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https://doi.org/10.1186/s12870-021-03383-x
RESEARCH ARTICLE
Identication ofPueraria spp. throughDNA
barcoding andcomparative transcriptomics
Laci M. Adolfo1†, Xiaolan Rao2† and Richard A. Dixon1*
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: Kudzu, DNA barcoding, Microsatellites, Comparative transcriptomics
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Summary
Various kudzu accessions were analyzed through barcod-
ing and comparative transcriptomics, generating tools for
identification and molecular pathway analysis.
Background
Kudzu has been used in traditional Chinese medicine
with the roots being considered the most valuable part of
the plant [1]. e high levels of isoflavones in the roots
are believed to be important for the medicinal properties
of kudzu [2]. Kudzu contains the same major isoflavones
that are found in other legumes, including the agly-
cones daidzein, genistein, and formononetin as well as
their O-glycosides daidzin, genistin, and ononin. How-
ever, kudzu also contains puerarin, the 8-C-glycoside of
daidzein [3]. Many of the health benefits of kudzu are
believed to come from puerarin, because the carbon-
carbon glycosidic bond in puerarin makes it resistant to
hydrolysis when ingested [2]. However, health benefits
have also been linked to daidzin and genistin, as well as
the methylated isoflavone formononetin and its glyco-
side, ononin. A Chinese pharmacopeia dating back to
200 B.C. mentions the roots of kudzu and their use in
various treatments. Kudzu was administered to help with
a range of ailments including inflammation, diarrhea,
and even alcoholism [4]. In its native habitat, Asia, kudzu
grows well with growth being controlled by pests and
Open Access
*Correspondence: Richard.Dixon@unt.edu
†Laci M. Adolfo and Xiaolan Rao contributed equally to this work.
1 BioDiscovery Institute and Department of Biological Sciences, University
of North Texas, 1155 Union Circle #305220, Denton, TX 76203-5017, USA
Full list of author information is available at the end of the article
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Page 2 of 18
Adolfoetal. BMC Plant Biology (2022) 22:10
climate. In the US, kudzu is an invasive weed, especially
in the southeast [5].
Mass planting of kudzu allowed it to spread rapidly
throughout the Southeast US, where the climate is per-
fect for it, with high temperatures and plenty of rainfall,
and natural predators are absent. Kudzu vines can grow
up to 12 in. a day. Kudzu out-competed native flora and
caused an economic burden as the vines crept up util-
ity poles and disrupted power [5]. e removal of kudzu
is a difficult process as simply removing the top foliage
does not stop the spread of the plant; kudzu’s extensive
root system includes a large tap root from which many
roots and vines sprout [6, 7]). e US federal govern-
ment declared kudzu a federal noxious weed in the mid
to late 1990’s. It was eventually removed from the federal
noxious weed list; however, it is still on the noxious weed
lists of several states, including Texas [7].
e taxonomy of kudzu is unclear, with multiple syno-
nyms and multiple varieties within species, such as Puer-
aria montana, P. thomsonii, and P. lobata which can also
be referred to as P. montana var. montana, P. montana
var. chinensis, and P. montana var. lobata, respectively.
e classification as different species and different vari-
ants has been confusing, especially as the morphologi-
cal characteristics of these individual varieties are highly
variable [8, 9].
e availability of established DNA barcodes that can
differentiate between different species/varieties would
allow for positive identification of kudzu in the wild, and
could aid ecological studies; for example, fecal samples
are often examined to determine the dietary behavior
of animals and insects [10–12]. Furthermore, DNA bar-
coding could facilitate quality control and assurance for
herbal supplements [13–15].
A previous study used kudzu accessions collected in the
field (Ardmore, OK) and obtained commercially (Kudzu
Kingdom, Kodak, TN) to interrogate puerarin biosyn-
thesis through differential expression analysis following
EST sequencing [16]. To aid the identification of these
and other kudzu accessions, we have generated barcodes
for the ITS2 and matK regions of three kudzu species/
varieties. We have also generated transcriptomic data of
the roots of the puerarin producing P. m. lobata and the
non-puerarin producing P. phaseoloides. e transcrip-
tomic data generated allows for differential gene expres-
sion analysis and also identifies simple sequence repeat
(SSRs) markers between the two kudzu species. ese
genomic resources will serve as references for identifying
kudzu species for eradication, harvesting of phytochemi-
cals, validation of supplements, and ecological research.
Additionally, the comparative transcriptomics provides a
molecular resource for exploring genes active in the syn-
thesis of valuable phytochemicals.
Results
Seed morphology
e origins of the kudzu accessions analyzed in the pre-
sent work are provided in the Methods. Wild kudzu col-
lected from Oklahoma and Texas, and USDA PI 434246
and PI 9227 all had kidney-shaped seeds. Most of the
seeds were dark brown with a few being lighter brown
to reddish. e seeds also had lighter colored striations.
ey measured approximately 3.2 mm in length (Fig.1A-
D). e Kudzu Kingdom, BRSEEDS, USDA PI 308576,
and USDA DLEG 890244 seeds were rectangular to
oblong. e seed colors ranged from maroon to orange
to golden yellow and were also approximately 3.2 mm in
length (Fig.1F-I). e USDA PI 298615 seeds were rec-
tangular to oblong, and dark to medium brown in color.
ey were smaller than the other seeds, measuring
approximately 2.1 mm in length (Fig.1E).
Plant morphology
All plants grew as vines with trifoliate leaves and tri-
chomes present on the leaves and stems/vines (Supple-
mental Fig. 1). DLEG 890244 (P. phaseoloides) did not
germinate so analysis of the whole plant, plant parts,
and roots was not possible. e wild kudzu accessions
as well as the P. m. lobata accessions all had prominent
trichomes as did the commercial and P. phaseoloides
accessions; however, the trichomes present on the P. m.
montana accession were less pronounced. e P. m. mon-
tana plants also had smaller, almond shaped leaves and
thinner vines as compared to the other plants (Fig. 2).
e thinner vines on P. m. montana made the vines more
malleable. e leaves of the commercial and the P. pha-
seoloides accessions were rounder than the P. m. mon-
tana accession. Interestingly, the leaves of the wild and
P. m. lobata accessions tended to vary even among the
same accession (Supplemental Fig.2). While some of the
P. m. lobata leaves were rounder, similar to that of the
commercial and P. phaseoloides accessions, others were
lobed. e lobing on the P. m. lobata leaves also var-
ied from slight to deep lobing. However, irrespective of
their overall shape, the leaves of the wild and P. m. lobata
accessions tended to come to a sharp point.
