Diversity within and between populations of two sympatrically distributed Hordeum species in Jordan

Summary The diversity of two sympatrically distributed barley species (Hordeum vulgare L. and Hordeum spontaneum C. Koch.) has been assessed for 7 morphometric and 13 qualitative traits. Phenotypically, Hordeum vulgare was more stable than Hordeum spontaneum for all morphometric traits; this is a reflection of domestication and selection. The overall diversity indexes were 0.546±0.125 and 0.502±0.113 for Hordeum vulgare and Hordeum spontaneum, respectively (P=0.095). However, Hordeum spontaneum expressed a higher level of diversity for most qualitative traits. The observed similarity for a number of diversity indexes is probably due to gene flow between the two species.     

Invasion of <Emphasis Type="Italic">Rhynchosporium commune</Emphasis> onto wild barley in the Middle East

Rhynchosporium commune was recently introduced into the Middle East, presumably with the cultivated host barley (Hordeum vulgare). Middle Eastern populations of R. commune on cultivated barley and wild barley (H. spontaneum) were genetically undifferentiated and shared a high proportion of multilocus haplotypes. This suggests that there has been little selection for host specialization on H. spontaneum, a host population often used as a source of resistance genes introduced into its domesticated counterpart, H. vulgare. Low levels of pathogen genetic diversity on H. vulgare as well as on H. spontaneum, indicate that the pathogen was introduced recently into the Middle East, perhaps through immigration on infected cultivated barley seeds, and then invaded the wild barley population. Although it has not been documented, the introduction of the pathogen into the Middle East may have a negative influence on the biodiversity of native Hordeum species.     

Islands and streams: clusters and gene flow in wild barley populations from the Levant

The domestication of plants frequently results in a high level of genetic differentiation between domesticated plants and their wild progenitors. This process is counteracted by gene flow between wild and domesticated plants because they are usually able to inter‐mate and to exchange genes. We investigated the extent of gene flow between wild barley Hordeum spontaneum and cultivated barley Hordeum vulgare, and its effect on population structure in wild barley by analysing a collection of 896 wild barley accessions (Barley1K) from Israel and all available Israeli H. vulgare accessions from the Israeli gene bank. We compared the performance of simple sequence repeats (SSR) and single nucleotide polymorphisms (SNP) marker data genotyped over a core collection in estimating population parameters. Estimates of gene flow rates with SSR markers indicated a high level of introgression from cultivated barley into wild barley. After removing accessions from the wild barley sample that were recently admixed with cultivated barley, the inference of population structure improved significantly. Both SSR and SNP markers showed that the genetic population structure of wild barley in Israel corresponds to the three major ecogeographic regions: the coast, the Mediterranean north and the deserts in the Jordan valley and the South. Gene flow rates were estimated to be higher from north to south than in the opposite direction. As has been observed in other crop species, there is a significant exchange of alleles between the wild species and domesticated varieties that needs to be accounted for in the population genetic analysis of domestication.     

Mitochondrial genome SNPs analysis of eight barley genotypes displaying hot spot regions,phylogenetic relationships and heteroplasmy

Abstract

Organellar genomes are small, circular entities that provide unique advantages as compared to the nuclear genome. The present study was aimed at evaluating the efficiency of utilizing mitochondrial single nucleotide polymorphisms (SNPs) approach in separating barley cultivars. Sequences generated via next-generation sequencing were further utilized to confirm the incidence of heteroplasmy in barley mitochondrial genome. The analysis involved seven cultivated barley (Hordeum vulgare subsp. vulgare) (VG) and one wild (H. vulgare subsp. spontaneum) (SP) genotypes. A total of 73 million paired-end reads per mitochondrial genomes across the eight barley genotypes were generated using Illumina HiSeq 2000 platform. Sequences of each genotype were separately aligned to the published barley mitochondrial reference genome, thus SNPs were detected. The overall results indicated the efficiency of using mitochondrial SNPs as a molecular marker in distinguishing among barley genotypes. Unique SNPs were determined in six out of the eight genotypes, where Giza131 and Giza129 had no specific mitochondrial SNPs, while Giza130 showed the largest number of unique mitochondrial SNPs. The phylogenetic tree indicated the close relationship between Giza129 and Giza130. Interestingly, SP was not clearly discriminated among genotypes.     

