Tonic Suppression of the Mesolimbic Dopaminergic System by Enhanced Corticotropin-Releasing Factor Signaling Within the Bed Nucleus of the Stria Terminalis in Chronic Pain Model Rats.

Although dysfunction of the mesolimbic dopaminergic system has been implicated in chronic pain, the underlying mechanisms remain to be elucidated. We hypothesized that increased inhibitory inputs to the neuronal pathway from the dorsolateral bed nucleus of the stria terminalis (dlBNST) to the ventral tegmental area (VTA) during chronic pain may induce tonic suppression of the mesolimbic dopaminergic system. To test this hypothesis, male Sprague Dawley rats were subjected to spinal nerve ligation to induce neuropathic pain and then spontaneous IPSCs (sIPSCs) were measured in this neuronal pathway. Whole-cell patch-clamp electrophysiology of brain slices containing the dlBNST revealed that the frequency of sIPSCs significantly increased in VTA-projecting dlBNST neurons 4 weeks after surgery. Next, the role of corticotropin-releasing factor (CRF) signaling within the dlBNST in the increased sIPSCs was examined. CRF increased the frequency of sIPSCs in VTA-projecting dlBNST neurons in sham-operated controls, but not in chronic pain rats. By contrast, NBI27914, a CRF type 1 receptor antagonist, decreased the frequency of sIPSCs in VTA-projecting dlBNST neurons in the chronic pain rats, but not in the control animals. In addition, histological analyses revealed the increased expression of CRF mRNA in the dlBNST. Finally, bilateral injections of NBI27914 into the dlBNST of chronic pain rats activated mesolimbic dopaminergic neurons and induced conditioned place preference. Together, these results suggest that the mesolimbic dopaminergic system is tonically suppressed during chronic pain by enhanced CRF signaling within the dlBNST via increased inhibitory inputs to VTA-projecting dlBNST neurons.SIGNIFICANCE STATEMENT The comorbidity of chronic pain and depression has long been recognized. Although dysfunction of the mesolimbic dopaminergic system has been implicated in both chronic pain and depression, the underlying mechanisms remain to be elucidated. Here, we show that the inhibitory inputs to the neuronal pathway from the dorsolateral bed nucleus of the stria terminalis (dlBNST) to the ventral tegmental area increase during chronic pain. This neuroplastic change is mediated by enhanced corticotropin-releasing factor signaling within the dlBNST that leads to tonic suppression of the mesolimbic dopaminergic system, which may be involved in the depressive mood and anhedonia under the chronic pain condition.

Activation of the neural pathway from the dorsolateral bed nucleus of the stria terminalis to the central amygdala induces anxiety-like behaviors.

The bed nucleus of the stria terminalis (BNST) and the central amygdala (CeA) comprise a forebrain unit that has been described as the "extended amygdala". These two nuclei send dense projections to each other and have been implicated in the regulation of negative emotional states, including anxiety and fear. The present study employed an optogenetic technique to examine whether stimulation of CeA-projecting dorsolateral BNST (dlBNST) neuron terminals would influence anxiety-like behaviors in male Sprague-Dawley rats. Photostimulation of CeA-projecting dlBNST neuron terminals produced anxiogenic effects in an elevated plus maze test. This finding is inconsistent with previous reports showing that optogenetic stimulation of BNST neurons projecting to the lateral hypothalamus (LH) and ventral tegmental area (VTA) produces anxiolytic rather than anxiogenic effects. To address this issue, electrophysiological analyses were conducted to characterize dlBNST neurons projecting to the CeA, LH, and VTA. dlBNST neurons can be electrophysiologically classified into three distinct cell types (types I-III) according to their responses to depolarizing and hyperpolarizing current injections. Whole-cell patch-clamp recordings revealed that more than 60% of the CeA-projecting dlBNST neurons were type II, whereas approximately 80% of the LH- and VTA-projecting dlBNST neurons were type III. These electrophysiological results will help elucidate the mechanisms underlying the heterogeneity of BNST neurons during the regulation of anxiety-like behaviors.

