Field Crops Research 127 (2012) 64–70
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Field Crops Research
journal homepage: www.elsevier.com/locate/fcr
Genetic diversity in tef (Eragrostis tef) germplasm using SSR markers
M. Zeid a,1 , K. Assefa b , A. Haddis c , S. Chanyalew b , M.E. Sorrells a,∗
a
b
c
Department of Plant Breeding and Genetics, 240 Emerson Hall, Cornell University, Ithaca, NY 14853, USA
Ethiopian Institute of Agricultural Research, Debre Zeit Agricultural Research Center, P.O. Box 32, Debre Zeit, Ethiopia
CSIRO Plant Industry, Bld 1-Dough Lab, Clunies Ross St., Acton, ACT 2601, Australia
a r t i c l e
i n f o
Article history:
Received 11 June 2011
Received in revised form 27 October 2011
Accepted 27 October 2011
Keywords:
Chloridoideae
Allelic diversity
Seed admixture
Lodging
a b s t r a c t
The Institute of Biodiversity Conservation (IBC) in Ethiopia curates a large number of tef accessions,
but there is little information about the genetic variation among the accessions. This study assessed
the genetic diversity and relationships among 326 cultivated tef accessions, 13 wild relatives, and four
commercial tef varieties from the U.S. using 39 SSR markers, 26 of which were flanking QTL intervals
for yield, lodging index and stem strength related traits. We estimated the allelic diversity and identified
markers associated with agronomic traits in this tef germplasm collection. Forty-seven loci were sufficient
to differentiate 80.8% of the tef accessions. In contrast to earlier studies, genetic similarity estimates
ranged from 0.21 to 0.99, indicating a high level of genetic diversity. In the course of this investigation, it
was discovered that seed admixture is a serious problem affecting the integrity of almost all released tef
varieties. Association was observed between the marker CNLTs540 and seed weight/plant. The majority
of the alleles detected were present in tef breeding lines and varieties suggesting that tef plant breeders
have been using a broad range of germplasm in their programs. The markers documented in this study
will be useful to identify and verify hybrids from crosses between promising lines that lack morphological
differences, an approach that was never attempted before in the tef breeding programs.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Tef, Eragrostis tef (Zucc.) Trotter, is an annual self-pollinated
grass species of the family Poaceae, subfamily Chloridoideae.
Among cultivated cereals, only finger millet belongs to the same
subfamily, thus the crop is only distantly related to the well-studied
cereal crops. Within the genus Eragrostis, tef is the only species
that is grown to produce grain (100 kernel weight = 0.18–0.38 mg)
for human consumption, while the straw is fed to livestock. The
crop is grown on a large scale in Ethiopia where it occupies 2.5
million hectares thereby ranking first among all cereals cultivated
in the country. The grains are ground into flour, fermented and
baked to produce flat pancake-like bread known as “injera”. Its
flour lacks gluten prompting its use in food products for individuals diagnosed with celiac disease (Spaenij-Dekking et al., 2005;
Hopman et al., 2006). Despite the high demand for tef in Ethiopia,
as observed from the doubling of the production area from 1.2 million ha in 1990 to 2.5 million ha in 2009, the yield of tef per unit area
has not changed for decades from an average of 1 ton/ha. Current
tef varieties can yield 3–4 times more than the country’s average
∗ Corresponding author. Tel.: +1 607 255 2180; fax: +1 607 255 6683.
E-mail address: mes12@cornell.edu (M.E. Sorrells).
1
Present address: Crop Science Department, Faculty of Agriculture, Alexandria
University, Alexandria, Egypt.
0378-4290/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.fcr.2011.10.013
under experimental conditions without lodging (Teklu and Tefera,
2005). However, both farmers’ production practices and lodging
are the two major limitations for yield improvement in tef nationwide. The current farmers’ production practices have not changed
much from those traditionally used generations ago as described
by Ketema (1997). In addition, lodging continues to be a serious
problem in tef production causing an estimated average yield loss
of 17% (Ketema, 1993). So far, it has not been possible to develop
lodging resistant varieties mainly because of the lack of variation
for lodging resistance within the available germplasm (Assefa et al.,
2010).
The fact that Ethiopia is considered to be the center of origin
and diversity of tef (Vavilov, 1951) implies that the indigenous
germplasm represents a rich resource base for plant breeders
to improve this crop. Collection and conservation of the available tef material and its wild relatives is an ongoing process in
Ethiopia because some of the regions have not yet been adequately represented in the germplasm collection (Demissie, 2001).
