Production of Two Elite Glutinous Rice Varieties by Editing Wx Gene

The waxy gene(Wx) in rice, which encodes the granule bound starch synthase enzyme, is responsible for amylose synthesis. Glutinous(sticky) rice has little or no amylose that can be used in various applications, such as brewing. In this study, knockout of the Wx gene with CRISPR/Cas9 technology was conducted in two elite japonica rice lines, Huaidao 5(HD5) and Suken 118(SK118), aiming to develop elite sticky rice varieties. We achieved six homozygous T_0 plants with more than 200 bp deletion in the Wx gene, as well as 36 wx-HD5 and 18 wx-SK118 homozygous transgene-free plants in the T_1 generation. The seeds of all the mutants were white and opaque, similar to those of sticky rice, and contained only 2.6%–3.2% amylose. Results of scanning electron microscopy showed that the quality of rice did not change. In conclusion, we successfully developed two elite sticky rice varieties.     

Analyses of albumins, globulins and amphiphilic proteins by proteomic approach give new insights on waxy wheat starch metabolism

Starch is composed of two types of glucose polymers: amylose and amylopectin. The Waxy (Wx) locus controls amylose synthesis in the wheat kernel. Hexaploid wheat has three Wx loci located on chromosomes 7A (Wx-A1), 4A (Wx-B1), and 7D (Wx-D1). Eight near isogenic lines (NILs) of Triticum aestivum cv. Tremie with one, two or three Wx null alleles were used. The albumin–globulin fraction, and amphiphilic proteins were separated using 2-dimensional electrophoresis (2DE) allowing the changes in the waxy kernel to be identified. Albumin–globulin fraction showed overexpression of sucrose synthases in the waxy NILs compared to the normal form of Tremie and a decrease in many proteins related to stress and defence metabolism such as serpins. A subunit of ADP-glucose pyrophosphorylase (AGPase), which is known to play a major role in starch synthesis, was also shown to be down regulated in the waxy NILs. Amphiphilic proteins confirmed the observations made on the albumin–globulin fraction with a decrease in a stress-related protein. These different regulations linked to observations made on wheat kernel (thousand kernel weight (TKW), protein amount per grain, size and distribution of the starch granules) led to formulation of the hypothesis that waxy endosperm does not reach maturity of the wild-type endosperm.     

Influence of amylopectin structure on rheological and retrogradation properties of waxy rice starches

A set of 13 waxy rice genotypes prepared by chemical-induced mutation of rice variety TNG67 and 7 waxy rice varieties widely grown in Taiwan were compared for structural, rheological and retrogradation characteristics of starches. Wide differences in retrogradation enthalpy (ΔH_ret), gel firmness and storage modulus (G’_ret) were observed for 2-week stored gels of 20 starches. Ratio of short-to-long amylopectin chains was significantly higher (p < 0.05) in starches from mutant genotypes than in commercial varieties. ΔH_ret and G’_ret of starch pastes stored over 4 weeks showed stronger correlation with amylopectin chain-profile compared to those stored for 2 weeks. Amount of long amylopectin chains was correlated positively (p < 0.05) with ΔH_ret and gel firmness. Overall, ratio of short-to-long amylopectin chains affected almost all the rheological and retrogradation parameters. Results of this study can be useful to plant breeders and food industry for quality improvement and selection of waxy rice mutants for various applications.     

Molecular marker assisted selection for improvement of the eating,cooking and sensory quality of rice (Oryza sativa L.)

