Application of ISSR-PCR to determine the genetic ... · Application of ISSR-PCR to determine the genetic relationship and genetic diversity among steudneri clover ... intercropping

AMERICAN JOURNAL OF SOCIAL AND MANAGEMENT SCIENCES ISSN Print: 2156-1540, ISSN Online: 2151-1559, doi:10.5251/ajbms.2014.4.1.1.14

2014, ScienceHu, http://www.scihub.org/AJSMS

Application of ISSR-PCR to determine the genetic relationship and genetic diversity among steudneri clover (Trifolium steudneri) and quartin clover

(Trifolium quartinianum) accessions of Ethiopia

Tadesse Abate1, Kassahun Tesfaye2, Edossa Fikru2 and Fentahun Mihret3

1Madawalabu University, School of Agriculture, Department of Animal and Range Science P.O. Box 247; Bale Robe; Ethiopia Email: [email protected]

2Addis Ababa University, College of Natural Science, Institute of Biotechnology, P.O. Box 1176; Addis Ababa, Ethiopia.

2Madawalabu University, School of Agriculture, Department of Plant Science P.O. Box 247; Bale Robe; Ethiopia.

3Bahirdar university, College of Agriculture and Environmental Science, Department of Animal Production and Technology, P.O. Box; Bahirdar, Ethiopia.

ABSTRACT

The objective of the present study was to determine the genetic diversity and relationship among Trifolium steudneri and Trifolium quartinianum accessions of Ethiopia using inter simple sequence repeat (ISSR) markers. A total of 72 accessions representing T. steudneri (48) and T. quartinianum (24) were used. DNA was extracted from a bulk of young leaf tissues from each accession using a modified Cetyl trimethyl ammonium bromide (CTAB) method. A total of 87 bands were amplified using four ISSR primers. Genetic diversity was high at the genus level (PPL = 100%, h = 0.25, I = 0.39) than at the species levels. Comparison of species-based genetic diversity showed that T. quartinianum was more diverse (PPL = 88.51%, h = 0.29, I = 0.44) than T. steudneri (PPL = 74.70%, h = 0.20, I = 0.30). AMOVA revealed the smaller molecular variance among species (28.93) than within species (71.07) which could be caused by the presence of several shared ISSR markers. UPGMA and PCO showed that most of the accessions from their respective species formed separate cluster. The study clearly indicates the ISSR-PCR method used was confirmed to be a reproducible and sensitive tool for the identification of Trifolium accessions and for determination of the correct starting material for plant breeding. Keywords: Genetic diversity, Genetic relationship, ISSR, T. quartinianum, T. steudneri

INTRODUCTION

Clover (Trifolium) is one of the largest genera belonging to the tribe Trifolieae under family Fabaceae (Nick et al., 2006). It is one of economically important genus due to the wide growing of at least 16 species as livestock forage and green manure crops (Gillett and Taylor, 2001) and over 125 species have the capacity to fix nitrogen through root nodulation by the bacterium Rhizobium leguminosarum (Sprent, 2001).

There are forty Trifolium species in sub-Saharan Africa, of which about 25% are endemic to Ethiopia (Thulin, 1982). About two-thirds of the clover species in Ethiopia are annuals and the others are perennials, while biennials are not found in Ethiopia. Even though the growing period of the annuals is shorter than that of perennials, their higher dry-matter yield (Akundabweni, 1984) could be of greater use when harvested and conserved to overcome seasonal feed

shortage. In addition, annual clovers can be planted on a range of soil types such as acid, saline, dry and wet soils (Lloveras and Iglesias, 2001). Furthermore, annual clovers could conveniently be integrated into the existing crop and livestock enterprise with greater management flexibility than perennial ley meadows.

Trifolium quartinianum A. Rich and Trifolium steudneri Schweinfur are annual clovers classified in section Vesicastrum (Ellison et al., 2006). They are diploid with 2n =16 (Badr, 1995) and self pollinated (ILCA, 1990).These two species are indigenous to the east African highlands that have potentially important forage legumes for grazing in native pastures with potential for use as an annual legume for pasture improvement and are suitable for hay and silage making. The two species are promising for hay production on bottom land sites with clay soil subject to seasonal water logging (Kahurananga and Asres Tsehay, 1984). Thus, they can be managed to

mailto:[email protected]

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

2

increase forage production without competing for land with food crops. These species are also used for intercropping with wheat or barley in tropical highlands to improve soil nitrogen and quality of residues for livestock feed. According to Zewdu Tesema (2004), T. quartinianum and T. steudneri can be undersown with barley simultaneously or at first weeding without affecting the grain and straw yield of barley but significantly increasing the total fodder yield. The two species are good seed producers and seed yields are in the range of 800 - 900 kg/ha (Akundabweni, 1984; Kahurananga and Asres Tsehay, 1991). Under appropriate temperature and soil aeration conditions, T. steudneri and T. quartinianum are a very good fixer of atmospheric nitrogen in symbiosis with the indigenous Rhizobium population and thus increase the yields of succeeding crops.