Isoavone content
An examination of the roots of all eight accessions
revealed that the Oklahoma and Texas collected mate-
rial and the P. m. lobata accessions all contained puera-
rin. In contrast, the commercial, P. phaseoloides, and P.
m. montana accessions did not contain puerarin (Fig.3).
In addition to puerarin, roots of the wild and P. m. lobata
accessions contained daidzin and daidzein. Other iso-
flavones, including genistein, genistin, and ononin were
present in reduced amounts in the wild and P. m. lobata
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Adolfoetal. BMC Plant Biology (2022) 22:10
accessions. e commercial and P. phaseoloides roots
contained a higher proportion of genistein, ononin, and
genistin than the Oklahoma and Texas material, P. m.
lobata, and P. m. montana roots. In fact, those three iso-
flavones were found in the highest proportion in roots of
the commercial and P. phaseoloides accessions. e P. m.
montana roots contained the least amount of isoflavones
based on HPLC peak areas, and these were mainly daid-
zin and daidzein (Fig.3C). While not containing puera-
rin, the commercial kudzu and P. phaseoloides had higher
percentages of daidzein and genistein aglycones among
their isoflavone complement (Supplemental Fig.3).
Internal transcribed spacer 2 sequencing
e internal transcribed spacer 2 (ITS2) region is gen-
erally between 200 and 250 bp. Given its small size, the
entire region was able to be captured using primers from
the 5.8S rRNA and 26S rRNA regions that flank the ITS2,
resulting in amplicons of 425–475 bp. An Illumina MiSeq
with paired end reads 2 × 300 was used, allowing for an
overlap in the middle of the sequence. Following trim-
ming and alignment, the whole sequenced amplicon was
468 bp for P. m. lobata, 449 bp for P. phaseoloides, and
436 bp for P. m. montana. e ITS2 region within the
whole amplicon sequence was 242 bp for P. m. lobata,
224 bp for P. phaseoloides, and 211 bp for P. m. montana.
ere were 80 nucleotide differences observed in com-
parisons between the P. m. lobata and the P. phaseoloides
groups in the ITS2 region (Supplemental Table1). Addi-
tional differences in the ITS2 regions were 18 nucleotide
insertions/deletions (indels) in the P. phaseoloides group
including one stretch of eight deleted nucleotides and
one stretch of ten nucleotides (Supplemental Table2).
Comparisons between the P. m. lobata and the P. m. mon-
tana groups revealed 55 nucleotide differences (Supple-
mental Table3) and 31 indels including one stretch of
19 deleted nucleotides in the P. m. montana group (Sup-
plemental Table4). e comparisons between P. phaseo-
loides and the P. m. montana groups had 51 nucleotide
differences (Supplemental Table5) and 17 indels (Supple-
mental Table6).
Maturase K (matK) sequencing
Of the ~ 1500 bp matK chloroplast gene, approximately
776 bp were amplified from the kudzu accessions using
primers suggested by Yu etal. (2011) [17] for having high
fidelity with angiosperms given the low nucleotide diver-
sity found in these regions. Given the length of the ampli-
con to be sequenced, Sanger sequencing was used.
Following trimming and alignment of the matK
sequences there were 17 single nucleotide polymor-
phisms (SNPs) identified between the P. phaseoloides and
P. m. lobata groups, 20 SNPs identified between the P. m.
lobata and P. m. montana groups, and 26 SNPs identified
between the P. phaseoloides and P. m. montana groups
(Table1). Given that matK is a coding region, the amino
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)
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Adolfoetal. BMC Plant Biology (2022) 22:10
acid substitutions that resulted from the SNPs were also
examined. ere were eight amino acid substitutions
between the P. phaseoloides and P. m. lobata groups, 12
between the P. m. lobata and P. m. montana groups, and
15 between the P. phaseoloides and P. m. montana groups
(Table2).
Phylogenetic analysis
A neighbor-joining phylogenetic tree was generated
using the ITS2 and matK sequences. For the ITS2 phy-
logenetic tree the generated sequences were combined
with sequences published in NCBI for kudzu species
as well as other legumes. e results in Fig.4 show that
the P. phaseoloides and commercial accessions clustered
together with a previously published P. phaseoloides ITS2
sequence from NCBI. Additionally, the P. m. lobata and
Texas and Oklahoma ITS2 sequences clustered with P.
m. lobata and P. montana sequences published at NCBI,
along with a singular P. m. thomsonii sequence. e P. m.
montana sequences clustered separately.
e phylogenetic tree for the matK sequences revealed
similar clustering as the ITS2 phylogenetic tree. e P.
phaseoloides and commercial kudzu matK sequences
clustered with published matK sequences for P. phaseo-
loides and N. phaseoloides (formerly P. phaseoloides). e
matK sequences of the P. m. lobata and Oklahoma and
Texas accessions were clustered with a few P. m. lobata
and P. montana sequences plus singular P. m. thomsonii
and P. pseudohirsuta sequences available NCBI. How-
ever, the P. m. lobata, Oklahoma, and Texas kudzu matK
sequences did not cluster as closely with many of the P.
m. lobata and P. montana matK sequences analyzed from
NCBI as they did in the ITS2 neighbor-joining tree. e
P. m. montana matK sequences also clustered separately
again, but this time they were grouped closer to other
species showing more similarity to the matK sequences
of Glycine spp (Fig.5).
Transcriptome sequencing andassembly
To obtain Pueraria root transcriptomes, RNA was
extracted and cDNA prepared from roots of Kudzu
Kingdom (P. phaseoloides) and Oklahoma (P. m. lobata)
accessions, and sequenced by the Illumina Hiseq2000
platform. e 100 bp paired-end Illumina reads were
trimmed with quality scores. Clean sequence reads
from P. phaseoloides and P. m. lobata were assembled
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)
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Adolfoetal. BMC Plant Biology (2022) 22:10
separately using a combination of the programs Velvet
[18] and Oases [19]. To optimize the assembly, Velvet/
Oases were run with different k-mer sizes (31, 43, 55, 67,
79 and 91 nt).
