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The rst draft genome of feather
grasses using SMRT sequencing
and its implications in molecular
studies of Stipa
Evgenii Baiakhmetov1,2*, Cervin Guyomar3,4, Ekaterina Shelest3,5, Marcin Nobis1,2* &
Polina D. Gudkova2,6
The Eurasian plant Stipa capillata is the most widespread species within feather grasses. Many taxa
of the genus are dominants in steppe plant communities and can be used for their classication and
in studies related to climate change. Moreover, some species are of economic importance mainly as
fodder plants and can be used for soil remediation processes. Although large-scale molecular data
has begun to appear, there is still no complete or draft genome for any Stipa species. Thus, here we
present a single-molecule long-read sequencing dataset generated using the Pacic Biosciences
Sequel System. A draft genome of about 1004 Mb was obtained with a contig N50 length of 351 kb.
Importantly, here we report 81,224 annotated protein-coding genes, present 77,614 perfect and
58 unique imperfect SSRs, reveal the putative allopolyploid nature of S. capillata, investigate
the evolutionary history of the genus, demonstrate structural heteroplasmy of the chloroplast
genome and announce for the rst time the mitochondrial genome in Stipa. The assembled nuclear,
mitochondrial and chloroplast genomes provide a signicant source of genetic data for further works
on phylogeny, hybridisation and population studies within Stipa and the grass family Poaceae.
In the year 2000, the Arabidopsis thaliana L. genome became the rst plant genome to be completely sequenced
and assembled1. Since then, many genomes from the plant kingdom have been sequenced, e.g. green algae2,3,
bryophytes4,5, ferns6, gymnosperms7,8 and angiosperms9,10. In the grass family (Poaceae) the reference assemblies
were primarily obtained for crops11–13 and model plants14–16. e advent of second-generation sequencing and
the subsequent decreasing of the overall sequencing costs have enabled the determination of whole genome
sequences in many non-model plant species17–20.
Recently, the 1KP project that was aiming to sequence 1,000 green plant transcriptomes21–23 has been followed
by the 10KP project24. e later initiative intends to sequence complete genomes from more than 10,000 plants
and protists. e project is supposed to be completed in 2023 and it presumes to provide family-level high-quality
reference genomes, ideally with chromosome-scale assemblies. Nevertheless, the data at the level of genera may
not be processed immediately24. In comparison to other kingdoms, plants have very large genomes13,25,26, high
ploidy level27 and the abundance of repetitive sequences28–30. Currently, to face these issues, the third-generation
sequencing has been applied. e so-called single-molecule real-time (SMRT) sequencing provided by Pacic
Biosciences (PacBio)31 and nanopore sequencing by Oxford Nanopore Technologies32 aord a range of benets,
including exceptionally long-read lengths (20kb or more), resolving extremely repetitive and GC-rich regions
and direct variant phasing32,33.
In the fossil record Stipa L., or a close relative genus, is known from about 34 Mya of the upper Eocene34,35.
For many decades, Stipa has been described as a genus with over 300 species common in steppe zones of Eurasia,
North Africa, Australia and the Americas36,37. According to the recent studies based on both morphological and
molecular data, the genus has been reduced and currently includes over 150 species geographically conned to
OPEN
Research
German
Institute for Genetics,
France. Department
*
doctoral.uj.edu.pl
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Europe, Asia and North Africa38–42. Most species of Stipa are dominants and/or subdominants in steppe plant
communities43–45 and can be used for their classication46. Moreover, some species are of economic importance
mainly as pasture and fodder plants, especially in the early phases of vegetation36,47, they can be used for soil
remediation processes48,49, in studies related to climate change50–52 and as ornamental plants (e.g. S. capillata L.,
S. pulcherrima K. Koch, S. pennata L.).
In recent years, large-scale molecular data began to appear for Stipa: de novo transcriptome assemblies of S.
purpurea Griseb.50,53, S. grandis P. A. Smirn.54 and S. lagascae Roem. & Schult.52, whole chloroplast genomes for
19 taxa57 and raw genomic data available via the NCBI Sequence Read Archive (SRA) for S. capillata58 and S.
breviora Griseb.59. In addition, nucleolar organising regions (NORs) were sequenced for six Stipa taxa60. Nev-
ertheless, no complete or dra genome assembly currently exists for any Stipa species. In order to ll this gap,
here we aim to: (1) present for the rst time a single-molecule long-read dataset (nuclear, mitochondrial and
chloroplast genomes) generated using the SMRT sequencing on the PacBio Sequel platform; (2) demonstrate
and discuss the potential usage of this data in further studies of Stipa.
For the goals of the study we chose to sequence the entire genome of S. capillata (Fig.1) as it is the most
widespread taxon within the genus, growing on sandy to loamy, nutrient poor soils in the dry grasslands of
Eurasia61. Currently, this species is increasingly attracting the interest of conservation biologists due to its large
distribution range, common occurrence in the Eurasian steppes and pseudosteppes, a limited number of refugia
in Europe and both great morphological and genetic variability within its range62–64.
