AFLP genetic polymorphism in wild barley (Hordeum spontaneum) populations in Israel

The genetic diversity produced by the amplified fragment length polymorphism (AFLP) method was studied in 94 genotypes of wild barley, Hordeum spontaneum (C. Koch) Thell., originating from ten ecologically and geographically different locations in Israel. Eight primer pairs produced 204 discernible loci of which 189 (93%) were polymorphic. Each genotype had a unique banding profile and the genetic similarity coefficient varied between 0.74 and 0.98. The phenogram generated from these similarities by the UPGMA method did not group genotypes strictly according to their geographical origin, which pattern was also seen in the principal coordinate (PCO) plot. Genetic diversity was larger within (69%) than among (31%) populations. Associations between ecogeographical variables and the mean gene diversity were found at one primer pair. The results are discussed and compared with data obtained by the simple sequence repeat (SSR) method.     

DNA sequence variation of wild barley Hordeum spontaneum (L.) across environmental gradients in Israel

Wild barley Hordeum spontaneum (L.) shows a wide geographic distribution and ecological diversity. A key question concerns the spatial scale at which genetic differentiation occurs and to what extent it is driven by natural selection. The Levant region exhibits a strong ecological gradient along the North–South axis, with numerous small canyons in an East–West direction and with small-scale environmental gradients on the opposing North- and South-facing slopes. We sequenced 34 short genomic regions in 54 accessions of wild barley collected throughout Israel and from the opposing slopes of two canyons. The nucleotide diversity of the total sample is 0.0042, which is about two-thirds of a sample from the whole species range (0.0060). Thirty accessions collected at ‘Evolution Canyon'' (EC) at Nahal Oren, close to Haifa, have a nucleotide diversity of 0.0036, and therefore harbor a large proportion of the genetic diversity. There is a high level of genetic clustering throughout Israel and within EC, which roughly differentiates the slopes. Accessions from the hot and dry South-facing slope have significantly reduced genetic diversity and are genetically more distinct from accessions from the North-facing slope, which are more similar to accessions from other regions in Northern Israel. Statistical population models indicate that wild barley within the EC consist of three separate genetic clusters with substantial gene flow. The data indicate a high level of population structure at large and small geographic scales that shows isolation-by-distance, and is also consistent with ongoing natural selection contributing to genetic differentiation at a small geographic scale.     

Adaptive microclimatic structural and expressional dehydrin 1 evolution in wild barley, Hordeum spontaneum, at 'Evolution Canyon', Mount Carmel, Israel

'Evolution Canyon' (ECI) at Lower Nahal Oren, Mount Carmel, Israel, is an optimal natural microscale model for unravelling evolution in action highlighting the twin evolutionary processes of adaptation and speciation. A major model organism in ECI is wild barley, Hordeum spontaneum , the progenitor of cultivated barley, which displays dramatic interslope adaptive and speciational divergence on the 'African' dry slope (AS) and the 'European' humid slope (ES), separated on average by 200 m. Here we examined interslope single nucleotide polymorphism (SNP) sequences and the expression diversity of the drought resistant dehydrin 1 gene ( Dhn1 ) between the opposite slopes. We analysed 47 plants (genotypes), 4–10 individuals in each of seven stations (populations) in an area of 7000 m², for Dhn1 sequence diversity located in the 5' upstream flanking region of the gene. We found significant levels of Dhn1 genic diversity represented by 29 haplotypes, derived from 45 SNPs in a total of 708 bp sites. Most of the haplotypes, 25 out of 29 (= 86.2%), were represented by one genotype; hence, unique to one population. Only a single haplotype was common to both slopes. Genetic divergence of sequence and haplotype diversity was generally and significantly different among the populations and slopes. Nucleotide diversity was higher on the AS, whereas haplotype diversity was higher on the ES. Interslope divergence was significantly higher than intraslope divergence. The applied Tajima D rejected neutrality of the SNP diversity. The Dhn1 expression under dehydration indicated interslope divergent expression between AS and ES genotypes, reinforcing Dhn1 associated with drought resistance of wild barley at 'Evolution Canyon'. These results are inexplicable by mutation, gene flow, or chance effects, and support adaptive natural microclimatic selection as the major evolutionary divergent driving force.     

