The complete mitogenome of Chrysopogon zizanioides (L.) Roberty (Poaceae), with its phylogenetic analysis.

Vetiver grass (Chrysopogon zizanioides), is a perennial and tussock C4 grass from the genus Chrysopogon of Poaceae, which has been widely used as a natural and inexpensive resource for multifarious environmental applications. The complete mitogenome of C. zizanioides was 551,622 bp in length, containing 40 protein-coding genes (PCGs), 19 transfer RNA genes (tRNAs), and six ribosomal RNA genes (rRNAs). All PCGs started with ATG and stopped with TNN (TAA, TAG, and TGA). The overall nucleotide composition is: 28.2% A, 28.2% T, 21.7% G, and 21.9% C, with a biased A + T content of 56.4%. Phylogenetic analysis using 14 PCGs of 22 species showed that C. zizanioides display a close relationship with Saccharum officinarum (LC107874) and Sorghum bicolor (DQ984518) in Poaceae.

Chromosome-scale genome assembly of Cucumis hystrix-a wild species interspecifically cross-compatible with cultivated cucumber.

Cucumis hystrix Chakr. (2n = 2x = 24) is a wild species that can hybridize with cultivated cucumber (C. sativus L., 2n = 2x = 14), a globally important vegetable crop. However, cucumber breeding is hindered by its narrow genetic base. Therefore, introgression from C. hystrix has been anticipated to bring a breakthrough in cucumber improvement. Here, we report the chromosome-scale assembly of C. hystrix genome (289 Mb). Scaffold N50 reached 14.1 Mb. Over 90% of the sequences were anchored onto 12 chromosomes. A total of 23,864 genes were annotated using a hybrid method. Further, we conducted a comprehensive comparative genomic analysis of cucumber, C. hystrix, and melon (C. melo L., 2n = 2x = 24). Whole-genome comparisons revealed that C. hystrix is phylogenetically closer to cucumber than to melon, providing a molecular basis for the success of its hybridization with cucumber. Moreover, expanded gene families of C. hystrix were significantly enriched in "defense response," and C. hystrix harbored 104 nucleotide-binding site-encoding disease resistance gene analogs. Furthermore, 121 genes were positively selected, and 12 (9.9%) of these were involved in responses to biotic stimuli, which might explain the high disease resistance of C. hystrix. The alignment of whole C. hystrix genome with cucumber genome and self-alignment revealed 45,417 chromosome-specific sequences evenly distributed on C. hystrix chromosomes. Finally, we developed four cucumber-C. hystrix alien addition lines and identified the exact introgressed chromosome using molecular and cytological methods. The assembled C. hystrix genome can serve as a valuable resource for studies on Cucumis evolution and interspecific introgression breeding of cucumber.

Candidate genes underlying the quantitative trait loci for root-knot nematode resistance in a Cucumis hystrix introgression line of cucumber based on population sequencing.

The southern root-knot nematode (RKN), Meloidogyne incognita (Kofoid & White) Chitwood, is one of most destructive species of plant parasitic nematodes, causing significant economic losses to numerous crops including cucumber (Cucumis sativus L. 2n = 14). No commercial cultivar is currently available with resistance to RKN, severely hindering the genetic improvement of RKN resistance in cucumber. An introgression line, IL10-1, derived from the interspecific hybridization between the wild species Cucumis hystrix Chakr. (2n = 24, HH) and cucumber, was identified with resistance to RKN. In this study, an ultrahigh-density genetic linkage bin-map, composed of high-quality single-nucleotide polymorphisms (SNPs), was constructed based on low-coverage sequences of the F2:6 recombinant inbred lines derived from the cross between inbred line IL10-1 and cultivar 'Beijingjietou' CC3 (hereinafter referred to as CC3). Three QTLs were identified accounting for 13.36% (qRKN1-1), 9.07% and 9.58% (qRKN5-1 and qRKN5-2) of the resistance variation, respectively. Finally, four genes with nonsynonymous SNPs from chromosome 5 were speculated to be the candidate RKN-resistant related genes, with annotation involved in disease resistance. Though several gaps still exist on the bin-map, our results could potentially be used in breeding programs and establish an understanding of the associated mechanisms underlying RKN resistance in cucumber.