Results
Assembled nuclear genome. e SMRT sequencing yielded in 23.16-fold genome coverage consisting of
25.84Gb sequence data with an N50 read length of 17,096bp (Supplementary TableS1). De novo assembling of
PacBio reads using Flye v.2.465,66 resulted in a genome size of 1,004 Mb67 with a contig N50 of 351kb and a GC
level of 45.97%. On the other hand, another de novo assembly performed with FALCON v.0.2.568 demonstrated
a smaller genome size of 773Mb with a GC level of 46.04%. However, the Flye assembly has a better N50 of
350,543 that is almost three times bigger than for FALCON. In case of applying Purge Haplotigs v1.1.169, the nal
genome size was reduced by 177Mb with an N50 of 381,155 (Table1) and a GC level of 45.82%.
Figure1. A representative individual of Stipa capillata.
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e subsequent analysis based on a benchmark of 4,896 conserved genes belonging to the Poales order
(dataset poales_odb10) revealed that the Flye assembly has 4,557 (93.10%) completed BUSCO (Benchmarking
Universal Single-Copy) genes and only 293 (6%) missing BUSCOs versus 2,765 (56.50%) and 1,945 (39.70%)
for the FALCON assembly. e Flye assembly aer Purge Haplotigs shows 4,304 (87.90%) completed BUSCOs
and 512 (10.50%) missing BUSCOs (Table2).
Scaolding of contigs. Nearly all contigs of S. capillata genome can be assigned to the reference chro-
mosomes of Brachypodium distachyon L., Hordeum vulgare L. and Aegilops tauschii Coss., whereas genomes of
Oryza sativa L. and especially Triticum aestivum L., have much less homology to the feathergrass assembly. In
particular, 95.16% contigs of S. capillata genome were assigned to seven chromosomes of A. tauschii genome,
94.68% to ve chromosomes of B. distachyon, 94.20% to seven chromosomes of H. vulgare, 89.92% to 12 chro-
mosomes of O. sativa and only 41.67% to 21 chromosomes of T. aestivum. e total length of non-assigned
contigs was reasonably low for A. tauschii (48.59Mb), B. distachyon (53.40Mb) and H. vulgare (58.17Mb),
whereas for O. sativa and T. aestivum it was about 101.15Mb and 585.40Mb, respectively (Table3). In addi-
tion, the RaGOO grouping condence and orientation condence scores per chromosome ranged from 57.81
to 76.11% and from 80.03 to 95.11%, respectively, indicating that the contigs could be placed on a chromosome
with an acceptable level of condence (Supplementary TableS2). e only exception is T. aestivum for which
scores ranged from 30.49 to 47.76% for the grouping condence score and from 57.81 to 70.19% for the orienta-
tion condence score. Nevertheless, based on the location condence score, the exact position of the contigs on
a chromosome could not be accurately estimated, reecting a low level of synteny to the reference genomes. In
Table 1. Statistics of the nuclear genome assemblies.
Metrics Flye assembly FALCON assembly Flye assembly aer Purge Haplotigs
Length of assembly, bases 1,003,531,354 773,212,558 826,891,869
Number of sequences 5,931 885 3,683
Largest length of a sequence, bases 2,321,367 590,564 2,321,367
Average length of sequences, bases 169,201 88,015 224,516
N50, bases 350,543 119,836 381,155
Number of sequences with N50 837 2,061 640
N100, bases 1,001 20,078 1,014
Table 2. BUSCO statistics.
Metrics Flye assembly FALCON assembly Flye assembly aer Purge Haplotigs
Complete BUSCOs 4,557 (93.10%) 2,765 (56.50%) 4,304 (87.90%)
Complete and single-copy BUSCOs 2,383 (48.70%) 2,408 (49.20%) 2,916 (59.60%)
Complete and duplicated BUSCOs 2,174 (44.40%) 357 (7.30%) 1,388 (28.30%)
Fragmented BUSCOs 46 (0.90%) 186 (3.80%) 80 (1.60%)
Missing BUSCOs 293 (6%) 1,945 (39.70%) 512 (10.50%)
Total BUSCO groups searched 4,896 (100%) 4,896 (100%) 4,896 (100%)
Table 3. RaGOO statistics.
Species Number of chromosomes (n) Number and the total length of contigs
assigned to the reference Number and the total length of non-
assigned contigs
B. distachyon70 54,061 (950.13Mb) 1,871 (53.40Mb)
94.68% 5.32%
H. vulgare71 74,036 (945.36Mb) 1,896 (58.17Mb)
94.20% 5.80%
A. tauschii72 74,161 (954.95Mb) 1,771 (48.59Mb)
95.16% 4.84%
O. sativa73 12 3,477 (902.39Mb) 2,455 (101.15Mb)
89.92% 10.08%
T. a est iv um74 21 2,434 (418.14Mb) 3,498 (585.40Mb)
41.67% 58.33%
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particular, the score was in a range of 31.30–43.66% for O. sativa, 26.06–39.13% for B. distachyon, 19.56–31.41%
for H. vulgare, 17.47–24.15% for A. tauschii and 10.30–38.23% for T. aestivum.
Transposable elements and nuclear genome annotation. Identication of transposable elements
(TEs) revealed that more than half of the S. capillata genome (57.68%) is occupied by repetitive sequences.
