Inter simple sequence repeat (ISSR) analysis of genetic diversity in tef [Eragrostis tef (Zucc.) Trotter]
Abstract
The DNA polymorphism among 92 selected tef genotypes belonging to eight origin groups was assessed using eight inter simple sequence repeat (ISSR) primers. The objectives were to examine the possibility of using ISSR markers for unravelling genetic diversity in tef, and to assess the extent and pattern of genetic diversity in the test germplasm with respect to origin groups. The eight primers were able to separate or distinguish all of the 92 tef genotypes based on a total of 110 polymorphic bands among the test lines. The Jaccard similarity coefficient among the test genotypes ranged from 0.26 to 0.86, and at about 60 % similarity level the clustering of this matrix using the unweighted pair-group method based on arithmetic average (UPGMA) resulted in the formation of six major clusters of 2 to 37 lines with further eight lines remaining ungrouped. The standardized Nei genetic distance among the eight groups of origin ranged between 0.03 and 0.32. The UPGMA clustering using the standardized genetic distance matrix resulted in the identification of three clusters of the eight groups of origin with bootstrap values ranging from 56 to 97. The overall mean Shannon Weaver diversity index of the test lines was 0.73, indicating better resolution of genetic diversity in tef with ISSR markers than with phenotypic (morphological) traits used in previous studies. This can be attributed mainly to the larger number of loci generated for evaluation with ISSR analysis as compared to the few number of phenotypic traits amenable for assessment and which are further greatly affected by environment and genotype×environment interaction. Analysis of variance of mean Shannon Weaver diversity indices revealed substantial (P≤0.05) variation in the level of diversity among the eight groups of origin. In conclusion, our results indicate that ISSR can be useful as DNA-based molecular markers for studying genetic diversity and phylogenetic relationships, DNA fingerprinting for the identification of varieties or cultivars, and also for genome mapping in tef.
Tef, Eragrostis tef (Zucc.) Trotter, is one of the most important cereals of Ethiopia. Estimates of the Central Statistical Authority (2000) indicate that tef accounts for about 31 % (2.11 million ha) of the total acreage and 22 % (1.68 million tons) of the gross grain production of all the seven major cereals cultivated in the country. The long sustained extensive cultivation of tef by the Ethiopian peasants has been accentuated by its versatile adaptation with good resilience to both drought and water logged soil conditions, preference in the national staple diet, cattle feed value of the straw, and high local market prices of both its grain and straw (Ketema 1993; Assefa et al. 1999, 2000, 2001a,b, 2002a,b).
Ethiopia is the center of both origin and diversity of tef (Vavilov 1951). In spite of the supreme importance of tef in Ethiopia, its productivity is relatively low. The national land unit average grain yield is about 0.8 t/ha (Central Statistical Authority 2000). Its low productivity has, amongst others, been due mainly to the low genetic yield potential of the types under cultivation coupled with traditional cultural practices, vulnerability to lodging especially under high input husbandry conditions, and other biotic and abiotic stresses.
The fact that tef is a crop unique to Ethiopia and that its cultivation as a cereal is little known elsewhere in the world implies that the genetic improvement of the crop relies heavily upon the indigenous germplasm resources. This, in turn, calls for systematic collection, characterization and evaluation, and conservation of the available tef germplasm resources.
Cognizant of this fact, some attempts have been made to investigate the genetic diversity of tef mainly on the basis of morphological, agronomic and phenologic traits and many studies have shown wide variation in the species (Mengesha et al. 1965; Ebba 1975; Costanza et al. 1979; Tefera et al. 1990; Ketema 1993; Tadesse 1993; Assefa et al. 1999, 2000, 2001a,b, 2002a,b). At present, the Institute of Biodiversity Conservation and Research of Ethiopia maintains ex situ reserves of 4395 tef germplasm accessions (Demissie 2001), and the National Tef Improvement Program of Debre Zeit Agricultural Research Center keeps short-term reserves of most of these collections for immediate breeding and other research uses.
