Molecular Phylogeny, DNA Barcoding, and ITS2 Secondary Structure Predictions in the Medicinally Important Eryngium Genotypes of East Coast Region of India.

Commercial interest in the culinary herb, Eryngium foetidum L., has increased worldwide due to its typical pungency, similar to coriander or cilantro, with immense pharmaceutical components. The molecular delimitation and taxonomic classification of this lesser-known medicinal plant are restricted to conventional phenotyping and DNA-based marker evaluation, which hinders accurate identification, genetic conservation, and safe utilization. This study focused on species discrimination using DNA sequencing with chloroplast-plastid genes (matK, Kim matK, and rbcL) and the nuclear ITS2 gene in two Eryngium genotypes collected from the east coast region of India. The results revealed that matK discriminated between two genotypes, however, Kim matK, rbcL, and ITS2 identified these genotypes as E. foetidum. The ribosomal nuclear ITS2 region exhibited significant inter- and intra-specific divergence, depicted in the DNA barcodes and the secondary structures derived based on the minimum free energy. Although the efficiency of matK genes is better in species discrimination, ITS2 demonstrated polyphyletic phylogeny, and could be used as a reliable marker for genetic divergence studies understanding the mechanisms of RNA molecules. The results of this study provide insights into the scientific basis of species identification, genetic conservation, and safe utilization of this important medicinal plant species.

Graph-based pan-genome reveals structural and sequence variations related to agronomic traits and domestication in cucumber.

Structural variants (SVs) represent a major source of genetic diversity and are related to numerous agronomic traits and evolutionary events; however, their comprehensive identification and characterization in cucumber (Cucumis sativus L.) have been hindered by the lack of a high-quality pan-genome. Here, we report a graph-based cucumber pan-genome by analyzing twelve chromosome-scale genome assemblies. Genotyping of seven large chromosomal rearrangements based on the pan-genome provides useful information for use of wild accessions in breeding and genetic studies. A total of ~4.3 million genetic variants including 56,214 SVs are identified leveraging the chromosome-level assemblies. The pan-genome graph integrating both variant information and reference genome sequences aids the identification of SVs associated with agronomic traits, including warty fruits, flowering times and root growth, and enhances the understanding of cucumber trait evolution. The graph-based cucumber pan-genome and the identified genetic variants provide rich resources for future biological research and genomics-assisted breeding.

Mechanism of phosphate sensing and signaling revealed by rice SPX1-PHR2 complex structure.

Phosphate, a key plant nutrient, is perceived through inositol polyphosphates (InsPs) by SPX domain-containing proteins. SPX1 an inhibit the PHR2 transcription factor to maintain Pi homeostasis. How SPX1 recognizes an InsP molecule and represses transcription activation by PHR2 remains unclear. Here we show that, upon binding InsP6, SPX1 can disrupt PHR2 dimers and form a 1:1 SPX1-PHR2 complex. The complex structure reveals that SPX1 helix α1 can impose a steric hindrance when interacting with the PHR2 dimer. By stabilizing helix α1, InsP6 allosterically decouples the PHR2 dimer and stabilizes the SPX1-PHR2 interaction. In doing so, InsP6 further allows SPX1 to engage with the PHR2 MYB domain and sterically block its interaction with DNA. Taken together, our results suggest that, upon sensing the surrogate signals of phosphate, SPX1 inhibits PHR2 via a dual mechanism that attenuates dimerization and DNA binding activities of PHR2.

Charge Segregation in the Intrinsically Disordered Region Governs VRN1 and DNA Liquid-like Phase Separation Robustness.

VERNALIZATION1 (VRN1) is a transcriptional repressor involved in plant vernalization that undergoes liquid-liquid phase separation (LLPS) with DNA. The naturally occurring VRN1-like proteins contain two B3 DNA binding domains connected by an intrinsically disordered region (IDR). The IDR length in VRN1-like proteins has a broad distribution, while the charge segregation pattern is largely conserved. We studied the effect of IDR length and charge segregation on DNA-induced VRN1 phase separation. When only neutral residues (Pro-Ser repeats) were used, the phase separation behavior is sensitive to IDR length, changing from gel-like aggregates (L = 40) to liquid-like droplets (L = 100-120) and clear solution (L = 160). When a pair of continuous patches of positive and negative residues were added to the IDRs, all the VRN1 variants formed robust and durable droplets with DNA independent of the IDR length. To test how robust the system is, we introduced folded green fluorescent protein or the enzyme GPX4 into VRN1 variants with charge segregation in IDR, the resulting proteins form LLPS with DNA as well. Our study implies that VRN1-like proteins use conserved charge segregation pattern to retain functional LLPS during evolution, and demonstrates the possibility of using this system to design novel biosensors or bio-factories by introducing various functional modules.

