Development of a CenH3-Based Haploid Inducer in Hexaploid Camelina sativa
Isabelle M. Henry* (email@example.com), Paul Osuna-Kleist, Helen Tsai, and Luca Comai
University of California–Davis
Camelina (Camelina sativa) is a promising oilseed crop that is particularly well suited for cultivation in the Northwest of the United States. The ECON project is an interdisciplinary project focused on two main objectives i) enhance nitrogen utilization efficiency and ii) boost oil yield. The long-term goals are to increase the economic profitability of camelina cultivation, by reducing the negative impact of nitrogen fertilization and increasing productivity competitiveness with other major oilseed crops such as canola. Approaches include characterizing genetic and genomic natural variation within camelina for the ability to absorb, translocate and assimilate nitrogen, and for recruiting beneficial rhizo-microbes to improve nitrogen acquisition. Researchers are also investigating the mechanisms underlying these differences to optimize yield potential by increasing seed size and enhancing oil synthesis. Within the ECON project, the laboratory aims at developing a haploid inducer line for camelina, a tool that will be instrumental for accelerating the breeding of several loci of interest in a polyploid background.
Haploid induction is powerful breeding tool. Amongst others, it allows the rapid production of complex genotypes when constructing experimental and breeding lines. This is particularly critical when dealing with polyploid genomes such allohexaploid C. sativa. For example, selfing a parent with 6 heterozygous non-linked loci is expected to result in 0.024% homozygous progeny. Crossing the same parent to a haploid inducer (HI) will produce 1.5% progeny with the desired genotype. Modification of the centromere-specific histone variant CENH3 engenders haploid induction in Arabidopsis thaliana. Specifically, crosses between a haploid inducer line carrying a mutated form of CENH3 and a wild-type line results in frequent elimination of the haploid inducer chromosomes and produces offspring of different types in similar numbers: paternal haploids, aneuploids, and diploids. Haploids are formed when all the HI chromosomes are lost and only the maternal WT chromosomes are retained.
The goal is to develop a cenH3-based haploid inducer in C. sativa. C. sativa is a very close relative of A. thaliana but the situation is complicated by the fact that the genome of camelina harbors three functional copies of the cenH3 genes, all of which need to be modified to produce a HI. A TILLING population of Camelina var. Ames 1043 was previously developed in the laboratory and more than 300 high reliability mutations were identified in the cenH3 genes. Through a series of crosses and selection steps, the following three mutations were combined into a single line, in the homozygous state: a nonsense mutation allele (genome 1), a missense mutation that is known to result in haploid induction in A. thaliana (genome 2), and a splice-site variant (genome 3). The resulting potential haploid inducer was crosses to WT Ames and the progeny screened for the presence of haploids. No haploid plant was recovered but >85% of the progeny lacked at least one chromosome, confirming that genome elimination is occurring in these crosses, albeit not sufficiently efficiently to eliminate all 20 chromosome of the haploid inducer parent. Interestingly, chromosomes from genome 2 were preferentially lost in the aneuploid progeny, consistent with previously documented relative expression dominance of the other two sub-genomes within the Camelina genome. Taken together, the results so far suggest that CENH3-based haploid induction is a feasible approach in camelina but complicated by the fact that a large percentage of aneuploid progeny survive, presumably because of the buffering effect of the polyploid background. Researchers are in the process of combining weaker CENH3 alleles in a new potential haploid inducer line in order to further increase the loss of HI chromosomes and hopefully obtain fully haploid lines.
This research is supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, Genomic Science program Award No. DE-SC0021369.