Expressed Centromere Specific Histone 3 (CENH3) Variants in Cultivated Triploid and Wild Diploid Bananas (Musa spp.)

Abstract

Centromeres are specified by a centromere specific histone 3 (CENH3) protein, which exists in a complex environment, interacting with conserved proteins and rapidly evolving satellite DNA sequences. The interactions may become more challenging if multiple CENH3 versions are introduced into the zygote as this can affect post-zygotic mitosis and ultimately sexual reproduction. Here, we characterize CENH3 variant transcripts expressed in cultivated triploid and wild diploid progenitor bananas. We describe both splice- and allelic-[Single Nucleotide Polymorphisms (SNP)] variants and their effects on the predicted secondary structures of protein. Expressed CENH3 transcripts from six banana genotypes were characterized and clustered into three groups (MusaCENH-1A, MusaCENH-1B, and MusaCENH-2) based on similarity. The CENH3 groups differed with SNPs as well as presence of indels resulting from retained and/or skipped exons. The CENH3 transcripts from different banana genotypes were spliced in either 7/6, 5/4 or 6/5 exons/introns. The 7/6 and the 5/4 exon/intron structures were found in both diploids and triploids, however, 7/6 was most predominant. The 6/5 exon/introns structure was a result of failure of the 7/6 to splice correctly. The various transcripts obtained were predicted to encode highly variable N-terminal tails and a relatively conserved C-terminal histone fold domain (HFD). The SNPs were predicted in some cases to affect the secondary structure of protein by lengthening or shorting the affected domains. Sequencing of banana CENH3 transcripts predicts SNP variations that affect amino acid sequences and alternatively spliced transcripts. Most of these changes affect the N-terminal tail of CENH3.

Keywords: CENH3; banana; centromere; genotype; histones; splice variants.

FIGURE 1

Phylogenetic tree of banana CENH3s. Unrooted Phylogenetic tree based on histone fold domain (HFD) and conserved CENH3 tail sections of six banana genotypes showing the MusaCENH3-1A, MusaCENH3-1B, and MusaCENH3-2 groups. Values at the root are bootstrap support values at 1000 replicates. The tree was drawn in MEGA 6 (Tamura et al., 2013).

FIGURE 2

Multiple alignment of banana CENH3s. The blue lines separate the different alignments: Block 1 is a MusaCENH3-1A alignment, block 2 is a MusaCENH3-1B, block 3 is MusaCENH3-2 and block 4 is an alignment to other monocots and dicots. The red highlights in the alignment are some amino acids substitutions observed in banana alleles within the HFD. Inset red box is the similarity index. Alignments were conducted in ClustalW (Larkin et al., 2007) as implemented in Geneious version 7.1 (Kearse et al., 2012).

FIGURE 3

Alignment of transcript variants to the reference transcript from diploid banana genotype ‘Calcutta 4’. Blocks (A–D) are alignments of genotypes ‘Gros Michel’, ‘Pisang Awak’, ‘Sukali Ndiizi’, and a combination of ‘Zebrina GF’ and species ‘Musa balbisiana’ to ‘Calcutta 4’, respectively. Inset in red is the nucleotide alignment similarity index.

FIGURE 4

Alignment of the CENH3 transcripts to genomic sequence of genotype ‘DH Pahang’ to identify splice mechanisms. The 7/6, 6/5, and 5/4 structures are represented intron/exon structures are represented. Alignment was performed using Splign (Kapustin et al., 2008).

FIGURE 5

Predicted secondary structures of banana CENH3. 1- MusaCENH3-1A, 2- MusaCENH3-1B, and 3- MusaCENH3-2. At the bottom are the different CENH3 domains, above the structures are the amino acids in logo format. Inset is the key to the secondary structures.