Vol. 8(40), pp. 5032-5046, 17 October, 2013

DOI: 10.5897/AJAR12.1013

ISSN 1991-637X ©2013 Academic Journals

http://www.academicjournals.org/AJAR

African Journal of Agricultural

Research

Full Length Research Paper

Assessment the genetic diversity of common bean Phaseolus vulgaris collection by microsatellite

SSR markers

Jawaher M. Shabib, Afaf I. Shehata*, Amal A. AL–Hazzani and Muna Al-rumaih

Department of Botany and Microbiology, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia.

Accepted 11 September, 2013

Common bean plants (Phaseolus vulgaris L.) are the most important edible beans for their high nutritional value as an important source of dietary protein. However, in spite of the importance of this crop species, its genetics has not been fully characterized. Advances in molecular biology techniques have provided the basis for uncovering virtually unlimited numbers of DNA markers. Polymerase Chain Reaction (PCR) -based markers are more convenient and accurate determination of the genetic relationships. The utility of PCR-DNA based marker is generally determined by the technology that is used to reveal DNA-based polymorphism between and within different organisms. Thus, the present investigation aimed to assess the genetic variability among the different varieties of common bean based on the genetic distances that obtained by simple sequence repeat (SSR) or microsatellite techniques. SSR-DNA markers will identify closely related genotypes of different varieties of common bean. In the present study, a total of eleven bean varieties, nine of P. vulgaris L., one of Phaseolus coccineus, and one of Phaseolus lunatus namely: Bean CORA, Judia cilena, Diamant, palati, Distinction, Contender, Fagiolo Nano, Ambra, Bronco, Merit, Lima bean were selected SSR markers that were relatively more efficient in detecting similarities among Pharsalus genotypes studied with similarities up to 95%. SSR profiling provides useful information on the level of polymorphism and genetic diversity in common bean. Key words: Phaseolus vulgaris L., common bean, polymerase chain reaction, dietary protein. ....

INTRODUCTION

Phaseolus vulgaris L. characterized by the classified genetic diversity (Sokal and Michener, 1998). P. vulgaris has a number of features that make it an attractive legume for genomic studies that include a relatively small genome, simple diploid genome, self pollinating reproduction and well-developed molecular maps (Gepts, 1993). The genetic diversity of common bean was the most useful part of bean biodiversity (Nicolè et al., 2011;

Freitas et al., 2011). However, more consistent agronomic and genetic knowledge about these collections is still lacking and it is a serious limitation to utilizing and managing (McClean et al., 2008). Only a few genetic studies on common bean using molecular techniques have been reported (McConnell et al., 2007). Verifiing the existence of polymorphism in different populations of common bean through AFLP procedure

*Corresponding author. E-mail: afafsh@ksu.edu.sa.

Shabib et al. 5033

Table 1. Eleven varieties of Phaseolus vulgaris L, Phaseolus coccineus and Phaseolus lunatus and their common name.

No Species Variety Common name

1 Phaseolus vulgaris L. Bean CORA Strik

2 Phaseolus vulgaris L. Judia cilena Bean pole

3 Phaseolus vulgaris L. Diamant Climbing French bean

4 Phaseolus vulgaris L. palati Dwarf French bean

5 Phaseolus vulgaris L. Distinction Romano

6 Phaseolus vulgaris L. Contender Bush bean

7 Phaseolus vulgaris L. Fagiolo Nano Snap bean

8 Phaseolus vulgaris L. Ambra Snap bean

9 Phaseolus vulgaris L. Bronco Snap bean

10 Phaseolus coccineus Merit Runner bean

11 Phaseolus lunatus Lima bean

(Juliana et al., 2011; Sarikamiş et al., 2009; Kumar et al., 2008), evaluations based on RAPD profiles would be suitable for providing such information due to the high level of polymorphism of this technique (Kwak and Gepts, 2009; Grisi et al., 2007). In recent years, fingerprinting system based on RAPD analysis have been increasingly utilized for detecting genetic polymorphism in several plant genera (Campos et al., 2010, 2007; Meghan et al., 2011).

Microsatellites have become the molecular markers of choice for a wide range of application in genetic mapping and genetic analysis, genotype identification and variety protection (Blair et al., 2008), seed purity evaluation and germplasm conservation and diversity studies (Acosta-Gallegos et al., 2007; Wen-Hsiung, 1979). Thus, the present investigation aimed to evaluate the use of SSR for the assessment the genetic diversity among Phaseouls varieties and also to compare the effectiveness of genotyping analyses in determining genetic relationships among the Phaseolus varieties. Therefore, the main objective of this study is to identify the different Phaseouls varieties using a unique PCR-DNA based markers SSR to prove the efficiency of SSR markers for discrimination of eleven varieties of Phaseouls. Evaluating the DNA-based markers will help find a wide range of their applications in gene mapping population genetics. MATERIALS AND METHODS

Plant materials

A total of eleven bean varieties, nine of P. vulgaris L., one of Phaseolus coccineus, and one of Phaseolus lunatus namely: Bean CORA, Judia cilena, Diamant, Palati, Distinction, Contender, Fagiolo Nano, Ambra, Bronco, Merit, Lima bean were used for the study (Table 1). All the eleven types of seeds of Phaseolus were obtained from the local companies (AsdcoAgro, Alznan Agricultural, and Green Field Agricultural: Riyadh, KSA) that were imported from the following countries (USA, France, Italy, Spain).

