Fragrance Gene Research Articles

Discovery of key volatile compound and regulatory gene governing the ‘taro-like’ aroma in wax gourd (Benincasa hispida)

The distinctive ‘taro-like’ aroma in wax gourd has become a crucial flavor characteristic, significantly enhancing its market value. Despite its importance, the volatile compounds responsible for this aroma and their genetic regulatory mechanisms have not been sufficiently studied. Utilizing headspace-gas chromatography-quadrupole time of flight-mass spectrometry (HS-GC-QTOF-MS), this research examined the volatile compounds in wax gourd germplasms with and without the ‘taro-like’ aroma, identifying 2-acetyl-1-pyrroline (2-AP) as the primary volatile responsible for this aroma. To uncover the genetic basis of this trait, segregating populations and recombinant inbred lines (RILs) were developed from MY-1 x GX-71 germplasms, which exhibit significant differences in aroma expression. Genetic analysis revealed that a single recessive gene governs the ‘taro-like’ aroma trait in wax gourd. Through bulked segregant analysis sequencing (BSA-seq) and quantitative trait loci (QTL) mapping, the candidate region for this aroma gene was localized to a 285.29 kb interval on Chromosome 2. Further examination of candidate gene expression and sequence variations highlighted the BhBADH gene's critical role in the formation of the ‘taro-like’ aroma. Additionally, RNA-seq analysis revealed significant enrichment of differentially expressed genes (DEGs) associated with metabolic pathways involved in environmental information processing, which contribute to 2-AP synthesis in wax gourd with a ‘taro-like’ aroma. Our research provides a theoretical foundation for the molecular breeding to enhance the aroma quality of wax gourd.

Selection of rice aroma from crossing aromatic rice x T250.7 mutant genotype using DNA marker

Abstract. Ishak I, Fedora F. 2024. Selection of rice aroma from crossing aromatic rice x T250.7 mutant genotype using DNA marker. Biodiversitas 25: 2191-2197. Breeding fragrant rice using conventional approach was considered ineffective due to time consuming, therefore molecular breeding using DNA markers was introduced. This research aimed to utilize marker-assisted selection (MAS) for detection of fragrance gene in rice related to molecular breeding. Crossing between aromatic rice (Sinar 1) and non-aromatic rice (T250.7 mutant genotype) showed the results at F1 plants and found the DNA fragment size of 257 bp for fragrance genes in rice was at TS1, TS2, TS3, TS4, TS5, and TS.7 genotypes, whereas, TS6 did not show a fragment DNA at 257 bp. It was predicted that the TS6 genotype did not cross between variety Sinar 1 with T250.7. The F2 seeds were selected and morphologically, they had slender seeds and continued planting for F2 plants. Based on DNA marker identification, it revealed that 75% of the rice plants carried aroma. Similarly, the selected F3 plants continued to be planted and molecular identification indicated that aroma segregation was the same as that in the previous generation. Plants with the number of accessions of 721, 761, and 7210 were predicted to have homogeneity in aroma after detection using molecular markers. The MAS in breeding programs can shorten the duration of rice selection.

Molecular basis of genetic improvement for key rice quality traits in Southern China

Grain qualities including milling quality, appearance quality, eating and cooking quality, and nutritional quality are important indicators in rice breeding. Significant achievements in genetic improvement of rice quality have been made. In this study, we analyzed the variation patterns of 16 traits in 1570 rice varieties and found significant improvements in appearance quality and eating and cooking quality, particularly in hybrid rice. Through genome-wide association study and allelic functional nucleotide polymorphisms analysis of quality trait genes, we found that ALK, FGR1, FLO7, GL7/GW7, GLW7, GS2, GS3, ONAC129, OsGRF8, POW1, WCR1, and Wx were associated with the genetic improvement of rice quality traits in Southern China. Allelic functional nucleotide polymorphisms analysis of 13 important rice quality genes, including fragrance gene fgr, were performed using the polymerase chain reaction amplification refractory mutation system technology. The results showed that Gui516, Gui569, Gui721, Ryousi, Rsimiao, Rbasi, and Yuehui9802 possessed multiple superior alleles. This study elucidates the phenotypic changes and molecular basis of key quality traits of varieties in Southern China. The findings will provide guidance for genetic improvement of rice quality and the development of new varieties.

