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Gao et al. BMC Genomics (2024) 25:277
https://doi.org/10.1186/s12864-024-10102-x BMC Genomics
*Correspondence:
Guohui Shen
zb5@saas.sh.cn
Zhihui Tian
tianzhihui@saas.sh.cn
Full list of author information is available at the end of the article
Abstract
Background Indian jointvetch (Aeschynomene indica) is a common and pernicious weed found in the upland direct-
seeding rice elds in the lower reaches of the Yangtze River in China. However, there are few reports on the degree
of harm, genetic characteristics, and management methods of this weed. The purpose of this study is to clarify the
harm of Indian jointvetch to upland direct-seeding rice, analyze the genetic characteristics of this weed based on
chloroplast genomics and identify its related species, and screen herbicides that are eective in managing this weed
in upland direct-seeding rice elds.
Results In a eld investigation in upland direct-seeding rice paddies in Shanghai and Jiangsu, we determined
that the plant height and maximum lateral distance of Indian jointvetch reached approximately 134.2cm and
57.9cm, respectively. With Indian jointvetch present at a density of 4/m2 and 8/m2, the yield of rice decreased by
approximately 50% and 70%, respectively. We further obtained the rst assembly of the complete chloroplast (cp.)
genome sequence of Indian jointvetch (163,613bp). There were 161 simple sequence repeats, 166 long repeats, and
83 protein-encoding genes. The phylogenetic tree and inverted repeat region expansion and contraction analysis
based on cp. genomes demonstrated that species with closer anity to A. indica included Glycine soja, Glycine max,
and Sesbania cannabina. Moreover, a total of 3281, 3840, and 3838 single nucleotide polymorphisms were detected
in the coding sequence regions of the cp. genomes of S. cannabina voucher IBSC, G. soja, and G. max compared with
the A. indica sequence, respectively. A greenhouse pot experiment indicated that two pre-emergence herbicides,
saufenacil and oxyuorfen, and two post-emergence herbicides, orpyrauxifen-benzyl and penoxsulam, can more
eectively manage Indian jointvetch than other common herbicides in paddy elds. The combination of these two
types of herbicides is recommended for managing Indian jointvetch throughout the entire growth period of upland
direct-seeding rice.
Complete chloroplast genome
and comparison of herbicides toxicity
on Aeschynomene indica (Leguminosae)
in upland direct-seeding paddy eld
YuanGao1, TianYuChen2, JiaqiLong2, GuohuiShen1* and ZhihuiTian1*
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Page 2 of 13
Gao et al. BMC Genomics (2024) 25:277
Background
e genus Aeschynomene in the family Leguminosae is
widely distributed in tropical and subtropical regions
of the world, mostly in America, Africa, and Asia, with
approximately 200 species identied to date [1–3].
Aeschynomene is an annual herbaceous plant that grows
as shrubs, with shield-shaped stipules, buttery-shaped
owers, and segmented leaves connected by diaphragms
[4]. Only one species of this genus has been found in
China to date, namely A. indica (Indian jointvetch) [2]. It
usually germinates in April and its seeds mature in Octo-
ber. e germination of Indian jointvetch seeds mainly
depends on the temperature during the day [5]. Research
has shown that the seeds of Indian jointvetch have dor-
mancy characteristics, which can survive for about six
months in the soil seed bank and mechanical scarica-
tion is an eective method to break dormancy [6].
Although this plant does not germinate under ooded
conditions, once it grows, Indian jointvetch indirectly
aects crop productivity by competing for resources,
hindering harvesting operations, and reducing grain
and seed quality [7–9]. Moreover, it has been conrmed
that mixing seeds of A. indica into rice as pig feed can
induce the development of pig diseases [1]. A. indica was
reported to be a weed in irrigated rice elds previously
[10, 11] and was considered the third most troublesome
weed after weedy rice and barnyard grass in certain rice-
producing areas [12]. However, A. indica was recently
found to impair the growth of upland direct-seeding
rice in the lower reaches of the Yangtze River in China
(Additional File 3, Figure S1). Because the planting area
of upland direct-seeding rice is increasing owing to the
global shortage of labor and water resources, more and
more attention has been paid to the serious weed prob-
lem [13–16]. Distinguishing plants of the genus Aeschyn-
omene is very dicult, and managing these weeds in rice
elds is also very challenging presently [3]. erefore,
further research is required to elucidate the characteris-
tics of this species, including the genetic characteristics,
degree of harm induced, and responses to available her-
bicides for eective management and control.
Chloroplasts are the main organelles involved in pho-
tosynthesis in photosynthetic plants or algae [17]. Chlo-
roplasts have their own distinct genetic material with
characteristics of non-recombination, haploid, and
single-parent inheritance, which leads to a highly con-
served genome; therefore, analysis of the chloroplast (cp.)
genome can provide very rich evolutionary information
[18–20]. Additionally, the cp. genome is small and easy to
obtain, oering unique research value in phylogeny, spe-
cies identication, and population genetics [21]. e cp.
genome has a typical tetrad structure, with large single
copy (LSC) and small single copy (SSC) regions sepa-
rated by inverted repeats (IRs) [22–24]. Previous studies
have shown that in the context of network evolution (i.e.,
hybridization) and polyploidy, analysis of the cp. genome
is particularly useful for characterizing the phylogenetic
and historical aspects of most plant lineages [25–27].