Comparative transcriptomics reveals suppressed expression of genes related to auxin and the cell cycle contributes to the resistance of cucumber against Meloidogyne incognita.

BACKGROUND: Meloidogyne incognita is a devastating nematode that causes significant losses in cucumber production worldwide. Although numerous studies have emphasized on the susceptible response of plants after nematode infection, the exact regulation mechanism of M. incognita-resistance in cucumber remains elusive. Verification of an introgression line, 'IL10-1', with M. incognita-resistance provides the opportunity to unravel the resistance mechanism of cucumber against M. incognita. RESULTS: In the present study, analyses of physiological responses and transcriptional events between IL10-1 (resistant line) and CC3 (susceptible line) were conducted after M. incognita infection. Physiological observations showed abnormal development of giant cells and M. incognita in IL10-1, which were the primary differences compared with CC3. Furthermore, Gene ontology (GO) analysis revealed that genes encoding cell wall proteins were up-regulated in IL10-1 and that the highly expressed lipid transfer protein gene (Csa6G410090) might be the principal regulator of this up-regulation. Simultaneously, analyses of gene expression profiles revealed more auxin-related genes were suppressed in IL10-1 than in those of CC3, which corresponded with the lower level of indole acetic acid (IAA) in the roots of IL10-1 than in those of CC3. Additionally, poor nucleus development as a clear indication of abnormal giant cells in IL10-1 was related to inhibition of the cell cycle. Of those genes related to the cell cycle, the F-box domain Skp2-like genes were down-regulated in IL10-1, whereas more of these genes were up-regulated in CC3. CONCLUSIONS: All of these findings indicate that suppressed expression of genes related to auxin and the cell cycle and highly expressed cell wall proteins play important roles in the abnormal development of giant cells, which hinders the development of M. incognita, thereby causing resistance to M. incognita in IL10-1. Knowledge from this research will provide a useful foundation for developing effective strategies in M. incognita-resistance breeding.

Identification of Four C40 Isomers by Means of a Theoretical XPS/NEXAFS Spectra Study.

XPS and NEXAFS spectra of four stable C40 isomers [29( C2), 31( C s), 38( D2), and 39( D5 d)] have been investigated theoretically. We combined density functional theory and the full core hole potential method to simulate C 1s XPS and NEXAFS spectra for nonequivalent carbon atoms of four stable C40 fullerene isomers. The NEXAFS showed obvious dependence on the four C40 isomers, and XPS spectra are distinct for all four isomers, which can be employed to identify the four stable structures of C40. Furthermore, the individual components of the spectra according to different categories have been investigated, and the relationship between the spectra and the local structures of C atoms was also explored.

Chromosome identification in Cucumis anguria revealed by cross-species single-copy gene FISH.

Cucumis anguria is a potential genetic resource for improving crops of the genus Cucumis, owing to its broad-spectrum resistance. However, few cytogenetic studies on C. anguria have been reported because of its small metaphase chromosomes and the scarcity of distinguished chromosomal landmarks. In this study, 14 single-copy genes from cucumber and rDNAs were used as probes for FISH to identify the individual chromosomes of C. anguria. The distinctive signal distribution patterns of the probes allowed us to distinguish each chromosome of C. anguria (A01-A12). Further, detailed chromosome characteristics were obtained through pachytene chromosome FISH. The lengths of pachytene chromosomes varied from 54.80 to 143.41 µm. The proportion of heterochromatin regions varied from 13.56% to 63.86%. Finally, the chromosomal homeologous relationship between C. anguria and cucumber (C1-C7) was analyzed. The results showed that A06 + A09, A03 + A12, A02 + A04, and A01 + A11 were homeologs of C1, C2, C3, and C6, respectively. Furthemore, chromosomes A08, A10, and A05 were homeologs of C4, C5, and C7, respectively. Chromosome identification and homeologous relationship analysis between C. anguria and cucumber lay the foundation for further research of genome structure evolution in species of Cucumis.

Organization and evolution of four differentially amplified tandem repeats in the Cucumis hystrix genome.

