Note to: Hox gene cluster of the ascidian, Halocynthia roretzi, reveals multiple ancient steps of cluster disintegration during ascidian evolution

Abstract Background In the previous paper published in 2017, we described the structure of Hox gene cluster of the ascidian, Halocynthia roretzi, and discussed the scenario for the disintegration of Hox gene clusters during evolution of ascidians. The description about the Hox gene cluster structure still represents the latest information, hence it has been left unchanged. In contrast, some points in Discussion, the description on the phylogenetic relationships among tunicates and the theoretical scenario for the disintegration of Hox gene cluster during evolution of ascidians, should be changed because the phylogenetic relationships among tunicates have recently been updated. The above mentioned points were made in accordance with the phylogenetic tree for tunicates based on the mitochondrial DNA sequences, which was the latest at the time of publication. In 2018, however, Kocot et al. and Delsuc et al. proposed new phylogenetic trees for tunicates based on a large number of nuclear gene sequences. The trees obtained by the two groups are essentially the same and different from the previous one in the phylogenetic positions of Appendicularia and Thaliacea, which leads to a change in the order of the emergence of ascidians and the Hox gene cluster disintegration during evolution of ascidians or tunicates. Results We add here a note to update the previous description on the phylogenetic relationships among tunicates and the theoretical scenario, including one Figure, so as to coincide with the new phylogenetic relationships among tunicates based on the nuclear gene sequences. Conclusion The previous summarized conclusion remains unchanged: we suggest that the Hox gene cluster of the ancestral ascidian experienced extensive genome shuffling during the course of evolution to Hr and Ci. Nevertheless, some features are shared in Hox gene components and gene organization on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to all ascidians and later lineage-specific events

Hox gene cluster of the ascidian, Halocynthia roretzi, reveals multiple ancient steps of cluster disintegration during ascidian evolution

Abstract Background Hox gene clusters with at least 13 paralog group (PG) members are common in vertebrate genomes and in that of amphioxus. Ascidians, which belong to the subphylum Tunicata (Urochordata), are phylogenetically positioned between vertebrates and amphioxus, and traditionally divided into two groups: the Pleurogona and the Enterogona. An enterogonan ascidian, Ciona intestinalis (Ci), possesses nine Hox genes localized on two chromosomes; thus, the Hox gene cluster is disintegrated. We investigated the Hox gene cluster of a pleurogonan ascidian, Halocynthia roretzi (Hr) to investigate whether Hox gene cluster disintegration is common among ascidians, and if so, how such disintegration occurred during ascidian or tunicate evolution. Results Our phylogenetic analysis reveals that the Hr Hox gene complement comprises nine members, including one with a relatively divergent Hox homeodomain sequence. Eight of nine Hr Hox genes were orthologous to Ci-Hox1, 2, 3, 4, 5, 10, 12 and 13. Following the phylogenetic classification into 13 PGs, we designated Hr Hox genes as Hox1, 2, 3, 4, 5, 10, 11/12/13.a, 11/12/13.b and HoxX. To address the chromosomal arrangement of the nine Hox genes, we performed two-color chromosomal fluorescent in situ hybridization, which revealed that the nine Hox genes are localized on a single chromosome in Hr, distinct from their arrangement in Ci. We further examined the order of the nine Hox genes on the chromosome by chromosome/scaffold walking. This analysis suggested a gene order of Hox1, 11/12/13.b, 11/12/13.a, 10, 5, X, followed by either Hox4, 3, 2 or Hox2, 3, 4 on the chromosome. Based on the present results and those previously reported in Ci, we discuss the establishment of the Hox gene complement and disintegration of Hox gene clusters during the course of ascidian or tunicate evolution. Conclusions The Hox gene cluster and the genome must have experienced extensive reorganization during the course of evolution from the ancestral tunicate to Hr and Ci. Nevertheless, some features are shared in Hox gene components and gene arrangement on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to tunicates as well as later ascidian lineage-specific events

Fluorescent in situ hybridization to ascidian chromosomes

The draft genome of the ascidian Ciona intestinalis has been sequenced. Mapping of the genome sequence to the Ciona 14 haploid chromosomes is essential for future studies of the genome-wide control of gene expression in this basal chordate. Here we describe an efficient protocol for fluorescent in situ hybridization for mapping genes to the Ciona chromosomes. We demonstrate how the locations of two BAC clones can be mapped relative to each other. We also show that this method is efficient for coupling two so-far independent scaffolds into one longer scaffold when two BAC clones represent sequences located at either end of the two scaffolds