Published March 20, 2015
Research
Analysis of Series of Cultivar Trials with
Perennial Grasses for Subdivided Target Regions
Thomas Eckl and Hans-Peter Piepho*
Abstract
Field trials with perennial grasses may often
be conducted at several locations with differ-
ent starting years. A key issue in the analysis of
such trials is the distinction between effects of
calendar years, which are associated with exter-
nal environmental variation, and harvest years,
which represent internal yield formation pro-
cesses of the perennial crop. Furthermore, anal-
ysis of field trials with perennial grasses needs to
account for serial correlation of observations on
the same plot from consecutive harvest years.
Such analyses are conveniently implemented
using mixed models. Here, we consider series
of trials when the target region is subdivided into
several zones. We show how cultivar yield means
per zone can be estimated borrowing strength
across zones. The proposed mixed models are
illustrated using simulated data generated by
employing variance component estimates from
real experiments. It is shown in simulations that
best linear unbiased prediction (BLUP) can pro-
vide more precise zone-specific mean estimates
than alternative methods.
crop science, vol. 55, march– april 2015  www.crops.org
T. Eckl, Bavarian State Research Center for Agriculture, Freising, Ger-
many. H-P. Piepho, Biostatistics Unit, Institute of Crop Science, Univ.
of Hohenheim, Germany. Received 28 Apr. 2014. *Corresponding
author (piepho@uni-hohenheim.de).
Abbreviations: AIC, Akaike Information Criterion; BLUE, best lin-
ear unbiased estimation; BLUP, best linear unbiased prediction; CS,
compound symmetry.
T he Bavarian State Research Center for Agriculture (Freising,
Germany) routinely conducts cultivar trials to provide crop
production advice and support to farmers. Field trials with peren-
nial crops such as ryegrass (Lolium perenne L.) involve repeated
measurements taken on the same plot on several occasions. Such
yield trials are frequently conducted at multiple locations to eval-
uate newly released ryegrass cultivars with annual yields recorded
per plot for three consecutive years. In Germany, each federal
state such as Bavaria used to present results of its conducted cul-
tivar trials separately. Thus, yield estimates were limited to trials
within administrative borders. To increase the precision and effi-
ciency of these trials, agroecological zones have recently been
defined across Germany, which differ in their habitat features and
their relevance for cultivar-specific reactions.
Trial locations are chosen to be representative of target zones
and thus are assigned to exactly one zone. The associated analysis
method allows combining yield trials of neighboring zones (Atlin
et al., 2000; Michel et al., 2007; Piepho and Möhring, 2005) to
provide yield estimates with high precision on a zonal basis. Up
to now, these types of multienvironment trials have only been
analyzed across zones for annual crops. The implementation and
Published in Crop Sci. 55:597–609 (2015).
doi: 10.2135/cropsci2014.04.0327
© Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA
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597
adaptation of this method has not yet been performed for
perennial forage crops. The statistical demands for peren-
nial crops are more intricate than for annual crops due
to the additional complexities arising from the repeated
measurements and the partial temporal overlap of trials
with different starting years.
A crucial point in the statistical analysis of trials with
perennial crops is that environmental influences have a
more permanent effect on perennial crops than they have
on annual crops. Crop yields from consecutive harvest
years are not independent from the conditions of the pre-
vious years. One further characteristic of perennial crops
is that a trial comprises multiple calendar years as well as
multiple harvest years but calendar year and harvest year
are not identical factors. Effects of calendar years repre-
sent the interaction of weather conditions and agricultural
management whereas effects of harvest years are based on
factors like aging of the sward and gaps in the plots.
The main goal of the analysis is to obtain the best esti-
mate of differences among cultivars for each given zone and
as a result gain well-founded information to support crop
production advice without having to increase trial costs.
Particular emphasis needs to be given to modeling the serial
correlation among consecutive measurements on the same
experimental unit as well as to separating the effect of cal-
endar year from harvest year in case of staggered starts.
This paper presents a general method to analyze trials
adapted for the division of a target region into zones
(Hartmann, 2010; Kleinknecht et al., 2013; Michel et al.,
2007), considering the repeated-measures structure aris-
ing in trials with perennial crops such as grasses. For more
complex data, we describe a three-stage analysis in which
cultivar means per trial and harvest year computed in the
first stage are analyzed across environments in two fur-
ther stages. Our approach to separating harvest year from
calendar year effects is similar in spirit to that used by
Casler (1999) and Conaghan et al. (2008). To exemplify
the models and methods, we created simulated data using
variance component estimates from a dataset on series of
regional yield trials with ryegrass.
Using these simulated data, the developed mixed
models for two-stage and three-stage analysis are compared
according to the resulting adjusted means and their pair-
wise correlation between analyses. At the end of our model
development, we show how to modify the models, by mod-
eling within-zone cultivar effects as random, to increase the
precision of the estimated means by combining information
of the target zone and the neighboring zones. Supplemental
Appendix S1 contains the SAS (Littell et al., 1998) program
code for implementation of our proposed models.
598 www.crops.org
MATERIALS AND METHODS
The derivation of the simulated dataset is described in the
following section. Thereafter, mixed models for unbiased esti-
mation of cultivar means across zones are developed. We show
the complexity of a single-stage model can be reduced by a
stagewise approach of the analysis. For this purpose, we pres-
ent appropriate two-stage and three-stage models, at first for a
simpler case with only one trial, followed by the more complex
case with several trials. At the end of our model development,
we show how to modify the models to increase the precision
of the estimated means by combining information of the target
zone and the neighboring zones.
The Data
Empirical Data
We used data from ryegrass cultivar trials conducted at 16 loca-
tions divided into six zones in southern Germany.
In total, 48 cultivars were tested between 2001 and 2011,
with first harvest years in 2001 to 2009. All trials were laid out
as randomized complete block designs. Depending on location
and growing conditions, the number of cuts ranged from three
to seven per year. The yield of all cuts for a given trial and har-
vest year were summed by plot and used as the response variable.
The resulting dataset was heavily unbalanced (i.e., there
were cultivars in each trial series, which could only be found
in a few trial years). The same also applied to trial locations.
Furthermore, some cultivars were targeted for specific zones
and hence were grown in only a subset of the trials. For a sin-
gle-stage analysis including every starting year, location, and
cultivar as shown in [24] on page 6 of Supplemental Appendix
S1, we ran into computational problems regarding computation
time, memory space, and convergence.
Simulated Data
Our main goal was to develop methods of analysis for which
complex data do not cause any of these problems. Single-stage
analysis can be considered as a gold standard against which to
compare stagewise methods (Piepho et al., 2012). The newly
developed methods should produce results similar to a full
single-stage analysis. Because of the extreme imbalances and
the resulting computational difficulties with the real data, we
decided to use simulated data for evaluation of alternative
approaches. Thus, we used variance component estimates from
the real data to create a simulated dataset that was smaller and
more balanced than the real dataset at hand. This gave us the
possibility to obtain results for a single-stage analysis and to
compare the developed two-stage and three-stage methods with
single-stage analysis and try different covariance structures.
