Volume 8, Number 7, July 2014 (Serial Number 75)
4. Summary
For the evaluation of the joint environmental impact, the dendroindication method is used. The spruce reference reaction in 20 years (from 1989 to 2008) due to environmental changes is expressed by the cumulative and current additional increment, which depends on the location of the stand and its morphometric characteristics. The Multiple Linear Regression model is developed, which describes the size of the environmental impact, as the arguments using indices inherent to all spruce stands of Kurzeme (stand age, average height, average diameter of the stand, the height above sea level and site index class). The unexplained information by the model expresses the specific impact value of each sampling plot. This category is represented by the effect of (local) factors specific only for the stand of each sampling plot (management regime, hydrological regime, soil characteristics, the location in the area, industrial pollution of the air, etc.). During the research planning, there was assumed that the essential impact of the polluted air comes from the industrial facilities located near Mažeikiai, Liep āja and Brocēni. The analysis of
the specific impact of the environment of each sampling plot allows a conclusion about a significant impact of the Mažeikiai Oil Refinery on the productivity decline of spruce stands of the vicinity in the period from 1989 to 2008.
References
[1] Council Directive 1999/30/EC of 22 April 1999. “Relating to Limit Values for Sulfur Dioxide, Nitrogen Dioxide and Oxides of Nitrogen, Particulate Matter and Lead in Ambient Air.” Official Journal of the European Communities L163: 41-60. [2] Лиепа И, Я. 1980. Динамика древесных ресурсов. Прогнозирование и экология (in Russian) (Stand Volume Dynamics. Forecasting and Ecology). Рига: Зинатне.
[3] Schweingruber, F. 1996. Tree Rings and Environment Dendroecology. Berne: Paul Haupt Verlag. [4] BALTI Group 2012. Latvia's Forests During 20 Years of
Independence. BALTI Group.
[5] NGO 2012. Forest Sector in Latvia. Za ļās mājas: NGO. [6] Sokal, R. R., and Rohlf, F. J. 1995. Biometry: The
Principles and Practice of Statistics in Biological Research. 3 rd edition. New York: W. H. Freeman and Company.
[7] Balmori, A. 2009. “Electromagnetic Pollution from Phone Masts. Effects on Wildlife.” Pathophysiology (16): 191-199.
[8] Balodis, V., Br ūmelis, G., Kalviškis, K., Nikodemus, O., Tjarve, D., and Znoti ņa, V. 1996. “Does the Skrunda Radiolocation Station Diminish the Radial Growth of Pine Trees?” The Science of the Total Environment 180 (1): 57-64.
[9] Liepa, I. 1996. Pieauguma M ācība (in Latvian) (Forest Increment Science). Jelgava: LLU. [10] Антанайтис, В., and Загреев, В. 1981. Прирост леса (in Russian) (Forest Increment). Москва: Лесная промышленность.
[11] Bi čevskis, M. 2005. “Egļu Astoņzobu Mizgrauzis, tā Izrais ītie Bojājumi un Ierobežošanas Metodes (in Latvian) (Spruce Bark Beetle, the Resulting Damage and Containment Methods).” R īga: Latvijas valsts meži.
[12] Bi čevskis M., 2006. Egļu Astoņzobu Mizgrauža Savairošan ās Ierobežošanas Sekmju Novērtējums AS LVM Vald ījumā Esošajos Mežos Pēc 2005. g. Janvāra
V ētras (in Latvian) (Spruce Bark Beetle (Ips typographus L.) Limiting Progress Assessment in Stock Company LVM Forests After the 2005th January Storms). Salaspils:
Latvijas Valsts Mežzin ātnes Institūts “Silava”.
July 2014, Vol. 8, No. 7, pp. 582-592
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
On-farm Evaluation of Effect of Organic and Mineral Fertilizers on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa
1 2 Hide Omae 3 , A. K. Saidou and Satoshi Tobita
1. Tropical Agriculture Research Front (TARF), Japan International Research Center for Agricultural Sciences (JIRCAS), 1091-1 Maezato-Kawarabaru, Ishigaki, Okinawa 907-0002, Japan 2. National Institute of Agronomic Research of Niger (INRAN), Niamey, PO Box 429, Niger 3. Crop, Livestock and Environment Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba,
Ibaraki 305-8686, Japan
Received: April 18, 2014 / Accepted: July 14, 2014 / Published: July 31, 2014.
Abstract: To determine the best combinations of organic and mineral fertilizer applications under resource-limited conditions in the Sahel, West Africa, on-farm experiments for two years in the Fakara region of western Niger is conducted. Ten treatments were tested; six of them received one of three organic fertilizers (none, millet husks, or manure), with or without mineral fertilizer; two grew millet/(dual-purpose) cowpea intercrops with mineral fertilizer in 1:1 or 4:4 row arrangement; one grew a millet/ (spreading) cowpea intercrop with manure; and one grew a millet/hibiscus intercrop with millet husks. Manure increased total biomass by 127%-147% (P < 0.001) and millet yield by 130%-184% (P < 0.01). Mineral fertilizer increased total biomass by 27% (P < 0.01) and millet yield by 24% (P < 0.05) in 2008. The N balance was greatest and positive in the manure treatment (P < 0.001), followed by millet husks. Mineral fertilizer on the intercropped dual-purpose cowpea increased the agronomic N use efficiency of millet by 329%-483% (P < 0.01) compared with manure. The use of small quantities of mineral fertilizer on the intercropped dual-purpose cowpea, therefore, is a best combination for limited N-resources-farmers. Single manure, millet husks plus mineral fertilizer, or year-alternative-application are also recommendable depends on farmers accessibility to the resources.
Key Words: Millet, cowpea, intercrop, manure, mineral fertilizer, millet husks, sandy soil, nitrogen use efficiency.
1. Introduction land productivity and soil fertility [3].
Pearl millet (Pennisetum glaucum), an important The soil in the Sahel, West Africa, is sandy, with a food crop in the rainfed Sahelian environment, low nutrient-holding capacity and low levels of P, N produces a moderately reliable grain yield [4]. Millet [1], and organic C. Additional environmental is traditionally cropped with cowpea (Vigna constraints, notably high temperatures and low annual unguiculata). Both crops are sown at very low rainfall (300-800 mm), force farmers to practice densities (< 5000 hills/ha) with no fertilizer [5]. low-input forms of agriculture such as shifting Although improved millet cultivars have been cultivation [2]. Recent decades have seen large developed, farmers still grow local landraces, which population increases, the breakdown of traditional provide grain for humans, fodder for livestock, and shifting cultivation systems, and a rapid decline of stalks for fencing, precluding their use as organic
fertilizer. Small doses of mineral fertilizer increased Corresponding author: Hide Omae, Ph.D., Senior
production [6, 7], but farmers cannot afford to buy researcher, research fields: agronomy, plant physiology, soil
fertility. E-mail: homae@affrc.go.jp. mineral fertilizer, and thus low-cost inputs must be
On-farm Evaluation of Effect of Organic and Mineral Fertilizers 583
on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa
found. Suitable alternatives include intercropping the biomass and yield of millet/cowpea intercrops, and and cereal-legume (or non-legume) rotation at
(2) to compare the best combination of organic and increased planting density, and improved cultivars of
mineral fertilizers with low-cost technologies such as cowpea [5, 8-14]. Different row arrangements in
the use of an improved cowpea cultivar with an cereal-legume intercrops also have also been optimum row arrangement in millet/cowpea and compared [15-17].
millet/non-legume intercropping. Among fertilizers, mineral fertilizer [18], manure
2. Materials and Methods
[19, 20], and both [21] have been tested. Mineral fertilizer appears to be the best means of restoring the
An on-farm experiments was conducted during the nutrient balance, increasing crop yields, and raising
2008 and 2009 cropping seasons in four villages rural incomes [3]. The addition of manure would
(Bokossay: 13°25.13 N, 2°47.27 E; Yerimadey: allow assessment of the dynamics of C and organic N
13°28.65 N, 2°42.25 E; Kodey: 13°23.50 N, and P in the evaluation of sustainable local 2°49.23 E; Mourey Koala Zeno: 13°35.02 N, agroecosystems [22].
