Jan and mini-Jan, a model system for potato functional genomics

Abstract

Potato (Solanum tuberosum) is the third most important food crop in the world. Although the potato genome has been fully sequenced, functional genomics research of potato lags relative to other major food crops due primarily to the lack of a model experimental potato line. Here, we present a diploid potato line, 'Jan', which possesses all essential characteristics for facile functional genomics studies. Jan has a high level of homozygosity after seven generations of self-pollination. Jan is vigorous and highly fertile with outstanding tuber traits, high regeneration rates, and excellent transformation efficiencies. We generated a chromosome-scale genome assembly for Jan, annotated genes, and identified syntelogs relative to the potato reference genome assembly DMv6.1 to facilitate functional genomics. To miniaturize plant architecture, we developed two "mini-Jan" lines with compact and dwarf plant stature using CRISPR/Cas9-mediated mutagenesis targeting the Dwarf and Erecta genes related to growth. Mini-Jan mutants are fully fertile and will permit higher-throughput studies in limited growth chamber and greenhouse space. Thus, Jan and mini-Jan provide an outstanding model system that can be leveraged for gene editing and functional genomics research in potato.

Figure 1.

Phenotypic characteristics of Jan. (a) Plant architecture. (b) Leaflets from a single compound leaf. (c) Flower. (d) Fruits. (e) Tubers from a single plant grown in a growth chamber.

Figure 2.

Allelic representation of DM and M6 in the Jan genome. Blocks of genomic sequence are in 100 kb resolution and color-coded by its parental origin: DM (blue), M6 (gold), or ambiguous (red) due to high sequence conservation between DM and M6.

Figure 3.

Diagrams of gRNAs and constructs for CRISPR/Cas9 experiments targeting the StD gene. (a) Illustration of the T-DNA region of the CRISPR/Cas9 construct. (b) Sequences and positions of the two gRNAs targeting the StD gene. Green color highlights “AG” represent the 3’ splicing site within intron 8. PAM sequences are highlighted in red. Bold letters represent sequence from exon 9.

Figure 4.

Genomic composition and phenotype of mini-Jan mutants from mutagenesis of the StD gene. (a) A single plant of Jan and four T0 mutants at 48 days after planting in a growth chamber. (b–f) Genomic DNA sequences, cDNA sequences, and predicted protein sequences of Jan (b), mutant i8–2 (c), mutant e9-2 (d), mutant i8-1 (e), and mutant e9-1 (f). The pre-mature stop codons are marked by magenta. The splicing AG sites are marked by green. The predicted protein sequences are in blue. The vertical blue line separates exon 9 from intron 8 sequence.

Figure 5.

The phenotypes of tissue culture plants of Jan and mini-Jan. (a) Tissue culture plants of Jan and mini-Jan^D after 25 days of culture. (b) Tissue culture plants of Jan and mini-Jan^E after 20 days of culture. Note: both mini-Jan^D and mini-Jan^E show a pronounced dwarf phenotype compared to the wild type Jan.

Figure 6.

Genomic composition and phenotype of mini-Jan mutants from mutagenesis of the StER gene. (a) Diagram of the gRNA for CRISPR/Cas9 experiments targeting the StER gene. (b) Sequences of Jan, er-1 and er-2 in the genomic regions associated with mutations of the StER gene. (c) A single plant of Jan, er-1 and er-2 at 28 days after planting in a growth chamber. (d) A single plant of Jan, er-1 and er-2 at 48 days after planting in a growth chamber. All vertical bars = 20 cm.