RAPD Analysis of the Genome in Species of the Genus Hordeum

Random amplification of polymorphic DNA (RAPD) was used to analyze six species, three populations, and seven regional cultivars of barley. A unique pattern of amplified DNA products was obtained for each species of the genus Hordeum.High polymorphism of barley species was revealed. Specific fragments were found in most RAPD patterns; the fragments can be used as molecular markers of corresponding species and subspecies. Several other DNA fragments were shown to serve as molecular markers of the H genome. Specific RAPD patterns were obtained for each population and each cultivar of H. vulgaresensu lato. In total, variation between the populations and between the cultivars was substantially lower than between species. Cluster analysis (UPGMA) was used to estimate genetic distances between theHordeumspecies, between the H. spontaneumpopulations, and between regional H. vulgarecultivars and a dendrogram was constructed.     

大麦6H染色体特异性标记的筛选和鉴定

从大麦、小麦和小麦－大麦６Ｈ染色体附加系ＲＡＰＤ分析筛选出对６Ｈ染色体特异的２个ＲＡＰＤ标记,转换为特异性ＰＣＲ标记,利用标记对不同植物材料进行ＰＣＲ扩增鉴定。表明凡含有大麦６Ｈ染色体的材料（Ｂｅｔｚｅｓ、Ｉｇｒｉ、ＣＳ６Ｈ附加系）均能扩增出特异带;而不含６Ｈ染色体的材料,包括小科、黑麦、长穗偃麦草、中间偃麦草、簇毛麦以及含有其他大麦染色体的小麦附加系均不主增出特异带。可见,２对ＰＣＲ引物具有大麦     

The use of RAPD markers in Hordeum phylogeny.

The phylogenetic relationships among 39 wild Hordeum species, subspecies, and cultivated barley were investigated using RAPD markers as discriminating characters. Seventy-six RAPD fragments were generated using 12 single decameric primers of arbitrary nucleotide sequences. Amplification reactions resulted in fragments ranging in length between 200 and 2000 bp. Clearly resolved bands were scored for their presence or absence in a binary matrix. Amplified products were treated as independent characters to generate a phenogram using the NTSYS-PC package. Tree topology was generally found to be consistent with those based on morphological treatments. However, a few species like H. erectifolium, H. jubatum and, to a lesser extent, H. bulbosum occupied a position different from previous classifications. The results demonstrated that RAPD technology represents a useful and reliable tool for detecting polymorphism for phylogenetic studies. Key words : RAPD analysis, molecular markers, phylogenetic studies, Hordeum species, barley.     

Genomic differentiation of Hordeum chilense from H. vulgare as revealed by repetitive and EST sequences

Hordeum vulgare, cultivated barley, and its wild relative, H. chilense, have several important traits that might be useful for wheat improvement. Here, in situ hybridization and barley expressed sequence tag (EST) markers were used to characterize and compare the chromosomes of H. chilense with those of H. vulgare. FISH with four repetitive DNA sequences, AG, AAG, 5S rDNA and 45S rDNA, was applied to the mitotic chromosomes of H. vulgare, H. chilense and available wheat-H. chilense addition and substitution lines. FISH with the AAG repeat differentiated the individual chromosomes of H. chilense and H. vulgare. The patterns of FISH signals in the two species differed greatly. The 45S rDNA signals were observed on two pairs of chromosomes in both species, while the 5S rDNA signals were observed on four pairs of chromosomes in H. vulgare and on one pair in H. chilense. The AG repeat showed FISH signals at the centromeric regions of all chromosomes of H. vulgare but none of the chromosomes of H. chilense. These results indicate that the chromosomes of the two species are highly differentiated. To study the homoeology between the two species, 209 EST markers of H. vulgare were allocated to individual chromosomes of H. chilense. One hundred and forty of the EST markers were allocated to respective chromosomes of H. chilense using the wheat-H. chilense addition and substitution lines. Twenty-six EST markers on average were allocated to each chromosome except to the chromosome 2H(ch)S, to which only 10 markers were allocated. Ninety percent of the allocated EST markers in H. chilense were placed on H. vulgare chromosomes of the same homo-eologous group, indicating that the expressed sequences of the two species were highly conserved. These EST markers would be useful for detecting chromatin introgressed from these species into the wheat genome.     

