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crop science, vol. 55, m ay–june 2015 www.crops.org 1
RESEARCH
Miscant hus is a perennial crop that can be cultivated for up
to 25 yr (Lewandowski et al., 2003b). Its biomass is pro-
duced in two phases: a yield-building phase, where the biomass
production gradually increases, and a plateau phase, where the
biomass production is maintained (Clifton-Brown et al., 2001a;
Clifton-Brown et al., 2008; Zub and Brancourt-Hulmel, 2010).
The plateau phase of biomass production, which corresponds
to the adult phase, can be attained at various ages according to
Clifton-Brown et al. (2008) and Miguez et al. (2008). Miscanthus
giganteus, the species most studied in long-term experiments,
can take up to 5 yr to reach the plateau phase of biomass produc-
tion depending on the environmental conditions and crop man-
agement practices.
Miscanthus biomass production is a complex trait involving
earliness and morphological traits such as canopy height, number
of stems per plant, and stem diameter (Robson et al., 2013; Zub
et al., 2011). During the yield-building phase, the canopy height,
stem number, and stem diameter, to a lesser extent, increase
Early Prediction of Miscanthus Biomass
Production and Composition Based on
the First Six Years of Cultivation
Stéphanie Arnoult,* Marie-Chantal Mansard, and Maryse Brancourt-Hulmel
ABSTRACT
Miscanthus is a promising feedstock for second-
generation bioethanol production. This perennial
crop produces its biomass in two phases: a yield-
building phase, where the biomass production
increases gradually, and a plateau phase, where
it is maintained. However, to target the breeding
of Miscanthus for second-generation bioethanol
production, the early selection of interesting traits
is critical. We therefore investigated the interan-
nual correlations within and among the traits
related to biomass production and composition.
We studied 21 clones belonging to M. gigan-
teus J. M. Greef & Deuter ex Hodk. & Renvoize,
M. sacchariflorus (Maxim.) Benth. & Hook. f. ex
Franch., and M. sinensis Andersson species cul-
tivated on plots from the second to the sixth year
at two harvest dates. The biomass production,
canopy height, plant stem number, and aboveg-
round plant volume index were better predicted
from the third year than from the second year
(minimum correlation coefcients of 0.76 and
0.67 respectively). The stem diameter was well
predicted from the second year (correlations
above 0.93). The canopy height and the aboveg-
round plant volume index determined in the sec-
ond and third year were the best predictors of
the biomass produced in the second, third, and
fourth year (minimum correlations of 0.77 against
0.52 for owering date or 0.64 for stem diameter).
For older crops, the canopy height measured in
the second and third year was the best predic-
tor of the biomass production (correlations above
0.70). The interannual correlations were lower for
the biomass composition-related traits than for
the production-related traits and uctuated over
time. These results showed that early prediction
of interesting traits is feasible to breed varieties
tailored for biofuel production.
S. Arnoult, INRA, UMR1281 SADV, 2 Chaussée Brunehaut, Estrées-
Mons, BP 50136, F-80203 Péronne Cedex, France; S. Arnoult (cur-
rent address), and M.C. Mansard, INRA, UE0972 GCIE Picardie,
2 Chaussée Brunehaut, Estrées-Mons, BP 50136, F-80203 Péronne
Cedex, France; M. Brancourt-Hulmel, INRA, UR1158 AgroImpact,
Site d’Estrées-Mons, 2 Chaussée Brunehaut, Estrées-Mons, BP 50136,
F-80203 Péronne Cedex, France. Received 14 July 2014. Accepted 24
Nov. 2014. *Corresponding author (Stephanie.Arnoult@mons.inra.fr).
Abbreviations: ADF, acid detergent ber; ADL, acid detergent lignin;
DM, dry matter; NDF, neutral detergent ber.
Published in Crop Sci. 55:1–13 (2015).
doi: 10.2135/cropsci2014.07.0493
© Crop Science Societ y of America | 5585 Guilford Rd., Madison, WI 53711 USA
All rig hts reserved. No part of this period ical may be reproduced or tra nsm itted in any
form or by a ny means, electronic or mechanical, includ ing photocopying, recording,
or any information storage and retr ieval system, without permission in wr iting from
the publisher. Per mission for printing and for reprinting the material contai ned herein
has been obta ined by the publisher.
2 www.c rops .org crop scie nce, v ol. 55, may–june 2015
gradually, similar to the biomass production ( Jezowski et
al., 2011; Zub et al., 2011). In addition to the eect of
the year of cultivation, the biomass production, ower-
ing date, canopy height, stem number, and stem diameter
vary according to the clone and the harvest date (Clif-
ton-Brown and Lewandowski, 2002; Gauder et al., 2012;
Larsen et al., 2014; Lewandowski and Kicherer, 1997).
Furthermore, biomass production is highly and positively
correlated with the owering date, canopy height, and
stem diameter (Zub et al., 2011). In addition, the aboveg-
round volume of the plant has been identied as a reliable
predictor of biomass production for the second and the
third years of cultivation (Zub et al., 2012a).
Moreover, the traits related to the biomass composi-
tion are more consistent throughout the years of cultivation
than the traits related to the biomass production (Allison et
al., 2011). The traits related to the biomass composition are
inuenced mainly by the harvest date and the Miscanthus
clone (Allison et al., 2011; Qin et al., 2012). The aboveg-
round biomass production is highly and positively associ-
ated with cellulose and lignin contents. It is negatively asso-
ciated with the contents of hemicellulose, soluble, and ash.
The second-generation bioethanol production indus-
try needs crops displaying high aboveground biomass pro-
duction as well as high cellulose and hemicellulose con-
tents and low lignin, soluble, and ash contents (van der
Weijde et al., 2013; Dumas and Navard, personal com-
munication, 2014).
To breed Miscanthus clones that are tailored for sec-
ond-generation bioethanol production through an e-
cient breeding strategy, the early selection of traits that
are related to the biomass production and composition in
the rst few years of cultivation is essential for this peren-
nial crop, that is, without having to wait for the adult
phase. For this purpose, biomass production and composi-
tion predictions are needed. Moreover, as a complex trait,
biomass production needs to be predicted with reliability
using nondestructive and easy-to-measure traits.
