Evaluation of Irradiated Okra based on Agronomical Traits ... · cording to Nei (1987). Also, using these distances, cluster analysis was performed by computational package MVSP (version

Assiut J. Agric. Sci., (48) No. (3) 2017 (81-96) ISSN: 1110-0486 Website: http://www.aun.edu.eg/faculty_agriculture E-mail: [email protected]

Evaluation of Irradiated Okra based on Agronomical Traits and RAPD Markers

Abd El-Aziz, M.H.; K.A. Zaied; Soher E.A. El-Gendy and N.A. Abd El- Gawad

1Genet. Dep. Fac. of Agric., Mansoura Univ., Mansoura, Egypt. 2Vegetable Res. Dept., Horticultural. Res. Inst., ARC, Egypt

*Corresponding author; E-mail: [email protected] Received on: 15/5 /2017 Accepted for publication on: 17/5/2017

Abstract This study was conducted on three inbred lines of okra Abelmoschus escu-

lents (C8, C9 and Cr) obtained from the Horticultural Research Institute, Agricul-tural Research Center. Seeds were irradiated with gamma rays at doses 10, 20 and 30 Kr. This study aimed to evaluate the effect of ionizing radiation on mo-lecular and phenotypic levels, using RAPD six out of 19 RAPD primers were succeeded in generating reproducible polymorphic amplicons among all irradi-ated plants and their control on each inbred line. All irradiated plants of soaked seeds showed the highest unique markers (21) compared with irradiated dry seeds which showed 11 unique markers. This indicated that gamma irradiation was effective for inducing higher rate of mutations in the soaked seeds. On the other hand, C8 gave the highest number of unique markers (10 negative and 4 positive). While, C9 displayed the highest percent of polymorphic amplicons (81.4%) and it was the only genotype which increased in number of complemen-tary sites with RAPD primers by irradiation treatments. Furthermore, other geno-types decreased in number of complementary sites with RAPD primers by treat-ments. Moreover, the correlation between molecular and phenotypic distances was positive and highly significant (r= 0.645) in Cr only, which may be ex-plained that 73.3% for Cr of amplicons were polymorphic from the genome areas which coded for studied traits. So, it is possible to reliance on the unique markers for this inbred line as markers assisted selection for the improved traits due to ir-radiation. In addition, Cr was the best in response to genetic improvement % by irradiation, especially 20/S treatment which led to significant improvement of yield and some of it’s component traits. The improvement percent, ranged from 21.4 to 152.0 % for number of leaves per plant (Nl/p), number of fruits/plant (Nf/p), number of seeds per pod (Ns/pod) and fruit yield per plant (FY/p) traits, was highly expected to be associated with four molecular markers. These mark-ers were three negative and one positive (485bp targeted by OP-A02 primer), which could be used as markers assisted selection to improve yield and its com-ponents in breeding programs and improvement of okra. Keywords: Okra, gamma radiation, RAPD, Molecular Distance, Phenotypic Distance

Introduction Okra, Abelmoschus esculentus L

is a member of the family Malvaceae order Malvales Kaur et al. (2013) and is considered as one of the most im-

portant vegetable crops in Egypt. Okra chromosome numbers is a var-ied and wide-ranging, where 2n = 72, 108, 120, 132 and 144 in regular se-ries of polyploidy with n = 12 kumar

Abd El-Aziz et al., 2017 http://ajas.js.iknito.com/

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et al. (2010). Cultivated area of okra in Egypt exceeded 13 thousand Fed-dan with productivity amounted to about 75 thousand tons (NARP, 1993). Therefore, there is a serious need to increase production of okra because of its highly important at nu-tritive and commercial levels. In this manner, geneticists and breeders who are interested in okra improvements used many different breeding pro-grams to increase production and im-prove the yield and its components. Mutation breeding as one of these programs has been widely used for the improvement of plant traits in various crops Micke (1988). Also, induced mutation has been benefited the plant breeders to explore genetic variations resulting from mutagens. Many reports are available for the successful use of mutation breeding in the production of new cultivars in many crops Hamzekhanlu et al. (2011). Gamma irradiation has been widely used to induce mutations and caused many and different changes on the morphological, cytological and molecular levels. Many phenotypic and biochemical effects caused by irradiation can be evaluated through direct identification of genotypes with DNA based assays Mengoni et al. (2000).

In this way, random amplified polymorphic DNA (RAPD) technique have proven to be the most useful to characterize identities and relation-ships of various crops Nair et al. (2013). The quick development of molecular techniques has allowed the analysis of large number of gene loci distributed throughout the plant ge-nome Singh and Kumar (2012). DNA and protein profiling techniques are

the most commonly used techniques for molecular diversity analysis Nwangburuka et al. (2011). Though, among the molecular markers, RAPD markers were helpful for evaluating genetic diversity and relationship in plants because it is easy handling, low cost and simple way Al adele et al. (2008). Also, RAPD analysis provided high level of polymorphism in the studied cultivar Uzun et al. (2012). The variation in DNA of the irradiated seeds in comparison to the control was successfully assessed us-ing RAPD technique El Sherif et al. (2011). This technique has been used to detect polymorphism and molecu-lar distance (MD) Kaur et al. (2013). Also, RAPD technique succeeded in detecting desirable traits linked to unique markers which could be sepa-rated in the future and used as as-sisted selected markers Hamzekhanlu et al. (2011). The desirable traits were the highest in some cases (plant high, number of leaves or fruits per plant and number of branches per plant )and the lowest in other cases( low fruit diameter and fruit length ) . In okra, low fruit diameter and fruit length were more economically fa-vorable for customers than the high diameter and length. In addition, ear-ly growing traits like number of first flower and date to first flower are more desirable than the late once. In agreed with this finding (Ndou et al., 2013; Nair and Mehta, 2014) studied induced desirable mutations in the two high and low improvement direc-tions resulting from radiation or chemical mutagens comparing with control.