Protein resources in wild barley,Hordeum spontaneum,in Israel: Predictive method by ecology and allozyme markers

Geographic variation in protein content of wild barley,Hordeum spontaneum, and the associations of protein content with ecological and allozyme markers were tested in an attempt to derive predictive guidelines for conservation and utilization in breeding programs. The study involved 195 genotypes of wild barley from 25 populations, 15 central and 10 marginal. These populations had been tested earlier for allozymic variation (Nevo & al. 1979 a, b). The results indicate that protein content varies both within, but particularly between populations. Notably, the 10 marginal populations exhibit high protein content but low kernel weight, as compared with the 15 central populations which displayed lower protein content but high kernel weight. Three variable combinations of climatic factors explain 40% of the variability in protein content among populations. Likewise, 3 variable combinations of allozyme allele frequencies explain a significant degree of spatial variance in protein content (R square = 0.63). — We conclude that natural populations of wild barley in Israel contain large amounts of yet untapped genes for protein content. These could be effectively screened and utilized for producing high protein cultivars of barley by following ecological and allozymic markers as predictive guidelines in screening natural populations of wild barley.     

Heritable somaclonal variation in wild barley (Hordeum spontaneum)

Summary Progenies of H. spontaneum plants regenerated from immature embryo derived calli were analysed for somaclonal variation on the following traits: (1) organization of the intergenic spacer of the rRNA genes; (2) B and C hordein pattern on SDS-PAGE; (3) genomic organization of the B and C hordein coding sequences; (4) mitochondrial DNA organization assayed by hybridization of Southern blots of total DNA with mitochondrial coding genes; (5) cytology. One out of twelve progeny plants was characterized as variant for two traits: (a) a loss of 1.8 and 2.5 kb Taq I intergenic rDNA spacer fragments and (b) a variant pattern of hordeins on 1-D SDS-PAGE. No numerical or structural chromosome variation was detected among the control plants therefore it is assumed that the variation was caused by the in vitro culture and transmitted, through sexual reproduction, to the analysed progeny.     

Ecological-genomic diversity of microsatellites in wild barley,Hordeum spontaneum,populations in Jordan

We analyzed the ecological-genomic diversity of microsatellites of wild barley, Hordeum spontaneum (C. Koch) Thell., at 18 loci in 306 individuals of 16 populations from Jordan across a southward transect of increasing aridity. The 18 microsatellites revealed a total of 249 alleles, with an average of 13.8 alleles per locus (range 3-29), with nonrandom distribution. The proportion of polymorphic loci per population averaged 0.91 (range 0.83-1.00); gene diversity, He, averaged 0.512 (range 0.38-0.651). We compared the number of alleles of the 18 loci to those found in Israel populations by Turpeinen et al. Out of the 280 alleles, 138 (49.3%) were unique (i.e. occurred in only one of the countries). The percentage of unique alleles in Jordan and Israel populations was 43.0% and 17.9%, respectively, suggesting that Jordan is an important center of origin and diversity of wild barley. Estimates of mean gene diversity were highest in the populations collected near the Golan Heights, such as Shuni North, Shuni South and Jarash. Sixty nine percent of the microsatellite variation was partitioned within populations and 31% between populations. Associations between ecogeographical values and gene diversity were established for eight microsatellite loci. The cluster produced by simple sequence repeat (SSR) data is mostly coincidence with the result of the dendrogram of the Spalax ehrenbergi superspecies of subterranean mole rats in Jordan based on allozyme gene loci. The major soil type in the wild barley habitat of each ecological group was different. Stepwise multiple regression analysis indicated that the variance of gene diversity was explained by altitude (R(2) = 0.362**). These observations suggest that microsatellites are at least partly adaptive and subject to natural selection.     

Genetic diversity and environmental associations of wild barley,Hordeum spontaneum,in Turkey

Genetic variability within and between populations of the wild progenitor of barley was studied electrophoretically. Thirty enzyme loci were assayed in 437 individuals representing 11 populations ofH. spontaneum in Turkey. The results indicated that: (a)H. spontaneum in Turkey is genetically rich in allozyme variation, but because of predominant self-pollination the variation is maintained as different homozygotes in the population; (b) genetic differentiation of populations includes clinal, regional, and local patterns, sometimes displaying sharp geographic differentiation over short distances; (c) overall indices of allozymic diversity and some allele frequencies of wild barley are significantly correlated with the environment and are predictable ecologically, chiefly by combinations of temperature and humidity variables; (d) a high percentage of alleles (66%) occur in local areas or are distributed sporadically rather than widespread; (e) Wright Fixation index was very high, F=0.995; (f) genetic distance was high (D=0.11, ranging from 0.031 to 0.288) between populations, and (g) average relative genetic differentiation was high among populations (Gst=0.47, ranging from 0.02 to 0.66). The spatial patterns and environmental correlates and predictors of genetic variation ofH. spontaneum in Turkey, indicated that genetic variation in wild barley populations is not only common, but also at least partly, adaptive. Therefore, a much fuller exploitation of these genetic resources by breeding is warranted.     

