Genetic Diversity Assessment Of Fenugreek Biology Essay

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Trigonella corniculata (Fenugreek) of Kasur, Pakistan, commonly known as "Kasuri Methi" is a Geographical Indicator of Pakistan, flowering annual herb, 30-60 cm height, with pale yellow flowers, and it is vital from medical and culinary aspects. The main objective of our research was to evaluate the potential of SDS-PAGE technique to assess the genetic diversity of Trigonella corniculata of Kasur with other landraces from all over the Pakistan. Genetic diversity of Trigonella corniculata of Kasur with other land races of Pakistan was evaluated by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Genetic diversity was assessed via UPGMA cluster analysis by constructing dendrogram of fractions of proteins, which were used for the calculation of similarity coefficients between these genotypes. The UPGMA cluster analysis showed that accessions from different geographical regions were distributed in different groups. Four major groups were observed on the dendrogram, where first and third group showed 100% similarity among themselves, while 23% deviation was observed in the second group. Whereas fourth group can be isolated (belongs to Kasur) as it is found different from all other groups. Results indicated that limited diversity was found at protein level. Although SDS- PAGE technique differentiated Trigonella corniculata of Kasur (Kasuri methi) from other landraces, which indicated the significance of geographical region. However, it is necessary to use more number of accessions from different geographical locations and to use other advanced techniques to confirm the available pattern.

Key words: Trigonella, Kasuri Methi, SDS-PAGE, Geographical Indicator

INTRODUCTION

Trigonella (fenugreek) is a flowering annual herb, belongs to family, Fabaceae, 30-60 cm height, with pale yellow or white flowers occasionally visited by insects, leaves are light green. There are two species of genus Trigonella which are of economic importance viz. Trigonella foenum graecum, the common Methi and Trigonella corniculata, the Kasuri Methi (Kumar et al., 1997; Warrier et al., 1995). Indigenous to countries on the eastern shores of Mediterranean, fenugreek is widely cultivated in India, Egypt, Ethiopia, Morocco and occasionally in England (Polhil and Ravin, 1981). Trigonella foenum-graecum is extensively grown in the tropical and subtropical regions of India. Different parts of the plant such as leaves and seeds are consumed in India. It is also used for medicinal purpose. According to ancient medicinal system, the Ayurveda, it is a herbal drugs that is bitter or pungent in taste. It is effective against anorexia and is a gastric stimulant (Rajagopalan, 2001). Fenugreek is becoming popular around the world with its extract used to flavor cheese in Switzerland, artificial maple syrup and bitter-run in Germany, roasted seeds as coffee-substitute in Africa, seed powder mixed with flour as fortification to make flat-bread in Egypt, as an anti-diabetic herb in Israel, whole seed and dried plant used as insect and pest repellent in grain storage, and oil used in perfumery in France (Rajagopalan, 1998). Research reports in recent years have indicated that fenugreek can be a remedy to diabetes by lowering blood sugar and cholesterol levels (Sharma, 1990).

Geographical Indicator (GI) is generally understood as a sign used on goods that have a specific geographical origin and possess qualities or a reputation that is due to that place of origin (Escudero, 2001). . In Pakistan some examples of GI products famous for their taste and quality are Basmati rice, Kasuri Methi, Multani Halwa, Sindhari Mangoes, Hunza Walnuts, Kinnos and many other varieties of agricultural and food products in foreign markets. We need to address the protection of GIs that we share with our neighboring countries through bilateral agreements such as with SAARC countries and Members of the ECO. Kasuri Methi is a Geographical Indicator (GI) of Pakistan, commonly grown in Kasur and there is need to for increased protection of geographical indicators of our agricultural products.

Within the boundaries of the European Union (EU), regulations like Regulation 2081/92 (European Communities, 1992) are economic in nature, primarily because agricultural products and foodstuffs play an important role in the Community economy and Common Agriculture Policy (CAP). GI also carries with itself the power of a monopoly (Papandreou, 1988).

Across the world only known and well-defined cultivars are grown in specific areas. Gene banks also harbor scanty germplasm collection of Trigonella species (Hymowitz, 1990). The neglected and the underuse status of these locally important crops indicates a risk of disappearance of important plant material developed over thousands of years of cultivation. One of the important factors restricting their large-scale production and development of better varieties is that very little information is available about their genetic diversity, inter and intraspecific variability and genetic relationship among these species. Therefore, attempts to analyze possible untapped genetic diversity become extremely essential for breeding and crop improvement.

The main objective of our research was to evaluate the potential of SDS-PAGE technique to assess the genetic diversity of Trigonella corniculata of Kasur a Geographical Indicator (GI) with accessions from all over the Pakistan and to develop an optimized and efficient operational system for their use.

MATERIALS AND METHODS

Seed Sample

Samples of Trigonella corniculata (Fenugreek) of Kasur and other landaraces were collected from different ecological regions of Pakistan. The samples were stored in labeled glass bottle to ensure safety, in the National Agricultural Research Centre (NARC), Islamabad, Pakistan (Table 1).