Several assembly-quality parameters were assessed,
including the ratio of using reads, median coverage
depth, the number of contigs, the number of tran-
scripts, the number of loci, average transcript length,
and the N50 values of contigs and transcripts (Supple-
mental Table 7, Supplemental Fig. 4). N50 represents
the sequence length L for which half of the bases in the
assembly are in sequences of length N > =L. [20–22] Of
the six k-mer tests in Velvet/Oases, a good balance for
the above parameters was found at k-mer 55 assembly,
resulting in 47,011 and 49,277 transcripts for P. phaseo-
loides and P. m. lobata, respectively. e full comparison
of the transcriptome data for P. phaseoloides and P. m.
lobata is given in Table3.
To further demonstrate the quality of the assembled
transcripts, the length distribution of the contigs in the
two transcriptomes is shown in Supplemental Fig. 5.
e N50 values of transcriptomes in P. phaseoloides and
P. m. lobata were 1988 and 1881 bp, respectively. For
further quality control, we mapped the assembled tran-
scriptomes to kudzu ESTs available from GenBank (6365
ESTs) and observed that 81% (5183 ESTs) and 96% (6110
ESTs) of known EST sequences were represented in our
transcriptome sets for P. phaseoloides and P. m. lobata,
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
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Adolfoetal. BMC Plant Biology (2022) 22:10
respectively. Kudzu ESTs were provided from a subtrac-
tive library with the P. phaseoloides root cDNA as the
driver and P. m. lobata root cDNA as the target [16]. It
is therefore reasonable that more kudzu ESTs are repre-
sented in the P. m. lobata root transcriptome set than in
the P. phaseoloides set.
Simple sequence repeats (SSRs) inthePueraria root
transcriptomes
Simple sequence repeats (SSRs) or microsatellites have
been broadly used as molecular markers in marker-
assisted selection for DNA fingerprinting [23, 24]. To
supply SSR markers for distinguishing between P. pha-
seoloides and P. m. lobata, we used the MISA scripts
program [25] to scan the Pueraria root transcrip-
tomes to identify gene-derived SSR markers. In total,
we detected 9220 and 6665 SSRs within 6729 and 5370
different transcripts from the P. phaseoloides and P.
m. lobata de novo assembled transcriptomes, respec-
tively. The putative SSRs are summarized in Supple-
mental Dataset 1. Excluding mono-repeats (3246 and
2625), 5974 and 4040 SSRs (dinucleotide to hexanu-
cleotide repeats) were identified within 4516 (13.6%)
and 3373 (9.7%) transcripts of P. phaseoloides and P.
m. lobata, respectively. The average frequency of SSRs
was one per 5.93 kb and 8.53 kb of the transcriptome
sequence in P. phaseoloides and P. m. lobata, respec-
tively. Among dinucleotide to hexanucleotide repeats,
the distribution of SSRs was as follows: di- (2143,
35.9% and 1138, 28.2%); tri- (3255, 54.5% and 2606,
64.5%); tetra- (204, 0.03% and 116, 0.03%); penta- (138,
0.02% and 80, 0.02%) and hexa- (234, 0.04% and 100,
0.02%) in P. phaseoloides and P. m. lobata transcripts,
respectively.
Table 1 Maturase K (matK) SNP analysis
Position SNP Type
P. phaseoloides P. m. lobata P. m. montana
562 C C A Transversion
569 C C G Transversion
581 G T C Variable
606 G T T Transversion
706 T T G Transversion
713–714 TT GC GC Transversion/Transition
780 T T C Transition
807 T T G Transversion
810 G T T Transversion
828 T T C Transition
846 A A C Transversion
891 G A A Transition
894 T C C Transition
905 C A A Transversion
917 A A G Transition
942 T A A Transversion
948 A G G Transition
954 T G T Transversion
966 A C A Transversion
990 G A G Transition
1012 C C T Transition
1014 A C A Transversion
1022 C C T Transition
1023 C A A Transversion
1044 G A G Transition
1045 C C A Transversion
1073 T T G Transversion
1090 A A C Transversion
1098 T A A Transversion
1118 C C T Transition
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Adolfoetal. BMC Plant Biology (2022) 22:10
Annotation, functional classication, mapping
andquantitation ofassembled transcripts
e transcriptome assembly from roots of P. phaseoloides
and P. m. lobata contains 47,011 and 49,277 transcript
isoforms, which represent a total of 33,221 and 34,677
distinct assembled loci, respectively. Each locus may
include several highly similar transcript isoforms, such as
splice variants, homologs and paralogs, and sequencing
errors [16, 22]. To reduce the degree of gene redundancy,
we chose the longest transcript to perform annotation as
the representative of the locus.
A homology search against NR resulted in 24,850
and 27,244 annotated genes in P. phaseoloides and P. m.
lobata, respectively. Among annotated genes, the most
abundant genes are involved in metabolic processes
according to their Gene Ontology (GO) categories using
Plant GOslim ancestor terms [26–28] (Fig.6A). Based on
top hits in the NR database, Pueraria transcripts have
strong homology to transcripts from soybean (Glycine
max), followed by green bean (Phaseolus vulgaris), con-
sistent with the close phylogenetic relationship between
kudzu and soybean [29] (Fig.6B).
To illustrate the coverage distribution of assembled
transcripts on Glycine max as the reference genome, we
aligned the transcripts to the 20 chromosomes in a 500 kb
interval (Fig. 7). Both P. phaseoloides and P. m. lobata
assembled transcripts covered all 20 soybean chromo-
somes without any large gap. e correlation between P.
phaseoloides and P. m. lobata transcriptome density was
0.74, indicating genetic divergence between these two
species. To pinpoint the location of polymorphisms, the
SSR-bearing transcripts were uniquely anchored to the
single best hit in the Glycine max genome. e inconsist-
ency in the SSR locations between P. phaseoloides and P.
m. lobata further indicates the genetic divergence of the
two accessions.
