无抗性选择标记转基因软米和糯稻新品系的选育及中间试验

直链淀粉含量是影响稻米品质的最重要因素之一。为培育直链淀粉含量较低的软米和糯稻新品系,在经共转化法获得的转反义Wx基因水稻自交后代中筛选获得国不含抗性选择标记基因的纯合体.对这些纯合转基因水稻的分析表明,其未成熟种子胚乳中Wx基因表达水平和成熟种子中Wx蛋白量较未转化对照有不同程度的降低。转基因水稻成熟种子中直链淀粉含量较未转化对照有不同程度的下降,2个转基因系降低至10%左右,相当于软米的直链淀粉含量,1个转基因系降到了2%以下,形成了糯稻新品系。转基因稻米中直链淀粉含量降低后,其胶稠度相应变软,而糊化温度未发生明显改变。田间试验结果表明,转基因水稻的大部分农艺性状与受体品种武香粳9号无显著差异。因此,本研究成功培育了无抗性选择标记且淀粉品质改良的转反义Wx基因水稻新品系。     

Evaluation of the waxy endosperm trait in proso millet (Panicum miliaceum)*

The entire USDA‐ARS maintained collection of 650 accessions of proso millet (Panicum miliaceum L.) was evaluated for the presence of accessions with waxy (amylose‐free) endosperm starch. Six accessions, five of which derived from mainland China, were identified. Segregation ratios for waxy endosperm were evaluated in F₂ and F₃ populations derived from crosses between two waxy accessions, PI 436625 (Lung Shu 16) and PI 436626 (Lung Shu 18), and several wild‐type accessions. The waxy trait was found to be under the control of duplicate recessive alleles at two loci, herein designated wx‐1 and wx‐2. Wild‐type alleles at these loci were designated Wx‐1 and Wx‐2. Iodine‐binding revealed a mean grain‐starch amylose concentration of 3.5% in waxy lines and 25.3% in wild‐type proso. Expression of the granule‐bound starch synthase (waxy protein) in waxy lines was reduced to approximately one‐tenth that of wild‐type accessions. The waxy accessions identified now are available for the introgression of this trait into breeding lines adapted to the Great Plains of North America.     

反义trxs基因导入对不同品质类型小麦产量和品质性状的影响

以转反义trxs基因小麦TY18和TY34及其相应未转基因对照为材料,于2007-2009年通过大田试验系统研究了反义trxs基因导入对两种品质类型小麦籽粒产量性状和加工品质指标的影响。结果表明,4个转基因株系的穗粒数和产量较对照显著提高,反义trxs基因对小麦籽粒淀粉品质有一定的正效应。4个转基因株系的淀粉含量、支链淀粉含量、峰值黏度、低谷黏度、最终黏度均显著提高,直/支比降低。反义trxs基因对小麦籽粒蛋白质品质的影响因品种类型而异,其中弱筋小麦的总蛋白、清蛋白、球蛋白、谷蛋白含量显著减少,醇溶蛋白含量显著增加,面粉的形成时间和稳定时间降低。强筋小麦除清蛋白、醇溶蛋白、谷蛋白含量减少外,面团的粉质和拉伸参数变化不明显。上述结果说明,反义trxs基因导入有利于两种品质类型小麦籽粒淀粉品质的改善,并在一定程度上改善了弱筋小麦的烘焙品质。     

QTL for rice grain quality based on a DH population derived from parents with similar apparent amylose content

A doubled haploid (DH)population consisting of 135 lines, derived from an indica (IR64) and a japonica (Azucena) rice with a similar apparent amylose content (AAC), was used to investigate the genetic factors affecting cooking and eating quality of rice. AAC,gelatinization temperature (GT), gel consistency (GC) and six starch pasting viscosity parameters were measured for quantitative trait loci (QTL) analysis using 193 molecular markers mapped on the DH population. A total of 17 QTLs were detected for the 9 traits, with at least one QTL and as many as 3 QTLs for each individual trait. No QTL for the measured parameters was found at the wx locus,possibly because of the similar AAC between the parents. Several QTLs with important effects on the variations in the measured parameters were detected in the present study which have not been found in earlier reports based on populations derived from parents with different AAC and wxgene alleles. Two interesting loci could be deduced from the present study according to the marker order compared with other genetic linkage maps. A QTL flanked by Amy2A and RG433 on the end of the long arm of chromosome 6, identified for GT, set back and consistency viscosity, might cover the gene encoding starch branching enzyme I. Similarly, a QTL flanked by RG139 and RZ58on chromosome 2, detected for hot paste viscosity and breakdown viscosity, might cover the gene encoding starch branching enzyme III. Generally, traits significantly correlated with each other shared identical QTL, but it was not true in some cases. The fine molecular mechanisms underlying these traits await further elucidation for the improvement of eating and cooking quality of rice. This revised version was published online in August 2006 with corrections to the Cover Date.     

