Fragrance or aroma in rice is considered as a special trait with huge economic importance that determines the premium price in global trade. With the availability of molecular maps and genome sequences, a major gene for fragrance (badh2) was identified on chromosome 8. An 8-bp deletion in the exon 7 of this gene was reported to result in truncation of betaine aldehyde dehydrogenase enzyme whose loss-of-function lead to the accumulation of a major aromatic compound, 2-acetyl 1-pyrroline (2AP) in fragrant rice. However, several studies have reported exceptions to this mutation and indicated the involvement of other genetic loci in controlling fragrance trait. These studies emphasize the need to characterize the fragrance and its underlying factors in a wide range of genetic resources available for this trait. This review summarizes the new insights gained on the genetic and molecular understanding of fragrance in rice.

The aroma of rice plays a role in its consumer acceptability. The popcorn‐like smell of aromatic rice stemming primarily from its 2‐ acetyl‐1‐pyrroline (2‐AP) content is considered desirable by many consumers. Conversely, hexanal has been correlated with off odors in rice that develop from lipid oxidation. A rapid method for 2‐AP and hexanal quantification suitable for use in breeding programs, large‐scale research efforts, and quality assurance programs is needed. While developing such a method, sample preparation (degree of milling, particle size), solvent extraction time and temperature, and gas chromatographic parameters were studied. Particle size had no influence on 2‐AP or hexanal recovered. One extraction solubilized ≈80% of the 2‐AP and 56% of the hexanal present in milled rice. The optimum extraction method was assessed to require 0.3 g of ground brown or milled rice in methylene chloride held at 85°C for 2.5 hr. The complete gas chromatographic run requires ≈25 min, and 50 samples can be analyzed per day. The optimized method's linear response (R2 = 0.99) and reproducibility was demonstrated. The stability of 2‐AP and hexanal in frozen milled rice and in refrigerated methylene chloride extracts was excellent for at least six months. Milled and unmilled commercial and breeders' aromatic rice samples contained 10–1,104 ng/g of 2‐AP and 148–2,541 ng/g of hexanal. Genotype had the greatest effect on the 2‐AP and hexanal content of two lines grown over four years and in four states.

A better understanding of the factors that contribute to the overall grain quality of rice (Oryza sativa) will lay the foundation for developing new breeding and selection strategies for combining high quality, with high yield. This is necessary to meet the growing global demand for high quality rice while offering producing countries additional opportunities for generating higher export revenues. Several recent developments in genetics, genomics, metabolomics and phenomics are enhancing our understanding of the pathways that determine several quality traits. New research strategies, as well as access to the draft of the rice genome, will not only advance our understanding of the molecular mechanisms that lead to quality rice but will also pave the way for efficient and targeted grain improvement.

MRQ74, a popular aromatic Malaysian landrace, allows for charging considerably higher prices than non-aromatic landraces. Thus, breeding this profitable trait has become a priority for Malaysian rice breeding. Despite many studies on aroma genetics, ambiguities considering its genetic basis remain. It has been observed that identifying quantitative trait loci (QTLs) based on anchor markers, particularly candidate genes controlling a trait of interest, can increase the power of QTL detection. Hence, this study aimed to locate QTLs that influence natural variations in rice scent using microsatellites and candidate gene-based sequence polymorphisms. For this purpose, an F2 mapping population including 189 individual plants was developed by MRQ74 crosses with 'MR84', a non-scented Malaysian accession. Additionally, qualitative and quantitative approaches were applied to obtain a phenotype data framework. Consequently, we identified two QTLs on chromosomes 4 and 8. These QTLs explained from 3.2% to 39.3% of the total fragrance phenotypic variance. In addition, we could resolve linkage group 8 by adding six gene-based primers in the interval harboring the most robust QTL. Hence, we could locate a putative fgr allele in the QTL found on chromosome 8 in the interval RM223-SCU015RM (1.63cM). The identified QTLs represent an important step toward recognition of the rice flavor genetic control mechanism. In addition, this identification will likely accelerate the progress of the use of molecular markers for gene isolation, gene-based cloning, and marker-assisted selection breeding programs aimed at improving rice cultivars. Copyright © 2014 Elsevier B.V. All rights reserved.