Pyramiding Rice Blast Resistance Gene Pi2 and Fragrance Gene badh2

Rice is a major food crop across the globe, but the frequent occurrence of rice blast in recent years has seriously affected the yield of rice. In addition, fragrance rice is becoming increasingly popular among consumers. In this study, the fragrant rice variety Wenxiang-1 was used as the donor of the fragrance gene badh2, and the rice variety R1179 was used as the donor of rice blast resistance gene Pi2. Plants that were homozygous for both Pi2 and badh2 were selected using marker-assisted selection (MAS) applied to the Wenxiang-1/R1179 F2 segregation population with the functional markers Pi2-1 and Badh2-1 as well as whole-genome-SNP-genotyping technology. Finally, “elite” rice varieties R365 and R403 that had both high levels of rice blast resistance (level 3 and 4) and fragrance (0.650 and 0.511 mg/kg) were bred. Genetic composition analysis indicated that 40.67% of the whole genome of R365 was inherited from Wenxiang-1, while 59.33% was inherited from R1179. Similarly, 46.26% of the whole genome of R403 was inherited from Wenxiang-1, while 53.74% was inherited from R1179. These new hybrid lines with R365 and R403 as the male parents also exhibit high yield per hectare, especially C815S/R365 and Yu03S/R403 F1, with yields per hectare of 9.93 ± 0.15 and 9.6 ± 0.17 tons. These plants also possess high levels of rice blast resistance (level 3 and 4) and fragrance (0.563 and 0.618 mg/kg).

Molecular Marker Assisted Breeding of a New <i>Japonica</i> Hybrid Rice

The development of Japonica  hybrid rice is of great significance to ensure supply of good quality Japonica  rice and food security in China. Aiming at the problems of late growth period, unstable yield, and the need to improve rice quality and seed production yield of Japonica  hybrid rice, a new Japonica  hybrid rice combination 'Shenyou28' with early maturity, good quality, blast resistance, stable and high yield and easy seed production was developed by using molecular breeding techniques including high-density rice gene chip. 'Shenyou28', bred by Shanghai Academy of Agricultural Sciences, is a three-line Japonica  hybrid rice with 'Shen21A' as sterile line and 'Shenhui26-28' as restorer line. For two-year regional trials from 2018 to 2019, the average output were 10 588.5 kg/hm 2 , which was 11.6% higher than the control variety 'Huayou14'. For the one-year production trial, the output reached 10 473.0 kg/hm 2 , which was 4.8% higher than the control variety 'Huayou14'. 'Shenyou28' has good quality, reaching the second grade of national standard, and has fragrance because it carries fragrance gene badh2-p-5 ’ UTR . It also has good resistance to rice blast and bacterial blight because it aggregates the rice blast resistance genes Pi2 , Pita , Pib , Pi9 , Pi54 , Pikm , Pit , and bacterial blight resistance gene Xa21 . It was approved by the Crop Variety Certification Committee of Shanghai in 2020. The breeding process, variety characteristics and key points of high yield mechanized seed production technology of 'Shenyou28' are introduced in this study.

Transcriptomic Insight into Aroma Pathway Genes and Effect of Ripening Difference on Expression of Aroma Genes in Different Mango Cultivars

Transcriptomic Insight into Aroma Pathway Genes and Effect of Ripening Difference on Expression of Aroma Genes in Different Mango Cultivars

Allelic diversity of the <i>FaOMT</i> gene (mesifurane biosynthesis) in promising strawberry cultivars and selected forms developed at the I.V. Michurin Federal Science Center