Marker‐assisted introgression of bacterial blight and blast resistance into IR 58025B,an elite maintainer line of rice

IR 58025A is a very popular wild‐abortive cytoplasmic male sterile (WA‐CMS) line of rice and is extensively used for hybrid rice breeding. However, IR 58025A and many hybrids derived from it possess mild aroma (undesirable in some parts of India) and are highly susceptible to bacterial blight (BB) and blast diseases. To improve IR 58025A for BB and blast resistance, we have introgressed a major dominant gene conferring resistance against BB (i.e. Xa21) and blast (i.e. Pi54) into IR 58025B, the maintainer line of IR 58025A. An introgression line of Samba Mahsuri (i.e. SM2154) possessing Xa21 and Pi54 genes in homozygous condition and fine‐grain type was used as donor parent, and backcross breeding strategy was adopted for targeted introgression of the resistance genes. PCR‐based molecular markers tightly linked to Xa21 and Pi54 were used for selection of BB‐ and blast‐resistant lines, while closely linked markers were used for identification of backcross‐derived plants devoid of Rf4 and aroma. At BC₂F₅, four backcross‐derived lines possessing resistance against BB and blast, devoid of aroma, high yield, short plant stature, long‐slender grain type and with recurrent parent genome recovery ranging from 88.8% to 98.6% were selected and advanced for further evaluation. The improved versions of IR 58025B, viz. SB54‐11‐143‐9‐44‐5, SB54‐11‐143‐9‐44‐98, SB54‐11‐143‐9‐44‐111 and SB54‐11‐143‐9‐44‐171, behaved as perfect maintainers when test‐crossed with WA‐CMS lines. Agronomically superior lines of improved IR 58025B are being converted to CMS line through backcrossing for developing high‐yielding and biotic stress‐resistant rice hybrids.     

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.     

水稻蛋白二硫键异构酶基因沉默载体构建及其转基因后代的高温结实特性分析

采用RT-PCR法亚克隆了PDI基因保守区内450 bp的靶标序列作为干扰区段, 构建了含有内含子hpRNA (ihpRNA)的双元表达载体pTCK303-RiOsPDI, 经农杆菌介导转化日本晴, 获得转基因植株; 通过在T₀代对其潮霉素(Hyg)抗性基因的PCR鉴定, 确定携带有干扰片段的T-DNA区已整合到水稻基因组中, 且在转基因T₁代符合3∶1的分离模式。半定量PCR和荧光定量PCR的检测结果表明, PDI基因沉默转基因阳性植株不同器官中的PDI表达量均显著降低, 尤其是其籽粒中表达量较微, 几乎能引起靶基因80%左右沉默。对转基因T₂代植株的高温结实特性和籽粒理化品质的检测结果, PDI基因沉默会引起高温胁迫处理下结实率的大幅度降低, 耐热性显著下降, 但其在常温处理下的结实率与对照之间无显著差异。此外, PDI基因沉默后, 稻米的透明度下降、垩白度增加, 但对籽粒粗蛋白总量和直链淀粉含量的影响不甚明显。     

不同粳稻品种和DH群体稻米淀粉RVA谱特征与蒸煮理化指标及相关分析

测定18个日本品种（系）、18个江苏品种（系）及3个DH群体粳稻稻米淀粉RVA谱特征值和蒸煮理化指标并分析了不同测定值间的相关性，结果表明如下：（1）江苏品种（系）峰值黏度（PKV）、热浆黏度（HPV）、冷胶黏度（CPV）、回复值（CSV）和直链淀粉含量（AC）均高于日本品种（系），糊化开始温度（PaT）低于日本品种（系），崩解值（BDV）、消减值（SBV）和胶稠度（GC）则差异不显著。根据RVA谱特征值将品种分为4种类型。（2）3个DH群体的RVA谱特征值和蒸煮理化指标均有广泛变异和超亲现象，其中以SBV变异系数最大。（3）各RVA谱特征值间有较好的相关性，PKV、HPV和CPV之间相互呈极显著正相关，PKV、HPV与BDV分别呈显著或极显著正相关，HPV、CPV、SBV与CSV分别呈极显著正相关，BDV与SBV呈极显著负相关，5个群体表现出比较好的一致性。另外，在3个DH群体中，CPV与BDV也呈现极显著正相关，PaT与PKV、BDV均呈显著或极显著正相关，PaT与SBV呈极显著负相关。（4）3个DH群体的蒸煮理化指标与RVA谱特征值的关系也很密切，AC与SBV显著或极显著正相关，与PaT显著或极显著负相关；GC与PKV、BDV、PaT极显著正相关，与SBV极显著负相关。用HPV、CPV、SBV、CSV和PaT5个特征值能较好反映稻米食味品质的优劣。     

