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. 2021 Jul 6;12(1):4141.
doi: 10.1038/s41467-021-24267-6.

Neofunctionalisation of the Sli gene leads to self-compatibility and facilitates precision breeding in potato

Ernst-Jan Eggers  1   2 , Ate van der Burgt  1 , Sjaak A W van Heusden  2 , Michiel E de Vries  1 , Richard G F Visser  2 , Christian W B Bachem  3 , Pim Lindhout  1
Affiliations

Neofunctionalisation of the Sli gene leads to self-compatibility and facilitates precision breeding in potato

Ernst-Jan Eggers et al. Nat Commun. .

Abstract

Genetic gain in potato is hampered by the heterozygous tetraploid genome of cultivated potato. Converting potato into a diploid inbred-line based F1-hybrid crop provides a promising route towards increased genetic gain. The introduction of a dominant S-locus inhibitor (Sli) gene into diploid potato germplasm allows efficient generation of self-fertilized seeds and thus the development of potato inbred lines. Little is known about the structure and function of the Sli locus. Here we describe the mapping of Sli to a 12.6 kb interval on chromosome 12 using a recombinant screen approach. One of two candidate genes present in this interval shows a unique sequence that is exclusively present in self-compatible lines. We describe an expression vector that converts self-incompatible genotypes into self-compatible and a CRISPR-Cas9 vector that converts SC genotypes into SI. The Sli gene encodes an F-box protein that is specifically expressed in pollen from self-compatible plants. A 533 bp insertion in the promotor of that gene leads to a gain of function mutation, which overcomes self-pollen rejection.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. QTL mapping and recombinant analysis in population 17SC11.
a Fluorescence microscopy of self-pollinated styles of SI (left) and SC (right) genotypes shows that self-pollen can grow through the styles of SC genotypes, while self-pollen tube growth is arrested in styles from SI plants. Although a few self-pollen tubes reach the end of the style in the SI genotype, these do not induce a berry set. These images are typical for SI and SC genotypes as evidenced in 50 or more similar genotypes. b The LOD profile resulting from QTL analysis in population 17SC11 shows a highly significant QTL on the distal end of chromosome 12. c Recombinant analysis suggests Sli must be located in an 12.6 kb interval, which contains two genes. The SC haplotype is shown in green, the SI haplotype is shown in red. Numbers above the haplotypes are the positions of the genotyped SNP markers on chromosome 12 of DM4.04. d Candidate gene PGSC0003DMG400016861 shows an SC-specific 533 nt insertion in the promoter, suggesting that the SC allele of PGSC0003DMG400016861 may show altered tissue-specific expression. The Sli-specific amino acid changes and their positions are shown below the gene model.
Fig. 2
Fig. 2. RNA sequencing of germinated pollen from SC and SI genotypes.
a Expression of several pollen-expressed genes on chromosome 12. Candidate gene PGSC0003DMG400016861 (black arrow) is expressed in pollen from SC genotypes, but not in pollen from SI genotypes, whereas other pollen-expressed genes show similar expression levels in SC and SI genotypes. The color scale is based on FPKM values relative to the highest expressing genotype, ranging from green (highest expression) to red (lowest expression). * For these two genes the RNA-seq reads were mapped to the corresponding tomato gene model. ** For this gene the RNA-seq reads were mapped to the corresponding pepper gene model. *** Gene model CA05g06620 was inferred from Pepper and lacks a predicted counterpart in the PGSC gene catalog. b Phylogenetic tree of the 533 bp insertion and homologous sequences in potato. The 533 bp insertion in the SC allele of Sli is indicated with the arrow.
Fig. 3
Fig. 3. Phenotypic analysis of Sli transgenic and CRISPR–Cas9 knock-out lines.
a Design of Sli pAGM:CRISPRΔSli construct. Four sgRNAs target the first exon of PGSC0003DMG400016861. Protospacer Adjacent Motifs are shown in red. b Fluorescence microscopy of self-pollinated styles from Sli transgenics, knock-outs, and untransformed controls. Wild-type B665 shows complete self-pollen tube growth through, but in knock-out line B665ΔSli-2 self-pollen tubes growth is arrested before the pollen tubes can reach the ovaries. Wild-type B666 and B667 show self-pollen tubes growth arrest, but transformation with pBINPLUS-Sli enables self-pollen tubes growth to the ovary. Each style in the image is composed of separate microscopy images of which the contrast and brightness levels were adjusted. The styles shown are representative of three independent experiments. c Pollen tube growth phenotypes of Sli transgenics, knock-outs and untransformed controls on a 0–4 scale: 0: no pollen tubes reach the ovary, 1: fewer than 25% of pollen tubes reach the ovary, 2: between 25 and 50% of pollen tubes reach the ovary, 3: between 50 and 75% of pollen tubes reach the ovary, and 4, more than 75% of pollen tubes reach the ovary. The boxes indicate the median and lower (25%) and upper (75%) quantiles, the whiskers indicate the smallest and largest observations. These data were generated in one greenhouse experiment with 3–5 clones of each genotype and are consistent in two independent experiments with the same genotypes. d PAGE analysis of PCR products from lines obtained after transformation of genotypes B663 and B665 with pAGM:CRISPRΔSli. Six regenerants show INDELs in the targeted area and are labeled B663ΔSli-1 and B665ΔSli-1 to B665ΔSli-5. The bands without labels are from regenerants in which pAGM:CRISPRΔSli did not induce INDELs. Additional lanes in the gel image were excised at the positions indicated by the vertical dashed lines. The horizontal dotted line represents a fragment size of 136 bp.
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