In recent years, a number of PCR-based molecular markers have been developed to evaluate genetic variation at the intraspecific and interspecific levels (Wolfe and Liston, 1998). But, it is important to understand that different markers have different properties and will reflect different aspects of genetic diversity (Karp and Edwards, 1995). Inter Simple Sequence Repeat Marker (ISSR) (Wolfe and Liston, 1998) without the requirement for prior knowledge of the genome sequence seems particularly suitable for germplasm comparison. ISSR markers were used for the first time to study the genetic relationship and genetic diversity of three South American and three Eurasiatic species (Rizza et al., 2007). It also used to asses genetic relationship and genetic diversity of four clover species from Europe (Dabkeviien et al., 2011). These research findings suggested that ISSR marker systems are suitable for clover DNA polymorphism studies.

Based on the above ground, inter simple sequence repeat (ISSR) marker was chosen for genetic diversity and relationship study of T. quartinianum and T. steudneri accessions of Ethiopia. Despite the importance of these clover species as a major livestock feed and contribution to soil fertility, so far, there were no published reports concerning ISSR method for analyzing the genetic diversity and relationship among the two species. Hence, the present study aimed to determine the genetic relationship and pattern of genetic variation between closely related species belonging to the same section using inter simple sequence repeat (ISSR) markers.

The study can give a base line information for efficient preservation, exploitation of the existing genetic resources and assist for germplasm management.

MATERIALS AND METHODS

Plant material: For the present study, 72 Trifolium accessions representing T. steudneri (48) and T. quartinianum (24) were used (Table 1).The accessions with the passport data were kindly provided by International Livestock Research Institute (ILRI) forage germplasm bank, Addis Ababa, Ethiopia. Seeds of each accession were sown in plastic pots and grown for three weeks in the greenhouse.

DNA extraction: Fresh young leaves from each accession were bulked and ground to a fine powder by pestle and mortar in liquid nitrogen. Total genomic DNA was extracted from the powder following the Cetyl Trimethyl Ammonium Bromide (CTAB) protocol established by Borsch et al., 2003 with slight modification. Finally pellets were dissolved in 100 l 1x TE solutions and kept in freezer at -20 for subsequent use.

PCR amplification and gel electrophoresis: A total of 13 ISSR primers were used for screening (Table 2). Four primers were selected and used for the analysis of 72 accessions of the two species based on the number of amplification products, the quality of the profiles, the level of polymorphism and the reproducibility of bands. Each PCR of ISSR markers had a final reaction volume of 25 l , containing 17 l ddH20, 0.8 l dNTPs (25 mM each ), 2.5 l of reaction buffer (10x taq polymerase buffer with 15 mM MgCl2), 3l MgCl2 (25mM), 0.4l primer (20 pmol/l) and 0.3l Taq Polymerase (1.5 unit) and1l diluted template DNA. Amplifications were performed in Biometra 2003 T3 Thermo cycler programmed to run the following temperature profile: a preheating and initial denaturation for 4 minutes at 94, then 40 x 15 seconds denaturation at 94, 1 minute primer annealing at 48, 1.30 minutes extension at 72 and the final extension for 7 minutes at 72. For each primer a negative control reaction was included. PCR product (11 l) and 1 l 6X loading dye were loaded into the agarose gel wells. The electrophoreses were done for about 3 hours at constant voltage of 80V. After electrophoresis, the gel was stained in ethidium bromide, distained with ddH2O, visualized under UV light, photographed and documented.

Table 1. Accessions of T. steudneri and T. quartinianum used for the study

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

3

Acc. No

species District Zone Latitude Longitude Altitude (m)

