High-density fluorescence in situ hybridization signal detection on barley (Hordeum vulgare L.) chromosomes with improved probe screening and reprobing procedures

The barley (Hordeum vulgare L.) genome was screened to identify sequences that could be used for fluorescence in situ hybridization (FISH). From 2000 transformed bacterium colonies carrying barley clones, 56 colonies were selected on the basis of the patterns that their PCR products produced when subjected to agarose gel electrophoresis. Among them, 42 (75%) exhibited fluorescent signals on barley chromosomes after in situ hybridization using the directly labeled PCR products. Sequencing revealed seven clones, pHv-365, pHv-177, pHv-1112, pHv-689, pHv-1476, pHv-1889, and pHv-1972, to be newly identified FISH-positive sequences. The remainder possess previously described sequences such as 5S, GAA microsatellite, centromere repeats, HVT01, and pHvMWG2315 (324 bp repeat). It is shown here that a combination of five probes, which produce strong signals on barley chromosomes, pHv-38 (5S), pHv-365, pHv-961 (HVT01), GAA, and TAG microsatellites, offer unequivocal recognition of each chromosome. The combination of three probes, i.e., pHv-1123 (barley 324 bp repeat), GAA, and TAG, decorated entire chromosomes with fine dotted signals and was useful for detecting the break points of aberrant chromosomes. The signals' distributions of pHv-177, pHv-1112, and TAG were highly polymorphic. An improved reprobing procedure and its usefulness are also discussed.     

Triple hybridization with cultivated barley (Hordeum vulgare L.)

Summary A crossing programme for trispecific hybridization including cultivated barley (Hordeum vulgare L.) as the third parent was carried out. The primary hybrids comprised 11 interspecific combinations, each of which had either H. jubatum or H. lechleri as one of the parents. The second parent represented species closely or distantly related to H. jubatum and H. lechleri. In trispecific crosses with diploid barley, the seed set was 5.7%. Crosses with tetraploid barley were highly unsuccessful (0.2% seed set). Three lines of diploid barley were used in the crosses, i.e. Gull, Golden Promise and Vada. Generally, cv Gull had high crossability in crosses with related species in the primary hybrid. It is suggested that Gull has a genetic factor for crossability not present in cv Vada and cv Golden Promise. One accession of H. brachyantherum used in the primary hybrid had a very high crossability (seed set 54.7%) in combination with cv Vada but no viable offspring was produced. In all, two trispecific hybrids were raised, viz. (H. lechleri x H. brevisubulatum) x Gull (2n=7–30) and (H. jubatum x H. lechleri) x Gull (2n=20–22). The first combination invariably had a full complement of seven barley chromosomes plus an additional chromosome no. 7, but a varying number of chromosomes (19–22) of the wild-species hybrid. The second combination had a full set of barley chromosomes. The meiotic pairing was low in both combinations.     

Visual screening of microspore-derived transgenic barley (Hordeum vulgare L.) with green-fluorescent protein

The green-fluorescent protein (GFP) gene from the Pacific Northwest jellyfish, Aequorea victoria, was used as a screenable marker in the production of transgenic barley plants. Isolated barley microspore culture was biolistically transformed with two synthetic forms of GFP, sgfp and pgfp. Thirty-seven fluorescing multicellular structures were isolated using epifluorescent microscopy. Sixteen structures developed shoots, but only five regenerated into green plants. Three events had been co-bombarded with #-glucuronidase (gus) and assayed positive for gus expression in the leaves, and all five events were positive for gfp expression. The expected transgene band size was PCR-amplified from all five plants, and Southern blots performed on three plants revealed unique patterns of gfp transgene integration. Fluorescent in situ hybridization also revealed the transgenic status and hemizygous nature of all the events. GFP-based visual screening provides a viable alternative method to chemical selection of transgenic plants from barley microspore culture.     

Association mapping of salt tolerance in barley (Hordeum vulgare L.)