II-32B is a key maintainer line used for hybrid rice breeding in China. However, it is of poor quality for most Chinese consumers because of its high apparent amylose content (AAC), high gelatinization temperature (GT) and non-fragrance. It is well known that the AAC, GT and fragrance traits are largely controlled by the Wx, starch synthase IIa (SSIIa), and fragrance (fgr) genes, respectively. With the aid of functional markers, we improved the quality of II-32B by introgressing the Wx, SSIIa, and fgr genes from Yixiang B, a fragrant maintainer line that has low AAC and low GT. The microsatellite allele (CT)₁₇ of Wx, the contiguous single nucleotide polymorphism TT allele of SSIIa and the 8-bp deletion allele of fgr were transferred to II-32B by two backcrosses and three selfings. Molecular marker assisted selection was applied in the series to select for individuals carrying Wx-(CT)₁₇, SSIIa-TT, and fgr-deletion alleles. According to the marker genotypes, seventeen homozygous lines for Wx-(CT)₁₇, SSIIa-TT, and fgr gene markers were finally selected. The improved II-32B lines were fragrant with reduced AAC and GT.     

Assessing the relationship between endosperm type and grain yield potential in sorghum (Sorghum bicolor L. Moench).

Sorghum hybrids with waxy endosperm (either homozygous or heterozygous waxy) have lower grain yield potential compared to non-waxy endosperm hybrids. The cause of this yield reduction is not known. From a genetic perspective, the yield reduction could be due to pleiotrophy or genetic linkage between the wx locus and other loci that influence grain yield. The specific cause of the relationship is important because an effective breeding program can alleviate the problem if it is due to linkage. The objective of this study was to determine whether linkage or pleiotrophy is causing the negative relationship between grain yield and waxy endosperm. From each of two F₂ breeding populations segregating for waxy endosperm, between 40 and 50 inbred lines were derived, with equal numbers of waxy and non-waxy endosperm lines. No selection for yield was practiced during the development of these lines. The lines from these two populations and a set of testcross hybrids (derived from one population) were evaluated in four environments in Texas from 1998 to 2000. Across all tests and environments, the combined yield of the waxy genotypes was 17% lower than non-waxy genotypes. While yields were lower in waxy genotypes, analysis of the individual inbred lines and hybrids revealed that several waxy inbred lines were not statistically different from the best non-waxy inbreds. These results imply that selection of high yielding waxy genotypes is possible, but a significant breeding emphasis on their development is required to effectively identify those genotypes.     

Wx基因对灌浆期小麦不同叶位叶片光合特性的影响

为了解Wx基因对小麦光合作用的影响,对8个小麦Wx近等基因系的不同叶位叶片光合参数进行了测定和分析。结果表明,小麦叶片净光合速率、气孔导度、胞间CO2浓度和蒸腾速率在叶位和基因型间均有极显著差异,缺失单个Wx基因使叶片光合参数下降,且降幅表现为Wx-D1Wx-A1Wx-B1;胞间CO2浓度不影响光合作用,不同叶位叶片光合参数明显呈旗叶倒二叶倒三叶,但WxABD(糯小麦)光合参数在叶位间没有显著差异;Wild type和WxABD总体光合能力和产量相对较高。以上结果说明,Wx基因的缺失会引起叶片光合能力下降,不利于小麦产量形成。     

Molecular marker for screening rice cultivars with intermediate amylose content in indica

The waxy (Wx) gene of rice encodes the granule-bound starch synthase(GBSS), which was required in the synthesis of amylose in endosperm. The anylose content (AC) of rice endosperm played an important role in grain yield,palatability, and processing quality.     

Molecular marker for screening rice cultivars with intermediate amylose content in indica

The waxy (Wx) gene of rice encodes the granule-bound starch synthase(GBSS), which was required in the synthesis of amylose in endosperm. The anylose content (AC) of rice endosperm played an important role in grain yield,palatability, and processing quality.     

