Chalkiness and premature controlled by energy homeostasis in OsNAC02 Ko-mutant during vegetative endosperm development.

BACKGROUND: Chalkiness is a common phenotype induced by various reasons, such as abiotic stress or the imbalance of starch synthesis and metabolism during the development period. However, the reason mainly for one gene losing its function such as NAC (TFs has a large family in rice) which may cause premature is rarely known to us. RESULTS: The Ko-Osnac02 mutant demonstrated an obviously early maturation stage compared to the wild type (WT) with 15 days earlier. The result showed that the mature endosperm of Ko-Osnac02 mutant exhibited chalkiness, characterized by white-core and white-belly in mature endosperm. As grain filling rate is a crucial factor in determining the yield and quality of rice (Oryza sativa, ssp. japonica), it's significant that mutant has a lower amylose content (AC) and higher soluble sugar content in the mature endosperm. Interestingly among the top DEGs in the RNA sequencing of N2 (3DAP) and WT seeds revealed that the OsBAM2 (LOC_Os10g32810) expressed significantly high in N2 mutant, which involved in Maltose up-regulated by the starch degradation. As Prediction of Protein interaction showed in the chalky endosperm formation in N2 seeds (3 DAP), seven genes were expressed at a lower-level which should be verified by a heatmap diagrams based on DEGs of N2 versus WT. The Tubulin genes controlling cell cycle are downregulated together with the MCM family genes MCM4 ( ↓), MCM7 ( ↑), which may cause white-core in the early endosperm development. In conclusion, the developing period drastically decreased in the Ko-Osnac02 mutants, which might cause the chalkiness in seeds during the early endosperm development. CONCLUSIONS: The gene OsNAC02 which controls a great genetic co-network for cell cycle regulation in early development, and KO-Osnac02 mutant shows prematurity and white-core in endosperm.

Production of grains with ultra-low heavy metal accumulation by pyramiding novel Alleles of OsNramp5 and OsLsi2 in two-line hybrid rice.

Ensuring rice yield and grain safety quality are vital for human health. In this study, we developed two-line hybrid rice (TLHR) with ultra-low grain cadmium (Cd) and arsenic (As) accumulation by pyramiding novel alleles of OsNramp5 and OsLsi2. We first generated low Cd accumulation restorer (R) lines by editing OsNramp5, OsLCD, and OsLCT1 in japonica and indica. After confirming that OsNramp5 was most efficient in reducing Cd, we edited this gene in C815S, a genic male sterile line (GMSL), and screened it for alleles with low Cd accumulation. Next, we generated R and GMSL lines with low As accumulation by editing OsLsi2 in a series of YK17 and C815S lines. When cultivated in soils that were heavily polluted with Cd and As, the edited R, GMSL, and TLHR plants showed significantly reduced heavy metal accumulation, while maintaining a relatively stable yield potential. This study provides an effective scheme for the safe production of grains in As- and/or Cd-polluted paddy fields.

The elite eating quality alleles Wxb and ALKb are regulated by OsDOF18 and coordinately improve head rice yield.

Head rice yield (HRY) measures rice milling quality and determines final grain yield and commercial value. Here, we report that two major quantitative trait loci for milling quality in rice, qMq-1 and qMq-2, represent allelic variants of Waxylv/Waxyb (hereafter Wx) encoding Granule-Bound Starch Synthase I (GBSSI) and Alkali Spreading Value ALKc/ALKb encoding Soluble Starch Synthase IIa (SSIIa), respectively. Complementation and overexpression transgenic lines in indica and japonica backgrounds confirmed that Wx and ALK coordinately regulate HRY by affecting amylose content, the number of amylopectin branches, amyloplast size, and thus grain filling and hardness. The transcription factor OsDOF18 acts upstream of Wx and ALK by activating their transcription. Furthermore, rice accessions with Wxb and ALKb alleles showed improved HRY over those with Wxlv and ALKc. Our study not only reveals the novel molecular mechanism underlying the formation of HRY but also provides a strategy for breeding rice cultivars with improved HRY.

Photosynthesis regulates tillering bud elongation and nitrogen-use efficiency via sugar-induced NGR5 in rice.