On the other side, phenotypic distances (PD) based on morphologi-


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cal or phenotypic traits were calcu-lated between genotypes according to (Excoffier et al. 1992), these dis-tances are commonly used to assess genetic variation which considered as an effective way to evaluate radia-tions effects (Beuningen & Busch, 1997).

Therefore, this study was fo-cused on the evaluate the effect of ionizing radiation on the seeds of three inbred lines of okra in terms of genetic improvement of some eco-nomical traits, as well as, evaluate ability of RAPD technique to target-ing molecular markers assisted selec-tion. Materials and Methods Plant material

Three inbred lines of okra Ab-elmoschus esculents (C8, C9 and Cr) obtained from the Horticultural Re-search Institute, Agricultural Re-search Center, Mansoura, Egypt were used in this study. Ionizing radiation

Dry seeds and soaked seeds (soaked seeds still in water one night before irradiated) treated with 10, 20, 30 Kr and control (un treated seeds) of gamma rays, source was Co60 at the National center for Radiation Re-search and Technology (NCRRT) (Iqbal et al., 1974). After irradiation seeds was planted directly in pots at rate of 2 seeds /pot using five repli-cates for each treatment Irradiation treatment

Seeds from each inbred line, were divided into seven groups for irradiation treatment, these groups were: [control (untreated seeds), 10, 20, 30/D (dry seeds irradiated with different doses of gamma rays (10, 20, 30 Kr) and 10, 20, 30/S (soaked

seeds with the tree different gamma doses)]. Molecular evaluation using RAPD analysis

Using DNeasy mini spin col-umns as described in detail by Wil-liams et al. (1990), bulked DNA ex-traction was performed from 100 mg of okra seeds collected from each treatment. Genomic DNA from each sample (inbred lines and their treat-ments) was used as a template for Po-lymerase Chain Reaction (PCR) and amplified using 19 RAPD primers. Amplification was carried out in a programmed PCR for 42 cycles as follows: 94°C/4 min (1 cycle), 94°C/1 min, 37°C/1 min, 72°C/2 min (40 cycles), 72°C/10 min (1 cycle) and 4°C (infinitive). Agarose (1.2%) was used for analyzing the PCR products. The DNA ladder (M: 100 bp DNA) was used (3 μl) for deter-mining the molecular size of the am-plicons (amplification products or bands from PCR). Molecular data from banding patterns of RAPD ex-periments were scanned by Gel Ana-lyzer 3 program which scoring clear amplicons as present (1) or absent (0) for each primer and entered in the form of a binary data matrix (Ciucã et al., 2004; Lisek et al., 2005). These data were used for estimating mo-lecular distances (MD) according to de Souza et al. (2012). Using these distances, cluster analysis was per-formed by computational package MVSP (version 3.1) using Nei & Li coefficients according to Nei & Li (1979).

Succeeded primer’s names and sequences


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Six primers succeeded to evalu-ate polymorphism in okra traits, those primers were: OPA-01(5´CAGGCCCTTC3`), OP-A02(5´CAATCGCCGT3`), OP-A18(5´TGGGGGACTC3), OP-B09 (5`TGGGGGACTC3`), OP-C04 (5`GGCTGTCCGT3`), OP-M01 (5`GTTGGTGGCT3`). Phenotypic evaluation

Phenotypic distances (PD) based on morphological traits were calculated using phenotypic data (da-ta not shown) which were estimated for 17 traits. These traits were : emergence percent (E %), seedling fresh weight (SFW g), seedling dry weight (SDW g), plant height (PH cm), number of leaves/plant (Nl/p), number of branches/plant (Nb/p), number of the first flowering (NFF), date of the first flower (DFF), Pollen grain viability % (PV %), number of ridges on fruits (Nr), fruit length (FL cm), fruit diameter (FD cm), number of fruits/plant (Nf/p), fruit weight (FW g), number of seeds/pod (Ns/pod), weight of 100 seeds (W100s) and fruit yield/plant (FY/p kg). These data were used for esti-mating phenotypic distances (PD) ac-cording to Nei (1987). Also, using these distances, cluster analysis was performed by computational package MVSP (version 3.1) using Nei & Li coefficients according to Kovach (2001).

Evaluate the effect of ionizing radiation on the relationship between molecular and phenotypic distances.

Simple correlations and regres-sion analyses were estimated using the computational software Minitab 17 according to Abd El-Aziz et al. (2016).

Estimation of significantly im-provement for economical studied traits

The significantly improvement percent (SI %) for all studied traits except emergence percent trait (this trait did not calculate LSD0.05 values) was calculated using the following formula:

SI % =

Where: mean performance of irra-diation trait which differed sig-nificantly from control.

mean performance of control trait. Results and Discussion Molecular evaluation using RAPD data

Molecular description of mu-tants might be useful for induced ef-ficient mutation induction protocol. DNA polymorphism detected by RAPD analysis gave a helpful mo-lecular marker for identification of mutants in gamma irradiated plants because that RAPD analysis was effi-cient in detecting gamma ray’s muta-tion (Lisek et al., 2005; Khan et al., 2010). In this study RAPD analysis was conducted using 19 arbitrary 10-mer primers. While only, six of these primers were succeeded in generating reproducible polymorphic amplicons among all irradiation treatments and their control in each inbred line (Fig-ure 1,2,3). From banding pattern pro-files of RAPD-analyses, highest changes between control and irradia-tion treatments were appeared in each inbred line. This demonstrated the success of RAPD markers in the de-tection of reproducible polymorphic patterns and confirmed to be valid in discriminating between control and their irradiation treatments of each


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inbred lines. These changes may be due to the structural rearrangements in DNA produced by different types of DNA damages which induced by radiation Hegazi and Hamideldin

(2010). The appearance of new am-plicons may be resulted from differ-ent DNA structural changes (Breaks, deletion, transpositions, etc.).

