Genetic variability evaluation in a Moroccan collection of barley, Hordeum vulgare L., by means of storage proteins and RAPDS

Article (PDF Available)inGenetic Resources and Crop Evolution 49:619-631 · March 2002with24 Reads
DOI: 10.1023/A:1021228730714
Abstract
The genetic variation existing in a set of barley (Hordeum vulgare L.) landrace samples recently collected in Morocco was estimated. Two kinds of genetic markers, seed storage proteins (hordeins) and random amplified polymorphic DNA (RAPD), were used. Only six out of 31 landraces were subjected to RAPD analysis. Both kinds of markers, RAPD and storage proteins, yielded similar results, showing that the level of variation observed in Moroccan barley was high: all landraces showed variability; 808 different storage protein patterns (multilocus associations) were observed among 1897 individuals (2.32 seeds per association, on average) with an average of 43 multilocus associations per accession. In general, genetic variation within accessions was higher than between accessions. The 100 polymorphic RAPD bands generated by 21 effective primers were able to generate enough patterns to differentiate between uniform cultivars and even between individuals in variable accessions. One of the aims of this work was to compare the effectiveness of RAPD versus storage protein techniques in assessing the variability of genetic resource collections. On average hordeins were more polymorphic than RAPDs: they showed more alternatives per band on gels and a higher percentage of polymorphic bands, although RAPDs supply a higher number of bands. Although RAPD is an easy and standard technique, storage protein analysis is technically easier, cheaper and needs less sophisticated equipment. Thus, when resources are a limiting factor and considering the cost of consumables and work time, seed storage proteins must be the technique of choice for a first estimation of genetic variation in plant genetic resource collections.
Genetic Resources and Crop Evolution 49: 619631, 2002.
619
2002 Kluwer Academic Publishers. Printed in the Netherlands.
Genetic variability evaluation in a Moroccan collection of barley,
Hordeum vulgare L., by means of storage proteins and RAPDs
121 1,
*
´´´
EL-Habib Dakir , Marıa-Luisa Ruiz , Pedro Garcıa and Marcelino Perez de la Vega
1 2
´
´´´´
Area de Genetica
,
Facultad de Biologıa
,
Universidad de Leon
, 24071
Leon
,
Spain
;
Instituto de Recursos
*
´´
Naturales
,
Universidad de Leon
, 24071
Leon
,
Spain
;
Author for correspondence
(
e-mail
:
degmpv
@
unileon
.
es
;
phone
: 134 987 291550;
fax
: 134 987 291501)
Received 7 August 2001; accepted in revised form 7 March 2002
Key words
:
Barley, Genetic variability, Hordeins, Hordeum vulgare, RAPD, Storage proteins
Abstract
The genetic variation existing in a set of barley (Hordeum vulgare L.) landrace samples recently collected in
Morocco was estimated. Two kinds of genetic markers, seed storage proteins (hordeins) and random amplified
polymorphic DNA (RAPD), were used. Only six out of 31 landraces were subjected to RAPD analysis. Both kinds
of markers, RAPD and storage proteins, yielded similar results, showing that the level of variation observed in
Moroccan barley was high: all landraces showed variability; 808 different storage protein patterns (multilocus
associations) were observed among 1897 individuals (2.32 seeds per association, on average) with an average of
43 multilocus associations per accession. In general, genetic variation within accessions was higher than between
accessions. The 100 polymorphic RAPD bands generated by 21 effective primers were able to generate enough
patterns to differentiate between uniform cultivars and even between individuals in variable accessions. One of the
aims of this work was to compare the effectiveness of RAPD versus storage protein techniques in assessing the
variability of genetic resource collections. On average hordeins were more polymorphic than RAPDs: they showed
more alternatives per band on gels and a higher percentage of polymorphic bands, although RAPDs supply a
higher number of bands. Although RAPD is an easy and standard technique, storage protein analysis is technically
easier, cheaper and needs less sophisticated equipment. Thus, when resources are a limiting factor and considering
the cost of consumables and work time, seed storage proteins must be the technique of choice for a first estimation
of genetic variation in plant genetic resource collections.
Introduction a significant level of genetic variability is still main-
tained in situ, with the advantage that these materials
The evaluation of the amount and the distribution of are adaptively responding to the environment.
the genetic variability are among the main issues in Biochemical and molecular markers are key tools
the conservation and utilization of plant genetic re- in the evaluation of genetic variability in both natural
sources. Another important issue is where to find populations and germplasm accessions, in determin-
genetic variability, in particular in relation to the ing duplications in germplasm collections, in
principal crops, since the new-bred cultivars are evolutionary and phylogenetic studies and in many
´
rapidly displacing landraces and local materials other studies (Perez de la Vega 1993; Kresovich et al.