Table 1. Accessions of Trigonella corniculata landraces of Pakistan studied

LABEL

ACCESSION

TOWN/CITY

1

PAK 020978

MAINWALI

2

PAK 021703

PAKPATTAN

3

PAK 022254

BAHAWALPUR

4

PAK 021131

FAISALABAD

5

PAK 021156

FAISALABAD

6

PAK 021330

SHORKOT

7

PAK 021675

NAROWAL

8

PAK 021986

KARACHI

9

PAK 021748

KARACHI

10

PAK 021711

NAROWAL

11

PAK 021696

OKARA

12

PAK 021117

FAISALABAD

13

PAK 022260

TALWANDI

14

PAK 022257

BAHAWALNAGAR

15

PAK 022219

DASKA

16

PAK 021908

SARGODHA

17

PAK 021900

SARGODHA

18

PAK 021889

SARGODHA

19

PAK 021882

FAISALABAD

20

DONATED

KASUR

21

DONATED

KASUR

22

DONATED

KASUR

SDS-PAGE Analysis

The variability of seed storage-proteins was analyzed by using SDS-PAGE (Damania et al., 1983). For extraction of protein, a single seed was ground to fine powder with mortar and pestle, Total 400 ul sample buffer was added to a 0.01g (10mg) seed powder and mixed thoroughly by vortex in an eppendorf tube (1.5ml) with a small glass rod. The extraction buffer contained the following final concentration: 0.5 M Tris-HCl (pH 6.8), 2.5% SDS, 10 % glycerol and 5% 2-mercaptoethanol, Kept overnight at 40 0C and centrifuged at 13000 rpm for 10 min. To monitor the movement of the protein in the gel, Bromophenol Blue (BPB) was used as a tracking dye. Seed protein was analyzed through slab-type SDS-PAGE using 7 % polyacrylamide gel. The molecular weights of the dissociated polypeptides were determined using protein standards (MW-SDS-70) of Sigma, USA. SDS-PAGE of total seed protein was carried out in a discontinuous buffer system following the method of Laemmli, 1970. The gels were stained with Coomassie Brilliant Blue (CBB) and destained till the background became transparent.

Data analysis

For each variety Electrophoregram were scored and the presence (1) or absence (0) of each band was noted. Presence and absence of data were entered in a binary data matrix. Based on electrophoretic band spectra, Jaccard's similarity index (JSI) was calculated by formula;

S= W/ (A+B-W)

Where W is the number of bands of common mobility, A the number of bands in type A and B is the number of bands in type B. All analysis was carried out using statistical software NTSYS-PC, version 2 (Rohlf, 1993). The genetic similarity coefficient matrix (Table 2) of 22 Trigonella landraces based on SDS-PAGE using UPGMA (Unweighted Pair Group Method with Arithmetic) (Nei and Lie, 1979) was used to construct a dendrogram using computer program NTSYS-PC version 2.0.

Table 2. Similarity coefficients among different accessions of Trigonella landraces from Pakistan

Acc.

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

1

1.00

2

1.00

1.00

3

1.00

1.00

1.00

4

1.00

1.00

1.00

1.00

5

1.00

1.00

1.00

1.00

1.00

6

1.00

1.00

1.00

1.00

1.00

1.00

7

1.00

1.00

1.00

1.00

1.00

1.00

1.00

8

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

9

0.84

0.84

0.84

0.84

0.84

0.84

0.84

0.84

1.00

10

0.77

0.77

0.77

0.77

0.77

0.77

0.77

0.77

0.93

1.00

11

0.84

0.84

0.84

0.84

0.84

0.84

0.84

0.84

0.93

0.9

1.00

12

0.80

0.80

0.80

0.80

0.80

0.80

0.80

0.80

0.83

0.90

0.8

1.00

13

0.88

0.88

0.88

0.88

0.88

0.88

0.88

0.88

0.97

0.90

1

0.87

1.00

14

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.84

0.77

0.8

0.80

0.88

1.00

15

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.84

0.80

0.8

0.80

0.88

1.00

1.00

16

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.84

0.77

0.8

0.80

0.88

1.00

1.00

1.00

17

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.84

0.77

0.8

0.80

0.88

1.00

1.00

1.00

1.00

18

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.84

0.77

0.8

0.80

0.88

1.00

1.00

1.00

1.00

1.00

19

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.84

0.77

0.8

0.80

0.88

1.00

1.00

1.00

1.00

1.00

1.00

20

0.67

0.67

0.67

0.67

0.67

0.67

0.67

0.67

0.77

0.69

0.7

0.64

0.74

0.67

0.67

0.67

0.67

0.67

0.67

1.00

21

0.67

0.67

0.67

0.67

0.67

0.67

0.67

0.67

0.77

0.69

0.7

0.64

0.74

0.67

0.67

0.67

0.67

0.67

0.67

1.00

1.00

22

0.69

0.69

0.69

0.69

0.69

0.69

0.69

0.69

0.64

0.56

0.6

583

0.69

0.69

0.69

0.69

0.69

0.69

0.69

0.76

0.76

1.00

RESULTS

Genetic Diversity Evaluation

The 22 accessions of Trigonella were used in present study showed various banding pattern using SDS-PAGE technique. In this study SDS-PAGE of grain proteins was performed in order to investigate genetic diversity among Trigonella accessions. Electrophoregram showing protein banding pattern of different accessions are given in Figures 1, 2, 3. A total of 16 bands were obtained, among them majority of bands were common in all accessions but other bands show variation.

