Pan-chloroplast genomes for accession-specific marker development in Hibiscus syriacus.

Hibiscus syriacus L. is a renowned ornamental plant. We constructed 95 chloroplast genomes of H. syriacus L. cultivars using a short-read sequencing platform (Illumina) and a long-read sequencing platform (Oxford Nanopore Technology). The following genome assembly, we delineate quadripartite structures encompassing large single-copy, small single-copy, and inverted repeat (IRa and IRb) regions, from 160,231 bp to 161,041 bp. Our comprehensive analyses confirmed the presence of 79 protein-coding genes, 30 tRNA genes, and 4 rRNA genes in the pan-chloroplast genome, consistent with prior research on the H. syriacus chloroplast genome. Subsequent pangenome analysis unveiled widespread genome sequence conservation alongside unique cultivar-specific variant patterns consisting of 193 single-nucleotide polymorphisms and 61 insertions or deletions. The region containing intra-species variant patterns, as identified in this study, has the potential to develop accession-specific molecular markers, enhancing precision in cultivar classification. These findings are anticipated to drive advancements in breeding strategies, augment biodiversity, and unlock the agricultural potential inherent in H. syriacus.

Cucurbitacin and volatile compound profiling reveals independent domestication of cucumber (Cucumis sativus L.) fruit.

Profiling of metabolites that confer bitter taste and flavor to cucumber products is necessary to produce preferred products. In this study, cucurbitacins A, B, C, D, E, and I and untargeted volatile compounds were analyzed using the fruit of 69 inbred cucumber of diverse cultivars. Only cucurbitacin C was detected in six inbreds. They were classified into four clusters based on the profiles of cucurbitacins and volatile compounds. Clusters 2 and 3 showed the largest difference with the highest and lowest volatile contents, respectively. Clusters 1 and 4 showed different fruit phenotypes of length and color. Fifteen F1 hybrids from selected inbreds were analyzed. Total volatile compound (TVC) content, especially for the alcohol content, was lower in the F1 hybrids than the mid-parent values, and the ratio of aldehyde to TVC was increased. This profiling will contribute to produce cucumber products with no bitter taste and improved flavor.

Admixture of divergent genomes facilitates hybridization across species in the family Brassicaceae.

Hybridization and polyploidization are pivotal to plant evolution. Genetic crosses between distantly related species are rare in nature due to reproductive barriers but how such hurdles can be overcome is largely unknown. Here we report the hybrid genome structure of xBrassicoraphanus, a synthetic allotetraploid of Brassica rapa and Raphanus sativus. We performed cytogenetic analysis and de novo genome assembly to examine chromosome behaviors and genome integrity in the hybrid. Transcriptome analysis was conducted to investigate expression of duplicated genes in conjunction with epigenome analysis to address whether genome admixture entails epigenetic reconfiguration. Allotetraploid xBrassicoraphanus retains both parental chromosomes without genome rearrangement. Meiotic synapsis formation and chromosome exchange are avoided between nonhomologous progenitor chromosomes. Reconfiguration of transcription network occurs, and less divergent cis-elements of duplicated genes are associated with convergent expression. Genome-wide DNA methylation asymmetry between progenitors is largely maintained but, notably, B. rapa-originated transposable elements are transcriptionally silenced in xBrassicoraphanus through gain of DNA methylation. Our results demonstrate that hybrid genome stabilization and transcription compatibility necessitate epigenome landscape adjustment and rewiring of cis-trans interactions. Overall, this study suggests that a certain extent of genome divergence facilitates hybridization across species, which may explain the great diversification and expansion of angiosperms during evolution.

Expanded transcriptomic view of strawberry fruit ripening through meta-analysis.

Strawberry is an important fruit crop and a model for studying non-climacteric fruit ripening. Fruit ripening and senescence influence strawberry fruit quality and postharvest storability, and have been intensively studied. However, genetic and physiological differences among cultivars preclude consensus understanding of these processes. We therefore performed a meta-analysis by mapping existing transcriptome data to the newly published and improved strawberry reference genome and extracted meta-differentially expressed genes (meta-DEGs) from six cultivars to provide an expanded transcriptomic view of strawberry ripening. We identified cultivar-specific transcriptome changes in anthocyanin biosynthesis-related genes and common changes in cell wall degradation, chlorophyll degradation, and starch metabolism-related genes during ripening. We also identified 483 meta-DEGs enriched in gene ontology categories related to photosynthesis and amino acid and fatty acid biosynthesis that had not been revealed in previous studies. We conclude that meta-analysis of existing transcriptome studies can effectively address fundamental questions in plant sciences.

