Fragrance Gene Research Articles

FUNCTIONAL CHARACTERIZATION OF TERPENE SYNTHASES OF 'AROMATIC' AND 'NON-AROMATIC' GRAPEVINE CULTIVARS

Grape-derived flavour compounds and some flavour precursors modified during fermentation, wine evolution and ageing, are fundamental in determining the organolectic parameters used to define wine quality. The knowledge of the biosyn-thesis of secondary metabolites that contribute to grape and wine flavour is not well understood and only a few genes involved in berry flavour biosynthesis pathways have been discovered and characterised. The aim of this research is the functional characterization of candidate genes thought to contribute to the volatile profiles of ?aromatic? (?Aleatico?, ?Moscato bianco?) and ?non-aromatic? (?Sangiovese?) cultivars. Samples of flowers and berries covering developmental stages from fruit set to technological ripening, of ?Aleatico?, ?Moscato bianco?, and ?Sangiovese? have been analysed for their aroma content and the expression of genes that are predicted to be involved in grape sesquiterpene and monoterpene biosynthesis. The aroma com-pounds have been extracted by SPE and SPME procedures and analysed by GS-MS, while the expression of characterised grape aroma genes and some candidate genes have been analysed by real-time RT-PCR. Some of the genes studied show correlations between their expression patterns and the accumulation of certain aroma compounds during berry development. These genes have been the focus of functional character¬ization studies and include a sesquiterpene synthase (which has an expression pattern that correlates with the production of farnesene in flowers buds and open flowers), putative monoterpene synthases that are expressed when linalool accumulates in flowers or during ripening, and putative linalool epoxidases that are expressed when trans-pyran-linalool oxide accumulates during berry ripening of all three grapevine cultivars studied. Some of these genes have been cloned in E. coli and the enzymatic activity characterized. The discovery and characterization of genes that encode enzymes from grapevine flavour biosynthesis pathways and knowledge of factors that influence their expression and activity during berry development would support decisions on management of genotype, environment and viticultural practices for improving grape flavour and aroma potential.

A proteomic study of rice cultivar TNG67 and its high aroma mutant SA0420

Fragrance is a very important economic trait for rice cultivars. To identify the aroma genes in rice, we performed a proteomics analysis of aroma-related proteins between Tainung 67 (TNG67) and its high aroma mutant SA0420. Seventeen of the differentially identified proteins were close related with the aroma phenotype of SA0420. Among them, 9 were found in leaves and 8 were found in grains. One protein (L3) was identified as the chloroplastic glyceraldehyde-3-phosphate dehydrogenase B (OsGAPDHB) which was less abundant in SA0420 than TNG67. Sequence analysis demonstrated that this protein in SA0420 carries a P425S mutation in the C-terminal extension domain, which might hinder the formation of holoenzyme, thereby changing the profile of aroma compounds. The protein profile of OsGAPDHB showed only a weak correlation to its transcription profile. This result indicated that the reduction of OsGAPDHB in SA0420 is regulated by post-translational processes and can only be analyzed by proteomics approach. Transgenic lines suppressing OsGAPDHB through RNAi harbored more fragrance than TNG67 but less than SA0420. With betaine-aldehyde dehydrogenase as the only fragrance gene identified in rice to date, OsGAPDHB may serve as the second protein known to contribute to the aroma phenotype.

Specific patterns of genetic diversity among aromatic rice varieties in Myanmar.

BackgroundAfter observing peculiar rice varieties in Myanmar, in terms of classification in varietal groups and of grain quality, we focused on Myanmar varieties and analyzed variations at 19 microsatellite loci as well as sequences of the aroma gene BADH2.ResultsMicrosatellites were able to retrieve the well-established classification into Indica (isozyme group 1), Japonica (group 6, comprising temperate and tropical forms) and specific groups from the Himalayan foothills including some Aus varieties (group 2) and some aromatic varieties (group 5). They revealed a new cluster of accessions close to, but distinct from, non-Myanmar varieties in group 5. With reference to earlier terminology, we propose to distinguish a group “5A” including group 5 varieties from the Indian subcontinent (South and West Asia) and a group “5B” including most group 5 varieties from Myanmar. In Myanmar varieties, aroma was distributed in group 1 (Indica) and in group 5B. New BADH2 variants were found. Some accessions carried a 43 bp deletion in the 3’ UTR that was not completely associated with aroma. Other accessions, all of group 5B, displayed a particular BADH2 allele with a 3 bp insertion and 100% association with aroma.ConclusionWith the new group and the new alleles found in Myanmar varieties, our study shows that the Himalayan foothills contain series of non-Indica and non-Japonica varietal types with novel variations for useful traits.Electronic supplementary materialThe online version of this article (doi:10.1186/1939-8433-5-20) contains supplementary material, which is available to authorized users.

