Chromosome Characteristics Of Solanum Species And Inter-specific Somatic Hybrids

Introgression of desired traits from wild potato species or other closely related Solanum species into potato cultivars by cell fusion is an applicable way to create new germplasms. Investigations into the genome compositions, chromosome rearrangement, and the association of trait variation with chromosome introgression of the somatic hybrids are of great significance for potato breeding and cytogenetic studies on the chromosomal level. Fluorescence in situ hybridization plays an important role in connecting DNA sequences and chromosome biology. Therefore, the present study established and optimized a fluorescence in situ hybridization system in potato, and further examined the characteristics of chromosomes of several related Solanum species that would probably being involved in potato genetic-resource creation. Additionally, chromosome analysis was performed with several somatic hybrids derived from S. tuberosum and S. melongena. The main results obtained are as following:1. The karyotype of potato chromosomes based on BAC-FISH markersFirst, a full set of12chromosomes of the wild potato species S. chacoense were identified through BAC-FISH with chromosome-specific BACs, including5from S. tuberosum and7from S. bulbocastanum. Then, the standard karyotype of12S. chacoense chromosomes was set up according to the genetic maps, the BAC linkage groups, as well as previously reported karyotype information, which would lay a foundation for follow-up study.2. Distribution characteristics of ITRs in potato, tomato, and eggplant chromosomesFISH hybridizations using telomeric probe were conducted in representative diploid Solanum species, including S. tuberosum (A genome), S. chacoense (A genome), S. bulbocastanum (B genome), S. pinnatisectum (B genome), S. paucissectum (P genome), S. lycopersicum, and S. melongena. The results showed that interstitial telomeric repeats (ITRs) had been undergone an intense amplification in centromeric regions of chromosomes in tested species. Dot blot analysis revealed basically the same trends between the increasing of the relative contents of telomeric sequences and the strengthening of FISH signals from telomeric probe in species assayed. Large amount of ITR FISH signals were located in primary constrictions of pachytene chromosomes in S. pinnatisectum (B genome). In addition, a pair of heterozygous ITR sites were detected in the pericentromeric regions on the long arms of chromosome12in potato A genome species S. chacoense. These results suggest that ITRs may be related to the functional centromeres.3. Distribution of rDNA in representative Solanum speciesThe5S rRNA genes and the5’ETS of45S rRNA gene spacer regions (IGS) were obtained by the PCR amplification in S. tuberosum, S. chacoense and S. melongena, respectively. Sequence analysis showed that the spacer regions of5S rRNA genes and also the5’ETS sequences between S. tuberosum and S. chacoense were highly homologous. However, the sequences of5S rRNA gene spacer regions and that of the5’ETS between S. tuberosum and S. melongena were diverged. The FISH assays were performed on chromosomes from seven potato, one tomato, and one eggplant species using S. tuberosum derived5S rDNA and25S rDNA probes. The results showed that5S rDNA signals were detected in only one set of homologous chromosome, while25S rDNA signals were detected in another set of homologous chromosome except for S. melongena in which25S rDNA signals presented on three pairs of homologous chromosomes. The5S rDNA and25S rDNA were found on distinct chromosomes when paring the homologous chromosomes. The strength of the5S rDNA or25S rDNA signals varied significantly between homologous chromosomes in S. tuberosum, S. berthaultii, S. bulbocastanum, S. acaule and S. melongena.4. Distribution of oligonucleotide SSRs on chromosomes in several Solanum speciesThe physical distributions of mono-, di-, and trinucleotide SSRs, including (A)n,(C)n,(CG)n,(AC)n,(TC)n,(AT)n,(AAC)n,(AAG)n,(AAT)n,(AGG)n,(CAC)n,(CAT)n,(CAG)n,(ACT)n,(ACG)n, and (GCC)n on metaphase chromosomes were investigated by FISH in potato cultivated species S. tuberosum and wild species S. chacoense. In general, all kinds of nucleotide SSRs were scattered on chromosomes. The signals of (A)n were mainly detected at the ends and parts of the centromeric regions of chromosomes in S. chacoense, but fewer signals were detected in S. tuberosum with random distributions. Signals from (AAT)n,(ACT)n, and (CAG)n were restricted at almost all the chromosomal ends in S. chacoense, while they were scattered throughout the chromosomes in S. tuberosum excluding a few located at chromosome ends. FISH mapping of the mixture of mono-, di-, and trinucleotide SSRs on mitotic metaphase chromosomes were conducted respectively in potato species S. chacoense, S. bulbocastanum, and&pinnatisectum, as well as in tomato. The distribution patterns of mono-and dinucleotide SSRs were similar between potato and tomato species. However, great discrepancy in distribution of trinucleotide SSRs was found between tomato and potato, almost all the trinucleotide SSRs signals were concentrated in or around the centromeric regions of tomato chromosomes with strong intensities while they were spotted principally at most chromosomal ends in potato species. These findings provide, to a certain extent, a basic information for screening differential SSRs among potato species and between potato and tomato.5. GISH analysis of chromosomes between S. tuberosum and several related speciesUsing the genomic DNA of cultivated potato S. tuberosum as a probe, GISH hybridizations were performed in species including S. tuberosum (A genome), S. chacoense (A genome), S. pinnatisectum (B genome), S. bulbocastanum (B genome), S. paucissectum (P genome), S. etuberosum (E genome), S. lycopersicum (L genome) and S. melongena. The results showed that S. tuberosum shared the highest homology of genomic DNA with S. chacoense and S. paucissectum, lower with S. etuberosum and S. pinnatisectum, lowest with S. bulbocastanum, S. lycopersicum and S. melongen. Therefore, if GISH is employed in discriminating the chromosomal compositions of somatic hybrids, it would be effective to discriminate S. tuberosum from S. bulbocastanum, S. lycopersicum and S. melongena, but it could be impractical when S. tuberosum is fused with S. chacoense and S. paucissectum.6. Identification of chromosome composition in the somatic hybridsGISH analysis with and without blocking were carried out separately with somatic hybrids derived from S. tuberosum and S. chacoense using S. chacoense genomic DNA as probe. The results demonstrated an infeasibility in identifying the chromosomal compositions of the hybrids owing to a high homology between the parents. For further tests, double-color GISH employing digoxin and biotin labeled genomic DNA probes from S. tuberosum and S. chacoense were conducted in somatic hybrids3C10-2(mixoploid),3C28-1(5x), and3C33-2(6x). The results showed that apart from a small number of chromosomal segments covered by apparent single color signals, most of the chromosomes were simultaneously painted on red or green signals from S. chacoense or S. tuberosum. It was illustrated impossible to discriminate the chromosomal compositions of the somatic hybrids between S. tuberosum and S. chacoense through GISH. The results indicate that GISH method is incapable of distinguishing chromosomal components in somatic hybrids derived from closely related species.Results from double-color GISH investigation of three somatic hybrids between potato and eggplant showed that the parental origins of the chromosomes, including whole chromosomes and rearranged chromosomes, were clearly identified. In combination with the rDNA and the telomere probe in further study, a variety of chromosomal rearrangements, involving end-to-end chromosome fusion and chromosome translocation containing the rDNA locus, were detected in the somatic hybrids. Two rearranged double-centromeric chromosomes, which might be resulted from telomere-to-telomere fusion and translocation, respectively, were found in somatic hybrid60-10. Additionally, ITR signal was found at the site where the rearrangement had occurred on a rearranged chromosome of60-13. The results demonstrated that the GISH detection method that assists with abundant cytogenetic markers serves as an effective tool in diagnosing the chromosome constituents of somatic hybrids.