Particularly, retrotransposons represent at least 16.12% and transposons are reaching no less than 7.22% of the
genome. Nonetheless, 34.34% of TEs are currently unclassied. Among classied repeats, long terminal repeats
(LTRs) were the most abundant elements within retrotransposons, whereas Tourist/Harbinger elements were
more common amid DNA-transposons. In total, 114,826 sequences were identied as simple repeats and occupy
0.57% of the genome. In addition, rolling-circles (0.28% of the genome) and low complexity sequences (0.11%
of the genome) were found (Table4).
e subsequent structural annotation of the masked genome revealed 53,535 nuclear genes (Supplementary
File 1). On the other hand, the unmasked genome has 154,755 structurally annotated genes and 94,237 of them
have BLAST hits in the NCBI non-redundant database. Nonetheless, among the 94,237 genes of the unmasked
genome, 12,094 sequences are related to transposable elements. In particular, 2,925 genes associated with trans-
posons, and 9,859 assigned to retrotransposons. In addition, 229 genes encode transposase-related proteins.
us, except transposable elements the unmasked genome has 81,224 genes that can be associated with already
known proteins (Supplementary File 2).
SSR markers. In total, 77,614 perfect repeat motifs were identied for the nuclear genome assembly using
Krait75 (Supplementary File 3). Within those, di- and tri-nucleotides were the most common types, accounting
28,365 (36.55%) and 25,794 (33.23%) repeats, respectively. Tetra-nucleotide motifs were the third most abundant
repeats with 9,777 SSRs (12.60%), followed by mono-nucleotides with 6,572 SSRs (8.47%) and penta-nucleotides
with 4,629 SSRs (5.96%). Hexa-nucleotides were the rarest motifs with 2,477 SSRs (3.19%). Only four mono-
nucleotide, four di-nucleotide and three tetra-nucleotide motifs were found in the mitochondrial and chloro-
plast genomes. However, a total length of those SSRs was in a range of 12–16bp. In addition, in total 58 unique
repeats present only in a single copy in a range 101–325bp were retrieved from the analysis of TEs. Within those
were four hexa-, 35 hepta-, nine octa-, ve nona- and ve deca- nucleotide motifs (Supplementary TableS3).
Divergence time of Stipa. e Bayesian phylogenetic reconstruction based on the ve loci within NORs
revealed the divergence time of Stipa from Brachypodium around 30.00–35.52 Mya and the putative origin of
feather grasses about 2.90–6.02 Mya (Fig.2). Although not all branches were well supported within the genus,
the current analysis conrmed the monophyly of Stipa and the general grouping of the analysed species regard-
ing their taxonomic positions. In particular, S. capillata and S. grandis represent the section Leiostipa Dumort; S.
magnica Junge, S. narynica Nobis, S. lipskyi Roshev. and S. caucasica Schmalh. belong to the section Smirnovia
Tzvelev. e remaining three groups include (1) S. orientalis Trin. and S. pennata L., (2) S. richteriana Kar. & Kir.,
S. lessingiana Trin. & Rupr., S. heptapotamica Golosk. and S. korshinskyi Roshev, (3) S. lagascae and S. breviora
currently have a discrepancy between morphological and molecular data. In addition, the divergence time esti-
mation indicates that the potential origin of the clade comprising S. capillata and S. grandis is in a range of 0.67–
2.93 Mya while the sister clade has the 95% credibility intervals for that parameter in a range of 2.38–4.78 Mya.
Furthermore, the lowest genetic divergence time was registered for S. lessingiana and S. richteriana (0.00–0.48
Mya) as well as for the split between S. heptapotamica and the two above-mentioned species (0.01–0.78 Mya).
e divergence times for the rest of taxa are present in Table5.
Table 4. Statistics of repetitive elements.
Type of repeats Number of elements Total (bp) % of genome
Class I: Retrotransposon: 123,524 161,756,598 16.12
SINEs 6,211 2,422,254 0.24
LINEs 26,453 19,189,619 1.91
LTR elements 90,860 140,144,725 13.97
Class II: DNA-transposon: 99,245 72,448,468 7.22
Hobo-Activator 6,824 3,826,368 0.38
Tc1-IS630-Pogo 619 500,988 0.05
PiggyBac 1 75 0.00
Tourist/Harbinger 11,326 3,980,231 0.40
Other 2 113 0.00
Unclassied 758,908 344,622,074 34.34
Total repeats 981,677 578,827,140 57.68
Rolling-circles 3,306 2,797,158 0.28
Low complexity 18,762 1,145,428 0.11
Simple repeats 114,826 5,716,291 0.57
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Figure2. Phylogeny and divergence time estimation by molecular clock analysis. Letters at each node refer
to Table5. Numbers in brackets represent the Bayesian posterior probabilities (BPP > 0.50 only). e blue
rectangles on the nodes indicate the 95% credibility intervals (CI) of the estimated posterior distributions of the
divergence times. e red circles indicate the presumed divergence time splits set as a reference. e scale on
the bottom shows divergence time in Mya. e gure was created using Figtree v1.4.4, https:// tree. bio. ed. ac. uk/
sow are/ gtr ee/.
Table 5. Node ages, BPP and CI related to Fig.2.
