Characterization of Centromeric Histone H3 (CENH3) Variants in Cultivated and Wild Carrots (Daucus sp.)

Abstract

In eukaryotes, centromeres are the assembly sites for the kinetochore, a multi-protein complex to which spindle microtubules are attached at mitosis and meiosis, thereby ensuring segregation of chromosomes during cell division. They are specified by incorporation of CENH3, a centromere specific histone H3 variant which replaces canonical histone H3 in the nucleosomes of functional centromeres. To lay a first foundation of a putative alternative haploidization strategy based on centromere-mediated genome elimination in cultivated carrots, in the presented research we aimed at the identification and cloning of functional CENH3 genes in Daucus carota and three distantly related wild species of genus Daucus varying in basic chromosome numbers. Based on mining the carrot transcriptome followed by a subsequent PCR-based cloning, homologous coding sequences for CENH3s of the four Daucus species were identified. The ORFs of the CENH3 variants were very similar, and an amino acid sequence length of 146 aa was found in three out of the four species. Comparison of Daucus CENH3 amino acid sequences with those of other plant CENH3s as well as their phylogenetic arrangement among other dicot CENH3s suggest that the identified genes are authentic CENH3 homologs. To verify the location of the CENH3 protein in the kinetochore regions of the Daucus chromosomes, a polyclonal antibody based on a peptide corresponding to the N-terminus of DcCENH3 was developed and used for anti-CENH3 immunostaining of mitotic root cells. The chromosomal location of CENH3 proteins in the centromere regions of the chromosomes could be confirmed. For genetic localization of the CENH3 gene in the carrot genome, a previously constructed linkage map for carrot was used for mapping a CENH3-specific simple sequence repeat (SSR) marker, and the CENH3 locus was mapped on the carrot chromosome 9.

The genome sequence ofAloe verareveals adaptive evolution of drought tolerance mechanisms

“…Arabidopsis thaliana as an outgroup (Figure 1). This phylogeny also corroborated with the earlier 312 reported phylogenies by Silvera et al, 2014, Dunemann et al, 2014, and Wang et al, 2016 were constructed using a limited number of genetic loci [64][65][66]. It is apparent from the phylogeny 314 that Dioscorea rotundata and Musa acuminata are the most closely related to Aloe vera, and share 315 the same clade (Figure 1).…”

State-of-the-art and novel developments of in vivo haploid technologies

“…In addition, if a two-step method is pursued, genetically engineered CENH3 variants or alien CENH3 genes have to be stably expressed to partially complement knockout mutations of the endogenous CENH3 . So far, CENH3 genes from several species have been cloned and characterized, e.g., rice (Hirsch et al 2009 ), tobacco (Nagaki et al 2009 ), soybean (Tek et al 2010 ), Brassica oleracea (Wang et al 2011 ), onion and garlic (Nagaki et al 2012 ), pea (Neumann et al 2012 ), common bean (Iwata et al 2013 ), carrot (Dunemann et al 2014 ), wheat (Yuan et al 2015 ) and rye (Evtushenko et al 2017 ). Nevertheless, to the best of our knowledge, protocols for haploid induction in other crop plants than maize, in which modifications within the CENH3-coding region were induced by mutagenesis, have not been published yet in the peer-reviewed literature.…”

Sunflower centromeres consist of a centromere-specific LINE and a chromosome-specific tandem repeat

“…Among the constitutive proteins of kinetochores, centromere-specific histone H3 (CENH3) acts as a base for assembling other kinetochore proteins, and its presence epigenetically determines the kinetochore position (Perpelescu and Fukagawa, 2011 ). The first identified CENH3 was CENP-A in humans (Earnshaw and Rothfield, 1985 ), and its orthologs have been isolated from 11 of 63 APG III orders, including Poales, Asparagales, Rosales, Fabales, Malpighiales, Malvales, Brassicales, Myrtales, Solanales, Asterales, and Apiales, in this decade (Talbert et al, 2002 ; Zhong et al, 2002 ; Nagaki et al, 2004 , 2005 , 2012b ; Nagaki and Murata, 2005 ; Sanei et al, 2011 ; Wang et al, 2011 ; Neumann et al, 2012 , 2015 ; Dunemann et al, 2014 ; Masonbrink et al, 2014 ; He et al, 2015 ; Maheshawari, 2015 ). However, no CENH3 has been isolated from Asterales species.…”