Genetic Analysis of Aromatic and Quality Rice Germplasm using Microsatellite Markers

Twenty rice genotypes were analysed with three SSR primers viz., RM223, RM515 and RM342 for detection of the fgr gene responsible for aroma. The primers RM223 and RM515 identified eight and seven genotypes, respectively as having the fgr locus. Presence of the specific allele of the RM342 amplicon, also correlated with strong aroma in two and with weak aroma in five genotypes. The banding pattern for the primer RM342 was different in genotypes with no aroma. Among the three primers (RM223, RM515 and RM342), RM223 detected the highest number of fgr loci in the 20 rice genotypes. In total 11 genotypes were identified as having strong aroma genes by the three primers. These accessions also showed high yields and therefore may prove to be a good source for producing aromatic rice. DOI: http://dx.doi.org/10.3329/ptcb.v22i1.11262Plant Tissue Cult. & Biotech. 22(1): 65-71, 2012 (June)

Introgression of bacterial leaf blight resistance and aroma genes using functional marker-assisted selection in rice (Oryza sativa L.)

Functional markers reduce the risk of false selection in marker-assisted selection (MAS), and recombination between the molecular marker and the gene of interest. The present study was conducted with the objective of combining the important basmati quality traits with resistance to bacterial leaf blight (BLB) by a combination of phenotypic selection and MAS. Screening of 29 recombinants for BLB resistant genes along with aroma (fgr) gene was performed using a suite of different markers. Twenty genotypes were found in homozygous condition for Xa21 and xa13 genes. Restriction digestion analysis with BsrI, gave nine resistant recombinants. Out of nine resistant recombinants, seven were in homozygous and two were in heterozygous conditions. Recombinants viz., IRS 5441-2-21, IRS 5441-2-79, IRS 5441-2-81, IRS 5441-2-85 and IRS 5441-2-91 possessed all the three BLB resistance genes and fgr in the homozygous condition. Recombinants with enhanced resistance to BLB, basmati quality and desirable agronomic traits were identified. This germplasm can be directly developed into commercial varieties or used as immediate potential donors of BLB resistance in basmati breeding programs.

Marker aided selection for aroma in F2 populations of rice

Marker aided selection (MAS) can be used for monitoring the presence or absence of genes in breeding populations and can be combined with conventional breeding approaches. In this study, crosses between two high yielding cultivars, Neda and Nemat with local aromatic varieties Sang tarom and Tarom deylamani were made followed by phenotypic selection for desirable individual plants in F 2 populations. Then, marker aided selection was applied to genotypic selection of aromatic plants using allele specific amplification (ASA) marker in these populations. Plants homozygous for aroma gene with good agronomic performance were identified. These plants can be used to develop new aromatic rice lines in the near future in Iran.

High‐throughput functional marker assay for detection of Xa/xa and fgr genes in rice (Oryza sativa L.)

We apply CE for high-throughput analysis of functional markers for marker-assisted selection in rice. The accuracy, throughput and reproducibility of CE analysis for sequence-tagged site (STS) and simple sequence repeat (SSR) markers for bacterial blight resistance and aroma genes are demonstrated by using a CE system. Multiplex PCR products displayed well-differentiated allelic variants using different STS and SSR markers for identification of xa13, Xa21 and fgr genes using the CE system compared to 1.2% agarose gel images. Moreover, consumption of PCR product is much less in the CE system compared to traditional agarose gel systems. Sample consumption is less than 0.1 μL per analysis, thereby conserving samples for further downstream analysis. Out of 29 genotypes in BC(1)F(3) generation, 16 plants were found homozygous for all the three genes, viz., xa13, Xa21 and fgr. These homozygous lines can be used as potential donors in rice breeding programmes.

