Msab 286

Somatic Mutation Analysis in Salix suchowensis Reveals
Early-Segregated Cell Lineages

Yifan Ren,†,1 Zhen He,†,1 Pingyu Liu,1 Brian Traw,1 Shucun Sun,2 Dacheng Tian,1 Sihai Yang,1 Yanxiao Jia,* ,3
and Long Wang * ,1
1
State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China
2
Department of Ecology, School of Life Science, Nanjing University, Nanjing, China
3
State Key Laboratory for Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China
†
These authors contributed equally to this work.
*Corresponding authors: E-mails: wanglong@nju.edu.cn; jiayanxiaozzu@163.com.
Associate editor: Gregory Wray
Downloaded from https://academic.oup.com/mbe/article/38/12/5292/6375452 by guest on 15 October 2023

Abstract
Long-lived plants face the challenge of ever-increasing mutational burden across their long lifespan. Early sequestration
of meristematic stem cells is supposed to efficiently slow down this process, but direct measurement of somatic
mutations that accompanies segregated cell lineages in plants is still rare. Here, we tracked somatic mutations in 33
leaves and 22 adventitious roots from 22 stem-cuttings across eight major branches of a shrub willow (Salix suchowensis).
We found that most mutations propagated separately in leaves and roots, providing clear evidence for early segregation
of underlying cell lineages. By combining lineage tracking with allele frequency analysis, our results revealed a set of
mutations shared by distinct branches, but were exclusively present in leaves and not in roots. These mutations were
likely propagated by rapidly dividing somatic cell lineages which survive several iterations of branching, distinct from the
slowly dividing axillary stem cell lineages. Leaf is thus contributed by both slowly and rapidly dividing cell lineages,
leading to varied fixation chances of propagated mutations. By contrast, each root likely arises from a single founder cell
within the adventitious stem cell lineages. Our findings give straightforward evidence that early segregation of meristems
slows down mutation accumulation in axillary meristems, implying a plant “germline” paralog to the germline of animals
through convergent evolution.
Key words: plant evolution, clonal variation, somatic mutation, Salix mutation, plant cell lineage.
Introduction 2004; Simberloff and Leppanen 2019). This hypothesis re-

Somatic mutations may arise every time a cell divides, and be ceived considerable attention in the past (Whitham and
Article
passed to the next generation if the cell harboring the muta- Slobodchikoff 1981; Whitham 1983; Antolin and Strobeck
tion becomes a germ cell. In animals, somatic mutations usu- 1985; Suomela and Ayres 1994; Simberloff and Leppanen
ally have degenerative effects and are associated with disease 2019).
and aging (Zhang and Vijg 2018). Owing to the early segre- By contrast, recent studies have focused more on the pre-
gation of germlines in most animals, the later developed so- cise detection of the plant somatic mutations as well as their
matic mutations have no chance to enter the germline (the potential inheritance (Watson et al. 2016; Schmid-Siegert et
germ-plasm theory [Weismann 1892]). In plants where germ- al. 2017; Plomion et al. 2018; Hanlon et al. 2019; Hofmeister et
line differentiates late, somatic mutations are supposed to act al. 2020; Wang et al. 2019; Orr et al. 2020; Yu et al. 2020).
as an important source of innovation for plant evolution Through measuring fixed somatic mutations in terminal
(Lanfear 2018; Plomion et al. 2018). They are frequently leaves from trees with lifespans of several centuries
used as source of genetic material in artificial breeding pro- (Schmid-Siegert et al. 2017; Plomion et al. 2018; Hanlon et
grams to create bud sport mutants among clonal descend- al. 2019; Hofmeister et al. 2020; Wang et al. 2019; Orr et al.
ants (Benedict 1923; Shamel and Pomeroy 1936; Roest et al. 2020), these studies found that old trees only accumulate very
1981; Foster and Aranzana 2018). Assuming ever-lasting ac- few somatic mutations, which is much lower than what has
cumulation and fixation of somatic mutations in long-lived been conjectured before (Sutherland and Watkinson 1986;
plants, the intraorganismal hypothesis supposes that they are Klekowski Jr and Godfrey 1989; O’Connell and Ritland 2004;
able to maximize the within-plant heterogeneity, allowing Yong 2012; Diwan et al. 2014). The low number of fixed
intraorganismal selection, and providing opportunities for mutations in old trees challenges the assumption of the intra-
plants to outmaneuver enemies or adapt to changing envi- organismal hypothesis, while implying an efficient strategy in
ronments (Whitham and Slobodchikoff 1981; Michel et al. plants to keep fixable mutations in check. Using time-lapse
ß The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://
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5292 Mol. Biol. Evol. 38(12):5292–5308 doi:10.1093/molbev/msab286 Advance Access publication September 25, 2021
Somatic Mutation Analysis in Salix suchowensis . doi:10.1093/molbev/msab286 MBE
imaging and computational modeling, Burian et al. (2016) sequencing (WGS) of multiple parts of a plant. In our prior
showed that this was likely achieved by setting aside the ax- study (Wang et al. 2019), we confirmed the usability of this
illary meristems early and thus reducing the number of stem approach by tracking somatic mutations in the woodland
cell divisions in shoot apical meristem (SAM) during plant strawberry. We found that several mutations were restricted
development (Groot and Laux 2016), a system analogous to to runners and were never been passed to daughter plants
the formation of germline in animals. (Wang et al. 2019), giving further evidence for the segregation
These novel findings emphasized the importance of of stem cell lineages.
timing of segregation for certain stem cell lineages, such Here, we interrogate somatic mutations to test for seg-
as the plant germline. There is a long-lasting debate on regated stem cell lineages in a perennial woody plant, the
whether plants have a segregated germline in determining shrub willow (Salix suchowensis spp.). Shrub willow has
the heritable somatic mutations (see Lanfear’s recent re- strong regeneration ability and is an important crop for
view [Lanfear 2018] on the historical background of re- both bioenergy and environmental engineering (Volk et al.
lated arguments). A late-segregated germline predicts 2004). Like many willows (Carlson 1938; Sennerby-Forsse

