Molecular and biochemical markers of some Vicia faba L. genotypes in response to storage insect pests infestation

Abstract

Storage insect pests cause serious losses to all legume crops in both quality and quantity. This study was conducted to study some morphological characters and biochemical components in eight genotypes of faba bean to determine biochemical and molecular markers for resistance and susceptibility to infestation with stored insects. The results showed that chlorophyll content in leaves, phenol, tannin, peroxidase (POD), polyphenol oxidase (PPO), and protein content in leaves and seeds of faba bean plants either significantly or non significantly increased the effect of insect infestation in the resistance genotypes (L.551, L.512, L.153, NA112, and L.1) as compared with the susceptible genotypes (L.16 and T.W). Protein electrophoresis showed a wide variation between genotypes and determined some biochemical markers (sodium dodecyl sulfate poly acrylamide gel electrophoresis, SDS-PAGE). In addition, molecular genetic markers for stored insects' resistance were obtained using inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) analyses.

Keywords:

• phenol
• tannin
• peroxidase
• polyphenol oxidase
• protein electrophoresis
• ISSR-PCR

Abbreviations
SDS-PAGE	=	sodium dodecyl sulfate poly acrylamide gel electrophoresis
ISSR-PCR	=	inter-simple sequence repeat polymerase chain reaction
POD	=	peroxidase
PPO	=	polyphenol oxidase

Introduction

Storage insect pests cause serious losses to all legume crops in both quality and quantity, particularly in the tropics and subtropics where temperatures and relative humidity are high. The most important species of storage insect pests of food legumes include: Callosobruchus chinensis, Callosobruchus maculatus, Callosobruchus analis, Acanthoscelides obtectus, and Bruchus incarnatus (Kashiwaba et al. 2003). In addition, Bruchus rufimanus, Bruchus dentipes, Bruchus quinqueguttatus, Bruchus emarginatus, Bruchus ervi, Bruchus lentis, and Bruchus pisorum may also cause significant losses in some legumes (Desroches et al. 1995). The seeds of legumes, once damaged by storage insects, are no longer fit for planting (due to poor germination) or for food or feed (due to spoilage and bad smell; Haile 2006).

Several environmental manipulations can be attained by employing a number of control measures like the use of chemical insecticides and cultural and physical control methods. Chemical pesticides are effectively used against storage insect pests but are inseparably associated with a number of drawbacks including high costs and concerns about environmental pollution and food safety. An effective and environment-friendly management option against storage insect pest in different legume crops could be achieved by improving the genetic resistance of the host plant (Somta et al. 2008).

Molecular tools give us an opportunity to develop genotypes that carry resistance traits (Ranjekar et al. 2003), and these tools have been utilized in DNA fingerprinting for identification of cultivars, marker-assisted selections and, to a limited extent, for genetic modification in breeding for insect resistance (Acosta-Gallegos et al. 2008).

Plant–insect interaction is a dynamic system, subjected to continual variation and change. In order to reduce insect attack, plants developed different defense mechanisms including chemical and physical barriers such as the induction of defensive proteins (Haruta et al. 2001), volatiles that attract predators of the insect herbivores (Birkett et al. 2000), secondary metabolites (Baldwin 2001), and trichome density (Fordyce & Agrawal 2001).

Chlorophyll content is one of the most important parameters in the relationships between plants and herbivores. Chlorophyll levels change during plant development (Costa et al. 2001). Chlorosis is the most obvious plant injury symptom on cabbage leaves after aphid feeding and is indicative of chlorophyll loss (Khattab 2007). Among the secondary metabolites, plant phenols constitute one of the most common and widespread group of defensive compounds, which play a major role in host plant resistance (HPR) against herbivores, including insects (Sharma et al. 2009). Tannins have a strong deleterious effect on phytophagous insects and affect the insect growth and development by binding to the proteins, reduce nutrient absorption efficiency, and cause midgut lesions (Barbehenn & Constabel 2011).

Oxidative state of the host plants has been associated with HPR to insects (He et al. 2011) which results in the production of reactive oxygen species (ROS) that are subsequently eliminated by antioxidative enzymes. Peroxidase (POD) constitutes one such group of enzymes which scavenges the ROS besides having other defensive roles. PODs are an important component of the immediate response of plants to insect damage (Gulsen et al. 2010; War et al. 2012). The polyphenol oxidase (PPOs) are also important enzymes in plants that regulate feeding, growth, and development of insect pests and play a leading role in plant defense against the biotic and abiotic stresses (He et al. 2011).

