Genome-wide identification, characterization, and genetic diversity of CCR gene family in Dalbergia odorifera

Abstract

Introduction: Lignin is a complex aromatic polymer plays major biological roles in maintaining the structure of plants and in defending them against biotic and abiotic stresses. Cinnamoyl-CoA reductase (CCR) is the first enzyme in the lignin-specific biosynthetic pathway, catalyzing the conversion of hydroxycinnamoyl-CoA into hydroxy cinnamaldehyde. Dalbergia odorifera T. Chen is a rare rosewood species for furniture, crafts and medicine. However, the CCR family genes in D. odorifera have not been identified, and their function in lignin biosynthesis remain uncertain.

Methods and results: Here, a total of 24 genes, with their complete domains were identified. Detailed sequence characterization and multiple sequence alignment revealed that the DoCCR protein sequences were relatively conserved. They were divided into three subfamilies and were unevenly distributed on 10 chromosomes. Phylogenetic analysis showed that seven DoCCRs were grouped together with functionally characterized CCRs of dicotyledons involved in developmental lignification. Synteny analysis showed that segmental and tandem duplications were crucial in the expansion of CCR family in D. odorifera, and purifying selection emerged as the main force driving these genes evolution. Cis-acting elements in the putative promoter regions of DoCCRs were mainly associated with stress, light, hormones, and growth/development. Further, analysis of expression profiles from the RNA-seq data showed distinct expression patterns of DoCCRs among different tissues and organs, as well as in response to stem wounding. Additionally, 74 simple sequence repeats (SSRs) were identified within 19 DoCCRs, located in the intron or untranslated regions (UTRs), and mononucleotide predominated. A pair of primers with high polymorphism and good interspecific generality was successfully developed from these SSRs, and 7 alleles were amplified in 105 wild D. odorifera trees from 17 areas covering its whole native distribution.

Discussion: Overall, this study provides a basis for further functional dissection of CCR gene families, as well as breeding improvement for wood properties and stress resistance in D. odorifera.

Keywords: Dalbergia odorifera; cinnamoyl-CoA reductase; forest breeding; genetic diversity; simple sequence repeat.

Figure 1

Chromosomal locations of CCR genes in D. odorifera. Each vertical bar indicates one chromosome. The chromosome number is shown at the left of each chromosome. Chromosomal locations of DoCCRs were mapped based on the D. odorifera genome. Red lines indicate tandem gene duplications.

Figure 2

Phylogenetic relationship, conserved motifs, conserved domains and gene structures of the DoCCR family. (A) The phylogenetic tree of all DoCCR proteins was constructed using neighbor-joining (NJ) method with 1,000 bootstrap. Different branch colors represent different groupings. (B) Conserved motifs in DoCCR proteins. The motifs, numbers 1-10, were displayed in different colored boxes. The green dashed box indicates the FR_SDR_e domain corresponding to the one in (D). (C) The conserved domain in the DoCCR proteins. Green boxes indicate FR_SDR_e domain, which identified by CDD database. The units on the bottom scale are the number of amino acids. (D) The exon-intron structure of DoCCR genes. Exons and introns were indicated by boxes and single lines. The thin blue boxes represent UTRs, yellow boxes represent CDSs and grey lines represent introns. Additionally, the red boxes represent SSR (excluding mononucleotide repeats).

Figure 3

Phylogenetic tree of DoCCRs and 28 charaterized CCRs from other plant species. The neighbor-joining (NJ) tree was constructed by MEGA11 with 1,000 bootstrap replicates. The purple shade indicates monocot CCRs, and other shades indicate dicot CCRs. The red star indicates the DoCCRs and the black circle indicates the functional CCRs from other plants. At, A. thaliana; Bp, B. platyphylla; Bn, B. napus; Ci, C. intermedia; Do, D. odorifera; Eg, E. gunnii; Gm, G. mexicanum; Ll, L. leucocephala; Lp, L. perenne; Mt, M. truncatula; Os, O. sativa; Pv, P. virgatum; Pd, P. dilatatum; Ph, P. hybrida; Ptre, P. tremuloides; Ptri, P. trichocarpa; Sl, S. lycopersicum; Sb, S. bicolor; Ta, T. aestivum; Zm, Z. mays. Detailed information of all CCRs from other plants are showed in Table S1 .

Figure 4

Syntenic relationships of DoCCR genes with other CCR genes in four representative plant species. Gray lines in the background indicated the collinear blocks within D. odorifera and other plant genomes. Red lines in highlight indicated the syntenic CCR gene pairs.

Figure 5

Representation of cis-acting elements in the promoter regions of DoCCR genes. (A) Distribution of different kinds of cis-elements in the upstream 2,000 bp sequences of the DoCCRs. Different color boxes correspond to the identity of cis-elements; some cis-elements may overlap with others. (B) The number of cis-elements in the promoters of the DoCCRs.

Figure 6

Expression profiles of DoCCR genes in different tissues and under abiotic stress. (A) The accumulation patterns of DoCCRs in different tissues, such as flower, seed, leaf, root, stem, top (Top VC), middle (Middle VC), and bottom (Bottom VC) of vascular cambium, sapwood (SW), transition zone (TZ). (B) The expression patterns of DoCCRs in stems after wounding treatment. Discolored (D), and healthy (H) zones of pruning-induced stems were assayed. Scale bars on the right represent log₂(TPM+1) transformed values.

Figure 7

Fingerprint analysis of 105 D. odorifera samples from SSR loci in DoCCCR5.