Association mapping of grain color, phenolic content, flavonoid content and antioxidant capacity in dehulled rice

Phytochemicals such as phenolics and flavonoids in rice grain are antioxidants that are associated with reduced risk of developing chronic diseases including cardiovascular disease, type-2 diabetes and some cancers. Understanding the genetic basis of these traits is necessary for the improvement of nutritional quality by breeding. Association mapping based on linkage disequilibrium has emerged as a powerful strategy for identifying genes or quantitative trait loci (QTL) underlying complex traits in plants. In this study, genome-wide association mapping using models controlling both population structure (Q) and relative kinship (K) were performed to identify the marker loci/QTLs underlying the naturally occurring variations of grain color and nutritional quality traits in 416 rice germplasm accessions including red and black rice. A total of 41 marker loci were identified for all the traits, and it was confirmed that Ra (i.e., Prp-b for purple pericarp) and Rc (brown pericarp and seed coat) genes were main-effect loci for rice grain color and nutritional quality traits. RM228, RM339, fgr (fragrance gene) and RM316 were important markers associated with most of the traits. Association mapping for the traits of the 361 white or non-pigmented rice accessions (i.e., excluding the red and black rice) revealed a total of 11 markers for four color parameters, and one marker (RM346) for phenolic content. Among them, Wx gene locus was identified for the color parameters of lightness (L*), redness (a*) and hue angle (H (o)). Our study suggested that the markers identified in this study can feasibly be used to improve nutritional quality or health benefit properties of rice by marker-assisted selection if the co-segregations of the marker-trait associations are validated in segregating populations.

Origin and Genetic Diversity of Aromatic Rice Varieties, Molecular Breeding and Chemical and Genetic Basis of Rice Aroma

Aroma is an important quality attribute of rice and is a key determinant of its market value. Among the different groups of aromatic rice varieties ‘Basmati’ from the Indian subcontinent and ‘Jasmine’ from Thailand occupy prime position in the international market. In addition, there are a large number of premium short-grain aromatic rice varieties cultivated by farmers in India and South-East Asia that have not been fully commercially utilised as yet. The origin and evolution of aromatic rice varieties is being unravelled by application of genomic tools. The common alleles of aroma gene seem to have their origin in the aromatic group of rice varieties native to the Sub-Himalayan region. Of more than two hundred volatile compounds present in the rice grain, 2-acetyl-l-pyrolline (2-AP) is considered as the key aroma compound present in almost all the aromatic rice varieties. However, there is significant variation in the type and intensity of aroma in the different groups of aromatic rice varieties suggesting involvement of additional chemical compounds in varying proportions. Studies have been undertaken to understand the genetics of rice aroma and to map the genes or quantitative trait loci (QTL) controlling aroma expression. Of the three mapped aroma QTL, qaro8.1 located on rice chromosome S is the most significant and it represents a non-functional allele of BADH2 gene coding for enzyme betaine aldehyde dehydrogenase. Functional allele of the BADH2 gene makes rice non-aromatic. Similarly, specific alleles of BADH1 gene located on rice chromosome 4 within the aroma QTL qaro4.1 show association with the aromatic rice varieties. The gene underlying QTL qaro3.1 on chromosome 3 has not yet been deciphered. Functional molecular markers have been developed for the major aroma QTL on chromosome S and marker-assisted breeding for high yielding aromatic rice varieties is now a reality. To safeguard the reputation of Basmati rice an international code of practice has been developed where DNA markers help check the purity of commercial samples. There is need to use advanced genomic and metabolomic approaches to further study the minor genes controlling rice aroma and understand the variation in type, intensity and stability of rice aroma. It is also required to improve the production and marketing of short grain aromatic rice varieties.

Delimitation of the fgr gene for rice fragrance to a 28-kb DNA fragment

The fragrance gene plays an important role in high-quality rice varieties and has been widely used in breeding programs. Using a random sample of 370 individuals from an F2 segregating population developed from a cross between a japonica rice variety 9407 with fragrant flavor and an indica variety IRBB60, the fgr locus was mapped on chromosome 8 between SSR markers, PSM465 and RM1109, with genetic distances of 0.3 cM and 0.1 cM to respective markers. These mapping efforts confirmed the previous mapping results. A large F3 mapping population with 7300 individuals was then developed from F2 plants, in which a small chromosomal region defined by the SSR markers, PSM465 and RM1109, was heterozygous. The analysis of recombinants in the fgr region anchored the gene locus to an interval of 28 kb flanked by the left marker NS9 and the right marker L06. Sequence analysis of this fragment predicted three open reading frames encoding putative 3-methylcrotonyl-CoA carboxylase, putative isoleucyl-tRNA synthetase, and betaine aldehyde dehydrogenase (BADH2). The latter was presumed to be the candidate gene for fragrance. This result will be very useful in molecular cloning of the fgr gene and marker-assisted transfer of the fgr gene in rice breeding programs.

Evaluation of the Fragrance Gene (fgr) in Self-Supplied Seed Lots of Black Rice (Oryza sativa L.) from Thailand and Laos

Evaluation of the Fragrance Gene (fgr) in Self-Supplied Seed Lots of Black Rice (Oryza sativa L.) from Thailand and Laos

Is there a second fragrance gene in rice?

