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A computational framework for mapping the timing of vegetative phase

change

Article  in  New Phytologist · March 2016

DOI: 10.1111/nph.13907

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 Research

Methods
A computational framework for mapping the timing of
vegetative phase change
Meng Xu1*, Libo Jiang2*, Sheng Zhu1*, Chunguo Zhou1, Meixia Ye2, Ke Mao2, Lidan Sun3, Xiaohua Su4,
Huixin Pan1, Shougong Zhang4, Minren Huang1 and Rongling Wu2,3
1
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China; 2Center for Computational Biology, College of Biological Sciences
and Technology, Beijing Forestry University, Beijing 100083, China; 3Center for Statistical Genetics, The Pennsylvania State University, Hershey, PA 17033, USA; 4Research Institute of
Forestry, Chinese Academy of Forestry, Beijing 100094, China

Summary
Author for correspondence:
Phase change plays a prominent role in determining the form of growth and development.
Rongling Wu Although considerable attention has been focused on identifying the regulatory control mecha-
Tel: +86 10 6233 6264
nisms of phase change, a detailed understanding of the genetic architecture of this phe-
Email: rwu@bjfu.edu.cn
nomenon is still lacking.
Received: 4 December 2015
We address this issue by deriving a computational model. The model is founded on the
Accepted: 17 January 2016
framework of functional mapping aimed at characterizing the interplay between quantitative
trait loci (QTLs) and development through biologically meaningful mathematical equations.
New Phytologist (2016)
A multiphasic growth equation was implemented into functional mapping, which, via a
doi: 10.1111/nph.13907 series of hypothesis tests, allows the quantification of how QTLs regulate the timing and pat-
tern of vegetative phase transition between independently regulated, temporally coordinated
Key words: functional mapping, growth processes. The model was applied to analyze stem radial growth data of an interspecific hybrid
equation, phase change, Populus, quantita- family derived from two Populus species during the first 24 yr of ontogeny. Several key QTLs
tive trait loci (QTLs). related to phase change have been characterized, most of which were observed to be in the
adjacent regions of candidate genes.

The identification of phase transition QTLs, whose expression is regulated by endogenous
and environmental signals, may enhance our understanding of the evolution of development
in changing environments.

such as Arabidopsis and maize, it was observed that microRNAs,

Introduction
To better respond to environmental signals for optimal survival
and reproduction, every organism would take a series of develop-
mental changes during its life cycle (Poethig, 2003; Baurle &
Dean, 2006; Huijser & Schmid, 2011). Some of these changes
occur in an abrupt and discontinuous fashion, leading to distinct
phases in morphology, physiology and anatomy (Poethig, 1990;
Bond, 2000; Chitwood et al., 2014). An example of discontinu-
ous phase changes is the transition of a plant from its vegetative
growth to flowering as the plant ages. The transition between dif-
ferent phases is controlled by developmental genetic programs
that are activated and mediated by both environmental and
endogenous stimuli (Rougvie, 2005; Huijser & Schmid, 2011;
Yang et al., 2013). Many studies have begun to reveal the molec-
ular mechanisms underlying phase changes in plants (Lauter
et al., 2005; Chuck et al., 2007). In annual herbaceous plants,
*These authors contributed equally to this work.
miR156 and miR172, through their sequential expression, serve
as the master regulators of phase change (Wu et al., 2009).
Recent studies have demonstrated that these microRNAs are also
responsible for vegetative changes from juvenile to adult phases
in perennial woody species (Wang et al., 2011).
Along with those discontinuous phase changes during devel-
opment, there are continuous patterns of developmental transi-
tions that pervade every aspect of growth and development at a
wide range of organizational levels from cells to organs to the
whole organisms (Poethig, 2003). Continuous phase transitions
take place gradually with a less conspicuous change, but they
may play a distinct role in assisting an organism to adjust its
growth form in a precise quantitative way to best adapt to envi-
ronmental perturbations (Huijser & Schmid, 2011). Albeit less
easily observed and characterized, such gradual phase changes
can be recognized by modeling the temporal pattern of growth
over time. Mathematical equations derived from either biologi-
cal or statistical perspective, or both, have proven of particular

