Vol.1, No.3, 131-142 (2010) Agricultural Sciences
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
Comparative evaluation of maize inbred lines (zea mays
l.) according to dus testing using morphological,
physiological and molecular markers
Vinod Kumar Yadav*, Indra Sen Singh
Department of Genetics and Plant Breeding, G.B. Pant University of Agriculture and Technology, Pantangar, India;
*Corresponding Author: vinodbreeder@in.com
Received 22 June 2010; revised 3 August 2010; accepted 7 August 2010.
A major challenge facing those involved in the
testing of new plant varieties for Distinctness,
Uniformity and Stability (DUS) is the need to
compare them against all those of ‘common
knowledge’. A set of maize inbred lines was
used to compare how morphological, physio-
logical characterization and RAPD molecular
marker described variety relationships. All the
inbred lines were confirmed as morphologically
and physiologically distinct. At morphological
level the maximum genetic distance (10.8) and
least genetic distance (1.6) were found. For
physiological characters distance varied from
0.35 to 1.92 and results from dendrogram, which
was made on the basis of dissimilarity matrix,
were grouped into five major clusters. From
RAPD, random primers provide polymorphic
amplification products; the distance varying
0.42 to 0.65 and dendrogram showed that these
lines formed close clusters due to the less
variation in these lines at molecular level. In the
present study, the molecular markers also ex-
posed useful genetic diversity and the visual
displays appeared to disperse the lines some-
what more evenly over the plot than the mor-
phological and physiological methods.
Key words: DUS; Inbred Lines; Maize; Markers;
Genetic Distance; Diversity
Maize is grown world wide on an approximately 161
million ha annually with a production of 685 million
metric tonnes [1]. It occupies an important position in
the world economy and trade as a food, feed and an in-
dustrial grain crop. Several million people in the devel-
oping world consume maize as a staple food and derive
their protein and calories requirement form it. Develop-
ment of high yielding hybrids is the most important ob-
jective of any maize breeder to enhance productivity. For
developing hybrids, better inbred lines with high mean
performance are required.
DUS Testing is one of the important criteria to test in-
bred lines for distinctness, uniformity and stability.
DUS Testing of cultivars is one of the requirements for
granting Plant Breeders Rights (PBR) and it is con-
ducted according to national guidelines prepared on the
basis of UPOV guidelines. The system accepted and in
operation in a large number of countries is as provided
by UPOV. Information is, thus, generated on the basis
of internationally accepted and followed norms,
thereby providing a basis for appropriate comparison of
materials identified under the national agricultural re-
search system (NARS) along side materials from other
Maize inbred lines represent a fundamental resource
for studies in genetics and breeding and are used exten-
sively in hybrid corn production [2,3]. Inbreds have also
been critical for molecular evaluation [4,5]. Knowledge
of genetic diversity in maize germplasm helps to ensure
that a broad genetic base of breeding materials is main-
tained, not just for sustaining genetic improvement but
also for reducing genetic vulnerability to pests and dis-
eases. This information may be obtained from pedigree
and test cross data, morphological and biochemical traits
or molecular markers and it is important for maximizing
heterosis because molecular markers can characterize
lines directly and precisely at the DNA level. They can
help maize breeders in efficiently assigning lines to het-
erotic groups and guide them in the choice of parents for
the development of new hybrids. Several types of mo-
V. K. YADAV et al. / Agricultural Sciences 1 (2010) 131-142
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
lecular markers are available for evaluating the extent of
genetic diversity in maize. These include (RFLP, RAPD,
AFLP, SSR, etc). The assessment of genetic diversity
within and among populations has been the concern of
several researchers in the past and it is especially impor-
tant for plant genetic resource management [6,7].
The objective of this study was to determine the po-
tential utility of morphological, physiological and RAPD
markers for application in research, product develop-
ment, seed production, intellectual property right (IPR),
and genetic resource conservation management in maize.