Node Node age (Mya) BPP 95% CI
A 48.59 1.00 44.53–52.78
B 32.77 1.00 30.00–35.52
C 4.39 1.00 2.90–6.02
D 3.55 0.40 2.38–4.78
E 3.02 0.28 1.95–4.14
F 2.26 0.40 1.21–3.40
G 2.15 0.63 1.05–3.32
H 2.04 1.00 1.15–3.02
I 1.77 0.85 0.76–2.87
J 1.73 1.00 0.67–2.93
K 1.56 0.96 0.81–2.38
L 0.91 0.67 0.28–1.60
M 0.71 1.00 0.11–1.46
N 0.33 0.39 0.01–0.78
O 0.16 0.28 0.00–0.48
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Assembled mitochondrial and chloroplast genomes. e resulting Flye assembly contained four
mitochondrial contigs with a total length of 438,037 bp76–79 represented by six edges and an entire 137,832bp-
long circular chloroplast genome combining a long single copy region (LSC) of 81,710bp, a short single copy
region (SSC) of 12,836bp and two inverted repeats (IR) of 21,643bp each (Fig.3). However, aer a manual
checking in IGV v.2.8.680 the nal size of the chloroplast genome was slightly reduced to 137,823bp. In addi-
tion, an analysis using Cp-hap81 detected two structural haplotypes of the chloroplast genome: haplotype A82
(LSC—IR, reverse-complement (rc)—SSCrc—IR) and haplotype B83 (LSC—IRrc—SSC—IR). We also obtained
one assembly using Unicycler v.0.4.884 resulted in 76 linear contigs from which 29 can be assigned to mitochon-
drial sequences with a total length of 1,668,569bp. Due to the Unicycler assembly being more complex and none
of the obtained contigs were likely to be circular in nature, for the downstream genome annotation we used the
Flye assembly.
In total, 112 and 133 genes were functionally annotated for mitochondrial and chloroplast genomes, respec-
tively. e mitochondrial annotation resulted in 78 protein-coding genes, 4 ribosomal RNA genes and 30 tRNA
genes. e chloroplast annotation contained 85 protein-coding genes, 8 ribosomal RNA genes and 40 tRNA
genes. e chloroplast genome size of 137,823bp generated with Flye and the number of annotated genes in the
current study were similar to the known assemblies for S. capillata obtained by Illumina sequencing57. However,
the previous genome assemblies were slightly longer, specically 137,830 bp86 and 137,835 bp87.
DArTseq markers. e DArT pipeline analysis resulted in 61,328 Silico markers and in 52,970 sequences
with SNPs. e BLAST process revealed 58,701 Silico markers and 52,252 sequences with SNPs that were suc-
cessfully mapped to 4,361 and 3,935 genome contigs, respectively. us, the current genome assembly has
95.72% of Silico markers and 98.64% of sequences with SNPs that are represented in 73.52% (the total length of
969.30Mb) and 66.34% (940.37Mb) of the contigs, respectively. In addition, we established that 50,953 Silico
markers and 47,181 sequences with SNPs were present only in a single copy in the genome. Finally, we identied
30 Silico markers and 10 sequences with SNPs aligned to the mitochondrial genome and only 2 Silico markers
and 4 sequences with SNPs that were found in the chloroplast genome.
Discussion
e number of sequenced plant genomes is rapidly increasing year by year serving as a fundamental resource
for various genomic studies. In the current work, we present a 1004Mb genome with the 23 × coverage of the
most widespread feather grass species, S. capillata, using SMRT PacBio sequencing. e current assembly com-
prises 5,931 sequences with a contig N50 length of 351kb (Table1). e BUSCO completeness score of 93.10%
(Table2), the observation of a large portion of TEs (57.68%, Table4) and the presence of Silico (95.72%) and
SNPs (98.64%) markers derived from the DArT platform indicate that the assembly is of high quality. Moreover,
the proportion of TEs has been reported for the rst time in the genus due to the previous de novo assemblies
which were performed exclusively based on transcriptomic data50,52,54. In addition, here we also attempted to
perform a reference-guided scaolding of the assembled contigs. Nevertheless, although nearly all contigs of
the S. capillata genome were assigned to the chromosomes of B. distachyon, H. vulgare and A. tauschii, it was
not possible to estimate their proper position on the reference with an acceptable level of condence (Table3
Figure3. Visualisation of the de novo mitochondrial and chloroplast genome assemblies using Bandage
v.0.8.185. (a) Contigs representing mitochondrion. (b) Contig representing chloroplast. Dierent colours
represent dierent contigs; length (in bp) and coverage (x) of edges within contigs are shown. e gure was
created using Bandage v.0.8.1, https:// rrwick. github. io/ Banda ge/.
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and Supplementary TableS2). In general, in the absence of a high-density genetic linkage map the task of recon-
structing pseudomolecules of chromosomes seems to be challenging. On the other hand, we believe that in order
to improve the contiguity of the long-read assembly the high-throughput chromosome conformation capture
(Hi-C)88 technique should be applied. Currently, many studies on non-model species successfully utilised a
combination of long-read techniques and Hi-C data to perform assemblies at chromosome scale89–91. Moreover,
an additional key for improving this genome assembly in the future is merely to get more sequencing reads.