that most somatic mutations are heritable, whereas an and Zsuffa 1995), shrub willow has strong resprouting
early-segregated germline will predict the contrary ability, and its stem-cuttings root very readily when culti-
(Lanfear 2018). The differences in timing of germline (or vated in favorable conditions. Histological analysis has
more broadly, stem cell lineages) segregation therefore revealed that most stem buds in willows are of axillary
create strong differences in the ability of these mutations origin (Fink 1983; Sennerby-Forsse and Zsuffa 1995),
to fuel genetic variation within populations and therefore whereas root buds are adventitious in origin (Carlson
contribute to evolution (Lanfear 2018; Plomion et al. 1938; 1950; Haissig 1970; Fjell 1985, 1987; Sennerby-
2018). However, whether the early segregation of the Forsse and Zsuffa 1995). Axillary buds/meristems are de-
stem cell lineage suggested by Burian et al. (2016) applies veloped in or near the leaf axils (Wang and Jiao 2018), and
to woody plants has yet to be confirmed. More impor- are presumably directly derived from the SAM with pos-
tantly, despite the achievements in measuring plant sible minimized number of cell divisions (Burian et al.
mutations, direct quantification of mutations for speci- 2016). By contrast, aboveground adventitious buds/mer-
fied cell lineages remains elusive in perennial woody istems most often initiate from cells neighboring vascular
plants. tissues (Bellini et al. 2014), and are likely derived from
Assessing the timing of segregation is partly viable through vascular cambial cell divisions (Steffens and Rasmussen
careful tracking of different cell lineages (Poethig 1989; Irish 2016) or by reprogramming differentiated somatic cells
1991). A wealth of information on tissue identity formation in (Dıaz-Sala 2014). Though both meristems originate from
plant development has come from studies of plant chimeras the SAM (Lucas et al. 2013; De Rybel et al. 2016), the pro-
(for reviews, see Poethig 1987, 1989; Szymkowiak and Sussex liferation distance from apical stem cells and cell division
1996; Frank and Chitwood 2016). Experiments using periclinal patterns leading to the formation of them are likely to be
and mericlinal chimeras have revealed in angiosperms that distinct. However, neither the relationship of their under-
the SAM is organized into three layers, termed as L1, L2, L3, lying cell lineages nor the patterns of corresponded so-
where each layer generally forms epidermal, subepidermal, matic mutations have been characterized previously in
and internal tissues, respectively (Irish 1991; Szymkowiak Salix, to the best of our knowledge.
and Sussex 1996). The cell fates in each layer are, however, We consider two models (fig. 1) that differ in whether
thought to be largely determined by physical location rather meristematic stem cell lineages segregate late (model 1)
than past lineage history (Irish 1991; Szymkowiak and Sussex or early (model 2) when forming the leaves and the ad-
1996). For example, gametes are usually observed to arise ventitious roots. In model 1, the leaves and adventitious
from L2 cells, but they can also be formed by L1 and L3 cells roots share same progenitor cell lineages until later dif-
(Szymkowiak and Sussex 1996). The adaptive significance of ferentiation. In this model, samples collected from the
this stratified structure has been discussed through mathe- same branch or same stem-cutting have closer genealogy
matical modeling (Klekowski and Kazarinova-Fukshansky (fig. 1e) and somatic mutations that predate the forma-
1984a, 1984b; Klekowski et al. 1985). With improved excision tion of this branch will be propagated to both organs. In
and imaging techniques, recent works have provided further model 2, the leaves and adventitious roots have distinct
insights on the gene regulatory programs within the meris- progenitor cell lineages from the very beginning. In this
tems (Wang et al. 2018; Kitagawa and Jackson 2019), espe- model, the genealogy of two organs is not correlated with
cially regarding their regeneration capability (Sena et al. 2009; their physical locations (fig. 1e), and somatic mutations
Rahni et al. 2016; Ikeuchi et al. 2019). are propagated separately by these cell lineages. The mod-
Mutations are widely recognized as valuable molecular els are tested by tracing somatic mutations in multiple
markers in these works for cell lineage tracing. Whereas tra- stem-cuttings of the same willow, with shoot/leaves rep-
ditional clonal analyses used induced mutations to mark cell resent for organs differentiated from axillary meristems
lineages (Poethig 1987), the use of irradiation may itself and adventitious roots represent for organs differentiated
change cell behavior and was difficult to control (Poethig from adventitious meristems (fig. 1a–d). Our aim is to
1987). This drawback could be overcome by directly tracking provide a landscape of somatic mutations in shrub willow
de novo somatic mutations through whole-genome and utilize these molecular markers to assess the
5293
Ren et al. . doi:10.1093/molbev/msab286 MBE

FIG. 1. Sampling strategies considering two possible segregation models. (a–d) Photos showing the two different meristem types. New leaves
formed by axillary buds/meristems (blue arrows) and can be viewed around 6–8 days post cut (dpc), whereas new roots formed by adventitious
buds/meristems (red arrows) can be viewed as early as 4 days post cut. For each cutting, three leaves from independent axillary buds (photo c) and
three roots from independent adventitious buds (photo d) are sampled. Scale bars, 0.2 cm for a–c and 0.5 cm for d. (e) Conjectured cell lineage
segregation models with expected presence of shared mutations. Model 1: late-segregated cell lineages in generating terminal leaves and adven-
titious roots. Under model 1, organs closer in physical locations tend to have closer genealogy therefore share more mutations arise predate their
formation. Model 2: the early segregation model. Under model 2, the physical locations of different organs do not predict their truly genealogy, and
mutations are separately transmitted by segregated cell lineage to each organ.
possibility of any early-segregated stem cell lineages. Our fixation in samples from both leaves and roots. In contrast,
results will contribute to the understanding of the role of support for model 2 will be found if the fixation of muta-
somatic mutations in plant evolution in a more general tions is mostly independent between leaves and roots.
sense than the intraorganismal hypothesis has To test between the models, we investigated somatic
conjectured. mutations in the 22 stem-cuttings (“Cut-122” in fig. 2a)
from eight branches (IVIII in fig. 2a). For 8 of 22 cuttings,
Results each cutting was sequenced for up to three independent
leaves, each from a separate axillary bud, and up to three
Sampling and Sequencing of the Genomes of a Shrub independent adventitious roots, each from a separate adven-
Willow titious bud (figs. 1a–d and 2b), yielding a total of 19 leaves and
Original stem-cuttings were collected from the shrub wil- 22 adventitious roots from those eight cuttings (supplemen-
low (S. suchowensis, individual ID “YAF1”) in 2016 after tary table S1, Supplementary Material online). For each of the
new shoots regenerated from its trunk which was cop- remaining 14 cuttings, a single leaf sample was collected for
piced in 2015 (supplementary fig. S1, Supplementary accurate tracing of the origins of mutations.
Material online). When each cutting was made, new In total, 33 leaves and 22 adventitious roots of the same
shoots/leaves were then generated from preformed axil- shrub willow were whole-genome sequenced (supplementary
lary buds/meristems (fig. 1a and c), whereas adventitious table S1, Supplementary Material online), yielding over 2,000-
roots were regenerated from preformed adventitious fold sequencing depth of a single tree (supplementary table
buds/meristems (fig. 1a, b, and d). The underlying cell S1, Supplementary Material online). Each leaf sample was
lineages forming leaves and roots could either segregate sequenced to an average of 126 million (M) cleaned reads
late upon differentiation, or at an early stage, correspond- (ranging from 92M to 163M), equivalent to around 44-fold
ing to our proposed segregation models #1 and #2, respec- raw depth of the 425 Mb estimated genome size (Dai et al.
tively (fig. 1e). Under model 1 in which no meristem is 2014). Each root sample was sequenced to around 119M
segregated until differentiation, mutations prior to the cleaned reads or approximately 42-fold depth (ranging from
segregation are expected to have the same chance of 92M to 161M).
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FIG. 2. Patterns of somatic mutations in the analyzed shrub willow. (a) Schematic of sampled branches and stem-cuttings. Eight major branches
(labeled I to VIII) were sampled and shown here. One cutting (15–20 cm) per branch was made in branches I, III, IV, VII, and VIII, whereas four, five,
and eight cuttings were made for branches II, V, and VI. Eight cuttings (labeled with superscript “M”) were measured for both leaf and adventitious
root samples, otherwise only one leaf was sampled. Approximate locations where a cutting was sampled are shown in yellow. Lowercase letters “a–
s” represent those BR-s mutations shared among different samples, which are colored by their appearance in leaves only (blue), roots only (red),
and both organs (purple with asterisk). (b) Details of eight stem-cuttings with both leaves and adventitious roots sequenced. Samples collected
from each cutting are enclosed within the black circle. Samples with shared BR-s mutations (lowercase letters) and a BR-m mutation (uppercase
letter “P”) are indicated and colored correspondingly. The one poor sequenced root sample is shown in gray.
Somatic Mutations Identified within the Single Shrub Material online); we keep calling them mutations as 1)
Willow they are more likely early mutations (see later for details)
After mapping and variant calling, we searched for candi- and 2) we only use them to track cell lineages so does not
date somatic variant sites across all samples (see Materials matter whether these somatic variants are from mutation
and Methods and supplementary fig. S2, Supplementary or recombination.
Material online, for details). For each candidate variant In total, we identified 199 reliable somatic mutations
site, the ancestral allele was inferred as the common allele across all analyzed samples, including 182 BR-s mutations
presents in samples from most major branches (i.e., >4 of and 17 BR-m mutations (table 1 and supplementary table
branches IVIII here). A mutation is called at the variant S2, Supplementary Material online). The 182 BR-s mutations
site where one major branch carries an allele differed from included 155 single-nucleotide variants (SNVs) and 27 inser-
the ancestral allele (hereafter denoted as BR-s mutations). tion/deletions (INDELs), of which 177 mutations were distrib-
The BR-s mutation therefore represents a somatic muta- uted in assembled chromosomes and five were located in
tion that arose after formation of that major branch. For unanchored scaffolds (fig. 3a). Consistent with their origina-
mutations raised before the formation of each major tion as de novo mutations, nearly all identified BR-s muta-
branch, samples from two or more major branches will tions (180 of 182) were heterozygous, of which 176 were
share the same allele which differs from the ancestral “homozygous->heterozygous” mutations (i.e., the ancestral
alleles (supplementary fig. S3, Supplementary Material on- genotype was homozygous, whereas the genotype of muta-
line, hereafter referred as BR-m mutations). The BR-m tion was heterozygous) and four were “heterozygous-
mutation lineage hence reflects the early stem cell lineages >heterozygous” mutations. The only two exceptions (two
which form multiple major branches. We note that a few “homozygous->homozygous” mutations in two root sam-
BR-m “mutations” may be generated by somatic recombi- ples) were high likely due to sequencing bias that only one
nation (i.e., gene conversion) in early cell lineages but not allele gets sequenced, as regions from which the two muta-
by mutation (supplementary fig. S3, Supplementary tions were called and were associated with low read-depths
5295
Table 1. Number of Mutations Identified in Shrub Willow YAF1.
Mutation Predicted Effects No. of BR-m Mutations No. of BR-s Mutations
Type
Sum Leafa Roota Both Sum Leaf Root Both Uncategorizedc
b
SNV Total 14 12 1 1 155 36(4 ) 72(12) 2 45(4)
Nonsynonymous 1 1 0 0 14 3 7(1) 0 4
Synonymous 0 0 0 0 7 1 4(1) 0 2
Intron 1 1 0 0 29 9(1) 14(3) 0 6(1)
Intergenic 12 10 1 1 105 23(3) 47(7) 2 33(3)
INDEL Total 3 3 0 0 27 3 14(1) 0 10(3)
Frameshift 1 1 0 0 2 0 1 0 1(1)
In-frame INDEL 0 0 0 0 2 0 1 0 1
Intron 0 0 0 0 6 1 3(1) 0 2
Intergenic 2 2 0 0 17 2 9 0 4(2)