Lab. Code

8471 T. steudneri Chebo and Gurage Shewa 0821N 03749E 1800 Ts Sh1

8489 T. steudneri Menagesha Shewa 0851N 03830E 2080 Ts Sh2 9720 T. steudneri Selale Shewa 0945N 03847E 2880 Ts Sh3

9966 T. steudneri Chebo and Gurage Shewa 0830 N 03757E 2000 Ts Sh4

10134 T. steudneri Jibat and Mecha Shewa 0903N 03811E 2220 Ts Sh5

10139 T. steudneri Menagesha Shewa 0902N 03818E 2240 Ts Sh6




7614 T. steudneri Selale Shewa 0953 N 03823E 2560 Ts Sh10

6221 T. steudneri Addis ababa Shewa 03857 E 1800 Ts Sh11



7620 T. steudneri Selale Shewa 0958 N 038 18 E 2550 Ts Sh14


6253 T. steudneri Menagesha Shewa 0902 N 03845 E 2380 Ts Sh16


8357 T. steudneri Jibat and mecha Shewa 0801N 03703E 2370 Ts Sh18



8087 T. steudneri Yerer and Kereyu Shewa 0835N 03908E 1780 Ts Sh21

8084 T. steudneri Yere and Kereyu Shewa 0839N 03904 E 1850 Ts Sh22

6222 T. steudneri Addis Ababa Shewa 0845 N 03857 E 1800 Ts Sh23


9452 T. steudneri Agew Midir Gojam 1057N 03632E 1850 Ts Goj25

9424 T. steudneri Debre Markos Gojam 1000N 03839E 2500 Ts Goj26

9456 T. steudneri Debre Tabor Gojam 1150 N 03735E 1950 Ts Goj27


2179 T. steudneri Debre Markos Gojam 1028 N 03809 E 2550 Ts Goj29

8520 T. steudneri Debre Markos Gojam 1030 N 03810E 2420 Ts Goj30

14602 T. steudneri Mota Gojam 1105N 03705E 2450 Ts Goj31

2177 T. steudneri Mota Gojam 1054 N 03802 E 2610 Ts Goj32


7697 T. steudneri Debre Markos Gojam 1016N 03749 E 2350 Ts Goj34

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

4

7647 T. steudneri Bichena Gojam 1041 N 038 11 E 2500 Ts Goj35


7658 T. steudneri Mota Gojam 1105 N 03753 E 2450 Ts Goj37


7645 T. steudneri Bichena Gojam 1037 N 03810 E 2520 Ts Goj39

7659 T. steudneri Bichena Gojam 1015N 03808E 2510 Ts Goj40

7652 T. steudneri Mota Gojam 1058N 03755E 2520 Ts Goj41

7667 T. steudneri Bichena Gojam 1037N 03801 E 2500 Ts Goj42

7671 T. steudneri Bichena Gojam 1027 N 03733 E 2610 Ts Goj43




2185 T. steudneri Debre Markos Gojam 1014 N 038 04 E 2420 Ts Goj47


9428 T.quartinianum Debre Markos Shewa 1008 N 037 58 E 2430 Tq Sh49

9975 T.quartinianum Chebo & Gurage Shewa 08 15 N 037 40 E 1840 Tq Sh50

8321 T.quartinianum Menagesha Shewa 0848 N 03854 E 1870 Tq Sh51

9968 T.quartinianum Chebo & Gurage Shewa 08 24 N 037 52 E 1870 Tq Sh52

6297 T.quartinianum Menagesha Shewa 0902 N 03845 E 2380 Tq Sh53

14408 T.quartinianum Shewa - - - Tq Sh54

8473 T.quartinianum Chebo & Gurage Shewa 0821 N 03749 E 1880 Tq Sh55

8464 T.quartinianum Chebo & Gurage Shewa 0817 N 037 47 E 1880 Tq Sh56

13716 T.quartinianum Shewa 10 02 N 038 14 E 2010 Tq Sh57

7675 T.quartinianum Debre Markos Gojam 1014 N 03806 E 2400 Tq Goj58

2049 T.quartinianum Debre Markos Gojam 1012 N 03752E 2400 Tq Goj59 2047 T.quartinianum Debre Markos Gojam 1015 N 03757 E 2500 Tq Goj60

8521 T.quartinianum Debre Markos Gojam 1126 N 037 36 E 2200 Tq Goj61

6277 T.quartinianum Bahir Dar Gojam 1140 N 03728 E 1900 Tq Goj62

9378 T.quartinianum Debre Markos Gojam 10 07 N 038 09 E 1980 Tq Goj63



8535 T.quartinianum Debre Markos Gojam 10 42 N 03704 E 2100 Tq Goj66 7771 T.quartinianum Libo Gondar 1203 N 037 44 E 1840 Tq Gon67 14586 T.quartinianum Gondar - - - Tq Gon68

13808 T.quartinianum Gondar - - 1950 TqGon69

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

5

7759 T.quartinianum Gondar Gondar 1222N 03717 E 1860 TqGon70

7746 T.quartinianum Debre Tabor Gondar 1153 N 03741 E 1860 Tq Gon71

7768 T. quartinianum Gonder Gondar 1227N 03731 E 1940 Tq Gon72

Acc. = Accession, E = East, N = North, m = meter, Lab. = Laboratory, TsSh = T. steudneri from Shewa, TsGoj = T. steudneri from Gojam, TqSh = T. quartinianum from Shewa, TqGoj = T. quartinianum from Gojam, TqGon = T. quartinianum from Gondar

Table 2. ISSR primers with motif, annealing temperature, amplification quality and motives screened for amplification of T. quartinianum and T. steudneri accessions

Code of primers

Primer motif T() Amplification quality Motives

880 (GGAGA)3 45 X Penta-nucleotide

818 (CA)8G 48 Polymorphic, Reproducible Dinucleotide

834 (AG)8YT 45 X Dinucleotide

841 (GA)8YC 48 Polymorphic, Reproducible Dinucleotide

826 (AC)8C 48 X Dinucleotide

873 (GACA)4 45 X * Tetra- nucleotide

844 (CT)8RC 48 Polymorphic, Reproducible Dinucleotide

824 (TC)8G 48 X * Dinucleotide

816 (CA)8T 48 X * Dinucleotide

848 (CA)8RG 48 Polymorphic, Reproducible Dinucleotide

812 (GA)8A 45 X Dinucleotide

813 (CT)8T 45 X * Dinucleotide

810 (AG)8T 45 X Dinucleotide

X: Less reproducible and less polymorphic, X*: no amplification, T (): annealing temperature

Band scoring and data analysis

Band scoring was performed manually for each primer based on presence (1) and absence (0) or as a missing observation (?), and each band was regarded as a locus. Only amplified bands that were clearly resolved were recorded, and a 0 and 1 data matrix was established. The resulting presence/absence data matrix of the ISSR phenotype was analysed using POPGENE version1.32 software (Yeh et al., 1999) to calculate percentage of polymorphic loci (PPL), gene diversity (h), and Shannons information index (I). Analysis of Molecular Variance (AMOVA) was carried out using the software ARLEQUIN version 3.01 (Excoffier et al., 2006) to estimate genetic variability within and among species. NTSYS- pc version 2.02 (Rohlf, 2000) and Free Tree 0.9.1.50 (Pavlicek et al., 1999) were used to calculate Jaccards similarity coefficient which is calculated with the formula:- Source: Primer kit 900 (UBC 900); Y = Pyrimidines (C

or T), R = purines (A or G)