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power in capturing continuous phase changes (Brody, 1945;
Koops, 1986). For example, a bi-logistic growth equation has
been shown to fit well the growth trajectory of above-ground
biomass that spans two distinct phases in sequence – the vegeta-
tive and reproductive (Sheehy et al., 2004). This growth pattern
of two phases, separated by flowering, was also found for a
dual-adapted tropical rice variety grown in both flooded and
aerobic environments (Clerget et al., 2014). The mathematical
equations allow the timing and nature of phase change to be
estimated precisely, providing a quantitative assessment on how
different phases contribute to end-point growth (Sun et al.,
2014). Similar mathematical relevance can be true for other
plants and organs, although no same pattern of vegetative juve-
nile–adult phases is held for all cases.
Different from previous studies purported to identify the tran-
scription factors related to phase change, this work focuses on
mapping the genetic architecture of this developmental phe-
nomenon by developing a computational framework. We reform
a mapping model – functional mapping – derived to identify
specific quantitative trait loci (QTLs) that govern the process and
pattern of development over time (Ma et al., 2002; Wu & Lin,
2006; He et al., 2010; Li & Wu, 2010; Jiang et al., 2015b), to
test how QTLs determine the timing and pattern of phase change
in development. The new model capitalizes on mathematical
derivations of QTL genotype-dependent growth parameters esti-
mated from functional mapping and formulates a testing proce-
dure for the genetic effect of QTLs on developmental timing.
Here, we show how the model can be used to map QTLs associ-
ated with the transition from a juvenile to adult phase of a woody
species, poplar. Given that ample genetic variation occurs in
phase change (Poethig, 1988; Wiltshire et al., 1998; Hudson
et al., 2014), the new model will provide a timely tool to map
phase change genes and understand the evolutionary significance
of these genes in the creation of phenotypic novelty.
Model
Multiphasic growth equation
The postembryonic growth of a plant can be broadly classified
into three phases, juvenile (before reproduction), mature (during
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which the plant flowers to produce seeds), and senescence
(decline in both growth and reproductive vigor) (Bond, 2000;
Fig. 1). According to Koops’ (1986) mathematical formulation,
we partition the overall growth, g(t), of the plant at age t into
three phases, expressed as:
g ðt Þ ¼ g1 ðt Þ þ g2 ðt Þ þ g3 ðt Þ
where g1(t), g2(t) and g3(t) form three components of the overall
growth corresponding to juvenile, mature and senescence phases,
respectively. The growth of each phase can be fitted by a logistic
function that has proven to be one of the universal biological
phenomena based on fundamental principles of biophysics and
biochemistry (West et al., 2001). Thus, we make the summation
of logistic functions to fit multiple (say K ) phases of growth
simultaneously, leading to a multiphasic growth equation,
expressed in hyperbolic tangent (tanh) form as:
X
K
g ðt Þ ¼ ½ak f1 þ tanhðbk ðt  ck ÞÞg; Eqn 1
k¼1
whose growth rate function is its first derivative, expressed as:
X
K   
g 0ðt Þ ¼ ak bk 1  tanh2 ðbk ðt  ck ÞÞ Eqn 2
k¼1
where k is the kth phase (k = 1, . . ., K ) with growth determined
by three characteristics: asymptotic value (2ak), duration (4/bk),
and age at maximum growth rate (inflection point) (ck). From these
parameters, we derived new parameters, visualized in Fig. 2, that
distinguish one phase from next (Koops, 1986). These parameters
are: the timing of the offset of one phase k, expressed as ck + 2/bk;
the timing of the onset of the next phase k + 1, expressed as ck+1 –
2/bk; the length of phase change from k to k + 1, expressed as (ck+1
– 2/bk+1) – (ck + 2/bk) = (ck+1 – ck) – 1/2 (4/bk+1 + 4/bk); and the
maximum growth rate in phase k, expressed as akbk.
Next, we implemented Eqn 1 into functional mapping to test
how a QTL controls overall growth curves. We will further for-
mulate a procedure for testing how the QTL regulates the timing
of phase change and phase-related differences in growth form.
Fig. 1 Form of height growth curve (thick red
line) undergoing different developmental
stages for a hypothesized woody plant.
Transition from the juvenile to the adult
stage usually occurs before reproductive
competence is acquired through the
maturation of sexual organs (flowers).
Senescent characteristics emerge as the tree
approaches maximum or asymptotic height.
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Fig. 2 Phase 1 (red) and phase 2 (blue) of a

growth curve (upper) and its growth rate
curve (lower) for a biphasic equation.
Asymptotic growth (2a), duration (4/b), and
age of maximum growth rate (c) for each
phase are shown, along with the length of
phase change s = (c2 – 1/2b2) – (c1 – 1/2b1).

