Potato

Rapid and Targeted Introgression of Traits into Potato via Genome Elimination

Participants

• Comai Lab: Luca Comai (PI), Han Tan (co-PI), Kirk Amundson, Michelle Fossi, Peter Lynagh, Livingstone Nganga, Benny Ordonez. Ek Han Tan was co-PI during 2015-2016 and is now at the University of Maine.
• Britt Lab: Anne Britt (co-PI), Sundaram Kuppu, Ross Johnson
• CIP, Peru: Hannele Lindqvist-Kreuze (co-PI),Merideth Bonierbale was co-PI and is now retired, Monica Santayana. Awais Khan was co-PI during 2015-2016 and is now at Cornell University.
• Collaborators: Ek Han Tan (University of Maine); Ravi Maruthachalam (IISER-TVM, India)

Description

A common plant breeding procedure involves moving valuable traits, such as disease resistance, from wild relatives to crop varieties. To achieve this, breeders cross a wild variety to a crop variety. The result is called a hybrid, and it mixes genes from both parents. Most of the genes from the wild variety, however, are unsuited for agriculture and must be cast away. This is done by repeated crossing to the crop variety, coupled to selection for the trait of interest, a process called backcrossing that often requires as many as 10 years. This constitutes a dramatic bottleneck to developing new varieties. Therefore, shortening the development time is particularly critical in the face of unexpected crop challenges when human sustenance depends on the rapid development of new, improved varieties. This project addresses this need. It aims at developing a method for rapid deployment of useful traits into crops, using potato as the experimental system. The collaboration between University of California-Davis and the International Potato Center will also train graduate and undergraduate students in genomic-based breeding. The method developed will be applicable in many crop species and will be useful for both basic and applied research.

Haploid induction, a process that generates plants with a single genome instead of the two parental ones, can significantly shorten the time it takes to make new varieties. However, no methods have resolved the problem of overcoming linkage drag, the common, negative association of deleterious genes with desirable ones. The project team discovered a method to facilitate the transfer of selected genomic regions to a target variety, providing a novel solution to make breeding faster and more efficient. The work plan combines rapid testing in the model plant Arabidopsis with exploration of natural and engineered haploid inducer systems in potato to develop methods for rapid and simple introgression of new traits in crops. The specific objectives are to 1) Exploit and develop haploid induction systems in potato, based on the S. tuberosum phureja haploid inducer as well as on manipulation of centromeric histone H3. 2) Engineer introgression of defined chromosomal segments from a donor to a recipient genome using different types of recombination mechanisms. Useful potato and Arabidopsis lines produced by this work will be deposited in, respectively, GRIN NRSP-6 (http://www.ars-grin.gov/nr6/) and the ABRC (https:/www.arabidopsis.org/) stock centers. Progress and resources will also be linked here.

Final report

This project addressed a major challenge of plant breeding: the long time it takes to develop a new variety due to the need to obtain precise combinations of parental genes. For example, when introducing a trait from a wild relative to an adapted variety it is difficult to avoid the accidental addition of undesirable genes. We proposed that this time could be shortened, and precision improved by developing and integrating two methods: haploid induction and genome editing. Haploid induction, a method to reduce genome complexity in half, can significantly shorten the time it takes to make new varieties. Genome editing could accelerate breeding by forcing recombination in specific sites, thus defining precise chromosomal intervals for introgression. We chose the important crop species potato to pursue these objectives. We have made several valuable contributions:

1. Protoplasts, plant cells stripped of their wall, are attractive because they uptake genome editing reagents much more easily than regular cells. In a paper published in plant physiology, we documented an important drawback: potato plants regenerated from protoplasts undergo frequent genomic changes and rearrangements. The finding validated reports dating back to the 1980’s providing exact molecular description of the changes and methodology for their detection (Fossi et al. 2019)
2. A long standing question in potato breeding is the amount of genomic contamination resulting from the use of haploid inducers. Breeders wish to have none, but multiple reports claimed the common presence of pieces of the haploid inducer genome left in the haploids. In a paper published in Genetics, we describe the genomic characterization of ~160 haploids of potato. We show that these are free of the haploid inducer contamination indicating that this is not a widespread problem. (Amundson et al. 2020)
3. Following up on the work reported under point 2, we scaled up our analysis to include ~1600 haploids. In 0.7% of these we documented the presence of haploid inducer DNA in the form of large chromosomal fragments. This level of contamination is acceptable and easily detected by the method we provide. Further, it provides an alternative method to introduce subgenomic quantities of DNA, when desirable. These findings have been reported in conferences and a manuscript in advanced preparation.
4. We developed a computational method that uses genome sequencing data from a population of potatoes to identify chromosomal translocations. We demonstrated the utility of this analysis in two populations of potato derived from well known varieties. The method is already available in Github and a manuscript describing these results is in advanced preparation.
5. Studying the plants produced in the work described in item 1 above, we found unusual rearrangements. Characterization of these rearrangements revealed the occurrence of unexpected recombination events between chromosomes. We detected the molecular signs of an homologous recombination mechanism called Break Induced Replication. This finding has evolutionary and applied significance. A manuscript is in preparation.
6. We have been pursuing methods for engineering large scale changes in chromosomes through genome editing and made significant progress in the model system arabidopsis. A manuscript is in preparation.