A spring barley collection of 192 genotypes from a wide geographical range was used to identify quantitative trait loci (QTLs) for salt tolerance traits by means of an association mapping approach using a thousand SNP marker set. Linkage disequilibrium (LD) decay was found with marker distances spanning 2–8 cM depending on the methods used to account for population structure and genetic relatedness between genotypes. The association panel showed large variation for traits that were highly heritable under salt stress, including biomass production, chlorophyll content, plant height, tiller number, leaf senescence and shoot Na⁺, shoot Cl^? and shoot, root Na⁺/K⁺ contents. The significant correlations between these traits and salt tolerance (defined as the biomass produced under salt stress relative to the biomass produced under control conditions) indicate that these traits contribute to (components of) salt tolerance. Association mapping was performed using several methods to account for population structure and minimize false-positive associations. This resulted in the identification of a number of genomic regions that strongly influenced salt tolerance and ion homeostasis, with a major QTL controlling salt tolerance on chromosome 6H, and a strong QTL for ion contents on chromosome 4H.     

Acrotrisomic analysis in linkage mapping in barley (Hordeum vulgare L.)

Summary Three acrotrisomic lines, Triplo IL^1S, 3L^3S, and 4L^4S, each carrying an extra acrocentric chromosome, were used for cytogenetic linkage mapping of barley chromosomes. The cytological structures of the acrocentric chromosome of the three acrotrisomic lines were studied with an improved Giemsa N-banding technique. The long (1L) and short arm (1S) of chromosome 1 had deficiencies of approximately 38% and 65%, respectively. The percentages of deficiencies were 0 and 77.8% for 3L and 3S, and 31.7 and 59.3% for 4L and 4S, respectively. All three genes tested (br, f _c , gs3) in 1S and all three genes tested, f8, n and 1k2 in 1L showed a disomic ratio indicating that they are located in the deficient segments. Two genes (a _c , yst2) located in the middle segment of 3S in linkage map showed a trisomic ratio, and two others a _n , x _s showed a disomic ratio. The only gene(f9) tested in 4L showed a trisomic ratio. Two genes (1g4, g1) located in the proximal segment of 4S in the linkage map showed a trisomic ratio, whereas two genes (br2, g13) located distally in 4S showed a disomic ratio, indicating that the breakage occurred between g1 and br2. This experiment demonstrates a new method for physical localization of genes on chromosome segments in material such as barley in which pachytene analysis can not be effectively used for accurate determination of break points in structural changes. Problems associated with this new technique are discussed.Contribution from the Department of Agronomy and published with the approval of the Director of Colorado State University Experiment Station as Scientific Series Paper No. 2823. Supported by USDA/SEA Competitive Research Grant Nos. 5901-0410-9-0334-0 and 82-CRCR-1-1020 and USDA-CSU Cooperative Research Grant 58-9AHZ-2-265     

Ring chromosomes in barley (Hordeum vulgare L.)

A plant with 2n = 14 + 1 ring chromosomes was obtained in the progeny of a primary trisomie for chromosome 7 of a two-rowed cultivar, Shin Ebisu 16. The morphological characteristics of the trisomic plants with an extra ring chromosome were similar to the primary trisomic for chromosome 7 (Semierect), which suggests that it originated from this chromosome. The ring chromosomes were not completely stable in mitotic cells because of abnormal behavior. Chromosome complements varied in different plants and in different roots within a plant. Root tip cells and spikes with 2n = 14 and 14 + 2 ring chromosomes were observed on plants with 14 + 1 ring chromosomes. Breakage-fusion-bridge cycle was inferred. The ring chromosome was associated with two normal homologues forming a trivalent in 17.6% sporocytes at metaphase I. The transmission of the extra ring chromosome was 23.1% in the progeny of the plant with 14 + 1 ring chromosomes. Trivalent formation may have been much higher at early prophase stages which were difficult to analyze in barley; only 4 of 120 sporocytes analyzed showed an isolated ring at pachytene. The ring chromosome moved to one pole without separation in 24.7% of the sporocytes at AI, and divided in 27.1% sporocytes giving rise to 8-8 separation. Only 10% of the sporocytes showed bridge formation at AI.     