Screening and incorporation of rust resistance from Allium cepa into bunching onion (Allium fistulosum) via alien chromosome addition.

Bunching onion (Allium fistulosum L.; 2n = 16), bulb onion (Allium cepa L. Common onion group), and shallot (Allium cepa L. Aggregatum group) cultivars were inoculated with rust fungus, Puccinia allii, isolated from bunching onion. Bulb onions and shallots are highly resistant to rust, suggesting they would serve as useful resources for breeding rust resistant bunching onions. To identify the A. cepa chromosome(s) related to rust resistance, a complete set of eight A. fistulosum - shallot monosomic alien addition lines (MAALs) were inoculated with P. allii. At the seedling stage, FF+1A showed a high level of resistance in controlled-environment experiments, suggesting that the genes related to rust resistance could be located on shallot chromosome 1A. While MAAL, multi-chromosome addition line, and hypoallotriploid adult plants did not exhibit strong resistance to rust. In contrast to the high resistance of shallot, the addition line FF+1A+5A showed reproducibly high levels of rust resistance.

Compartmentation and localization of bioactive metabolites in different organs of Allium roylei.

To investigate the involvement of Allium roylei metabolites in the plant's defenses, a comprehensive analysis of the content of cysteine sulfoxides, flavonols, polyphenols, ascorbic acid, and saponins was carried out in the various organs of this species. Metabolomics high performance liquid chromatography (HPLC), spectral-based analysis, and histochemcial studies have given important insight to the validity of saponins as a key component involved in plant protection. The root-basal stem, bulb, and leaf extracts exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity with inhibition concentration (IC(50)) ranging from 0.649 to 0.757 mg/mL. The antimicrobial properties of the saponin and flavonoid crude extracts were evaluated. The saponin extracts demonstrated significant antifungal activity depending on the applied concentration, and the growth inhibition rate of the tested fungal pathogens ranged from 1.07 to 47.76%. No appreciable antibacterial activity was recorded in the same sample.

Biochemical analyses of the antioxidative activity and chemical ingredients in eight different Allium alien monosomic addition lines.

We measured the antioxidant contents and antioxidative activities in eight Allium fistulosum-shallot monosomic addition lines (MAL; FF+1A-FF+8A). The high antioxidative activity lines (FF+2A and FF+6A) showed high polyphenol accumulation. These additional chromosomes (2A and 6A) would therefore have anonymous genes related to the upregulation of polyphenol production, the antioxidative activities consequently being increased in these MALs.

Alien genes introgression and development of alien monosomic addition lines from a threatened species, Allium roylei Stearn, to Allium cepa L.

To produce alien monosomic addition lines (AMALs) of Allium cepa (genomes CC, 2n = 2x = 16) carrying extrachromosomes from Allium roylei (RR, 2n = 2x = 16), reciprocal backcrossing of allotriploids (2n = 24, CCR) with diploids (2n = 16, CC) and selfing of a single allotriploid were carried out. The chromosome numbers in the BC(2)F(1) and BC(1)F(2) progenies ranged from 16 to 32. Forty-eight plants were recorded to possess 2n = 17 among a total of 169 plants in observation. Through the analyses of isozymes, expressed sequence tag (EST) markers, and karyotypes, all eight possible types of A. cepa-A. roylei monosomic addition lines (CC+1R-CC+8R) could be identified. Seven types of representative AMALs (without CC+2R) were used for the GISH analysis of somatic chromosomes. Except for CC+6R, all AMALs showed an entire (unrecombined) extrachromosome from A. roylei in the integral diploid background of A. cepa. A single recombination between A. cepa and A. roylei was observed on the extrachromosome in the remaining type. All alloplasmic AMALs possessing A. roylei cytoplasm showed high or complete pollen sterility. Only the autoplasmic CC+4R with A. cepa cytoplasm possessed relatively high pollen fertility. The bulbs of CC+4R displayed the distinct ovoid shape that discriminates them from spherical or oval ones in other AMALs. Downy mildew screening in the field showed higher resistance in A. roylei, a hypo-allotriploid (CCR-nR, 2n = 23), and an allotriploid (CCR, 2n = 24). Meanwhile, no complete resistance was found in some AMALs examined. This was the first trial toward the establishment of a complete set of A. cepa-A. roylei monosomic additions.

