Genetic variation and structure of complete chloroplast genome in alien monoecious and dioecious Amaranthus weeds

doi:10.21203/rs.3.rs-509003/v1

Amaranthus is a complex taxon with economic importance as well as harmful weeds. We studied the genetic variation and structure of the chloroplast genomes of 22 samples from 17 species of three subgenera. It was found that the length of the chloroplast genome of Amaranthus varied from 149,949 bp of A. polygonoides to 150,757 bp of A. albus. The frequencies of SNPs and InDels in chloroplast genomes were 1.79 % and 2.86 %, and the variation mainly occurred in the non-coding regions. The longest InDel was 387 bp, which occurred on ycf2, followed by 384 bp InDel on psbM-trnD. Two InDels in ndhE-I on the SSC make the three subgenera clearly distinguished. In LSC, SSC and IRs regions, there were four 30 bp forward and reverse repeats, and the repeats in SSC and LSC were in nearly opposite positions in circular genome structure, and almost divided the circular genome into symmetrical structures. In the topological tree constructed by chloroplast genome, species in subgen. Amaranthus and subgen. Acnida form monophyletic branches separately and cluster together. A. albus, A. blitoides and A. polygonoides were separated from subgen. Albersia, and the rest of subgen. Albersia were clustered into a monophyletic branch. The rpoC2, ycf1, ndhF-rpl32 were good at distinguishing most amaranths. The trnk-UUU-atpF, trnT-UGU-atpB, psbE-clpP, rpl14-rps19, and ndhF-D can distinguish several similar species. In general, the chloroplast genome is of certain value for the identification of the similar species of Amaranthus, which provides more evidence for clarifying the phylogenetic relationships within the genus.

Evolutionary Genetics

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

Molecular Biology

Amaranthus

three subgenera

Chloroplast genome

Genetic variation

Hotspots

The genus of Amaranthus includes 74 species, of which 55 species native to the Americas and the rest originated from the Euraisa, South Africa and Austrilia / Oceania^1-2. The genus contains pseudocereals crops such as A. caudatus L., A. cruentus L., and A. hypochondriacus L., leaf vegetables A. tricolor and A. blitum, endangered plants A. pumulis, and agricultural weeds³. The Flora of China (2003) recorded 15 species and two varieties, all of which were alien except for A. tricolor⁴. Since then, some authors have successively found new alien amaranths: A. bouchonii⁵, A. tenuifolius⁶, A. palmeri⁷, A. standelyansus⁸, A. powellii⁵, A. dubius⁹ from the collected specimens in China. In port monitoring regions, A. tuberculatus, A. arenicola, A. crispus etc. were newly intercepted and controlled (Xu, unpublished). Among them, A. arenicola and A. crispus were transient colonization (Xu, unpublished).

According to inflorescences position, the number of perianth segments and urticle dehiscent / indehiscent^2,10, as well as dioecious or monoecious, are divided into three subgenera: Amaranthus subgen. Amaranthus, Amaranthus subgen. Acnida (L.) Aellen ex K.R.Robertson, and Amaranthus subgen. Albersia (Kunth) Gren. & Godr.^10-11. Of these, 9 species were listed as “introduced, invasive and noxious plants” in the USDA Plants Database (Southern Weed Science Society 1998), and 21 species as“agricultural weeds” in the Global Compendium of Weeds¹². The genus is the focus of weed scientific research¹³, because of these amaranth weeds posed a certain threat to agricultural ecology in the new habitat. A. palmeri and A. tuberculatus invade gradually into the new continents out of their origins, and were detected their resistant biotypes¹⁴. Accurate identification of these species is the basis of weed prevention and control. However, the taxonomy of Amaranthus has always been difficult, especially because of the large number of complex taxa which are difficult to define due to the interspecific hybridization and gene introgression.