Aromatic rice is highly prized by most rice consumers, and many countries cultivate traditional and improved aromatic varieties. 2-Acetyl-1-pyrroline (2AP) is the major aromatic compound in rice, and is believed to accumulate because of an eight-base-pair (8-bp) deletion in an allele at the fragrance locus. In this study, 2AP was quantified and the presence or absence of the fragrance allele (fgr) was determined in 464 samples of traditional varieties of rice from the T.T. Chang Genetic Resources Centre at the International Rice Research Institute. It was shown that a number of aromatic varieties, primarily from South and South-East Asia, do not carry the 8-bp deletion, but 2AP was identified in both raw and cooked rice of these varieties. We suggest that the 8-bp deletion in fgr is not the only cause of aroma, and at least one other mutation drives the accumulation of 2AP. The amount of 2AP in most uniform fgr genotypes was not significantly different from that in aromatic nfgr genotypes, but several fgr genotypes, primarily from South Asia, reproducibly accumulated exceptionally large amounts of 2AP. We suggest that the mutation leading to 2AP in aromatic nfgr varieties possibly originated several times and, through either domestication or evolution, the fgr gene and other alleles leading to 2AP have combined in South Asia, leading to several highly aromatic traditional varieties. The identification of multiple mutations for 2AP will enable rice breeding programmes to select actively for multiple genetic sources of 2AP, leading to the development of highly aromatic and, consequently, high-quality varieties of rice.

Genetic analysis and gene fine mapping of aroma in rice (Oryza sativa L. Cyperales, Poaceae)

Abstract Weinvestigatedinheritanceandcarriedoutgenefinemappingofaromaincrossesbetweenthearomaticelitehybridrice Oryza sativa indicavariety Chuanxiang-29B (Ch-29B) and the non-aromatic rice O. sativa indicavariety R2 andO. sativa japonicaLemont (Le). The F 1 grains and leaves were non-aromatic while the F 2 non-aroma to aroma segre-gation pattern was 3:1. The F 3 segregation ratio was consistent with the expected 1:2:1 for a single recessive aromagene in Ch-29B. Linkage analysis between simple sequence repeat (SSR) markers and the aroma locus for the aro-matic F 2 plants mapped the Ch-29B aroma gene to a chromosome 8 region flanked by SSR markers RM23120 at0.52 cM and RM3459 at 1.23 cM, a replicate F 2 population confirming these results. Three bacterial artificial chromo-some(BAC)clonescoverchromosome8markersRM23120andRM3459.Ourmolecularmappingdatafromthetwopopulations indicated that the aroma locus occurs in a 142.85 kb interval on BAC clones AP005301 or AP005537,implying that it might be the same gene reported by Bradbury et al(2005a; Plant Biotec J. 3:363-370). The flankingmarkers Aro7, RM23120 and RM3459 identified by us could greatly accelerate the efficiency and precision of aro-matic rice breeding programs.

A single nucleotide polymorphism (SNP) marker linked to the fragrance gene in rice ( Oryza sativa L.)

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.

Trisomic genetic analysis of aroma in three Japanese native rice varieties (Oryza sativa L.)

Information on the genetics of aroma in rice facilitates breeding and selection of new aromatic varieties with high yield and good quality. Objective of the present study was to make clear the number of genes controlling aroma, and the allelism of aroma genes and the location of aroma gene(s) on the chromosome in three Japanese native aromatic rice varieties (Kabashiko, Shiroikichi and Henroyori). Lack of leaf aroma in all F1 plants of non-aromatic/aromatic crosses indicated the recessive nature of aroma, and the segregation ratios (3:1) of non-aromatic to aromatic plants in its F2 populations from Nipponbare/aromatic varieties crosses revealed that each of the three aromatic varieties contains a single recessive gene for aroma. Through trisomic analysis, the segregation of non-aromatic and aromatic plants in all F2 populations from the crosses between trisomics lines NT8, with an extra chromosome 8, and aromatic varieties deviated significantly from disomic segregation of 3:1 ratios, and fitted to trisomic segregation, however, in other F2 populations derived from other 7 types of trisomic F1 plants, the segregation ratios of non-aromatic to aromatic were 3:1, indicating that the single recessive aroma gene was located on chromosome 8 in three aromatic varieties.

PCR-based molecular markers for the fragrance gene in rice (Oryza sativa. L.)