Discovery of a new source of resistance to Fusarium oxysporum, cause of Fusarium wilt in Allium fistulosum, located on chromosome 2 of Allium cepa Aggregatum group.

This study was carried out to evaluate the antifungal effect of Allium cepa Aggregatum group (shallot) metabolites on Fusarium oxysporum and to determine the shallot chromosome(s) related to Fusarium wilt resistance using a complete set of eight Allium fistulosum - shallot monosomic addition lines. The antifungal effects of hexane, butanol, and water extraction fractions from bulbs of shallot on 35 isolates of F. oxysporum were examined using the disc diffusion method. Only hexane and butanol fractions showed high antifungal activity. Shallot showed no symptom of disease after inoculation with F. oxysporum f. sp. cepae. The phenolic content of the roots and the saponin content of root exudates of inoculated shallot increased to much higher levels than those of the control at 3 days after inoculation. Application of freeze-dried shallot root exudates to seeds of A. fistulosum soaked in a spore suspension of F. oxysporum resulted in protection of seedlings against infection. Among eight monosomic addition lines and A. fistulosum, FF+2A showed the highest resistance to Fusarium wilt. This monosomic addition line also showed a specific saponin band derived from shallot on the thin layer chromatography profile of saponins in the eight monosomic addition lines. The chromosome 2A of shallot might possess some of the genes related to Fusarium wilt resistance.

Chronic pain-induced neuronal plasticity in the bed nucleus of the stria terminalis causes maladaptive anxiety.

The comorbidity of chronic pain and mental dysfunctions such as depression and anxiety disorders has long been recognized, but the underlying mechanisms remain poorly understood. Here, using a mouse model of neuropathic pain, we demonstrated neuronal plasticity in the bed nucleus of the stria terminalis (BNST), which plays a critical role in chronic pain-induced maladaptive anxiety. Electrophysiology demonstrated that chronic pain increased inhibitory inputs to lateral hypothalamus (LH)-projecting BNST neurons. Chemogenetic manipulation revealed that sustained suppression of LH-projecting BNST neurons played a crucial role in chronic pain-induced anxiety. Furthermore, using a molecular genetic approach, we demonstrated that chronic pain elevated the excitability of a specific subpopulation of BNST neurons, which express cocaine- and amphetamine-regulated transcript (CART). The elevated excitability of CART-positive neurons caused the increased inhibitory inputs to LH-projecting BNST neurons, thereby inducing anxiety-like behavior. These findings shed light on how chronic pain induces psychiatric disorders, characterized by maladaptive anxiety.

Molecular and biochemical identification of alien chromosome additions in shallot (Allium cepa L. Aggregatum group) carrying extra chromosome(s) of bunching onion (A. fistulosum L.).