Efficient in vitro plant regeneration and generation of transgenic plants in barley (Hordeum vulgare L.) using particle bombardment

Plant regeneration from immature embryos of 15 Indian barley genotypes (Hordeum vulgare L.) was examined. Highest percent callus induction and number of regenerated plantlets were obtained in cultivars BL 2 (94.4 %; 12.1), RD 2668 (92.6 %; 9.1) and RD 2552 (90.8 %; 7.8). The highly responding cultivar BL 2 was selected for further development of transformation protocol. The plasmid DNA isolated from pCAMBIA1381 harbouring hptII gene as selectable marker and gusA gene as reporter was used. Particle bombardment was used for transformation of immature embryos and regeneration of transgenic plants in Indian barley genotype for the first time. Transformation experiments were carried out using different parameters and optimum conditions for DNA delivery was standardized. The transient expression of gusA gene was investigated as a preliminary test of optimum DNA delivery and for selecting the most appropriate bombardment parameters. The optimum conditions were: gold microparticles (diameter 1.0 μm) shot with 1,100 psi rupture disc pressure. The 3 cm distance between rupture disk and macrocarrier and 9 cm target tissue distance yielded high transient GUS expression. The immature embryos were bombarded twice to increase area for efficient gene delivery. Osmotic medium optimization with 0.4 M sorbitol and preculture of immature embryos for 5 days prior to bombardment resulted into efficient gene transfer in barley. Selection of transformed tissue was performed after 7 days resting step on selection medium containing 50 mg?l^?1 hygromycin. After two more selection steps, green shoots were rooted on MSB₅ medium with 50 mg?l^?1 hygromycin. PCR analysis using primers specific for hptII and gusA genes and Southern blot analysis with hptII probes confirmed the stable integration of transgene in barley genome. Molecular analysis of T₁ generation plantlets revealed the amplification of selectable marker hptII gene in the progeny.     

Complex interspecific hybridization in barley (Hordeum vulgare L.) and the possible occurrence of apomixis

Summary Several complex hybrids were produced from the combination [(Hordeum lechleri, 6x xH. procerum, 6 x) × H. vulgare, 2 x]. Crosses with six diploid barley lines resulted in triple hybrids, most of which had a full complement of barley chromosomes (no. 1–7), but were mixoploid with respect to alien chromosomes (19–22). In one combination, chromosome no. 7 was duplicated. Meiosis in triple hybrids showed low, but variable pairing (1.3–5.5 chiasmata per cell). The syndesis probably did not include the barley chromosomes. Direct back-crosses to di- and tetraploid barley lines were unsuccessful. Chromosome doubling of the triple hybrid based on cv Pallas resulted in a plant with 2n = 53–56, which had an increased fertility. Backcrosses to one di- and one tetraploid barley line resulted in offspring. The cross made with the tetraploid line (Haisa II), produced a 28-chromosomic plant in which the male parental genome was absent. We suspect that this plant may have arisen through parthenogenetic development of a reduced female gamete. The other cross with a diploid line (9208/9) resulted in plant with 2n = 51–53. The most likely explanation for this second plant is that an unreduced gamete from the amphiploid was fertilized by a normal gamete from the backcross parent, and during early embryo development, some chromosomes were eliminated.     

Intergeneric somatic hybridization of rice (Oryza sativa L.) and barley (Hordeum vulgare L.) by protoplast fusion

An intergeneric somatic hybrid was obtained upon fusion of protoplasts of rice and barley. Protoplasts isolated from suspension cultures of rice cells were fused by electrofusion with protoplasts that had been isolated from young barley leaves. Some of the resultant calli formed green spots and shoots. Only one shoot formed roots, and it was subsequently successfully transferred to soil in a greenhouse. Its morphology closely resembled that of the parental rice plant. Cytological analysis indicated that the plant had both small chromosomes from rice and large chromosomes from barley. Southern hybridization analysis with a fragment of the tryptophan B (trpB) gene revealed both a rice-specific band and a barley-specific band. Mitochondrial (mt) and chloroplast (cp) DNAs were also analyzed using the same method. The plant was shown to contain novel mitochondrial and chloroplast sequence rearrangements that were not detected in either of the parents. Received: 5 March 1997 / Revision received: 4 September 1997 / Accepted: 13 September 1997     

Production of fertile transgenic barley (Hordeum vulgare L.) plant using the hygromycin-resistance marker

