Yonghua Han

An Integrated Genetic and Cytogenetic Map of the Cucumber Genome

The Cucurbitaceae includes important crops such as cucumber, melon, watermelon, squash and pumpkin. However, few genetic and genomic resources are available for plant improvement. Some cucurbit species such as cucumber have a narrow genetic base, which impedes construction of saturated molecular linkage maps. We report herein the development of highly polymorphic simple sequence repeat (SSR) markers originated from whole genome shotgun sequencing and the subsequent construction of a high-density genetic linkage map. This map includes 995 SSRs in seven linkage groups which spans in total 573 cM, and defines ∼680 recombination breakpoints with an average of 0.58 cM between two markers. These linkage groups were then assigned to seven corresponding chromosomes using fluorescent in situ hybridization (FISH). FISH assays also revealed a chromosomal inversion between Cucumis subspecies [C. sativus var. sativus L. and var. hardwickii (R.) Alef], which resulted in marker clustering on the genetic map. A quarter of the mapped markers showed relatively high polymorphism levels among 11 inbred lines of cucumber. Among the 995 markers, 49%, 26% and 22% were conserved in melon, watermelon and pumpkin, respectively. This map will facilitate whole genome sequencing, positional cloning, and molecular breeding in cucumber, and enable the integration of knowledge of gene and trait in cucurbits.

Centromere repositioning in cucurbit species: Implication of the genomic impact from centromere activation and inactivation

The centromere of an eukaryotic chromosome can move to a new position during evolution, which may result in a major alteration of the chromosome morphology and karyotype. This centromere repositioning phenomenon has been extensively documented in mammalian species and was implicated to play an important role in mammalian genome evolution. Here we report a centromere repositioning event in plant species. Comparative fluorescence in situ hybridization mapping using common sets of fosmid clones between two pairs of cucumber (Cucumis sativus L.) and melon (Cucumis melo L.) chromosomes revealed changes in centromere positions during evolution. Pachytene chromosome analysis revealed that the current centromeres of all four cucumber and melon chromosomes are associated with distinct pericentromeric heterochromatin. Interestingly, inactivation of a centromere in the original centromeric region was associated with a loss or erosion of its affixed pericentromeric heterochromatin. Thus, both centromere activation and inactivation in cucurbit species were associated with a gain/loss of a large amount of pericentromeric heterochromatin.

Distribution of the tandem repeat sequences and karyotyping in cucumber (Cucumis sativus L.) by fluorescence in situ hybridization

We analyzed repeat sequences composition in the genome of cucumber inbred line 9930 using whole-genome shotgun reads. The analysis showed that satellite DNA sequences are the most dominant components in the cucumber genome. The distribution pattern of several tandem repeat sequences (Type I/II, Type III and Type IV) on cucumber chromosomes was visualized using fluorescence in situ hybridization (FISH). The FISH signals of the Type III and 45S rDNA provide useful cytogenetic markers, whose position and fluorescence intensity allow for easy identification of all somatic metaphase chromosomes. A karyotype showing the position and fluorescence intensity of several tandem repeat sequences is constructed. The establishment of this FISH-based karyotype has created the basis for the integration of molecular, genetic and cytogenetic maps in Cucumis sativus and for the ultimate genome sequencing project as well.

Chromosome-Specific Painting in Cucumis Species Using Bulked Oligonucleotides

Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a single chromosome of cucumber were identified using a newly developed bioinformatic pipeline and then massively synthesized de novo in parallel. The synthesized oligos were amplified and labeled with biotin or digoxigenin for use in fluorescence in situ hybridization (FISH). We developed three different probes with each containing 23,000–27,000 oligos. These probes spanned 8.3–17 Mb of DNA on targeted cucumber chromosomes and had the densities of 1.5–3.2 oligos per kilobases. These probes produced FISH signals on a single cucumber chromosome and were used to paint homeologous chromosomes in other Cucumis species diverged from cucumber for up to 12 million years. The bulked oligo probes allowed us to track a single chromosome in early stages during meiosis. We were able to precisely map the pairing between cucumber chromosome 7 and chromosome 1 of Cucumis hystrix in a F1 hybrid. These two homeologous chromosomes paired in 71% of prophase I cells but only 25% of metaphase I cells, which may provide an explanation of the higher recombination rates compared to the chiasma frequencies between homeologous chromosomes reported in plant hybrids.

A high-resolution cucumber cytogenetic map integrated with the genome assembly

BackgroundHigh-resolution cytogenetic map can provide not only important biological information on genome organization but also solid foundation for genetic and genomic research. The progress in the molecular and cytogenetic studies has created the basis for developing the cytogenetic map in cucumber (Cucumis sativus L.).ResultsHere, the cytogenetic maps of four cucumber chromosomes (chromosomes 1, 3–5) were constructed by fluorescence in situ hybridization (FISH) analysis on cucumber pachytene chromosomes. Together with our previously constructed cytogenetic maps of three cucumber chromosomes (chromosomes 2, 6–7), cucumber has a complete cytogenetic map with 76 anchoring points between the genetic, the cytogenetic and the draft genome assembly maps. To compare our pachytene FISH map directly to the genetic linkage and draft genome assembly maps, we used a standardized map unit—relative map position (RMP) to produce the comparative map alignments. The alignments allowed a global view of the relationship of genetic and physical distances along each cucumber chromosome, and accuracy and coverage of the draft genome assembly map.ConclusionsWe demonstrated a good correlation between positions of the markers in the linkage and physical maps, and essentially complete coverage of chromosome arms by the draft genome assembly. Our study not only provides essential information for the improvement of sequence assembly but also offers molecular tools for cucumber genomics research, comparative genomics and evolutionary study.