conditional probability of this QTL genotype given the adjacent

Functional mapping of phase change QTLs
marker genotype of progeny i (Wu et al., 2007); and fj(yi) is the
Because of tremendous efforts by geneticists worldwide, a vast multivariate normal distribution density function with genotype-
amount of mapping materials used to map complex traits for a dependent mean vector expressed:
wide range of species have been established (Cheverud et al.,
1995; Peiffer et al., 2014; Rebetzke et al., 2014; Jiang et al., lj ¼ ðlj ð1Þ; . . .; lj ðT ÞÞ Eqn 4a
2015a). Consider such a mapping population available to map
phase change. We assume that it is composed of n progeny, each and (T 9 T) covariance matrix
genotyped for a panel of molecular markers from which to con- 0 2 1
r ð1Þ    r1T
struct a genetic linkage map that covers the entire genome. Each X B
¼ @ ... .. .. C
progeny is measured for a growth trait, such as plant height or . . A Eqn 4b
diameter, at a series of T time points spanning its life cycle. rT 1    r2 ðT Þ
Let yi = (yi(1), . . ., yi(T)) denote a vector of time-dependent
growth trait values for progeny i. By hypothesizing that multi- The elements in mean vector (Eqn 4a) are the expected
phasic growth curves are controlled by a set of QTLs located on means of the growth trait for genotype j from time 1 to T,
the linkage map, functional mapping formulates a mixture likeli- respectively, and those in matrix (Eqn 4b) are the time-
hood model expressed as: dependent variances (on diagonal) and covariances of differ-
ent pairs of time points (off diagonal). A full expression of
Y
n   Eqns 4a and 4b is specified by T and ½T(T – 1) parame-
LðyÞ ¼ x1ji f1 ðy 1 Þ þ . . . þ xJ ji fJ ðy 1 Þ Eqn 3
i¼1 ters, respectively.
Compared with traditional static mapping models, func-
where xj|i is the proportion of the jth mixture proportion (j = 1, tional mapping shows two distinct features. First, it models
. . ., J), corresponding to the jth QTL genotype, described by the and estimates genotypic means for each QTL genotype j by

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a mathematical equation. For example, functional mapping
does not directly estimate T expected genotypic means in
equation (Eqn 4a), rather than models these means by multi-
phase growth Eqn 1. Specifically, we express genotype-
dependent means as:
lj ¼ ðlj ð1Þ; . . .; lj ðT ÞÞ
XK  
 
¼ ajk 1 þ tanh bjk ð1  cjk Þ ; . . .;
Eqn 5
k¼1 !
X
K   
½ajk 1 þ tanh bjk ðT  cjk Þ 
k¼1
through a set of parameters (ajk, bjk, cjk) (j = 1, . . ., J for geno-
types; k = 1, . . ., K for phases). Thus, by comparing genotype-
dependent differences in this parameter set, we can determine
whether this QTL affects growth curves.
Second, functional mapping chooses and uses a cost-effective
statistical model to structure the longitudinal covariance Σ by
a particular set of parameters. Such a model can be parametric,
such as autoregressive (Ma et al., 2002), antedependence (Zhao
et al., 2005) and autoregressive moving average (Li et al.,
2010), nonparametric (Yap et al., 2009), or semiparameteric
(Das et al., 2012), depending on data types. Estimating fewer
parameters, these models display increasing statistical power for
QTL detection. Of these models, antedependence is suggested
to be the most parsimonious because it uses fewer parameters
to capture the complex structure of a matrix. Zhao et al.
(2005) provides a detail on how to derive the first-order struc-
tured antedependence (SAD(1)) model for matrix structure by
defining two parameters, innovation variance (m2) and first-
order antendependence parameter (/), that is, error in the cur-
rent time point only depends on that of its immediately pre-
ceding time point. If even time intervals are assumed, we
obtain the residual variance at time t and covariance between
time t and t0 as:
1u 2 2t
r2 ðt Þ ¼ m ; t ¼ 1; . . .; T Eqn 6a
1  u2
1  u2t 2
rtt 0 ¼ ut 0t m ; t 0 [ t ¼ 1; . . .; T Eqn 6b
1  u2
Thus, by implementing these expressions in Eqns 6(a) and
6(b) into the matrix in Eqn 4a, functional mapping estimates the
parsimonious structure of the matrix without losing much infor-
mation.
Many algorithms have been developed to estimate the
parameters contained in likelihood (Eqn 3). Ma et al. (2002)
implemented the Expectation Maximization algorithm to esti-
mate the QTL positions, growth parameters in the mean
vectors and matrix-structuring parameters by taking advantage
of closed forms of the inverse and determinant of the
autoregressive matrix. Liu & Wu (2009) incorporated the
Bayesian approach for parameter estimation, improving
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computational efficiency. A more sophisticated Bayesian pro-
cedure was devised to estimate the longitudinal structure of
covariance via semiparametric models. A computer technique,
simplex algorithm derived from the optimization principle,
has been widely applied to estimate growth parameters con-
stituting nonlinear equations and matrix-structuring parame-
ters (Zhao et al., 2005). In this study, we deployed the
simplex algorithm to obtain the estimation of all parameters
contained in Eqns 5, 6(a) and 6(b).
Hypothesis tests
The existence of a significant QTL can be tested by formulating
the following hypothesis tests:
 
