Characteristics of Amorphophallus konjac as indicated by its genome

Abstract

Amorphophallus konjac, belonging to the genus Amorphophallus of the Araceae family, is an economically important crop widely used in health products and biomaterials. In the present work, we performed the whole-genome assembly of A. konjac based on the NovaSeq platform sequence data. The final genome assembly was 4.58 Gb with a scaffold N50 of 3212 bp. The genome includes 39,421 protein-coding genes, and 71.75% of the assemblies were repetitive sequences. Comparative genomic analysis showed 1647 gene families have expanded and 2685 contracted in the A. konjac genome. Likewise, genome evolution analysis indicated that A. konjac underwent whole-genome duplication, possibly contributing to the expansion of certain gene families. Furthermore, we identified many candidate genes involved in the tuber formation and development, cellulose and lignification synthesis. The genome of A. konjac obtained in this work provides a valuable resource for the further study of the genetics, genomics, and breeding of this economically important crop, as well as for evolutionary studies of Araceae family.

Full text

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Characteristics of Amorphophallus
konjac as indicated by its genome
Lifang Li
1,6, Min Yang
1,6, Wei Wei
1, Jianrong Zhao
1, Xuya Yu
2, Rarisara Impaprasert
3,
Jianguang Wang
4, Jiani Liu
1, Feiyan Huang
1, George Srzednicki
5* & Lei Yu
1*
Amorphophallus konjac, belonging to the genus Amorphophallus of the Araceae family, is an
economically important crop widely used in health products and biomaterials. In the present work, we
performed the whole-genome assembly of A. konjac based on the NovaSeq platform sequence data.
The nal genome assembly was 4.58 Gb with a scaold N50 of 3212 bp. The genome includes 39,421
protein-coding genes, and 71.75% of the assemblies were repetitive sequences. Comparative genomic
analysis showed 1647 gene families have expanded and 2685 contracted in the A. konjac genome.
Likewise, genome evolution analysis indicated that A. konjac underwent whole-genome duplication,
possibly contributing to the expansion of certain gene families. Furthermore, we identied many
candidate genes involved in the tuber formation and development, cellulose and lignication
synthesis. The genome of A. konjac obtained in this work provides a valuable resource for the further
study of the genetics, genomics, and breeding of this economically important crop, as well as for
evolutionary studies of Araceae family.
e genus Amorphophallus1, a member of the Araceae family, is a perennial, herbaceous plant (Fig.1a). It is
estimated that it includes over 170 species occurring from West Africa, through subtropical and tropical Asia
and further south in the tropical regions of the western Pacic and north-eastern Australia2. e Amorphophal-
lus plants store their reserve polysaccharides, starch and glucomannan, in underground tubers. Some of these
species contain considerable amounts of konjac glucomannan (KGM). e species producing glucomannan are
generally known by the common name ‘konjac’ and are economically important as a raw material for food and
pharmaceutical products worldwide3. is common name comes from Amorphophallus konjac, species that has
been used widely in China and Japan for commercial KGM production. KGM it is used in products ranging
from emulsiers to weight loss supplements, in addition to its long-standing usage as a food and traditional
medicine. China is both, a center of diversity for Amorphophallus and one of the major producers of this plant
worldwide. It is also, along with Japan, one of the leading producers of KGM derived products. A. konjac is a
diploid species (2n = 13) and is one of the important commercial crops cultivated in the central and western
regions of China because it is the only plant species which is rich in KGM concentration4. KGM is a water-soluble,
neutral polysaccharide with a high molecular weight5,6. KGM is a β-1, 4 linked polysaccharide composed of a
-glucose (G) and -mannoses (M) backbone7. e KGM backbone possesses 5–10% acetyl-substituted residues
and the presence of substituted group benets KGM for the solubility in aqueous solution, leading to high vis-
cosity that forms a thick hydrocolloid even when used at low concentrations8. is property makes it one of the
most versatile and economically useful hydrocolloids with industrial applications including the manufacture of
foods, pharmaceuticals and chemicals. KGM is used in a wide range of commercial products throughout Asia
and increasingly throughout the rest of the world6. us, the high quality and purity of KGM obtained from A.
konjac make it the most abundant cultivated Amophophallus species in China, especially in Yunnan. Daguan
county is one of the largest plantation areas of A. konjac in Yunnan and the local A. konjac as an economically
important crop for rural revitalization in this region. en, the representative landrace A. konjac in this region
was used for whole genome sequencing.
Given the economic potential of KGM, a number of studies have been conducted on Amorphophallus species
producing this biopolymer6,9–11. e researchers focused on the relationship between genetic markers and KGM
biosynthesis in A. konjac, and adopted a transcriptomics approach to identify potentially useful regions in the
genome. ey also studied several other KGM producing Amorphophallus species. ese studies are on-going in
OPEN
1College of Agronomy, Yunnan Urban Agricultural Engineering and Technological Research Center, Kunming
University, Kunming, China. 2Faculty of Life Science and Technology, Kunming University of Science and
Technology, Kunming, China. 3Department of Microbiology, King Mongkut’s University of Technology Thonburi,
Bangkok, Thailand. 4School of Life Sciences, Yunnan University, Kunming, China. 5Food Science & Technology,
School of Chemical Engineering, The University of New South Wales, Sydney, Australia. 6These authors contributed
equally: Lifang Li and Min Yang. *email: georgesrz@yahoo.com; yulei0425@163.com
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order to better understand the association between genetic diversity and KGM content in a broader population
of Amorphophallus species.
e main species of Amorphophallus genus have been studied and described in relation to their morphol-
ogy and palynology12–15. Since the morphological and palynological characters are highly variable, a number
of molecular markers have been employed to determine relationships in the genus. ese markers include the
LEAFY (FLint2) gene and the chloroplast regions rbcL, matK and trnL16–19. Since phylogenetic studies based on
these regions do not produce consistent cladograms (due to a high level of conicting signals in the informative
characters), further variable regions and also other non-sequencing molecular methods are needed to establish
the evolutionary history of Amorphophallus. e transcriptomics approach may lead to useful insights into
important traits such as KGM production, tuber formation and development and other characteristics.