To accomplish this goal, we report molecular profile and
pedigree data for a set of 30 inbred lines. We assessed
the discrimination ability of data obtained from mor-
phology, physiology and RAPD; and compared inbred
lines that are revealed by these data with association that
would be expected on the basis of known pedigrees. We
also discuss the cost of effectiveness of acquiring RAPD
data with respect to the potential use of this technology
by breeders and conservators.
Morpho-agronomic studies and RAPD characteriza-
tion of maize inbred lines on UPOV harmonized charac-
teristics, generally as per DUS test guideline [8,9] were
1) Seed material used and test conditions
The experimental material used for the present study
comprised 30 maize inbreeds (data not shown) devel-
oped under the auspices of GBPUA&T, Pantnagar and
used in hybrid breeding programme, were grown at the
Crop Research Centre, G. B. Pant University of Agricul-
ture and Technology, Pantnagar. Two evaluation trials
were conducted during Kharif, 2002 and Kharif, 2003.
In addition, these lines were selfed and selfed seed was
harvested separately, for molecular analysis. Recom-
mended package of agronomic practices and plant pro-
tection measures were adopted.
2) Characteristics used for morphological and physio-
logical evaluation
UPOV’s DUS test guidelines [10,11] were generally
followed beginning from the trial layout to recording of
the last field related observation. In UPOV [11] 34 mor-
phological and physiological characteristics to be re-
corded in maize at different stages of plant growth are
given. Out of these, 12 characteristics are marked with
asterisk (*), which means that characteristics have to be
compulsorily observed in every environment. Keeping
this in view, a total number of 27 characteristics were
selected for observations. Characters considered for
testing of inbred lines [12] are given in Table 1.
3) Molecular characterization
The same set of 30 inbred lines used in the morpho-
logical & physiological study, were used for molecular
characterization by RAPD analysis. Leaves from 6-8
plants (3-4 leaf stage) were bulked and ground to a fine
powder with liquid nitrogen using a mortar and pestle.
DNA was extracted using a modified CTAB procedure
[13]. Chemicals, glasswares and instruments used for
this study were as per standardized source.
2.1. Amplification and Detection
Approximately 10 ng of DNA was used as template
for PCR in 25 µl reaction containing 1X PCR buffer, 200
µM each dNTPs, 0.76 U Tag polymerase and 30 ng of
primer (PCR buffer, dNTPmix, Taq polymerase and
primers were from Bangalore Genei Pvt. Ltd.). The de-
tails of lot number and sequence of primers are given in
Table 2. Amplification was carried out using eppendorf
thermocycler by the following amplification conditions
i.e., initial denaturation at 94°C (5 min.); 36 cycles of
94°C (30 sec.) denaturation, 34°C (1min) annealing, and
72°C (1 min) extension; and then a final extension at
72°C (5 min). 2% Agarose gel was prepared for frac-
tionation of RAPD markers. During preparation of Aga-
rose gel 5 µl Ethidium bromide stack (10 mg/ml) was
added. DNA template and dye were loaded in 5:1 ratio
and electrophoresis was done at 60 V for 4-5 hrs in 1X
TBE buffer.
2.2. Gel Scoring
The amplification products were scored separately for
each primer. The bands observed on the gel were com-
pared across the lanes for products with similar molecu-
lar weight, based on specific molecular weight marker.
The bands were scored for the presence or absence by
binary coding, i.e., assigning a value of 1 for presence
and 0 for absence in a lane.
2.3. Data Analysis
To determine the relationships among lines, we calcu-
lated similarities (dissimilarities) between inbred lines
from different data sets as follows:
1) Pair-wise comparisons for understanding ‘clear
distinguishability’ among inbreds:
The single values obtained for each characteristic for
each plant were compared with each other inbred over
the set of seven characteristics. A characteristic was con-
sidered to be clearly distinct between the pair of inbreds
under comparison, if there was no overlapping of the
description and there was a clear difference of at least
V. K. YADAV et al. / Agricultural Sciences 1 (2010) 131-142
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Table 1. Characters used in DUS testing of inbred lines.