Improved tef varieties are not replacing local landraces as fast
as in the case of wheat, however, this might change soon, since
tef production is becoming a more market-driven process where
white-seeded varieties are favored over darker-seeded types (Belay
et al., 2008). The fear for genetic erosion of tef diversity as observed
in tetraploid wheat landraces (Tsegaye and Berg, 2007) due to the
expansion of improved varieties is the main reason for collection
and conservation of tef germplasm. The Institute of Biodiversity
M. Zeid et al. / Field Crops Research 127 (2012) 64–70
Conservation (IBC) in Ethiopia has 4395 tef accessions (Demissie,
2001) that are shared with research institutes and tef breeders in
Ethiopia, but most of the tef collection is lacking adequate passport
data necessary for proper germplasm utilization. Several studies
have examined the diversity available in some of the available
tef germplasm using morphological and agronomic traits (Tefera
et al., 1990; Assefa et al., 1999, 2000, 2001a,b,c, 2002a,b, 2003b;
Ayele et al., 1999; Adnew et al., 2005). The results of all these
studies generally agreed on the existence of high levels of variation for many of the phenotypic traits investigated. The most
extensive study, however, was conducted by Ebba (1975), where
more than 1000 field and market collections from tef were morphologically characterized for inter and intra-sample variation.
Based on results of that study, 35 distinct landrace collections of
tef were selected to represent the entire variation in the indigenous germplasm. Similar attempts were also undertaken at the
DNA level using random amplified polymorphic DNA (RAPD) (Bai
et al., 2000), amplified fragment length polymorphism (AFLP) (Bai
et al., 1999b; Ayele and Nguyen, 2000). Those studies focused
mainly on the genetic diversity among the 35 landrace collections
of Ebba (1975), in addition to a few pure line accessions from
the IBC, and accessions of Eragrostis pilosa and Eragrostis curvula
were included for comparison. In those studies, it was possible to
differentiate cultivated tef from its wild relatives and accessions
within tef, however, the genetic similarity estimates among the
tef accessions were generally high (0.73–0.98). It was concluded
that the diversity in cultivated tef was very low and the authors
stressed the need for inter-specific crosses with E. pilosa to overcome this problem. Since the low level of variation at the DNA
level could not explain the large morphological variation observed
among tef accessions, it was assumed that only a few genes controlled this broad morphological variation (Bai et al., 2000). The
utilization of inter-simple sequence repeats (ISSR) markers (Assefa
et al., 2003a) provided, for the first time, much lower estimates
for genetic similarity (0.26–0.86) among 92 tef accessions including most of the accessions screened in the previous studies. These
results suggested that the low level of diversity observed earlier
was marker related and that microsatellites would provide better
markers for future diversity studies in tef. This assumption was
validated by Zeid et al. (2010), where 53% of the tef-specific SSR
markers developed proved to be polymorphic between the parental
lines of an inter-specific cross between E. tef (Kaye Murri) and E.
pilosa (30–5). In addition, a number of SSR markers were screened
on a limited number of tef accessions and a promising level of
polymorphism among those accessions was observed. These markers have been used successfully for genetic map development
and QTL analysis in tef for yield, lodging and related traits (Zeid
et al., 2010) and were proposed as a useful tool for tef germplasm
organization and fingerprinting. SSR markers have been very useful for studying the genetic diversity of self-pollinated cereals
including wheat (Khlestkina et al., 2007; Mohammadi et al., 2009)
and rice (Garris et al., 2005; Chao et al., 2007; de Oliveira Borba
et al., 2009) and for studying the genetic integrity of collections
in gene banks (Hirano et al., 2009). Furthermore, they were also
employed for verifying pure line and hybrid seed identity in hybrid
rice programs (Nandakumar et al., 2004; Sundaram et al., 2008).
The objectives of this study were to (i) estimate the genetic
diversity and the phylogenetic relationship among tef breeding
lines in the core germplasm used in the tef breeding programs
in Ethiopia and their wild relatives and some commercial tef
varieties from the U.S.; (ii) fingerprint all released Ethiopian tef
varieties; (iii) study the effect of seed admixture on the integrity
of the released varieties; and (iv) to examine the allelic diversity
in a set of QTL-flanking SSR markers in the tef germplasm and
their potential association with stem lodging and related traits
in tef.