Vicia faba is the most important legume crop for human and livestock in Egypt. It makes an important contribution to the diet of people in many countries. It represents a very interesting class of food crops due to its high protein content (30%; Gaber et al. 2000). It can grow successfully in different soil types and it increases soil fertility.

The present study aimed to study the effect of stored insects on morphological, physiological, and molecular changes in eight genotypes of Vicia faba and to obtain molecular and biochemical markers for resistance and susceptibility to stored insects.

Materials and methods

The present investigation was carried out during two growing seasons (2010–2012) at Bhtem Research Station, Egypt. Eight faba bean genotypes were utilized in this study (Table 1).

Table 1. The name of faba bean genotypes and its origin.

Parent	Pedigree
L.441	Landraces in Benisuf
L.512	Landraces in Benisuf
L.153	Landraces in Assiut
L.1	Landraces in Assiut
Triple white (T.W)	Individual plant selections from Sudanese materials
NA112	Individual plant selections from Pakistan
Giza 3	Egypt
L.16	Landraces in Aswan

Preliminary study

Tannin contents in seeds, the preference of stored insects to host seeds, the ability to complete development and the susceptibility index in host seeds were used to determine the resistance of faba bean genotypes to stored insects.

Preference experiment: Three cylinders were divided into 48 equal compartments to represent three replicates. Each genotype was represented by 2 g of seeds which were placed on each compartment. Each experiment cylinder was provided with 75 pairs of newly emerged adults. Preference percentage was measured as the following equation:

Mean development period (MDP): Number of days from time of egg laying up to adult emergence from the seeds was counted.

Field experiments: Seeds of eight faba bean genotypes were planted after screening in a randomized block design with three replications. The experimental plant consisted of three ridges 3.0-m long, 60-cm apart with single-seeded hills, 20-cm apart on both sides of ridges. Three plants of each genotype were collected after 45 days (vegetative stage) to determine chlorophyll content, phenol, tannin, POD, PPO, total protein, protein electrophoresis, and DNA isolation. At the end of growing season, the plants were collected to measure morphological criteria and yield.

Determination of photosynthetic pigments

Chlorophyll a, Chlorophyll b, and carotenoids were determined in faba bean leaves. The spectrophotometric method recommended by Vernon and Seely (1966) was used. The pigment contents were calculated as mg g^–1 fresh weight of leaves.

Determination of total phenols

Levels of soluble phenols in faba bean leaves were determined in accordance with Dihazi et al. (2003). The absorbance of the developed blue color was read at 725 nm. Tannic acid was used as the standard and the amount of soluble phenols was expressed as mg tannic acid g^–1 dry weight.

Determination of tannin content

Tannin was determined using vanillin hydrochloric acid method as described by Burn (1971). The absorbance of samples was measured at 500 nm using spectrophotometer. Pure catechin was used for standard curve.

Extraction of enzymes and total soluble protein

Two grams of fresh sample were homogenized in cold phosphate buffer (0.05 M at pH 6.5). The homogenate was centrifuged at 10,000g for 10 minutes. The pigments were removed from the supernatant by adsorbing on activated charcoal and filtered. The filtrate was completed to a known volume and used to determine enzymes and total soluble protein.

Assay of enzymes activity

POD (EC 1.11.1.7) and PPO (EC 1.14.18.1) was assayed following the method of Kar and Mishra (1976). The sample was read at 430 nm and the enzyme activity was expressed as enzyme activity/gram fresh weight/h.

Estimation of total soluble protein

The total soluble protein content in the supernatant was determined according to Lowry et al. (1951). The quantity of total soluble protein was calculated according to the standard curve of Bovine Serum Albumin and expressed as mg g⁻¹ fresh weight.

Protein electrophoresis

Gel electrophoresis sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE) was carried out with gel slabs according to the method of Laemmli (1970). Polypeptide pattern was analyzed on 12% SDS polyacrylamide gels. Protein subunit bands were stained with Coomassie blue R-250 by standard techniques. The gel was scanned using the Gel-Pro analyzer. The molecular weights were calculated by Gel-Pro analyzer program according to the molecular weight marker.