We hypothesized that (i) the traits that are related to
the biomass production and composition of a mature crop
can be predicted in the early stages, and (ii) an index com-
bining several simple traits can improve the reliability of
the predicted biomass production.
Therefore, for breeding purpose, the aims of this study
were (i) to investigate the potential to predict the traits of
interest that are related to biomass production and compo-
sition in the early stages of the crop and (ii) to determine
reliable predictors using simple traits or an index to esti-
mate biomass production.
To meet these objectives, we studied 21 Miscanthus
clones that were cultivated for ve consecutive years
with two harvest dates (autumn and winter har vests).
These two harvest dates are potentially interesting for
diverse end uses. We studied the interannual correlations
involving the biomass production, the traits related to bio-
mass production (i.e., the owering date, canopy height,
number of stems per plant, and stem diameter), and the
traits related to the biomass composition (i.e., the cellu-
lose, hemicellulose, lignin, soluble, and ash contents in the
aboveground biomass). This paper will provide guidelines
for the early selection of Miscanthus clones that are tailored
for second-generation bioethanol production.
MATERIALS AND METHODS
Experimental Site and Climatic Conditions
The experimental site is located in the Picardie region of
Northern France (4953 N, 300 E) at the French National
Institute for Agricultural Research (IN RA) experimental unit
in Estrées-Mons, France. The soil is a deep loam soil (Ortic
Luvisol, FAO classication) and the climate is oceanic. The
rainfall and temperature data were collected for the duration of
the trial by a local meteorological station located at a distance
of 1 km from the experimental site.
Experimental Design
The experiment was designed as a randomized complete block
comprised of six blocks, each containing 21 clones. The trial
contained a total of 126 plots, each plot measuring 16 m2.
Among the 21 Miscanthus clones planted, four were identied
as M. giganteus clones, 15 as M. sinensis clones, and two as M.
sacchariorus clones (Table 1). Among the four M. giganteus
clones, H8 was considered as a M. giganteus clone as it was
a hybrid between M. sacchariorus and M. sinensis. The clone
named Flo was identied as belonging to the M. giganteus
species using nuclear DNA analysis with amplied fragment
length polymorphism markers (Rambaud, personal communi-
cation, 2013) and cpDNA analysis (Feng et al., 2014).
In addition to the various clones, the harvest date was
studied in this experiment. Two harvest dates were tested: an
autumn harvest and a winter harvest. The autumn harvest was
performed at the end of the growing period according to the
earliness of each clone, that is, 200 degree-days after the ow-
ering time of each clone. The winter harvest was performed at
the end of winter at overmaturit y (in February). This harvest
date appeared to be more suitable for second-generation bio-
ethanol production (Cadoux et al., 2014).
Within the six blocks, the autumn harvest was consistently
assigned to the same three blocks and the winter harvest was
always performed on the three remaining blocks.
Each 16-m2 plot consisted of four rows of eight plants. A
border row that surrounded the plots was planted with the same
clone, that is, M. sinensis ‘M a le par t us’.
Trial Management
The trial was planted by hand in spring 2007 at a density of two
plants per square meter. The plants were watered immediately
after planting to ensure good root contact with the soil. No
irrigation was applied during the following years of cultivation.
No fertilizer was applied during the entire experiment. The
weeds were controlled by hand during the two rst years and
by machine hoeing for the subsequent years.
crop science, vol. 55, may–jun e 2015 www.crops.org 3
sample of approximately 500 g of fresh matter was randomly
selected from each plot and weighed. Each sample was dried at
65C for 4 d in a well-ventilated oven. The moisture content
of each sample was determined and used to calculate the dry
weight and moisture content of the total plot biomass. The dry
weight (referred to as the aboveground biomass production) was
expressed as tons of dr y matter (DM) per hectare (t DM ha−1).
Biomass Components
The canopy height, stem number per plant, and stem diameter
were recorded in each plot before each har vest. The canopy
height corresponded to the distance from the ground to the
ligule of the last ligulated leaf. In each plot, the canopy height
and the stem number per plant were measured on three median
plants. These three plants were chosen as they reected the
median values of the canopy height and the stem number per
plant. These median values were based on the measures of all
the plants of each plot. The average canopy height and stem
number per plant were calculated from these three individual
values. The stem diameter was measured at a height of 5 cm
above the ground on 12 randomly selected stems per plot and
the average stem diameter was calculated.
Calculation of the Aboveground Plant
Volume Index
The aboveground plant volume index (expressed in cm3) was
calculated as a function of the canopy height, stem number per
plant, and stem diameter according to Zub et al. (2012a):
Measurements and Calculations
The owering date and the aboveground biomass production and
its components (i.e., canopy height, stem number per plant, and
stem diameter) were measured each year from the second to the
sixth year after planting (2008–2012) and for each harvest date.
Flowering Date
The owering date corresponded to the date on which 50% of
the plants in each plot had their owers open and when anthers
were rst extruded. This date was recorded for each plot by
counting the number of plants for which at least one single
ower was open. This count was performed each year during
July, August, September, and October, corresponding to the
owering period as a function of the earliness of the clones. The
record was performed every 2 d. Some clones (Flo, GiB, GiD,
and H5) did not ower in the Picardie region because the ow-
ering stage was not reached. These clones were of particular
interest in our study because of their high aboveground biomass
production. Therefore, in the absence of visible owering, the
owering date was recorded as the rst frost day of each year so
all clones could be included in the analysis. The rst frost day
was based on the average daily temperature below 0C. The
owering date was expressed in degree-days using the same
base temperature of 10C as Jensen et al. (2013).
Aboveground Biomass Production
Each plot was harvested using a reed harvester. The plants were
cut at a height of approximately 5 cm above the ground. The
total aboveground fresh matter of each plot was weighed. A
Table 1. Description of the 21 Miscanthus clones studied in the experiment, including the species, ploidy level, name, code,
and acquisition.