Seed preparation Germinated seeds were collected when the seedling had developed to a stage at which coleoptile had expanded to 5 to 7 cm. Small plants were washed by distelled water and dried and saved in plastic bags at -20°C. Then, the young leaves of these seedlings were used in DNA assays and screening for the genotypes by SSR technique.

DNA extraction

Approach and solutions of Qiagens were used to extract DNA from young, tender leaves collected separately from each individual. Grounded powder was transferred to Buffer ATL, the tissue lysis buffer, Proteinase K was added, vortexed, and incubated at 56°C for 1 h, until the tissue were completely lysed. At the end, RNase (10 μg /ml) was added into each tube to kill RNA present.

DNA quality

Genequant (Amersham pharmcia Biotec, USA) enihcam was used for determining the purity of DNA and its evaluation was determined according to evnancnnncum Analysis Beer and Lambert naincvincna Manual (WPA Beer/Lambert law). The absorbance of DNA was determined by using long vector 260 nm, while its absorbance for its protein was determined on 280 nm long, and the purity of the DNA was calculated by the ratio of the absorbance at 260 by 280 nm. The DNA samples were considered pure when the evaluation estimated between 1.5 and 2%. The samples were also loaded onto agarose gel to visualize integrity and purity of DNA.

Microsatellites or simple sequence repeats (SSR) amplification

SSR-PCR reactions were conducted according to Williams et al. (???) (Koornneef et al., 2002). Five Operon primers (Table 2) were used. SSR reaction were performed in a 25 µl volume, containing 20 ng template DNA, 10 pmol\µl of primer, 2.5 mM dNTPs, 10x PCR buffer, 2U Taq DNA polymerase, sterilized distilled water and MgCl2. The amplifications were conducted with a Perkin-Elmer 9700 Thermal Cycler (Applied iosystems, Foster City, CA, USA), with an initial denaturation at 94°C for 5 min for 1 cycle followed by 35 cycles of 94°C for 1 min, an annealing phase of between 50 to

5034 Afr. J. Agric. Res.

Table 2. Nucleotide sequences of five selected SSR primers.

Name Sequence Percentage of GC%

AG1 5'-CATGCAGAGGAAGCAGAGTG-3' 55

AF483839 5'-GAGCGTCGTCGTTTCGAT-3' 55.6

GATS11 5'-CACATTGGTGCTAGTGTCGG-3' 55

AF483840 5'-AGCGCAATGCTACTCGAAAT-3' 45

GATS91 5'-GAGTGCGGAAGCGAGTAGAG-3' 60

10097

87

77 77

70

5753

43

33

10

Figure 1. Germination percentages of Phaseolus varieties.

59°C depending on the primers used for 1 min, and an extension at 72°C for 1 min, and a final post-extension at 72°C for 5 min. Electrophoresis The products of the reaction were electrophoresed on 1.4% agarose gels in 1% TBE buffer containing 0.2 μg/μL of ethidium bromide photographed under U. V. for analyzing DNA fragents of PCR. Data analysis Fragments amplified with microsatellite and RAPD primers were scored as presence (+) and absence (-). Genetic diversity was calculated using formula by Nei and Li (1979) and Makan et al. (2009). That obtained by software – Hercules software, version… (BioRad, ……).

RESULTS Germination percentage Germination percentage of Phaseolus seeds varied

among eleven genotypes as revealed by data recorded on germination 15 days after plantation, and following images (Figure 1) show the growth of different Phaseolus types. Germination percentage varied from 10 to 100%. Bronco and Merit obtained the highest and lowest germination percentage of 100 and 10%, respectively. Few genotypes did not germinate well as their germination percentage was below 50%, for example, Fagiolo Nano and Cilena. Results of germination percentage of Phaseolus genotypes are presented in Figure 1. Polymorphism detected by (SSR) analysis DNA extracted from 11 common beans was examined for the SSR pattern. Five SSR primers were used to investigate eleven common beans. Amplifications of the five SSR primers produced a total of 331 clearly detectable amplified products, 200 of them were polymorphic pattern and 121 of them were monomorphic pattern. The SSR primer AF483839 generated the highest number of bands (103 bands) (Figure 8) and

Shabib et al. 5035

Table 3. Quantities and lengths of DNA bands resulting from reaction of primer SSR-AF483840 with DNA extracted from eleven Phaseolus varieties.

Band length

(bp) Distinction

Cora Bean

Lima Ambra Merit Palati Judia Cilena

Bronco Diamant Contender Fagiolo Nano

2.642 + - - + + + + + + + +

1.6 + + + + + + + + + + +

1.5 + + + + + + + + + + +

1.4 + - - - - - - - + + -

1.3 - - - - - - - - - - -

1.2 - - - - - - - - + - -

1.1 + + + + + + + + + + +

1.0 + + + + + - - + + + +

900 + + + + - - - - + + -

800 + + + + + + + + + + -

700 + + + - + + + + + + +

600 + + + + + + + + + + +

500 + + + + + - - - + + -

400 + - - - - - - - - - -

300 - - - - - - - - + + +

200 - - - - - - - - - - -

100 - - - - - - - - - - -

NB 12 9 9 9 9 7 7 8 13 12 8

TBL 13142 8700 8700 10642 10442 8942 8942 9942 14242 13042 9442

+ indicates existence of bands, - indicates non-existence, NB is band number, TBL is total band length.