throughout the world. Barley, Hordeum vulgare L., is 1997). During the 70’s and 80’s the electrophoresis of
one of the main crops and this species is well repre- both storage proteins and isozymes were widely used
sented in many Germplasm Bank collections, never- in these kinds of analyses, being gradually substituted
theless it is worth evaluating the level of genetic in many studies by DNA markers during the 80’s and
variability maintained in small scale farming in a faster during the 90’s. In particular random amplified
developing country such as Morocco, that is, whether polymorphic DNA markers (RAPDs) obtained by
620
means of the polymerase chain reaction (PCR) have expanded cotyledons or seedling leaves are used as
become one of the most widely used marker system in DNA sources. Thus, both techniques are non-destruc-
studies related to plant genetic resources. The advan- tive and mature plants can develop from the sampled
tages and limitations of this technique have been individuals. Another common characteristic is that
extensively discussed (Karp and Edwards 1997). both markers can be analyzed without previous
Currently, amplified fragment length polymorphisms knowledge of their genetic control, as dominant
(AFLPs) are increasing their application in basic and markers (presence-absence) when considering each
applied plant genetics. But the suitability of a marker band as a different genetic locus.
system is not only based in its ability to discriminate The prolamin fraction of barley storage proteins is
between individuals and the number of loci which can known as hordeins. They are located in the endosperm
be assayed simultaneously, but also by the total cost as a heterogeneous mixture of polypeptides described
(research time and consumables) of the technique. as belonging to four classes, known as the B- C- D-,
Genetic variability evaluation involves analyzing sev- and g- or A-hordeins. B- and C-hordeins express a
eral loci in relatively large samples of individuals, wide variation among different samples of wild and
natural populations or accessions. Therefore, the cost cultivated barley. D-hordeins, the high molecular
of techniques could be the limiting factor when large weight fraction, are less variable than B- and C-
collections have to be analyzed and /or when the hordeins. Hordein genes are clustered into multigene
economic resources available are scarce. This may be families and each group of structurally similar hordein
the case in many developing countries rich in genetic polypeptides is encoded by one or more complex loci
resources. Consequently, relatively cheap techniques and four loci have been well characterized and chro-
that demand less sophisticated laboratory equipment mosomally located (Shewry et al. 1987; Vapa and
´
could be the appropriate choice (Aman 1997). Radovic 1998).
The electrophoresis of storage proteins is one of the The aims of this work were to estimate the genetic
most widely applied technique for obtaining genetic variation existing in a set of barley (Hordeum vulgare
markers in plant species, in particular polyacrylamide L.) landraces recently collected in Morocco and to
gel electrophoresis in presence of sodium dodecyl evaluate the effectiveness of two marker techniques,
´
sulphate (SDS-PAGE) (Perez de la Vega 1993). In seed storage proteins and random amplified polymor-
most cases, seed storage proteins are used, although in phic DNA, to estimate this variation. The levels of
suitable species other storage proteins such as tuber variation observed at both the collection level and
proteins have been used. Most electrophoretic tech- within most of the accessions were high, so much so
niques yield a high number of discrete bands on gels, that in general the genetic variation within accessions
thus, polyacrylamide gel electrophoresis of storage was higher than between accessions. The results
proteins has been considered the most powerful single indicate that the genetic variation conserved in
system for identifying genetically uniform plant va- Moroccan barley is still high and could help in
rieties (Nielsen and Johansen 1986). As previously designing the strategies to conserve Moroccan barley
mentioned, RAPD markers are becoming generally resources.
used in many genetic studies, they also yield a high
number of discrete bands, but, when comparing with
other marker systems, such as storage proteins or Materials and methods
isozymes, RAPDs have the advantage that, thanks to
the numerous primers available, the number of Sample collection
markers which are usually obtained is much higher.
Both kinds of markers, RAPD and storage proteins, Barley (Hordeum vulgare L.) seed samples were
share some similar characteristics. Both techniques collected by one of the authors (EHD, Moroccan
need only a small amount of tissue to obtain sufficient citizen) in small farmer-markets in central Morocco
material for analysis. For those species with relatively (Figure 1) directly from farmers selling a few hun-
large seeds, a part of the endosperm or the cotyledons dreds kilograms of barley. The farmers were asked to
can be used to extract proteins and the rest of the seed be sure they were small-scale farmers and the samples
saved for further use, or alternatively seed halves can had come from materials they have been sown for
be differentially extracted. Seeds can yield enough years from their own seeds or supplemented with
DNA for RAPDs, but usually a small amount of seeds from other farmers, that they do not buy seeds
621
to sow from big seed dealers, that they came from macia-Biotech LMW Calibration Kit was used. Elec-
villages near the market place, etc. For this study, 31 trophoresis was carried out at 25 mA for 5 h and
samples were chosen; hereafter these samples will be cooled by means of circulating water at approximately
designated as accessions. The cultivar Hassan was 10 8C. Gels were stained for 48 h with a mixture of
used as control material for marker analyses. 0.02% Comassie Brilliant Blue R-250, 5% ethanol
and 6% trichloroacetic acid, distained for 24 h in a
Storage protein analysis solution of 10% methanol and 7% acetic acid, rinsed
in tap water and then transferred to a conservation
Sixty seeds per accession were excised into two solution (8% acetic acid, 10% glycerol and 25 %
halves, the half containing only endosperm was ethanol), after an hour gels were covered with pre-
weighed, crushed, and the proteins were extracted by wetted cellophane and slowly dried between filter
adding 25 mL /mg of extraction solution for 1 h: 4 mL paper. Gels can be conserved in this form. Proteins
of distilled water, 0.3 mL of 2-mercaptoethanol and were named using a letter (B, C or D) indicating the
1.7 mL of a previously prepared solution (12.5 mL of hordein group followed by a number and / or letter in
Tris-HCl 1M, pH 6.8 buffer, 20 mL of glycerol, 24.1 order of decreasing mobility on gels, according to
mL of distilled water, 4 g of SDS and 20 mg of previous nomenclatures (Shewry et al. 1978a, 1978b;
pyronin-G). Tubes were then immersed in boiling Bernardo et al. 1997).