a
BR-m mutations present exclusively in leaves or in adventitious roots.
b
BR-s mutations shared between multiple leaves or between multiple roots; the number of sample-specific mutations could be obtained by subtracting this number from the
total number (number outside the parenthesis).
c
For cuttings with only a single leaf was sequenced, the origin of these mutations could not be firmly assessed, so are left as uncategorized.
(10 reads). The proportion of “heterozygous- might arise when dealing with BR-m mutations (supplemen-
>heterozygous” BR-s mutations (4/182 ¼ 2.20%) was higher tary fig. S3, Supplementary Material online).
than the estimated heterozygosity (0.394%) of the genome We find more BR-s mutation events in chromosomes
(Fisher’s exact test, P ¼ 0.006521), implying possible muta- with larger size (Spearman’s Rho ¼ 0.5568, P ¼ 0.01328),
genic effects of heterozygosity per se, as has been reported with Chr01, the largest chromosome, having the most
in other studies (Amos 2010; Yang et al. 2015). BR-s mutations (24 events identified). After accounting
The 17 BR-m mutations included 14 SNVs and 3 INDELs, of for chromosome size, the mutations are roughly distrib-
which 16 mutations were distributed in assembled chromo- uted evenly across all chromosomes, with only a few
somes and one was located in an unanchored scaffold (fig. regions (fewer than 9 Mb in size overall) as candidates
3a). Patterns of these 17 BR-m mutations and their implica- to be mutation hotspots (supplementary table S4,
tions are discussed in more detail below. Supplementary Material online, permutation test with
We selected 35 mutations, including all 17 BR-m muta- 10,000 randomizations in 1 Mb windows, P < 0.05).
tions and 18 arbitrarily selected BR-s mutations, for Most of the mutations are found within noncoding
Polymerase Chain Reaction (PCR) amplification followed by regions, whereas 21 SNVs and 4 INDELs reside in coding
Sanger sequencing (supplementary table S3, Supplementary regions (table 1). The number of mutations in coding and
Material online). A total of 26 of these mutations (15 BR-m noncoding regions are within the expectation from ge-
and 11 BR-s) were confirmed by Sanger sequencing (supple- nomic coding areas (expected ¼ 14.72% based on the
mentary table S3, figs. S4 and S5, Supplementary Material coding regions of the reference genome, observ-
online). The remaining nine nonanalyzable cases included ed ¼ 13.55% for SNVs, Chi-squared with Yates
six that failed PCR amplification (generally due to unavailabil- correction ¼ 0.1682, P ¼ 0.6817; observed ¼ 14.81% for
ity of suitable primers), and three that yielded poor Sanger INDELs, Chi-squared with Yates correction ¼ 0.0002132,
sequencing signals (supplementary table S3 and fig. S4, P ¼ 1), suggesting no apparent selection. Over 60% of
Supplementary Material online). The absence of mutation mutations are transitions dominated by C->T (or
alleles in control samples (e.g., some root samples were sev- G->A) changes and enriched in CpG sites (table 2 and
ered as control when testing BR-m leaf-exclusive mutations) fig. 3b), suggesting a transition bias when compared with
is also confirmed (supplementary table S3, figs. S4 and S5, the genomic expectation (genomic GC con-
Supplementary Material online). Given a likelihood that the tent ¼ 34.4%). This transition bias is frequently ob-
next mutation would fail validation, we estimated a false served for spontaneous mutations in many plant
positive rate (FPR) of no more than (1/27 =) 3.70%. Note species (Yang et al. 2015; Watson et al. 2016; Xie et al.
that the FPR was even lower for mutations shared by two 2016; Schmid-Siegert et al. 2017; Wang et al. 2019).
or more samples, for which the likelihood was less than 1%, The generation of adventitious root is accompanied by
the square of 3.70%, for a mutation being not validated in two callus formation (fig. 1b and d), which often introduces a
or more independent samples. large number of mutations when cultured in vitro
(Phillips et al. 1994; Jiang et al. 2011; Zhang et al. 2014;
Wang et al. 2019). Is callus-induced mutagenesis also hap-
Genomic Landscape and Profile of Somatic Mutations pening in vivo and so might introduce some uncertainties
in Shrub Willow here? The eight leaf-cutting groups allow us to directly
The identified mutations allow us to depict the somatic ge- assess this possibility. A BR-s mutation identified specific
nomic landscape of this woody plant. We mainly focus on BR- to a single leaf or an adventitious root (denoted as
s mutations here (representing 91% of all identified muta- “sample-specific” mutation hereafter) is most probably
tions) to remove any uncertainty in ancestral inference that only raised during the latest stem cell divisions. If these
5296

FIG. 3. Properties of shrub willow somatic mutations. (a) Distribution of somatic mutations across the 19 chromosomes. Mutations found in one or
more leaves (circles), in one or more roots (rectangles), or in both leaves and roots (stars) are distinguished. Uncategorized mutations are marked
by triangles. SNV and INDEL mutations are colored in blue and red, respectively. BR-m mutations and BR-s mutations are labeled using uppercase
and lowercase letters, respectively. (b) Triplet nucleotide context of sample-specific mutations. The triplet mutation rate per bp per sample is
calculated as the number of mutated triplets (along with their complements, including mutation at first, second, and third positions) normalized
by the overall abundances of the triplets in the reference genome. The rates are sorted by highest (leftmost) to lowest (rightmost) based on the
overall sample-specific triplet mutation rates.
sample-specific mutations in adventitious roots have dif- P ¼ 1). Both leaf and root mutations have a bias favoring
ferent profiles compared with those in leaves (Jiang et al. transitions (65%) over transversions (35%), with most
2011; Zhang et al. 2014), the process of callus formation is mutations as C->T or G->A changes (table 2). No signif-
likely to introduce substantial numbers of mutations in icant differences in mutation spectra are observed be-
roots. tween leaf and root mutations (two-sided Fisher’s exact
We find 108 BR-s sample-specific mutations within the test, P ¼ 0.4756). Further investigation of triplet nucleo-
eight leaf-root groups, including 35 leaf mutations and 73 tide context reveals that both leaf and root mutations are
root mutations (table 1 and supplementary table S2, enriched in CpG sites (fig. 3b). There seems to be no sig-
Supplementary Material online). Of the 35 sample- nificant difference with respect to per triplet mutation
specific leaf mutations, four are in coding sequences rate between leaf and root mutations (two-sided Fisher’s
(CDS), whereas 11 of the 73 sample-specific root muta- exact test, P ¼ 0.5477), suggesting similar influences of
tions are in CDS (table 1). These fractions of CDS numbers trinucleotide context on mutations from both organs. A
are not significantly different between two organs (two- caveat here is that we have very limited number of muta-
sided Fisher’s exact test, P ¼ 0.7697). Similarly, the non- tions for each comparison, so more data are demanded
synonymous/synonymous ratios of two organs (excess of for a solid conclusion. Nonetheless, at least we see no
nonsynonymous mutations over synonymous ones in one large difference in mutation profiles between leaf and
organ could indicate strong selection) are also not signif- root, implying no strong evidence for callus-induced mu-
icantly different (table 1, two-sided Fisher’s exact test, tagenesis here.
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Table 2. Spectra of BR-s SNV Mutation Identified in Leaves and Adventitious Roots.
Type of Mutation Overall (Fraction) Sample-Specific
Leaf (Fraction) Root (Fraction)