M 1 2 3 4 5 6 7 8 9 10 11 M

2.642

1.500

1.000

500

400

300

Figure 2. Genomic DNA amplification pattern in eleven Phaseolus verities with SSR-AF483840 primer. 100 bp ladder was used as a standard size marker (M).

seems most effective than other primers used in the present study. The SSR primer AG1 produced the lowest number of bands (41 bands) (Figure 4). Results on total number of bands (TNB), number of polymorphic bands (NPB), number of monomorphic bands (NMB), percentage of total number of bands (B%), percentage of polymorphic bands (P%) and percentage of mono-morphic bands (M%) are summarized in Table 3. The

primer AF483840 generated the highest number of polymorphic bands (57 bands) (Figure 2), the primer GATS11 generated the lowest number of polymorphic bands (14 bands) (Figure 10). The primer AF483840 generated the highest number of monomorphic bands (44 bands) (Figure 2). The primer AG1 and primer AF483839 generated the lowest number of monomorphic bands (Kumar et al., 2006) (Figures 4 to 8). Primer AF483840

5036 Afr. J. Agric. Res.

Phaseolus varieties

Tota

l num

be

r of

base p

air (

bp)

Figure 3. Number of base pairs (bp) resulted for eleven Phaseolus varieties using SSR-AF483840 primer.

produced 103 amplification products out of which 57 were polymorphic for each varieties (55.3%), 2 were monomorphic for each varieties (1.9%), and 44 were monomorphic for all varieties (42.7%) (Table 3, Figures 2 and 3). Most polymorphic bands were shown at band length of 2.642, 1.4, 1.0, 900, 800, 700, 500 and 300; monomorphic bands for each varieties were observed at 1.2, 400 bp and monomorphic bands for all varieties were observed at 1.6, 1.5, 1.1 and 600 bp.

The highest number of band recorded was 13 for the type Diamant where eight bands were olymorphic and five monomorphic bands recorded at the length of 1.6, 1.5, 1.2, 1.1 and 600 kp. Detection of highest number of bands and monomorphic band at 1.2 bp may serve as a marker to differentiate Diamant from other types in the present study. Polymorphic bands were recorded at the lengths of 2.642, 1.4, 1.0, 900, 800, 700, 500 and 300 kp. The number of bands for the type Distinction and Contender was 12; for the type Distinction, seven bands were polymorphic and five bands were monomorphic; for the type Contender, eight bands were polymorphic and four bands were monomorphic. Presence of a monomorphic band of 400 bp in type Distinction may be a useful marker for its differentiation from other varieties. Number and nature of bands observed in the types Lima bean and Bean cora was found similar and these bands were observed at the lengths of 1.6, 1.5, 1.1, 1.0, 900, 800, 700, 600 and 500 kp. Bands of similar nature and similar in number were also detected in types Judia cilena and Palati at the lengths of 2.642, 1.6, 1.5, 1.10, 800, 700 and 600 kp. A band of length 2.642 bp was found common in most types except Bean CORA and Lima bean. Bands of length 1.6, 1.5, 1.1 and 600 kp were common in all bean types. A band of length 1.4 bp was found common in types Distinction, Diamant and

Contender. Judia and Palati types varied from most other types by not having a band of length 1.0 kp. Absence of a band of 1.0 kp in types Judia and Palati may serve as a marker to distinguish them from other types being studied.

A band of length 900 bp was found common in the types Distinction, Bean CORA, Lima bean, Ambra, Diaman and Contender. Type Fagiolo Nano differed from other types by not having a band of length 800 bp. A band of length 500 bp was found common in the types Distinction Bean CORA, Lima bean, Ambra, Merit and Diamant Contender. A band of length 300 bp was found common in types Diaman, Contender and Fagiolo Nano. The primer AG1 produced 41 amplification products out of which 16 were polymorphic for each varieties (39%), 3 were monomorphic for each varieties (7.3%) and 22 were monomorphic for all varieties (53.6%) (Table 4, Figures 4 and 5). Most polymorphic bands were observed at lengths of 5000, 4900, 1300 bp, and monomorphic bands were observed at the lengths of 2500, 2000, 1500, 1400 and 1000 bp. The highest number of bands recorded was six in the type Fagiolo Nano, two were polymorphic and four were monomorphic bands recorded at the lengths of 2500, 2000, 1500 and 1400 bp. Detection of highest number of bands and presence of two monomorphic bands (2000 and 1400 bp) in type Fagiolo Nano may provide some evidence of existence of differences between this type and other types being studied. Polymorphic bands for the said type were recorded at the lengths of 5000 and 4900 bp. Number of bands for the type Distinction was five; two bands were polymorphic recorded at the lengths of 5000, 4900 bp; and three bands were monomorphic recorded at the lengths of 2500, 1500 and 1000. Number of bands for the types Bean CORA, Lima bean, Ambra and Judia cilena was

Shabib et al. 5037

Table 4. Quantities and lengths of DNA bands resulting from reaction of primer SSR-AG1 with DNA extracted from eleven Phaseolus varieties.