Cross-species transcriptomic comparisons have been
shown to be feasible [30, 31]. erefore, to obtain a
comparative gene expression pattern between the two
Pueraria accessions, we aligned the sequencing reads to
Glycine max as the reference genome [32]. Overall, 65
and 66% of the cleaned reads from P. phaseoloides and
P. m. lobata were mapped to the Glycine max protein
database, respectively, and 84% of Glycine max proteins
were covered with at least one mapped read (Supplemen-
tal Table8). For each Gmax protein code, the number of
matching reads was counted and the hit count was then
transformed to RPKM (the reads per kilobase of tran-
script per million) to normalize for the number of reads
available for each line [30]. e coverage of the functional
classes between P. phaseoloides and P. m. lobata were
similar (Supplemental Fig.6A). e majority of gene cat-
egorieswere well represented by more than 70% of genes
in each class for both mappings. Among them, 87 and
91% of genes classified in secondary metabolism were
detected in P. phaseoloides and P. m. lobata, respectively.
e average RPKM values for each accession were 19.9
and 20.3, respectively. To define “differentially expressed
genes”, we used the criterion of 2-fold difference in RPKM
value with the filter of RPKM value above 20 between the
two RNA samples. By these criteria, 1631 and 1675 genes
were considered as differentially expressed in P. phaseo-
loides and P. m. lobata, respectively. Overall, genes classi-
fied in photosynthesis (PS), oxidative pentose phosphate
pathway (OPP), major and minor carbohydrate (CHO)
metabolism, and secondary metabolism were enriched
in P. m. lobata, whereas genes classified in C1-metabo-
lism, S-assimilation, and DNA and RNA metabolism
were more represented in P. phaseoloides (Supplemen-
tal Fig. 6B). A detailed comparison for genes enriched
in secondary metabolism is shown in Supplemental
Fig.6C. It is clear that the transcriptome of P. m. lobata
is enriched in genes encoding proteins involved in flavo-
noid biosynthesis.
Discussion
Identication ofkudzu species using barcoding
With the verified samples provided by GRIN-Global,
the wild collected and commercial kudzu accessions
compared previously for puerarin production [16] were
identified as P. montana lobata and P. phaseoloides,
respectively. e ITS2 and matK sequences for the P. m.
Table 2 Maturase K (matK) amino acid substitutions
Position Amino acid substitutions
P. phaseoloides P. m. lobata P. m.
montana
188 L L I
190 T T S
194 W L S
202 R S S
236 Y Y D
238 L R R
269 N K K
270 E D D
302 S Y Y
306 Y Y C
318 H Q H
322 L F L
341 S S L
349 Q Q K
358 M M R
364 I I L
373 S S L
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Adolfoetal. BMC Plant Biology (2022) 22:10
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 phylo geny. fr and visualized in Mega 11. (Details for pipeline in Methods)
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Adolfoetal. BMC Plant Biology (2022) 22:10
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 phylo geny. fr and visualized in Mega 11. (Details for pipeline in Methods)
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Page 10 of 18
Adolfoetal. BMC Plant Biology (2022) 22:10
lobata and Oklahoma kudzu accessions matched one
another and had clear differences from the P. phaseo-
loides and commercial kudzus, which also matched one
another, and had clear differences from the P. m. montana
kudzu. e seed morphology of the P. m. montana and P.
phaseoloides was most similar in shape while the seeds of
P. m. lobata and P. phaseoloides were most similar in size.
e plant morphology of the P. m. lobata and the P. pha-
seoloides was most similar with thicker vines and larger
leaves. e P. m. lobata and wild kudzu accessions were
the only plants analyzed that contained puerarin. e
puerarin content for these accessions is consistent with
previous reports [33].
e use of ITS2 and matK combined proved ben-
eficial in strengthening the identification of the different
Pueraria species. Although the ITS2 region analyzed was
smaller than the matK region analyzed, there were more
nucleotide differences found in the ITS2 region, presum-
ably because it is a non-coding region. e ITS2 region
varied in size for all three kudzu species analyzed, from
211 bp to 242 bp. e primers used included a plant-
specific forward primer located in the 5.8S RNA and a
universal reverse primer located in the 26S RNA. e
plant-specific forward primer offers benefits by reduc-
ing the unintended amplification of other organisms such
as fungi. Using the primers in the 5.8S and 26S regions
resulted in an amplicon size between 450 and 500 bp.
is amplicon size was perfect for using next generation
sequencing (NGS). e use of NGS helps reduce noise
that can be generated from amplification and sequenc-
ing bias by allowing for greater depth of coverage. e
greater coverage depth also allows for any incorrect
sequences to be muffled by the true sequence. is noise
was further reduced by using low cycle numbers in the
amplification prior to sequencing. e difference in size
can make alignment difficult; however, using primers in
the relatively conserved 5.8S and 26S regions helps over-
come alignment and amplification problems [34]. In con-
trast, despite the reduced number of nucleotide changes,
the matK region aligned perfectly across all three species
analyzed. e ease of alignment for matK is common
given that it is a coding region of the chloroplast [17].
Table 3 Statistics of the transcriptome data
Data P. phaseoloides P.m. lobata
Raw reads 38,381,722 33,214,058
Clean reads 38,014,210 32,891,280
Assembled transcripts 47,011 49,277
Percent assembled 87.8 82.9
Assembled depth 11.9 10.3
Mean length 1320 1239
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
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 11 of 18
Adolfoetal. BMC Plant Biology (2022) 22:10
e use of published ITS2 and matK sequences from
other plants and Pueraria species in a neighbor-joining
tree with the sequences generated showed clear cluster-
ing of the P. phaseoloides and commercial kudzus with P.
phaseoloides plants published in NCBI. e P. m. lobata
and wild-collected Oklahoma and Texas kudzu clustered
with the other P. m. lobata and P. montana sequences
while the P. m. montana sequences clustered separately.
e neighbor-joining trees for both genes resulted in
similar clades encompassing the different accessions
analyzed along with the published sequences in NCBI.
Although a single concatenated tree that included both
genes could have provided additional resolving power to
show the relatedness of all the accessions analyzed, there
was a lack of ITS2 and matK sequences in NCBI from the
same samples of kudzu and other legumes, making such
analysis not possible.