Figure 1. Banding patterns of RAPD -PCR products for okra inbred line C8 and their treatments pro-

duced by six arbitrary 10-mer primers (OP-A01, OP-A02, OP-A18, OP-B09, OP-M01 and OP-C04).

Figure 2. Banding patterns of RAPD -PCR products for okra inbred line C9 and their treatments pro-



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Figure 3. Banding patterns of RAPD-PCR products for okra inbred line Cr and their treatments pro-


In addition, it is clear from Ta-ble 1 that the six primers out of 19 succeeded in detecting large number of amplicons A (+) and A (−) which differ among irradiation treatments and their control of each inbred line. For inbred line C8 and their treat-ment, all primers successfully showed multiple band profiles (Figure 1) with several amplicons varied from 4 (for OP-A01) to 11 (for OP-A18) (Ta-ble1). In total, 40 amplicons were found, 30 (75.0%) of them were po-lymorphic; of which were 14 (35%) unique amplicons (UA), four UA (+) and ten UA (−). The primer OP-A01 did not target any UA (− or +). The

highest number of UA were targeted by primer OP-C04 with two UA (−) and two UA (+).

Also, all primers were success-fully showed multiple band profiles for C9 (Figure 2), with number of amplicons varied from 4 (for OP-A01) to 9 (for OP-A18 and OP-B09) (Table1). In total 43 amplicons were found 35 (81.4%) of them were po-lymorphic. Seven out of 35(16.3%) amplicons were UA (two negative and five positive UA). The primer OP-A18 detected the highest number of UA (one negative and two positive UA), while the primers OP-A01 and OP-A02 do not targeted any UA.


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Table 1. List of RAPD primers, number of total amplicons, number of polymor-phic amplicons, unique amplicons and percentage of polymorphism obtained by analyzing three inbred lines C8, C9 and Cr with their treatments.

C8 C9 Cr A (−) A (+) A (−) A (+) A (−) A (+)

UA (−) UA (+) UA (−) UA (+) UA (−) UA (+)

Primer Name TA PA

(−) No. MS- t PA (+) No. MS- t

Poly. %

T.A. PA (−) No. MS- t

PA (+) No. MS- t

Poly. %

T.A. PA (−) No. MS- t

PA (+) No. MS- t

Poly. %

OP-A01` 4 1 0 --- 1 0 --- 50.0 4 2 -- --- 1 -- --- 75.0 9 2 -- --- -- 3 445-10/S 550-10/S 590-10/S

55.6

OP-A02` 6 1 2 105 - 30/S 380 - 10/D

0 1 140 - 10/D 66.7 6 2 -- --- 3 -- --- 83.3 6 3 1 220-10/S -- 1 485-20/S 83.3

OP-A18 11 7 2 120 - 30/S 215 - 30/S 1 1 50 - 20/S 100 9 3 1 175-10/S 2 2 45-10/D

480-30/D 88.9 8 3 1 135-20/S 1 2 1060-10/S 1200-10/S 87.5

OP-B09` 5 1 1 320 - 30/S 0 0 --- 40.0 9 2 -- --- 3 1 180-30/S 66.7 8 4 1 310-20/S 1 1 280-20/D 87.5

OP-C04 8 2 2 290 - 20/S 500 - 10/S 0 2

170 - 10/D

560 - 30/D

75.0 7 -- 1 380-10/D 4 1 415-30/S 85.7 7 3 1 210-20/S -- -- --- 57.1

OP-M01` 6 1 3 245 - 20/S 330 - 10/D 420 - 20/D

1 0 --- 66.7 8 3 -- --- 3 1 320-20/D 87.5 7 3 -- --- 2 -- --- 71.4

13 10 3 4 12 2 16 5 18 4 4 7 Total 40

23 (57.5%) 7 (17.5%) 75.0 43

14 (32.6%) 21 (48.8%) 81.4 45

22 (48.9%) 11 (24.4 %) 73.3

T.A: Total amplicons; A (−): Amplicons, appear in control and absent in one or more of the treatments; PA (−): Polymorphic A (-); UA (−): Unique negative amplicon, absent in one treatment; A (+): Ampli-cons, absent in control and appear in one or more of the treatments; PA (+): Polymorphic A (+); UA (+): Unique positive amplicon, appear in one treatment; MS: Molecular size (bp) for unique amplicon; t: control or treatments; Poly. %: Polymorphism percent.

Moreover, in Cr, six primers amplified 45 amplicons, 33 (73.3%) of them were polymorphic and 11 (24,4) were UA (4 negative and 7 positive) (Table 1). The highest num-ber of UA were produced by the pri-mers OP-A01 and OP-A18, while the primer OP-M01do not succeeded in showing any UA.

