24种香稻品种甜菜碱醛脱氢酶2基因突变位点的分析及分子标记开发

根据香型与非香型水稻甜菜碱醛脱氢酶2基因(badh2)在第2、第4内含子、第7外显子3处序列差异和第2外显子1处序列差异,分别设计了两类检测badh2第7和第2外显子突变的功能性分子标记引物M7和M2;利用两类引物,分别对属于第7外显子突变的香型水稻W香99075和第2外显子突变的香型水稻武香14、非香型水稻以及两种香稻分别与非香稻杂交的F1植株基因组DNA进行PCR检测后发现,M7和M2引物完全能够分别被用于以第7和第2外显子突变的香稻作为亲本,进行分子标记辅助培育香稻新品种的研究.M7引物综合考虑了badh2内含子和外显子两方面突变情况而设计的.以非香稻261S、分别发生第7和第2外显子突变的香稻品种W香99075和武香14为对照,使用M7和M2引物,对本实验室收集的另外22份香稻品种进行badh2突变位点检测,结果可将这些香稻分为badh2第2外显子突变类型、第7外显子突变类型和外显子未发生突变类型,同时明确了大多目前在上海等周边地区种植的香稻品种的badh2所属的突变位点.开展本研究为利用分子标记辅助选育香型水稻新品种研究奠定了重要的基础.     

Genotypes Analyses of Ten Genes Related to Eating Quality of Twenty-three Fragrant Rice Cultivars by Molecular Marker

Fragrance is a very important quality traits of rice, fragrant rice research has been an important research issue in rice research fields in nowadays. In our study, using the detection method of molecular markers, genotypes of the 10 genes (Wx, SSII 3, SBE3, AGPiso, SSIII 2, AGPlar, PUL, SSI, ISA, SSIV 2) associated rice eating quality were analyzed in the 23 fragrant rice cultivars. The result indicated that the “2845”rice cultivar possesses the best combination of genotypes related to the highest eating quality, and the next best rice cultivars were “Songxiangzaojing”, ”Suhuxiangjing”, “B1”, “Wuyun2645”, “Tongyunjing”, “Yinxiang28”, “Xiangjing49②”, “99983”, “Wxiang99075”, “07-08”, “Yunjingyou15”, “29185”, “Nanhai318”. Other 9 rice cultivars (“Dahuaxiangjing”, “Wuxiang14”, “Xiangjing”, “Della”, “Dalixiang”, “Taiguoxiangdao”, “Cxiang517”, “Xiangdao No1”, “Zhongxiang No1”) obtain some of bad genotypes which may have adverse effect on rice eating quality. This study was helpful to establish an overall understanding of the genotypes of these ten eating quality related genes in the 23 tested fragrant rice cultivars, and provides the important genotype information in selecting and cultivating new fragrant rice with good eating quality in future via molecular markers assisted selection method.     

水稻香味基因分子标记的开发及应用

根据香稻材料在Badh2基因(甜菜碱醛脱氢酶基因)第7外显子的突变位点,设计了分子标记YY5-YY8用来区分这种突变类型的香稻材料。同时结合已报道的针对Badh2在第2、4、5外显子处突变设计的功能标记InDel-E2和FMbadh2-E4-5,对80份不同生态区香稻材料进行了标记检测。结果表明,26份材料属于第7外显子突变类型,37份材料属于第2外显子突变类型,无第4、5外显子突变类型材料。该研究构建了针对Badh2突变的新的分子标记YY5-YY8,并且鉴定了上述香稻材料的突变类型,为优质香稻分子育种奠定基础。     

Discovery of a new fragrance allele and the development of functional markers for the breeding of fragrant rice varieties

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. Weiwei Shi and Yi Yang contributed equally to this work.     