Recently, it was shown that contig length metrics are positively correlated with both read length and sequence
coverage. Specically, long-read assemblies in maize demonstrated that the highest contig N50 of 24.54Mb was
reached with a subread N50 of 21,166bp and a 75-fold depth of coverage while the longest contig of 79.68Mb
was observed with the same subread N50 but with a 60-fold depth92.
e newly generated genome has a GC content of 45.97% that is similar to the known estimates for species
in Stipa varying in a range of 46.61–49.05%93, and more broadly to grasses ranging from 43.57% in O. sativa to
46.90% in Z. mays94. Recently, it was shown that a higher GC content in monocots is associated with adaptation
to extremely cold and/or dry climates95. e genus Stipa highly supports this hypothesis due to the fact that
all feather grasses are adapted to temperate, dry climates36. In addition, a positive correlation between the GC
content and genome size was established96 suggesting insertion of LTR retrotransposons as a potential driving
force of genome enlargement97. Similarly, here we showed that the expansion of the S. capillata genome also
resulted from insertions of repetitive sequences that occupy 57.68% of the genome including LTR retrotrans-
posons (13.97%). However, among all repetitive sequences around 34.34% are currently unclassied (Table4).
Nonetheless, the total proportion of TEs in S. capillata in comparison to other species within the Poaceae family
is close to Oryza minuta J. Presl (58.35%) and O. alta Swallen98 (57.54%), bigger than in B. distachyon99 (28.10%)
and O. sativa100 (45.52%) and smaller than in O. granulata Nees & Arn.101 (67.96%), Avena sativa L.102 (69.47%)
and T. aestivum103 (84.67%).
Importantly, the presented genome size is roughly twice smaller than the expected size of 2,355Mb and twice
bigger than the expected monoploid size of 589Mb estimated using ow cytometry93. Considering that we were
unable to remove redundant sequences due to possible heterozygosity and the number of duplicated BUSCOs
(Tables1 and 2), it may be presumed that the current genome assembly combines two very distinct genomes.
To the current knowledge, the vast majority of Stipa species have 44 (2n = 4x) chromosomes and are supposed
to be tetraploids41,104. In addition, recently it was shown that a single-copy region ACC1 and a low-copy nuclear
gene At103 have two dierent copies in Stipa104,105. us, it may suggest that S. capillata, and the genus Stipa in
general, has arisen through hybridisation between genetically distant diploid species (2n = 22) and the subsequent
allopolyploidisation via whole genome duplication (WGD) rather than via one WGD event of an ancestral spe-
cies. Well-documented examples of natural allopolyploid taxa in the Pooideae subfamily are Triticum turgidum
L. (2n = 4x = 28, genome constitution AABB) and T. a estivum (2n = 6x = 42, AABBDD) formed through hybridi-
sation and successive chromosome doubling of ancestral diploid species T. urartu (2n = 2x = 14, AA), Aegilops
speltoides Tausch. (2n = 2x = 14, BB) and A. tauschii (2n = 2x = 14, DD)106. Moreover, in the tribe Stipeae based on
the At103 gene allopolyploidy was reported for the genus Patis Ohwi (2n = 46, 48)105. Heretofore, at least three
hypotheses were considered regarding the base chromosome number in Stipeae: x = 7107, x = 11108,109 and x = 12110.
Recently, it was suggested that the latter two are more plausible41,104. us, in order to better assemble the S. capil-
lata genome and verify if Stipa is an allopolyploid genus we suggest sequencing at chromosome level the close
relative diploid species (2n = 22) from genera representing, e.g. Ptilagrostis Griseb., Achnatherum P. Beauv., e.g.
A. calamagrostis L. (2n = 22 + 0‒2B), or Piptatheropsis Romasch., P. M. Peterson & Soreng (2n = 20, 22, 24)41,104.
In general, the number of genes in Poaceae varies from 28,835 in the smallest known genome, Oropetium
thomaeum Trin. (2n = 20; genome size of 245Mb)111, to 107,891 in T. aestivum (2n = 42; 14,547Mb)112. Here, we
reported 53,535 nuclear genes that were structurally annotated for the masked genome assembly. Such a num-
ber of genes was roughly 1.8 and 1.6 times smaller than previously determined for S. grandis (94,674 genes)54
and S. purpurea (84,298 genes)50, respectively. On the other hand, the annotation analysis of the unmasked
genome resulted in 81,224 genes associated with already known proteins. In comparison, only 65,047 function-
ally annotated genes were reported for S. grandis while S. purpurea had 58,966. Nonetheless, as RNA-seq data is
currently unavailable for S. capillata, we believe that the current version of the genome annotation demands a
further investigation to properly characterise the genes sets when the appropriate information will be available.
SSR markers are widely distributed across the genome and they are commonly applied in establishing genetic
structure in Stipa. Previously, polymorphic microsatellite primers were reported in populations of S. purpurea
(11113, 15114 and 29115 loci), S. pennata (7 loci116), S. breviora (21 loci117) and S. glareosa (9 loci118). In the pre-
sent study, we identied 77,614 perfect SSR markers (Supplementary File 3) and 58 imperfect repeat motifs
presented only in a single copy (Supplementary TableS3). Although we did not test them on the population
level we are condent that such a number of new loci will be a valuable source for the farther development of
SSR markers in S. capillata, and more generally in the genus Stipa. Additionally, the revealed loci could be used
for the designing dominant inter simple sequence repeat (ISSR) markers119. Recently, the usefulness of applying
ISSRs were shown for studies in S. bungeana120, S. ucrainica and S. zalesskii121, S. tenacissima122 and the hybrid
complex S. heptapotamica123.