However, information on the composition, structure,
interspecic dierences, and evolutionary relationships
of A. indica based on its cp. genome is limited. erefore,
exploring the cp. genomic information of A. indica can
provide a theoretical basis for the management or utiliza-
tion of this species and predict the possibility of evolu-
tion of related species into paddy weeds.
Current weed control methods in upland direct-seed-
ing rice elds mainly include cultural weed management
practices and physical methods such as deep plowing,
germination, mechanical weeding, and hand-pulling
weeding, along with chemical methods using herbicides
[28–30]. Chemical methods for weed control in upland
direct-seeding rice elds have specic advantages of
obtaining higher yields with relatively lower labor costs
[30–32]. Aeschynomene weeds are often controlled using
a combination of imazapyr and imazapic herbicides [3].
However, this genus of weed has been found to exhibit
imazapic resistance [33]. In addition, this herbicide is
typically considered to be unsafe for rice growth. To our
knowledge, there have been no systematic studies on
the toxicity of common herbicides used for A. indica in
rice elds. erefore, screening for suitable herbicides
to eectively control A. indica is of great signicance to
achieve a high and optimal yield of upland direct-seeding
rice.
To address these questions, we performed an in-depth
analysis of the genetic characteristics and herbicide
response of A. indica growing in the upland direct-seed-
ing rice elds of Shanghai, China. We studied the mor-
phological characteristics of this weed in rice elds and
its eect on rice yield. e cp. genome of A. indica was
then sequenced and compared to those of other species
to analyze their genetic characteristics and evolution-
ary relationships. Finally, the toxicities of common her-
bicides currently used in rice elds in China against A.
indica were evaluated in a greenhouse pot experiment
to determine a reasonable chemical control method. Our
Conclusions This study provides molecular resources for future research focusing on the identication of the
infrageneric taxa, phylogenetic resolution, and biodiversity of Leguminosae plants, along with recommendations for
reliable management methods to control Indian jointvetch.
Keywords Indian jointvetch, Chloroplast genome, Related species, Upland direct-seeding rice, Herbicide
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Page 3 of 13
Gao et al. BMC Genomics (2024) 25:277
study thus provides a theoretical and practical founda-
tion for gaining a better understanding of the damage of
the troublesome dicotyledonous weed A. indica imposes
in upland direct-seeding rice elds and facilitating strate-
gies for its eective management.
Methods
Plant materials
Indian jointvetch seeds were collected from upland
direct-seeding rice elds in Jinshan District, Shanghai,
China (N 30.81°, E 121.18°) in October 2021. e seeds
were dried to constant weight after collection and stored
at 4°C until planted.
Determination of eld traits
Plant height and the maximum lateral distance of Indian
jointvetch growing in upland direct-seeding rice elds in
Shanghai and Jiangsu Province were measured in Octo-
ber 2021. More than 20 plants were randomly selected;
however, no more than three plants were sampled from
each paddy eld. Seeds were collected, dried to a con-
stant weight, and weighed. A total of 294 upland direct-
seeding rice elds were investigated, elds with Indian
jointvetch were recorded, and their frequency was
calculated.
Subsequently, 0, 1, 2, 3, and 4 Indian jointvetch seed-
lings grown in a greenhouse previously were trans-
planted into a 1 m2 upland direct-seeding rice planting
area and all other weeds were manually removed. When
the upland direct-seeding rice had matured, the eective
spike number, grains per spike, and 1000-seed weight of
upland direct-seeding rice seeds were measured. Each
treatment contains three biological replicates and the
entire experiment was repeated twice. Data of percent-
ages of fresh weight compared to the control (none trans-
planted Indian jointvetch) were subjected to ANOVA.
For comparison of the dierences in the rice yield indica-
tors among the ve groups, the Duncan’s Multiple Range
Test (P < 0.05) was used. ANOVA was conducted using
SPSS version 20 (SPSS, Chicago, IL, USA).
Construction of the A. indica cp. genome
DNA sequencing and genome assembly
Total genomic DNA from ten Indian jointvetch at the
seedling stage was extracted using a modied cetyltri-
methylammonium bromide method and applied to con-
struct a 500-bp paired-end library using the NEBNext
Ultra DNA Library Prep Kit (New England Biolabs,
Ipswich, MA, USA) for next-generation sequencing on
the Illumina NovaSeq 6000 platform (Berry Genom-
ics Co., Ltd., Beijing, China). Approximately 2.35 Gb of
raw data from Indian jointvetch were generated with
150-bp paired-end read lengths. De novo assembly was
performed using NOVOPlasty v4.2 software (https://
github.com/ndierckx/NOVOPlasty, accessed March 24,
2023). Detailed sequencing and assembly methods are
described in our previous study [34].