Fertile transgenic barley (Hordeum vulgare L.) plants were obtained by high velocity particle bombardment. The plasmid pBCl was used to deliver the selectable hph gene and reporter Gus gene into immature embryo. After the selection culture 18 hygromycin resistant plants were obtained. Samples for Southern hybridization and enzymatic Gus assay were obtained from 11 plants. Southern hybridization confirmed the presence of the hph gene in the 11 hygromycin resistant plants(T₀). Enzymatic assay indicated that all the t₀ plants that showed hph positive in Southern analysis possessed detectable amount of Gus activity. To date all the 11 t₀ plants reached maturity and mature seeds were obtained Transmission of the hph gene to progeny(T₁) of two independent t₀ plants was confirmed by Southern hybridization.Abbreviations Adh Alcohol Dehydrogenase - BA 6-Benzylaminopurine - cv cultivar - 2,4-D 2,4-Dichlorophenoxyacetic Acid - Gus -Glucuronidase - hph Hygromycin Phosphotransferase - 4MU 4-Methyl-umbelliferone     

Estimation of genetic parameters in barley (Hordeum vulgare L.)

Summary Four exotic and four indigenous strains of barley were used for making diallel crosses. The sets of parents and crosses making full, half and quarter diallel were analysed in a randomized block design for plant height, number of effective tillers, ear length, grain yield per plant, 100 grain weight and number of grains per ear.The three alternatives of diallel were similar with respect to the estimates of degree of dominance, general combining ability and specific combining ability, indicating that all these three methods of diallel were equally efficient. However, as the number of entries are minimum in quarter diallel, it would be economical in terms of cost, time and labour to estimate genetic parameters by this method. Average degree of dominance was found in the range of overdominance. The ranking of parents on the basis of their array mean was similar to the ranking based on gca effects. Similarly, the ranking of crosses on the basis of per se performance was similar to the ranking based on sca effects. This suggests that the selection of best general combiner or best cross combinations may be easier and more effective through array mean for per se performance rather than through high gca and sea effects, respectively. From among 56 crosses, IB-226 X X C-164 was the one which showed superiority for maximum number of characters followed by AB-12/59 X PTS-57. High sea effect for plant height, ear length, grain yield, 100 grain weight and number of grains per ear was the result of cross between parents having high X low general combining ability, indicating additive X dominance type of gene interaction. For number of effective tillers, high sca was produced by low x low general combiners, indicating dominance x dominance gene interaction.Part of a Ph. D. thesis submitted by senior author to Haryana Agricultural University, Hissar.     

Estimation of genetic parameters in barley (Hordeum vulgare L.)

Summary Four different sets of partial diallels were analysed for their relative efficiencies for estimating the genetic parameters in barley: (1) partial diallel with 12 parents, each involved in only 5 crosses; (2) partial diallel with 12 parents, each involved in only 3 crosses; (3) partial diallel with 8 parents, each involved in only 5 crosses; and (4) partial diallel with 8 parents, each involved in only 3 crosses. In partial diallel experiments, the estimates of gca effects were higher than in those of full diallel. Ranking pattern of the parents on the basis of gca effects in partial diallels deviated considerably from the ranking in full diallel. With decreasing s per parent, the deviation in ranking was also more. This clearly suggests the unsuitability of partial diallel analysis for screening high general combiners. Selection of best cross combinations is also not possible because only a sample of crosses (s out of n) is analysed under partial diallel so that there is every possibility of the best cross being excluded from the sample. In general, overdominance was exhibited, indicating that there is ample scope for heterosis breeding in barley.     