ISSR-PCR of genomic DNA

Seeds of faba bean plants (100 mg) were ground under liquid nitrogen to a fine powder to extract DNA as described by Dellaporta et al. (1983). Inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) was conducted using seven 10-mer arbitrary primers with the sequences shown in Table 2. The DNA amplifications were performed in an automated thermal cycle (model Techno 512) programmed for one cycle at 94°C for 4 min followed by 45 cycles of 1 min at 94°C, 1 min at 57°C, and 2 min at 72°C. The reaction was finally stored at 72°C for 10 min. PCR products were run at 100 V for 1 hour on 1.4% agarose gels to detect polymorphism between various genotypes under study. After electrophoresis, the ISSR patterns were visualized with UV transilluminator. Gels were photographed using a Polaroid camera.

Table 2. List of primer names and their nucleotide sequences used in the study for ISSR procedure.

No.	Name	Sequence
1	14 A	5′ CTC TCT CTC TCT CTC TTG 3′
2	44B	5′ CTC TCT CTC TCT CTC TGC 3′
3	HB-09	5′ GTG TGT GTG TGT GC 3′
4	HB-11	5′ GTG TGT GTG TGT TGT CC 3′
5	HB-13	5′ GAG GAG GAG GC 3′
6	HB-14	5′ CTC CTC CTC GC 3′
7	HB-15	5′ GTG GTG GTG GC 3′

Statistical analysis

All data were subjected to statistical analysis and means were compared by Duncan's multiple range test using Mstat C computer package.

Results and discussion

Effect on morphological criteria

Nine traits were measured on eight genotypes of faba bean: plant height, number of branches, first fertile nodes, number of seeds per pod, number of pods per plant, number of seeds per plant, seed weight per plant, 100 seeds’ weight, and pod length. Mean comparison for the yield and seed quality traits were done according to Duncan's multiple range test as shown in Table 3. These mean comparisons revealed highly significant and nonsignificant differences between the investigated genotypes. Number of pods per plant and number of seeds per plant significantly increased in the tolerant genotypes. These results are in accordance with El-Sayed (2006).

Table 3. Effect of infestation with stored insects on morphological criteria in susceptible and tolerant genotypes of Vicia faba plants.

Genotypes	Susceptibility index	Plant height (cm)	No. of branches	First fertile nodes (cm)	No. of seeds/pods	No. of pods/plant	No. of seeds/plant	Seed weight/plant (g)	100 seed weight (g)	Pod length (cm)
L.551	0.00	117.0A	3.0C	4.0CD	4.0A	21.0BCD	70.2ABC	57.0AB	80.3AB	10.0AB
L.512	0.00	92.0D	4.0BC	7.0A	4.0A	22.0BC	57.2CDE	49.5BC	81.4A	8.0C
L.153	0.00	105.0BC	4.0BC	5.0B	3.0BC	17.0E	49.2E	41.2C	84.0A	10.0AB
L.1	0.00	112.5AB	4.0BC	4.0CD	4.0AB	24.0AB	73.2AB	65.2A	88.6A	12.0A
NA112	0.00	45.0E	5.0A	4.0CD	3.0C	26.0A	80.0A	19.7D	24.5C	5.0D
T.W	6.12A	83.8D	3.0D	2.0E	3.0C	22.0BC	61.2BCD	45.5BC	74.6B	7.0C
Giza3	2.15B	117.0A	4.0AB	3.0DE	4.0AB	18.0DE	64.7BCD	57.2AB	87.4A	9.0BC
L.16	5.88A	102.5C	3.0C	4.0BC	3.0BC	19.0CDE	52.5DE	45.7BC	87.4A	9.0BC
LSD at 5%	0.60	9.45	0.803	1.008	0.711	3.66	13.98	13.01	8.47	1.82

Note: Different letters indicate significant difference according to Duncan's multiple range test.

Effect on photosynthetic pigments and biochemical components

Chlorosis of Vicia faba leaves which results from stored insects attack may be explained by the significant reduction in total photosynthetic pigments content observed in the infested leaves of the susceptible genotypes (L.16 and T.W) as compared with the other genotypes and the resistance genotypes (L.551, L.512, L.153, NA112, and L.1; Table 4). These results are in accordance with El-Khawas (2012) who found that significant reduction in chlorophyll a and b levels in Pisum sativum leaves was attacked by leaf miners as well as the significant increase in carotenoids was observed in the infested leaves as compared with the healthy ones. The decrease in the photosynthetic pigments may be due to the inhibition of pigment biosynthesis due to Mg deficiency which is a constituent of chlorophyll or due to damage of palisade tissue which contains the chloroplast or lack of assimilates which drain toward the insect or to the effect of ROS on these pigments (Stacey & Keen 1996; Khattab 2007).