Biochemical assessment using chromatography of leaf phenolics and electrophoresis of seed proteins revealed great variation in tef (Bekele and Lester 1981). Similarly, analysis of seed storage proteins using SDS-PAGE procedure and 1-D isoelectric focusing showed that all the albumin, globulin and prolamin fractions were highly polymorphic, with the differences in the prolamin fraction alone allowing the segregation of the 37 tested tef genotypes into seven groups (Bekele et al. 1995). However, flavonoid patterns, and caryopsis and pollen morphology did not show significant variation among tef genotypes (Bekele 1986). Similarly, no intraspecific variation among eight studied varieties was noted in molecular genetic sequence analysis of the non-coding regions of chloroplast DNA, 18S rDNA and the transcription factor VP1 (Espelund et al. 2000).
Tef is an allotetraploid with 2n=4X=40 chromosomes. Compared to most other cereals, the genome size of tef is relatively small. The 1C genome size in different cultivars ranged from 714–733 Mbp (Ayele et al. 1996). Genetic diversity studies using random amplified polymorphic DNA (RAPD) (Bai et al. 2000) and amplified fragment length polymorphism (AFLP) (Ayele et al. 1999; Bai et al. 1999a; Ayele and Nguyen 2000) markers revealed a relatively low level of polymorphism in tef. In these studies, the similarity coefficient among the studied tef genotypes ranged between 84 % and 96 % for RAPD and between 73 % and 99 % for AFLP. However, considerable size variation of rDNA repeats was noted among tef accessions, and this is suggested to provide an effective tool for screening and selecting genetically diverse accessions of tef for breeding (Pillay 1997).
In this study, inter simple sequence repeat (ISSR) amplification was employed to assess the genetic diversity in tef germplasm. This PCR-based DNA marker system relies on the abundance of simple sequence repeats (SSRs) or microsatellites in the eukaryotic genomes (Lagercrantz et al. 1993). The method involves PCR amplification of regions between two adjacent and inversely oriented microsatellites using a single, usually 16–25 base pair long (Reddy et al. 2002) SSR-containing primer anchored at the 3′- or 5′-end by two to four arbitrary, often degenerate, nucleotides (Fang et al. 1997). The primer can be based on any of the SSR motifs (di-, tri-, tetra- or penta-nucleotides) found at microsatellite loci. The technique combines the advantages of AFLP and microsatellite (SSR) analysis to the taxonomic universality of RAPD (Zietkiewicz et al. 1994). Unlike SSR analysis, it does not require prior sequence information for primer design, and it can overcome some of the technical limitations of RFLP and RAPD (Nagaoka and Ogihara 1997; Ratnaparkhe et al. 1998).
ISSR has been used to study the genetic diversity in rice (Oryza sativa) (Blair et al. 1999; Virk et al. 2000), trifoliate orange (Poncirus trifoliata) germplasm (Fang et al. 1997), common bean (Phaseolus vulgaris) (Galvan et al. 2003), lupin (Lupinus albus) (Gilbert et al. 1999), grain sorghum (Sorghum bicolor) and diploid (AA) banana (Musa acuminata) (Godwin et al. 1997), wheat (Triticum spp.) (Nagaoka and Ogihara 1997), hop (Humulus lupulus) (Patzak 2001), potato (Solanum tuberosum) (Prevost and Wilkinson 1999), and wild barley (Hordeum vulgarae subsp. spontaneum) (Tanyolac 2003). Nevertheless, the ISSR technique has not so far been employed in tef genetic diversity studies. The objectives of the present study were, therefore, to examine the possibility of using ISSR analysis for discerning genetic diversity in tef, and to assess the extent and pattern of genetic diversity in tef germplasm with respect to origin groups.
MATERIAL AND METHODS
Plant material
Ninety-two tef germplasm lines were used in this study. The lines were grouped into eight classes of origin as improved varieties and seven regions of origin from which the germplasm materials were initially collected (Table 1). The class of improved varieties included the ten improved tef varieties released from Debre Zeit Agricultural Research Center until the year 1995 (Tefera et al. 1995). The localization of the other seven regions of origin of the germplasm lines can be seen from the map of Ethiopia provided by Assefa et al. (2001a, 2002a). The test genotypes also included the 35 tef cultivars established and described by Ebba (1975), and designated as “Cv.” preceding the names in Table 1.
