文章介绍了江苏省农业科学院国 家水稻改良中心南京分中心近十年来在粳稻品质、抗性和产量潜力改良方面的理论研究与育种成果。实践证明,选用综合性状优良、配合力高的亲本配组,对遗传力 高的性状进行早代选择,可显著提高育种效果;对鉴定难度大的性状利用主基因功能标记和紧密连锁的分子标记进行辅助选择,可大大提高选择效率,加快多基因聚 合速度。产量和株型性状的选择应注意产量构成因素与株型性状间的相互协调,特别要重视稳产性、广适性、抗倒性和后期熟相的选择。

Abstract The concentration of 2-acetyl-1-pyrroline has been determined in the steam volatile oils of 10 different varieties of rice. From these data the amount present in the cooked rice was calculated. This varied from less than 0.006 parts per million (ppm) for Calrose rice to 0.09 ppm for Malagkit Sungsong variety rice based on the dry weight of the rice. Odor panel evaluation described the odor of 2-acetyl-1-pyrroline as "popcorn"-like. Odor evaluation of the amount of popcorn-like odor in the different rice varieties ranked them in the general order of the concentration of this compound. Other odor quality evaluation tests confirmed the importance of this compound to the aroma of the more aromatic rice varieties.

We report here the identification of a DNA marker closely linked to a gene for aroma in rice. The DNA marker was identified by testing 126 mapped rice genomic, cDNA , and oat cDNA , clones as hybridization probes against Southern blots, consisting of DNA from a pair of nearly isogenic lines (NILs) with or without the aroma gene. Chromosomal segments introgressed from the donor genome were distinguished by RFLPs between the NILs. Linkage association of the clone with the gene was verified using an F 3 segregating for aroma. Cosegregation of the scented phenotype and donor-derived allele indicated the presence of linkage between the DNA marker and the gene. RFLP analysis showed that the gene is linked to a single-copy DNA clone, RG28 , on chromosome 8, at a distance of 4.5 cM. The availability of a linked DNA marker may facilitate early selection for the aroma gene in rice breeding programs.

A new approach was developed which succeeded in tagging for the first time a major gene and two QTLs controlling grain aroma in rice. It involved a combination of two techniques, quantification of volatile compounds in the cooking water by gas chromatography, and molecular marker mapping. Four types of molecular marker were used (RFLPs, RAPDs, STSs, isozymes). Evaluation and mapping were performed on a doubled haploid line population which (1) conferred a precise character evaluation by enabling the analysis of large quantities of grains per genotype and (2) made possible the comparison of gas chromatography results and sensitive tests. The population size (135 lines) provided a good mapping precision. Several markers on chromosome 8 were found to be closely linked to a major gene controlling the presence of 2-acetyl-1-pyrroline (AcPy), the main compound of rice aroma. Moreover, our results showed that AcPy concentration in plants is regulated by at least two chromosomal regions. Estimations of recombination fractions on chromosome 8 were corrected for strong segregation distortion. This study confirms that AcPy is the major component of aroma. Use of the markers linked to AcPy major gene and QTLs for marker-assisted selection by successive backcrosses may be envisaged.

The whole rice genome sequence was used to assist in the identification of a single nucleotide polymorphism (SNP) marker linked to the fragrance gene ( fgr) in rice. Genes flanked by restriction fragment length polymorphism and microsatellite markers known to be linked to the fragrance gene were identified by DNA sequence alignment of EST sequences against BAC clones covering this region of chromosome eight. Re-sequencing and comparison of parts of these genes derived from a fragrant and a non-fragrant cultivar revealed only one SNP (a C/T transition) in more than 6 kbp of sequence from 14 genes. Ten of eleven fragrant genotypes and six of 14 non-fragrant genotypes tested carried the C allele. This approach indicated a generally low level of SNP polymorphism in cultivated rice suggesting that association of SNP with phenotypes should be an efficient path to gene discovery in cultivated rice.