65
2. Materials and methods
2.1. Plant material
In this study, a total of 343 Eragrostis germplasm accessions were
assayed. The Debre Zeit Agricultural Research Center (DZARC) in
Ethiopia provided all of the accessions except for four that were provided by The Teff Company, Caldwell, Idaho, U.S. (Supplementary
Table 1). The germplasm accessions provided by DZARC included
13 wild Eragrostis species and 271 tef breeding accessions that are
actively being evaluated and used in the tef breeding program.
Twenty-one of those breeding accessions had the same selection
number with different suffix (Supplementary Table 1). In addition,
33 landraces that had been described earlier by Ebba (1975) to represent the entire tef variation grown in Ethiopia and 22 improved
varieties that were developed between 1995 and 2007 were also
included.
2.2. DNA extraction and genotyping
DNA was extracted from bulked leaves of five seedlings from
each accession based on the method of Tai and Tanksley (1990).
Because of the tiny seed size of tef, seed admixture is a common
problem. To study the effect of seed admixture on the identity of the most recent varieties, 12 lines were genotyped from
seed lots provided by DZARC in 2003 and were compared to
those of seed lots from 2009. Similarly, fingerprints of seedlings
from the cultivar Kaye Murri and the wild accession E. pilosa
(30–5) from three different seed lots were compared. Internal
checks consisting of aliquots of DNA extracted from six accessions assigned to two different wells across the four 96-well plates
assayed were employed as controls for any errors during sample
handling.
Sixty eight of the tef SSR primer-pairs developed by Zeid et al.
(2010) were screened on a subset of 37 tef genotypes (19 of
the most recent improved varieties and 18 landraces) in addition to E. pilosa (30–5) using the same protocol described by the
authors (Supplementary Table 2). The PCR amplification products were separated on 4% polyacrylamide gels and visualized
after staining with silver nitrate. Based on results of this preliminary screening, 13 primer-pairs of high polymorphic information
content values (Botstein et al., 1980) that were also capable of
differentiating the screened genotypes were selected for assaying the germplasm studied here (Supplementary Table 2). This
set of 13 selected primer-pairs in addition to 26 other primers
that were flanking QTL for various traits in tef including 100 seed
weight, plant height, and lodging index in the study by Zeid et al.
(2010) were labeled using fluorescent dye and used to fingerprint the entire germplasm collection under study (Table 1). SSR
fragments were analyzed on the ABI PRISM 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and visualized and
scored using Genographer 2.1.4 software (Benham et al., 1999).
Scoring of products either from polyacrylamide gels or from the
digital visualization from Genographer was performed by numbering the products of each primer-pair based on the number
of alleles detected for that locus. The software NTSYSpc v2.21h
(Rohlf, 2009) was used to calculate the shared band similarity coefficient (Lynch, 1990) and the UPGMA clustering method
(Sneath and Sokal, 1973) was applied to the calculated similarity matrix. Relationships among accessions were analyzed using
the principal coordinate analysis (PCoA) method (Gower, 1966)
in NTSYSpc. Power Marker v3.25 (Liu and Muse, 2005) was used
to calculate the number of alleles per locus, Wright’s fixation
index (Fst) and polymorphism information content (PIC) for each
locus.
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M. Zeid et al. / Field Crops Research 127 (2012) 64–70
Table 1
SSR primer pairs used for genetic diversity and association analysis.