Sij= a

a+b+c

Where, a is the total number of bands shared between individuals i and j, b is the total number of bands present in individual but not in individual j and c is the total number of bands present in individual but not in individual i. The unweighted pair group method with arithmetic average (UPGMA) (Sneath and Sokal, 1973) was used in order to determine the genetic relationship among accessions of the two species and generates phenogram using NTSYS- pc version 2.02 (Rohlf, 2000).To further examine the patterns of variation among individual samples, a principal coordinated analysis (PCO) was performed based on Jaccards coefficient (Jaccard, 1908). The calculation of Jaccards coefficient was made with PAST software

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

6

version 1.18 (Hammer et al., 2001).The first three axes were later used to plot with STATISTICA version 6.0 software (Hammer et al., 2001; Statistica soft, Inc.2001).

RESULTS

ISSR primers and banding patterns

All the selected four primers have dinucleotide repeat motives. A total, 87 ISSR bands

were amplified for the two Trifolium species. The number of bands for individual primer ranged from 20 to 23, while the average number of bands per primer was 22.5. The highest number of bands was amplified with primers 841 and 848, while the lowest number was amplified with 844 (Table 3). The fragment size amplified with these primers was in the range of 200 to 1000bp. Representative gels illustrating the amplification profiles produced by the ISSR marker assay by employing primer 844 are shown in figure 1. Species specific ISSR bands All ISSR primers, except primer 818 in T. steudneri, showed species specific bands (Table 3). A total of nine bands specific to T. steudneri were generated. Primer 841 showed the highest number of T. steudneri specific bands (4 bands). The number of bands amplified by the four primers specific to T. quartinianum was 21. The highest number of bands were amplified by 844 (6 bands), while the lowest were produced by 818,841 and 848 (5 bands) (Table 3). Genetic diversity: The four ISSR primers detected 20 to 23 polymorphic loci in 72 accessions of the two Trifolium species. All four primers generated a total of 87 scorable bands, all of which were polymorphic i.e., the percentage of polymorphic loci (PPL) was 100%. Furthermore, each individual primer also possessed the highest degree of polymorphism (100%). The overall gene diversity (h) by the four primers was 0.25, while Shannons information index (I) was 0.39 (Table 4). The highest gene diversity (0.29) and Shannons information index (0.43) were obtained for primer 818, while primer 848 showed the least for both indices 0.22 and 0.35, respectively (Table 4) At the genus level, T. quartinianum was more polymorphic (77 bands; 88.51 %), than T. steudneri (65 bands; 74.70 %). T. quartinianum also showed the

highest gene diversity (0.29) and Shannons information index (0.44) than T. steudneri (Table 5).

Table 4. Level of genetic diversity revealed by four ISSR primers in T. steudneri and T. quartinianum

ISSR primer

NPL PPL h I

818 21 100 0.29 0.43

844 20 100 0.26 0.40 841 23 100 0.23 0.37

848 23 100 0.22 0.35

Over all 87 100 0.25 0.39

NPL = Number of polymorphic Loci; PPL = Percent of polymorphic Loci; h = Gene diversity; I = Shannons information index

Table 5. Genetic diversity at the genus and species level

Species NPL PPL h I

T. steudneri 65 74.70 0.20 0.30

T. quartinianum 77 88.51 0.29 0.44

Average

Genus level

71

87

81.60

100

0.25

0.25

0.37

0.39

Analysis of Molecular Variance (AMOVA): An AMOVA was conducted on the ISSR data scores of the four dinucleotide ISSR primers from the combination of the two species. AMOVA result obtained showed that most of the total genetic variation (71.07%) was found within species, whereas only 28.93% occurred among species (Table 6).

Clustering analysis

Dendrogram was constructed using unweighted pair group method with arithmetic average (UPGMA) to investigate the relationship among the 72 accessions of the two species. The UPGMA dendrogram formed five major clusters at about 44% similarity level with similarity value ranged from 23-97%. The first major cluster (I) consisted of 44 accessions (42 from T. steudneri and two from T. quartinianum), the second major cluster (II) was formed by three accessions of T. quartinianum, the third major cluster (III) was formed by four accessions of T. quartinianum, the fourth major cluster (IV) was formed by seven accessions of T. quartinianum and the fifth major cluster (V) was formed by four accessions of T. quartinianum.

Table 3. List of ISSR primers used and overview of their banding pattern

ISSR

Primers

Repeat motif Annealing temperature ()

No of scorable bands No of species specific bands

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

8

T. steudneri T.quartinianum

818 (CA)8G 48 21 0 5

844 (CT)8RC 48 20 2 6

841 (GA)8YC 48 23 4 5

848 (CA)8RG 48 23 3 5

Total 87 9 21

Average 22.5 2.25 5.25

Y = (C, T); R = (A, G).

Fig. 1. Banding pattern of primer 844 in T. steudneri and T. quartinianum accessions. M represents a 1000 bp DNA ladder; S and Q, respectively stand for T. steudneri and T. quartinianum

Table 6. Analysis of molecular variance (AMOVA)

Source of variation d.f Sum of

squares

Variance

components

Percentage

of variation

P-Value*

Among species 1 117.16 3.40 28.93 P< 0.001

Within species 70 584.73 8.35 71.07 P< 0.001

Total 71 701.89 11.75

d.f = degree of freedom, * significance tests after 1023 permutations

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

9

Fig. 2. UPGMA dendrogram based on Jaccard similarity coefficient among 72 accessions of the two species.

