Exploring Modification Of Centromeric Histone 3 Cenh3 As A Strategy For Haploid Induction In Carrot Daucus Carota PDF Download

Precision editing of plant genomes has potential to accelerate crop improvement and advance functional genomics. One application of this technology to advance carrot breeding is the use of gene editing to create an in vivo haploid inducer for hybrid carrot production. Carrot (Daucus carota) is an economically important vegetable crop grown in many regions of the world and a major contributor of vitamin A to the human diet. The traditional method for producing carrot hybrid cultivars is a time and resource intensive process. An alternative to the traditional breeding method is the use of doubled haploids as inbred parents. Double haploids are created by the induction of haploids followed by subsequent doubling of the chromosomes. Doubled haploids can be achieved in one generation, which would significantly reduce the amount of time to make inbred parents.Recently, studies in Arabidopsis, wheat (Triticum aestivum), and maize (Zea mays) have demonstrated that modification of centromeric histone H3 (CENH3) can lead to the production of haploid plants. A variety of strategies have been employed to in these studies manipulate CENH3 that have induced haploids, with varying rates of success. Since the function of CENH3 is highly conserved across plant species, modification of CENH3 could lead to the development of an in vivo haploid inducer in any plant. A method for generating gene-edited carrot plants was developed in order to modify the carrot genome. This method utilizes transient transformation of protoplasts followed by regeneration of plants from protoplasts. This method proved efficient at generating a large number of gene-edited, transgene-free carrot plants. An important consideration of performing genomic modification of plants is the potential of inducing unintended changes the genome. To explore if large-scale chromosome aberrations are present in the plants regenerated from protoplasts, whole genome sequencing was performed on these plants. Read depth data from this whole genome sequence data was used for a dosage analysis to detect the presence of numerical or stuctural abberations. Using this analysis, we did not identify the presence chromosome aberrations in the plants regenerated from protoplasts. To determine if mutations in CENH3 can result in uniparental genome elimination, the basis of haploid induction, a variety of carrot plants with amino acid substitutions in the region of CENH3 encoding the histone fold domain were created. Nineteen of these cenh3 mutant plants were crossed with wild-type plants. A progeny of one of these crosses was identified as putative tetraploid that was likely a haploid during its genesis. Therefore, modification of CENH3 in carrot has the potential to induce ploidy changes in carrot.