To determine parameter values for the data simulation, we
had to reduce the original dataset. A core set of four cultivars
which were tested in most of the trials was included, yield-
ing a dataset of cultivar trials conducted within 10 yr at seven
locations split into three zones, with four cultivars laid out as a
randomized complete block design with three replicates. Using
the reduced data, the covariance parameter estimates needed
for the simulation were obtained with the single-stage mixed
model shown in Model [24] in Supplemental Appendix S1.
crop science, vol. 55, march– april 2015
 With the covariance parameters estimates obtained from
the reduced, original dataset, we simulated a new, more bal-
anced dataset for further analysis. This simulated dataset was
generated applying the method described below. An Excel file
with the simulated data is provided as Supplemental Appendix
S2. The dataset simulates trials conducted within 5 yr in four
zones, each containing four locations with 10 cultivars laid out
as a randomized complete block design with three replicates. We
simulated a response for each plot. Observations are correlated
due to the random effects of the assumed linear mixed models.
The general form of the model is given by
y = Xb  Zu  e,
where X is the design matrix for the fixed effects, Z is the design
matrix for the random effects, b is the vector of fixed effects
(associated with factors GEN, HAR, and ZON), u is the vector
of random effects, and e is a residual error term. Random effects
(u, associated with factors YR, LOC, TRL, and BLK) and errors
(e) are assumed to be independent with zero mean vectors and
variance–covariance matrices G and R, respectively. Hence, the
data y follows a multivariate normal distribution with mean Xb
and variance–covariance matrix V = ZGZT + R.
To create correlated data y for the assumed design follow-
ing the single-stage mixed model shown in the Supplemental
Appendix S1 in Model [24], we made use of the Cholesky
decomposition of the variance-covariance matrix of the data
(V). For this procedure, we first estimated the variance compo-
nents from the reduced dataset described above. These estimates
were then used as parameter values (PARMS statement in SAS)
in a single-stage mixed model to calculate a variance-covariance
matrix V of the data for the assumed design of the simulated
trials. The computed matrix V was then factored uniquely into
a product V = CCT with the Cholesky decomposition of V,
where C, an upper triangular matrix, is called the Cholesky
root and CT is the transpose of C.
A random vector z T = {z1 ...z n }, z ~ N (0, I ) of indepen-
dent standard normal deviates was generated to calculate the
correlated vector of standard normal deviates x = Cz so that x
~ N(0,V) with Var(Cz) = CICT. Consequently, the simulated
data y = x + 1μ, where μ is the intercept, are normally distrib-
uted with the mean E(y) = 1μ, where 1 is a vector of ones.
Definition of Variables for Analysis
The simulated dataset contains the variables listed below in
Table 1, which will be used to formulate mixed models in the
subsequent sections. We classified the variables as treatment,
block, and repeated factors (Piepho and Eckl, 2014). Treatment
factors are selected by the experimenter to study and compare
their effect on given response variables. Its levels are randomly
assigned to experimental units. Block factors, on the other hand,
are used to identify experimental units and arrange them into
homogenous groups or blocks (Milliken and Johnson, 1992,
Section 4.2). The block factors can be used to set up a model
that holds when there are no treatment effects. This model may
also be referred to as block model (Piepho et al., 2003) or design
structure (Milliken et al., 2010). Repeated measurements taken
on the same experimental unit are characterized by a repeated
factor such as harvest year (Piepho et al., 2004).
crop science, vol. 55, march– april 2015  www.crops.org
Table 1. Structure of factors zone (ZON) and location (LOC).
ID Explanation
Response variable
YLD Cumulative yield per plot and year
(sum of all cuts in a given harvest year)
Treatment factor
GEN Cultivar
Blocking factors
ZON Zone; see Fig. 1
LOC Location; nested within ZON
TRL Trial; this identifies an experiment running up to
three consecutive years nested within LOC
YR Calendar year (i.e., year a given trial was established)
BLK Block of randomized complete block design;
nested within TRL
PLT Plot, nested within BLK
Repeated factor
HAR Harvest year (i.e., current year a trial is harvested)
For clarity, a brief description of the factors is given here.
A trial (TRL) with a perennial crop corresponds to a single
experiment at a single location, which, in our case, is conducted
during three harvest years. Thus, a trial with a perennial crop
is introduced as a multi–calendar year (YR) entity consisting of
consecutive harvest years (HAR) and is nested within locations
(LOC). A harvest year typically comprises three cuts within the
current calendar year. The number of cuts sometimes goes up
to seven per year. The yields of all cuts in a year are summed
per plot and used as response variable (YLD) in our models. At
a location, several trials may be run simultaneously in the same
calendar year. These trials usually are staggered in the sense that
they have different start years. Trial locations are chosen to be
representative of target zones, assigned to exactly one zone and
thus nested within zones (ZON).
Model Derivation for Unbiased Estimation
of Cultivar Means per Zone
In this section we develop models that allow estimation of cul-
tivar means per zone, taking the cultivar effects as fixed. These
models essentially exploit only the information per zone for
estimating cultivar yields per zone. The method of estimation is
best linear unbiased estimation (BLUE). In the next section, we
consider a modification of this approach, based on random cul-
tivar effects, that allows borrowing strength from other zones.
The method of estimation used for that approach will be BLUP.
Here, we first consider the case of a single trial conducted
at several locations and then extend it to the case of several
trials with different start years. In either of these cases, we first
present a two-stage approach and then a three-stage approach.
Several Locations, One Trial
For the development of a suitable model, we first consider the
simple case, where trials are started in the same year and thus also
have the same first and subsequent harvest years. For now, we
consider only one trial per location. A separation of the effects
of calendar year and harvest year is not yet necessary in this case.
When the trials are completed, in our case after three calendar
years, a new set of trials with new cultivars could begin.
599
Figure 1. Division of the target region, Deutschland-Mitte-Süd, into zones: 6, sommertrockene Lagen (dry summer zone); 7, günstige
Übergangslagen (favorable transition zone); 8, Hügelländer Süd (hilly country–South); 9, Mittelgebirgslagen West (uplands–West); 10,
Mittelgebirgslagen Ost (uplands–East); and 11, Voralpengebiet (prealpine region).

A straightforward analysis may be performed when all trials Table 2. Structure of factors zone (ZON), location (LOC)
have the same start year and the same number of harvest years. nested within zone as indicated by consecutive numbering,
The total yield across years may then be fitted by a mixed model calendar year (YR), and harvest year (HAR) when trials with
analyzing the sum of all yields per plot, avoiding any modeling start year 2008 are considered.
of serial correlation of repeated measures. However, when the Calendar year (YR)
number of harvest years is not constant in all trials and some trials ZON LOC 2008 2009 2010
have staggered starts, as will be considered in more detail later, 1 1 HAR = 1 HAR = 2 HAR = 3
more complex modeling is needed. The simulation assumes trials 1 2 HAR = 1 HAR = 2 HAR = 3
conducted in four zones each comprising four trial locations. 4 15 HAR = 1 HAR = 2 HAR = 3
All trials were simulated as a randomized complete block design 4 16 HAR = 1 HAR = 2 HAR = 3
with three complete blocks (BLK). We assumed the first harvest
year to be 2008, and there were three consecutive harvest years
(Table 2). A total of 10 cultivars (GEN) were simulated and the
YLD = GEN + FER + GEN·FER: BLK +
simulated yield per plot was used as response variable.