Primer
Linkage group
Gene diversity
PIC
CNL 53
CNLT 12
CNLTs11
CNLTs17a
CNLTs17b
CNLTs85
CNLTs95
CNLTs96
CNLTs102a
CNLTs102b
CNLTs117
CNLTs136a
CNLTs136b
CNLTs205a
CNLTs205b
CNLTs220
CNLTs244
CNLTs255
CNLTs275a
CNLTs295
CNLTs334
CNLTs338
CNLTs380
CNLTs392
CNLTs416a
CNLTs416b
CNLTs438
CNLTs455a
CNLTs455b
CNLTs485
CNLTs498
CNLTs540
CNLTs33
CNLTs42
CNLTs60
CNLTs91
CNLTs116
CNLTs133
CNLTs150a
CNLTs150b
CNLTs157
CNLTs216
CNLTs315
CNLTs328
CNLTs538
CNLTs484a
CNLTs484b
13
9
6
–
13
1
9
24
6
–
6
3
–
3
–
5
3
3
15
19
2
1
23
9
1
–
2
15
–
7
9
9
–
–
0.07
0.58
0.89
0.03
0.12
0.61
0.05
0.48
0.22
0.13
0.52
0.81
0.89
0.61
0.06
0.64
0.52
0.87
0.12
0.81
0.54
0.17
0.84
0.40
0.78
0.84
0.81
0.84
0.84
0.04
0.24
0.50
0.75
0.91
0.87
0.64
0.75
0.89
0.78
0.64
0.79
0.80
0.72
0.58
0.88
0.73
0.88
0.07
0.49
0.88
0.03
0.12
0.53
0.05
0.36
0.19
0.13
0.40
0.79
0.88
0.53
0.06
0.59
0.41
0.86
0.12
0.79
0.44
0.17
0.82
0.32
0.75
0.82
0.78
0.82
0.82
0.04
0.23
0.40
0.73
0.90
0.86
0.58
0.71
0.88
0.76
0.58
0.77
0.78
0.69
0.53
0.87
0.68
0.87
–
–
–
–
–
–
–
–
–
2.3. Phenotyping and association analysis
Phenotypic data on traits mainly related to stem characteristics and lodging were measured on 271 accessions (Table 2). The
experiment was conducted at DZARC in the central highlands of
Ethiopia during the 2001 main season. Seeds (0.75 g plot−1 ) were
sown in single 1 m long rows with 20 cm spacing between rows in
a randomized complete block design with three replicates. Traits
measured prior to harvesting on plot basis included days to heading, days to maturity, and lodging (scored on a 0–5 scale, where
“0” represented upright standing plants and “5” for plants lying
flat on the ground). Plant yield (seed weight/plant in g), and post
harvest measures for stem characteristics including; penetrometer
reading on the first, second and third internodes (measured by a
digital force gauge, model MG10- 0.002 kgf, as the force needed to
penetrate the stem at the internodes position), length (cm) and
diameter (mm) of the first and second internodes, length (cm)
and diameter (mm) of the peduncle and culm length (cm), were
all measured as the mean of five plants per plot. The association
between best linear unbiased predictors of the three replicates for
the studied traits and alleles from all 39 SSR markers employed
were analyzed using the mixed linear model (MLM) in TASSEL v2.1
(http://ww2.maizegenetics.net/) taking the average relationship
between accessions (kinship matrix) into account. A false discovery
rate cutoff was determined from the observed p-value distribution,
FDR = 0.05.
3. Results
All 68 SSR primer-pairs successfully amplified products for all
of the 37 tef accessions in addition to E. pilosa (30–5) and the markers differentiated all 37 tef lines. The PIC values for the markers
ranged from 0.05 to 0.86 (Supplementary Table 2). Eighteen markers amplified only two alleles, one specific to tef lines and the
second specific to E. pilosa including the two markers (CNLTs37
and 68) with the lowest PIC values, 0.05 and 0.09, respectively.
Thirteen primer-pairs out of the 69 screened were then selected
to study the genetic diversity among the entire germplasm collection. The selection was based on both their PIC values and ability to
differentiate the closely related lines as observed on silver nitrate
stained polyacrylamide gels.
The process of scoring alleles of a single marker on different gels
is very difficult and error prone (Zeid et al., 2003), consequently the
13 selected primer-pairs for their high PIC values and the 26 other
primer-pairs that were flanking QTL position in the study of Zeid
et al. (2010) were fluorescent labeled and scored using the software
Genographer v2.1.4. Fingerprints from each of the two aliquots of
the six internal checks employed as controls for error during sample handling were identical for all 47 loci that were amplified from
the 39 primer-pairs. Similarly, fingerprints from the three different
seed lots for the landrace Kaye Murri were identical. On the other
Table 2
Minimum, maximum, mean phenotypic values and standard deviation of 272 tef lines for the different traits.