e genomes of two important monocotyledonous species in the order of Alismatales namely Spirodela
polyrhiza20 and Zostera marina21 have been sequenced and their characteristics have been described by the
authors of these papers. Although A. konjac as a glucomannan-producing cash crop in many Asian countries,
there have been no any genomic information reports on A. konjac before we conducted whole-genome sequenc-
ing on this species. erefore, we sequenced the whole genome of A. konjac, and the data was submitted to the
NCBI database in 2020. Although Gao etal. subsequently provided a high-quality chromosome-level genome
of A. konjac22, our results can also enrich the genomic information of Amorphophallus to a certain extent. In this
study, we performed a series of genomic analyses on A. konjac including assembly, annotations, identication
of phylogenetic relationship, gene family analysis, divergence time estimation. We also identied cellulose and
lignication synthesis genes, and tuber formation and development genes. e results will provide important
insights as well as resources for future study of A. konjac.
130.7
124.6
86.2 47.5 0
Million years ago
Gene families
Expansion
Contraction
+1119
-2621
+587
-1829
+1647
-2685
+52
-874
+0
-3
+3302
-674
+534
-2580
+814
-349
MRCA
(11659)
Combined Change
Across Lineages
Z. marina
S. polyrhiza
A. konjac
Z. mays
O. sativa
124.6
124.6
86.2
86.2
130.7
130.7
47.5
47.5
A. konjac
Z. marina
O. sativa
S. polyrhize
Z. mays
0
8,000
16,000
24,000
32,000
40,000
48,000
Single-copy orthologs
Multiple-copy orthologs
Unique paralogs
Other orthologs
Unclustered genes
Number of genes
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2
Substitutions per synonymous site (Ks)
Percentage of gene pairs
All
Tandem
Corrected
b
c
d
a
Figure1. Overview for evolutionary analysis of A. konjac. (a) Images of the sequenced A. konjac. (b) Ortholog
clustering analysis of the protein-coding genes in the A. konjac genome. (c) Phylogenetic tree and divergence
time of A. konjac and four plant species. Phylogenetic tree was generated from the single-copy orthologs using
the maximum-likelihood method. e divergence time range is shown by red blocks. e predicted divergence
time is shown as number inside the pink blocks. e pie charts show the proportion of expanded/contracted
gene families in each plant species. (d) Distribution of substitutions per synonymous site (Ks) in A. konjac.
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Results
Genome assembly and annotation
e DNA sequencing data (1119.58Gb, average 110× coverage) of the A. konjac sample were obtained using the
Illumina Hiseq 2500 sequencer. A summary of the sequence data used for the assembly is presented in TableS1.
e estimated genome size is 4,512,012,462bp using 19-mer frequency distribution based on the paired-end
sequenceing data (Fig.S1), which is consistent with measurement by ow cytometry (Fig.S2). Based on the
Illumina sequencing data, 2.99Gb contigs were assembled using SOAPdenovo223 (TableS2). Aer construct-
ing scaolds and lling gaps, the 4.58Gb A. konjac reference genome was assembled, and this resulted in the
7,423,768 scaolds with a scaold N50 of 3212bp (Tables1, S2). e A. konjac genome shows signicant genomic
synteny with S. polyrhiza. e assembly performed in this study captured 75.81% (188 of 248) of core eukaryotic
genes (TableS3) and captured 624 complete BUSCOs v5.2.2 (TableS4) using core eukaryotic genes mapping
approach soware (CEGMA) and BUSCO soware24, respectively.
Combination of de novo prediction and homology-based search resulted in identication of 3,289,511,160bp
repetitive elements in A. konjac genome (TableS5), make up about 71.75% of the assembled genomes (TableS5).
Most of the repeats were de novo predicted (70.98%), the repeats detected by homologous method were relatively
few (TableS5). Among the repeats in the A. konjac genome, 69.16% were transposable elements (TEs), of which
52.06% were long terminal repeats (LTR), including 31.42% Gypsy LTRs and 11.6% Copia LTRs (TableS6).
A total of 39,241 protein-coding genes were predicted in assembled genomes following a combination of
homology and abinitio methods, with an average coding length of 1372.75bp and a mean of 2.29 exons per
gene, respectively (Table1, Fig.S3, TableS7), the gene number and average gene length is close to that of S.
polyrhiza and the average gene is longer than that of Oryza sativa and Zea mays (Fig.S4, TableS7). Moreover,
an average of 92.22% of the RNA sequencing (RNA-seq) reads of the four A. konjac tissues (leaf, stem, root and
tuber) could be mapped to the genome. In addition, 65.26% of the predicted genes (25,725/39,241) showed
expression levels (FPKM > 0.05) by aligning leaf, stem, root and tuber RNA-seq data to the set of protein-coding
genes using TopHat225, and estimating expression values based on the resulting alignments using Cuinks26. In
total, 26,456, 26,512, 25,797 and 33,715 of the predicted genes were assigned with a functional annotation in the
Swiss-Prot, KEGG, InterProScan, and Trembl databases, respectively (TableS8), a total of 34,126 of the predicted
genes (87%) were assigned with a functional annotation in at least one database (TableS8).
An overview of annotated ncRNA is shown in TableS9. 1078 miRNAs, 761 tRNAs, 2894 rRNAs and 1553
snRNAs were predicted in A. konjac.
Table 1. Summary of genome assembly and annotation.