Coefficient

0.24 0.42 0.61 0.79 0.97

TsSh1 TsSh8 TsSh9 TsSh6 TsSh7 TsSh12 TsSh11 TsSh3 TsSh4 TsSh5 TsSh14 TsSh16 TsSh19 TsSh15 TsSh24 TsSh23 TsSh21 TsSh22 TsSh17 TsSh20 TsSh2 TsSh13 TsGoj27 TsGoj29 TsGoj30 TsGoj34 TsGoj35 TsGoj32 TsGoj33 TsGoj36 TsGoj37 TsGoj31 TsGoj28 TsGoj39 TsGoj40 TsGoj42 TsGoj41 TsGoj45 TsGoj46 TsGoj47 TsGoj48 TsGoj38 TsGoj43 TqSh52 TqGoj60 TqSh50 TqSh55 TqSh54 TsSh10 TsSh18 TqSh49 TqSh53 TqGoj58 TqGoj59 TqGoj61 TsGoj25 TqSh56 TqGoj62 TqGon71 TqGon72 TqGon67 TqGon68 TqGon69 TqGon70 TqGoj63 TqGoj64 TqGoj66 TqGoj65 TsGoj26 TqSh57 TqSh51 TsGoj44

Key:

TqGoj = T. quartinianum Gojam

TqGon = T. quartinianum Gondar

TqSh = T. quartinianum Shewa

TsGoj = T. steudneri Gojam

TsSh = T. steudneri Shewa

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

10

Fig. 3. Two-dimensional plot obtained from principal coordinate analysis of using 87 ISSR bands with Jaccard's coefficient similarity.

Ts Sh1Ts Sh2Ts Sh3

Ts Sh4

Ts Sh5

Ts Sh6Ts Sh7

Ts Sh8Ts Sh9

Ts Sh10

Ts Sh11

Ts Sh12

Ts Sh13

Ts Sh14

Ts Sh15

Ts Sh16

Ts Sh17

Ts Sh18

Ts Sh19

Ts Sh20

Ts Sh21Ts Sh22Ts Sh23

Ts Sh24

Ts Goj25

Ts Goj26

Ts Goj27

Ts Goj28

Ts Goj29Ts Goj30

Ts Goj31Ts Goj32Ts Goj33Ts Goj34

Ts Goj35Ts Goj36Ts Goj37

Ts Goj38Ts Goj39

Ts Goj40Ts Goj41

Ts Goj42

Ts Goj43

Ts Goj44

Ts Goj45Ts Goj46

Ts Goj47Ts Goj48

Tq Sh49

Tq Sh50

Tq Sh51

Tq Sh52

Tq Sh53

Tq Sh54

Tq Sh55

Tq Sh56

Tq Sh57

Tq Goj58Tq Goj59

Tq Goj60

Tq Goj61

Tq Goj62

Tq Goj63

Tq Goj64

Tq Goj65

Tq Goj66

Tq Gon67

Tq Gon68

Tq Gon69Tq Gon70

Tq Gon71

Tq Gon72

-0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4Coordinate 1

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

Co

ord

ina

te 2

T. steudneri

T. quartinianum

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

11

Fig. 4. Three-dimensional plot obtained from principal coordinate analysis of using 87 ISSR bands with Jaccard's coefficient similarity.

T. steudneri

T.quartinianum

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

11

Principal Coordinate (PCO) analysis: Principal coordinate (PCO) analysis was carried out on all the ISSR data obtained from 72 accessions from the two species. The first three coordinates of the PCo had Eigen values of 8.33, 3.52 and 3.00 with percentage of variation 18.19%, 7.69% and 6.56%, respectively, were used to show the grouping of all of the accessions from the two species using two and three coordinates (Figure and ). From Figure 12, it was observed that the two dimensional PCoA plot separated all the accessions into two major clusters. All the T. quartinianum accessions appeared at the left as a separate major cluster whereas the majority of the T. steudneri accessions appeared at the right. Three-dimensional plot plot also showed grouping of most of the accessions in to respective species (Figure 4).

DISCUSSION

Applications of ISSR markers in Trifolium species diversity study: Inter-simple sequence repeat (ISSR) markers have become widely used in population studies because they have been found to be highly variable, to require less investment in time, money and labor than other methods (Wolfe and Liston, 1998), and to have the ability to be inherited (Gupta et al., 1994; Tsumura et al., 1996). Different studies showed the usefulness of inter simple sequence repeat markers for resolving polymorphism and determine inter- and intra- genomic diversity across species (Souframanien and Gopalakrishna, 2004; Balasaravanan et al., 2005; Wang et al., 2012; Singh et al., 2011). Several researchers also used ISSR markers to study genetic variability within and among populations of forage species (Bolourchian et al., 2013; Jonaviclene et al., 2009; Zarrabian et al., 2013; Shirvani1 et al., 2013). Better reproducibility of products of ISSR bands compared to other markers such as RAPD could be due to its longer SSR-based primers with higher annealing temperatures (Huangfu et al., 2009). Moreover, as microsatellites are frequent and widely distributed throughout the genome, the ISSR targets are abundant. Compared with SSR markers, where the flanking regions of the SSR motifs have to be known in advance, ISSR amplification takes advantage of the fact that no prior sequence information is required, and the results are therefore obtained more rapidly and cost effectively (Wang et al., 2008; Yang et al., 1996; Bornet and Branchard, 2001).