a
b
Trait
Abbreviation
Minimum
Maximum
Mean (SD)
Lodginga (0–5 score)
Days to headinga (days)
Days to maturitya (days)
Seed weight/plantb (g)
Culm lengthb (cm)
Peduncle lengthb (cm)
Peduncle diameterb (cm)
Length of first internodeb (cm)
Length of second internodeb (cm)
Culm diameter at first internodeb (cm)
Culm diameter at second internodeb (cm)
Penetrometer reading at first internodeb (kgf)
Penetrometer reading at second internodeb (kgf)
Penetrometer reading at third internodeb (kgf)
Lodg
DH
DM
SW
CL
PdL
PdD
IL1
IL2
CD1
CD2
Pr1
Pr2
Pr3
1.94
27.06
107.07
1.31
23.93
12.17
1.08
4.95
6.84
1.20
1.11
0.942
0.69
0.47
2.07
44.31
113.18
2.79
47.2
20.20
1.85
5.98
8.31
2.35
2.21
2.42
1.82
1.09
2.02
34.83
110.05
1.73
36.26
16.00
1.45
5.34
7.50
1.73
1.70
1.61
1.20
0.76
Measurements were scored on a plot basis.
Measurements were based on the mean of 5 randomly selected plants/plot.
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.02
3.50
0.98
0.23
4.05
1.37
0.14
0.14
0.25
0.21
0.20
0.29
0.21
0.12
M. Zeid et al. / Field Crops Research 127 (2012) 64–70
hand, only one line (DZ-Cr-82), of the recently developed varieties,
had identical fingerprints for both the 2003 and 2009 seed lots,
while the other 11 lines showed variations in their fingerprints
and consequently their genetic similarity estimates (GS) ranged
between 0.45 and 0.98 with a mean GS of 0.79. Genetic similarity between seeds from the three seed lots of the wild accession E.
pilosa (30–5) varied between GS = 0.45 and GS = 0.67 with a mean
GS of 0.55.
The 47 loci scored were able to clearly differentiate the 277
accessions (80.8%) while 27 accessions showed similarity levels of
GS = 1 to at least one other accession and up to five out of the 343
accessions under study (Supplementary Table 3). After excluding
duplicate accessions, the genetic similarity estimates between tef
accessions ranged between 0.20 and 0.99. A dendrogram was generated to summarize the relationship between all 343 accessions
included in this study (Supplementary Fig. 1). All wild Eragrostis
species, except for the E. cilianensis accession, clustered together
separately from cultivated tef accessions. The dendrogram revealed
a small number of closely clustered accessions. Graphical representation of the relationships between accessions in the PCoA showed
a good differentiation of the tef breeding lines on the first coordinate that explained 7.5% of the variance. The third coordinate (4.3%
of the variance), on the other hand, clearly differentiated the tef
accessions from their wild relatives (Fig. 1). As shown in this figure,
all the 33 landraces and the four U.S. varieties were differentiated
from one another.
3.1. Allelic diversity and association analysis
The number of alleles amplified per locus across all 343 accessions ranged from 8 to 23 in the case of those markers selected for
their high PIC value and from 2 to 23 for the QTL-flanking markers chosen from previous studies (Fig. 2). Although the tef breeding
lines and varieties harbored the majority of alleles (85%) observed
in this study (Fig. 2), a number of unique alleles were amplified
from wild relatives of tef (9.9%), tef landraces (3.8%) and the U.S.
lines (1.3%). Those unique alleles could be valuable for tef breeders
in cases where the number of alleles available in the tef accessions and varieties is very low as observed for QTL-flanking loci
in the left part of the histogram of Fig. 2. Association between stem
related traits in addition to lodging and yield/plant and the 47 loci
were analyzed for the 271 unique tef accessions. The marker locus
CNLTs540 was significantly associated with seed weight/plant but
no other significant associations were observed.
4. Discussion
Molecular characterization of germplasm accessions is a useful
tool for studying and managing the genetic diversity available in the
genebanks. This tool becomes even more valuable when passport
data is very scarce or lacking. In this study, microsatellite markers were used to characterize the core collection of tef breeding
germplasm available in Ethiopia including breeding lines, released
varieties and wild species, some of which have been employed
for producing inter-specific crosses. The 271 breeding lines studied here along with the 33 landrace collections are the primary
germplasm resources used by breeders for tef improvement. Contemporary tef breeding programs have shifted from mass selection
within landraces towards targeted inter- and recently intra-specific
crosses since the proper hybridization methods were established
by Berhe (1975) according to Assefa et al. (2010).