Assembly
Assembled genome size (bp) 4,584,988,971
Genome-sequencing depth (×) 244.18
No. of scaolds 7,423,768
N50 of scaolds (bp) 3212
Longest scaold (bp) 85,347
GC content of the genome (%) 45.71
N length (bp) 887,681,325
Annotation
Percentage of repeat sequences (%) 71.75
Repeat sequence length (bp) 3,289,511,160
No. of predicted protein-coding genes 39,241
Percentage of average gene length (bp) 1,372.75
Average exon length (bp) 257.08
Average exon per gene 2.29
Total intron length (bp) 30,870,726
tRNAs 761
rRNAs 2894
snRNAs 1553
miRNAs 1078
Family number 13,190
Genes in families 22,730
Average genes per family 1.72
Unique families 3001
Un-clustered genes 16,691
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Gene family cluster
Based on pair-wise protein sequence similarity, the gene family clustering analysis of ve species genes, Z.
marina, O. sativa, S. polyrhiza, Z. mays and A. konjac has been carried out. A total of 22,730 genes in A. konjac
were clustered into 13,190 gene families, however, A. konjac has 16,691 unclustered genes and 3001 unique gene
families (Table1,Fig.1b, Fig.S5A, TableS10), that is more than other four species, and the number of single-
copy orthologs genes in A. konjac is 4509. e Venn diagram (Fig.S5a) shows that ve species share a common
core set of 6438 gene families.
e unique gene families in A. konjac were enriched in nucleobase-containing compound biosynthetic
process, nucleobase-containing compound catabolic process, regulation of nucleobase-containing compound
metabolic process, aromatic compound biosynthetic process, heterocycle catabolic process, negative regula-
tion of growth, 1,3-beta--glucan synthase complex, cytoskeleton organization, membrane, molecular function
regulator, peptidase regulator activity, 1,3-beta--glucan synthase activity and so on (Fig.S5B). Moreover, the
unique gene families containa large number of unique paralogous genes (7847 genes) that are not orthologous
to any known genes in other four species, which were enriched in 1,3-beta--glucan synthase complex, a series
of related components of vesicle membrane and so on in cellular component. e 1,3-beta--glucan synthase
complex can catalyse the transfer of a glucose group from UDP-glucose to a (1→3)-beta--glucan chain, which
may be related with the high starch content in tuber and the fast-growing trait in A. konjac.
Evolution, expansion and contraction
To systematically study the evolutionary dynamics of Alismatales species, species phylogeny was performed
utilizing single-copy orthologous genes among ve species, which included 4509 single-copy orthologous genes
in A. konjac. As illustrated in Fig.1c, the estimated divergence time is 130.7 (124.6–139.9) million years ago
(MYA) between Alismatales and Poaceae, Araceae and Zosteraceae separated at about 124.6 (115.3–131.9) MYA,
the divergence time is 86.2 (78.2–96.0) MYA between S. polyrhiza and A. konjac (Fig.1c). is result based on
genomic data will provide a phylogenetic framework for interpreting the evolutionary events of the family.
Comparative analysis of the gene family expansion and contraction showed that 1647 gene families have
expanded and 2685 contracted in the A. konjac genome (Fig.1c). Based on the InterProScan functional annota-
tion, the expansive genes in A. konjac were enriched in iron coordination entity transport, vitamin E metabolic
process, vitamin E biosynthetic process, cofactor transport, heme transport and so on in the biochemical pro-
cesses (p-value < 0.05) (Fig.S6). Furthermore, the gene families that had undergone contraction in A. konjac were
enriched in reproduction, pollination, pollen-pistil interaction, multi-sprout formation, reproductive process,
cell recognition and various biochemical processes (p-value < 0.05) (Fig.S7), which may suggest that the mode
of reproduction is asexual reproduction principally in A. konjac, and the occurrence of sexual reproduction
needs particular conditions.
Whole-genome duplication (WGD) followed by gene loss has been found in most eudicots and is regarded
as the major evolutionary force that gives rise to gene neofunctionalisation in both plants and animals27. Syn-
onymous substitution rates showed a unimodal distribution, indicating that the WGD of A. konjac occurred
recently (Fig.1d), it needs better reference genome to identify that whether or not it corresponds to the ⍺SP/
βSP WGDs in Alismatales20.
Detection of positively selected genes
Positive selection was proposed to contribute to tness. Respectively 686 and 122 genes of A. konjac were deter-
mined as positively selected genes and compared with S. polyrhiza and Z. marina (TablesS11, S12). GO enrich-
ments showed that more positively selected genes in A. konjac in comparison with S. polyrhiza were involved in
RNA biosynthetic process, regulation of biosynthetic process, regulation of gene expression, protein modication
process, cell wall organization or biogenesis, transcription, DNA-templated cell synthesis, cell growth and so
on (Fig.S8). Moreover, the positively selected genes in A. konjac were more involved than those in Z. marina in
leucine biosynthetic process, regulation of signal transduction, regulation of cell communication, regulation of
signaling, regulation of response to stimulus and so on (Fig.S9).
Analysis of transcription factor families
Transcription factors regulate gene expression and protein kinases regulate cellular activities by phosphorylat-
ing target proteins in response to internal or external signals. is study identied a total of 1275 transcription
factors and 345 transcriptional regulators in A. konjac (TableS13). e number of transcription factors in A.
konjac is more than in S. polyrhiza (1115 genes), and the number of transcriptional regulators in A. konjac is
more than in both, S. polyrhiza and Z. marina (271 and 307 genes, respectively), but fewer than that in maize
(573 genes). e AP2/ERF-ERF, GRAS, HSF, SBP, ULT transcription factors are more abundant in A. konjac in
comparison with S. polyrhiza and Z. marina, as well as the AUX/IAA, mTERF, and SNF2 transcriptional regula-
tors. is dierence may be caused by dierent growth environment, A. konjac is a terrestrial plant, while other
two are hydrophilous plants. In addition, the number of BBR-BPC and ULT genes in A. konjac is higher than in
maize. In co-transfection experiments, BBR activates (GA/TC)-containing promoters27, and its overexpression in
tobacco leads to a pronounced leaf shape modication28. In Arabidopsis, the ULTRAPETALA1 (ULT1) gene is a
key negative regulator of cell accumulation shoot and oral meristems, and the mutations in ULT1 can cause the
enlargement of inorescence and oral meristems, the production of supernumerary owers and oral organs,
and a delay in oral meristem termination, downregulation of both ULT genes can lead to shoot apical meristem
arrest shortly aer germination, revealing a requirement for ULT activity in early development29.