The present study employed ISSR markers to assess genetic relationship and genetic diversity of T. steudneri and T. quartinianum accessions from Ethiopia. In this study the dinucleotide ISSR primers

818 and 848 with CA repeat and 844 and 841 with CT and GA motives, respectively detected genetic diversity at the genus and species levels. Reddy et al., (2002) reported that generally primers with (AG), (GA), (CT), (TC), (AC), (CA) repeats show higher polymorphism in plants than primers with other di-, tri- or tetra nucleotides. Moreover, the choice of appropriate primer motives in ISSR fingerprint is critical to detect high polymorphism and reveal relationship. The four di-nucleotide ISSR primers chosen for this study amplified large number of loci, varying from 20-23 fragments per primer displaying 100% polymorphism with high mean gene diversity (h= 0.25) and Shannons information index (I= 0.39). This indicates the existence of high level of genetic diversity within the two Trifolium species in Ethiopia. Among the two species, T. quartinianum was more divers and variable. This may be due to geographical and altitudinal differences in which the accessions collected. Although there was no research conducted at the genus level using any of the molecular markers, Arslan and Tamkoc, (2009) studied the genetic diversity of two species of forage grasses (P. trivialis and P. angustifolia) in the Poa genus using ISSR markers. These researchers used 11 accessions belonging to P. trivialis and three accessions of P. angustifolia for their study, and reported high level of inter-species genetic polymorphism (90.52%) comparable with the present result. The high diversity associated with these forage species could be due to lack of selection and strict domestication under low overgrazing of forage crops.

Both T. steudneri and T. quartinianum maintained a higher genetic diversity at the species level when compared to the average percentage (70%) observed in higher plants (Zou et al., 2001). Up to now, there are no published reports concerning ISSR method for analyzing the genetic diversity of T. steudneri and T. quartinianum. However, Rizza et al., (2007) studied the genetic diversity of 34 genotypes from six Trifolium species using ISSR marker and found 89.5% polymorphism in T. medium, 0.0% in T. pretense, 52.4% in T. repens, 92.9% in T. argentinense, 46.4% in T. polymorphum and 78.0% in T. riograndense. The level of polymorphism in T. argentinense and T. medium were comparable with the present result, while the other four Trifolium species showed a lower level of polymorphism than the present result.

Dabkeviien et al., (2011) used ISSR marker to assess the level and pattern of genetic diversity in four Trifolium species represented by two varieties, one breeding sample and two wild population of T.

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

12

pratense, four wild populations of T. medium, two varieties and one population of T. resupinatum, and two varieties and three wild populations of T. repense. They observed 69.5% polymorphism in T. medium, 68.9% in T. resupinatum, 76.2% in T. pretense and 73.6% in T. repense. Recently, Aryanegad et al., (2013) also studied the genetic diversity of 14 accessions of three species of Trifolium using ISSR markers and found 60% polymorphism in T. fragiferum, 58.67% in T. hybridum and 77.32% in T. pratense. The genetic diversity investigated in the present study, T. steudneri and T. quartinianum was higher than the ones reported by Dabkeviien et al., (2011) and Aryanegad et al., (2013).

Moreover, Ulloa et al., (2003) studied the genetic diversity of 20 breeding populations and cultivars of red clover (Trifolium pratense) using RAPD marker and reported 74.2% polymorphism, while Arzani and Samei, (2004) studied the genetic diversity of 20 Persian clover (T. resupinatum) with RAPD marker and found 80% polymorphism. The genetic diversity investigated in the present study was higher than the ones reported by Ulloa et al., (2003) and Arzani and Samei, (2004), despite of the fact that these researchers employed RAPD markers for their study. Generally, the higher level of genetic variation found in this study may be due to the fact that geographically isolated populations in certain geographic locations could accumulate genetic differences and evolve unique genotypes as they adapt to different environment (Souframanien and Gopalakrishn, 2004).

Genetic structure and portioning of genetic diversity: In the present study, the results of AMOVA at genus level showed that 71.07% of the total variation results from differentiation within species of T. steudneri and T. quartinianum. The smaller molecular variance between T. steudneri and T. quartinianum revealed that they had a common ancestor. Aryanegad et al., (2013) reported lower genetic variation among the three Trifolium species (29%) which is in agreement with the present result.

Genetic relationships among species: UPGMA and PCO analysis were used to observe the genetic relationship among the total accessions from the two species. Except few accessions remaining distinct and ungrouped, at 44% similarity level, the UPGMA dendrogram constructed at genus level showed that most of the accessions from the same species grouped together. Moreover, the range of genetic similarity among the total accessions was in the range of 23-97%. Higher genetic similarity was observed among T. steudneri accessions than T. quartinianum

accessions. Two accessions of T. steudneri (TsSh21 and TsSh22), collected from central highland showed the highest level of genetic similarity (97%). There are few ungrouped accessions of T. quartinianum and T. steudneri in figure 8 which showed clearly different genetic amplification pattern from other individuals of the population. In this case, a study with different primers and the inclusion of more individuals would be necessary to verify the clustering. The UPGMA tree topology was also further confirmed by the two and three dimensional PCO plot, in which most individuals of the two species formed a separate cluster unique to each species.