Unlike with animals where the cytochrome oxidase I
(COI) gene of the mitochondria is considered the gold
standard for species differentiation, plants do not cur-
rently have a specific region that is accepted as having
good discriminatory value. However, several regions have
been proposed as well as the use of two regions together
[35, 36]. e ITS2 region has been shown to have high
discriminatory power in both Fabaceae genera and angi-
osperms [37–43]. In Vignaspecies, coupling matK and
ITS2 increased the resolving power of the barcodes com-
pared to using them individually [40].
e use of the ITS2 and matK regions can success-
fully differentiate species of the genus Pueraria as well
as variants of the same species. e ITS2 and matK for
P. m. lobata and P. phaseoloides were generated from
four different populations of the respective species. e
sequences for the populations of each species matched
one another as well as from samples within the popula-
tions. is shows that for the kudzu species analyzed,
ITS2 and matK have enough nucleotide exchange to dif-
ferentiate the different species but do not segregate out
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)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 12 of 18
Adolfoetal. BMC Plant Biology (2022) 22:10
different populations of the same species. e ability of
these two regions to not set apart different populations
of the same species is extremely important in allowing for
clear identification of kudzu species regardless of where
the plant originated. e ITS2 and matK sequences
generated have been uploaded to BOLD (Barcode of
Life Database) [44] to be available to other researchers
attempting to identify plants whether directly or through
the examination of plant material present in supplements
or even in the feces of organisms to understand their diet
as done by Yamamoto and Uchida (2018) [12].
Interestingly, the seed and plant morphology, barcod-
ing sequence differences, and phylogenetic separation
between P. montana montana and P. montana lobata
would suggest that these plants are more than mere vari-
eties of the same species as suggested by van der Maesen
[8]. e differences present at both a phenotypic and gen-
otypic level for these plants align with their being sepa-
rate species as previously suggested by Ohashi etal. [45].
Ohashi etal. suggest the presence of two species, P. mon-
tana and P. lobata, where P. lobata has the subspecies P.
l. lobata and P. l. thomsonii. A comprehensive analysis of
P. l. thomsonii (also known as P. thomsonii and P. m. chin-
ensis) as done here for P. m. lobata, P. m. montana, and P.
phaseoloides could discern whether P. l. thomsonii is best
categorized as a subspecies of Pueraria lobata or as its
own species.
Summary ofthetranscriptome dataset
e rapid development of next-generation sequenc-
ing (NGS) technologies has enabled discovery of novel
genes by using the RNA-seq approach [46, 47]. To pro-
vide a basis for a better understanding of the bioactive
natural products in kudzu, we have performed a com-
parative whole root transcriptome analysis. ree other
reports have generated transcriptomes for different tis-
sue types of P. m. lobata [48–50], and more recently,
for different tissues of P. thomsonii and P. candollei var.
mirifica [51, 52]. However, none of these analyses exam-
ine two different kudzu species for comparative gene
expression. A previous phylogenetic study showed 80%
of US kudzu analyzed had matching genotypes with one
or more samples from the same population [53]. is
suggest that the transcriptome generated from kudzu
from Oklahoma (P. m. lobata) could be a representa-
tive genomic resource for this noxious weed that domi-
nates throughout the Southeastern US. In Oklahoma
alone a report suggests a loss of almost $168 million in
the lumber industry over 5 years [54]. Knowledge of its
transcriptome can lead to development of methods of
biological eradication.
It is challenging to perform de novo assembly of tran-
scriptomes in non-model organisms lacking a reference
genome. Early studies demonstrated that optimization of
the transcriptome assembly using various k-mer lengths
is highly desirable for de novo assemblies [22, 55, 56].
In the present study, various parameters were analyzed
with a combination of Velvet and Oases. Velvet/Oases
start by constructing de Bruijn graphs directly from
sequencing reads, remove errors, and then resolve each
de Bruijn graph to extract transcripts for each connected
component (called “loci”) in the graph [18, 19, 22]. Vel-
vet/Oases allow a range of k-mer sizes to accommodate
variation in read coverages among genes. Longer k-mers
lead to more specificity, with lower coverage and sensi-
tivity. Assembly quality decreases towards both lower
and higher k values [18, 19, 22]. Assembly quality tests
were performed to determine the most suitable param-
eter; the usage ratio of reads, depth, length, and number
of assembled transcripts [22, 55, 56]. e Velvet/Oases
k–mer 55 assembly was selected as the representative
for the Pueraria root transcriptomes, resulting in 47,011
and 49,277 transcripts with 33,221 and 34,677 loci,
respectively. is is consistent with the gene number
for the majority of sequenced plant genomes of between
20,000 and 40,000 [21].
Dierentiation ofPueraria species
Simple sequence repeats (SSRs) markers have been
widely used in plant genetic studies because of their
tendency toward being multiallelic, expression of both
parental alleles, quantity, and vast coverage in genomes
[57]. Genic SSRs (derived from genes, ESTs, or cDNA
clones) have some advantages over genomic SSRs includ-
ing being easily generated, characterized, and possessing
transferability between different species [58].
Previous markers identified to distinguish kudzus
included 13 allozyme loci, 11–49 randomly amplified
polymorphic DNAs (RAPDs), and 13–15 microsatellite
locations [9, 53, 59–62]. Most recently, genic SSRs were
identified from P. m. montana and P. phaseoloides [63].
Some of these reports used other kudzu species or varie-
ties; however, the goal of all of them was beyond identifi-
cation and focused more on population/genetic diversity
and origin of kudzu’s introduction. Here we identified
9220 and 6665 genic SSRs from the assembled tran-
scripts from P. phaseoloides and P. m. lobata, respectively.
Excluding mono-SSRs, 5974 and 4040 genic SSRs were
detected in 13.6 and 9.7% of the transcripts with the fre-
quency of one SSR per 5.93 kb and 8.53 kb in the P. pha-
seoloides and P. m. lobata transcriptomes, respectively.
Frequencies of genic SSRs were reported as 1 per 3.92 kb
or 8.63 kb from de novo assembled transcriptomes in the
legume species lentil and chickpea, respectively [56, 64].
Additionally, the genic SSR frequency in Chinese sweet-
gum was 1 per 5.12 kb [65].
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 13 of 18
Adolfoetal. BMC Plant Biology (2022) 22:10
Factors affecting the frequency and types of SSRs
include the taxon, the genomic make-up, and the SSR
mining length used for analysis [66]. Here we applied
the same parameters for mining microsatellites in the P.
phaseoloides and P. m. lobata transcriptomes, so the dif-
ferences in SSR frequency likely indicate differences in
genomic composition. Except for mono-repeats, the most
abundant SSRs were tri-nucleotide repeats (54.5 and
64.5%), then di-nucleotide repeats (35.95 and 28.2%) in
P. phaseoloides and P. m. lobata transcripts, respectively.
is is consistent with the observation that tri-SSRs are
generally the most frequently occurring SSRs found in
genic SSRs, followed by di-SSRs [58, 67]; however, there
are exceptions as with Camellia japonica [68]. Among all
the tri-nucleotides, AAG/CTT was found to be the most
frequent motif, consistent with recent studies [69–71].