Species Name Code Ploidy level†Acquired from
M. giganteus M. giganteus UK GiB 3xUnited Kingdom, ADAS
M. giganteus DK GiD 4xDenmark, Nordic Biomass
M. giganteus Floridulus Flo‡3x France, Nursery Chombart
M. sacchariflorus x M. sinensis Hybrid 8 H8§2xDanish Institute Of Agricultural Science, Aarhus
M. sacchariflorus M. sacchariflorus Sac 2xFrance, Nursery Chombart
M. sacchariflorus Hybrid 5 H5§4xDanish Institute Of Agricultural Science, Aarhus
M. sinensis M. sinensis August Feder Aug 2xFrance, Nursery Chombart
M. sinensis Ferner Osten Fer 2xBelgium, Nursery Bruckeveld
M. sinensis Flamingo Fla 2xFrance, Nursery Chombart
M. sinensis Goliath Gol 4xFrance, Nursery Chombart
M. sinensis Goliath DK GoD 4xDenmark, Nordic Biomass
M. sinensis Graziella Grz 2xFrance, Nursery Chombart
M. sinensis Herman Müssel Her 2xBelgium, Nursery Bruckeveld
M. sinensis Hybrid 6 H6§4x ¶Danish Institute Of Agricultural Science, Aarhus
M. sinensis Malepartus Mal 2xFrance, Nursery Chombart
M. sinensis Punktchen Pun 2xBelgium, Nursery Bruckeveld
M. sinensis Purpurescens Pur 2xBelgium, Nursery Bruckeveld
M. sinensis Rotsilber Rot 2xFrance, Nursery Chombart
M. sinensis Silberspinne Sil 2xFrance, Nursery Chombart
M. sinensis Strictus Str 2xFrance, Nursery Chombart
M. sinensis Yak u Ji m a Yak 2xBelgium, Nursery Bruckeveld
† From Zub et al. (2012b).
‡ The clon e named “Flo” was determined as a clone be longin g to the M. giganteus species (Feng et al., 2014; Rambaud, personal communication, 2013).
§ Clones p revious ly studied by Clif ton-Brown et al. (2001b) and Lewand owski et al. (2003a).
¶ The H6 clo ne was determined a s a tetraplo id clone (4x) (Rambaud, personal communication, 2013).
4 www.c rops .org crop scie nce, v ol. 55, may–june 2015
Aboveground plant
volume index = canopy height
stem number per plant
(stem diameter/2)2
Chemical Analyses
of the Biomass Composition
The biomass composition of the total aboveground biomass was
determined using chemical analyses from samples harvested in
the third, fourth, and fth years (i.e., 2009–2011).
Sample Preparation
Dried aboveground biomass samples were coarsely ground
using a crusher mill (Viking, GE 220 model) and then ground
using a hammer crusher (Gondard Productions) to pass through
a 1-mm screen as recommended for subsequent ber analysis by
Van Soest and Wine (1967).
Determination of the Cellulose, Hemicellulose,
Lignin, and Soluble Contents
The ground samples were analyzed by the LANO laboratory
(Laboratoire Agronomique de Normandie, France) for soluble,
neutral detergent ber (NDF), acid detergent ber (ADF), and
acid detergent lignin (ADL) according to a protocol adapted
from the Van Soest method (Van Soest and Wine, 1967).
Briey, the NDF fraction corresponded to the ash-corrected
residue remaining after reuxing for 60 min in a neutral-bu-
ered detergent solution. The ADF fraction corresponded to the
ash-corrected residue remaining after reuxing the samples in a
solution of hexadecyltrimethylammonium bromide in 0.5 mol
L−1 sulfuric acid. The ADL fraction was obtained by treating
the ADF with 72% sulfuric acid.
The cellulose, hemicellulose, and lignin contents of each
sample were calculated by subtracting the corresponding values
from the NDF, ADF, and ADL fractions (expressed as % DM):
(i) the NDF consists of cellulose, hemicelluloses, and lignins;
(ii) the ADF consists of cellulose and lignin, and (iii) the ADL
consists of lignin. The cellulose, hemicellulose, and lignin con-
tents of each sample were calculated according to Eq. [1–3]:
Cellulose content (cellulose) = ADF − ADL [1]
Hemicellulose content
(hemicellulose) = NDF − ADF [2]
Lignin content (lignin) = ADL [3]
The soluble content (soluble) was expressed as percentage
dry matter (% DM), calculated according to Eq. [4], which cor-
responds to the NDF soluble fraction as follows:
Soluble = 100 − NDF [4]
Determination of the Ash Content
The ash content was expressed as percentage dry matter (%
DM) and corresponds to the dry matter remaining after calci-
nation of the samples at 550C for 12 h.
Statistical Analysis
The experimental dataset was analyzed through correlations
between the traits using the Pearson correlation coecients of
the CORR procedure (SAS Institute Inc., 2000). In addition,
simple linear regression was performed for the analysis of the
aboveground biomass production, the log (aboveground plant
volume index), and the cellulose content.
For the traits related to aboveground biomass production,
we used 19 clones corresponding to the 21 clones that were
used in our experiment except for clones H5 and Sac, for which
data were not available for the stem number, stem diameter,
and aboveground volume. For the traits that are related to the
biomass composition, we used 19 clones corresponding to the
21 clones that were used in our experiment except for clones
Her and Pun, for which data were not available.
We calculated the correlations for each year and each har-
vest date separately, considering each of the three blocks.
RESULTS
Evolution of Biomass Production-
and Composition-Related Traits
The average values for the aboveground biomass produc-
tion increased gradually from the second to the fourth year
of cultivation, and subsequently, were more consistent up
to the sixth year (Table 2a). Additionally, the average values
for the aboveground biomass production from the winter
harvest of the fourth year were particularly high (Table 2a);
this was attributed to more favorable climatic conditions in
this year (data not shown). A peak in the biomass produc-
tion was clearly visible for the two harvest dates during
the fourth year. It appeared that the biomass production
plateau was attained in this year in the autumn harvest,
whereas the winter yields were more variable (Fig. 1).