The treatment of 10 kr for soaked seeds (10/S) was the highest in showing UA (8) for the overall in-bred lines. Likewise, all irradiation treatments for soaked seeds were the highest in showing UA (21) than all irradiation treatments for dry seeds (11 UA). The results indicated that gamma irradiation was an effective tool in inducing mutations especially with the soaked seeds. This illustrated that most RAPD markers used in this study were effective in showing unique markers to be reliable in the election of beneficial mutations.

These results agreed with those ob-tained by Hamzekhanlu et al. (2011), who concluded that RAPD markers were effective in distinguishing the great level of mutation as a result of polymorphism and variation due to gamma radiation in soybean between the irradiated and non-irradiated lines.

On the other hand, from Table 1, C8 showed the highest number of UA with 14 (10 negative and 4 posi-tive), as well as, Cr showed 11 UA (4 negative and 7 positive) and C9 showed 7 (2 negative and 5 positive). Also, the primer OP-A18 succeeded to show 3 unique markers for each inbred line. These primers showed the highest number of amplicons with highest percentage of polymorphism and suggesting that this primer was the best in illustrate the variations in-duced by radiation in okra.


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Besides, from data of A (−) and A (+) which refers to the effect of treatments on the susceptibility of studied inbred lines for compatibility with RAPD primers, these results showed that the percentage of A (+) was greater than A(−) in C9 (A+%=48.8, A−%=32.6) and vice versa in C8 and Cr (A+%=17.5, A−%=57.5 and A+% = 24.4, A−%=48.9, respectively). This indi-cated that C9 was the only genotype which increased in number of com-plementary sites with RAPD primers by treatments, while other genotypes decreased in number of complemen-tary sites with RAPD primers by

treatments. These results were in harmony with Hegazi and Hamidel-din (2010), who found that the high doses of okra gamma radiation gave high changes in DNA pattern. Cluster analysis based on MD and PD

Using MD and PD (Data not shown), cluster analysis was per-formed for appearance the effect of gamma radiation on the molecular and phenotypic levels and the rela-tionship between them (Figure 4,5 and 6). The UPGMA dendrograms based on MD and PD representing the cluster analysis for control and irradiation treatments.

Figure (4). Dendrograms for C8 showing the relationship among control and their irradiation treatments

based on MD and PD.

Figure (5). Dendrograms for C9 showing the relationship among control and their irradiation treatments

based on MD and PD.

Figure (6). Dendrograms for Cr showing the relationship among control and their irradiation treatments

based on MD and PD.


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These dendrograms succeeded in illustrating the degree of genetic divergence induced by radiation in each inbred line through molecular and phenotypic distances. This agreed with Aladele et al. (2008), who found that RAPD succeeded in differentiat-ing okra genotypes. Also, Khan et al. (2010) reported that RAPD can be used as a respectable technique in distinguish plants genotypes.

These cluster analyses, showed true variation among control and irra-diation treatments for each inbred line. This variation proved that irra-diation treatments applied in this study was succeeded in induced ge-netic variations among molecular and phenotypic levels of inbred lines. Also, this variation can be useful in okra breeding programs by selection. This agreed with, Singh and Datta (2009), who demonstrated that gamma irradiation encouraged pheno-typic changes in wheat, whereas total leaf mass, plant mass and the number tillers increased 3 times more than control as a result of gamma irradia-tion. Also, Sangsiri et al. (2005) found that with 500 gy dose, mutant characters appeared, demonstrated that gamma irradiation encouraged phenotypic changes.

As shown in Figure (6), the rela-tionships between (MD) and (PD) for

C8, C9 were negative correlation, where the correlation values were r=-0.335, r=-0.021 respectively. While, Cr was highly significant positive correlation (r= 0.645). The negative correlation values between MD and PD for C8 and C9 inbred lines may be explained by the fact that 75.0% for C8 and 81.4% for C9 of ampli-cons were polymorphic from any area of genome. These areas may not nec-essarily be coded for any morpho-logical trait. This explained that 73.3% for Cr of amplicons were po-lymorphic from the genome areas which coded for morphological traits.

The negative-correlation among two types of genetic distances for C8 and C9 can be explicated by the fact all treatments had been evaluated us-ing one molecular technique at one location and one season. To deal with the poor correlation between molecu-lar and phenotypic distances in C8 and C9, it may be better using many more numbers of RAPD primers or other molecular techniques to evalu-ate the treatments at more than loca-tion or allocation under different en-vironments. These agreed with Abd El-Aziz et al. (2016), who found that the same relationship of MD for six inbred lines of maize were not corre-lated significantly with PD.

0.350.300.250.200.150.10

300

250

200

150

100

50

S 61.5287R-Sq 11.2%R-Sq(adj) 6.5%

GD

PD

C8PD = 232.7 - 311.9 GD

Pearson correlation of PD and GD = -0.335 ns 0.50.40.30.20.1

200

175

150

125

100

75

50

S 43.1629R-Sq 0.0%R-Sq(adj) 0.0%

GD

PD

C9PD = 130.1 - 8.19 GD

Pearson correlation of PD and GD = -0.021 ns

0.50.40.30.20.1

500

400

300

200

100

0

S 123.640R-Sq 41.6%R-Sq(adj) 38.5%

GD

PD

CrPD = - 73.60 + 1046 GD

Pearson correlation of PD and GD = +0.645 ** Figure 7. Relationship between MD and PD for three inbred lines C8, C9 and Cr.