BLK·GEN·FER,
To formulate a mixed model, we used the framework
described by Piepho et al. (2003, 2004). We want to point
out that we find the used model syntax helpful because mixed where the fixed effects are stated before the colon and random
models are described with a notation that is close to that used effects after the colon. The effect BLK·GEN·FER is under-
with linear mixed model statistical software packages. More- scored to indicate that it is the residual. The dot operator is
over, the structure of random effects with a repeated-measures used to define crossed effects. It corresponds to the asterisk (*)
correlation structure can be specified more clearly. operator in SAS.
As an example, a linear two-way factorial mixed model Thus, our syntax is close to the statements needed for this
with yield coded as YLD, cultivar coded by GEN, fertilizer model in linear model packages such as the MIXED procedure
coded by FER, and random block coded by BLK written as of the SAS system:

600 www.crops.org crop science, vol. 55, march– april 2015

 Model YLD = GEN FER GEN*FER;
Random BLK;
Note that the residual BLK·GEN·FER will be fitted automatically.
From theoretical considerations, single-stage analysis
is regarded as preferable and is considered the gold standard.
Nevertheless, because of both its simplicity and computational
efficiency (Piepho and Michel, 2000) and because single-stage
and stagewise analysis often yield very similar results (Möhring
and Piepho, 2009; Piepho and Eckl, 2014), we also consider
models for two-stage and three-stage analysis.
In Piepho and Eckl (2014), we compared several stagewise
approaches to account for the repeated-measures structure,
including modeling serial correlation at the plot level, which
appears to be the most obvious method. However, plot level
modeling (Method b in Piepho and Eckl, 2014) turned out to
be computationally much more demanding than modeling the
serial correlation at the level of cultivar means per harvest year.
As both methods were very highly correlated with each other
and were both highly correlated with single-stage analysis
results, we prefer to model serial correlation at the cultivar-
mean level for computational efficiency (Method a). For this
reason, we here only present results based on that method.
Two-Stage Analysis
A two-stage analysis is the standard procedure in the analysis
of official cultivar trials in Germany since it has the practical
advantage that trials can be analyzed individually in the first
stage. This makes it easier to consider any specific features
of both the experimental design and the preferred model for
within-trial analysis. The two-stage method to analyze sev-
eral locations and several trial years where serial correlation is
modeled in stage two as presented in Piepho and Eckl (2014,
Electronic Appendix S1, Fig. 14) can easily be expanded to
account for the division of a target region into zones.
Model for First Stage Analysis. In the first stage, we
compute cultivar means for each harvest year in separate analy-
ses for each trial. A model for first-stage analysis laid out in a
randomized complete block design with fixed effects for cultivar
and block can be found in Supplemental Appendix S1 on page
3 (also see Piepho and Eckl, 2014). This is fitted separately for
every year. We then use a mixed model, to be described below,
to analyze and estimate the serial correlation between cultivar
means in different harvest years in the same trial in stage two.
Model for Second Stage Analysis. To develop a model
for the second stage, we start with the model that would be used
to analyze a single level of the repeated factor (Piepho et al.,
2003). The mixed model can be formulated as follows, using
the framework described in Piepho et al. (2003, 2004):
GEN + ZON + ZON·GEN:
ZON·LOC + ZON·LOC·GEN, [1]
where fixed effects (GEN + ZON + ZON·GEN) precede random
effects (ZON·LOC + ZON·LOC·GEN) and are separated from
these by a colon. The dot (·) is an operator for concatenating fac-
tors or effects (corresponding to the asterisk * in SAS).
crop science, vol. 55, march– april 2015  www.crops.org
To complete the model, correlation structures for random
effects associated with repeated factors need to be accommo-
dated. In repeated-measures models, subjects correspond to
statistically independent units on which repeated measures
are taken. In our case, the random effects ZON·LOC and
ZON·LOC·GEN of the block model for a single level of the
repeated factor now become subjects. These secondary subjects
correspond to means across several plots, the plots being the
primary subjects of the design. The important point here is that
taking averages per cultivar across plots generates a new type of
subject at a higher level of aggregation, which inherits any serial
correlation on the primary subjects. Different observations on
the same subject taken in different harvest years represent the
same treatment whereas levels of treatments can change only
between subjects. Errors from different secondary subjects
(means across plots) (ZON·LOC·GEN) are independent and
need to be distinguished from the part of the effects identify-
ing the different observations from the same experimental unit.
These effects pertaining to the repeated measurements (HAR)
may be serially correlated (Piepho and Eckl, 2014).
To clarify the structure of random effects with a repeated-
measures correlation structure, we expand this model by
concatenating the interaction ZON·LOC·GEN as well as the
interaction ZON·LOC with the repeated factor HAR using the
dot operator (·) and fully crossing the remaining (fixed) effects
with HAR using the crossing operator (×). Each random effect of
the block model and the interaction is modeled as a serially cor-
related effect. Subjects are boldfaced, the repeated factor HAR in
these effects is boldfaced and italicized (Piepho and Eckl, 2014).
In our model, there is a single observation (mean) per culti-
var–location combination for every harvest year. Consequently,
the random effect ZON·LOC·GEN represents the lowest-level
“subject” on which repeated measures on HAR are available
and the effect will thus be written as ZON·LOC·GEN·HAR.
The same reasoning applies to the effect ZON·LOC·HAR,
which absorbs the serially correlated block effects (Piepho and
Eckl, 2014). We fit the following model in the second stage:
GEN × HAR × ZON:
ZON·LOC·HAR + ZON·LOC·GEN·HAR [2]
Resolving the factorial structure, this expands as
GEN + HAR + ZON + GEN·HAR +
GEN·ZON + ZON·HAR + GEN·ZON·HAR:
ZON·LOC·HAR + ZON·LOC·GEN·HAR [3]
Three-Stage Analysis. The two-stage approach sug-
gested above can be extended to three stages in a straightforward
way (Piepho et al., 2012). It should be stressed, however, that
serial correlation will need to be taken into account throughout
all three stages and thus models used in stage two and three
must be as close as possible to the Model [19] used for single-
stage analysis described in the Supplemental Appendix S1 on
page 3; otherwise, important variances and covariances will not
be taken into account.
