Creation of fragrant peanut using CRISPR/Cas9

Targeted knockout of the betaine aldehyde dehydrogenase genes AhBADH1 and AhBADH2 using CRISPR/Cas9 produced peanut mutant lines with significantly elevated 2-acetyl-1-pyrroline levels and a strong aroma, marking the first creation of fragrant peanut lines. Peanut (Arachis hypogaea L.) is an important food and oil crop valued for its oil-rich kernel, high protein content, and diverse nutritional components, making it a staple raw material in the food industry. Fragrance is an essential trait influencing both the initial appeal and flavor quality of food products. For instance, fragrant rice has gained global popularity due to its appealing aroma. The fragrance trait in rice is regulated by the BADH2 gene, which encodes betaine aldehyde dehydrogenase 2 (BADH) (Chen et al., 2008). In non-fragrant rice, BADH2 catalyzes the conversion of γ-aminobutyraldehyde (GABald) to γ-aminobutyric acid. When BADH2 function is disrupted, GABald is redirected to form Δ-1-pyrroline and the subsequent 2-acetyl-1-pyrroline (2-AP), the key compound contributing to rice fragrance (Okpala et al., 2019). The advent of precise genome editing tools, especially the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas (CRISPR-associated) system, has enabled targeted manipulation of BADH2. Successful development of fragrant lines has been achieved by deleting BADH2 in crops such as maize (Wang et al., 2021), sorghum (Zhang et al., 2022), foxtail millet (Zhang et al., 2023), and soybean (Xie et al., 2024). However, no fragrant peanut cultivar has yet been reported. Given peanut's global significance as both an oil crop and a popular food source, we aimed to investigate whether BADH2 is conserved in peanut and whether fragrant peanut lines can be produced through targeted gene disruption. To identify BADH2 homologs, we used both the amino acid sequence of OsBADH2 and OsBADH1 for blast searches against the peanut reference genome (https://legacy.peanutbase.org/gbrowse_peanut1.0). Four putative AhBADH genes located on chromosomes 1, 5, 11, and 15 showed over 73% sequence similarity with either OsBADH2 or OsBADH1. Phylogenetic analysis indicated that the genes on chromosomes 1 and 11 (97% sequence homology) formed a clade with GmBADH2 and were designated as AhBADH2A and AhBADH2B, respectively. The genes on chromosomes 5 and 15 (96% sequence homology) clustered with GmBADH1 and were thus named AhBADH1A and AhBADH1B, respectively (Figure S1). Quantitative reverse transcriptase polymerase chain reaction analysis showed that AhBADH1 was primarily expressed in flowers, with minimal expression in stems, leaves and kernels and almost no expression in roots. AhBADH2 exhibited the highest expression in flowers, with moderate levels in kernels, roots, stems, and leaves (Figure S2). To understand the polymorphisms of AhBADHs in peanut germplasm, we scanned the AhBADH1A, AhBADH1B, AhBADH2A, and AhBADH2B sequences of 353 peanut landraces and cultivars (Zheng et al., 2024), and focused on variations in the exon regions. A total of six single nucleotide polymorphisms were found that resulted in single amino acid substitutions (Table S1). All accessions harboring those polymorphisms were then subjected to fragrance sensory test. However, none were found to be fragrant (Table S1), indicating the lack of phenotypic variation of this trait in peanut. In light of this, we aimed to create new AhBADH alleles by directly targeting the genes with CRISPR/Cas9 in pursuit of fragrant peanut lines. Given that BADH1, alongside BADH2, has also been implicated in fragrance generation (Amarawathi et al., 2008; Singh et al., 2010), both AhBADH1 and AhBADH2 were investigated in this study. Two single guide RNAs (sgRNAs) were designed: one targeting the conserved region of the first exons of AhBADH1A and AhBADH1B, and the other targeting the equivalent region of AhBADH2A and AhBADH2B. A CRISPR/Cas9 expression vector (Figure 1A) was constructed and used to transform the peanut variety Yuhua 9326 (YH9326) via particle bombardment. A total of 490 hygromycin-resistant lines were obtained, of which 270 