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Highly significant positive cor-relation obtained between MD and PD in Cr cultivar may be explained with the high relation between the two types of distances, this high cor-relation proved that gamma rays suc-ceeded in changing the characters of Cr trait on the two phenotypic and molecular levels. This was in har-mony with what was illustrated pre-viously in okra by Martinello et al. (2001), who explained that MD based on RAPD data, average distance, from the morphological and quantita-tive data has similar dendrogram pat-terns. Also, Barazani et al. (2003) confirmed the importance of combin-ing molecular markers with morpho-logical data. All those authors, indi-cates that it is possible to reliance on the unique amplicons in this inbred line as markers assisted selection for the improved traits due to irradiation.

Assessment of height and de-crease in the studied traits resulting from irradiation treatments.

For assessment of irradiation ef-fect on phenotypic traits, in Table 2, it was clear that irradiation treatments lead to significant changes in all stud-ied traits. Many of these changes

were economically desirable and some were not.

The economically desirable im-provement was in some traits as fol-lows: 7 traits for C8 (Nl/p, Nb/p, NFF, Nf/p, Ns/pod, W100s and FY/p kg), 4 traits for C9 (Nb/p, Nr, FD cm, and W100s) and 9 traits for Cr (Nl/p, Nb/p, Nr, FL cm, Nf/p, FW g, Ns/pod, W100s and FY/p kg). It could be noticed that most improved traits were very important as it were economical and productive traits.

The improvement in C8 traits mostly resulted from the irradiation treatment 30/D which led to im-provement of yield and some yield component traits. While, C9 was the lowest inbred line in response to im-provement by irradiation treatments. As for Cr, was the best in response to improvement by irradiation treat-ment, especially 20/S which led to significant improvement of yield and some yield component traits. This could be attributed to the high corre-lation between changes under mo-lecular and phenotypic levels which reflected that gamma irradiation suc-ceeded in improving the most traits in Cr.


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Table 2. Mean performance of morphological and yield for three inbred lines. C8 C9 Cr

Treatments Treatments Treatments Trait DI Con. Low High LSD0.05Con. Low High

LSD0.05 Con. Low High LSD0.05

SFW g In. 115.8 5.1* (30/S)

155.5 (20/D)

74.5 78.6 30.1* (30/S)

118.2 (10/D)

47.2 100.7 56.3 (30/S)

135.6 (20/D)

57.8

SDW g In. 12.5 0.6* (30/S)

16.8 (20/D) 8.0 9.1 4.2

(30/S) 9.6

(10/D) 5,6 9.7 1.8* (20/S)

13.6 (10/D) 6.7

PH cm In. 282 143* (10/S)

261 (10/D) 56 279 146*

(20/S) 249

(20/D) 69 256 155* (30/S)

221 (10/D) 48

Nl/p In. 334 277* (30/S)

483* (30/D)

57 349 249 (30/S)

392 (20/S)

116 322 286 (10/D)

636* (20/S)

115

Nb/p In. 5.33 5.33 (10/D,30/S)

7.50* (10,20/S)

1.12 5.00 5.00 (30/S)

6.67* (20/S)

1.28 6.83 5.83 (30/S)

9.33* (10/S)

1.53

NFF De. 3.17 3.17 (20/D)

5.00* (20/S) 0.99 3.33 3.33

(30/S) 4.50

(20/S) 1.53 5.67 4.67 (20/S)

7.00 (10/S) 1.86

DFF De. 56.2 55.8 (10/D)

72.5* (30/S) 2.3 56.7 57.0

(10/S) 65.0* (30/S) 5.2 62.0 60.7

(10/D) 66.2* (30/S) 3.0

PV % In. 88.1 48.2* (30/D, S)

72.7* (10/S) 9.1 81.7 49.9*

(30/S) 73.1* (10/D) 3.1 78.6 46.4*

(30/D) 53.1

(10/D) 4.3

Nr In. 5.83 5.00* (20,30/S)

5.50 (20/D)

0.55 6.33 5.00* (20/D,20/S,30/S)

8.50* (10/S)

0.92 6.17 6.00 (`10/D)

8.00* (20/D)

1.08

FL cm De. 4.10 3.12* (10/S)

3.93 (30/S) 0.85 4,18 3.11*

(30/S) 5.05

(10/S) 1.00 4.01 3.46 (20/D)

4.92* (30/S) 0.88

FD cm De. 1.03 0.94 (30/D)

1.15 (10/S) 0.24 1.22 0.99*

(30/D) 1.26

(20/S) 0.19 1.09 0.98 (20/D)

1.55* (10/S) 0,25

FW g De. 4.83 3.84* (20/D)

5.40 (10/S)

0.85 6.48 4.20 (30/S)

7.74 (10/S)

1.47 5.31 4.51 (10/D)

9.84* (10/S)

1.98

Nf/p In. 326 270 (30/S)

474* (30/D) 60 342 241

(30/S) 383

(20/S) 112 332 274 (10/D)

627* (20/S) 113

Ns/pod In. 73.2 74.2 (10/D)

96.2* (30/D) 7.2 101.7 83.8*

(30/D) 93.5

(10/S) 11.0 96.2 98.8 (10/D)

116.8* (20/S) 12.4

W100s In. 4.51 4.46 (10/D)

4.83* (30/S) 0.24 4.22 4.26

(10/D) 4.79*

(20,30/S) 0.34 4.67 4.74 (10/D)

5.09* (30/S) 0.19

FY/p kg In. 1.60 1.26 (30/S)

2.31* (30/D)

0.57 2.21 1.00* (30/S)

2.67 (20/S)

0.77 1.77 1.22 (10/D)

4.46* (20/S)

1.53

* Significant at 0.05, DI: Desirable improvements by increasing (In.) or decreasing (De.) compared with genetic origin (Con.)

Molecular markers related to sig-nificant economic improved traits.

