Overcoming Self-Incompatibility in Diploid Potato Using CRISPR-Cas9

Abstract

Potato breeding can be redirected to a diploid inbred/F1 hybrid variety breeding strategy if self-compatibility can be introduced into diploid germplasm. However, the majority of diploid potato clones (Solanum spp.) possess gametophytic self-incompatibility that is primarily controlled by a single multiallelic locus called the S-locus which is composed of tightly linked genes, S-RNase (S-locus RNase) and multiple SLFs (S-locus F-box proteins), which are expressed in the style and pollen, respectively. Using S-RNase genes known to function in the Solanaceae gametophytic SI mechanism, we identified S-RNase alleles with flower-specific expression in two diploid self-incompatible potato lines using genome resequencing data. Consistent with the location of the S-locus in potato, we genetically mapped the S-RNase gene using a segregating population to a region of low recombination within the pericentromere of chromosome 1. To generate self-compatible diploid potato lines, a dual single-guide RNA (sgRNA) strategy was used to target conserved exonic regions of the S-RNase gene and generate targeted knockouts (KOs) using a Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (Cas9) approach. Self-compatibility was achieved in nine S-RNase KO T₀ lines which contained bi-allelic and homozygous deletions/insertions in both genotypes, transmitting self compatibility to T₁ progeny. This study demonstrates an efficient approach to achieve stable, consistent self-compatibility through S-RNase KO for use in diploid potato breeding approaches.

Keywords: CRISPR-Cas9; S-RNase; diploid potato; gene editing; self-incompatibility.

FIGURE 1

S-RNase gene structure and allelic variants in diploid potato and related species. (A) S-RNase predicted amino-acid sequence alignment of the DM (S_p5) and RH (S_t5 and S_t6) alleles. Underlined regions in red represent the typical five conserved regions (C1 to C5) and two hypervariable regions (HVa and HVb) of the S-RNase gene family. Exon/intron boundary is indicated with a filled triangle within the HVa region (B) S-RNase gene structure. The S-RNase open reading frame is composed of two exons separated by one small intron. Zoomed-in regions are shown within dotted lines indicating the intronic and exonic regions used for RH-specific primer design within the reported S-RNase alleles. (C) Phylogenetic tree constructed using the Neighbor Joining method based in the proportion of S-RNase amino acid differences. S-RNase from Nicotiana sylvestris was used as out-group. Numbers above each branch represent bootstrapping percentages from1000 replications. (D) Pairwise amino acid similarity of S-RNase in Solanum species and detected S-RNase alleles. S-RNase alleles are represented first by a species name abbreviation followed by S and the allele number for Tuberosum (t) or Phureja (p) group in Solanum tuberosum. For other species, a similar pattern is used, and allele numbers or letters (i.e., Sx for P. hybrida) are added if reported. S. tub: S. tuberosum, S. chc: S. chacoense, S. neo: S. neorickii, P. int: P. integrifolia, P. hyb: P. hybrida, N. syl: N. sylvestris.

FIGURE 2

S-RNase gene mapping in diploid potato. (A) RH S-RNase (S. tub_S_t6) allelic screening on the DRH F1 population using the S-RNase and housekeeping Sucrose synthase 3 gene (Sus-3) primers. DM and RH parental lines are shown in the first two lanes followed by the negative control (C), and the RH-S-RNase segregation pattern of 11 F1-derived lines. (B) The S-RNase gene mapped to 16.3 cM on the short arm near the centromeric region of chromosome 1 (red). (C) Marey map of physical (Mb) versus genetic (cM) distances from chromosome I showing the S-RNase gene within a low-recombination region (red box). Asterisks within the red box represent SNPs spanning the region between to 6.1 and 18.9 Mb in the potato physical map (solcap_snp_c2_27882 and solcap_snp_c1_16425 markers, respectively).

FIGURE 3

CRISPR/Cas9 mediated mutagenesis in two self incompatible diploid potato lines. (A) Single-guide RNAs (sgRNAs) designed to target the S-RNase exon1 (sgRNA 1) and exon 2 (sgRNA 2). Zoomed-in regions within the dotted lines show sgRNA and PAM sequences. (B) Seven and (C) three bi-allelic S-RNase knockouts (KOs) DRH-195 and DRH-310 T₀ lines, respectively, detected by insertion/deletion polymorphisms compared to wild-type (WT) and negative controls (C). All DRH-195 KOs exhibit polymorphic deletions and DRH-310 monomorphic deletions (lines 195–105, 195–137, 195–142, and 195–160 presented a faint mutated or WT-like bands that is not observed in the figure). (D) Different mutation types detected by amplicon sequencing in selected KOs for DRH-195, and (E) DRH-310 T₀ lines respectively. Wild-type S-RNase exhibiting selected sgRNAs (sgRNA1 and sgRNA2) and PAM sequences are shown at the top of each alignment. Different types of mutations including deletions (–), insertions (+), and inversions (i) detected in DM (DM) and RH (RH) S-RNase alleles of each T₀ KO DRH-derived lines shown as ‘195-’ or ‘310-.’

FIGURE 4

S-RNase expression and KO phenotype in self incompatible diploid potato lines. (A) Fruits obtained after 5 weeks of self-pollination in an S-RNase DRH-195-derived T₀ mutant line. (B) Dropped flowers after self-pollination in the WT DRH-195. (C) Semi-quantitative reverse transcription PCR (RT-PCR) in DRH-195 and DRH-310 self-pollinated WT and KO T₀ lines (DRH-195.158 and DRH-310-21, respectively). RNA was isolated from pistils 24 h after self-pollination revealing S-RNase expression in WT but not in KO lines as compared with the housekeeping gene control (EF1α). (D) T₁ plants derived from the DRH-195.158 T₀ line were screened with the S-RNase primers. WT-like bands with 1 bp deletion on each target site (causing frameshift leading to a premature stop codon, Supplementary Figure S1) are observed in T₀ and T₁ lines. Cas9 gene did not transmit to T₁ line 5 (lane 5). A previously undetected band observed in lane 6 is potentially the result of transgenerational CRISPR/Cas9 activity. T₀: DRH-195.158, C: Negative control. The red box is showing a T₁ line segregating out Cas9 while maintaining the S-RNase KO.

FIGURE 5

Fruit formation in WT and T₁ S-RNase KO diploid potatoes lines. (A). Fruit setting arrest 2 weeks after self-pollination in the WT DRH-310. (B) Fruits obtained after 4 weeks of self-pollination in an S-RNase DRH-195-derived T₁ mutant line.