The effects of puroindoline b on the ultrastructure of endosperm cells and physicochemical properties of transgenic rice plant

Endosperm texture is an important factor governing the end-product quality of cereals. The texture of wheat (Triticum aestivum L.) endosperm is controlled by puroindoline a and b genes which are both absent in rice (Oryza sativa L.). It has been reported that the endosperm texture of rice can be modified by puroindoline genes. The mechanism, however, by which puroindolines affect the ultrastructure of rice endosperm cells remains to be investigated. In this study, we observed the ultrastructure of endosperm cells and the morphology of isolated starch granules of the transgenic rice expressing the puroindoline b gene. SEM and TEM observations indicated that compound starch granules were embedded within the matrix material in non-transgenic rice, Nipponbare, whereas they were surrounded by spaces in the transgenic rice. The morphology and size of each starch granule were not different between non-transgenic and the transgenic rice. However, the transgenic rice flour showed smaller particle size, higher starch damage, and lower viscosity during gelatinization than that of non-transgenic rice. These results confirm that puroindoline b reduces the grain hardness in rice. Moreover, the results also suggest that puroindoline b functions at the surface of compound starch granules, and not on polygonal starch granules in rice endosperm.     

Agronomic,molecular and antioxidative characterization of red- and purple-pericarp rice (Oryza sativa L.) mutants in Taiwan

Pigmentation of rice grain is controlled by Ra, Rc and Rd genes, and the expressions of these genes differ among red-, purple- and white-pericarp varieties. The present study examined the grain yield and the expression of Ra, Rc and Rd genes in non-waxy white-pericarp rice SA418 and waxy white-pericarp rice SA419 and their respective red-pericarp and purple-pericarp mutants with waxy or non-waxy endosperm. Significant variations in 100-grains weight and grain yield were observed among the tested mutants. The Ra was expressed in purple-pericarp mutants, while the Rc was expressed in red-pericarp mutants. The total phenolics, total flavonoids, total anthocyanins and total proanthocyanidins contents and antioxidant activities in the bran part also differed among the mutants. Non-waxy red-pericarp mutant M-69 had heavier 100-grains weight (2.86 g), contained more total phenolics (49.37 mg g⁻¹ bran dry weight) and produced higher grain yield (6.93 t ha⁻¹) than white-pericarp rices SA418 (2.43 g, 2.89 mg g⁻¹ bran dry weight and 2.80 t ha⁻¹, respectively) and SA419 (2.62 g, 2.20 mg g⁻¹ bran dry weight and 6.73 t ha⁻¹, respectively). Thus, the polished rice grain of M-69 can be used for staple food consumption, and its bran parts are useful for producing health-promoting by-product.     

杂交糯稻不育系嘉农wx6A的选育及应用

采用0．35kGy 60Co-γ射线辐照诱变杂交稻保持系中9B干种子约0．5kg,于M2带胚糙米群体获得糯质基因突变体嘉农wx6B,通过与中9A的杂交、回交,育成糯稻不育系嘉农wx6A。除胚乳性状差异显著外,嘉农wx6A与中9A生育期相同,且农艺性状、穗部性状、花器性状、花粉不育特性和配合力均十分相似。嘉农wx6A于2005年9月通过福建省技术成果鉴定,利用其配制的杂交糯稻组合嘉糯6优3号（嘉农wx6A／蜀恢527wxR）具有高产、优质、适应性广等特点。     

Gluten free rice cookies with resistant starch ingredients from modified waxy rice starches: Nutritional aspects and textural characteristics

Experimental gluten-free (GF) rice cookies were formulated with 100% rice flour (CTR) or by substituting 50% of rice flour with native waxy rice starch (WRS) or with three different resistant starch (RS) ingredients obtained from debranched, annealed or acid and heat-moisture treated WRS (RS_a, RS_b and RS_c, respectively). Chemical composition, in vitro starch digestibility and physical and textural characteristics were carried out. Among cookies, RS_a-cookies had the highest total dietary fibre content, the lowest rapidly digestible starch and the highest RS contents. All the three RS preparations have proved effective in increasing the proportion that tested as RS with respect to native WRS. However, the estimated RS loss for each applied RS ingredients caused by the baking process followed the order of RS_a < RS_c < RS_b. Last, the lowest vitro glycaemic index value was measured for RS_a-cookies. Among cookies, differences in colour and hardness were reported. The partial replacement of commercial rice flour with RS_a could contribute to formulate GF cookies with higher dietary fibre content and likely slowly digestible starch properties more than equivalent amounts of RS_b and RS_c.     

