Yuichi Uno

Fragrance is among the most valuable traits of high-quality crops and influences consumer preferences. Although 2-acetyl-1-pyrroline (2AP) is a key component of fragrant cultivars in several crops, its genetic mechanism in lettuce ( Lactuca sativa L.) remains poorly understood. The betaine aldehyde dehydrogenase ( BADH ) gene has been identified as causative for 2AP-derived fragrance in rice and soybean cultivars. Hence, we conducted a linkage analysis using an F ₂ population derived from a cross between 'Kukichisya' (fragrant) and 'Rennet' (non-fragrant) for three candidate genes of BADH orthologs in the lettuce genome. Analysis linked LOC111877932 located in LG4 to the fragrance trait, and it was designated LsBADH1 . Comparison among 'Kukichisya', 'Salinas', and candidate BADH of sunflower ( Helianthus annuus L.) revealed three non-synonymous single-nucleotide polymorphisms (nsSNPs) in exons 1, 2, and 9, and suggested that nsSNP in exon 9 was strongly correlated with fragrance in 'Kukichisya.' A premature stop codon introduced in exon 5 of LsBADH1 using Target-AID base-editing technology resulted in truncated BADH1 and higher 2AP levels. Our results indicated that LsBADH1 is responsible for the 2AP-derived fragrance. Our findings can be applied to select cultivars based on a novel concept for the cooking process, providing a transformative platform to breed fragrant lettuce as a high-value-added product.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of small conserved eukaryotic proteins that mediate fusion between organelles and the plasma membrane. Prior to fusion, complementary SNAREs, such as syntaxin, synaptosome-associated protein (SNAP), and synaptobrevin, assemble between membranes with the aid of accessory proteins that provide a scaffold to initiate SNARE zippering, pulling the membranes together, and mediating fusion. SNAP-29 and Syntaxin 6 from Bombyx mori were expressed in Escherichia coli and purified using affinity chromatography. We then produced antibodies against SNAP-29, and Syntaxin 6 of Bombyx mori in rabbits, which were used for immunohistochemistry. Immunohistochemistry results revealed that the expression of SNAP-29 was restricted to neurons in the pars intercerebralis (PI) and dorsolateral protocerebrum (DL) of the brain. The amount of SNAP-29 in the brain increases with hunger. Syntaxin 6 was restricted to neurons in the PI, the central complex (CX) and ventro-median protocerebrum (VP). SNAP-29 co-localized with SNAP-25 and Syntaxin 1A in the PI, and with Syntaxin 1A in the DL. Syntaxin 6 co-localized with SNAP-25 and Syntaxin 1A in the PI and with SNAP-25 in the CX. Bombyxin-immunopositive neurons of the brain occurred in SNAP-29 and Syntaxin 6-IRs. PTTH- and period-immunopositive neurons of the brain occurred in SNAP-29-IRs. Syntaxin 6 is present in EH secretory neurons of the brain.

Carnations (Dianthus caryophyllus L.) are amongst the three most commercially valuable cut flowers worldwide. However, traditional breeding methods are often time-consuming and labor-intensive. Although genome editing is used as an alternative method for creating new varieties, the high heterozygosity of carnations inhibits the ability to maintain varietal characteristics in null segregants except for target-derived traits. The use of homozygous lines is a possible solution. Therefore, this study aimed to establish regeneration, transformation, and genome editing methods using seed-carnation varieties. The effects of four auxins (indole-3-butyric acid, IBA; a-naphthaleneacetic acid, NAA; 2,4-dichlorophenoxyacetic acid, 2,4-D; and 3-indoleacetic acid, IAA) and five cytokinins (6-benzyladenine, BA; thidiazuron, TDZ; kinetin, KT; zeatin, ZT; and N⁶-2-isopentenyl adenine, 2IP) on callus and shoot induction were evaluated. The combination of 0.05 mg/l 2,4-D and 4 mg/l TDZ had the highest shoot formation rate at 28%. In addition, shoot hyperhydricity was reduced by increasing the size of culture vessels. Sucrose, agar, and AgNO₃ concentrations, as well as pH, were optimized to facilitate regeneration. Hygromycin at 12.5 mg/l was subsequently used as the selection agent after Agrobacterium-mediated transformation. Finally, the phytoene desaturase gene was knocked out using the CRISPR/Cas9 system. The obtained albino shoot had a one-base deletion or two-base insertion in the genome sequence. To our knowledge, this is the first study to establish a system for genome editing of callus-derived shoots from a homozygous seed-propagated carnation, which may contribute to the rapid breeding of the new varieties.