were randomly chosen for mutation detection by Sanger sequencing. Results showed that 242 lines, accounting for 89.6% of all lines genotyped, exhibited edits at one or more target sites, with editing efficiencies of 83.7%, 85.2%, 86.7%, and 84.1% for AhBADH1A, AhBADH1B, AhBADH2A, and AhBADH2B, respectively. Creation of fragrant peanut with elevated 2-acetyl-1-pyrroline (2-AP) content through clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated disruption of AhBADH1 and AhBADH2 (A) Design of two single guide RNAs (sgRNAs) targeting the homoeoallele pairs, with a schematic diagram of the T-DNA region of the CRISPR/Cas9 vector. (B) Mutation analysis of lines #47-1, #151-1, and #233-1. The protospacer adjacent motif sequence and nucleotide insertions are indicated in orange and blue, respectively. "‒" indicates a single nucleotide deletion. N.d.: not determined, indicating inserted sequence only partially verified. Two genotypes separated by "/" indicate simultaneous deletion and insertion. (C) Representative chromatograms for 2-AP and 2, 4, 6-trimethyl pyridine (TMP) (as internal standard) in the seeds of ahbadh1/2 mutants and wild-type YH9326. (D) 2-AP content in dry seeds of ahbadh1/2 mutants and YH9326, shown as means ± SD (n = 3), with P-values from a one-way analysis of variance test (*P < 0.05. **P < 0.01). (E) Photograph of fresh kernels of YH9326, lines #47-1, #151-1, and #233-1. Scale bar = 2 cm. To decipher roles of AhBADH1 and AhBADH2 in fragrance generation, we selected 24 mutant lines for regeneration, including mono-mutants, double mutants of AhBADH1A and AhBADH1B, double mutants of AhBADH2A and AhBADH2B, and tetra-mutants of all four genes (Table S2). T0 plants were cultivated in a greenhouse with a 16 /8 h light/dark cycle at 28°C/25°C. However, only 10 lines successfully produced seeds, likely due to the extended tissue culture period, which lasted nearly 10 months prior to regeneration. Notably, among the T0 plants, only those with mutations in all four AhBADH alleles emitted a strong fragrance, suggesting that disruption of all four genes may be necessary to achieve the desired phenotype in peanut. From the 10 fertile lines, we selected lines #47, #151, and #233, each harboring three or four edited alleles, to advance to the next generation. The T1 seeds of the three mutant lines were planted and genotyped, confirming that all the edits were successfully transmitted to the T1 generation (Table S3). Lines #47-1, #151-1, and #233-1 (genotypes shown in Figure 1B) were selected for further investigation. Seeds from these mutant lines and the wild-type YH9326 were analyzed for 2-AP measurement using gas chromatography-mass spectrometry. As seen in Figure 1C, a 2-AP peak was detected in all three mutant lines but was absent in YH9326. Quantification of 2-AP levels indicated that all mutant lines had elevated 2-AP levels compared to YH9326, with line #151-1 exhibiting the highest concentration, followed by lines #47-1 and #233-1 (Figure 1D). Differences in 2-AP accumulation are likely due to the "defective" mutations in lines #47-1 and #233-1 as compared to #151-1 (Figure 1B). Line #47-1 retained a functional AhBADH1A, potentially allowing low but continuous GABald consumption (Bradbury et al., 2008), reducing the 2-AP pool; line #233-1 had a 3-bp deletion in AhBADH2A, removing the sixth amino acid proline and possibly retaining partial protein function. Notably, only lines #151-1 and #47-1 had significantly higher 2-AP levels than YH9326, while line #233-1 did not, suggesting AhBADH2 as a key enzyme in the 2-AP biosynthesis pathway in peanut, with disruption of both AhBADH2 alleles essential for fragrance generation in peanut. The highest 2-AP content in line #151-1 was consistent with the observations in T0 and T1 plants, where distinct fragrance was only present in plants with deletions in all four AhBADHs, indicating that both AhBADH1 and AhBADH2 contributed to 2-AP accumulation and fragrance generation in peanut. A similar conclusion was reached by Xie et al. (2024) in their study on GmBADHs (Xie et al., 2024). Notably, the seeds of line #151-1 exhibited a darker