Our results suggest that the SSRs identified here are reli-
able and can be useful tools for assaying genetic variation
in Pueraria populations.
Cross-species mapping in protein space is a viable
strategy to compare different species when an equidis-
tant reference is available [30]. rough mapping reads
by alignment on the soybean protein sequence, we quan-
tified transcript abundance in P. phaseoloides and P. m.
lobata. Transcripts catalogued in photosynthesis, major
CHO metabolism and minor CHO metabolism were
enriched in the wild-collected, invasive P. m. lobata com-
pared with the commercial species P. phaseoloides. is
is consistent with the competitive ability of P.m. lobata
for fixing carbon [72]. Transcripts classified in secondary
metabolism were also enriched in P. m. lobata, particu-
larly genes involved in flavonoid biosynthesis.
Conclusions
Puerarin is found in some but not all species of Pueraria.
Here we have identified the ITS2 and matK barcodes as
sufficient to differentiate between three kudzu species
(P. montana, P. lobata, and P. phaseoloides), and in so
doing identified the wild and commercial kudzu species
used previously for preliminary gene identification in the
puerarin pathway [16]. We have also provided molecular
tools for more in-depth differential expression analysis of
natural product pathways between transcriptomes of P.
m. lobata and P. phaseoloides, as well as the identification
of microsatellites for further use to aid in identification of
the two species.
Methods
Chemicals
Daidzin, genistein, and genistin were purchased from
Cayman Chemical Company (Ann Arbor, MI). All other
standards were purchased from Indofine Chemical
Company (Hillsborough, NJ). HPLC solvents were from
FisherSci (Walthanm, MA). Other chemicals were pur-
chased from Sigma-Aldrich (St. Louis, MO) unless oth-
erwise indicated.
Seeds
Oklahoma wild kudzu seeds were collected (under Texas
Department of Agriculture permit no 14-NIPP-01) from
P street SE, near the junction with Springdale Road, in
Ardmore, OK (34.159, − 97.108). e kudzu from Okla-
homa had previously been identified as P. montana [73].
Kudzu Kingdom seeds were ordered from Kudzu King-
dom, a division of SunTop Inc., in Kodak, TN. Texas wild
kudzu seeds were collected (under Texas Department of
Agriculture permit no 19-NIPP-01) off Copeland road
under Batman the ride at Six Flags Over Texas in Arling-
ton, TX (32.759, − 97.067). e kudzu from Texas had
previously been identified as P. m. lobata and validated
by Texas Invaders (Site Record 19,737). BR seeds were
ordered from the company BRSeeds in Araçatuba, São
Paulo, Brazil as P. phaseoloides. P. montana (Lour.) Merr.
var. lobata (Willd.) collected in the United States (PI
434246); P. montana (Lour.) Merr. var. lobata (Willd.) col-
lected in Kanagawa, Japan (PI 9227); P. montana (Lour.)
Merr. var. montana donated from Taiwan (PI 298615);
Neustanthus phaseoloides (Roxb.) Benth. (formerly P.
phaseoloides (Roxb.) Benth.) collected in Venezuela (PI
308576) were ordered through USDA Grin Global from
the Plant Genetic Resources Conservation Unit in Grif-
fin, GA (under Texas Department of Agriculture permit
no 19-NIPP-01 where applicable). N. phaseoloides (DLEG
890244) seeds collected from an unknown location were
ordered through USDA Grin Global from the Desert
Legume Program in Tucson, AZ. Seeds ordered through
USDA Grin Global were verified by an ARS Systematic
Botanist and are publicly available.
Seed sterilization, germination, andplant growth
conditions
Seeds were scarified in sulfuric acid for 20 min (BR seeds,
Kudzu Kingdom seeds, USDA P. phaseoloides, and USDA
P. montana var. montana seeds), or 45 min (Texas, Okla-
homa, and USDA P. montana lobata (Origins Japan and
US). ey were then rinsed with copious amounts of
water three times, dried and sterilized in 20% (v/v) bleach
for 5 min. e seeds were allowed to dry before being
plated on water agar. e plates were placed in the dark
at 4 °C for 5 days, then moved to a 24 °C light chamber
and monitored for germination. Once germinated the
seeds were placed in a greenhouse with temperature set-
tings from 20 °C–28 °C and at least 14 h of light.
For root isoflavone analysis a young vine was cut from
the main plant and the cut tip dipped in IBA (indole
3-butyric acid) before being placed in damp soil. e
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 14 of 18
Adolfoetal. BMC Plant Biology (2022) 22:10
cuttings were monitored and after 4 weeks were repotted.
After 8 weeks the roots were washed of excess soil and
harvested for isoflavone analysis.
DNA isolation
Tissues, including leaves and seeds, were collected and
placed in 2 mL Eppendorf tubes with a single ball bear-
ing. e tubes were placed in liquid nitrogen and the tis-
sue was ground using a Retsch Mixer Mill 400 at 30 Hz
for 15 s. e samples were then checked for degree of
grinding and placed in liquid nitrogen. If the tissue was
not thoroughly ground, it was run on the Retsch Mill
again until efficient tissue grinding was achieved.
Tissue was suspended in 500 μL of 2X CTAB extrac-
tion buffer, vortexed for 5 s to mix and placed in a 60 °C
oven for 30 min with occasional mixing. Tissue was cen-
trifuged at room temperature at 16,000 x g for 5 min. e
upper liquid was transferred to a new tube being careful
to avoid the tissue debris. An equal volume of cold chlo-
roform was added to the tubes, which were then vor-
texed for 5 s and centrifuged at 4 °C for 10 min at 12,000
x g. e upper aqueous phase was carefully transferred
to a new tube, an equal volume of cold chloroform was
added, the mixture vortexed for 5 s and then centrifuged
at 4 °C for 10 min at 12,000 x g. e upper aqueous phase
was collected, an equal volume of cold isopropanol was
added, the tube incubated at room temperature for
10 min, and then centrifuged at 4 °C for 10 min at 12,000
x g. e liquid was carefully poured off and 1 mL of 70%
(v/v) ethanol was added to the tube, which was centri-
fuged for 1 min at room temperature at 12,000 x g. e
liquid was again poured off, the tube re-centrifuged for
10 s and the remaining liquid carefully removed avoiding
the pellet. e tube was briefly placed in a centrifuge with
a cold trap (SpeedVac) to remove any residual ethanol.
e pellet was resuspended in 50 μL ddH2O. e DNA
concentration was calculated on a NanoDrop™ 2000.