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Contractive cellulose and lignication synthesis genes
Amorphophallus konjac is a lodging plant a trait that is consistent with a reduction of genes involved in cell wall
biosynthesis and lignication. According to InterProScan annotation, 50 cellulose synthase (CesA) and cellu-
lose synthase-like (Csl) genes were identied in A. konjac (Table2), which is obviously fewer than in the woody
bamboo species. Lignin, a major component of secondary cell wall, plays an important role for support, water
transport and stress responses in vascular plants19. A total of 20 genes involved in the lignin biosynthesis pathway
were detected in A. konjac (Table2), which contained 6 lignin biosynthesis gene families out of 10 families (PAL,
4CL, HCT, CCR , F5H, CAD but not C4H, C3H, CCoAMT, COMT). Overall, the absolute copy number of both
cellulose- and lignin-related genes decreased in A. konjac compared with woody species. e expression of CesA
and Csl genes also showed two dierent proles (Fig.2a), of which the expression of most genes (Cluster I and
Cluster II) was higher in tuber, bre and stem than in leaf, and expression of six genes (cluster III) were higher
in leaf than in tuber, bre and stem. For the expressed prole of lignin-related genes, the leaf and stem showed
distinct dierence against bre and tuber (Fig.2b).
Tuber formation and development genes
Sucrose metabolism is considered important for the development of a plant sink organ. In most plants, assimi-
lated carbon in source leaves is transported as sucrose into sink organs, including roots, tubers, fruit, and seeds30.
e present study investigated the genes related to starch and sucrose metabolism pathway and found that the
expressed prole of most genes in bre and tuber showed distinct dierence against the leaf and stem, which
were consistently high expression (Fig.3, TableS14). To utilise sucrose, this bond should be cleaved to generate
the two monosaccharides. Sucrose synthase (SUS) is the key enzyme that catalyzes both the synthesis and the
cleavage of sucrose30. SUS is a glycosyl transferase, which converts sucrose into UDP-glucose and fructose in
the presence of uridine diphosphate (UDP). SUS shows consistently high expression patterns in bre and tuber,
whereas low expression was observed in leaf and stem (Fig.3). On the other hand, SPS plays a major role in
photosynthetic sucrose synthesis by catalysing the rate-limiting step of sucrose biosynthesis from UDP-glucose
and fructose-6-phosphate. e expression of sucrose-phosphate synthase (SPS) gene was higher in leaf (Fig.3),
which was consistent with the role played as a limiting factor in the export of photoassimilates out of the leaf.
ese results suggest that sucrose synthase specically facilitates the storage and maturation of sinks.
Sucrose generated from photosynthates in source organs is transported to sink organs and is then converted
into starch. Plants store sugar as polymerised starch, enabling the storage of a larger amount of sugar without
problems caused by osmotic pressure30. In A. konjac, the starch synthase (glgA), granule-bound starch synthase
(WAXY), and glucose-1-phosphate adenylyltransferase (glgC) showed high expression patterns in bre and tuber
(Fig.3), which catalyse precursor substances to synthesise starch. Specially, the expression of 1,4-alpha-glucan
branching enzyme (GBE1) gene was slightly higher in leaf when comparing the three tissues. GBE catalyzes
the formation of α-1,6 branching points in starch and plays a key role in synthesis31. In general, starch synthe-
sized and accumulated directly from the products of photosynthesis in the leaf during the daytime, and is then
degraded into sugars as an energy source for the following night32. erefore, the high expression of GBE1 in
leaf may be related to the synthesis of starch through photosynthesis.In addition, 59 putative genes involved in
Table 2. Copy number variations of cellulose synthase (CesA), cellulose synthase-like (Csl), and lignication
synthesis related genes between 12 plants. PAL: Phenylalanine ammonia lyase; C4H: Cinnamate-4-
hydroxylase; C3H: ρ-Coumaroyl 3′-hydroxylase/Coumaroyl 3-hydroxylase; 4CL: 4-Coumarate CoA Ligas;
HCT: Hydroxycinnamoyl-CoA: shikimate/quinate hydroxycinnamoyltransferase; CCR: Cinnamoyl-CoA
reductase; CCoAOMT: Trans-caeoyl-CoA 3-O-methyltransferase; CAD: Cinnamyl alcohol dehydrogenase;
F5H: Ferulate 5-hydroxylase; COMT: Caeic acid 3-O-methyltransferase. Akon: Amorphophallus konjac; Bam:
Bonia amplexicaulis; Ped: Phyllostachys edulis; Ola: Olyra latifolia; Rgu: Raddia guianensis; Bdi: Brachypodium
distachyon; Osa: Oryza sativa; Zma: Zea mays; Sbi: Sorghum bicolor; Ath: Arabidopsis thaliana; Ptr: Populus
trichocarpa; Spir: Spirodela polyrhiza. a Data from Guo etal.70. b data from Wang etal.20.