Many authors have studied on the taxonomy and evolution of the genus. The latest taxonomy revision of monoecious species was completed by Bayón (2015)², and the comprehensive dioecious taxonomy was Sauer’s monographs¹. Waselkov et al. (2018) conducted the phylogenetic analyses of 58 species based on three low-copy nuclear genes and two chloroplast regions¹⁵. Stetter and Schmid (2017) inferred the phylogeny of 35 amaranths using genotyping by sequencing (GBS)¹⁶. Xu et al. (2020) analyzed ITS, ALS (domain C, A and D) and ALS (domain B and E) and constructed topological trees¹⁴. In addition to the part incorrect conclusions drawn by Stetter and Schmid (2017) due to missampling, all of them concluded that the classification boundaries between subgenera are not very clear because of some kinds of amaranths, such as A. palmeri and A. spinosus, A. arenicola and subgen. Amaranthus, as well as A. tuberculatus. And the monophyletic branches of the subgen. Albersia have insufficient bootstrap support value.

Additionally, chloroplast genome-related studies include: Chaney et al. (2016) first reported full chloroplast genomes of A. hypochondriacus, A. cruentus, A. caudatus and their hypothetical wild ancestor species A. hybridus, and found 210 single nucleotide polymorphisms (SNPs) and 122 insertion/deletion polymorphisms (InDels) compared to the reference chloroplast genome¹⁷. Viljoen et al. (2018) studied chloroplast genomes and matK, rbcL, ITS in 59 accessions of 9 species of subgen. Amaranthus and 4 species of subgen. Albersia, and mainly focused on the genetic relationship between wild and domesticated grain amaranths¹⁸. At present, studies on the chloroplast genome of Amaranthus are mainly focused on the grain amaranths, and there is a lack of overall studies on the three subgenera.

In this paper, combined with the problems existing in the classification and evolution of the genus, and the research need of the genetic variation of alien invasive amaranths, we collected alien species of 3 subgenera, and perform complete analysis of the chloroplast genome, in order to further understand the feature of the chloroplast genomes of Amaranthus, and the critical regions of chloroplast genomes used to explain the phylogenetic relationship of the genus, especially the evolution of A. palmeri and A. spinosus, A. tuberculatus and A. arenicola, A. spinosus and A. dubius. The results will provide a new basis for the taxonomic revision, phylogenetic evolutionary, weed evolutionary biology and the development of genetic resources.

Genomic features

The quadripartite structure of 22 samples of 17 species in Amaranthus consists of a large single-copy region (LSC with 83, 382 – 84, 062 bp), a small single-copy region (SSC with 17, 937 – 18, 124 bp), and a pair of inverted repeat regions (IRs with 23, 964–24, 357 bp). The full length of the 22 cp genomes ranges from 149,949 bp in A. polygonoides to 150, 756 bp in A. albus (Table 1). The chloroplast genome sequences were deposited in GenBank (Table 1).

The total GC content was 36.5% to 36.6%, only A. albus, A. blitoides and A. polygonoides have a GC content of 36.5% (Table 1). The chloroplast genome contains a total of 133 genes, including 88 protein-coding genes, 37 tRNA genes, and 8 rRNA genes, 18 of which were duplicated in the inverted repeat regions (Table S2). The gene rps12 was trans-spliced; the 50-end exon was located in the LSC region, whereas the 30- intron and exon were duplicated and located in the inverted repeat regions. The partial duplicate of rps19 and ycf1 genes appeared as pseudogenes as they lost their protein-coding ability. 16 genes have introns.

Variants of cp genomes

The length of the SSC region was conserved among the subgenera by comparing the length of the chloroplast genomes of 22 individuals from 17 species. A. palmeri, A. tuberculatus and A. arenicola in subgen. Acnida were 18027 - 18042 bp in length (average value 18038.5 ± 5.3151), the SSC length of 5 species of subgen. Amaranthus was 17937-17948 bp (average value 17941 ± 3.3665), and the SSC length of 8 species of subgen. Albersia was 18057 - 18124 bp (average value 18076.3 ± 22.6806) (Table 1; Figure 1). At SSC, there were about 77 bp InDels in ndhE-G and 180bp InDels in ndhG-I, which induced the variation of SSC length among subgenera (Figure 2). The frequencies of SNPs and InDels in the chloroplast genomes of the 17 species were 1.79% and 2.86%, respectively (Table 2). The frequencies of SNPs and InDels in the genes were 1.22% and 1.14%, and the frequencies of SNPs and InDels in the intergenic spacer were 3.25% and 7.32%, respectively (Table 2). In general, the variation mainly occurred in the intergenic spacer region, and InDels mainly occurred in the non-coding region (Table 2). The longest InDel was 387 bp, which occurred on ycf2, followed by 384 bp InDel on psbM-trnD.

