Figure 1.
Growth phenotype of amiRNA lines at 14–16°C and RPP1-like gene expression.
(A) Growth phenotype of 5-week old Ler/Kas-2 NIL plants transformed with amiRNAs effective (KB209 and KB212) or not effective (KB228) suppressing incompatibility. (B) Expression of individual RPP1-like Ler genes determined by qRT-PCR in suppressed (compatible) amiRNA KB209 lines (KB209-1, KB209-2, KB209-3), KB212 (KB212-1, KB212-2, KB212-3) and non-suppressed (incompatible) amiRNA lines KB212 (KB212-4, KB212-5), KB228 (KB228-1, KB228-2, KB228-3). Values are relative to Ler and the mean ± SD of three biological replicates each using three technical replicates. cNIL (complemented NIL), NIL (incompatible Ler/Kas-2 near-isogenic line) [23], [31]. Significant differences in gene expression between Ler and other genotypes using Student's t-test are indicated by asterisks: *P<0.05, **P<0.01, ***P<0.005.
Figure 2.
Transgene and PR-1 expression in ColRPP1 lines.
Expression of RPP1-like Ler R1, R2, R3, R4, R5, R7 and R8 transgenes (left axis) and PR-1 (right axis) determined in individual homozygous ColRPP1 lines, Ler and NIL plants grown at 14–16°C by qRT-PCR. Values are relative to Ler and the mean ± SD of three biological replicates each using three technical replicates. NIL (incompatible Ler/Kas-2 near-isogenic line [31]). Significant differences in gene expression between Ler and different ColRPP1 lines using Student's t-test are indicated by asterisks: *P<0.05, **P<0.01, ***P<0.005.
Figure 3.
Effects of R3 expression with different allelic combinations at SRF3 and QTL5.
Expression of RPP1-like Ler genes determined by qRT-PCR in different Ler/Kas-2 recombinant inbred lines (RIL) carrying homozygous allelic combinations at RPP1-like/SRF3/QTL5 loci: Ler/Ler/Kas-2 (AAB), Ler/Kas-2/Ler (ABA), Ler/Kas-2/Kas-2 (ABB). AAB and ABA represent compatible allele combinations, whereas ABB triggers incompatibility. The ABB eds1-2 line carrying an EDS1 loss-of-function mutation is described in [31]. Values are relative to Ler and the mean ± SD of three biological replicates each using three technical replicates. Significant differences in the gene expression between Ler and different genotypes using Student's t-test are indicated by asterisks: *P<0.05, **P<0.01, ***P<0.005.
Figure 4.
Growth phenotype at 14–16°C of ColRPP1/Kas-2 plants and RPP1-like Ler hemizygous lines.
(A) Growth phenotype of representative F3 plants derived from the cross of ColRPP1 R1, R2, R3, R4, R5, R7 and R8 transgenic lines with Kas-2, which carry their respective transgenes in combination with incompatible alleles at interacting loci. (B) Growth phenotype of the F1 progeny derived from the cross of genotypes in (A) with the incompatible Ler/Kas-2 NIL [31]. Genotypes are shown in the lower panel (A′ and B′).
Figure 5.
Estimated population structure (A) and principal component analysis, PCA (B), derived from the analysis of 149 genome-wide SNP in genetically distinct individuals in the Gorzów population (n = 44) and accessions from neighboring countries (Czech Republic and Austria, n = 33; Germany, n = 88) or more distant regions (Netherlands, n = 21; Russia and former Soviet Union, n = 27; Central Asia, n = 61). Each accession is colored in segments depicting individual's estimated membership fractions in six main clusters (optimal K = 6, for lower K values see S10 Figure). Gw individuals carrying (Gw+) or not (Gw−) the RPP1-like Ler haplotype are represented in different colors in the PCA plot.
Figure 6.
Genetic diversity of the RPP1-like locus in the Gorzów population.
(A) Neighbor-joining tree showing the genome-wide genetic diversity among Gw accessions, estimated from a set of 149 genome-wide SNPs. Boxes represent genes within the RPP1-like haplotype (R1 to R8, with R8 the closest to the tree) conserved with Ler (red) differing from Ler (gray), or absent (white), based on amplification and sequencing of RPP1-like genes with specific primers designed for the RPP1-like Ler cluster. Accessions carrying the RPP1-like Ler haplotype are highlighted in red. Accessions used for crosses with Kas-2 or Kond and their compatibility/incompatibility outcome are indicated. (B) Neighbor-joining tree of RPP1-like genes in Gw− and Kas-2 showing extensive allelic variation. Phylogeny is based in sequencing RPP1-like genes in Gw− individuals (Gw-31, Gw-44, Gw-55 and Gw-99) and Kas-2.
Figure 7.
Incompatible phenotypes derived from the cross of Gw+/Kas-2 and Gw+/Kond accessions.
Gw+ individuals were crossed to Kas-2 and Kond and their F2 progenies screened for the occurrence of incompatible phenotypes at 14–16°C (see S5 Table). Dwarf plants on the left of each cross carry homozygous Gw (Ler) alleles at RPP1-like locus and homozygous SRF3 Kas-2 or Kond alleles, which are not present in normal sized sister F2 plants from the same cross (right). Incompatibility is absent in the cross of Gw− accessions with Kas-2 or Kond (Gw-160 as representative; see S5 Table).