Distinct structural variants and repeat landscape shape the genomes of the ancient grapes Aglianico and Falanghina.

Mounting evidence recognizes structural variations (SVs) and repetitive DNA sequences as crucial players in shaping the existing grape phenotypic diversity at intra- and inter-species levels. To deepen our understanding on the abundance, diversity, and distribution of SVs and repetitive DNAs, including transposable elements (TEs) and tandemly repeated satellite DNA (satDNAs), we re-sequenced the genomes of the ancient grapes Aglianico and Falanghina. The analysis of large copy number variants (CNVs) detected candidate polymorphic genes that are involved in the enological features of these varieties. In a comparative analysis of Aglianico and Falanghina sequences with 21 publicly available genomes of cultivated grapes, we provided a genome-wide annotation of grape TEs at the lineage level. We disclosed that at least two main clusters of grape cultivars could be identified based on the TEs content. Multiple TEs families appeared either significantly enriched or depleted. In addition, in silico and cytological analyses provided evidence for a diverse chromosomal distribution of several satellite repeats between Aglianico, Falanghina, and other grapes. Overall, our data further improved our understanding of the intricate grape diversity held by two Italian traditional varieties, unveiling a pool of unique candidate genes never so far exploited in breeding for improved fruit quality.

Autopolyploid inheritance and a heterozygous reciprocal translocation shape chromosome genetic behavior in tetraploid blueberry (Vaccinium corymbosum).

Understanding chromosome recombination behavior in polyploidy species is key to advancing genetic discoveries. In blueberry, a tetraploid species, the line of evidences about its genetic behavior still remain poorly understood, owing to the inter-specific, and inter-ploidy admixture of its genome and lack of in depth genome-wide inheritance and comparative structural studies. Here we describe a new high-quality, phased, chromosome-scale genome of a diploid blueberry, clone W85. The genome was integrated with cytogenetics and high-density, genetic maps representing six tetraploid blueberry cultivars, harboring different levels of wild genome admixture, to uncover recombination behavior and structural genome divergence across tetraploid and wild diploid species. Analysis of chromosome inheritance and pairing demonstrated that tetraploid blueberry behaves as an autotetraploid with tetrasomic inheritance. Comparative analysis demonstrated the presence of a reciprocal, heterozygous, translocation spanning one homolog of chr-6 and one of chr-10 in the cultivar Draper. The translocation affects pairing and recombination of chromosomes 6 and 10. Besides the translocation detected in Draper, no other structural genomic divergences were detected across tetraploid cultivars and highly inter-crossable wild diploid species. These findings and resources will facilitate new genetic and comparative genomic studies in Vaccinium and the development of genomic assisted selection strategy for this crop.

A high-quality carrot genome assembly provides new insights into carotenoid accumulation and asterid genome evolution.

We report a high-quality chromosome-scale assembly and analysis of the carrot (Daucus carota) genome, the first sequenced genome to include a comparative evolutionary analysis among members of the euasterid II clade. We characterized two new polyploidization events, both occurring after the divergence of carrot from members of the Asterales order, clarifying the evolutionary scenario before and after radiation of the two main asterid clades. Large- and small-scale lineage-specific duplications have contributed to the expansion of gene families, including those with roles in flowering time, defense response, flavor, and pigment accumulation. We identified a candidate gene, DCAR_032551, that conditions carotenoid accumulation (Y) in carrot taproot and is coexpressed with several isoprenoid biosynthetic genes. The primary mechanism regulating carotenoid accumulation in carrot taproot is not at the biosynthetic level. We hypothesize that DCAR_032551 regulates upstream photosystem development and functional processes, including photomorphogenesis and root de-etiolation.

Evidence for emergence of sex-determining gene(s) in a centromeric region in Vasconcellea parviflora.