Repeat and SSR analyses

Each species has 28 to 38 repeats, distributed in 30 locations, including 11 to 14 forward repeats, 11 to 17 palindromic repeats, and 6 to 8 reverse repeats ranging from 30 to 64 bp in length. There were 19 common repeats locations, of which 11 had no variation and 8 had variation in length. The R3, R8, R11 and R13 had the most abundant variation (Figure 3). The R12 (forward and reverse repeats) was distributed in LSC, IRa, SSC and IRb. The R12 on SSC is almost opposite to R12 on LSC, dividing the entire circular genome into two parts of nearly equal length. The repeats on LSC were mainly concentrated near Repeat 12 (loci 29572-46282), loci 8166-8327, loci 29572 and loci 75230. The repeats on IRs are constant within the genus. There were two common repeats in SSC, and one was a palindrome sequence shared by subgen. Acnida, subgen. Amaranthus, and A. albus.

MISA analysis showed that each cp genome of Amaranthus contained 29-39 SSRs (Table 3). On average, the number of SSR types from more to less was mono-, tetra-, di-, tri-, penta- and hexa-nucleotides in order (Table 3). About 55.56% of those SSRs were composed of A or T bases. Among all SSRs, most loci located in LSC (77.78 %) and IGS (71.91%). About 12 repeat motifs were shared by all species in the genus while the remaining motifs were species-specific or subgenus-specific (Table 3). Different combinations of SSR markers could distinguish all species except A. standleynaus and A. crispus, A. dubius and A. spinosus (Table 3).

Phylogenetic trees of whole chloroplast genomes

The results obtained in this study in limited samples were basically consistent with previous studies based on chloroplast gene sequences. A. palmeri, A. arenicola and A. tuberculatus in subgen. Acnida clustered together (BS/PP=100/1) (Figure 4). A. hybridus, A. hypochondriacus, A. dubius, A. spinosus, A. retroflexus clustered together (BS/PP=100/1) (Figure 4). And the above two clades were very close (BS/PP=100/1) (Figure 4). A. albus and A. blitoides were clustered (BS/PP=35/0.84) and separated from subgen. Albersia and were closely related to subgen. Amaranthus and subgen. Acnida (BS/PP=58/0.99) (Figure 4). A. polygonoides become a single basal branch. The rest of subgen. Albersia were clustered into one branch (BS/PP=100/1) (Figure 4).

Hotspots for Amaranthus

The partially qualified fragment regions searched by exhaustive method were overlapped, and the overlapped regions were combined together as a hotspot region. Finally, 16 hotspot fragments with a length of 737 to 2818 bp were obtained, and the SNP variation frequency ranged from 0.78% to 1.49% (Table S3). The topological trees constructed by the alignments of these 17 hot fragments and the topological trees constructed by the alignment sequences of each gene and intergenic spacer were consistent with the chloroplast genome topological tree, namely, the hotspots with more than 90% bootstrap value support for the subgen. Amaranthus, subgen. Acnida and subgen. Albersia branch (excluding A. albus, A. polygonoides, and A. blitoides) were ndhF-rpl32, ycf1 and rpoC2 (Figure S1).

In several similar taxa, there were 25 InDels and 11 SNPs between A. tunetanus and A. standleyanus. A. crispus and A. standleyanus had no difference. There are 46 SNPs and 144 InDels between A. arenicola and A. tuberculatus. By sequence alignment and variation analysis, it was found that trnK-UUU-atpF, trnT-UGU-atpB, psbE-clpP, rpl14-rps19, ndhF-D could be used to distinguish A. tunetanus from A. standleyanus, A. crispus, and A. arenicola from A. tuberculatus.

In this study, the SSC length of A. hybridus and other species in subgen. Amaranthus was basically consistent with previous studies on four grain amaranths¹⁷ (Chaney et al., 2016). Chaney et al. (2016) reported that the chloroplast genome of Amaranthus contained 111 genes, while Viljoen et al. (2018) reported that A. tricolor contained 140 genes ^17-18. Data in both studies showed minor errors and duplications. After repeated data proofreading in this study, 133 genes were confirmed in the chloroplast genome of Amaranthus. In addition, due to the inclusion of more amaranths than the former sample, the number of loci polymorphisms found increased to 2735 SNPs and 4363 InDels.