Transitions (Total) 106 (0.627) 22 (0.688) 39 (0.650)
A->G/T->C 17 (0.101) 7 (0.219) 5 (0.083)
G->A/C->T 89 (0.527) 15 (0.469) 34 (0.567)
Transversions (Total) 49 (0.290) 10 (0.313) 21 (0.350)
A>T/T->A 11 (0.065) 3 (0.094) 4 (0.067)
A->C/T->G 11 (0.065) 2 (0.063) 3 (0.050)
G->T/C->A 23 (0.136) 5 (0.156) 12 (0.200)
G->C/C->G 4 (0.024) 0 (0) 2 (0.033)
NOTE.—The fractions of each change are given in parentheses.

Leaves and Adventitious Roots Are Likely Segregated Second, we consider a shared de novo mutation that we
Early observe in roots of cuttings from two branches. This muta-
To test between model 1 and model 2 (fig. 1e), we take ad- tion (“P” in fig. 4) is found in roots from branches II and V,
vantage of the somatic mutations that are shared among whereas it is absent in all leaves (referred to as “root-exclusive”
samples as molecular markers. mutation hereafter). For mutation “P” to exclusively propa-
First, the 17 BR-m mutations allow us to trace back to the gate through adventitious roots but no leaves across
stage prior to the branching events that forming the eight branches, the expected chance is low (<0.0352) under model
sampled branches (IVIII). We find from the 15 BR-m muta- 1 (i.e., the chance that none of the 15 leaves in branches II and
tions (mutations “AO” in fig. 4) that the “mutation alleles” V would have this mutation). Thus, this root-exclusive mu-
propagate exclusively in leaves across all branches (referred to tation “P” is also not consistent with mode 1.
as “leaf-exclusive” mutation hereafter). At these mutation Third, we consider more recent mutations which are de
sites, all sequenced roots have homozygous genotypes, novo to specific major branches. Of the 182 BR-s mutations,
whereas most leaves have heterozygous genotypes (over 14 19 mutations, which are from cuttings with both leaves and
leaf-exclusive mutations [“AN”] were present in 28 of 33 roots sequenced, are shared among different samples in a
sequenced leaf samples). Given the low heterozygosity branch (fig. 4 and supplementary table S2, Supplementary
(0.394%) estimated for the genome, we are reasonable to Material online). There are four BR-s mutations (“ad” in
consider them as leaf de novo mutations (e.g., a A/A->A/T branch VI) present in multiple leaves and propagated
mutation in leaf) rather than root mutations/recombination through two or more cuttings (usually consecutive cuttings),
(e.g., a A/T->A/A mutation or gene conversion in root, sup- indicating their origination prior to the formation of the
plementary fig. S3, Supplementary Material online). This ob- corresponded cuttings. None of the four mutations is found
servation suggests that the leaves and roots are initiated from in roots within the same cutting, which is with low expecta-
a cluster of multiple founder cells, as these 15 mutations tion under model 1 (<0.168 for all three roots within the
should already present in the initial founder cell niche. The same cutting to lack this mutation).
near-fully absence of these mutation in roots could hardly be Fourth, another 13 BR-s shared mutations (mutations
explained by their overall lower-sequencing coverage (supple- “eg” in branch II and “hq” in branch VI) are de novo to
mentary note S1, Supplementary Material online). Further roots of cuttings within a branch, of which 12 (“eg” and
Sanger sequencing accompany PCR amplification confirmed “iq”) with the exception of “h” are present only in single
their absences in roots (supplementary note S1, table S3, figs. cuttings (figs. 2a, 2b, and 4). This contrasts with the leaf
S4 and S5, Supplementary Material online). The chance of specific mutations “ad,” which are all present in multiple
seeing 15 such mutations which present in nearly all 33 leaves cuttings (fig. 4), suggesting that these root mutations possibly
but absent in any of the 22 root samples is essentially nearly arise later. As before, the finding that all 13 root de novo
zero under model 1, after correcting for the putative influence mutations would not be found in leaves has a very low prob-
of lower root coverage (see Materials and Methods for ability under model 1 (<0.055).
details), providing strong evidence for rejecting model 1. In the above, we ignore three mutations, one BR-m
These 15 mutations suggest that at least one cell lineage mutation (“Q”) and two BR-s mutations (“r” and “s”),
(the lineage propagate mutations “AO”) forming the leaves which are found both in leaves and roots (fig. 4).
is segregated from the adventitious stem cell lineage forming Mutation “Q” is present in nearly all sequenced leaf and
the roots and that this segregation predates the formation of root samples from branches III, VI, VII, and VIII, but is
all sampled branches (fig. 5a and b). The relatively large num- absent from all sequenced samples from branches I, II,
ber of mutations further leads us to speculate that the muta- IV, and V. This mutation implies a possible closer relation-
tions “AO” mark putative rapidly dividing cell lineages, also ship of branches III, VI, VII, and VIII to each other relative
distinct from the stem cell lineages of the axillary meristem to the other four branches considering the extremely low
(see Discussion for details). possibility that we would see the same mutation
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FIG. 4. Reconstructed ontogenetic tree of sequenced samples. The right four panels show the presence of 17 BR-m (“AQ”), 19 BR-s shared
(“as”), and 108 BR-s sample-specific mutations in each sequenced sample. Mutations in each sample are marked by different colors
following: blue—mutation only observed in leaf, red—mutation only observed in adventitious root, purple (also marked by asterisk)—
mutation observed in both leaf and root, and gray—ancestral alleles without mutation. Uncategorized mutations are not shown here. VAF
of each mutation in each sample is indicated by the saturation of the color. The leftmost panel is a maximum-likelihood ontogenetic tree
which is constructed using base mutations identified. The black triangle represents the inferred ancestral sequence. Only bootstrap values
over 0.6 are shown (1,000 replicates bootstrap test). Corresponding sample IDs are given between two parts with the format: “Branch ID”
(e.g., Br-I, Br-II, . . .), “Cutting ID” (e.g., Cut1, Cut2, . . .), and “Sample ID” (ID with “L” initial represents leaf, ID with “R” initial represents
adventitious root).
independently arise in four branches. The mutation “r” Leaf Might Be Differentiated from Multiple Founder
appears in one leaf and two roots from a single cutting Cells whereas Root Is Differentiated from One
(fig. 2b), and the mutation “s” presents in two nearby Are leaves and adventitious roots differentiated from multiple
cuttings and are found in two leaves of cutting-19 while founder cells or single founder cell? A mutation presents
in one leaf and two roots of cutting-20 (fig. 2b). Given the within a single founder cell will be passed to nearly all cells
fact that the dominate part of mutations favors model 2 whose DNA were sequenced, whereas a mutation from one
over model 1, observation of these three mutations in of multiple founder cells will be passed to only part of the
both leaves and adventitious roots is less likely to suggest sequenced cells, leading to varied allele frequencies (fig. 6a).
any possibility of model 1. Rather, these three mutations Assuming a low chance of somatic recombination, all somatic
are more likely to imply putative cell lineages, which could mutations are expected to remain in a heterozygous state
replenish both leaf and root cell lineages after their early during propagation (i.e., allele frequency ¼50%). We consider
segregation (fig. 5). the variant allele frequency (VAF, estimated as “read-depth of
In summary, the majority of shared BR-m (16 of 17) and the mutation allele”/“overall read-depth at this site” for a
BR-s (17 of 19) mutations that propagated separately be- certain mutation of each sample) as a surrogate of the allele
tween leaves and adventitious roots gives clear evidence frequency of mutation in terminal cell population of different
that there exist different cell lineages between two organs organs (Yu et al. 2020). In real sequencing, the VAF is
segregated prior to the formation of all branches. The con- expected to follow a binomial distribution (fig. 6a) deter-
structed ontogenetic tree using somatic mutations (fig. 4) mined by both allele frequency and the read-depth at a given
matches well with the expectation of model 2. Beyond the locus (Yu et al. 2020). The distribution of VAF for mutations
segregation, the three mutations shared between leaves and from single founder cell is predicted to be similar to that
roots also imply the existence of shared replenishing cell derived from the intrinsic heterozygous sites (i.e., pre-
