The vegetable soybean (Glycine max L. Merr.) plant is commonly consumed in Southeast Asian countries because of its nutritional value and desirable taste. A “pandan-like” aroma is an important value-added quality trait that is rarely found in commercial vegetable soybean varieties. In this study, three novel aromatic soybean cultivars with a fragrant volatile compound were isolated. We confirmed that the aroma of these cultivars is due to the potent volatile compound 2-acetyl-1-pyrroline (2AP) that was previously identified in soybean. A sequence comparison of GmBADH1/2 (encoding an aminoaldehyde dehydrogenase) between aromatic and non-aromatic soybean varieties revealed a mutation with 10 SNPs and an 11-nucleotide deletion in exon 1 of GmBADH2 in Quxian No. 1 and Xiangdou. Additionally, a 2-bp deletion was detected in exon 10 of GmBADH2 in ZK1754. The mutations resulted in a frame shift and the introduction of premature stop codons. Moreover, genetic analyses indicated that the aromatic trait in these three varieties was inherited according to a single recessive gene model. These results suggested that a mutated GmBADH2 may be responsible for the aroma of these three aromatic soybean cultivars. The expression and function of GmBADH2 in aromatic soybean seeds were confirmed by qRT-PCR and CRISPR/Cas9. A functional marker developed on the basis of the mutated GmBADH2 sequence in Quxian No. 1 and Xiangdou was validated in an F₂ population. A perfect association between the marker genotypes and aroma phenotypes implied that GmBADH2 is a major aroma-conferring gene. The results of this study are potentially useful for an in-depth analysis of the molecular basis of 2-AP formation in soybean and the marker-assisted breeding of aromatic vegetable soybean cultivars.

Aroma is an important economic trait of vegetable soybeans, which greatly influences their market value. The 2-acetyl-1-pyrroline (2AP) is considered as an important substance affecting the aroma of plants. Although the 2AP synthesis pathway has been resolved, the differences of the 2AP synthesis in the aromatic and non-aromatic vegetable soybeans are unknown. In this study, a broad targeted metabolome analysis including measurement of metabolites levels and gene expression levels was performed to reveal pathways of aroma formation in the two developmental stages of vegetable soybean grains [35 (S5) and 40 (S6) days after anthesis] of the ‘Zhexian No. 8’ (ZX8, non-aromatic) and ZK1754 (aromatic). The results showed that the differentially accumulated metabolites (DAMs) of the two varieties can be classified into nine main categories including flavonoids, lipids, amino acids and derivatives, saccharides and alcohols, organic acids, nucleotides and derivatives, phenolic acids, alkaloids and vitamin, which mainly contributed to their phenotypic differences. Furthermore, in combination with the 2AP synthesis pathway, the differences of amino acids and derivatives were mainly involved in the 2AP synthesis. Furthermore, 2AP precursors’ analysis revealed that the accumulation of 2AP mainly occurred from 1-pyrroline-5-carboxylate (P5C), not 4-aminobutyraldehyde (GABald). The quantitative RT-PCR showed that the associated synthetic genes were 1-pyrroline-5-carboxylate dehydrogenase (P5CDH), ∆¹-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PRODH) and pyrroline-5-carboxylate reductase (P5CR), which further verified the synthetic pathway of 2AP. Furthermore, the betaine aldehyde dehydrogenase 2 (GmBADH2) mutant was not only vital for the occurrence of 2AP, but also for the synthesis of 4-aminobutyric acid (GABA) in vegetable soybean. Therefore, the differences of 2AP accumulation in aromatic and non-aromatic vegetable soybeans have been revealed, and it also provides an important theoretical basis for aromatic vegetable soybean breeding.