Reduced fertility caused by meiotic defects and micronuclei formation during microsporogenesis in xBrassicoraphanus.

BACKGROUND: Hybridization and polyploidization events are important driving forces in plant evolution. Allopolyploids formed between different species can be naturally or artificially created but often suffer from genetic instability and infertility in successive generations. xBrassicoraphanus is an intergeneric allopolyploid obtained from a cross between Brassica rapa and Raphanus sativus, providing a useful resource for genetic and genomic study in hybrid species. OBJECTIVE: The current study aims to understand the cause of hybrid sterility and pollen abnormality in different lines of synthetic xBrassicoraphanus from the cytogenetic perspective. METHODS: Alexander staining was used to assess the pollen viability. Cytogenetic analysis was employed to monitor meiotic chromosome behaviors in pollen mother cells (PMCs). Origins of parental chromosomes in xBrassicoraphanus meiocytes were determined by genome in situ hybridization analysis. RESULTS: The xBrassicoraphanus lines BB#4 and BB#6 showed high rates of seed abortion and pollen deformation. Abnormal chromosome behaviors were observed in their PMCs, frequently forming univalents and inter-chromosomal bridges during meiosis. A positive correlation also exists between meiotic defects and the formation of micronuclei, which is conceivably responsible for unbalanced gamete production and pollen sterility. CONCLUSION: These results suggest that unequal segregation of meiotic chromosomes, due in part to non-homologous interactions, is responsible for micronuclei and unbalanced gamete formation, eventually leading to pollen degeneration and inferior fertility in unstable xBrassicoraphanus lines.

Comparative transcriptome and metabolome analyses of two strawberry cultivars with different storability.

Postharvest storability is an important trait for breeding strawberry (Fragaria × ananassa Duch.). We evaluated the postharvest fruit quality of five strawberry cultivars ('Durihyang', 'Kingsberry', 'Maehyang', 'Seolhyang', and 'Sunnyberry') and identified differences in their fruit ripening during the transition from the big-green to fully-red stage between two cultivars with the highest ('Sunnyberry') and lowest ('Kingsberry') storability, using comparative transcriptome and -metabolome analysis. The differentially expressed genes revealed transcriptome changes related to anthocyanin biosynthesis and cell walls. Consistently, the metabolites of both cultivars showed general changes during ripening along with cultivar-specific characteristics in sugar and amino acid profiles. To identify the genes responsible for storability differences, we surveyed the expression of transcription factors, and found that the expression levels of WRKY31, WRKY70, and NAC83 correlated with delayed senescence and increased storability. Among them, the expression levels of NAC83, and its downstream target genes, in the five cultivars suggested that NAC83 expression can be used to predict postharvest strawberry fruit storability.

Jasmonic acid and ERF family genes are involved in chilling sensitivity and seed browning of pepper fruit after harvest.

Pepper (Capsicum annuum L.) fruit is sensitive to temperatures below 10 °C, which severely diminish fruit quality during cold chain distribution. Seed browning was a major chilling symptom in 36 genotypes of C. annuum fruit screened after storage at 2 °C for 3 weeks. Among them, pepper fruits of chilling-insensitive 'UZB-GJG-1999-51' and -sensitive 'C00562' were treated at 2 °C for 0 or 24 h, respectively. Analyses of integrated transcriptome-metabolome and relative gene expression in seeds obtained from the two genotypes were conducted to identify key factors involved in the seed browning induced by chilling. The relative contents of branched-chain amino acids such as leucine, isoleucine, and valine were significantly increased after chilling. Transcriptome identification showed 3,140 differentially expressed genes (log twofold change > 1.0 and FDR-corrected p value < 0.05) affected by chilling between the two genotypes. Particularly, genes related to jasmonic acid synthesis and signaling were differentially expressed. A regulatory network of jasmonic acid synthesis and signaling, and regulation of ERF family genes might contribute to chilling response in pepper fruit. The results of this study may help facilitate further studies to develop chilling-insensitive peppers and could be a basis for improving postharvest fruit quality.