Sl. No. Characters and code Stage of observation Expression Score
A. Physiological characteristics
Poor 1
Good 2
1. Early plant vigour PLT_VGR To be recorded after 25 days of sowing
Very good 3
Lax 3
Medium 5 2. Tassel texture, TAS_TXT To be recorded after tasselling
Dense 7
Pink 1
Green 2
Light purple 3
3. Tassel anther glume colour, ANT_GCLR -do-
Purple 4
Absent 0
4. Tassel-glume base colour, GLUM_CLR -do-
Present 1
Green 1
Pink 2
Red 3
5. Silk colour at emergence, SILK_CLR To be recorded 5-6 days after silking
Purple 4
Yellowish green 1
Light green 2
Green 3
6. Leaf colour, LF_CLR To be recorded at full-foliage stage
Dark green 4
Erect 1
7. Leaf orientation, LF_ORI -do-
Drooping 2
Absent 0
8. Leaf pubescence, LF_PUB -do-
Present 1
Smooth 1
Leathery 2 9. Leaf texture, LF_TEXT -do-
Normal 3
Absent 0
10. Anthocyanin pigmentation, ANTH_PIG -do-
Present 1
Poor 3
Intermediate 5 11. Husk cover, HUSK_CVR To be recorded using ears on five random
Good 7
Cylindrical 1 12. Ear shape, EAR_SHP To be recorded after harvesting
Cylindrical-Conical 2
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Conical 3
Round 4
Regular 1
Irregular 2
Straight 3
13. Kernel row arrangement, KER_ARR -do-
Spiral 4
14. Kernel colour, KER_CLR -do- White 1
Yellow 2
Variegated 3
Orange 4
15. Grain shape, GRN_SHP -do- Shrunken 1
Round 2
Indented 3
Pointed 4
16. Grain size, GRN_SIZ -do- Small 3
Medium 5
Bold 7
B. Morphological/Quantitative characters
17. Days to tasseling, DAY_TASS
To be recorded as number of days from
sowing to when 50% of the plants have
shed pollen. Pollen shading on the central
axis is recorded as tassel emergence
18. Days to silking, DAS_SILK
Number of days from sowing to when silks
have emerged on 50% of the plants. Silk
emergence in plants is recorded as days to
19. Tassel branching, TASS_BRN To be recorded after tasseling
20. Plant height (cm), PLT_HGT To be measured from ground level to the
base of the tassel (after milk stage)
21. Ear height (cm), EAR_HGT To be measured from base of the plant to
the point bearing the first ear
22. Ear length (cm), EAR_LT To be measured as distance from the base
of the tip of the ear
23. Ear width (cm), EAR_WD
To be measured at the central part of the
upper most ear as maximum girth of the
24. Number of kernel rows, KER_ROW To be recorded as number of kernel – rows
in the central part of the uppermost ear
25. Number of kernels/row, KER_PROW To be recorded as average number of ker-
nels/five – rows of five respective ears
26. 100 seed weight (g), SED_WGT To be recorded after harvesting
27. Grain yield / plant (g), YLD_PLT Average yield of five random plants are
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Table 2. Primer codes and sequence.
Sl. No. Primer code Sequence
one character-state, another criterion for clear distin-
guishability in UPOV guidelines [10].
2) Morphological and physiological comparison of
inbred lines:
The mean values for eleven morphological characters
and scaling values for physiological characters were
used to assess dissimilarity between inbred lines. The
matrix of all the characteristics was standardized and
used to calculate euclidean distances among the inbreds.
A dendrogram was constructed using Sequential Ag-
golomerative Hierarchical and Nested clustering Analy-
sis (SAHN) to provide a general visualization of rela-
tionship between inbreds on morphological and physi-
cogical characteristics.
3) RAPD analysis to establish distinctness:
From the presence/absence of bands, matrix of data on
the basis of their Rm values, similarity coefficients
among the inbreds were calculated following [14]. As
shown in (1).