Flavonoid extraction
Root tissue was collected from plants and placed in a
2 mL Eppendorf tube with a single ball bearing. e tis-
sue was placed in liquid nitrogen before being lyophi-
lized on a Labconco freeze dryer for 3 days. e tube was
then placed in liquid nitrogen and ground on a Retsch
Mixer Mill 400 at 30 Hz for 15 s. Twenty mg of tissue was
transferred to a new tube and remaining tissue stored at
-80 °C. e 20 mg of tissue was resuspended in 1.5 mL of
80% (v/v) methanol and sonicated for 1 h in an ice water
ultrasonic bath (Branson, Danbury, CT). Following soni-
cation, the tubes were placed on an end-over-end rotator
at 4 °C overnight, then centrifuged for 20 min at 12,000 x
g. e supernatant was transferred to a new tube being
careful to avoid the tissue debris pelleted at the bottom of
the tube. e tubes were placed on a nitrogen evaporator
(Organomation Associates Inc., Berlin, MA) to dry under
a stream of air/nitrogen. After the contents of the tubes
had dried, 250 μL of ddH2O was added and the tubes
placed on an end-over-end rotor at 4 °C for 1 h.
Ethyl acetate extraction of flavonoids was performed
twice by adding 2 times the volume of ethyl acetate to
the tube, inverting to mix, and centrifuging at 12,000 x
g for 10 min at 4 °C. e top layer was transferred to a
new tube and dried under a stream of air/nitrogen on an
Organomation nitrogen evaporator. e contents of the
tubes were resuspended in 150 μL of 100% methanol. e
samples were then analyzed by HPLC.
ITS2 metagenomic sequencing
e ITS2 region was sequenced in collaboration with the
BioDiscovery Institute (BDI) Genomics Center (Denton,
TX) and Salient Genomics LLC (Krum, TX). Total DNA
was used to amplify the ITS2 regions with barcode and
index adapters attached to ITS2 primer sequences ITS-
p3/ITS-u4 [34]. e samples were prepped and run on
an Illumina MiSeq (Illumina, Inc., San Diego, CA). Prior
to sequencing, DNA from every accession was amplified
with the ITS-p3/ITS-u4 primers to check amplicon size
[34]. When run on a 1% agarose gel, all of the amplicons
ran just under the 500 bp band of the ladder, consistent
with the expected amplicon size of around 450 bp. How-
ever, the size of the P. m. montana amplicon was slightly
lower than that of the other accessions consistent with
the sequencing results.
matK Sanger sequencing
DNA samples were amplified with matK primers [17]
using NEB’s Q5 Hot-start polymerase following the man-
ufacturer’s instructions including extension time. e
annealing temperature was calculated using NEB’s Tm
calculator. Following amplification, the samples were sent
to Eurofins Genomics (Louisville, KY) for PCR clean-up
and one-pass Sanger method sequencing. To confirm the
amplicons prior to sequencing, they were run on a 1%
agarose gel. All the amplicons ran between the 500 bp and
1000 bp band of the ladder, consistent with the expected
amplicon length of around 775 bp.
Barcode sequence analysis
Barcoding sequences were analyzed using Geneious
Prime (San Diego, CA). Once the sequences were
imported in Geneious Prime they were paired and
trimmed using the BBDuk plugin to remove Illumina
adapters as well as low quality (below 30) and short (less
than 100 bp) reads (for ITS2 sequences). e forward and
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 15 of 18
Adolfoetal. BMC Plant Biology (2022) 22:10
reversed reads were merged together using BBMerge.
Merged sequences with a length between 430 and 480 bp
were extracted (for ITS2 sequences). e reads were
assembled de novo using the Geneious assembler and a
consensus sequence was generated for each sample. e
samples were aligned for each amplicon group to identify
SNPs and Indels between the three accessions.
Barcoding phylogenetic trees
e phylogenetic trees were made using a pipeline built
with phylogeny.fr. e pipeline settings used MUSCLE
for the sequence alignment, Gblocks for the alignment
curation, and ProtDist/FastDist + BioNJ for building
the phylogenetic tree with a bootstrap value of 1000.
e phylogenetic tree was viewed and edited with
Mega 11 [74–81].
HPLC analysis
Twenty μL samples were injected on an Agilent 1220
Infinity II with a C18 reverse phase column. e 50 min
run used the solvents 0.1% (v/v) formic acid (A) and ace-
tonitrile (B) with a gradient as follows: 0–5 min, 95% A;
5–10 min, 85% A; 10–25 min, 77% A; 25–30 min, 67% A;
30–35 min, 60% A; 35–40 min, 0% A; 40–45 min, 0% A;
45–50 min, 95% A with a flow rate of 1 mL/ min. Absorp-
tion was measured at 254 nm.
RNA extraction, cDNA library construction andIllumina
sequencing
As described [82], each RNA-library was prepared from
1 μg of total RNA isolated from one sample each of Kudzu
Kingdom (P. phaseoloides) and Oklahoma (P.m. lobata)
roots using TruSeq RNA Sample Prep Kits v2 (Illumina
Inc., San Diego, CA), according to the manufacturer’s
instructions, at the Genomics Core Facility at the Noble
Foundation. e prepped samples with individual indexes
were pooled together to run on one Hiseq2000 lane tar-
geting 100 bp paired reads. e Hiseq2000 run was con-
ducted at the Genomics Core Facility of the Oklahoma
Medical Research Foundation, Oklahoma City.
Short read de novo assembly oftranscriptomes
Processing of the 100 bp paired-end Illumina reads began
by interleaving the read mates for each sample into a
single file and trimming bases with quality scores of 20
or less from the end of each read. Reads less than 40 bp
long after trimming were discarded along with their
mates [82]. Each of the Pueraria root Illumina libraries
was assembled separately using a combination of Vel-
vet 1.2.10 [18] and Oases 0.2.08 [19]. To optimize the
assembly towards higher contiguity and specificity, Vel-
vet was run using different hash lengths (k-mers 31, 43,
55, 67, 79 and 91) with an average insert length of 300 bp.
e results of the Velvet assemblies were then run
through Oases using an insert length of 300 bp. Other
parameters of Velvet and Oases were set as default.