Akon BamaPedaOlaaRguaBdiaOsaaZmaaSbiaAthaPtraSpirb
CesA 15 27 26 12 10 19 11 20 12 10 18 10
Csl 35 55 51 40 35 24 34 33 37 29 37 21
PAL 3 13 8 6 7 8 9 10 9 4 5 3
C4H 0 6 4 1 2 3 4 4 3 1 2 3
4CL 7 11 6 4 5 5 5 3 5 4 5 9
HCT 1 5 4 2 2 2 2 2 2 1 2 20
C3H 0 3 3 1 2 1 2 2 2 3 3 1
CCoAOMT 0 2 2 2 1 1 1 2 1 1 2 1
CCR 3 7 5 5 2 2 2 1 2 2 7 21
F5H 4 3 3 2 2 2 3 2 2 2 4 3
COMT 0 2 1 2 1 1 1 1 1 1 2 5
CAD 2 2 3 5 1 1 1 1 1 2 1 4
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Leaf
Stem
Fibr
e
T
uber
C424552781.1
scaffold570138.1
scaffold1428569.1
scaffold1231807.1
scaffold1294268.1
scaffold172367.1
scaffold793354.2
scaffold477935.2
scaffold1416963.1
scaffold778347.1
scaffold567120.2
scaffold477935.1
scaffold442503.1
scaffold1474830.1
scaffold1368005.1
scaffold1333147.1
scaffold1282474.2
C427401444.1
C427193443.1
C427100123.1
C426578615.1
C427076827.1
scaffold560982.1
scaffold662248.1
C426434765.1
scaffold1498400.1
scaffold1178008.1
C426594413.1
C427108391.1
C426912747.1
C427253058.1
scaffold1370363.1
scaffold1378781.1
C426910267.1
scaffold1320605.1
scaffold940359.1
scaffold490911.2
scaffold525577.1
C427166595.1
C427243432.1
scaffold1383231.1
scaffold1457361.1
scaffold1223788.1
scaffold823186.1
scaffold397646.1
scaffold1253996.1
scaffold337472.1
C426321109.1
scaffold470710.1
scaffold470711.1
0
0.5
1
1.5
2
2.5
Cluster I
Cluster II
Cluster III
a
Leaf
Stem
Fibre
Tuber
C427350524.1
scaffold713276.1
scaffold623084.1
scaffold234563.2
scaffold1191157.1
C426494589.1
scaffold1269903.1
scaffold1279511.1
scaffold1325657.1
scaffold1330045.1
scaffold1119499.1
scaffold234563.1
scaffold1488998.1
scaffold649897.1
scaffold1324338.2
scaffold1362070.1
C426742117.1
scaffold1174802.1
scaffold1347659.1
scaffold1235466.1
0
0.
5
1
1.
5
2
2.
5
3
b
Figure2. Heatmaps of gene expression. (a) Heatmap depicting the expressed prole of CesA and Csl genes; (b)
Heatmap depicting the expressed prole of lignin-related genes.
D-Fructose-6P
D-Fructose-6P
UDP-glucose
Sucrose
Sucrose-6P
α-D-Glucose-1P
D-Glucose-6P
Celldextrin
ADP-glucoseAm
y
lose
Dextrin
D-Glucose
Trehalose-6Po Trehalose
Cellulose
Cellobiose
β-D-Glucoside
1,3-β-Glucan
Sucrose (extracellular)
D-Fructose
D-Glucose-6P
Maltose
Trehalose (extracellular)
PYG
SUS
SPS
GBE1
malQ
HK
HK
FRK
glgC
AMY
AMY
BMY
BMY
EG
EG
malZ
malZ
bglU
INV
INV
ENPP1_3
GPI
pgm
bglX
bglX
bglB
bglU
bglX
bglB
SPP
WAXY
glgA
TPS
otsB
TPS
GN
Fibre
Tuber
Leaf
Stem
0
2
4
6
8
10
D-Glucose
bglU
bglB
Starch;Glycogen
SPS
WAXY
EG
GN
Figure3. e expression proles in FPKM (fragments per kilobase per million reads mapped) of genes
involved in the starch and sucrose metabolism pathway in the four tissues (tuber, bre, stem and leaf) from
7-month-old plant of A. konjac. Data are plotted as log10 values. PYG: glycogen phosphorylase; SUS: sucrose
synthase; GBE1: 1,4-alpha-glucan branching enzyme; glgA: starch synthase; malQ: 4-alpha-glucanotransferase;
HK: hexokinase; FRK: fructokinase; glgC: glucose-1-phosphate adenylyltransferase; otsB: trehalose 6-phosphate
phosphatase; AMY: alpha-amylase; BMY: beta-amylase; EG: endoglucanase; malZ: alpha-glucosidase; bglU:
beta-glucosidase; INV: beta-fructofuranosidase; ENPP1_3: ectonucleotide pyrophosphatase/phosphodiesterase
family member 1/3; GPI: glucose-6-phosphate isomerase; pgm: phosphoglucomutase; bglX: beta-glucosidase;
bglB: beta-glucosidase; SPP: sucrose-6-phosphatase; WAXY: granule-bound starch synthase; TPS: trehalose
6-phosphate synthase/phosphatase; GN: included GN1_2_3 (glucan endo-1,3-beta-glucosidase 1/2/3), GN4
(glucan endo-1,3-beta-glucosidase 4) and GN5_6 (glucan endo-1,3-beta-glucosidase 5/6).
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the pathway wrere identied (Fig.4) according the previous studies on glucomannan biosynthesis22,33, and most
of them also were highly expressed in bre and tubers.
Discussion
As a major provider of KGM, A. konjac is abundant in southern China and Japan. e dierent species of genus
Amorphophallus show high genetic diversity. A. konjac is classied as a species with high KGM content. Its tubers
contain between 40 and 70% KGM33. In the natural habitat, fruiting eciency of A. konjac is less than 1% through
sexual reproduction. Although breeding strategies for A. konjac comprise asexual and sexual reproduction, sexual
reproduction happens on the condition of cross-pollination. Increasingly agricultural studies reported that special
structure of inorescence in A. konjac can facilitate the cross-pollination process and possibly increase diversity
of KGM-biosynthetic gene pool. However, genomic background of many traits of A. konjac is little known.
Here, we report the earliest sequenced A. konjac genome, which was sequenced by our research team in 2018
and uploaded to the ncbi database. e genome assembly of A. konjac exhibited a total size of 4.58Gb, which
was smaller than the another genome of A. konjac (5.60Gb) was assembled by Gao etal. using a combination of
Illumina, PacBio, and Hi-C technology22. Meanwhile, Gao etal. also identied 80.6% of the assembled sequences
as repetitive sequences, and 75.6% were transposable elements (TEs)22. Among the various TEs, long terminal
repeats (LTRs, 74.04%), especially Gypsy (40.28%) and Copia (9.58%) type, were remarkably prevalent in the
genome22. Nevertheless, we found that A. konjac genome comprised of 71.75% repeat sequences and 69.16%
were TEs, including 31.42% Gypsy LTRs and 11.6% Copia LTRs. A potential reason for the smaller genome size
and fewer repetitive sequences may be related to the second-generation sequencing data used in the present
study. e second-generation sequencing technologies are dicult to get the large repetitive sequences and lead
to incomplete assemblies34,35. Strong correlation between genome size and the proportion of TEs (especially
LTR-Copia and LTR-Gypsy) has been reported in many studies34,36, 37. In addition, previous studiep also found
that the A. konjac and the S. polyrhiza shared a recent WGD event, which is consistent with the results of this
study21. is study employed the genome analysis to characterise genetic traits of A. konjac. e results implied
that A. konjac possesses 3001 unique families and 4509 single-copy orthologs in a total of 13,190 identied
genes in comparison with the other four species (Z. marina, O. sativa, S. polyrhiza and Z. mays). In addition,
time-tree based on phylogenetic analysis showed that a more closely genetic relationship was found between S.