Table 4. Effect of infestation with stored insects on photosynthetic pigments and biochemical components in susceptible and tolerant genotypes of Vicia faba leaves and seeds.

		Phenol (mg tannic acid/g fresh weight)		Tannin (mg catechin acid/100g fresh weight)		POD (enzyme activity/g fresh weight/h)		PPO (enzyme activity/g fresh weight/h)		Soluble protein (mg/g fresh weight)
Genotypes	Total pigments (mg/g fresh weight)	Leaves	Seeds	Leaves	Seeds	Leaves	Seeds	Leaves	Seeds	Leaves	Seeds
L.551	13.68BC	4.04A	1.41B	55.23A	18.29B	23.27ABC	34.76A	49.00AB	9.65B	72.9A	22.03BC
L.512	16.54A	3.56AB	1.59AB	52.83A	23.77A	24.30AB	32.43A	39.23CD	15.27A	74.0A	33.63A
L.153	16.53A	2.87B	2.01A	36.80B	22.96A	18.70BC	26.30B	39.13CD	13.91A	73.87A	25.03B
L.1	14.75AB	2.77B	1.55B	34.07BC	23.07A	25.37A	33.14A	55.10A	7.69BC	75.2A	24.73B
NA112	13.30BC	0.90C	1.47B	27.67BC	24.77A	25.53A	25.41B	44.73BC	8.17BC	71.93A	19.33C
T.W	10.36DE	2.88B	0.697C	24.63BCD	13.51C	9.05D	16.03C	34.00DE	7.61BC	44.40C	14.23D
Giza3	12.13CD	4.32A	1.69AB	23.50CD	20.00B	17.33C	15.85C	28.20E	6.42CD	64.9AB	13.97D
L.16	8.50E	1.54C	0.593C	13.76E	14.03C	8.47D	16.33C	15.90F	3.91D	54.30BC	14.57D
LSD at 5%	2.28	0.97	0.43	1.24	2.42	6.07	5.37	6.29	3.13	15.28	3.94

Note: Different letters indicate significant difference according to Duncan's multiple range test.

The obtained results in Table 4 revealed that total phenols content was significantly accumulated in leaves and seeds of all Vicia faba genotypes as compared with the susceptible genotypes (L.16 and T.W). These results are in harmony with El-Khawas (2012) who found that the total phenols in Pisum sativum leaves attacked by leaf miners showed highly significant increments of infested host plants as compared with the healthy ones. In addition, Perveen et al. (2001) reported that resistant and semi-resistant varieties of cotton attacked by insects possessed significantly greater phenolic content than susceptible varieties. The greater phenol concentration in resistant genotypes may contribute to the inhibited herbivory due to the toxic or phagodeterrent effect of phenolics toward insects. The elevation of phenols can be explained as a mechanism of defense that acts as a barrier to insect feeding. These phenolic compounds are known to inhibit the larval development and growth by acting as feeding deterrents. Therefore, the activity of phenols is specific to the plant and pest interaction. The feeding deterrence can be due to the generation of the ROS in the insect's digestive tract, particularly in the midgut. They aid in the oxidative damage of the midgut lipids and proteins, resulting in death of the insect. As such, it may be the main reason for the enhanced phenolic acid levels in the plant as a defense strategy.

Data obtained in Table 4 demonstrate significant increment in the tannin content in seeds of all Vicia faba genotypes as being compared with the susceptible genotypes (L.16 and T.W). The tolerant genotypes recorded the maximum accumulation in tannin content. In addition, tannin content increased significantly in leaves of two resistant genotypes (L.551 and L.512) but showed nonsignificant effects in the other genotypes as compared with the susceptible genotypes. These results are in accordance with Panda and Khush (1995) who found that the biosynthesis of secondary metabolites (like lignins, tannins, etc.) plays an important role in the seed defense against insects such as repellents, feeding inhibitors, and anti-nutritional factors. The increase in tannin content in tolerant genotypes may be due to the toxic effect on insects because they bind to salivary proteins and digestive enzymes including trypsin and chymotrypsin resulting in protein inactivation. Insect herbivores that ingest high amounts of tannins fail to gain weight and may eventually die. In addition, the tannin content in seeds of all Vicia faba genotypes showed significant increase as compared with susceptible genotypes except triple white genotype.

