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Karyotype dynamics driven by complex chromosome rearrangements constitute a fundamental issue in evolutionary genetics. The evolutionary events underlying karyotype diversity within plant genera, however, have rarely been reconstructed from a computed ancestral progenitor. Here, we developed a method to rapidly and accurately represent extant karyotypes with the genus, Cucumis, using highly customizable comparative oligo-painting (COP) allowing visualization of fine-scale genome structures of eight Cucumis species from both African-origin and Asian-origin clades. Based on COP data, an evolutionary framework containing a genus-level ancestral karyotype was reconstructed, allowing elucidation of the evolutionary events that account for the origin of these diverse genomes within Cucumis. Our results characterize the cryptic rearrangement hotspots on ancestral chromosomes, and demonstrate that the ancestral Cucumis karyotype (n = 12) evolved to extant Cucumis genomes by hybridizations and frequent lineage- and species-specific genome reshuffling. Relative to the African species, the Asian species, including melon (Cucumis melo, n = 12), Cucumis hystrix (n = 12) and cucumber (Cucumis sativus, n = 7), had highly shuffled genomes caused by large-scale inversions, centromere repositioning and chromothripsis-like rearrangement. The deduced reconstructed ancestral karyotype for the genus allowed us to propose evolutionary trajectories and specific events underlying the origin of these Cucumis species. Our findings highlight that the partitioned evolutionary plasticity of Cucumis karyotype is primarily located in the centromere-proximal regions marked by rearrangement hotspots, which can potentially serve as a reservoir for chromosome evolution due to their fragility., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Allopolyploids undergo "genomic shock" leading to significant genetic and epigenetic modifications. Previous studies have mainly focused on nuclear changes, while little is known about the inheritance and changes of organelle genome in allopolyploidization. The synthetic allotetraploid Cucumis ×hytivus , which is generated via hybridization between C. hystrix and C. sativus , is a useful model system for studying cytonuclear variation. Here, we report the chloroplast genome of allotetraploid C. ×hytivus and its diploid parents via sequencing and comparative analysis. The size of the obtained chloroplast genomes ranged from 154 673 to 155 760 bp, while their gene contents, gene orders, and GC contents were similar to each other. Comparative genome analysis supports chloroplast maternal inheritance. However, we identified 51 indels and 292 SNP genetic variants in the chloroplast genome of the allopolyploid C. ×hytivus relative to its female parent C. hystrix . Nine intergenic regions with rich variation were identified through comparative analysis of the chloroplast genomes within the subgenus Cucumis . The phylogenetic network based on the chloroplast genome sequences clarified the evolution and taxonomic position of the synthetic allotetraploid C. × hytivus . The results of this study provide us with an insight into the changes of organelle genome after allopolyploidization, and a new understanding of the cytonuclear evolution.

The importance of allopolyploidy in plant evolution has been widely recognized. The genetic changes triggered by allopolyploidy, however, are not yet fully understood due to inconsistent phenomena reported across diverse species. The construction of synthetic polyploids offers a controlled approach to systematically reveal genomic changes that occur during the process of polyploidy. This study reports the first fully sequenced synthetic allopolyploid constructed from a cross between Cucumis sativus and C. hystrix , with high-quality assembly. The two subgenomes are confidently partitioned and the C. sativus -originated subgenome predominates over the C. hystrix -originated subgenome, retaining more sequences and showing higher homeologous gene expression. Most of the genomic changes emerge immediately after interspecific hybridization. Analysis of a series of genome sequences from several generations (S 0 , S 4 -S 13 ) of C . × hytivus confirms that genomic changes occurred in the very first generations, subsequently slowing down as the process of diploidization is initiated. The duplicated genome of the allopolyploid with double genes from both parents broadens the genetic base of C . × hytivus , resulting in enhanced phenotypic plasticity. This study provides novel insights into plant polyploid genome evolution and demonstrates a promising strategy for the development of a wide array of novel plant species and varieties through artificial polyploidization., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Long non-coding RNAs (lncRNAs) play critical regulatory roles in various biological processes. However, the presence of lncRNAs and how they function in plant polyploidy are still largely unknown. Hence, we examined the profile of lncRNAs in a nascent allotetraploid Cucumis hytivus (S 14 ), its diploid parents, and the F 1 hybrid, to reveal the function of lncRNAs in plant-interspecific hybridization and whole genome duplication. Results showed that 2206 lncRNAs evenly transcribed from all 19 chromosomes were identified in C. hytivus , 44.6% of which were from intergenic regions. Based on the expression trend in allopolyploidization, we found that a high proportion of lncRNAs (94.6%) showed up-regulated expression to varying degrees following hybridization. However, few lncRNAs (33, 2.1%) were non-additively expressed after genome duplication, suggesting the significant effect of hybridization on lncRNAs, rather than genome duplication. Furthermore, 253 cis-regulated target genes were predicted for these differentially expressed lncRNAs in S 14 , which mainly participated in chloroplast biological regulation (e.g., chlorophyll synthesis and light harvesting system). Overall, this study provides new insight into the function of lncRNAs during the processes of hybridization and polyploidization in plant evolution.