According to the previous studies, based on three chloroplast loci124 and four chloroplast loci and one nuclear
region105, it was shown that the origin of Stipeae can be estimated in a range of 30.60–47.30 Mya and 21.20–39
Mya, respectively. Here, based on the ve loci within NORs we demonstrated that the potential split between
Stipa representing the tribe Stipeae and Brachypodium (the tribe Brachypodieae) took place approximately
30–35.52 Mya that supports the previous ndings105,124,125. e present results also suggest that the genus Stipa
likely originated ca. 4.39 (2.90–6.02) Mya. On the other hand, one previous study indicated the origin of feather
grasses at about 12.90 Mya124 while another one showed dierent estimates based on chloroplast loci (21.20
Mya, 13–22) and the At103 region105. Specically, two copies of At103 had the following suggested ages: 15.78
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(6.30–26.60) Mya for the Eurasian Stipeae lineage and 5.62 (0–6.50) Mya for the American Stipeae lineage105.
us, the latter estimate is close enough to the origin-age calculated in the current study. In addition, our data
on the divergence time among S. richteriana, S. lessingiana and S. heptapotamica (Fig.2 and Table5) conforms
to the previous ndings on the ongoing hybridisation among these taxa123 suggesting NORs as a useful tool
for revealing species of putative hybrid origin. Nonetheless, we believe that the current and previous estimates
regarding the origin of Stipa should be treated with caution. Firstly, to our knowledge, there is still no available
fossil data for any Stipa species from the Old World that can properly calibrate the historical diversication in the
genus. Currently, the earliest denite Stipa caryopses were found in central Poland and are dated ca. 4,000 BC126.
Secondly, available data demonstrate incongruence between chloroplast and nuclear loci analyses. In further
studies we suggest utilising single-copy nuclear genes derived from whole genome sequencing projects. irdly,
dierent sets of species and parameters used for inferring diversication dates may result in dierent estimates127.
Finally, we report a 137,823bp chloroplast genome that is similar to the known assemblies in Stipa and spe-
cically in S. capillata57. Here we highlight the applicability of a long-read sequencing technology like PacBio
for the straightforward assembling of plastomes using Flye67,68. In addition, due to the long-reads we were able
to identify two haplotypes presented in S. capillata. is result supports the previous ndings in Poaceae81 sug-
gesting that plastome structural heteroplasmy can be a common state in feather grasses. Moreover, for the rst
time in the genus Stipa, here we present a 438,037bp mitochondrial genome. e current size of this genome
is close to Alloteropsis semialata (R.Br.) Hitchc. (442,063bp)128, T. aestivum (452,526bp)129, Sorghum bicolor
L. (468,628bp)130 and A. speltoides (476,091bp)131. Nevertheless, the present version of the genome is consti-
tuted by four contigs rather than one circular sequence. Although the general acceptance among mitochondrial
biologists is that plant mitochondrial genomes have a variety of congurations132–134, in order to verify if a more
accurate assembly could be performed, we suggest reusing our data for a more comprehensive analysis of the
mitochondrial structures within Stipa.
Materials and methods
Plant material and DNA extraction. Our research complies with relevant institutional, national, and
international guidelines and legislation. A S. capillata sample from Kochkor River Valley, central Kyrgyzstan
(Supplementary TableS4), was selected for genome sequencing. e sample was stored in silica gel at ambient
temperature until DNA extraction was performed. Total genomic DNA was isolated from dried leaves aer a
six-month storage period using a CTAB large-scale DNA extraction protocol (Supplementary information S1,
describedin Supplementary File 6). DNA extraction was performed by SNPsaurus (USA). In addition, we iso-
lated DNA from dried leaves using a Genomic Mini AX Plant Kit (A&A Biotechnology, Poland). Subsequently,
quality check, quantication and concentration adjustment were accomplished using a NanoDrop One (ermo
Scientic, USA) and agarose gel electrophoresis visualisation. e concentration of the sample was adjusted to
50ng/μL. e puried DNA sample (1μg) was sent to Diversity Arrays Technology Pty Ltd (Canberra, Aus-
tralia) for sequencing and DArT marker identication. Moreover, to test the phylogenetic power of NORs in
Stipa, we supplemented the study with ve specimens of S. richteriana Kar. & Kir, three of S. lessingiana Trin. &
Rupr., four of S. heptapotamica Golosk. and four of S. korshinskyi Roshev. (Supplementary TableS4). e isola-
tion of genomic DNA was performed from dried leaf tissues using a modied CTAB method135.
Library construction and sequencing. In total, 5 ug of S. capillata genomic DNA were used to construct
a PacBio library according to the 20kb PacBio template preparation protocol omitting a shearing step. e size
selection cut-o was set at 15kb. e library preparation followed by sequencing on three PacBio Sequel SMRT
cells (Pacic Biosciences, Menlo Park, CA, USA) was carried out by SNPsaurus, LLC. Prior to the assembly,
reads from each SMRT cell were inspected and quality metrics were calculated using SequelQC v.1.1.0136. A
high-density assay using the DArT complexity reduction method for S. capillata was performed according to a
previously reported procedure137.