The activity of POD in leaves and seeds of all Vicia faba genotypes was found to be significantly increased as compared with susceptible genotypes (Table 4). These results are in accordance with He et al. (2011) who found that POD activity in three chrysanthemum cultivars ‘Keiun’, ‘Han6’, and ‘Jinba’ were enhanced by aphid herbivory. POD is one such enzyme of prime importance in plant defense that eliminates the ROS besides its other defensive roles. It has been implicated as an important component of the immediate response of plants to insect damage (Rani & Jyothsna 2010). Production of phenoxy and other oxidative radicals by the PODs in association with phenols directly deter the feeding by insects and/or produces toxins that reduce the plant digestibility, which in turn leads to nutrient deficiency in insects with drastic effects on their growth and development (Zhang et al. 2008). In addition, PODs have been reported to have direct toxicity in guts of herbivores (Zhu-Salzman et al. 2008).

Infestation with stored insects caused either significant increase in some resistant genotypes or nonsignificant effect in the others resistance genotypes in PPO in leaves and seeds of Vicia faba genotypes as compared with the susceptible one (Table 4).These results are similar to Rani and Pratyusha (2013) who reported that pest infestation in cotton plants elevated the PPO levels, which may be due to the reduced nutritional quality of plant and indigestibility to the insect. In addition, He et al. (2011) found that PPO activity in three chrysanthemum cultivars, ‘Keiun’, ‘Han6’, and ‘Jinba,’ were enhanced by aphid herbivory. In several studies, higher levels of PPO activity have been associated with the resistance of plants to insects (Chen et al. 2006). The PPO's role in reduction of nutrient quality, digestibility, and palatability of plant tissues to insects, and the production of quinones by catalyzing phenolic oxidation is highly appreciating defensive response against herbivory (Bhonwong et al. 2009).

The results in Table 4 indicated that the total soluble protein in leaves and seeds of the resistance genotypes of Vicia faba (L.551, L.512, L.153, L.1, and NA112) significantly increased as compared with susceptible genotypes (L.16 and T.W). These results are similar to Rani and Pratyusha (2013) who found that infested cotton plant expressed higher levels of proteins than normal plant. Also, War et al. 2012 found that proteins content were increased in insect damaged three groundnut genotypes as compared to uninfested control plants. In general, the induction was greater in the insect-resistant genotypes than in the susceptible one. Increase in the protein concentration may be due to the generation of defense-related proteins after stored insect's infestation. Plants defend themselves by producing these defense-related proteins at high concentrations (Lawrence & Koundal 2002).

Protein electrophoresis

Variation in SDS-PAGE banding patterns of protein extracted from Vicia faba leaves in response to infestation with stored insects are shown in Table 5 and Figure 1. Three types of modifications are observed in the protein patterns of Vicia faba leaves: some protein bands disappeared, other proteins were selectively increased; and synthesis of a new set of protein was induced. These responses were observed under infestation with stored seeds. It is clearly shown that the total number of protein bands in all faba bean genotypes under infestation with stored insects ranged between 27 and 34 bands. The protein banding patterns of seedlings in all genotypes comprise 12 major bands. These bands were recorded at the molecular weights of 111.9, 105.3, 68.2, 63.1, 35.5, 31.2, 27.8, 22.4, 16.8, 14.9, 14.3, and 13 kDa.

Figure 1. Electrophoretic banding pattern in leaves and seeds of eight Vicia faba genotypes under infestation with stored insects.

Note: 1 – L.551, 2 – L.512, 3 – L.153, 4 – L.1, 5 – T.W, 6 – NA112, 7 – Giza3, 8 – L.16.

Table 5. Molecular weight and intensities (%) of protein banding patterns extracted from leaves of eight Vicia faba genotypes infested with stored insects.

