A case of spontaneous coronary artery dissection that later turned out to be Takayasu arteritis.

Spontaneous coronary artery dissection (SCAD) is a rare cause of acute coronary syndrome (ACS), which typically occurs in women at low risk of atherosclerosis. We herein report a case of SCAD in a 57-year-old man who later developed Takayasu arteritis. The patient presented to our hospital complaining of chest pain and was diagnosed with unstable angina. Emergent coronary angiography was performed, and optical coherence tomography revealed that ACS was caused by SCAD. The patient was treated medically without further ballooning or stenting. Because there was a bilateral difference in blood pressure, the systemic artery was screened by contrast-enhanced computed tomography, which showed left subclavian artery occlusion, proximal stenosis of the superior mesenteric artery, right common iliac artery dissection, and left external iliac artery dissection. Based on these results and 18F-fluorodeoxyglucose positron emission tomography findings, we diagnosed Takayasu arteritis. Prednisolone and tocilizumab were selected for medical treatment, and the patient was in a good condition at one year after the diagnosis. Takayasu arteritis can cause dissection of various arteries and should be suspected when atypical SCAD or multiple dissections are present. Early initiation of immunosuppressive therapy can control disease activity. Learning objective: Spontaneous coronary artery dissection (SCAD) is an important cause of acute coronary syndrome. In this case, we experienced a case of SCAD which turned out to be the first symptom of Takayasu arteritis. Immunosuppressive therapy was effective for both coronary lesion and systemic vasculitis. Not only fibromuscular dysplasia, but also various types of vasculitis should therefore be considered in the differential diagnosis when encountering atypical SCAD cases.

Genetic Cause of Hybrid Lethality Observed in Reciprocal Interspecific Crosses between Nicotiana simulans and N. tabacum.

Hybrid lethality, a type of postzygotic reproductive isolation, is an obstacle to wide hybridization breeding. Here, we report the hybrid lethality that was observed in crosses between the cultivated tobacco, Nicotiana tabacum (section Nicotiana), and the wild tobacco species, Nicotiana simulans (section Suaveolentes). Reciprocal hybrid seedlings were inviable at 28 °C, and the lethality was characterized by browning of the hypocotyl and roots, suggesting that hybrid lethality is due to the interaction of nuclear genomes derived from each parental species, and not to a cytoplasmic effect. Hybrid lethality was temperature-sensitive and suppressed at 36 °C. However, when hybrid seedlings cultured at 36 °C were transferred to 28 °C, all of them showed hybrid lethality. After crossing between an N. tabacum monosomic line missing one copy of the Q chromosome and N. simulans, hybrid seedlings with or without the Q chromosome were inviable and viable, respectively. These results indicated that gene(s) on the Q chromosome are responsible for hybrid lethality and also suggested that N. simulans has the same allele at the Hybrid Lethality A1 (HLA1) locus responsible for hybrid lethality as other species in the section Suaveolentes. Haplotype analysis around the HLA1 locus suggested that there are at least six and two haplotypes containing Hla1-1 and hla1-2 alleles, respectively, in the section Suaveolentes.

Mechanisms of Very Late Stent Thrombosis in Japanese Patients as Assessed by Optical Coherence Tomography.

BACKGROUND: Although very late stent thrombosis (VLST) remains an important concern, the underlying etiology and clinical characteristics are not fully elucidated in Japanese patients who undergo intravascular imaging-guided percutaneous coronary intervention (PCI) regularly. METHODS: We identified 50 VLST lesions (bare-metal stent [BMS] [n = 16], first-generation drug-eluting stent [DES] [n = 14] and newer-generation DES [n = 20]) in patients managed in our institutes. The underlying mechanism of VLST was assessed by optical coherence tomography (OCT), and the major etiology of each lesion was determined. The aim of this study was to explore the mechanisms of VLST of BMSs and DESs in Japanese patients. RESULTS: The median duration since stent implantation was 10 years (range: 1-20). The most frequent etiology of VLST was neoatherosclerotic rupture (44%), followed by neointimal erosion (24%). Edge disease (10%) and evagination (10%) were similarly observed. Malapposition (8%) was deemed to be acquired late by looking at intravascular imaging from the index procedure. Uncovered struts (2%) and in-stent calcified nodule (2%) were the least frequent etiologies. Regardless of etiology, signs of neoatherosclerosis were present in most lesions (82%). Most patients received single (68%) or dual (8%) antiplatelet therapy or oral anticoagulation alone (4%), whereas a considerable proportion of patients discontinued medication (20%). Regarding the treatment strategy, drug-coated balloon was the most frequent strategy (56%), followed by implantation of newer DESs (34%). CONCLUSIONS: Various mechanisms have been identified in Japanese patients with VLST. In these patients, biological responses seemed to be more relevant than the index procedure-related factors.