Endosperm Structure of White-Belly and White-Core Rice Grains Shown by Scanning Electron Microscopy

Abstract:

White-belly and white-core are the major two types of grain chalkiness in japonica rice. This study aims to compare the morphological features of white-belly and white-core using a scanning electron microcope (SEM). A japonica rice cultivar Wuyujing3 and its mutants were used as materials. Nearly 1000 SEM images were observed, and 12 representative photos were selected. SEM images showed contrasting differences between white-belly and white-core in endosperm microstructure including the shape of endosperm cell, the size distribution of starch granules, and the amount of protein bodies. White-belly and white-core also varied markedly in morphological features of the cracked compound starch granules. Our findings should help to advance our understanding of the multi-faceted nature of grain chalkiness from the perspective of starch and protein accumulation, and should be of value for future work on rice grain chalkiness.     

Retrogradation of waxy and normal corn starch gels by temperature cycling

Gelatinized waxy and normal corn starches at various concentrations (20–50%) in water were stored under temperature cycles of 4°C and 30°C (each for 1 day) up to 7 cycles or at a constant temperature of 4°C for 14 days to investigate the effects of temperature cycling on the retrogradation of both starches. Compared to starches stored only at 4°C, both starches stored under the 4/30°C temperature cycles exhibited smaller melting enthalpy for retrogradation (ΔH_r), higher onset temperature (T_o), and lower melting temperature range (T_r) regardless of the starch concentration tested. Fewer crystallites might be formed under the temperature cycles compared to the isothermal storage, but the crystallites formed under temperature cycling appeared more homogeneous than those under the isothermal storage. The effect of starch content on the retrogradation was greater when the starch gels were stored under cycled temperatures. The reduction in ΔH_r and the increase in conclusion temperature (T_c) by retrogradation under 4/30°C temperature cycles became more apparent when the starch concentration was lower (20 or 30%). Degree of retrogradation based on melting enthalpy was greater in normal corn starch than in waxy corn starch when starch content was low.     

Characterization of physicochemical qualities and starch structures of two indica rice varieties tolerant to high temperature during grain filling

An extreme high temperature during grain filling is an important environmental factor that reduce the yield and quality of rice. In this study, we compared the grain appearance, composition and starch structure of four rice varieties response to high temperature during grain filling. The results obviously revealed that two indica AUS varieties Halwa and Jamir showed more tolerance to high temperature during grain filling than either the japonica Nipponbare or another AUS rice DJ24. The data showed that Halwa and Jamir presented less chalkiness, less grain weight loss, less change of endosperm components as well as starch structure than Nipponbare and DJ24 under high temperature treatment. Hence, the results implied that Halwa and Jamir might be useful targets to further identify genetic mechanism response to high temperature, at least during grain filling and endosperm development. Moreover, these AUS varieties have potential application value in further breeding of rice with good quality and heat tolerance.     