lineages for both organs (fig. 5). existing heterozygous variants) in the genome, whereas
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FIG. 5. A proposed model for development of leaves and adventitious roots in shrub willow. (a, b) Progenitor cells (purple squares) of axillary
meristems (arrowhead) are segregated (dashed lines) early in SAM from rapidly dividing cells (blue squares). The progenitor cells (red squares) of
adventitious meristems (asterisks) are likely derived from the segregated axillary meristems. Each leaf is differentiated from the leaf primordia (LP)
which are formed by a chimeric cluster of rapidly dividing cells (blue squares aside each leaf, numbers are not scaled to actual proportions of
different sources). Each adventitious root is differentiated from a single founder cell within the adventitious meristems (red squares aside the
asterisks). (c, d, e) Three branching events are indicated (①→②, ②→③, ③→④). The earliest rapidly dividing cells marked by mutation “A”
are pushed away together with the meristematic stem cells and are recruited to form leaves after iterative branching. The axillary meristems
replenish new progenitor cells for leaf (e.g., blue with “Q”) and adventitious meristems (e.g., red with “Q”). Mutations could happen frequently
within rapid dividing cell lineages (e.g., mutation “a”), but will only present with low VAF in leaves. Only mutations arise in meristematic cells (e.g.,
mutation “Q” and “r”) could reach a high VAF in leaves of subsequent branches. Cell divisions from some progenitor cells are indicated by dashed
lines with arrows.
mutations from multiple founder cells tend to have VAFs easily fixed in root than in leaf owing to the cell population
50% (fig. 6b). “bottleneck.” Therefore, we expect higher observable muta-
We found most leaf mutations have VAFs <30% (figs. 4 tion rate in root than in leaf. The prior-mentioned 108 BR-s
and 6b), apparently deviating from the distribution of VAF for mutations specific to each leaf or adventitious root allowed us
pre-existing heterozygous variants (unequal variances t-test, to test this prediction. The sample-specific mutations share
P < 2.2e-16). It’s noteworthy that the leaf-exclusive BR-m the same timespan between leaf and root during differentia-
mutations generally have lowest VAFs (mean ¼10.6%, tion from their latest progenitor cells. Based on these sample-
SD ¼ 6.42%, fig. 6b), as validated by PCR-Sanger results specific mutations, we estimated a normalized per site per
(mean ¼ 11.2%, SD ¼ 8.39%, supplementary figs. S4 and S5, sample (see Discussion on interpretation of the unit) rate of
Supplementary Material online), suggesting these mutations 4.32 109 (60.786 109 SEM) for leaf SNV mutations,
only present in a limited portion of cells within each leaf. and 8.15 109 (61.42 109 SEM) for root SNV muta-
Contrary to leaf, the distribution of VAF for root mutations tions. The INDEL mutation rate is 4.05 1010
matches well with the distribution of pre-existing heterozy- (62.21 1010 SEM) per site per sample for leaf and
gous variants (unequal variances t-test, P ¼ 0.056) with a peak 1.76 109 (60.501 109 SEM) per site per sample for
around 50% (figs. 4 and 6b). The distinct distributions be- root. The observed somatic mutation rate in root is around
tween leaf mutations and root mutations suggest that leaf 2-fold of that in leaf (two-sided Brunner–Munzel [BM] test,
samples are more likely derived from multiple founder cells P ¼ 0.033 for SNV mutations and P ¼ 0.008 for INDEL muta-
(fig. 6a and b), whereas an adventitious root may be differ- tions), confirming the prediction.
entiated from a single founder cell. Both findings are consis- Given the same read-depth cutoff for mutation calling and
tent with the previous chimera studies (Marcotrigiano and both organs are sequenced at 40-fold, an issue here is that
Stewart 1984; Broertjes and van Harten 1985; Poethig 1987; more leaf mutations will be below the read-depth threshold
Furner and Pumfrey 1992; Irish and Sussex 1992). (and become undetectable) if the leaf is initiated from mul-
Secondly, the different number of founder cells in initiating tiple founder cells although root is only from a single founder
leaf and adventitious root predicts that mutations are more cell (fig. 6a). This offers another validation of whether the
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FIG. 6. Estimated VAFs from somatic mutations for different developmental stages. (a) Read-depth fractions as the indicator of mutation allele
frequency from single or multiple founder cells. Here “A” represents the ancestral allele, whereas “T*” stands for de novo mutation presents in one
recent founder cell. Only when mutation “T*” is from a single founder cell can it be propagated to all later cells, leading to a binomial distribution of
VAF peaked at 50%, otherwise the VAF distribution will be skewed toward a lower peak depending on the initial fraction of mutation “T*” in
founder cells. (b) Distributions of VAFs for leaf and root mutations. The gray histograms are drawn from pre-existing variant sites randomly
sampled (n ¼ 10,000) from fully heterozygous sites across all sequenced leaf and root samples to measure the variance of VAFs owing to
sequencing bias. Only mutation and variant sites with read-depth no less than 10 were presented here to reduce the bias in low-depth regions.
The peaks contributed by BR-m “AO” mutations (around 12.5%) and BR-s sample-specific mutations (around 25%) in leaves are marked by
dashed circles. (c) Distribution of VAFs estimated for root mutations from woody species Prunus mume and Prunus persica. Data collected from
Wang et al. (2019). For all figures, “adv. root” is abbreviation for adventitious root.
higher mutation rate observed in adventitious root is due to a SEM) per site per sample for SNV and INDEL mutations,
higher fixation rate (i.e., because root is from a single founder respectively, which is similar to that of the leaf’s (two-sided
cell, whereas leaf is from multiple founder cells) or a more BM Test, P ¼ 0.4104 for SNVs and P ¼ 0.6639 for INDELs). The
mutagenic process (e.g., more mutations are induced in roots similar mutation rate of two organs at more comparable
through callus formation). Once we use a fairly depth cutoff depth cutoff confirms that the fixation chance is the major
for two organs, that is, lower cutoff in leaf than in root, we factor leading to the observed higher mutation rate in adven-
expect to see similar mutation rates if the fixation chance is titious root, and further confirms that root forms from fewer
the main factor, whereas persistent higher mutation rate in founder cells than leaf.
root, if any mutagenic process, is the main factor. Therefore,
we require 10 supporting reads for root mutations although
keeps requiring only 5 supporting reads for leaf mutations Discussion
(2-fold requirement for root mutations compared with leaf The canonical plant development model posits that SAM
mutations considering leaf is from at least two founder cells). contains a self-replenishing population constituted by a few
The re-estimated root mutation rate is 3.53 109 slowly diving initial cells whereas some of their descendants
(61.08 109 SEM) and 2.72 1010 (61.87 1010 divide actively when differentiating into leaves (Evans and
5301
Barton 1997). The initial cells only divide when forming new newly arisen mutations (Whitham and Slobodchikoff 1981;
axillary meristems upon iterative branching (Burian et al. Simberloff and Leppanen 2019), which in principal requires
2016). It is less clear however whether a leaf in a high-order that 1) the new mutation is directly selection-favored within
branch also contains cells from the descendants of the earliest the meristem to ensure its fixation in later development and
SAM or is solely derived from the descendants of the latest 2) the new mutation is advantageous in the heterozygous
axillary meristems. Our results suggest that both the descend- state and provides phenotypic effects at both moderate-
ants from earliest SAM (rapidly dividing “somatic” cell lineage and low allele frequency. However, these assumptions are
propagate mutations “AO”) and the replenishing cell line- poorly supported by our results, as we found that the chance
ages (slowly dividing stem cell lineages propagate mutations for a somatic mutation to be fully fixed in branch is generally
“Q,” “r,” and “s”) contribute to the formation of the leaf (fig. very low as supposed previously (Burian et al. 2016). This is
5). Considering that adventitious meristems/buds mostly evidenced by: 1) no BR-s mutations were detected in either all
form from cambial cell divisions (Steffens and Rasmussen leaves or all roots within a major branch in this study, suggest-
2016), which are also derived from SAM (Nieminen et al. ing later mutations can rarely fully fix within a whole branch;