Rice tillering is one of the most important agronomical traits largely determining grain yield. Photosynthesis and nitrogen availability are two important factors affecting rice tiller bud elongation; however, underlying mechanism and their cross-talk is poorly understood. Here, we used map-based cloning, transcriptome profiling, phenotypic analysis, and molecular genetics to understand the roles of the Decreased Tiller Number 1 (DTN1) gene that encodes the fructose-1,6-bisphosphate aldolase and involves in photosynthesis required for light-induced axillary bud elongation in rice. Deficiency of DTN1 results in the reduced photosynthetic rate and decreased contents of sucrose and other sugars in both leaves and axillary buds, and the reduced tiller number in dtn1 mutant could be partially rescued by exogenous sucrose treatment. Furthermore, we found that the expression of nitrogen-mediated tiller growth response 5 (NGR5) was remarkably decreased in shoot base of dtn1-2, which can be activated by sucrose treatment. Overexpression of NGR5 in the dtn1-2 could partially rescue the reduced tiller number, and the tiller number of dtn1-2 was insensitive to nitrogen supply. This work demonstrated that the sugar level regulated by photosynthesis and DTN1 could positively regulate NGR5 expression, which coordinates the cross-talk between carbon and nitrate to control tiller bud outgrowth in rice.

An orchestrated ethylene-gibberellin signaling cascade contributes to mesocotyl elongation and emergence of rice direct seeding.

A mechanized direct seeding of rice with less labor and water usage, has been widely adopted. However, this approach requires varieties that exhibit uniform seedling emergence. Mesocotyl elongation (ME) offers the main drive of fast emergence of rice seedlings from soils; nevertheless, its genetic basis remains unknown. Here, we identify a major rice quantitative trait locus Mesocotyl Elongation1 (qME1), an allele of the Green Revolution gene Semi-Dwarf1 (SD1), encoding GA20-oxidase for gibberellin (GA) biosynthesis. ME1 expression is strongly induced by soil depth and ethylene. When rice grains are direct-seeded in soils, the ethylene core signaling factor OsEIL1 directly promotes ME1 transcription, accelerating bioactive GA biosynthesis. The GAs further degrade the DELLA protein SLENDER RICE 1 (SLR1), alleviating its inhibition of rice PHYTOCHROME-INTERACTING FACTOR-LIKE13 (OsPIL13) to activate the downstream expansion gene OsEXPA4 and ultimately promote rice seedling ME and emergence. The ancient traits of long mesocotyl and strong emergence ability in wild rice and landrace were gradually lost in company with the Green Revolution dwarf breeding process, and an elite ME1-R allele (D349H) is found in some modern Geng varieties (long mesocotyl lengths) in northern China, which can be used in the direct seeding and dwarf breeding of Geng varieties. Furthermore, the ectopic and high expression of ME1 driven by mesocotyl-specific promoters resulted in rice plants that could be direct-seeded without obvious plant architecture or yield penalties. Collectively, we reveal the molecular mechanism of rice ME, and provide useful information for breeding new Green Revolution varieties with long mesocotyl suitable for direct-seeding practice.

Transcriptome and Proteome Analysis Revealed That Hormone and Reactive Oxygen Species Synergetically Regulate Dormancy of Introgression Line in Rice (Oryza sativa L.).

Dormancy is a complex agronomy phenotype controlled by multiple signaling and a key trait repressing pre-harvest sprouting (PHS). However, the signaling network of dormancy remains unclear. In this study, we used Zhonghua11 (ZH11) with a weak dormancy, and Introgression line (IL) with a strong dormancy to study the mechanism of hormones and reactive oxygen species (ROS) crosstalk regulating rice dormancy. The germination experiment showed that the germination rate of ZH11 was 76.86%, while that of IL was only 1.25%. Transcriptome analysis showed that there were 1658 differentially expressed genes (DEGs) between IL and ZH11, of which 577 were up-regulated and 1081 were down-regulated. Additionally, DEGs were mainly enriched in oxidoreductase activity, cell periphery, and plant hormone signal transduction pathways. Tandem mass tags (TMT) quantitative proteomics analysis showed 275 differentially expressed proteins (DEPs) between IL and ZH11, of which 176 proteins were up-regulated, 99 were down-regulated, and the DEPs were mainly enriched in the metabolic process and oxidation-reduction process. The comprehensive transcriptome and proteome analysis showed that their correlation was very low, and only 56 genes were co-expressed. Hormone content detection showed that IL had significantly lower abscisic acid (ABA) contents than the ZH11 while having significantly higher jasmonic acid (JA) contents than the ZH11. ROS content measurement showed that the hydrogen peroxide (H2O2) content of IL was significantly lower than the ZH11, while the production rate of superoxide anion (O2.-) was significantly higher than the ZH11. These results indicate that hormones and ROS crosstalk to regulate rice dormancy. In particular, this study has deepened our mechanism of ROS and JA crosstalk regulating rice dormancy and is conducive to our precise inhibition of PHS.