In rice (Oryza sativa), the presence of a dominant Badh2 allele encoding betaine aldehyde dehydrogenase (BADH2) inhibits the synthesis of 2-acetyl-1-pyrroline (2AP), a potent flavor component in rice fragrance. By contrast, its two recessive alleles, badh2-E2 and badh2-E7, induce 2AP formation. Badh2 was found to be transcribed in all tissues tested except for roots, and the transcript was detected at higher abundance in young, healthy leaves than in other tissues. Multiple Badh2 transcript lengths were detected, and the complete, full-length Badh2 transcript was much less abundant than partial Badh2 transcripts. 2AP levels were significantly reduced in cauliflower mosaic virus 35S-driven transgenic lines expressing the complete, but not the partial, Badh2 coding sequences. In accordance, the intact, full-length BADH2 protein (503 residues) appeared exclusively in nonfragrant transgenic lines and rice varieties. These results indicate that the full-length BADH2 protein encoded by Badh2 renders rice nonfragrant by inhibiting 2AP biosynthesis. The BADH2 enzyme was predicted to contain three domains: NAD binding, substrate binding, and oligomerization domains. BADH2 was distributed throughout the cytoplasm, where it is predicted to catalyze the oxidization of betaine aldehyde, 4-aminobutyraldehyde (AB-ald), and 3-aminopropionaldehyde. The presence of null badh2 alleles resulted in AB-ald accumulation and enhanced 2AP biosynthesis. In summary, these data support the hypothesis that BADH2 inhibits 2AP biosynthesis by exhausting AB-ald, a presumed 2AP precursor.

稻米香味是水稻的重要食味品质之一, 其主要受第8染色体上编码甜菜碱脱氢酶基因Badh2控制, 该基因突变可导致香味物质2-乙酰-1-吡咯啉(2-AP)的含量增加从而促进香味的产生。本研究以三明市农业科学研究院自主选育的优质籼型杂交稻保持系明太B为受体, 利用CRISPR-Cas9技术对其Badh2基因进行编辑和敲除。获得2个T₀代转基因纯合突变体植株并对其衍生的48个T₁代单株进行鉴定和分析, 获得1个不含转基因载体骨架且在第2外显子插入单个碱基T的纯合突变体株系明太B-badh2。利用半定量PCR和qRT-PCR技术以及气相色谱质谱联用仪(GC-MS)检测Badh2基因相对表达量和2-AP含量; 同时采用农业行业标准(NY/T 1433-2014)推荐的48对水稻SSR引物进行指纹图谱分析。结果表明, 该株系Badh2基因RNA表达水平显著下调; 籽粒中香味物质2-AP的含量显著增加; 指纹图谱分析发现, 仅1对引物Rm571在野生型和突变体之间鉴定到等位变异, 两组材料遗传差异较小。此外, 本研究还对野生型和突变体T₂代植株表型性状、稻米蒸煮食味品质和外观品质指标进行了考察和测定分析。结果表明, 所有指标在两组材料间均无显著差异。进一步采用测交和回交转育方法并结合Badh2位点测序分析, 成功选育获得了其对应的纯合香型三系不育系明太A-badh2。通过与恢复系明恢703、明恢3009测配, 其组合产量与国家审定品种明太优703、明太优3009相近且表现出较强的超标优势。此外, 通过与香型恢复系明恢1831测配后发现其组合籽粒中香味物质2-AP含量极显著高于对照组合明太A/明恢1831。因此, 利用CRISPR-Cas9基因编辑技术, 可对水稻香味基因Badh2进行精准定向编辑和敲除, 实现对水稻香味性状的改良, 为创制香型籼稻不育系提供理论指导, 从而加快香型杂交稻育种进程。

CRISPR/Cas9基因编辑技术已经成为水稻育种新型手段，香稻育种一直是水稻育种行业的研究热点，水稻香味主要由8号染色体上的甜菜碱脱氢酶基因Badh2控制。为了改良原有品种的香味性状，促进香稻育种的发展，以非香型粳稻品种东农425为试验材料，构建了2个CRISPR/Cas9敲除载体对Badh2基因进行定点编辑，第1个载体（pYLCRISPR/Cas9-B1-gRNA）的2个靶点分别位于第2和第3外显子上，第2个载体（pYLCRISPR/Cas9-B2-gRNA）的2个靶点均在第2外显子上。采用农杆菌介导法对东农425进行遗传转化，成功获得了T₀纯合植株，并对其衍生出的T₁植株进行T-DNA元件检测，共获得8个突变类型不同且不含有转基因成分的纯合突变株系。同时采用咀嚼法和氢氧化钾浸泡法对这8个纯合突变株系的水稻籽粒进行香味检测，结果表明，8个Badh2株系均具有不同程度的香味，总体上载体pYLCRISPR/Cas9-B1-gRNA编辑的3个株系的香味更为浓郁。对这8个纯合突变株系的主要农艺性状进行考察，发现突变株系的穗数、穗粒数、结实率及千粒质量与野生型相比均没有明显差异。综上，本研究对东农425的Badh2基因成功地进行了编辑，并获得了香味显著提高且无转基因成分的纯合突变体材料，为加快香型粳稻品种的培育提供了技术支持。