Different DNA repair pathways are involved in single-strand break-induced genomic changes in plants.

In nature, single-strand breaks (SSBs) in DNA occur more frequently (by orders of magnitude) than double-strand breaks (DSBs). SSBs induced by the CRISPR/Cas9 nickase at a distance of 50-100 bp on opposite strands are highly mutagenic, leading to insertions/deletions (InDels), with insertions mainly occurring as direct tandem duplications. As short tandem repeats are overrepresented in plant genomes, this mechanism seems to be important for genome evolution. We investigated the distance at which paired 5'-overhanging SSBs are mutagenic and which DNA repair pathways are essential for insertion formation in Arabidopsis thaliana. We were able to detect InDel formation up to a distance of 250 bp, although with much reduced efficiency. Surprisingly, the loss of the classical nonhomologous end joining (NHEJ) pathway factors KU70 or DNA ligase 4 completely abolished tandem repeat formation. The microhomology-mediated NHEJ factor POLQ was required only for patch-like insertions, which are well-known from DSB repair as templated insertions from ectopic sites. As SSBs can also be repaired using homology, we furthermore asked whether the classical homologous recombination (HR) pathway is involved in this process in plants. The fact that RAD54 is not required for homology-mediated SSB repair demonstrates that the mechanisms for DSB- and SSB-induced HR differ in plants.

Pathway conversion enables a double-lock mechanism to maintain DNA methylation and genome stability.

The CMT2 and RNA-directed DNA methylation (RdDM) pathways have been proposed to separately maintain CHH methylation in specific regions of the Arabidopsis thaliana genome. Here, we show that dysfunction of the chromatin remodeler DDM1 causes hundreds of genomic regions to switch from CMT2 dependency to RdDM dependency in DNA methylation. These converted loci are enriched at the edge regions of long transposable elements (TEs). Furthermore, we found that dysfunction in both DDM1 and RdDM causes strong reactivation of TEs and a burst of TE transposition in the first generation of mutant plants, indicating that the DDM1 and RdDM pathways together are critical to maintaining TE repression and protecting genomic stability. Our findings reveal the existence of a pathway conversion-based backup mechanism to guarantee the maintenance of DNA methylation and genome integrity.

The plastome sequence of Bactris gasipaes and evolutionary analysis in tribe Cocoseae (Arecaceae).

The family Arecaceae is distributed throughout tropical and subtropical regions of the world. Among the five subfamilies, Arecoideae is the most species-rich and still contains some ambiguous inter-generic relationships, such as those within subtribes Attaleinae and Bactridineae. The hypervariable regions of plastid genomes (plastomes) are interesting tools to clarify unresolved phylogenetic relationships. We sequenced and characterized the plastome of Bactris gasipaes (Bactridinae) and compared it with eight species from the three Cocoseae sub-tribes (Attaleinae, Bactridinae, and Elaeidinae) to perform comparative analysis and to identify hypervariable regions. The Bactris gasipaes plastome has 156,646 bp, with 113 unique genes. Among them, four genes have an alternative start codon (cemA, rps19, rpl2, and ndhD). Plastomes are highly conserved within tribe Cocoseae: 97.3% identity, length variation of ~2 kb, and a single ~4.5 kb inversion in Astrocaryum plastomes. The LSC/IR and IR/SSC junctions vary among the subtribes: in Bactridinae and Elaeidinae the rps19 gene is completely contained in the IR region; in the subtribe Attaleinae the rps19 gene is only partially contained in the IRs. The hypervariable regions selected according to sequence variation (SV%) and frequency of parsimony informative sites (PIS%) revealed plastome regions with great potential for molecular analysis. The ten regions with greatest SV% showed higher variation than the plastid molecular markers commonly used for phylogenetic analysis in palms. The phylogenetic trees based on the plastomes and the hypervariable regions (SV%) datasets had well-resolved relationships, with consistent topologies within tribe Cocoseae, and confirm the monophyly of the subtribes Bactridinae and Attaleinae.

