The large single-copy (LSC) region functions as a highly effective and efficient molecular marker for accurate authentication of medicinal Dendrobium species

Abstract

Having great medicinal values, Dendrobium species of "Fengdou" (DSFs) are a taxonomically complex group in Dendrobium genus including many closely related and recently diverged species. Traditionally used DNA markers have been proved to be insufficient in authenticating many species of this group. Here, we investigated 101 complete plastomes from 23 DSFs, comprising 72 newly sequenced and 29 documented, which all exhibited well-conserved genomic organization and gene order. Plastome-wide comparison showed the co-occurrence of single nucleotide polymorphisms (SNPs) and insertions/deletions (indels), which can be explained by both the repeat-associated and indel-associated mutation hypotheses. Moreover, guanine-cytosine (GC) content was found to be negatively correlated with the three divergence variables (SNPs, indels and repeats), indicating that GC content may reflect the level of the local sequence divergence. Our species authentication analyses revealed that the relaxed filtering strategies of sequence alignment had no negative impact on species identification. By assessing the maximum likelihood (ML) trees inferred from different datasets, we found that the complete plastome and large single-copy (LSC) datasets both successfully identified all 23 DSFs with the maximum bootstrap values. However, owing to the high efficiency of LSC in species identification, we recommend using LSC for accurate authentication of DSFs.

Keywords: Authentication; Dendrobium species of “Fengdou”; Large single-copy (LSC); Plastomic comparison.

Graphical abstract

Figure 1

Plastome map of Dendrobium species of “Fengdou”. The genes inside and outside the circle are transcribed clockwise and counterclockwise, respectively. Genes from different functional groups are shown in different colors. The thick lines represent the inverted repeat regions (IR_A and IR_B) that separate the plastome into large single-copy (LSC) and small single-copy (SSC) regions.

Figure 2

Comparison of IR/SC junction regions among the plastomes of 20 Dendrobium species of “Fengdou”.

Figure 3

Percentages of SNPs, indels, and repeats and GC content in 260 nonoverlapping bins of 600 bp each through the alignment of 101 complete plastomes of 23 Dendrobium species of “Fengdou”.

Figure 4

Relationships between SNP density and the distance to indels at the levels of the complete plastome and coding regions.

Figure 5

Comparisons of the discriminatory power of 16 datasets for 23 Dendrobium species of “Fengdou”. (A) Maximum likelihood (ML) tree of 23 Dendrobium species of “Fengdou” inferred from the strictly filtered alignment of LSC dataset. All individuals of each species formed a monophyletic clade, but only one individual per species is shown for simplicity. Bootstrap support values >50% are shown above branches. (B) Identification results of different datasets for 23 Dendrobium species of “Fengdou” (Figs. S1–S5). The identification results of the complete plastome, LSC, IR and SSC were derived from their strictly filtered datasets. Different datasets are shown in different colors and shapes. For each dataset, the solid figures represent successfully identified species, whereas the hollow ones stand for unsuccessfully identified species (I, ITS; I2, ITS2; M, matK; R, rbcL; T, trnH-psbA; A, atpF-atpH; P, psbK-psbI; TT, trnT-trnL; R32, rpl32-trnL; C, clpP-psbB; TI, trnL intron; R16, rps16-trnQ).