A major part of the nuclear genome of most plants is composed of different repetitive DNA elements. Studying these sequence elements is essential for our understanding of the nature and consequences of genome size variation between different species, and for studying the large-scale organization and evolution of plant genomes. Sugar beet (Beta vulgaris L.) is an important crop and a suitable model for such investigations: with a genome size of 0-8 pg C (760 Mbp) it contains significant amounts of all major groups of repetitive sequences among its nine chromosome pairs, but analysis is not complicated by polyploidy or the huge size of some genomes, and there are valuable genetic data, recombinant DNA libraries and wild relatives to complement studies of sequence contribution to genome size in sugar beet. A sophisticated understanding of the structure of the genome will provide valuable data about the major factors responsible for genome size variation, useful aids in the development of a molecular understanding of genome evolution, and perhaps indicate strategies for crop improvement. Using molecular and cytological approaches, we have characterized a range of differentially organized repetitive DNA sequence elements from the genomes of cultivated and wild beet species, leading to an extensive model of the repetitive DNA, its organization and evolution. © 1998 Annals of Botany Company

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Repetitive DNA Elements as a Major Component of Plant Genomes

Tetraploid cultivated potato (Solanum tuberosum) is the World's fourth most important crop and has been subjected to much breeding effort, including the incorporation of resistance to viruses. Several new approaches, ideas and technologies have emerged recently that could affect the future direction of virus resistance breeding. Thus, there are new opportunities to harness molecular techniques in the form of linked molecular markers to speed up and simplify selection of host resistance genes. The practical application of pathogen-derived transgenic resistance has arrived with the first release of GM potatoes engineered for virus resistance in the USA. Recently, a cloned host virus resistance gene from potato has been shown to be effective when inserted into a potato cultivar lacking the gene. These and other developments offer great opportunities for improving virus resistance, and it is timely to consider these advances and consider the future direction of resistance breeding in potato. We review the sources of available resistance, conventional breeding methods, marker-assisted selection, somaclonal variation, pathogen-derived and other transgenic resistance, and transformation with cloned host genes. The relative merits of the different methods are discussed, and the likely direction of future developments is considered.

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Breeding virus resistant potatoes ( Solanum tuberosum ): a review of traditional and molecular approaches

In recent years, the rapid development of somatic cell genetics has made possible the transfer of alien genes over wide taxonomic distances by somatic hybridization. In this review, the potential of somatic hybridization in the breeding of crops within the Brassicaceae, Fabaceae, Poaceae and Solanaceae is discussed. It is evident from these studies that many hybrids, either symmetric or asymmetric, which are fertile have the potential to be used as a bridge between the alien species and the crop. Progeny analysis of some hybrid combinations also reveals intergenomic translocations which may lead to the introgression of the alien genes. Furthermore, fusion techniques enable the resynthesis of allopolyploid crops to increase their genetic variability and to restore ploidy level and heterozygosity after breeding at reduced ploidy level in polyploid crops.

The potential of somatic hybridization in crop breeding

Somatic fusion of mesophyll protoplasts was used to produce hybrids between the frost-tolerant species Solanum commersonii (2n=2x=24) and dihaploid S. tuberosum (2n=2x=24). This is a sexually incompatible combination due to the difference in EBN (Endosperm Balance Number, Johnston et al. 1980). Species with different EBNs as a rule are sexually incompatible. Fifty-seven hybrids were analysed for variation in chromosome number, morphological traits, fertility and frost tolerance. About 70% of the hybrids were tetraploid, and 30% hexaploid. Chloroplast counts in stomatal guard cells revealed a low frequency of cytochimeras. The frequency of aneuploids was relatively higher at the hexaploid level (hypohexaploids) than at the tetraploid level (hypotetraploids). The somatic hybrids were much more vigorous than the parents, and showed an intermediate phenotype for several morphological traits and moderate to profuse flowering. Hexaploid hybrid clones were less vigorous and had a lower degree of flowering than the tetraploid hybrid clones. All of the hybrids were female fertile but male sterile except for one, which was fully fertile and self-compatible. Many seeds were produced on the latter clone by selfing and on the male-sterile clones by crossing. The somatic hybrid plants showed an introgression of genes for frost tolerance and an adaptability to cold from S. commersonii. Therefore, the use of these somatic hybrids in breeding for and in genetic esearch on frost tolerance and cold-hardening is suggested.