Band length (bp)

Distinction Cora Bean

Lima Ambra Merit Palati Judia Cilena

Bronco Diamant Contender Fagiolo Nano

10000 - - - - - - - - - - -

8000 - - - - - - - - - - -

7000 - - - - - - - - - - -

6000 - - - - - - - - - - -

5000 + + + + - - - - - - +

4900 + + + + - - + + + + +

4000 - - - - - - - - - - -

3000 - - - - - - - - - - -

2500 + + + + + + + + + + +

2000 - - - - - - - - - - +

1500 + + + + + + + + + + +

1400 - - - - - - - - - - +

1300 - - - - - + + - - - -

1200 - - - - - - - - - - -

1100 - - - - - - - - - - -

1000 + - - - - - - - - - -

NB 5 4 4 4 2 3 4 3 3 3 6

TBL 14900 13900 13900 13900 4000 5300 10200 8900 8900 8900 17300

+ indicates existence of bands, - indicates non-existence, NB is band number, TBL is total band length.

M 1 2 3 4 5 6 7 8 9 10 11 M

1000

800 700 600

500 400

300

200

Figure 4. Genomic DNA amplification pattern in eleven Phaseolus varieties with primer SSR-AG1. 1 Kb ladder was used as a standard size marker (M).

four; two bands were polymorphic, two bands were monomorphic.

The lowest number of monomorphic bands recorded was two and it was observed in type Merit at the lengths of 1.500 and 2500 bp. This may indicate that Merit is different from other types and may provide some basis for differences among different species of genus Phaseolu as Merit is P. coccineus. The type’s Lima bean, Bean CORA and Ambra showed similarity in terms of

having similar nature and number of bands observed at the lengths of 5000, 4900, 2500 and 1500 bp. Bands of lengths 4900, 2500, 1500 bp were also found common in the types Bronco, Diamant and Contender. A band of length 5000 bp was found common in types Distinctio Bean CORA, Lima bean, Ambra and Fagiolo Nano. Most types exhibited a similarity by having a band of length of 4900 bp except the types Palati and Merit. Absence of band of length 4900 bp may provide evidence that Palati

5038 Afr. J. Agric. Res.

Phaseolus varieties

Tota

l num

be

r of

base p

air (

bp)

Phaseolus varieties

Tota

l num

be

r of

base p

air (

bp)

Figure 5. Number of base pairs (bp) resulted for eleven Phaseolus varieties using SSR-AG1 primer.

and Merit differ from other types being studied. Two bands of lengths 2500 and 1500 bp were found common in all types. The types Judia cilena and Palati were similar in the existence of one band of the length 1300 bp. Primer GATS91 produced 58 amplification products. 56 of these products were polymorphic (96.6%) and 2 were monomorphic (3.4%) (Table 5, Figures 6 and 7). Most polymorphic bands were detected at the lengths of 3100, 3000, 2900, 2800, 2300, 2200, 2100, 2000, 19000, 1800, 1700, 1600, 1500, and monomorphic were found at the lengths of 1000 and 2700 bp. In the type Palati, the number of polymorphic bands were found to be the highest with nine bands and these bands were recorded at the lengths of 3100, 2900, 2300, 2100, 2000, 1800, 1700, 1600 and 1500 bp.

Detection of highest number of bands in type Palati may be an indicative of its distinctness from other types and smaller differences had already been detected due to the reaction of SSR primer AG1. In the type, Judia cilena, eight polymorphic bands were revealed at the lengths of 3100, 2300, 2100, 2000, 1900, 1800, 1600 and 1500 bp. Seven polymorphic bands were found in the types Distinction, Bean CORA and Merit, while type Distinction had one monomorphic band. Presence of a monomorphic band of 1000 bp in Distinction may serve as a marker to differentiate it from other types. The lowest number of two bands was recorded in the type Bronco, one of these band recorded at the length of 1800 bp was polymorphic while the other recorded at the length of 2700 bp was monomorphic. Detection of lowest band number in

Bronco may serve as a marker to differentiate it from other types and already lowest number of bands has been detected for this type through the reaction of RAPD primer OPP-120. No bands were observed in the type Ambra. A band of length 3100 bp was found common in types Palati and Judia cilena. A band of length 3000 bp was found common in types Bean CORA, Lima bean, Merit and Diamant. A band of length 2900 bp was found common in types Distinction, Lima bean and Palati. A band of length 2800 bp was found common in types Distinction, Bean CORA and Merit Diamant. The types Palati, Judia cilena, Contender and Fagiolo Nano showed similarity by having a common band of length 2300 bp. Similar pattern was detected in types Lima Bean and Diamant as these two types possessed a band of length 2200 bp.

A band of length 2100 bp was found common in types Bean CORA, Merit, Palati and Judia cilena. A band of length 1900 bp was found common in types Judia cilena, Contender naa Fagiolo Nano. In the types, Bean CORA, Merit, Palati, Judia cilena and Bronco, a band having length of 1800 bp was found common. Most types of bean showed a similarity by having a band with length of 1700 bp except Ambra, Judia cilena and Bronco. The types Distinction, Bean CORA, Merit, Palati, Judia cilena and Diamant possessed a common band having length of 1600 bp. A band of length 1500 bp was found in all bean types except Lima bean, Ambra and Bronco. The primer AF483839 gave 80 amplification products, out of these, 56 were polymorphic for each varieties (70%), 2 were

Shabib et al. 5039

Table 5. Quantities and lengths of DNA bands resulting from reaction of primer SSR-GATS91 with DNA extracted from eleven Phaseolus varieties.