In addition, the range in the clonal variation for the
aboveground biomass production increased with the
number of years of cultivation for each harvest date; the
minimum values were especially consistent for this trait,
while the maximum values increased with the number of
years of cultivation (Table 2a).
The canopy height, stem number per plant, and aboveg-
round plant volume index followed the same trend as the
aboveground biomass production over the years of cultiva-
tion (Table 2a). In contrast, the average of the stem diameter
and owering date were consistent over time (Table 2a).
Regarding the clonal variation, the observation of the
minimum and maximum values showed that the range in
the canopy height, stem number per plant, and aboveg-
round plant volume index increased with the number
of years of cultivation for each harvest date (Table 2a).
However, the ranges corresponding to the owering date
crop science, vol. 55, may–jun e 2015 www.crops.org 5
Table 2. Description of the dataset of the Miscanthus clones grown in northern France and harvested on two harvest dates
(autumn and winter) from the second to the sixth year of cultivation for the traits related to the biomass production (a) and
composition (b).
(a)
Variable
(Unit)
(Number of
clones)
Year of
cultivation 2 3 4 5 6
Harvest
date Autumn Winter Autumn Winter Autumn Winter Autumn Winter Autumn Winter
Aboveground
biomass
production
(t DM† ha−1)
(19 clones)
Mean SE§2.7 0.3 2.2 0.2 8.0 0.9 8.7 0.7 14. 2 1.2 24.7 1. 8 12. 8 1.3 14. 5 1.1 14.4 1. 3 17. 3 1. 2
Min 0.1 0 .1 0.3 0.6 1.2 4.7 0.9 3.4 0.0 2.4
Max 10.6 5.8 22.7 1 7. 2 36.4 52.0 44.9 32.4 38.2 36.9
Flowering date
(DD‡ ba se 10)
(19 clones)
Mean SE 864 17 NA¶934 21 888 25 888 16 891 17 880 23 869 25 904 14 886 14
Min 535 NA 635 639 724 726 603 586 718 74 4
Max 1061 NA 118 7 118 7 108 9 1089 12 08 12 08 103 8 103 8
Canopy
height (cm)
(19 clones)
Mean SE 96 4 100 5 130 7 144 8 148 8 161 9 132 9 156 10 15 5 10 180 8
Min 45 36 58 68 72 85 53 72 77 90
Max 190 166 229 24 3 273 292 295 310 320 328
Stem number
per plant
(19 clones)
Mean SE 23 2 19 2 49 5 57 6 118 13 109 10 10 9 12 104 10 NA NA
Min 6 4 13 11 36 28 41 29 NA NA
Max 73 90 198 208 425 330 410 347 NA NA
Stem diameter
(mm)
(19 clones)
Mean SE NA 5.8 0.3 5.0 0.3 5.3 0.3 5.2 0.3 5.6 0.3 5.1 0.3 6.0 0.4 5.4 0.3 5.9 0.4
Min NA 2.8 1.4 2.6 2.3 2.5 2. 2 3.0 2.1 2.5
Max NA 9.5 10.0 9.8 10.3 10.0 10.7 12.0 10 .9 11.7
Aboveground
plant volume
index
(cm3)
(19 clones)
Mean SE NA 588 72 1291 181 1784 212 32 51 409 3792 366 2758 408 4221 575 NA NA
Min NA 14 30 161 656 656 308 669 NA NA
Max NA 195 5 6 416 6 612 13 86 1 9910 1175 2 19 605 NA NA
† DM, dry matter.
‡ DD, degree-days.
§ SE, standa rd error.
¶ NA, data not available.
Table 2. Continued.
(b)
Variable
(Unit)
(Number of clones)
Year of
cultivation 345
Harvest date Autumn Winter Autumn Winter Autumn Winter
Cellulose
(% DM†)
(19 clones)
Mean SE‡35.7 0.3 43.4 0.5 35.8 0.4 42.3 0.4 34.3 0.3 41.0 0.5
Min 31.3 34.2 26.2 3 7.1 30.3 33.6
Max 40.6 49.5 41. 4 4 8 .1 39.3 48.0
Hemicelluloses
(% DM)
(19 clones)
Mean SE 34.2 0.3 35.4 0.5 31.9 0.3 33.9 0.4 35.6 0.3 36.2 0.4
Min 29.4 2 7.7 26.6 2 7. 3 31.5 29.8
Max 38.4 40.4 35.9 39.2 39.3 40.6
Lignins
(% DM)
(19 clones)
Mean SE 5.3 0.2 7.7 0.3 5.5 0.2 7. 9 0.2 4.0 0.2 5.8 0.2
Min 3.5 4.0 3.0 5.7 2.2 4.2
Max 9.2 12 .1 9.1 11. 5 6.3 8.4
Solubles
(% DM)
(19 clones)
Mean SE 24. 8 0.3 13.6 0.3 26.9 0.4 16 .0 0.3 26.2 0.2 17.0 0.3
Min 19.8 9.8 21.9 11. 6 23.0 13. 2
Max 29.5 20.6 35.3 2 1.9 29.9 25.8
Ash
(% DM)
(19 clones)
Mean SE 4.3 0 .1 3.0 0.1 4.7 0.1 3.4 0 .1 5.0 0 .1 3.5 0.2
Min 1.8 1. 2 2.6 1. 5 2.8 1. 5
Max 5.9 5.5 6.2 5.2 6.4 10 .3
† DM, dry matter.
‡ SE, standa rd error.
6 www.c rops .org crop scie nce, v ol. 55, may–june 2015
and stem diameter remained relatively consistent over the
years of cultivation (Table 2a).
The average values of the traits related to the biomass
composition were more consistent than the aboveground
biomass production-related traits; for each harvest date,
the average values of the cellulose, hemicellulose, lignin,
soluble, and ash contents in the total aboveground biomass
were consistent from the third to the fth year of cultivation
(Table 2b). In contrast with the traits related to the biomass
production, the range in the clonal variation for all the traits
related to biomass composition were consistent throughout
the years of cultivation for each harvest date (Table 2b).