Production of aromatic three-line hybrid rice using novel alleles of BADH2.

Aroma is a key grain quality trait that directly influences the market price of rice globally. Loss of function of betaine aldehyde dehydrogenase 2 (OsBADH2) affects the biosynthesis of 2-acetyl-1-pyrroline (2-AP), which is responsible for aroma in fragrant rice. The current study was aimed at creating new alleles of BADH2 using CRISPR/Cas9 gene editing technology under the genetic background of the japonica Ningjing 1 (NJ1) and indica Huang Huazhan (HHZ) varieties. Sensory evaluation and analysis using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) showed that the grains of the four homozygous T1 lines with new alleles of BADH2 (nj1-cr BADH2 -1, nj1-cr BADH2 -2, hhz-cr BADH2 -1 and hhz-cr BADH2 -2) produced moderate fragrance and had significantly increased 2-AP content compared with wild-types. Moreover, there were no significant differences in the amylose content and gelatinization temperature among the four lines with new alleles of BADH2 to the wild-types. Thereafter, we crossed the HHZ background new alleles of BADH2 with CMS line Taonong 1A (TN1A) to produce a three-line hybrid variety B-Tao-You-Xiangzhan (BTYXZ) with increased grain aroma. The 2-AP content in grains of the improved BTYXZ-1 and BTYXZ-2 reached at 26.16 and 18.74 µg/kg, and the gel consistency of BTYXZ-1 and BTYXZ-2 increased significantly by 9.1% and 6.5%, respectively, compared with the wild-type Tao-You-Xiangzhan (TYXZ). However, the γ-aminobutyric acid (GABA) content in the improved three-line hybrid rice BTYXZ-1 (5.6 mg/100 g) and BTYXZ-2 (10.7 mg/100 g) was significantly lower than that of the TYXZ. These results demonstrated that CRISPR/Cas9 gene editing technology could be successfully utilized in improving aroma in non-fragrant japonica and indica varieties. In addition, the newly developed BADH2 alleles provided important genetic resources for grain aroma improvement in three-line hybrid rice.

Genome-Wide Analysis and Functional Characterization of Pyruvate Kinase (PK) Gene Family Modulating Rice Yield and Quality.

Pyruvate kinase (PK) is one of the three rate-limiting enzymes of glycolysis, and it plays a pivotal role in energy metabolism. In this study, we have identified 10 PK genes from the rice genome. Initially, these genes were divided into two categories: cytoplasmic pyruvate kinase (PKc) and plastid pyruvate kinase (PKp). Then, an expression analysis revealed that OsPK1, OsPK3, OsPK4, OsPK6, and OsPK9 were highly expressed in grains. Moreover, PKs can form heteropolymers. In addition, it was found that ABA significantly regulates the expression of PK genes (OsPK1, OsPK4, OsPK9, and OsPK10) in rice. Intriguingly, all the genes were found to be substantially involved in the regulation of rice grain quality and yield. For example, the disruption of OsPK3, OsPK5, OsPK7, OsPK8, and OsPK10 and OsPK4, OsPK5, OsPK6, and OsPK10 decreased the 1000-grain weight and the seed setting rate, respectively. Further, the disruption of OsPK4, OsPK6, OsPK8, and OsPK10 through the CRISPR/Cas9 system showed an increase in the content of total starch and a decrease in protein content compared to the WT. Similarly, manipulations of the OsPK4, OsPK8, and OsPK10 genes increased the amylose content. Meanwhile, the grains of all CRISPR mutants and RNAi lines, except ospk6, showed a significant increase in the chalkiness rate compared to the wild type. Overall, this study characterizes the functions of all the genes of the PK gene family and shows their untapped potential to improve rice yield and quality traits.

Modification of starch composition, structure and properties through editing of TaSBEIIa in both winter and spring wheat varieties by CRISPR/Cas9.