color, resembling roasted peanuts, in contrast to the pink hue observed in YH9326, lines #47-1, and #233-1 (Figure 1E). A similar change in seed coat color was also seen in another mutant line, #233-2, which had disruptions in all four AhBADH alleles resulting from continued editing in the T0 generation (Table S3). To assess the agronomic impacts of disrupting all four AhBADH alleles in peanut, major traits of lines #151-1 and #233-2 were further analyzed. Results showed there were no significant differences between the mutant lines and YH9326 in main shoot length, lateral shoot length, pod number per plant, or seed yield per plant. However, both mutant lines accumulated significantly higher oil content compared to YH9326. Additionally, changes were observed in the contents of several fatty acid components (Tables S4, S5, Figure S3). The specific mechanism by which AhBADH deletions affect oil content, fatty acid composition, and seed coat color in peanut remains to be elucidated. Whole-genome resequencing of four T2 plants of line #151-1 showed that no off-target effects occurred at predicted off-target sites (Table S6), with each plant still carrying two to three copies of the transgenes. Interestingly, all transgenes were inserted precisely at the Cas9 cleavage site (3 bp upstream of the protospacer adjacent motif) within the target loci in all four plants (Figure S4), suggesting efficient target DNA recognition and cleavage, likely due to the transient expression of the editing reagents at those sites. The findings further revealed that line #151-1 continued to exhibit biallelic mutations, with both transgene insertion (Figure S4A, C) and small insertions and deletions (Figure 1B) at the AhBADH2A and AhBADH1B loci arising from the previous generation. Homologous transgene insertion in AhBADH1A in line #151-1 (Figure S4B), although undesirable, could potentially be addressed by incorporating the AhBADH1A allele from line #233-1 in future breeding programs. In conclusion, we successfully generated novel alleles for AhBADH1A, AhBADH1B, AhBADH2A, and AhBADH2B through CRISPR/Cas9, marking the first creation of fragrant peanut lines with promising potential for the breeding of high-value fragrant peanut cultivars. This study was supported by grants from the National Key Research and Development Program of China (2022YFD1200400), the Key Project of Science and Technology of Henan Province (201300111000, 221100110300), the Earmarked Fund (CARS-13), and the Henan Province Agriculture Research System (S2012-5). The authors declare no conflicts of interest. L.S. and X.Y.Z. conceived the study. L.L.X., P.Y.Q., H.H.Z., H.L., B.Y.H., X.B.W., Z.X.Z., X.D.D., L.Q., and W.Z.D. performed the experiments. L.L.X. wrote the manuscript. L.S. and X.Y.Z. revised the manuscript. All authors read and approved the final manuscript. The raw sequencing reads for T2 plants of mutant line #151-1 have been submitted to the National Genomics Data Center database under project accession number PRJCA035307. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13864/suppinfo Figure S1. Phylogenetic analysis of betaine aldehyde dehydrogenase (BADH) proteins across various plant species Figure S2. Relative transcription levels of AhBADH1 and AhBADH2 in various tissues Figure S3. Oil content of YH9326, lines #151-1 and #233-2, shown as means ± SD (n = 3), with P-values from a one-way analysis of variance test (*P < 0.05. **P < 0.01) Figure S4. Whole-genome resequencing analysis of transgene locations in four T2 plants of line #151-1 by Integrative Genomics Viewer (IGV) Table S1. Genetic variations of AhBADHs from 353 cultivars and landraces and sensory test result Table S2. Mutation analysis of the T0 lines regenerated Table S3. Genetic transmission of edited alleles in lines #47, #151, and #233 Table S4. Investigation of major agronomical traits in YH9326 and line #151-1 Table S5. Investigation of major agronomical traits in YH9326 and line #233-2 Table S6. Analysis of off-target effects in four T2 plants from line #151-1 Table S7. Primers used in this study Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.