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Phenotyping for QTL Mapping

Article · January 2002

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5.2 Phenotyping for QTL Mapping
S Chandra1 and F R Bidinger2

Abstract
The reliability of QTL mapping results depends in a major way on the
achieved level of accuracy and precision of field phenotyping of mapping
population individuals. The accuracy of phenotyping determines how realistic
the QTL mapping results are. Increased precision of phenotyping increases
heritability which, in turn, increases the statistical power of QTL detection.
Using appropriate incomplete block designs and biometric analysis techniques
that effectively account for extraneous variation in the field can increase the
accuracy and precision of field phenotyping. Randomization of mapping
population individuals to field plots, although it should be faithfully followed,
may not alone guarantee bias-free phenotyping. This is due to the use of
typically closely laid out small plot sizes in field phenotyping, which induces
spatial correlation among observations from nearby plots, introducing bias in
phenotyping. Based on our experience, we strongly recommend using spatial
statistical methods to account for this spatial correlation in order to achieve
bias-free and precise phenotyping.

Introduction
Mapping of quantitative trait loci (QTL) is predicated on detecting a
significant statistical association between the phenotypes and the marker-
genotypes of individuals of a mapping population. Data on both phenotypic
value or performance and marker-genotyping of mapping population
individuals are therefore required. Phenotyping data contain information on
segregation and genetic effects of QTL. Marker-genotyping data provide
information on site-specific segregation of putative QTL on the genome. The
level of accuracy and precision of both the phenotyping and the marker-

1. Senior Scientist (Statistics), International Crops Research Institute for the Semi-Arid Tropics,
Patancheru 502 324, AP, India.
2. Principal Scientist (Physiology), International Crops Research Institute for the Semi-Arid Tropics,
Patancheru 502 324, AP, India.

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genotyping data determines the level of reliability of results from QTL
mapping. It is therefore important, to achieve a high level of reliability of QTL
mapping results, so that highly accurate and precise phenotyping and marker-
genotyping data are generated using efficient phenotyping and genotyping
protocols. This chapter presents a discussion of phenotyping protocols to
enhance the reliability and relevance of QTL mapping results.
An efficient suite of phenotyping protocols is one that, for the targeted
environmental conditions, delivers highly accurate and precise assessment of
phenotypic value or performance of mapping population individuals for the
agroeconomic traits of interest. Given a random/representative sample of ng
mapping population individuals to be phenotyped, with ng chosen to be large
enough (≈500) to achieve high statistical power for QTL detection, the
required components of the suite of phenotyping protocols are:
• A representative sample of ne environments and their optimal location;
• Number of replications nr per individual in each environment;
• An experimental field with np=ngnr plots in each environment;
• An experimental design to effectively account for extraneous variation in
experimental field; and
• Appropriate biometric techniques for efficient analysis of data.
Before any discussion on these issues, it is useful to first clarify the
meaning of the key concepts of precision and accuracy of estimates.

Accuracy and Precision

The basic phenotypic data required for QTL analyses are the estimates of
phenotypic performance of individuals in single environments and/or across
environments. The total uncertainty/error in the estimated phenotypic
performance m of any individual, with m being its corresponding true,
unknown phenotypic performance, is quantified by its mean square error
MSE(m) = E(m-m)2 = E{m-E(m)}2+{E(m)-m}2 where E is the average
value. The term E{m-E(m)}2 is the error variance of m, the square root of
which is the standard error (SE). The second term represents the square of the
bias in m. There are thus two component errors that make up the total error in
m – the SE that measures the imprecision in m, and the bias that measures the
inaccuracy in m. The smaller the SE, the higher the precision of m. The
smaller the bias, the more accurate m is as an estimate of m. It is the accuracy

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of m that is the more critical factor for realistic QTL mapping results. In
contrast, a smaller SE increases the heritability of the trait, which in turn
enhances the power of QTL detection.
The suite of phenotyping protocols to be used should be designed so as to
minimize both SE and bias in m. It is often taken for granted that the physical
act of randomization of individuals to field plots will provide unbiased/
accurate estimates of m. While this argument is mathematically valid, it
provides no guarantee of achieving bias-free estimates. The randomization
argument only allows us to proceed with data analysis as if the phenotypic
expression of an individual in a given field plot is independent of the
phenotypic expression of other individuals falling on other field plots in the
experimental area. This is highly unlikely to be the case for typical small plot
phenotyping trials for QTL mapping, however refined the randomization
scheme may be. Due to the small size of closely laid out plots, there is a
distinct possibility, despite randomization, of the phenotypic expressions of
individuals in nearby plots affecting each other and hence being biased.
Nevertheless, randomization should still be followed to avoid personal bias,
but data analysis, where appropriate, should consider accounting for the
possibility of correlated phenotypic expression of individuals in nearby plots in
order to obtain maximally bias-free estimates of m.