The intersection of DNA replication with antisense 3' RNA processing in Arabidopsis FLC chromatin silencing.

How noncoding transcription influences chromatin states is still unclear. The Arabidopsis floral repressor gene FLC is quantitatively regulated through an antisense-mediated chromatin silencing mechanism. The FLC antisense transcripts form a cotranscriptional R-loop that is dynamically resolved by RNA 3' processing factors (FCA and FY), and this is linked to chromatin silencing. Here, we investigate this silencing mechanism and show, using single-molecule DNA fiber analysis, that FCA and FY are required for unimpeded replication fork progression across the Arabidopsis genome. We then employ the chicken DT40 cell line system, developed to investigate sequence-dependent replication and chromatin inheritance, and find that FLC R-loop sequences have an orientation-dependent ability to stall replication forks. These data suggest a coordination between RNA 3' processing of antisense RNA and replication fork progression in the inheritance of chromatin silencing at FLC.

A reference-grade genome identifies salt-tolerance genes from the salt-secreting mangrove species Avicennia marina.

Water scarcity and salinity are major challenges facing agriculture today, which can be addressed by engineering plants to grow in the boundless seawater. Understanding the mangrove plants at the molecular level will be necessary for developing such highly salt-tolerant agricultural crops. With this objective, we sequenced the genome of a salt-secreting and extraordinarily salt-tolerant mangrove species, Avicennia marina, that grows optimally in 75% seawater and tolerates >250% seawater. Our reference-grade ~457 Mb genome contains 31 scaffolds corresponding to its chromosomes. We identified 31,477 protein-coding genes and a salinome consisting of 3246 salinity-responsive genes and homologs of 614 experimentally validated salinity tolerance genes. The salinome provides a strong foundation to understand the molecular mechanisms of salinity tolerance in plants and breeding crops suitable for seawater farming.

Revealing atomic-scale molecular diffusion of a plant-transcription factor WRKY domain protein along DNA.

Transcription factor (TF) target search on genome is highly essential for gene expression and regulation. High-resolution determination of TF diffusion along DNA remains technically challenging. Here, we constructed a TF model system using the plant WRKY domain protein in complex with DNA from crystallography and demonstrated microsecond diffusion dynamics of WRKY on DNA by employing all-atom molecular-dynamics (MD) simulations. Notably, we found that WRKY preferentially binds to one strand of DNA with significant energetic bias compared with the other, or nonpreferred strand. The preferential DNA-strand binding becomes most prominent in the static process, from nonspecific to specific DNA binding, but less distinct during diffusive movements of the domain protein on the DNA. Remarkably, without employing acceleration forces or bias, we captured a complete one-base-pair stepping cycle of the protein tracking along major groove of DNA with a homogeneous poly-adenosine sequence, as individual hydrogen bonds break and reform at the protein-DNA binding interface. Further DNA-groove tracking motions of the protein forward or backward, with occasional sliding as well as strand crossing to minor groove of DNA, were also captured. The processive diffusion of WRKY along DNA has been further sampled via coarse-grained MD simulations. The study thus provides structural dynamics details on diffusion of a small TF domain protein, suggests how the protein approaches a specific recognition site on DNA, and supports further high-precision experimental detection. The stochastic movements revealed in the TF diffusion also provide general clues about how other protein walkers step and slide along DNA.

Maize Endochitinase Expression in Response to Fall Armyworm Herbivory.

A large percentage of crop loss is due to insect damage, especially caterpillar damage. Plant chitinases are considered excellent candidates to combat these insects since they can degrade chitin in peritrophic matrix (PM), an important protective structure in caterpillar midgut. Compared to chemical insecticides, chitinases could improve host plant resistance and be both economically and environmentally advantageous. The focus of this research was to find chitinase candidates that could improve plant resistance by effectively limiting caterpillar damage. Five classes of endochitinase (I-V) genes were characterized in the maize genome, and we isolated and cloned four chitinase genes (chitinase A, chitinase B, chitinase I, and PRm3) present in two maize (Zea mays L.) inbred lines Mp708 and Tx601, with different levels of resistance to caterpillar pests. We also investigated the expression of these maize chitinases in response to fall armyworm (Spodoptera frugiperda, FAW) attack. The results indicated that both chitinase transcript abundance and enzymatic activity increased in response to FAW feeding and mechanical wounding. Furthermore, chitinases retained activity inside the caterpillar midgut and enzymatic activity was detected in the food bolus and frass. When examined under scanning electron microscopy, PMs from Tx601-fed caterpillars showed structural damage when compared to diet controls. Analysis of chitinase transcript abundance after caterpillar feeding and proteomic analysis of maize leaf trichomes in the two inbreds implicated chitinase PRm3 found in Tx601 as a potential insecticidal protein.

