Stomata variation in the process of polyploidization in Chinese chive (Allium tuberosum).

BACKGROUND: Stomatal variation, including guard cell (GC) density, size and chloroplast number, is often used to differentiate polyploids from diploids. However, few works have focused on stomatal variation with respect to polyploidization, especially for consecutively different ploidy levels within a plant species. For example, Allium tuberosum, which is mainly a tetraploid (2n = 4x = 32), is also found at other ploidy levels which have not been widely studied yet. RESULTS: We recently found cultivars with different ploidy levels, including those that are diploid (2n = 2x = 16), triploid (2n = 3x = 24), pseudopentaploid (2n = 34-42, mostly 40) and pseudohexaploid (2n = 44-50, mostly 48). GCs were evaluated for their density, size (length and width) and chloroplast number. There was no correspondence between ploidy level and stomatal density, in which anisopolyploids (approximately 57 and 53 stomata/mm2 in triploid and pseudopentaploid, respectively) had a higher stomatal density than isopolyploids (approximately 36, 43, and 44 stomata/mm2 in diploid, tetraploid and pseudohexaploid, respectively). There was a positive relationship between ploidy level and GC chloroplast number (approximately 44, 45, 51, 72 and 90 in diploid to pseudohexaploid, respectively). GC length and width also increased with ploidy level. However, the length increased approximately 1.22 times faster than the width during polyploidization. CONCLUSIONS: This study shows that GC size increased with increasing DNA content, but the rate of increase differed between length and width. In the process of polyploidization, plants evolved longer and narrower stomata with more chloroplasts in the GCs.

RAD-Seq analysis of wild Japanese garlic (Allium macrostemon Bunge) growing in Japan revealed that this neglected crop was previously actively utilized.

Allium macrostemon Bunge, commonly referred to as "no-biru" in Japan, is a widespread wild onion species found across the country. Despite being deeply entwined in ancient Japanese culture, it remains an underutilized crop in Japan. Determining the origins of its domestic populations and understanding their genetic composition is crucial to highlighting the plant's historical significance in Japan. This study aims to bridge this knowledge gap by examining the genetic diversity of 47 A. macrostemon samples from various regions in Japan using RAD-Seq. Our analyses distinguished unique population structures, dividing the samples into three distinct groups: A, B, and C. Notably, groups A and B showed clear evidence of bulb propagation, while group C did not. Group C formed four subgroups: C1, C2, C3, and C4. Hybridization between subgroup C1 and either group A, B, or both, resulted in the emergence of subgroups C2, C3, and C4. Thus, groups A, B, and C1 are posited as the ancestral populations. Additionally, our morphological observations indicated distinct differences among these three groups. Our findings also suggest that human migration may have influenced the plant's distribution, hinting at active usage in the past that later waned, causing its current underutilized status.

Biosynthesis and Metabolism of Garlic Odor Compounds in Cultivated Chinese Chives (Allium tuberosum) and Wild Chinese Chives (Allium hookeri).

Chinese chives is a popular herb vegetable and medicine in Asian countries. Southwest China is one of the centers of origin, and the mountainous areas in this region are rich in wild germplasm. In this study, we collected four samples of germplasm from different altitudes: a land race of cultivated Chinese chives (Allium tuberosum), wide-leaf chives and extra-wide-leaf chives (Allium hookeri), and ovoid-leaf chives (Allium funckiaefolium). Leaf metabolites were detected and compared between A. tuberosum and A. hookeri. A total of 158 differentially accumulated metabolites (DAM) were identified by Gas Chromatography-Mass Spectrometry (GC-MS) and Liquid Chromatography-Mass Spectrometry (LC-MS), among which there was a wide range of garlic odor compounds, free amino acids, and sugars. A. hookeri contains a higher content of fructose, garlic odor compounds, and amino acids than A. tuberosum, which is supported by the higher expression level of biosynthetic genes revealed by transcriptome analysis. A. hookeri accumulates the same garlic odor compound precursors that A. tuberosum does (mainly methiin and alliin). We isolated full-length gene sequences of phytochelatin synthase (PCS), γ-glutamyltranspeptidases (GGT), flavin-containing monooxygenase (FMO), and alliinase (ALN). These sequences showed closer relations in phylogenetic analysis between A. hookeri and A. tuberosum (with sequence identities ranging from 86% to 90%) than with Allium cepa or Allium sativum (which had a lower sequence identity ranging from 76% to 88%). Among these assayed genes, ALN, the critical gene controlling the conversion of odorless precursors into odor compounds, was undetected in leaves, bulbs, and roots of A. tuberosum, which could account for its weaker garlic smell. Moreover, we identified a distinct FMO1 gene in extra-wide-leaf A. hookeri that is due to a CDS-deletion and frameshift mutation. These results above reveal the molecular and metabolomic basis of impressive strong odor in wild Chinese chives.

Characterization of antibiotic-resistant, coagulase-negative staphylococci from fresh produce and description of Staphylococcus shinii sp. nov. isolated from chives.