In addition, Chaney et al. (2016) found 29-37 SSRs in four grain amaranths¹⁷. In our study, 29 to 39 SSRs were identified. After statistical analysis and labeling of the SSRs from each sample, different combinations of SSR markers were found to be able to distinguish the similar species: A. arenicola and A. tuberculatus, A. standleyanus and A. tunetanus. In terms of repeats, Chaney et al. (2016) reported 34 to 37 repeats, including 14 to 16 forward repeats and 20 to 21 palindromes¹⁷. This study found 28 to 38 repeats, 11 to 14 forward repeats, 11 to 17 palindromic repeats, and 6 to 8 reverse repeats. Moreover, the distribution of repeats on the chloroplast genome of Amaranthus is found to be regular, such as the distribution of R12. This symmetrical structure should play an important role in the recombination or stabilization of Amaranthus chloroplast genes.

The topological tree constructed from the chloroplast genome is basically consistent with the phylogenetic results of Waselkov et al. (2018) using the chloroplast sequence of matK/trnK-UUU and trnL-UAA¹⁵. Namely, A. albus, A. blitoides, A. polygonoides from subgen. Albersia points out to become a separate branch. A. palmeri and A. spinosus belong to the original subgenus. The chloroplast capture event speculated to occur in Waselkov et al. (2018) was further confirmed. In combination with nuclear gene studies, the relationship between A. palmeri and A. spinosus^15-16,19, and A. palmeri is one of the few species in the dioecious subgenera (A. watsonii and A. arenicola) that have the characteristics of five perianth segments, suggestting that the hybridization of a species of subgen. Acnida and A. spinosus in the earlier stage may have led to the chloroplast capture event, which eventually resulted in the formation of A. palmeri.

In combination with previous studies, we found that rpoC2, ycf1 and ndhF-rpl32 sequences can be used for phylogenetic and taxonomic identification of Amaranthus, according to the principle of similar topological tree branches with the whole chloroplast genome. However, these three sequences cannot effectively distinguish the similar species. In previous studies on the ITS and chloroplast genes matK/trnK-UUU and trnL-UAA of A. arenicola and A. tuberculatus, the two species were almost indistinguishable^14-15. In this study, it was found that there was only one SNP site difference in matK/trnK-UUU between A. arenicola and A. tuberculatus, while their ITS^{14, 20} and trnL-UAA sequences showed no difference²⁰. In contrast, there are 46 SNPs and 144 InDels between A. arenicola and A. tuberculatus on chloroplast genomes. The ITS sequences of A. crispus and A. tunetanus were the same, with only one base difference from A. standleyanus¹⁴. However, there were 25 InDels and 11 SNPs in the chloroplast genomes of A. tunetanus and A. standleyanus. The five newly discovered regions, trnK-UUU-atpF, trnT-UGU-atpB, psbE-clpP, rpl14-rps19, and ndhF-D, have enough parsimony information sites to distinguish several similar species.

In conclusion, the chloroplast genome is of some significance to the phylogenetic study of Amaranthus. However, the study of interspecific and intraspecific gene variation had better be combined with the morphological characteristics of the samples. For species whose morphology is difficult to define, identification errors often occur in samples, and thus the results of molecular analysis are correspondingly wrong. Additionally, the inconsistency of phylogenetic relationships between the chloroplast genome and the nuclear gene sequence of Amaranthus may provide new evidence for the evolution and origin of some species.

Plant samples, DNA extraction, and sequencing

In this experiment, 21 samples from 16 species of Amaranthus and three species as outgroups were used for chloroplast genome analysis (Table S1). All samples were collected from the wild population around the processing plants, wastelands, wharfs in the port supervision area except A. deflexus. A. deflexus is a common weed collected from the wasteland near the Spanish fields. The specimen collection team was composed of officials of the National Port Weed Monitoring Office of CIQ. The samples collected were approved by the customs and other plant quarantine authorities, and complied with relevant regulations. Habitat and biodiversity were not damaged, and endangered species were not involved. The specimens were deposited at the plant inspection and quarantine institute of Chinese Academy of Inspection and Quarantine (CAIQ) (Beijing, China). All samples were mature plants with flowers and fruits, and identified according to the classification monographs of Amaranthus by Sauer (1972)²¹, Mosyakin and Robertson (2003)³, and Bayón (2015)². Total genomic DNA was extracted from the silica-dried leaf tissues using Plant Genomic DNA Kit (Tiangen Biotech Co., China). Genomic DNA of each individual was indexed by a barcode and then pooled together with other samples for sequencing in one lane of HiSeq 2500 (Illumina) (Novogene, Beijing, China).