PCR amplification data from the samples that were
inconsistent were not included in the final statistical
analysis. The similarity matrix was also subjected to
SAHN clustering analysis to construct dendrogram.
All the numerical taxonomic analyses with respect to
morpho-agronomic and RAPD were performed using the
SAS software, and NTSYS-pc software [15].
3.1. Pair-Wise Comparison for Understanding
‘Clear Distinguishability’ among Inbreds
Data on each plant from different inbred lines were
subjected to measure clear distinguishability by a dif-
ference of at least one character-state in different pos-
sible pairs of 30 inbreds. Table 3 shows number of in-
bred pairs in which different characteristics appeared to
contribute towards ‘clear distinguishability’ provid- ing
the final ranking of different characteristics for dis-
crimination among different inbreds. There was a wide
range of distinguishability across the inbred lines. Tas-
sel branching, which is the key characteristic for use of
different inbreds as male parents in hybrid production
occupied first position. Other characteristics, such as
plant height, kernels/row, ear height, ear length and ear
width for which good degree of uniformity has been
maintained also occupied good position in ‘clear distin-
3.2. Morphological Characters
The mean values for 11 morphological characters were
subjected to dissimilarity analysis. The dissimilarity ma-
trix Table 4 and on the basis of matrix dendrogram was
constructed Figure 1 to provide general visualization.
Given this diversity, all 30 lines were found to differ
Table 3. Importance of different characteristics in terms of
their contribution towards ‘clear distinguishbility’.
NO. Characteristics Number of pairs
separated rank
1. TASS_BRN 396 1
2. PLT_HGT 384 2
3. EAR_HGT 367 4
4. EAR_LT 364 5
5. EAR_WD 333 6
6. KER_ROW 311 7
7. KER_PROW 383 3
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o. ofmatchingbandsintwolanescompared
Similarity Index =Total number ofbands (1)
from each other industrial. The genetic distance between
inbred 2 and inbred 12 was the least (1.6). The maxi-
mum genetic distance was noted between inbred 17 and
25 followed by inbred 10 and 17; and 15 and 17. Den-
drogram resulting from cluster analysis of 30 inbred
lines could be primarily divided into three major groups.
3.3. Physiological Characters
For physiological characters different scaling values
were used to assess dissimilarity between inbred lines.
The results obtained by this analysis is presented in Ta-
ble 5 (dissimilarity matrix) and Figure 2. (dendrogram)
respectively. Genetic distance varied from 0.35 to 1.92
for 30 inbred lines. The minimum distance indicating
closely related inbred lines was between inbred 13 and
23 (0.35). Two inbred lines that highly differed from
each other were 2 and 20 (1.92). Dendrogram made on
the basis of dissimilarity matrix showed five main clus-
ters, and within these clusters different number of lines
were present.
3.4. Feasibility to Establish Distinctness
Using Rapd
The bands generated from the 10 primer combinations
across 30 inbred lines were used to work out genetic
distance. Random primers provide highly polymorphic
amplification product. The distance varied from 0.42 to
0.65 Table 6. Between inbred lines 27 and 29 it was the
least (0.42). The farthest genetic distance was found
between inbred lines 2 and 20; 2 and 28; and 3 and 16.
In general, inbred 2 was genetically more distinct and
diverse from other lines under study.
The dendrogram of 30 lines is shown in Figure 3.
These lines formed closed clusters due to reduced
amount of variation between them. Lines did not cluster
according to source population, which has been reported
in other studies also showing large amount of diversity
within the source populations relative to between popu-
lations [16]
3.5. Discussion
The question of Plant Variety Protection (PVP) has
been brought into worldwide focus by the agreement on
Trade Related Aspects of Intellectual Property Right
(TRIPS), which is a part of GATT (General Agreement
on Tariffs and Trade) Agreement establishing the World
Trade Organization (WTO) in 1995. The PBR concept is
based on the realization that if commercial plant breed-
ing is to be encouraged for the benefit of agriculture and
society, measures have to be taken to allow breeders to
profit from their product [17]. Further, it makes possible
to define a plant grouping with sufficient specificity to
allow the unambiguous assignment and enforcement of
property rights.