Background. Fruit aroma is an important consumer trait of strawberry varieties. Identification of genotypes carrying the genes of the aromatic complex in fruits is an important stage in the development of new cultivars with aromatic fruits. The purpose of this study was to analyze the allelic state of the FaOMT fruit aroma gene in strawberry cultivars and selected forms developed at the I.V. Michurin Federal Science Center to identify promising genotypes for use in breeding for fruit aroma.Materials and methods. The research materials were strawberry cultivars and promising selected forms developed at the I.V. Michurin FSC using the methods of introgressive and intervarietal hybridization. Allelic state of the FaOMT gene was identified with the codominant marker FaOMT-SI/NO.Results and conclusion. The FaOMT fruit aroma gene is represented in the studied strawberry varieties and forms by three combinations of alleles: the homozygous state of the functional allele, the heterozygous combination of the alleles, and the homozygous state of the nonfunctional allele. Cvs. ‘Rubinovy kaskad’, ‘Privlekatelnaya’ and ‘Feyerverk’ are characterized by a heterozygous combination of alleles of the FaOMT gene. Cvs. ‘Lastochka’, ‘Pamyati Zubova’, ‘Flora’, ‘Yarkaya’ and ‘Urozhaynaya CGL’ are characterized by the homozygous state of the FaOMT+ allele. Selected strawberry forms 928-12, 26-5, 72-71 and 62-41 have the heterozygous genotype; hybrids 298-19-9-43 and 932-29 are characterized by the homozygous state of the FaOMT+ allele; selected forms 61-12 and 65-26 are characterized by the homozygous state of the nonfunctional FaOMT– allele. Strawberry forms promising for breeding, with the homozygous state of the functional FaOMT+ allele, were identified: cvs. ‘Lastochka’, ‘Pamyati Zubova’, ‘Flora’, ‘Yarkaya’ and ‘Urozhaynaya CGL’, and selected forms 298-19-9-43 and 932-29.

Genetic Analysis of a Collection of Rice Germplasm (Oryza sativa L.) through High-Density SNP Array Provides Useful Information for Further Breeding Practices

Traditional breeding strategies mainly focus on the evaluation of trait performance, but pay less attention to the changing genetic background. A comprehensive understanding of the genetic diversity of germplasms is crucial for the deliberate improvement of specific traits. A collection of 154 highland rice varieties were collected as the initial genetic resource in our breeding program to improve the pathogen resistance and eating and cooking qualities. These varieties were analyzed using a whole-genome SNP array and were clustered into three groups. Further analysis revealed that the favorable alleles of pathogen resistance genes are mostly absent in our collected varieties. However, it showed that most varieties possess favorable alleles of Waxy (Wx) and ALKALI DEGENERATION (ALK), which are able to enhance the eating and cooking qualities. Moreover, only about one fifth of all varieties harbors favorable the allele of fragrance gene Betaine aldehyde dehydrogenase (BADH2). Together, these results give an overall view of the genetic constitution of the target traits, which provide useful information for future genetic improvement in breeding practices.

Molecular analysis of aroma gene (BADH2) in Biriyanicheera: a tropical aromatic rice genotype from Kerala, India.

The aromatic rice cultivars sometimes show variation in aroma when they are grown in regions other than their normal traditional growing regions. An early maturing selection from a Kerala aromatic local landrace with short grains, named 'Biriyanicheera', when grown in normal tropical conditions was sufficiently fragrant. The present study focused on the analysis of aroma in 'Biriyanicheera' rice genotype through molecular methods. The seeds of two aromatic rice varieties viz., Biriyanicheera andGandhakasala (aromatic check) along with one non-aromatic rice variety Triveni (control) were used for the study. The BADH2 gene was amplified in all the three rice varieties. Upon sequencing the amplified PCR products of genomic DNA, the mutation in BADH2 gene was detected. The sequencing results of aromatic rice varieties revealed the presence of 8 base pair mutation in exon 7 in Biriyanicheera and Gandhakasaala, whereas this mutation was absent in the non-aromatic variety Triveni. Aroma production in Biriyanicheera variety is observed to be due to the similar mutation in BADH2 gene as that of the popular scented rice Basmati.