Genetic diversity among wild and cultivated barley as revealed by RFLP

Genetic variability of cultivated and wild barley, Hordeum vulgare ssp. vulgare and spontaneum, respectively, was assessed by RFLP analysis. The material consisted of 13 European varietes, single-plant offspring lines of eight land races from Ethiopia and Nepal, and five accessions of ssp. spontaneum from Israel, Iran and Turkey. Seventeen out of twenty-one studied cDNA and gDNA probes distributed across all seven barley chromosomes revealed polymorphism when DNA was digested with one of four restriction enzymes. A tree based on genetic distances using frequencies of RFLP banding patterns was estimated and the barley lines clustered into five groups reflecting geographical origin. The geographical groups of land-race lines showed less intragroup variation than the geographical groups of spontaneum lines. The group of European varieties, representing large variation in agronomic traits, showed an intermediate level. The proportion of gene diversity residing among geographical groups (F_ST) varied from 0.19 to 0.94 (average 0.54) per RFLP pattern, indicating large diversification between geographical groups.     

Seminal roots of wild and cultivated barley differentially respond to osmotic stress in gene expression,suberization, and hydraulic conductivity

Wild barley, Hordeum vulgare spp. spontaneum, has a wider genetic diversity than its cultivated progeny, Hordeum vulgare spp. vulgare. Osmotic stress leads to a series of different responses in wild barley seminal roots, ranging from no changes in suberization to enhanced endodermal suberization of certain zones and the formation of a suberized exodermis, which was not observed in the modern cultivars studied so far. Further, as a response to osmotic stress, the hydraulic conductivity of roots was not affected in wild barley, but it was 2.5-fold reduced in cultivated barley. In both subspecies, osmotic adjustment by increasing proline concentration and decreasing osmotic potential in roots was observed. RNA-sequencing indicated that the regulation of suberin biosynthesis and water transport via aquaporins were different between wild and cultivated barley. These results indicate that wild barley uses different strategies to cope with osmotic stress compared with cultivated barley. Thus, it seems that wild barley is better adapted to cope with osmotic stress by maintaining a significantly higher hydraulic conductivity of roots during water deficit.     

Repeated DNA sequences isolated by microdissection. II. Comparative analysis in Hordeum vulgare and Triticum aestivum

The genomic organization of two satellite DNA sequences, pHvMWG2314 and pHvMWG2315, of barley (Hordeum vulgare, 2n=14, HH) was studied by comparative in situ hybridization (ISH) and PCR analysis. Both sequences are members of different RsaI families. The sequence pHvMWG2314 is a new satellite element with a monomer unit of 73 bp which is moderately amplified in different grasses and occurs in interstitial clusters on D-genome chromosomes of hexaploid wheat (Triticum aestivum, 2n=42, AABBDD). The 331-bp monomer pHvMWG2315 belongs to a tandemly amplified repetitive sequence family that is present in the Poaceae and preferentially amplified in Aegilops squarrosa (2n=14, DD), H. vulgare and Agropyron elongatum. (2n=14, EE). The first described representative of this family was pAs 1 from Ae. squarrosa. Different sequences of one satellite DNA family were amplified from Ae. squarrosa, A. elongatum and H. vulgare using PCR. Characteristic differences between members of the D and H genome occurred in a variable region which is flanked by two conserved segments. The heterogeneity within this element was exploited for the cytogenetic analysis of Triticeae genomes and chromosomes. Comparative ISH with pHvMWG2315 identified individual wheat and barley chromosomes under low (75%) and high (85%) hybridization stringency in homologous and heterologous systems. We propose the designation Tas330 for the Triticeae amplified sequence (Tas) satellite family with a 330 bp average monomer length.     

