Towards Efficient Doubled Haploid Production in Perennial Ryegrass (Lolium perenne L.)
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Date
2017Type
- Doctoral Thesis
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Abstract
The ability to produce doubled haploid plants has found broad application in fundamental and applied research as well as in practical plant breeding. In maize (Zea mays L.) and barley (Hordeum vulgare L.), for instance, routine, large-scale doubled haploid production in a single generation has replaced the 5-7 cycles of repeated self-fertilization formerly required to obtain sufficiently homozygous material. Doubled haploid lines have been invaluable for robust, multi-year phenotyping in quantitative trait loci mapping studies and their complete homozygosity has simplified genome sequence assembly efforts. Furthermore, doubled haploids are directly released as stable, homogeneous cultivars or used as parents of F1 hybrid seed to exploit the phenomenon of heterosis. At this time, however, neither homozygous line nor doubled haploid production can be efficiently applied in the important forage crop perennial ryegrass (Lolium perenne L.). An effective self-incompatibility system as well as a marked sensitivity to inbreeding depression hamper iterations of self-fertilization, while low and highly genotype-specific responses render the available in vitro doubled haploid induction methods unproductive. Major benefits associated with easy access to homozygous germplasm, particularly the straightforward production of high yielding hybrid varieties, are therefore unavailable to those working with this economically significant species.
This thesis documents the initiation of a modern reboot of the development of an efficient doubled haploid production system for perennial ryegrass. The main goal was to obtain a level of understanding of the genetic factors, as well as their genomic locations, governing the response to in vitro doubled haploid induction, that would enable rapid and effective introduction of high levels of androgenic ability into recalcitrant germplasm. Four distinct phases of the research are reported in chapters 2 through 5, which are summarised below.
First of all, a comprehensive overview of the ways in which haploid and doubled haploid techniques may be exploited to reduce the time, space and investment associated with perennial ryegrass research and breeding, is given in chapter 2. The potential of doubled haploid induction to 1) purge deleterious alleles from germplasm intended for breeding, 2) develop mapping populations for genetic and genomic studies, 3) simplify haplotype mapping, 4) fix transgenes and mutations for functional gene validation and molecular breeding, and 5) hybrid cultivar development is discussed. It is concluded that relatively minor improvements to existing in vitro doubled haploid induction protocols of perennial ryegrass should make some exceedingly useful applications available to the forage grass community.
A pilot study to characterize the responses of putative highly androgenic germplasm to a decades old in vitro anther culture protocol is presented in chapter 3. The method proved to be efficient and large numbers of microspore-derived embryo-like structures as well as green and albino plantlets could be recovered. In addition, significant genotype-dependent variation was observed between the individual anther donors, confirming the usefulness of the plant material for further investigations into the androgenic ability of perennial ryegrass.
Chapter 4 describes the results of the subsequent large, two year screen of the response to the in vitro anther culture protocol of nine distinct, bi-parental populations, obtained by crossing two genotypes with contrasting androgenic capacities. The variation in embryo production, plant regeneration and green plant production observed between and within the populations was large, and its pattern indicated the presence of different genes and alleles involved in the control of the component traits of androgenic ability. An insignificant association of embryo production with plant regeneration, as well as a low correlation between green and albino plant yield, suggested that distinct genes influence these traits. Furthermore, the environment was found to affect the incidence of albinism to a larger degree than genetic components. Lastly, it was concluded that the evaluated populations could provide rare, beneficial alleles for the introgression of high levels of androgenic capacity into recalcitrant material.
Finally, chapter 5 addresses the identification of genetic loci associated with androgenic ability, which were determined via a powerful genotyping-by-sequencing-based genome-wide association study using the plant material and phenotypic data of the previous chapter. Between 1 and 10 quantitative trait loci were identified for anther response, embryo and total plant production, green and albino plant production and regeneration. Interestingly, a locus with a major effect on green plant regeneration was identified on perennial ryegrass linkage group 5 which may prove to be orthologous to loci detected at a similar genomic location in four related Poaceae species. In addition, two intriguing candidate genes, encoding chromatin binding domains of the developmental phase transition regulator, Polycomb Repressive Complex 2, were identified and merit further investigation. Ultimately, these results currently enable the development of molecular markers to rapidly introgress androgenic capacity into recalcitrant perennial ryegrass germplasm.
It is hard to overstate the importance of doubled haploid techniques to advance research and increase the speed of genetic gain of breeding programmes. Here, I have demonstrated the effectivity of an in vitro anther culture method for doubled haploid production in perennial ryegrass, a forage species of global importance. In addition, plant material carrying valuable alleles as well as the molecular tools to identify them are now available, so that efficient doubled haploid production may soon be added to the arsenal of research and breeding tools of the perennial ryegrass community. My work will significantly accelerate forage grass breeding and constitute a key step towards the realization of a grass hybrid breeding programme. Finally, the results obtained here may prove to be of significant benefit to research and breeding efforts in related Poaceae species as well. Show more
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https://doi.org/10.3929/ethz-b-000218913Publication status
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ETH ZurichSubject
Doubled haploid (DH); Perennial ryegrass (Lolium perenne L.); FORAGE GRASSES (FORAGE CROPPING); GWASOrganisational unit
03969 - Studer, Bruno / Studer, Bruno
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