Genetic and geographic variation of bulbous barley (Hordeum bulbosum L.) assessed by rapd markers

Estimated the genetic relationships among 21 barley accessions from 17 bulbous barley (H. bulbosum L.), 4 cultivated barley (H. vulgare L.) collected from different part of Turkey were investigated using Random Amplified Polymorphic DNA (RAPD). Eleven informative primers amplified 111 markers of which 98 (89.8%) were polymorphic. A dendogram was constructed using the UPGMA method based on the RAPD markers. The range of genetic similarity was from 0.111 to 0.815. The accessions were grouped into two main clusters based on the molecular data. The H. vulgare and H. bulbosum separated into two groups in the Principle Component Analysis.     

Identification of wild and cultivated Hordeum species using two-primer RAPD fragments

Random amplified polymorphic DNAs (RAPD) analysis has been adapted to assess the degree of RAPD polymorphism within the genus Hordeum to determine if this approach can distinguish wild and cultivated species. Nineteen wild and seven cultivated accessions were evaluated using 4 random 10-mer primers. The potential of the RAPD assay was further increased by combining two primers in a single polymerase chain reaction (PCR). RAPD fragments generated by two pairs of arbitrary 10-mer primers discriminated six wild species and one cultivated species by banding profiles. The size of the amplified DNA fragments ranged from 150 to 2300 base pairs. 33 %percent of the fragments were common to both wild and cultivated species; 67% were specific to either wild or cultivated species. The average difference in fragments was less within the species than among the species. By comparing RAPD fingerprints of wild and cultivated barley, markers were identified among the set of amplified DNA fragments which could be used to distinguish wild and cultivated Hordeum species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Polymorphic chloroplast simple sequence repeat primers for systematic and population studies in the genus Hordeum

In this study we report the development of primers to amplify polymorphic chloroplast simple sequence repeats in the genus Hordeum , which includes cultivated barley ( H. vulgare ssp. vulgare ) and its wild progenitor H. vulgare ssp. spontaneum . Polymorphic products were amplified in a wide range of Hordeum spp. and intraspecific variation was detected in both cultivated and wild barley. A decrease in cytoplasmic diversity was observed between sspp. spontaneum and vulgare as well as between ssp. vulgare landraces and cultivars, which is characteristic of domestication processes in many crop species. We also observed possible evidence for reticulate evolution of H. brachyantherum polyploids, with apparent multiple cytoplasmic introgressions during successive polyploidization events.     

大麦1H特异性CAPs标记和ASA标记的创制

选取大麦1H染色体的STS标记MWG913特异性扩增小麦,把得到的片段进行克隆.用Taq酶切分类并测序,把得到的序列同大麦的序列进行比较.依据比较结果,选取对大麦特异的内切酶,用该酶来酶切大麦、小麦、黑麦、长穗偃麦草、中间偃麦草、簇毛麦的MWG913扩增产物,获得对大麦1H染色体特异的CAPs标记.同时,依据酶切位点碱基的差异设计引物对扩增的产物进行第二次扩增,得到该位点的一对染色体特异性ASA标记.     

Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

A software tool was developed for the identification of simple sequence repeats (SSRs) in a barley ( Hordeum vulgare L.) EST (expressed sequence tag) database comprising 24,595 sequences. In total, 1,856 SSR-containing sequences were identified. Trimeric SSR repeat motifs appeared to be the most abundant type. A subset of 311 primer pairs flanking SSR loci have been used for screening polymorphisms among six barley cultivars, being parents of three mapping populations. As a result, 76 EST-derived SSR-markers were integrated into a barley genetic consensus map. A correlation between polymorphism and the number of repeats was observed for SSRs built of dimeric up to tetrameric units. 3'-ESTs yielded a higher portion of polymorphic SSRs (64%) than 5'-ESTs did. The estimated PIC (polymorphic information content) value was 0.45 +/- 0.03. Approximately 80% of the SSR-markers amplified DNA fragments in Hordeum bulbosum, followed by rye, wheat (both about 60%) and rice (40%). A subset of 38 EST-derived SSR-markers comprising 114 alleles were used to investigate genetic diversity among 54 barley cultivars. In accordance with a previous, RFLP-based, study, spring and winter cultivars, as well as two- and six-rowed barleys, formed separate clades upon PCoA analysis. The results show that: (1) with the software tool developed, EST databases can be efficiently exploited for the development of cDNA-SSRs, (2) EST-derived SSRs are significantly less polymorphic than those derived from genomic regions, (3) a considerable portion of the developed SSRs can be transferred to related species, and (4) compared to RFLP-markers, cDNA-SSRs yield similar patterns of genetic diversity.     