Ecotypes and genetic divergence among sympatrically distributed populations of Hordeum vulgare and Hordeum spontaneum from the xeric region of Jordan

Summary The progeny of paired samples of Hordeum vulgare L. and Hordeum spontaneum C. Koch, collected from Jordan's xeric region was used in this study. Statistical analyses of seven easily measured morphometric traits were used to elucidate the relationships and distances between populations of both species, to detect any ecogeographical races, and to study the interrelationships and adjustments in the morphometric traits under study. Flag leaf area and plant height were the two most important discriminating variables which totally separated Hordeum vulgare from Hordeum spontaneum and accounted for 85.3% of total phenotypic variance in the collection. Cluster analysis indicated that the level of divergence among populations of both species was considerably different. Populations of Hordeum vulgare clustered at a maximum Euclidean distance of 2.08, while the maximum distance at which populations of Hordeum spontaneum clustered was 1.49. Three ecotypes each of Hordeum vulgare and Hordeum spontaneum were identified. These ecotypes corresponded to the environmental range of the collection sites. The interrelationships between the seven morphometric traits were adjusted in different ways as revealed by the principal components analysis. Sampling from the different clusters identified in this analysis is expected to increase the allelic diversity for selection and breeding purposes.     

Trends in comparative genetics and their potential impacts on wheat and barley research

We review some general points about comparative mapping, the evolution of gene families and recent advances in the understanding of angiosperm phylogeny. These are considered in relation to studies of large-genome cereals, particularly barley (Hordeum vulgare) and wheat (Triticum aestivum), with reference to methods of gene isolation. The relative merits of direct map-based cloning in barley and wheat, utilization of the smaller genome of rice (Oryza sativa) and gene homology methods that utilize information from model species such as Arabidopsis thaliana are briefly discussed.     

Genetic Diversity Analysis of Tibetan Wild Barley Using SSR Markers

One hundred and six accessions of wild barley collected from Tibet, China, including 50 entries of the two-rowed wild barley Hordeum vulgare ssp. spontaneum (HS), 29 entries of the six-rowed wild barley Hordeum vulgare ssp. agriocrithon (HA), and 27 entries of the six-rowed wild barley Hordeum vulgare ssp. agriocrithon var. lagunculiforme (HL), were analyzed using 30 SSR markers selected from the seven barley linkage groups for studying genetic diversity and evolutionary relationship of the three subspecies of Tibetan wild barley to cultivated barley in China. Over the 30 genetic loci that were studied, 229 alleles were identified among the 106 accessions, of which 70 were common alleles. H. vulgare ssp. spontaneum possesses about thrice more private alleles (2.83 alleles/locus) than HS (0.93 alleles/locus), whereas almost no private alleles were detected in HL. The genetic diversity among-subspecies is much higher than that within-subspecies. Generally, the genetic diversity among the three subspecies is of the order HS > HL > HA. Phylogenetic analysis of the 106 accessions showed that all the accessions of HS and HA was clustered in their own groups, whereas the 27 accessions of HL were separated into two groups (14 entries with group HS and the rest with group HA). This indicated that HL was an intermediate form between HS and HA. Based on this study and previous works, we suggested that Chinese cultivated barley might evolve from HS via HL to HA.     

The draft genome of a wild barley genotype reveals its enrichment in genes related to biotic and abiotic stresses compared to cultivated barley

Wild barley (Hordeum spontaneum) is the progenitor of cultivated barley (Hordeum vulgare) and provides a rich source of genetic variations for barley improvement. Currently, the genome sequences of wild barley and its differences with cultivated barley remain unclear. In this study, we report a high‐quality draft assembly of wild barley accession (AWCS276; henceforth named as WB1), which consists of 4.28 Gb genome and 36 395 high‐confidence protein‐coding genes. BUSCO analysis revealed that the assembly included full lengths of 95.3% of the 956 single‐copy plant genes, illustrating that the gene‐containing regions have been well assembled. By comparing with the genome of the cultivated genotype Morex, it is inferred that the WB1 genome contains more genes involved in resistance and tolerance to biotic and abiotic stresses. The presence of the numerous WB1‐specific genes indicates that, in addition to enhance allele diversity for genes already existing in the cultigen, exploiting the wild barley taxon in breeding should also allow the incorporation of novel genes. Furthermore, high levels of genetic variation in the pericentromeric regions were detected in chromosomes 3H and 5H between the wild and cultivated genotypes, which may be the results of domestication. This H. spontaneum draft genome assembly will help to accelerate wild barley research and be an invaluable resource for barley improvement and comparative genomics research.     

