Phylogenetic Relationships Between Mediterranean and Middle-asian Wild Species of the Genus Hordeum L. As Revealed by Biochemical and Molecular Markers
H. El Rabey,
The phylogenetic relationships of 60 accessions of the genus Hordeum (29 Mediterranean and 20 middle-Asian wild accessions, together with nine American accessions and two of unknown origin), representing together nine species, were investigated by AFLP markers. Three hundred sixty six AFLP fragments were used for studying the molecular genetic diversity among the studied species, 339 out of them were polymorphic. Forty seven protein bands were obtained from the water soluble and the water insoluble seed storage protein by SDS-PAGE electrophoresis of 18 accessions representing nine species (two accessions/species). One band was common to all species and the other 46 bands were polymorphic. The phylogenetic tree deduced from AFLP analysis is concordant to a large extent with that deduced from seed storage protein. Highly significant cophenetic correlation coefficient was obtained between both AFLP (0.96) and seed storage protein (0.89) indicating the reliability of the results. The studied taxa were clustered according to their genome type. All Mediterranean and middle-Asian wild accessions could be integrated into the existing phylogenetic scheme.
to cite this article:
H. El Rabey, K.F. Abdellatif, M.K.H. Ebrahim, N. Abbas, J.A. Khan and E. Komor, 2013. Phylogenetic Relationships Between Mediterranean and Middle-asian Wild Species of the Genus Hordeum L. As Revealed by Biochemical and Molecular Markers. Pakistan Journal of Biological Sciences, 16: 168-174.
Received: December 07, 2012;
Accepted: February 13, 2013;
Published: March 16, 2013
The genus Hordeum contains ca 32 species and ca 45 taxa occurring in
temperate areas of Eurasia, North and South Africa and Central and South America.
The evolutionary pattern in this genus is complex, including different breeding
systems and various forms of polyploidy (Von Bothmer et
al., 2003). Aberg, 1940 recognized four Hordeum
sections, Stenostachys (Nevski) and Bulbohordeum (Nevski)
for perennial species and section Campestria (Ands) and Cerealia
(Ands) for annual ones, whereas Nevski (1941) recognized
five sections. Love, 1984 separated the genus
Hordeum into two genera based on genome structure namely Hordeum sensu
stricto, including both Hordeum vulgare and Hordeum bulbosum
and Critesion including the other species. Jaaska
(1992) and Jorgensen (1986) studied interspecific
relationships in the genus on the basis of the electrophoretic variation of
isozymes. Von Bothmer et al. (1986, 1987)
defined four basic genomes according to the meiotic behavior of different interspecific
hybrids and 32 species, genome I for H vulgare and H. bulbosum,
Y for H. murinum, X for H marinum and H for the remaining Hordeum
species, H. halophilum was added in section Critesion. The recent
molecular techniques supported mostly the above developed schemes using repetitive
DNA sequences, molecular hybridization, RFLP and in situ hybridization (Molnar
et al., 1989; Molnar and Fedak, 1989; Gonzalez
and Ferrer, 1993; Svitashev et al., 1994).
Blattner (2004) analysed 91 accessions representing
all Hordeum species. This analysis confirmed the previously formed four
clades now named as H, I, Xa and Xu. El
Rabey and Al-Maliki, 2011 compared the phylogenetic relationships of the
genus Hordeum based on 5 AFLP primer combinations (E37/M33, E37/M38,
E41/M33, E41M40 and E42M38) and ITS sequences of the ribosomal RNA genes. The
AFLP markers turned out as a convenient tool to reveal the interspecific genetic
diversity in the genus Hordeum and the result was concordant with previous
studies. The aim of the present study was to reveal the genetic diversity and
the phylogenetic relationships between 60 wild accessions of Hordeum
mostly from Mediterranean and Asian origin which had not been analyzed so far,
using AFLP markers and, partly, seed storage protein patterns.
MATERIALS AND METHODS
Plant material: A total of 60 accessions belonging to nine Hordeum
species either directly sampled from the Egyptian flora or supplied by different
gene banks were used for this study as shown in Table 1. The
accessions were chosen in order to represent the four main sections according
to Von Bothmer et al. (1995) and the Old and New
World's flora and focused on Mediterranean and middle-Asian origin.