water for 9 min and afterwards centrifuged at 10,000
rpm for 5 min. Twenty
m
L of the supernatant were RAPD analysis
inserted into each sample well. Proteins were elec-
trophoresed following the Laemmli (1970) method Genomic DNA was individually extracted from 14-
with minor modifications in 12% SDS-PAGE vertical day-old seedling leaves following the Dellaporta et al.
slab gels in a Bio-Rad Protean II tray cell. For the (1983) method with some modifications described by
estimation of protein molecular weight, the Phar- Saghai-Maroof et al. (1984). Twenty seedlings from
Figure
1.
Barley collection sites in Morocco. From north to south and west to east: 29, Tlat Ain Aouda (Rabat); 34, Rhamna (Casa Blanca); 31,
Rommani; 37, Oulad Mdakra; 28, Echaouia; 39, Lamdakra; 32, Jmis Zhailiga; 27, Tlat Sidi Bennour (El Jadida); 38, Oulad Hrir; 35, Oulad
Sidi; 46, Amzab; 43, Oulad Mrah; 5 and 7, Jmis Beni Chagdal; 1, 2, 3, 4, 14, 19, 24 and 26, Fquih Ben Salah; 15 and 40 Souk Al Ahad; 45,
ˆ
Tazemmit; 47 Beni Mellal; 9, 10 and 12, Souk Sebt; 30, Arbiaa Sjor; 36, Marrakech
622
each of six accessions and the cultivar Hassan were Results
analyzed. DNA was quantified by means of a Gene
Quant (Pharmacia) spectrophotometer. DNA was am- Morphological data
plified from approximately 30 ng of genomic DNA by
means of the polymerase chain reaction (PCR) using Field plots of 1.5 3 1.5 m per sample were sown with
as single primers 10-mer oligonucleotides obtained seeds spaced 10 cm from each other. In addition to
from Operon Technologies Inc. (31 primers were Hassan, Albacete and Zaida cultivars were also in-
used), and Taq DNA polymerase (Promega). DNA cluded as controls, and wheat was used in order to
extracted from single seedlings was enough for the 31 separate plots and as border. Morphological charac-
reactions. Samples were incubated in a thermal cycler ters such as row number, plant height, ear length,
(Perkin Elmer Mod 480) programmed as follow: number of tillers, weight of 100 seeds, etc., were
initial strand separation at 94 8C (1 min), 45 cycles at measured from 30 plants chosen at random. Since the
´
36 8C (1 min)/72 8C (2 min)/ 94 8C (1 min), final climatic conditions in Leon (in the northwest of the
extension at 72 8C (10 min). Amplification products Spanish Meseta, and about 850 m above sea level) are
were electrophoresed in 1.5% agarose gels in TAE very different from the original conditions in Moroc-
buffer for 4 h at 25 8C and detected by staining with co, the results were probably heavily influenced by
ethidium bromide (10
m
g/ ml). The 1 kb Plus DNA these circumstances and they are not included in this
Ladder from GIBCO-BRL was used as standard in paper. However, they were useful to observe: 1) a
gels, and a sample of the amplified fragments from the general higher variance within accessions in com-
cultivar Hassan was always included in gels. For parison to control cultivars; 2) that all accessions
RAPD analysis, gel images captured under UV light except 37 and 38 were six-row accessions (accessions
were recorded by means of an image analyzer (UVP 14, 29 and 36 were mixed with a low percentage of
Image Storer) and printed on paper. two-row plants); and 3) that the morphological
characteristics of most Moroccan accessions differ
from modern bred barley cultivars, for instance, they
Variability evaluation and statistical analyses
were taller, with fewer and thicker tillers per plant.