polyrhiza and A. Konjac (divergent time, 86.2 million years) than another three species (divergent time, over 100
million years between A. konjac and Z. marina, O. sativa and Z. mays). Moreover, the data of this study further
illustrated that some contracted genes in A. konjac genome are involve in pollination, pollen-pistil interaction
and reproductive process, which may oer genomic hints for sexual reproduction of A. konjac.
Positive selection was proposed to contribute to tness. e ratio of non-synonymous to synonymous sub-
stitutions (Ka/Ks), is widely used for the estimation of positive selection at the amino-acid site38. Analysis of the
ratios of Ka/Ks between Chrysanthemum morifolium and C. boreale two Chrysanthemum species, indicating
that 107 genes experienced positive selection, with Ka/Ks more than one, which may have been crucial for the
adaptation, domestication, and speciation of Chrysanthemum39. In current study, we identied 625 and 111
genes in A. konjac were detected under positive selection compared to S. polyrhiza and Z. marina, respectively.
Enrichment analysis suggested that those genes under positive selection are involved in biosynthetic process of
RNA and other organic substances, regulatory process of biogenesis, cellular organization and cell growth. ese
results support the fact that diverse genes were under positive selection in A. konjac, which might inuence the
FRK
Starch
GDP-mannose
UGP
Glucomannan
CSLA
CSLD
Sucrose
glucose
HXK
UDP-glucose
SUS
fructose
HXK
Fructose-6-
phospate
glucose-6-
phospate
glucose-1-
phospate
PGI PGM
Mannose-6-phospate
PMI
PMM
Mannose-1-phospate
GMPP
GDP-glucose
SS
ADP-glucose
AGP
Amylose
SBE
GMPP
INV
-1.5
-1.0
-0.5
0.0
0.5
1.0
L
S
F
T
L
S
F
T
L
S
F
T
L
S
F
T
L
S
F
T
L
S
F
T
L
S
F
T
L
S
F
T
L
S
F
T
L
S
F
T
L
S
F
T
L
S
F
T
L
S
F
T
L
S
F
T
L
S
F
T
Figure4. Putative biosynthetic pathway of KGM. SUS: Sucrose synthase, INV: invertase, PGI: phosphoglucose
isomerase, PGM: phosphoglucomutase\, PMI: phosphomannose isomerase, PMM: phosphomannomutase,
SS: starch synthase, GMPP: GDP-mannose pyrophosphorylase, UGP: UDP-glucose pyrophosphorylase, AGP:
ADP-glucose pyrophosphorylase, FRK: fructokinase, HXK: hexokinase, SBE: starch branching enzyme, CSLA:
cellulose synthase-like A, CSLD: Cellulose synthase-like D. L: Leaf, S: Stem, F: Fibre, T: Tuber.
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adaptation and evolution of A. konjac. Some genes under positive selection can be used as potential biomarkers
for breeding outcrossing species. So far, asexual reproduction of tubers is widely used for breeding A. konjac in
traditional agriculture. However, many problems are related to asexual breeding process, such as low breeding
eciency, long cultivation cycle, high risk of infectious diseases, and breeding degeneration. Genome analysis
in the present study partially demonstrates evolutionary scenario of A. konjac undergoing articial breeding,
and helps to screen outcrossing populations with high KGM content.
Additionally, the analysis of the data collected in the present study suggested that a total of 20 genes were
observed to act in biosynthetic pathways of lignin, which might help cells of A. konjac adapt in habitats suitable
for fast-growing.
Over a few decades, puried KGM from tubers of A. konjac, a dietary bre composed of hydro-colloidal
polysaccharide, was used widely as food additive as well as dietary supplement in many countries. Results
from nutritional studies indicated that KGM can decrease the levels of triglycerides, glucose, cholesterol, and
blood pressure, and prevent many chronic diseases through wide-ranging regulation of metabolism40. Other
studies suggested that KGM content over 50% dry matter should be used to obtain high-purity glucomannan
for development of additives and supplements since high-purity glucomannan can easily form transparent and
odourless gel with high viscosity. e cultivated A. konjac was reported to be major source of high KGM content
material (KGM content over 45% dry matter). Apart from environmental factors and cultivation conditions,
genetic factors are presumed to contribute to productive eciency of high KGM content. However, it is still not
clear which genes of A. konjac genome are involved in regulatory process of KGM biosynthesis in tubers. In
this study, genomic and transcriptomic analysis has been applied to characterise the metabolic process of starch
and sucrose in A. konjac. Previous studies have demonstrated that polysaccharide metabolism is essential both
for formation of tuber sink and biosynthetic source of KGM in A. konjac. Transcriptomic analysis of A. konjac
in the present study suggested that expression patterns of starch and sucrose metabolism diered between
tubers and leaf or stem, and sucrose metabolism related genes maintained consistently higher expression level in
tubers than in leaf and stem. For example, starch synthase (glgA), granule-bound starch synthase (WAXY), and
glucose-1-phosphate adenylyltransferase (glgC) are more expressed in tubers and bres than in leaf and stem.