2015), we can guess the replenishing cell lineages most likely and 2) most mutations may only have a chance to be fixed
correspond to the axillary meristems (fig. 5). Our results hence within a single-cell lineage derived from meristems, as the
confirm that the axillary meristems (together with the adven- majority of mutations detected in this study are sample-
titious meristems) are segregated from other rapidly dividing specific mutations; and 3) though early mutations may
cell lineages (e.g., the cell linage marked by “AO”) as early as have higher chance to be propagated to more branches,
the cell divisions prior to the formation of all major branches the iterative replenishing process by low-dividing axillary mer-
(Klekowski et al. 1985; Poethig 1987; Irish 1991; Szymkowiak istems might eventually reduce their fixation proportion as
and Sussex 1996; Evans and Barton 1997; Burian et al. 2016), seen for BR-m mutations (fig. 6b). The replenishing process
with the state-of-the-art WGS-based clonal analysis. seems to introduce very limited number of mutations to
Histological analysis in shrub willow shows that the apical axillary meristems, as witnessed by only three mutations
meristem is surrounded by multiple putative leaf primordia shared between leaves and roots, adding further support
(supplementary fig. S6, Supplementary Material online), a for the notion that plants protect their meristems from mu-
structure similar to many plants including other willow spe- tational burden by segregating these meristems early (Burian
cies (Berggren 1984) and Arabidopsis (Evans and Barton 1997; et al. 2016; Schmid-Siegert et al. 2017; Lanfear 2018; Plomion
Burian et al. 2016). Since the axillary meristems arise later et al. 2018).
from the axils of leaves developed from these primordia We note here that our resolution in mutation detection is
(fig. 5), it is highly likely that the axillary meristems have limited by moderate sequencing depth (40-fold) so only
already been specified or segregated there (Burian et al. mutations with VAF no less than 12.5% (fig. 6b) could be
2016). However, only with marker-based analysis could we reliably detected (five supporting reads/40-fold). Thereby,
confirm this segregation. the mutations that accompany rapidly dividing cells might
Beyond the early segregation, our results further reveal that be much more than those we detected. The rate estimated
these rapidly dividing “somatic” cell lineages (carrying BR-m from the sample-specific mutations is likely a more accurate
mutations AO) can survive leaf and branch formation, and estimator. Since these mutations are detected at moderate
are present even after several iterative branching without sequencing depth, only early mutations from one or a few
being replaced by other lineages (fig. 5). The persistent pres- early cell divisions within the meristem could reach this high
ence of these BR-m mutations with low VAFs implies that the fraction in the terminal cell population. Thereby, the unit
independent branches are likely recruited from a chimeric might be most suitably interpreted as “per site per (few)
cluster of cells where only a portion of the cells retained these cell division(s)” instead of “per site per sample.” Given the
mutations. Furthermore, the cells containing these BR-m same reason, the “per site per sample” rate is possibly not
mutations may be predetermined to contribute exclusively directly comparable to those “per site per year” rates esti-
to the formation of leaves in the new branch, but not the mated for many old trees (Schmid-Siegert et al. 2017; Plomion
growth of stems and vascular tissues. Therefore, initiation of et al. 2018; Hanlon et al. 2019; Hofmeister et al. 2020; Orr et al.
axillary meristem from SAM seemingly involves concomi- 2020), as these “per site per year” rates represent “observable”
tantly cell divisions of both slowly and rapidly dividing cells (or fixable) mutations after many years of accumulation,
(fig. 5). Preservation of such rapidly diving cells along with rather than the yearly mutation rate.
iterative branching provides another possible strategy to re- The low fixation chance but not necessarily low rate of
duce the number of cell divisions in meristematic stem cells somatic mutations we see here is most possibly a direct con-
when forming new leaves in subsequent branches. sequence of early segregation of axillary meristems, a conjec-
The intraorganismal selection hypothesis posits that so- ture that has been proposed for a century (Laux 2003; Lanfear
matic mutations may be selectively advantageous in gener- 2018). The early-segregated meristems also suggest the pos-
ating “mosaic sectors” against natural enemies, such as sibility of an early-segregated plant germline (Lanfear 2018).
herbivores and pathogens (Whitham and Slobodchikoff The estimated mutation rate for axillary meristems is much
1981; Whitham 1983; Antolin and Strobeck 1985; Suomela lower than that of other cell lineages, which is likely consistent
and Ayres 1994; Simberloff and Leppanen 2019). This hypoth- with the prior observation that the somatic reversion rate is
esis assumes that selective pressures could help to fix the 4-fold lower in the commonly assumed gamete-bearing L2
5302
layer than in L1 layer in peach (Chaparro et al. 1995). Tracking year 2014 and was coppiced in 2015 to stimulate new shoots
heritable somatic mutations may provide more clues as to in 2016 (supplementary fig. S1, Supplementary Material on-
which cell lineage direct contributes to the formation of line). The sampling was performed in 2016. A total of 22 stem-
gametes in woody plants. cuttings from eight major branches were sampled from this
Does adventitious root contain another independent cell tree (fig. 2a). The stem-cuttings we collected are from differ-
lineage, similar to the one marked by “AO” in leaf, besides ent developmental stages (fig. 2a), including postcoppice
the replenishing cell lineages? Currently, we found no strong stem tissue (e.g., cuttings 6, 8, 13), sylleptic (e.g., cuttings 3,
evidence. The root BR-m mutation “P” is present in all three 15), and doubly sylleptic branches (e.g., cuttings 19, 20), to
root samples from cutting-2 but only one of three root sam- represent iterative branching processes. The collected cut-
ples from cutting-8 (fig. 2b). Therefore, the mutation “P” tings were first cultivated in 1/2 Murashige and Skoog (MS)
could have occurred in a cell downstream of its original mer- fluid medium for 1 week to obtain sufficient nutrients and
istematic stem cell but ended up in a limited number of were then transplanted into fresh water and cultivated under
closely related adventitious meristems, distinct from muta- 25 C room temperature. Leaf and root DNA were