Study of the backcross progeny of barley-wheat hybrids by the RAPD and RAMPO methods

The genomes of alloplasmic wheat lines were analyzed by PCR-based methods: random amplified polymorphic DNA (RAPD) and random amplified microsatellite polymorphism (RAMPO). Lines L-16(1) and L-17(2) were obtained by three backcrosses and line L-79(10), by four backcrosses of the barely-wheat hybrid Hordeum vulgare (2n = 14) (variety Nepolegayuschii) x Triticum aestivum (2n = 42) (variety Saratovskaya 29) with different common wheat varieties. These lines proved to be euploid (2n = 42). The aneuploid line L-9 (2n = 43 + t) was obtained after a second backcross of the hybrid H. geniculatum All. (2n = 28) x T. aestivum (2n = 42) (Pyrotrix 28) with the variety Pyrotrix 28. The RAPD patterns of L-16(1) and L-17(1) contained fragments present only in the patterns of the parental wheat varieties and, in addition, fragments absent from the latter. This fragment from the pattern of L-16(1) was cloned. Analysis of its primary structure showed that the difference between L-16(1) and the parental wheat genotypes may be related to a mutation that had occurred during the development of the alloplasmic line at the binding site of an arbitrary primer. The genomes of plants of the lines L-79(10) and L-9 contain, in addition to the RAPD fragments of wheat, those characteristic of barley. RAMPO revealed higher polymorphism level among wheat varieties than that detected by RAPD. The hybridization patterns of the lines L-16(1), L-17(1), and L-79(10) contained fragments specific for wheat, and the patterns of L-9 contained both wheat and barley fragments.     

The development and application of molecular markers for abiotic stress tolerance in barley

This article represents some current thinking and objectives in the use of molecular markers to abiotic stress tolerance. Barley has been chosen for study as it is an important crop species, as well as a model for genetic and physiological studies. It is an important crop and, because of its well-studied genetics and physiology, is an excellent candidate in which to devise more efficient breeding methods. Abiotic stress work on cultivated gene pools of small grain cereals frequently shows that adaptive and developmental genes are strongly associated with responses. Developmental genes have strong pleiotropic effects on a number of performance traits, not just abiotic stresses. One concern is that much of the genetic variation for improving abiotic stress tolerance has been lost during domestication, selection and modern breeding, leaving pleiotropic effects of the selected genes for development and adaptation. Such genes are critical in matching cultivars to their target agronomic environment, and since there is little leverage in changing these, other sources of variation may be required. In barley, and many other crops, greater variation to abiotic stresses exists in primitive landraces and related wild species gene pools. Wild barley, Hordeum spontaneum C. Koch is the progenitor of cultivated barley, Hordeum vulgare L. and is easily hybridized to H. vulgare. Genetic fingerprinting of H. spontaneum has revealed genetic marker associations with site-of-origin ecogeographic factors and also experimentally imposed stresses. Genotypes and collection sites have been identified which show the desired variation for particular stresses. Doubled haploid and other segregating populations, including landrace derivatives have been used to map genetically the loci involved. These data can be used in molecular breeding approaches to improve the drought tolerance of barley. One strategy involves screening for genetic markers and physiological traits for drought tolerance, and the associated problem of drought relief-induced mildew susceptibility in naturally droughted fields of North Africa.     

Comparative high resolution map of the six-rowed spike locus 1 (vrs1) in several populations of barley, Hordeum vulgare L

Multiple alleles at the vrs1 locus control the development and fertility of the lateral spikelets of barley (Hordeum vulgare L.), which is a key character in the study of yield, utilization and domestication. In this study, six linkage maps of the vrs1 locus were constructed, using different mapping populations developed from nine different barley cultivars (H. vulgare subsp. vulgare) or mutant and wild barley (H. vulgare subsp. spontaneum). A total of 8387 chromosomes (gametes) were sampled for analysis based on a hypothesis that orders of marker loci were the same over the different parental lines. The results showed that four markers and the vrs1 locus in all cases were arranged in the same order, which was in a good agreement with the hypothesis. This makes the linkage maps suitable for the positional cloning of the alleles at the vrs1 locus.     