Sex chromosomes have been studied in many plant and animal species. However, few species are suitable as models to study the evolutionary histories of sex chromosomes. We previously demonstrated that papaya (Carica papaya) (2n = 2x = 18), a fruit tree in the family Caricaceae, contains recently emerged but cytologically heteromorphic X/Y chromosomes. We have been intrigued by the possible presence and evolution of sex chromosomes in other dioecious Caricaceae species. We selected a set of 22 bacterial artificial chromosome (BAC) clones that are distributed along the papaya X/Y chromosomes. These BACs were mapped to the meiotic pachytene chromosomes of Vasconcellea parviflora (2n = 2x = 18), a species that diverged from papaya ∼27 million years ago. We demonstrate that V. parviflora contains a pair of heteromorphic X/Y chromosomes that are homologous to the papaya X/Y chromosomes. The comparative mapping results revealed that the male-specific regions of the Y chromosomes (MSYs) probably initiated near the centromere of the Y chromosomes in both species. The two MSYs, however, shared only a small chromosomal domain near the centromere in otherwise rearranged chromosomes. The V. parviflora MSY expanded toward the short arm of the chromosome, whereas the papaya MSY expanded in the opposite direction. Most BACs mapped to papaya MSY were not located in V. parviflora MSY, revealing different DNA compositions in the two MSYs. These results suggest that mutation of gene(s) in the centromeric region may have triggered sex chromosome evolution in these plant species.

Copy number variation in potato - an asexually propagated autotetraploid species.

Copy number variation (CNV) has been revealed as a significant contributor to the genetic variation in humans. Although CNV has been reported in several model animal and plant species, the presence of CNV and its biological impact in polyploid species has not yet been documented. We conducted a fluorescence in situ hybridization (FISH)-based CNV survey in potato, a vegetatively propagated autotetraploid species (2n = 4x = 48). We conducted FISH analysis using 18 randomly selected potato bacterial artificial chromosome (BAC) clones in a set of 16 potato cultivars with diverse breeding backgrounds. Six BACs (33%) with insert sizes of 137-145 kb were found to be associated with large CNV events detectable at the cytological level. We demonstrate that the large CNVs associated with two specific BACs (RH102I10 and RH83C08) were widespread among potato cultivars developed in North America and Europe. We measured the transcript abundance of four genes associated with the CNV spanned by BAC RH102I10. All four genes displayed a dosage effect in transcription. Although potato is vegetatively propagated, we observed that female gametes lacking the RH102I10-associated CNV were inferior to those with at least one copy of this CNV, indicating that the RH102I10-associated CNV can impact on the growth and development of the potato plants. Our results show that CNV is highly abundant in the potato genome and may play a significant role in genetic variation of this important food crop.

Repeatless and repeat-based centromeres in potato: implications for centromere evolution.

Centromeres in most higher eukaryotes are composed of long arrays of satellite repeats. By contrast, most newly formed centromeres (neocentromeres) do not contain satellite repeats and instead include DNA sequences representative of the genome. An unknown question in centromere evolution is how satellite repeat-based centromeres evolve from neocentromeres. We conducted a genome-wide characterization of sequences associated with CENH3 nucleosomes in potato (Solanum tuberosum). Five potato centromeres (Cen4, Cen6, Cen10, Cen11, and Cen12) consisted primarily of single- or low-copy DNA sequences. No satellite repeats were identified in these five centromeres. At least one transcribed gene was associated with CENH3 nucleosomes. Thus, these five centromeres structurally resemble neocentromeres. By contrast, six potato centromeres (Cen1, Cen2, Cen3, Cen5, Cen7, and Cen8) contained megabase-sized satellite repeat arrays that are unique to individual centromeres. The satellite repeat arrays likely span the entire functional cores of these six centromeres. At least four of the centromeric repeats were amplified from retrotransposon-related sequences and were not detected in Solanum species closely related to potato. The presence of two distinct types of centromeres, coupled with the boom-and-bust cycles of centromeric satellite repeats in Solanum species, suggests that repeat-based centromeres can rapidly evolve from neocentromeres by de novo amplification and insertion of satellite repeats in the CENH3 domains.

Comparative FISH mapping of Daucus species (Apiaceae family).

The cytogenetic characterization of the carrot genome (Daucus carota L., 2n = 18) has been limited so far, partly because of its somatic chromosome morphology and scant of chromosome markers. Here, we integrate the carrot linkage groups with pachytene chromosomes by fluorescent in situ hybridization (FISH) mapping genetically anchored bacterial artificial chromosomes (BACs). We isolated a satellite repeat from the centromeric regions of the carrot chromosomes, which facilitated the study of the pachytene-based karyotype and demonstrated that heterochromatic domains were mainly confined to the pericentromeric regions of each chromosome. Chromosome-specific BACs were used to: (1) physically locate genetically unanchored DNA sequences, (2) reveal relationships between genetic and physical distances, and (3) address chromosome evolution in Daucus. Most carrot BACs generated distinct FISH signals in 22-chromosome Daucus species, providing evidence for syntenic chromosome segments and rearrangements among them. These results provide a foundation for further cytogenetic characterization and chromosome evolution studies in Daucus.