To develop the bunching onion (Allium fistulosum L.; genomes, FF) chromosome-specific genetic markers for identifying extra chromosomes, eight shallot (A. cepa L. Aggregatum group; genomes, AA)--A. fistulosum monosomic addition plants (AA+nF) and 62 shallot--A. fistulosum single-alien deletion plants (AAF-nF) were analyzed by 23 different chromosome-specific genetic markers of shallot. The eight monosomic addition plants consisted of one AA+2F, two AA+6F, and five AA+8F. Of the 62 single-alien deletion plants, 60 could be identified as six different single-alien deletion lines (AAF-1F, -3F, -4F, -6F, -7F, and -8F) out of the eight possible types. Several single-alien deletion lines were classified on the basis of leaf and bulb characteristics. AAF-8F had the largest number of expanded leaves of five deletion plants. AAF-7F grew most vigorously, as expressed by its long leaf blade and biggest bulb size. AAF-4F had very small bulbs. AAF-7F and AAF-8F had different bulbs from those of shallot as well as other types of single-alien deletion lines in skin and outer scale color. Regarding the sugar content of the bulb tissues, the single-alien deletion lines showed higher fructan content than shallot. Moreover, shallot could not produce fructan with degree of polymerization (DP) 12 or higher, although the single-alien deletion lines showed DP 20 or higher. The content of S-alk(en)yl-L-cysteine sulfoxide (ACSO) in the single-alien deletion lines was significantly lower than that in shallot. These results indicated that chromosomes from A. fistulosum might carry anonymous factors to increase the highly polymerized fructan production and inhibit the synthesis of ACSO in shallot bulbs. Accordingly, alien chromosomes from A. fistulosum in shallot would contribute to modify the quality of shallot bulbs.

Central Histamine Boosts Perirhinal Cortex Activity and Restores Forgotten Object Memories.

BACKGROUND: A method that promotes the retrieval of lost long-term memories has not been well established. Histamine in the central nervous system is implicated in learning and memory, and treatment with antihistamines impairs learning and memory. Because histamine H3 receptor inverse agonists upregulate histamine release, the inverse agonists may enhance learning and memory. However, whether the inverse agonists promote the retrieval of forgotten long-term memory has not yet been determined. METHODS: Here, we employed multidisciplinary methods, including mouse behavior, calcium imaging, and chemogenetic manipulation, to examine whether and how the histamine H3 receptor inverse agonists, thioperamide and betahistine, promote the retrieval of a forgotten long-term object memory in mice. In addition, we conducted a randomized double-blind, placebo-controlled crossover trial in healthy adult participants to investigate whether betahistine treatment promotes memory retrieval in humans. RESULTS: The treatment of H3 receptor inverse agonists induced the recall of forgotten memories even 1 week and 1 month after training in mice. The memory recovery was mediated by the disinhibition of histamine release in the perirhinal cortex, which activated the histamine H2 receptor. Histamine depolarized perirhinal cortex neurons, enhanced their spontaneous activity, and facilitated the reactivation of behaviorally activated neuronal ensembles. A human clinical trial revealed that treatment of H3 receptor inverse agonists is specifically more effective for items that are more difficult to remember and subjects with poorer performance. CONCLUSIONS: These results highlight a novel interaction between the central histamine signaling and memory engrams.

Biochemical and genetic analysis of carbohydrate accumulation in Allium cepa L.

Onion and shallot (Allium cepa L.) exhibit wide variation in bulb fructan content, and the Frc locus on chromosome 8 conditions much of this variation. To understand the biochemical basis of Frc, we conducted biochemical and genetic analyses of Allium fistulosum (FF)-shallot (A. cepa Aggregatum group) alien monosomic addition lines (AALs; FF+1A-FF+8A) and onion mapping populations. Sucrose and fructan levels in leaves of FF+2A were significantly lower than in FF throughout the year, and the springtime activity of acid invertase was also lower. FF+8A showed significantly higher winter sucrose accumulation and sucrose phosphate synthase (SPS) activity. Inbred high fructan (Frc_) lines from the 'W202Ax Texas Grano 438' onion population exhibited significantly higher sucrose levels prior to bulbing than low fructan (frcfrc) lines. Sucrose synthase (SuSy) activity in these lines was correlated with leaf hexose content but not with Frc phenotype. Markers for additional candidate genes for sucrose metabolism were obtained by cloning a major SPS expressed in onion leaf and exhaustively mining onion expressed sequence tag resources. SPS and SuSy loci were assigned to chromosome 8 and 6, respectively, using AALs and linkage mapping. Further loci were assigned, using AALs, to chromosomes 1 (sucrose phosphate phosphatase), 2 (SuSy and three invertases) and 8 (neutral invertase). The concordance between chromosome 8 localization of SPS and elevated leaf sucrose levels conditioned by high fructan alleles at the Frc locus in bulb onion or alien monosomic additions of chromosome 8 in A. fistulosum suggest that the Frc locus may condition variation in SPS activity.