Development of New Rice (Oryza. sativa L.) Breeding Lines through Marker-Assisted Introgression and Pyramiding of Brown Planthopper, Blast, Bacterial Leaf Blight Resistance, and Aroma Genes

Brown planthopper, blast, and bacterial blight are the main biotic stressors of rice and can cause a massive loss in rice production. Aroma is an important character of rice quality. It is of far-reaching significance to breed resistant and high-quality varieties using germplasms with objective genes. In this study, the introgression and pyramiding of brown planthopper (BPH), blast, and bacterial leaf blight (BLB) resistance genes and aroma genes into elite rice maintainers and restorers were conducted through conventional cross-breeding coupled with the marker-assisted selection (MAS) breeding method. Single-plant selection was performed from F2 onwards to select desirable recombinants possessing alleles of interest with suitable phenotypes. Respective linked markers were used in each generation from intercrossing to the F7 generation for tracking the presence of targeted genes. A total of 74 improved lines (ILs) have been developed which possess a combination of 1 to 4 genes for BPH, blast, and BLB resistance and aroma. These ILs showed moderate to high resistance to multiple biotic stresses (BPH, blast and BLB) or aromatic fragrance without obvious negative effects on agronomic traits. As multiple resistance and aromatic traits have become significant objectives in rice breeding, these resistance and/or aroma gene introgressed or pyramided lines have important application prospects. Core ideas: (1) marker-assisted breeding was used to pyramid multiple genes for an elite breeding line; (2) improved lines with the introgression of 1–4 genes were developed to achieve high resistance against various biotic stresses and aroma; (3) new lines were used as donor parents to introgress multiple genes in other genetic backgrounds.

Haplotype Analysis of BADH1 by Next-Generation Sequencing Reveals Association with Salt Tolerance in Rice during Domestication.

Betaine aldehyde dehydrogenase 1 (BADH1), a paralog of the fragrance gene BADH2, is known to be associated with salt stress through the accumulation of synthesized glycine betaine (GB), which is involved in the response to abiotic stresses. Despite the unclear association between BADH1 and salt stress, we observed the responses of eight phenotypic characteristics (germination percentage (GP), germination energy (GE), germination index (GI), mean germination time (MGT), germination rate (GR), shoot length (SL), root length (RL), and total dry weight (TDW)) to salt stress during the germination stage of 475 rice accessions to investigate their association with BADH1 haplotypes. We found a total of 116 SNPs and 77 InDels in the whole BADH1 gene region, representing 39 haplotypes. Twenty-nine haplotypes representing 27 mutated alleles (two InDels and 25 SNPs) were highly (p < 0.05) associated with salt stress, including the five SNPs that have been previously reported to be associated with salt tolerance. We observed three predominant haplotypes associated with salt tolerance, Hap_2, Hap_18, and Hap_23, which were Indica specific, indicating a comparatively high number of rice accessions among the associated haplotypes. Eight plant parameters (phenotypes) also showed clear responses to salt stress, and except for MGT (mean germination time), all were positively correlated with each other. Different signatures of domestication for BADH1 were detected in cultivated rice by identifying the highest and lowest Tajima’s D values of two major cultivated ecotypes (Temperate Japonica and Indica). Our findings on these significant associations and BADH1 evolution to plant traits can be useful for future research development related to its gene expression.

Genomic Characterization of the Fruity Aroma Gene, FaFAD1, Reveals a Gene Dosage Effect on γ-Decalactone Production in Strawberry (Fragaria × ananassa).

Strawberries produce numerous volatile compounds that contribute to the unique flavors of fruits. Among the many volatiles, γ-decalactone (γ-D) has the greatest contribution to the characteristic fruity aroma in strawberry fruit. The presence or absence of γ-D is controlled by a single locus, FaFAD1. However, this locus has not yet been systematically characterized in the octoploid strawberry genome. It has also been reported that the volatile content greatly varies among the strawberry varieties possessing FaFAD1, suggesting that another genetic factor could be responsible for the different levels of γ-D in fruit. In this study, we explored the genomic structure of FaFAD1 and determined the allele dosage of FaFAD1 that regulates variations of γ-D production in cultivated octoploid strawberry. The genome-wide association studies confirmed the major locus FaFAD1 that regulates the γ-D production in cultivated strawberry. With the hybrid capture-based next-generation sequencing analysis, a major presence–absence variation of FaFAD1 was discovered among γ-D producers and non-producers. To explore the genomic structure of FaFAD1 in the octoploid strawberry, three bacterial artificial chromosome (BAC) libraries were developed. A deletion of 8,262 bp was consistently found in the FaFAD1 region of γ-D non-producing varieties. With the newly developed InDel-based codominant marker genotyping, along with γ-D metabolite profiling data, we revealed the impact of gene dosage effect for the production of γ-D in the octoploid strawberry varieties. Altogether, this study provides systematic information of the prominent role of FaFAD1 presence and absence polymorphism in producing γ-D and proposes that both alleles of FaFAD1 are required to produce the highest content of fruity aroma in strawberry fruit.