In order to analyze both sets of data, hordeins and
Storage proteins
RAPDs, in the same way, the results presented in this
work were obtained considering each band, either
The technique used allowed for a clear observation of
hordein or RAPD, as an independent marker with two
B, C and D hordeins. A hordeins were poorly resolved
alternatives, presence vs. absence; although it is
and, therefore, they were not scored. A total of 27
known that some hordein bands are controlled by
bands were scored in all the seed assayed: 13 (ordered
alleles (C4-C5, locus Hor-
1
; B7-B8, locus Hor-
2
,
from lower to higher molecular weight, B0, B1, B2,
D1-D2, locus Hor-
3
; B4b-B4c, locus Hor-4). When
B3, B4a, B4b, B4c, B4d, B5, and B6 from 20 kDa to
the allelic nature of these bands was considered (that
40 kDa, and B7, B8, and B9, from 43 to 46 kDa) in the
is, scoring 23 markers instead of 27) the results on
B hordein zone, nine (C0, C1 C2, C3, Ca, and Cb,
variability, genetic distances and phenograms were
from 50 kDa to 62 kDa and C4, C5 and C6, from 67 to
similar. The diversity index of each accession was
79 kDa) in the C hordein zone, and five (D0, D1, D2,
m2
1
2
]
calculated as o 12ox , where x are the fre-
SD
D3, and D4,) in the D hordein zone from 94 kDA to
ii
m
11
quencies of each of the two alternatives (presence vs. 120 kDa. Table 1 shows some data on these hordein
absence) per band site and m is the number of sites bands. Their frequencies in the whole set of seed
scored. This diversity index would be equal to the analyzed vary widely: from 1.0 (B0, B1, B2, B3, and
average heterozygosity if the populations were pan- D4 fixed in these Moroccan materials) to 0.001. Their
mictic and each band was controlled by a locus. presence in the different accessions was also highly
Similarity indices (Hedrick 1971) were used to obtain variable. The F parameters give a different indica-
ST
the phenograms. Similar results were obtained when tion of this variation. F values range from 1 to 0,
ST
using Hedrick or Nei’s unbiased genetic identity (Nei values close to 1 indicate that most of the genetic
1978). Phenograms were obtained by the UPGMA variation is due to differences between populations or
method (Sneath and Sokal 1973) or the Neighbor- accessions, values close to 0 indicate that most of it is
joining method (Saitou and Nei 1987). due to variation within accessions. These bands with
623
the higher F values are good for differentiating accession, i.e. taking into account each different band
ST
between accessions. The average F values over all combination as a different association. It can be
ST
polymorphic markers was 0.268 which indicates that observed (Table 2) that, with the exception of acces-
most of the hordein variation was within accessions in sions 37 and 38 and the control variety Hassan,
this Moroccan collection. Moroccan barley accessions showed a high level of
The variability parameters scored were: 1) Average variation for hordeins: on average 60.4% of the bands
of alternatives per band (presence or absence) in each were polymorphic, 43 multilocus phenotypes were
accession, which would be equivalent to the number observed in each accession and the diversity index
of alleles per locus if the alternatives presence or was of 0.202.
absence were controlled by dominant and recessive A total of 808 multilocus associations were ob-
alleles, respectively. 2) Percentage of polymorphic served in the 1897 seeds analyzed. Each of the three
bands per accession, considering a site polymorphic if most frequent associations is characteristic of a single
the most frequent alternative had a frequency not monomorphic or nearly monomorphic accession. All
higher than 0.99 in the considered accession. 3) The the seeds of accession 37 were fixed for association A
diversity index, which would be equal to the expected which was also observed in a single seed of two
heterozygosity under random mating conditions. 4) additional accessions. Association B was observed in
The number of multilocus associations present in each all seeds from the variety Hassan and C in most of the
seeds from accession 38. The rest of the relatively
frequent associations (observed in at least 10 out of
the 1897 seeds) were present in several accessions
ranging from 11 seeds per accessions to one seed per
Table
1.
Hordein bands considered.
accession. However, most of the multilocus associa-
b
Hordein Molecular Frequency F
ST
a
tions were very infrequent since 523 were observed in
band weight
a single seed in all the seed analyzed and 119 in two
in kDa
seeds.
D4 116.8 1.000 0.000
The phenograms obtained from hordein data (Fig-
D3 112.8 0.001 0.016
ure 2) showed three clear groups of accessions. The
D2 106.8 0.394 0.797
D1 105.3 0.606 0.793
first group included 29 accessions, the second group
D0 93.2 0.937 1.000
accession 37 and the variety Hassan and the third one
C6 78.9 0.919 0.413
the accession 38, which was clearly discriminated
C5 68.5 0.450 0.208
from all other accessions. In the first group two
C4 66.5 0.547 0.209
subgroups were observed including 18 and 11 acces-
Cb 61.5 0.731 0.161
Ca 58.8 0.581 0.141
sions, respectively. No relationship between the geog-
C3 56.0 0.678 0.105
raphic origin of the accession and the cluster in which
C2 54.5 0.916 0.047
each one was included was observed. For instance,
C1 53.5 0.190 0.196
accessions 1, 2, 3, 4, 14, 19, 24, and 26 were collected
C0 50.1 0.015 0.053
in Fquin Ben Salah, an inland town (Figure 1), but
B9 45.9 0.307 0.270
B8 44.6 0.354 0.126
they are included in different clusters in the two
B7 43.7 0.646 0.126
subgroups mixed with samples collected in coastal
B6 37.8 0.845 0.237
towns. The separation of accessions 37 and 38 from
B5 37.3 0.887 0.062
all other Moroccan accessions on the basis of hordein
B4d 36.1 0.050 0.614
patterns agrees with the two-row nature of these two
B4c 35.4 0.436 0.154
B4b 35.0 0.439 0.142
accessions.