Chloroplast and Mitochondrial DNA Variation in HORDEUM VULGARE and HORDEUM SPONTANEUM

A survey of restriction fragment polymorphism in Hordeum vulgare and Hordeum spontaneum was made using 17 and 16 hexanucleotide restriction endonucleases on chloroplast (cp) and mitochondrial (mt) DNA, respectively. The plant accessions originated from various places throughout the Fertile Cresent and Mediterranean. The types of changes in cpDNA consisted of nucleotide substitutions and insertions and deletions on the order of 100 base pairs. In contrast, mtDNA has most likely undergone larger insertions and deletions of up to 20 kilobase pairs in addition to rearrangements. Grouping of mtDNA fragment data showed that in some cases geographical affinities existed between the two species, whereas in others there were no clear affinities. Nucleotide diversity estimates derived from the restriction fragment data were used in a number of comparisons of variability. Comparisons of overall mtDNA variability (nucleotide diversity = 9.68 x 10^-4) with cpDNA variability (nucleotide diversity = 6.38 x 10^-4 ) indicated that the former are somewhat more variable. Furthermore, there was no indication that the wild H. spontaneum (cpDNA diversity = 5.57 x 10^-4; mtDNA diversity = 6.04 x 10 ^-4) was more variable than the land races of H. vulgare (cpDNA diversity = 5.88 x 10^-4; mtDNA diversity = 9.79 x 10^-4). In fact, on the basis of mtDNA diversity, H. vulgare was the more variable species. Comparison of organelle nucleotide diversity estimates with an estimate of nuclear nucleotide diversity derived from existing isozyme data provided evidence that both organelle genomes are evolving at a slower rate than the nuclear genome.     

Allele-Dependent Barley Grain β-Amylase Activity

The wild ancestor of cultivated barley, Hordeum vulgare subsp. spontaneum (K. Koch) A. & Gr. (H. spontaneum), is a source of wide genetic diversity, including traits that are important for malting quality. A high β-amylase trait was previously identified in H. spontaneum strains from Israel, and transferred into the backcross progeny of a cross with the domesticated barley cv Adorra. We have used Southern-blot analysis and β-amy1 gene characterization to demonstrate that the high β-amylase trait in the backcross line is co-inherited with the β-amy1 gene from the H. spontaneum parent. We have analyzed the β-amy1 gene organization in various domesticated and wild-type barley strains and identified three distinct β-amy1 alleles. Two of these β-amy1 alleles were present in modern barley, one of which was specifically found in good malting barley cultivars. The third allele, linked with high grain β-amylase activity, was found only in a H. spontaneum strain from the Judean foothills in Israel. The sequences of three isolated β-amy1 alleles are compared. The involvement of specific intron III sequences, in particular a 126-bp palindromic insertion, in the allele-dependent expression of β-amylase activity in barley grain is proposed.     