Production of somatic hybrids between frost-tolerant Solanum commersonii and S. tuberosum: characterization of hybrid plants.

Potato is the third most important global food crop and the most widely grown noncereal crop. As a species highly amenable to cell culture, it has a long history of biotechnology applications for crop improvement. This review begins with a historical perspective on potato improvement using biotechnology encompassing pathogen elimination, wide hybridization, ploidy manipulation and applications of cell culture. We describe the past developments and new approaches for gene transfer to potato. Transformation is highly effective for adding single genes to existing elite potato clones with no, or minimal, disturbances to their genetic background and represents the only effective way to produce isogenic lines of specific genotypes/cultivars. This is virtually impossible via traditional breeding as, due to the high heterozygosity in the tetraploid potato genome, the genetic integrity of potato clones is lost upon sexual reproduction as a result of allele segregation. These genetic attributes have also provided challenges for the development of genetic maps and applications of molecular markers and genomics in potato breeding. Various molecular approaches used to characterize loci, (candidate) genes and alleles in potato, and associating phenotype with genotype are also described. The recent determination of the potato genome sequence has presented new opportunities for genomewide assays to provide tools for gene discovery and enabling the development of robustly unique marker haplotypes spanning QTL regions. The latter will be useful in introgression breeding and whole-genome approaches such as genomic selection to improve the efficiency of selecting elite clones and enhancing genetic gain over time.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.12099

Applications of biotechnology and genomics in potato improvement

Somatic hybrids between Solanum tuberosum L. cv. Gracia (2n=4x=48) and Solanum brevidens Phil. (2n=2x=24) were produced via fusion of mesophyll protoplasts. Selection of the protoplast derived putative hybrid calli was based on their vigorous growth. Additive isozyme patterns and chromosome numbers as well as the expression of parental morphological characters have proved the hybrid origin of the selected regenerants. Extensive chromosome loss during the regeneration process resulted in aneuploid hybrids with high frequency. Genomic instability could not be detected in these plants during the period of vegetative propagation. Regenerants from hybrid tissues exhibited wide morphological variation especially in tuber formation. The detailed morphological analysis based on the use of multivariate method (principal component analysis, PCA) enabled to identify morphological groups among the hybrid clones. The positioning of hybrid clones in the PCA space could not be correlated with chromosome numbers. The genomic ratio represented by the tetraploid and diploid parents influenced the morphology of somatic hybrid population according to the applied analytical system. Two selected hybrid clones have exhibited an intermediate degree of frost tolerance compared to the parents, based on the recovery of plants from lower buds after freezing of potted plants.

Characterization of morphological variation and cold resistance in interspecific somatic hybrids between potato (Solanum tuberosum L.) and S. brevidens Phil.

Asymmetric somatic hybrids between Solanum tuberosum L. and S. brevidens Phil. have been obtained via the fusion of protoplasts from potato leaves and from cell suspension culture of S. brevidens. The wild Solanum species served as donor after irradiation of its protoplasts with a lethal X-ray dose (200 Gy). Selection of the putative hybrids was based on the kanamycin-resistance marker gene previously introduced into the genome of Solanum brevidens by Agrobacterium-mediated gene transfer. Thirteen out of the 45 selected clones exhibited reduced morphogenic potential. The morphological abnormalities of the regenerated plantlets were gradually eliminated during the extended in vitro culture period. Cytological investigations revealed that the number of chromosomes in the cultured S. brevidens cells used as protoplast source ranged between 28–40 instead of the basic 2n=24 value. There was a high degree of aneuploidy in all of the investigated hybrid clones, and at least 12 extra chromosomes were observed in addition to the potato chromosomes (2n=48). Interand intraclonal variation and segregation during vegetative propagation indicated the genetic instability of the hybrids, which can be ascribed to the pre-existing and X-ray irradiation-induced chromosomal abnormalities in the donor S. brevidens cells. The detection of centromeric chromosome fragments and long, poly-constrictional chromosomes in cytological preparations as well as non-parental bands in Southern hybridizations with restriction fragment length polymorphism (RFLP) markers revealed extensive chromosome rearrangements in most of the regenerated clones. On the basis of the limited number of RFLP probes used, preferential loss of S. brevidens specific markers with a non-random elimination pattern could be detected in hybrid regenerants.