Neointimal characteristic changes following drug-coated balloons in lesions with repeated revascularization.

BACKGROUND: Since their emergence, drug-coated balloons (DCBs) have been used widely to treat in-stent lesions with coronary artery disease (CAD). However, despite their superior efficacy to balloon angioplasty, how DCBs affect neointimal characteristics is poorly understood. OBJECTIVES: We aimed to assess the neointimal characteristic changes following DCB treatment. METHODS: Using optical frequency domain imaging (OFDI), we serially observed the in-stent lesion site just after and 1 year after DCB angioplasty in 12 lesions of 11 patients with repeated revascularization. Neoatherosclerosis was defined as lipid-laden neointima with or without calcification in the stented lesion. Progression or regression of neoatherosclerosis, newly formed neointimal calcification, newly formed uncovered strut and newly formed evagination were assessed. Tiny tissue protrusion was also recorded as mushroom-like protrusion. RESULTS: Underlying stents were first-generation (n = 5) or newer (n = 7) drug-eluting stents (DESs) with implantation durations ranging from 1 to 15 years (median 8 years). Surprisingly, two-thirds of the lesions (67%, 8 of 12) showed progression of neoatherosclerosis, while a quarter of lesions (25%, 3 of 12) showed regression of neoatherosclerosis. The maximal lipid arc increased from 122° to 174°. Newly formed neointimal calcification was observed in 2 of 12 lesions (16%). Newly formed uncovered struts (33%; 4 of 12) and newly formed evaginations (33%; 4 of 12) were not rare. Mushroom-like protrusion was found in a quarter of lesions (25%; 3 of 12). CONCLUSION: Our study demonstrated that a considerable number of lesions showed varied neointimal characteristic changes in a small number of patients. Further studies in a larger population are needed to understand the clinical impact of these findings.

Understanding and overcoming hybrid lethality in seed and seedling stages as barriers to hybridization and gene flow.

Hybrid lethality is a type of reproductive isolation barrier observed in two developmental stages, hybrid embryos (hybrid seeds) and hybrid seedlings. Hybrid lethality has been reported in many plant species and limits distant hybridization breeding including interspecific and intergeneric hybridization, which increases genetic diversity and contributes to produce new germplasm for agricultural purposes. Recent studies have provided molecular and genetic evidence suggesting that underlying causes of hybrid lethality involve epistatic interaction of one or more loci, as hypothesized by the Bateson-Dobzhansky-Muller model, and effective ploidy or endosperm balance number. In this review, we focus on the similarities and differences between hybrid seed lethality and hybrid seedling lethality, as well as methods of recovering seed/seedling activity to circumvent hybrid lethality. Current knowledge summarized in our article will provides new insights into the mechanisms of hybrid lethality and effective methods for circumventing hybrid lethality.

Angioscopic Findings of Stenosis Versus Occlusion in Femoropopliteal Artery Disease.

BACKGROUND: Despite the increase in the number of patients with peripheral artery disease (PAD), the pathophysiology is not fully elucidated. Recently, angioscopy with a 0.48-megapixel equivalent resolution camera became available for patients with PAD. We aimed to compare the plaque component between native stenosis and occlusion in the femoropopliteal artery using this modality. MATERIALS AND METHODS: Thirty-two consecutive patients who underwent endovascular treatment for native femoropopliteal artery disease with angioscopy were studied. The major angioscopic classifications of each lesion were defined as follows: atheromatous plaque (AP) was defined as luminal narrowing without any protrusion, calcified nodule (CN) was defined as a protruding bump with surface irregularity, a mainly reddish thrombus was defined as organizing thrombus (OG), and organized thrombus (OD) was defined by more than half of the thrombus showing a whitish intima-like appearance. RESULTS: A total of 34 lesions (stenosis, n=18; occlusion, n=16) from 32 patients were included. All stenotic lesions showed AP or CN (n=8 [44%], n=10 [56%], respectively), whereas all occluded lesions showed OG or OD (n=5 [31%], n=11 [69%], respectively), which amounted to a statistically significant difference (p<0.001). In occluded lesions, stiff wires (>3 g) were required to cross all lesions classified as OD, whereas this was not always necessary for lesions classified as OG (11 [100%] of 11, 1 [25%] of 5, respectively; p=0.04). Yellow color plaques were observed to a similar degree in all angioscopic classifications. Major adverse limb events, defined as amputation and any reintervention at 12 months, were highly variable, depending on the angioscopic findings, and tended to be more frequently observed in CN and OD (13% in AP, 40% in CN, 0% in OT, and 36% in OD, p=0.25). CONCLUSION: Angioscopy revealed varying components in stenosis and occlusion with different degrees of clinical impact. This may provide new information on the pathophysiology of PAD.