A study of gene action for yield and quality traits of Basmati rice (Oryza sativa L.) following Hayman's graphical approach

The present study was conducted on 36 crosses obtained by carrying out a crossing program in nine Basmati rice genotypes following half-diallel mating design. Following, Hayman's graphical and numerical approach to comprehend the genetic system involved in the inheritance of traits, it was concluded that, out of 18 traits, only two traits viz., 100-grain weight, and aroma fulfilled the adequacy of additive-dominance model indicating absence of epistasis. Further, Vr/Wr graph was plotted and genetic components w ere estimated for these two traits only. By plotting Vr/Wr graph for 100-grain weight and aroma, partial dominance and over-dominance were observed in the inheritance, respectively. The ratio of KD/Kr implied influence of more of recessive alleles in inheritance of 100-grain weight among parents and dominant alleles for aroma genes among parents. Estimates of genetic components indicated the role of both additive and dominance effect in the inheritance of 100-grain weight and aroma. High heritability in narrow sense was observed for the traits.

GENETIC BASIS OF RICE AROMA GENE AND ITS APPLICATION IN RICE GENETICS AND BREEDING: A REVIEW

GENETIC BASIS OF RICE AROMA GENE AND ITS APPLICATION IN RICE GENETICS AND BREEDING: A REVIEW

Tibet Identification of the Fragrance Allele and Development of the Functional Markers for Fragrance in Taiguoxiaoxiangzhan

The fragrant rice breeding has become an important direction for hybrid rice breeding program. The selection and utilization of parent materials with ideal plant type, fine grain quality, strong resistance and other excellent agronomic characteristics with strong fragrance, especially the utilization and cultivation of the fragrant rice restorer lines, is of great significance to further promote the fragrant hybrid rice breeding. Taiguoxiaoxiangzhan is a restorer line with excellent grain quality, strong resistance and fragrance, which has been widely used in fragrant rice breeding in China. In order to trace the fragrance in Taiguoxiaoxiangzhan, we identified the type of the fragrance gene in Taiguoxiaoxiangzhan was&nbsp; badh2-E7&nbsp; which has an 8 bp deletion and three single nucleotide polymorphisms (SNPs) in exon 7, and this gene is a non-functional&nbsp; badh2 &nbsp;allele and that the functional&nbsp; Badh2 &nbsp;allele encoding betaine aldehyde dehydrogenase (BADH2) could render rice non-fragrant. Furthermore, we found that the Taiguoxiaoxiangzhan increased 2-acetyl-1-pyrroline (2-AP) content (0.108 mg/kg) compared with the non-fragrant rice varieties of Nipponbare and 9311 (&lt;0.01 mg/kg) by the GC-MS method. We also proved that FMbadh2-E7 and InDel-E7, two molecular markers reported earlier, can be applied in different genotypes, including&nbsp; Badh2/Badh2 &nbsp;(non-fragrance homozygote),&nbsp; Badh2/bahd2 &nbsp;(non-fragrance heterozygote) and&nbsp; badh2/badh2&nbsp; (fragrance homozygote). In addition, based on sequence divergence amongst the functional&nbsp; Badh2 &nbsp;and null&nbsp; badh2-E7 &nbsp;allele, we developed two functional markers, FMbadh2-E7A and FMbadh2-E7B, which can be easily used to distinguish non-fragrant from fragrant rice. Genetic analysis shows that the segregation of&nbsp; Badh2/Badh2 ,&nbsp; Badh2/badh2&nbsp; and &nbsp;badh2/badh2 &nbsp;in the F 2 &nbsp;population derived from crosses between Taiguoxiaoxiangzhan and different restorer lines fitted a theoretical ratio of 1:2:1, indicating that the fragrance of Taiguoxiaoxiangzhan is controlled by a single nuclear recessive gene ( bahd2 ). In this study, the identification and genetic analysis of the fragrance gene in Taiguoxiaoxiangzhan and the development of functional markers for fragrance have been studied, which provides a theoretical foundation for the further breeding of fragrant hybrid rice with excellent quality, high yield and strong resistance by molecular marker-assisted selection technology.