An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2

BackgroundIntegration of molecular, genetic and cytological maps is still a challenge for most plant species. Recent progress in molecular and cytogenetic studies created a basis for developing integrated maps in cucumber (Cucumis sativus L.).ResultsIn this study, eleven fosmid clones and three plasmids containing 45S rDNA, the centromeric satellite repeat Type III and the pericentriomeric repeat CsRP1 sequences respectively were hybridized to cucumber metaphase chromosomes to assign their cytological location on chromosome 2. Moreover, an integrated molecular cytogenetic map of cucumber chromosomes 2 was constructed by fluorescence in situ hybridization (FISH) mapping of 11 fosmid clones together with the cucumber centromere-specific Type III sequence on meiotic pachytene chromosomes. The cytogenetic map was fully integrated with genetic linkage map since each fosmid clone was anchored by a genetically mapped simple sequence repeat marker (SSR). The relationship between the genetic and physical distances along chromosome was analyzed.ConclusionsRecombination was not evenly distributed along the physical length of chromosome 2. Suppression of recombination was found in centromeric and pericentromeric regions. Our results also indicated that the molecular markers composing the linkage map for chromosome 2 provided excellent coverage of the chromosome.

Karyotyping in Melon (Cucumis melo L.) by Cross-Species Fosmid Fluorescence in situ Hybridization

Chromosome identification is critical for cytogenetic research and will accelerate studies on genetic variation and breeding, especially for those species with relatively little sequence information. So far, no reliable cytological landmarks have been developed to distinguish individual chromosomes in melon. In this study, using FISH (fluorescence in situ hybridization) combined with comparative genome information, we selected 21 cucumber fosmids anchored by SSR markers as chromosome-specific cytological markers for melon chromosomes. Moreover, with the help of melon centromeric satellite DNA repeats CentM, 45S rDNA and 5S rDNA, sequential FISH with 3 sets of multi-fosmid cocktails were conducted on the same metaphase cell, which allowed us to simultaneously identify each of the 12 metaphase chromosomes of melon and a standardized melon karyotype of somatic metaphase chromosomes was constructed. Finally, we compared the distribution of 21 FISH-mapped fosmids between melon and cucumber chromosomes, which allows a better understanding of the evolutionary process shaping these 2 species. Our study provides a basis for cytological characterization of the melon genome and comparative genomics of Cucurbitaceae.

Chromosome painting and comparative physical mapping of the sex chromosomes in Populus tomentosa and Populus deltoides

Dioecious species accounted for 6% of all plant species, including a number of crops and economically important species, such as poplar. However, sex determination and sex chromosome evolution have been studied only in few dioecious species. In poplar, the sex-determining locus was mapped to chromosome 19. Interestingly, this locus was mapped to either a peritelomeric or a centromeric region among different poplar species. We developed an oligonucleotide (oligo)-based chromosome painting probe based on the sequence of chromosome 19 from Populus trichocarpa. We performed chromosome painting in P. tomentosa and P. deltoides. Surprisingly, the distal end on the short arm of chromosome 19, which corresponds to the location of the sex-determining locus reported in several species, was not painted in both species. Thus, the DNA sequences associated with this region have not been anchored to the current chromosome 19 pseudomolecule, which was confirmed by painting of somatic metaphase chromosome 19 of P. trichocarpa. Interestingly, the unpainted distal ends of the two chromosome 19 did not pair at the pachytene stage in 22–24% of the meiotic cells in the two species, suggest that these regions from the sex chromosomes have structurally diverged from each other, resulting in the reduced pairing frequency. These results shed light on divergence of a pair of young sex chromosomes in poplar.

Genomic Abundance Is Not Predictive Of Tandem Repeat Localization In Grass Genomes

Highly repetitive regions have historically posed a challenge when investigating sequence variation and content. High-throughput sequencing has enabled researchers to use whole-genome shotgun sequencing to estimate the abundance of repetitive sequence, and these methodologies have been recently applied to centromeres. Previous research has investigated variation in centromere repeats across eukaryotes, positing that the highest abundance tandem repeat in a genome is often the centromeric repeat. To test this assumption, we used shotgun sequencing and a bioinformatic pipeline to identify common tandem repeats across a number of grass species. We find that de novo assembly and subsequent abundance ranking of repeats can successfully identify tandem repeats with homology to known tandem repeats. Fluorescent in-situ hybridization shows that de novo assembly and ranking of repeats from non-model taxa identifies chromosome domains rich in tandem repeats both near pericentromeres and elsewhere in the genome.