| Origin group | Total No. | List of genotypes (DZ-01-numbers or variety cross numbers or cultivar names) |
|---|---|---|
| Varieties (Improved) | 10 | DZ-01-99, DZ-01-196, DZ-01-354, DZ-01-787, DZ-01-974, DZ-Cr-37, DZ-Cr-44, DZ-Cr-82, DZ-Cr-255 and DZ-Cr-358 |
| Shewa | 36 | cv. Ada, cv. Addissie, cv. Balami, cv. Beten, cv. Enatite, cv. Fesho, cv. Kaye Agachem, cv. Manya, cv. Purpurea, cv. Rubicunda, cv. Rosea, cv. Viridis, DZ-01-30, DZ-01-237, DZ-01-247, DZ-01-291, DZ-01-305, DZ-01-306, DZ-01-647, DZ-01-663, DZ-01-667, DZ-01-691, DZ-01-695, DZ-01-725, DZ-01-751, DZ-01-801, DZ-01-855, DZ-01-886, DZ-01-1015, DZ-01-1056, DZ-01-1268, DZ-01-1276, DZ-01-1277, DZ-01-1278, DZ-01-1281 and DZ-01-1288 |
| Wellega | 14 | cv. Adoensis, cv. Alba, cv. Bunniye, cv. Dschanger, cv. Gea Lammie, cv. Karadebi, cv. Kaye Murri, cv. Tullu Nasy, cv. Variegata, DZ-01-553, DZ-01-558, DZ-01- 569, DZ-01-581 and DZ-01-587 |
| Harergie | 7 | cv. Burssa, cv. Denkeye, cv. Gorradie, cv. Hamrawe Murri, cv. Hatalla, DZ-01-9 and DZ-01-10 |
| Gojam | 6 | cv. Curati, cv. Dabbi, cv. Janno, cv. Murri, cv. Trotteriana and cv. Zuccagniana |
| Keffa | 3 | cv. Gofarie, cv. Gommadie and cv. Shawa Gemerra |
| Wello | 13 | DZ-01-478, DZ-01-481, DZ-01-483, DZ-01-504, DZ-01-512, DZ-01-513, DZ-01-517, DZ-01-518, DZ-01-527, DZ-01-530, DZ-01-542, DZ-01-544 and DZ-01-1243 |
| Tigray | 3 | DZ-01-1218, DZ-01-1225 and DZ-01-1234 |
DNA isolation
Genomic DNA was isolated from leaves of three- to four-week old young seedlings grown in the greenhouse. Leaf samples were harvested and homogenized to fine powder in liquid nitrogen (−80°C) using mortar and pestle. The DNA extraction was made using a modification of the CTAB method (Wang et al. 1996). To approximately 300 mg of homogenized leaf tissue, 750 μl of extraction buffer (0.1 M Tris (pH 7.5), 50 mM EDTA and 500 mM NaCl) and 100 μl of 10 % (w/v) sodium dodecyl sulfate (SDS) were added, and incubated at 65°C for 20 min. After adding 250 μl of 5 M potassium acetate, the mix was incubated on ice for at least 30 min, and centrifuged at 14 000 rpm for 15 min to collect the supernatant. This was followed by precipitation with an equal volume of cold (−20°C) iso-propanol and centrifuging at 14 000 rpm for 10 min. The resulting pellet was dissolved in 250 μl of 1×TE (10 mM Tris-HCl pH 7.6 and 1 mM EDTA), and then, 250 μl of buffer (pH 7.5) composed of 0.2 M Tris-HCl, 50 mM EDTA, 2 M NaCl and 2 % hexadecyltrimethylammonim bromide (CTAB) was added followed by incubation in water bath at 65°C for 15 min. After addition of an equal volume of phenol:chloroform (1:1) mixture, the supernatant was collected following centrifugation at 14 000 rpm for 5 min. This procedure was repeated using chloroform, and the supernatant collected was precipitated by the addition of an equal volume of cold (−20°C) iso-propanol followed by centrifugation at 14 000 rpm for 10 min. Finally, the pellet was air-dried and then dissolved in 200 μl of 1×TE overnight. From this, the RNA was removed through digestion with 2 μl of 1 mg ml−1 ribonuclease A for 200 μl DNA-TE mix and incubation at 37°C in water bath for 30 min. The resulting DNA solution was stored in freezer (−20°C) for later use.
The quality of the DNA samples was assessed using 1 % agarose gel electrophoresis and ethidium bromide visualization. The DNA concentration was determined by fluorochrome spectrophotometery using Hoechst 33258 dye with calf thymus DNA as standard (Brunk et al. 1979).