For the rest of the specimens used in the current study, the quality control using a uorometer (PerkinElmer
Victor3, USA) and gel electrophoresis, library construction using a TruSeq Nano DNA Library kit (350bp
insert size; Illumina, USA) and sequencing using 100bp paired-end reads on an Illumina HiSeq 2500 platform
(Illumina, USA) were performed by Macrogen Inc. (South Korea).
Nuclear genome assembly and validation. e execution of this work involved using many soware
tools, whose versions, settings and parameters are described in Supplementary information S2(available in Sup-
plementary File 6). e de novo assembly of the PacBio data was performed using Flye v.2.465,66. e dra assem-
bly was cleaned by running BLASTn v.2.10.0138 against the NCBI nucleotide database v.5, and subsequently
sending each BLAST hit to the JGI taxonomy server (https:// taxon omy. jgi- psf. org/) with a downstream step of
keeping only plant contigs. ereaer, Qualimap v.2.2.2139 was used to identify mean coverage for each contig. In
the nal assembly we kept only contigs with an average coverage of more than 10x. In addition, overrepresented
contigs (> 60x) were BLASTed against the NCBI nucleotide database v.5 and sequences assigned to chloroplasts
and mitochondria were removed.
Due to the nal assembly performed with Flye v.2.4 being roughly twice bigger than an expected monoploid
genome size of 589 Mb93, we accomplished an additional assembly with FALCON v.0.2.568 and applied Purge
Haplotigs v1.1.169 to lter redundant sequences due to possible heterozygosity. e assemblies’ statistics were
analysed using assembly-stats v.1.0.1140. In addition, in order to assess the completeness of the genome assemblies,
we investigated the presence of highly conserved orthologous genes using BUSCO v.4.0.6141.
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Scaolding of contigs. Due to there being no reference genome for any Stipa species, here we applied
RaGOO v.1.1142 to verify if a reference-guided scaolding can be performed for the dra genome contigs based
on four genomes from the Pooideae subfamily (B. distachyon70, H. vulgare71, A. tauschii72, T. aestivum 74) and one
genome from the Oryzoideae subfamily (O. sativa73). e subsequent assessment of the scaolding accuracy
was based on three parameters: (1) location condence score, (2) orientation condence score and (3) grouping
condence score142.
Repeat prediction and nuclear genome annotation. e repeat prediction for S. capillata was per-
formed using a de novo transposable element (TE) family identication and modeling package RepeatModeler
v.2.0.1143 which includes three repeat nding programs; RECON144, RepeatScout145, and TRF146. e resulting
TE library was supplemented by the transposable elements database (Release 19, http:// botse rv2. uzh. ch/ kelld
ata/ trep- db/).147 Subsequently, the genome assembly was masked for TEs regions by RepeatMasker v.4.1.0148
(http:// repea tmask er. org) with the search engine RMBlast v.2.9.0 + 149 and the custom library created in the pre-
vious step. Next, gene and protein sequences were predicted using Augustus v.3.2.3 with the unmasked and
v.3.3.3150 with the masked genome assemblies. e predicted protein sequences of the unmasked assembly were
then BLASTed against the NCBI protein database v.5 and the subsequent BLAST hit descriptions were added to
GFF (General Feature Format) les.
Genome-wide identication of microsatellite markers. e unmasked nuclear genome, chloroplast
and mitochondrial genome assemblies were screened for perfect mono-, di-, tri-, tetra-, penta- and hexa-nucleo-
tide repeat motifs using Krait v.1.3.375. We applied the following criteria: mono-nucleotide repeat motifs contain
at least 12 repeats, di-nucleotide repeat motifs contain at least seven repeats, tri-nucleotide repeat motifs contain
at least ve repeats, tetra-, penta- and hexa-nucleotide repeat motifs contain at least four repeats.
Divergence time of Stipa. In order to estimate the divergence between S. capillata and other Stipa species
we used the nucleolar organising regions. Firstly, we prepared a set of reference sequences including S. lipskyi
Roshev.151, S. magnica Junge152, S. narynica Nobis153, S. caucasica Schmalh.154, S. orientalis Trin.155 and S. pen-
nata L.156. Secondly, we mapped raw reads of S. capillata, S. richteriana, S. lessingiana, S. heptapotamica and S.
korshinskyi (Supplementary TableS2) as well as S. grandis55, S. breviora59, S. lagascae157 to the reference set
using Minimap2 v.2.17-r941158 with keeping only uniquely mapped reads by Samtools v.1.9159. irdly, the de
novo assembly of the NORs was performed using Canu v.2.0160 for S. capillata and SPAdes v.3.14.1161 for the rest
of Stipa species. Additionally, we added to the analysis B. distachyon162 as an ingroup member of the Pooideae
subfamily and O. sativa163 as an outgroup representing the Oryzoideae subfamily within the Poaceae family.