B4a 34.4 0.462 0.162
B3 33.0 1.000 0.000
B2 31.9 1.000 0.000
RAPD markers
B1 29.1 1.000 0.000
B0 27.4 1.000 0.000
Six barley accessions were chosen for this analysis,
a
Approximate since they were estimated according the mobility of
four representing samples with higher or intermediate
the bands on gels in relation to the mobility of molecular weight
levels of variability for hordeins and two representing
markers.
b
Among all 1897 seeds analyzed. the accessions with the lowest level of variability for
624
Table
2.
Hordein variability parameters in Moroccan barley accessions.
a
Accession Sample size Average of alternatives Percentage of Diversity index Number of
per band polymorphic bands (standard error) multilocus associations
Hassan 60 1.0 00.0 0.000 (0.000) 1
01 60 1.6 63.0 0.237 (0.042) 42
02
60 1.7 70.4 0.198 (0.040) 47
03 60 1.6 63.0 0.262 (0.044) 56
04 60 1.7 70.4 0.244 (0.041) 53
05 58 1.6 63.0 0.222 (0.040) 45
07 57 1.6 59.3 0.227 (0.041) 50
09 60 1.6 63.0 0.205 (0.039) 51
10 60 1.6 63.0 0.226 (0.041) 49
12 58 1.6 63.0 0.226 (0.042) 45
14 58 1.7 70.4 0.280 (0.041) 48
15 60 1.6 63.0 0.232 (0.043) 51
19 58 1.7 70.4 0.246 (0.040) 50
24 60 1.7 66.7 0.207 (0.041) 45
26 60 1.7 70.4 0.243 (0.041) 53
27 60 1.6 59.3 0.170 (0.037) 38
28 60 1.6 59.3 0.221 (0.041) 53
29
60 1.7 66.7 0.267 (0.042) 57
30 59 1.6 59.3 0.175 (0.037) 36
31 58 1.7 66.7 0.264 (0.041) 49
32 60 1.6 55.6 0.218 (0.042) 46
34
60 1.7 70.4 0.216 (0.037) 44
35 60 1.7 66.7 0.217 (0.040) 52
36
60 1.7 74.1 0.188 (0.037) 42
37
60 1.0 00.0 0.000 (0.000) 1
38
58 1.5 48.1 0.034 (0.008) 8
39 60 1.6 59.3 0.202 (0.041) 49
40 60 1.7 70.4 0.218 (0.041) 46
43 60 1.7 66.7 0.189 (0.036) 39
45 58 1.7 66.7 0.221 (0.040) 45
46 58 1.6 63.0 0.208 (0.038) 47
47 57 1.6 63.0 0.219 (0.041) 48
b
Average 59 1.6 60.4 0.202 (0.041) 43
a
All the collected landraces are six-row barley, except 37 and 38, which are two-row, and accessions 14, 29 and 36, which include a low
percentage of two-row plants. Accessions in italics were subjected to RAPD analysis.
b
Without Hassan.
this kind of marker (Table 2). The cultivar Hassan accessions than within accessions. This F contrasts
ST
was again used as control. to the F value obtained for hordeins in the whole set
ST
Of the 31 primers used, 21 yielded a positive and of accessions (0.268) but it is similar to the value for
repeatable pattern of amplification products. A total of the same set of these six accessions (0.555). These
160 reproducible fragments (bands) ranging from results point out that the F differences observed are
ST
approximately 300 bp to 1,800 bp were observed in probably due to the election of the sample (a lower
these patterns. Band polymorphism was observed for number of accessions with high and low internal
all the 21 positive primers with a total of 100 variation) than to the kind of genetic marker studied.
(562.5%) polymorphic bands. On average, each posi- The comparative analysis of six accessions and the
tive primer generates 7.62 bands, ranging from three cultivar Hassan showed that on average hordeins had
to twelve, and 4.76 polymorphic bands, ranging from more polymorphism than RAPDs: more alternatives
one to eleven (Table 3). The average F value of all per band on gels and a higher percentage of poly-
ST
the polymorphic RAPD markers was 0.642, which morphic bands (Table 4) although RAPDs supply a
indicates that RAPD variation was higher between higher number of markers even with the relatively low
625
Figure
2.
Phenogram of the Moroccan barley accessions based on hordein patterns (27 bands). Distances were estimated by the Hedrick index
and the phenogram obtained by the UPGMA method. Underlined accessions were subjected to RAPD analysis
number of primers used in this study. The cultivar other patterns in the remaining seeds), was mono-
Hassan was monomorphic for both kinds of markers, morphic for RAPDs. This result could be due to the
hordeins and RAPDs. Accession 38, which showed a lower sample size used for the RAPD analysis. On the
low level of polymorphism when hordeins were con- other hand, accession 37, which was monomorphic
sidered (diversity index 0.034; 50 out of 58 seeds for hordeins, showed three different RAPD
showed the same hordein pattern in addition to seven phenotypes (Figure 3). In all other accessions tested,
626
Table
3.