The thick aleurone1 Gene Encodes a NOT1 Subunit of the CCR4-NOT Complex and Regulates Cell Patterning in Endosperm.

Maize (Zea mays) thick aleurone1 (thk1-R) mutants form multiple aleurone layers in the endosperm and have arrested embryogenesis. Prior studies suggest that thk1 functions downstream of defective kernel1 (dek1) in a regulatory pathway that controls aleurone cell fate and other endosperm traits. The original thk1-R mutant contained an ∼2-Mb multigene deletion, which precluded identification of the causal gene. Here, ethyl methanesulfonate mutagenesis produced additional alleles, and RNA sequencing from developing endosperm was used to identify a candidate gene based on differential expression compared with the wild-type progenitor. Gene editing confirmed the gene identity by producing mutant alleles that failed to complement existing thk1 mutants and that produced multiple-aleurone homozygous phenotypes. Thk1 encodes a homolog of NEGATIVE ON TATA-LESS1, a protein that acts as a scaffold for the CARBON CATABOLITE REPRESSION4-NEGATIVE ON TATA-LESS complex. This complex is highly conserved and essential in all eukaryotes for regulating a wide array of gene expression and cellular activities. Maize also harbors a duplicate locus, thick aleurone-like1, which likely accounts for the ability of thk1 mutants to form viable cells. Transcriptomic analysis indicated that THK1 regulates activities involving cell division, signaling, differentiation, and metabolism. Identification of thk1 provides an important new component of the DEK1 regulatory system that patterns cell fate in endosperm.

Meiotic Chromosome Stability and Suppression of Crossover Between Non-homologous Chromosomes in xBrassicoraphanus, an Intergeneric Allotetraploid Derived From a Cross Between Brassica rapa and Raphanus sativus.

Hybridization and polyploidization are major driving forces in plant evolution. Allopolyploids can be occasionally formed from a cross between distantly related species but often suffer from chromosome instability and infertility. xBrassicoraphanus is an intergeneric allotetraploid (AARR; 2n = 38) derived from a cross between Brassica rapa (AA; 2n = 20) and Raphanus sativus (RR; 2n = 18). xBrassicoraphanus is fertile and genetically stable, while retaining complete sets of both B. rapa and R. sativus chromosomes. Precise control of meiotic recombination is essential for the production of balanced gametes, and crossovers (COs) must occur exclusively between homologous chromosomes. Many interspecific hybrids have problems with meiotic division at early generations, in which interactions between non-homologous chromosomes often bring about aneuploidy and unbalanced gamete formation. We analyzed meiotic chromosome behaviors in pollen mother cells (PMCs) of allotetraploid and allodiploid F1 individuals of newly synthesized xBrassicoraphanus. Allotetraploid xBrassicoraphanus PMCs showed a normal diploid-like meiotic behavior. By contrast, allodiploid xBrassicoraphanus PMCs displayed abnormal segregation of chromosomes mainly due to the absence of homologous pairs. Notably, during early stages of meiosis I many of allodiploid xBrassicoraphanus chromosomes behave independently with few interactions between B. rapa and R. sativus chromosomes, forming many univalent chromosomes before segregation. Chromosomes were randomly assorted at later stages of meiosis, and tetrads with unequal numbers of chromosomes were formed at completion of meiosis. Immunolocalization of HEI10 protein mediating meiotic recombination revealed that COs were more frequent in synthetic allotetraploid xBrassicoraphanus than in allodiploid, but less than in the stabilized line. These findings suggest that structural dissimilarity between B. rapa and R. sativus chromosomes prevents non-homologous interactions between the parental chromosomes in allotetraploid xBrassicoraphanus, allowing normal diploid-like meiosis when homologous pairing partners are present. This study also suggests that CO suppression between non-homologous chromosomes is required for correct meiotic progression in newly synthesized allopolyploids, which is important for the formation of viable gametes and reproductive success in the hybrid progeny.