单向导RNA (sgRNA)是CRISPR/Cas9基因组编辑技术体系的重要元件之一。然而研究显示, 很多sgRNA不能有效工作, 因此需要对多个设计的候选sgRNA进行筛选, 以验证它们的有效性。早期对sgRNA有效性的验证采用的是完整编辑载体瞬时转化原生质体或者叶片的方法。这些方法费时费力, 成功率不高, 尤其是对于原生质体制备效率比较低的棉花。本研究针对GhMAPKKK2和GhAE基因分别设计靶序列, 构建了只转录sgRNA的载体: GhU6-5P::MAPKKK2-sgRNA-1300和GhU6-5P::AE-sgRNA-1300, 并通过农杆菌注射YZ-1 Cas9转基因棉花植株叶片; 与此同时, 构建了对应完整的CRISPR/Cas9 基因组编辑载体: GhU6-5P::MAPKKK2-sgRNA-Cas9和GhU6-5P::AE-sgRNA-Cas9, 并通过农杆菌注射YZ-1野生型棉花植株的叶片。另外, 针对GhPDS、GhCLA1、GhMAPKKK2和GhAE基因分别设计靶序列并构建了GhU6-5P-2::PDS-sgRNA-CLCrVA、GhU6-5P-2::CLA1- sgRNA-CLCrVA、GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA和GhU6-5P-2::AE-sgRNA-CLCrVA病毒投送载体, 通过农杆菌注射YZ-1 Cas9转基因棉花植株叶片。以上试验均以转化对应空载体的植株为对照。对转化后的棉花叶片基因组DNA进行PCR扩增后酶切, 并对未完全消化的PCR产物进行克隆测序, 结果显示, 转化GhU6-5P::AE-sgRNA- 1300、GhU6-5P::MAPKKK2-sgRNA-Cas9、GhU6-5P::AE-sgRNA-Cas9载体的棉花植株均未检测到靶基因突变, 而转化GhU6-5P::MAPKKK2-sgRNA-1300、GhU6-5P-2::PDS-sgRNA-CLCrVA、GhU6-5P-2::CLA1-sgRNA-CLCrVA、GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA和GhU6-5P-2::AE-sgRNA-CLCrVA载体的Cas9转基因阳性植株基因序列发生了改变, 突变类型包括碱基替换、碱基缺失和碱基插入。表明以Cas9转基因阳性植株为转化受体的策略可以高效真实地验证sgRNA的有效性, 排除了因转化效率低而带来的假阴性的结果, 且病毒载体投送sgRNA的策略更高效、更准确。该sgRNA高效验证体系的建立, 为棉花功能基因组学研究提供了重要的技术基础。

【目的】 为了促进耐盐香稻育种，利用CRISPR/Cas9系统对粳稻品种连粳11的Badh2和OsRR22基因进行编辑，以期快速获得一批不含有转基因成分且具有耐盐性和香味的纯合水稻材料。【方法】 根据Badh2和OsRR22基因序列中编辑位点的敲除效率设计靶位点，构建pH-Ubi-Cas9-Badh2-OsRR22敲除载体，利用农杆菌介导法转入受体品种连粳11中。对转基因后代进行潮霉素和Cas9标记PCR检测以及靶基因测序，获得无外源基因插入的badh2-osrr22纯合株系，并对后代种子性状和苗期耐盐性进行分析。【结果】 T₂代成熟种子中2-AP含量较背景材料连粳11显著增加，千粒重、粒长、粒宽无明显变化；128 mmol/L氯化钠处理14 d后株系21-30较连粳11苗高增加15.2%，苗鲜质量增加45.2%，苗干质量增加13.2%。【结论】 利用CRISPR/Cas9技术对Badh2和OsRR22基因编辑获得能稳定遗传且无外源基因插入的耐盐香稻材料，加快了水稻多性状聚合的选育进程。

The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has been demonstrated to be a robust genome engineering tool in a variety of organisms including plants. However, it has been shown that the CRISPR/Cas9 system cleaves genomic DNA sequences containing mismatches to the guide RNA strand. We expected that this low specificity could be exploited to induce multihomeologous and multiparalogous gene knockouts. In the case of polyploid plants, simultaneous modification of multiple homeologous genes, i.e. genes with similar but not identical DNA sequences, is often needed to obtain a desired phenotype. Even in diploid plants, disruption of multiparalogous genes, which have functional redundancy, is often needed. To validate the applicability of the CRISPR/Cas9 system to target mutagenesis of paralogous genes in rice, we designed a single-guide RNA (sgRNA) that recognized 20 bp sequences of cyclin-dependent kinase B2 (CDKB2) as an on-target locus. These 20 bp possess similarity to other rice CDK genes (CDKA1, CDKA2 and CDKB1) with different numbers of mismatches. We analyzed mutations in these four CDK genes in plants regenerated from Cas9/sgRNA-transformed calli and revealed that single, double and triple mutants of CDKA2, CDKB1 and CDKB2 can be created by a single sgRNA.© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.