Oligonucleotides used as primers in RAPD assays, number
barley landraces show that in small scale farming
of fragments observed and number of polymorphic fragments.
six-row materials are still prevalent, like in other
Primer N.F. P Primer N.F. P
North African countries (Yasuda et al. 1993), and that
the amount of genetic variability conserved in this
OPA01 10 4 OPB11 7 3
OPA03 9 9 OPB18 7 4 kind of materials in Morocco is still very high. For
OPA04 4 3 OPC05 11 6
instance, it was higher than in a sample of Ethiopian
OPA07 8 2 OPC15 10 6
barley landraces (Demissie and Bjornstad 1997): the
OPA09 3 1 OPG11 7 5
percentage of polymorphic loci at the 95% level was
OPA18 4 1 OPG19 8 7
49.6% in our hordein sample and 35.3% in the Ethio-
OPA19 12 10 OPH08 12 11
OPA20 8 6 OPH14 7 4 pian sample, while the diversity indexes were of 0.202
OPB05 10 7 OPP06 4 1
and 0.134, respectively. The lowest polymorphism
OPB07 10 7 OPW06 4 1
values observed in the barley collection from
OPB08 5 2 Average 7.62 4.76
Ethiopia, which is considered a secondary center of
N.F. 5 Number of amplified fragments, P 5 Number of poly-
diversity for barley (Bekele 1983), could be also due
morphic fragments Primers A16, B01, C13, C14, C16, D10, D14,
to the smaller number of individual analyzed per
G20, H05, and W12 produced faint bands or no band at all.
sample for Ethiopian landraces (5) and the loci ana-
lyzed, 15 isozyme loci and two hordein loci (Demissie
each seedling showed a unique multilocus set of and Bjornstad 1997). The other type of marker used,
RAPD bands (Figure 3). Seedlings from accessions RAPD, also showed a high variability in Moroccan
37 and 38 and the cultivar Hassan were clustered in a samples: the average of polymorphic bands per primer
single cluster each. However, seedlings from the other (4.76 considering all the seed sample) was similar to
accessions were mixed in each main cluster (Figure the average found in old French six-rowed varieties
3). A similar result was obtained for hordeins when (Bahram et al. 1999), higher than in modern barley
individuals instead accessions were compared (data cultivars (1.6) (Tinker et al. 1993) or even in some
not shown). samples of wild barley (3.6) (Dawson et al. 1993).
The data on Moroccan barley agree with many other
reports on the importance of landraces and old va-
rieties as stocks and sources of genetic variability.
Discussion The high level of polymorphism in Moroccan
barley would agree with the suggestion that Morocco
The data observed in this collection of Moroccan is a possible domestication center of this crop (Molina
Table
4.
Variability parameters for hordeins and RAPDs in a subsample of barley accessions.
Accession Sample size Average of alternatives Percentage of Diversity index
per band polymorphic bands (standard error)
02 Hordeins 60 1.7 70.4 0.198 (0.040)
RAPDs 20 1.3 30.0 0.094 (0.024)
29 Hordeins 60 1.7 66.7 0.267 (0.042)
RAPDs 20 1.4 37.5 0.112 (0.028)
34 Hordein 60 1.7 70.4 0.216 (0.037)
RAPDs 20 1.4 38.7 0.124 (0.027)
36 Hordeins 60 1.7 74.1 0.188 (0.037)
RAPDs 20 1.3 34.4 0.102 (0.024)
37 Hordeins 60 1.0 0.00 0.000 (0.000)
RAPDs 20 ,1.1 2.50 0.009 (0.014)
38 Hordeins 58 1.5 48.1 0.034 (0.008)
RAPDs 20 1.0 0.00 0.000 (0.000)
Hassan Hordeins 60 1.0 0.00 0.000 (0.000)
RAPDs 20 1.0 0.00 0.000 (0.000)
Average Hordeins 1.5 47.1 0.129 (0.026)
Average RAPDs 1.2 20.4 0.063 (0.016)
627
Figure
3.
Dendrogram of the 140 seedlings based on RAPD polymorphisms. Genetic distances obtained from the Jaccard index and the
Neighbor-joining method were used. The first two numbers in the series of four indicate the accession number and the other two the individual
number within each accession. HS-01 to 20 (Hassan) and 38-01 to 20 indicate that the 20 seedlings of each of these two accessions showed the
same multilocus phenotype
628
Cano et al. 1982, 1987). Relative high levels of storage proteins have been related to adaptive roles
genetic variability for isozyme and hordein have also (Nevo and Payne 1987; Nevo et al. 1983, 1986), and
been reported in barley landraces from the nearby even RAPD distribution showed a strong association
Spain (Bernardo et al. 1997), but also in landraces with microsite edaphic variables as the multilocus
from other countries, such as Finland (Ahokas and organization revealed soil-specific multilocus-geno-
Poukkula 1999), which can hardly be considered a types (Owuor et al. 1999). However, in this Moroccan
domestication center. Recent papers based on the use sample there is no evident association in the dis-
of molecular markers (RAPDs or AFLPs) and the tribution of hordein patterns; both single and mul-
analysis of allelic sequences point to a monophyletic tilocus, and the geographical origin of samples. Such
origin of cultivated barley (Badr et al. 2000; Blattner high genetic variation within accessions could be due
´
and BadaniMandez 2001), thus making a secondary to one or more of these causes: response to micro-
domestication in Morocco unlikely, and suggesting environmental variables (mostly edaphic), seed mix-
alternative hypotheses for the high level of polymor- tures by farmers, and segregation from heterozygous
phism of Moroccan populations (Blattner and plants originated by infrequent outcrossings (we have
´
BadaniMandez 2001). detected a 0.3% of heterozygous plants in our sample
Considering the information from hordeins, which scoring the presence of alleles for Hor-
1
to Hor-
4
have been analyzed in more accessions and more loci).