Analyses of targeted/untargeted metabolites and reactive oxygen species of pepper fruits provide insights into seed browning induced by chilling.

Hot peppers are sensitive to low temperature, and seed browning significantly reduces the fruit quality. This study aims to clarify the mechanisms of seed browning in terms of metabolite changes. Metabolites were analysed during a 30-day-storage period at 2 °C and 10 °C. Gamma-aminobutyric acid, tyrosine, phenylalanine, and isoleucine concentrations were significantly higher at 2 °C storage than at 10 °C. Reactive oxygen species (ROS) generation was associated with seed browning. Transcription of jasmonic acid synthesis and ROS scavenging genes were higher in hot peppers stored at 2 °C than those stored at 10 °C. This study elucidated the mechanisms underlying seed browning and chill damage in hot peppers during storage at low temperatures and our findings may help improve hot peppers' quality following harvesting.

Timing and Pattern of Anthocyanin Accumulation during Grain Filling in Purple Waxy Corn (Zea mays L.) Suggest Optimal Harvest Dates.

Purple-corn kernels contain anthocyanins, a group of antioxidants proposed to be beneficial to human health. This study investigated the concentrations of anthocyanins and amino acids and the composition of fatty acids in the kernels of purple waxy corn (Zea mays L.) "Heukjinjuchal" during grain filling to determine when the grain nutritional value is at its highest. During grain filling, anthocyanin contents increased as the kernel color darkened. Among the anthocyanins measured, cyanidin-3-ß-O-glucoside reached the highest contents, 57.0-409.1 mg kg-1 fresh weight in raw kernels and 1027.6 mg kg-1 in dry seeds. Pelargonidin-3-ß-O-glucoside and malvidin-3-ß-O-glucoside became detectable at 21 days after silking; they occurred in the second- and third-highest amounts, respectively, among anthocyanins in the purple-corn cultivars tested. The anthocyanin accumulation pattern was strongly associated with physicochemical properties and partly associated with amino acid content. Anthocyanin contents increased in a stepwise rather than linear fashion. This study showed that kernels undergo dramatic changes that affect the nutritional value of fresh corn.

Revealing biomass heterosis in the allodiploid xBrassicoraphanus, a hybrid between Brassica rapa and Raphanus sativus, through integrated transcriptome and metabolites analysis.

BACKGROUND: Heterosis is biologically important but the molecular basis of the phenomenon is poorly understood. We characterized intergeneric hybrids between B. rapa cv. Chiifu and R. sativus cv. WK10039 as an extreme example of heterosis. Taking advantage of clear heterosis phenotypes and the genetic distance between parents, we performed transcriptome and metabolite analysis to decipher the molecular basis of heterosis. RESULTS: The heterosis was expressed as fresh weight in the field and as inflorescence stem length in the glass house. Flowering time, distributed as a normal segregating population, ranged from the early flowering of one parent to the late flowering of the other, in contrast to the homogeneous flowering time in a typical F1 population, indicating unstable allelic interactions. The transcriptome and metabolome both indicated that sugar metabolism was altered, suggesting that the change in metabolism was linked to the heterosis. Because alleles were not shared between the hybridized genomes, classic models only partly explain this heterosis, indicating that other mechanisms are involved. CONCLUSION: The differential expression of genes for primary and secondary metabolism, along with the altered metabolite profiles, suggests that heterosis could involve a change in balance between primary and secondary metabolism.

A genetic characterization of Korean waxy maize (Zea mays L.) landraces having flowering time variation by RNA sequencing.

Maize is the second-most produced crop in the Korean peninsula and has been continuously cultivated since the middle of the 16th century, when it was originally introduced from China. Even with this extensive cultivation history, the diversity and properties of Korean landraces have not been investigated at the nucleotide sequence level. We collected 12 landraces with various flowering times and performed RNA-seq in the early vegetative stage. The transcriptomes of 12 Korean landraces have been analyzed for their genetic variations in coding sequence and genetic relationships to other maize germplasm. The Korean landraces showed specific genetic characteristics and were closely related to a Chinese inbred line. Flowering-time related gene profiles pointed to multiple causes for the variation of flowering time within Korean landraces; the profiles revealed significant positive and negative correlations among genes, allowing us to infer possible mechanisms for flowering time variation in maize. Our results demonstrate the value of transcriptome-based genetic and gene expression profiles for information on possible breeding resources, which is particularly needed in Korean waxy landraces.