Characterization of genetic diversity in core collection accessions of wild barley,Hordeum vulgare ssp. spontaneum

Genetic variability in the 143 core accessions of wild barley, Hordeum vulgare ssp. spontaneum, was assessed by allozyme analysis. A total of 34 alleles were detected at ten isozyme loci. All loci were polymorphic except Pgd-1, which was monomorphic. Est-2 and Est-4 were the most diverse loci, with genetic diversity values of 0.747 and 0.686, respectively. The comparison of the results with those of previous studies indicates that all alleles occurring in cultivated and wild barley are observed in this set of the wild Barley Core Collection. Only one allele (Pgd-1 Tj) was absent. It is noteworthy that one new allele at the Ndh-2 locus and another new allele at Aco-2 locus were first detected in the present study. Nine of the 34 alleles were rare and detected only in one to four accessions. The genetic similarities among the 143 accessions ranged from 0.18 to 1.00. Data analysis based on clustering and principal coordinate analysis showed that a high level of genetic variability exists in this set of core accessions, and indicated that some duplication probably exists in this set core based on the present study.     

Microgeographic edaphic differentiation in allozyme polymorphisms of wild barley (Hordeum spontaneum,Poaceae)

Allozymic variation in proteins encoded by 22 loci was analyzed electrophoretically in 278 individual plants of wild barley,Hordeum spontaneum, the progenitor of cultivated barley, in four 100 meter transects, in Israel, each equally subdivided into basalt and terra rossa soil types. Significant differentiation according to soil was found in 9 alleles. Our results suggest that allozyme polymorphisms in wild barley are at least partly adaptive, and differentiate by edaphic natural selection rather than by stochastic processes, and/or neutrality of allozymic variants.     

Genetic diversity and environmental associations of wild barley,Hordeum spontaneum (Poaceae), in Iran

Genetic diversity and structure of populations of the wild progenitor of barleyHordeum spontaneum in Iran was studied by electrophoretically discernible allozymic variation in proteins encoded by 30 gene loci in 509 individuals representing 13 populations of wild barley. The results indicate that: a)Hordeum spontaneum in Iran is extremely rich genetically but, because of predominant self-pollination, the variation is carried primarily by different homozygotes in the population. Thus, genetic indices of polymorphismP-1% = 0.375, range = 0.267–0.500, and of genetic diversity,He = 0.134, range = 0.069–0.198, are very high. b) Genetic differentiation of populations includes clinal, regional and local patterns, sometimes displaying sharp geographic differentiation over short distances. The average relative differentiation among populations isGst = 0.28, range = 0.02–0.61. c) A substantial portion of the patterns of allozyme variation in the wild gene pool is significanctly correlated with the environment and is predictable ecologically, chiefly by combinations of temperature and humidity variables. d) The natural populations studied, on the average, are more variable than two composite crosses, and more variable than indigenous land races of cultivated barely,Hordeum vulgare, in Iran. — The spatial patterns and environmental correlates and predictors of genetic variation ofH. spontaneum in Iran indicate that genetic variation in wild barley populations is not only rich but also at least partly adaptive. Therefore, a much fuller exploitation of these genetic resources by breeding for disease resistance and economically important agronomic traits is warranted.     

Haploid plants regenerated via anther culture in wild barley (Hordeum spontaneum C. Kock)

Summary Androgenic plants have been obtained via anther culture in four natural populations of Hordeum spontaneum. Microscopic observations revealed that androgenesis started with the formation of two vegetative-type nuclei derived from the mitotic division of the uninucleate microspores. In this species androgenesis was affected by the type and concentration of the sugars added to the culture medium: the highest response (17% of callusing anthers) was observed on media containing 80 g l^–1 maltose. The highest production of androgenic plants (per 100 anthers, 5.9 green and 4.3 albino plants) was obtained from callus grown on these same media. About half of the green plants regenerated were haploid, while the others were diploid and set seed.Abbreviations IAA indolacetic acid - BAP 6-benzylaminopurine     

High crossability of wild barley (Hordeum spontaneum C. Koch) with bread wheat and the differential elimination of barley chromosomes in the hybrids