Genome assembly and annotation

The paired-end sequencing data (2 × 150 bp) were used to assemble its complete chloroplast genome. Sequencing adapters and barcodes were trimmed and low quality reads with Q value ≤ 30 removed. Trimmed paired end reads were mapped to the chloroplast sequence of A. hypochondriacus (GenBank accession: MG 836505), with default parameters. The reads were assembled using the Geneious Prime v. 2020.1.2 (Biomatters, Auckland, New Zealand). The consensus chloroplast sequence of Amaranthus spp. was retrieved separately and used as a reference for several rounds of mapping of itself reads in order to validate its consensus chloroplast sequence. All trimmed and quality-filtered sequence reads have been deposited in Genbank of NCBI. Non-mapped reads, which are assumed to be of non-plastid origin, were excluded from further analysis. The complete chloroplast genome sequence was annotated using the Geneious Prime v. 2020.1.2 (Biomatters, Auckland, New Zealand) by comparing with the genome of A. hypochondriacus (GenBank accession: MG 836505). The assembled and annotated Amaranthus spp. chloroplast genome sequence was deposited at NCBI (Table 1).

Genome comparative analysis

A comparative plot of full alignment with annotations of the 22 chloroplast genomes was produced and the nucleotide variability was calculated by Geneious prime v. 2020.1.2 (Biomatters, Auckland, New Zealand) to analyze the total number of mutations. The comparative analysis included the reference sequence A. hypochondriacus.

Characterization of repeat sequences and SSRs

We used REPuter²² to identify the position and size of repeat sequences, which included forward, palindromic, reverse, and complement repeats in the chloroplast genomes of Amaranthus. The sequence identity and minimum length of repeat size was set to > 90% and 30 bp. MISA perl script (Thiel et al. 2003) was used to detect the simple sequence repeats (SSRs) in the chloroplast genomes²³. The thresholds for mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide SSRs were 10, 5, 4, 3, 3, and 3 repeat units, respectively.

Phylogenetic trees

All phylogenetic analyses were undertaken by the Geneious Prime v. 2020.1.2 software (Biomatters, Auckland, New Zealand), based on the chloroplast genomes of 25 sequences of 20 species (Table S1), including the reference chloroplast genome A. hypochondriacus, and three outgroups, Celosia trigyna (Genbank Accession: MN057637), Alternanthera philoxeroides (Genbank Accession: MK795965) and Froelichia latifolia (Genbank Accession: MH286309). The 25 chloroplast genome sequences were aligned using MAFFT²⁴. The DNA substitution model (GTR+I+G model) was chosen using jModelTest 2.1.6²⁵, and used in maximum likelihood (ML) analysis and Bayesian inference. ML analysis was conducted using RAxML version 8.2.11²⁶ on the Geneious Prime v. 2020.1.2 (Biomatters, Auckland, New Zealand). Bayesian inference was conducted using MrBayes 3.2.6²⁷ with Ngen=1 000 000, Samplefreq=200, and Burninfrac=0.25.

Search for hotspots

Two methods were used to select suitable regions: (1) search with SNP sites greater than 10 per 1000bp based on exhaustive method by Microsoft Excel 2010; (2) the gene and gene spacer were analyzed one by one manually. Finally, a topological tree was constructed for the searched region and compared with the chloroplast genome topological tree to test the resolution authenticity of this region. The topology tree construction method is consistent with the method in 2.6.

Acknowledgements

This work was supported by the Basic Scientific Research Program of CAIQ (2020JK036, 2014JK010，2017JK038), National agricultural standardization Project (NBFW-14-2018). The work involved in this study is part of the above projects on pest identification techniques.

Author Contributions

Han Xu: study design, data interpretation, manuscript writing. Ning Xiang: study design. Junhua Zhang: study design. Yongjiang Zhang: study design, conducting the study.

Competing Interests

The authors declare no competing financial and/or non-financial interests in relation to the work described.