水稻香味的遗传机制比较复杂,从1930年开始,就对香味的遗传机理进行过大量的研究,但不同的研究者所得出的结果不同。随着现代生物技术的发展,水稻香味基因的分子标记定位方面的研究报道不少,但目前所找到的PCR标记与香味基因间的遗传距离偏大,不利于有效地开展香味性状的分子标记辅助选择。为此,进一步对水稻香味性状的遗传及其基因的精细定位是十分必要的。本研究以爆玉米花香型水稻品系粤丰B和无香味品种320B为材料,研究了水稻粤丰B的香味遗传机制,并利用SSR标记对控制香味性状的基因进行了标记定位。结果表明,水稻香味受一对隐性基因控制,有香为隐性,无香为显性;在纯合基因型中水稻叶片的香味与米粒的香味呈高度的一致性;但在杂合的基因型中,叶片无香的单株,其米粒有不香与有香的分离;并将隐性香味基因(fgr)定位于第8染色体上,位于SSR标记GR01和RM223之间,与两标记间的遗传距离分别为3.3cM和5.7cM。

对四川省新选育的 7个香稻保持系和 1个引进改良的香稻品种的香味遗传和等位性进行了分析 ,同时 ,利用微卫星 DNA标记对 D香 2 B和泸香 90的香味基因进行了初步定位。结果显示 ,所有香稻品系 (品种 )与非香稻杂交 ,F1 植株的叶片均无香味 ,显示出香味由隐性基因控制 ;从香与香杂交 F1 植株、回交 BC1 F1 和 F2 群体分析看 ,D香 1B、D香 2 B、内香 2 B、内香 4 B、绵香 2 B、绵香 3B、宜香 1B的香味受单隐性基因所控制 ;泸香 90的香味可能受一对抑制基因和一对香味基因控制。初步将 D香 2 B和泸香 90的香味基因定位在第八染色体上 ,D香 2 B

随着现代生物技术的发展,越来越多的报道证明,香稻的香味物质为2-乙酰-1-吡咯啉,水稻的香味性状受位于水稻第8染色体上的1对隐性基因控制。本研究以11个香稻品种和4个非香稻品种为材料,分析了香味基因的遗传;以茉莉花香型的粳稻品种粳香米和非香品种MR63的F2群体为材料,对控制香味性状的基因进行了精细定位。结果表明,水稻香味受1对隐性基因控制,并将香味基因定位在水稻第8染色体上,位于InDeL标记AP05537-17和AP004463-13之间,与标记AP004463-13间的距离为0.4cM,与标记AP05537-17间的距离为1.6cM,在物理图谱上的物理距离大约252kb。分析发现BAC克隆AP004463最有可能包含该基因。最终在该区域内预测有15个基因可能与该香味基因相关。

The flavour or fragrance of basmati and jasmine rice is associated with the presence of 2-acetyl-1-pyrroline. A recessive gene ( fgr ) on chromosome 8 of rice has been linked to this important trait. Here, we show that a gene with homology to the gene that encodes betaine aldehyde dehydrogenase (BAD) has significant polymorphisms in the coding region of fragrant genotypes relative to non-fragrant genotypes. The accumulation of 2-acetyl-1-pyrroline in fragrant rice genotypes may be explained by the presence of mutations resulting in a loss of function of the fgr gene product. The allele in fragrant genotypes has a mutation introducing a stop codon upstream of key amino acid sequences conserved in other BADs. The fgr gene corresponds to the gene encoding BAD2 in rice, while BAD1 is encoded by a gene on chromosome 4. BAD has been linked to stress tolerance in plants. However, the apparent loss of function of BAD2 does not seem to limit the growth of fragrant rice genotypes. Fragrance in domesticated rice has apparently originated from a common ancestor and may have evolved in a genetically isolated population, or may be the outcome of a separate domestication event. This is an example of effective human selection for a recessive trait during domestication.

Rice ( Oryza sativa ) has two betaine aldehyde dehydrogenase homologs, BAD1 and BAD2, encoded on chromosome four and chromosome eight respectively. BAD2 is responsible for the characteristic aroma of fragrant rice. Complementary DNA clones of both BAD1 and BAD2 were isolated and expressed in E. coli . BAD2 had optimum activity at pH 10, little to no affinity towards N-acetyl-γ-aminobutyraldehyde (NAGABald) with a Km of approximately 1002mM and moderate affinity towards γ-guanidinobutyraldehyde (GGBald) and betaine aldehyde (bet-ald) with Km values of approximately 26002μM and 6302μM respectively. A lower Km of approximately 902μM was observed with γ-aminobutyraldehyde (GABald), suggesting BAD2 has a higher affinity towards this substate in02vivo. The enzyme encoded on chromosome four, BAD1, had optimum activity at pH 9.5, showed little to no affinity towards bet-ald with a Km of 302mM and had moderate affinity towards GGBald, NAGABald and GABald with Km values of approximately 545, 420 and 49702μM respectively. BAD1 had a half life roughly double that of BAD2. We discuss the implications of these findings on the pathway of fragrance generation in Basmati and Jasmine rice and the potential of rice to accumulate the osmoprotectant glycine betaine.