DNA isolation: Plants were grown in the greenhouse. About 20 seeds were
sown and young leaves of 3-5 representative plants were collected in sterilized
50 mL polypropylene tubes and lyophilized in a Christ PG 30 freeze-dryer machine.
Leaves were ground and kept at -70°C until use. DNA was extracted according
to a modified CTAB method (Saghai-Maroof et al.,
AFLP markers: AFLP markers were developed according to Vos
et al. (1995) with following minor modifications. Briefly, the genomic
DNA was restricted using EcoRI as rare cutter and MseI as frequent cutter. Double
stranded EcoRI and MseI adapters were constructed by MWG-Biotech GmbH, Germany,
according to Vos et al. (1995) and were ligated
to the restricted DNA. The sequences of these adapters are as follows: MseI-adapters:
92A18 (5-GACGATGAGTCCTGAG) and 92A19 (TACTCAGGACTCAT-5), EcoRI-adapters: 91M35
(5-bio-CTCGTAGACTGCGTACC) and 91M36 (CTGACGCATGGTTAA-5). The two primer combinations
E40/M38 and E42/M38 were constructed by MWG-Biotech GmbH, (Germany) and used
in fingerprinting the studied taxa (Table 2).
Storage protein markers: Both water soluble and water insoluble proteins
were extracted from the seeds of 18 accessions (Table 1) that
were selected to represent the different four barley genomes.
|| Barley accessions used for AFLP and storage protein analyses
|*: Accessions used for storage protein, SDS: PAGE analysis
Protein extraction and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was
performed according to the method of Laemmli (1970) to
study the genetic diversity among the different genomes.
Data analysis: Both AFLP and protein gels were scored as 0/1 for absence/presence
of the bands, respectively. Number and percentage of the polymorphic bands were
calculated. Similarity coefficient matrices were calculated using Dice similarity
algorithm (Dice, 1945) for both markers (AFLP and protein).
Phenograms were constructed using the UPGMA method (Unweighted Pair-Group Method
with arithmetical algorithms Averages (Sneath and Sokal, 1973)
and the correlation cophenetic coefficients were calculated. For the above mentioned
analysis, the NTSYS PC2.0 software was used (Rohlf, 1998).
AFLP analysis: Altogether 366 bands were obtained from the AFLP analysis,
339 (93%) out of them were polymorphic (Table 2), 189 bands
out of 209 were polymorphic for the first AFLP primer combination (E42-M38)
while 150 bands out of 157 were polymorphic for the second primer combination
The AFLP markers (Fig. 1) and the dendrogram (Fig. 2) separated the barley accessions according to their genome type. All the accessions of H. vulgare spontaneum and H. bulbosum had the H genome type, whereas the H. murinum accessions with genome type Xu were like a subgroup of H. The second group in this cluster with genome type I was also divided into two groups, the first contains all species that have the genome I (i.e., H. bogdanii, H. brevisbulatum, H. chilense, H. jubatum and H. pusillum) while the second group contains H. marinum which has the Xa genome (Fig. 2). A highly significant correlation cophenetic coefficient was obtained with the dendrogram of the AFLP (r = 0.96) which proved the reliability of the results.
Protein analysis: A high percentage of polymorphism was obtained from
the protein analysis, where 46 polymorphic bands out of 47 were scored representing
98% (Table 2, Fig. 3). All the bands obtained
from the water soluble protein were polymorphic, whereas 26 out of 27 bands
were polymorphic for the water insoluble protein (Table 2).
|| Sequences and polymorphic bands of AFLP and storage protein
markers for the 60 barley accessions
|| Part of AFLP banding pattern (E42-M38 primer combination)
of accessions of the different genome types
||Dendrogram of the 60 barley accessions using AFLP data based
on Dices similarity coefficient and the UPGMA method
The protein banding pattern of the H-genome accessions was different from the
pattern of the I-genome accessions. According to the dendrogram produced from
the analysis of both water soluble and water insoluble protein, the barley accessions
were divided into two clusters similarly to the AFLP results (Fig.
||Part of water insoluble seed storage protein banding pattern
of barley accessions representing both Old and New Worlds species
||Dendrogram of 18 barley accessions using both water soluble
and water insoluble protein data based on Dices similarity coefficient
and the UPGMA tree building method
The first cluster contains the accessions with genome H (H. bulbosum
and H. vulgare spontaneum) and genome Xu (H. murinum)
like a subgroup of H. The second cluster consists of the accessions with the
genome I (H. bogdanii, H. brevisbulatum, H. chilense, H. jubatum and H. pusillum)
and the genome Xa (H. marinum) like a subgroup of I (Fig.