2°38.78 E) in the Fakara commune, Dantiandou The objectives of this study were (1) to study the
district, Tillaberi prefecture, western Niger, about 50 effect of low-cost organic and mineral fertilizers on
km northeast of Niamey, the capital (Fig. 1).
Fig. 1 Location of the experimental plots.
On-farm Evaluation of Effect of Organic and Mineral Fertilizers
on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa
The experimental plots were treated as mother weeding, respectively with micro-dosing technique [6, fields of mother-baby trials [23]. There were 61
7]. Each experiment used a local millet cultivar, households in Maourey Kouara Zeno, 90 in Yerima
“Haïni tchirey” (120 days to harvest). The experiments Dey, 51 in Bokossay, and 105 in Ko Dey (2009, pers.
were conducted in a strip-plot design with four comm.). The Zarma are the principal ethnic group of
replications (one per village). Table 1 shows details. this region. They are agriculturalists engaged mainly
The millet was sown at 1.0 m × 1.0 m, and thinned in rainfed production of millet (Pennisetum glaucum
to three plants per hill after 2 weeks. It was planted in (L.) R. Br.) and cowpea (Vigna unguiculata (L.)
June and harvested in October each year. Cowpea was Walp.). The prevailing soil type in the region is
sown at 1.0 m × 1.0 m. It was planted in July and Psammentic Paleutalfs, with a high sand fraction and
harvested in October each year. At physiological typical characteristics of an infertile soil [24, 25]. The
maturity, millet plants in each plot were harvested and rainfall starts from June until September, with a total
partitioned into ears and stalks. After drying, the ears rainfall of about 550 mm, which peaks in August [26].
were threshed and the total biomass was determined. From 2001 to 2007, the annual average rainfall at
Cowpea plants were also harvested at physiological Kodey was 435 mm. In 2008, the annual rainfall was
maturity, partitioned into fodders and pods. After 442.2 mm within 38 days of rain; in 2009, it was
drying, the pods were threshed. All aboveground crop 515.6 mm within 45 days of rain. The mean annual
residues were removed from the plots at end of each temperature ranged from 22.8 °C to 31.2 °C in 2008,
cropping season.
from 23 °C to 36 °C in 2009. Soil samples were taken from the top 15 cm, In 2008, in each mother field in each village, ten
air-dried, crushed, and sieved (2-mm mesh) for plots (20 m × 20 m) were established. Six of them
chemical analysis. Following wet digestion with received one of six combined fertilizer treatments:
salicylic acid-thiosulfate, the total N content was three forms of organic fertilizer (none, millet husks, or
measured at 660 nm with a Technicon Auto-Analyzer manure), with or without mineral fertilizer, and grew a
II (Pulse Instrumentation Ltd., Saskatoon, SK, Canada) millet/cowpea intercrop, using a local cowpea [28]. After extraction with Bray No. 1 solution, landrace.
available P was measured by the molybdenum blue Two of them grew millet/cowpea intercrops in 1:1
method [29]. Organic C content was determined by or 4:4 row arrangements, using a dual-purpose,
the Walkley-Black method [30]. medium-maturity cowpea line (85 days), TN256-87 [2,
The air-dry plant samples were ground 3 months 27], with mineral fertilizer. One grew a spreading
after the harvest; 200 mg of each sample was digested cowpea line, IT99-213-11-1 [2], with manure. And
with salicylic acid-thiosulfate, and the total N content one grew a millet/hibiscus (Hibiscus sabdariffa,
was measured at 660 nm with the Technicon ‘Wankoye’) intercrop, with millet husks. In 2009, the
Auto-Analyzer II. N uptake was determined by ten plots were each divided into two (9.5 m × 20 m);
multiplying the N concentration by the plant biomass, half of each was fertilized as in 2008, and the other
and the N balance was calculated as atmospheric N half was not fertilized. Millet received 9 t/ha of cow
minus N uptake. The ratio of atmospheric N in dung manure, 6 t/ha of millet husks which were
cowpea was taken from Yakubu et al. [31]. derived from refining process of millet head. For
The agronomic nitrogen uptake efficiency (ANUPE, application of mineral fertilizer, 3 g of compound
kg of aboveground biomass/kg of N applied) and the
agronomic nitrogen use efficiency (ANUE, kg of of urea per hill were applied at sowing and at first
mineral fertilizer (N: P 2 O 5 :K 2 O = 15:15:15), and 2 g
grain/kg of N applied) were determined as [32]:
On-farm Evaluation of Effect of Organic and Mineral Fertilizers 585
on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa
Table 1 Combination of treatment in experimental plots, Fakara, western Niger.
Application Intercrop
Cowpea or
Ro
history Millet/Cowpea
arrangement
Application
Hibiscus cultivar
- Millet/Cowpea
local 1:1
No
2008 Millet/Cowpea
local 1:1
Manure
2008 Millet/Cowpea
local 1:1
Millet husks
2008 Millet/Cowpea
local 1:1
Manure + Mineral fertilizer
2008 Millet/Cowpea
local 1:1
Millet husks + Mineral fertilizer
2008 Millet/Cowpea
local 1:1
Mineral fertilizer
2008 Millet/Cowpea
TN256-87
Mineral fertilizer
2008 Millet/Cowpea
TN256-87
Mineral fertilizer
2008 Millet/Hibiscus
IT99-213-11-1 1:1
Manure
2008 Millet/Cowpea
Wankoye
Millet husks
2008 & 09 Millet/Cowpea
2008 & 09 Millet/Cowpea
local
Millet husks
2008 & 09 Millet/Cowpea
local
Manure + Mineral fertilizer
2008 & 09 Millet/Cowpea
local
Millet husks + Mineral fertilizer
2008 & 09 Millet/Cowpea
local
Mineral fertilizer
2008 & 09 Millet/Cowpea
TN256-87
Mineral fertilizer
2008 & 09 Millet/Cowpea
TN256-87
Mineral fertilizer
2008 & 09 Millet/Hibiscus
IT99-213-11-1
Millet husks
ANUPE = (UB N − UB 0 )/F N 135%-152% (P < 0.001), cowpea biomass by ANUE = (Y N −Y 0 )/F N 91%-111% (P < 0.01), and millet grain yield by
where UB N is aboveground biomass of N-fertilized 130%-184% (P < 0.01). Mineral fertilizer increased plot (kg/ha), UB 0 is aboveground biomass of total biomass by 27% (P < 0.01), millet biomass by control plot (kg/ha), F N is amount of fertilizer 34% (P < 0.01), cowpea biomass by 14% (P < 0.05), N (kg/ha applied), Y N is grain yield of N-fertilized
and millet grain yield by 24% (P < 0.05) in 2008. plot (kg/ha), and Y 0 is grain yield of control plot
Repeat application of fertilizers in 2009 increased (kg/ha).
total biomass by 28% (P < 0.05) and millet biomass The results were tested by analysis of variance
by 31% (P < 0.05) compared to the application only in (ANOVA) followed by student’s t-test in JMP version
2008. Interactions among fertilizers and application
9.0.0 software (SAS Institute, Cary, NC, USA). history were not significant except for that between organic and inorganic fertilizers on millet grain yield
3. Results and Discussion
in 2008 (P < 0.05). Relative to the control, the ratio of
3.1 Effect of Organic and Mineral Fertilizer millet biomass to total biomass was higher in manure application in 2009 (P < 0.05) and in mineral fertilizer With organic fertilizer, the total (millet + cowpea),
application in both years (P < 0.05). The differences millet, and cowpea biomass and the millet grain yield
in biomass among fertilizer treatments were due to decreased in the order of manure > millet husks >
nutrient availability [33].
control in both 2008 and 2009 (Table 2). Relative to As a result of greater N input and uptake, the N the control, manure increased total biomass by
balance was greatest and positive in the manure
127%-147% (P < 0.001), millet biomass by treatment (P < 0.001), followed by millet husks (Table
On-farm Evaluation of Effect of Organic and Mineral Fertilizers
on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa
Table 2 Effect of organic and in-organic fertilizer and application history on biomass and grain of millet / cowpea intercrop at experimental fields, Fakara, Niger in 2008 and 2009.