PCR amplification and electrophoresis
Nine primers (six from the Biotechnology Laboratory, University of British Columbia (UBC), USA, and three from DNA Technology A/S, Denmark) were tested on selected phenotypically contrasting tef genotypes, and eight primers that showed polymorphism and good resolution were selected for further analysis (Table 2).
| Primer | No. bands generated | No. lines separated | H′** (Mean±SE) | PIC** (Mean±SE) | ||
|---|---|---|---|---|---|---|
| Code | Sequence (5′ to 3′)* | Total | Polymorphic | |||
| 855 | ACA CAC ACA CAC ACA CYT | 40 | 15 | 73 | 0.64±0.03 d | 0.29±0.02 b |
| 888 | BDB CAC ACA CAC ACA CA | 52 | 15 | 75 | 0.70±0.02 bcd | 0.32±0.01 b |
| 889 | DBD ACA CAC ACA CAC AC | 42 | 14 | 49 | 0.66±0.04 cd | 0.31±0.02 b |
| 890 | VHV GTG TGT GTG TGT GT | 41 | 15 | 63 | 0.72±0.02 bcd | 0.33±0.01 b |
| 891 | HVH TGT GTG TGT GTG TG | 42 | 15 | 84 | 0.83±0.02 a | 0.48±0.01 a |
| 827 | ACA CAC ACA CAC ACA CG | 48 | 12 | 71 | 0.80±0.02 ab | 0.45±0.01 a |
| 811a | GAG AGA GAG AGA GAG AC | 46 | 12 | 79 | 0.76±0.02 abc | 0.44±0.01 a |
| 825a | ACA CAC ACA CAC ACA CT | 35 | 12 | 75 | 0.72±0.03 bcd | 0.42±0.01 a |
| Mean | 43.25 | 13.75 | 71.13 | 0.73±0.03 | 0.38±0.01 | |
- aProducts of DNA Technology A/S, while the others are that of GIBCOBRL®
- *Y=Pyrimidine (C or T)
- B=Non-A (i.e. C, G or T)
- D=Non-C (i.e. A, G or T)
- H=Non-G (i.e. A, C or T)
- V=Non-T (i.e. A, C or G)
- **Means within a column followed by different letters are significantly different at P≤0.05 (t-test)
The PCR mix (25 μl) for ISSR analysis included 10 ng template tef genomic DNA, 1× PCR buffer (75 mM Tris-HCl of pH 8.8, 20 mM (NH4)2SO4, 0.01 % (v/v) Tween 20), 2 mM MgCl2, 0.2 mM of each dNTPs, 2 % formamide, 0.198 μM primer of UBC or 0.396 μM primer of DNA Technology, and 0.04 U/μl or 0.06 U/μl of Taq polymerase (SIGMA) for UBC and DNA Technology primers, respectively. As a filler of the components in the reaction, 1 ml diethyl pyrocarbonate (DEPC) in 1 l of double distilled water was used. Amplification was performed in a GENE AMP PCR System thermocycler (HITACHI Ltd., Tokyo, Japan) programmed with the following temperature profiles: initial denaturation at 94°C for 1 min and final extension at 72°C for 5 min with the intervening 35 cycles of 94°C for 1 min, drop to 55°C at ramp rate of 0.5°C s−1, 55°C for 2 min, rise to 72°C at ramp rate of 1.3°C s−1, 72°C for 0.5 min and rise to 94°C at ramp rate of 1.3°C s−1.
Electrophoretic separation of the PCR products was done on a polyacrylamide gel (CleanGel 48S, Amersham Pharmacia Biotech AB) which was first rehydrated for at least one hour face down in a rehydrating buffer (0.112 Tris adjusted to pH 6.4 with acetic acid). The sample loading buffer consisted of 20 % (w/v) sucrose, 10 % (w/v) Ficoll, 0.005 % (w/v) bromophenol blue, 5 M urea and 1 mM EDTA. Loading buffer (5 μl) was added to each PCR product, and 6 μl of the mix was finally loaded in each slot of the gel. Electrode strips were soaked with 25 ml of electrode buffer (0.2 M Tris pH 8.0, 0.2 M Tricine and 0.55 % SDS) and placed in contact with the gel at the top and bottom or cathodal and anodal ends of the gel. The gel was run on a horizontal Multiphor II Electrophoresis Unit at 450 V and 20 mA current for 25 min and then at 50 mA current of similar voltage for 2 h. Electrophoresed PCR products were visualized by silver staining using the Hoefer Automated Gel Stainer (Pharmacia Biotech). A 100-base-pair ladder was loaded on either side of the gel for use as a standard for the estimation of the sizes of bands.