601
 To set up a three-stage analysis, the hierarchy of the design Table 3. Classification of factors trial (TRL), calendar year
needs to be split in three stages. The first stage is the same as (YR), and harvest year (HAR) when trials with several start
before, such that we first compute the cultivar means per trial years are considered.
and harvest year. Since we are mainly interested in estimating the Entries Calendar Year (YR)
Trial
cultivar means, we will be averaging across environment effects No. Common Unique 2008 2009 2010 2011 2012
in the second stage. Care should be taken that the environmental
1 1–3 804–810 HAR = 1 HAR = 2 HAR = 3
covariance and the genetic covariance do not get intermingled
2 1–3 904–910 HAR = 1 HAR = 2 HAR = 3
and remain separable in the third stage of the analysis. There-
3 1–3 1004–1010 HAR = 1 HAR = 2 HAR = 3
fore, in the second stage, we estimate GEN·HAR-means within
zones, which fulfills the conditions stated above. For each zone
(ZON), we fit the following model in the second stage: we identify the individual trials using the factor TRL. This
leads to having both a main effect for YR and a main effect
GEN·HAR: for TRL, which are fundamentally different (Piepho and Eckl,
LOC·HAR + LOC·GEN·HAR [4] 2014). First, we look at the model across zones within a single
harvest year. We fit the following model in the second stage:
The computed GEN·HAR-means can then be evaluated across
zones in stage three with the following model. (GEN + ZON + GEN·ZON:

GEN + HAR + ZON + GEN·HAR + YR + GEN·YR + ZON·YR + ZON·GEN·YR +

GEN·ZON + ZON·HAR: ZON·LOC + ZON·LOC·YR +
ZON·LOC·GEN + ZON·LOC·TRL +
ZON·GEN·HAR [5] ZON·LOC·GEN·YR + ZON·LOC·TRL·GEN [6]

In the previous section, we expanded each random effect of the

Several Locations, Several Trials
Yield trials for perennial crops such as ryegrass conducted by the
Bavarian Research Institute have staggered starts. Specifically,
new trials are laid out each year to accommodate new culti-
vars entering the system and thereby being able to give timely
advice to producers. To compare cultivars that entered trials in
different years, a joint analysis is needed. Therefore, we now
consider the case of several trials at the same location that do not
necessarily have the same first harvest year. For every one of the
four zones of the previous simulated dataset, we simulated two
additional trials for starting years 2009 and 2010. For every new
trial, a core set of three cultivars was kept in each trial, whereas
the remaining seven cultivars were replaced by seven new cul-
tivars. The core set is comprised of Cultivars 1, 2, and 3. The
seven cultivars tested in 2008 are numbered 804 to 810. Culti-
vars for 2009 and 2010 are numbered accordingly (Table 3). This
approach makes it possible to combine the information on the
core set of cultivars across the three trials and also to compare
cultivars not tested in the same trial. This staggered arrangement
of trials has the advantage that the performance of the tested
cultivars can be assessed within a short period of time. However,
besides the modeling of the serial correlation among consecutive
measurements, effects for calendar year and harvest year now
need to be separated, which requires a more refined approach.
In the following, we present models for two-stage and
three-stage analysis. A suitable model for single-stage analysis is
presented in the Supplemental Appendix S1 on page 6.
Two-Stage Analysis. Again, to develop the model for
the second-stage analysis, we initially focus on a single level
of the repeated factor. Several trials can now simultaneously
be run at a location. Moreover, these trials may usually have
different start years and so the same harvest year can occur in
several years. To distinguish block effects from different trials,
block model and the interaction as a serially correlated effect for
the repeated factor HAR. The factor YR, although part of the
block model, does not correspond to a physical unit on which
repeated measures are taken. Therefore, any effects involving
year will not be expanded as serially correlated.
Expanding the model by concatenating the random block
effects as well as the interactions with HAR and fully crossing
the remaining effects with HAR leads to:
GEN + ZON + GEN·ZON:
YR + GEN·YR + ZON·YR + ZON·GEN·YR
+ ZON·LOC·YR + ZON·LOC·GEN·YR)
× HAR + ZON·LOC·HAR +
ZON·LOC·GEN·HAR + ZON·LOC·TRL·HAR +
ZON·LOC·TRL·GEN·HAR [7]
When several harvest years are analyzed, the effects
ZON·LOC·YR·HAR and ZON·LOC·TRL·HAR
and also the effects ZON·LOC·GEN·YR·HAR and
ZON·LOC·GEN·TRL·HAR become fully confounded (Piepho
and Eckl, 2014). This means that the effects ZON·LOC·YR·HAR
and ZON·LOC·TRL·HAR produce identical columns in the
design matrix. Thus the two effects are indistinguishable and
cannot be separated. To resolve the problem, we drop one of
each of these pairs of confounded terms from the model. It will
not make any difference which one is dropped.
Resolving the factorial structure and dropping the con-
founded effects, the model expands as:

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 GEN + ZON + HAR + GEN·HAR + GEN·ZON +
ZON·HAR + GEN·ZON·HAR:
YR + GEN·YR + YR·HAR + GEN·YR·HAR
+ ZON·YR + ZON·GEN·YR + ZON·YR·HAR
+ ZON·GEN·YR·HAR + ZON·LOC·YR +
ZON·LOC·GEN·YR + ZON·LOC·HAR +
ZON·LOC·GEN·HAR +
ZON·LOC·TRL·HAR +
ZON·LOC·TRL·GEN·HAR [8]
Three-Stage Analysis. Similar to the three-stage approach
for only one trial, the model for several trials can be derived
from the two-stage method shown above. As before, we first
compute the cultivar means per trial and harvest year in the first
stage of the analysis and average across environment effects in
the second stage. Now greater caution needs to be taken not to
mix up the environmental covariance and the genetic covari-
ance between zones in the third stage of the analysis.
As mentioned before, we cannot simply estimate cultivar
means within zones because doing this would make the cultivar–
year effects inseparable from genetic covariance between zones
in the next step. We therefore estimate GEN·YR·HAR-means
within zones in the second stage, which keeps calendar year
(YR) as well as harvest year (HAR) separate from each other and
thus avoids any commingling in the third stage. For each zone
(ZON), we fit the following model in the second stage:
GEN + HAR + GEN·HAR:
YR + YR·HAR + GEN·YR + GEN·YR·HAR
+ LOC·YR + LOC·GEN·YR + LOC·YR·HAR
+ LOC·GEN·YR·HAR + LOC·HAR +
LOC·GEN·HAR + LOC·TRL·HAR +
LOC·TRL·GEN·HAR [9]
Now that we estimate GEN·YR·HAR-means, the effect
GEN·YR·HAR needs to be taken fixed and the random
effects YR, YR·GEN, and YR·HAR can be dropped because
they are contained in the fixed three-way effect. Further-
more, dropping the confounded effects LOC·YR·HAR and
LOC·GEN·YR·HAR leads to the model:
GEN·YR·HAR:
LOC·YR + LOC·GEN·YR + LOC·HAR
+ LOC·GEN·HAR + LOC·TRL·HAR +
LOC·TRL·GEN·HAR [10]
The computed GEN·YR·HAR-means can then be evaluated
across zones in stage three with the following model.