H0 : ajk ; bjk ; cjk  ðak ; bk ; ck Þ for j ¼ 1; . . .; J ; k ¼ 1; . . .; K
H1: At least one of the equalities above does not hold,
under each of which we obtain the maximum likelihood esti-
mates of all growth parameters and matrix-structuring
parameters and then plug these estimates into the correspond-
ing likelihoods. By calculating the log-likelihood ratio (LR) of
the two hypotheses and comparing it with a critical threshold,
we determine whether a QTL exists to affect growth curves.
The threshold can be empirically determined from permuta-
tion tests by reshuffling the phenotypic data 1000 times
(Churchill & Doerge, 1994). At each time, an LR value was
calculated and the top 5% of LR values among 1000 times
was then chosen as the critical threshold at the 5% signifi-
cance level.
After a QTL is observed to be significant, we will further test
how this QTL control growth form by regulating phase change.
We will focus on testing the genetic control of the timing of
phase change and its relative contribution to the overall growth.
We formulate the following null hypotheses:
H0 : cjk  2=bjk  ck  2=bk ; Eqn 7
H0 : cjk þ 2=bjk  ck þ 2=bk ; Eqn 8
  
H0 : cj ðkþ1Þ  2=bj ðkþ1Þ  cjk þ 2=bjk
 ðck þ1  2=bk þ1 Þ  ðck þ 2=bk Þ; Eqn 9
for j = 1, . . ., J, to test whether this QTL affects the timing of the
onset and offset of any phase k (k = 1, . . ., K) and the length of
phase change from k to k + 1, respectively. We can also test
whether the QTL controls the asymptotic growth and maximum
growth rate at a particular phase k (k = 1, . . ., K) by formulating
the null hypotheses:
H0 : ajk  ak ; Eqn 10
H0 : ajk bjk  ak bk ; Eqn 11
for j = 1, . . ., J, respectively.
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In practice, it may be interesting to test how two adjacent
phases are different in terms of growth form. These tests can be
formulated by defining the following null hypotheses:
H0 : ajðkþ1Þ  ajk  akþ1  ak ; Eqn 12
H0 : 4=bjðkþ1Þ  4=bjk  4=bðkþ1Þ  4=bk ; Eqn 13
H0 : ajðkþ1Þ bjðkþ1Þ  ajk bjk  akþ1 bkþ1  ak bk ; Eqn 14
for j = 1, . . ., J, which allows the genetic control of differences in
asymptotic growth, duration and maximum growth rate between
any two adjacent phases, k and k + 1 (k = 1, . . ., K  1).
Parameter estimates under the null hypotheses (Eqns 7–14)
can be obtained from the same algorithm derived for the alter-
native hypotheses, but with a constraint posed by each of
these null hypotheses. The test statistics for these hypothesis
tests are expressed as the LRs of the null and alternative
hypotheses. These ratios are thought of as being chi-square-
distributed with the degrees of freedom equal to the difference
in the number of parameters to be estimated under the alter-
native and null hypotheses. Alternatively, the critical thresholds
for these tests can be determined from simulation studies. By
simulating raw phenotypic data under the constraint of each
null hypothesis, the model was used to analyze such data to
obtain the LR values. The top 5% of LR values from at least
1000 simulation replicates was used as the threshold of this
test. When all these tests (Eqns 7–14) are undertaken, multiple
corrections should be made to control the family-wise error
rate.
Application
Plant materials
Because of their prolonged duration of various phases in shoot
development, woody plants have been used as a model system to
study phase change (Assmann, 1970; Greenwood, 1989; Poethig,
2010; Wang et al., 2011). We applied the new model to map
QTLs for the timing of vegetative phase change in a forest tree –
poplar. In the 1970s, eastern cottonwood (Populus deltoides), nat-
urally distributed in the central United States, was introduced to
China (Wu et al., 1992). It grows rapidly in a large range of area
from the Yangtze River to the Huang River, with a similar climate
to its native habitat. In the early 1980s, artificial hybridization was
made using a Populus deltoides clone, I-69, as the mother, and an
Euramerican poplar (P. 9 euramericana) clone, I-45, as the father,
in a hope to find superior hybrids that combine the strengths of
Table 1 Means of monthly temperature and rainfall during 1987–2010 at Xuzhou, Jiangsu, China
Jan Feb Mar Apr May
Average temperature (°C) 0.83 3.85 8.75 15.61 21.02
Average rainfall (mm) 20.63 23.08 35.41 36.39 65.17
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the mother’s fast growth and the father’s cold and drought hardi-
ness. From this hybridization, a full-sib population of 450
progeny, along with I-69, was planted in a randomized block
design with four replicates using rametes at a spacing of 6 9 6 m
in a uniform land at Zhangji Forest Farm (34.14°N, 117.38°W)
in Jiangsu Province, China, in 1987. The trial site is situated in a
monsoon-influenced humid subtropical climate. Table 1 gives the
averages of monthly temperature and monthly precipitation in
1987–2010. For some reason, another parent I-45 was planted in
a different but macroenvironmentally similar land at the same
farm, which will be used for approximate comparison.
We randomly chose 64 of these progeny as a pilot study of
genetic mapping. They, along with two parents, I-69 and I-45,
were genotyped genome-wide by single nucleotide polymor-
phisms (SNPs) using the Applied Biosystems (Foster City, CA,
USA) QuantStudio 12K Flex Real-Time PCR System. SNP
genotypes were characterized through stringent quality-control
filters. In all, a total of 156 362 SNPs were available, of which
94 591 and 61 771 belongs to testcross and intercross markers,
respectively (Lu et al., 2004). Testcross markers are those that are
segregating only one heterozygous parent, whereas the segregation
of intercross markers results from the heterozygosity of both par-
ents. Stem analysis was conducted for one tree from each progeny,
which measured annual growth data of stem height and diameter
at the stem base during the first 24 yr of growth from 1987 to
2010 (Supporting Information Fig. S1). Stem height is defined as
the length of the main stem from stem–root connection to the tip.
Although stem diameter was widely used as a measure of stem
growth in forestry, it is misleading to assess the net amount of
radial growth from year to year. For example, a tree can grow
3 cm yr1 in diameter at its juvenile stage, but may grow only
1 cm yr1 as it gets older. However, the amount of carbon accu-
mulated at older ages is much larger than that at younger ages.
For this reason, we used annual stem cross-sectional area (CSA) at
breast height as a measure of radial growth for QTL mapping.
QTL detection by functional mapping
By plotting CSA over age for each progeny, we found that radial
growth curve can be well fitted by tanh (Eqn 1) (with adjusted
R2 > 0.99; Fig. S2). Using estimated curve parameters (aik, bik,
cik), each curve was partitioned into its two underlying logistic
components, each representing a different phase of growth, juve-
nile or early adult (Fig. S3). We used the tanh to fit the mean
growth curve of all progeny (Fig. 3a), from which to identify a
juvenile–adult phase transition of stemwood radial growth. The
juvenile phase started at year 1 and ends at year 10.5  1.2,
whereas the ages of the onset and offset of the early adult phase
Jun Jul Aug Sep Oct Nov Dec
25.46 27.47 26.41 22.30 16.27 8.90 3.02
125.05 244.20 144.05 65.97 34.98 30.38 15.65
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(a) (b)