Genome component analysis and gene annotation
Genes encoding proteins, tRNAs, and rRNAs in the cp.
genome of Indian jointvetch were predicted using GeSeq
software (https://chlorobox. mpimp-golm. mpg. de/
geseq.com ml/, accessed March 24, 2023). e protein
sequences of cp. genes were compared with known pro-
teins in databases using BLASTP (https://ncbiinsights.
ncbi.nlm.nih.gov/tag/blastp/, accessed March 24, 2023)
(e-value < 1 × 10− 5). Because there may be more than one
alignment result for each sequence, only one optimal
alignment result was reserved for database alignment for
the given gene to ensure its biological signicance. e
amino acid sequences of Indian jointvetch were aligned
with sequences included in the Non-Redundant Pro-
tein Sequence (http://www.ncbi.nlm.nih.gov/, accessed
March 24, 2023), Swiss-Prot (http://www.ebi.ac.uk/uni-
prot, accessed March 24, 2023), Clusters of Orthologous
Groups, Kyoto Encyclopedia of Genes and Genomes
(KEGG; http://www.genome.jp/kegg/, accessed March
24, 2023), and Gene Ontology (GO; http://geneontology.
org/, accessed March 24, 2023) databases to obtain func-
tional annotation information for the coding genes.
Analysis of genetic relationships and identication
characteristics
Phylogenetic analysis
Nineteen cp. genomes of common plants, including the
model plant of dicotyledons Arabidopsis thaliana, com-
mon plants growing in rice elds (Oryza sativa Indica, O.
sativa Tropical Japonica, Echinochloa oryzoides, Eclipta
prostrata, Ammannia arenaria, Ammannia multiora,
Cyperus iria, Cyperus diormis), and common Legumi-
nosae plants (Vicia sepium, Sesbania cannabina, Medi-
cago sativa, Medicago truncatula, Medicago polymorpha,
Glycine max, Glycine soja, Astragalus sinicus), were
downloaded from the National Center for Biotechnology
Information (NCBI) database (see Additional File 1, Table
S1 for accession numbers) and subject to phylogenetic
analysis to determine their evolutionary relationships
with Indian jointvetch. e sequences were aligned using
ClustalW (v2.0.12) (http://www.clustal.org/clustal2/,
accessed April 4, 2023) with default settings. e DNA
substitution model was utilized based on the Akaike
information criterion [35]. e phylogenetic tree was
constructed by the maximum-likelihood (ML) method
using PhyML v3.0 (htp://ww.atgc-montpeller. fr/phyml/,
accessed April 4, 2023) and bootstrap values were calcu-
lated for 1000 replicates [36, 37]. e tree building model
was nally evaluated using jModelTest 2.1.10 (https://
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Page 4 of 13
Gao et al. BMC Genomics (2024) 25:277
github.com/ddarriba/jmodeltest2, accessed April 4,
2023), with the best model “GTR + I + G .”
Contraction and expansion analysis of IR regions
We performed IR contraction and expansion analy-
sis for the cp. genomes of Indian jointvetch and species
with closer genetic relationships selected based on the
phylogenetic analysis. e four quadripartite struc-
tures (LSC, SSC, and two IR repeat regions) of each cp.
genome were compared, and changes in the copy num-
ber of related genes caused by contraction and expansion
of the IR or pseudogenes, resulting in boundary regions,
were analyzed. Genes that crossed or were adjacent to
these boundaries were identied. In addition, the length
and distance from the boundaries of these genes were
analyzed.
Single nucleotide polymorphism (SNP) analysis
Using MUMmer software (http://mummer.sourceforge.
net/, accessed April 4, 2023), the cp. genome sequences
of Indian jointvetch (as the reference) and closely related
species, based on phylogenetic and contraction/expan-
sion IR analyses, were completely aligned, and prelimi-
nary ltering was performed to detect potential SNP
sites. Sequences of 100bp on both sides of the candidate
SNP site of the reference sequence were extracted and
aligned with the assembly results using BLAT v35 soft-
ware (http://hgdownload.soe.ucsc.edu/admin/exe/linux.
x86_64/blat/, accessed April 4, 2023) to verify the SNP
site. If the alignment length was less than 101bp, it was
considered an unreliable SNP and was removed; if the
alignment was repeated multiple times, the SNP was
considered a repetitive region and was removed, result-
ing in only reliable SNPs.
Herbicide sensitivity assays
We determined the susceptibility of Indian jointvetch
to common herbicides applied in rice elds using a
pot experiment in the greenhouse. e soil was a mid-
dle loam obtained from a farmland in the suburbs of
Shanghai, China, where herbicides had not been used.
Five seeds were sown in each plastic cup (7 × 7 × 7 cm),
water was added until saturation, a ne layer of soil was
added, and the seeds were treated with herbicides (i.e.,
pre-emergence treatment) using a 3WP-2000 walking-
type spray system (Nanjing Agricultural Mechanization
Research Institute of the Ministry of Agriculture, China).