MAIN CONCLUSION: Three subtelomeric satellites and one interstitial 5S rDNA were characterized in Cucumis hystrix, and the pericentromeric signals of two C. hystrix subtelomeric satellites along C. sativus chromosomes supported the hypothesis of chromosome fusion in Cucumis. Tandem repeats are chromosome structural fractions consisting of highly repetitive sequences organized in large tandem arrays in most eukaryotes. Differentiation of tandem repeats directly affects the chromosome structure, which contributes to species formation and evolution. Cucumis hystrix (2n = 2x = 24) is the only wild Cucumis species grouped into the same subgenus with C. sativus (2n = 2x = 14), hence its phylogenetic position confers a vital role for C. hystrix to understand the chromosome evolution in Cucumis. However, our knowledge of C. hystrix tandem repeats is insufficient for a detailed understanding of the chromosome evolution in Cucumis. Based on de novo tandem repeat characterization using bioinformatics and in situ hybridization (ISH), we identified and characterized four differentially amplified tandem repeats, Cucumis hystrix satellite 1-3 (CuhySat1-CuhySat3) located at the subtelomeric regions of all chromosomes, and Cucumis hystrix 5S (Cuhy5S) located at the interstitial regions of one single chromosome pair. Comparative ISH mapping using CuhySat1-3 and Cuhy5S revealed high homology of tandem repeats between C. hystrix and C. sativus. Intriguingly, we found signal distribution variations of CuhySat2 and CuhySat3 on C. sativus chromosomes. In comparison to their subtelomeric signal distribution on C. hystrix chromosomes, CuhySat3 showed a pericentromeric signal distribution and CuhySat2 showed both subtelomeric and pericentromeric signal distributions on C. sativus chromosomes. This detailed characterization of four C. hystrix tandem repeats significantly widens our knowledge of the C. hystrix chromosome structure, and the observed signal distribution variations will be helpful for understanding the chromosome evolution of Cucumis.

Identification of all homoeologous chromosomes of newly synthetic allotetraploid Cucumis × hytivus and its wild parent reveals stable subgenome structure.

Allopolyploidy and homoeologous recombination are two important processes in reshaping genomes and generating evolutionary novelties. Newly formed allopolyploids usually display chromosomal perturbations as a result of pairing errors at meiosis. To understand mechanisms of stabilization of allopolyploid species derived from distant chromosome bases, we investigated mitotic stability of a synthetic Cucumis allotetraploid species in relation to meiosis chromosome behavior. The Cucumis × hytivus is an allotetraploid synthesized from interspecific hybridization between cucumber (Cucumis sativus, 2n = 14) and its wild relative Cucumis hystrix (2n = 24) followed by spontaneous chromosome doubling. In the present study, we analyzed the wild parent C. hystrix and the latest generation of C. hytivus using GISH (genomic in situ hybridization) and cross-species FISH (fluorescence in situ hybridization). The karyotype of C. hystrix was constructed with two methods using cucumber fosmid clones and repetitive sequences. Using repeat-element probe mix in two successive hybridizations allowed for routine identification of all 19 homoeologous chromosomes of allotetraploid C. hytivus. No aneuploids were identified in any C. hytivus individuals that were characterized, and no large-scale chromosomal rearrangements were identified in this synthetic allotetraploid. Meiotic irregularities, such as homoeologous pairing, were frequently observed, resulting in univalent and intergenomic multivalent formation. The relatively stable chromosome structure of the synthetic Cucumis allotetraploid may be explained by more deleterious chromosomal viable gametes compared with other allopolyploids. The knowledge of genetic and genomic information of Cucumis allotetraploid species could provide novel insights into the establishment of allopolyploids with different chromosome bases.

Comparative chromosomal localization of 45S and 5S rDNAs and implications for genome evolution in Cucumis.

Ribosomal DNAs are useful cytogenetic markers for chromosome analysis. Studies investigating site numbers and distributions of rDNAs have provided important information for elucidating genome organization and chromosomal relationships of many species by fluorescence in situ hybridization. But relevant studies are scarce for species of the genus Cucumis, especially in wild species. In the present study, FISH was conducted to investigate the organization of 45S and 5S rDNA among 20 Cucumis accessions, including cultivars and wild accessions. Our results showed that the number of 45S rDNA sites varied from one to five pairs in different accessions, and most of these sites are located at the terminal regions of chromosomes. Interestingly, up to five pairs of 45S rDNA sites were observed in C. sativus var. sativus, the species which has the lowest chromosome number, i.e., 2n = 14. Only one pair of 5S rDNA sites was detected in all accessions, except for C. heptadactylus, C. sp, and C. spp that had two pairs of 5S rDNA sites. The distributions of 5S rDNA sites showed more variation than 45S rDNA sites. The phylogenetic analysis in this study showed that 45S and 5S rDNA have contrasting evolutionary patterns. We find that 5S rDNA has a polyploidization-related tendency towards the terminal location from an interstitial location but maintains a conserved site number, whereas the 45S rDNA showed a trend of increasing site number but a relatively conserved location.