GEN × HAR ×YR × ZON [11]
crop science, vol. 55, march– april 2015  www.crops.org
Resolving the factorial structure and taking the calendar year
(YR) as a random factor, this expands as:
GEN + HAR + GEN·HAR + ZON + GEN·ZON +
HAR·ZON + GEN·HAR·ZON: YR +
YR·HAR + YR·GEN + YR·GEN·HAR +
YR·ZON + YR·ZON·HAR + YR·GEN·ZON +
YR·GEN·ZON·HAR [12]
Model Derivation for Prediction of Cultivar
Means per Zone
Our main goal of the analysis is to be able to compute the best
estimate for the cultivar yield within every zone. Up to now,
we have taken the cultivar factor (GEN) as fixed. Applying this
model with fixed cultivar effects will lead to a BLUE of cultivar
means in a zone using only data from that specific zone. These
estimates, however, do not use the information of the neighbor-
ing zones. If there is a high correlation in the ranking of the
cultivars between the zones, several methods exist to increase
the precision of the estimated zone-specific means by borrow-
ing strength across zones. To optimize the estimation for the
cultivar yield within one zone, the information of the target
zone and the neighboring zones need to be combined. An easy
way to do this is to compute an unweighted mean for every
cultivar across the zones. It is an unweighted mean in the sense
that each zone contributes the same amount of information to
the cultivar mean across zones. Such an unweighted mean can
be calculated with a model that handles GEN as a fixed factor.
Another approach is to combine the observations of the target
zone and the neighboring zones by a weighted estimator. The
problem to solve is to find the optimal weights for the model. It
is easily noticed that some zones, especially neighboring zones,
are often agroecologically more similar to the targeted zone than
other zones. Moreover, the means computed per zone may differ
in precision between zones (e.g., depending on the number of
trials per zone). This suggests that the derived two-stage and
three-stage models should be changed in a way such that the
genetic correlation between zones, as well as the precision of
means per zone, is used to calculate weights for a weighted mean
across zones. It is shown in Piepho and Möhring (2005) that
this can be accomplished by using cultivar BLUPs (Searle et al.,
1992) based on a suitable mixed model. The modification will
lead to an optimal weighted mean which minimizes the error of
prediction for that zone and requires that cultivar is modeled as
a random factor in stage three. In the preceding stages, however,
all cultivar effects not involving other random factors must still
be modeled as fixed (Piepho et al., 2012).
The assumption of random cultivars may appear difficult to
justify for cultivars that are the subject of selection. While in a
Bayesian framework, essentially all parameters are random vari-
ables, in a frequentist setting, the notion of randomness involves
the assumption of random sampling from some parent popula-
tion. In the context of cultivar testing, we may consider the
cultivars under test as a random sample from the potential set of
cultivars that could have been obtained by repeatedly running
the same kind of breeding programs that generated the entries
under consideration (Piepho and Möhring, 2005). We would
also like to point out that the assumption of random cultivars is
603
standard in the routine analysis of cultivar trials in some coun-
tries, including Australia (Smith et al., 2005). The assumption
of random genotypic effects is also becoming very common in
the analysis of plant breeding trials (Bernardo, 2010), where the
continuing selection raises the same questions with respect to
the definition of random sampling. For a further discussion and
motivation of the use of mixed models for cultivar evaluation
under selection, see Piepho and Möhring (2006).
Several Locations, One Trial
As before, we first consider trials with the same first harvest
year (Table 2). We will start with the derivation of the model
for the two-stage analysis. The analysis uses the genotypic cor-
relations of the investigated zones, which is a measure for the
similarity of the zones, to calculate the optimal weights.
Two-Stage Analysis. We will refer back to the two-stage
Model [3] (see Model derivation for unbiased estimation, Sev-
eral locations one trial, Two stage analysis) for several locations
and one trial where cultivar BLUEs are calculated. Now, to
estimate cultivar BLUPs, all effects in model [3] involving the
factor GEN are regarded as random. We fit the following model
in the second stage:
HAR + ZON + ZON·LOC + ZON·HAR+
ZON·LOC·HAR:
GEN + GEN·HAR + GEN·ZON +
GEN·ZON·HAR + ZON·LOC·GEN +
ZON·LOC·GEN·HAR [13]
With the effects GEN, GEN·ZON, and GEN·HAR,
GEN·ZON·HAR taken as random, this model implies a com-
pound symmetry (CS) structure for the genetic correlation
between zones. That way, a constant genetic correlation between
zones is assumed for composite effects (GEN + GEN·ZON)
and (GEN·HAR + GEN·ZON·HAR). Using the same syntax
as with repeated measures, we could express this correlation
structure taking ZON as the repeated factor and GEN and
GEN·HAR as subjects (i.e., we would represent these effects by
GEN·ZON and GEN·HAR·ZON). One could use more com-
plex structures like UN or UN(1) to model the genetic correla-
tion between zones, allowing for heterogeneity between zones.
In our experience, however, more complex covariance struc-
tures often do not converge or result in similar results as data
gets sparse (Kleinknecht et al., 2013), so we here stick with the
simpler CS model and retain the simple random effects specifi-
cation in [13] for simplicity and computational efficiency.
Furthermore, all fixed effects except the effect with the
highest-order interaction can be dropped since they will be
absorbed by the latter effect. This leads to the following model.
ZON·LOC·HAR:
GEN + GEN·HAR + GEN·ZON +
GEN·ZON·HAR + ZON·LOC·GEN +
ZON·LOC·GEN·HAR [14]
604 www.crops.org
Three-Stage Analysis. Since in the second-stage, estimates
are produced by zone, the adaptation for estimating weighted
means need only be performed for the third stage of the analy-
sis. Effects HAR and ZON can be dropped and only the fixed
effect with the highest-order interaction, ZON·HAR, remains.
We fit the following model in the third stage:
ZON·HAR:
GEN + GEN·ZON + GEN·HAR
+ GEN·ZON·HAR [15]
Several Locations, Several Trials
Again we consider trials which do not necessarily have the same
first harvest year (Table 3). The derivation of the model will
be similar to the case with only one trial, such that all effects
involving the factor GEN are regarded as random and all fixed
effects except the effect with the highest order interaction can
be dropped because the higher-order fixed effect absorbs all
contained lower-order effects.
Two-Stage Analysis. To derive the model for estimat-
ing cultivar BLUPs, we will change the stage-two Model [8]
for several locations and several trials where cultivar BLUEs are
calculated and take all effects in the model involving the factor
GEN as random. We fit the following model in the second stage:
ZON·LOC·TRL·HAR:
GEN + GEN·HAR + GEN·ZON +
GEN·ZON·HAR + GEN·YR + GEN·YR·HAR
+ GEN·YR·ZON + GEN·YR·ZON·HAR +
GEN·YR·ZON·LOC + ZON·LOC·GEN +
ZON·LOC·GEN·HAR + ZON·LOC·GEN·TRL+
ZON·LOC·GEN·TRL·HAR [16]
Again, we keep the simpler CS variance–covariance structure
and model all cultivar-zone effects with simple random effects
and drop all fixed effects except the effect with the highest-
order interaction ZON·LOC·TRL·HAR.