individuals than RAPDs, genetic variation in this set The phenetic analyses carried out (Nei and Hedrick
of barley landraces is mainly distributed within acces- distances and UPGMA and Neighbor-joining analy-
sions. A similar result was described for Sardinian ses) on the set of samples analyzed for both hordeins
landraces in which 84% of the genetic variation for and RAPDs resulted in trees with similar topologies
isozymes and 89% for RAPDs was found located compatible in their major features. Therefore, only the
within populations (Papa et al. 1998). A high degree Neighbor-joining trees using Nei distances are shown
of genetic heterogeneity within populations is com- (Figure 4). Accessions 37 and 38 and the cultivar
mon in wild populations and in landraces of autogam- Hassan are relatively distant from the other Moroccan
ous plants species and crops (Nevo 1992; Parzies et accessions which are separated by very short genetic
´
al. 2000; Perez de la Vega et al. 1994); while the distances. The similar topologies obtained when using
opposite is usually observed in modern bred cultivars either storage proteins or RAPDs points out that both
or varieties, thus, for instance, White and Cooke kind of markers are useful to obtain similar genetic
(1992) only found a 9.92% of heterogeneous samples relationships among intraspecific plant samples in
for hordeins among 353 barley cultivars. Another spite of the fact that each one covers different part of
proof of the high variation within accessions and the genome: the first one only covers the variation in a
populations is the high number of multilocus associa- few loci coding storage proteins while the other
tions. The average number of multilocus associations arbitrarily covers large parts of the entire genome.
per Moroccan accession was 43, with a maximum of Most of the accessions collected in Morocco agree
57 in accession number 29 (Table 2); therefore, in with the characteristics of crop landraces: they show
several accessions almost any of the 60 seeds assayed genetic variation within samples, and they agree with
had a set of hordein bands which were different from morphological characteristics of old cereal varieties
any other seed of the same accession, or even of the and landraces in which straw production was im-
whole collection, since 818 different associations portant for feeding livestock. The exceptions are
were observed in the 1897 seeds assayed (2.32 seeds accessions number 37 and 38, which showed the
per association, on average). A high level of diversifi- lowest levels of polymorphism among all the acces-
cation of the hordein patterns in landraces has also sions and most likely came from bred uniform cul-
been described in other landraces (Ahokas and Pouk- tivars with mixtures of other seeds. Accession 37 was
kula 1999). There is evidence that multilocus associa- uniform for hordeins and showed a relatively low
tions (mainly isozyme data) play an adaptive role in variation for RAPDs due to the variation of only four
´
many plant species and crops (Allard 1999; Perez de bands out of 160 RAPD bands. On the other hand,
´´
la Vega 1997; Perez de la Vega and Garcıa 1997), this accession 38 was uniform for RAPDs but showed an
fact is more easily testable in autogamous species in intermediate variation for hordeins (Table 4). Fifty
which multilocus associations are particularly well out of the 58 seeds analyzed for hordeins of accession
maintained owing to self-pollination. Likewise, seed 38 showed the same multilocus pattern, while in the
629
remaining eight seeds a total of another seven mul- 6,01 g while the averages of the other accessions
tilocus associations were observed; it is possible that ranged from 4.09 to 5.26). Thus, it seems that bred
the smaller sample of the 20 seedlings analyzed for cultivars are being incorporated into small scale farm-
RAPDs did not included any individual different from ing in Morocco and therefore they could displace
the majority genotype. Accessions 37 and 38 were the landraces in the near future, although conscious or
only samples of two-row barley, and while accession unconscious mixtures of seeds in this non-technologi-
38 showed a general plant morphology which was cal farming could contribute to maintaining in situ
more similar to other Moroccan accessions (with the genetic variability for a period of time.
exception of two-row phenotype), accession 37 ag- The assessment of genetic variation in large sam-
reed with the characteristics of a modern cultivar, for ples of plant genetic resources represents a high cost
instance, significantly shorter than all other accessions in time and value of consumables. Thus, reliable,
or controls (an average of 84.2 cm while the average affordable and economical techniques should be pre-
of other Moroccan accessions, including number 38, ferred, at least in a first screening of genetic vari-
was between 107.3 and 126.0), the shortest awns, and ability. Electrophoresis of seed storage proteins is one
a significantly higher weight of 100 seeds (average of of these techniques capable of giving an estimate of
the genetic variation within and between accessions
due to the relatively high number of genetics markers
which can be scored. Furthermore, since endosperm
half seeds are used, the technique is not destructive.