Each treatment was performed with 30 mL of liquid
(450 L ha− 1 water) using a fan-shaped nozzle. Control
plants were sprayed with the same amount of water as
the herbicide for the treated plants. e seedlings were
then placed in a greenhouse for cultivation. When the
seeds of the control group germinated and the treatment
group showed a signicant gradient, the Indian joint-
vetch plants were cut and weighed. Five seeds were sown
and cultivated at the three-leaf stage for post-emergence
herbicide application using the same method. After 21
days, the above-ground Indian jointvetch in each cup
was cut and weighed. e dose and details of the herbi-
cides are listed in Table1. e experiment included three
to four biological replicates and the entire experiment
was repeated twice. e eective rate of each herbicide
causing 50% reduction in plant growth (GR50) was deter-
mined using the four-parameter logistic function with
the “drc” add-on package [38] in the R 3.1.3 Language
and Environment for Statistical Computing [39]. is
model is dened as follows:
Y=c+{(d−c)/(1 +exp(b(logx −loge )) )}
where parameter e represents GR50 as the dose produc-
ing a response halfway between the upper limit d and the
lower limit c, and parameter b denotes the relative slope
around e.
Table 1 Information of the herbicides used in this study
Type Herbicides Manufacturer Dose (g a.i. ha− 1)
Pre-emergence Bensulfuron-methyl Zhejiang Tianyi Biotechnology Co.,Ltd., Shaoxing, Zhejiang, China 0, 5.625, 11.25, 22.5, 45, 90, 180
Butralin Shield Corporation, Fuzhou, Jiangxi, China 0, 135,270, 540, 1080, 2160, 4320
Oxyuorofen Yifan Biotechnology Group Co., Ltd., Wenzhou, Zhejiang, China 0, 11.25, 22.5, 45, 90, 180, 360
Saufenacil BASF SE, Ludwigshafen, Rheinland-Pfalz, Germany 0, 0.984375, 1.96875, 3.9375,
7.875, 15.75, 31.5
Post-emergence Pyrazosulfuron-ethyl Liben Crop Technology Co., Ltd., Liangyungang, Jiangsu, China 0, 0.9375, 1.875, 3.75, 7.5, 15, 30
2-methyl-4-chloro-
phenoxyacetic acid
Jiangsu Jian Gu Chemical Industry Co., Ltd., Suqian, Jiangsu Prov-
ince, China
0, 24.375, 48.75, 97.5, 195, 390,
780
Penoxsulam Corteva Agriscience, Wilmington, DE, USA 0, 0.9375, 1.875, 3.75, 7.5, 15, 30
Quinclorac Jiangsu Repont Agrochemical Co., Ltd., Changzhou, Jiangsu, China 0, 18.75, 37.5, 75, 150, 300, 600
Florpyrauxifen-benzyl Corteva Agriscience, Wilmington, DE, USA 0, 0.28125, 0.5625, 1.125, 2.25,
4.5, 9
Pyraquinate Shandong Cynda (Chemical) Co., Ltd., Jinan, Shandong, China 0, 4.6875, 9.375, 18.75, 37.5, 75,
150
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Gao et al. BMC Genomics (2024) 25:277
Results
Impact of A. indica on rice growth
We rst investigated the morphological features of Indian
jointvetch during seed maturity in upland paddy elds in
the lower reaches of the Yangtze River in China. e aver-
age height of Indian jointvetch was 134.2 ± 3.7 cm, which
was higher than that of the rice (approximately 90cm).
e mean maximum lateral distance was 57.9 ± 2.1 cm.
e seeds presented a kidney shape (Additional File 3,
Figure S2) with a 1000-seed weight of 10.39 ± 0.055g. e
frequency of occurrence of this weed in upland paddy
elds in our survey was 13.85 ± 2.22%. From the perspec-
tive of rice damage, as the number of Indian jointvetch
plants increased, the eective spike number and grains
per spike of upland direct-seeding rice exhibited a sig-
nicant downward trend. Rice seed weights also showed
an overall downward trend with an increasing number of
Indian jointvetch plants. Specically, when the number of
Indian jointvetch was 0, 2, 4, 6, and 8, the yields of upland
direct-seeding rice were 8019.3, 5534.4, 3783.2, 3611.4,
and 2092.5kg/ha, respectively (Fig.1).
Characteristics of the A. indica cp genome
Cp genome map
After trimming low-quality fragments from the raw data,
33,624,994 clean reads with a GC content of 35.93% were
mapped to the complete cp. genome of Indian joint-
vetch. De novo assembly resulted in a circular genome of
163,613bp in length (Fig.2). e raw reads were depos-
ited in the NCBI GenBank database (accession number:
PRJNA963187). e complete cp. genome displayed the
typical quadripartite structure of most angiosperms,
including an LSC region, SSC region, and pair of IRs (IRa
and IRb). e lengths of the LSC, SSC, and IR regions
were 88,562, 19,801, and 27,625 bp, respectively, and
the intergenic region length was 86,150bp. e Indian
jointvetch cp. genome contains 83 protein-coding genes
(Table2).