Number of Environments and Replications

With ng individuals, each phenotyped with nr replications in each of the ne
environments, the error variance of the observed average phenotypic
performance m of an individual is given by
sm2= (sGE2/ne)+{se2/(nenr)} (1);
where sGE2 and se2 are, respectively, the genotype-environment interaction
variance and the pooled error variance. Increasing nr only reduces the second
term, while increasing ne affects a reduction in both terms on the right-hand
side of (1). For a fixed manageable number of plots, the maximal reduction in
sm2 is theoretically achieved with nr=1, with a corresponding increase in ne. At
least ne=3 environments should be considered, selected in a manner so as to
represent one intermediate and two extreme points on the scale of expected
variation in the targeted set of environmental conditions. Though nr=1 is
optimal, it is advisable to take nr=2 so as to get an internal estimate of error

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variance in each environment and to enable estimation of sGE2, which is
important in obtaining a more realistic multienvironment estimate of trait
heritability h2 = sG2/(sG2+sm2).

Experimental Design
A statistically sound design for an experiment requires three basic ingredients
– replication and randomization of individuals, and local control of error arising
from interplot variation. These ingredients, when properly used, have three
benefits: they allow separation of signal from noise, needed to obtain unbiased
estimates of differences in phenotypic performance m of individuals; they
maximize the signal-to-noise ratio; and they deliver a valid and unbiased
estimation of level of noise/uncertainty in results. The signal is the true
differences among individuals and the noise/error is due to interplot variation.
Replication is necessary to obtain an internal estimate of experimental
error variance and to permit separation of sGE2 from error variance. Multiple
observations from within a plot do not constitute replication. Randomization
provides statistical validity to results and protection from bias. Local control of
error can be physically achieved by proper blocking of plots in a manner that
maximizes interblock and minimizes intrablock variation. It is the physical
device of blocking that, if properly carried out, helps reduce experimental
error. However, no matter how effective the blocking is, there is always some
variation left uncontrolled within blocks. As the experiment progresses with
time, it is also possible that the continuously changing nature of extraneous
environmental factors will induce additional intrablock variations in field
phenotyping experiments, if the block size is large. It is therefore safer to use
blocks of not more than 8–10 plots. Orientation of the blocks, as far as
possible, should be perpendicular to the expected gradient in the
experimental field, glasshouse bench, etc.

Replication versus Blocking

Replication simply indicates the number of plots (nr) assigned to an individual.
The basic function of replication is to deliver an estimate of error mean square
se2; it is not a device to reduce se2. Increased nr only helps in obtaining a better
estimate of se2. In attempting to reduce SE of a mean SEm=√(se2/nr), the first
thought that comes to mind is to increase nr. This increases the cost of
experimentation. In contrast, blocking is a physical method to reduce se2.

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Rarely is enough thought given to reducing SEm by reducing se2, by attempting
effective physical blocking of plots in terms of proper orientation and size.
Attempts should be made, where appropriate, to use covariance/spatial
analysis techniques for a possible additional reduction in se2.
Generalized lattice (also known as alpha) designs are suitable for
phenotyping large numbers of individuals. They are more flexible than lattice
designs that need ng to be necessarily a perfect square of some integral
number. Alpha designs also offer greater convenience for management of the
experiment and better choices to conform to expected interplot variation in
the field. For ng=300 individuals, some possible design choices are: 3 × 100,
5 × 60, 6 × 50, 10 × 30, and 15 × 20, where the first number is the block size
and the second number is the number of (incomplete) blocks per replication.
Alpha designs may also allow better use of available experimental area, as
small blocks allow much more flexibility in the layout of an experiment than
do larger blocks or classical replications.

Biometric Analysis of Data

Having generated and entered the data, we are often eager to quickly get our
results. While this is natural, haste may result in the waste of time and effort,
because a sequential plan for data analysis has not been properly thought out.
A plan for data analysis, made before undertaking actual data analysis, should
consist of the following six steps:
• First , screen and validate the data for correctness.
• Second , bring the data into a format required by the software to be used for
analysis.
• Third , understand the treatment and block-structure of the data.
• Fourth , understand the nature of the data (discrete, continuous,
percentages, etc.).
• Fifth , determine the nature of experimental and environmental factors –
fixed or random.
• Sixth , build an analysis model according to the structure and nature of data
and the nature of experimental and environmental factors.
It is only at the end of the sixth step that the actual data analysis can be
effectively and confidently begun.
In the context of phenotyping for QTL mapping, the effects of mapping
population individuals should be considered as random, with their average