Foods high in amylose content and resistant starch (RS) offer great potential to improve human health and lower the risk of serious noninfectious diseases. Common wheat (Triticum aestivum L.) is a major staple food crop globally. However, the RS contents in the grains of modern wheat varieties are low. Here, we report the generation of high-amylose wheat through targeted mutagenesis of TaSBEIIa in a modern winter wheat cv Zhengmai 7698 (ZM) and a spring wheat cv Bobwhite by CRISPR/Cas9, respectively. We generated a series of transgene-free mutant lines either with partial or triple-null TasbeIIa alleles in ZM and Bobwhite, respectively. Analyses of starch composition, structure and properties revealed that the effects of partial or triple-null alleles were dosage dependent with triple-null lines demonstrated more profound impacts on starch composition, fine structures of amylopectin and physiochemical and nutritional properties. The flours of triple-null lines possessed significantly increased amylose, RS, protein and soluble pentosan contents which benefit human health. Baking quality analyses indicated that the high-amylose flours may be used as additives or for making cookies. Collectively, we successfully modified the starch composition, structure and properties through targeted mutagenesis of TaSBEIIa by CRISPR/Cas9 in both winter and spring wheat varieties and generated transgene-free high-amylose wheat. Our finding provides deep insights on the role of TaSBEIIa in determining starch composition, structure, properties and end-use quality in different genetic backgrounds and improving RS content with multiple breeding and end-use applications in cereal crop species through genome editing for health benefits.

CDE4 encodes a pentatricopeptide repeat protein involved in chloroplast RNA splicing and affects chloroplast development under low-temperature conditions in rice.

Pentatricopeptide repeat (PPR) proteins play important roles in the post-transcriptional modification of organellar RNAs in plants. However, the function of most PPR proteins remains unknown. Here, we characterized the rice (Oryza sativa L.) chlorophyll deficient 4 (cde4) mutant which exhibits an albino phenotype during early leaf development, with decreased chlorophyll contents and abnormal chloroplasts at low-temperature (20°C). Positional cloning revealed that CDE4 encodes a P-type PPR protein localized in chloroplasts. In the cde4 mutant, plastid-encoded polymerase (PEP)-dependent transcript levels were significantly reduced, but transcript levels of nuclear-encoded genes were increased compared to wild-type plants at 20°C. CDE4 directly binds to the transcripts of the chloroplast genes rpl2, ndhA, and ndhB. Intron splicing of these transcripts was defective in the cde4 mutant at 20°C, but was normal at 32°C. Moreover, CDE4 interacts with the guanylate kinase VIRESCENT 2 (V2); overexpression of V2 enhanced CDE4 protein stability, thereby rescuing the cde4 phenotype at 20°C. Our results suggest that CDE4 participates in plastid RNA splicing and plays an important role in rice chloroplast development under low-temperature conditions.

GBSS-BINDING PROTEIN, encoding a CBM48 domain-containing protein, affects rice quality and yield.

The percentage of amylose in the endosperm of rice (Oryza sativa) largely determines grain cooking and eating qualities. Granule-bound starch synthase I (GBSSI) and GBSSII are responsible for amylose biosynthesis in the endosperm and leaf, respectively. Here, we identified OsGBP, a rice GBSS-binding protein that interacted with GBSSI and GBSSII in vitro and in vivo. The total starch and amylose contents in osgbp mutants were significantly lower than those of wild type in leaves and grains, resulting in reduced grain weight and quality. The carbohydrate-binding module 48 (CBM48) domain present in the C-terminus of OsGBP is crucial for OsGBP binding to starch. In the osgbp mutant, the extent of GBSSI and GBSSII binding to starch in the leaf and endosperm was significantly lower than wild type. Our data suggest that OsGBP plays an important role in leaf and endosperm starch biosynthesis by mediating the binding of GBSS proteins to developing starch granules. This elucidation of the function of OsGBP enhances our understanding of the molecular basis of starch biosynthesis in rice and contributes information that can be potentially used for the genetic improvement of yield and grain quality.

Generation of a new thermo-sensitive genic male sterile rice line by targeted mutagenesis of TMS5 gene through CRISPR/Cas9 system.