Cp genome components
e cp. genome of Indian jointvetch contained 39 tRNA
genes and eight rRNA genes (Table3). ere were 70
protein-coding and 26 tRNA genes located within the
LSC; 10 protein-coding genes, 10 tRNA-coding genes,
and four rRNA-coding genes located within IRb or IRa;
and 13 protein-coding genes and one tRNA gene located
within the SSC region (Fig. 2). All 83 genes encoding
proteins in the cp. genome of Indian jointvetch were
functionally annotated, which mainly belonged to the
photosynthesis and self-replication categories. e gene
names, groups, and categories are listed in Table4. e
genes were mainly associated with GO biological pro-
cesses (Additional File 3, Figure S3), and were associ-
ated with KEGG energy production and conversion,
translocation, ribosomal structure, and biogenesis path-
ways (Additional File 3, Figure S4). In total, 161 simple
sequence repeats (SSRs) were identied in the Indian
jointvetch cp. genome. ere were eight SSRs on IRa or
IRb, 114 on the LSC region, and 31 on the SSC region.
In total, 166 long repeats (LRs) were identied in the cp.
genome of Indian jointvetch (Table5).
Genetic similarity analysis
Phylogenetic analysis
Bayesian inference of 19 complete cp. genomes showing
the same topology demonstrated that Indian jointvetch
clustered into a single clade (Fig.3). Indian jointvetch,
A. sinicus, M. polymorpha, G. soja, and G. max have the
most recent common ancestor (MRCA) (BS = 100 for the
ML tree), and this group has an MRCA with S. cannabina
Fig. 1 Eect of the number of Indian jointvetch plants on rice yield in the eld. The standard errors of the means are described by vertical bars. ANOVA
signicance groupings are shown as a, b, c, and d (P < 0.05)
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Page 6 of 13
Gao et al. BMC Genomics (2024) 25:277
voucher IBSC voucher (BS = 100 for the ML tree). e
closest relatives to these plants were E. prostrata, O.
sativa Indica, O. sativa Tropical Japonica, and E. oryzi-
cola (BS = 100 for the ML tree). In addition, the group
formed by S. cannabina-1, M. sativa, M. polymorpha, A.
thaliana, C. iria, C. diormis, A. arenaria, and A. multi-
ora was closely related to the Indian jointvetch group.
e most distantly related plant to Indian jointvetch was
V. sepium.
IR expansion and contraction
To further observe the potential genetic relationships
among the most closely related species to Indian joint-
vetch, namely S. cannabina voucher IBSC, G. soja, and
G. max, based on the contraction and expansion of
IR regions, the gene variations at the IR/SSC and IR/
LSC boundary regions of the four species were com-
pared (Fig.4); A. sinicus and M. polymorpha do not have
complete IR regions and were therefore excluded from
this analysis. e genes rps19/ rpl2, trnN/ ndhF, ycf1/
trnN, and rpl2/ trnH were identied at the junctions of
Fig. 2 Assembly, size, and features of the chloroplast genome of Indian jointvetch. The genes outside the circle are transcribed in the counterclockwise
direction and the genes inside the circle are transcribed in the clockwise direction. Dierent colors of genes represent dierent functions. The dark gray
area and light gray area of the inner circle represent the GC and AT content of the genome, respectively
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Page 7 of 13
Gao et al. BMC Genomics (2024) 25:277
the LSC/IRb, IRb/SSC, SSC/IRa, and IRa/LSC regions,
respectively, in Indian jointvetch. Among them, ndhF
crosses IRb/SSC and ycf1 crosses SSC/IRa. Among all
tested species, the closest to Indian jointvetch was S. can-
nabina voucher IBSC with respect to the expansion and
contraction of IR regions; these two species exhibited
identical boundary genes with the only dierence being
the length of the genes or the distance between the genes
and the boundary.
SNP analysis
SNP analysis was performed to compare the dieren-
tiation among Indian jointvetch, S. cannabina voucher
IBSC, G. soja, and G. max. A total of 4659 SNPs were
detected in S. cannabina voucher IBSC, including 1378
SNPs in the intergenic spacer regions and 3281 SNPs in
the coding sequence regions (Table6; Additional File 2,
Table S2). e nonsynonymous to synonymous substitu-
tion ratio was 0.626. Two SNPs were detected at the start
codon and eight SNPs were detected at the stop codon.