Next, all sequences were aligned using MAFFT v.7.471164. Subsequently, the aligned sequences were visualised
in AliView v.1.26165 and divided in ve loci: (1) 18S ribosomal RNA, (2) Internal Transcribed Spacer 1 (ITS1),
(3) 5.8S ribosomal RNA, (4) Internal Transcribed Spacer 2 (ITS2) and (5) 26S ribosomal RNA (Supplementary
File 4). Estimation of divergence times was performed in BEAST2 v.2.6.3166 using the 121,321 substitution model
determined by bModelTest167. We used the following constraints for time calibrations: 38–48 million years ago
(Mya) for the Brachypodium-Oryza split101 and 33–39 Mya for the potential origin and divergence of Stipa34,35.
en, the divergence time was estimated using the strict clock model and the Yule prior. In total, we ran the
analysis three times independently, 50 million Markov chain Monte Carlo (MCMC) generations for each run.
e log and tree les were combined using LogCombiner v.2.6.3 (a part of the BEAST package) with the rst ve
million generations discarded as burn-in from each run. Next, Tracer v.1.7.1168 was used to check the log les
regarding Eective Sample Size (ESS) values. As all ESSs exceeded 200, we summarised the nal maximum clade
credibility tree (Supplementary File 5) in TreeAnnotator v.2.6.3 (a part of the BEAST package). e nal tree was
visualised and edited using FigTree v.1.4.4169.
Mitochondrial and chloroplast genomes assembly, annotation and validation. Prior to assem-
bly, we mapped raw reads to 11 reference mitochondrial genomes of species belonging to the Poaceae family
(Supplementary TableS5) using Minimap2 v.2.17-r941158. Only uniquely mapped reads were kept by Samtools
v.1.9159 for the next step. De novo mitochondrial assembly of the 4.08Mb data was performed using Flye v.2.7.1-
b1590.
In the next step, we BLASTed the resulting contigs against the NCBI nucleotide database v.5, and sequences
assigned to mitochondria were kept. en, the PacBio subreads were mapped onto the kept contigs using Mini-
map2, and only uniquely mapped reads were retained by Samtools. A new de novo assembly of the 15.51Mb
data was performed using Flye. In order to check if the mitochondrial contigs obtained by Flye could be merged
into larger scaolds we applied Circlator v.1.5.5170. However, the resulting sequences were identical to the Flye
contigs. In addition, we used Unicycler v.0.4.884 with reads that were mapped onto the Flye contigs as a reference.
Further, to detect all possible structural haplotypes of the chloroplast genome we applied Cp-hap81. Next, we
mapped raw reads onto the resulting mitochondrial contigs and the chloroplast genomes to manually check in
IGV v.2.8.680 if any potential SNPs or indels are present. Eventually, annotations of the nal mitochondrial con-
tigs of 438,037bp and the chloroplast genomes of 137,823bp were performed using Geneious Prime v.2021.1.1
(https:// www. genei ous. com) based on 85% and 95% similarities to the reference genomes of mitochondria and
chloroplasts, respectively (Supplementary TableS5).
In Silico mapping of DArT marker sequences. Since the DArT markers are designed to target active
regions of the genome171, here we use them to validate the completeness of the nuclear genome assembly and
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improve the accuracy of data ltering in further genomic studies on Stipa. Two data types, Silico and SNPs mark-
ers, were mapped to the nuclear genome using BLASTn v.2.10.0. As a query we used trimmed DArT sequences
in a range of 29–69bp with the percent identity values to the reference genome of 95% or greater and removing
alignments below 95% of a query.
Data availability
e raw PacBio reads are available at NCBI Sequence Read Archive172. e nal genome assemblies are deposited
into NCBI Assembly database under the following Accession Numbers: nuclear assembly (JAGXJF000000000)67;
mitochondrion assembly, contig 1 (MZ161090)76, contig 2 (MZ161091)77, contig 3 (MZ161093)78 and contig 4
(MZ161092)79; chloroplast assemblies, haplotype A (MZ146999)82 and haplotype B (MZ145043)83. e masked
and the unmasked versions of the nuclear genome annotation are presented in the Supplementary File 1 and
the Supplementary File 2, respectively.
Received: 17 November 2020; Accepted: 24 June 2021
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Acknowledgements
We would like to express our gratitude to Eric Johnson from SNPsaurus, Artem Kasianov from Institute for Infor-
mation Transmission Problems of the Russian Academy of Sciences (Moscow, Russia) and Igor A. Shmakov from
Altai State University (Barnaul, Russia) for their valuable assistance in the genome assembling. We also thank the
iDiv High-Performance Computing cluster for providing computing resources for this paper. Finally, we thank
two anonymous reviewers for providing valuable comments on the manuscript. e study was supported by the
Russian Science Foundation (grant no.19-74-10067). E.B. was supported via the RSF (grant no.19-74-10067) and
a DS grant of the Jagiellonian University (DS/D/WB/IB/2/2019). M.N. was supported by the National Science
Centre, Poland (grant no. 2018/29/B/NZ9/00313). P.D.G. was supported by the RSF (grant no.19-74-10067).
e open‐access publication of this article was funded by the BioS Priority Research Area under the program
"Excellence Initiative – Research University" at the Jagiellonian University in Krakow.
Author contributions
E.B., P.D.G., M.N. planned the study. E.B. supervised the research. M.N. and P.D.G. identied and collected
biological samples. E.B., C.G., E.S. performed the nuclear genome assembly. E.B. performed the remaining bio-
informatic analyses and wrote the manuscript. All authors revised the dra, provided comments and approved
the nal 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- 021- 94068-w.
Correspondence and requests for materials should be addressed to E.B.orM.N.
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