Millet biomass ratio Treatment
Biomass (kg/ha)
Grain (kg/ha)
Millet + Cowpea
Cowpea to total biomass
62 50 0.63 0.92a Organic matter
(O) Millet husks
88 31 0.60 0.84b Non
72 28 0.65 0.87b Mineral fertilizer
69 30 0.68a 0.89a (MF)
76 42 0.59b 0.87b Application
32 - 0.90 history (AH)
Applied in 2008-09
42 - 0.86 O
Applied in 2008
n.s. n.s. * MF
n.s. ** *
n.s. n.s. n.s. O X MF
AH n.s.
n.s. n.s. n.s. O X AH
n.s. n.s. n.s. MF X AH
n.s. n.s. n.s. O X MF X AH
n.s. n.s. n.s. *, **, *** indicate significantly different at 0.05, 0.01 and 0.001 level, respectively while n.s. indicates no significantly different. Different alphabets indicate statistically significant at 0.05 level (by Student t test).
Table 3 Effect of organic and in-organic fertilizer and application history on N balance in millet / cowpea intercropping at experimental fields, Fakara, Niger in 2008 and 2009.
N balance N uptake / N input Treatment
N input by the
N input from
application
atmosphere
N uptake by millet
N uptake by cowpea
(kg N/ha)
(kg N/ha)
(kg N/ha)
(kg N/ha)
(kg N/ha)
32.8a 0.4b 6.3 Organic matter
(O) Millet husks
8.1b 0.6b 6.2 Non
-7.2c 1.6a 6.3 Mineral fertilizer
1.9 -7.4c
14.0a 0.7b 6.6 (MF)
10.8b 1.0a 6.0 Application
25.2 2.0 38.8 39.7a 0.8 1.6b history (AH)
Applied in 2008-09
59.5 59.5 17.4 1.4 12.9 19.2a
25.2 2.5 38.8 -15.2b 0.8 10.9a O
Applied in 2008
59.5 0.0 17.4 1.7 12.9 14.5b
*** *** n.s. MF
* ** n.s.
*** n.s. *** O X MF
n.s. *** n.s. O X AH
*** n.s. ** MF X AH
** n.s. * O X MF X AH
n.s. n.s. n.s. *, **, *** indicate significantly different at 0.05, 0.01 and 0.001 level, respectively while n.s. indicates no significantly different. Different alphabets indicate statistically significant at 0.05 level (by Student t test).
3). There was a significant difference between organic 9% compared to millet husks (P < 0.01). Total soil N application and no application in N uptake/input in
and P contents increased between years (P < 0.001). 2008. Repeat application in 2009 increased N input
Manure made the N balance positive. Lack of and uptake by millet by 32% (P < 0.05), and resulted
nutrient availability makes it negative [33]. There is in a bigger positive N balance and a smaller N
much evidence that N and P availability sets crop uptake/input ratio. Use of mineral fertilizer increased
yields in West Africa [34], but past experience shows N input and uptake by cowpea, which contributed to
that their supply to plants should be organically increase N balance by 9% (P < 0.05) and lower N
mediated [35]. In sandy soils, it thus appears uptake/input ratio by 30% (P < 0.01) in 2008.
fundamental to manage all components that affect soil Manure increased total soil N by 11% (P < 0.05,
fertility. Biological processes are crucial to sustaining Table 4). No application increased soil P content by
the fertility of sandy soils, as they control N flux [36].
On-farm Evaluation of Effect of Organic and Mineral Fertilizers 587
on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa
Table 4 Effect of organic and in-organic fertilizer and application history on total soil N, Bray I-P and organic C in millet / cowpea intercrop at experimental fields, Fakara, Niger in 2008 and 2009.
Total N
Bray I-P
Organic C
Treatment
(mg/kg)
(mg/kg)
Organic matter
Millet husks
fertilizer (MF)
Application Applied in 2008-09
history (AH)
Applied in 2008
Year (Y)
AH n.s.
*, **, *** indicate significantly different at 0.05, 0.01 and 0.001 level, respectively while n.s. indicates no significantly different. Different alphabets indicate statistically significant at 0.05 level (by Student t test).
As in other pedoclimatic zones, the assessment of N nitrogen use efficiency (hereafter, ANUE) of millet by and P in agricultural systems on tropical sandy soils is
90%-288% (n.s.) compared with organic fertilizers
a useful tool for defining sustainable intensification (manure and millet husks), followed by organic plus plans necessary to respond to population increases and
mineral fertilizers (Table 5). Millet husks plus mineral global change issues [37, 38].
fertilizer increased ANUPE of total biomass by 117%, Mineral fertilizer increased agronomic nitrogen
that of millet biomass by 107%, that of cowpea uptake efficiency (hereafter, ANUPE) of total (millet
biomass by 177%, and ANUE of millet grain by 171% and cowpea) biomass by 107%-213% (n.s.), that of
compared with millet husks alone, although the millet biomass by 142%-273% (n.s.), and agronomic
differences were not significant. In contrast, manure
On-farm Evaluation of Effect of Organic and Mineral Fertilizers
on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa
Table 5 Agronomic N uptak e and N use efficiency in different application of organic and in-organic fertilizer at experimental fields, Fak ara, Niger in 2008 and 2009.
ANUE (kg/N kg) Treatment
ANUPE (kg/N kg)
Millet Cowpea
20.7 17.6 3.1 2.4 0.3 Organic / In-
Millet husks
organic Manure + Mineral fertilizer 33.5 27.9 5.7 4.1 0.0 fertilizer (O)
Millet husks + Mineral fertilizer 45.0 36.4 8.6 6.5 -0.2
9.3 -1.5 Application
Mineral fertilizer
64.8 65.4 -0.6
Applied in 2008, 2009 31.3 28.4 2.9 6.0 -0.2 history (AH)
Applied in 2008
n.s. n.s.
n.s. n.s. O X AH
AH n.s.
n.s.
n.s.
n.s. n.s. *, **, *** indicate significantly different at 0.05, 0.01 and 0.001 level, respectively while n.s. indicates no significantly different. Different alphabets indicate statistically significant at 0.05 level (by Student t test).
plus mineral fertilizer did not increase either ANUPE fertilizer in 2009 (Table 6). Relative to millet husks, or ANUE compared with manure alone.
manure also increased total biomass by 67%-120% (P The use of crop residues plus fertilizer quadrupled
< 0.001), millet biomass by 69%-176% (P < 0.001), grain yield relative to the control, and the use of
millet yield by 106%-231% (P < 0.001) in 2009, and fertilizer doubled millet yield relative to the control.
cowpea yield by 74%-1550% (P < 0.01) in 2008. Crop residues can significantly improve the efficiency
When compared within same fertilizer applications, of use of the applied fertilizer [39]. The differences
mineral fertilizer on intercropped cowpea line between manure and millet husks when mineral
TN256-87 in the 4:4 row arrangement reduced the fertilizer is added might be due to differences in N
millet biomass ratio to total biomass in 2009 (P < mineralization.