Previously, some physiological tests suggested the role of sucrose-phosphate synthase (SPS) as exporting factor
of photoassimilates out ohe leaf. Down regulation of SPS can specically help A. konjac facilitate storage and
maturation of polysaccharides in tubers. e ndings in the present study partially clarify versatile functions
of polysaccharide metabolism specic to tubers of A. konjac, and thus potentially help to study biosynthetic
mechanism of formation of KGM.
Conclusions
In this study, we sequenced, assembled, annotated, and analysed the genome of the A. konjac, which belongs to
the genus Amorphophallus of the family Araceae. e 4.58Gb A. konjac genome encoded 39,421 protein-coding
genes and 3,289,511,160bp repetitive sequences, accounting for 71.75% of the genome sequences. Whole-genome
duplication event has been observed within the A. konjac genome. In addition, the sequencing of A. konjac
genome revealed the evolution and the gene expressed dierence in tuber formation and provided a genomic
resource for further study of Amorphophallus genus. Comparative genomics analyses identied the contraction
of gene families associated with reproduction and also genes related with cellulose and lignication synthesis.
e knowledge of the genomic sequences may help in improvement of A. konjac germplasm and facilitate further
studies on KGM synthesis.
Methods
DNA isolation and sequencing
Amorphophallus konjac was obtained from the Daguan county (one of the main plantation areas of A.konjac in
Yunnan), and cultivated in the glasshouse of Kunming University in Yunnan. Fresh leaves were collected from
mature A. konjac plants and frozen in liquid nitrogen. en genomic DNA was extracted from leaves using the
cetyltrimethylammonium bromide (CTAB) method41. e integrity of the extracted DNA was checked by 0.75%
agarose gel electrophoresis. e quantity and quality of the DNA were detected using a NanoDrop ND-2000
(NanoDrop products, Wilmington, DE, USA) and Qubit 2.0 Fluorometer (Invitrogen Ltd, Paisley, UK). Paired-
end libraries with insert sizes of 325bp, 434bp, 529bp, and 647bp were constructed using NEBNext Ultra II
DNA Library Prep Kit for Illumina (NEB, USA), and mate pair libraries with insert sizes of 3kb, 7kb, 12kb,
and 16kb were constructed using Illumina Nextera Mate Pair Library Preparation Kit (Illumina, USA). All the
constructed libraries were sequenced on a NovaSeq platform (Illumina, USA) using PE-150 module. In total,
about 1119.58Gb of data were generated on Illumina platforms.
All reads were preprocessed for quality control and ltered using the in-house Perl script. e raw data were
ltered by removing reads with more than 5% N or more than 40bp low-quality bases called below Q30. e
redundant reads resulting in duplicate base calls were ltered; only one copy of any duplicated paired-end reads
was retained. e yielded clean data were used for de novo assembly.
Genome size estimation
Before genome assembly, we used Illumina short reads to estimate the genome size using a k-mer based method.
An optimal k-mer value was obtained by Jellysh42, and genome size was estimated using GenomeScope v2.043
based on the 19-mer frequency distribution data. A 19-mer was the k-mer length recommended for use with the
GenomeScope 2.0 program and was not adjusted because we had high coverage and a low error rate. e genome
size was also estimated by ow cytometry using Z. mays as internal standard and propidium iodide as the stain.
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Assembly
e ltered reads were used to perform assembly with SOAPdenovo223 developed by BGI. First, the contigs
were constructed with k-mer = 47 using pair-end data, and the scaolds were assembled with k-mer = 33 using
both mate-pair and pair-end data. e nal assembly was generated aer gap lling with Gapcloser v1.12 in
SOAPdenovo package23.
Repeats annotation
First, the research team searched for tandem repeats across the genome using the program Tandem Repeat Finder
(TRF)44. e transposable elements (TEs) in the genome were identied by a combination of homology-based
and de novo approaches. For homolog-based prediction, known repeats were identied using RepeatMasker45
and RepeatProteinMask45 against Repbase16.1046. RepeatMasker was applied for DNA-level identication using
a custom library. At the protein level, RepeatProteinMask was used to perform an RMBLAST search against the
TE protein database. For de novo prediction, RepeatModeler (http:// repea tmask er. org/) and LTR FINDER47
were used to identify de novo evolved repeats inferred from the assembled genome.
Gene prediction and functional annotation
e research team employed EVidence Modeler (EVM)48 to consolidate RNA-seq, protein alignments with
abinitio gene predictions and homologous method annotation into a nal gene set. For transcriptome, reads
were cleaned with Trimmomatic Version 0.3249. is step removed reads containing adapter, reads containing
poly-N and low-quality reads from the raw data and yielded clean data for downstream analysis. en, the reads
were aligned to the genome with HISAT2 Version: 2.1.050. Alignments were then assembled independently with
StringTie Version: v1.3.3b51. Protein sequences of ve plant species: Arabidopsis thaliana52, Oryza sativa53, Zea
mays54, Zostera marina21 and Spirodela polyrhiza20 were used for the homology-based method. First, the tblastn
was performed with e-value cuto 1e-5, blast hits with low quality in the genome were discarded. en predicted
regions were extended by 2000bp both upstream and downstream, and aligned against protein sequence using
GeneWise55 to identify gene structure. e soware AUGUSTUS56, GenScan57, GlimmerHMM58 and SNAP59
were used for abinitio gene prediction, AUGUSTUS and GenScan prediction used the gene model parameters
trained on maize, but GlimmerHMM and SNAP prediction used gene model parameters trained on rice. All
lines of evidence were then fed into EVM using intuitive weighting (RNAseq > cDNA/protein > ab initio gene
predictions).
Gene functions were assigned according to the best match alignment using Blastp against Swiss-Prot,
TrEMBL and KEGG databases. InterProScan functional analysis and Gene Ontology IDs were obtained using
InterProScan60.
e GO enrichment was done with Ontologizer 2.061 by using one-sided Fisher’s exact test, the Parent–Child-
Union method, with a p-value cut-o of 0.05.
Genes related to cellulose synthase (CesA), cellulose synthase-like (Csl) were identied according to the
InterProScan annotation, and the genes related to phenylpropanoid-lignin biosynthesis and starch and sucrose
metabolism pathway were identied according to the KEGG annotation. Furthermore, the genes with alignment
hits covering over 200 amino acids and at least 50% protein sequence identity were considered to be candidate
genes.