The genomic DNA clone RG28, linked to the major fragrance gene of rice (fgr), was assessed for polymorphism in order to produce a PCR-based marker for fragrance. A small mono-nucleotide repeat, that was polymorphic between a pair of fragrant and non-fragrant cultivars, was identified and developed into a co-dominant PCR-based marker. The polymorphism-information-content determinations for three microsatellite markers, that have been genetically mapped near RG28, are also presented. These PCR-based markers will be highly useful in distinguishing fragrance-producing alleles from non-fragrance-producing alleles at the fgr locus.

Inheritance of Aroma in Six Rice Cultivars

‘Jasmine 85’, a soft‐cooking aromatic rice (Oryza sativa L.) cultivar, was released in the USA with the objective of displacing imported Thai Jasmine rice in local markets. However, the reduced aroma and off‐white color of its milled rice limit its marketability. The present objective was to determine the number of genes controlling aroma in four rice lines (Jasmine 85, ‘Amber’, a dwarf mutant from ‘Basmati 370’ (PI 457917), and ‘Dragon Eyeball 100’), and to evaluate the allelism of aroma in these four lines and with the single recessive gene known to be in both ‘A‐301’ and Della‐X2. Lack of leaf aroma in aromatic/non‐aromatic F1 hybrids indicated the recessive nature of the aroma in all studied rices. F2 segregation ratios revealed that Jasmine 85 and PI 457917 each contain a single gene for aroma, and the aromatic nature of the F1 leaves from crosses with each other and with A‐301 and Della‐X2 revealed that the aroma genes in all four cultivars are allelic. Amber and Dragon Eyeball 100 each contain two aroma genes, a novel gene plus one allelic to the gene in A‐301, Della‐X2, Jasmine 85, and PI 457917. Incorporation of a novel aroma gene into Jasmine 85 could potentially increase its marketability. No linkage was detected between the two aroma genes in Dragon Eyeball 100 and five morphological characters. High levels of sterility observed in Amber F2 populations may hinder the use of its novel aroma gene.

Inducible overproduction of the Aspergillus nidulans pentafunctional AROM protein and the type-I and -II 3-dehydroquinases from Salmonella typhi and Mycobacterium tuberculosis.

The aroQ gene of Mycobacterium tuberculosis, encoding a type-II 3-dehydroquinase, and the aroD gene of Salmonella typhi, encoding a type-I 3-dehydroquinase, have been highly overexpressed in Escherichia coli using the powerful trc promoter contained within the expression vector pKK233-2. The M. tuberculosis type-II 3-dehydroquinase has been purified in bulk from overproducing strains of E. coli to greater than 95% homogeneity. The protein is extremely heat-stable, is active as a homododecamer and has the lowest reported Km value of any type-II 3-dehydroquinase. The pentafunctional aromA gene of Aspergillus nidulans has been overexpressed more than 120-fold in an A. nidulans aromA- qutB- double mutant from a truncated quinate-inducible qutE promoter, such that the AROM protein is visible as a significant fraction (approx. 6%) in cell-free crude extracts. The M. tuberculosis aroQ gene has been fused to the same truncated qutE promoter and shown to encode quinate-inducible 3-dehydroquinase activity that allows a qutE- mutant strain of A. nidulans to utilize quinate as sole carbon source.

RFLP tagging of a gene for aroma in rice

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 F3 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.

Overproduction in Escherichia coli of the dehydroquinate synthase domain of the Aspergillus nidulans pentafunctional AROM protein

The pentafunctional AROM protein of Aspergillus nidulans is encoded by the complex aromA locus and catalyses steps 2-6 in the synthesis of chorismate, the common precursor for the aromatic amino acids and p-aminobenzoic acid. DNA sequences encoding the 3-dehydroquinate synthase (DHQ synthase) and 3-dehydroquinase domains of the AROM protein have been amplified with the inclusion of a translational stop codon at the C-terminus by PCR technology. These amplified fragments of DNA have been subcloned into the prokaryotic expression vector pKK233-2 and expressed in Escherichia coli. As a result, the DHQ synthase domain is overproduced in E. coli, forming 30% of total cell protein, and can be purified to greater than 80% homogeneity by a simple two-step protocol. The 3-dehydroquinase domain is produced at a specific activity 8-fold greater than the corresponding activity encoded by the aromA gene in A. nidulans. The qutB gene of A. nidulans encoding quinate dehydrogenase has similarly been subjected to PCR amplification and expression in E. coli. The quinate dehydrogenase is not overproduced, but is active in E. coli as a shikimate dehydrogenase, as the presence of the qutB gene allows the growth of an E. coli mutant strain lacking shikimate dehydrogenase on minimal medium lacking aromatic-amino-acid supplementation.