Band length

(bp) Distinction

Cora Bean

Lima Ambra Merit Palati Judia Cilena

Bronco Diamant Contender Fagiolo Nano

10000 - - - - - - - - - - -

8000 - - - - - - - - - - -

7000 - - - - - - - - - - -

6000 - - - - - - - - - - -

5000 - - - - - - - - - - -

4000 - - - - - - - - - - -

3100 - - - - - + + - - - -

3000 - + + - + - - - + - -

2900 + - + - - + - - - - -

2800 + + - - + - - - + - -

2700 - - - - - - - + - - -

2600 - - - - - - - - - - -

2300 - - - - - + + - - + +

2200 - - + - - - - - + - -

2100 - + - - + + + - - - -

2000 + - - - - + + - - - -

1900 - - - - - - + - - + +

1800 - - - - - - - - - - -

1700 - + - - + + + + - - -

1600 + + - - + + + - + - -

1500 + + - - + + + - + + +

1000 + - - - - - - - - - -

NB 7 7 4 0 7 9 8 2 6 4 4

TBL 13500 14500 9800 0 14500 19000 16300 4500 12800 7400 7400

+ indicates existence of bands, - indicates non-existence, NB is band number, TBL is total band length.

M 1 2 3 4 5 6 7 8 9 10 11 M

1000 800 700 600 500 400

300

200

Figure 6. Genomic DNA amplification pattern in eleven Phaseolus varieties with SSR-GATS91 primer. 1 kb ladder was used as a standard size marker (M).

5040 Afr. J. Agric. Res.

Phaseolus varieties

Tota

l num

be

r of

base p

air (

bp)

Phaseolus varieties

Tota

l num

be

r of

base p

air (

bp)

Figure 7. Number of base pairs (bp) resulted for eleven Phaseolus varieties using SSR-GATS91 primer.

Table 6. Quantities and lengths of DNA bands resulting from reaction of primer SSR-AF483839 with DNA extracted from eleven Phaseolus varieties.

Band length

(bp) Distinction

Cora Bean

Lima Ambra Merit Palati Judia Cilena

Bronco Diamant Contender Fagiolo Nano

10000 - - - - - - - - - - -

8000 - - - - - - - - - - -

7000 - - - - - - - - - - -

6000 - - - - - - - - - - -

5000 - - - - - - - - - - -

4000 - - - - - - - - - - -

3100 + + + + - - - - - - -

3000 - - - - + + + + + + +

2900 + - - - - - - - - - -

2800 - + + + + + + - - - -

2700 - + + + - - - + + + +

2600 + + - + + + + + + + +

2000 + + + + - - - - - - -

1900 + + + + + + + + + + +

1800 + + + + + + + + + + +

1700 + + + + - - - + + + +

1600 + + - + - - - + + + +

1500 - - - - - - - - - - -

1400 - - - - - - - - - - -

1300 - - - - - - - - - - -

1200 - - - - - - - - - - -

1100 + - - - - - - - - - -

1000 + + + + - - - + + + +

NB 9 9 7 9 6 6 6 7 7 7 7

TBL 18700 20200 16000 20200 13100 13100 13100 15300 15300 15300 15300

+ indicates existence of bands, - indicates non-existence, NB is band number, TBL is total band length.

monomorphic for each varieties (2.5%) and 22 were monomorphic for all varieties (27.5%) (Table 6, Figures 8

and 9). Most polymorphic bands were observed at 3100, 3000, 2800, 2700, 2600, 2000, 1700, 1600, 1000 bp and

Shabib et al. 5041

Figure 8: Genomic DNA amplification pattern in eleven Phaseolus varieties with SSR-

AF483839 primer. 1Kb ladder was used as a standard size marker(M).

1000 800

700

600

500 400

300

200

M 1 2 3 4 5 6 7 8 9 10 11 M

Figure 8: Genomic DNA amplification pattern in eleven Phaseolus varieties with SSR-

AF483839 primer. 1Kb ladder was used as a standard size marker(M).

1000 800

700

600

500 400

300

200

M 1 2 3 4 5 6 7 8 9 10 11 M

Figure 8. Genomic DNA amplification pattern in eleven Phaseolus varieties with SSR-AF483839 primer. 1 kb ladder was used as a standard size marker (M).

Phaseolus varieties

Tota

l num

be

r of

base p

air (

bp)

Figure 9. Number of base pairs (bp) resulted for eleven Phaseolus varieties using SSR-AF483839 primer.

monomorphic bands were observed at 2900, 1900, 1800 and 1100 bp. The highest number of bands recorded was nine in the types Distinction, Bean CORA and Ambra. For the types Bean CORA and Ambra, seven bands were polymorphic and two bands were monomorphic. For the types Distinction, five bands were polymorphic and four bands were monomorphic. Distinction seems to be a slightly different type than others in the present study as monomorphic bands at 2900 and 1100 bp are usually detected for this type and these can serve as markers to differentiate it from other types Bands having the lengths of 3100, 2800, 2700, 2000, 1900, 1800 and 1700 bp were

found common among types such as Bean CORA and Ambra.

Types Bronco, Diamant, Contender and Fagiolo Nano had common bands having lengths of 3000, 2700, 2600, 1900, 1800, 1700 and 1600 bp. Types Merit, Palati and Judia cilena were found to be similar by having same number and nature of bands of lengths 3000, 2800, 2600, 1900, 1800 and 1000 bp. A band having length of 3000 bp was found common among types namely, Merit, Palati, Judia cilena, Bronco, Diamant, Contender and Fagiolo Nano. A band of length 2800 bp was found common among types such as Bean CORA, Lima bean,

5042 Afr. J. Agric. Res.

Table 7. Quantities and lengths of DNA bands resulting from reaction of primer SSR-GATS11 with DNA extracted from eleven Phaseolus varieties.