0.001) relative to 1:1 row arrangement. Manure on the The single application of fertilizers in 2008
intercropped cowpea line IT99K-213-11-1 reduced increased ANUPE of the total biomass by 45%,
cowpea yield by 89% (P < 0.01) compared to that on increased that of millet biomass by 42%, and
the intercropped local cowpea in 2008. Millet husks increased that of cowpea biomass by 72%, but
on intercropped hibiscus reduced the millet biomass decreased ANUE of millet by 20%, although no
ratio compared to that on intercropped local cowpea differences were significant.
(P < 0.01). Repeat application of fertilizers increased total biomass by 31% (P < 0.05), millet biomass by
3.2 Combination Effect of Intercrop, Cowpea Cultivar 35% (P < 0.05), millet yield by 38% (P < 0.05), and and Row Arrangement increased millet biomass ratio (P < 0.05) compared to
With organic fertilizer, manure increased total
single application.
biomass by 53%-133% (P < 0.001), millet biomass by Different combinations of millet and cowpea 52%-173% (P < 0.001), and millet yield by cultivars gave different yield performances [9], and 77%-183% (P < 0.001) compared with mineral
the selection of genotypes with a suitable combination
On-farm Evaluation of Effect of Organic and Mineral Fertilizers 589
on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa
Table 6 Effect of intercrop, cowpea cultivar, row arrangement and application on biomass and grain of millet / cowpea intercrop at experimental fields, Fakara, Niger in 2008 and 2009
Biomass (kg/ha)
Grain (kg/ha)
Cowpea or Millet biomass ratio Combination of treatments (C)
Hibiscus to total biomass Intercrop
Millet + Cowpea or
Millet
Cowpea or
2008 2009 2008 2009 Millet / Cowpea
Cowpea or Row Hibiscus cultivar arrangement
88ab 34 0.65 0.92a Millet / Cowpea
TN256-87
Mineral fertilizer
88ab 65 0.71 0.79b Millet / Cowpea
TN256-87
Mineral fertilizer
64ab 21 0.71 0.91a Millet / Cowpea IT99K-213-11-1
local
Mineral fertilizer
6c 12 0.55 0.92a Millet / Cowpea
57b 55 0.66 0.92a Millet / Hibiscus
- 89 0.60 0.76b Millet / Cowpea
Wankoye
Millet husks
Millet husks
99a 33 0.57 0.87a
- 44 - 0.89a Application history (AH)
Applied in 2008-09
Applied in 2008
- 45 - 0.85b
C n.s.
** n.s. n.s. ***
n.s. n.s. n.s. * C X AH
AH n.s.
n.s. n.s. n.s. n.s. *, **, *** indicate significantly different at 0.05, 0.01 and 0.001 level, respectively while n.s. indicates no significantly different. Different alphabets indicate statistically significant at 0.05 level (by Student t test).
Table 7 Agronomic N uptake and N use efficiency in different combination of intercrop, cowpea cultivar, row, organic and in-organic fertilizer application at experimental fields, Fakara, Niger in 2008 and 2009.
Combination of treatment (C)
ANUPE (kg/N kg)
ANUE (kg/N kg)
Cowpea Intercrop
Millet +
Cowpea
Millet or Hibiscus cultivar arrangement
Cowpea or
Row
Application
Cowpea or
Millet
or
Hibiscus Millet / Cowpea
Hibiscus
Hibiscus
13.8 21.0a 0.5 Millet / Cowpea
TN256-87
Mineral fertilizer
107.1a
93.3a
13.7 15.3ab 2.2 Millet / Cowpea
TN256-87
Mineral fertilizer
70.5ab
56.8abc
9.3bc -1.5 Millet / Cowpea IT99K-213-11-1
local
Mineral fertilizer
8.6 3.6c -0.7 Millet / Cowpea
4.2 4.9c 0.0 Millet / Hibiscus
5.4 0.6c 1.2 Millet / Cowpea
Wankoye
Millet husks
Millet husks
37.8 32.9 4.9 8.1 0.2 Application history (AH)
Applied in 2008, 2009
Applied in 2008
** n.s.
AH n.s.
n.s.
n.s.
n.s. n.s.
n.s. n.s. *, **, *** indicate significantly different at 0.05, 0.01 and 0.001 level, respectively while n.s. indicates no significantly different. Different alphabets indicate statistically significant at 0.05 level (by Student t test).
of traits with a strong relationship with and a direct space, supporting photosynthesis, despite at before the effect on yield at an appropriate density increased the
risk of insect attack.
productivity of cowpea [40]. Oso et al. [13] studied Relative to manure, mineral fertilizer on the two row spacing arrangements (2:3 and 1:1 maize to
intercropped cowpea line TN256-87 in the 1:1 row cowpea) and concluded that the planting pattern in
arrangement increased ANUPE of the total biomass intercropping appears to influence leaf infestation by
by 242%-263% (P < 0.01), increased that of millet beetles. Nambiar et al. [41] ascribed the effect of the
biomass by 246%-346% (P < 0.01), and increased intercropping system to the shading of legumes by the
ANUE of millet by 329%-483% (P < 0.01, Table 7). cereal and the consequent decrease in photosynthesis.
Relative to millet husks, mineral fertilizer on the The 4:4 row arrangement may allow cowpea more
intercropped cowpea line TN256-87 increased
On-farm Evaluation of Effect of Organic and Mineral Fertilizers
on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa
ANUPE of the total biomass by 417%-676% (P <
the global scale.
0.01), that of millet biomass by 430%-1011% (P < As conclusion, the use of small quantities of 0.01), and increased ANUE of millet by 775%-3400%
mineral fertilizer, and its combination with the (P < 0.01). There were no significantly differences
intercropped dual-purpose cowpea gave the best observed when compared within the same fertilizer
performance on ANUPE and ANUE. Therefore, this applications. Single fertilizer application in 2008
can be recommendable for poor N resources farmers increased ANUPE of the total biomass by 52% (n.s.),
like in the Sahel, West Africa. In addition, single that of millet biomass by 48% (n.s.), and that of
application of manure, millet husks plus mineral cowpea biomass by 78% (n.s.).
fertilizer, or year-alternative-application of fertilizer Variation in N uptake and in N use efficiency
are also recommendable depends on the farmers depends on N fertilization [42]. The variation in N
accessibility to the resources.
uptake was higher at low N than at high N ANUE in
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July 2014, Vol. 8, No. 7, pp. 593-602
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Detection and Protein Modelling for Consensus Region of hsp70 Gene Family in Egyptian Arundo donax
1 2 Mohamed A. Ezz 3 , Mona I. Salah and Abdullah I. Ammoura 1. GEBRI, Bioinformatics Department, Sadat University, P.O. Box 79/22857, Egypt
2. College of Biotechnology, Misr University for Scienceand Technology, 6th of October City, October 12451, Egypt 3. Molecular Genetics Lab, College of Biotechnology, Misr University for Scienceand Technology, 6th of October City, October
12451, Egypt
Received: April 18, 2014 / Accepted: July 14, 2014 / Published: July 31, 2014.