MYB1 transcription factor is a candidate responsible for red root skin in radish (Raphanus sativus L.).

Root skin color is one of the economically important traits in radish (Raphanus sativus), and the pigmentation in red skin varieties is largely attributable to anthocyanin accumulation. Pelargonidin was found as a major anthocyanin pigment accumulated in the sub-epidermal layer of red radish roots. In the 20 F2 population generated from the F1 with red root skins, root skins with red and white colors segregated in a 3:1 ratio. Additionally, a test cross between a red F3 individual and a white skin individual gave rise to 1:1 segregation of red and white, indicating that the root skin color of radish is determined by a single locus and red color is dominant over white. We performed association mapping for root skin color using SNPs obtained from RNA-seq analysis. Segregation analysis on the 152 F3 test-cross population revealed an RsMyb1 transcription factor as a candidate gene to determine root skin color. A PCR marker based on the polymorphism within 2 kb of RsMyb1 was developed and tested on 12 and 152 individuals from F2 and F3 test cross populations, respectively, and red and white root skin colors were completely distinguished corresponding to the genotypes. Expression levels of RsMyb1 in red or purple root cultivars were significantly higher than in white root cultivars. These findings suggest that RsMyb1 is a crucial determinant for anthocyanin biosynthesis in radish roots, and the molecular marker developed in this study will be useful for marker-assisted selection for red skin individuals at early seedling stages.

Root Glucosinolate Profiles for Screening of Radish (Raphanus sativus L.) Genetic Resources.

Radish (Raphanus sativus L.), a root vegetable, is rich in glucosinolates (GLs), which are beneficial secondary metabolites for human health. To investigate the genetic variations in GL content in radish roots and the relationship with other root phenotypes, we analyzed 71 accessions from 23 different countries for GLs using HPLC. The most abundant GL in radish roots was glucoraphasatin, a GL with four-carbon aliphatic side chain. The content of glucoraphasatin represented at least 84.5% of the total GL content. Indolyl GL represented only 3.1% of the total GL at its maximum. The principal component analysis of GL profiles with various root phenotypes showed that four different genotypes exist in the 71 accessions. Although no strong correlation with GL content and root phenotype was observed, the varied GL content levels demonstrate the genetic diversity of GL content, and the amount that GLs could be potentially improved by breeding in radishes.

The naked endosperm genes encode duplicate INDETERMINATE domain transcription factors required for maize endosperm cell patterning and differentiation.

The aleurone is the outermost layer of cereal endosperm and functions to digest storage products accumulated in starchy endosperm cells as well as to confer important dietary health benefits. Whereas normal maize (Zea mays [Zm]) has a single aleurone layer, naked endosperm (nkd) mutants produce multiple outer cell layers of partially differentiated cells that show sporadic expression of aleurone identity markers such as a viviparous1 promoter-ß-glucuronidase transgene. The 15:1 F2 segregation ratio suggested that two recessive genes were involved, and map-based cloning identified two homologous genes in duplicated regions of the genome. The nkd1 and nkd2 genes encode the INDETERMINATE1 domain (IDD) containing transcription factors ZmIDDveg9 and ZmIDD9 on chromosomes 2 and 10, respectively. Independent mutant alleles of nkd1 and nkd2, as well as nkd2-RNA interference lines in which both nkd genes were knocked down, also showed the nkd mutant phenotype, confirming the gene identities. In wild-type kernels, the nkd transcripts were most abundant around 11 to 16 d after pollination. The NKD proteins have putative nuclear localization signals, and green fluorescent protein fusion proteins showed nuclear localization. The mutant phenotype and gene identities suggest that NKD controls a gene regulatory network involved in aleurone cell fate specification and cell differentiation.

A putative plant organelle RNA recognition protein gene is essential for maize kernel development.

