Exploring the use of wild relatives in potato breeding through integrated cytogenetic and genomic approaches

Cultivated potato and its wild relatives represent a more diverse and accessible germplasm resource than that of any other crop. Diversity coming from wild relatives can be used to introduce specific traits into the cultivated background. Breeders have long used introgressive hybridization, in which chromatin carrying a gene of interest from a wild relative is integrated into the genome of the crop by interspecific hybridization. During the subsequent backcrossing generations, genes of interest are incorporated into the crop chromosomes by homeologous recombination. The offspring are then selected for the desired trait while the original cultivated genetic background is recovered by backcrossing and selection as far as possible. The efficient use of wild relatives now requires extensive knowledge of their allelic variation and genomic structure, including the screening for desirable traits. To minimize the occurrence of linkage drag when introgressed chromatin still contains closely linked wild traits from the ancestral donor, knowledge on the genomic structure of crop and donor species is indispensable. In this work I have presented an overview of genetic, cytogenetic and genomic characteristics of Solanum commersonii, a South American wild relative of potato. The main aim was to create scientific tools to distinguish the genomes of S. commersonii and S. tuberosum (cultivated potato), along with S. chacoense, and trace their genomes in introgressive hybridization breeding programmes. The application of these tools to pinpoint past introgressions into cultivated potato and to plan introgressive hybridization schemes is reviewed in Chapter 2, with an emphasis on the need to make these technologies part of the routine toolkit for pre-breeding in order to exploit potato wild relatives at their fullest. A variety of questions related to the use of particular potato wild relatives in introgression are addressed using different cytogenetic and genomic technologies. In Chapter 3 pairing of the homoeologous S. commersonii genomes and S. tuberosum Group Phureja chromosomes in F1 interspecific triploid hybrids was analyzed, observing an almost autotriploid behaviour. Genome painting was used to follow the fate of specific chromosomes in these hybrids and backcross derivatives. Homoeologues could not be distinguished by fluorescence signals, so this approach did not provide tools to select against wild chromatin in the advanced materials nor pinpoint the chromosomal exchanges between homoeologues. However, the results suggest a low degree of genomic divergence between the species involved in terms of repetitive sequences and highlight their potential for homoeologous pairing. A comparative cytogenetic mapping analysis was performed in Chapter 4, to test collinearity among genomes of the different species and thus anticipate the existence of either suppression of recombination or linkage drag. Solanum commersonii and S. chacoense are collinear with cultivated S. tuberosum on the whole chromosome scale. Some differences observed in microscopic distances between the signals suggest either small-scale rearrangements or reduction/amplification of repetitive sequences. No major rearrangements have been found, making these amenable species for efficient introgressive hybridization breeding. In Chapter 5 we looked at the repetitive fraction of the genomes of potato and its wild relatives compared to tomato and its wild relatives, to elucidate if the differences in genome differentiation observed among the clades can be correlated to different dynamics in repetitive elements. Using a clustering approach with short read data, we found that the classes of repetitive elements across the clades are largely conserved, but their abundances are different and the repeat profiles allowed us to discriminate the two clades. We also found that the repeat content of S. etuberosum is more similar overall to the potato clade. All these tools provide information about possible crossability among these species in terms of their genome similarity and collinearity in chromosome structure. However, the possibility of small-scale differences required a look at synteny at the sequence level. The main challenge to be addressed in Chapter 6 was to achieve a contiguous de novo assembly that would allow for structural comparison, from the genome of a highly heterozygous plant. This was solved by sequencing a haploid clone obtained through in vitro culture of microspores and by taking a hybrid approach to the assembly, integrating short Illumina reads with much longer PacBio sequence data. A genetic map was developed using SNP data generated through GBS from a biparental segregating population. Then the hybrid assembly was anchored to the linkage map into 12 pseudomolecules built independently from any reference genome. The 12 pseudomolecules showed high overall homology with the DM potato and M6 S. chacoense pseudomolecules, with the order of scaffolds homologous among the three species. However, contiguity is lost in the pericentromere region and therefore, lack of collinearity is observed here. Small-scale rearrangements could not be conclusively distinguished from assembly or anchoring artifacts, with a few exceptions. In general terms, there is need for further improvement of contiguity and manual correction of the orientation of scaffolds in order to unequivocally detect rearrangements at the microsynteny scale. This can be achieved by using Bionano genome mapping and/or chromatin conformation capture technologies in the near future. To apply these technologies, extremely pure High Molecular Weight (HMW) DNA is necessary, so in Chapter 7 we adapted a pre-existing HMW DNA isolation protocol to optimize it for Solanum species. The results presented here address the doubts that have discouraged potato pre-breeders to use Solanum commersonii and S. chacoense in introgressive hybridization breeding. These species can be crossed with diploid potato producing viable offspring that can be used in backcrosses with cultivated potato. Wild chromosomes pair and recombine with their cultivated homoeologues, making introgression possible. These species are highly collinear at the large and small scale and their genomes have not diverged much both at the level of repetitive sequences and at the level of synteny. With the advent of diploid potato breeding, these species are particularly promising because of their traits, diversity and their adaptability and hardiness, but also because of their high similarity with potato from the genetic, cytogenetic and genomic points of view.</p

