The PET2-cytoplasm represents a well characterized new source of cytoplasmic male sterility (CMS) in sunflower. It is distinct from the PET1-cytoplasm, used worldwide for commercial hybrid breeding, although it was, as PET1, derived from an interspecific cross between Helianthus. petiolaris and H. annuus. Fertility restoration is essential for the use of CMS PET2 in sunflower hybrid breeding. Markers closely linked to the fertility restorer gene are needed to build up a pool of restorer lines. Fertility-restored F1-hybrids RHA 265(PET2) × IH-51 showed pollen viability of 98.2% ± 1.2, indicating a sporophytic mode of fertility restoration. Segregation analyses in the F2-population of the cross RHA 265(PET2) × IH-51 revealed that this cross segregated for one major restorer gene Rf-PET2. Bulked-segregant analyses investigating 256 amplified fragment length polymorphism (AFLP) primer combinations revealed a high degree of polymorphism in this cross. Using a subset of 24 AFLP markers, three sequence-tagged site (STS) markers and three microsatellite markers, Rf-PET2 could be mapped to the distal region of linkage group 13 between ORS1030 and ORS630. Three AFLP markers linked to Rf-PET2 were cloned and sequenced. Homology search against the sunflower genome sequence of HanXRQ v1r1 confirmed the physical location of Rf-PET2 close to the restorer gene Rf1 for CMS PET1. STS markers were mapped that can now be used for marker-assisted selection.

Hybrid breeding in sunflowers based on CMS PET1 requires development of restorer lines carrying, in most cases, the restorer gene Rf1. Markers for marker-assisted selection have been developed, but there is still need for closer, more versatile, and co-dominant markers linked to Rf1. Homology searches against the reference sunflower genome using sequences of cloned markers, as well as Bacterial Artificial Chromosome (BAC)-end sequences of clones hybridizing to them, allowed the identification of two genomic regions of 30 and 3.9 Mb, respectively, as possible physical locations of the restorer gene Rf1 on linkage group 13. Nine potential candidate genes, encoding six pentatricopeptide repeat proteins, one tetratricopeptide-like helical domain, a probable aldehyde dehydrogenase 22A1, and a probable poly(A) polymerase 3 (PAPS3), were identified in these two genomic regions. Amplicon targeted next generation sequencing of these nine candidate genes for Rf1 was performed in an association panel consisting of 27 maintainer and 32 restorer lines and revealed the presence of 210 Single Nucleotide Polymorphisms (SNPs) and 67 Insertions/Deletions (INDELs). Association studies showed significant associations of 10 SNPs with fertility restoration (p-value < 10−4), narrowing Rf1 down to three candidate genes. Three new markers, one co-dominant marker 67N04_P and two dominant markers, PPR621.5R for restorer, and PPR621.5M for maintainer lines were developed and verified in the association panel of 59 sunflower lines. The versatility of the three newly developed markers, as well as of three existing markers for the restorer gene Rf1 (HRG01 and HRG02, Cleaved Amplified Polymorphic Sequence (CAPS)-marker H13), was analyzed in a large association panel consisting of 557 accessions.

In sunflower, two approaches have been followed to isolate genes: map-based-cloning and candidate gene approaches. Map-based cloning of nuclear male sterility and fertility restoration genes, as well as, genes conferring resistance to downy mildew so far has been unsuccessful. This is primarily due to the large genome size, the high content of repetitive elements, the lack of publicly available large mapping populations and large insert genomic libraries. On the other hand, candidate gene approaches in the areas of developmental biology (e.g., embryogenesis, non-dormancy, pollen and pistil-specific genes), abiotic stress (e.g., heat-shock and drought-related genes), disease resistance and quality traits (e.g., fatty acid biosynthesis or tocopherol biosynthesis) have proven to be very successful. However, in sunflower even if a gene was identified, the lack of a good transformation system hampers verification of the candidate gene. Fortunately, heterologous systems and mutant analyses have proven to be helpful for gene verification and characterization. In spite of these problems, enormous progress has been made on the cloning of genes in sunflower. The current expansion of genomic resources in sunflower promises to accelerate the process of isolation and characterization of agronomically important genes.