Organization and evolution of subtelomeric satellite repeats in the potato genome.

Subtelomeric domains immediately adjacent to telomeres represent one of the most dynamic and rapidly evolving regions in eukaryotic genomes. A common feature associated with subtelomeric regions in different eukaryotes is the presence of long arrays of tandemly repeated satellite sequences. However, studies on molecular organization and evolution of subtelomeric repeats are rare. We isolated two subtelomeric repeats, CL14 and CL34, from potato (Solanum tuberosum). The CL14 and CL34 repeats are organized as independent long arrays, up to 1-3 Mb, of 182 bp and 339 bp monomers, respectively. The CL14 and CL34 repeat arrays are directly connected with the telomeric repeats at some chromosomal ends. The CL14 repeat was detected at the subtelomeric regions among highly diverged Solanum species, including tomato (Solanum lycopersicum). In contrast, CL34 was only found in potato and its closely related species. Interestingly, the CL34 repeat array was always proximal to the telomeres when both CL14 and CL34 were found at the same chromosomal end. In addition, the CL34 repeat family showed more sequence variability among monomers compared with the CL14 repeat family. We conclude that the CL34 repeat family emerged recently from the subtelomeric regions of potato chromosomes and is rapidly evolving. These results provide further evidence that subtelomeric domains are among the most dynamic regions in eukaryotic genomes.

Evolution of chromosome 6 of Solanum species revealed by comparative fluorescence in situ hybridization mapping.

Comparative genetic linkage mapping using a common set of DNA markers in related species is an important methodology in plant genome research. Here, we demonstrate a comparative fluorescence in situ hybridization (FISH) mapping strategy in plants. A set of 13 bacterial artificial chromosome clones spanning the entire length of potato chromosome 6 was used for pachytene chromosome-based FISH mapping in seven distantly related Solanum species including potato, tomato, and eggplant. We discovered one paracentric inversion and one pericentric inversion within specific lineages of these species. The comparative FISH mapping data revealed the ancestral structure of this chromosome. We demonstrate that comparative FISH mapping is an efficient and powerful methodology to study chromosomal evolution among plant species diverged for up to 12 million years.

Chromatin structure and physical mapping of chromosome 6 of potato and comparative analyses with tomato.

Potato (Solanum tuberosum) has the densest genetic linkage map and one of the earliest established cytogenetic maps among all plant species. However, there has been limited effort to integrate these maps. Here, we report fluorescence in situ hybridization (FISH) mapping of 30 genetic marker-anchored bacterial artificial chromosome (BAC) clones on the pachytene chromosome 6 of potato. The FISH mapping results allowed us to define the genetic positions of the centromere and the pericentromeric heterochromatin and to relate chromatin structure to the distribution of recombination along the chromosome. A drastic reduction of recombination was associated with the pericentromeric heterochromatin that accounts for approximately 28% of the physical length of the pachytene chromosome. The pachytene chromosomes 6 of potato and tomato (S. lycopersicum) share a similar morphology. However, distinct differences of heterochromatin distribution were observed between the two chromosomes. FISH mapping of several potato BACs on tomato pachytene chromosome 6 revealed an overall colinearity between the two chromosomes. A chromosome inversion was observed in the euchromatic region of the short arms. These results show that the potato and tomato genomes contain more chromosomal rearrangements than those reported previously on the basis of comparative genetic linkage mapping.

Analysis of 90 Mb of the potato genome reveals conservation of gene structures and order with tomato but divergence in repetitive sequence composition.