Non-coding gene annotation
Soware tRNAscan-SE62 is specied for Eukaryotic tRNA and was deployed for tRNA annotation. e research
team used homologous method to identify rRNA. e rRNA sequence data downloaded from Rfam database63
was used as a reference. INFERNAL64 was used to identify snRNA.
Gene family cluster
To identify dierent sets of gene clusters, protein-coding genes sequences of O. sativa53, Z. mays54, Z. marina21and
S. polyrhiza20 were used to locate gene clusters. Aer pairwise aligning using Blastp with an e-value cuto of 1e-5
had been conducted, OrthoMCL package65 was performed to identify the gene family clusters using the Blastp
output with default parameters, nal paralogous and orthologous genes were dened using MCL soware in
OrthoMCL.
Phylogenetic tree construction
Single-copy orthologous genes dened by OrthoMCL65 were formed, and then multiple single-copy genes were
aligned using Muscle66 and the aligned sequences were extracted to feed to MrBayes (http:// mrbay es. sourc
eforge. net) to infer the species phylogeny using a maximum likelihood (ML) approach under the best-t model
GTR + G from ModelFinder. Z. mays and O. sativa were used as outgroups. To estimate the divergence time of
each species, the information about the already known divergence time data between these species from http://
www. timet ree. org/ were collected. e topology of the ML tree was fed to MCMCTREE in paml version 4.467 for
constructing a divergence time tree and calculate the divergence time. Based on the calculated phylogeny and
the divergence time, CAFÉ (Computational Analysis of Gene Family Evolution, version 2.1)68, a tool based on
the stochastic birth and death model for the statistical analysis of the evolution of gene family size, was applied
to identify gene families that had undergone expansion and/or contraction.
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Detection of positively selected genes
To detect genes under positive selection, Blastn was performed to align the coding sequence (CDS) libraries of
Z. marina21and S. polyrhiza20 against the A. konjac CDS library, respectively, in order to nd the gene pairs with
the best alignments. e resulting orthologous gene pairs were aligned again with the default parameters as a
preparation for KaKs_Calculator 1.269 which nally yielded a dataset of each gene pair’s Ka/Ks ratio, and the Ka/
Ks ratio > 1 was dened as a positively selected gene (signicance, P-value < 0.05).
RNA-seq
Four tissues (namely tubers, bres, stems and leaves) of A. konjac were harvested from the same 7-month-old
plant, and three biological replicates for each tissue of living plants were collected. Total RNA was extracted
from these tissues using the RNAprep pure plant kit (Tiangen). 3g of total RNA per sample were used as input
material for the RNA sample preparation. Beads with oligo (dT) were used to isolate poly (A) mRNA from total
RNA. RNA sequencing libraries were constructed from these mRNA using the TruSeq RNA Sample Preparation
Kit (Illumina, San Diego, USA). Briey, the Elution 2-Frag-Prime (94°C for 8min, 4°C hold) was used to elute,
fragment and prime the mRNA with Elute, Prime, Fragment Mix (Illumina). First strand cDNA synthesis was
performed with First Strand Master Mix and SuperScript II mix (ratio: 1l SuperScript II/7l First Strand Master
Mix) (Invitrogen). e second strand was synthesized with Second Strand Master Mix (Illumina) and Ampure
XP beads (Illumina) were used to separate the double-stranded (ds) cDNA from the 2nd strand reaction mix.
Aer end repair and the addition of a 3’-dA overhang, the cDNA was ligated to Illumina PE adapter oligo mix
(Illumina), and size-selected for 350 ± 20bp fragments by gel purication. Aer 15 cycles of PCR amplication,
the 350bp paired-end libraries were sequenced using the paired-end sequencing module (150bp at each end)
of the Illumina HiSeq 4000 platform.
e corresponding trimmed clean reads were aligned to the related reference genome employing TopHat224
soware with default settings. Calculation of gene expression level was conducted using Cuinks v2.2.125. Frag-
ments per kilobase of exon per million fragments mapped (FPKM) were used to normalize RNA-seq fragment
counts and estimate the relative abundance of each gene. e DEGs were decided based on a P-value < 0.05 and
at least a twofold change between the two FPKMs.
Ethical approval
We conrm that all the experimental research and eld studies on plants (either cultivated or wild), including
the collection of plant material, complied with relevant institutional, national, and international guidelines and
legislation. e tuber of A. konjac was collected from Daguan county, and was cultured in the green house. All
the material is owned by the authors and/or no permissions are required.
Data availability
Accession numbers: e genome sequence of A. konjac has been deposited in DDBJ/EMBL/GenBank nucleo-
tide core database under accession code SUB7124908 (https:// www. ncbi. nlm. nih. gov/ sra/ PRJNA 608095). e
sequencing reads of Illumina sequencing libraries have been deposited under NCBI Sequence Read Archive with
Project ID PRJNA608095. e Project ID of all the RNA-seq data is SRP251185.
Received: 1 June 2023; Accepted: 14 December 2023
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Acknowledgements
is study was supported by Yunnan Province Youth Talent Support Program (Grant No.YNWR-QNBJ-2018-32);
Yunnan Fundamental Research Projects (Grant NO. 202101BA070001-163); Yunnan Education Department
Research Project (Grant No. 2022J0644, 2023J0827); Yunnan Provincial Science and Technology Department
(No. 2019FH001-008, 2019FH001-051). e funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Author contributions
L.L. and L.Y. conceived the project and its components and wrote the manuscript. W.W., J.L. and J.Z. prepared
the sample material for sequencing. L.L., L.Y., M.Y., X.Y. and R.I. conducted the genome sequencing and assem-
bling. L.L., G.S., L.Y. and M.Y. performed the data analysis. M.Y., R.I. and F.H. prepared the gures and tables.
All authors reviewed the manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 023- 49963-9.
Correspondence and requests for materials should be addressed to G.S.orL.Y.
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