Recent Progress of Basic Studies of Natural Products and Their Dental Application.

The present article reviews the research progress of three major polyphenols (tannins, flavonoids and lignin carbohydrate complexes), chromone (backbone structure of flavonoids) and herbal extracts. Chemical modified chromone derivatives showed highly specific toxicity against human oral squamous cell carcinoma cell lines, with much lower toxicity against human oral keratinocytes, as compared with various anticancer drugs. QSAR analysis suggests the possible correlation between their tumor-specificity and three-dimensional molecular shape. Condensed tannins in the tea extracts inactivated the glucosyltransferase enzymes, involved in the biofilm formation. Lignin-carbohydrate complexes (prepared by alkaline extraction and acid-precipitation) and crude alkaline extract of the leaves of Sasa species (SE, available as an over-the-counter drug) showed much higher anti-HIV activity, than tannins, flavonoids and Japanese traditional medicine (Kampo). Long-term treatment with SE and several Kampo medicines showed an anti-inflammatory and anti-oxidant effects in small size of clinical trials. Although the anti-periodontitis activity of synthetic angiotensin II blockers has been suggested in many papers, natural angiotensin II blockers has not yet been tested for their possible anti-periodontitis activity. There should be still many unknown substances that are useful for treating the oral diseases in the natural kingdom.

Correction: RNA-sequencing-based transcriptome and biochemical analyses of steroidal saponin pathway in a complete set of Allium fistulosum-A. cepa monosomic addition lines.

[This corrects the article DOI: 10.1371/journal.pone.0181784.].

Effect of reactive oxygen species on quality maintenance of broccoli florets with electrostatic atomized water particle treatment.

The effect of electrostatic atomized water particle (EAWP) treatment on quality maintenance during postharvest storage was determined to clarify a physiological role of reactive oxygen species in broccoli florets. The EAWP treatment suppressed the floret yellowing of broccoli during storage. The hydrogen peroxide content increased immediately after EAWP treatment but then remained at a low level during storage. Catalase (CAT) activity increased rapidly with EAWP treatment and was higher than that in the control for the first 4days of storage. The BoCAT gene expression level with EAWP treatment also showed a high trend as compared to the control. The gene expressions of BoRboh, BoCu/ZnSOD, and BoAPX showed almost the same tendency with or without EAWP treatment. These findings suggested that hydrogen peroxide formed by EAWP treatment and regulated by CAT could be relevant to maintaining the quality of broccoli.

RNA-sequencing-based transcriptome and biochemical analyses of steroidal saponin pathway in a complete set of Allium fistulosum-A. cepa monosomic addition lines.

The genus Allium is a rich source of steroidal saponins, and its medicinal properties have been attributed to these bioactive compounds. The saponin compounds with diverse structures play a pivotal role in Allium's defense mechanism. Despite numerous studies on the occurrence and chemical structure of steroidal saponins, their biosynthetic pathway in Allium species is poorly understood. The monosomic addition lines (MALs) of the Japanese bunching onion (A. fistulosum, FF) with an extra chromosome from the shallot (A. cepa Aggregatum group, AA) are powerful genetic resources that enable us to understand many physiological traits of Allium. In the present study, we were able to isolate and identify Alliospiroside A saponin compound in A. fistulosum with extra chromosome 2A from shallot (FF2A) and its role in the defense mechanism against Fusarium pathogens. Furthermore, to gain molecular insight into the Allium saponin biosynthesis pathway, high-throughput RNA-Seq of the root, bulb, and leaf of AA, MALs, and FF was carried out using Illumina's HiSeq 2500 platform. An open access Allium Transcript Database (Allium TDB, http://alliumtdb.kazusa.or.jp) was generated based on RNA-Seq data. The resulting assembled transcripts were functionally annotated, revealing 50 unigenes involved in saponin biosynthesis. Differential gene expression (DGE) analyses of AA and MALs as compared with FF (as a control) revealed a strong up-regulation of the saponin downstream pathway, including cytochrome P450, glycosyltransferase, and beta-glucosidase in chromosome 2A. An understanding of the saponin compounds and biosynthesis-related genes would facilitate the development of plants with unique saponin content and, subsequently, improved disease resistance.