Abstract : Degenerate primers are particularly useful in amplifying homologous genes from different organisms. This paper describes a method for designing degenerate primers for a given multiple alignment of DNA sequences of hsp70 gene family using ClustalW algorithm and detect the consensus region in gene family of hsp70 in a plant species which have not any recorded information about hsp70 gene family in the Genbank of National Centre of Biotechnology Information (NCBI) like Arando donax .The sequenced consensus sequence of Arando donax is considered a gene marker in building the genome map sequence. The BLASTn program is used to find a homology between more than one accession numbers of DNA sequences, (X67711.2) was for Oryza sativa (hsp70), (AY372071.1) was for Nicotiana tabacum (hsp70) and (L41253.2) was for Lycopersicon esculentum (Hsc70). In silco PCR module was performed to detect the melting temperatures (Tm) and predicted the PCR product size (783 bp).The result of designed degenerate primers showed that there was a homology founded among the designed primers and the DNA templates of the recorded sequences (AY372071.1, X67711.2 and L41253.2) with at least 80% identity. The designed degenerate primers were used to isolate a consensus region of hsp70 gene family of Arando donax at the expected molecular weight (783 bp). The isolated PCR product, (783 bp) of Arando donax was sequenced and submitted to the Database of Japan (DDBJ) with accession number AB819871. The ORF finder tool translated the accession number AB819871 and gave a selected frame which used to build 3D structure model. In conclusion, this study focused on the importance of designing the degenerate primers to isolate the gene family and predict the 3D structure of gene family depending on the ORF finder tool of Genebank.
Key words : ClustalW tool, degenerate PCR, different melting temperatures, in silico PCR, SWISS-MODEL workspace.
1. Introduction blocks of conserved regions in multiple global alignments. Therefore, alignment quality should be
The Hsp70s are found in all major cellular
very high [4].
compartments of eukaryotes and every bacterium Degenerate PCR is a PCR method that uses examined to date, bind partially unfolded proteins. degenerate primers to amplify unknown DNA They appear to bind nascent chains in the process of sequences that are related to a known DNA sequence protein synthesis and completed polypeptides upon or to amplify a mixture of related sequences in one release from ribosome [1]. Classical methods for PCR reaction. Degenerate primers are just a mix of degenerate primer design include software applications primers with similar sequences. Degenerate PCR is such as CODEHOP [2] or PrimaClade [3]. These useful for identify new members of a gene family or methods usually rely on the identification of clear orthologous genes from different organisms (http://
www.protocolonline.org/prot/Molecular_Biology/PCR/ Corresponding author: Mohamed A. Ezz, M.Sc., Ph.D. Candidates, research filed: molecular genetics. E-mail:
Degenerate_PCR___Degenerate_Primer). Degenerate ezz111@yahoo.com.
Detection and Protein Modelling for Consensus Region of hsp70
Gene Family in Egyptian Arundo donax
primers are particularly useful in amplifying (L41253.2), (AY372071.1), (X67711.2) and homologous genes from different organisms [5, 6]. In
(XM_003563568.1). The MegAlign module used the the “candidate gene approach”, known genes that
ClustalW algorithm to align more than one sequence affect similar processes in one organism could have
and produced the conserved blocks, which had been their homologues amplified in other related organisms
used to design the degeneracy primers. The setting by the use of a well-designed degenerate primer pair
parameters of ClustalW were adjusted (gap penalty [7]. The authors determined which parts are conserved
equal to 15, gap length penalty was equal to 6.66, and which are variable in the conserved blocks which
DNA transition weight equal to 0.6 and DNA weight produced by the ClustalW2 algorithm to design the
matrix was ClustalW). The definition of ClustalW degenerate primers to detect a consensus region of
parameters
hsp70 gene in Arando donax. The authors report a (1) Gap Penalty: the amount deducted from the new accession number for Arando donax,
alignment score for each gap in the alignment and AB819871.1 and identify all open reading frames for
Gaps of different sizes carry the same penalty. the accession number AB819871.1 by ORF finder tool
(2) Gap Length Penalty: the value deducted from and select the PROSITE pattern to retrieve an
the alignment score after first multiplying it by the alignment of UniProtKB/Swiss-Prot presented true
length of gaps. Longer gaps have a greater penalty positive hits with the selected frame. Authors predict
than shorter gaps.
the 3D structure of the selected frame. (3) For ClustalW only, you may specify values for Delay Divergent Seqs (%): alignment of any
2. Material and Methods
sequences with lower percent identities than this
2.1 Blasting by the Query Sequence threshold, with respect to other sequences in your The DNA sequence Oryza sativa hsp70 gene was
project, is delayed until after alignment of sequences retrieved from the sequence database of NCBI with
with higher percent identities.
gene information number gi|21664286 and accession (4) DNA Transition Weight: transitions are number (X67711.2), the FASTA file format was used
purine-purine (A-G) and pyrimidine-pyrimidine (C-T) as a query sequence to hit the database of Gene bank
substitutions. In DNA alignments, matches are usually and the category of Database that was chosen a (nt/nr)
scored 1, mismatches 0. The transition weight—allows nucleotide collection non redundant database and the
you to specify a score for transitions anywhere algorithm type of the BLAST (Basic Local Alignment
between 0 and 1. Increasing the transition weight Search Tool) was BLAST-nucleotide (BLASTn). The
towards 1 may help to make finer distinctions between three accession numbers which retrieved from closely related sequences. For distantly-related BLASTn and selected were (X67711.2) of Oryza
sequences, you may want to set this value to 0. sativa hsp70 gene, (AY372071.1) of Nicotiana
2.3 The Module of Primer Select
tabacum (HSP70-3) gene, (L41253.2) of Lycopersicon esculentum (Hsc70).
The module of primer select of DNASTAR lasergene program, version 7.0 was used to locate the
2.2 Detect the Conserved Block Sequence to Design specific primers. The location of consensus sequence the Degenerate Primers was assigned to be from the nucleotide position 2913
The MegAlign module of DNASTAR lasergene to 4557 (extracted conserved blocks from MegAlign version 7.0 was used to detect the consensus sequence
module) and the PCR product length was adjusted to between the previous four accessions numbers
be from 690 bp to 800 bp. All the parameter of primer
Detection and Protein Modelling for Consensus Region of hsp70
Gene Family in Egyptian Arundo donax
characteristics were adjusted from the primer locate tabacum (Samsun), Oryza sativa (Sakha102), menu of the program then the scoring option was
Lycopersicon esculentum (Castlerock) and leaves selected to adjust the values of set control to be in
samples of Arando donax were collected from intermediate including the following parameters,
Alexanderia, Egypt. The genomic DNA was isolated melting temperature (Tm), the primer length, hairpin
by the method CTAB (Cetyltrimethyl ammonium loop, overall stability and primer stability of 3’ end.
bromide according to the method [9]) for the three The default value of primer stability of 3’ end was
plants (Oryza, Tomato, Nicotina and Arando). The (-8.5 kCal/mol) is the average of all nearest neighbor
total volume of PCR reaction was equal to 25 µL (1.5 pentamer interactions, Primers with pentamer ΔG (the
µL of DNA with O. D 50-100 ng/µL), 0.5 µL for each amount of required energy to break the secondary
primer (Master Mix 12.5 µL of DreamTaq PCR structure in hairpin loop) more stable than -8.5
Master Mix (2X) and 10 µL free nucleases water). kCal/mol (more negative) have a tendency to false
The primers were synthesized at prime and are more likely to form hairpins and self
metabion-international. The PCR reactions were dimmers.