Fig. 3 Stem growth trajectories in cross-sectional area at breast height of an interspecific full-sib family of Populus during the first 24 yr in an experimental
plantation. (a) Raw growth data of 64 hybrids whose mean curve was fitted by a tanh. (b) An overall growth curve (black) is composed of two distinct
phases of growth, juvenile and early adult. Curves in the first and second phase, indicated by red and green, respectively, differ dramatically in shape. The
dot part of growth curves is the prediction of growth. The timings of onsets and offsets of each phase are indicated beneath the x-axis. The durations of
each phase are also indicated, along with the length of phase change (slash lines). The asymptotic growth of each phase is shown on the y-axis.

(a) (b)

Fig. 4 Manhattan plots of significance tests for all testcross (a) and intercross single nucleotide polymorphisms (SNPs) (b) across 19 chromosomes of the
Populus genome plus unanchored SNPs by functional mapping of 24-yr growth curves of stem cross-sectional area in a full-sib family of poplar. Red
horizontal lines are the critical thresholds at the 5% significance level obtained after Bonferroni correction. The abbreviations shown in the significant
region are those genes with known biological function.

were 8.9  0.9 and 30.6  2.1 yr, respectively. The phase change
from the juvenile to early adult occurred from age 8.9  0.7
to 10.5  0.9 yr, experiencing 1.6  0.2 yr (Fig. 3b). The juve-
nile and early adult phases reached aysmptotic growth at
379.8  41.3 and 560.9  59.1 cm2 within a duration of
9.5  0.8 and 21.7  3.2 yr, respectively, implying that the
former had a higher maximum growth rate
(39.98  4.5 cm2 yr1) than the latter (25.85  2.9 cm2 yr1).
As expected, in the same macroenvironment, clone I-45 dis-
plays smaller stem radial growth than clone I-69. There are pro-
nounced transgressive segregants towards the better parent I-69,
that is, extreme phenotypes compared with phenotypes observed
in the two parental clones (Fig. 3a). A great variability was
detected in growth curves of CSA among the progeny, suggesting
the possible existence of growth QTLs. By incorporating tanh
(Eqn 1) into the likelihood (Eqn 3), functional mapping identi-
fied 100 testcross QTLs and 118 intercross QTLs (Fig. 4). Over
two-thirds of testcross QTLs and over a half of intercross QTLs
were within or adjacent to candidate genes that have been
observed to function in known plant growth-related pathways
(Table S1). It is likely that a diverse range of genes were involved
in genetic variation for stem diameter growth occurring as a result
of the expansion of vascular cambium. Notably, most of testcross
QTLs were located on chromosomes 5 and 14 (Fig. 4a). Of these
QTLs, 75% were segregating because of the heterozygous geno-
type of mother I-69, suggesting that the I-69 allele contributes
more than the I-45 allele to variation in growth form. It appears
that intercross QTLs were distributed much more sporadically
throughout the Populus genome, although there were more
QTLs on some chromosomes than on others (Fig. 4b).
We chose five most significant testcross QTLs and five most
significant intercross QTLs, all of which are annotatable, for an
in-depth analysis (Table 2). SNP chr1/3652886 was found to
be in a region of the PKFP gene that encodes sugar (fructose)
metabolism. SNP chr14/1662924 resides in the QWRF2 gene
related to microtubule severing activity, which plays an impor-
tant role in intercellular spreading. SNP chr13/269980 was
observed to be in the region of the RING/U-box gene that
serves a range of functions from self-incompatibility and hor-
mone responses to abiotic stress responses. SNP chr5/4707454