Basal endosperm transfer layer (BETL) cells are responsible for transferring apoplastic solutes from the maternal pedicel into the endosperm, supplying the grain with compounds required for embryo development and storage reserve accumulation. Here, we analyze the maize (Zea mays L.) empty pericarp6 (emp6) mutant, which causes early arrest in grain development. The Emp6+gene function is required independently in both the embryo and endosperm. The emp6 mutant causes a notable effect on the differentiation of BETL cells; the extensive cell wall ingrowths that distinguish BETL cells are diminished and BETL marker gene expression is compromised in mutant kernels. Transposon tagging identified the emp6 locus as encoding a putative plant organelle RNA recognition (PORR) protein, 1 of 15 PORR family members in maize. The emp6 transcript is widely detected in plant tissues with highest levels in embryos and developing kernels. EMP6-green fluorescent protein (GFP) fusion proteins transiently expressed in Nicotiana benthamiana leaves were targeted specifically to mitochondria. These results suggest that BETL cell differentiation might be particularly energy intensive, or alternatively, that mitochondria might confer a developmental function.

Maize opaque5 encodes monogalactosyldiacylglycerol synthase and specifically affects galactolipids necessary for amyloplast and chloroplast function.

The maize (Zea mays) opaque5 (o5) locus was shown to encode the monogalactosyldiacylglycerol synthase MGD1. Null and point mutations of o5 that affect the vitreous nature of mature endosperm engendered an allelic series of lines with stepwise reductions in gene function. C(18:3)/C(18:2) galactolipid abundance in seedling leaves was reduced proportionally, without significant effects on total galactolipid content. This alteration in polar lipid composition disrupted the organization of thylakoid membranes into granal stacks. Total galactolipid abundance in endosperm was strongly reduced in o5(-) mutants, causing developmental defects and changes in starch production such that the normal simple granules were replaced with compound granules separated by amyloplast membrane. Complete loss of MGD1 function in a null mutant caused kernel lethality owing to failure in both endosperm and embryo development. The data demonstrate that low-abundance galactolipids with five double bonds serve functions in plastid membranes that are not replaced by the predominant species with six double bonds. Furthermore, the data identify a function of amyloplast membranes in the development of starch granules. Finally, the specific changes in lipid composition suggest that MGD1 can distinguish the constituency of acyl groups on its diacylglycerol substrate based upon the degree of desaturation.

The thick aleurone1 mutant defines a negative regulation of maize aleurone cell fate that functions downstream of defective kernel1.

The maize (Zea mays) aleurone layer occupies the single outermost layer of the endosperm. The defective kernel1 (dek1) gene is a central regulator required for aleurone cell fate specification. dek1 mutants have pleiotropic phenotypes including lack of aleurone cells, aborted embryos, carotenoid deficiency, and a soft, floury endosperm deficient in zeins. Here we describe the thick aleurone1 (thk1) mutant that defines a novel negative function in the regulation of aleurone differentiation. Mutants possess multiple layers of aleurone cells as well as aborted embryos. Clonal sectors of thk1 mutant tissue in otherwise normal endosperm showed localized expression of the phenotype with sharp boundaries, indicating a localized cellular function for the gene. Sectors in leaves showed expanded epidermal cell morphology but the mutant epidermis generally remained in a single cell layer. Double mutant analysis indicated that the thk1 mutant is epistatic to dek1 for several aspects of the pleiotropic dek1 phenotype. dek1 mutant endosperm that was mosaic for thk1 mutant sectors showed localized patches of multilayered aleurone. Localized sectors were surrounded by halos of carotenoid pigments and double mutant kernels had restored zein profiles. In sum, loss of thk1 function restored the ability of dek1 mutant endosperm to accumulate carotenoids and zeins and to differentiate aleurone. Therefore the thk1 mutation defines a negative regulator that functions downstream of dek1 in the signaling system that controls aleurone specification and other aspects of endosperm development. The thk1 mutation was found to be caused by a deletion of approximately 2 megabases.

Regulation of aleurone development in cereal grains.

The aleurone layer of cereal grains is important biologically as well as nutritionally and economically. Here, current knowledge on the regulation of aleurone development is reviewed. Recent reports suggest that the control of aleurone development is more complex than earlier models portrayed. Multiple levels of genetic regulation control aleurone cell fate, differentiation, and organization. The hormones auxin and cytokinin can also influence aleurone development. New technical advances promise to facilitate future progress.