分子标记分析二十三种香型水稻十个食味品质相关基因的基因型

选取23种香型水稻品种为供试材料,利用分子标记检测方法,进行了10个与稻米食味品质相关基因：耽、SSII-3、SBE3、AGPiso、SSIII-2、AGPlar、P比、SSI、ISA、SSIV-2的基因型分析。结果发现,含有最好食味品质基因型的水稻是“2845”;其次是“松香早粳”、“苏沪香粳”、“B1”、“武运2645”、“通运粳”、“银香28”、“香粳49②”、“99983”、“W香99075”、“07-08”、“云粳优15”、“29185”、“南海318”：另外9种香稻,“大华香粳”、“武香14”、“香粳”、“Della光身稻”、“大粒香”、“泰国香稻”、“C香517”、“香稻1号”、“中香1号”都分别含有一些可能会影响稻米食味品质的基因型。开展本研究不仅能够使人们对这些香稻食味品质基因型有一个全面的认识,还可为今后利用分子标记辅助培育优良食味品质香稻新品种和亲本的选择提供重要的基因型信息。     

Development of a simple functional marker for fragrance in rice and its validation in Indian Basmati and non-Basmati fragrant rice varieties

Fragrance development in rice has been reported due to a 8-bp deletion in the exon 7 of badh2 gene located on Chromosome 8S. Multiplex markers targeting the functional InDel polymorphism was earlier reported for genotyping fragrance trait, but the marker was observed to be inconsistent and difficult to use. We have developed a simple, co-dominant, functional marker for fragrance trait, which can be resolved in an agarose gel and validated in Basmati and non-Basmati aromatic rice varieties and in a mapping population segregated for fragrance trait. The marker targets the InDel polymorphism in badh2 gene and amplifies 95 and 103 bp fragments in fragrant and non-fragrant genotypes, respectively. The newly developed marker was highly efficient in discriminating all fragrant and non-fragrant genotypes and showed perfect co-segregation with the trait of fragrance in the mapping population. We recommend the use of this simple, low-cost marker in routine genotyping for fragrance trait in large scale breeding materials and germplasm.     

The genetic origin of fragrance in NERICA1

In this study, we investigated the cause and origin of fragrance in NERICA1, a fragrant rice inbred line developed from an interspecific cross between two non-fragrant parents. The genetic cause of fragrance in NERICA1 was found to be due to a previously reported mutation in the BADH2 gene, the same allele responsible for the majority of modern fragrant rice varieties. Haplotype analysis around the BADH2 gene in NERICA1, its parents, and 95 other varieties carrying the badh2.1 allele identified the source of the badh2.1 allele in NERICA1 was a fragrant tropical japonica variety, WAB638-1, which had been growing in the vicinity of the NERICA1 nursery during varietal development. The allele-specific marker for the badh2.1 allele consistently predicted fragrance in the diverse African germplasm tested, making it very useful for marker-assisted breeding of fragrant rice varieties in Africa.     

Genetic and molecular basis of fragrance in rice

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 dehydrogenease 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.     

Discovery of a new fragrance allele and development of functional markers for identifying diverse fragrant genotypes in rice

Discovery of new fragrance alleles provides important genetic resources for breeding fragrant rice. In this study, a hybrid complementation test demonstrated the association of a new fragrance allele without mutation in the coding region with flavor formation in a fragrant rice variety Nankai 138. The new allele (badh2-p-5′UTR) has a 3-bp deletion in the 5′ untranslated region and an 8-bp insertion in the promoter (?1,314 site upstream from the initiation codon). Surprisingly, we found that there is also an 8-bp insertion in the promoter of the badh2-E7 allele. We developed a new sequence tagged site functional marker to identify the badh2-p-5′UTR and badh2-E7 alleles according to the 8-bp insertion in their promoters. A cleaved amplified polymorphic sequence (AluI) functional marker targeting a common base substitution in the intron 2 of three badh2 alleles, viz. badh2-p-5′UTR, badh2-E7 and badh2-E2, was developed to identify diverse genotypes for fragrance in rice. Based on the results of sequence alignments among the three badh2 alleles, we suggest that the badh2-E7 and badh2-p-5′UTR alleles may have the same genetic origin. In addition, the genetic distance between the badh2-E7 and badh2-p-5′UTR alleles may be closer than that between the badh2-E2 and the badh2-p-5′UTR alleles, or between the badh2-E2 and the badh2-E7 alleles.     