e numbers of SNPs in the cp. genomes of G. soja and
G. max were much higher than that for S. cannabina
Table 2 Summary of chloroplast genome features in Indian
jointvetch
Genome Features Indian jointvetch
Genome size (bp) 163,613
LSC length (bp) 88,562
SSC length (bp) 19,801
IR length (bp) 27,625
Intergenic region Length (bp) 86,150
Overall GC content (%) 35.54
GC content of LSC (%) 32.77
GC content of SSC (%) 28.71
GC content of IR (%) 42.41
Number of Protein-coding genes 83
Table 3 Non-coding RNAs in the Indian jointvetch chloroplast
genome
Type ncRNA number Total length
(bp)
Average
length (bp)
Length/
Genome
(%)
tRNA 39 2943 75 1.8
rrn23 2 5630 2815 3.44
rrn4.5 2 208 104 0.13
rrn16 2 2982 1491 1.82
rrn5 2 242 121 0.15
Table 4 Annotated genes of the Indian jointvetch chloroplast genome
Category Groups Genes
Photosynthesis Subunits_of_photosystem_I psaA, psaB, psaC, psaI, psaJ
Subunits_of_photosystem_II psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbJ, psbK, psbL,
psbM, psbN, psbT, psbZ
Subunits_of_NADH_dehydrogenase ndhA, ndhB, ndhB, ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ,
ndhK
Subunits_of_cytochrome_b/f_complex petA, petB, petD, petG, petL, petN
Subunits_of_ATP_synthase atpA, atpB, atpE, atpF, atpH, atpI
Large_subunit_of_Rubisco rbcL
Self-replication Large_subunits_of_ribosome rpl14, rpl16, rpl2, rpl2, rpl20, rpl23, rpl23, rpl32, rpl33, rpl36
Small_subunits_of_ribosome rps11, rps12, rps12, rps14, rps15, rps16, rps18, rps19, rps19, rps2, rps3,
rps4, rps7, rps7, rps8
DNA-dependent_RNA_polymerase rpoA, rpoB, rpoC1, rpoC2
Ribosomal_RNAs 8 rRNA
Transfer_RNAs 39 tRNAs
Other genes Maturase matK
Protease clpP1
Envelope_membrane_protein cemA
Acetyl-CoA_carboxylase accD
C-type_cytochrome_synthesis_gene ccsA
Translation_initiation_factor
protochlorophillide_reductase_subunit
Genes unknown Proteins_of_unknown_function Ycf1, ycf2, ycf3, ycf4
Table 5 Single sequence repeats (SSRs) and long repeats (LRs) in
the Indian jointvetch chloroplast genome
Type of repeats Distribution Number
SSR Genome 161
Coding 22
IRa/b 8
LSC 114
SSC 31
LR Hamming Distance = 0 22
Hamming Distance = 1 6
Hamming Distance = 2 28
Hamming Distance = 3 110
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Gao et al. BMC Genomics (2024) 25:277
Fig. 4 Comparison of large sequence copy (LSC), inverted repeat (IRb, IRa), and small sequence copy (SSC) border regions of the chloroplast genomes
of Indian jointvetch and related species
Fig. 3 Phylogenetic tree constructed using the maximum-likelihood method based on alignments of complete chloroplast genome sequences. The
numbers at the nodes indicate bootstrap values from 1000 replicates. If the bootstrap value is 100, this number is not shown on the nodes. The species
newly sequenced in this study is shown in bold
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Page 9 of 13
Gao et al. BMC Genomics (2024) 25:277
voucher IBSC compared to the Indian jointvetch cp.
genome.
Chemical herbicide eects
Toxicity of pre-emergence herbicides
In pre-emergence application, as the doses of oxyuo-
rofen and saufenacil increased, Indian jointvetch was
signicantly inhibited on the 14th day after herbicide
treatment and showed a gradient decrease pattern there-
after. However, upon treatment with the other two herbi-
cides, bensulfuron-methyl and butralin, Indian jointvetch
did not show signicant growth inhibition until 21 days
after treatment (Fig.5). e toxicity calculation showed
that the GR50 value of oxyuorofen on Indian jointvetch
was 34.9g a.i. ha− 1 and the GR90 value was 157.5g a.i.
ha− 1; the GR50 value of saufenacil on Indian jointvetch
was 1.3g a.i. ha− 1 and the GR90 value was 10.3g a.i. ha− 1
(Fig.5).
Toxicity of post-emergence herbicides
With post-emergence application, with the increase in
the doses of orpyrauxifen-benzyl, pyrazosulfuron-ethyl,
and penoxsulam, Indian jointvetch was signicantly
inhibited on the 21st day after herbicide treatment and
showed a decreasing gradient thereafter; the other three
herbicides, quinclorac, 2-methyl-4-chlorophenoxy-
acetic acid, and pyraquinate, did not have a signicant
inhibitory eect on the growth of Indian jointvetch.
From the toxicity calculation, the GR50 value of orpy-
rauxifen-benzyl on Indian jointvetch was 0.3g a.i. ha− 1
and the GR90 value was 1.3g a.i. ha− 1. e GR50 value of
penoxsulam on Indian jointvetch was 4.0g a.i. ha− 1 and
the GR90 value was 16.8 g a.i. ha− 1. e GR50 value of
pyrazosulfuron-ethyl on Indian jointvetch was 11.9g a.i.
ha− 1 and the GR90 value was g a.i. ha− 1 737.5, but this far
exceeds its safe usage limit (45.0g a.i. ha− 1) (Fig.6).
