Genetic dissection of self-compatibility in perennial ryegrass (Lolium perenne L.) and its exploitation for breeding

In plants, reproductive strategies determine the degree of genetic variation of a species and influence the procedures to develop new cultivars. Self-compatibility (SC), defined as the ability for a plant to self-fertilize, is a reproductive trait evolved through the loss of ancestral self-incompatibility (SI) during the evolution of many plant species. Studying the molecular mechanisms underlying the transition from SI to SC and its consequences for plant adaptation and speciation has engaged the scientific community for decades. For some crops, SC is a tool to efficiently produce homozygous plant material by self-pollination, which breeders have used to generate parental lines for hybrid breeding schemes. This makes SC not only a trait of fundamental importance for plant biology but also of great agricultural interest to produce high yielding grass hybrids. This thesis has investigated genetic causes of SC in perennial ryegrass (Lolium perenne L.), one of the world’s most important forage crops. Perennial ryegrass is characterized by a SI system that prevents inbreeding and makes it a strictly allogamous species. Naturally occurring sources of SC in perennial ryegrass have been documented, however their regulation is just starting to be understood at the genetic level. Moreover, a general utilization of SC for breeding is lacking. The goal of this thesis was to advance the current understanding of the genetic architecture of SC in perennial ryegrass by identifying genomic regions and molecular determinants responsible for SC. The research described in chapters 2-5 and here summarized, provide both biological as well as practical insights for the application of SC in forage grass breeding. After a general introduction, chapter 2 reviews the state of the art about the SI system in grasses and describes in detail known causes of SC. Strategies for the discovery of novel SC sources are discussed, together with a review of the possibilities that SC presents for forage grass breeding. Specifically, the importance of using SC to purge deleterious alleles and to fix genetic variation in heterotic groups in order to implement hybrid breeding strategies is addressed. Moreover, the perspectives of using SC as a tool to develop immortalized mapping populations for the genetic dissection of agronomic traits are discussed. Chapter 3 describes a fine mapping experiment aimed at narrowing down a genomic region harboring a SC causal gene in a perennial ryegrass population. Previous classical genetic studies on this population mapped a SC locus on linkage group (LG) 5. However, the region identified was still considerably large, making it difficult to suggest candidate genes. With a combination of quantitative trait locus (QTL) mapping and analysis of segregation distortion based on a genome-wide linkage map, we confirmed the presence of the QTL on LG 5. In addition, we further fine mapped the locus to a 0.26 cM region, considerably improving the mapping resolution achieved in previous studies. Lastly, projecting the interval onto a genome assembly, we identified potential candidate genes. Chapter 4 reports the development and genetic characterization of a perennial ryegrass population segregating for SC. A segregating population was developed starting from germplasm provided by the seed company Deutsche Saatvederelung AG (DSV) and consisted of S1 and S2 lines originating from two founder self-compatible genotypes. To investigate structure and genetic diversity within the population, SNP markers were developed from a genotyping-by-sequencing (GBS) library. Combining 69,002 SNPs with SC phenotypic variation in a genome-wide association study, we identified SNPs with a significant association. Significant SNPs mapped on chromosome 2 and 7 and indicated the presence of three candidate QTL, revealing novel sources of SC and providing a fundamental resource for SC diagnostic markers in marker-assisted selection programs. Lastly, a pilot study described in chapter 5 investigates the occurrence of a temperature-induced SC in perennial ryegrass, a phenomenon called pseudo-self-compatibility (PSC). A set of 16 self-incompatible genotypes selected from the DSV perennial ryegrass germplasm was incubated at 30 °C during flowering and pollen tube growth was assessed using in vitro self-pollinations. The PSC response was found to be highly genotype-dependent, revealing the presence of variation for this trait. This study paves the way to further genetic mapping studies aimed at determining the genetic control of PSC. In conclusion, chapter 6 discusses the findings showed in the previous chapters in a broader context. Also, future perspectives are addressed, describing possible further experimental designs to complement the results presented in this thesis