Enhancement of Ultrasonic Seed Treatment on Yield,Grain Quality Characters,and 2-Acetyl-1-Pyrroline Biosynthesis in Different Fragrant Rice Genotypes

Fragrant rice is popular for the good grain quality and special aroma. The present study conducted a field experiment to investigate the effects of ultrasonic seed treatment on grain yield, quality characters, physiological properties and aroma biosynthesis of different fragrant rice genotypes. The seeds of three fragrant rice genotypes were exposed to 1 min of ultrasonic vibration and then cultivated in paddy field. The results of present study showed that ultrasonic seed treatment increased grain yield of all fragrant rice genotypes but the responses of yield formation to ultrasonic were varied with different genotypes. Compared with control, ultrasonic seed treatment increased grain 2-acetyl-1-pyrroline (2-AP, the key component of fragrant rice aroma) content by 13.40%–44.88%. Ultrasonic seed treatment also reduced the crude protein contents in grains. The head rice rate, rice length, chalky rice rate, and chalkiness degree were influenced by ultrasonic for one or two fragrant rice genotypes. The activities of peroxidase and superoxide dismutase were also enhanced due to ultrasonic seed treatment. In conclusion, ultrasonic seed treatment increased grain, regulated grain aroma and quality, and improved stress resistance of fragrant rice varieties.     

水稻不育系261S矮化突变体及其F_1‘新闵优香粳’杂交稻的性状分析

以两系杂交稻'闵优香粳'品种的父本W香99075和母本不育系261S及其261S矮化突变株系为材料,对两种母本及其F1植株分别进行形态特征、产量和F2稻米品质比较分析,以明确不育系261S矮化突变株系的育种应用价值.结果显示:(1)不育系261S矮化突变株系与原不育系261S相比,花粉不育性相似,株高、穗长、千粒重分别极显著降低13.8 cm、4.5 cm、5.2 g,但有效穗比原不育系261S极显著增加62.8%;(2)261S矮化突变株系×W香99075组合的F1"新闵优香粳"植株高度较'闵优香粳'极显著降低11 cm,而单株产量两者差异不显著;(3)两组合F2稻米胶稠度和碱消值几乎没有变化,但"新闵优香粳" 比'闵优香粳'的蛋白质含量提高9.57%、直链淀粉含量极显著降低10.17%,稻米品质较优.研究表明,以植株较矮、分蘖性较强、单株有效穗多的261S矮化突变株系为母本与W香99075杂交,可在不影响杂交稻产量表现的基础上有效降低原品种'闵优香粳'植株的高度,同时稻米品质也得到改善;说明261S矮化突变株系可作为水稻两系杂交育种的新不育系材料.     

Characterization of the major fragance gene from an aromatic japonica rice and analysis of its diversity in Asian cultivated rice

In Asian cultivated rice (Oryza sativa L.), aroma is one of the most valuable traits in grain quality and 2-ACP is the main volatile compound contributing to the characteristic popcorn-like odour of aromatic rices. Although the major locus for grain fragrance (frg gene) has been described recently in Basmati rice, this gene has not been characterised in true japonica varieties and molecular information available on the genetic diversity and evolutionary origin of this gene among the different varieties is still limited. Here we report on characterisation of the frg gene in the Azucena variety, one of the few aromatic japonica cultivars. We used a RIL population from a cross between Azucena and IR64, a non-aromatic indica, the reference genomic sequence of Nipponbare (japonica) and 93-11 (indica) as well as an Azucena BAC library, to identify the major fragance gene in Azucena. We thus identified a betaine aldehyde dehydrogenase gene, badh2, as the candidate locus responsible for aroma, which presented exactly the same mutation as that identified in Basmati and Jasmine-like rices. Comparative genomic analyses showed very high sequence conservation between Azucena and Nipponbare BADH2, and a MITE was identified in the promotor region of the BADH2 allele in 93-11. The badh2 mutation and MITE were surveyed in a representative rice collection, including traditional aromatic and non-aromatic rice varieties, and strongly suggested a monophylogenetic origin of this badh2 mutation in Asian cultivated rices. Altogether these new data are discussed here in the light of current hypotheses on the origin of rice genetic diversity.     