Transferring desired genes from wild species to cultivars through alien addition lines (AALs) has been shown to be an effective method for genetic improvement. Cucumis hystrix Chakr. (HH, 2 n = 24) is a wild species of Cucumis that possesses many resistant genes. A synthetic allotetraploid species, C. hytivus (HHCC, 2 n = 38), was obtained from the cross between cultivated cucumber, C. sativus (CC, 2 n = 14), and C. hystrix followed by chromosome doubling. Cucumis sativus - C. hystrix AALs were developed by continuous backcrossing to the cultivated cucumbers. In this study, 10 different types of AALs (CC-H01, CC-H06, CC-H08, CC-H10, CC-H12, CC-H06+H09, CC-H06+H10, CC-H06+H12, CC-H08+H10, CC-H01+H06+H10) were identified based on the analysis of fluorescence in situ hybridization (FISH) and molecular markers specific to C. hystrix chromosomes. And the behavior of the alien chromosomes in three AALs (CC-H01, CC-H06+H10, CC-H01+H06+H10) at meiosis was investigated. The results showed that alien chromosomes paired with C. sativus chromosome in few pollen mother cells (PMCs). Further, disomic alien addition lines (DAALs) carrying a pair of C. hystrix chromosome H10 were screened from the selfed progenies of CC-H10. Chromosome pairing between genomes provides cytological evidence for the possible introgression of alien chromosome segments. The development of AALs could serve as a key step for exploiting and utilizing valuable genes from C. hystrix .

Background: Meiosis of newly formed allopolyploids frequently encounter perturbations induced by the merging of divergent and hybridizable genomes. However, to date, the meiotic properties of allopolyploids with dysploid parental karyotypes have not been studied in detail. The allotetraploid Cucumis ×hytivus (HHCC, 2n = 38) was obtained from interspecific hybridization between C. sativus (CC, 2n = 14) and C. hystrix (HH, 2n = 24) followed by chromosome doubling. The results of this study thus offer an excellent opportunity to explore the meiotic properties of allopolyploids with dysploid parental karyotypes., Results: In this report, we describe the meiotic properties of five chromosomes (C5, C7, H1, H9 and H10) and two genomes in interspecific hybrids and C. ×hytivus (the 4th and 14th inbred family) through oligo-painting and genomic in situ hybridization (GISH). We show that 1) only two translocations carrying C5-oligo signals were detected on the chromosomes C2 and C4 of one 14th individual by the karyotyping of eight 4th and 36 14th plants based on C5- and C7-oligo painting, and possible cytological evidence was observed in meiosis of the 4th generation; 2) individual chromosome have biases for homoeologous pairing and univalent formation in F 1 hybrids and allotetraploids; 3) extensive H-chromosome autosyndetic pairings (e.g., H-H, 25.5% PMCs) were observed in interspecific F 1 hybrid, whereas no C-chromosome autosyndetic pairings were observed (e.g. C-C); 4) the meiotic properties of two subgenomes have significant biases in allotetraploids: H-subgenome exhibits higher univalent and chromosome lagging frequencies than C-subgenome; and 5) increased meiotic stability in the S 14 generation compared with the S 4 generation, including synchronous meiosis behavior, reduced incidents of univalent and chromosome lagging., Conclusions: These results suggest that the meiotic behavior of two subgenomes has dramatic biases in response to interspecific hybridization and allopolyploidization, and the meiotic behavior harmony of subgenomes is a key subject of meiosis evolution in C. ×hytivus. This study helps to elucidate the meiotic properties and evolution of nascent allopolyploids with the dysploid parental karyotypes.

Key message A single recessive gene for complete resistance to powdery mildew and a major-effect QTL for partial resistance to downy mildew were co-localized in a Cucumis hystrix introgression line of cucumber. Downy mildew (DM) and powdery mildew (PM) are two major foliar diseases in cucumber. DM resistance (DMR) and PM resistance (PMR) may share common components; however, the genetic relationship between them remains unclear. IL52, a Cucumis hystrix introgression line of cucumber which has been reported to possess DMR, was recently identified to exhibit PMR as well. In this study, a single recessive gene pm for PMR was mapped to an approximately 468-kb region on chromosome 5 with 155 recombinant inbred lines (RILs) and 193 F 2 plants derived from the cross between a susceptible line 'changchunmici' and IL52. Interestingly, pm was co-localized with the major-effect DMR QTL dm5.2 confirmed by combining linkage analysis and BSA-seq, which was consistent with the observed linkage of DMR and PMR in IL52. Further, phenotype-genotype correlation analysis of DMR and PMR in the RILs indicated that the co-localized locus pm/dm5.2 confers complete resistance to PM and partial resistance to DM. Seven candidate genes for DMR were identified within dm5.2 by BSA-seq analysis, of which Csa5M622800.1, Csa5M622830.1 and Csa5M623490.1 were also the same candidate genes for PMR. A single nucleotide polymorphism that is present in the 3' untranslated region (3'UTR) of Csa5M622830.1 co-segregated perfectly with PMR. The GATA transcriptional factor gene Csa5M622830.1 may be a likely candidate gene for DMR and PMR. This study has provided a clear evidence for the relationship between DMR and PMR in IL52 and sheds new light on the potential value of IL52 for cucumber DMR and PMR breeding program.