Data Availability

The data generated and analyzed in this study are available from the authors on request.

Ethics declarations

Not applicable.

Collection statement

The samples collected were approved by the customs and other plant quarantine authorities, and complied with relevant regulations. Habitat and biodiversity were not damaged, and endangered species were not involved.

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Table 1 Summary information for the chloroplast genomes of Amaranthus.

No.	Species	Comparison of genome length (bp)				GC content	Genbank Accession
No.	Species	Total	LSC	SSC	IRs	GC content	Genbank Accession
1	Amaranthus retroflexus	150244	83605	17937	24351	36.6%	MN091971
2	Amaranthus dubius	150524	83880	17940	24352	36.6%	MN091972
3	Amaranthus spinosus 113	150523	83879	17940	24352	36.6%	MT526784
4	Amaranthus spinosus 11902	150524	83880	17940	24352	36.6%	MT526783
5	^*Amaranthus hypochondriacus	150523	83878	17941	24352	36.6%	^*MG836505
6	Amaranthus hybridus	150690	84062	17948	24340	36.6%	MT559305
7	Amaranthus palmeri	150731	84010	18027	24347	36.6%	MN091990
8	Amaranthus arenicola JSTZ	150632	83901	18039	24346	36.6%	MN091969
9	Amaranthus arenicola HBTS	150630	83899	18039	24346	36.6%	MZ152791
10	Amaranthus tuberculatus GZW	150679	83945	18042	24346	36.6%	MT559304
11	Amaranthus tuberculatus 11994	150695	83961	18042	24346	36.6%	MN091967
12	Amaranthus tuberculatus 12194	150696	83962	18042	24346	36.6%	MN091968
13	Amaranthus blitum	150621	83806	18057	24379	36.6%	MT526777
14	Amaranthus crispus	150567	83793	18060	24357	36.6%	MT526778
15	Amaranthus standleyanus 11960	150567	83793	18060	24357	36.6%	MT526781
16	Amaranthus standleyanus 7433	150568	83794	18060	24357	36.6%	MT526782
17	Amaranthus tunetanus	150581	83805	18062	24357	36.6%	MT526780
18	Amaranthus deflexus	150256	83489	18065	24351	36.6%	MT526776
19	Amaranthus capensis	150707	83928	18075	24352	36.6%	MT526779
20	Amaranthus blitoides	150667	83878	18089	24350	36.5%	MT526786
21	Amaranthus albus	150756	83943	18111	24351	36.5%	MT526785
22	Amaranthus polygonoides	149948	83896	18124	23964	36.5%	MT472619

Note: ^*Chloroplast genomic data for Amaranthus hypochondriacus were obtained from GenBank.

Table 2 Variation of the chloroplast genomes in Amaranthus.

Region	Length (bp)	SNPs		InDels
Region	Length (bp)	Numbers	Frequency (%)	Numbers	Frequency (%)
Consensus sequence	152519	2735	0.0179	4363	0.0286
Gene	110128	1354	0.0123	1258	0.0114
CDS	80201	1034	0.0129	862	0.0107
tRNA	2780	9	0.0032	0	0
rRNA	9042	6	0.0007	0	0
Intron	18105	305	0.0168	396	0.0219
IGS	42391	1381	0.0326	3105	0.0732

Table 3-1 Distribution of SSRs in the chloroplast genome of Amaranthus.

Region

Locus

SSR type

pol

alb

blo

blu

cap

def

cri

sta 2

sta 7433

sta 11960

ret

hyb

hyp

dub

spi

spi 11902

pal

are

tub 11994

tub 12194

tub GZW

LSC

IGS

trnK-UUU-rps16

4457-4468

(AAGA)3

*

4719-4730

(AT)6

*

*

4743-4806

(ATT)4

*

*

4762-4782

(AT)8

*

*

4782-4793

(TTA)4

*

4795-4806

(ATT)4

*

4796-4807

(TTA)4

*

4878-4892

(TAT)5

*

4880-4894

(TTA)4,5

*

*

*

4882-4911

(ATT)5

*

4913-4924

(TTA)4

*

*

4929-4943

(TAT)5

*

Intron

rps16

5798-5809

(T)12

*

*

IGS

rps16-trnQ-UUG

6789-6805

(A)13

*

IGS

trnQ-UUG-psbK

7257-7269

(T)12

*

IGS

psbK-psbI

7851-7863

(T)12,13

(T)

