International Journal of Advancements in Research & Technology, Volume 2, Issue5, May-2013
ISSN 2278-7763
186
MORPHOLOGICAL AND GENETIC DIVERSITY OF PAKISTANI WHEAT GERMPLASM UNDER DROUGHT
STRESS
Ammar Ali1, Nawab Ali2, Imran Ali2, Muhammad Adnan3, Nimat Ullah2, Zahoor Ahmed Swati1
1
Institute of Biotechnology and Genetic Engineering, KPK Agriculture University Peshawer, Pakistan.
Department of Biotechnology and Genetic Engineering, Kohat University of Science & Technology, Kohat,
Pakistan.
3
Department of Botany, Kohat University of Science & Technology, Kohat, Pakistan.
2
Email: drammar.qureshi@gmail.com
ABSTRACT
Drought stress is a threat to agricultural productivity especially wheat (Triticum aestivum L.), a staple food in
Pakistan. In the present study, the morphological and RAPD-based molecular characteristics of twelve varieties
of Pakistani wheat were investigated as a function of drought stress. All the twelve genotypes were grown under
four irrigation treatments (T1-380 ml, T2-190 ml, T3-126 ml and T4-95 ml) in the green house of IBGE, Khyber Pakhtunkhwa Agricultural University, Peshawar and morphological parameters were studied after maturation of genotypes. The differences among the morphological parameters were found to be significant for treat-
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ments, genotypes and interaction of water regimes. Increasing level of water stress decreased the values for all
characters while only Tatara, Ghaznavi-98, ZAS-08 and ZAS-42 showed good results in water stress conditions
that are to be recognized as drought tolerance varieties. The genetic distance was calculated by unweighted pair
group of arithmetic means (UPGMA). The cluster analysis was performed and the resulted dendogram revealed
that wheat genotypes such as Tatara, 38-IBWSN-1052 and 38-IBWSN-1059 have high genetic similarity (76%)
in comparison to ZAS34 and ZAS67, which were genetically very distant (52%). The information regarding the
drought tolerant and genetically distant varieties explored and classified in this study will be a useful tool for
choosing genotypes in the wheat breeding towards a superior genotype of high productivity and stress tolerance.
Key words: Triticum aestivum L., Drought stress, RAPD markers, genetic diversity.
1 INTRODUCTION
Wheat (Triticum aestivum L.) is the most important
one of the most important factors limiting crop yields
and widely cultivated staple food crop in the world.
around the world. The drought stress negatively dis-
The wheat has been reported to have second position tress plant growth and development leading to sharp
after rice regarding the production of proteins and cal-
decrease in plants productivity
ories which are almost 72% in the average diet
resource of the world is arid or semi-arid. In Pakistan,
[1] .
[2] .
Most of the land
Although wheat can be produced in high amount, the drought is the most serious constraint for crop producdrought stress in most of the rural areas of Pakistan tion. The problem of drought is acute in the developresults in huge reduction of the expected yield.
ing countries of the world where about 37% of the
Drought is a complex phenomenon, and is considered wheat growing areas are semi-arid with low moisture
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International Journal of Advancements in Research & Technology, Volume 2, Issue 3, March-2013
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as a limiting factor for higher yield
[3] .
In Pakistan,
187
main objective of this work was to investigate the ge-
nearly 80% of agricultural land is irrigated, while the netic variations in the wheat genotypes using RAPDrest is rain fed. In case of Khyber Pakhtoonkhwa, a PCR markers and to recognize better-quality, yield and
province in Pakistan, 68.5% wheat is grown on rain drought tolerant genotypes.
fed areas and 31.5% on irrigated areas
[4] .
Under such
situation, the wheat breeders are working hard to pro- 2 MATERIALS AND METHODS
duce new genotypes, which could tolerate soil and atmospheric moisture stress at different stages of plant
2.1 MORPHOLOGICAL STUDIES
Twelve wheat genotypes including ten advanced
development.
wheat lines and two checks (Table 1) were selected to
The breeding approaches to develop new or improved
investigate their performance against drought stress.
cultivars against stress need a thorough understanding
The different wheat genotypes were cultivated in dif-
of the reactions of plant tissues or organs against the
ferent pots and were treated with different levels of
specific stress. Selection of four traits such as high water stress after every 15 days interval using four irspikes, high 100 grains weight, high weight of rigation treatments such as T1-380 ml, T2-190 ml, T3-
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seeds/spike and high biological yield are useful for
126 ml and T4-95 ml. Once the plants reached at ma-
high yield potential in wheat drought breeding pro- ture stage, seven morphological traits such as plant
grams [5] . The molecular marker has been widely used height, number of productive tillers, spike length,
in the previous studies in order to understand the number of grains per spike, yield per plant, one thouwheat genetic diversity. Several molecular markers sands grains weight and root/shoot ratio were recordhave been used in order to characterize the genetic ed. The wheat genotypes were grown in pots at the
materials in the wheat breeding programs. The use of green house of the Institute of Biotechnology and Gemolecular markers such as Random Amplified Poly- netic Engineering (IBGE), KP Agricultural University
morphic DNA (RAPD), Simple Sequence Repeats Peshawar and were exposed to various levels of water
(SSR) and Amplified Fragment Length Polymorphism stress using four irrigation treatments (T1-380 ml, T2(AFLP) is very important to estimate the genetic di-
190 ml, T3-126 ml and T4-95 ml) with 15 days inter-
versity in the agronomically important crops
val in each case. After maturation, the aforementioned
[6], [7], [8],
[9] .