Creation of novel alleles of fragrance gene OsBADH2 in rice through CRISPR/Cas9 mediated gene editing.

Fragrance in rice grains is a key quality trait determining its acceptability and marketability. Intensive research on rice aroma identified mutations in betaine aldehyde dehydrogenase (OsBADH2) leading to production of aroma in rice. Gene editing technologies like CRISPR/Cas9 system has opened new avenues for accelerated improvement of rice grain quality through targeted mutagenesis. In this study, we have employed CRISPR/Cas9 tool to create novel alleles of OsBADH2 leading to introduction of aroma into an elite non-aromatic rice variety ASD16. PCR analysis of putative transformants using primers targeting the flanking regions of sgRNA in the 7th exon of OsBADH2 identified 37.5% potential multi-allelic mutations in T0 generation. Sensory evaluation test in the leaves of T0 lines identified thirteen lines belonging to five independent events producing aroma. Sequence analysis of these aromatic T0 lines identified 22 different types of mutations located within -17 bp to +15bp of sgRNA region. The -1/-2 bp deletion in the line # 8-19 and -8/-5 bp deletion in the line # 2-16 produced strong aroma and the phenotype was stably inherited in the T1 generation. Comparative volatile profiling detected novel aromatic compounds viz., pyrrolidine, pyridine, pyrazine, pyradazine and pyrozole in the grains of T1 progenies of line # 8-19. This study has demonstrated the use of CRISPR/Cas9 in creating novel alleles of OsBADH2 to introduce aroma into any non-aromatic rice varieties.

Allelic variations in aroma gene in cultivated rice varieties

Germplasm is a valuable source of genetic diversity that supports crop improvement efforts in any breeding programme but it must first be fully characterised for economically valuable traits before it can be effectively utilised. In rice (Oryza sativa), the development of new varieties with improved aroma requires correct phenotyping and prior knowledge of the available genes and alleles governing the aroma trait in the gene pool. Correct phenotyping and genotyping can be achieved using sensory methods and functional markers associated with polymorphisms that define the aroma genes. The objective of this study was to evaluate the aroma status of rice accessions and to assess for the various alleles of badh2 gene using functional markers. A total of 56 rice accessions were evaluated at National Crops Resources Research Institute (NaCRRI) in Uganda for their aroma using sensory methods and a molecular marker to differentiate between aromatic and non- aromatic accessions. The aromatic accessions were then evaluated for variations within the betaine aldehyde dehydrogenase2 (badh2) gene responsible for aroma in rice using functional markers. Sensory evaluation of aroma identified 23 accessions to be aromatic; while 33 were non-aromatic. Molecular results identified 20 accessions as aromatic; while 36 accessions were non-aromatic. Functional marker analysis indicated the presence of badh2-E7 allele in 20 aromatic accessions within this collection that could be employed in the breeding programme for the rice aromatic trait.&#x0D; Key words: badh2 alleles, functional markers, Oryza sativa

Characterization of Haplotype Diversity in the BADH2 Aroma Gene and Development of a KASP SNP Assay for Predicting Aroma in U.S. Rice