Interannual Correlations for Aboveground
Biomass Production and Related Traits
The aboveground biomass production in the winter har-
vest was highly and signicantly correlated between the
years of cultivation, with correlation coecients ranging
from 0.69 to 0.95 (Table 3a). Interestingly, the aboveg-
round biomass production was highly and signicantly
correlated between two subsequent years (in the diago-
nal of the matrix), with correlation coecients ranging
from 0.87 to 0.95 (Table 3a). In contrast, the correlations
between nonsequential years were lower and a mini-
mum correlation coecient of 0.69 was observed (Table
3a). In addition, the correlation coecients gradually
decreased when the interval between the years increased;
the aboveground biomass harvested in the sixth year was
highly correlated with the aboveground biomass produced
in the fourth and fth year (correlation coecients above
0.85), whereas it was less correlated with the aboveground
biomass produced in the second and third year (Table 3a).
Interestingly, the relative contribution of the indi-
vidual genotypes was similar throughout the years for
these correlations as illustrated in Fig. 2. In particular, the
clones Flo, GiB, and GiD, which clearly stood out on the
plots, maintained their relative contribution (Fig. 2).
Regarding the traits related to the biomass produc-
tion, they were always correlated between the years of
cultivation, with correlation coecients ranging from
0.59 to 0.99 in the winter harvest (Tables 3b–f ). The
owering date generally displayed the highest correlation
coecients between years followed by the stem diame-
ter, canopy height, aboveground plant volume index, and
stem number per plant (Tables 3b–f ).
Nevertheless, these traits were more or less correlated
depending on the interval between the years under con-
sideration. The correlation coecients decreased when the
interval between the years increased for the canopy height
and stem number per plant (Tables 3c, e). In contrast, the
owering date and stem diameter displayed consistent
Table 3. Interannual correlations between the second, third,
fourth, fifth, and sixth years of cultivation in the winter harvest
for (a) the aboveground biomass production and its related
traits: (b) the flowering date, (c) canopy height, (d) stem diam-
eter, (e) stem number per plant, and (f) aboveground plant
volume index of 19 Miscanthus clones. All correlation coef-
ficient values significant at the 0.05 probability level.
Pearson correlation coefficient
Yea r 2 Year 3 Yea r 4 Yea r 5 Year 6
(a) Aboveground biomass production (t DM† ha−1)
Yea r 2 0.88 0.78 0.72 0.69
Yea r 3 0.87 0.81 0.76
Yea r 4 0.90 0.85
Yea r 5 0.95
(b) Flowering date (DD‡ base 10)
Yea r 2 NA§NA NA NA
Yea r 3 0.99 0.97 0.96
Yea r 4 0.98 0.96
Yea r 5 0.94
(c) Canopy height (cm)
Yea r 2 0.90 0.79 0.72 0.67
Yea r 3 0.95 0.91 0.83
Yea r 4 0.97 0.93
Yea r 5 0.93
(d) Stem diameter (mm)
Yea r 2 0.94 0.95 0.94 0.93
Yea r 3 0.93 0.93 0.95
Yea r 4 0.93 0.94
Yea r 5 0.96
(e) Stem number per plant
Yea r 2 0.84 0.68 0.59 NA
Yea r 3 0.92 0.86 NA
Yea r 4 0.89 NA
Yea r 5 NA
(f) Aboveground plant volume index (cm3)
Yea r 2 0.80 0.70 0.65 NA
Yea r 3 0.79 0.88 NA
Yea r 4 0.67 NA
Yea r 5 NA
† DM, dry matter.
‡ DD, degree-days.
§ NA, data not available.
Figure 1. Average aboveground biomass production of 19 Miscan-
thus clones har vested in autumn and winter of the second to the
sixth year of cultivation. The errors bars represent the standard error.
crop science, vol. 55, may–jun e 2015 www.crops.org 7
correlation coecients between the years of cultivation
(Tables 3b, d). Lastly, the coecients were more variable
for the aboveground plant volume index (Table 3f).
All these interannual correlations observed in the
winter harvest for the aboveground biomass production
and its related traits were similar to those observed in the
autumn harvest (Supplemental Table S1a–f ). In addition,
these correlations showed a certain predictive capabil-
ity, which was assessed by a cross-validation. Compar-
ing the correlations obtained from one block of the trial
to the correlations of the two remaining blocks, a high
consistency of the correlations was found between the two
groups (Supplemental Table S2).
These results indicate that the aboveground biomass
production and its related traits observed in the sixth year,
which is considered as an adult age of the crop, were better
predicted from the third year than from the second year.
Correlations Between Autumn and Winter
Harvest Dates for Aboveground Biomass
Production and Related Traits
The aboveground biomass production and its related traits
were signicantly correlated between the autumn and
winter harvests, with correlation coecients ranging from
0.52 to 0.99 (Tables 4a–f ). The stem diameter and owering
date generally displayed the highest correlation coecients
between the harvest dates; the coecients varied from 0.84
to 0.99. These traits were followed by the canopy height,
aboveground biomass production, aboveground plant
volume index, and stem number per plant (Tables 4a–f ).
The correlation coecients between the second and
other years were generally lower than those from the third
year (Tables 4a–f ).
These results indicate that the aboveground biomass
production and its related traits were well correlated
between the autumn and winter harvests, especially from
the third year of growth.
Reliable Predictors of Adult Aboveground
Biomass Production
The aboveground biomass production was highly and
positively correlated with the owering date, canopy
height, stem diameter, and aboveground plant volume
index, with correlation coecients varying from 0.52
to 0.94 in the winter harvest (Table 5). In contrast, the
correlations between the aboveground biomass produc-
tion and the stem number per plant were nonsignicant
or weakly negative (Table 5). Nevertheless, the values of
these coecients varied over time. The correlations of the
aboveground biomass production with the canopy height
and aboveground plant volume index decreased from the
second to the sixth year. In contrast, the correlations with
the owering date and stem diameter increased (Table 5).