Four bread wheat (Triticum aestivum L.) cultivars, Aobakomugi, Chinese Spring, Norin 61 and Shinchunaga, were pollinated with five barley lines/cultivars consisting of three cultivated barley (Hordeum vulgare L.) lines, Betzes, Kinai 5 and OHL089, and two wild barley (Hordeum spontaneum C. Koch) lines, OUH602 and OUH324. Crossability, expressed as the percentage of embryo formation, varied from 0 to 55.4% among the cross combinations. The two wild barley lines generally had a higher crossability than the previously reported best pollinator, Betzes, and some Japanese wheat cultivars were better as the female parent than Chinese Spring. Ninety four hybrid plants were obtained from 250 embryos cultured, and their somatic chromosome numbers ranged from 21 to 36. Eighteen plants were mosaic in chromosome number. Twenty one-chromosome plants appeared most frequently (45.7%) followed by 28-chromosome plants (14.9%). C-banding analysis revealed that elimination of barley chromosomes was mainly responsible for the occurrence of aneuploid plants. In hypoploids derived from Betzes-crosses, chromosome 5 was preferentially eliminated as previously reported, while in hypoploids derived from OUH602-crosses, chromosome 4 was preferentially eliminated. The wild barley line OUH602 may be a useful parent for producing a new wheat-barley addition set because of its high crossability with wheat and a different pattern of chromosome elimination.     

Natural selection causing microsatellite divergence in wild emmer wheat at the ecologically variable microsite at Ammiad, Israel

Genetic diversity at 28 microsatellite loci was studied in a natural population of Triticum dicoccoides at the Ammiad microsite, north of the Sea of Galilee, Israel. This microsite was subdivided into four major habitats, North, Valley, Ridge and Karst, and further subdivided into nine subhabitats. The units thus defined showed strong and highly significant differentiation in ecological factors; in particular with respect to cover, proximity and height of rocks, and surface soil moisture after early rains. The results showed that allele distributions at microsatellite loci were nonrandom and associated with habitats. Significant genetic differentiation and variation in repeat number were found among subpopulations in the four major habitats and nine subhabitats. Habitat-specific and -unique alleles and linkage disequilibria were observed in the Karst subpopulation. The subpopulations dwelling in drier habitats and subhabitats showed higher genetic diversities at microsatellite loci. These results suggest that natural selection, presumably through aridity stress, acts upon microsatellite divergence predominantly on noncoding sequences, thereby contributing to differences in fitness. Received: 9 September 1999 / Accepted: 16 September 1999     

Differential selection of growth rate-related traits in wild barley, Hordeum spontaneum, in contrasting greenhouse nutrient environments

Across-species comparisons show that inherent variation in relative growth rate (RGR) and its underlying traits are correlated with habitat productivity. In this study, we test the hypothesis that growth rate-related traits confer differential selective effects in contrasting nutrient environments. We specifically test whether high RGR is targeted by selection in nutrient-rich environments whereas low values of traits that underlie RGR [specific leaf area (SLA), leaf mass fraction and leaf area ratio (LAR)] confer a direct fitness advantage in nutrient-poor environments, resulting in selection of low RGR as a correlated response. We measured RGR, its underlying component traits, and estimated fitness in a range of wild barley (Hordeum spontaneum) accessions grown under high and low nutrient conditions. Selection on component traits differed between the two environments, while total selection of RGR was not significant. Using multiple regression and path analysis to estimate direct fitness effects, a selective advantage of high LAR and SLA was demonstrated only under nutrient-rich conditions. While supporting the view that observed associations between habitat richness and some RGR-component traits reflect adaptation to differing nutrient regimes, our data suggest that direct selection targets component traits rather than RGR itself.     

Edaphic microsatellite DNA divergence in wild emmer wheat, Triticum dicoccoides, at a microsite: Tabigha, Israel

Twenty eight microsatellite markers were used to analyze genetic divergence in tandem dinucleotide repeated DNA regions between two edaphic subpopulations of Triticum dicoccoides growing on the contrasting terra rossa and basalt soilsfrom a microsite at Tabigha, north of the Sea of Galilee, Israel. The terra rossa soil niche was drier and more stressful than the basalt throughout the growing season (November to May). Significant microsatellite divergence in allele distribution, repeat length, genetic diversity, and linkage disequilibria were found between emmer wheat from the two soil types over two short transects of 100 m each. Soil-specific and -unique alleles and linkage disequilibria were observed in the terra rossa and basalt subpopulations. A permutation test showed that the effects of random genetic drift were very low for the significant genetic diversity at microsatellite loci between the two subpopulations, suggesting that an adaptive molecular pattern derived by edaphic selection may act upon variation of the microsatellites. Received: 4 February 2000 / Accepted: 31 March 2000<@head-com-p1a.lf>Communicated by H.F. Linskens     