Chromosomal structures and repetitive sequences divergence in Cucumis species revealed by comparative cytogenetic mapping.

BACKGROUND: Differentiation and copy number of repetitive sequences affect directly chromosome structure which contributes to reproductive isolation and speciation. Comparative cytogenetic mapping has been verified an efficient tool to elucidate the differentiation and distribution of repetitive sequences in genome. In present study, the distinct chromosomal structures of five Cucumis species were revealed through genomic in situ hybridization (GISH) technique and comparative cytogenetic mapping of major satellite repeats. RESULTS: Chromosome structures of five Cucumis species were investigated using GISH and comparative mapping of specific satellites. Southern hybridization was employed to study the proliferation of satellites, whose structural characteristics were helpful for analyzing chromosome evolution. Preferential distribution of repetitive DNAs at the subtelomeric regions was found in C. sativus, C hystrix and C. metuliferus, while majority was positioned at the pericentromeric heterochromatin regions in C. melo and C. anguria. Further, comparative GISH (cGISH) through using genomic DNA of other species as probes revealed high homology of repeats between C. sativus and C. hystrix. Specific satellites including 45S rDNA, Type I/II, Type III, Type IV, CentM and telomeric repeat were then comparatively mapped in these species. Type I/II and Type IV produced bright signals at the subtelomeric regions of C. sativus and C. hystrix simultaneously, which might explain the significance of their amplification in the divergence of Cucumis subgenus from the ancient ancestor. Unique positioning of Type III and CentM only at the centromeric domains of C. sativus and C. melo, respectively, combining with unique southern bands, revealed rapid evolutionary patterns of centromeric DNA in Cucumis. Obvious interstitial telomeric repeats were observed in chromosomes 1 and 2 of C. sativus, which might provide evidence of the fusion hypothesis of chromosome evolution from x = 12 to x = 7 in Cucumis species. Besides, the significant correlation was found between gene density along chromosome and GISH band intensity in C. sativus and C. melo. CONCLUSIONS: In summary, comparative cytogenetic mapping of major satellites and GISH revealed the distinct differentiation of chromosome structure during species formation. The evolution of repetitive sequences was the main force for the divergence of Cucumis species from common ancestor.

Single-copy gene-based chromosome painting in cucumber and its application for chromosome rearrangement analysis in Cucumis.

Chromosome painting based on fluorescence in situ hybridization (FISH) has played an important role in chromosome identification and research into chromosome rearrangements, diagnosis of chromosome abnormalities and evolution in human and animal species. However, it has not been applied widely in plants due to the large amounts of dispersed repetitive sequences in chromosomes. In the present work, a chromosome painting method for single-copy gene pools in Cucumis sativus was successfully developed. Gene probes with sizes above 2 kb were detected consistently. A cucumber karyotype was constructed based on FISH using a cocktail containing chromosome-specific gene probes. This single-copy gene-based chromosome painting (ScgCP) technique was performed by PCR amplification, purification, pooling, labeling and hybridization onto chromosome spreads. Gene pools containing sequential genes with an interval less than 300 kb yielded painting patterns on pachytene chromosomes. Seven gene pools corresponding to individual chromosomes unambiguously painted each chromosome pair of C. sativus. Three mis-aligned regions on chromosome 4 were identified by the painting patterns. A probe pool comprising 133 genes covering the 8 Mb distal end of chromosome 4 was used to evaluate the potential utility of the ScgCP technique for chromosome rearrangement research through cross-species FISH in the Cucumis genus. Distinct painting patterns of this region were observed in C. sativus, C. melo and C. metuliferus species. A comparative chromosome map of this region was constructed between cucumber and melon. With increasing sequence resources, this ScgCP technique may be applied on any other sequenced species for chromosome painting research.