Three-Stage Analysis. The adaptation for estimating
weighted means need only be performed for the third stage
of the analysis. For the second stage, Model [10] (see Model
derivation for unbiased estimation, Several locations, several
trials, Three stage analysis) can be used. While taking GEN
and its interactions as random, all effects not involving GEN are
regarded as fixed. Dropping all fixed effects except the effect
with the highest order interaction, ZON·HAR·YR, will result
in the model shown below.
ZON·HAR·YR:
GEN + GEN·HAR + GEN·YR + GEN·HAR·YR +
GEN·HAR·ZON + GEN·YR·ZON + GEN·ZON +
ZON·HAR·GEN·YR [17]
Again, for the reasons stated above, complex structures for cor-
relation between zones are not used for this model. The CS
structure is modeled with simple random effects.
crop science, vol. 55, march– april 2015
RESULTS
In accord with the structure in the Materials and Methods
section, we present results for the developed mixed models
for unbiased estimation of cultivar means per zone and for
prediction of cultivar means per zone. We make compari-
sons among the stagewise approaches as well as between
the two models for unbiased estimation and prediction of
cultivar means according to the resulting adjusted means
and their pairwise correlation between analyses.
Unbiased Estimation of Cultivar Means
per Zone
Several Locations, One Trial
Two-Stage Analysis. There is a wide range of covariance
structures that can be chosen to model serial correlations.
Thus, it is important to explore different variance–covari-
ance structures when using mixed models. Penalized fit
statistics, which include a term for penalizing overfitting
such as the Akaike Information Criterion (AIC), can indi-
cate how well each model fits the data compared with
other models. The model with the smallest AIC value is
considered to have the best tradeoff between the goodness
of fit and the complexity of the model and is therefore
preferred. For our data, we chose to compare three differ-
ent structures. The TOEP(1) structure (banded Toeplitz
matrix structure with off-diagonal bands set to zero) is
used for specifying the same variance component for each
harvest year and no serial correlation between harvest
years. The AR(1) model (first-order autoregressive struc-
ture) is popular in longitudinal data analysis with equally
spaced time intervals in cases where serial correlation
decreases with distance in time (Potthoff and Roy, 1964).
With only three harvest years, the pattern of covariances
or correlations for different time lags may be nearly con-
stant (Piepho and Eckl, 2014). The CS structure assumes
that the serial correlations are constant for all time lags. In
SAS, the structure of the covariance matrix can be speci-
fied using the “TYPE = covariance-structure” option.
The CS model, however, can also be implemented by fit-
ting simple random effects (see Supplemental Appendix
S1). Model fits for several locations and a single trial (first
harvest year 2010) are shown in Table 4.
In contrast to the TOEP(1) structure, the AR(1) and
CS structures model a serial correlation between har-
vest years. Thus, the high AIC values in Table 4 for the
TOEP(1) structure indicate serial correlation between
harvest years. Since the AIC values suggest that the
homogenous CS model fits best, we prefer this model for
computing cultivar means. This model has the additional
advantage that it can be fit by simple random effects. As an
example, we rewrite Eq. [3] with simple random effects,
which will result in the following expression.
crop science, vol. 55, march– april 2015  www.crops.org
Table 4. Model fit statistics for simulated data with several
locations and a single trial with homogeneous variances
between locations.
Model† Akaike Information Criterion
CS 2884.7
AR(1) 2891.3
TOEP(1) 2959.7
†
AR(1), first order autoregressive structure; CS, compound symmetry structure;
TOEP(1), banded Toeplitz matrix structure with off-diagonal bands set to zero.
Table 5. Second stage variance parameter estimates and
their standard errors for the two-stage analysis of simulated
data for a single trial and several locations.
Effect Estimate SE
ZON·LOC 166.37 93.18
ZON·LOC·HAR 157.18 46.79
ZON·LOC·GEN 46.13 8.64
Residual 48.93 4.71
GEN + HAR + ZON + GEN·HAR +
GEN·ZON + ZON·HAR + GEN·ZON·HAR:
ZON·LOC + ZON·LOC·HAR +
ZON·LOC·GEN + ZON·LOC·GEN·HAR [18]
For the analysis we used Model [18], which has a homogeneous
compound symmetry structure, between harvest years, imple-
mented by fitting simple random effects. Variance parameter
estimates and their standard errors are shown in Table 5.
This two-stage analysis, however, requires long com-
puting times and high memory usage as datasets get large.
We therefore introduce a three-stage analysis, which
entails a second level of approximation (Piepho et al., 2012)
but makes it possible to analyze rather bulky datasets in a
straightforward way and still deal with unbalanced data.
Three-Stage Analysis. For analysis, the simulated
data were assumed to have a homogeneous compound
symmetry structure, implemented by fitting simple
random effects. The variance parameter estimates and
their standard errors for the analysis with models [4] and
[5] are shown in Table 6.
Several Locations, Several Trials
Two-Stage Analysis. When we tried this model in a
two-stage analysis using all the zones, locations, and trials
of the dataset gained from the German cultivar trials, our
computer, a desktop PC with an Intel Core2 Duo E8400
processor and 4GB of RAM, operating on Windows XP,
ran out of memory. To evade such problems, we use the
three-stage Model [17].
For the simulated data, the variance parameter esti-
mates and their standard errors for the two-stage analysis
are shown in Table 7. The model assumes a homogeneous
compound-symmetry structure, implemented by fit-
ting simple random effects. For comparison, the variance
605
Table 6. Variance parameter estimates and their standard Table 8. Variance parameter estimates and their standard
errors for the three-stage analysis of simulated data for a errors for the three-stage analysis of simulated data with
single trial and several locations. three trials and several locations.
Second stage results Simu- Second-stage results
Effect Zone 1 SE Zone 2 SE lated
Effect Data Zone 1 SE Zone 2 SE
LOC 56.37 134.85 283.60 256.25
LOC·YR 168 404.09 174.00 146.10 78.85
LOC·HAR 263.12 154.71 75.96 46.10
GEN·YR·LOC 16 30.77 8.47 14.01 7.27
LOC·GEN 68.90 23.34 30.61 12.10
LOC 227 2.56 91.13 243.32 240.78
Residual 48.29 9.29 38.74 7.46
LOC·HAR 6 10.51 11.37 8.02 20.59
Effect Zone 3 SE Zone 4 SE LOC·GEN 18 29.79 13.10 8.02 8.98
LOC 156.63 146.78 168.87 213.42 LOC·GEN·HAR 12 1.48 5.24 11.01 6.30
LOC·HAR 49.27 31.75 240.38 141.78 TRL·LOC 65 35.23 28.03 37.71 40.40
LOC·GEN 43.45 17.37 41.55 16.36 TRL·LOC·HAR 41 9.75 9.70 32.88 31.59
Residual 56.81 10.93 51.89 9.99 GEN·TRL·LOC 30 19.61 9.50 34.89 10.19
Third stage results Residual 24 13.30 8.19 15.55 6.56
Effect Estimate SE Effect Zone 3 SE Zone 4 SE
Residual 44.70 8.60 LOC·YR 168 69.54 39.94 159.50 74.05
GEN·YR·LOC 16 15.34 6.44 29.94 13.06
LOC 227 140.22 151.39 15.24 65.76
Table 7. Variance parameter estimates and their standard LOC·HAR 6 5.89 14.19 2.25 7.88
errors for the two-stage analysis of simulated data with three LOC·GEN 18 0 . 20.51 13.94
trials and several locations. LOC·GEN·HAR 12 22.45 6.27 18.51 12.62
Single stage Two-stage TRL·LOC 65 67.36 51.46 74.63 49.01
Simu-
lated analysis analysis TRL·LOC·HAR 41 24.82 23.30 7.63 6.93
Effect Data Estimate SE Estimate SE GEN·TRL·LOC 30 50.25 10.45 17.99 9.99
YR 219 305.86 318.84 304.39 319.01 Residual 24 14.24 6.75 7.17 4.14
GEN·YR 18 52.46 38.77 52.19 38.77 Third-stage results
HAR·YR 3 17.68 33.13 17.59 33.11 Estimate SE
GEN·HAR·YR 21 27.06 20.85 26.94 20.85
YR 219 314.08 320.96
YR·ZON 429 303.05 152.07 301.58 152.07
GEN·YR 18 53.04 38.30
GEN·YR·ZON 13† 0 . 0 .