The recessive fgr gene on chromosome 8 is associated with rice fragrance. It has been reported that this gene is a non-functional badh2 allele and that the functional Badh2 allele encoding putative betaine aldehyde dehydrogenase (BADH2) could render rice non-fragrant. Here we report the discovery of a new badh2 allele and the development of functional markers for the badh2 locus. A total of 24 fragrant and ten non-fragrant rice varieties were studied and sequenced for their Badh2/badh2 loci. Of the 24 fragrant rice varieties, 12 were found to have the known badh2 allele ( badh2-E7 ), which has an 8-bp deletion and three single nucleotide polymorphisms ( SNPs ) in exon 7; the others had a novel null badh2 allele ( badh2-E2 ), which has a sequence identical to that of the Badh2 allele in exon 7, but with a 7-bp deletion in exon 2. Both null badh2 alleles are responsible for rice fragrance. Based on sequence divergence amongst the functional Badh2 and two null badh2 alleles, we developed functional markers which can be easily used to distinguish non-fragrant from fragrant rice and to differentiate between two kinds of fragrant rice. These functional markers will find their usefulness in breeding for fragrant rice varieties via marker-assisted selection.

水稻甜菜碱醛脱氢酶2基因(Badh2)第7外显子和第2外显子的突变是导致稻米变香的主要原因。本研究分别对包含第7外显子或第2外显子的PCR产物进行测序分析,以明确27种适宜海南省种植的香稻品种的Badh2的突变序列。研究结果表明:海香5701、山栏香糯、海香6309等20个品种Badh2基因的突变类型为第7外显子发生了8 bp的缺失和3个碱基的突变,所有27个水稻香稻品种第2外显子序列都未发生改变。本研究结果可为这些香稻品种的保护与鉴定、及作为香味基因供体改良其他优良品种奠定基础。

pAbstract/p pBackground/p pAromatic rice is popular worldwide because of its characteristic fragrance. Genetic studies and physical fine mapping reveal that a candidate gene (itfgr/it/itOsBADH2/it) homologous to itbetaine aldehyde dehydrogenase /itis responsible for aroma metabolism in fragrant rice varieties, but the direct evidence demonstrating the functions of itOsBADH2 /itis lacking. To elucidate the physiological roles of itOsBADH2/it, sequencing approach and RNA interference (RNAi) technique were employed to analyze allelic variation and functions of itOsBADH2 /itgene in aroma production. Semi-quantitative, real-time reverse transcription-polymerase chain reaction (RT-PCR), as well as gas chromatography-mass spectrometry (GC-MS) were conducted to determine the expression levels of itOsBADH2 /itand the fragrant compound in wild type and transgenic itOsBADH2/it-RNAi repression lines, respectively./p pResults/p pThe results showed that multiple mutations identical to itfgr /itallele occur in the 13 fragrant rice accessions across China; itOsBADH2 /itis expressed constitutively, with less expression abundance in mature roots; the disrupted itOsBADH2 /itby RNA interference leads to significantly increased 2-acetyl-1-pyrroline production./p pConclusion/p pWe have found that the altered expression levels of itOsBADH2 /itgene influence aroma accumulation, and the prevalent aromatic allele probably has a single evolutionary origin./p

2-acetyl-1-pyrroline (2AP) is the principal compound responsible for grain fragrance in rice. In fragrant rice cultivars, BADH2 (betaine-aldehyde dehydrogenase 2) is inactivated. Here, we describe the effect of amiRNA (artificial microRNA) transgenesis targeted at BADH2 in rice. BADH2 (but not BADH1) expression was down-regulated in transgenic lines where the amiRNA was driven by the maize ubiquitin promoter, and in these lines, grain 2AP content was also significantly elevated; meanwhile, 2AP could not be detected in the grain of wild-type lines. The leaf proline content in the transgenics was increased through the simultaneous down-regulation of PRODH and up-regulation of P5CS. In transgenic lines where the same amiRNA was driven by the endosperm-specific promoter GluC, no effect on BADH2 expression was detectable, and the content of grain 2AP did not differ from that present in wild-type grains.