Phenotypic landscapes: phenological patterns in wild and cultivated barley

Phenotypic variation in natural populations is the outcome of the joint effects of environmentally induced adaptations and neutral processes on the genetic architecture of quantitative traits. In this study, we examined the role of adaptation in shaping wild barley phenotypic variation along different environmental gradients. Detailed phenotyping of 164 wild barley (Hordeum spontaneum) accessions from Israel (of the Barley1K collection) and 18 cultivated barley (H. vulgare) varieties was conducted in common garden field trials. Cluster analysis based on phenotypic data indicated that wild barley in this region can be differentiated into three ecotypes in accordance with their ecogeographical distribution: north, coast and desert. Population differentiation (Q_ST) for each trait was estimated using a hierarchical Bayesian model and compared to neutral differentiation (F_ST) based on 42 microsatellite markers. This analysis indicated that the three clusters diverged in morphological but not in reproductive characteristics. To address the issue of phenotypic variation along environmental gradients, climatic and soil gradients were compared with each of the measured traits given the geographical distance between sampling sites using a partial Mantel test. Flowering time and plant growth were found to be differentially correlated with climatic and soil characteristic gradients, respectively. The H. vulgare varieties were superior to the H. spontaneum accessions in yield components, yet resembled the Mediterranean types in vegetative characteristics and flowering time, which may indicate the geographical origin of domesticated barley.     

The Horn of Africa as a centre of barley diversification and a potential domestication site

According to a widely accepted theory on barley domestication, wild barley (Hordeum vulgare ssp. spontaneum) from the Fertile Crescent is the progenitor of all cultivated barley (H. vulgare ssp. vulgare). To determine whether barley has undergone one or more domestication events, barley accessions from three continents have been studied (a) using 38 nuclear SSR (nuSSRs) markers, (b) using five chloroplast SSR (cpSSR) markers yielding 5 polymorphic loci and (c) by detecting the differences in a 468 bp fragment from the non-coding region of chloroplast DNA. A clear separation was found between Eritrean/Ethiopian barley and barley from West Asia and North Africa (WANA) as well as from Europe. The data from chloroplast DNA clearly indicate that the wild barley (H. vulgare ssp. spontaneum) as it is found today in the “Fertile Crescent” might not be the progenitor of the barley cultivated in Eritrea (and Ethiopia). Consequently, an independent domestication might have taken place at the Horn of Africa. Jihad Orabi and Gunter Backes have contributed equally to this work.     

Estimating the outcrossing rate of barley landraces and wild barley populations collected from ecologically different regions of Jordan

The results of previous studies conducted at the University of Hohenheim and the International Center for Agricultural Research in the Dry Areas (ICARDA) indicated that the yielding ability and stability of barley (Hordeum vulgare L.) could be improved in environments with drought stress by increasing the level of heterozygosity. This would require increasing the outbreeding rate of locally adapted breeding materials. As a first step, we estimated the outcrossing rate of 12 barley landraces (Hordeum vulgare ssp. vulgare, in short H. vulgare) and 13 sympatrically occurring populations of its wild progenitor [Hordeum vulgare ssp. spontaneum (C. Koch), in short H. spontaneum] collected from semi-arid localities in Jordan during the 1999/2000 growing season. In each H. vulgare or H. spontaneum population 28–48 spikes were sampled, and up to six offspring (seeds) per spike (called a family) were used for PCR analyses. Collection sites covered high–low transects for rainfall and altitude in order to detect possible environmental effects on the outcrossing rate. Four microsatellite markers located on different chromosomes were used to genotype the samples for estimating the outcrossing rate. Low season-specific multilocus outcrossing rates (t_m) were found in both cultivated and wild barley, ranging among populations from 0–1.8% with a mean of 0.34%. Outcrossing rates based on inbreeding equilibrium (t_e), indicating outcrossing averaged across years, were two- to threefold higher than the season-specific estimates. Under high rainfall conditions somewhat higher—though not significantly higher—outcrossing rates were observed in H. spontaneum than in H. vulgare. The season-specific outcrossing rate in H. spontaneum was positively correlated (r=0.67, P=0.01) with average annual precipitation and negatively correlated (r=0.59, P=0.05) with monthly average temperature during flowering. The results suggest that outcrossing may vary considerably among seasons and that high precipitation and cool temperatures during flowering tend to enhance outcrossing. The rather low levels of outcrossing detected indicate that increased vigour due to heterozygosity has not been a major fitness advantage in the evolution and domestication of H. spontaneum and H. vulgare, respectively. Stable seed production to secure survival under extreme heat and drought stress may have been more important. Cleistogamy may be considered as an effective mechanism to warrant pollination even in drought-stunted plants with non-extruding spikes.     