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Table 2 Physical positions, segregation types, effect types and curve heritabilities of 10 significant single nucleotide polymorphisms (SNPs), detected by a
high-dimensional modeling framework of variable selection and functional mapping, that affect stem cross-sectional growth at stem base in Populus trees
and predicted functions or homology of adjacent candidate genes to these SNPs

Segregating Physical SNP

type SNP Line no. Chr position P-value Allele Heritability classification Protein ID Domain Annotation

Testcross 117 854 1323941 14 1662924 8.61 9 1025 C/T 15.10 Intron 18222631 PF04484 QWRF2
111 190 1249601 13 269980 2.66 9 1023 A/C 10.14 Intron 18220676 PF00096 RING/U-box
118 150 1328076 14 2428884 2.24 9 1022 A/T 1.50 Exon 18224346 PF01535 Vid27
48 726 548063 5 4707454 6.08 9 1022 C/T 2.62 Exon 18207567 PF01301 MUM2
49 080 551232 5 5314544 1.67 9 1021 G/T 1.59 Intron 18208080 PF00005 PXA1
Intercross 1799 18156 1 3652886 9.78 9 1026 C/T 6.03 Intron 18239306 PF00069 PKFP
110 720 1244495 12 14207793 2.38 9 1024 C/G 6.87 Intron 18228971 PF00999 KEA5
48 794 548688 5 4804826 3.50 9 1023 C/G 1.95 Exon 18208487 PF00571, CBS
PF00564
86 049 965518 9 8420519 1.07 9 1022 C/T 17.21 Intron 18228214 PF00400 WD-40
93 597 1051864 10 12831172 6.46 9 1022 A/G 2.78 Intron 18240595 PF11976 ULSP

is within the MUM2 gene that encodes a b-galactosidase
required for cell wall modification. Other significant SNPs can
also be similarly annotated.
For each significant QTL, we estimated genotype-dependent
growth parameters for two different phases, with which to esti-
mate the dynamic pattern of genetic effects exerted by each
locus. In general, the magnitudes of genetic effects (i.e. additive
effects for testcross QTLs and additive and dominant effects for
intercross QTLs) increased with age, although some QTLs dis-
played a larger extent of increase than others (Fig. 5a,b). Dra-
matic variation in the temporal pattern of heritability explained
by a locus was observed (Fig. 5c), which can be roughly sorted
into three types: heritabilities increase monotonously with age;
heritabilities decrease consistently with age; and heritabilities
increase rapidly and then decrease gradually, with a peak at
years 3–7.
Genetic control of phase change
The advantage of functional mapping lies in its capacity to test
whether a significant QTL determines the onset and offset of a
phase, its duration and maximum growth rate, and the length of
phase change. Genetic control of each of these phase change-
related parameters by individual QTLs can be tested per hypothe-
ses (Eqns 7–14). Here, we interpreted the results from two repre-
sentative loci, that is, testcross QTL chr14/1662924 and
intercross QTL chr1/3652886. Both loci affect the timing and
nature of phase change, but with different patterns (Fig. 6). Dur-
ing the juvenile phase, genotype TC at chr14/1662924 has a
slightly earlier onset (year 0.5 vs 1.2) but a slightly later offset
(year 11.2 vs 10.1) than genotype CC, thus the former displays
an extended period of juvenile growth than the latter (8.9 vs
10.7) (Fig. 6a). An inverse pattern was found between the two
genotypes in these parameters for the early adult phase. Ulti-
mately, genotype CC is relatively short in the length of phase
change (1.2 vs 2.0) than its counterpart TC.
Three genotypes at chr1/3652886 differ in the onset, offset
and duration of each growth phase (Fig. 6b), suggesting that this
QTL affects not only overall growth but also the timing of devel-
opment. From the two QTLs considered, it appears that none of
these parameters is consistently related with the overall growth at
age 24 yr. However, the duration of phase change seems to be

(a) (b) (c)

Fig. 5 Temporal pattern of genetic effects and heritability for stem cross-sectional area in an interspecific full-sib family of Populus. (a) Additive effects of
five chosen testcross quantitative trail loci (QTLs) (red) and five chosen intercross QTLs (blue). (b) Dominant effects of the intercross QTLs. (c) Heritability
explained by each of these chosen QTLs.