BackgroundAroma is an important grain quality trait in rice, controlled by mutations within the BADH2 gene. The trait is simply inherited, and its importance in variety development makes it a practical target for marker-assisted selection in applied breeding programs. The predominant functional mutation within BADH2, an 8-bp indel, can be reliably detected using a PCR-based assay, but the available assays and associated genotyping platforms are insufficient for large-scale applied molecular breeding applications and are not compatible with outsourcing genotyping services.ResultsWe first characterized SNP diversity across the BADH2 gene in a collection of 2932 rice varieties to determine the number of gene haplotypes in O. sativa. Using 297 gene-based SNPs, 11 haplotype groups were detected, and subsequently identified a minimal set of nine informative SNPs that uniquely identified the BADH2 haplotypes. These nine SNPs were developed into KASP assays and used to examine a panel of 369 U.S. rice accessions. The panel represented modern breeding germplasm and included all known aroma pedigree sources in U.S. rice. Six haplotypes were detected within the U.S. panel, of which two were found in majority (85%) of varieties. A representative set of 39 varieties from all haplotype groups was evaluated phenotypically to distinguish aromatic from non-aromatic lines.ConclusionOne haplotype (Hap 6) was found to be perfectly associated with the aromatic phenotype. A single KASP SNP unique to Hap 6 was demonstrated to reliably differentiate aromatic from non-aromatic rice varieties across U.S. germplasm.

Uncovering mechanisms of greengage wine fermentation against acidic stress via genomic, transcriptomic, and metabolic analyses of Saccharomyces cerevisiae.

Acid stress is one of the most common adverse conditions during fermentation of fruit wines, and the acid tolerance of yeasts is, therefore, critical for fruit wine production. However, the biological mechanism underlying the acquired tolerance of yeasts against acid stress is poorly understood. We have previously obtained an evolved Saccharomyces cerevisiae strain ET008-c54 with increased tolerance against acid stress, and potentially, it serves as a promising yeast strain for greengage wine fermentation. In the current study, we further revealed the alterations responsible for the adaptation of ET008-c54 to low pH by whole-genome re-sequencing, transcriptomic, and metabolic analyses. Results confirmed the outstanding fermenting performance of ET008-c54 at low pH as compared with the parental ET008. More specifically, the growth rate of ET008-c54 at low pH was increased by 6.24 times and the fermentation time was shortened by 70%. Differences were also observed in the physiology of the strains through ergosterol, H+-ATPase activity, and aroma determinations. By integrating both RNA-seq and whole-genome re-sequencing data, we demonstrated some metabolic pathways in ET008-c54, namely ergosterol synthesis and ferrous iron uptake, in which several acid-responsive genes were involved being upregulated. Also, upregulation of the pathways responsible for aroma compound formation, including fatty acid ethyl ester synthesis and aromatic amino acid biosynthesis, was identified. Thus, the enhanced fermentation ability of ET008-c54 at low pH should be, at least partly, contributed by the altered gene expressions associated with the aforementioned pathways. By elucidating the biological mechanism of yeasts against acid stress, this current study allows better-defined targets for future studies of genetic improvement of wine yeasts and enhancement of the fermentation processes. KEY POINTS: • Metabolic analysis confirmed the excellent fermentation performance of ET008-c54. • Acid tolerance genes for ergosterol synthesis and ferrous iron uptake were upregulated. • Aroma genes for fatty acid ethyl ester and aromatic amino acid synthesis were upregulated.

A key \u2018foxy\u2019 aroma gene is regulated by homology-induced promoter indels in the iconic juice grape \u2018Concord\u2019

‘Concord’, the most well-known juice grape with a parentage of the North American grape species Vitis labrusca L., possesses a special ‘foxy’ aroma predominantly resulted from the accumulation of methyl anthranilate (MA) in berries. This aroma, however, is often perceived as an undesirable attribute by wine consumers and rarely noticeable in the common table and wine grape species V. vinifera. Here we discovered homology-induced promoter indels as a major genetic mechanism for species-specific regulation of a key ‘foxy’ aroma gene, anthraniloyl-CoA:methanol acyltransferase (AMAT), that is responsible for MA biosynthesis. We found the absence of a 426-bp and/or a 42-bp sequence in AMAT promoters highly associated with high levels of AMAT expression and MA accumulation in ‘Concord’ and other V. labrusca-derived grapes. These promoter variants, all with direct and inverted repeats, were further confirmed in more than 1,300 Vitis germplasm. Moreover, functional impact of these indels was validated in transgenic Arabidopsis. Superimposed on the promoter regulation, large structural changes including exonic insertion of a retrotransposon were present at the AMAT locus in some V. vinifera grapes. Elucidation of the AMAT genetic regulation advances our understanding of the ‘foxy’ aroma trait and makes it genetically trackable and amenable in grapevine breeding.