Random Amplified Polymorphic DNA Analysis of Heterodera cruciferae and H. schachtii populations

Heterodera schachtii and H. cruciferae are sympatric in California and frequently occur in the same field upon the same host. We have investigated the use of polymerase chain reaction (PCR) amplification of nematode DNA sequences to differentiate H. schachtii and H. cruciferae and to assess genetic variability within each species. Single, random oligodeoxyribonucleotide primers were used to generate PCR-amplified fragments, termed RAPD (random amplified polymorphic DNA) markers, from genomic DNA of each species. Each of 19 different random primers yielded from 2 to 12 fragments whose size ranged from 200 to 1,500 bp. Reproducible differences in fragment patterns allowed differentiation of the two species with each primer. Similarities and differences among six different geographic populations of H. schachtii were detected. The potential application of RAPD analysis to relationships among nematode populations was assessed through cluster analysis of these six different populations, with 78 scorable markers from 10 different random primers. DNA from single cysts was successfully amplified, and genetic variability was revealed within geographic populations. The use of RAPD markers to assess genetic variability is a simple, reproducible technique that does not require radioisotopes. This powerful new technique can be used as a diagnostic tool and should have broad application in nematology.     

Characterization of Hordeum vulgare - Hordeum bulbosum chromosome substitution lines by restriction fragment length polymorphism analysis.

Plants obtained from crosses between Hordeum vulgare and H. bulbosum were previously analyzed cytologically and for isozyme composition. They were identified as possessing substitutions of one or more H. vulgare chromosomes by their H. bulbosum homoeologues. To confirm their constitution and assess the merits of molecular techniques, chromosome-specific probes developed for the Triticeae were hybridized to Southern blots of DNA extracted from these plants and their parents. The hybridization patterns in the substitution plants confirmed that particular chromosomes of H. vulgare were replaced by their H. bulbosum homoeologues. For most probes, heterozygosity between pairs of H. bulbosum chromosomes was recorded. A possible duplication involving H. bulbosum homoeologues of barley chromosomes 4 and 7 was observed. Although molecular and cytological methods for analyzing chromosomally engineered plants are complementary, molecular probes may uncover differences not discernible using light microscopy or isozyme analysis.     

Development and molecular cytogenetic identification of new winter wheat--winter barley ('Martonvásári 9 kr1' - 'Igri') disomic addition lines.

This paper describes a series of winter wheat - winter barley disomic addition lines developed from hybrids between winter wheat line Triticum aestivum L. 'Martonvásári 9 kr1' and the German 2-rowed winter barley cultivar Hordeum vulgare L. 'Igri'. The barley chromosomes in a wheat background were identified from the fluorescent in situ hybridization (FISH) patterns obtained with various combinations of repetitive DNA probes: GAA-HvT01 and pTa71-HvT01. The disomic addition lines 2H, 3H, and 4H and the 1HS isochromosome were identified on the basis of a 2-colour FISH with the DNA probe pairs GAA-pAs1, GAA-HvT01, and pTa71-HvT01. Genomic in situ hybridization was used to confirm the presence of the barley chromosomes in the wheat genome. The identification of the barley chromosomes in the addition lines was further confirmed with simple-sequence repeat markers. The addition lines were also characterized morphologically.     

Rapid estimation of genetic relatedness among heterogeneous populations of alfalfa by random amplification of bulked genomic DNA samples

A procedure which involves the use of RAPD markers, obtained from bulked genomic DNA samples, to estimate genetic relatedness among heterogeneous populations is demonstrated in this study. Bulked samples of genomic DNA from several alfalfa plants per population were used as templates in polymerase chain reactions with different random primers to produce RAPD patterns. The results show that the RAPD patterns can be used to determine genetic distances among heterogeneous populations and cultivars which correspond to their known relatedness. The results also indicate that, by using ten primers with bulked DNA samples from ten individuals, 18–72 populations or cultivars can be distinguished from each other on the basis of at least one unique RAPD marker. We anticipate that DNA bulking and methods for comparing RAPD patterns will be very useful for identifying cultivars, for studying phylogenetic relationships among heterogeneous populations and for selecting parents to maximize heterosis in crosses.     