Capsicum annuum with causal allele of hybrid weakness is prevalent in Asia.

Reproductive isolation, including hybrid weakness, plays an important role in the formation of species. Hybrid weakness in Capsicum, the cessation of plant growth, is caused by two complementary dominant genes, A from C. chinense or C. frutescens and B from C. annuum. In the present study, we surveyed whether 94 C. annuum accessions had B or b alleles by crossing with C. chinense having the A allele. Of the 94 C. annuum accessions, five had the B allele, three of which were native to Latin America and two were native to Asia. When combined with previous studies, the percentage of B carriers was 41% in Japan, 13% in Asia excluding Japan, 6% in Latin America, and 0% in Europe and Africa. In addition, 48 accessions of C. annuum from various countries were subjected to SSR analysis. Clades with high percentages of B-carriers were formed in the phylogenetic trees. In the principal coordinate analysis, most B-carriers were localized in a single group, although the group also included b-carriers. Based on these results, we presumed that the B allele was acquired in some C. annuum lines in Latin America, and B-carriers were introduced to the world during the Age of Discovery, as along with the b-carriers.

Parental Genome Imbalance Causes Hybrid Seed Lethality as Well as Ovary Abscission in Interspecific and Interploidy Crosses in Nicotiana.

Enhanced ovary abscission after pollination and hybrid seed lethality result in post-zygotic reproductive isolation in plant interspecific crosses. However, the connection between these barriers remains unclear. Here, we report that an imbalance in parental genomes or endosperm balance number (EBN) causes hybrid seed lethality and ovary abscission in both interspecific and intraspecific-interploidy crosses in the genus Nicotiana. Auxin treatment suppressed ovary abscission, but not hybrid seed lethality, in an interspecific cross between Nicotiana suaveolens and N. tabacum, suggesting that ovary abscission-related genes are located downstream of those involved in hybrid seed lethality. We performed interploidy crosses among N. suaveolens tetraploids, octoploids, and neopolyploids and revealed hybrid seed lethality and ovary abscission in interploid crosses. Furthermore, a higher maternal EBN than paternal EBN caused these barriers, as previously observed in N. suaveolens × N. tabacum crosses. Altogether, these results suggest that maternal excess of EBN causes hybrid seed lethality, which in turn leads to ovary abscission through the same mechanism in both interspecific and interploidy crosses.

The hybrid lethality of interspecific F1 hybrids of Nicotiana: a clue to understanding hybrid inviability-a major obstacle to wide hybridization and introgression breeding of plants.

Reproductive isolation poses a major obstacle to wide hybridization and introgression breeding of plants. Hybrid inviability in the postzygotic isolation barrier inevitably reduces hybrid fitness, consequently causing hindrances in the establishment of novel genotypes from the hybrids among genetically divergent parents. The idea that the plant immune system is involved in the hybrid problem is applicable to the intra- and/or interspecific hybrids of many different taxa. The lethality characteristics and expression profile of genes associated with the hypersensitive response of the hybrids, along with the suppression of causative genes, support the deleterious epistatic interaction of parental NB-LRR protein genes, resulting in aberrant hyper-immunity reactions in the hybrid. Moreover, the cellular, physiological, and biochemical reactions observed in hybrid cells also corroborate this hypothesis. However, the difference in genetic backgrounds of the respective hybrids may contribute to variations in lethality phenotypes among the parental species combinations. The mixed state in parental components of the chaperone complex (HSP90-SGT1-RAR1) in the hybrid may also affect the hybrid inviability. This review article discusses the facts and hypothesis regarding hybrid inviability, alongside the findings of studies on the hybrid lethality of interspecific hybrids of the genus Nicotiana. A possible solution for averting the hybrid problem has also been scrutinized with the aim of improving the wide hybridization and introgression breeding program in plants.

Genetic Mapping of the HLA1 Locus Causing Hybrid Lethality in Nicotiana Interspecific Hybrids.