carried out using a DNA thermal cycler (Techne-312
2.4 Check for the Specificity and Uniqueness of the and Biometra-unoII) for every plant with different Degenerate Primers
melting temperatures Oryza sativa (94 o
C 2 min then
C 30 s, Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/
40 r as follows 94 o C 30 s, Tm 45 C to 47
C 10 min), Lycopersicon tools/primer-blast) [8], was used to detect the esculentum (94 o
72 o
C 1 min followed by 72 o
C 30 s, homology between the designed primers and targeted
C 2 min then 40 as follows 94 o
C 10 templates in the gene bank database and also the
Tm 45.3 o
C 30 s, 72 o
C 1 min followed by 72 o
C 2 min then 40 as position of degeneracy nucleotides and the uniqueness
min) and Arando donax (94 o
C 1 min for the forward and reverse primers. The parameters
follows 94 o C 30 s, Tm 45.0 C 30 s, 72
C 10 min). Amplified products were for Primer-BLAST were adjusted to be default. The
followed by 72 o
analyzed on a 2% agarose gel containing ethidium forward and reverse primers were putted through the
bromide. 100 bp DNA ladder have loaded, Biolabs. input text box of the Primer-BLAST homepage page
2.7 Sequencing the Expected Size of hsp70 Gene of (http://www.ncbi.nlm.nih.gov/tools/primer-blast).
Arando donax
2.5 PCR In silco Module to Predict and Produce All the True Condition of PCR Program
The PCR product was clarifid and sequencd using Genetic Analyzer (23 ABI 3730XLs) by the The PCR in silico module is a tool which
degenerate primers forward and reverse primers. The implemented the FAST PCR program by database search was performed using BLASTn
Ruslan-Kalendar version 4.0.8 university of Helsinki, program (http://blast.ncbi.nlm.nih.gov/BlastAlign. cgi). Finland and the PCR in silico module was used to
2.8 Detection of (ORF) by Using ORF Finder Tool for produce all the details of the PCR program like Tm
Accession Number AB819871.1
for the forward and reverse primer and the predict PCR product and informed the positions of primers
Analysis tool which finds all open reading frames which were aligned on the template sequences
of a selectable minimum size in a user’s sequence or (L41253.2, AY372071.1, X67711.2 and AB819871.1).
in a sequence already in the database. This tool
2.6 Genomic DNA Isolation and PCR Reaction identifies all open reading frames using the standard
or alternative genetic codes. The deduced amino acid The cultivars were grown in black soil Nicotiana
sequence can be saved in various formats and
Detection and Protein Modelling for Consensus Region of hsp70
Gene Family in Egyptian Arundo donax
searched against the sequence database using the
2.10 Retrieving PROSITE Documentations Analysis BLAST server (www.ncbi.nlm.gov/projects/gorf/).
and Producing Multiple Sequence Alignment The submitted accession number AB819871.1 of
The selected frame (frame -1, ORF from 72..602) Arundo donax was entered in the form of the ORF
sequence was used as query sequence to hit by the finder tool to find the best translated frame and run subjected family signatures of SCANPROSITE Database. BlASTp (Blast protien) to find the related protein Then selected PROSITE pattern to retrieve an domain family to construct the caldeogram depending alignment of UniProtKB/Swiss-Prot true positive hits on the BLASTp results. with the selected frame (frame -1, ORF from 72..602).
2.9 Submit Protein Sequences to Scan Them against the PROSITE Collection of Motifs
2.11 Predicting the 3D Structure of ORF Finder Results
PROSITE currently contains patterns and profiles The 3D structure of the selected frame (frame -1,
specific for more than a thousand protein families or ORF from 72..602) was predicted by using the server
domains. Each of these signatures comes with documentation providing background information on
Swiss-model (http://swissmodel.expasy.org/ interactive), which is a fully automated protein structure homology
the structure and function of these proteins (http://www.expasy.org/tools/scanprosite/scanprosite-
modelling server, accessible via the ExPASy web server, or from the program DeepView (Swiss
doc). PROSITE is a method of determining what is
Pdb-Viewer).
the function of uncharacterized proteins translated from genomic or cDNA sequences. It consists of a
3. Results and Discussion
database of biologically significant sites and patterns
3.1 Retrieving the Homologous Sequence by the formulated in such a way that with appropriate
BLAST Tool
computational tools it can rapidly and reliably identify which known family of protein (if any). The new
The BLAST program (Basic Local Alignment sequence belongs to (http://www.expasy.org/prosuser
Search Tool) produced a local alignment between the html#general). In some cases the sequence of an
query sequence (L41253.2) of Lycopersicon unknown protein is too distantly related to any
esculentum and the subjected records in the database protein of known structure to detect its resemblance
(AY372071.1) for Nicotiana tabacum, (X67711.2) for by overall sequence alignment, but it can be Oryza sativa. Each pairwise alignment (local identified by the occurrence in its sequence of a
alignment) had a bit score (normalized score for the particular cluster of residue types which is variously
score of the alignment) and the optimal score of known as a pattern, motif, signature, or fingerprint.
alignment (OPS). The distance of similarity between These motifs arise because of particular requirements
the query sequence and the targeted sequences had a on the structure of specific region(s) of a protein which
significance value or not, that depending on the may be important, for example, for their binding
E-value for every bit scores of pairwise alignment. properties or for their enzymatic activity The (E-value) reflected the significance of similarity (http://www.expasy.org/prosuser. html#general). The
between the query sequence and the targeted or accession number AB819871.1 of Arundo donax was
subjected sequences (Table 1). The homology results entered in the form of the link between the query sequence accession number (http://prosite.expasy.org/scanprosite) and the option
(L41253.2) of Lycopersicon esculentum and the
of submit protein sequences was selected to scan them subjected records in the database. The subjected
against the PROSITE collection of motifs. records which were selected (AY372071.1) for
Detection and Protein Modelling for Consensus Region of hsp70
Gene Family in Egyptian Arundo donax
Table 1 Sequences produce significant alignments.
Accession Description Max score E-value Max identity L41253.2
(query) Lycopersicon esculentum Hsc70 (Hsc70) gene, complete cds
0 100% X67711.2
0 80% AY372071.1 Nicotiana tabacum heat shock protein 70-3 (HSP70-3) gene, complete cds
Oryza sativa hsp70 gene for heat shock protein 70
and had some degeneracy nucleotides in the position from the position 2913 to 4557 that range was used to produce the degenerated primers pairs.
3.3 The Module of Primer Select The module of primer select of DNASTAR
Lasergene version 7 had analyzed the consensus sequence and the characteristics of primer design to produce a report of amplification summery which had obtained as shown in the (Fig. 1). The amplification
Fig. 1 The report of amplification summary.
summery described and documented the information about the designed primers, forward primer Nicotiana tabacum and (X67711.2) for Oryza sativa
GCTGCTGAAGAAATCTCCTCCAT and the reverse were homologous to query sequence and they were
primer AGCACCATAAGCAACAGCCTCAT where selected depending on their similarity of E-values
C and which were equal to zero for all the three accession
the (Tm) of forward primer was equal to 55.6 o
C. The numbers (L41253.2, AY372071.1 and X67711.2).
the (Tm) of reverse primer was equal to 56.8 o
primer Tm difference was equal to 1.2 o C.
3.4 Check for the Quality and Uniqueness of the Region
3.2 The Results of MegAlign Tool to Detect Conserved
Selected Primers and the Homology between the The homologous sequences were retrieved from the
Template Sequences and Primers
database we downloaded the FASTA format of the The quality and uniqueness of the forward and reverse three accession numbers (L41253.2, AY372071.1 and
primers were checked by using the Primer-BLAST X67711.2) in one text file and then the text file was
tool (http//www.ncbi.nlm.nih.gov/tools/primer-blast). processed by MegAlign tool of DNASTR Lasergene
The best alignment of forward and reverse primers version 7.0 to produce the multiple sequence alignment
with the three accession numbers (L41253.2, between the three accession numbers (L41253.2,
AY372071.1 and X67711.2). The Primer-BLAST tool AY372071.1 and X67711.2).Variant degrees of was used to check the uniqueness of the selected
conservation were found and the highly conserved primers and produced a summary table of melting blocks (Red Bar) was detected from the histogram bar
temperatures as shown in (Table 2). The analysis which indicated for the degree of the conservation in
number (3.4.1) showed the results of using colour metric scale. The red bar indicated for the best
Primer-BLAST2. The primer pair aligned and hit 180 positions of conserved sequences which were assigned
subjects, which recorded in the gene bank as hsp70 gene from the nucleotide position 3557 to 4491. The
or Hsc70 gene. The taxonomy of the recorded subjects, detected block was produced the consensus sequence
were distributed between monocots and dicots plants.