4). The reliability of these results was proven by the highly significant
correlation of cophenetic coefficient for the protein dendrogram (r = 0.89).
The genus Hordeum has generally been considered as a well defined and
easily recognized monophyletic plant group which is characterized by three one-flowered
spikelets at each rachis node, the two lateral ones are either rudimentary or
sterile and the central one is fertile in the two-rowed barley, or both of them
are fertile in the six-rowed barley. Earlier authors considered all wild species
to be fairly closely related to cultivated barley so they thought that all these
species constitute genetic resources for breeding purposes, even though rather
strong sterility barriers were found to operate (Von Bothmer
et al. (1995). Von Bothmer et al. (2003)
reported that triticeae represents a highly successful evolutionary branch in
the grass family (poaceae) and comprises a vast number of species and genera
and the numerous wild species are thus potential gene sources for cereal breeding.
Twenty-nine Mediterranean and 20 middle-Asian wild barley accessions were analysed
to reveal their location in the barley phylogeny. The phylogenetic analyses
based on the 339 AFLP bands and the 46 protein bands divided the studied taxa
into two main groups representing the H and the I genome type. It was also noted
that accessions of the same species were clustered together. These results are
consistent with the recently developed phylogenetic system (reviewed by Blattner,
2009), except that Xu and Xa are more clearly separated
there from the H, respectively I group than in our results. The four Hordeum
genome groups (H, I, Xa, Xu) are monophyletic and contain several allo- and
autopolyploidic species. The accessions in our study are mostly diploid, except
H. jubatum which is tetraploid and H. procerum which is hexaploid,
Both AFLP data and seed storage protein analyses succeeded in discriminating
the accessions according to the genome type and both methods came to the same
results. Thus, there are no basic differences between the phylogeny based on
AFLP or seed proteins, but there are small differences in the degree of relationships
when compared to the scheme by Blattner (2009). The
genetic diversity between the studied Mediterranean accessions was only half
of that found by Liu et al. (2002) with analysis
of ten allozymes. Zimmer and Wen (2012) reviewed the
current state of low and single-copy nuclear markers that have been applied
successfully in plant phylogenetics. They advocate the potential of massively
parallel high throughput or Next-Generation Sequencing approaches for future
molecular phylogenetic and evolutionary investigations.
The present study focussed especially on barley species and accessions which came from the Mediterranean area. We integrated them into the previously established phylogenetic tree, thus broadening the knowledge of the gene pool which is present in wild barley species from the North African to middle Asian origin.
This study was supported by Minufiya University, Sadat City, Minufiya, Egypt and King Abdulaziz University, Jeddah, KSA. The help in obtaining the samples from seed banks is gratefully acknowledged.
1: Aberg, E., 1940. The taxonomy and phylogeny of Hordeum L. sec. Cerealia ands, with special reference to Tibetan barleys. Symbolae Botanicae Upsalienses, 4: 1-156.
2: Blattner, F.R., 2004. Phylogenetic analysis of Hordeum (Poaceae) as inferred by nuclear rDNA ITS sequences. Mol. Phylogenet. Evol., 33: 289-299.
CrossRef | PubMed |
3: Blattner, F.R., 2009. Progress in phylogenetic analysis and a new infrageneric classification of the barley genus Hordeum (Poaceae: triticeae). Breed. Sci., 59: 471-480.
PubMed | Direct Link |
4: Von Bothmer, R., N. Jacobsen, C. Baden, R.B. Jargensen and I. Linde-Laursen, 1995. An Ecogeographical Study of the Genus Hordeum. 2nd Edn., International Plant Genetic Resources, Rome.
5: Von Bothmer, R., J. Flink and T. Landstrom, 1986. Meiosis in interspecific Hordeum hybrids. I. Diploid combinations. Can. J. Genet. Cytol., 28: 525-535.