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8 Research
(a) (b)
associated with overall growth. For example, genotypes CC, TT
and CT at chr1/3652886 use 1.0, 2.6 and 4 yr to complete phase
transition, while their 24 yr overall growths are 1189.2, 866.7
and 859.9 cm2, respectively. A similar phenomenon was observed
for all other significant QTLs detected.
As expected, maximum growth rate is a determinant of overall
growth; a larger growth rate is positively associated with a larger
amount of growth. However, maximum rate of growth in differ-
ent phases may contribute differently to the overall growth,
which is under genetic control. At QTL chr14/1662924, two
genotypes differ more dramatically in maximum growth rate dur-
ing the juvenile phase than during the early adult phase (Fig. 7a).
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Fig. 6 Growth curves of stem cross-sectional
area for different genotypes at two chosen
loci, testcross quantitative trail locus (QTL)
chr14/1662924 with two genotypes, CC and
TC (a), and intercross QTL chr1/3652886
with three genotypes, CC, TT and CT (b), in
an interspecific full-sib family of Populus.
Upper panel, the fitness of genotypic mean
curves (thick line) to raw growth data (thin
line) by tanh. Middle and lower panels,
growth curves of each genotype are
dissected into juvenile (red) and early adult
phases (green). The estimates of key
parameters, including the onset, offset and
duration of each phase, are given. The length
of phase change for each genotype is
indicated by a grained region.
At QTL chr1/3652886, genotype CC and TT are closer to each
other than to the third genotype CT in the maximum growth rate
of the juvenile phase, but the pattern of such genotypic differ-
ences changes during the early adult phase (Fig. 7b). Genotypes
TT and CT are very close, but both differ dramatically from
genotype CC in the maximum growth rate of the early adult
phase.
Computer simulation
To show how the new model reveals the reality, we mimicked the
earlier example to simulate a full-sib family of 64 progeny
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(a)
(b)
Fig. 7 Curves of growth rate (cm2 yr1) of the juvenile (left) and early
adult phase (right) for two genotypes, CC and TC, at testcross quantitative
trait locus (QTL) chr14/1662924 (a) and three genotypes, CC, TT and CT,
at intercross QTL chr1/3652886 (b) in an interspecific full-sib family of
Populus. Maximum growth rates for each genotype are given.
containing 10 000 intercross markers. Phenotypic data of a
growth trait over 24 yr were simulated by time-varying genotypic
values defined by tanh (Eqn 1) plus a SAD(1)-structured covari-
ance matrix. We used the heritability at the offset point of the
juvenile stage as a surrogate for the heritability of the growth
curve. Thus, by adjusting values of innovation variance, we
obtained two different levels of curve heritability, 0.05 and 0.10.
Fig. 8 illustrates the estimated growth curves from the simulated
data, in a comparison with true curves. In general, genotype-
dependent curves can well be estimated from our current small
sample size. As expected, estimation precision increases with
increasing heritability. When sample size increases to 200, the
precision of curve estimation has been much improved even with
Fig. 8 Estimated mean curves of stem cross-sectional area for three different genotypes, AA, Aa and aa, at a simulated quantitative trait locus explaining a
curve heritability of 0.05 (slash line) or 0.10 (dot line), in a comparison with the true curves (solid line).
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Research 9
a small heritability. We also estimated the empirical power of
detecting a growth QTL based on 1000 simulation replicates. It
can be seen that such power is quite high (> 0.85) even with a
small sample size when curve heritability is modest. With increas-
ing heritability, the power of QTL detection increases dramati-
cally.
Discussion
As an intrinsic mechanism for an organism to approach its opti-
mal survival and reproduction, the time at which the organism
decides to change developmental phase is regulated by complex
interactions between endogenous and environmental factors,
such as transcriptional expression, nutrition, temperature, or
photoperiod (Poethig, 2003; Rougvie, 2005; Baurle & Dean,
2006; Huijser & Schmid, 2011; Yang et al., 2013). Classic quan-
titative genetic studies and those based on genetic mutations sug-
gested that genes may play a pivotal role in many developmental
transitions (Poethig, 1988; Wiltshire et al., 1998; Hudson et al.,
2014). Our work extends these classical studies by reforming a
genetic mapping approach – functional mapping – to identify
specific QTLs involved in the timing and nature of phase change.
The uniqueness of this model has two folds. First, despite its per-
vasion in every aspect of biological processes, continuous phase
transition cannot be observed easily from its subtle change from
time to time. The new model is equipped with a capacity to iden-
tify the timing of continuous phase change based on mathemati-
cal equations of biological relevance and quantify the temporal
regulation of QTLs on when and how a phase change takes place
gradually.
Second, the new model allows a genome-wide search for the
distribution of all possible phase change QTLs by incorporating
it into a genome-wide association study (GWAS) setting. GWAS
has emerged as a routine approach to illustrate a comprehensive
picture of the genetic control mechanisms underlying complex
traits or diseases (http://www.genome.gov/gwastudies/). By mea-
suring phenotypic values of complex traits repeatedly during a
time course, GWAS may produce an important by-product, that
is, the identification of phase change QTLs, by using our