Discussion
According to the results of our on-site investigation,
Indian jointvetch causes serious harm to upland direct-
seeding rice elds in the lower reaches of the Yang-
tze River in China. Upland direct-seeding rice is an
important food crop grown in areas with limited water
resources presently. However, rampant weeds are an
obstacle to the cultivation of upland direct-seeding rice
[14–16]. e rough management practice of the upland
direct-seeding rice eld system has further aggravated
weed ravages. Although studies have shown that Indian
jointvetch can be considered a green fertilizer that is
benecial for rice growth [40], we obtained contrasting
results. In fact, the tall Indian jointvetch plant type has
reduced the survival resources of rice, thereby seriously
restricting its yield. However, research on Indian joint-
vetch in rice elds is limited.
Table 6 Single nucleotide polymorphisms (SNPs) in S. cannabina voucher IBSC, G. soja, and G. max chloroplast genomes compared to
the Indian jointvetch chloroplast genome
Species Start Stop Synonymous Nonsynonymous CDS Intergenic Total SNPs
S. cannabina voucher IBSC 2 8 2000 1252 3281 1378 4659
G. soja 1 7 2344 1469 3840 1595 5435
G. max 1 7 2342 1470 3838 1594 5432
CDS, coding sequence
Fig. 5 Dose–response analyses for the response of Indian jointvetch to pre-emergence herbicides. The X-axis represents the dose (g a.i. ha− 1) and the
Y-axis represents the percentage of fresh weight to that of the untreated control. “Bm,” “Sf,” “Of,” and “Bl” represent the herbicides bensulfuron-methyl,
saufenacil, oxyuorofen, and butralin, respectively
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Gao et al. BMC Genomics (2024) 25:277
Given the serious harm caused by Indian jointvetch in
rice elds, genetic research is important to gain a deeper
understanding of this weed and predict its evolution
in rice elds in the future. Because the cp. genome has
unique research value in phylogeny, species identica-
tion, and population genetics [21], we sequenced the
cp. genome of Indian jointvetch for the rst time in this
study, demonstrating a typical tetrad structure, including
genes encoding proteins, tRNAs, and rRNAs, similar to
the cp. genomes reported in other plants [22, 34, 41]. e
complete cp. genome of Indian jointvetch was 163,613bp
long, which is larger than that of other common plants
found in rice elds with reported cp. genomes, includ-
ing those of the genera Oryza [42], Echinochloa [41], and
Ammannia [34].
Repeating sequences are unique labels of a species,
including SSRs and LRs. SSRs, composed of 1–6 nucle-
otide repeat units, are widely distributed molecular
genetic markers in the cp. genome of plants [43, 44]. We
identied 161 SSRs in the cp. genome of Indian joint-
vetch, which can be used for population genetics analy-
ses and plant genotyping [45–48]. We also identied 166
LRs in the Indian jointvetch cp. genome. LRs are a spe-
cial type of DNA repeat sequence that typically occupy
a large proportion of the genome [49]. ese repetitive
structures exhibit diversity in the cp. genome and pro-
mote molecular recombination [50], which has important
molecular and biological signicance in the study of plant
evolution [51]. us, the SSRs and LRs detected in this
study oer important biological information resources
for the identication of Indian jointvetch and to facilitate
genetic diversity and population structure research of
Aeschynomene.
In order to explore the closely related plants of Indian
jointvetch, we conducted phylogenetic tree analysis, IR
expansion and contraction comparisons, and SNP stud-
ies based on the cp. genome. Genome assembly and
characterization provides great value in dening species,
revealing phylogenetic relationships, and determining
taxonomic status [52–55]. Phylogenetic analysis based on
the cp. genomes showed that species with a close genetic
relationship to Indian jointvetch included A. sinicus, M.
polymorpha, G. soja, G. max, and Sesbania cannabina
voucher IBSC. Notably, the genetic relationship between
the two species of S. cannabina registered in the NCBI
database is not close, suggesting that they may in fact
represent dierent species or diverged due to geographi-
cal isolation and local adaptation. IR expansion and con-
traction in the cp. genome are common phenomena in
plants [18], mainly occurring at the IR/SC junction [56],
which is a key driving force in plant evolution [57–60].
e results of IR expansion and contraction analyses
further indicated that the genetic relationship between
Indian jointvetch and S. cannabina voucher IBSC was
closer. Because their boundary genes are completely
identical, except for dierences in length of the genes or
the distance between the genes and the boundary (Fig.4).
SNPs are important indicators of evolutionary dierences
between closely related species. ese direct molecular
markers can clearly display the exact nature and location
of allele variations [61]. Using the cp. genome of Indian
jointvetch as reference, we identied 3281, 3840, and
3838 SNPs in the cp. genomes of S. cannabina voucher
IBSC, G. soja, and G. max, respectively. e fewer SNPs
detected represents the fewer variations in single nucleo-
tides. is result further indicated that the Indian joint-
vetch and S. cannabina voucher IBSC have a relatively
close genetic relationship, which is consistent with the
phylogeny and IR expansion and contraction results.
ese SNPs can be used as important dierential nucleo-
tide databases to distinguish the species.