Summary Fragrant rice is favoured worldwide because of its agreeable scent. The presence of a defective badh2 allele encoding betaine aldehyde dehydrogenase (BADH2) results in the synthesis of 2-acetyl-1-pyrroline (2AP), which is a major fragrance compound. Here, transcription activator-like effector nucleases (TALENs) were engineered to target and disrupt the OsBADH2 gene. Six heterozygous mutants (30%) were recovered from 20 transgenic hygromycin-resistant lines. Sanger sequencing confirmed that these lines had various indel mutations at the TALEN target site. All six transmitted the BADH2 mutations to the T1 generation; and four T1 mutant lines tested also efficiently transmitted the mutations to the T2 generation. Mutant plants carrying only the desired DNA sequence change but not the TALEN transgene were obtained by segregation in the T1 and T2 generations. The 2AP content of rice grains of the T1 lines with homozygous mutations increased from 0 to 0.35 0.75 mg/kg, which was similar to the content of a positive control variety harbouring the badh2-E7 mutation. We also simultaneously introduced three different pairs of TALENs targeting three separate rice genes into rice cells by bombardment and obtained lines with mutations in one, two and all three genes. These results indicate that targeted mutagenesis using TALENs is a useful approach to creating important agronomic traits.

【目的】香稻作为一类特殊的水稻群体,以其清香可口的品质特性备受消费者的欢迎。到目前为止,水稻中的香味主要受第8染色体上编码甜菜碱醛脱氢酶基因Badh2控制。【方法】通过CRISPR/CAS9技术对中花11的香味基因Badh2进行编辑。【结果】获得转基因T_0代植株并对其所衍生的T_1代20个单株进行了鉴定分析,获得了一个剔除了载体骨架且第1外显子上插入一个碱基T的突变体材料。该材料中Badh2 RNA水平显著下调;利用GC-MS技术测定野生型及突变体材料籽粒2-乙酰-1-吡咯啉含量,结果表明突变体材料中的香味物质显著增加;此外,我们还对野生型及T_2代香型植株水稻产量及稻米蒸煮食味品质进行了考查及测定分析,发现除分蘖数及结实率呈现出显著差异外(P0.05),其余各项指标在两组材料间都无显著差异。【结论】通过CRISPR/CAS9技术成功地对水稻香味基因进行了编辑,可为香稻育种提供丰富的理论指导,加快香稻的育种进程。

Background To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. Results We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. Conclusions We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

Allele specific amplification (ASA) is a low-cost, robust technique that can be utilised to discriminate between alleles that differ by SNP's, insertions or deletions, within a single PCR tube. Fragrance in rice, a recessive trait, has been shown to be due to an eight02bp deletion and three SNP's in a gene on chromosome 8 which encodes a putative betaine aldehyde dehydrogenase 2 (BAD2). Here we report a single tube ASA assay which allows discrimination between fragrant and non-fragrant rice varieties and identifies homozygous fragrant, homozygous non-fragrant and heterozygous non-fragrant individuals in a population segregating for fragrance. External primers generate a fragment of approximately 58002bp as a positive control for each sample. Internal and corresponding external primers produce a 35502bp fragment from a non-fragrant allele and a 25702bp fragment from a fragrant allele, allowing simple analysis on agarose gels.

香型稻米因其在蒸煮和食用时都会有特殊的香味受到人们的喜爱．研究中，分别以实验室培育的香型正常胚水稻和非香巨胚水稻“上师大5号”作为亲本进行杂交，结合水稻香味基因分子标记辅助常规育种成功选育出香型巨胚水稻“上师大8号”．比较分析“上师大5号”和“上师大8号”两种巨胚水稻主要农艺性状和产量性状．结果显示：虽然“上师大5号”水稻平均每穗实粒数极显著地超过“上师大8号”水稻，但是“上师大8号”有效穗数略多于“上师大5号”水稻，而且千粒重也略高于“上师大5号”水稻，由此两者平均单株重接近，分别为29．10g和28．92g，统计分析差异不显著．两种巨胚水稻胚性状分析显示：“上师大8号”巨胚水稻的胚重量以及胚体积都与“上师大5号”巨胚水稻统计分析无显著差异，而且两种巨胚水稻的胚与糙米重量比以及胚与糙米体积比，统计分析都没有显著差异．开展此研究，为今后香型巨胚水稻的市场开发应用奠定了重要基础．