	Genotypes
Marker (mol. wt.) Kda	L.551	L.512	L.153	L.1	NA112	T.W	Giza 3	L.16
120.3							0.7
111.9	0.3	0.1	0.3	0.1	0.2	0.2	0.2	0.0
105.3	0.1	0.2	0.1	0.1	0.0	0.0	0.1	0.1
101.5	0.0			0.1	0.1	0.2		0.2
97.2		0.1	0.1	0.1	0.1	0.1	0.1
85.6		0.2	0.2	0.2	0.2	0.2		0.1
80.6	0.6	0.3	0.7	0.2	0.2
74.3	0.5	1.0		0.4	0.4	0.8	0.7	0.3
68.2	0.8	0.8	0.5	0.7	0.8	0.9	1.2	0.2
63.1	0.8	0.4	0.4	0.9	0.8	0.9	0.7	0.4
60.3			0.1	0.5	0.7	0.6	0.7	1.2
57.3	2.1	1.5	1.3	0.2	0.3	0.3	2.3
55.6				1.1	1.1	1.7		0.3
51.9	0.5	0.8	0.2	1.0	0.3	1.2	0.7
48.3	0.8	0.7	0.7	0.4		1.1	0.4	1.5
44.4	0.9	1.1	0.7	1.0	1.1		1.3
40.7							5.1	1.8
37.5		1.3	9.4	4.5	5.4	9.2	5.9	8.8
35.5	12.3	2.4		4.4	5.1	4.7	3.2	4.4
31.2	2.7	9.7	17.6	7.8	7.5	14.1	11.5	5.0
27.8	1.5	1.8	3.2	2.5	2.4	2.5	2.4	1.8
25.6	2.0	1.3			3.5	3.2
24.3	0.8	2.1	2.0	4.1		0.8	2.9	1.9
22.4	4.0	4.2	3.0	3.8	3.7	2.7	2.9	4.7
21.1	4.8	3.5					1.4
20.7	1.4		3.5	3.5	3.6	2.8	1.5	3.8
19.7			1.2	1.4	0.2	1.5	1.3
19.1	1.9				0.6
18.5		2.2	2.0		1.5	1.8	2.3	2.0
18.1	2.2	2.6	2.1	2.4	2.2		0.7
17.5				1.0	0.5	1.9
16.8	4.6	4.6	4.8	5.5	5.1	4.4	4.4	6.7
16.2	1.7	1.1		1.5	1.3	1.3	0.9	0.8
15.7		1.7	1.6		1.1	1.1	0.9	0.9
15.3	1.1	1.0	0.4	0.4			0.6
14.9	2.1	2.0	3.1	1.5	1.6	1.7	1.4	2.5
14.3	2.3	2.6	2.7	2.0	1.6	3.3	1.8	2.4
13.9	2.7	1.8	2.0	1.8	2.3	2.0		1.5
13.5	2.0		2.7		1.8		1.2	2.0
13.0	17.7	26.1	16.6	23.5	22.5	22.1	28.5	28.2
No. of bands	29	30	30	32	34	29	32	27
Sum	75.2	79.0	83.2	78.6	79.6	89.2	89.9	83.5

Infestation with stored insects induced a considerable variation in the protein patterns of all genotypes of faba bean leaves. These results are similar to Khattab (2007) who found that the infested and the control healthy cabbage leaves exhibited similar protein profiles but quantitative differences in some polypeptide chains were observed. The electrophoretic patterns of infested cabbage leaves showed an increase in polypeptide chains and a decrease in the amount of other polypeptide chains compared with their respective control. Changes in protein synthesis under infestation of stored insects may be due to changes in the efficiency of mRNA translation or the regulation of RNA transcription transport and stability. Also, biotic stress (insects) leads to difference in gene expressions where alterations in protein could be due to alteration in regulation of transcription, mRNA processing, or due to altered rates of protein degradation.

The total number of bands in leaves of sensitive genotypes (L. 16 and T.W) was decreased as being compared with the tolerance genotypes, but the intensity of protein of these genotypes increased. These results indicated that the decrease in the protein level in biotic stressed plants might be attributed to a decrease in protein synthesis, the decrease availability of amino acids and the denaturation of enzymes involved in amino acid and protein synthesis. These results, in accordance with Rafie et al. (1996) and Jerez (1998), demonstrated changes in protein profiles in resistant plants after insect feeding. It was suggested that synthesis or increased expression of specific plant proteins may serve to enhance the plant resistance to stresses (Ni et al. 2001).

Four bands can be considered as negative markers associated with tolerance to stored insects infestation. These bands were found in all tolerance genotypes (L.551, L.512, L.153, L.1, and NA112) and disappeared in the resistance genotypes (L.16 and T.W). These bands have molecular weights of 80.6, 44.4, 18.1, and 15.3 kDa.

The results of SDS-PAGE of proteins extracted from seeds of eight faba bean revealed a total number of 17 bands with molecular weights ranging from about 76.3 to 16.5 kDa which were not necessarily present in all genotypes. Data showed three common bands which have molecular weights 34.5, 28.0, and 20.1 kDA (Table 6 and Figure 1). The genotypes exhibited a different pattern in the presence of the bands. The maximum number of bands (17 bands) was recorded in the tolerant genotypes (NA112). The band which has a molecular weight 43.2 kDa can be considered as a negative molecular marker for tolerance to stored insects. These results are similar to the results obtained by El-Sayed (2006) who found that protein electrophoresis of two genotypes of faba bean infested with bruchid detected distinct bands which differentiated the most susceptible from the most resistant.