Cluster analysis on the basis of SDS-PAGE

The UPGMA cluster analysis showed that accessions from different geographical regions were distributed in different groups. The result of cluster analysis is given in dendrogram (Figure 4) on the basis of similarity coefficient. Four major groups were observed on the dendrogram. Group I consisted of maximum number of 14 genotypes, followed by group II and III with 5 and 2 genotypes, respectively. First ( PAK 020978, PAK 021703, PAK 022254, PAK 021131, PAK 021156, PAK 021330, PAK 021675, PAK 022257, PAK 022219, PAK 021908, PAK 021900, PAK 021889, PAK 021882, PAK 021986) and third group (20 (Donated from Kasur), 21 (Donated from Kasur) showed 100% similarity among themselves while 23% deviation was observed in the second group (PAK 021748, PAK 021711, PAK 021696, PAK 021117, PAK 022260), whereas fourth group can be isolated (belongs to Kasur 22) as it is found different from all other groups.

Figure 1. Electrophoregram showing protein banding pattern of different accessions of

Trigonella corniculata landraces (passport data is given in the Table 1).

Figure 2. Electrophoregram showing protein banding pattern of different accessions of

Trigonella corniculata landraces (passport data is given in the Table 1).

Figure 3. Electrophoregram showing protein banding pattern of different accessions of

Trigonella corniculata landraces (passport data is given in the Table 1).

Coefficient of Similarity

0.67

0.75

0.83

0.92

1.00

10MW

1

2

3

4

5

19

18

17

16

15

14

6

7

8

9

13

11

10

12

20

21

22

Figure 4. UPGMA cluster analysis showing the diversity and relationship of Pakistani landraces of Trigonella corniculata based on SDS-PAGE

Table 3. Groups observed on dendrogram

GROUPS

ACCESSIONS

Group I

PAK 020978, PAK 021703, PAK 022254, PAK 021131, PAK 021156, PAK 021330, PAK 021675, PAK 021986, PAK 022257, PAK 022219, PAK 021908, PAK 021900, PAK 021889, PAK 021882

Group II

PAK 021748, PAK 021711, PAK 021696, PAK 021117, PAK 022260

Group III

20 (Donated from Kasur), 21 (Donated from Kasur)

Group IV

22 (Donated from Kasur)

DISCUSSION

In the present study an attempt has been made to examine the level of genetic diversity within accessions of Trigonella corniculata. Although variation in storage protein banding pattern was revealed by SDS-PAGE, however, its magnitude was low. A low level of genetic diversity may be attributed to narrow genetic base. The dendrogram as a whole revealed low genetic diversity at protein levels because most varieties are in the same cluster. Fufa et al. (2005) reported that the genetic diversity estimates based on seed storage protein were lowest because they were the major determinants of end-use quality, which is a highly selected trait. Genetic diversity of Trigonella corniculata land races revealed by SDS-PAGE of different accessions of germplasm with the Kasuri Methi showed that limited interspecific diversity was found at protein level. Four major groups were observed on the dendrogram, wherease first and third group showed 100% similarity among themselves while 23% deviation was observed in the second group whereas fourth group can be isolated (belongs to Kasur 22) as it is found different from all other groups. The genotypes collected from different geographical locations grouped (I and III) in the same clusters, which might be due to human selection. Separation of group four from other groups indicated the significance of geographical region. However, it is necessary to use more number of accessions from different geographical locations to confirm the available pattern.

CONCLUSION

From the present investigation, it was concluded that Trigonella corniculata landraces displayed limited diversity at inter-specific level. Although SDS- PAGE technique differentiated Trigonella corniculata of Kasur (Kasuri methi) from other landraces, which indicated the significance of geographical region. However, it is necessary to use more number of accessions from different geographical locations and to use other advanced techniques to confirm the available pattern.

ACKNOWLEDGMENTS

We express our genuine thanks to the Federal Seed Certification and Registration Department (FSC & RD), Islamabad, Pakistan, for providing the seed material of wheat genotypes and to the National Agriculture Research Centre (NARC), Islamabad, Pakistan for providing research facilities. We also indebted to the Ministry of Food Agriculture and Livestock (MINFAL) for providing financial support for this work under the project Strengthening of Intellectual Property Rights System in Ministry of Food Agriculture and Livestock .

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