As the aboveground biomass production was highly
correlated with the canopy height and aboveground plant
volume index, they appeared to be the best predictors of
the aboveground biomass production (Table 5). Neverthe-
less, the log (aboveground plant volume index) increased
the correlation coecients with the aboveground bio-
mass production mainly during the rst years (Fig. 3a–h).
Therefore, the canopy height and the log (aboveground
plant volume index) were the best predictors of the
aboveground biomass production.
The correlations between these two best predic-
tors and the aboveground biomass produced from the
Figure 2. Contribution of the individual Miscanthus clones to the
interannual correlations between (a) the third and fourth years
of cultivation, (b) the third and fifth years, and (c) the third and
sixth years for the aboveground biomass production in the winter
harvest. A total of 19 Miscanthus clones were used for these plots.
The filled squares represent the M. giganteus clones, and the
filled triangles represent the M. sinensis clones. For details on the
clones, see Table 1. An asterisk represents significant correlations
at the 0.05 probability level; r, Pearson correlation coefficient.
8 www.c rops .org crop scie nce, v ol. 55, may–june 2015
second to the sixth year were higher for the predictors
in the third and fourth years compared with the second
year (Table 5). The aboveground biomass produced in
the second to the fourth year was slightly more correlated
with the log (aboveground plant volume index) than with
the canopy height observed in the second or third year
(Table 5). The correlations between the canopy height and
the aboveground biomass produced in the fth and sixth
years were slightly higher than the log (aboveground plant
volume index) (Table 5). Similar trends were observed in
the autumn harvest (Supplemental Table S3).
Interannual Correlations for Biomass
Composition-Related Traits
The correlation coecients between the years of culti-
vation were positive and signicant for all the biomass
composition traits in the winter harvest (Tables 6a–e).
However, the correlation coecients ranged from 0.38 to
0.85 and were generally lower than those obtained for the
aboveground biomass production and its related traits.
The cellulose, hemicellulose, and lignin contents in
the aboveground biomass displayed generally high inter-
annual correlation coecients, with values ranging from
0.69 to 0.85 (Tables 6a–c). In contrast, the correlations
were lower for the soluble and ash contents (Tables 6d–e).
For a given trait, these coecients varied among the
years. They were low in some years, as was the case for
the cellulose content measured between the third and the
fth years (Table 6a) and for the lignin content measured
Table 5. Correlations between the aboveground biomass
production (predicted variable) of the second, third, fourth,
fifth, and sixth years of cultivation using the following predic-
tors: the flowering date, canopy height, stem diameter, stem
number, and aboveground plant volume index using untrans-
formed and log-transformation data observed in the second,
third, and fourth years of cultivation. These correlations were
performed using 19 Miscanthus clones observed during the
winter har vest. All correlation coefficient values significant at
the 0.05 probability level.
Predictor Year
Predicted variable: Aboveground
biomass production
Yea r 2 Yea r 3 Ye ar 4 Year 5 Year 6
Flowering date Year 2 NA†NA NA NA NA
Yea r 3 0.52 0.71 0.78 0.77
Yea r 4 0.75 0.79 0.79
Canopy height Ye a r 2 0.91 0.90 0.79 0.76 0 .70
Yea r 3 0.87 0.82 0.89 0.86
Yea r 4 0.84 0.94 0.93
Stem diameter Ye a r 2 0.72 0.72 0 .74 0.81 0.77
Yea r 3 0.64 0.72 0.84 0.83
Yea r 4 0.75 0.86 0.83
Stem number per
plant
Yea r 2 NS‡NS NS NS NS
Yea r 3 NS NS NS NS
Yea r 4 NS −0.33 −0.32
Aboveground
plant volume
index
Yea r 2 0.9 0 0.83 0.82 0.75 0.68
Yea r 3 0.77 0.85 0.91 0.90
Yea r 4 0.78 0.85 0.78
Log (Aboveground
plant volume
index)§
Yea r 2 0.95 0.92 0.86 0.72 0.63
Yea r 3 0.90 0.91 0.86 0.83
Yea r 4 0.83 0.90 0.82
† NA, data not available.
‡ NS, Not sign ificant at the 0.05 proba bilit y level.
§The correlations were calculated using the log (aboveground biomass production).
Table 4. Correlations between the autumn and winter harvest
dates within and between the second, third, fourth, fifth, and
sixth year of cultivation for the traits related to the aboveg-
round biomass production from 19 Miscanthus clones: (a)
aboveground biomass production, (b) flowering date, (c)
canopy height, (d) stem diameter, (e) stem number per plant,
and (f) aboveground plant volume index. All correlation coef-
ficient values significant at the 0.05 probability level.
Pearson correlation coefficient
Yea r 2 Yea r 3 Yea r 4 Yea r 5 Ye a r 6
Autumn harvest
(a) Aboveground biomass production (t DM† ha−1)
Winter
harvest
Yea r 2 0.78 0.80 0.77 0.72 0. 74
Yea r 3 0.77 0.82 0.81 0 .76 0.76
Yea r 4 0.68 0.79 0.84 0.82 0.8 0
Yea r 5 0.68 0.85 0.92 0.91 0.87
Yea r 6 0.63 0.84 0.92 0.90 0.89
(b) Flowering date (DD‡ base 10)
Winter
harvest
Yea r 2 NA§NA NA NA NA
Yea r 3 0.90 0.96 0.98 0.96 0.88
Yea r 4 0.91 0.96 0.99 0.97 0.90
Yea r 5 0.90 0.96 0.97 0.97 0.88
Yea r 6 0.87 0.94 0.95 0.94 0.87
(c) Canopy height (cm)
Winter
harvest
Yea r 2 0.83 0.85 0.78 0.70 0.72
Yea r 3 0.82 0.96 0.93 0.88 0.89
Yea r 4 0.73 0.95 0.97 0.9 6 0.95
Yea r 5 0.67 0.92 0.97 0.98 0.96
Yea r 6 0.63 0.88 0.93 0.95 0.94
(d) Stem diameter (mm)
Winter
harvest
Yea r 2 NA 0.91 0.84 0.92 0.93
Yea r 3 NA 0.93 0.91 0.95 0.95
Yea r 4 NA 0.91 0.86 0.93 0.94
Yea r 5 NA 0.95 0.90 0.95 0.95
Yea r 6 NA 0.94 0.92 0.97 0.97
(e) Stem number per plant
Winter
harvest
Yea r 2 0 .71 0.64 0.57 0.52 NA
Yea r 3 0.76 0.86 0.86 0.83 NA
Yea r 4 0.75 0.85 0.91 0.8 6 NA
Yea r 5 0.67 0.83 0.92 0.92 NA
Yea r 6 NA NA NA NA NA
(f) Aboveground plant volume index (cm3)
Winter
harvest
Yea r 2 NA 0.73 0.57 0.70 NA
Yea r 3 NA 0.85 0.80 0.90 NA
Yea r 4 NA 0.75 0.64 0.73 NA
Yea r 5 NA 0.73 0.77 0.87 NA
Yea r 6 NA NA NA NA NA
† DM, dry matter.