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phenotypic values derived as best linear unbiased predictions (BLUPs) using
restricted maximum likelihood (ReML). The BLUPs differ from the usual
(generalized) least square means in that the former show a smaller range
among the phenotypic values of the individuals than the latter. BLUPs are
expected to represent more realistic differences among individuals’
phenotypic values as extreme phenotypic values, which might arise by chance
from a fortuitous interplay of external factors, are forced to shrink towards the
general mean. Treating individuals’ effects as random is also consistent with
the fact that the mapping population individuals constitute a representative
sample from the underlying mapping population. Considering the effects of
individuals as random is anyway necessary for a valid estimation of genetic
variance and heritability, which is required to assess the prospects for marker-
assisted selection. The effects of (incomplete) blocks should be treated as
random. The effects of environments should be considered as random if more
than eight environments are used for phenotyping, and fixed if less than eight
environments are used. As a result of genotype effects being random and
environment effects being fixed/random, the effects of genotype-environment
interactions (GEI) will be random. Analysis can be done for each environment
separately, or jointly across-environments, depending on the approach to be
used for QTL analyses. The ReML BLUPs of individuals and of GEI from an
across-environments analysis, however, may be more appropriate to use for
QTL mapping, as this will more objectively allow detecting QTL for GEI also
(Yan et al. 1998). This is because an across-environments analysis will
properly separate the effects of individuals from those of GEI.

Effect of Alpha Blocking and Spatial Analysis on

Heritability and QTL Mapping
As a result of effective blocking a decrease in error variance, and therefore an
increase in heritability of a trait, can be expected. An increase in heritability, as
noted earlier, effects an increase in the power of QTL mapping. Table 5.2.1
presents results of two analyses, one based on excluding and other on including
the effects of blocks in a 9x18 alpha design trial on pearl millet at Patancheru
conducted in 2000. Accounting for block effects (a) consistently resulted in an
increase in heritability, (b) the number of detected QTL either remained the
same or increased, and (c) LOD scores and R2 values of detected QTL
generally increased.

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Table 5.2.1. Effect of Alpha-blocking (9 plots/block × 18 blocks/replication) on
phenotyping of 160 testcrossed pearl millet mapping populations in irrigated control
and early-onset terminal drought stress environments, Patancheru dry season, 2000.
REML analyses were done with and without the block effect (+ and – block) in the
model. Numbers of QTL detected are those with LOD scores > 2.0, from a combined
model to obtain cumulative LOD score and R2 values using interval mapping in
MAPMAKER.
Cumulative LOD (R2)
Plot mean heritability Number of QTL scores of detected
(%) detected putative QTL
Variable –block + block –block + block –block + block
Irrigated control
Days to flowering 72.9 75.9 4 4 15.8 (41.1) 21.7 (51.2)
Stover yield m-2 56.2 57.3 2 4 7.9 (24.7) 13.1 (36.3)
Grain yield m-2 31.4 37.5 1 1 4.3 (13.8) 4.7 (15.2)
Biomass yield m-2 44.2 46.9 2 2 8.1 (26.4) 8.3 (26.8)
Harvest index 63.9 67.6 3 5 17.7 (45.2) 26.2 (57.7)
Early-onset stress
Days to flowering 72.0 79.3 3 4 17.7 (45.2) 22.2 (53.5)
Stover yield m-2 54.7 57.3 3 4 11.6 (33.7) 12.2 (36.0)
Grain yield m-2 53.4 58.9 4 4 17.2 (46.7) 17.3 (48.7)
Biomass yield m-2 39.7 43.8 2 4 6.0 (19.0) 9.3 (29.1)
Harvest index 68.6 71.2 5 5 27.2 (59.3) 26.2 (57.7)

In another detailed study of the effects of spatial analysis on QTL

mapping results in pearl millet with 149 F2 intercross mapping population
individuals laid out in an alpha design, it was found that strong spatial
variability existed along rows and/or columns of the trial fields. Spatial
adjustment substantially decreased the error variance and bias, and increased
heritability, the latter up to 100% for days to flowering at Nagaur in Rajasthan.
Relative ranking of spatially adjusted means for all traits analyzed was
substantially different from unadjusted alpha-design-based means. Use of
spatially adjusted means substantially increased LOD scores of detected QTL,
the increase being as much as 100% for days to flowering QTL at Nagaur. This
was accompanied by a correspondingly proportionate increase in R2 values of
detected QTL. A number of additional QTL of large effects, not detectable in
analysis based on unadjusted alpha means, were also detected. Spatial analysis,
compared to alpha blocking, provided more realistic and powerful QTL

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 mapping resulted, from use of bias-free and more precise estimates of
phenotypic values.

Bibliography
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Moreau, L., Monod, H., Charcosset, A., and Gallais, A. (1999). Marker-assisted
selection with spatial analysis of unreplicated field trials. TAG 98:234-242.
Yan, J., Zhu, J., He, C., Benmoussa, M., and Wu, P. (1998). Molecular dissection of
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