Opportunities of marker‐assisted selection for rice fragrance through marker–trait association analysis of microsatellites and gene‐based markers

Developing fragrant rice through marker‐assisted/aided selection (MAS) is an economical and profitable approach worldwide for the enrichment of an elite genetic background with a pleasant aroma. The PCR‐based DNA markers that distinguish the alleles of major fragrance genes in rice have been synthesised to develop rice scent biofortification through MAS. Thus, the present study examined the aroma biofortification potential of these co‐dominant markers in a germplasm panel of 189 F₂ progeny developed from crosses between a non‐aromatic variety (MR84) and a highly aromatic but low‐yielding variety (MRQ74) to determine the most influential diagnostic markers for fragrance biofortification. The SSRs and functional DNA markers RM5633 (on chromosome 4), RM515, RM223, L06, NKSbad2, FMbadh2‐E7, BADEX7‐5, Aro7 and SCU015RM (on chromosome 8) were highly associated with the 2AP (2‐acetyl‐1‐pyrroline) content across the population. The alleles traced via these markers were also in high linkage disequilibrium (R² > 0.70) and explained approximately 12.1, 27.05, 27.05, 27.05, 25.42, 25.42, 20.53, 20.43 and 20.18% of the total phenotypic variation observed for these biomarkers, respectively. F₂ plants harbouring the favourable alleles of these effective markers produced higher levels of fragrance. Hence, these rice plants can be used as donor parents to increase the development of fragrance‐biofortified tropical rice varieties adapted to growing conditions and consumer preferences, thus contributing to the global rice market.     

Grain Yield,Quality and 2-Acetyl-1-Pyrroline of Fragrant Rice in Response to Different Planting Seasons in South China

Climate conditions is an important factor affected the fragrant rice growth and development. In order to study the effects of different planting seasons on fragrant rice performance in South China, present study was conducted with three planting seasons (early season (April to July), middle season (June to September) and late season (August to November)) and three fragrant rice cultivars, ‘Basmati-385’, ‘Meixiangzhan-2’ and ‘Xiangyaxiangzhan’. The results showed that the highest grain yield and grain 2-acetyl-1-pyrroline (2-AP, key component of fragrant rice aroma) content were both recorded in late season treatment while the fragrant rice in middle season treatment produced the lowest grain yield, grain filling percentage, 1000-grain weight and gain 2-AP content. The highest contents of precursors (proline, pyrroline-5-carboxylic acid and 1-pyrroline) which related to 2-AP biosynthesis were recorded in late season treatment compared with early season treatment and middle season treatment. The highest activities of enzymes (proline dehydrogenase, pyrroline-5-carboxylic acid synthetase and ornithine transaminase) which involved in 2-AP biosynthesis were also observed in late season treatment. Moreover, the fragrant rice cultivars in late season possessed the lowest chalk rice rate, chalkiness as well as the highest brown rice rate, head rice and protein content. Thus, the optimal season for fragrant rice production in South China is the late season.     