Direct Link |
6: Von Bothmer, R., J. Flink and T. Landstrom, 1987. Meiosis in interspecific Hordeum hybrids. II. Triploid combinations. Evolut. Trends Plants, 1: 41-50.
7: Von Bothmer, R., K. Sato, T. Komatsuda, S. Yasuda and G. Fischbeck, 2003. The Domestication of Cultivated Barley. In: Diversity in Barley (Hordeum Vulgare), Von Bothmer, R., T. van Hintum, H. Knupffer and K. Sato (Eds.). Elsevier Science B.V., Amsterdam, Netherlands, PP: 9-27.
8: Dice, L.R., 1945. Measures of the amount of ecological association between species. Ecology, 26: 297-302.
Direct Link |
9: Jaaska, V., 1992. Isoenzyme variation in barley (Hordeum L.). Aspartate aminotransferase and 6-phosphoglyconate dehydrogenase. Hereditas, 116: 29-35.
10: Jorgensen, R.B., 1986. Relationships in the barley genus (Hordeum): An electrophoretic examination of proteins. Hereditas, 104: 273-291.
11: Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685.
CrossRef | Direct Link |
12: Liu, F., G.L. Sun and B. Salomon, 2002. Characterization of genetic diversity in core collection accessions of wild barley ( Hordeum vulgare sp. spontaneum ). Hereditas, 136: 67-73.
PubMed | Direct Link |
13: Love, A., 1984. Conspectus of the triticeae. Feddes Rep., 95: 425-521.
Direct Link |
14: Molnar, S.J., P.K. Gupta, G. Fedak and R. Wheatcroft, 1989. Ribosomal DNA repeat unit polymorphism in 25 Hordeum species. Theor. Applied Genet., 78: 387-392.
15: Molnar, S.J. and G. Fedak, 1989. Polymorphism in ribosomal DNA repeat units of 12 Hordeum species. Genome, 32: 1124-1127.
Direct Link |
16: Nevski, S.A., 1941. Beitrage zur kenntnis der wildwachsenden gersten in zusammenhang mit der Frage den Ursprung von Hordeum vulgare L. und Hordeum distichon L. zu klaren (Versuch einer Monographie der Gattung Hordeum Trudy). Botanski Inst. Akad. Nauk SSSR Ser., 15: 255-264.
17: Rohlf, F.J., 1998. NTSYS-pc. Numerical Taxonomy and Multivariate Analysis System. Exeter Software, Setauket, New York.
18: Saghai-Maroof, M.A., K.M. Soliman, R.A. Jorgensen and R.W. Allard, 1984. Ribosomal DNA spacer length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dynamics. Proc. Natl. Acad. Sci., 81: 8014-8019.
Direct Link |
19: Sneath, P.H.A. and R.R. Sokal, 1973. Numerical Taxonomy. WH Freeman and Co., San Francisco, USA.
20: Svitashev, S., T. Bryngelsson, A. Vershinin, C. Pedersen, T. Sall and R. von Bothmer, 1994. Phylogenetic analysis of the genus Hordeum using repetitive DNA sequences. Theor. Applied Genet., 89: 801-810.
Direct Link |
21: Vos, P., R. Hogers, M. Bleeker, M. Reijans and T. van de Lee et al., 1995. AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res., 23: 4407-4414.
CrossRef | PubMed | Direct Link |
22: Zimmer, E.A. and J. Wen, 2012. Using nuclear gene data for plant phylogenetics: Progress and prospects. Mol. Phylogenet. Evol., 65: 774-785.
CrossRef | PubMed |
23: El Rabey, H. and A.L. Al-Maliki, 2011. Application of randomly amplified polymorphic DNA (RAPD) markers and polyphenol oxidases (PPO) genes for distinguishing between the diploid (Glaucum) and the tetraploid (Leporinum) accessions in Hordeum murinum complex. Afr. J. Biotechnol., 10: 13064-13070.
Direct Link |
24: Gonzalez, J.M. and E. Ferrer, 1993. Random amplified polymorphic DNA analysis in Hordeum species. Genome, 36: 1029-1031.
PubMed | Direct Link |