Complete assignment of structural genes involved in flavonoid biosynthesis influencing bulb color to individual chromosomes of the shallot (Allium cepa L.).

We analyzed Japanese bunching onion (Allium fistulosum L.) - shallot (Allium cepa L. Aggregatum group) alien chromosome addition lines in order to assign the genes involved in the flavonoid biosynthesis pathway to chromosomes of the shallot. Two complete sets of alien monosomic additions (2n = 2x + 1 = 17) were used for determining the chromosomal locations of several partial sequences of candidate genes, CHS, CHI, F3H, DFR, and ANS via analyses of PCR-based markers. The results of DNA marker analyses showed that the CHS-A, CHS-B, CHI, F3H, DFR, and ANS genes should be assigned to chromosomes 2A, 4A, 3A, 3A, 7A, and 4A, respectively. HPLC analyses of 14 A. fistulosum - shallot multiple alien additions (2n = 2x + 2 - 2x + 7 = 18 - 23) were conducted to identify the anthocyanin compounds produced in the scaly leaves. A direct comparison between the genomic constitution and the anthocyanin compositions of the multiple additions revealed that a 3GT gene for glucosylation of anthocyanidin was located on 4A. Thus, we were able to assign all structural genes involved in flavonoid biosynthesis influencing bulb color to individual chromosomes of A. cepa.

Control of isoperoxidases involved in chlorophyll degradation of stored broccoli (Brassica oleracea) florets by heat treatment.

Changes in isoperoxidases involved in chlorophyll (Chl) degradation of stored broccoli (Brassica oleracea L.) florets and their control by heat treatment (HT) were determined. Chl a and b contents in non-heat-treated broccoli florets decreased greatly after 2 days at 15 degrees C, whereas the contents in heat-treated florets (50 degrees C for 2 h) showed almost no change. Three isoperoxidases involved in Chl degradation were detected by means of molecular exclusion chromatography and the molecular weights of those isoperoxidases were about 95 (Type I), 67 (Type II) and 56 (Type III) kDa, respectively. Only Type I was detected in broccoli florets immediately after harvest, and its activity in non-heat-treated broccoli increased greatly during storage. Both Type II and Type III were present in non-heat-treated broccoli with floret senescence. HT suppressed the enhancement of all of the isoperoxidase activities. Cycloheximide treatment also effectively retarded the increase in Types I, II and III isoperoxidase activities concomitant with the suppression of floret yellowing. The K(m) values corresponding to Chl a of Type II and Type III were lower than Type I, and the V(max)/K(m) values corresponding to Chl a of Type II and Type III were higher than Type I. This suggests that both Types II and III could be closely associated with Chl degradation in broccoli florets and that HT might inhibit floret senescence by suppression of isoperoxidase activities.

Diversity evaluation based on morphological, physiological and isozyme variation in genetic resources of garlic (Allium sativum L.) collected worldwide.