Band length (bp)

Distinction Cora Bean

Lima Ambra Merit Palati Judia Cilena

Bronco Diamant Contender Fagiolo Nano

10000 - - - - - - - - - - -

8000 - - - - - - - - - - -

7000 - - - - - - - - - - -

6000 - - - - - - - - - - -

5000 - - - - - - - - - - -

4000 - - - - - - - - - - -

3100 - - - - - - - - - - -

3000 - - - - - - - - - - -

2600 + - - - - - - + + + +

2400 + + + + + + + + + + +

2200 + + + + + + + + + + +

2000 + + + - + - - - - - -

1800 - - - - - - - - - - -

1600 + + + - - - - - - - -

1500 - - - - + - - - - - -

1400 + - + - - - - - - - -

1200 - - - - + - - - - - -

1000 + + + + + + + + + + +

NB 7 5 6 3 6 3 3 4 4 4 4

TBL 13200 9200 10600 5600 10300 5600 5600 8200 8200 8200 8200

+ indicates existence of bands, - indicates non-existence, NB is band number, TBL is total band length.

Ambra, Merit, Palati and Judia cilena. Types Bean CORA, Lima bean, Ambra, Bronco, Diamant, Contender and Fagiolo Nano exhibited similarity by having a common band of length 2700 bp. A band of length 2600 bp was found in all bean types except Lima bean. Types Distinction, Bean CORA, Lima bean and Ambra had one band common among them and length of this band was 2000 bp. Two bands having lengths of 1800 and 1900 bp were found in all types. Most bean types had bands of length 1600 and 1700 bp except Merit, Palati and Judia cilena. Absence of bands of 1600 and 1700 bp may serve as a basis to differentiate Merit, Palati and Judia cilena from other types being studied. The primer GATS11 produced 49 amplification products and 14 of these products were polymorphic for each varieties (28.6%), 2 were monomorphic for each varieties (4%) and 33 were monomorphic for all varieties (67.3) (Tables 25, 48, 49 and Figure 22).

Most polymorphic bands were shown at lengths such as 2600, 2000, 1600, 1400, while monomorphic bands were seen at the lengths of 2400, 2200, 1500, 1200 and 1000 bp. The highest number of bands seven was recorded at the type Distinction, four bands were polymorphic recorded at the lengths of 2600, 2000, 1600, 1400 bp and three bands were monomorphic recorded at the lengths of 2400, 2200 and 1000 bp. The number of bands for the type Lima bean and Merit was six. For the type Lima bean, three bands were polymorphic and three

bands were monomorphic. For the type, Merit, one band was polymorphic and five were monomorphic. The type Merit (P. coccineus) seems to be distinct from other types, mostly P. vulgaris by having two distinct monomorphic bands. These differences in lengths of bands observed may serve as a basis to differentiate among different species of Phaseolus. Types Bronco, Diamant, Contender and Fagiolo had common bands of lengths 2600, 2400, 2200, 1000 bp while types Ambra, Palati and Judia cilena had common bands of lengths 2400, 2200 and 1000 bp.

A band having length 2600 bp was observed among each of the types Distinction, Bronco, Diamant, Contender and Fagiolo Nano. Bands having lengths of 2400, 2200 and 1000 bp were found among all types studied. The types Distinction, Bean CORA, Lima bean and Merit possessed common bands of length 1600 and 2000 bp. A band of length 1400 bp was found in each of the type Distinction and Lima bean. The results from primer GATS11 are presented in Table 7, Figures 10 and 11. Similarity coefficients and dendrogram for SSRs The highest similarity percentage, 95.2% was found between types Distinction and Fagiolo Nano while lowest similarity percentage, 55.6% was found between types

Shabib et al. 5043

M 1 2 3 4 5 6 7 8 9 10 11 M

1000 800 700 600 500 400

300

200

Figure 10. Genomic DNA amplification pattern in eleven Phaseolus varieties with SSR-GATS11 primer. 1 kb ladder was used as a standard size marker (M).

Phaseolus varieties

Tota

l num

be

r of

base p

air (

bp)

Figure 11. Number of base pairs (bp) resulted for eleven Phaseolus varieties using SSR-GATS11 primer.

Distinction and Judia Cilena. Similarity percentages among various Phaseolus varieties are shown in Table 8. The dendogram constructed from SSR markers clustered together the genotypes having the same genetic background. Degrees of similarity among various Phaseolus varieties are indicated in Figure 12. Analysis of dendogram from SSR markers divided the Phaseolus varieties into two groups. The first group included type Judia cilena. The second group was divided into two sub-groups; the first sub-group had one bean type (Palati)

and second sub-group had nine Phaseolus varieties that were further divided into two ranks. The first rank included two types Distinction and Fagiolo Nano. Other seven types from the second rank were further divided in two sub-ranks. The first sub-rank had three Phaseolus varieties Bean CORA, Lima bean and Contender; the second sub-rank had four types that were divided into two species; the first specie included two types, Ambra and Merit. SSR markers were relatively efficient markers in detecting similarities among Phaseolus varieties

5044 Afr. J. Agric. Res.

Table 8. Similarity percentages among various Phaseolus varieties by SSR.