Hybrid lethality, a postzygotic mechanism of reproductive isolation, is a phenomenon that causes the death of F1 hybrid seedlings. Hybrid lethality is generally caused by the epistatic interaction of two or more loci. In the genus Nicotiana, N. debneyi has the dominant allele Hla1-1 at the HLA1 locus that causes hybrid lethality in F1 hybrid seedlings by interaction with N. tabacum allele(s). Here, we mapped the HLA1 locus using the F2 population segregating for the Hla1-1 allele derived from the interspecific cross between N. debneyi and N. fragrans. To map HLA1, several DNA markers including random amplified polymorphic DNA, amplified fragment length polymorphism, and simple sequence repeat markers, were used. Additionally, DNA markers were developed based on disease resistance gene homologs identified from the genome sequence of N. benthamiana. Linkage analysis revealed that HLA1 was located between two cleaved amplified polymorphic sequence markers Nb14-CAPS and NbRGH1-CAPS at a distance of 10.8 and 10.9 cM, respectively. The distance between these markers was equivalent to a 682 kb interval in the genome sequence of N. benthamiana.

Temperature-dependent sugar accumulation in interspecific Capsicum F1 plants showing hybrid weakness.

In plants, F1 hybrids showing hybrid weakness exhibit weaker growth than their parents. The phenotypes of hybrid weakness are often suppressed at certain temperatures. However, it is unclear whether hybrid weakness in Capsicum annuum × C. chinense is temperature-dependent or not. Our study showed that Capsicum hybrid weakness was suppressed at 30 and 35 °C and was induced at 15, 20, and 25 °C. Moreover, we investigated the time course of hybrid weakness in cell death, metabolite content, and gene expression in leaves of plants transferred to 20 °C after growing at 30 °C for 21 days. The expression of pathogen defense-related genes was upregulated at 1 day after transfer to 20 °C (DAT). Cell death was detected at 7 DAT, plant growth had almost stopped since 14 DAT, and sugars were accumulated at 42 DAT in hybrid plants. The study revealed that some sugar transporter genes, which had been upregulated since 7 DAT, were involved in sugar accumulation in Capsicum hybrid weakness. Thus, our results demonstrated that gene expression changes occur first, followed by physiological and morphological changes after induction of hybrid weakness. These responses observed in this study in Capsicum hybrid weakness are likely to be owed to plant defense responses-like reactions.

Two Nicotiana occidentalis accessions enable gene identification for Type II hybrid lethality by the cross to N. sylvestris.

Hybrid lethality, meaning the death of F1 hybrid seedlings, has been observed in many plant species, including Nicotiana. Previously, we have revealed that hybrids of the selected Nicotiana occidentalis accession and N. tabacum, an allotetraploid with S and T genomes, exhibited lethality characterized by the fading of shoot color. The lethality was suggested to be controlled by alleles of loci on the S and T genomes derived from N. sylvestris and N. tomentosiformis, respectively. Here, we extended the analysis of hybrid lethality using other two accessions of N. occidentalis identified from the five tested accessions. The two accessions were crossed with N. tabacum and its two progenitors, N. sylvestris and N. tomentosiformis. After crosses with N. tabacum, the two N. occidentalis accessions yielded inviable hybrid seedlings whose lethality was characterized by the fading of shoot color, but only the T genome of N. tabacum was responsible for hybrid lethality. Genetic analysis indicated that first-mentioned N. occidentalis accession carries a single gene causing hybrid lethality by allelic interaction with the S genome.

The role of chaperone complex HSP90-SGT1-RAR1 as the associated machinery for hybrid inviability between Nicotiana gossei Domin and N. tabacum L.

Two cultured cell lines (GTH4 and GTH4S) of a Nicotiana interspecific F1 hybrid (N. gossei × N. tabacum) were comparatively analyzed to find genetic factors related to hybrid inviability. Both cell lines proliferated at 37 °C, but after shifting to 26 °C, GTH4 started to die similar to the F1 hybrid seedlings, whereas GTH4S survived. As cell death requires de novo expression of genes and proteins, we compared expressed protein profiles between the two cell lines, and found that NgSGT1, a cochaperone of the chaperone complex (HSP90-SGT1-RAR1), was expressed in GTH4 but not in GTH4S. Agrobacterium-mediated transient expression of NgSGT1, but not NtSGT1, induced cell death in leaves of N. tabacum, suggesting its possible role in hybrid inviability. Cell death in N. tabacum was also induced by transient expression of NgRAR1, but not NtRAR1. In contrast, transient expression of any parental combinations of three components revealed that NgRAR1 promoted cell death, whereas NtRAR1 suppressed it in N. tabacum. A specific inhibitor of HSP90, geldanamycin, inhibited the progression of hypersensitive response-like cell death in GTH4 and leaf tissue after agroinfiltration. The present study suggested that components of the chaperone complex are involved in the inviability of Nicotiana interspecific hybrid.