Identification of intergenomic translocations involving wheat,Hordeum vulgare and Hordeum chilense chromosomes by FISH

Intergenomic translocations between wheat, Hordeum chilense and Hordeum vulgare have been obtained in tritordeum background. Advanced lines from the crosses between three disomic chromosome addition lines for chromosome 2Hv, 3Hv, and 4Hv of barley (Hordeum vulgare) in Triticum aestivum cv. Chinese Spring (CS) and hexaploid tritordeum (2n = 6x = 42, AABBHchHch) were analyzed. Multicolor FISH using both genomic DNA from H. chilense and H. vulgare were used to establish the presence and numbers of H. vulgare introgressions into tritordeum. Interspecific H. vulgare/H. chilense and intergeneric wheat/H. vulgare and wheat/H. chilense translocations were identified. Frequencies of plants containing different kinds of intergenomic translocations between chromosome arms are presented. These lines can be useful for introgressing into tritordeum characters of interest from H. vulgare.     

Haplotype characterization and markers at the barley Mlo powdery mildew resistance locus as tools for marker-assisted selection.

Recessive mlo alleles of the barley Mlo gene confer resistance to almost all known isolates of the powdery mildew fungal pathogen targeting barley (Hordeum vulgare). To characterize haplotypes present in the Mlo chromosomal region of cultivated Mlo and mlo barley genotypes, we conducted a polymorphism search in 3 predicted low-copy sequence regions adjacent to the Mlo gene by examining a sample of 4 Mlo and 3 mlo cultivars. Eight single-nucleotide polymorphisms (SNPs) and 1 insertion-deletion (indel) were detected, and easy to use PCR-based markers were developed for typing the SNPs. The PCR markers were used to characterize a collection of 46 Mlo and 25 mlo barley cultivars, identifying 3 distinct mlo-11 haplotypes, 1 mlo-9 haplotype, and 4 Mlo haplotypes. We summarized the haplotype and marker information obtained here and in a previous study to help breeders identify strategies for mlo marker-assisted selection. The ability of the markers to identify mlo-resistant genotypes in segregating populations was demonstrated using 2 resistance-characterized F2 populations derived by 3-way crosses.     

Inheritance and RAPD tagging of multiple genes for resistance to net blotch in barley.

A doubled haploid barley (Hordeum vulgare L.) population that was created from a cross between cultivars 'Léger' and 'CI 9831' was characterized by RAPD (random amplified polymorphic DNA) markers for resistance to isolate WRS857 of Pyrenophora teres Drechs. f. sp. maculata Smedeg., the causal agent of the spot form of net blotch. Resistance, which initially appeared to be conferred by a single gene from the approximate 1:1 (resistant : susceptible) segregation ratio of the doubled-haploid (DH) progeny, was found to be associated with three different genomic regions by RAPD analysis. Of 500 RAPD random primers that were screened against the parents, 195 revealed polymorphic bands, seven showed an association to the resistance in bulks, and these seven markers were mapped to three unlinked genomic regions. Two of these regions, one of which was mapped to chromosome 2, have major resistance genes. The third region has some homology to the chromosome 2 region. This study demonstrates the simultaneous location of markers for more than one gene governing a trait by using RAPD and bulked segregant analysis (BSA).     

Genetic structure and linkage disequilibrium in landrace populations of barley in Sardinia

Multilocus digenic linkage disequilibria (LD) and their population structure were investigated in eleven landrace populations of barley (Hordeum vulgare ssp. vulgare L.) in Sardinia, using 134 dominant simple-sequence amplified polymorphism markers. The analysis of molecular variance for these markers indicated that the populations were partially differentiated (F(ST) = 0.18), and clustered into three geographic areas. Consistent with this population pattern, STRUCTURE analysis allocated individuals from a bulk of all populations into four genetic groups, and these groups also showed geographic patterns. In agreement with other molecular studies in barley, the general level of LD was low (13% of locus pairs, with P < 0.01) in the bulk of 337 lines, and decayed steeply with map distance between markers. The partitioning of multilocus associations into various components indicated that genetic drift and founder effects played a major role in determining the overall genetic makeup of the diversity in these landrace populations, but that epistatic homogenising or diversifying selection was also present. Notably, the variance of the disequilibrium component was relatively high, which implies caution in the pooling of barley lines for association studies. Finally, we compared the analyses of multilocus structure in barley landrace populations with parallel analyses in both composite crosses of barley on the one hand and in natural populations of wild barley on the other. Neither of these serves as suitable mimics of landraces in barley, which require their own study. Overall, the results suggest that these populations can be exploited for LD mapping if population structure is controlled.