DIS. COR. LIM. AMB. MER. PAL. JUD. BRO. DIM. CON. FAG.

DIS. 100.0

COR. 76.2 100.0

LIM. 76.2 100.0 100.0

AMB. 73.7 88.9 88.9 100.0

MER. 76.2 80.0 80.0 88.9 100.0

PAL. 70.0 84.2 84.2 82.4 84.2 100.0

JUD. 55.6 70.6 70.6 80.0 70.6 62.5 100.0

BRO. 73.7 77.8 77.8 87.5 88.9 70.6 80.0 100.0

DIM. 80.0 73.7 73.7 82.4 84.2 66.7 75.0 94.1 100.0

CON. 73.7 88.9 88.9 87.5 77.8 70.6 80.0 87.5 82.4 100.0

FAG. 95.2 80.0 80.0 77.8 80.0 73.7 58.8 77.8 84.2 77.8 100.0

Figure 12. Dendrogram of SSR analysis representing similarity among various Phaseolus varieties.

studied up to 95%. This can be probably due to the dominant nature of SSR markers and also because these markers are reproducible. SSR seem more efficient markers because they showed relatively higher levels of polymorphism (28.6 to 96.6). Thus, it is essential to recognize differences between quantitative characteristics. DISCUSSION Phaseolus genotypes exhibited wide variation in their germination percentage ranging from 10 to 100%. As the same constant temperature of 20°C was provided to all genotypes during the process of germination, these differences can be attributed to difference in genetic

make up of genotypes, differences in seed structure, origin of genotypes or history of post harvest handling of genotypes (Zhang et al., 2008). Gel electrophoresis revealed a differential and comparable band pattern for genotypes which indicates that the genotypes differ in their genetic make up to some extent. This finding can have implications for detailed studies on genetic diversity among genotypes (Masi et al., 2009). Molecular markers such as SSR can be used to study genetic diversity among genotypes (Kumar et al., 2006; Menz et al., 2004; Tams et al., 2005). In the present study, SSR used to define genetic relationship among 11 common bean genotypes and to investigate which marker system is more efficient in detecting similarities and differences among common bean genotypes. The findings of higher polymorphism detected by SSR are in agreement with

Shabib et al. 5045

Table 9. The efficiency of SSR markers.

Molecular marker Similarity

percentage (%) Percentage of polymorphism

Total number of bands from five primers used

SSR 55.6 - 95.2 28.6 - 96.6 331

earlier findings of Menz et al. (2004), Badjakov et al. (2006), Tams et al. (2005) and Blair et al. (2007). SSR are characterized by their hyper variability, abundance, reproducibility, Mendelian mode of inheritance and co-dominant nature (Sicard et al., 2005). Amplification success and polymorphism declines with increased genetic distance (Hanai et al., 2007). The high- level of polymorphism of micro-satellite markers and their wide cross-species transportability make these new markers useful for mapping and molecular characterization of Phaseolus species (Sicard et al., 2005). The relativity tree divided Phaseolus types into two groups. Recent studies indicate that molecular markers like RAPD and SSR have the potential to provide evidence regarding intra-specific diversity within the genus (Hanai et al., 2007).

Dendogram from SSR markers put the common bean genotypes in slightly different groups, thereby, indicating this marker vary in their efficiency in extracting similarities and differences among these types (Table 9). The similarity values from SSR markers was higher (in most cases > 40%); it indicates that genetic diversity among studied genotypes was lower.

In conclusion, SSR profiling technique may provide useful information on the level of polymorphism and diversity in common bean, thereby, indicating their utilization in the characterization of common bean genotypes. The informative primers identified in this study will be useful in genetic analysis of bean accessions in germplasm holdings. In addition, the putative cultivar-specific bands may be used for genotype characterization and grouping germplasm accessions. Estimates of inter and intra cultivars genetic diversity provide plant breeders with relevant background data that can be utilized in the development of new cultivars with a broader genetic base. These findings can be of high significance for management of germplasm as genotypes may vary in their response to biotic and abiotic stresses and yield potential.

ACKNOWLEDGEMENT

This research project was supported by a grant from the Research Centre, of the Centre for Female Scientific and Medical Colleges, Deanship of Scientific Research, King Saud University. REFERENCES Acosta-Gallegos JA, Kelly JD, Gepts P (2007). Prebreeding in common

Bean and Use of Genetic Diversity from Wild Germplasm. Crop Sci. 47:S-44-59.

Blair MW, Buendía HF, Giraldo MC, Métais I, Peltier D (2008). Characterization of AT-rich microsatellites in common bean (Phaseolus vulgaris L.). Theor. Appl. Genet. 118:91-103.

Badjakov I, Todorovska E, Kondakova V, Boicheva R, Atanassov A (2006). Assessment the genetic diversity of Bulgarian raspberry germplasm collection by microsatellite markers. J. Fruit. Ornamental. Plant Res. 14:61-76.

Blair MW, Dìaz JM, Hidalgo R, Dìaz LM, Duque MC (2007). Microsatellite characterization of Andean races of common bean (Phaseolus vulgaris L.). Theor. Appl. Genet. 116:29-43.

Campos T, Oblessuc PR, Sforça DA, Cardoso JMK, Baroni RM, Sousa ACB, Carbonell SAM, Chioratto AF, Garcia AAF, Rubiano LB (2010). Inheritance of growth habit detected by genetic linkage analysis using microsatellites in the common bean (Phaseolus vulgaris L.). Mol Breed, Epub.