Similar result was reported by Aryanegad et al., (2013) who analysed 14 accessions of three Trifolium species Viz T. fragiferum, T. hybridum and T. pretense by using 10 ISSR primers. Using UPGMA and two dimensional plot, most of the accessions belonging to the same species were grouped together. Arslan and Tamkoc, (2009) also analysed 11 accessions of two forage grasses species Viz P. trivialis and P. angustifolia by using 20 ISSR primers. Using UPGMA dendrogram, the accessions belonging to the same species were clustered together. In general, this indicates that ISSR markers have the power to distinguish accessions of closely related species.

CONCLUSIONS

The present study is the first report on genetic diversity and relationships of T. steudneri and T. quartinianum using ISSR marker. The genetic diversity data generated by four ISSR primers revealed that high genetic diversity exists at the genus than at species level. T. quartinianum showed higher genetic diversity than T. steudneri. The smaller molecular variance and several shared ISSR fragments between the two species indicated that they had a common ancestor. UPGMA cluster analysis and PCO supported the grouping of accessions to the respective species in the analysis of the total accessions of the two species. The findings of this study indicates that ISSR markers could be good tools to assess the genetic diversity and relationship of T. steudneri and T. quartinianum accessions.

REFERENCES

Akundabweni L. (1984). Forage potential of some annual native Trifolium species in the Ethiopian highlands. PhD thesis, South Dakota State University, Brookings.

Arslan E, Tamkoc A (2009). The application of ISSR-PCR to determine the genetic relationship and genetic diversity between narrow leaved blue grass (Poa angustifolia) and reoughblue grass (Poa trivialis)

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

13

accessions. Turk J Biol. 35: 415-423.

Aryanegad P, Farshadfar E, Safari H, Shirvani H. (2013). Application of ISSR molecular markers in genetic diversity of three Trifolium species. J. Bio. & Env. Sci. 3 :78-84

Arzani A, Samei K (2004). Assessment of genetic diversity among Persian clover cultivars as revealed by RAPD markers. In : Genetic Variation for Plant Breeding. Proc. of the 17th EUCARPIA Gen. Congr, Pp.85-88, (Vollmann, J., Grausgruber, H. and Ruckenbauer, P., eds.). EUCARPIA (European Association for Research on Plant Breeding) & BOKU.

Badr A. (1995). Electrophoretic studies of seed proteins in relation to chromosomal criteria and the relationships of some taxa of Trifolium. Taxon 44: 183- 191.

Balasaravanan T, Chezhian P, Kamalakannan R, Ghosh M,Yasodha R,Varghese M, Gurumurthi K. (2005). Determination of inter-and intra-species genetic relationships among six Eucalyptus species based on

inter simple sequence repeats (ISSR). Tree Physiology 25: 1295-1302.

Bolourchian F, Farshadfar M, Safari H, Shirvani H. (2013). Evaluation of genetic diversity in different genotypes of (Medicago sativa L.) using ISSR markers. Inti. J. Farm and Alli Sci. 2: 955-960.

Bornet B, Branchard M. (2001). Nonanchored inter simple sequence repeat (ISSR) markers: Reproducible and specific tools for genome fingerprinting. Plant Mol. Biol. Report 19: 209215.

Borsch T, Hilu K, Quandt, Wilde V, Neinhuis C, Barthlott W. (2003). Noncoding plastid trnT-trnF sequences

reveal a well resolved phylogeny of basal angiosperms. J . Evol. Biol. 16:558576.

Dabkeviien G, Paplauskien V, Vilinskas E. (2011). Assessment of genetic diversity in Trifolium spp. using ISSR and RAPD markers. Science and Technology 9: 210 - 214 .

Ellison N, Liston A, Steiner J, Williams W, Taylor N. (2006). Molecular phylogenetics of the clover genus (Trifolium-Leguminosae). Mol Phylogenet Evol. 39: 688-705.

Excoffier L, Laval G, Schneider S. (2006). An Integrated Software Package for Population Genetics Data Analysis. Computational and Molecular Population Genetics Lab (CMPG), Institute of Zoology, University

of Berne, Switzerland.

Gillett J, Taylor N. (2001). The World of Clovers. Iowa State University Press, Ames, Iowa, USA.

Gupta M, Chyi Y, Romero-Severson J, Owen J. (1994). Amplification of DNA markers from evolutionarily diverse genomes using single primers of simple-sequence repeats. Theor. Appl. Genet. 89: 998-1006.

Hammer O, Harper D, Ryan P. (2001). PAST :

Paleontological statistics software package for education and data analysis. Palaeontologia electronic 4: 9, http//palaeo electronica.org/2001_1/past/issue1-01.htm.

Huangfu C, Song X, Qiang S. (2009). ISSR variation within and among wild Brassica juncea populations: implication for herbicide resistance evolution. Genet. Resour. Crop Evol. 56: 913-924.

International Livestock Centre for Africa (ILCA). (1990). Annual report and programme highlights. ILCA, Addis Ababa, Ethiopia. 28Pp.

Jaccard P. (1908). Nouvelles rescherches Sur la distribution florale. Bull. Soc. Vaud. Sci. Nat. 44: 223-270.