In the broader context, the work described here involved the training of five graduate students and about twenty undergraduates who participated in the research. In the Summer of 2018, we provide a biotechnology workshop for high school students targeting under-represented groups. In this 2-day event 17 highschool students from low performing schools in the Sacramento area came to UC Davis to interact with science, scientists and the biotechnology industry. Students interacted with scientists from diverse backgrounds both regarding their field of studies and their personal identities and histories. Students were able to perform scientific experiments and see themselves as researchers. In other activities. In collaboration with the International Potato Center, we have imported Planta Quatro, an improved haploid inducer and are finalizing its deposit in the GRIN germplasm repository. In response to our work on genome stability and haploid genome structure we have received complimentary feedback from breeding companies to which our findings and methods are highly significant.

Citations

1. Amundson, Kirk R., Benny Ordoñez, Monica Santayana, Ek Han Tan, Isabelle M. Henry, Elisa Mihovilovich, Merideth Bonierbale, and Luca Comai. 2020. “Genomic Outcomes of Haploid Induction Crosses in Potato (Solanum Tuberosum L.).” Genetics 214 (2): 369–80. https://doi.org/10.1534/genetics.119.302843.
2. Fossi, Michelle, Kirk Amundson, Sundaram Kuppu, Anne Britt, and Luca Comai. 2019. “Regeneration of Solanum Tuberosum Plants from Protoplasts Induces Widespread Genome Instability.” Plant Physiology 180 (1): 78–86. https://doi.org/10.1104/pp.18.00906.

Data

We have sequenced Solanum tuberosum phureja haploid inducers IVP-35, IVP-48, IVP-101 and Planta Quatro (Pl-4), S. tuberosum Alca Tarma and Desiree, and three populations of dihaploids produced at CIP (LOP and MM populations, Lima, Peru) and at Davis (BB population). Here are the available sequences:

• Deposited sequence, BioProject PRJNA408137:
  • WGS of IVP-101 and PL-4
  • WGS of LOP-868
  • Low pass WGS of LOP dihaploid population
• Deposited sequence, BioProject PRJNA510212. These are in support the Fossi et al. paper (Plant Phys. in press):
  • Low pass WGS of CenH3 transgenic lines, Desiree background
  • Low pass WGS of protoplast-regenerated lines, Desiree background

Material

We have imported the phureja haploid inducer Planta 4 (Pl-4). Pl-4 has cleared the Plant Germplasm Quarantine Program. As soon as possible, Pl-4 will be deposited in the GRIN-run NRSP-6-United States Potato Gene bank where it will be available through the CIP standard SMTA agreement which protects national resources according to the Multilateral System for Access and Benefit Sharing of the International Treaty for Food and Agriculture. Additional material form our program will be deposited in the GRIN system. This includes:

• Sequence characterized protoplast regenerants from Desiree
• Sequence characterized Phureja genomic introgressions into dihaploids of assorted tetraploid varieties of tuberosum

---

• 

• 

Publications

In peer-review journals

• Amundson, Kirk R., Benny Ordoñez, Monica Santayana, Ek Han Tan, Isabelle M. Henry, Elisa Mihovilovich, Merideth Bonierbale, and Luca Comai. 2020. “Genomic Outcomes of Haploid Induction Crosses in Potato (Solanum Tuberosum L.).” Genetics 214 (2): 369–80. https://doi.org/10.1534/genetics.119.302843.
• Fossi, Michelle, Kirk Amundson, Sundaram Kuppu, Anne Britt, and Luca Comai. 2019. “Regeneration of Solanum Tuberosum Plants from Protoplasts Induces Widespread Genome Instability.” Plant Physiology 180 (1): 78–86. https://doi.org/10.1104/pp.18.00906.

Others

• 1205-TU. DECIPHERING THE MECHANISM INVOLVED IN FORMATION OF HAPLOID POTATO PROGENY IN THE SOLANUM TUBEROSUM GROUPS TUBEROSUM X PHUREJA CROSSES. Nganga L., Tan E.K.1, Ordonez B., Amundson K., Santayana M., Khan A., Bonierbale, M., Comai L. SolGenomics2016
• 307-TH. GENOME DOSAGE CHARACTERIZATION OF SOLANUM TUBEROSUM HAPLOIDS Amundson K.R., Tan E.H.1, Ordoñez B., Santayana M., Bonierbale M., Khan A., Comai L. SolGenomics2016
• 506-TH. DEVELOPMENT OF A PROTOPLAST SYSTEM FOR NON-TRANSGENIC, TARGETED GENOME EDITING IN SOLANUM TUBEROSUM. Fossi M.M., Amundson K.R., Jinata S.N.1, Comai L. SolGenomics2016
• Amundson K. 2017 UC Davis Plant Sciences Symposium, Davis CA, USA
• Amundson K. 2019 ASPB, San Jose, CA, USA
• Amundson K. 2019 Gene School Workshop, Kasetsart University, Bangkok, Thailand (invited talk)
• Amundson K. 2019 Potato Association of America Meeting, Winnipeg, MB, CA
• Amundson K. 2019 ASPB, San Jose, CA, USA
• Isabelle M Henry, Michelle Fossi, Weier Guo, Kirk Amundsen, Benny Ordonez, Sundaram Kuppu, Anne Britt and Luca Comai, “Widespread Chromosome Instability in Regenerated Potato Plants,” Plant & Animal Genomes XXVII Conference, January 2018, San Diego, CA.

Funding

NSF, IOS, Division Of Integrative Organismal Systems. Plant Genome Program Award Number:1444612