tions “AO.” This partly explains the contradictory observa- extracted using the Cetyltrimethyl Ammonium Bromide
tion that there are many BR-m mutations present in nearly all method (Clarke 2009) after growing for about 3 weeks.
leaves and absent in all roots (e.g., mutations “AO”), but no Quality of DNA samples was tested by microplate reader
BR-m mutation is present in most adventitious roots and and agarose gel electrophoresis to ensure sufficient quantity
absent in all leaves. and integrity for whole-genome sequencing. Qualified DNA
The low fixation chance seems contrary to the empirical samples were fragmented into an insert size of about 300–
prevalence of bud sport mutants (Shamel and Pomeroy 1936; 350 bp by sonication and sequenced on the Illumina
Foster and Aranzana 2018). However, our finding that the Hiseq4000 platform with 150-bp paired-end reads. All quality
adventitious root is likely differentiated from a single founder testing, library construction, and sequencing steps were per-
cell (fig. 6a) suggests a possibility that some mutations can be formed at BGI-Shenzhen. Each sample was sequenced to
fixed through a cellular population “bottleneck” effect within around 18 Gb (averaged 44, supplementary table S1,
certain meristems. By establishing an organ from a single an- Supplementary Material online) after cleaning for low quality
cestral cell, a mutation that predates this ancestral cell can be reads, either >5 bp Ns or more than 30% base calls with
fully fixed within the organ. Furthermore, this process seems quality score below 20.
to be a universal phenomenon for the development of plant
roots, as the VAF distribution of somatic mutations identified Alignment and Initial Variant Discovery
in underground roots (i.e., roots differentiated from root api- Detailed procedures for read alignments and initial variant
cal meristems) of some plants also peaked at around 50% (fig. discovery followed the same pipelines described in Wang et
6c). The varied fixation chance of somatic mutations within al. (2019). In brief, cleaned reads were mapped to the
different organ emphasizes a strategy that not only helps pseudomolecule-level assembly of S. suchowensis (Dai et al.
reducing somatic mutations fixed in reproduction-related 2014) downloaded from PopGenIE database (ftp://plantge-
meristems but also maintains a substantial level of somatic nie.org/Data/PopGenIE/Salix-suchowensis, v4.1) using BWA-
mosaicisms within other meristems. mem algorithm (Li 2013). This assembly contains 229-Mb
Our study also demonstrates the power of somatic muta- sequences anchored in 19 chromosomes, with an additional
tion as a hallmark in elucidating cell lineages (Wang et al. 6.7-Mb sequences assembled into large scaffolds longer than
2019). However, compared with the standard methods using 10 kb. The BWA (version 0.7.10-r789) was run with option “-
chimeras which operate at the level of the cell (Poethig 1987; M” to keep the resulted SAM file (Li et al. 2009) compatible
1989; Szymkowiak and Sussex 1996; Frank and Chitwood with downstream processes. Picard package version 1.114
2016), our approach has limitations in explicitly determining (https://broadinstitute.github.io/picard/, last accessed
which cell lineage corresponds to the specified cell layer of the September 30, 2021) was used to mark noninformative PCR
SAM, since the somatic mutations are called from bulk se- duplicates in mapping results by MarkDuplicates function
quencing of multiple cell populations (Wang et al. 2019). bundled inside. GATK package (version 3.5) was further
Future studies combining single-cell profiling (Woodworth used to perform local realignment using
et al. 2017) as well as DNA barcoding technologies RealignerTargetCreator and IndelRealigner functions to min-
(Kebschull and Zador 2018) will give a more complete picture imize false variant calls due to alignment errors around INDEL
about the cellular development of woody plants. locations (DePristo et al. 2011).
After mapping to the reference assembly, the leaf sam-
Materials and Methods ples covered 90.9% of the reference genome with at
least one reliable read (i.e., reads with mapping quality
Sample Collection, DNA Extraction, and Whole- score [MAPQ] 20, indicating a mismapping rate 1%),
Genome Resequencing from the lowest 87.0% to the highest 91.3% (supplemen-
The shrub willow (S. suchowensis) YAF1 (male) was kindly tary table S1, Supplementary Material online). This pro-
provided by Jiangsu Academy of Forestry, China, and was portion dropped slightly to 81.0% in average when only
transplanted in Nanjing University. This tree was started as considering genomic regions covered by at least ten reli-
a sapling from cutting breeding (single cutting in origin) in able reads (supplementary table S1, Supplementary
5303
Material online). The root samples contained slightly all leaves/roots according to when they are generated, sup-
lower coverage than leaf samples, ranging from 78.6% to plementary fig. S3b and d, Supplementary Material online).
90.7% (86.6% in average, supplementary table S1, To detect BR-m mutations under the first situation, we
Supplementary Material online), but the reduction was compared all combinations of seven branches against the
stronger when only considering regions with 10 reliable remaining branch and ensuring that the mutation was absent
reads (52.8% in average, supplementary table S1, in the one branch but present in more than one of the seven
Supplementary Material online). The low coverage in branches (supplementary note S3, Supplementary Material
root samples is due to higher sequencing yield loss caused online). Note that this corresponds to all situations (i.e.,
by putative bacterial contaminants in roots (supplemen- m ¼ 7, 6, 5, 4, 3, 2) as we do not require the mutations to
tary note S2 and fig. S7, Supplementary Material online), be present in all seven branches. For mutated branches with
especially in one sample “Cut18-R2,” which yielded a cov- multiple samples sequenced (branches II, V, and VI), these
erage of 1.4% with over ten reliable reads. Therefore, we mutations are expected to be present in all of them as men-
excluded this sample from identification of mutations, tioned above. However, amplification or sequencing bias

but used it for confirmation of mutations identified in could cause the mutated allele to be missed in a few samples.
other samples. We also paid special attention to remove Therefore, we used a lenient requirement that the mutation
any putative contaminants when performing mutation should be present in at least 90% of the samples in these three
calling (supplementary note S2 and fig. S2, branches, that is, at least 8 samples in branch II, 13 samples in
Supplementary Material online). branch V, and 22 samples in branch VI (supplementary note
Variants, including SNVs and small-sized INDELs (1–100 S3 and table S5, Supplementary Material online). To detect
bp), were called for all samples using two algorithms, BR-m mutations under the second situation, we directly com-
UnifiedGenotyper (UG) and HaplotypeCaller (HC), both pared leaves with roots. A mutation was determined to be
implemented in GATK. A union set of called variants from exclusive to leaves if it was only present in leaves from two or
both algorithms were used for downstream mutation identi- more branches but was absent in all roots. A mutation was
fication to reduce the false negative rate (FNR; Xie et al. 2016; determined to be exclusive to roots if it was only present in
Wang et al. 2019). Only reads with MAPQ over 20 (Phred roots from two or more branches but was absent from all
scaled, equivalent to less than 1% mismapping rate) were leaves.
used for variant calling here. Variants that resided in short All mutation candidates were called first using a parallel
scaffolds (size <10 kb) were discarded for further mutation comparison strategy (Sung et al. 2012; Wang et al. 2019) to
calling, as these unanchored fragments (average size ¼666 bp, remove sequencing or mapping errors which show up re-
median size ¼372 bp) were generally highly repetitive in peatedly between focal samples which are supposed to carry
nature. the mutation and control samples which are supposed not
to carry the mutation (Li and Stoneking 2012). Candidate
Somatic Mutation Calling mutations were further filtered by 1) removing sites with a
Since spontaneous somatic mutations generally arise and are low variant quality score (<50) or with many noninforma-
fixed during cell divisions, the distribution of mutations is tive calls (>5 samples applied here as sites failed this crite-
expected to reflect their historical origin in the plant’s devel- rion were frequently associated with other sequencing or
opment, that is, follow the ontogeny. For example, a mutation mapping issues); 2) requiring 5 supporting reads for the
only present in a single branch (denoted as a BR-s mutation) mutation in at least one focal sample, and requiring at least
most likely emerged during or after the separation of that one reliable read carrying the identical mutation (with base
branch. In contrast, a mutation present in multiple branches quality 30 to avoid sequencing errors) for the remaining
(denoted as a BR-m mutation) would indicate an origination focal samples; and 3) requiring presence of both forward
predating the separation of these branches (the underlying and reverse strands for the supporting reads, which mini-
ontogeny not observed). The BR-s mutations could be easily mizes mismapping caused by homology sequences (Xie et
identified through comparing one branch against all other al. 2016; Wang et al. 2019). For BR-m mutations under sit-
branches. For BR-m mutations, we considered two possible uation 1, more rigorous criteria were used to reduce FPR
situations (supplementary fig. S3, Supplementary Material after extensive assessment (supplementary note S3 and ta-
online): 1) part (denoted as “m,” m > 1 and m < 8) of the ble S5, Supplementary Material online). A flowchart of the
eight sampled branches was originated from a single branch mutation screening is included to show the number of
(supplementary fig. S3a and c, Supplementary Material on- candidates at each step (supplementary fig. S2,
line), for which we should expect mutations present in m Supplementary Material online). All filtered mutations
branches but absent in all other branches (since these muta- were assessed manually using Integrative Genomics Viewer
tions are earlier than the separation of the m branches, we (Thorvaldsdottir et al. 2013) to remove ambiguous cases
should also expect them to be present in nearly all samples such as mutations found in regions with extremely high-
obtained from the m branches); (2) if the early segregation sequencing errors, mutations from possible exogenous con-
model (model 2) is correct, that is, the two organs could have taminations, or mutations from reads which are poorly
segregated cell lineages at the beginning, there might exist aligned. Only candidates that passed all criteria were
some mutations present exclusively in leaves or in roots (sim- retained for the analyses (supplementary table S2,
ilarly, the mutations are also expected to be present in nearly Supplementary Material online).
5304
Evaluation of Identified Somatic Mutations amplified independently. For each sample, more than 32
The overall strategy and criteria have been extensively tested monoclonal colonies were selected and validated by colony
across various plant taxa and were estimated previously to PCR. Positive clones were subsequently sequenced by Sanger
have a FPR <5% within callable regions when sufficient con- reaction, yielding a total of 362 analyzable results from 20
trol samples (5) are provided (Wang et al. 2019). For exam- mutated samples (supplementary table S3 and fig. S5,
ple, when calling BR-s mutations in one branch, all other Supplementary Material online). The mutation alleles were
branches serve as control samples, whereas when calling verified in all 20 samples (at least one clone contains the
BR-m mutations exclusively present in leaves (i.e., situation mutation alleles for each sample), with 40 of 362 clones (i.e.,
2), all root samples serve as controls. The analytical artifacts overall mutation allelic fraction ¼11.11%) contain the mu-
could thus be removed efficiently as the majority of them tation alleles (supplementary fig. S5, Supplementary Material
have the same chance to be found in focal and control sam- online).
ples (Li and Stoneking 2012). For the remaining two BR-m mutations (“G” and “O”), no
Further, we assessed all 17 BR-m mutations and a ran- suitable primer is available (supplementary fig. S4,