Grain protein variability among populations of wild barley (Hordeum spontaneum C. Koch.) from Jordan

Summary Protein content, kernel weight, and genetic diversity in the storage protein hordein, encoded by the Hor 1 and Hor 2 loci, were assessed in 12 populations of wild barley (Hordeum spontaneum C. Koch.) collected from central, peripheral, and marginal areas of its distribution in Jordan. Protein content ranged from 106.3 to 239.1 g kg^-1, and kernel weight ranged from 21.17 to 31.8 mg. Populations with high protein content and heavy kernels have been identified. Electrophoretic analysis of the storage protein hordein showed that the two hordein loci, Hor 1 and Hor 2, are highly polymorphic, having 34 and 38 alleles, respectively. Polymorphism (H_e) was highest in central populations (H_e Hor 1=0.859, H_e Hor 2=0.782), intermediate in peripheral populations (H_e Hor 1=0.566, H_e Hor 2=0.509), and lowest in marginal populations (H_e Hor 1=0.392, H_e Hor 2=0.349). Geographical distances between populations were not indicative of Nei's genetic similarity (NI). NI values averaged 0.209 and ranged from 0.0 to 0.83, supporting the hypothesis of an island population model for the species. The high proportion of allelic diversity, apportioned among populations for Hor 1 (0.584) and Hor 2 (0.495) loci, indicates that these natural populations are a rich reserve of genetic variability for protein. This variability is readily exploitable in breeding.     

Genetic analysis of the accumulation of COR14 proteins in wild (Hordeum spontaneum) and cultivated (Hordeum vulgare) barley

The cold-regulated (COR14) protein of 14 kDa is a polypeptide accumulated under low-temperature conditions in the chloroplasts of barley leaves. In H. vulgare the COR14 antibody cross-reacts with two proteins, with a slightly different relative molecular weight around the marker of 14.4 kDa, referred to as COR14a and COR14b (high and low relative molecular weight, respectively). In a collection of H. spontaneum genotypes a clear polymorphism was found for the corresponding COR proteins. While some accessions showed the same COR pattern as cultivated barley, in 38 out of 61 accessions examined the COR14 antibody cross-reacted with an additional coldregulated protein with a relative molecular weight of about 24 kDa (COR24). The accumulation of COR24 was often associated with the absence of COR14b; the relationship between the COR14b/COR24 polymorphism and the adaptation of H. spontaneum to different environments is discussed. By studying COR14 accumulation in cultivated barley we have found that the threshold induction-temperature of COR14a is associated with the loci controlling winter hardiness. This association was demonstrated by using either a set of 30 cultivars of different origin, or two sets of frost-tolerant and frost-sensitive F1 doubled-haploid lines derived from the cross Dicktoo (winter type) x Morex (spring type). These results suggest that the threshold induction-temperature of COR14a can be a potential biochemical marker for the identification of superior frostresistant barley genotypes.     

Detection and quantification of Paenibacillus polymyxa in the rhizosphere of wild barley (Hordeum spontaneum) with real-time PCR

Aim:  To detect and quantify the plant drought tolerance enhancing bacterium Paenibacillus polymyxa in a collection of 160 Hordeum spontaneum rhizosphere samples at the 'Evolution Canyon' ('EC'), Israel.
Methods and Results:  PCR primers and a FAM-TAMRA probe (6-carboxyfluorescein, 6-carboxy-tetramethyl-rhodamine) targeting 16S rRNA genes were designed and used to detect and quantify the target strain. Two commercial kits, Bio101 Fast Spin and Mo Bio Ultra Clean Soil DNA, were tested for DNA isolation from the rhizosphere and surrounding soil. Population densities of P. polymyxa were studied in the rhizosphere of wild barley and surrounding soil from the contrasting climatic slopes at the 'EC' using the real-time PCR and culture based methods.
Conclusion:  Paenibacillus polymyxa is one of the best established species in wild barley rhizosphere at the 'EC' slopes. With the real-time PCR assay we are able to detect 1 pg of DNA per PCR corresponding to 100 cells per ml. The results at the 'EC' correlate well to bacterial estimations by culture based methods.
Significance and Impact of the Study:  Significantly higher P. polymyxa cell number was detected in the rhizosphere of arid 'African' microclimate indicating possible role of adaptive co-evolution with plants.