The histone variant H2A.W and linker histone H1 co-regulate heterochromatin accessibility and DNA methylation.

In flowering plants, heterochromatin is demarcated by the histone variant H2A.W, elevated levels of the linker histone H1, and specific epigenetic modifications, such as high levels of DNA methylation at both CG and non-CG sites. How H2A.W regulates heterochromatin organization and interacts with other heterochromatic features is unclear. Here, we create a h2a.w null mutant via CRISPR-Cas9, h2a.w-2, to analyze the in vivo function of H2A.W. We find that H2A.W antagonizes deposition of H1 at heterochromatin and that non-CG methylation and accessibility are moderately decreased in h2a.w-2 heterochromatin. Compared to H1 loss alone, combined loss of H1 and H2A.W greatly increases accessibility and facilitates non-CG DNA methylation in heterochromatin, suggesting co-regulation of heterochromatic features by H2A.W and H1. Our results suggest that H2A.W helps maintain optimal heterochromatin accessibility and DNA methylation by promoting chromatin compaction together with H1, while also inhibiting excessive H1 incorporation.

Determination of soluble and insoluble-bound phenolic compounds in dehulled, whole, and hulls of green and black lentils using electrospray ionization (ESI)-MS/MS and their inhibition in DNA strand scission.

The soluble and insoluble-bound phenolic fractions of hull, whole, and dehulled black and green lentil extracts were identified and quantified using electrospray ionization (ESI)-MS/MS. Several in vitro antioxidant tests and inhibition of DNA strand scission were conducted to assess different pathways of activity. The most abundant phenolics in the soluble fractions were caffeic acid (412.2 µg/g), quercetin, (486.5 µg/g) quercetin glucoside (633.6 µg/g) luteolin glucoside (239.1 µg/g) and formononetin (920 µg/g), while myricetin (534.1 µg/g) and catechin (653.4 µg/g) were the predominant phenolics in the insoluble bound fraction. Hulls of both lentil cultivars had the highest phenolic content and the strongest antioxidant activity followed by whole and dehulled samples. Thus, lentil hulls would serve as an excellent source for the production of functional foods. Moreover, ESI-MS/MS (direct infusion) analysis was the rapid and high-throughput approach for the determination of bioactives in lentils by reducing the analysis time.

Physiological and genetic characterization of heat stress effects in a common bean RIL population.

Heat stress is a major abiotic stress factor reducing crop productivity and climate change models predict increasing temperatures in many production regions. Common bean (Phaseolus vulgaris L.) is an important crop for food security in the tropics and heat stress is expected to cause increasing yield losses. To study physiological responses and to characterize the genetics of heat stress tolerance, we evaluated the recombinant inbred line (RIL) population IJR (Indeterminate Jamaica Red) x AFR298 of the Andean gene pool. Heat stress (HS) conditions in the field affected many traits across the reproductive phase. High nighttime temperatures appeared to have larger effects than maximum daytime temperatures. Yield was reduced compared to non-stress conditions by 37% and 26% in 2016 and 2017 seasons, respectively. The image analysis tool HYRBEAN was developed to evaluate pollen viability (PolVia). A significant reduction of PolVia was observed in HS and higher viability was correlated with yield only under stress conditions. In susceptible lines the reproductive phase was extended and defects in the initiation of seed, seed fill and seed formation were identified reducing grain quality. Higher yields under HS were correlated with early flowering, high pollen viability and effective seed filling. Quantitative trait loci (QTL) analysis revealed a QTL for both pod harvest index and PolVia on chromosome Pv05, for which the more heat tolerant parent IJR contributed the positive allele. Also, on chromosome Pv08 a QTL from IJR improved PolVia and the yield component pods per plant. HS affected several traits during the whole reproductive development, from floral induction to grain quality traits, indicating a general heat perception affecting many reproductive processes. Identification of tolerant germplasm, indicator traits for heat tolerance and molecular tools will help to breed heat tolerant varieties to face future climate change effects.