HAR·YR 3 15.42 29.54
HAR·YR·ZON 17 7.21 12.81 7.17 12.81
GEN·HAR·YR 21 24.39 20.11
GEN·HAR·YR·ZON 12 17.55 6.53 17.51 6.53
YR·ZON 429 352.65 150.51
YR·ZON·LOC 168 193.96 44.73 192.99 44.73
GEN·YR·ZON 13† 0 .
GEN·YR·ZON·LOC 16 18.01 3.94 18.21 3.94
HAR·YR·ZON 17 9.16 11.04
ZON·LOC 227 104.72 68.89 104.22 68.89
Residual 29.08 6.86
HAR·ZON·LOC 6 6.87 7.02 6.80 7.02
†
Variance component estimated to be zero. Hence, no standard error could be
GEN·ZON·LOC 18 12.08 6.46 11.79 6.46 computed.
GEN·HAR·ZON·LOC 12 16.77 3.78 16.87 3.78
TRL·ZON·LOC 65 48.30 18.62 50.69 18.62
TRL·HAR·ZON·LOC 41 18.38 8.75 18.40 8.75 The AIC values of model fits for different covariance
GEN·TRL·ZON·LOC 30 32.39 5.90 32.50 5.90 structures assuming homogeneity of variance among loca-
Residual 24 28.31 4.99 13.29 3.42 tions are shown in Table 9. For this dataset, we found the
†
Variance component estimated to be zero. Hence, no standard error could be model fit to be similar for the AR(1) model and the CS model,
computed.
indicating small heterogeneity in correlations for different
time lags. It is valuable to model serial correlation between
components used for the simulated dataset are included in harvest years, as can be seen from the high AIC values for
the table. the TOEP(1) structure. This is expected, as we simulated the
Three-Stage Analysis. For analysis of the simulated data to be correlated according to a CS structure.
data, we assumed a homogeneous compound symmetry
structure between harvest years, implemented by fitting Comparison of Single-Stage, Two-Stage,
simple random effects. Table 8 shows variance parame- and Three-Stage Analysis
ter estimates and their standard errors for the three-stage It was mentioned before that two-stage analysis requires
analysis of the simulated data as well as variance compo- long computing times and high memory usage when data-
nents used for the simulated dataset for comparison. sets get large. Besides the important advantage that trials
can be analyzed individually, two-stage analysis compared

606 www.crops.org crop science, vol. 55, march– april 2015

Table 9. Second-stage Model [10] fit statistics for each zone
(ZON) for three-stage analysis using simulated data with sev-
eral trials and several locations applying different covariance
structures. Bold indicates the lowest Akaike Information
Criterion (AIC) value of the applied covariance structures in
each zone.
AIC
Model †
Zone 1 Zone 2 Zone 3
CS 2145.2 2126.4 2168.1
AR(1) 2139.9 2128.8 2171.8
TOEP(1) 2203.2 2190.2 2227.9
†
AR(1), first order autoregressive structure; CS, compound symmetry structure;
TOEP(1), banded Toeplitz matrix structure with off-diagonal bands set to zero.
Table 10. Correlations for GEN·HAR mean estimates for sim-
ulated data with three trials using single-, two- and three-
stage analysis assuming homogeneity of variance between
locations.
Method
Method Two-stage Three-stage
Single-stage 1.0000
Two-stage
with a single-stage analysis can save some memory space
and computing time as well. For the simulated data with
three trials and several locations, the running time for the
single-stage analysis with PROC MIXED was 5 h and 40
min and 4 h and 36 min for the two-stage analysis using
a desktop PC with an Intel Core2 Duo E8400 processor
and 4GB of RAM, operating on Windows XP. Since plot
data is modeled in the first stage, a bigger dataset could
have been used for the two-stage analysis, whereas the
single-stage analysis was at its limits concerning memory
space. Our main goal was to develop methods of analysis
for which computation time, memory space, and con-
vergence will not be problematic for complex data. The
computing time for the three-stage analysis with PROC
MIXED was 15 s. The division of the analysis into three
parts makes the individual models smaller and less com-
plex. Single-stage and two-stage mean estimates (BLUE)
are very highly correlated. The correlation of adjusted
GEN·HAR means was also high between single-stage
and three-stage analysis and also between two-stage and
three-stage analysis (Table 10).
Prediction of Cultivar Means per Zone
All analyses presented in this section are based on a three-
stage approach.
Several Locations, One Trial
We wanted to compare the performance of BLUE and
BLUP as estimators of the cultivar effect per zone and
see what methodology for our derived three-stage models
will compute the better estimate for the cultivar–zone
yields (Table 11). To do so, we added random cultivar
crop science, vol. 55, march– april 2015  www.crops.org
Table 11. Correlation of true values, best linear unbiased
predictions (BLUPs), and best linear unbiased estimations
(BLUEs) GEN·ZON means for simulated data with one trial
and several locations. Results were obtained by a three-
stage approach.
BLUP Simulated
BLUE 0.6209 0.5453 b†
Zone 4 BLUP 0.7462 a
2154.5 †
Mean correlations (based on 100 simulation runs) in this column followed by a
2156.1 common letter are not significantly different according to a paired t test at the
5% level using a linear model with fixed effects for simulation run and estimation
2193.4 method.
effects for the simulation and thus obtained the true culti-
var values for every zone. The advantage of knowing the
true cultivar values in the simulation is that not only were
we able to compare the values of BLUE and BLUP but we
could also correlate these estimators with the true values.
For the simulated data with one trial and several locations,
we used Models [4]/[5] to obtain GEN·ZON BLUEs. The
BLUPs of GEN·ZON were estimated using Models [4]/
0.9997
[15] by a three-stage approach.
0.9997
The results show that BLUPs are more highly corre-
lated with the true (simulated) values than BLUEs.