Campos T, Benchimol LL, Carbonell SAM, Chioratto AF, Formighieri EF and Souza AP (2007). Microsatellites for genetic studies and breeding programs in common bean. Pesq. Agropec. Bras. 42:401-408.

Freitas G, Ganança J, Nóbrega H, Nunes É, Costa G, Slaski J, Carvalho M (2011). Morphological evaluation of common bean diversity on the Island of Madeira. Genet. Resour. Crop Evol. 58(6):861-874(14).

Grisi MCM, Blair MW, Gepts P, Brondani C, Pereira PAA, Brondadi RPV (2007). Genetic mapping of a new set of microsatellite markers in a reference common bean (Phaseolus vulgaris) population BAT93 x Jalo EEP558. Genet. Mol. Res. 3:691-706.

Gepts P (1993). The use of molecular and biochemical markers in crop evolution studies. Evol. Biol. 27:51-94.

Hanai LL, Campos T, Camargo LEA, Benchimol LL, de Souza AP, Melotto M, Carbonell SA, Chioratto AF, Consoli L, Formighieri EF (2007). Development, characterization and comparative analysis of polymorphism at common bean- SSR loci isolated from genic and genomic sources. Genome 50:266-277.

Juliana MKCP, Alisson FC, Maria Imaculada Z, Carlos AC, Sérgio AMC, Jorge MCM, Rodrigo G, Antonio AFG, Tatiana de C, Anete de PS, Luciana BR (2011). Genetic diversity in cultivated carioca common beans based on molecular marker analysis. Genet. Mole. Biol. 34(1):88-102.

Kumar V, Sharma S, Kero S, Sharma S, Sharma K, KumarMand Bhat KV (2008). Assessment of genetic diversity in common bean (Phaseolus vulgaris L.) germplasm using amplified fragment length polymorphism (AFLP). Sci. Hort. 116:138-143.

Kwak M Gepts P (2009). Structure of genetic diversity in the two major gene pools of common bean (Phaseolus vulgaris L., Fabaceae). Theor. Appl. Genet. 118:979-992.

Koornneef M, Bentsink L, Hilhorst H (2002). Seed dormancy and germination. Curr. Opin. Plant Biol. 5:33-36.

Kumar J, Vijeshwar V, Shahi AK, Qazi GN (2006). Balyan HS, Development of simple sequence repeat markers in Cymbopogon species. Planta Medica. 73:262-266.

McClean PE, Lavin M, Gepts P, Jackson SA (2008). Phaseolus vulgaris: A diploid model for soybean. In G. Stacey (Eds), Soybean Genomics. Springer, Berlin. pp 55-78.

McConnell MD, Mamidi S, Rossi M, Lee RK, McClean PE (2007). A gene-based linkage map of common bean (Phaseolus vulgaris L.). Plant and Animal Genome Abstracts. pp. 15-20.

Makan HK, Ravikumar Patil HS, Abhilash M, Mohan Kumar HD (2009). Genetic diversity in commercial varieties of chilli as revealed by RAPD method. Ind. J. Sci. Technol. 2(4):91-94.

Meghan MM, Vanessa KF, Elizabeth PR, Matthew RL, Henry JT, Mark

5046 Afr. J. Agric. Res.

AB (2011). Evaluation of diversity among common beans (Phaseolus vulgaris L.) from two centers of domestication using 'omics' technologies. BMC Genomics 11:686.

Masi P, Logozzo G, Donini P, Spagnolleti P (2009). Analysis of genetic structure in wedely distributed common bean landraces with different plant growth habits using SSR and AFLP markers. Crop Sci. 49:187-199.

Menz MA, Klein RR, Unruh 2NC, Rooney WL, Klein PE, Mullet JE (2004). Genetic diversity of public inbreds of Sorghum determined by mapped AFLP and SSR markers. Crop Sci. 44:1236-1244.

Nicolè S, Erickson DL, Ambrosi D, Bellucci E, Lucchin M, Papa R, Kress WJ, Barcaccia G (2011). Biodiversity studies in Phaseolus species by DNA barcoding. Genome 54(7):529-45.

Sokal RR, Michener CM (1998). A Family Fabaceae. Univ. Kansas Sci. 28:1409-1438.

Sarikamiş G, Yasar F, Bakir M, Kazan K (2009). Genetic characterization of green bean (Phaseolus vulgaris) genotypes from eastern Turkey. Genet. Mol. Res. 8:880-887.

Sicard D, Nanni L, Porfiri O, Bulfon D, Papa R (2005). Genetic diversity

of Phaseolus vulgaris L. and P. coccineus L. landraces in central Italy. Plant Breed. 124:464-472.

Tams SH, Melchinger AE, Bauer E (2005). Genetic similarity among European winter triticale elite germplasms assessed with AFLP and comparisons with SSR and pedigree data. Plant Breed. 124:154-160.

Wen-Hsiung L (1979). A simulation study on Nei and Li's model for estimating DNA divergence from restriction enzyme maps. J. Mole. Evol. 17(4):251-255.

Zhang X, Blair MW, Wang S (2008). Genetic diversity of Chinese common bean (Phaseolus vulgaris L.) landraces assessed with simple sequence repeat markers. Theor. Appl. Genet. 117:629-640.