As shown in Table 3, most irra-diation treatments were led to signifi-cant economic improvement in sev-eral traits could be linked to the mo-lecular.

For C8, it was clear that the number of improved traits that could be linked to molecular markers were eight traits, the percent of improve-ment for these traits ranged from 7.1 to 45.4 % for W100S and N.f.p re-spectively, which could be associated with ten molecular markers. While, the desirable improvement was only for four traits in C9, which were Nr, FD, FL and W100 with 13.5, 18.9, 25.6 and 34.3 improvement %, re-

spectively. The improvement in each one of these traits, could be associ-ated with one molecular marker as described in Table 3.

More precisely, the inbred line Cr showed significant improvement in six traits, which is highly expected to be associated with 11 molecular markers due to the high correlation between the MD and PD in this line. These molecular markers were seven positive and four negative, one of them (280 bp targeted by OP-B09) could be associated with the im-provement in Nr trait by 10/S, six of them could be associated with the improvement in Nb/P and FW g traits by 20/D treatment. As well as, Cr was the best in response to genetic im-


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provement %, especially by 20/S, which led to significant improvement of yield and some yield component with SI% was ranged from 21.4 to 152.0 % in Nl/P, Nf /p, Ns/pod and FY/p kg traits, which were associated

significantly with four molecular markers, three negative and one posi-tive (485bp targeted by OP-A02 pri-mer). These primers could be used as a marker to improve one or more traits of yield and yield components.

Table 3. Compatibility among molecular markers and desirable improvement of

some economical studied traits. DI* Markers (M.)

DI* % Traits Total No. of

M. Type of

M. MS (bp) Primer Treatment Cultivar

23.9 40.7

FL Nb/P

1 − 500 OP-C04 10/S

20.5 FW 1 − 420 OP-M01 20/D

40.7 Nb/P 3 + − −

50 290 245

OP-A18 OP-C04 OP-M01

20/S

44.9 45.4 31.4 44.4

Nl/P Nf/p

Ns/pod FY/p kg

1 + 560 OP-C04 30/D

7.1 W100s 4 − − −

105 215, 120

320

OP-A02 OP-A18 OP-B09

30/S

C8

34.3 N.r 1 − 175 OP-A18 10/S 18.9 FD 1 + 480 OP-A18 30/D 25.6 13.5

FL W100s

2 + 180 415

OP-B09 OP-C04

30/S C9

36.6 Nb/P 6 + − +

590,550,445 220

1200,1060

OP-A01 OP-A02 OP-A18

10/S

29.7 Nr 1 + 280 OP-B09 20/D 97.5 88.9 21.4 152.0

Nl/P Nf\p

Ns/pod FY/p kg

4

+ − − −

485 135 310 210

OP-A02 OP-A18 OP-B09 OP-C04

20/S

Cr

M.: Molecular markers [+: UA (+) or – UA (-)]; MS: Molecular size (bp), DI* % significantly desirable improvement percent.

Results obtained from the table

indicated that, most of irradiation treatments were led to desirable im-provement of many studied traits in associated with molecular markers via RAPD technique. These markers could be used as markers assisted se-lection for these traits in breeding and improvement of okra. These results agreed with Dubey et al. (2007), who found that irradiated okra seeds with

different doses of gamma rays caused an improvement in number of yield and yield component traits. predicted by Hamzekhanlu et al. (2011), who indicated that RAPD markers in fu-ture researches could be very impor-tant in detecting mutation that associ-ated with desirable traits which could use as marker assisted selection. To achieve this importance in our study, DNA sequencing must be done for


93

these molecular markers between ir-radiation lines and their original sources to identify DNA markers as-sist selection of desirable traits. This was the aim to achieve in future stud-ies. Conclusion

In conclusion, gamma rays were succeeded for inducing desirable changes in okra at the two phenotypic and molecular levels. The inbred line Cr was the highest one in improved traits by radiation and it was the only one giving high positive significant correlation between molecular and phenotypic distances. In addition, RAPD technique proved to be a good method in detecting different molecu-lar markers which could be used as markers assisted selection for im-proved traits by irradiation treatments in programs of breeding and im-provement of okra. References Abd El-Aziz, M. H.; A. N. Attia; M. S.

Sultan; M. A. Badawi and A. R. M. Al-Rawi (2016). Phenotypic and genetic diversity and their re-lationship to f1 performance for yield traits in some maize inbred lines. J. Agric. Chem. and Bio-techn, Mansoura Univ., 7(3): 96:104.

Aladele, S. E.; O. J. Ariyo and Robert De Lapena (2008). Genetic rela-tionships among West African okra (Abelmoschus Caillei) and Asian genotypes (Abelmoschus es-culentus) using RAPD. African Journal of Biotechnology,7 (10): 1426-1431.

Barazani, O.; A. Atayev; B. Yakubov; V. Kostiukovsky; K. Popov and A. G. Goldhirsh (2003). Genetic vari-ability in Turkmen populations of Pistacia Vera L. Genet. Resour. Crop Evol. 50: 383-389.

Beuningen, V. L. T. and R. H. Busch (1997). Genetic diversity among North American spring wheat cul-tivars: III. Cluster analysis based on quantitative morphological traits. Crop Sci., 37: 981–988.

Ciucã, M.; M. Pãcureanu and M. Iuora. (2004). RAPD markers for poly-morphism identification in para-sitic weed Orobanche Cumana wallr. Agricultural research and development institute (A.R.D.I.), 915200 Fundulea, Calarasi Coun-ty.