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.

The important role of polyploidy in plant evolution is widely recognized. However, many questions remain to be explored to address how polyploidy affects the phenotype of the plant. To shed light on the phenotypic and molecular impacts of allopolyploidy, we investigated the leaf development of a synthesized allotetraploid (Cucumis × hytivus), with an emphasis on chlorophyll development. Delayed leaf maturation was identified in C. × hytivus, based on delayed leaf expansion, initial chlorophyll deficiency in the leaves and disordered sink-source transition. Anatomical observations also revealed disturbed chloroplast development in C. ×hytivus. The determination of chlorophyll biosynthesis intermediates suggested that the chlorophyll biosynthesis pathway of C. × hytivus is blocked at the site at which uroporphyrinogen III is catalysed to coproporphyrinogen III. Three chlorophyll biosynthesis-related genes, HEMA1, HEME2 and POR, were significantly repressed in C. × hytivus. Sequence alignment showed both synonymous and non-synonymous substitutions in the HEMA1, HEME2 and POR genes of the parents. Cloning of the chlorophyll biosynthetic genes suggested the retention of homoeologs. In addition, a chimeric clone of the HEMA1 gene that consisted of homologous genes from the parents was identified in C. × hytivus. Overall, our results showed that allopolyploidization in Cucumis has resulted in disturbed chloroplast development and reduced chlorophyll biosynthesis caused by the repressed expression of duplicated homologous genes, which further led to delayed leaf maturation in the allotetraploid, C. × hytivus. The preferential retention/loss of certain types of genes and non-reciprocal homoeologous recombination were also supported in the present study, which provides new insights into the impact of allopolyploidy., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

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.

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.

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.

Allopolyploidization is considered an essential evolutionary process in plants that could trigger genomic shock in allopolyploid genome through activation of transcription of retrotransposons, which may be important in plant evolution. Two retrotransposon-based markers, inter-retrotransposon amplified polymorphism and retrotransposon-microsatellite amplified polymorphism and a microsatellite-based marker, inter simple sequence repeat were employed to investigate genomic changes in early generations of a newly synthesized allotetraploid Cucumis × hytivus Chen & Kirkbride (2n = 4x = 38) which was derived from crossing between cultivated cucumber C. sativus L. (2n = 2x = 14) and its wild relative C. hystrix Chakr. (2n = 2x = 24). Extensive genomic changes were observed, most of which involved the loss of parental DNA fragments and gain of novel fragments in the allotetraploid. Among the 28 fragments examined, 24 were lost while four were novel, suggesting that DNA sequence elimination is a relatively frequent event during polyploidization in Cucumis. Interestingly, of the 24 lost fragments, 18 were of C. hystrix origin, four were C. sativus-specific, and the remaining two were shared by both species, implying that fragment loss may be correlated with haploid DNA content (genome size) of diploid parents. Most changes were observed in the first generation after polyploidization (S(1)) and stably inherited in the subsequent three generations (S(2)-S(4)), indicating that genomic changes might be a rapid driving force for the stabilization of allotetraploids. Sequence analysis of 11 of the 28 altered DNA fragments showed that genomic changes in the allotetraploid occurred in both coding and non-coding regions, which might suggest that retrotransposons inserted into genome randomly and had a genome-wide effect on the allotetraploid evolution. Fluorescence in situ hybridization (FISH) analysis revealed a unique distribution of retrotransposon and/or microsatellite flanking sequences in mitotic and meiotic chromosomes, where the preferential FISH signals occurred in the centromeric and telomeric regions, implying that these regions were the possible hotspots for genomic changes.

We used a newly synthesized allotetraploid between C. sativus (2n = 2x = 14, n gametic chromosome number, x haploid chromosome number) and C. hystrix (2n = 2x = 24) to study the genomic events in its early generations. Results from cytological characterization of the F(1) and the allotetraploid progenies showed that the rate of bivalents in meiotic metaphase I of the F(1) was greatly improved by chromosome doubling, and further improved during the selfing process of allopolyploid resulting into relatively diploid-like meiosis. Extensive genomic changes were detected by amplified fragment length polymorphism analysis. The changes mainly involved loss of parental restriction fragments and gaining of novel fragments. The total detectable changes were from 11.1 to 32.1%, and the frequency of losing parental fragments was much higher than that of gaining novel fragments. Some of the changes were initiated as early as in the F(1) hybrid, whereas others occurred after chromosome doubling (polyploid formation). No significant differences were detected in the reciprocal F(1) hybrids and S(0) generations. But the data showed that the frequency of sequence losing in C. sativus was about two times higher than in the C. hystrix. Our results demonstrated that the sequence elimination was the major event of genomic changes, and it might provide the physical basis for the diploid-like meiotic behavior in the diploidization of the newly formed allopolyploids. Moreover, the results suggest that the sequence elimination was not caused by cytoplasmic factors, and might relate to genomic recombination and to the numbers of parental chromosome.