Evolutionary modes of emergence of short interspersed nuclear element (SINE) families in grasses

Short interspersed nuclear elements (SINEs) are non‐autonomous transposable elements which are propagated by retrotransposition and constitute an inherent part of the genome of most eukaryotic species. Knowledge of heterogeneous and highly abundant SINEs is crucial for de novo (or improvement of) annotation of whole genome sequences. We scanned Poaceae genome sequences of six important cereals (Oryza sativa, Triticum aestivum, Hordeum vulgare, Panicum virgatum, Sorghum bicolor, Zea mays) and Brachypodium distachyon to examine the diversity and evolution of SINE populations. We comparatively analyzed the structural features, distribution, evolutionary relation and abundance of 32 SINE families and subfamilies within grasses, comprising 11 052 individual copies. The investigation of activity profiles within the Poaceae provides insights into their species‐specific diversification and amplification. We found that Poaceae SINEs (PoaS) fall into two length categories: simple SINEs of up to 180 bp and dimeric SINEs larger than 240 bp. Detailed analysis at the nucleotide level revealed that multimerization of related and unrelated SINE copies is an important evolutionary mechanism of SINE formation. We conclude that PoaS families diversify by massive reshuffling between SINE families, likely caused by insertion of truncated copies, and provide a model for this evolutionary scenario. Twenty‐eight of 32 PoaS families and subfamilies show significant conservation, in particular either in the 5′ or 3′ regions, across Poaceae species and share large sequence stretches with one or more other PoaS families.     

TEnest: automated chronological annotation and visualization of nested plant transposable elements

Organisms with a high density of transposable elements (TEs) exhibit nesting, with subsequent repeats found inside previously inserted elements. Nesting splits the sequence structure of TEs and makes annotation of repetitive areas challenging. We present TEnest, a repeat identification and display tool made specifically for highly repetitive genomes. TEnest identifies repetitive sequences and reconstructs separated sections to provide full-length repeats and, for long-terminal repeat (LTR) retrotransposons, calculates age since insertion based on LTR divergence. TEnest provides a chronological insertion display to give an accurate visual representation of TE integration history showing timeline, location, and families of each TE identified, thus creating a framework from which evolutionary comparisons can be made among various regions of the genome. A database of repeats has been developed for maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), and barley (Hordeum vulgare) to illustrate the potential of TEnest software. All currently finished maize bacterial artificial chromosomes totaling 29.3 Mb were analyzed with TEnest to provide a characterization of the repeat insertions. Sixty-seven percent of the maize genome was found to be made up of TEs; of these, 95% are LTR retrotransposons. The rate of solo LTR formation is shown to be dissimilar across retrotransposon families. Phylogenetic analysis of TE families reveals specific events of extreme TE proliferation, which may explain the high quantities of certain TE families found throughout the maize genome. The TEnest software package is available for use on PlantGDB under the tools section (http://www.plantgdb.org/prj/TE_nest/TE_nest.html); the source code is available from (http://wiselab.org).     

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.     

A barley RFLP map: alignment of three barley maps and comparisons to Gramineae species

Several gene linkage maps have been produced for cultivated barley. We have produced a new linkage map for barley, based on a cross between Hordeum vulgare subsp. spontaneum and Hordeum vulgare subsp. vulgare (Hvs x Hvv), having a higher level of polymorphism than most of the previous barley crosses used for RFLP mapping. Of 133 markers mapped in the Hvs x Hvv F₂ population, 69 were previously mapped on other barley maps, and 26 were mapped in rice, maize, or wheat. Two known gene clones were mapped as well as two morphological markers. The distributions of previously mapped markers were compared with their respective barley maps to align the different maps into one consensus map. The distributions of common markers among barley, wheat, rice and maize were also compared, indicating colinear linkage groups among these species.To be considered dual first authorsPublished with the approval of the Director of the Colorado State University/Agricultural Experiment Station.