscosity different fromthe normal wheat. Other analyses to evaluate the rheological characteristicsof the partial-waxy genotypes are under way and a breedingprogramme to select new waxy wheat varieties is in progress     

Genotypical differences in nitrate content inLactuca sativa L. and related species and correlation with dry matter content

Summary Nitrate content was measured in 135 genotypes of cultivated lettuce (Lactuca sativa L.) and 21 genotypes of wildLactuca spp. grown in two experiments. In Experiment 1 plants were grown on nutrient film. In Experiment 2 plants were grown in large pots of potting soil. Within cultivated lettuce five plant types were distinguished and within each of them genotypes were found with low nitrate content. The coefficient of variation in the experiment with plants growing in large pots was not larger than in the experiment with plants growing on nutrient film. In butterhead genotypes nitrate content was negatively correlated with dry matter content and positively with plant fresh weight.     

Influence of single-nucleotide polymorphisms in the gene encoding granule-bound starch synthase I on amylose content in Vietnamese rice cultivars

Amylose content is one of the most important factors influencing the physical and chemical properties of starch in rice. Analysis of 352 Vietnamese rice cultivars revealed a wide range of variation in apparent amylose content and the expression level of granule-bound starch synthase. On the basis of single-nucleotide polymorphisms (SNP) at the splicing donor site of the first intron and in the coding region of the granule-bound starch synthase I gene, Waxy gene, alleles can be classified into seven groups that reflect differences in apparent amylose content. The very low and low apparent amylose content levels were tightly associated with a G to T in the first intron whereas intermediate and high amylose was associated with a T genotype at SNP in exon 10. The correlation between the combination of T genotype at SNP in the first intron, C in exon 6, or C in exon 10 was predominant among low amylose rice varieties. Our analysis confirmed the existence of Wx^op allele in Vietnamese rice germplasm. The results of this study suggest that the low amylose properties of Vietnamese local rice germplasm are attributable to spontaneous mutations at exons, and not at the splicing donor site.     

Genetic studies of speciation in cultivated rice. 5. Inter- and intraspecific differentiation in the waxy gene expression of rice

Summary To get an insight in the gene regulation at the waxy locus of rice, the Wx gene product (Wx protein) controlling the synthesis of amylose was examined by electrophoretic techniques. Among nonwaxy rice strains, two different alleles, Wx ^a and Wx ^b, were found at the waxy locus. Wx ^a drastically enhanced the quantitative level of Wx protein as well as the amylose content in endosperm starch as compared with Wx ^b. The alleles acted additively in triploid endosperms. This implies that regulatory elements responsible for the Wx gene expression are on the same chromosome. The distribution patterns of Wx ^a and Wx ^b in five species of Oryza revealed that the regulatory changes are closely related to racial differentiation within a common rice species (O. sativa), suggesting that Wx ^b might have been selected for through the difference in grain quality during domestication.     

An alanine to threonine change in the Wx-D1 protein reduces GBSS I activity in waxy mutant wheat

The Wx-D1 protein (granule-bound starch synthase) of Kanto 107, Tanikei A6099 (low amylose line), and Tanikei A6599-4 (waxy line) has been analyzed by SDS-PAGE, peptide mapping and DNA sequencing. Kanto 107 and Tanikei A6099 have the same amino acid sequences in the mature protein, but amino acid substitution (alanine to threonine) occurs at position 258 in the mature protein in Tanikei A6599-4. A comparison of deduced amino acid sequences of the mature Wx-D1 protein in these lines indicates that point mutation in the Wx-D1 gene of Tanikei A6599-4 is responsible for its waxy character.This mutant waxy wheat does not show a reduction in amylose content identicalto other waxy wheats, which probably reduces activity of the GBSS I enzyme but does not to a complete loss of activity. We discuss the function of the mutant Wx-D1 protein in starch synthesis. This revised version was published online in August 2006 with corrections to the Cover Date.     

小麦籽粒灌浆过程中有关淀粉合成酶的活性及其效应

在不同土壤肥力条件下, 对不同品质类型小麦品种的籽粒淀粉积累及淀粉合成过程中的关键酶-ADPG焦磷酸化酶、可溶性淀粉合成酶与淀粉粒结合态淀粉合成酶活性变化进行了研究. 结果表明, ADPG焦磷酸化酶、可溶性淀粉合成酶与总淀粉和支链淀粉积累速率呈极显著正相关; 淀粉粒结合态淀粉合成酶与直链淀粉积累速率呈极显著正相关,     

不同小麦品种籽粒淀粉组分及相关酶活性的差异

根据小麦成熟期籽粒淀粉组分的差异,用聚类分析的方法,将籽粒总淀粉含量相近、直链淀粉和支链淀粉含量存在差异的9个小麦品种分为3组：低直链淀粉组、中直链淀粉组和高直链淀粉组。研究籽粒灌浆过程中,直链淀粉与支链淀粉比值（直/支比值）的变化动态、淀粉合成相关酶活性的变化动态及其与淀粉组分积累的关系。结果表明：     

The G locus in Petunia hybrida Vilm: negative relationship to endogenous chlorophyll,starch and sugar

Suumary Three sets of Petunia hybrida Vilm. breeding lines with each set comprised of three genotypes, multiflora (gg), grandiflora (GG), and the heterozygote (Gg) were used in biochemical analyses to determine the possible mode of action of alleles at the G locus and their pleiotropic effects. Chlorophyll extractions did not correlate with visual observations that seedlings of the gg genotype have darker green foliage than seedlings of the GG genotypes. Likewise, chlorophyll, starch and sugar content were not related to greater seedling vigor, an apparent pleiotorpic effect to the g allele. Inconsistent differences in total soluble sugars and starch content were found with respect to the three genotypes. A qualitative difference in leaf sugar profiles as revealed by thin-layer and gasliquid chromatography separation was found. The Gg and gg genotypes consistently exhibited two extra bands at R_f 0.01 and at 0.52 to 0.56 which were absent in extracts from the GG genotype. The unknown compounds did not co-chromatograph with several sugar standards.