Several Locations, Several Trials
The comparison of the performance of BLUE and BLUP
estimators was performed analyzing the simulated data
with the added random cultivar effects using a three-stage
approach. For the data with several locations and several
trials, we used Models [10]/[12] to obtain GEN·ZON
BLUEs. Estimating GEN·ZON BLUPs with Models [10]/
[17] was not that straightforward this time. Due to the
small number of cultivars in the dataset and the replace-
ment of 7 out of 10 old cultivars for newer ones every
new trial, the necessary estimates for cultivar variances
were lacking in precision. Imprecise variance estimates
result in poor estimates for cultivar BLUPs. An analysis
using BLUPs to estimate cultivar performance is therefore
recommended only when having a substantial number of
cultivars in the dataset. To compensate for this drawback
of our data and to explore the potential gain in precision
by BLUP when variance components are estimated with
good precision, we ran an additional analysis in which
the genotypic variances in Model [17] were fixed to the
value of the true genotypic variances we simulated the
data with. To confirm the difference of estimates com-
puted with BLUE and BLUP, we created 100 simulated
datasets and calculated GEN·ZON BLUPs and BLUEs for
each dataset accordingly. The correlations between the
true values and the three estimators, BLUE, BLUP with
fixed genetic variances, and BLUP, were then compared.
To do so, we set up a linear model (PROC GLM) with
effects for replicate and method and used Tukey’s test for
multiple comparisons between the three estimators.
607
Table 12. Correlation of true values, best linear unbiased pre-
dictions (BLUPs), best linear unbiased estimations (BLUEs),
and BLUPs with fixed genetic variances for simulated data
with several trials and several locations. Results were
obtained by a three-stage approach.
BLUP BLUP fixed
BLUE 0.6835 0.6889
BLUP 0.9871
BLUP fixed
†
Mean correlations (based on 100 simulation runs) in this column followed by a
common letter are not significantly different according to a Tukey-test at the 5%
level using a linear model with fixed effect for simulation run and estimation method.
We found significant differences in the correlation
averages among the three methods (Table 12). The mean
correlation using BLUE was considerably smaller com-
pared with an analysis with BLUP. Although estimates
for BLUP with estimated variance components and BLUP
with fixed variances are highly correlated, fixing variances
leads to estimates closer to the true genotypic values. This
shows that BLUP can lead to more precise mean estimates
than using BLUE, especially when cultivar variances can
be estimated precisely.
Results also indicate that BLUP outperforms BLUE.
Also, BLUP with fixed variance components is slightly
more closely correlated with the true genotypic values
than is BLUP when variance components are estimated
from the same data.
Discussion
This research was initiated as a result of efforts to adapt meth-
ods for combining information across regional yield trials
from several zones in Germany (Michel et al., 2007) to the
repeated-measures structure as arising in trials with peren-
nial crops such as grasses. A previous article (Piepho and
Eckl, 2014) focused on the analysis of the repeated-measures
structure. The present paper now accounts for the division
of a target region into zones, extending earlier proposals for
annual crops (Michel et al., 2007). Such an extension is the
subject of current research (Hartmann, 2010).
This paper presented different methods to analyze trials
with perennial species adapted for the division of a target
region into zones. Methods for the analysis of a repeated-
measures structure arising in trials with perennial crops
were already discussed in Piepho and Eckl (2014), where
the main challenge was to handle the complexity of the
datasets. For the situation when several trials with staggered
starts were performed side by side, problems with comput-
ing time and memory space arose with single-stage and
two-stage models. A three-stage analysis, in which cultivar
means per trial and harvest year computed in the first stage
are analyzed across environments in two further stages, was
derived to cope with computational problems.
To be able to compare the variance component
estimates and adjusted means for cultivar–harvest year
608
combinations of the three-stage, two-stage, and single-
stage analysis without running into convergence prob-
lems, we created simulated data using variance component
estimates from a dataset on series of regional yield trials
Simulated
with ryegrass. We demonstrated that a three-stage analysis
0.5274 c†
leads to results (BLUE) similar to single-stage or two-stage
0.7115 b analysis. Computing time and memory (RAM) required
0.7378 a were substantially smaller for the three-stage analysis. In
addition, convergence issues were not as problematic for
complex data. We therefore recommend the three-stage
method to analyze perennial crop trials on a zonal basis.
There is a wide range of covariance structures that can be
chosen to account for serial correlation that arises in peren-
nial crop trials. We compared different variance–covariance
structures, such as the TOEP(1) structure (banded Toeplitz
matrix structure with off-diagonal bands set to zero), the
AR(1) structure (first-order autoregressive structure), and
the CS structure, using AIC. The CS model, which we pre-
ferred for our dataset, can be fitted by simple random effects
and could be used in place of more complex modeling when
explicitly fitting repeated measures covariance structures.
In our analyses, we found the CS model to perform well.
When applying our framework in other areas, fit of alter-
native covariance structures should always be explored. In
particular, it is appropriate to consider models that allow for
heterogeneity of variance between years, including CSH
and ARH(1) (both are available in SAS).
In this paper, at first we have taken the cultivar factor as
fixed, which will lead to an unbiased estimation of cultivar
means (BLUE). Our main analysis goal was the ability to
compute the best estimate of cultivar yield within every
zone. If there is a high correlation in ranking of cultivars
between zones, the precision of the estimated means may
increase when the information of the target zone and the
neighboring zones are combined. If the number of cultivars
is large, we recommend using cultivar BLUPs, which use
the genetic correlation between zones, as well as the preci-
sion of means per zone to calculate weights for a weighted
mean across zones. Therefore, the models with fixed effects
were modified to calculate BLUPs by regarding cultivar
effects as random. It is shown that BLUPs lead to a better
estimate for the cultivar yield if estimates of variance com-
ponents for cultivars are precise. In the case for the simu-
lated dataset, the number of cultivars was small and esti-
mates of variance components for cultivars turned out to
be poor, adversely affecting the performance of BLUP. We
fixed the genotypic variances in the applied model to the
value of the true genotypic variances the data was simulated
with. The results affirmed our suggestion that the method
of choice for trials with a large enough number of cultivars
is to use BLUP. In trials with ryegrass cultivars, the number
of cultivars is notoriously small. For making use of BLUP,
one might also consider using long-term variance estimates
based on a larger number of cultivars, which may be more
www.crops.org crop science, vol. 55, march– april 2015
stable and precise than estimates based on a small number
of cultivars tested in a given series of trials.
Supplemental Information Available
Supplemental information is included with this article.
Supplemental Appendix S1. We here describe the
implementation of our proposed models and methods
using the SAS procedure MIXED.
Supplemental Appendix S2. This comprises a SAS file
and Excel file.
Acknowledgments
The authors wish to thank the Arbeitskreis Mitte-Süd for con-
ducting the trials and the working group IPZ 4b of the Bavar-
ian Research Institute (Freising, Germany) for coordinating
the trials and collating the data. This work was funded by the
Bavarian State Ministry of Food, Agriculture and Forestry
(A/11/03). We would like to thank three anonymous reviewers
for very helpful comments.
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