Characterization of chromosome instability in interspecific somatic hybrids obtained by X-ray fusion between potato (Solanum tuberosum L.) and S. brevidens Phil

An early identification of fusion products was based on the presumed vigorous growth of hybrid calluses after fusion between Solanum brevidens and S. tuberosum leaf protoplasts. The S. brevidens protoplasts were unable to form multicellular colonies under the applied culture conditions. Three size groups of calluses were separated and analyzed at two different early phases of culture period. “Squash blot” hybridization with a S. brevidens specific repetitive DNA probe showed that the group of the largest calluses consisted of putative somatic hybrids with a frequency of 80–100% in three independent experiments. Furthermore, approximately 80–95% of the middle sized calluses and 33–90% of the smallest ones were shown to be hybrid. The unexpectedly high percentage of fusion products, even in the case of the smallest calluses, may result from the suppression of the development of parental potato colonies in cultures with mixed cell population. Till this time 120 independent colonies selected as putative hybrids have been regenerated into plants. All of them exhibited hybrid phenotype, and their hybrid origin was proved by cytological and restriction fragment length polymorphism analyses.

Vigorous growth of fusion products allows highly efficient selection of interspecific potato somatic hybrids: molecular proofs.

A species-specific repetitive DNA fragment has been isolated from a genomic library of Solanum brevidens. Sequence analysis revealed a regular organization of three non-homologous subrepeats forming tandemly-arranged composite repetitive units. Interpretation of Southern hybridization patterns based on the known sequence data suggests that the isolated sequence element represents an abundant organization type, although the presence of simple tandem arrays of the subrepeats is also indicated. Seventy-four percent sequence similarity was found between one of the S. brevidens subrepeats (Sb4AX) and a satellite DNA (TGRI) localized as a subtelomeric repeat on almost all Lycopersicon esculentum chromosomes. Insitu hybridization indicated that, similarly to TGRI, the S. brevidens-specific repeats are located at the ends of the arms of several chromosomes. On the basis of the data obtained, a common ancestral sequence can be proposed for the tomato (TGRI) and the S. brevidens (Sb4AX) repeat however, the molecular organization of this element in these two species evolved in a basically different manner.

Organization of a Solatium brevidens repetitive sequence related to the TGRI subtelomeric repeats of Lycopersicon esculentum.

As an alternative to Agrobacterium-mediated gene transfer, direct transformation of potato (Solanum tuberosum L.) cultivars was achieved by using the Mg2+/PEG protocol (Negrutiu et al. 1987) to stimulate DNA uptake into leaf protoplasts. The frequency of kanamycin-resistant clones varied between 1–12% from experiment to experiment independently of the genotype used. A deleterious effect of heat shock treatment (45°C for 5 min.) has been found on colony formation during optimization of the transformation procedure. The Mg2+ ion concentration (15–35 mM), the denaturation (100°C, 10 min. right before the treatment) and the form of the plasmid DNA (linear or circular) had no significant effect on the efficacy of the transformation. Application of denatured calf thymus DNA as carrier molecules (at concentration of 50 μg ml-1, however, resulted in a significant decrease in the number of the resistant cell colonies). Sixty to 80 percent of the kanamycin-selected callus tissues regenerated shoots. The presence and expression of the introduced neomycin phosphotransferase neo gene in regenerants with normal morphology and tetraploid chromosome number was proved by biological tests based on rooting and callus formation in the presence of the antibiotic and by NPT enzyme activity assay. Southern analysis revealed the integration of the introduced DNA molecules carrying the neo marker gene, into the genome of potato.

J. Preiszner

Papers

Characterization of morphological variation and cold resistance in interspecific somatic hybrids between potato (Solanum tuberosum L.) and S. brevidens Phil.

Characterization of chromosome instability in interspecific somatic hybrids obtained by X-ray fusion between potato (Solanum tuberosum L.) and S. brevidens Phil

Vigorous growth of fusion products allows highly efficient selection of interspecific potato somatic hybrids: molecular proofs.

Organization of a Solatium brevidens repetitive sequence related to the TGRI subtelomeric repeats of Lycopersicon esculentum.

PEG-mediated transformation of leaf protoplasts of Solanum tuberosum L. cultivars