Aquaporin-facilitated water uptake in barley (Hordeum vulgare L.) roots

It is not known to what degree aquaporin-facilitated water uptake differs between root developmental regions and types of root. The aim of this study was to measure aquaporin-dependent water flow in the main types of root and root developmental regions of 14- to 17-d-old barley plants and to identify candidate aquaporins which mediate this flow. Water flow at root level was related to flow at cell and plant level. Plants were grown hydroponically. Hydraulic conductivity of cells and roots was determined with a pressure probe and through exudation, respectively, and whole-plant water flow (transpiration) determined gravimetrically in response to the commonly used aquaporin inhibitor HgCl(2). Expression of aquaporins was analysed by real-time PCR and in situ hybridization. Hydraulic conductivity of cortical cells in seminal roots was largest in lateral roots; it was smallest in the fully mature zone and intermediate in the not fully mature 'transition' zone along the main root axis. Adventitious roots displayed an even higher (3- to 4-fold) cortical cell hydraulic conductivity in the transition zone. This coincided with 3- to 4-fold higher expression of three aquaporins (HvPIP2;2, HvPIP2;5, HvTIP1:1). These were expressed (also) in cortical tissue. The largest inhibition of water flow (83-95%) in response to HgCl(2) was observed in cortical cells. Water flow through roots and plants was reduced less (40-74%). It is concluded that aquaporins contribute substantially to root water uptake in 14- to 17-d-old barley plants. Most water uptake occurs through lateral roots. HvPIP2;5, HvPIP2;2, and HvTIP1;1 are prime candidates to mediate water flow in cortical tissue.     

Advanced backcross QTL analysis in barley (Hordeum vulgare L.)

This paper reports on the first advanced backcross-QTL (quantitative trait locus) project which utilizes spring barley as a model. A BC(2)F(2) population was derived from the initial cross Apex ( Hordeum vulgare ssp. vulgare, hereafter abbreviated with Hv) x ISR101-23 ( H. v. ssp. spontaneum, hereafter abbreviated with Hsp). Altogether 136 BC(2)F(2) individuals were genotyped with 45 SSR (simple sequence repeat) markers. Subsequently, field data for 136 BC(2)F(2) families were collected for 13 quantitative traits measured in a maximum of six environments. QTLs were detected by means of a two-factorial ANOVA with a significance level of P < 0.01 for a marker main effect and a marker x environment (M x E) interaction, respectively. Among 585 marker x trait combinations tested, 86 putative QTLs were identified. At 64 putative QTLs, the marker main effect and at 27 putative QTLs, the M x E interaction were significant. In five cases, both effects were significant. Among the putative QTLs, 29 (34%) favorable effects were identified from the exotic parent. At these marker loci the homozygous Hsp genotype was associated with an improvement of the trait compared to the homozygous Hv genotype. In one case, the Hsp allele was associated with a yield increase of 7.7% averaged across the six environments tested. A yield QTL in the same chromosomal region was already reported in earlier barley QTL studies.     

Nuclear genes affecting percentage of green plants in barley (Hordeum vulgare L.) anther culture

Summary The genetics behind response in barley anther culture was studied with 22 reciprocal and one single: cross between three varieties with high and four varieties with low capacity for green plant formation. Effects of genotypes dominated embryo formation and percentages of green plants, accounting for 62 and 76% of total variation, respectively, with almost no genetic effect on the ability to regenerate plants from pollen embryos. Nuclear genes could explain all genotype effects in this plant material, since no reciprocal effects were indicated. The three parents with high and the four parents with low capacity for green plant formation formed two phenotypically homogeneous groups, producing 27–52% and 0–7% green plants, respectively. Genetic variation within hybrids for both embryo and green plant formation could be explained completely by general combining ability (GCA). The results are discussed with respect to a previous similar study in hexaploid wheat and the reported existence of DNA deletions in the plastid genomes in albino plants from anther culture of wheat and barley.     

Chromium uptake and transport in barley seedlings (Hordeum vulgare L.)

Summary Potassium chromate is more toxic to the growth of barley in solution culture than chromic chloride, though apparent uptake of the latter is much faster. Inhibitor studies indicate that CrO₄ ^2- uptake is active whereas Cr³⁺ uptake is passive, demonstrating that the two forms do not share a common uptake mechanism. Studies on the form of Cr inside root cells show that in plants fed CrO₄ ^2- the Cr remains largely unchanged whereas in plants fed Cr³⁺ a little CrO₄ ^2- (0.5 per cent) is produced. This conversion is dependent on the presence of living material and is probably enzymatic. Chromate uptake follows Michaelis-Menten kinetics at low concentration and is competitively inhibited by sulphate. Transport of chromium up the root is very slow, accounting for the low levels of Cr in the shoots. Chromate is transported better than Cr³⁺ though still to a very limited extent. These experiments provide a physiological basis for previous observations.     