The aim of this study was to obtain primary information about the global diversity of garlic (Allium sativum L.) by evaluating morphological, physiological and isozyme variation. A total of 107 garlic accessions collected worldwide were grown in Yamaguchi, Japan. Five morphological traits (bulb weight, bulb diameter, number of cloves per bulb, number of bulbils and scape length) and one physiological trait (bolting period) of the collected garlic showed wide variation. Meanwhile, a total of 140 garlic accessions, including the 107 mentioned above, were characterized by leucine aminopeptidase (LAP) and phosphoglucoisomerase (PGI) isozyme analyses; they clearly showed polymorphisms in putative isozyme loci (Lap-1, Lap-2 and Pgi-1). Allelic frequencies were estimated in each group of accessions categorized by their geographical origin, and the observed (Ho) and expected (He) heterozygosities were calculated. The allelic frequencies differed between groups. A principal component analysis based on morpho-physiological data indicated a grouping of the garlic accessions into Central Asian and Northern Mediterranean groups as well as others. We discuss the roles of artificial and natural selection that may have caused differentiation in these traits, on the assumption that ancestral domesticated garlic populations have adapted in various regions using standing variation or mutations that accumulated during expansion, and have evolved along with human-preferred traits over a long history of cultivation.

Tomatidine and lycotetraose, hydrolysis products of alpha-tomatine by Fusarium oxysporum tomatinase, suppress induced defense responses in tomato cells.

Many fungal pathogens of tomato produce extracellular enzymes, collectively known as tomatinases, that detoxify the preformed antifungal steroidal glycoalkaloid alpha-tomatine. Tomatinase from the vascular wilt pathogen of tomato Fusarium oxysporum f. sp. lycopersici cleaves alpha-tomatine into the aglycon tomatidine (Td) and the tetrasaccharide lycotetraose (Lt). Although modes of action of alpha-tomatine have been extensively studied, those of Td and Lt are poorly understood. Here, we show that both Td and Lt inhibit the oxidative burst and hypersensitive cell death in suspension-cultured tomato cells. A tomatinase-negative F. oxysporum strain inherently non-pathogenic on tomato was able to infect tomato cuttings when either Td or Lt was present. These results suggest that tomatinase from F. oxysporum is required not only for detoxification of alpha-tomatine but also for suppression of induced defense responses of host.

Production and characterization of alien chromosome additions in shallot (Allium cepa L. Aggregatum group) carrying extra chromosome(s) of Japanese bunching onion (A. fistulosum L.).

First and second backcrosses of amphidiploid hybrids (2n = 4x = 32, genomes AAFF) between shallot (Allium cepa Aggregatum group) and A. fistulosum were conducted to produce A. cepa - A. fistulosum alien addition lines. When shallot (A. cepa Aggregatum group) was used as a pollinator, the amphidiploids and allotriploids set germinable BC(1) and BC(2) seeds, respectively. The 237 BC(1) plants mainly consisted of 170 allotriploids (2n = 3x = 24, AAF) and 42 hypo-allotriploids possessing 23 chromosomes, i.e., single-alien deletions (2n = 3x-1 = 23, AAF-nF). The single-alien deletions in the BC(1) progeny showed dwarfing characteristics and were discriminated from the allotriploids (2n = 24) and hyper-allotriploids (2n = 25) by means of flow cytometric analysis. The chromosome numbers of 46 BC(2) seedlings varied from 16 to 24. Eight monosomic additions (2n = 2x+1 = 17, AA+nF) and 20 single-alien deletions were found in these BC(2) seedlings. Consequently, six kinds of A. cepa - A. fistulosum alien chromosome additions possessing different chromosome numbers (2n = 17, 18, 20, 21, 22, 23) were recognized in the BC(1) and BC(2) populations. A total of 79 aneuploids, including 62 single-alien deletions, were analyzed by a chromosome 6F-specific isozyme marker (Got-2) in order to recognize its existence in their chromosome complements. This analysis revealed that two out of 62 single-alien deletions did not possess 6F. One (AAF-6F) out of the possible eight single-alien deletions could be identified at first. The present study is a first step toward the development of a useful tool, such as a complete set of eight different single-alien deletions, for the rapid chromosomal assignment of genes and genetic markers in A. fistulosum.