Table 6. Molecular weight and intensities (%) of protein banding patterns extracted from seeds of eight Vicia faba genotypes infested with stored insects.

	Genotypes
Marker (mol. wt.) KDa	L.551	L.512	L.153	L.1	NA112	T.W	Giza 3	L.16
76.3	2.5	1.5	0.7		0.7	1.7	1.8	1.5
70.4		3.2	1.7	1.2	1.0			0.5
63.6				1.3				0.2
58.1	2.6	5.5
51.9					0.5	0.6	0.4	0.5
45.6			3.6	1.0		3.3	2.1	2.3
43.2	5.1	6.3	5.0	2.4	3.5		0.9
39.9	5.2		4.8	0.8
38.0				0.5
34.5	9.6	11.0	7.1	8.5	7.8	7.6	5.6	6.6
28.0	6.7	5.8	7.8	6.0	5.8	7.0	5.4	6.9
25.1	5.5		5.5		4.4	3.2	3.4	4.1
24.4		5.6		4.2	0.9	1.7	2.0	1.0
28.3	4.1			0.8	2.3	2.0	1.9	4.1
22.9			1.9	3.0
21.5	14.8	9.7	14.3			10.1	24.1	22.4
21.2				20.8	13.0	6.5
20.1	6.1	4.7	4.8	7.0	6.7	7.0	10.8	9.1
19.3	3.3	2.1	1.1		1.9	1.7		7.2
18.7		1.8	0.6	4.1	2.3	4.2	4.9
18.2			0.8	1.5	1.6
17.7	2.7	2.5			3.1	5.6	5.4	3.8
17.3			1.7	4.5	3.4
16.5					4.0			3.9
No. of bands	12	12	15	16	17	14	13	15
Sum	68.1	59.7	61.3	67.6	62.9	62.2	68.8	74.3

Molecular markers

ISSR markers are useful in gene tagging and can be used for detecting markers linked to the gene of interest. In the present study, seven primers for ISSR were used to identify the molecular markers for resistance and susceptibility of Vicia faba genotypes to infestation with stored insects. All selected primers developed PCR products with a variable number of bands (Table 7).

Table 7. DNA polymorphism of eight genotypes of Vicia faba infested with stored insects.

