Marker-Assisted Gene Pyramiding and the Reliability of Using SNP Markers Located in the Recombination Suppressed Regions of Sunflower (Helianthus annuus L.)

Abstract

Rust caused by the fungus Puccinia helianthi and downy mildew (DM) caused by the obligate pathogen Plasmopara halstedii are two of the most globally important sunflower diseases. Resistance to rust and DM is controlled by race-specific single dominant genes. The present study aimed at pyramiding rust resistance genes combined with a DM resistance gene, using molecular markers. Four rust resistant lines, HA-R3 (carrying the R₄ gene), HA-R2 (R₅), HA-R8 (R₁₅), and RHA 397 (R_13b), were each crossed with a common line, RHA 464, carrying a rust gene R₁₂ and a DM gene Pl_Arg. An additional cross was made between HA-R8 and RHA 397. Co-dominant simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers linked to the target genes were used to discriminate between homozygotes and heterozygotes in F₂ populations. Five pyramids with different combinations of rust resistance genes were selected in the homozygous condition through marker-assisted selection, and three of them were combined with a DM resistance gene Pl_Arg: R₄/R₁₂/Pl_Arg, R₅/R₁₂/Pl_Arg, R_13b/R₁₂/Pl_Arg, R₁₅/R₁₂, and R_13b/R₁₅. The pyramiding lines with the stacking of two rust and one DM genes were resistant to all known races of North American sunflower rust and all known races of the pathogen causing DM, potentially providing multiple and durable resistance to both rust and DM. A cluster of 12 SNP markers spanning a region of 34.5 Mb on chromosome 1, which co-segregate with Pl_Arg, were tested in four populations. Use of those markers, located in a recombination suppressed region in marker selection, is discussed.

Keywords: downy mildew; gene pyramiding; resistance; rust; sunflower.

Figure 1

PCR gel image of single nucleotide polymorphism (SNP) markers for testing homozygous triple-resistant F₂ plants from HA-R3/RHA 464. (a) SFW05240 linked to R₄, (b) NSA_001570 linked to R₁₂, (c,d) NSA_002851 and NSA_006530 linked to Pl_Arg. 1: HA-R3, 2: RHA 464, 3–6: Homozygous triple-resistant plants for R₄/R₁₂/Pl_Arg.

Figure 2

PCR gel image of SSR markers for testing the homozygous double-resistant F₂ plants from RHA397/HA-R8. (a,b) ORS316 and HT382 linked to R_13b. (c,d) SUN398 and SUN406 linked to R₁₅. 1: RHA 397, 2: HA-R8, 3–6: Homozygous double-resistant plants for R_13b/R₁₅.

Figure 3

PCR gel image of SNP markers linked to Pl_Arg indicates recombination between NSA_008037 and NSA_007595 in selected F₂ plants from RHA 397/RHA 464. (a) NSA_005423, (b) NSA_008037, (c) NSA_007595, and (d) NSA_001835. 1: RHA 397, 2: RHA 464, 3: 14-21-319, 4: 14-21-413, 5: 14-21-129. 14-21-129 was homozygous at the NSA_005423 and NSA_008037 loci but heterozygous at the NSA_007595 and NSA_001835 loci.

Figure 4

PCR gel image of SNP markers linked to Pl_Arg indicates recombination between NSA_005063 and NSA_002851 in the selected F₂ plants from HA-R8/RHA 464. (a) NSA_002208, (b) NSA_005063, (c) NSA_002851, and (d) NSA_001835. 1: HA-R8, 2: RHA 464, 3: 16-46-202, and 4: 16-46-329. 16-46-202 and 16-46-329 did not have Pl_Arg SNP alleles at the NSA_002208 or NSA_005063 loci but had Pl_Arg SNP alleles at the NSA_002851 and NSA_001835 loci.

Figure 5

A crossover occurred between marker E and F and changed the linkage phase of Pl_Arg with the markers. A to N represent 14 SNP markers listed in Table 2. Lower case letters represent the HA-R8 SNP allele and the upper case letters represent the RHA 464 SNP allele. Pl_Arg co-segregated with the first five markers.