The RAPD polymerase chain reaction emerged as seven morphological traits were recorded.
an effective and easier technique to trace chromosomal
segments in the translocation lines
[10] .
The investiga-
tion by RAPD primer provides practically different
number of markers to evaluate individual genotypes
and to carry out the large scale screening of genetically diverse cultivars for their better breeding
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[11] .
The
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188
minutes), the pellet was washed with 70% ethanol. The
pellet was dried for an hour at room temperature and
re-suspended in 40 microlitre TE buffer (10 mM Tris,
1 mM Na2 EDTA, pH = 8.0). The sample was incubated for 1 h in RNAase (40 mg) to remove RNA. The
pure DNA samples were stored in refrigerator for further use [12] .
2.2.2 PCR CONDITIONS AND GEL ELECTROPHORESIS
A total of five Randomly Amplified Polymorphic
DNA (RAPD) primers (obtained from GeneLInk
Inc. NY 10532, USA) were used to detect genetic
polymorphism at DNA level during this study (see
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Table 2). All the PCR reactions were carried out in
25 µl containing 200 ng total genomic DNA, 0.25
2.2 MOLECULAR STUDIES
µM of each primer, 200 µM of dNTPs, 50 mM
KCl, 10 mM Tris, 1.5 mM MgCl2 and 2.5 units of
2.2.1 DNA EXTRACTION
Taq DNA polymerase
[13] .
Amplification condi-
Three to four fresh leaves were collected from plants
tions were; an initial denaturation (94oC, 5 min),
and DNA was extracted by a slight modification in the
followed by 30 cycles each consisting of a dena-
CTAB method
The leaf material (100 mg) was
turation step (94oC, 1 min), annealing temperature
crushed with knitting needle to make fine powder and
according to primers Tm for 1 minute and an ex-
homogenized in 200 µl extraction buffer (1% SDS,
tension (72oC, 2 min). The last cycle was followed
100 mM NaCl, 100 mM Tris, 100 mM Na2 EDTA, pH
by 10 minutes extension at 72oC. All the amplifi-
= 8.5 by HCl). Equal volume (500 µl) of phenol, chlo-
cation reactions were performed using GeneAmp
roform and isoamyl alcohol (25:24:1) was added, the
PCR System 2700 (Applied Biosystem) program-
homogenate was gently mixed and centrifuged at
mable thermocycler. The amplification products
15,000 rpm for 5 minutes. The upper aqueous phase
were electrophoresed on 1.5% agarose/TBE gel,
(200 µl) was transferred to a new tubes and DNA was
and visualized by staining with Ethidium Bromide
precipitated with one tenth volume (20 µl) of 3 M So-
under U.V light and were photographed using gel
dium acetate (pH 4.8) and 2.5 volume (500 µl) of iso-
documentation system “Uvitec”.
[12] .
prophenol. After centrifugation (15,000 rpm for 5
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189
height values observed in decreasing order were
69.734 cm (T1), 55.348 cm (T2), 48.811 cm (T3) and
42.152 cm (T4). By increasing the water stress, the
plant height was decreased for all the genotypes under
study. This kind of reduction in plant height under
drought stress was also reported by Shalaby et al
(1982)
[15] .
An important feature of the development
of wheat plant is its ability to produce tillers i.e. lateral
branches which enables the plant to respond to variation in density of sowing. Similarly, in response to wa2.3 STATISTICAL ANALYSIS
ter stress, numbers of tillers decreased, that is support-
The data collected for morphological parameters were
analyzed by applying F test and differences among varieties, treatments and interaction of treatments with
ed by by Keim and Kronstad (1981)
[16] .
Tatara,
Ghaznavi-98, ZAS-08, ZAS-42 were highly tolerant
wheat genotypes as they produced high number of
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genotypes were tested at 5% probability.
productive tillers. The drought stress was reported to
The bands detected by each primer pair was interpret-
have a negative effect on the grains per spike and
ed as a genetic locus. The locus was scored 1 as pre-
spike length
[17] .
In present study, the mean spike
sent and 0 as absent. The bi-variate 1-0 data matrix length was decreased with the increase in drought
was developed. The molecular data was analyzed us-
stress. The highest mean spike length was reported for
ing Nie and Lie formula for the calculation of genetic
Tatara (8.675 cm) and smallest mean spike length for
distance using the unweighted pair group of arithmetic
ZAS-34 (5.625 cm).
mean (UPGMA) procedure, while a dendrogram was
The yield per plant under drought stress was high for
constructed using Genetyx win software program [14] .
