Identification of Pueraria spp. through DNA barcoding and comparative transcriptomics

Abstract

Background: Kudzu is a term used generically to describe members of the genus Pueraria. Kudzu roots have been used for centuries in traditional Chinese medicine in view of their high levels of beneficial isoflavones including the unique 8-C-glycoside of daidzein, puerarin. In the US, kudzu is seen as a noxious weed causing ecological and economic damage. However, not all kudzu species make puerarin or are equally invasive. Kudzu remains difficult to identify due to its diverse morphology and inconsistent nomenclature.

Results: We have generated sequences for the internal transcribed spacer 2 (ITS2) and maturase K (matK) regions of Pueraria montana lobata, P. montana montana, and P. phaseoloides, and identified two accessions previously used for differential analysis of puerarin biosynthesis as P. lobata and P. phaseoloides. Additionally, we have generated root transcriptomes for the puerarin-producing P. m. lobata and the non-puerarin producing P. phaseoloides. Within the transcriptomes, microsatellites were identified to aid in species identification as well as population diversity.

Conclusions: The barcode sequences generated will aid in fast and efficient identification of the three kudzu species. Additionally, the microsatellites identified from the transcriptomes will aid in genetic analysis. The root transcriptomes also provide a molecular toolkit for comparative gene expression analysis towards elucidation of the biosynthesis of kudzu phytochemicals.

Keywords: Comparative transcriptomics; DNA barcoding; Kudzu; Microsatellites.

Fig. 1

Morphology of seeds from each kudzu accession. A Oklahoma (wild); B Texas (wild); C PI 9227 (P. m. lobata); D PI 434246 (P. m. lobata); E PI 298615 (P. m. montana); F Kudzu Kingdom (commercial); G BRSEEDS (commercial); H PI 308576 (P. phaseoloides); I DLEG 890244 (P. phaseoloides)

Fig. 2

Images of vines, leaves, and trichomes for each plant accession. A-B Oklahoma (wild); C-D, Texas (wild); E-F PI 9227 (P. m. lobata); G-H PI 434246 (P. m. lobata); I-J, PI 298615 (P. m. montana); K-L Kudzu Kingdom (commercial); M-N BRSEEDS (commercial); O-P PI 308576 (P. phaseoloides)

Fig. 3

Isoflavone profiles of the roots of the eight accessions examined. A HPLC chromatogram showing the isoflavone profiles of the wild and P. m. lobata roots (a. PI 9227, b. PI 434246, c. Oklahoma, d. Texas); B The isoflavone profiles of the commercial and P. phaseoloides roots (a. Kudzu Kingdom, b. BRSEEDS, c. PI 308576); C The isoflavone profile of the P. m. montana roots (PI 298615); D Isoflavone standards. mAU is milli-absorbance units. 1. Puerarin, 2. Daidzin, 3. Genistin, 4. Ononin, 5. Daidzein, 6. Genistein, 7. Formononetin

Fig. 4

Phylogenetic tree of ITS2 sequences from the Pueraria accessions in the present work (colored in blue (wild and P. m. lobata), maroon (P. m. montana), and green (commercial and P. phaseoloides)) and those published in NCBI. The scale bar indicates the length of 0.1 substitutions. The pipeline was created using phylogeny.fr and visualized in Mega 11. (Details for pipeline in Methods)

Fig. 5

Phylogenetic tree of matK sequences from the Pueraria accessions in the present work (colored in blue (wild and P. m. lobata), maroon (P. m. montana), and green (commercial and P. phaseoloides)) and those published in NCBI. The scale bar indicates the length of 0.06 substitutions. The pipeline was created using phylogeny.fr and visualized in Mega 11. (Details for pipeline in Methods)

Fig. 6

Gene ontology classification and homology characteristics of Pueraria root transcript sequences. A Gene ontology analysis of the assembled transcripts. B Species distribution of homology search of Pueraria transcriptomes against the NR database

Fig. 7

Distribution of the assembled Pueraria transcripts mapped to the soybean genome. External track shows the density of P. m. lobata transcripts aligned to the Gmax genome, in both + (outside) and – (inside) strands in purple. The middle track shows the density of P. phaseoloides transcripts aligned to the Gmax genome, in both + (outside) and – (inside) strands in blue. Inner track show the SSR-bearing transcripts aligned to the Gmax genome sequence, with P. m. lobata strands in orange (outside) and P. phaseoloides strands in green (inside)