INT-Hi-C reveals distinct chromatin architecture in endosperm and leaf tissues of Arabidopsis.

Higher-order chromatin structure undergoes striking changes in response to various developmental and environmental signals, causing distinct cell types to adopt specific chromatin organization. High throughput chromatin conformation capture (Hi-C) allows studying higher-order chromatin structure; however, this technique requires substantial amounts of starting material, which has limited the establishment of cell type-specific higher-order chromatin structure in plants. To overcome this limitation, we established a protocol that is applicable to a limited amount of nuclei by combining the INTACT (isolation of nuclei tagged in specific cell types) method and Hi-C (INT-Hi-C). Using this INT-Hi-C protocol, we generated Hi-C data from INTACT purified endosperm and leaf nuclei. Our INT-Hi-C data from leaf accurately reiterated chromatin interaction patterns derived from conventional leaf Hi-C data. We found that the higher-order chromatin organization of mixed leaf tissues and endosperm differs and that DNA methylation and repressive histone marks positively correlate with the chromatin compaction level. We furthermore found that self-looped interacting genes have increased expression in leaves and endosperm and that interacting intergenic regions negatively impact on gene expression in the endosperm. Last, we identified several imprinted genes involved in long-range and trans interactions exclusively in endosperm. Our study provides evidence that the endosperm adopts a distinct higher-order chromatin structure that differs from other cell types in plants and that chromatin interactions influence transcriptional activity.

Ultra-long DNA molecule isolation from plant nuclei for ultra-long read genome sequencing.

Long and ultra-long read DNA sequencing technologies require high molecular weight DNA with high quality and sufficient quantity, which could be challenging to obtain from recalcitrant plant tissues. We describe a protocol to isolate ultra-long DNA from 12 species for ultra-long read genome sequencing. A suitable nuclei lysis buffer is critical for DNA quality and yield. This protocol will enable individual labs to isolate high molecular weight DNA at a rapid pace with low cost from a variety of plant species. For complete information on the use and execution of this protocol, please refer to: Zhang et al. (2020).

SDC mediates DNA methylation-controlled clock pace by interacting with ZTL in Arabidopsis.

Molecular bases of eukaryotic circadian clocks mainly rely on transcriptional-translational feedback loops (TTFLs), while epigenetic codes also play critical roles in fine-tuning circadian rhythms. However, unlike histone modification codes that play extensive and well-known roles in the regulation of circadian clocks, whether DNA methylation (5mC) can affect the circadian clock, and the associated underlying molecular mechanisms, remains largely unexplored in many organisms. Here we demonstrate that global genome DNA hypomethylation can significantly lengthen the circadian period of Arabidopsis. Transcriptomic and genetic evidence demonstrate that SUPPRESSOR OF drm1 drm2 cmt3 (SDC), encoding an F-box containing protein, is required for the DNA hypomethylation-tuned circadian clock. Moreover, SDC can physically interact with another F-box containing protein ZEITLUPE (ZTL) to diminish its accumulation. Genetic analysis further revealed that ZTL and its substrate TIMING OF CAB EXPRESSION 1 (TOC1) likely act downstream of DNA methyltransferases to control circadian rhythm. Together, our findings support the notion that DNA methylation is important to maintain proper circadian pace in Arabidopsis, and further established that SDC links DNA hypomethylation with a proteolytic cascade to assist in tuning the circadian clock.

Historical biogeography of Pomaderris (Rhamnaceae): Continental vicariance in Australia and repeated independent dispersals to New Zealand.