‡ DD, degree-days.
§ NA, data not available.
crop science, vol. 55, may–jun e 2015 www.crops.org 9
Figure 3. Pairwise plots of traits between the aboveground biomass production of the third, fourth, fifth, and sixth years and the aboveground plant volume index of the third year for
untransformed (a, b, c, d) and log-transformed (e, f, g, h) data; data collected in the winter harvest. A total of 19 Miscanthus clones were used for these plots. The filled squares represent
the M. giganteus clones, and the filled triangles represent the M. sinensis clones. For details on the clones, see Table 1. An asterisk represents significant correlations at the 0.05
probability level; r, Pearson correlation coefficient.
10 www.c rops .org crop scie nce, v ol. 55, may–june 2015
between the fth and fourth years (Table 6c). Similarly, the
ash content also displayed low correlation coecients, espe-
cially between the third and the fth years and between the
fourth and the fth years (Table 6e). Similar trends were
observed in the autumn harvest (Supplemental Table S4a–e).
Despite these low correlations, the most productive
clones were consistently ranked throughout the years with
respect to the cellulose, hemicellulose and lignin contents
(Fig. 4 for cellulose content).
DISCUSSION
This study of the prediction of the traits related to Mis-
canthus biomass production and composition in the young
growth stages of the crop partially addressed our hypoth-
eses: (i) The traits related to the biomass production were
predicted with robustness in the early growth stages of
the crop, but the traits related to the biomass composition
were not well predicted; moreover, (ii) the canopy height
and the log (aboveground plant volume index) were reli-
able predictors of the biomass production.
We therefore discuss two main points directly related
to our results: (i) the potential to predict Miscanthus biomass
production and its related traits from the young stages of the
crop, (ii) the traits that are reliable predictors of the biomass
production, and (iii) the signicance of our results for the
early selection of promising Miscanthus clones as feedstock
for second-generation bioethanol production.
Prediction of Biomass Production
and Related Traits in Miscanthus from
Young Crop Stages
Clifton-Brown et al. (2001b) reported that the third-year
biomass production of 15 Miscanthus clones at ve sites in
Europe was better correlated with the second-year bio-
mass production than with the biomass produced in the
rst year. In addition, Clifton-Brown and Lewandowski
(2002) studied the same clones in southern Germany
for the rst 3 yr of cultivation with autumn and winter
Figure 4. Contribution of the individual Miscanthus clones to the
interannual correlations between (a) the third and fourth years of
cultivation and (b) the third and fifth years of cultivation for the
cellulose content observed in the winter harvest. A total of 19 Mis-
canthus clones were used for these plots. The filled squares rep-
resent the M. giganteus clones, the filled circles represent the
M. sacchariflorus clones, and the filled triangles represent the M.
sinensis clones. For details on the clones, see Table 1. An aster-
isk represents significant correlations at the 0.05 probability level;
r, Pearson correlation coefficient.
Table 6. Interannual correlations between the third, four th,
and fifth years of cultivation in the winter har vest for the
traits related to the biomass composition from 19 Miscan-
thus clones: (a) the cellulose, (b) hemicellulose, (c) lignin, (d)
soluble, and (e) ash contents. All correlation coefficient val-
ues significant at the 0.05 probability level.
Pearson correlation coefficient
Yea r 3 Year 4 Year 5
(a) Cellulose (% DM†)
Yea r 3 0.80 0.69
Yea r 4 0.85
(b) Hemicelluloses (% DM)
Yea r 3 0.83 0.83
Yea r 4 0.82
(c) Lignins (% DM)
Yea r 3 0.83 0.82
Yea r 4 0. 74
(d) Solubles (% DM)
Yea r 3 0.61 0.50
Yea r 4 0.68
(e) Ash (% DM)
Yea r 3 0.73 0.38
Yea r 4 0.47
† DM, dry matter.
crop science, vol. 55, may–jun e 2015 www.crops.org 11
harvests and reported that the biomass production in the
rst year was not always related to the biomass produced
in subsequent years. These authors concluded that the
selection of Miscanthus clones showing optimal biomass
production should be based on at least 2 yr of eld trials.
Our results agree with these observations.
Similar trends have been described in the literature
for the traits related to the biomass production, particu-
larly for the plant height and number of stems per plant.
A moderate regression coecient (r = 0.67) was observed
between Miscanthus height in the rst and third year of
cultivation (Clifton-Brown and Lewandowski, 2002).
In addition, these authors showed that the shoot density
measured at the end of the rst growing season, which
could be associated with our measure of the stem number
per plant, was poorly correlated with the shoot density
measured at the end of the third growing season. We stud-
ied an extended time period, that is, longer than the three
rst years, and found lower correlations between traits
measured in the second year compared with subsequent
years. These results suggest that the plant height and stem
number per plant were predicted with more reliability
from the third year of cultivation than from the rst 2 yr.
In addition, our results indicated that the stem diameter
consistently displayed high correlation coecients between
the second to the sixth year, which was similar to the
results reported for poplar (Populus spp.), a woody perennial
crop of particular interest for the second-generation bio-
ethanol production (Kaczmarek et al., 2013). Furthermore,
the aboveground plant volume index was well correlated
between years, which is similar to the results reported for
poplar using a comparable index (Kaczmarek et al., 2013).