BACKGROUND: Two-line hybrid rice with high yield potential is increasingly popular and the photo- and temperature-sensitive male sterile line is one of the basic components for two-line hybrid rice breeding. The development of male sterile lines through conventional breeding is a lengthy and laborious process, whereas developing thermo-sensitive genic male sterile (TGMS) lines for two-line hybrid breeding by editing a temperature-sensitivity gene by CRISPR/Cas9 is efficient and convenient. RESULTS: Here, thermo-sensitive genic male sterility (TGMS) was induced by employing the CRISPR/Cas9 gene editing technology to modify the gene TMS5. Two TGMS mutants, tms5-1 and tms5-2, both lacking any residual T-DNA, were generated in the indica rice cultivar Zhongjiazao17 (cv. YK17) background. When grown at a sub-optimal temperature (22 °C), both mutants produced viable pollen and successfully produced grain through self-fertilization, but at temperatures 24 and 26 °C, their pollen was sterile and no grain was set. F1 hybrids derived from the crosses between YK17S (tms5-1) and three different restorer lines outperformed both parental lines with respect to grain yield and related traits. CONCLUSION: The YK17S generated by CRISPR/Cas9 system was proved to be a new TGMS line with superior yield potential and can be widely utilized in two-line hybrid breeding of indica rice.

NF-YB1-YC12-bHLH144 complex directly activates Wx to regulate grain quality in rice (Oryza sativa L.).

Identification of seed development regulatory genes is the key for the genetic improvement in rice grain quality. NF-Ys are the important transcription factors, but their roles in rice grain quality control and the underlying molecular mechanism remain largely unknown. Here, we report the functional characterization a rice NF-Y heterotrimer complex NF-YB1-YC12-bHLH144, which is formed by the binding of NF-YB1 to NF-YC12 and then bHLH144 in a sequential order. Knock-out of each of the complex genes resulted in alteration of grain qualities in all the mutants as well as reduced grain size in crnf-yb1 and crnf-yc12. RNA-seq analysis identified 1496 genes that were commonly regulated by NF-YB1 and NF-YC12, including the key granule-bound starch synthase gene Wx. NF-YC12 and bHLH144 maintain NF-YB1 stability from the degradation mediated by ubiquitin/26S proteasome, while NF-YB1 directly binds to the 'G-box' domain of Wx promoter and activates Wx transcription, hence to regulate rice grain quality. Finally, we revealed a novel grain quality regulatory pathway controlled by NF-YB1-YC12-bHLH144 complex, which has great potential for rice genetic improvement.

OsPK2 encodes a plastidic pyruvate kinase involved in rice endosperm starch synthesis, compound granule formation and grain filling.

Starch is the main form of energy storage in higher plants. Although several enzymes and regulators of starch biosynthesis have been defined, the complete molecular machinery remains largely unknown. Screening for irregularities in endosperm formation in rice represents valuable prospect for studying starch synthesis pathway. Here, we identified a novel rice white-core endosperm and defective grain filling mutant, ospk2, which displays significantly lower grain weight, decreased starch content and alteration of starch physicochemical properties when compared to wild-type grains. The normal starch compound granules were drastically reduced and more single granules filled the endosperm cells of ospk2. Meanwhile, the germination rate of ospk2 seeds after 1-year storage was observably reduced compared with wild-type. Map-based cloning of OsPK2 indicated that it encodes a pyruvate kinase (PK, ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40), which catalyses an irreversible step of glycolysis. OsPK2 has a constitutive expression in rice and its protein localizes in chloroplasts. Enzyme assay showed that the protein product from expressed OsPK2 and the crude protein extracted from tissues of wild-type exhibits strong PK activity; however, the mutant presented reduced protein activity. OsPK2 (PKpα1) and three other putative rice plastidic isozymes, PKpα2, PKpß1 and PKpß2, can interact to form heteromer. Moreover, the mutation leads to multiple metabolic disorders. Altogether, these results denote new insights into the role of OsPK2 in plant seed development, especially in starch synthesis, compound granules formation and grain filling, which would be useful for genetic improvement of high yield and rice grain quality.

White Leaf and Panicle 2, encoding a PEP-associated protein, is required for chloroplast biogenesis under heat stress in rice.

The plastid-encoded RNA polymerase (PEP) plays an important role in the transcription machinery of mature chloroplasts, yet details of its function remain elusive in rice. Here, we identified a novel PEP-associated protein (PAP), WLP2, based on its two allelic white leaf and panicle mutants, wlp2s and wlp2w. The two mutants were albino lethal at high temperatures and showed decreased chlorophyll accumulation, abnormal chloroplast ultrastructure, and attenuated photosynthetic activity. Map-based cloning suggested that WLP2 encodes a putative pfkB-type carbohydrate kinase family protein, which is homologous to fructokinase-like 1 (AtFLN1) in Arabidopsis. WLP2 is mainly expressed in green tissues and its protein localizes in chloroplasts. Expression levels of PEP-encoded genes, chloroplast development genes and photosynthesis-related genes were compromised in wlp2 mutants, indicating that WLP2 is essential for normal chloroplast biogenesis. Moreover, WLP2 and its paralog OsFLN2 can physically interact with thioredoxin OsTRXz to form a TRX-FLN regulatory module, which not only regulates transcription of the PEP-encoded genes but also maintains the redox balance in chloroplasts under heat stress. Furthermore, the wlp2w mutant gene represents a potential advantage in enhancing seed purity and high-throughput breeding. Our results strongly indicate that WLP2 protects chloroplast development from heat stress via a TRX-FLN regulatory module in rice.

