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ReproTag - Targeting reproductive traits for more efficient forage grass breeding

Final Report Summary - REPROTAG (ReproTag - Targeting reproductive traits for more efficient forage grass breeding)


Grasslands are key components of a diverse but productive European agroecosystem. Permanent and temporary pastures and meadows cover around 36% of the cultivated area in Europe, and up to 70% in the UK and Switzerland. Grasslands account for a large proportion of the annual production of meat and milk, thereby contributing to sustainable feed and food production on both local and global levels. Moreover, grasslands provide essential ecosystem services such as regulation of carbon sequestration and nutrient cycling, preservation of biodiversity and provision of attractive landscapes for recreation and tourism.
High yielding forage grass species are major components of permanent and temporary grasslands. Perennial ryegrass (Lolium perenne L.), as many of the important forage grass species, is characterized by an effective self-incompatibility (SI) system that promotes cross-pollination. Such allogamous species are genetically improved as population and synthetic varieties, only partially exploiting the available heterosis. However, changing climatic and socioeconomic environments call for varieties with improved yield potential, quality and resistance or tolerance to biotic and abiotic stresses. To maintain and improve the productivity and sustainability of grasslands, novel tools and innovative breeding approaches based on SI and self-fertility (SF) are needed to fix heterotic combinations. Hence, hybrid forage grass varieties have the potential to outperform current population and synthetic varieties. However, the ability of inbreeding through repeated self-pollination, a prerequisite for hybrid breeding, is hampered by SI. Moreover, a practical and economic system to produce hybrid seed in commercial quantities is missing in forage crop species, calling for novel strategies based on biological mechanisms to efficiently control pollination ensuring hybrid seed production.
The overall goal of the FP7-MC-CIG project "ReproTag - Targeting reproductive traits for more efficient forage grass breeding" was the identification, characterization and utilization of biological mechanisms in forage grasses to control pollination, which can – among a broad range of other applications – be exploited in hybrid breeding schemes. In particular, the proposed research focused on the two-locus SI system, molecular processes leading to SF, doubled haploid induction and male sterility mechanisms in perennial ryegrass, which will serve as a diploid model for other major grass species.

Self-incompatibility (SI)
The grass family (Poaceae), the fourth largest family of flowering plants, encompasses the most economically important cereal, forage, and energy crops, and exhibits a unique gametophytic SI mechanism that is controlled by at least two multiallelic and independent loci, S and Z. Despite intense research efforts over the last six decades, the genes underlying S and Z remain uncharacterized. We have applied a fine-mapping approach to identify the S-locus in perennial ryegrass and provide multiple evidence that a domain of unknown function 247 gene (hereinafter referred to as LpSDUF247) is involved in the recognition of self and nonself pollen. Using a total of 10,177 individuals from seven different mapping populations segregating for S, we narrowed the S-locus to a genomic region containing eight genes, the closest recombinant marker mapping at a distance of 0.016 cM. Of the eight genes cosegregating with the S-locus, a highly polymorphic gene encoding for a protein containing a DUF247 domain, was fully predictive of known S-locus genotypes at the amino acid level in the seven mapping populations. Strikingly, this gene showed a frameshift mutation in self-compatible darnel (Lolium temulentum L.), whereas all of the self-incompatible species of the Festuca-Lolium complex were predicted to encode functional proteins.
Similar as for the S locus, a map-based cloning strategy was applied to identify the Z components of the SI system in perennial ryegrass: Using a total of more than 10,000 individuals from two independent mapping populations segregating for Z, the pollen and stigma components were pinpointed to a single BAC clone containing four positional candidate genes. Further validation on the basis of haplotype sequencing and nucleotide diversity analyses has been used to determine the Z locus. The genes of the putative pollen and stigma components of the gametophytic SI system in Poaceae are currently being functionally described in more detail.

Self-fertility (SF)
With the aim to characterize and utilize SF as an important tool for more efficient forage grass breeding, we have established different SF sources from perennial ryegrass and created segregating populations to genetically and functionally characterize these SF sources. For two independent populations, the genetic locations leading to SF has been determined. Additional plant material and genomics resources can now be developed to target the causative genes for SF and to deliver molecular markers for targeted breeding. This will facilitate the introgression of this trait into advanced breeding germplasm, and allow the development of inbred lines for powerful grass hybrids.

Doubled haploid (DH) induction
A complementary approach to efficiently produce inbred lines by repeated selfing on the basis of SF is doubled haploid (DH) induction. We have developed an efficient DH induction protocol to obtain homozygous lines in perennial ryegrass. By means of anther culture, completely homozygous lines can be obtained within one generation cycle. Genotypes with high responsiveness to anther culture have been identified and it has been shown that the trait can be crossed into breeding material. Moreover, the genetic analysis of the response to DH induction will now allow the development of a molecular marker system to select for high responsiveness and to facilitate the introgression of this trait into advanced breeding germplasm.

Cytoplasmic male sterility (CMS) and its restoration
Cytoplasmic male sterility (CMS) is a widely applied mechanism to control pollination for commercial hybrid seed production and although CMS systems have been identified in perennial ryegrass, they are yet to be fully characterised. We have established a bioinformatics pipeline for efficient identification of candidate restorer of fertility (Rf) genes for CMS. From a high-quality genomic draft of the perennial ryegrass genome, 373 pentatricopeptide repeat (PPR) genes were identified and classified, further identifying 25 restorer of fertility-like PPR (RFL) genes through a combination of DNA sequence clustering and comparison to known Rf genes. This extensive gene family was targeted as the majority of Rf genes in higher plants are RFL genes. These RFL genes were further investigated by phylogenetic analyses, identifying three groups of perennial ryegrass RFLs. These three groups likely represent genomic regions of active RFL generation and identify the probable location of perennial ryegrass PPR-Rf genes.
This pipeline allows for the identification of candidate PPR-Rf genes from genomic sequence data and can be used in any plant species. Functional markers for PPR-Rf genes will facilitate map-based cloning of Rf genes and enable the use of CMS as an efficient tool to control pollination for hybrid crop production.

The achieved results, as specified above, have significantly increased our understanding of the genetic mechanisms underlying grass reproductive traits and how these mechanisms can be used for efficient hybrid breeding in forage and turf grasses.