Detection and Protein Modelling for Consensus Region of hsp70
Gene Family in Egyptian Arundo donax
Table 2 Products on intended targets.
Sequence(5' →3') Length Tm GC% Forward primer
GCTGCTGAAGAAATCTCCTCCAT 24 61.48 41.67% Reverse primer
AGCACCATAAGCAACAGCCTCAT
3.4.1 The Results of Using Primer BLAST2
Template 1176 .........G.............. 1154
> X67711.2 Oryza sativa hsp70 gene for heat
3.5 Define the Degeneracy of the Nucleotide in the
shock
DNA Sequence
protein 70 product length = 783
The degenerate sequences in the aligned sequences
Forward primer 1
was obtained by the primer blast tool for the four
GCTGCTGAAGAAATCTCCTCCAT 23
accession numbers (X67711.2, L41253.2,
Template 2979 .....C.....G........... 3001
AY372071.1 and XM_003563568.1) and reported in
Reverse primer 1
the analysis number (3.4.1) then the mismatched bases
AGCACCATAAGCAACAGCCTCAT 23
between the primer and the templates were indicated
Template 3761 ....................... 373
for the degenerate bases and highlighted in gray
> L41253.2 Lycopersic esculentum (Hsc70) gene,
colour and reported in the analysis number (3.4.1).
complete cds
The colour of the degenerate bases were red
product length = 783
F-HSP70-AD KCWGCYGAAGARATYTCYTCYAT
Forward primer 1
R-HSP70-AD AGCWCCATARGCAACAGCCTCRT
GCTGCTGAAGAAATCTCCTCCAT 23
where K(G/T), W(T/A), R(A/G) and Y(T/C).
Template 2779 T...................T.. 2801
3.6 The Result of PCR In silico Module
Reverse primer 1 AGCACCATAAGCAACAGCCTCAT 23
The expected PCR product which had been
Template 3561 ...T.................G. 3539
predicted was equal to 783 bp for all the three
> AY372071.1 Nicotiana tabacum heat shock
accession numbers by the program Fast PCR version
protein 70-3 (HSP70-3) gene, complete cds
4.0. The PCR in silico module of Fast PCR program
product length = 783
was used to detect the true positions of the aligned
Forward primer 1
primers with the template sequences (L41253.2,
GCTGCTGAAGAAATCTCCTCCAT 23
AY372071.1, and X67711.2). Fast PCR program was Template 1419 .................T..... 1441 used to detect the true PCR Condition like Tm and the
Reverse primer 1
“%” of similarity between the primer pairs and the
AGCACCATAAGCAACAGCCTCAT 23
three accessions (L41253.2, AY372071.1 and
Template 2201 .........G............. 217
X67711.2). The “f” is for forward and the “r” is for
> XM_003563568.1 Brachypodium distachyon
reverse as shown in (Fig. 2a), (Fig. 2b) and (Fig. 2c).
heat shock cognate 70 kDa protein 1-like, transcript
3.7 Loading the PCR Products
product length = 783 Forward primer 1
The products of PCR for three plants (Tomato, Rice
GCTGCTGAAGAAATCTCCTCCAT 23
and Arando) had been detected approximately at M.W
Template 394 T.A...........T..T..... 416
size 783 bp as in (Fig. 3), DNA ladder from 100 bp to
Reverse primer 1
2000 bp had been loaded. Some bands are not the
AGCACCATAAGCAACAGCCTCAT 23
specific band that was referred to the designed low
Detection and Protein Modelling for Consensus Region of hsp70
Gene Family in Egyptian Arundo donax
result of ORF finder detection tool for the accession number (AB819871) achieved five frames. The selected frame (frame -1, ORF from (72..602)) gave a translated frame with Length: 176 aa which hit the subjected records in the Genbank and found distribution of 100 Blast hits (related to hsp70) on the query sequence and hit on the conserved domain
PTZ00009 [PTZ00009], heat shock 70 kDa protein
Fig. 2a PCR in silico result for Lycopersic esculentum—(L41253.2).
and achieved a significance E-value 2.44e-100.
3.9 Retrieving Prosite Documentations and Analyzing Family Signature 2 of Heat Shock hsp70 Proteins
The first technical section of the PROSITE documentation was selected, HSP70_2, PS00297 of heat shock hsp70 proteins family signature 2. The results were described as the PROSITE methods (with tools and information) and were covered in Swiss-Prot true The ClustalW format in condensed colour was
Fig. 2b PCR in silico result for Nicotiana
selected to see the degree of the conservation on the
tabacum—(AY372071.1).
motif HSP70_2, PS00329 of heat shock hsp70 proteins family. The retrieved results of HSP70_2 motif was [IFDLGGGTfdvSLL] as shown in the (Fig. 4a) and (Fig. 4b).
3.10 From Amino Acids Sequence to the 3D Structure The server was used to build an expected model
(http://swissmodel.expasy.org/interactive). The server was used to detect the high score of pair wise
alignment to compare the query sequence to the
curated 3D structure of the recorded protein in the
Fig. 2c PCR in silico result for Oryza sativa—(X67711.2).
protein data bank (PDB). Then calculated the
melting temperature from which were from 45 o
C to
secondary structure prediction of the amino-acids
47 o C Tm. sequence and finally to the right expected structure. The query sequence of amino acid (176 aa) was one of
3.8 Finding the Subjected Homologous Records to the the translated frame (frame -1, and ORF from
Accession Number (AB819871) (72..602)) of the accession number AB819871.1. The
The expected band of Arando donax was sequenced calculation of similarity among the query sequence of and submitted to the Database of Japan DDBJ and had
frame-1 (176 aa) of Arando donax and the targeted 3D the accession number (AB819871). The length of the
structures using the matrix of Blosum 62 and BLAST submitted nucleotide sequence was 764 and molecule
program were carried out to find the optimal score of type which was sequenced is genomic DNA. The
pair wise alignment, E-value and the overlap regions
Detection and Protein Modelling for Consensus Region of hsp70
Gene Family in Egyptian Arundo donax
Fig. 3 Amplified PCR of Genomic Products by using the designed degenerate Primer pairs to detect the gene of hsp70. Lane (2) product of Lycopersicon esculentum, Lane (3) product of Oryza sativa, Lane (4) and Lane (5) products of Arando donax. The expected sizes of PCR products at 783 bp as the red arrow assigned.
the score(GMQE) was 0.87 as shown in the (Fig. 5). GMQE (Global Model Quality Estimation) is a quality
estimation which combines properties from the
target-template alignment. The resulting GMQE score is expressed as a number between zero and one,
Fig. 4a The retrieved results of HSP70_2 motif was
reflecting the expected accuracy of a model built with
[IFDLGGGTfdvSLL].
that alignment and template. Higher numbers indicate
Fig. 4b The translated frame (-1) was scanned to detect the motif signal of HSP70_2, PS00329.
of pair wise alignment. The PDB number of 1kaz.1.A was one of the highest and the best score of pair wise
Fig. 5 The model_01 description and used the template
alignment between the PDB records and the query
1kaz.1 and query sequence achieved a high GMQE (0.87)
amino acid sequence (176 aa) and the significance of
for the expected template as the red circle indicated.