12

(T)

13

(T)

12

(T)

13

(T)

13

(T)

13

(T)

12

(T)

12

(T)

12

(T)

12

(T)

12

(T)12

IGS

psbI-trnS-GCU

8172-8184

(T)12,13

(T)

12

(T)

12

(T)

12

(T)

12

(T)

13

(T)

13

(T)13

tRNA

trnS-GCU

8214-8226

(A)12

*

*

*

IGS

trnS-GCU-trnG-UCC

8654-8666

(A)12,13

(A)13

(A)12

Intron

trnG-UCC

9466-9484

(T)12

*

IGS

atpA-atpF

11720-11734

(T)13,14,15

(T)

14

(T)

13

(T)

15

(T)

15

(T)

15

(T)

13

(T)

13

(T)

13

(T)

14

(T)

13

IGS

atpF-atpH

13305-13319

(TTTAT)3

*

13459-13470

(GGAA)3

*

CDS

rpoC2

16683-16696

(T)14

*

18568-18580

(T)13

*

Intron

rpoC1

22993-23008

(A)12,13,14,15,16

(A)15

(A)16

(A)16

(A)14

(A)13

(A)16

(A)13

(A)13

23015-23028

(T)12

*

*

*

*

IGS

rpoB-trnC-GCA

28048-28080

(A)12

*

*

IGS

petN-psbM

29550-29563

(A)12,14

(A)14

(A)12

IGS

psbM-trnD-GUC

30072-30084

(A)12

*

*

30127-30318

(T)12

*

30358-30371

(A)12

*

*

30742-30753

(A)12

*

*

Notes: Except for “blo”, which stands for A. blitoides, and “blu”, which stands for A. blitum, the other species are represented by the first three letters of their species names. “*” indicates that SSR markers at this locus are the same in different species, and blank indicates that SSR markers at this locus are not present in this species.

Table 3-2 Distribution of SSRs in the chloroplast genome of Amaranthus.

Region

Locus

SSR type

pol

alb

blo

blu

cap

def

cri

sta 2

sta 7433

sta 11960

ret

hyb

hyp

dub

spi

spi 11902

pal

are

tub 11994

tub 12194

tub GZW

LSC

IGS

trnD-GUC-trnY-GUA

31288-31299

(T)12

*

IGS

trnE-UUC-trnT-GGU

32019-32035

(A)16

*

IGS

trnT-GGU-psbD

32638-32653

(T)12,14

(T)14

(T)12

(T)12

33128-33139

(TCTT)3

*

IGS

trnG-GCC-trnfM-CAU

37604-37615

(T)12

*

*

37622-37641

(TCAAAA)3

*

*

37631-37646

(AAAC)3

*

IGS

rps4-trnT-UGU

47256-47276

(TA)6,7,9

(TA)6

(TA)6

(TA)7

(TA)9

(TA)6

47597-47608

(A)12

*

47604-47619

(AT)6,8

(AT)6

(AT)7

(AT)6

(AT)8

(AT)6

47619-47632

(TA)6,7

(TA)6

(TA)7

(TA)7

IGS

trnT-UGU-trnL-UAA

48127-48138

(A)12

*

Intron

trnL-UAA

48605-48618

(A)12,13,14

(A)13

(A)13

(A)14

(A)13

(A)12

IGS

ndhK-ndhC

51542-51553

(T)12

*

*

*

IGS

ndhC-trnV-UAC

52311-52322

(TTTC)3

*

IGS

trnV-UAC-trnM-CAU

53534-53545

(ATCT)3

*

*

IGS

trnM-CAU-atpE

53706-53721

(T)14,15,16

(T)15

(T)14

(T)15

(T)16

(T)15

(T)16

(T)15

IGS

atpB-rbcL

55745-55757

(A)12,13

(A)12

(A)12

(A)13

(A)12

IGS

accD-psaI

60133-60157

(T)12,15,16,17

(T)12

(T)15

(T)16

(T)17

(T)16

60267-60281

(A)15

*

CDS

psaI

60733-60744

(TTTA)3

*

IGS

petL-petG

66751-66770

(T)16,17,18,19,20

(T)20

(T)19

(T)18

(T)16

(T)17

(T)18

(T)19

(T)20

(T)18

(T)17

IGS

rpl33-rps18

68574-68586

(T)13

*

68665-68679

(TATTA)3

*

68678-68689

(TA)6

*

IGS

rpl20-rps12

69940-69953

(T)14

*

70437-70450

(T)13

*

Notes: Except for “blo”, which stands for A. blitoides, and “blu”, which stands for A. blitum, the other species are represented by the first three letters of their species names. “*” indicates that SSR markers at this locus are the same in different species, and blank indicates that SSR markers at this locus are not present in this species.