Data collection and analysis
Each ISSR band was considered as an independent character or locus, and polymorphic bands were scored visually as either absent (“0”) or present (“1”) for each of the 92 genotypes. Qualitative differences in band intensity were not considered. Three independent scorings were made on each gel, and only those bands consistently scored were considered for analysis.
Pair-wise genetic similarity matrix was generated among the 92 tef genotypes using Jaccard similarity coefficient (Jaccard 1908). For comparison, the Nei (1972) genetic distance matrix was also generated for the 92 test genotypes.
Using the Jaccard similarity and Nei genetic distance (dissimilarity) matrices cluster analyses were performed and corresponding phenograms generated for the 92 genotypes using the unweighted pair-group method with arithmetic averages (UPGMA) (Sneath and Sokal 1973). A cophenetic matrix was computed from the clustering matrix in order to assess the goodness of fit of the phenograms obtained by comparing the cophenetic value matrix with the initial similarity matrix using the Mantel's (1967) statistic Z. Principal coordinate analysis (PCA) was done based on Nei (1972) genetic distance. All these analyses were done using the NTSYS-pc package (Rohlf 2000).
The analysis of genetic diversity pattern among the eight groups of origin was done using the Genetic Distance and Phylogenetic Analysis (Dispan 1993) pc package using the standard genetic distances (Nei 1972) to construct UPGMA (Sneath and Sokal 1973) dendrogram with bootstrap tests (Felsenstein 1985) for the clustering tree generated.
The standardized (relative) Shannon Weaver diversity indices (H′) were computed for each primer following the procedure used by Kefyalew et al. (2000) and Assefa et al. (2002a). Analysis of variance of Shannon Weaver diversity indices was performed to examine the pattern of diversity among the origin groups, and pair-wise t-test was used to compare the mean Shannon Weaver diversity levels of the origin groups.
RESULTS AND DISCUSSION
The eight ISSR primers generated a total of 346 scorable bands (average ca 43/primer and range 35–52) of which 110 (32 %) were polymorphic in the 92 tef genotypes examined. The number of polymorphic bands generated by a primer varied between 12 and 15. The size of the bands ranged from less than 100 to 3 500 bp. In previous tef DNA polymorphism studies, the number of scorable bands generated varied from 15 to 46 (Bai et al. 1999a) or 38 to 110 (Ayele and Nguyen 2000) for each AFLP primer pair, and from 2 to 13 per primer in RAPD analysis (Bai et al. 2000).
The Jaccard similarity coefficient ranged from 0.26 between the most distant (dissimilar) lines cv. Enatite and DZ-01-1056 both from Shewa to 0.86 between the closest (most similar) lines DZ-01-751 and DZ-01-801 both of which are again from Shewa. The eight ISSR primers enabled the separation of all the 92 test lines. Examination of the band differences between even the most similar (Jaccard similarity coefficient=0.86) lines DZ-01-751 and DZ-01-801 (Fig. 1) indicated that they differ in seven (=6 %) of the 110 fragments or bands. Across all genotypes, the Nei genetic distance ranged from 0.07 between DZ-01-751 and DZ-01-801 to 0.89 between cv. Enatite and DZ-01-1056. In comparison, in the assessment of RAPD polymorphism in some tef germplasm lines the genetic distance for all the test accessions fell in the range of 0.84–0.96, indicating low polymorphism (Bai et al. 2000). Although 42 of the 47 lines used in the latter study were also included in the present study, our results, however, showed a wider range of polymorphism which could be attributed to the greater resolution of diversity with ISSR than with RAPD marker systems and also to the differences in the genotypes included.
UPGMA phenogram of 92 tef lines using Jaccard similarity of ISSR band profiles. Please supply better quality artwork for figure 1 and 2.
At about 60 % similarity level, the phenogram generated using UPGMA based on the Jaccard similarity coefficient resulted in the formation of six major clusters of 2 to 37 lines, with eight lines remaining distinct and ungrouped (Fig. 1). Clustering of the 92 tef genotypes was also done using the Nei genetic distance or dissimilarity matrix and the UPGMA dendrogram (not shown) generated using this method showed an identical pattern of clustering to that obtained using the Jaccard similarity matrix.