No.	Mol. size (bp)	L.551	L.512	L.153	L.1	NA112	T.W	Giza3	L.16
44 B
1	1157	−	+	−	−	−	−	−	−
2	956	−	+	+	−	−	−	−	−
3	770	+	+	+	+	−	−	−	−
4	671	+	+	+	−	−	−	−	−
5	577	+	−	−	+	−	−	+	−
6	547	−	+	−	−	−	−	−	−
7	503	+	−	+	−	+	−	−	−
8	454	−	+	−	−	−	+	−	−
9	387	+	+	+	+	+	+	+	+
10	316	−	−	−	−	−	−	+	+
Total		5	7	5	3	2	2	3	2
14 A
1	1480	−	+	−	−	−	−	−	−
2	1041	−	−	−	−	−	−	+	−
3	939	−	−	−	−	−	−	−	+
4	665	−	−	−	+	−	−	−	−
5	603	+	+	+	−	−	−	−	−
6	528	+	+	+	+	+	+	+	−
7	478	−	−	−	−	+	−	+	−
8	444	+	−	+	+	−	+	−	+
9	402	−	+	−	−	+	−	+	−
10	356	−	−	−	−	+	+	+	+
11	297	−	−	−	−	−	+	+	+
12	245	−	−	−	−	−	−	−	+
Total		3	4	3	3	4	4	6	5
HB-9
1	967	−	+	+	−	+	−	−	+
2	900	−	−	−	+	−	−	−	−
3	631	+	−	+	−	+	−	+	+
4	542	+	+	+	+	+	+	+	+
5	493	+	+	−	−	+	−	−	+
6	460	−	−	−	+	−	+	−	−
7	412	+	−	+	+	+	−	+	+
8	367	+	+	+	−	+	+	+	−
9	303	−	−	−	+	−	−	−	+
10	287	+	+	+	+	+	+	+	−
11	245	−	−	−	−	−	+	−	−
Total		6	5	6	6	7	5	5	6
HB-11
1	967	−	−	−	−	−	−	+	+
2	880	+	+	+	−	+	+	−	−
3	784	−	+	−	+	−	+	+	+
4	724	−	+	+	+	+	−	−	−
5	661	−	−	−	+	+	−	−	−
6	593	+	−	−	−	−	+	+	+
7	548	−	+	−	−	+	−	−	−
8	500	+	−	+	+	+	+	+	+
9	458	−	+	+	−	−	−	−	−
10	402	+	+	+	+	+	+	+	+
11	359	−	−	−	−	+	+	+	+
12	307	+	+	+	+	−	+	+	+
13	266	+	+	+	+	+	−	−	−
Total		6	8	7	7	8	7	7	7
HB-13
1	1103	+	+	+	+	−	−	−	−
2	889	−	−	−	−	−	−	+	−
3	663	−	+	−	−	+	−	+	−
4	598	−	+	+	+	−	−	−	−
5	546	−	−	−	−	+	−	+	+
6	386	+	+	+	+	+	+	+	+
7	284	+	+	+	+	+	+	+	−
8	204	+	+	+	+	+	+	+	+
Total		4	6	5	5	5	3	6	3
HB-14
1	1045	−	−	+	+	−	−	−	−
2	891	+	−	−	−	−	−	−	−
3	701	+	−	+	+	+	−	−	−
4	635	−	+	−	+	−	−	+	−
5	570	+	−	+	−	+	+	−	+
6	533	−	+	−	−	−	−	−	−
7	464	+	+	+	+	+	+	+	+
8	383	−	−	+	−	−	+	+	+
9	326	−	−	−	−	−	−	−	+
Total		4	3	5	4	3	3	3	4
HB-15
1	860	+	+	+	+	−	−	−	−
2	740	−	−	−	−	−	−	+	+
3	520	+	+	+	+	+	+	+	+
4	400	+	+	+	+	+	+	+	+
5	360	+	+	+	+	+	+	+	+
6	280	+	+	+	+	+	+	+	+
Total		4	5	5	5	4	4	5	5

The data showed that 71 DNA bands were detected among all genotypes of Vicia faba leaves, of which 62 bands were polymorphic (87.3%) (Table 8 and Figure 2). The highest number of ISSR bands was detected for primer HB-11 (13 bands), while the lowest was scored for HB-15 (6 bands). Table 8 shows the polymorphic bands were generated from each primer. Three primers gave three molecular markers (negative markers) associated with tolerance to stored insects infestation (Table 9). The results of ISSR analysis using the seven primers indicated the appearance and disappearance of DNA polymorphic bands at all genotypes.

Figure 2. DNA polymorphism using ISSR-RAPD in Vicia faba leaves.

Table 8. Number and types of the amplified DNA bands as well as the percentage of the total polymorphism generated by seven ISSR primers in eight genotypes of Vicia faba.

		Polymorphic band
Primer code	Monomorphic bands	Shared bands	Unique bands	Total bands	Polymorphism (%)
44 B	1	7	2	10	90.0
14 A	–	7	5	12	100
HB-9	1	8	2	11	90.9
HB-11	1	12	–	13	92.3
HB-13	2	5	1	8	75.0
HB-14	1	6	2	9	88.9
HB-15	4	2	–	6	33.3
Total	9	49	13	71	87.3

Table 9. ISSR markers for the tolerance assessment to infestation with stored insects.

Primer code	No. of marker/primer	Mol. size (bp)	Marker type
HB-11	1	266	Positive marker
14 A	1	297	Negative marker
HB-14	1	383	Negative marker

At the level of ISSR molecular markers, four primers showed specific markers for the studied traits. These results confirmed the useful application of ISSR-PCR analysis to detect the genetic variability between genotypes, similar to with Joshi et al. (2000) who demonstrated that ISSR markers are a valuable method for detecting genetic variability among rice varieties and for rapidly identifying cultivars. Also, Abo El-kheir et al. (2010) detected some specific ISSR molecular markers associated with Orobanche infestation tolerance in some faba bean cultivars.

Conclusion

The present data suggest that five faba bean genotypes may be resistant genotypes and can be used through breeding programs. In general, the eventual incorporation of yield traits, the biochemical and the molecular genetic markers for the selected faba bean genotypes are efficient tools to be applied as marker-assisted selection closely linked to important traits which greatly contribute to practical crop improvement programs.