Detection and Protein Modelling for Consensus Region of hsp70
Gene Family in Egyptian Arundo donax
Fig 6 The alignment between the model_01 and the template 1Kaz.1.A and the ligand of (k+) ion in contact with chain A: D34, G36, T39, D41 as the red line indicated.
by a query sequence, the second step was used the BLASTn programme of NCBI (http://blast.ncbi.nlm. nih.gov/Blast.cgi) to find the homology between the query sequence and the subjected records which were homologous to the query sequence and also in the same gene family category, the third step was used the ClustalW algorithm to produce a multiple sequence alignment and the authors found the conserved block which had the reduced number of degenerate
nucleotides , the fourth step found and checked for the
Fig. 7 The model_01 was built with ProMod Version 3.70.
specificity of the degenerate primers which hit only higher reliability. Once a model is built, the GMQE
the subjected record of hsp70 gene family only and gets updated for this specific case by also taking into
finally the PCR in silco was performed and the account the QMEAN4 score of the obtained model in
selected melting temperature Tm was calculated and order to increase reliability of the quality estimation.
adjusted to be the annealing temperature which used and depending on the quality of GMQE the model
in the running of thermal cycler. The amino acids template—alignment was 1kaz.1.A which was sequence (176 aa) was one of the translated frame producing significant alignment and the expected
(frame -1) of the accession number, AB819871.1 and secondary structure of query sequence could be had
produced by using the ORF finder tool. The PDB ID the approximate secondary structure of the model
number, 1Kaz.1.A was homologous to the translated crystal structure 1kaz.1.A in PDB as shown in (Fig.
frame (frame -1) because the (GMQE) score of 6).The expected model_01 is a part of the chain A in
alignment between the expected model_01 and the template model 1Kaz.1.A as shown in the (Fig 7).
1Kaz.1.A template was a significance value and equal to 0.87. Depending on the score of Global Model
Quality Estimation (GMQE) the model_01 was the The authors report the use of such degenerate
4. Conclusion
expected secondary structure of the translated frame primers to detect the genes of hsp70 and from the
(frame -1). Model_01 was expected to be the same multiple DNA sequences, authors determined which
secondary structure of the crystallized 3D structure parts are conserved and which are variable. Based on
1Kaz.1.A.
this information, the conserved DNA motif used as
References
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Biogenesis.” Microbiol Rev. 57 (2): 402-414.
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Detection and Protein Modelling for Consensus Region of hsp70
Gene Family in Egyptian Arundo donax
[2] Rose, T. M., Henikoff, J. G., and Henikoff S. 2003. Arabidopsis Thaliana.” Mol. Plant Microbe Interact 11 “CODEHOP (COnsensus-DEgenerate hybrid
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B., Stelly, D. M., and Michelmore, R. W. 1998. [3] Gadberry, M. D., et al. 2005. “Primaclade—a Flexible
“Resistance Gene Candidates Identified by PCR with Tool to Find Conserved PCR Primers Across Multiple
Degenerate Oligonucleotide Primers map to Clusters of species.” Bioinformatics 21 (7): 1263-1264.
Resistance Genes in Lettuce.” Mol. Plant Microbe [4] Gorrón, E., et al. 2010. “A New Method for Designing
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[8] Rozen, S., and Skaletsky, H. 2000. “Primer3 on the [5] Aarts, M. G., Lintel Hekkert, B. te, Holub, E. B., Beynon,
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July 2014, Vol. 8, No. 7, pp. 603-604
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
The Emergence of Hybrid Seeds and Polyembryony in Some Citrus Cultigens
Nino Davit Kipiani Department of Agricultural Sciences, Kutaisi State University, Kutaisi, 4600, Georgia
Received: April 18, 2014 / Accepted: July 14, 2014 / Published: July 31, 2014.
Abstract: An experiment was conducted and through distant hybridization a new promising forms of citruses were obtained. Namely, after hybridization of lemon Georgian and broad-leaved mandarin unshiu (mother components) with early-ripening trifoliata and a hybrid of citrus ichangensis—“Caucasus” (father components) a wide range of nucellar seedlings were obtained. It should be noted that nucellar seedlings of hybrid nature are practically closer to distant hybrids of sexual origin that are sharply inclined to the side of the mother component and produce good quality fruit. Unfortunately, such hybrids are either very rare or cannot be obtained at all. While, in the case of the study, through using new combinations in hybridization, the possibility of getting nucellar seedlings of the hybrid nature is much bigger. Distant hybridization turned out to be particularly interesting because of the polyembryony of citrus plants. Due to this, it gives opportunity to get new promising forms not only from the egg cell but from nucellar cells as well.
Key words: Distant hybridization, polyembryony, nucellar and hybrid seedlings.
1. Introduction through distant hybridization.
In order to get nucellar seedlings of hybrid nature, Citruses are the best objects for studying the events the flowers of lemon Georgian and broad-leaved of polyembryony. As it is known that citruses and mandarin unshiu were pollinated with the pollen from other citrus plants are characterised by the trifoliata (early-ripening) and a hybrid of citrus polyembryony, i.e., when in one ovule two or more ichangensis—“Caucasus”. Hybrid seeds were sown embryos are developed. Consequently, from one seed and the obtained hybrid seedlings were genetically two or more plants appear that is connected to the studied from the moment of the emergence of hybrid development of additional embryo in nucellar cells. seedlings to the end of two vegetation periods. [2]. It Embryos developed from nucellar cells are called should be noted that it is possible to distinguish the nucellar embryos and the plants grown from such seedlings of sexual hybrids from nucellar ones by embryos are referred as plants of nucellar origin or external phenotypic signs: leaf shape, size and its nucellar seedlings [1]. down, the smell of a leaf, etc. These signs are
2. Materials and Methods
phenotypic but they are genotypic (inherited) as well. These were the characteristic features used when
The experiment was conducted in Laboratory of comparing the initial seedlings with their parents. The Citrus Genetics and Selection within the framework of seedlings which have the above mentioned male-parent the national scientific grant project. The aim of the phenotypic characteristics were considered to be project was to obtain new promising forms of citruses sexual hybrids and were grouped separately while the
rest (similar to a mother) were referred as nucellar Corresponding author: Nino Davit Kipiani, Ph.D., assistant
professor, research fields: Agriculture, Potential Possibilities of seedlings and were grouped separately. The results of Distant Hybridization in Citruses. E-mail: the study are given in the Table 1 below:
nino.kipiani74@yahoo.com.
The Emergence of Hybrid Seeds and Polyembryony in Some Citrus Cultigens
Table 1 The Emergence of hybrid seeds and polyembryony.
The number of emerged
Including
The number of
# Combination
seedlings
sown seeds
Hybrid seedlings
Nucellar seedlings
pieces % 1. Broad-leaved Mandarin Unshiu × Trifoliata (early-ripening) 100
pieces
pieces %
62 48 77.4 14 22.6 Broad-leaved Mandarin Unshiu
2. × Citrus
73 58 79.5 15 19.5 ichangensis—“Caucasus” 3. Georgian Lemon × Trifoliata (early-ripening)
90 68 75.6 22 24.4 4. Georgian Lemon × Citrus ichangensis—“Caucasus”
The data given in the table clearly show that the seedlings is higher than in case of other combinations. possibility of obtaining nucellar seedlings in citruses
The percentage difference between combinations to through the distant hybridization is generally low and
obtain nucellar seedlings is not very big but it's it varies according to the hybridization combinations.
necessary to constate it since the possibilities of The analysis of the material enabled to detect a
obtaining nucellar seedlings are limited and apparently, common pattern. Although there is no big difference
are conditioned by the biological characteristics between them, but the ability of seeds to emerge from
specific to species.
hybrid seeds in both combinations of Citrus