Cophenetic value matrix was computed from the clustering tree matrix and comparison of the resulting matrix with the initial similarity matrix revealed normalized Mantel's (1967) Z statistic cophenetic correlation coefficient (r)=0.74, indicating relatively good fit of the cluster analysis performed with the initial similarity matrix. Similar comparison of the genetic distance matrix with the corresponding cophenetic value matrix generated from the dissimilarity based clustering matrix showed a relatively low value of r=0.66. This indicates better fitness to each other of the former two matrices than the latter two genetic dissimilarity based matrices. However, comparison of the Jaccard similarity and Nei genetic distance matrices revealed an almost perfect fit (r=−0.98). The negative value of r in the latter comparison is because one of the pair of matrices compared is based on similarity while the other one in contrast is based on dissimilarity.
Compared to previous phenotypic evaluation studies with various tef germplasm materials, the formation of six major complexes of the genotypes in the present study is more in agreement with the grouping of the 35 tef cultivars established by Ebba (1975) into six major groups than with the clustering of 36 tef and two other related Eragrostis species accessions into three groups (Costanza et al. 1979). Our results are also in line with the identification of a number of clusters of tef germplasm materials evaluated for heterogeneity of seed protein prolamin fraction (Bekele et al. 1995), and various pheno-morphic and agronomic traits (Assefa et al. 1999, 2000, 2001a,b). In contrast, UPGMA clustering of different tef germplasm materials using Jaccard similarity matrix showed only a single group for RAPD (Bai et al. 2000), and two groups for AFLP (Ayele and Nguyen 2000; Bai et al. 1999a) marker band profiles. The differences can be attributed to the variation in the type and number of genotypes, and the techniques employed.
All of the ten improved tef varieties released until 1995 (Tefera et al. 1995) aggregated in cluster I (Fig. 1 and Table 3). The Jaccard similarity coefficient among the ten varieties ranged from 0.58 between DZ-01-787 and DZ-Cr-82 to 0.80 between DZ-01-196 and DZ-01-354. This generally indicates that the ten improved tef varieties included in this study are relatively similar, although it was possible to differentiate all of them by the use of the eight ISSR primers. Our findings, however, illustrate that ISSR can be used for DNA fingerprinting for the identification of tef cultivars or varieties.
| Origin group | Cluster | Total | ||||||
|---|---|---|---|---|---|---|---|---|
| I | II | III | IV | V | VI | Solitary | ||
| Varieties | 10 | 0 | 0 | 0 | 0 | 0 | 0 | 10 |
| Shewa | 12 | 12 | 1 | 1 | 5 | 0 | 5 | 36 |
| Wellega | 5 | 8 | – | – | – | 1 | – | 14 |
| Harergie | 4 | 1 | 1 | – | – | – | 1 | 7 |
| Gojam | 3 | 2 | – | – | – | 1 | – | 6 |
| Keffa | 2 | – | – | – | – | – | 1 | 3 |
| Wello | 1 | 10 | – | 1 | – | – | 1 | 13 |
| Tigray | – | 3 | – | – | – | – | – | 3 |
| Total | 37 | 36 | 2 | 2 | 5 | 2 | 8 | 92 |
Of the 35 cultivars established by Ebba (1975), 19 grouped in cluster I, 10 in cluster II and two in cluster VI, while four of them remained solitary without grouping. The Jaccard similarity coefficient among these 35 tef cultivars varied from 0.33 between cv. Beten and cv. Enatite to 0.78 between cv. Adoensis and cv. Purpurea. Three of the clusters (i.e. III, V and VI) did not contain any of the 35 cultivars, and apart from the ungrouped four cultivars (i.e. Hatalla, Manya, Gommadie and Enatite) there were four solitary germplasm lines (i.e. DZ-01-30, DZ-01-504, DZ-01-663 and DZ-01-1015). This indicates the presence of tef genotypes that are genetically different from the 35 cultivars earlier established and described by Ebba (1975) and confirmed the results of previous pheno-morphic and agronomic trait evaluation studies which also suggested the existence of tef types distinct from the established cultivars (Assefa et al. 1999, 2000, 2001a). The variety DZ-01-196 and cv. Manya are often taken as synonymous, and the same is true for variety DZ-01-354 and cv. Enatite. But, in the present study, the Jaccard similarity coefficients were 0.52 between DZ-01-196 and Manya, and 0.37 between variety DZ-01-354 and cv. Enatite. The Nei genetic distances for these two pairs of genotypes were 0.34 and 0.55, respectively.