Mapping QTLs for grain quality using an introgression line population from a cross between <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Emphasis Type="Italic">O. rufipogon</Emphasis>

Quantitative trait loci (QTL) associated with four milling recovery properties, two chemical properties, six paste properties of grain, and six textural parameters of cooked rice were identified using an introgression line (IL) population of rice developed from an interspecific cross over two years. The IL population consisted of 121 lines from a cross between wild rice (O. rufipogon Griff.) and a japonica cultivar. A total of 28 QTLs were identified for the 14 traits using inter mapping. Of these, 10 were significant over two years indicating that these QTLs are stable across years and environments. For eight (21%) of the QTLs identified, the O. rufipogonderived alleles contributed a desirable effect on amylose content, protein content, minimum viscosity, final viscosity, and consistency. Among these, pc3 for protein content and ac7 for amylose content were significant in both years and showed an R² value of 25.5 and 30.9%, respectively. The markers closely associated with these useful alleles can be used to trace the inheritance of specific chromosome segments in the IL population and also offer a starting point for map-based cloning of genes underlying these traits.     

结实期水分胁迫对水稻反义Wx基因转化系主要米质性状及米饭质地的影响

以反义Wx基因的转化系及其受体亲本武运粳7号(9522)为材料,研究了结实期水分胁迫对主要品质性状及米饭质地的影响。结果表明,与水层灌溉处理相比,结实期水分胁迫使整精米率和垩白粒率提高;崩解值明显降低,而最终黏度和消减值却明显提高;米饭的硬性增加,黏附性减小,稻米的蒸煮食味品质有着变劣的趋势。     

Identification and gene cloning of a male‐sterile mutant Oswbc11 in rice

Commercial application of nuclear genetic male sterility has been improved recently by a novel technique, Seed Production Technology (SPT), which incorporates transgenic maintainer lines capable of propagating non‐transgenic nuclear male‐sterile lines for use as female parents in hybrid production. Here, we identified a rice nuclear male‐sterile mutant, Oswbc11, with abnormal pollen development and lipid transport. We finely mapped the Oswbc11 gene into a 12.5‐kb region on chromosome 10 and found one candidate gene, which had a base substitution (C to T) resulting in a premature stop codon and was functionally confirmed by CRISPR/Cas9 technology. Gene OsWBC11 encodes a sub‐family member of adenosine triphosphate‐binding cassette transporter, which participates in the active transport of a wide range of molecules across membranes. Moreover, the agronomic traits of Oswbc11 mutants showed no significant differences compared to the wild‐type control except for the seed setting rate. These results indicated that Oswbc11 gene could be used in rice hybrid breeding as a recessive nuclear male‐sterile gene combined with CRISPR/Cas9 and the SPT technology and applied in different rice varieties.     

Comparative Study of CaMsrB2 Gene Containing Drought‐Tolerant Transgenic Rice (Oryza sativa L.) and Non‐Transgenic Counterpart

Concerns regarding the safety of transgenic foods have been raised because of possibility of undesirable effects development during genetic engineering. Analysis of phenotypic traits can increase the likelihoods of identifying those unintended effects in dietary composition of the GM crops. Objective of this study was to compare the transgenic lines with their non‐transgenic counterpart. Different vegetative and reproductive traits as well as antioxidant properties were considered to evaluate the transgenic (HV8 and HV23) lines containing CaMsrB2 gene and their non‐transgenic (Ilmi) parent line. Grain size and weight, seed germination, root length, root and shoot dry weight, length and width of flag leaf, plant height, and ligule, stamen and carpel length were not significantly different. Onset and completion of heading in each line occurred almost during the same period. The antioxidant properties in terms of DPPH (1,1‐diphenyl‐2‐picrylhydrazyl) radical scavenging activity and polyphenol content were not statistically different under same treatment condition. The results suggested that the transgenic rice lines containing CaMsrB2 gene were equivalent to their non‐transgenic counterpart without any visible unintended effects.     