From the perspective of crop protection, it is neces-
sary to study reasonable measures to eliminate Indian
jointvetch from paddy elds. e advantages of chemical
methods for weed control in upland direct-seeding rice
elds have been reported for decades in terms of higher
Fig. 6 Dose –response analyses for response of Indian jointvetch to post-emergence herbicides. The X-axis represents the dose (g a.i. ha− 1) and the Y-axis
represents the percentage of fresh weight to that of the untreated control. “Fb,” “Ps,” “MCPA,” “Qc,” “Pq,” and “Pz” represent the herbicides orpyrauxifen-
benzyl, penoxsulam, 2-methyl-4-chlorophenoxyacetic acid, quinclorac, pyraquinate, and pyrazosulfuron-ethyl, respectively
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Page 11 of 13
Gao et al. BMC Genomics (2024) 25:277
yields and lower labor costs [30–32]. However, the chem-
ical control of Indian jointvetch is currently very chal-
lenging [3]. Our results also indicated that the availability
of herbicides for Indian jointvetch control is limited. Her-
bicides commonly used to control broadleaf weeds in
rice elds in China, such as bensulfuron-methyl and
2-methyl-4-chlorophenoxyacetic acid [62–64], had poor
eects on inhibiting the germination of Indian jointvetch.
Nevertheless, we found that among the 10 herbicides
tested, four met the requirements, including two pre-
emergence herbicides (saufenacil and oxyuorfen) and
two post-emergence herbicides (orpyrauxifen-benzyl
and penoxsulam). Given that mixing Indian jointvetch
seeds with rice seriously reduces the quality of the rice
[1], we strongly recommend using herbicides contain-
ing these four components to control Indian jointvetch,
which is rampant in rice elds; however, further research
is needed to establish eective control strategies.
It should be pointed out that the understanding of the
genetic characteristics of Indian jointvetch and the pre-
diction of the evolution of related plants into rice eld
weeds in this study are only based on the chloroplast
genome. Further research should be based on broader
morphological and whole-genome. Additionally, this
study only provides suggestions for chemical control of
Indian jointvetch. Further research is needed on control
strategies that are more conducive to biodiversity and
even the value of resource utilization of this weed.
Conclusions
In this study, we obtained the rst complete cp. genome
of Indian jointvetch (A. indica), a common weed found
in upland direct-seeding rice elds in the lower reaches
of the Yangtze River in China. Overall, our study indi-
cates that species with closer anity to Indian jointvetch
include (but are not limited to) Glycine soja, Glycine
max, and Sesbania cannabina based on the cp. genome.
erefore, it is necessary to be vigilant about these
plants becoming farmland weeds (especially S. canna-
bina), entering rice elds, and causing harm to rice. Note
that because A. sinicus and M. polymorpha do not have
complete IR regions, further analyses of these two spe-
cies were not conducted in this study, which may war-
rant further exploration of their potential harm in upland
direct-seeding rice elds. Toxicity evaluation of common
herbicides currently used in rice elds indicate the need
for the combination of herbicides to achieve eective
control of Indian jointvetch and improve the yield and
quality of upland direct-seeding rice.
Abbreviations
cp Chloroplast
GO Gene Ontology
GR50 The herbicide dose needed to cause 50% growth reduction of plant
IBSC South China Botanical Garden, Chinese Academy of Sciences
IR Inverted Repeat
KEGG Kyoto Encyclopedia of Genes and Genomes
LR Long repeat
LSC Large single copy
ML Maximum likelihood
MRCA Most recent common ancestor
NCBI National Center for Biotechnology Information
SSC Small single copy
SNP Single nucleotide polymorphism
SSR Simple sequence repeat
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12864-024-10102-x.
Additional File 1
Additional File 2
Additional File 3
Acknowledgements
We thank Shanghai BIOZERON Biotechnology Co., Ltd. for performing the
high-throughput sequencing.
Author contributions
Study design, Y.G.; concept, Y.G., Z.T., and G.S.; data collection, Y.G., T.C., and J.L.;
analysis, Y.G., T.C., and J.L.; manuscript writing, Y.G; review, Z.T., and G.S.
Funding
This research was funded by Shanghai Agriculture Applied Technology
Development Program, China (Grant number T20210104).
Data availability
The datasets generated and/or analyzed during the current study are available
in the [National Center for Biotechnology Information] repository, [Accession
number: PRJNA963187].
Declarations
Ethics approval and consent to participate
The seeds of Aeschynomene indica collected in this study were approved by
the landowner.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1Eco-Environmental Protection Research Institute, Shanghai Academy of
Agricultural Sciences, 201403 Shanghai, China
2School of Chemical and Environmental Engineering, Shanghai Institute
of Technology, 201418 Shanghai, China
Received: 7 December 2023 / Accepted: 8 February 2024
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