Genetic Diversity of Global Aromatic Rice Varieties

Genetic diversity of 434 rice accessions collected from 16 countries, including 345 fragrance rice varieties and 89 non fragrance rice varieties, have been analyzed. A total of 573 alleles were detected by using 77 simple sequence repeats (SSR) primer pairs covering all rice 12 chromosomes. The value of allelic polymorphism information content (PIC) ranged from 0090 to 0845, with an average of 0516 per locus; Gene diversity (GD) varied from 0091 to 0859, with an average of 0573; The mean value of major allele frequencies (MAF) was 0540, covering from 0251 to 0953. In addition, 434 rice accessions are divided into three sub populations by cluster and population structure analysis, and FST between sub populations showed a mean of -0116, ranging from -0623 to 0494; The score of genetic distance calculated by Nei′s method appeared from 0207 to 0355. Major allele frequencies within each sub population distributed from 0408 to 0746, and gene diversity level from 0354 to 0699, while PIC from 0320 to 0658. Sequencing 6 mitochondrion genes in 18 rice varieties exhibited no different in 5 genes, whereas Mit4 contains a 3 SNPs in the gene body, which acts as an important marker to understanding the relationship between Indica/Japonica differentiation and the evolution of fragrant gene. Finally, genetic diversity and mitochondrion gene sequencing would help to know about the origin of fragrant resource and benefit rice breeding.     

Haplotype variation at Badh2, the gene determining fragrance in rice

Fragrance is an important component of end-use quality in rice. A set of 516 fragrant rice accessions were genotyped and over 80% of them carried the badh2.7 allele. A subset of 144 mostly fragrant accessions, including nine of Oryza rufipogon, was then subjected to a detailed diversity and haplotype analysis. The level of linkage disequilibrium in the Badh2 region was higher among the fragrant accessions. Re-sequencing in the Badh2 region showed that badh2.7, badh2.2 and badh2.4–5 all arose in the japonica genepool, and spread later into the indica genepool as a result of deliberate crossing. However, loss-of-function alleles of Badh2 are also found in the indica genepools, and then transferred into japonica. Evidence for three new possible FNPs was obtained from the Badh2 sequence of 62 fragrant accessions. Based on these data, we have elaborated a model for the evolution of Badh2 and its participation in the rice domestication process.     

A PCR-based marker for a locus conferring the aroma in Myanmar rice (Oryza sativa L.)

Aromatic rice is an important commodity for international trade, which has encouraged the interest of rice breeders to identify the genetic control of rice aroma. The recessive Os2AP gene, which is located on chromosome 8, has been reported to be associated with rice aroma. The 8-bp deletion in exon 7 is an aromatic allele that is present in most aromatic accessions, including the most popular aromatic rice varieties, Jasmine and Basmati. However, other mutations associated with aroma have been detected, but the other mutations are less frequent. In this study, we report an aromatic allele, a 3-bp insertion in exon 13 of Os2AP, as a major allele found in aromatic rice varieties from Myanmar. The insertion is in frame and causes an additional tyrosine (Y) in the amino acid sequence. However, the mutation does not affect the expression of the Os2AP gene. A functional marker for detecting this allele was developed and tested in an aroma-segregating F(2) population. The aroma phenotypes and genotypes showed perfect co-segregation of this population. The marker was also used for screening a collection of aromatic rice varieties collected from different geographical sites of Myanmar. Twice as many aromatic Myanmar rice varieties containing the 3-bp insertion allele were found as the varieties containing the 8-bp deletion allele, which suggested that the 3-bp insertion allele originated in regions of Myanmar.     

野生稻DNA导入水稻后代的SSR检测分析

以宁夏地区水稻主栽品种‘宁粳16号’、‘宁粳23号’为受体,普通野生稻(Oryza rufipogon,2n=2x=24, AA)为供体,利用花粉管通道导人法与茎注射法相结合的导入方法,将普通野生稻DNA导入宁夏水稻栽培品种, 对筛选出的21份形态、农艺性状典型变异的D2代材料及其对照利用SSR分子标记进行分子鉴定,结果表明,有 17份变异材料扩增出野生稻特有的DNA片段,而2个对照均不能扩增出野生稻特异的DNA片段,证明这些材料就是野生稻DNA片段导入的后代。另外有些材料出现受体DNA条带的减少,可能是由于野生稻DNA片段整合到水稻基因组中该引物结合位点或附近,原有的结合位点破坏而造成的。     

Effect of 5'' splice site mutations on splicing of the preceding intron.

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.