Cytoplasmic male sterility (CMS) plays an important role in the production of hybrid seeds. In sunflower, commercial hybrid breeding is based on a single CMS-inducing cytoplasm, PET1. In this particular male sterility, which was derived from an interspecific cross between Helianthus petiolaris Nutt and Helianthus annuus L., CMS is caused by the expression of an aberrant mitochondrial gene that prevents the development of viable pollen. The restoration of pollen fertility in the presence of the PET1cytoplasm has been reported to be controlled by two dominant nuclear genes (Rf1and Rf2). In a previous work, a linkage map has been constructed around the Rf1 gene that consisted of 35 AFLP (amplified fragment length polymorphism) markers, 7 RAPD (random amplified polymorphic DNA) markers, and 1 SSR (simple sequence repeat) marker. These markers represent a good opportunity and an excellent basis for a map-based cloning approach. Markers tightly linked to the restorer locus Rf1 have been used as overgo probes to be hybridized against sunflower BAC libraries. Positive BAC clones were identified and a putative closed contig around Rf1 was constructed by fingerprinting. Here we report on: (i) the verification of the identified BAC clones near the Rf1 gene and (ii) the inability of the DNA fingerprinting methodology alone for identifying overlapping DNA fragments that can be assembled into contigs, showed by using different restriction enzymes, and (iii) the description of a fast and efficient method to clone BAC ends into a high copy number vector based on double antibiotics selection.

Knowledge about the forces generating and conserving linkage disequilibrium (LD) is important for drawing conclusions about the prospects and limitations of association mapping. The objectives of our research were to examine the importance of (1) selection, (2) mutation, and (3) genetic drift for generating LD in a typical maize breeding program. We conducted computer simulations based on genotypic data of Central European maize open-pollinated varieties which have played an important role as founders of the European flint heterotic group. The breeding scheme and the dimensioning underlying our simulations reflect essentially the maize breeding program of the University of Hohenheim. Results suggested that in a plant breeding program of the examined dimension and breeding scheme, genetic drift and selection are major forces generating LD. The currently used population-based association mapping tests do not explicitly correct for LD caused by these two forces. Therefore, increased type I error rates are expected if these tests are applied to plant breeding populations. As a consequence, we recommend to use family-based association tests for association mapping approaches in plant breeding populations.

It has been claimed that the system that delivers the products of plant breeding reduces the diversity of cultivated varieties leading to an increased genetic vulnerability. The main goal of our study was to monitor the temporal trends in genetic diversity over the past five decades among maize cultivars with the largest acreage in Central Europe. Our objectives were to (1) investigate how much of the genetic diversity present in important adapted open-pollinated varieties (OPVs) has been captured in the elite flint germ plasm pool, (2) examine changes in the genetic diversity among the most important commercial hybrids as well as in their dent and flint parents, (3) analyze temporal changes in allele frequencies between the dent and flint parental inbreds, and (4) investigate linkage disequilibrium (LD) trends between pairs of loci within the set of parental dent and flint lines. We examined 30 individuals of five prominent OPVs from Central Europe, 85 maize hybrids of economic importance, and their dent and flint parental components with 55 SSRs. LD was significant at probability level P=0.01 for 20.2% of the SSR marker pairs in the 82 dent lines and for 17.2% in the 66 flint lines. The dent and flint heterotic groups were clearly separated already at the beginning of hybrid breeding in Central Europe. Furthermore, the genetic variation within and among varieties decreased significantly during the five decades. The five OPVs contain numerous unique alleles that were absent in the elite flint pool. Consequently, OPVs could present useful sources for broadening the genetic base of elite maize breeding germ plasm.

Landraces of maize represent a valuable genetic resource for breeding and genetic studies. Using simple sequence repeat (SSR) markers, we analysed five flint maize populations from Central Europe that had played an important role in the pre-hybrid era in Germany. Our objectives were to (1) investigate the molecular genetic diversity within and among the populations based on the SSR analysis of individuals, (2) compare these results of the SSR analysis based on individuals with those based on bulks, (3) examine genotype frequencies for deviations from Hardy–Weinberg equilibrium (HWE) at individual loci, and (4) test for linkage disequilibrium (LD) between pairs of loci within populations. Thirty individuals and their bulked DNA per population were fingerprinted with 55 SSR markers. Across all populations, 46.7% of the SSR markers deviated significantly from HWE, with an excess of homozygosity in 97% of the cases. This excess of homozygosity can largely be explained by experimental errors during the amplification of SSRs apart from genuine genetic causes. Allele frequencies of the SSR analyses of individuals and bulks were significantly correlated (r=0.85, P< 0.01), suggesting that SSR analysis of bulks is very cost-effective for large-scale molecular characterisation of germplasm collections. No evidence for genome-wide LD among pairs of loci was observed, indicating that the populations are well suited for high resolution association mapping studies.