Tatara, Ghaznavi-98, ZAS-08, and ZAS-42 in comparison to other genotypes. The poor yield of ZAS-34,
ZAS-67, 38-IBWSN-1077 and 26-ESWYT-124 under
3 RESULTS AND DISCUSSION
3.1 MORPHOLOGICAL STUDIES
drought stress can be related to their inability to avoid
or tolerate stress. The mean data pertaining to grain
yield per plant is given in Table 3, which revealed sig-
The present investigation revealed that the varieties
and treatments differences for plant height were significant (Table 3). Tatara exhibited the tallest mean
plant height (66.987 cm) while 26ESWYT-124 was
nificant differences in the yielding potentiality of various varieties under study. The reduction in grains yield
due to drought stress has been supported by
Gebeyehou and Knot
[18], and
Hooker et al [19] .
the shortest (47.860 cm). In case of treatments, the
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to be significant for treatments, genotypes and interaction of water regimes. The increasing level of water
stress decreased the values for all the studied morphological parameters. The greater values were recorded
for T 1 followed by T 2 , T 3 and T 4, respectively. The
ranking of genotypes based on morphological characteristics was recorded as Tatara, ZAS-08, ZAS-42, 38IBWSN-1052, SCO-27, Ghaznavi-98, 26-ESWYT124, 38-IBWSN-1059, 38-IBWSN-1077, ZAS-67,
ZAS-70 and ZAS-34.
3.2 MOLECULAR STUDIES
The genotypic differences for 1000 grain weight were All the genotypes revealed various levels of genetic
significant and these differences may be due to genetic polymorphism for the loci detected by using five
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diversification among the genotypes. As for as the
RAPD primers. A total of 134 bands (alleles) were ob-
treatments are concerned, it is evident that treatments
served in twelve genotypes giving an average of 26.8
significantly affected the grain weight of various vari-
bands per genotype. Some primers are highly poly-
eties. Most probably, it can be assumed that optimum morphic among different wheat genotypes [23] . Acsoil moisture at T1 resulted in increased spike length cording to this work, among the five primers, the priwhile under the reduced moisture level in T2, T3 and
mer GLF10 yielded an average maximum number of
T4, due to stunted growth smaller grain weight with bands (2.50) per genotype, while primer GLG20
smaller spike length was obtained. This is also sup- yielded lowest number of bands on average (1.84) per
A most
genotype. The Nei’s and Lei’s coefficient similarity
common observation concerning roots under drought
matrix was used to estimate the genetic distance
stress is the increase in root/shoot dry matter weight
among wheat genotypes. It was observed that the most
ratio
In the present study root/shoot ratio was in-
similar genotypes were SCO-27 and ZAS-70 (86%)
creased with increase in drought stress. The increase
while the most dissimilar genotypes were 26-ESWYT-
in ratio results from the relatively greater decrease in
124 and Ghaznavi-98 (17%) as shown in (Table 4).
shoot growth than in root growth under drought stress.
Furthermore, breeders during breeding programs usu-
ported by the findings of of Swati et al
[21] .
[20] .
However, in some rare cases root weight increased in ally share breeding material and use genetically similar parents which may lead to a concern of lack of geabsolute terms under drought stress .
[22]
The differences for all of these characters were found
netic diversity
[24], [25] .
The pyramiding crosses are
recommended to increase the genetic diversity in difCopyright © 2013 SciResPub.
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191
ferent populations and will be useful tool for choosing
genotypes in wheat breeding programs [26] .
4 CONCLUSION
It has been observed that drought stress imposed at
different growth phases leads to different effects on
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wheat cultivars. In the present study, Tatara, Ghaznavi-98, ZAS-08 and ZAS-42 wheat genotypes showed
maximum drought tolerance in comparison to ZAS-
The cluster analysis was carried out and dendogran
was generated using Genetyx Win software and the
cluster analysis of genetic similarity matrices revealed
that the wheat genotypes under study could be divided
into two main groups (Figure 1). Ghaznavi-98 and Tatara were clustered in same group showing more genetic similarity (76%) between each other, while ZAS34 and ZAS-67 wheat genotypes were genetically very
distant i.e. with 52% genetic similarity. It has been reported that molecular marker technology enables the
identification and characterization of stress tolerance
34, ZAS-67, 38-IBWSN-1077 and 26-ESWYT-124. In
similar way, the wheat genotypes such as ZAS-34,
ZAS-67, 26-ESWYT-124 and Ghaznavi-98 were
found genetically very distant in comparison to Tatara,
38-IBWSN-1052,
38-IBWSN-1059,
SCO-27
and
ZAS-70. The high drought tolerant and genetically
very distant varieties reported in this work can be used
in future breeding programs to increase the genetic
variability in Pakistani wheat germplasm and produce
a new breed with high productivity and stress resistance.
varieties during different phases of plant development
and determination of genetic relationship among toler-
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