Phenylalanine ammonia-lyase and phenolic compounds are related to hybrid lethality in the cross Nicotiana suaveolens×N. tabacum.

Hybrid lethality observed in hybrid seedlings between Nicotiana suaveolens and N. tabacum is characterized by browning, initially of the hypocotyls and eventually of entire seedlings. We investigated the mechanism underlying this browning of tissues. A phenylalanine ammonia-lyase (PAL) gene codes an enzyme involved in a pathway producing phenolic compounds related to the browning of plant tissues. The expression of PAL rapidly increased with the induction of hybrid lethality. Phenolic compounds were observed to be accumulated in whole parts of hybrid seedlings. Treatment of hybrid seedlings with L-2-aminooxy-3-phenylpropionic acid (AOPP), an inhibitor for PAL, suppressed browning and decreased the phenolic content of hybrid seedlings. Although programmed cell death (PCD) was involved in hybrid lethality, AOPP treatment also suppressed cell death and enhanced the growth of hybrid seedlings. These results indicated that PAL is involved in hybrid lethality, and phenolic compounds could be the cause of hybrid lethality-associated tissue browning.

A hypersensitive response-like reaction is involved in hybrid weakness in F1 plants of the cross Capsicum annuum × Capsicum chinense.

Hybrid weakness in Capsicum is characterized by the termination of leaf differentiation after the development of several leaves. F1 plants in some crosses between Capsicum annuum and Capsicum chinense show weakness; this phenomenon has not been investigated in detail since first reported. In the present study, we characterized morphologically and physiologically hybrid weakness in Capsicum. F1 plants did not show weaker growth than their parents 20 days after germination (DAG), but at 40 DAG, the hybrid weakness phenotype was evidenced by almost complete arrest of new leaf formation, delayed increase in plant height, and reduced upper internode length. The shoot apical meristem (SAM) of F1 plants exhibited delayed development and an abnormal structure characterized by a flat shape and the presence of fuzzy cell layers on the surface. These abnormal SAMs of F1 plants may lead to dwarfism. Dead cells and accumulation of H2O2 were visually detected in leaves of F1 plants, and cell death was considered to be programmed, as it was accompanied by internucleosomal fragmentation of DNA. The expression of immunity marker genes PR1 and PR2 was upregulated in leaves of F1 plants. These results suggest that a hypersensitive response-like reaction is involved in Capsicum hybrid weakness.

A high maternal genome excess causes severe seed abortion leading to ovary abscission in Nicotiana interploidy-interspecific crosses.

Seed abortion and ovary abscission, two types of postzygotic reproductive barriers, are often observed in interspecific and/or interploidy crosses in plants. However, the mechanisms underlying these reproductive barriers remain unclear. Here, we show that the distinct types of seed developmental abnormalities (type I and type II seed abortion) occur in a phased manner as maternal to paternal genome dosage increases and that type II seed abortion is followed by ovary abscission. We revealed that these two types of seed developmental abnormalities are observed during seed development in the interploidy-interspecific crosses of Nicotiana suaveolens and N. tabacum. Moreover, in the cross showing type II seed abortion, several events, such as changes in abscission-related gene expression and lignin deposition, occurred in the ovary abscission zone, eventually leading to ovary abscission. Notably, successive increases in maternal ploidy using ploidy manipulated lines resulted in successive type I and type II seed abortions, and the latter was accompanied by ovary abscission. Conversely, both types of seed abortion and ovary abscission could be overcome with a ploidy manipulation technique that balances parental ploidy levels. We thus concluded that a high maternal genome excess cross may cause severe seed developmental defects and ovary abscission. Based on our findings, we propose a model explaining the abortion phenomena, where an interaction between the promotive and inhibitive effects of the parental genomes determines the developmental destiny of seeds. SIGNIFICANCE STATEMENT: We demonstrate that a stepwise increase in maternal ploidy results in a stepwise increase in seed abortion severity, leading to ovary abscission in plants. We propose a model explaining the abortion phenomena, where an interaction between the promotive and inhibitive effects of the parental genomes determines the developmental destiny of seeds.