De Souza, S. G. H.; V. C. Pípolo; D. D. Garbúglio; N. S. F. Júnior; C. F. Ruas and P. M. Ruas (2012). Mo-lecular distance estimated by RAPD markers and performance of top cross hybrids in Popcorn. American J. of Plant Sciences, 3: 1666-1673.

Dubey A. K.; J. R. Yadav and B. Singh (2007). Studies on induced muta-tions by gamma irradiation in okra (Abelmoschus esculentus (L.) Monch). Progressive Agric., 7 (1/2): 46-48.

El Sherif, F.; S. Khattab; E. Ghoname; N. Salem and K. Radwan (2011). Effect of gamma irradiation on en-hancement of some economic traits and molecular changes in Hibiscus Sabdariffa L. Life Science Journal, 8(3):220-229.

Excoffier, L.; P. Smouse and J. Quattro (1992). Analysis of molecular va-riance inferred from metric dis-tances among DNA haplotypes: application to human mitochon-drial DNA restriction data. Genet-ics. 131: 479-491.

Hamzekhanlu, M.Y.; A. I. Darbandi; N. P. Beiranv; M. T. Hallajian and A. Majdabadi (2011). Phenotypic and molecular analysis of M7 genera-tion of soybean mutant lines through random amplified poly-morphic DNA (RAPD) marker and


94

some morphological traits. African Journal of Agricultural Research, 6(7):1779-1785.

Hegazi A. Z. and N. Hamideldin (2010). The effect of gamma irradiation on enhancement of growth and seed yield of okra [Abelmoschus escu-lentus (L.) Monech] and associated molecular changes. Journal of Hor-ticulture and Forestry, 2(3): 038-051.

Iqbal, J.; M. Kutaček and V. Jiraček. (1974). Effects of acute gamma ir-radiation on the concentration of amino acids and protein-nitrogen in Zea mays. Radiation Botany, 14(3):165-172.

Kaur, K.; Mamta Pathak; Satinder Kaur; Dharminder Pathak and Neena Chawla. (2013). Assessment of morphological and molecular di-versity among okra [Abelmoschus esculentus (l.) Moench]. Germ-plasm,12 (21):3160-3170.

Khan, A. I.; F. S. Awan; B. Sadia; R. M. Rana and I. A. Khan (2010). Ge-netic diversity studies among col-oured cotton genotypes by using RAPD markers. Pak. J. Bot., 42(1): 71-77.

Kovach, W. (2001). Multi-variate statis-tical package 3.12G. Kovach Computing Services, Aberystwyth, Wales, UK

Kumar S., Sokona Dagnoko, Adamou Haougui, Alain Ratnadass, Dov Pasternak and Christophe Kouame (2010). Okra (Abelmoschus spp.) in West and Central Africa: Poten-tial and progress on its improve-ment. African Journal of Agricul-tural Research 5(25):3590-3598.

Lisek, A.; Małgorzata Korbin and El żbieta Rozpara (2005). Using sim-ply generated RAPD markers to distinguish between sweet cherry (prunus Avium l.) Cultivars. Fruit ornam. Plant res., 14: 53-59.

Martinello, G. E.; N. R. Leal; A. T. Jr. Amaral; M. G. Pereira and R. F. Daher (2001). Comparison of morphological characteristics and RAPD for estimating genetic di-versity in Abelmoschus spp. Acta Hort., (ISHS) 546:101-104.

Mengoni A.; Gori A. and Bazzicalupo M. (2000). Use of RAPD and mi-crosatellite (SSR) variation to as-sess genetic relationships among populations of tetra ploid alfalfa (Medicago sativa). Plant Breeding, 119: 311-17.

Micke, A. (1988). Genetic improvement of grain legumes using induced mutations. An overview in: Im-provement of Grain Legume Pro-duction Using Induced Mutations. IAEA, Vienna, 1-51.

Nair, B. R.; R. G. Vadhyar; M. R.M; R. P Chandran; S.R Resmi; J. Sreeja and G. Valsaladevi (2013). Identi-fication of molecular markers in species of Abelmoschus medic. (Malvaceae) for inter- and intra-specific discrimination. Indian Journal of Fundamental and Ap-plied Life Sciences, 3 (1): 52-61

Nair, R.; and A.K. Mehta (2014). in-duced mutagenesis in cowpea [Vi-gna Unguiculata (L.) Walp] Var. Arka Garima. Indian J. Agric. Res., 48 (4):247 – 257.

NARP (1993). National Agricultural Re-search Project, Horticultural crops. Vol. 3, July. NARP, CSIR, Accra.

Ndou, V. N.; H. Shimelis, A. Odindo and A. T. Modi.(2013). Response of selected wheat genotypes to ethyl methane sulphonate concen-tration, treatment temperature and duration. Scientific Research and Essays, 8(4):189 -196.

Nei, M. (1987). Molecular Evolutionary Genetics. (Chapter 9). New York: Columbia University Press.es, UK.

Nei, M. and W. H. Li (1979). Mathe-matical model for studying genetic


95

variation in terms of restriction en-donucleases. Proc. Natl. Acad. Sci., USA, 76:5269-5273.

Nwangburuka, C. C.; O. B. Kehinde; D. K. Ojo; A. R. Popoola; O. Odu-waye; O. A. Denton and M. Ade-koya (2011). Molecular characteri-zation of twenty-nine okra acces-sions using the random amplified polymorphic DNA (RAPD) mo-lecular marker. actaSATECH 4(1): 1 – 14.

Sangsiri, C.; W. Sorajjapinun, and P. Srinives (2005). Gamma Radiation Induced Mutations in Mung bean. Science Asia, 31: 251-255.