Annotation
e assembled transcript isoforms were searched
against the NCBI NR database using blastx alignment
(1e-6) [83], and further annotated with default param-
eter values using Blast2Go [84]. After the Blast2Go
mapping process, EC numbers from the KEGG pathway
[85] and GO terms were generated.
SSR detection
In a pre-process step, poly-T (poly-A) stretches from
the 5′ (3′) were removed by EST-trimmer scripts
[86]. Parameters were set as removing (T)5 or (A)5
in a range of 50 bp on the 5′- or 3′-end, respectively.
Sequences of less than 100 bp were discarded and
sequences larger than 3000 bp were clipped at their
3′ side [30]. Then trimmed sequences were analyzed
using MISA scripts [30] to identify Simple Sequence
Repeats (SSRs). Mono-, di, tri-, tetra-, penta- and
hexanucleotide repeats with a minimum of 10, 7, 5, 5,
5, and 5 subunits were regarded as SSRs, respectively.
Mapping andquantication ofsequence reads
As described [30], the Illumina sequence reads were
mapped onto coding sequences of the Glycine max
genome (version Gmax_275_Wm82.a2.v1 download
from Phytozome website) by blastx [83] with threshold
as 1e-6. To reduce multiple-mapping problems, cod-
ing sequences from primary transcripts without alter-
native splice sites in the Glycine max genome were
used [32]. The blastx output was parsed with in-house
PERL scripts to count the number of reads mapped to
each Glymax protein and then to calculate the RPKM
value for every Glymax protein in each library.
Abbreviations
BLAST: Basic local alignment search tool; BOLD: Barcode of life database;
CHO: Carbohydrate; COI: Cytochrome oxidase I; CTAB: Cetyltrimethylam-
monium bromide; DNA: Deoxyribonucleic acid; EC: Enzyme nomen-
clature; EST: Expressed sequence tag; GO: Gene ontology; GPS: Global
positioning system; GRIN: Germplasm resource information network;
HPLC: High performance liquid chromatography; IBA: Indole 3-butyric
acid; ITS: Internal transcribed spacer; ITS2: Internal transcribed spacer
2; KEGG: Kyoto encyclopedia of genes and genomes; matK: Maturase
K; mAU: Milli-absorbance units; MISA: Microsatellite identification tool;
NCBI: National center for biotechnology information; NGS: Next genera-
tion sequencing; NR: Non-redundant protein; OPP: Oxidative pentose
phosphate; PCR: Polymerase chain reaction; PERL: Practical extraction
and reporting language; PS: Photosynthesis; RNA: Ribonucleic acid;
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 16 of 18
Adolfoetal. BMC Plant Biology (2022) 22:10
RPKM: Reads per kilobase of transcript per million; SNP: Single nucleo-
tide polymorphism; SRA: Sequence read archive; SSR: Simple sequence
repeat; USDA: United States Department of Agriculture.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s12870- 021- 03383-x.
Additional le1: Supplemental Figure1. Images of vines and whole
plant morphology. Supplemental Figure2. Leaves from USDA PI 9227
P. m. lobata plants. Supplemental Figure3. Percent composition of six
common isoflavones in each of the seven accessions. Supplemental
Figure4. Quality measurements for Velvet/Oases assemblies. Supple-
mental Figure5. Length distribution of the assembled transcripts in P.
phaseoloides and P. m. lobata. Supplemental Figure6. Pathway repre-
sentation analysis of the soybean transcripts mapped by Pueraria reads.
Supplemental Table1. ITS2 nucleotide changes between P. m. lobata and
P. phaseoloides. Supplemental Table2. ITS2 insertions/deletions between
P. m. lobata and P. phaseoloides. Supplemental Table3. ITS2 nucleotide
changes between P. m. lobata and P. m. montana. Supplemental Table4.
ITS2 insertions/deletions between P. m. lobata and P. m. montana. Sup-
plemental Table5. ITS2 nucleotide changes between P. phaseoloides and
P. m. montana. Supplemental Table6. ITS2 insertions/deletions between
P. phaseoloides and P. m. montana. Supplemental Table7. Assembly
statistics (Velvet/Oases) for P. phaseoloides and P. m. lobata. Supplemental
Table8. Statistics of Pueraria reads mapped to soybean by BLAST.
Additional le2: Supplemental Dataset 1. Putative SSRs from tran-
scripts of P. phaseoloides and P. m. lobata.
Acknowledgements
We thank the Desert Legume Program and the Plant Genetic Resources
Conservation Unit, Griffin, GA in connection with GRIN-Global for supplying
seeds. We thank Awinash Bhatkar of the Texas Department of Agriculture for
supplying the kudzu transportation permit. We thank Sebastien Santini (CNRS/
AMU IGS UMR7256) and the PACA Bioinfo platform (supported by IBISA) for
the availability and management of the phylo geny. fr website used to build
neighbor-joining phylogenetic trees for barcode sequence comparison.
Authors’ contributions
LMA performed DNA barcoding analysis. XR performed RNA-seq analysis. RAD
conceived experiments, and funded and guided research. All authors read and
approved the final manuscript.
Funding
This work was supported by the University of North Texas using start-up funds
awarded to Dr. Richard Dixon. The funding body played no role in the design
of the study and collection, analysis, and interpretation of data and in writing
the manuscript.
Availability of data and materials
The DNA barcoding sequences are available on the BOLD system
with the processIDs KUDZU002–21 to KUDZU046–21. Sequence data
from this article can be found in the NCBI Sequence Read Archive
(SRA) repository, NCBI SRA accession No. SRX768865. The assembled
transcriptomes can be found at NCBI, accession numbers 10672212
and 10671973.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 BioDiscovery Institute and Department of Biological Sciences, University
of North Texas, 1155 Union Circle #305220, Denton, TX 76203-5017, USA. 2 Col-
lege of Life Sciences, Hubei University, Wuhan 430068, Hubei Province, China.
Received: 12 July 2021 Accepted: 5 December 2021
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