dom subset of 18 BR-s mutations with PCR amplification Supplementary Material online). We did not further assess
followed by Sanger sequencing (supplementary table S3, those two mutations after failing several rounds of trials.
Supplementary Material online). Samples for validation The FPR was estimated as:
were randomly picked from those with sufficient DNA. of validated mutations
PCR amplification and Sanger sequencing were performed FPR ¼ 1 – no: ;
ðno: of analyzable mutations for validation þ 1Þ
for each sample and each mutation independently. The
VAFs of BR-m mutations were assessed using two
assuming if we verify one more mutation by Sanger sequenc-
approaches. The first approach, for each BR-m mutation
ing, it might be failed to validate.
in “A, C, D, F, H, J, K, L, N, P, Q,” including roughly ten
The FNR was estimated using a simulation method de-
mutated samples (samples supposed to carry a certain mu-
scribed before (Keightley et al. 2015; Wang et al. 2019). Briefly,
tation) and four control samples, was PCR amplified (sup-
a total of 1,000 synthetic mutation sites were generated from
plementary table S3 and fig. S4, Supplementary Material
the same sequencing data by replacing 1,000 randomized sites
online). The PCR products (representing pooled cell popu-
in them. The read-depth of each synthetic mutation was
lations) for each sample were Sanger sequenced indepen-
sampled from the distribution of the real mutations identi-
dently, yielding a total of 110 and 41 analyzable results for
fied. The leaf and root samples were simulated separately
mutated and control samples, respectively. The obtained
considering the differences in their coverages and read-
Sanger chromatogram traces were subsequently decom-
depth distributions. The FNR was calculated as:
posed using Indigo (https://www.gear-genomics.com/in-
digo/, last accessed September 30, 2021) to calculate the No: of uncallable mutations
allelic fractions of mutations (Rausch et al. 2020). This ap- FNR ¼ ;
No: of valid synthetic mutations
proach verified all 11 BR-m mutations here and confirmed
the mutations in 105 of the 110 samples (supplementary Based on the simulation results, around 79.4% and 71.5%
table S3 and fig. S4, Supplementary Material online). The (calculated as “100% - FNR”) of the reference genome were
five unconfirmed cases (two from mutation “A” and three estimated to be callable for leaf and root samples, respectively
from mutation “J”) were those with no reliable signal found (supplementary table S6, Supplementary Material online).
for the mutation allele. These are not unexpected due to The normalized per site per sample somatic mutation rate
the low frequency presence of these mutations. Consistent (s) is calculated as:
with this, we also observed two cases where the mutation s ¼ m=½G ð1 FNRÞ 2 S ð1 – FPRÞ;
allele could be detected by PCR but not previously reported
by WGS analysis (supplementary fig. S4, Supplementary where m is the number of somatic mutations called in a
Material online, mutation “K” and “L” in sample Cut22-L1 certain organ, G is the haploid genome size, and S is the
which has lowest average depth in leaves). All 41 control number of analyzed samples. Only those samples from cut-
samples were also confirmed to not contain the mutations tings with both leaf and root sequenced were analyzed here,
(supplementary table S3 and fig. S4, Supplementary Material hence S ¼ 19 for leaf samples, S ¼ 21 for root samples.
online).
In second approach, the PCR products were first cloned
using TA cloning (also known as rapid cloning) prior to Downstream Analysis
Sanger sequencing. The PCR products amplified by DNA For prediction of effects of identified mutations, we used
polymerase (P312-01; Vazyme Biotech Co.) were integrated SnpEff version 4.0 (De Baets et al. 2012) based on the gene
into the pMD20-T vector (6028; Takara Bio Inc.) by ligation models of S. suchowensis (v4.1, also downloaded from the
reaction. Then, the plasmids were transformed into compe- PopGenIE database). BM test was performed using R (R
tent cells of Escherichia coli. For each BR-m mutation in “B, Development Core Team 2013) package “lawstat” (Hui et
D, E, F, I, J, M, N,” two or three mutated samples were PCR al. 2008).
5305
Heterozygosity of the genome was estimated as Hf/Gi, mutations, Pmax =(1 – 0.2)21 ¼ 9.22 103. The chance we
where Hf is the number of fully heterozygous variants (i.e., see over ten such mutation sites is essentially near zero
variants with heterozygous genotype across all called samples, [(9.22 103)10 ¼ 4.46 1021]. This again suggested that
given as heterozygous genomic differences in supplementary these mutations cannot be explained by imbalance of
fig. S2, Supplementary Material online) and Gi is the overall mutation-detection power. The chances of other mutations
size of informative genomic regions used for variants calling, could be derived using the same approach with different n
which is equivalent to the size of the analyzed genomic and m numbers.
regions covered by no fewer than five reads (supplementary
table S1, Supplementary Material online). Supplementary Material
To assess whether somatic mutations are evenly distrib- Supplementary data are available at Molecular Biology and
uted along the genome, we divided the whole genome in Evolution online.
nonoverlapping 1 Mb windows and counted the mutation
events for each window (observations). All windows were Acknowledgments

then tested using a Monte Carlo process, with 10,000 ran-
This work was supported by grants from the National Natural
domizations of shuffling all mutation events across the whole
Science Foundation of China (Grant Nos. 31970236,
genome to derive the expectations. The unbiased estimation
91631104, 31970517, and 31900203). The funders had no
of empirical P (expected type I error rate) was derived as
role in study design, data collection and analysis, decision
(n þ 1)/(m þ 1) for each window, where n is the number of
to publish, or preparation of the manuscript.
seeing more in randomization than observation and m is the
number of randomization (North et al. 2003). Regions with
P < 0.05 were defined as hotspot regions, which will indicate
Author Contributions
nonrandom occurrences of somatic mutations within these L.W., Y.J., D.T., and S.Y. planned and designed the research.
regions. Y.R., Z.H., P.L., and L.W. performed experiments, conducted
An ontogenetic tree was constructed using an approxi- fieldwork, and analyzed data. L.W., Y.J., S.Y., S.S., and B.T. wrote
mately maximum-likelihood approach implemented by the manuscript. Y.R. and Z.H. contributed equally.
FastTree (Price et al. 2010) (version 2.1.10) with generalized
time-reversible model. Interactive Tree Of Life (Letunic and Data Availability
Bork 2019) was used to annotate and display the tree. Plot of The sequencing reads have been submitted to the National
chromosome distributions was generated using RIdeogram Center for Biotechnology Information (NCBI) under
package (Hao et al. 2019). BioProject PRJNA497989. Codes for resequencing preprocess-
To derive the expected chance of leaf-exclusive or root- ing, mutation calling as well as downstream analysis are avail-
exclusive mutations under model 1, we consider “p” as the able at GitHub page https://github.com/ArthurSilver/willow-
real chance we can finally see a mutation in a particular se- workflows (last accessed September 30, 2021). Original Sanger
quenced sample (since p is a probability here, 0 <p 1). sequencing results (in ab1 format) and other related miscel-
Under model 1, the propagation of a mutation to each sam- laneous data have been deposited in https://doi.org/10.6084/
ple is completely random, so p is the same in both leaf and m9.figshare.14746179.
root (meaning a mutation has the same independent chance
to be detected in any of the samples formed after this mu-
tation). Considering the possibility that the lower coverage
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Somatic Mutation Analysis in Salix suchowensis Reveals

Early-Segregated Cell Lineages

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Introduction 2004; Simberloff and Leppanen 2019). This hypothesis re-

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Leaf (Fraction) Root (Fraction)

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