Singh, B. and P.S. Datta (2009). Gamma irradiation to improve plant vigour, grain development, and yield at-tributes of wheat. Radiation Phys-ics and Chemistry, 79(2): 139-143.

Singh, V. and V. Kumar (2012). An op-timized method of DNA isolation from highly mucilage-rich okra (Abelmoschus esculentus l.) for PCR analysis. Advances in Ap-plied Science Research, (3):1809-1813.

Uzun A.; U. Seday and C. Turkay (2012). Molecular marker based analysis of genetic diversity and relationships in some Turkish and foreign olive cultivars and acces-sions. Sabrao Journal of Breeding and Genetics 44 (1): 177-188.

Williams, J.K.; A. R. Kubelisk; K. J. Livak; J.A. Rafalski and S.V. Tin-gey (1990). DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research, 18: 6531–6535.


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ات علي الصفات المحصولية وواسمتقييم البامية المعاملة باالشعاع بناء RAPD ونيرة على عبدالجواد سهير السيد عبده الجندي، خليفة عبدالمقصود زايد، محمد حسن عبدالعزيز

. مصر– جامعة المنصورة – كلية الزراعة –قسم الوراثة ١ . مصر– المنصورة –ية مركز البحوث الزراع- معهد بحوث البساتين ٢

:الملخص C8 ، C9 ، Cr هـي داخلياً من البامية سالالت مرباهذه الدراسة على ثالثة هأجريت

تم تقـسيم بـذور . مركز البحوث الزراعيةـ والتي تم الحصول عليها من معهد بحوث البساتين تـم موعـات هـذه المج حيـث ). منقوعة أخرىمجموعة جافة و(مجموعتين إلى السالالتهذه

تقيـيم تـأثير لك بهـدف ذو كيلو راد ٣٠ و ٢٠ و ١٠ متدرجة شعة جاما بجرعات أل تعريضهاصـفة ١٧تقدير ومن خالل RAPDبواسطة تقنية اإلشعاع على المستوى الجزيئي والمظهري

وقد نجح ستة مـن . )المعاملةالنباتات غير (الكنترول مقارنة مع بالعة ع المش الفردية على النباتات فـي الكنترولومعامالت البين جميع تباينت تضاعف تتابعات من الجينوم استهداف في بادئ ١٩واسمات جزيئية هي األعلى في إظهار كانت المنقوعةجميع معامالت التشعيع للبذور . ساللةكل

١١ أعطـت والتـي للبذور الجافـة معامالت اإلشعاع جميع مقارنةً ب ) واسمة ٢١بواقع (ة متفرد الطفـرات اسـتحداث كانت أداة فعالة فـي جاما يدل على أن أشعة هذا .ةمتفرديئية واسمة جز

من الواسمات الجزيئية أعلى عدد C8ت الساللة أعط أخرىمن ناحية . معاملة النقع وخاصة مع أعلـى نـسبة C9 ت الساللةأظهرفي حين ). إيجابية ٤ سلبية و ١٠ ( واسمة ١٤ بواقع المتفردة

التتابعـات عـدد يهف ازدادت الوحيد الذي الوراثيالتركيب كان حيث ٪ ٨١,٤ تباين حزم بواقع في حـين انخفـضت ،بتأثير معامالت التشعيع المختلفة المستخدمة RAPDبوادئ مع لمتكاملةا

بتـأثير معـامالت RAPDبـوادئ مع لمتكاملة ا التتابعات في عدد التراكيب الوراثية األخرى يـة بين المـسافات الجزيئيـة والمظهر االرتباطلك، كانت عالقة عالوة على ذ. التشعيع المختلفة

فيمما يوضح أن نسبة تباين الحزم ، فقط Cr الساللة في) r = 0.645 (موجبة وعالية المعنوية هـذا يـشير . للصفات المدروسةالمشفرةالجينوم تتابعات ربما تكون من ) ٪ ٧٣,٣( الساللةهذا االنتخابعلى تساعد جزيئية عالماتك الساللة هلهذلواسمات الجزيئية ا على االعتماد إمكانيةإلى

للتحسن األفضل استجابةً Cr كانت الساللة ، باإلضافة إلى ذلك . التشعيع نتيجةللصفات المحسنة تأد والتـي للبذور المنقوعـة كيلو راد ٢٠المعاملة ، خاصة بواسطة معامالت التشعيع الوراثي

بـين حيث تراوحت نسب التحسين . المحصول وبعض صفات مكوناته صفةإلى تحسن كبير في بالنبات وعدد البـذور بـالقرن ) القرون(وراق وعدد الثمار ألعدد ا لصفات ٪١٥٢,٠ و ٢١,٤

واسـمات أربع مع تكون مرتبطة أن يفترض بشكل كبير والتي، ومحصول النبات بالكيلو جرام زوج مـن ٤٨٥ الجزيئـي حجمهـا [ موجبةا كانت واحدة منه و منها كانت سالبة ثالثة جزيئية

ـ ايمكن اسـتخدامه هذه الواسمات. ])OP-A02( بالبادئاستهدفهاوتم النيوكليوتيدات ات كعالم باسـتعمال ومكوناتـه لمحصول ا أو أكثر من صفات صفةلتحسين االنتخابعلى تساعد جزيئية

.يةفي برامج التربية والتحسين في البام الوراثيالتركيب هذا

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Evaluation of Irradiated Okra based on Agronomical Traits ... · cording to Nei (1987). Also, using these distances, cluster analysis was performed by computational package MVSP (version