BACKGROUND: The Solanaceae family contains a number of important crop species including potato (Solanum tuberosum) which is grown for its underground storage organ known as a tuber. Albeit the 4th most important food crop in the world, other than a collection of approximately 220,000 Expressed Sequence Tags, limited genomic sequence information is currently available for potato and advances in potato yield and nutrition content would be greatly assisted through access to a complete genome sequence. While morphologically diverse, Solanaceae species such as potato, tomato, pepper, and eggplant share not only genes but also gene order thereby permitting highly informative comparative genomic analyses. RESULTS: In this study, we report on analysis 89.9 Mb of potato genomic sequence representing 10.2% of the genome generated through end sequencing of a potato bacterial artificial chromosome (BAC) clone library (87 Mb) and sequencing of 22 potato BAC clones (2.9 Mb). The GC content of potato is very similar to Solanum lycopersicon (tomato) and other dicotyledonous species yet distinct from the monocotyledonous grass species, Oryza sativa. Parallel analyses of repetitive sequences in potato and tomato revealed substantial differences in their abundance, 34.2% in potato versus 46.3% in tomato, which is consistent with the increased genome size per haploid genome of these two Solanum species. Specific classes and types of repetitive sequences were also differentially represented between these two species including a telomeric-related repetitive sequence, ribosomal DNA, and a number of unclassified repetitive sequences. Comparative analyses between tomato and potato at the gene level revealed a high level of conservation of gene content, genic feature, and gene order although discordances in synteny were observed. CONCLUSION: Genomic level analyses of potato and tomato confirm that gene sequence and gene order are conserved between these solanaceous species and that this conservation can be leveraged in genomic applications including cross-species annotation and genome sequencing initiatives. While tomato and potato share genic features, they differ in their repetitive sequence content and composition suggesting that repetitive sequences may have a more significant role in shaping speciation than previously reported.

Major cytogenetic landmarks and karyotype analysis inDaucus carota and other Apiaceae.

Karyotype analysis provides insights into genome organization at the chromosome level and into chromosome evolution. Chromosomes were marked for comparative karyotype analysis using FISH localization of rDNA genes for the first time in Apioideae species including taxa of economic importance and several wild Daucus relatives. Interestingly, Daucus species did not vary in number of rDNA loci despite variation in chromosome number (2n = 18, 20, 22, and 44) and previous publications suggesting multiple loci. All had single loci for both 5S and 18S-25S (nucleolar organizing region) rDNA, located on two different chromosome pairs. The 5S rDNA was on the short arm of a metacentric chromosome pair in D. crinitus (2n = 22) and D. glochidiatus (2n = 44) and on the long arm of a metacentric pair in other Daucus species, suggesting possible rearrangement of this chromosome. For other Apiaceae, from two (Apium graveolens), to three (Orlaya grandiflora), to four (Cuminum cyminum) chromosomes had 18S-25S rDNA sites. Variability for number and position of the 5S rDNA was also observed. FISH signals enabled us to identify 20-40% of the chromosome complement among species examined. Comparative karyotype analysis provides insights into the fundamental aspects of chromosome evolution in Daucus.

Nuclear and cytoplasmic genome composition of Solanum bulbocastanum (+) S. tuberosum somatic hybrids.

Somatic hybrids between the wild incongruent species Solanum bulbocastanum (2n = 2x = 24) and S. tuberosum haploids (2n = 2x = 24) have been characterized for their nuclear and cytoplasmic genome composition. Cytologic observations revealed the recovery of 8 (near-)tetraploid and 3 hexaploid somatic hybrids. Multicolor genomic in situ hybridization (GISH) analysis was carried out to study the genomic dosage of the parental species in 5 somatic hybrids with different ploidy. The GISH procedure used was effective in discriminating parental genomes in the hybrids; most chromosomes were unambiguously colored. Two (near-)tetraploid somatic hybrids showed the expected 2:2 cultivated-to-wild genomic dosage; 2 hexaploids revealed a 4:2 cultivated-to-wild genomic dosage, and 1 hexaploid had a 2:4 cultivated-to-wild genomic dosage. Characterization of hybrid cytoplasmic genomes was performed using gene-specific primers that detected polymorphisms between the fusion parents in the intergenic regions. The analysis showed that most of the somatic hybrids inherited the plastidial and mitochondrial DNA of the cultivated parent. A few hybrids, with a rearranged mitochondrial genome (showing fragments derived from both parents), were also identified. These results confirmed the potential of somatic hybridization in producing new variability for genetic studies and breeding.