Reliable Predictors of Biomass Production
Jezowski (2008) reported that the biomass production of
six Miscanthus clones harvested in winter was highly cor-
related with the canopy height in the second and third
years of cultivation. In addition, Clifton-Brown et al.
(2001b) showed that the height of 15 Miscanthus clones was
well correlated with the biomass harvested in autumn of
the third year. Gauder et al. (2012) reported that the plant
height at senescence was a reliable parameter to estimate
the biomass harvested in winter. Furthermore, Robson
et al. (2013) observed that the canopy height of 244 Mis-
canthus clones was better correlated with the biomass
harvested in winter of the third year than the stem diam-
eter or the maximum canopy height. Our results veried
these observations and conrmed that the canopy height
was generally better correlated with the biomass produc-
tion than other morphological traits.
Clifton-Brown et al. (2001b) reported that the shoot
density, which is similar to our measurement of the number
of stems per plant, was poorly correlated with the aboveg-
round biomass harvested in autumn of the third year. In
contrast, Gauder et al. (2012) reported a relatively high and
negative correlation between the yield and the shoot den-
sity in a study performed on 14 consecutive years. Our
results agree with both studies; there were no correlations
between the second- and third-year data, whereas we
observed negative correlations in the fourth year irrespec-
tive of the harvest dates. These results showed that the cor-
relation between the stem number and the biomass pro-
duction increased over time and suggested that the stem
number did not contribute to the yield during the early
growth stages compared with the later stages. Moreover,
the contribution of the stem number to the biomass pro-
duction could be compensated by other variables such as
the height of the plants and the stem diameter. With a sim-
ilar height of the plants between all the genotypes, it would
be expected a better contribution of the stem number.
In addition, Zub et al. (2012a) reported that the aboveg-
round plant volume index of Miscanthus was a reliable pre-
dictor of the biomass produced in the second and third
years. Our results conrmed these ndings over a longer
time period (from the second to the sixth year). However,
we showed that the aboveground plant volume index was
a less reliable predictor of the biomass produced in the fth
and sixth years, compared with the canopy height. This
may be due to the diculty to determine the stem number
per plant for older plants in comparison to younger plants.
Therefore, we can suggest that this decreased the aboveg-
round plant volume index reliability to predict the biomass
production of older Miscanthus crops.
In a poplar study, Rae et al. (2004) found that the
stem volume index was the best predictor of the biomass
production, followed by the total basal area and the tree
height at harvest, which were used to calculate the stem
volume index. In addition, Scaracia-Mugnozza et al.
(1997) reported that the stem volume growth of poplar
well reected the increase in tree biomass. These observa-
tions conrmed our Miscanthus results.
Breeding Implications for the Early Selection
of Interesting Miscanthus Clones
Selecting clones based on the aboveground biomass pro-
duced in the rst and second years would be less eective
than a selection based on the third year data; however, an
initial screening of the most productive Miscanthus clones
from the rst and second years could be feasible because
Clifton-Brown and Lewandowski (2002) showed that
the most productive Miscanthus clones identied in the
rst year were generally also the most productive in the
subsequent years. However, our results showed that the
screening has to be continued for at least 3 yr.
Because of the high correlations between the two har-
vest dates, the clone selection can be performed using a
single harvest date. Under the tested conditions, this would
12 www.c rops .org crop scie nce, v ol. 55, may–june 2015
allow the autumn harvest to be selected indirectly from the
winter harvest, which is the most common practice.
In addition, the canopy height and the aboveground
volume index were useful criteria to make an early selec-
tion of the most productive Miscanthus clones. Given the
very small dierences in predictive power between these
two predictors, breeders should use the canopy height to
accurately identify the most productive clones, as it is an
easy-to-measure trait. Nevertheless, we explored here a
great variability regarding the height of the plants in com-
parison to the other traits. For a lower variability, it should
be expected a greater impact of the stem number as found
in switchgrass (Panicum virgatum L.) (Price and Casler, 2014).
This would lead to a superiority of the aboveground plant
volume index to predict the subsequent years of production
in comparison to the plant height and this would require a
better assessment of the stem number per plant. This is par-
ticularly interesting as the canopy height and aboveground
plant volume index were nondestructive measurements.
Furthermore, the prediction of the traits related to the
biomass composition was less reliable when using the rst
years of cultivation in comparison with the aboveground
biomass production-related traits under the tested con-
ditions. However, an initial screening can be attempted
during the rst few years among the most productive
clones with respect to the cellulose, hemicellulose, and
lignin contents because we observed that the contribution
of the most productive clones were consistent over time.
CONCLUSIONS
This study investigated the possibility of early prediction
of the traits related to Miscanthus biomass production and
composition on a single-site testing.
The biomass production and its related traits, more
particularly, the stem diameter, owering date, and
canopy height, displayed high correlations between the
years. These traits were better predicted from the third
year of cultivation than from the second year. Moreover,
the correlations between the two harvest dates were high.
Therefore, to early select these traits in Miscanthus in the
rst few years of cultivation, it is more reliable to screen
the genotypes for at least 3 yr and it is possible to limit the
evaluation to a single harvest date.
In addition, the biomass produced in the rst years (i.e.,
the second to fourth year) was reliably predicted using the
log (aboveground plant volume index) of the second and
third years; however, the biomass produced in later years
(i.e., in the fth and sixth years) was better predicted using
the canopy height of the second and third year.
Additionally, a rst screening of the cellulose, hemi-
cellulose, and lignin contents can be attempted among the
most productive genotypes during the early stages of the
crop as the most productive clones were consistently ranked
throughout the years with respect to these variables.
Acknowledgments
The authors would like to thank the FUTUROL project funded
by Oseo, which supported this study. Particular thanks to Benoit
Decaux and the sta of the UE GCIE for their valuable help in the
records, harvests, and maintenance of the trial. Thanks to Yves
Barrière, Jacques Le Gouis, and François-Xavier Oury for their
valuable recommendations during the preparation of this text.
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