Comparative Genomics and Phylogenetic Analysis Revealed the Chloroplast Genome Variation and Interspecific Relationships of Corylus (Betulaceae) Species

Abstract

Corylus L. is an economically and phylogenetically important genus in the family Betulaceae. Taxonomic and phylogenetic relationships of Corylus species have long been controversial for lack of effective molecular markers. In this study, the complete chloroplast (cp) genomes of six Corylus species were assembled and characterized using next-generation sequencing. We compared the genome features, repeat sequences, sequence divergence, and constructed the phylogenetic relationships of the six Corylus species. The results indicated that Corylus cp genomes were typical of the standard double-stranded DNA molecule, ranging from 160,445 base pairs (bp) (C. ferox var. thibetca) to 161,621 bp (C. yunnanensis) in length. Each genome contained a pair of inverted repeats (IRs), a large single-copy (LSC) region and a small single-copy (SSC) region. Each of the six cp genomes possessed 113 unique genes arranged in the same order, including 80 protein-coding, 29 tRNA, and 4 rRNA genes. C. yunnanensis contained the highest number of repeat sequences, and the richest SSRs in six cp genomes were A/T mononucleotides. Comparative analyses of six Corylus cp genomes revealed four hotspot regions (trnH-psbA, rpoB-trnC, trnF-ndhJ, and rpl32-trnL) that could be used as potential molecular markers. Phylogenetic analyses of the complete chloroplast genomes and 80 protein-coding genes exhibited nearly identical topologies that strongly supported the monophyly of Corylus and simultaneously revealed the generic relationships among Betulaceae. The availability of these genomes can offer valuable genetic information for further taxonomy, phylogeny, and species delimitation in Corylus or even Betulaceae plants.

Keywords: Corylus; chloroplast genome variation; comparative genomics; interspecific relationships; phylogenetic analysis.

FIGURE 1

Gene maps of the six Corylus chloroplast genomes. Genes shown outside the outer circle are transcribed clockwise and those inside are transcribed counterclockwise. Genes belonging to different functional groups are color-coded. Dashed area in the inner circle indicates the GC content of the chloroplast genome.

FIGURE 2

The comparison of the LSC, IR, and SSC border regions among the six Corylus chloroplast genomes.

FIGURE 3

The type and distribution of repeated sequences and SSRs in the cp genome of six Corylus species. (A) Number of five repeat types; (B) Number of repeat sequences by length; (C) Number of six SSR types; (D) Number of identified SSR motifs in different repeat class types.

FIGURE 4

MAUVE Alignment of the six Corylus chloroplast genomes. The C. yunnanensis genome is shown at top as the reference genome. Within each of the alignments, local collinear blocks are represented by blocks of the same color connected by lines.

FIGURE 5

Sliding-window analysis of the whole cp genomes of six Corylus species. Window length: 600 bp; step size: 200 bp. X-axis: position of the midpoint of a window. Y-axis: nucleotide diversity of each window.

FIGURE 6

Phylogenetic tree of Corylus and closely related species in Betulaceae using Maximum likelihood (ML) and Bayesian inference (BI) methods based on: (A) the complete chloroplast genome sequences, (B) 80 protein-coding genes. Six genera designated in this study are highlighted with different vertical bars in different colors on the cladogram. Bootstrap values (BS) ≥ 70% in ML analysis and posterior probabilities (PP) ≥ 0.95 in BI analysis are listed above the branches (BS/PP). The hyphen refers to BS ≤ 70% in ML or PP ≤ 0.95 in BI.