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. 2022 Mar 5;13(3):465.
doi: 10.3390/genes13030465.

KASP Markers Specific for the Fertility Restorer Locus Rf1 and Application for Genetic Purity Testing in Sunflowers (Helianthus annuus L.)

Aleksandra Radanović  1 , Yves Sprycha  2 , Milan Jocković  1 , Monja Sundt  2 , Dragana Miladinović  1 , Constantin Jansen  3 , Renate Horn  2
Affiliations

KASP Markers Specific for the Fertility Restorer Locus Rf1 and Application for Genetic Purity Testing in Sunflowers (Helianthus annuus L.)

Aleksandra Radanović et al. Genes (Basel). .

Abstract

Single nucleotide polymorphisms (SNPs) were significantly associated with fertility restoration of cytoplasmic male sterility (CMS) PET1 by the restorer gene Rf1. For these SNPs, four Kompetitive allele-specific PCR (KASP) markers were successfully designed. The KASP markers cover the fertility restorer locus Rf1, spanning about 3 Mb, and clearly differentiate restorer and maintainer lines. For genetic purity testing in sunflower hybrid production, the efficiency for detecting contaminations in samples was simulated using mixtures of hypocotyls or leaves. Contaminations of restorer lines with 1%, 3%, 5%, 10%, and 50% of maintainer lines were screened with all four KASP markers. Contaminations of 10% could be clearly detected in pools of 100 plants. Contaminations below this level require detection on a single plant level. For single plant detections, ethyl methanesulfonate-treated sunflower F1 hybrids, which had been phenotypically evaluated for male sterility (potential mutation in the Rf1 gene) were screened. Nine identified either partially male-sterile or male-sterile plants were analyzed with all four KASP markers and only one proved to be a hybrid with a mutation, seven were male-sterile contaminants in the F1 seeds used (1.6%) and one a recombinant plant. The four KASP markers should be valuable tools for marker-assisted selection (MAS) in sunflower breeding regarding the restorer locus Rf1.

Keywords: CMS PET1 cytoplasm; EMS mutagenesis; KASP marker; candidate gene; genetic purity; hybrid breeding; marker-assisted selection; restorer gene Rf1; single nucleotide polymorphism; sunflower.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Locations of SNPs significantly associated with the fertility restorer locus Rf1 in the new sunflower genome assembly HanXRQr2. SNPs successfully used for KASP marker design are shown in bold.
Figure 2
Figure 2
Grouping obtained by KASP markers (a) 621.5 and (b) 621.11. The latter splits the restorer lines into two groups. However, the outgroup of restorer lines (marked by an oval) can still be separated from their crosses to the CMS tester (also marked by an oval). NC negative control.
Figure 3
Figure 3
Grouping obtained by KASP markers (a) 841.38 and (b) 861.19. NC negative control.
Figure 4
Figure 4
Genetic purity testing for hybrid breeding using hypocotyls and KASP marker 621.5. (a) Contaminations of restorer UGA-SAM1-136 with maintainer UGA-SAM1-109 were simulated and (b) Contaminations of restorer UGA-SAM1-191 with maintainer UGA-SAM1-109.
Figure 5
Figure 5
Genetic purity for hybrid breeding using leaves and KASP marker 621.5. (a) Contaminations of restorer UGA-SAM1-136 with maintainer UGA-SAM1-136 were simulated and (b) Contaminations of restorer UGA-SAM1-191 with maintainer UGA-SAM1-082.
Figure 6
Figure 6
Investigation of the restorer locus Rf1 in EMS-treated plants (M1 generation) by application of two KASP markers. (a) Screening with KASP 621.5, (b) Screening with KASP 621.11.
Figure 7
Figure 7
Investigation of the restorer locus Rf1 in EMS-treated plants (M1 generation) by application of two KASP markers. (a) Screening with KASP 841.38 and (b) Screening with KASP 861.19.
Figure 8
Figure 8
Phenotypic evaluation of the partially male sterile EMS-treated F1 plants Mut 1 (a) und Mut 10 (b) in comparison to the male fertile, untreated F1 hybrid cmsHA342 × RHA325 (c).
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