Coagulase-negative Staphylococcus (CoNS) species may possess antibiotic resistance genes and have been associated with nosocomial infections. In this study, 91 CoNS with decreased susceptibility to oxacillin were isolated from fresh produce using oxacillin containing agar plates. Their antibiotic resistances were determined phenotypically and all isolates were identified by rep-PCR, 16S rRNA and rpoB gene sequencing. Furthermore, the genomes of representative strains were sequenced in order to confirm species identification by phylogenomics. The majority (64 of 91) of the CoNS strains could be identified as Mammaliicoccus (M.) fleurettii, while 13 were identified as M. sciuri, 8 as M. vitulinus, 2 as Staphylococcus (S.) epidermidis and single strains each as S. warneri, S. xylosus, Staphylococcus spp. and S. casei. Most of the strains were generally susceptible to clinically-relevant antibiotics, but only few (< 7%) strains possessed multiple resistances. Both oxacillin and cefoxitin resistant isolates were considered to be presumptive methicillin-resistant CoNS. From whole genome sequencing data of 6 representative strains, the mecA gene, accessory genes and the SCC loci were compared, which revealed high variability between some of the strains. The major fatty acids of K22-5MT strain included anteiso-C15:0, iso-C15:0, iso-C17:0, anteiso-C17:0, C18:0, and C20:0. Average nucleotide identity and digital DNA-DNA hybridization values indicated that Staphylococcus strain K22-5MT was below the species delineation cutoff values for ANI (less than 91%) and DDH (less than 44.4%), with the most closely related species being the S. pseudoxylosus S04009T type strain. Thus, strain K22- 5MT (=DSM 112532T, =LMG 32324T) represents a novel species, for which the name Staphylococcus shinii sp. nov. is proposed.

Determination of reliable reference genes for gene expression studies in Chinese chive (Allium tuberosum) based on the transcriptome profiling.

Chinese chive (Allium tuberosum) is widely cultivated around the world for its unique flavor, nutrient, and medicinal values, yet its molecular mechanism on flavor formation and other metabolic pathways remains intangible. The elucidation of these complex processes begins with investigating the expression of the genes of interest, however the appropriate reference genes (RGs) for normalizing the gene expression are still unavailable in A. tuberosum. To fill this lacuna, transcriptome-wide screening was undertaken to identify the most stable genes according to the analysis of their FPKM values. The expression stability of the RGs was further evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The comprehensive analysis showed that GLY1 and SKP1, instead of two traditionally used RGs (eIF1α and ACT2), were the most stable genes across diverse A. tuberosum tissues, indicating the necessity to carefully validate the stability of RGs prior to their use for normalizations. As indicated by geNorm, the normalizations with at least two RGs could give more accurate results. qRT-PCR experiments were conducted with randomly selected genes, demonstrating that normalization with a combination of GLY1 and SKP1 resulted in reliable normalization results. Our finding represents the first attempt toward establishing a standardized qRT-PCR analysis in this economically important vegetable.

Transcriptome landscapes of multiple tissues highlight the genes involved in the flavor metabolic pathway in Chinese chive (Allium tuberosum).

The unique flavor of Allium tuberosum is primarily associated with the hydrolysis of a series of organosulfur compounds, S-alk(en)yl cysteine sulphoxides (CSOs), upon tissue bruising or maceration. To obtain the tissue-specific transcriptomes, 18 RNA-Seq libraries representing leaf, root, stem, mature flower, inflorescence, and seed tissues of A. tuberosum were sequenced, finally yielding 133.7 Gb clean reads. The de novo assembled transcriptomes enabled the identification of 223,529 unigenes, which were functionally annotated and analyzed for the gene ontology and metabolic pathways. Furthermore, to reveal the flavor metabolic pathways, a total of 205 unigenes involved in the sulfur assimilation and CSO biosynthesis were identified, and their expression profiles were analyzed by RNA-Seq and qRT-PCR. Collectively, this study provides a valuable resource for in-depth molecular and functional researches especially on flavor formation, as well as for the development of molecular markers, and other genetic studies in A. tuberosum.

Host- and stage-dependent secretome of the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Arbuscular mycorrhizal fungi form the most wide-spread endosymbiosis with plants. There is very little host specificity in this interaction, however host preferences as well as varying symbiotic efficiencies have been observed. We hypothesize that secreted proteins (SPs) may act as fungal effectors to control symbiotic efficiency in a host-dependent manner. Therefore, we studied whether arbuscular mycorrhizal (AM) fungi adjust their secretome in a host- and stage-dependent manner to contribute to their extremely wide host range. We investigated the expression of SP-encoding genes of Rhizophagus irregularis in three evolutionary distantly related plant species, Medicago truncatula, Nicotiana benthamiana and Allium schoenoprasum. In addition we used laser microdissection in combination with RNA-seq to study SP expression at different stages of the interaction in Medicago. Our data indicate that most expressed SPs show roughly equal expression levels in the interaction with all three host plants. In addition, a subset shows significant differential expression depending on the host plant. Furthermore, SP expression is controlled locally in the hyphal network in response to host-dependent cues. Overall, this study presents a comprehensive analysis of the R. irregularis secretome, which now offers a solid basis to direct functional studies on the role of fungal SPs in AM symbiosis.