两个垩白突变体的鉴定及突变基因的图位克隆

【目的】研究两个水稻垩白突变体胚乳垩白的形成机制,为稻米品质改良提供理论基础。【方法】以从中花11的EMS突变体库中筛选出的两个稳定遗传的垩白突变体eb6、eb7为材料,对其进行农艺性状、稻米理化性质和遗传学分析,并利用eb6与南京11衍生的F2群体对控制垩白的基因进行图位克隆。同时对候选基因的表达模式及淀粉合成相关基因在突变体及野生型中表达情况进行了分析。【结果】与野生型相比,两个突变体胚乳中央部位呈现白色且不透明,淀粉复合颗粒形状不规则且排列疏松,而突变体胚乳边缘部位与野生型无异,都为半透明状,淀粉复合颗粒呈多面体且排列致密。相对于野生型,突变体eb6、eb7成熟种子中的直链淀粉含量和胶稠度显著降低,蛋白质含量显著升高。RVA谱分析显示突变体淀粉黏滞性明显低于野生型。同时,支链淀粉聚合度分析显示突变体eb6中聚合度(DP)小于16的短链显著增加,DP为16~23的中长链显著减少。遗传分析表明突变体eb6、eb7胚乳垩白表型由单隐性核基因控制,并且它们为一对等位突变体。利用突变体eb6与南京11杂交衍生的F2群体将突变体基因定位在第1染色体长臂上物理距离86.6 kb的区间内。该区间包含22个开放阅读框(ORF),其中ORF13编码腺苷二磷酸葡萄糖焦磷酸化酶大亚基2(Os AGPL2)。序列分析发现eb6与eb7分别在Os AGPL2第3、第7外显子上发生1个单碱基替换,并分别导致一个氨基酸替换。RT-PCR及原位杂交结果显示,Os AGPL2主要在水稻发育中的籽粒中表达。同时Os AGPL2的突变导致了多个淀粉合成相关基因在水稻籽粒灌浆过程中的表达模式发生改变。【结论】突变体eb6、eb7籽粒胚乳出现严重垩白表型为Os AGPL2突变所致。本研究进一步证明了Os AGPL2在调控水稻籽粒胚乳中淀粉的合成、淀粉复合颗粒的形成及稻米理化性质的平衡中起着重要的作用。     

Effects of Variations in Starch Synthase on Starch Properties and Eating Quality of Rice

Abstract

We evaluated the effects of functional variation in three starch synthases in rice (Oryza sativa L.)?granule-bound starch synthase I (GBSSI, wx), starch synthase I (SSI, SSI), and starch synthase IIa (SSIIa, alk)?between indica cultivar Kasalath and japonica cultivar Nipponbare on starch properties and eating quality. We used three near-isogenic lines?NIL(Wx^a), NIL(SSI ^k), and NIL(Alk)?containing chromosomal segments of Kasalath on a Nipponbare genetic background. The Wx^a allele explained most of the difference in amylose content between the two cultivars, and decreased the peak viscosity and breakdown to less than half of those of Nipponbare. These changes reduced the quality of cooked rice both just after cooking and after storage at 5ºC. The variation in SSIIa also affected the eating quality after storage of cooked rice at 5ºC : NIL(Alk) became harder and less sticky than Nipponbare, although the rices were comparable just after cooking. Differential scanning calorimetry revealed faster retrogradation of the once-gelatinized starch in NIL(Alk). The variation in SSI alleles hardly affected these properties.     

Expression Profiles and Protein Complexes of Starch Biosynthetic Enzymes from White-Core and Waxy Mutants Induced from High Amylose Indica Rice

Physicochemical properties of endosperm starches in milled rice determine its cooking and eating quality. Amylose is synthesized by granule-bound starch synthase I (GBSSI), whilst amylopectin is synthesized by the synergistic activities of starch synthases (SSs), branching enzymes (BEs) and debranching enzymes (DBEs). However, the complexes formed by starch biosynthetic enzymes are not well characterized. Gene expression profiles and protein complexes were determined in white-core (GM645) and waxy (GM077) mutants derived from a high amylose indica rice Guangluai 4 (GLA4). In GM645, genes including AGPS1, GBSSI, SSIIa, BEI, BEIIa, BEIIb, PUL, ISA1 and SP were significantly downregulated during seed development. In GM077, the expression levels of AGPL2, AGPS1, AGPS2b, SSIIIa, BEI, PUL and ISA1 were significantly upregulated. Co-immunoprecipitation assays revealed interactions of SSs-BEs, SSs-PUL and BEs-PUL in developing seeds. However, weak SSI-SSIIa interaction was detected in GM077, whilst SSI-PUL interaction was absent. Weak interaction signals for SSI-SSIIa, SSIIa-BEI, SSIIa-BEIIb, BEI-BEIIb and SSI-BEI were also observed in GM645. These results suggest that the protein-protein interactions for starch biosynthesis are modified in mutants, which provides insight into the mechanisms of starch biosynthesis, particularly in indica rice.