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The specific DNA barcodes based on chloroplast genes for species identification of Theaceae plants

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Abstract

More than 600 species in over 40 genera have been identified in family Theaceae worldwide. The accurate identification of Theaceae plants can ensure the market economic order, and it plays a vital role in achieving the sustainable utilization of germplasm resources. DNA barcoding, one of the most potential species identification technologies at present, has advanced in the rapid, accurate and repetitive discrimination of species. In this study, matK + ndhF + ycf1 was observed as the optimal combined candidate gene sequence of DNA barcodes by analyzing genetic information of four single chloroplast DNA sequences, including matK, rbcL, ndhF and ycf1, as well as six combined gene sequences. Subsequently, the experiments were performed on phylogenetic analysis based on genetic distance to study the phylogenetic relationship of Theaceae plants and evaluate the species identification accuracy of matK + ndhF + ycf1. Lastly, the species-specific DNA barcodes were designed by searching the variable sites (one type of single nucleotide polymorphism sites) for the accurate identification of Camellia amplexicaulis, Franklinia alatamaha, Gordonia brandegeei and Stewartia micrantha. The previous methods of screening and testing candidate gene sequences were optimized, and innovation was made in the above methods. The process of making visual DNA barcodes was standardized. Besides, DNA barcoding technology increased the accuracy of species identification and DNA barcoding was analyzed in accordance with the theories of population genetics (e.g., neutral theory of molecular evolution). The results of the study will lay a basis for the identification and protection of Theaceae species and germplasm resources.

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Data availability

The data supporting the finding of this study is provided in the manuscript and its supplementary material.

Abbreviations

cpDNA:

Chloroplast DNA

C sites:

Conserved sites

V sites:

Variable sites

Pi sites:

Parsimony informative sites

S sites:

Singleton sites

SNP sites:

Single nucleotide polymorphism sites

ii:

Identical Pairs

si:

Transitionsal pairs

sv:

Transversional pairs

R value:

Transitionsal pairs/Transversional pairs 

θ:

Nucleotide substitution rate

Hd:

Haplotype diversity

S:

Segregating sites

π:

Nucleotide diversity

Fu’s Fs :

Representative value of neutrality tests

Tajima’s D :

Representative value of neutrality tests

P value:

Hypothetical probability

ML tree:

Maximum likelihood tree

NJ tree:

Neighbor joining tree

Ca :

Camellia amplexicaulis

Fa :

Franklinia alatamaha

Gb :

Gordonia brandegeei

Sm :

Stewartia micrantha

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Acknowledgements

The authors are grateful Dr. Shuai Hu, School of life sciences, Tsinghua University for critical review of this manuscript.

Funding

Authors would like to acknowledge the funding received from Key Research & Development Project of Hunan Provincial Department of Science and Technology (2019NK2081), National Natural Science Foundation of China (31872866) and National Key Research and Development Program of China (2017YFF0210301) to carry out this assignment.

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Author notes

  1. Shuai Jiang and Fenglin Chen have contributed equally to this work.

Authors and Affiliations

  1. College of Biology, Hunan University, Changsha, 410082, China

    Shuai Jiang, Fenglin Chen, Pai Qin, Hai Xie, Guo Peng, Yongliang Li & Xinhong Guo

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  1. Shuai Jiang
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  3. Pai Qin
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Contributions

XHG and YLL designed the research. SJ, FLC, PQ, HX, GP and YLL collected and analyzed data. SJ, FLC, YLL and XHG wrote the main manuscript text. SJ prepared all figures and tables. All authors read and approved the manuscript.

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Correspondence to Yongliang Li or Xinhong Guo.

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Supplementary Information

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Fig. S1

The ML tree of Theaceae plants coming from matK + rbcL + ycf1 sequences based on the K2P model. The bootstraps of tree branches were all greater than 75%. Triangle marks (color in red) on branches represent bootstrap values; the bigger the triangles, the higher the bootstrap values would be. At the same time, displaying branch lengths on the main branches, namely the length of evolutionary distance, which retained four significant digits. (TIF 2747 kb)

Fig. S2

The Bayes tree of Theaceae plants coming from matK + rbcL + ycf1 sequences based on the K2P model. The bootstraps of tree branches were all greater than 75%. Triangle marks (color in red) on branches represent bootstrap values; the bigger the triangles, the higher the bootstrap values would be. At the same time, displaying branch lengths on the main branches, namely the length of evolutionary distance, which retained four significant digits. (TIF 3597 kb)

Fig. S3

Combinatorial super DNA barcodes employed as substitute sequences including one-dimensional and two-dimensional DNA barcodes of Theaceae plants based on matK, ndhF and ycf1. In one-dimensional DNA barcodes, base A, T, C, G in green, red, blue and black respectively. (TIF 6484 kb)

Table S1

Accession numbers, version numbers, accepted names, synonyms and definition of matK (single gene sequence) in Theaceae plants on the NCBI online database. (XLSX 19 kb)

Table S2

Accession numbers, version numbers, accepted names, synonyms and definition of rbcL (single gene sequence) in Theaceae plants on the NCBI online database. (XLSX 17 kb)

Table S3

Accession numbers, version numbers, accepted names, synonyms and definition of ndhF (single gene sequence) in Theaceae plants on the NCBI online database. (XLSX 19 kb)

Table S4

Accession numbers, version numbers, accepted names, synonyms and definition of ycf1 (single gene sequence) in Theaceae plants on the NCBI online database. (XLSX 17 kb)

Table S5

Accession numbers, version numbers, accepted names, synonyms and definition of matK + rbcL (combined gene sequence) in Theaceae plants on the NCBI online database. (XLSX 17 kb)

Table S6

Accession numbers, version numbers, accepted names, synonyms and definition of matK + ndhF (combined gene sequence) in Theaceae plants on the NCBI online database. (XLSX 16 kb)

Table S7

Accession numbers, version numbers, accepted names, synonyms and definition of matK + ycf1 (combined gene sequence) in Theaceae plants on the NCBI online database. (XLSX 16 kb)

Table S8

Accession numbers, version numbers, accepted names, synonyms and definition of rbcL + ndhF (combined gene sequence) in Theaceae plants on the NCBI online database. (XLSX 16 kb)

Table S9

Accession numbers, version numbers, accepted names, synonyms and definition of rbcL + ycf1 (combined gene sequence) in Theaceae plants on the NCBI online database. (XLSX 16 kb)

Table S10

Accession numbers, version numbers, accepted names, synonyms and definition of ndhF + ycf1 (combined gene sequence) in Theaceae plants on the NCBI online database. (XLSX 16 kb)

Table S11

Average AT and GC content at different coding positions of codons in Theaceae plants. (DOCX 17 kb)

Table S12

The mean number of identical pairs, base transition and transversion at different coding positions of codons and ratio of transitionsal pairs to transversional pairs (R value) of Theaceae plants. (DOCX 21 kb)

Table S13

Accession numbers, version numbers, accepted names, synonyms and definition of matK + ndhF + ycf1 (combined gene sequence) in Theaceae plants on the NCBI online database (XLSX 15 kb)

Table S14

NCBI Blast output of alternative candidate gene sequence segments. (DOCX 18 kb)

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Jiang, S., Chen, F., Qin, P. et al. The specific DNA barcodes based on chloroplast genes for species identification of Theaceae plants. Physiol Mol Biol Plants 28, 837–848 (2022). https://doi.org/10.1007/s12298-022-01175-7

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