Key message: We identified LsMybW as the allele responsible for the shift in color from black to white seeds in wild ancestors of lettuce to modern cultivars. Successfully selected white seeds are a key agronomic trait for lettuce cultivation and breeding; however, the mechanism underlying the shift from black—in its wild ancestor—to white seeds remains uncertain. We aimed to identify the gene/s responsible for white seed trait in lettuce. White seeds accumulated less proanthocyanidins than black seeds, similar to the phenotype observed in Arabidopsis TT2 mutants. Genetic mapping of a candidate gene was performed with double-digest RAD sequencing using an F₂ population derived from a cross between “ShinanoPower” (white) and “Escort” (black). The white seed trait was controlled by a single recessive locus (48.055–50.197 Mbp) in linkage group 7. Using five PCR-based markers and numerous cultivars, eight candidate genes were mapped in the locus. Only the LG7_v8_49.251Mbp_HinfI marker, employing a single-nucleotide mutation in the stop codon of Lsat_1_v5_gn_7_35020.1, was completely linked to seed color phenotype. In addition, the coding region sequences for other candidate genes were identical in the resequence analysis of “ShinanoPower” and “Escort.” Therefore, we proposed Lsat_1_v5_gn_7_35020.1 as the candidate gene and designated it as LsMybW (Lactuca sativaMyb White seeds), an ortholog encoding the R2R3-MYB transcription factor in Arabidopsis. When we validated the role of LsMybW through genome editing, LsMybW knockout mutants harboring an early termination codon showed a change in seed color from black to white. Therefore, LsMybW was the allele responsible for the shift in seed color. The development of a robust marker for marker-assisted selection and identification of the gene responsible for white seeds have implications for future breeding technology and physiological analysis.

We analyzed FLOWERING LOCUS T (FT) orthologs to elucidate the regulatory mechanisms of flowering in carnations. There are six FT candidate genes in the carnation genome. Phylogenetic analysis and amino acid alignment suggested that four genes were FT-like genes involved in promoting flowering. Of these, Dca19666.1 had the all conserved amino acids necessary for florigen activity. Therefore, this gene was designated as DcFT1 and used for further analysis. DcFT1 transcript levels increased as the plants developed from the vegetative to the reproductive growth stages. DcFT1 was unevenly expressed in the leaves, and was more abundant in young leaves. A splice variant was identified; however, its relationship with flowering could not be determined. The early blooming cultivar showed an early elevation of DcFT1 compared to the late-blooming cultivar. Treatment to accelerate flowering by cooling at the end of the day also accelerated the increase in DcFT1 expression. The cloned DcFT1 was overexpressed in Arabidopsis for functional characterization. The resulting transgenic plants began to bolt earlier than the control plants transformed with the empty vector. qRT-PCR analysis of the DcFT1-overexpressor showed that several genes related to flowering were upregulated. These results suggest that DcFT1 is an important regulator of carnation flowering.

Prickly lettuce ( Lactuca serriola ), which is considered the wild ancestor of lettuce, has black seeds, whereas the major seed color of domesticated lettuce is black or white. The successfully-selected white seed trait is a key domestication trait for lettuce cultivation and breeding; however, the mechanism underlying the shift from black to white seeds remains to be clarified. We aimed to identify the gene/s responsible for white seed trait in lettuce. Genetic mapping of a candidate gene was performed with double-digest RAD sequencing using an F ₂ population derived from a cross between ‘ShinanoPower’ (white seed) and ‘Escort’ (black seed). The white seed trait was controlled by a single recessive locus (48.055–50.197 Mbp) in linkage group 7. Narrowing down using five PCR-based markers and 84 cultivars, eight candidate genes were mapped in the locus. Only the LG7_v8_49 . 251Mbp_HinfI marker, which employs a single nucleotide mutation in the stop codon of Lsat_1_v5_gn_7_35020 . 1 was completely linked to the seed color phenotype. In addition, the sequences of the coding region for candidate genes except for Lsat_1_v5_gn_7_35020 . 1 were identical in the resequence analysis of ‘ShinanoPower’ (white seed) and ‘Escort’ (black seed). Therefore, we proposed Lsat_1_v5_gn_7_35020 . 1 , a gene located in the locus, as the candidate gene and designated it as LsTT2 , an ortholog encoding the R2R3 MYB transcription factor in Arabidopsis . When we validated the role of LsTT2 in seed color through genome editing, LsTT2 knockout mutants harboring an early termination codon showed a change in seed color from black to white. White seeds accumulated less proanthocyanidins than black seeds, which was similar to the phenotype observed in Arabidopsis TRANSPARENT TESTA 2 ( TT2 ) mutants. Therefore, LsTT2 was the allele responsible for the shift in seed color from black to white. The development of a robust marker for marker-assisted selection and identification of the gene responsible for white seeds has implications for future breeding technology and physiological analysis.

Rab, a low molecular weight GTP-binding protein, regulates the transmission of intracellular proteins. Insect neuropeptides that are directly involved in growth, development, reproduction, homeostasis, courtship, feeding, circadian rhythm and many other physiological processes are synthesized in neurons and ganglia in the brain and secreted by specific neurosecretory cells in tissues such as the corpus allatum and frontal ganglion. To clarify the relationship between Rabs, two neuropeptides, Allatotropin (AT) and Prothoracicotropic hormone (PTTH) and the circadian clock protein, Period (PER), were used to determine the locations of nine Rabs (Rab1, Rab3, Rab6, Rab7, Rab11, Rab14, Rab19, Rab21 and RabX4). Rab6-, Rab11-, Rab14- and Rab21-immunohistochemical reactivities (IRs) partially overlapped AT-IR in the brain in B. mori. Rab3-, Rab6-, Rab11-, Rab14-, and Rab21-IRs overlapped AT-IR in the frontal ganglion. Of the seven Rabs, only Rab11-IR overlapped PTTH-IR in the brain. Rab1-, Rab3-, Rab11-, Rab14-, Rab19-, Rab21-, RabX4- and NUF (nuclear fallout, an effector of Rab11)-IRs, overlapped PER-IR in the brain. Therefore, Rab may regulate the exocytosis of PTTH, AT and a protein associated with the circadian rhythm.