The first set of 26 SSR markers employed here were distributed
across 12 of the 30 linkage groups identified by Zeid et al. (2010) and
were flanking QTL positions for various traits in tef. An additional
set of 13 markers with high PIC values that were not previously
67
mapped were selected from 68 markers. These 39 markers differentiated 80.8% of the accessions assayed and identified the rest as
redundant accessions (Supplementary Table 3 and Fig. 1). When the
fingerprints of either of the two sets of markers were used to assess
the variation in the 20 tef varieties from the 2009 seed lot, all varieties, except DZ-Cr-255 and DZ-Cr-82 were differentiated, and the
correlation between the GS estimates for the two marker sets was
r = 0.71. These markers thus, established the first tef fingerprints
that could be used for variety identification and protection.
Because of the scarcity of passport data on the tef accessions
studied here, identical (redundant) accessions were expected. We
identified 27 cases where accessions were identical to one or more
other accessions (Supplementary Table 3). Only one accession representing those redundant accessions was used in further analysis,
while the rest (N = 39) were excluded. On the other hand breeding accessions carrying the same selection number with a different
suffix (N = 21) as seen in Supplementary Table 1, were shown to
be dissimilar, and the basis for the addition of the suffix was
not documented. Knowing that farmers traditionally grow tef in
mixtures even with contrasting seed colors (Assefa et al., 2002a),
the most probable explanation is that plant materials collected
from the same farmer’s field during germplasm acquisition were
assigned the same selection number, and a different suffix was
attached if morphological differences were apparent. This identification method is an indication that the methodologies employed
during tef germplasm acquisition were efficient and successful in
recognizing the available variation within a specific collection area.
After excluding 39 redundant accessions, the genetic similarity estimates among the remaining tef accessions ranged between
0.20 and 0.99 revealing a broad base of genetic diversity available
in tef. These results indicated that the high GS estimates reported
in previous studies (Ayele et al., 1999; Bai et al., 1999b; Ayele and
Nguyen, 2000; Bai et al., 2000) that use the same plant material
(landraces) included in this study, was a marker dependent issue
rather than an inherent low polymorphism in tef as previously suggested. These results are in agreement with Assefa et al. (2003a),
where estimates of GS based on eight ISSR markers ranged from
0.26 to 0.86 among a smaller set (92) of tef accessions, most of
which were included in the present study as well. Furthermore,
the present findings also showed that the 33 landraces, that were
originally selected by Ebba (1975) from farmer’s fields, based solely
on morphological traits to represent the entire variation in the tef
germplasm were equivalent in their range of variation for the GS
values (0.20–0.99) to that of the 271 breeding accessions. However,
significant differences were detected between the gene diversity
for the breeding lines (Fst = 0.60) and landraces (Fst = 0.07). These
results indicate the importance of the breeding line accessions as a
major source of genetic diversity for the tef breeding programs.
In this study, 70% of the markers were polymorphic between
the two landrace accessions, Kaye Murri and Fesho. These two
accessions were the parents of the first population used to construct a linkage map in tef (Bai et al., 1999a), using AFLP markers
of which only 6.1% were polymorphic. The high level of polymorphism observed between the two landraces as shown in our study
indicates the potential of those SSR markers for future mapping
work utilizing intra-specific crosses and for combining the results
obtained from the inter-specific cross E. tef × E. pilosa reported by
Zeid et al. (2010). The four U.S. commercial varieties studied here
had a mean GS value of 0.41 to the other tef accessions and a
maximum GS value of GS = 0.69, suggesting that those are unique
lines that were carefully selected and/or bred independent of the
DZARC germplasm collection. Those four lines could be evaluated
for their performance in Ethiopia for any beneficial traits they might
possess.
All 13 wild Eragrostis accessions studied here except E. cilianensis were differentiated from the cultivated tef accessions in
68
M. Zeid et al. / Field Crops Research 127 (2012) 64–70
PCo3
0.7
Breeding lines
0.6
Landraces
0.5
Wild accessions
US lines
Kaye Murri
0.4
Varieties
0.3
0.2
E. unioloides-46-1
E. cilianensis
0.1
PCo1
0
-0.6
-0.4
-0.2
-0.1
0
0.2
0.4
0.6
0.8
-0.2
-0.3
-0.4
Fig. 1. Principal co-ordinate analysis of 326 tef accessions (breeding lines, landraces and varieties) and 13 wild Eragrostis accessions representing the entire breeding program
of tef in Ethiopia in addition to 4 U.S. commercial lines based on 47 loci. The first principal coordinate explained 7.5% of the variation, while the third coordinate explained
4.3% of the variation.