Exploring the use of wild relatives in potato breeding through integrated cytogenetic and genomic approaches

Cultivated potato and its wild relatives represent a more diverse and accessible germplasm resource than that of any other crop. Diversity coming from wild relatives can be used to introduce specific traits into the cultivated background. Breeders have long used introgressive hybridization, in which chromatin carrying a gene of interest from a wild relative is integrated into the genome of the crop by interspecific hybridization. During the subsequent backcrossing generations, genes of interest are incorporated into the crop chromosomes by homeologous recombination. The offspring are then selected for the desired trait while the original cultivated genetic background is recovered by backcrossing and selection as far as possible. The efficient use of wild relatives now requires extensive knowledge of their allelic variation and genomic structure, including the screening for desirable traits. To minimize the occurrence of linkage drag when introgressed chromatin still contains closely linked wild traits from the ancestral donor, knowledge on the genomic structure of crop and donor species is indispensable. In this work I have presented an overview of genetic, cytogenetic and genomic characteristics of Solanum commersonii, a South American wild relative of potato. The main aim was to create scientific tools to distinguish the genomes of S. commersonii and S. tuberosum (cultivated potato), along with S. chacoense, and trace their genomes in introgressive hybridization breeding programmes. The application of these tools to pinpoint past introgressions into cultivated potato and to plan introgressive hybridization schemes is reviewed in Chapter 2, with an emphasis on the need to make these technologies part of the routine toolkit for pre-breeding in order to exploit potato wild relatives at their fullest. A variety of questions related to the use of particular potato wild relatives in introgression are addressed using different cytogenetic and genomic technologies. In Chapter 3 pairing of the homoeologous S. commersonii genomes and S. tuberosum Group Phureja chromosomes in F1 interspecific triploid hybrids was analyzed, observing an almost autotriploid behaviour. Genome painting was used to follow the fate of specific chromosomes in these hybrids and backcross derivatives. Homoeologues could not be distinguished by fluorescence signals, so this approach did not provide tools to select against wild chromatin in the advanced materials nor pinpoint the chromosomal exchanges between homoeologues. However, the results suggest a low degree of genomic divergence between the species involved in terms of repetitive sequences and highlight their potential for homoeologous pairing. A comparative cytogenetic mapping analysis was performed in Chapter 4, to test collinearity among genomes of the different species and thus anticipate the existence of either suppression of recombination or linkage drag. Solanum commersonii and S. chacoense are collinear with cultivated S. tuberosum on the whole chromosome scale. Some differences observed in microscopic distances between the signals suggest either small-scale rearrangements or reduction/amplification of repetitive sequences. No major rearrangements have been found, making these amenable species for efficient introgressive hybridization breeding. In Chapter 5 we looked at the repetitive fraction of the genomes of potato and its wild relatives compared to tomato and its wild relatives, to elucidate if the differences in genome differentiation observed among the clades can be correlated to different dynamics in repetitive elements. Using a clustering approach with short read data, we found that the classes of repetitive elements across the clades are largely conserved, but their abundances are different and the repeat profiles allowed us to discriminate the two clades. We also found that the repeat content of S. etuberosum is more similar overall to the potato clade. All these tools provide information about possible crossability among these species in terms of their genome similarity and collinearity in chromosome structure. However, the possibility of small-scale differences required a look at synteny at the sequence level. The main challenge to be addressed in Chapter 6 was to achieve a contiguous de novo assembly that would allow for structural comparison, from the genome of a highly heterozygous plant. This was solved by sequencing a haploid clone obtained through in vitro culture of microspores and by taking a hybrid approach to the assembly, integrating short Illumina reads with much longer PacBio sequence data. A genetic map was developed using SNP data generated through GBS from a biparental segregating population. Then the hybrid assembly was anchored to the linkage map into 12 pseudomolecules built independently from any reference genome. The 12 pseudomolecules showed high overall homology with the DM potato and M6 S. chacoense pseudomolecules, with the order of scaffolds homologous among the three species. However, contiguity is lost in the pericentromere region and therefore, lack of collinearity is observed here. Small-scale rearrangements could not be conclusively distinguished from assembly or anchoring artifacts, with a few exceptions. In general terms, there is need for further improvement of contiguity and manual correction of the orientation of scaffolds in order to unequivocally detect rearrangements at the microsynteny scale. This can be achieved by using Bionano genome mapping and/or chromatin conformation capture technologies in the near future. To apply these technologies, extremely pure High Molecular Weight (HMW) DNA is necessary, so in Chapter 7 we adapted a pre-existing HMW DNA isolation protocol to optimize it for Solanum species. The results presented here address the doubts that have discouraged potato pre-breeders to use Solanum commersonii and S. chacoense in introgressive hybridization breeding. These species can be crossed with diploid potato producing viable offspring that can be used in backcrosses with cultivated potato. Wild chromosomes pair and recombine with their cultivated homoeologues, making introgression possible. These species are highly collinear at the large and small scale and their genomes have not diverged much both at the level of repetitive sequences and at the level of synteny. With the advent of diploid potato breeding, these species are particularly promising because of their traits, diversity and their adaptability and hardiness, but also because of their high similarity with potato from the genetic, cytogenetic and genomic points of view