Table 3-3 Distribution of SSRs in the chloroplast genome of Amaranthus.

Region

Locus

SSR type

pol

alb

blo

blu

cap

def

cri

sta 2

sta 7433

sta 11960

ret

hyb

hyp

dub

spi

spi 11902

pal

are

tub 11994

tub 12194

tub GZW

LSC

Intron

clpP Intron

71472-71483

(AAAT)3

*

71537-71551

(AAAAT)3

*

72068-72079

(T)12

*

*

72227-72244

(A)12,14,15,16

(A)15

(A)16

(A)12

(A)16

(A)15

(A)14

(A)12

IGS

psbH-petB

75854-75865

(TTTC)3

*

Intron

petD

77815-77828

(AT)6,7

(AT)6

(AT)7

CDS

rpoA

78957-78969

(T)13

*

IGS

rps11-rpl36

80525-80536

(T)12

*

*

Intron

rpl16

82886-82910

(T)12,13,14,15

(T)15

(T)12

(T)14

(T)13

82950-82971

(A)12,13,14

(A)13

(A)14

(A)12

CDS

rpl22

85134-85147

(T)14

*

*

IRa

IGS

rps19-rpl2

85622-85633

(T)12

*

CDS

ycf2

90647-90658

(CTT)4

*

rRNA

rrn23

105401-105412

(AGGT)3

*

IGS

rrn4.5-rrn5

106884-106895

(CCCT)3

*

SSC

IGS

ndhF-rpl32

112060-112074

(T)13

*

112936-112948

(AAT)4

*

IGS

rpl32-trnL-UAG

113619-113632

(TA)6,7

(TA)6

(TA)7

(TA)6

(TA)6

113867-113882

(T)16

*

114113-114125

(T)13

*

114129-114150

(A)13,14,15

(A)13

(A)14

(A)13

(A)15

(A)14

IGS

trnL-UAG-ccsA

114635-114650

(T)12,13,14,15,16

(T)12

(T)13

(T)15

(T)14

(T)16

CDS

ndhD

116051-116062

(AATA)3

*

IGS

psaC-ndhE

117765-117782

(TCTAGT)3

*

CDS

ndhE

118073-118095

(TATT)3

*

*

Intron

ndhA

121028-121039

(T)12

*

IGS

rps15-ycf1

127822-127833

(TCTT)3

*

IRb

IGS

rrn4.5-rrn5

131038-131049

(AGGG)3

*

rRNA

rrn23

132517-132528

(CTAC)3

*

CDS

ycf2

147273-147284

(AAG)4

*

IGS

rpl2-rps19

152298-152309

(A)12

*

Notes: Except for “blo”, which stands for A. blitoides, and “blu”, which stands for A. blitum, the other species are represented by the first three letters of their species names. “*” indicates that SSR markers at this locus are the same in different species, and blank indicates that SSR markers at this locus are not present in this species.

No competing interests reported.

FigureS1ndhFycf1rpoC2.jpg
Figure S1 Three maximum likelihood topological trees based on ndhF-rpl32, ycf1 and rpoC2 of Amaranthus and three outgroups. Values at each node indicate maximum likelihood bootstrap support (BS) / Bayesian inference posterior probability (PP) value.
SupplementaryinformationTableS14.xlsx
Table S1 Materials information of Amaranthus. Table S2 Genes contained in the sequenced amaranths chloroplast genomes. Table S3 The hotspot regions obtained based on the exhaustive method.

Genetic variation and structure of complete chloroplast genome in alien monoecious and dioecious Amaranthus weeds

Status:

Version 1

Abstract

Figures

Introduction

Results

Discussion And Conclusion

Materials And Methods

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1