Jonaviclene K, Paplausklene V, Brazauskas G. (2009). Isozymes and ISSR markers as a tool for the assessment of genetic diversity in Phleum spp. Zemdirbyste-Agriculture 96: 47-57.

Kahurananga J, Asres Tsehay, (1991). Variation in flowering time, dry matter and seed yield among Trifolium species, Ethiopia. Tropical Grasslands. 25: 20-25.

Karp A, Edwards K. (1995). Molecular techniques in the analysis of the extent and distribution of genetic diversity. IPGRI Workshop on Molecular Genetic Tools in Plant Genetic Resources, 911 October, Rome, IPGRI.

Lloveras J, Iglesias I. (2001). Morphological development and forage quality changes in crimson clover (Trifolium incarnatum L.). Grass and Forage Science 56:395-404.

Nick W, Aaron L, JeVrey J, Warren M, Norman L. (2006). Molecular phylogenetics of the clover genus (Trifolium-Leguminosae). Molecular Phylogenetics and Evolution 39: 688705.

Pavlicek A, Hrda S, Flegr J. (1999). Free-tree-soft ware program for construction of phylogenetic trees on the basis of distance data and bootstrap/jackknife analysis of the tree robustness. Application in the RAPD analysis of genus Frenkelia. Folia Biol. (Praha). 45: 97-99.

Reddy M, Sarla N, Siddiq E. (2002). Inter simple sequence repeat (ISSR) polymorphism and its application in plant breeding. Euphytica 128: 9 17.

Rizza M, Real D, Reyno R, Porro V, Burgueco J, Errico E, Quesenberry K. (2007). Genetic diversity and DNA content of three South American and three Eurasiatic Trifolium species. Brasilian J. Genetics and Molecular Biology 30: 1118-1124.

Rohlf F. (2000). NTSYS-pc ver 2.11T. Exter Software, Setauket, New York.

Shirvani1 H, Etminan A, Safari H. (2013). Evaluation of genetic diversity within and between populations for Agropyron trichophorum by ISSR markers. Intl J Farm

Am. J. Soc. Mgmt. Sci., 2014, 4(1): 1-14

14

& Alli Sci. 2 : 949-954.

Singh D, Srivastava A, Srivastava A, Srivastava R. (2011). Genetic diversity among three Morinda species using

RAPD and ISSR markers. Indian Journal of Biotechnology 10: 285-293.

Sneath P, Sokal R. (1973). Numerical Taxonomy. Freeman, Sanfrancisco.

Souframanien J, Gopalakrishna T. (2004). A comparative analysis of genetic diversity in blackgram genotypes using RAPD and ISSR markers. Theoretical and Applied Genetics 109: 1687-1693.

Sprent J. (2001). Nodulation in Legumes. Royal Botanic Gardens, Kew.

Statistica Stat Soft, Inc.2001. STATISTICA (data analysis software system) Version 6.0. www.statsoft.com.

Thulin M. (1982). Legumes in Ethiopia: An illustrated guide. Opera Bot. 83.

Tsumura Y, Kawahara T, Wickneswari R, Yoshimura K. (1996). Molecular phylogeny of Dipterocarpaceae in Southeast Asia using RFLP of PCR-amplified chloroplast genes. Theor. Appl. Genet. 93: 22-29.

Ulloa O, Ortega F, Campos H. (2003). Analysis of genetic diversity in red clover (Trifolium pretense) breeding

populations as revealed by RAPD genetic markers. Genome 46: 529-35.

Wang C, Zhang H, Qian Z, Zhao G. (2008). Genetic differentiation in endangered Gynostemma pentaphyllum (Thunb.) Makino based on ISSR

polymorphism and its implications for conservation. Biochem. Syst. Ecol. 36: 699705.

Wang X, Yang R, Feng S, Hou X, Zhang Y, Li Y, Ren Y. (2012 ). Genetic Variation in Rheum palmatum and Rheum tanguticum (Polygonaceae), two medicinall and endemic species in China using ISSR markers. PLoS ONE 7: 1-10.

Wolfe A, Liston A. (1998). Contribution of PCR based methods to plant systematic and evolutionary biology. In: Molecular Biology of Plants II DNA Sequencing ,Pp.

43-86, (Soltis, D. E., Soltis, P. S., Doyle, J.J., eds.). Kluwer Acad. Publ., Boston, Dordrecht, London.

Yang W, de Oliveira A, Godwin I, Schertz K, Bennetzen J. (1996). Comparison of DNA marker technologies in characterizing plant genome diversity: Variability in Chinese sorghums. Crop Sci. 36: 16691676.

Yeh F, Yang R, Boyle T. (1999). Population genetic analysis of codominant markers and qualitative traits. Belgian J. Bot. 129:157.

Zarrabian M, Majidi M, Ehteman M, Hossein M. (2013). Genetic diversity in worldwide collection of Sainfoin using morphological, anatomical and molecular markers. Crop Sci. 53: 2483-2496.

Zewdu Tesema (2004). Grain and straw yield of barley as influenced by undersowing time of annual forage legumes and fertilization. Trop. Sci. 44: 8588.

Zou Y, Ge S, Wang X. (2001). Molecular Markers in One System and Evolutionary Biology. Beijing, China: Science Press.

Documents

Application of ISSR-PCR to determine the genetic ... · Application of ISSR-PCR to determine the genetic relationship and genetic diversity among steudneri clover ... intercropping