reformed functional mapping model. Specifically, these QTLs
determine the timing of development from one phase to next,
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10 Research
the length of times between developmental landmarks of differ-
ent phases, and differences in relative growth rate, the time of
maximum growth rate, the time of maximum acceleration, the
time of maximum deceleration, and the duration of linear growth
between different phases (Sun et al., 2014). Genetic variants that
drive phenotypic variation in phase change are integrated with
recent discoveries of regulatory genes and pathways related to this
phenomenon (Wu et al., 2009; Wang et al., 2011; Yang et al.,
2013), which will certainly lead to a clear understanding of how
the organism changes its growth and developmental form to
adapt its environment.
Although different phases present somewhat distinct changes
in organ morphology and growth form, a modification of the
fundamental pattern of organ development in one phase can be
imposed on the organ development of other phases, which makes
successive phases change in a graded fashion (Poethig, 1990).
The regulatory mechanism underlying one phase interacts with
those of other phases in ways that can be mediated by a multitude
of factors. It remains to be determined if these key factors are
implemented in the model. In flowering plants, a group of plant-
specific transcription factors, such as SBP/SPL proteins, have
been observed to participate in the formation of vegetative phase
change through the regulation of miR156 and miR157 during
the juvenile phase (Wu et al., 2009; Yamaguchi et al., 2009).
Thus, the construction of a regulatory network from these
miRNAs to SBP/SPL is crucial for understanding the mechanistic
causes of phase change, and, furthermore, the integration of such
a network into functional mapping (Wu & Lin, 2006) can strik-
ingly advance the level of such an understanding by revealing the
organization and function of the pathways constituting the net-
work from so-called expression QTLs and protein QTLs and
their interactions.
Our model provides a tool to characterize the molecular basis
for variation in the sequential expression of juvenile and adult
forms of growth. The model was used to analyze real data from
the genome-wide genetic mapping study of growth curves in a
woody plant, poplar. It has been shown to perform much better
in inferring a comprehensive view of genetic architecture than
traditional static regression analysis. Of the 15 significant QTLs
detected, a large portion can be annotated to function-known
candidate genes for regulating plant growth, development and
adaptation (Table 2), suggesting the biological relevance of our
model. All these loci are subject to further analysis in order to
investigate their impact on the timing and nature of phase change
in stem diameter growth of poplar. Through a detailed testing
procedure, as shown in Eqns 7–14, we have illustrated a clear pic-
ture of the pattern of genetic control by each of these QTLs over
the onset and offset of a particular phase, the timing and length
of phase change, and growth and maximum growth rate in each
phase. Such information has not been made available before the
development of our model, but it can clearly gain additional
insight into the genetic architecture of growth toward the poten-
tial increase of tree breeding.
There is no doubt that the results from our model can help to
explore the genetic mechanisms that cause the evolution of phase
change (and therefore developmental pattern) when the model is
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extended to jointly map phase change QTLs in multiple related
taxa. Sun et al. (2014) has for the first time integrated functional
mapping into a phylogenetic study to characterize specific QTLs
for heterochronic variation. Analogously, such an extension can
be straightforward for the current model to map phylogenetic
QTLs for phase change. The computer code used in this study is
available at http://ccb.bjfu.edu.cn/program.html.
Acknowledgements
We thank Cong Xu for drawing Fig. 1. This research was sup-
ported by the ‘Twelfth Five-Year’ National Science and Technol-
ogy Support Program (2012BAD01B03), National Basic
Research Program of China (2009CB119100), the China Post-
doctoral Science Foundation Grant (2014T70530), Special Fund
for Forest Scientific Research in the Public Welfare (201404102),
Changjiang Scholars Award, and ‘Thousand-person Plan’ Award.
Author contributions
M.X. collected the data. L.J. performed data analysis and com-
puter simulation. S.Z. collected the data. C.Z. performed stem
analysis. M.Y. participated in data analysis. K.M. interpreted the
results. L.S. interpreted the data and participated in the design of
data analysis. X.S., H.P., S.Z. and M.H. produced the poplar
population. R.W. conceived of the idea, designed the model and
wrote the manuscript.
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Supporting Information
Additional supporting information may be found in the online
version of this article.
Fig. S1 The goodness of fit of tanh to individual tree growth.
Fig. S2 Overall growth composed of juvenile and early-mature
phases for individual trees.
Fig. S3 Overall growth equation that is partitioned into two
phases, juvenile and early mature, for stem cross-sectional area
during the first 24 yr of growth for a tree from each progeny of
fullsib family derived from Populus deltoides clone, I-69, as the
mother, and an Euramerican poplar (P. 9 euramerican’s) clone,
I-45, as the father.
Table S1 Gene annotations of significant SNPs detected by the
new model
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directed to the New Phytologist Central Office.
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