the cluster analysis (Supplementary Fig. 1) and also on the third
PCoA coordinate (Fig. 1). Previous studies employing molecular
markers (Ayele et al., 1999; Bai et al., 1999b; Ayele and Nguyen,
2000; Bai et al., 2000) differentiated the three species; E. tef, E.
pilosa and E. curvula, however, this is the first time that other
wild species have been compared to a large number of tef accessions. Although E. cilianensis has been used as a cereal crop in
parts of West Africa (Dalziel, 1937) and was suggested in many
studies to be the progenitor of tef (Ingram and Doyle, 2003), our
data suggested that an error due to mislabeling is the reason for
a presumed close relationship to tef accessions. This, however,
needs to be verified by screening another source of seed from that
species.
Mislabeling and lack of passport data are major problems for the
tef germplasm collection and its wild relatives in Ethiopia and this
study is a first step towards identifying and resolving this problem.
Most of the wild accessions preserved at DZARC are the same accessions described by Jones et al. (1978), however, other accessions
such as Dakota-50-1, Kiubins-20-1 and Oferifiana (Supplementary
Table 1) are examples of cases where information is lacking except
for being described as wild Eragrostis species, a description that is
clearly supported by our results presented here (Fig. 1).
Fig. 2. Frequency of alleles detected using (A) 32 loci flanking QTL positions and (B) 15 loci of high PIC values, for 326 tef accessions (breeding lines, landraces and varieties),
13 wild Eragrostis accessions and 4 U.S. commercial lines.
M. Zeid et al. / Field Crops Research 127 (2012) 64–70
Tef is a predominantly self-pollinated crop, and for that reason any change in the genetic integrity of a specific line could be
attributed to seed admixture as pollen flow is very limited in selfpollinated cereals (Matus-Cadiz et al., 2004; Wang et al., 2009). To
address this issue and its effects on preserving line identity, fingerprints of two different seed lots from 2003 and 2009, of 12 tef lines
were compared. Only one (DZ-Cr-82) of the 12 lines showed identical fingerprints between the seeds lots, and three lines shared
a GS of less than 0.70, thereby signifying the seriousness of the
seed admixture problem. Seed admixture is a common problem in
small grain cereals and can easily be detected using SSR markers
(Donini et al., 1998; Khlestkina et al., 2007). Tef breeders generally depend on morphological traits, especially those of the panicle
form and lemma color, to differentiate lines in the field, and this
task is becoming even more difficult, since all new lines are of the
loose panicle type that is associated with higher yields. In addition the tiny seeds of tef make it very hard to maintain truly pure
seed stocks because admixture can occur during any step of the
seed production and handling process unless extreme care is practiced. For example, the close relationship between the line DZ-Cr-37
from the 2003 seed lot with DZ-Cr-385 (GS = 0.89 (a line derived
from the cross of DZ-01-2785 × E. pilosa (30–5)) and HO-Cr-136
(GS = 0.87) (of unknown origin) was unexpected. However, two different seed lots of the same line DZ-Cr-37 were also different with
GS = 0.86. Another clear case of seed admixture was observed with
the accession E. pilosa (30–5), where the average GS between the
three fingerprinted seed lots was GS = 0.55. These discrepancies in
relationships between lines and seed lots of the same line are evidence of loss of integrity that could be attributed to seed admixture.
In contrast, identical fingerprints for all three seed lots Kaye Murri
were observed. This could be attributed to the unique morphology
of that line, as it is characterized by its thick culms, compact and
long panicles, red lemmas and white grains that make it easy to
distinguish. This line has been involved in marker development,
linkage mapping and QTL analysis (Zhang et al., 2001; Yu et al.,
2006a,b, 2007) and is used in the current tef breeding program as
a donor parent for strong culms. Interestingly, our results (Fig. 1)
showed that this line was positioned closer to the wild Eragrostis
species than the majority of tef accessions on the third principal
coordinate. The fingerprint of that line substantiates the unique
morphology because the mean GS between Kaye Murri and all tef
accessions is equal to the mean GS between that line and all the
wild species (GS = 0.42).