Recombinant inbred lines of the intraspecific cross between two phenotypically contrasting tef cultivars Kaye Murri×Fesho were used in molecular AFLP marker based mapping, which generated 25 linkage groups (Bai et al. 1999b). In the present study, cv. Kaye Murri and cv. Fesho clustered in two different groups, namely cluster VI and II, respectively (Fig. 1). The Jaccard similarity coefficient between them was about 0.57, while the genetic distance between them was 0.32. This indicates that, especially considering the absence of any DNA marker information by that time, the choice of the two cultivars as divergent parents for the generation of mapping population was generally fair and optimal. With the accumulation of DNA polymorphism information, more appropriate and contrasting parents can be selected. On the other hand, our results indicate that the genetic map generated using recombinant inbred lines of the above two cultivars can be enriched by using ISSR markers.
Examination of the distribution of the lines among the clusters (Table 3) did not result in comprehensively distinct clustering patterns based on group of origin. Similar results have previously been reported with studies of phenotypic traits of other tef germplasm materials (Assefa et al. 2001b, 2003), but the absence of clustering patterns showing trends of grouping based on origin is not in agreement with the findings of Ayana and Bekele (1998) with Ethiopian sorghum germplasm.
Nine principal components (PCs) with eigenvalues greater than unity extracted a cumulative of 73 % of the gross variance of the test tef genotypes (data not shown). Of these, the first three PCs having eigenvalues ranging from 2.63 to 51.97 accounted for about 64 % of the total variance, and each one of them from the first to the third PC explained about 57 %, 5 % and 3 % of the overall variation among the test genotypes. By comparison, previous studies involving evaluation of various tef germplasm materials for different pheno-morphic traits depicted the involvement of four to five important PCs in explaining much of the gross phenotypic variance (Assefa et al. 1999, 2000, 2001a,b, 2003), thus, indicating the complexity of the species.
Standardized genetic distances among the eight groups of origin of the tef test lines ranged from 0.03 between Shewa and Wellega to 0.32 between Keffa and Tigray (Table 4).
| Origin group | Varieties | Shewa | Wellega | Harergie | Gojam | Keffa | Wello | Tigray |
|---|---|---|---|---|---|---|---|---|
| Varieties | 0 | |||||||
| Shewa | 0.1285 | 0 | ||||||
| Wellega | 0.1128 | 0.0333 | 0 | |||||
| Harergie | 0.1025 | 0.0690 | 0.0521 | 0 | ||||
| Gojam | 0.1109 | 0.0721 | 0.0493 | 0.0488 | 0 | |||
| Keffa | 0.1242 | 0.1545 | 0.1248 | 0.1229 | 0.1415 | 0 | ||
| Wello | 0.2508 | 0.0638 | 0.0998 | 0.1826 | 0.1827 | 0.3034 | 0 | |
| Tigray | 0.2608 | 0.0949 | 0.1393 | 0.2058 | 0.1910 | 0.3205 | 0.1192 | 0 |
The UPGMA clustering using the standardized genetic distances resulted in the identification of three groups of origin with bootstrap values ranging from 56 to 97 (Fig. 2). The first group consisted of Shewa, Wellega, Harergie, Gojam and improved varieties. The second cluster contained Wello and Tigray, while Keffa stood solitary without grouping with the others. The pattern of clustering and the variation in genetic distance appeared to follow proximity based trend thus implying material exchange more among neighbouring regions than among those farther apart. This holds for the clustering of Tigray and Wello together in one class and that of Shewa, Gojam and Wellega together. But the grouping of Harergie together with Gojam and Wellega can not be explained by proximity-based material exchange. It presumably seems that since Harergie is not a major tef growing area, the introduction of tef to this region could have been made by the people who have migrated from the northern and central parts of Ethiopia and brought with them tef seeds of their own.
UPGMA dendrogram of eight groups of origin using standard genetic distances for ISSR band profiles of 92 tef germplasm lines (numbers near branches are bootstrap values).