HWA1- and HWA2-Mediated Hybrid Weakness in Rice Involves Cell Death, Reactive Oxygen Species Accumulation, and Disease Resistance-Related Gene Upregulation.

Hybrid weakness is a type of reproductive isolation in which F1 hybrids of normal parents exhibit weaker growth characteristics than their parents. F1 hybrid of the Oryza sativa Indian cultivars 'P.T.B.7' and 'A.D.T.14' exhibits hybrid weakness that is associated with the HWA1 and HWA2 loci. Accordingly, the aim of the present study was to analyze the hybrid weakness phenotype of the 'P.T.B.7' × 'A.D.T.14' hybrids. The height and tiller number of the F1 hybrid were lower than those of either parent, and F1 hybrid also exhibited leaf yellowing that was not observed in either parent. In addition, the present study demonstrates that SPAD values, an index correlated with chlorophyll content, are effective for evaluating the progression of hybrid weakness that is associated with the HWA1 and HWA2 loci because it accurately reflects degree of leaf yellowing. Both cell death and H2O2, a reactive oxygen species, were detected in the yellowing leaves of the F1 hybrid. Furthermore, disease resistance-related genes were upregulated in the yellowing leaves of the F1 hybrids, whereas photosynthesis-related genes tended to be downregulated. These results suggest that the hybrid weakness associated with the HWA1 and HWA2 loci involves hypersensitive response-like mechanisms.

Morphological changes during juvenile-to-adult phase transition in sorghum.

MAIN CONCLUSION: Morphological and genetic markers indicate that in sorghum, the juvenile-to-adult phase transition occurs during the fourth and fifth leaf stages. This timing differs from those reported for other plants. The juvenile-to-adult (JA) phase transition is an important event for optimizing vegetative growth and reproductive success in plants. Among the Poaceae crops, which are a vital food source for humans, studies of the JA phase transition have been restricted to rice and maize. We studied the morphological and genetic changes that occur during the early development of sorghum and found that dramatic changes occur in shoot architecture during the early vegetative stages. Changes were observed in leaf size, leaf shape, numbers of trichomes, and size of the shoot apical meristem. In particular, the length/width ratios of the leaf blades in the fifth and upper leaves were completely different from those of the second to fourth leaves. The fifth and upper leaves have trichomes on their adaxial sides, which were absent on the lower leaves. We also analyzed expression of two microRNAs that are known to be molecular markers of the JA phase transition and found that expression of miR156 was highest in the second to fourth leaves and then was gradually down-regulated, whereas miR172 expression followed the opposite pattern. These results suggest that in sorghum, the second and third leaves represent the juvenile phase, the fourth and fifth leaves are in the transition stage, and the sixth and upper leaves are in the adult phase. Thus, the JA phase transition occurs during the fourth and fifth leaf stages. These findings are expected to be useful for understanding the early development of sorghum.

Nicotiana suaveolens accessions with different ploidy levels exhibit different reproductive isolation mechanisms in interspecific crosses with Nicotiana tabacum.

Reproductive isolation, including prezygotic and postzygotic barriers, is a mechanism that separates species. Many species in the Nicotiana section Suaveolentes exhibit reproductive isolation in crosses with Nicotiana tabacum. In this study, we investigated whether the chromosome numbers and ploidy levels of eight Nicotiana suaveolens accessions are related to the reproductive isolation after crosses with N. tabacum by flow cytometry and chromosome analyses. Additionally, the internal transcribed spacer (ITS) regions of the eight N. suaveolens accessions were sequenced and compared with the previously reported sequences of 22 Suaveolentes species to elucidate the phylogenetic relationships in the section Suaveolentes. We revealed that four N. suaveolens accessions comprised 64 chromosomes, while the other four accessions carried 32 chromosomes. Depending on the ploidy levels of N. suaveolens, several types of reproductive isolation were observed after crosses with N. tabacum, including decreases in the number of capsules and the germination rates of hybrid seeds, as well as hybrid lethality and abscission of enlarged ovaries at 12-17 days after pollination. A phylogenetic analysis involving ITS sequences divided the eight N. suaveolens accessions into three distinct clades. Based on the results, we confirmed that N. suaveolens accessions vary regarding ploidy levels and reproductive isolation mechanisms in crosses with N. tabacum. These accessions will be very useful for revealing and characterizing the reproductive isolation mechanisms in interspecific crosses and their relationships with ploidy levels.