4.1. Allelic diversity and association analysis
The phenotypic variation in this study did not reach the levels of
variation reviewed earlier by Assefa et al. (2001a) despite the fact
that there was overlap of accessions in both studies. Culm length,
for example, a trait of major interest for better lodging resistance
in tef, ranged from 23.93 to 47.2 cm in the present study (Table 2),
while Assefa et al. (2001a) reported a range of 11–82 cm. Nevertheless, environmental effects influence these traits limiting the
value of the comparison. The penetrometer reading adopted in this
study as a measurement of stem strength at the lowest 3 internodes
was difficult to measure because of the extremely thin stems of tef
as compared to maize. Furthermore, no correlation was observed
between lodging and the penetrometer readings at any of the three
internodes (data not shown).
There were no significant associations observed between the
SSR markers and any of the traits except for the marker CNLTs540
previously reported by Zeid et al. (2010) to be flanking a QTL for
lodging index. In the current study, however, this marker was associated with seed weight/plant rather than the lodging score. In
general, lodging index has been shown to be positively correlated
with seed weight/plant (Yu et al., 2007; Zeid et al., 2010), but in
69
the study by Zeid et al. (2010), no QTL for seed weight co-localized
with the QTL for lodging index. In this work, lodging score using 0–5
scale rather than the lodging index (Caldicott and Nuttall, 1979) was
employed, however, neither of the scales was correlated with any
of the stem related traits. These observations suggest the need for
an improved measurement method if lodging improvement in tef
is to be achieved. In rice for example, lodging is known to be a complex trait that is the product of many stem and root characteristics.
Pushing resistance, which is an index of resistance to stem bending and root lodging, was adopted as a more reliable measure for
lodging resistance (Kashiwagi et al., 2008). This method employs a
prostrate tester (Daiki Rika Kogyou Co., Tokyo, Japan) and could be
explored in tef. Nevertheless, our study revealed much allelic diversity for some of the loci that were associated with agronomic traits
in previous studies. The accessions in this study were selected from
farmers’ landraces through pure-line selection (Assefa et al., 2010)
and therefore, they represent the alleles harbored by those lines
(Fig. 2). Thus the germplasm collection and conservation efforts
were successful in capturing a large amount of the available diversity while enabling plant breeders to access this diversity. Other
alleles that were only present in landraces or U.S. lines could be
easily integrated into the breeding pipeline if they prove to be
of value to the breeding program. A more difficult option would
be to introgress useful alleles from wild relatives of tef. In both
cases, however, molecular markers would be useful for monitoring
this process and ensure a faster and more efficient transfer of the
desired alleles.
The tef breeding program has been successful in producing
seven improved varieties through hybridization methods (Assefa
et al., 2010), while relying only on morphological traits to ensure
that the cross was successful. Those morphological traits have been
the bases for selecting the parental lines of the cross, verifying the
success of the hybridization process and for selecting among lines
of the segregating populations. The most recent and successful
selections, Quncho and Gamachis (Supplementary Table 1), were
derived from the same cross (DZ-01-974 × DZ-01-196). While the
former was selected for its desired white seed color, the latter was
selected for its early maturity which is beneficial for moisturestress prone areas (Belay et al., 2008). Our marker results are in
agreement with the pedigree relationship between the parental
lines and the two selected lines indicating the SSR markers used
in this study provide an unprecedented tool for improving the efficiency of the tef breeding program once implemented. Molecular
markers have been used for assessing seed purity in hybrid seed
lots in rice (Nandakumar et al., 2004; Sundaram et al., 2008). In
tef however, these markers would also give breeders the chance
to identify and verify hybrids from the cross between two promising lines that lack clear morphological differences. This will allow
many new cross combinations that may combine short culm length
and high yield, a combination that was not achieved with the cross
between Kaye Murri and E. pilosa due to the weak stem and low
yield potential of the wild parent (Tefera et al., 2003; Yu et al.,
2007). The markers could also be used to organize the entire tef
germplasm collection, reducing the number of duplicate accessions and grouping accessions based on their genetic relationship.
The SSR markers presented here are reliable PCR-based markers
that are easy to reproduce and once implemented in the tef breeding program, marker-assisted breeding and gene introgression will
become feasible.
Acknowledgements
We gratefully acknowledge financial support from the McKnight
Foundation’s Collaborative Crop Research Program for the African
Chloridoid Cereals project (Grant No. 06-448). Thanks are due to all
70
M. Zeid et al. / Field Crops Research 127 (2012) 64–70
technicians at the Debre Zeit Agricultural Research Center, Ethiopia
for their help collecting the field data.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.fcr.2011.10.013.
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