The RAPD technique is certainly highly useful for
this purpose since it affords us with an great number
of polymorphic markers which allows us to discrimi-
nate between uniform cultivars (Selbach and Cavalli-
Molina 2000; Kraic et al. 1998) or even individuals in
heterogeneous accessions (Figure 3), but, although
RAPD is currently an easy and standard technique and
the laboratory equipment is economical, storage pro-
tein analysis is technically easier, cheaper and needs
less sophisticated equipment. Considering the cost of
consumables in Spain, for us the analysis of the 1897
seed for hordeins had a similar cost to that of the 140
seedlings for RAPDs. Therefore, if resources are the
main limiting factor, we find that seed storage pro-
teins must be the technique of choice for a first
estimation of genetic variation in plant genetic re-
source collections.
Acknowledgements
This work was supported by funds from the Uni-
´
versidad de Leon and a personal grant to EHD from
the International Centre for Advanced Mediterranean
Agronomic Studies Mediterranean Agronomic In-
stitute of Zaragoza, Spain.
Figure
4.
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    • "Another important issue is where to find genetic variability. Barley (Hordeum vulgare L.) is one of the main cereal crops worldwide (Dakir et al. 2002). Large germplasm collections of cultivated (Hordeum vulgare subsp. "
    Full-text · Article · Oct 2015
    • "Another important issue is where to find genetic variability. Barley (Hordeum vulgare L.) is one of the main cereal crops worldwide (Dakir et al. 2002). Large germplasm collections of cultivated (Hordeum vulgare subsp. "
    [Show abstract] [Hide abstract] ABSTRACT: Fifteen barley landraces were collected from different localities in the West-Bank,-Palestine during 2009. A field experiment was conducted at the Faculty of Agriculture-An Najah National University to evaluate several agronomical traits of these landraces in 2010-2011 growing season. Cluster analysis was performed using the complete-linkage method, genotypic coefficient of variation (GCV), phenotypic coefficient of variation (PCV), broad sense heritability (H2), and genetic advance (GA) were calculated for the quantitative traits. Significant diversity was exhibited among the landraces regarding days to 90% heading, 100- grain weight, number of grains per spike, spike length, and awns length. The Cluster analysis showed high genetic diversity among the collected landraces with dissimilarity ranging from 0.26 to 0.75. The fifteen landraces were grouped into four clusters. Genotypic coefficient of variation ranged from 6.1 to 22.9, whereas phenotypic coefficient of variation ranged from 6.6 to 41.8 with maximum phenotypic and genotypic variability observed for number of fertile tellers, number of grains per spike and spike length. Moderate to high heritability (broad sense) estimates (70-87%) were found for most of the characters. The genetic advance was highest for number of grains per spike (39.4%), followed by spike length (37.2%). High positive significant correlations were found among the different studied traits with correlation coefficient ranging from 0.395 to 0.536. The results of this study indicated high genetic diversity among barley landraces in Palestine, which make them potential sources for selection and hybridization programmes. © 2015, Pakistan Agricultural Scientists Forum. All rights reserved.
    Full-text · Article · Oct 2015
    • "Another important issue is where to find genetic variability. Barley (Hordeum vulgare L.) is one of the main cereal crops worldwide (Dakir et al. 2002). Large germplasm collections of cultivated (Hordeum vulgare subsp. "
    [Show abstract] [Hide abstract] ABSTRACT: Fifteen barley landraces were collected from different localities in the West-Bank,-Palestine during 2009. A field experiment was conducted at the Faculty of Agriculture-An Najah National University to evaluate several agronomical traits of these landraces in 2010-2011 growing season. Cluster analysis was performed using the complete-linkage method, genotypic coefficient of variation (GCV), phenotypic coefficient of variation (PCV), broad sense heritability (H2), and genetic advance (GA) were calculated for the quantitative traits. Significant diversity was exhibited among the landraces regarding days to 90% heading, 100- grain weight, number of grains per spike, spike length, and awns length. The Cluster analysis showed high genetic diversity among the collected landraces with dissimilarity ranging from 0.26 to 0.75. The fifteen landraces were grouped into four clusters. Genotypic coefficient of variation ranged from 6.1 to 22.9, whereas phenotypic coefficient of variation ranged from 6.6 to 41.8 with maximum phenotypic and genotypic variability observed for number of fertile tellers, number of grains per spike and spike length. Moderate to high heritability (broad sense) estimates (70-87%) were found for most of the characters. The genetic advance was highest for number of grains per spike (39.4%), followed by spike length (37.2%). High positive significant correlations were found among the different studied traits with correlation coefficient ranging from 0.395 to 0.536. The results of this study indicated high genetic diversity among barley landraces in Palestine, which make them potential sources for selection and hybridization programmes.
    Article · Jan 2015
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