De Novo Assembly and Annotation of the Chinese Chive (Allium tuberosum Rottler ex Spr.) Transcriptome Using the Illumina Platform.

Chinese chive (A. tuberosum Rottler ex Spr.) is one of the most widely cultivated Allium species in China. However, minimal transcriptomic and genomic data are available to reveal its evolution and genetic diversity. In this study, de novo transcriptome sequencing was performed to produce large transcript sequences using an Illumina HiSeq 2000 instrument. We produced 51,968,882 high-quality clean reads and assembled them into 150,154 contigs. A total of 60,031 unigenes with an average length of 631 bp were identified. Of these, 36,523 unigenes were homologous to existing database sequences, 35,648 unigenes were annotated in the NCBI non-redundant (Nr) sequence database, and 23,509 unigenes were annotated in the Swiss-Prot database. A total of 26,798 unigenes were assigned to 57 Gene Ontology (GO) terms, and 13,378 unigenes were assigned to Cluster of Orthologous Group categories. Using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, we mapped 21,361 unigenes onto 128 pathways. Furthermore, 2,125 sequences containing simple sequence repeats (SSRs) were identified. This new dataset provides the most comprehensive resource currently available for gene expression, gene discovery, and future genomic research on Chinese chive. The sequence resources developed in this study can be used to develop molecular markers that will facilitate further genetic research on Chinese chive and related species.

[Cytogenetic effects in Allium schoenoprasum growing on the anthropogenically contaminated soil].

Cytogenetic effects in Allium schoenoprasum meristematic root tip cells grown for a year on the territory contaminated with 235U, 238U and 232Th decay series radionuclides, heavy metals and As were studied. The area is characterized with different concentrations of chemical compounds in soil affecting a toxic element migration in biocoenosis. Analysis of the chromosome aberration spectrum showed an ambiguous cell response to soil contamination. Within the weighted absorbed dose range up to 1.2 Gy the higher the dose the aberrant cell frequency increase was shown. But further increase in the dose resulted in a genotoxic effect decrease due to high toxic effects of heavy metals and radionuclides in soil. This was registered as a mitotic index decrease that can provoke a chromosome aberration frequency underestimation and result in erroneous conclusions about genotoxic effects in A. schoenoprasum used as a bioindicator.

Genotoxicity and cytotoxicity assay of water sampled from the underground nuclear explosion site in the north of the Perm region (Russia).

The results of our study revealed a local biologically relevant surface water contamination in the radionuclide anomaly in the north of Russia (Perm region) by means of Allium schoenoprasum L. anaphase-telophase chromosome aberration assay. This radionuclide anomaly was formed in 1971 as a result of an underground nuclear explosion with soil excavation. Specific activities of main dose-forming radionuclides in all examined reservoirs are below intervention levels officially adopted in Russia for drinking water. We found that (90)Sr significantly contributes to induction of cytogenetic disturbances. Our previous data and the data described here suggest that metal and radionuclide combined exposure (with the dose below permissible exposure limits for human) may cause substantial biological effects. These effects are in part due to synergic response. The findings described here indicated that development of a new concept of radiation protection for humans and biota should be based on the clear understanding of biological effects of low doses of radiation in chronic exposure to multi-pollutant mixtures.

The fertility restorer genes X and T alter the transcripts of a novel mitochondrial gene implicated in CMS1 in chives (Allium schoenoprasum L.).

A chimeric mitochondrial gene configuration, mainly derived from sequences associated with the essential genes atp9 and atp6, was isolated from the sterility-inducing cytoplasm of the CMS1 system in chives (Allium schoenoprasum L.). This sequence is not found in four other cytoplasm types from chives; however, two copies are present in the mitochondrial DNA of CMS1-inducing cytoplasm, whose 5'-sequences are homologous to those of the atp9 gene. We provide evidence to show that one of the two CMS1-specific copies is actively transcribed, and two transcripts which terminate at the same position but differ in their 5'initiation sites were localized using the RACE technique. These transcripts of 942 and 961 nt, respectively, were confirmed to be the major products of this gene in CMS1 plants by Northern hybridization. However, smaller transcripts were found to accumulate in plants in which fertility had been restored. Restoration of fertility was induced either by the gene X, or the gene T at high temperatures. In (S1) X. genotypes a transcript with an estimated size of 440 nt was detected in all tissues examined. An additional hybridization signal with an estimated size of approximately 850 nt is expressed in temperature-sensitive plants [(S1) xxT.], and the intensity of a minor 350-nt transcript is enhanced. These latter alterations, conditioned by the gene T, occur independently of the growth temperature, but are limited to the flowers; they were not observed in leaves. The CMS1 transcripts are edited at seven positions and contain an ORF with a maximum coding capacity of 780 nt (containing the start codon derived from the atp9 gene in-frame). Use of the third in-frame start codon would result in the synthesis of a protein of a size very close to that of a previously described CMS1-specific protein, which has an apparent molecular weight of 18 kDa. The coding sequence that begins at this third in-frame start codon is also present in the sterility-inducing cytoplasms (S) and (T) in the onion, and absent in (N) cytoplasm.