不同生态条件下籼稻米饭质地和淀粉RVA谱的特性

研究了6个籼稻品种在大范围不同的生态环境条件下,米饭质地和淀粉RVA特征谱的变化。结果表明,供试样品的米饭质地指标硬度和黏度存在极显著环境主效应,环境与品种互作效应在硬度、黏附性和黏度3个性状中均极显著。6项RVA特征值（峰值黏度、热浆黏度、最终黏度、崩解值、消减值和回复值）均存在极显著环境主效应;环境与品种互作效应在上述性状中,均极显著;且生态条件对RVA谱的影响因品种不同而异。6个试验点中,米饭质地指标黏附性的变异系数最小;RVA的热浆黏度、最终黏度、峰值黏度、崩解值和回复值以及米饭质地的硬度、黏度值的变异系数较小;RVA的消减值的变异系数最大。     

Breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer and herbicide

In this paper, we described the breeding of transgenic rice restorer line for multiple resistance against bacterial blight, striped stem borer (SSB) and herbicide by conventional crossing of two transgenic parental lines transformed independently with different genes. Two stable transgenic rice lines used as donor parents were developed, one was Zhongguo91 which contained cry1Ab gene (for insect resistance) and bar gene (for tolerance of herbicide), and the other was Yujing6 which contained Xa21 gene (resistance to bacterial blight). The elite restorer line Hui773 was used as recipient and crossed with the two stable transgenic rice lines. Then five successive backcrosses were made using Hui773 as recurrent parent. Two rice elite restorers, T773-1 expressing cry1Ab and bar genes and T773-2 expressing Xa21 gene, were obtained, which were confirmed by PCR analysis and testing selectable marker genes in the hybrid progenies. The cross was made between T773-1 and T773-2 to select stable restorer line carrying Xa21, cry1Ab and bar genes. Finally, we obtained transgenic restorer line T773 with good agronomic traits and obvious multiple resistance to Xanthomonas oryzae pv. oryzae, striped stem borer (Chilo suppressalis) and herbicide. The hybrid F₁ generation produced from the cross between transgenic restorer line T773 and a corresponding male sterile line Zaohua2A maintained obvious resistance to rice bacterial blight, rice leaffolder and striped stem borer, and showed significant heterosis. Our results indicate that it is feasible to develop transgenic hybrid rice cultivar through breeding transgenic restorer lines.     

Molecular detection of WA‐CMS restorers from tropical japonica‐derived lines,their evaluation for fertility restoration and adaptation

Hybrid rice based on wild‐abortive cytoplasmic male sterility (WA‐CMS) is important in boosting rice production, which requires diverse parents to harness heterosis. For this, exploiting the diversity of japonica through tropical japonica (TRJ) lines is an excellent route. In this study, 310 TRJ‐based new plant type (NPT) lines were developed and evaluated for Rf3 and Rf4 genes. Gene‐based (DRRM‐Rf3‐5 and DRRM‐Rf3‐10) and functional marker (RMS‐SF21‐5) targeted Rf3 locus, while gene‐linked (RM6100) and functional marker (RMS‐PPR9‐1) targeted the Rf4 locus. The frequency of the restorer allele of Rf3 gene was lower when compared to that of Rf4. Combined phenotypic and molecular screening using gene‐based and functional markers identified 42 lines that carried Rf3 and/or Rf4 genes. All the selected lines produced fertile F₁s when crossed to a WA‐CMS line, “Pusa 6A”, but with varying levels of spikelet fertility. This is the first report of a marker‐cum‐phenotype‐based restorer selection using TRJ‐derived lines. Multilocation evaluation of these lines at three locations indicated better adaptation for grain yield in some of the lines.     