Across all the eight primers, the mean Shannon Weaver diversity indices (H′) of the lines ranged from 0.47 for DZ-01-99 to 0.96 for DZ-01-291. The overall mean H′ was 0.73 (Table 5). This indicates that as compared to the intermediate (0.52 or 0.54) H′ estimates obtained in evaluation of qualitative and phenologic characters in other tef germplasm (Kefyalew et al. 2000; Assefa et al. 2002a), the ISSR technique was effective in resolving about 20 % more genetic diversity in the germplasm. In spite of the differences in the absolute values, the relative Nei variation index computed for each primer using the formula given by Hennink and Zeven (1991) showed similar patterns to that of the H′ values with an almost perfect correlation of r=0.99. The overall mean Nei variation index was 0.67.
| Origin group | Shannon Weaver diversity index | |
|---|---|---|
| Mean* | SE (±) | |
| Varieties (Improved) | 0.6377b | 0.0587 |
| Shewa | 0.7691a | 0.0516 |
| Wellega | 0.6799b | 0.0783 |
| Harergie | 0.7163ab | 0.0993 |
| Gojam | 0.6497b | 0.1190 |
| Keffa | 0.6917ab | 0.1320 |
| Wello | 0.7630a | 0.0715 |
| Tigray | 0.8141a | 0.2670 |
| Overall mean | 0.7275 | 0.0108 |
- *Means followed by the same letter are not significantly different as judged by t-test at P≤0.05.
Analysis of variance of the mean H′ values and the relative Nei variation indices (data not shown) showed significant (P≤0.01) variation among the eight origin groups. Similarly, significant regional variance was noted in seed color and days to maturity in tef germplasm populations from central and northern parts of Ethiopia (Kefyalew et al. 2000), and in lemma color, number of culm internodes, and counts of basal and middle spikelet florets in accessions from western and southern parts of Ethiopia (Assefa et al. 2002a).
Pair-wise t-test comparison of the mean H′ values depicted three classes of the eight origin groups (Table 5). Mean Shannon Weaver diversity indices of improved varieties, Wellega and Gojam as a group ranged from 0.63 to 0.68 and were significantly (P≤0.05) lower than that of Shewa, Wello and Tigray which in contrast as a group exhibited high values between 0.76 and 0.81. As the third class, Harergie (0.72) and Keffa (0.69) exhibited intermediate mean H′ values that were not significantly different than that of all the remaining origin groups.
In an attempt to compare the effectiveness of the different primers in studying genetic diversity in tef, marker index and resolving power were computed following the method of Prevost and Wilkinson (1999), and polymorphism information content (PIC) was computed using the formula provided by Smith et al. (1997). However, none of these measures were found to be significantly correlated with each other, and with number of genotypes separated by each primer (Table 2), or the mean Shannon Weaver and Nei variation indices of the primers. In contrast, Prevost and Wilkinson (1999) found a strong and seemingly linear relationship between resolving power and ability to distinguish genotypes (r2=0.98). But, in line with our findings, they reported failure of marker index to correlate with genotype diagnosis.
In the present study, the number of genotypes separated by the eight ISSR primers varied from 49 to 84 with an average of 71 per primer (Table 2). As shown in the same table, the analyses of variance showed significant (P≤0.05) differences among the eight primers in Shannon Weaver diversity index and PIC. The former ranged from 0.64 for primer 855 to 0.83 for primer 891. The PIC values varied from 0.29 for primer 855 to 0.48 for primer 891. Mean PIC values (0.42–0.48) of primers 891, 827, 811 and 825 were substantially (P≤0.05) higher than those of the other four primers which exhibited comparable PIC values of 0.29–0.33.
Overall, our findings demonstrated that ISSR markers would be promising for the assessment of genetic diversity and phylogenetic relationships, DNA fingerprinting for the identification of cultivars or varieties and for genome mapping in tef. The use of different ISSR primers for PCR amplification and various tef germplasm materials would enable the realization of the potential of the technique in discerning variation in the species. In addition, the molecular genetic maps so far produced using AFLP (Bai et al. 1999b) and RFLP (Zhang et al. 2001) can be enriched to produce denser genetic map by the complementary use of ISSR marker analysis on the mapping populations.
Acknowledgements
The study was financed by the Swedish International Development Agency (Sida/SAREC) for which we are very grateful. The senior author would thank Mrs. Britt Green and Mrs. Ann-Sofie Fält for their assistance in the laboratory analysis, and Mr. Linus Masumbuko for his technical assistance in the laboratory work and data analysis. I sincerely appreciate Professor Tomas Bryngelsson for reviewing the original manuscript and forwarding valuable comments and suggestions.