GRAIN INCOMPLETE FILLING 2 regulates grain filling and starch synthesis during rice caryopsis development.

Rice grain filling determines grain weight, final yield and grain quality. Here, a rice defective grain filling mutant, gif2, was identified. Grains of gif2 showed a slower filling rate and a significant lower final grain weight and yield compared to wild-type. The starch content in gif2 was noticeably decreased and its physicochemical properties were also altered. Moreover, gif2 endosperm cells showed obvious defects in compound granule formation. Positional cloning identified GIF2 to encode an ADP-glucose pyrophosphorylase (AGP) large subunit, AGPL2; consequently, AGP enzyme activity in gif2 endosperms was remarkably decreased. GIF2 is mainly expressed in developing grains and the coded protein localizes in the cytosol. Yeast two hybrid assay showed that GIF2 interacted with AGP small subunits OsAGPS1, OsAGPS2a and OsAGPS2b. Transcript levels for granule-bound starch synthase, starch synthase, starch branching enzyme and starch debranching enzyme were distinctly elevated in gif2 grains. In addition, the level of nucleotide diversity of the GIF2 locus was extremely low in both cultivated and wild rice. All of these results suggest that GIF2 plays important roles in the regulation of grain filling and starch biosynthesis during caryopsis development, and that it has been preserved during selection throughout domestication of modern rice.

The rice nuclear gene WLP1 encoding a chloroplast ribosome L13 protein is needed for chloroplast development in rice grown under low temperature conditions.

Plastidial ribosome proteins (PRPs) form the major component of the plastidial ribosome. Here we describe a rice mutant named wlp1 (white leaf and panicles 1) selected from a population of tissue culture regenerants. The early seedling leaves of the mutant were albino, as was the immature panicle at heading, and the phenotype was more strongly expressed in plants exposed to low temperature conditions. Changes in the leaf pigmentation of the mutant were due to altered chlorophyll content and chloroplast development. Positional cloning of WLP1, followed by complementation and knock-down experiments, showed that it encodes a 50S ribosome L13 protein. The WLP1 protein localized to the chloroplast. WLP1 was mainly transcribed in green tissues and particularly abundantly in the early seedling leaves. In addition, the expression level of WLP1 was induced by the low temperature. The transcription pattern of a number of genes involved in plastidial transcription/translation and in photosynthesis was altered in the wlp1 mutants. These results reveal that WLP1 is required for normal chloroplast development, especially under low temperature conditions. This is the first report on the function of PRPs in rice.

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.

Rapid identification of fragrant rice using starch flavor compound via NIR spectroscopy coupled with GC-MS and Badh2 genotyping.

The identification of fragrant rice varieties using near-infrared reflectance spectroscopy (NIRS) models has attracted extensive attention from regulatory authorities worldwide. In this study, 138 fragrant and 54 nonfragrant rice varieties were planted in the same region and distinguished using sensory evaluation, gas chromatography-mass spectrometry analysis, and betaine aldehyde dehydrogenase 2 (Badh2) genotyping. Then, the 2-acetyl-1-pyrroline (2-AP) content was assessed based on partial least-squares discriminant (PLS-DA) models generated after 2nd individually or combined with SNV/MSC/smoothing preprocessing successfully classified fragrant rice both in the calibration and predictive sets. Moreover, design of experiments (DoE)-based preprocessing selection was employed as an effective strategy to optimize the calibration models compared with the one variable at a time (OVAT) method. Further, Badh2 genotype sample screening assisted with identifying authentic fragrant rice and guaranteed the NIRS model's prediction accuracy in identifying fragrant rice. In conclusion, the high throughput PLS-DA multivariate method coupled with NIRS data was applied to identify fragrant rice varieties in routine monitoring and was effective, accurate, and rapid.