反义Trxs基因导入对弱筋小麦豫麦18淀粉积累及淀粉合成关键酶表达的影响

以转反义硫氧还蛋白基因小麦TY18-99和TY18-100及其相应未转基因对照为材料,于2007-2009年通过盆栽试验系统研究了反义Trxs基因(anti-Trxs)导入对弱筋小麦豫麦18籽粒灌浆过程中淀粉积累、淀粉合成关键酶活性以及淀粉合成酶基因表达的影响。结果表明,反义Trxs基因对小麦籽粒淀粉积累有一定的正效应。2个转基因株系的总淀粉和支链淀粉的积累速率较对照显著提高;在整个籽粒形成过程中,反义Trxs基因导入显著提高了淀粉分支酶(SBE)活性。在籽粒灌浆中期和后期,反义Trxs基因导入显著提高了腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)和可溶性淀粉合酶(SSS)活性,降低了颗粒束缚型淀粉合酶(GBSS)活性。实时荧光定量RT-PCR分析表明,反义Trxs基因促进了AGPase、SBE I和SSS (SSS I、SSS II和SSS III)基因的表达,抑制了GBSS I基因的表达。上述结果说明,反义Trxs基因导入后促进了AGPase、SBE I和SSS基因的转录,从而使籽粒灌浆期间的淀粉积累速率发生了改变,最终显著提高了总淀粉和支链淀粉的含量,降低了直链淀粉含量,进而提高了淀粉的支/直比,这可能是转基因小麦淀粉品质改善和产量提高的主要原因。     

Identification of allelic variations of puroindoline genes controlling grain hardness in wheat using a modified denaturing PAGE

The amount of long chains (LC) of amylopectin in high-amylose rice is thought to be one of the important determinants of its quality when cooked. A wide range of differences in LC content have been reported in rice varieties, which can be clearly divided into four classes based on LC and apparent amylose content: namely, amylose and LC-free, low or medium-amylose and low-LC, high-amylose and medium-LC, high-amylose and high-LC. However, genetic factors controlling LC content have not been fully understood. Here, we performed quantitative trait loci (QTL) analysis of LC content using 157 recombinant inbred lines (RILs) derived from a cross of a low-LC cultivar, Hyogokitanishiki, and a high-LC line, Hokuriku 142. By analyzing randomly selected 15 RILs, it was shown that high LC content (≥11%) was associated with high setback viscosity (≥200 RVU), and that low LC (≤ 3%) was associated with low setback viscosity (≤ 130 RVU), as measured by a Rapid Visco Analyzer. With setback viscosity as an indicator for LC content, QTL analysis was conducted using 60 DNA markers including a CAPS marker that distinguished Wx a and Wx b alleles coding for granule-bound starch synthase I (GBSSI or Wx protein), the enzyme working for amylose biosynthesis. Only one QTL with a peak log of likelihood score at the wx locus was detected, and no line showing setback viscosity corresponding to the medium-LC class appeared. The fact that wx mutants of Hokuriku 142 lacked LC in their rice starch supports the view that the functional Wx allele is indispensable for LC synthesis in addition to amylose synthesis in rice endosperm. We suggest three possible reasons why no line with medium-LC content was observed. First, the locus (loci) responsible for generation of medium-LC may be located very close to the wx locus and not able to be dissected by the population and DNA markers we used. Second, there may be special QTLs for medium-LC cultivars that do not exist in low- or high-LC cultivars. Third, medium-LC cultivars may have an as-yet unidentified Wx allele with lower capability in LC synthesis compared to the Wx allele in high-LC cultivars.     

Pasting properties of colored rice starch by ball-mill treatment

Physically modified rice starch by ball-mill is important for making good quality bread, noodles, and several processed foods. The production of rice flour was done by ball-mill in an industrial scale. The effects of ball-mill treatment on the physicochemical properties of rice starch were investigated. The gel pasting properties of Joseongheugchal employed to ball-mill treatment showed that pasting time was 0.44 min shorter than that of the control and it also decreased peak viscosity (PKV), hot peak viscosity (HPV), and cold paste viscosity (CPV). In Hongjinju rice, RVA (rapid visco analyzer) pasting trace was characterized with high peak viscosity (3,448 RVU: rapid visco unit), and cold paste viscosity (2,975 RVU), also with high breakdown viscosity (1,597 RVU). The onset, peak, and conclusion gelatinization temperatures of ball-milled Josengheugchal rice starch were 57.4, 64.2, and 76.5°C, respectively, which were significantly higher than those of the control. The parameters of DSC (Differential scanning calorimetry) of ball-milled Hongjinju rice were 57.9, 65.1, and 77.3°C, respectively. The mean particle size of starch in both Josengheugchal and Hongjinju by ball-milling was decreased. The distribution of mean particle size in ball-milled rice starch was smaller in Josengheugchal than in Hongjinju compared to the native starch. As rice grains were ball-milled, the regions of amorphism were becoming larger and larger at the expense of crystalline regions, and diffraction peak patterns were decreased commonly in two colored rice cultivars. The ¹³C FT-solid state NMR patterns of starches showed dramatic chemical shifts of the ¹³C resonances by ball-milled treatment.