Analysis of genetic diversity and of relationship
among the elite breeding materials can significantly aid
in crop improvement [18]. In maize, this information is
useful in planning for hybrid and line development, as-
signing lines to heterotic groups and in plant variety
protection [19], molecular markers are more powerful in
assessing genetic diversity in comparison with the mor-
phological data, pedigree data and biochemical data,
because these markers reveal differences at the level of
DNA [20]. The lines used in this study were a small but
representative sample of existing commercial hybrids,
and so typified the kind of diversity encountered by the
testing authorities conducting registration tests. They
were all morphologically and physiologically distinct, as
would be expected.
In the present study, the molecular markers also ex-
posed useful genetic diversity, and the visual displays
appeared to disperse the line somewhat more evenly
over the plot than the morphological and physiological
method. However, there was little agreement on variety
relationships between the morphology, physiology and
the molecular methods. Other workers have reported a
distances [21-24]. Lines that display high phenotypic
dissimilarity need not be genetically dissimilar. The pur-
pose of pre-screening would be to subdivide candidate
varieties into groups, so reducing the number of controls
and pair-wise comparisons that have to be examined in
the morphology test. However, this process assumes that
the pre-screening characters guarantee that varieties
placed in different groups are distinct in the morpho-
logical characters used for registration. Clearly, this
would not be the case as the present study showed that
molecular and morphological differences were not cor-
related. Therefore, using molecular markers as grouping-
characters would by default, require acceptance of their
use as a distinguishing characters, at least for the most-
divergent inbred lines. An alternative way to deal with
the poor correlation between genetic and morphological
distances could be to select only molecular markers
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Table 4. Pair-wise dissimilarity matrix of 30 inbred lines based upon morphological variables.
V. K. YADAV et al. / Agricultural Sciences 1 (2010) 131-142
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Table 5. Pair-wise dissimilarity matrix of 30 inbred lines based upon physiological variables.
V. K. YADAV et al. / Agricultural Sciences 1 (2010) 131-142
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Table 6. Pair-wise dissimilarity matrix based upon Jaccard’s coefficient of 30 inbred lines by RAPD analysis.
V. K. YADAV et al. / Agricultural Sciences 1 (2010) 131-142
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Figure 1. Dendrogram of 30 inbred lines based upon mean of 11 morphological variables.
Figure 2. Dendrogram of 30 inbred lines based upon mean of 16 physiological variables.
V. K. YADAV et al. / Agricultural Sciences 1 (2010) 131-142
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Figure 3. Dendrogram of 30 inbred lines based upon RAPD analysis.
linked to phenotypic traits in DUS testing.
The diversity patterns of the inbred lines revealed a
large amount of diversity that did not allow a clear-cut
distinction between groups. This case is similar to that of
the CIMMYT populations, which served as germplasm
sources for many of the Asian lines [16], where a large
amount a diversity within, relative to between, source
populations was observed. On the other hand, the het-
erotic groups in the US and European temperate maize
were clearly differentiated in previous studies using
RFLPs and SSRs [23-27].
This study is an initial attempt to characterize the
breadth of germplasm diversity, from which we con-
cluded that breeding activity at Pantnagar has not caused
a decline in the overall amount of diversity in the inbred
lines. In sum and substance, it can be stated that al-
though the work had concentrated on DUS testing, it is
myth and less a reality. There are only small number of
descriptors available in released and notified cultivars in
India and their parental lines. If an attempt is made by
considering a large number of descriptors, establishment
of ‘clear distinguishability for each material may not be
difficult. Morphological markers and molecular markers
with insufficient primers do not generate sufficient di-
versity in the population. So sufficient primers which
cover whole genome should be used in the further stud-
ies on DUS testing.
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