本研究根据香味基因(badh2-E2)与非香基因(Badh2)在第2外显子7个脱氧核苷酸序列缺失差异,建立了一个新的STS分子标记和检测方法;并根据badh2-E2与Badh2+310 bp位点脱氧核苷酸的差异性,建立一个新的CAPS(EheⅠ)分子标记和检测方法。结果显示:建立的STS分子标记与前人的报道相比扩增的条带更小,只有100 bp左右,能够更加清晰地鉴别出香与非香基因型之间的差异;而建立的CAPS(EheⅠ)分子标记,纯合非香稻可获得长度为292 bp和375 bp两条带,纯合香稻可获得长度为667 bp一条带,杂合F1植株可获得长度为292 bp、375 bp和667 bp3条带,对于不同基因型检测结果的判断效果更明显。采用本研究建立的方法能够提高水稻badh2-E2类型香味基因选择效率。

香味是稻米的重要品质特性之一,在组成香味的100多种化合物中,2-乙酰基-1-吡咯啉(2AP)是最主要的成分。研究表明:位于水稻第8号染色体的一对隐性基因(fgr)控制着2AP的合成。本研究利用和fgr基因连锁的SSR标记RM8264、RM515、RM1109、RM223、RM23147和SNP标记,采用常规PCR法和巢式PCR法,以非香品种黄花占、茉莉香型的香稻特优米、香稻宜香A、宜香A×黄花占为对照,对陕西省水稻种质资源进行香味分析。结果显示:SSR分子标记RM23147没有扩增出片段,RM8264、RM1109在香稻和非香稻品种间没有多态性,RM515、RM223作为水稻香味基因的特异标记鉴定香味效果较为精确,而用在fgr基因区域开发的SNP标记鉴定水稻品种中fgr基因的存在与否更准确。

According to the mutation in exon 7 of 2 (Betaine aldehyde dehydrogenasegene) in fragrant rice,we designed a functional marker YY5-YY8,which can be used to check rice materials with this mutation or not.Other two reported markers for exon 2-mutated and exon 4 or 5-mutated types of 2,InDel-E2 and FMbadh2-E4-5,were also used to analyze the mutations of 2 in 80 fragrant rice materials from different ecological regions.It showed that 26 fragrant rice materials were of the exon 7-mutated type,37 fragrant rice materials were of exon 2-mutated type and none of them was found to be of exon 4 or 5-mutated type.This study constructed a new molecular marker YY5-YY8 for checking the mutation of 2 and identified the mutation types of these fragrant rice varieties for breeding high quality fragrant rice.

香稻是一种珍贵的具有天然香味的功能稻和特种稻,具有食味品质 好、营养保健、经济价值高等优点,因而深受消费者青睐,是当前水稻研究的热点问题.近年来大量的研究揭示了香稻香味的遗传特点及遗传多样性,并进一步对香 稻香味基因进行了精细定位与克隆,这对香稻的育种研究具有重要的指导意义.同时,研究者在香稻的保香栽培方面也取得了积极进展,优质香稻的产业化、无公 害、绿色生产将是未来香稻发展的方向.本文综述了香稻在香味遗传机理、国内外育种状况、香稻选育方法、影响香味的环境因素、保香栽培要点等方面的研究,并 对今后香稻的研究趋势进行了展望,这将为今后香稻的进一步研究提供参考.

以杂交香稻组合培杂软香和常规香稻品种桂香占为材料，采用盆栽试验，研究了施用锌、铁、镧肥 对香稻米质和产量的影响。结果表明，氯化锌基施和喷施及氯化镧基施处理均能显著地提高培杂软香和桂香占的整精米率，6个锌、铁、镧肥施用处理均能显著地增 加桂香占的整精米率而降低其垩白粒率，氯化铁基施和喷施处理亦能显著地降低培杂软香的垩白度；氯化锌喷施处理能显著地提高培杂软香和桂香占的结实率和产 量，氯化镧和氯化铁喷施处理亦能显著提高桂香占的产量。

本文研究了京香1号的挥发物质的构成,并阐明2—乙酰—1—吡咯啉是水稻中的一种重要的香味成分;开发质量检测的快速方法—顶空分析;对不同品种的该物质浓度作了比较;另外还做了 N、Zn 肥对香味效应的研究。

香气是稻米的重要品质特征之一。近20余年来，随着人民生活水平的提高，香米的消费量正在逐年增加。与之相适应，一些农学家和谷物化学家对香米中香气的研究也日趋深入。本文就香气的成分，香气的检定方法、香气的遗传特性以及香气与环境等诸方面的研究成果作一综述。