AIM: Gondwanan biogeographic patterns include a combination of old vicariance events following the breakup of the supercontinent, and more recent long-distance dispersals across the southern landmasses. Floristic relationships between Australia and New Zealand have mostly been attributed to recent dispersal events rather than vicariance. We assessed the biogeographic history of Pomaderris (Rhamnaceae), which occurs in both Australia and New Zealand, by constructing a time-calibrated molecular phylogeny to infer (1) phylogenetic relationships and (2) the relative contributions of vicariance and dispersal events in the biogeographic history of the genus. LOCATION: Australia and New Zealand. METHODS: Using hybrid capture and high throughput sequencing, we generated nuclear and plastid data sets to estimate phylogenetic relationships and fossil calibrated divergence time estimates for Pomaderris. BioGeoBEARS and biogeographical stochastic mapping (BSM) were used to assess the ancestral area of the genus and the relative contributions of vicariance vs dispersal, and the directionality of dispersal events. RESULTS: Our analyses indicate that Pomaderris originated in the Oligocene and had a widespread Australian distribution. Vicariance of western and eastern Australian clades coincides with the uplift of the Nullarbor Plain c. 14 Ma, followed by subsequent in-situ and within-biome diversification with little exchange across regions. A rapid radiation of southeastern Australian taxa beginning c. 10 Ma was the source for at least six independent long-distance dispersal events to New Zealand during the Pliocene-Pleistocene. MAIN CONCLUSIONS: Our study demonstrates the importance of dispersal in explaining not only the current cross-Tasman distributions of Pomaderris, but for the New Zealand flora more broadly. The pattern of multiple independent long-distance dispersal events for Pomaderris, without significant radiation within New Zealand, is congruent with other lowland plant groups, suggesting that this biome has a different evolutionary history compared with the younger alpine flora of New Zealand, which exhibits extensive radiations often following single long distance dispersal events.

DNA-binding properties of the MADS-domain transcription factor SEPALLATA3 and mutant variants characterized by SELEX-seq.

KEY MESSAGE: We studied the DNA-binding profile of the MADS-domain transcription factor SEPALLATA3 and mutant variants by SELEX-seq. DNA-binding characteristics of SEPALLATA3 mutant proteins lead us to propose a novel DNA-binding mode. MIKC-type MADS-domain proteins, which function as essential transcription factors in plant development, bind as dimers to a 10-base-pair AT-rich motif termed CArG-box. However, this consensus motif cannot fully explain how the abundant family members in flowering plants can bind different target genes in specific ways. The aim of this study was to better understand the DNA-binding specificity of MADS-domain transcription factors. Also, we wanted to understand the role of a highly conserved arginine residue for binding specificity of the MADS-domain transcription factor family. Here, we studied the DNA-binding profile of the floral homeotic MADS-domain protein SEPALLATA3 by performing SELEX followed by high-throughput sequencing (SELEX-seq). We found a diverse set of bound sequences and could estimate the in vitro binding affinities of SEPALLATA3 to a huge number of different sequences. We found evidence for the preference of AT-rich motifs as flanking sequences. Whereas different CArG-boxes can act as SEPALLATA3 binding sites, our findings suggest that the preferred flanking motifs are almost always the same and thus mostly independent of the identity of the central CArG-box motif. Analysis of SEPALLATA3 proteins with a single amino acid substitution at position 3 of the DNA-binding MADS-domain further revealed that the conserved arginine residue, which has been shown to be involved in a shape readout mechanism, is especially important for the recognition of nucleotides at positions 3 and 8 of the CArG-box motif. This leads us to propose a novel DNA-binding mode for SEPALLATA3, which is different from that of other MADS-domain proteins known.

Comparative analysis of nuclear, chloroplast, and mitochondrial genomes of watermelon and melon provides evidence of gene transfer.

During plant evolution, there is genetic communication between organelle and nuclear genomes. A comparative analysis was performed on the organelle and nuclear genomes of the watermelon and melon. In the watermelon, chloroplast-derived sequences accounted for 7.6% of the total length of the mitochondrial genome. In the melon, chloroplast-derived sequences accounted for approximately 2.73% of the total mitochondrial genome. In watermelon and melon, the chloroplast-derived small-fragment sequences are either a subset of large-fragment sequences or appeared multiple times in the mitochondrial genome, indicating that these fragments may have undergone multiple independent migration integrations or emerged in the mitochondrial genome after migration, replication, and reorganization. There was no evidence of migration from the mitochondria to chloroplast genome. A sequence with a total length of about 73 kb (47%) in the watermelon chloroplast genome was homologous to a sequence of about 313 kb in the nuclear genome. About 33% of sequences in the watermelon mitochondrial genome was homologous with a 260 kb sequence in the nuclear genome. A sequence with a total length of about 38 kb (25%) in the melon chloroplast genome was homologous with 461 sequences in the nuclear genome, with a total length of about 301 kb. A 3.4 Mb sequence in the nuclear genome was homologous with a melon mitochondrial sequence. These results indicate that, during the evolution of watermelon and melon, a large amount of genetic material was exchanged between the nuclear genome and the two organelle genomes in the cytoplasm.