Transgenic barley (Hordeum vulgare L.) by electroporation of protoplasts

Summary Protoplasts isolated from calli derived from cultured microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite cultivar) were transformed with the neomycin phosphotransferase marker gene (nptII) by electroporation. Screening of the regenerated plants for the NPTII activity by gel assay resulted in three positive signals. Southern blot analysis and NPTII assays of second and third generation plants confirmed the genomic integration of the transferred gene and that the new trait was inherited by the progeny.     

Identification and mapping of a new leaf stripe resistance gene in barley (Hordeum vulgare L.)

Pyrenophora graminea is the seed-borne pathogen causal agent of barley leaf stripe disease. Near-isogenic lines (NILs) carrying resistance of the cv ”Thibaut” against the highly virulent isolate Dg2 were obtained by introgressing the resistance into the genetic background of the susceptible cv ”Mirco”. The segregation of the resistance gene was followed in a F₂ population of 128 plants as well as on the F₃ lines derived from the F₂ plants; the segregation fitted the 1:2:1 ratio for a single gene. By using NILs, a RAPD marker associated with the resistance gene was identified; sequence-specific (STS) primers were designed on the basis of the amplicon sequence and a RILs mapping population with an AFLP-based map were used to position this molecular marker to barley chromosome 1 S (7HS). STS and CAPS markers were developed from RFLPs mapped to the telomeric region of barley chromosome 7HS and three polymorphic PCR-based markers were developed. The segregation of these markers was followed in the F₂ population and their map position with respect to the resistance gene was determined. Our results indicate that the Thibaut resistance gene, which we designated as Rdg2a, maps to the telomeric region of barley chromosome 7HS and is flanked by the markers OPQ-9₇₀₀ and MWG 2018 at distances of 3.1 and 2.5 cM respectively. The suitability of the PCR-based marker MWG2018 in selection- assisted barley breeding programs is discussed. Received: 22 June 2000 / Accepted: 16 October 2000     

Cytogenetics of in vitro cultured somatic cells and regenerated plants of barley (Hordeum vulgare L.)

Summary Cytogenetic analysis of immature embryoderived calli and regenerated plants of barley has demonstrated high heterogeneity of callus cultures and significant differences in cytogenetic processes between different callus lines. Regenerated plants usually have a normal chromosome complement (2n=14). Tetraploid plaints occur with a frequency of 1%. No chromosome aberrations have been detected by Feulgen staining. The phenomenon of chromosome stickiness recorded from the 2nd day of culture was discovered in a majority of callus lines as well as the phenomena of chromatin hypercondensation and chromosome supercoiling. A possible contribution of cytogenetic and molecular processes to somaclonal variation is discussed.     

Quantitative trait loci for salinity tolerance in barley (Hordeum vulgare L.)

Salinity stress is a major limitation in barley production. Substantial genetic variation in tolerance occurs among genotypes of barley, so the development of salt-tolerant cultivars is a potentially effective approach for minimizing yield losses. The lack of economically viable methods for screening salinity tolerance in the field remains an obstacle to breeders, and molecular marker-assisted selection is a promising alternative. In this study, salinity tolerance of 172 doubled-haploid lines generated from YYXT (salinity-tolerant) and Franklin (salinity-sensitive) was assessed in glasshouse trials during the vegetative phase. A high-density genetic linkage map was constructed from 76 pairs of simple sequence repeats and 782 Diversity Arrays Technology markers which spanned a total of 1,147 cM. Five significant quantitative trait loci (QTL) for salinity tolerance were identified on chromosomes 1H, 2H, 5H, 6H and 7H, accounting for more than 50% of the phenotypic variation. The tolerant variety, YYXT, contributed the tolerance to four of these QTL and Franklin contributed the tolerance to one QTL on chromosome 1H. Some of these QTL mapped to genomic regions previously associated with salt tolerance in barley and other cereals. Markers associated with the major QTL identified in this study have potential application for marker-assisted selection in breeding for enhanced salt tolerance in barley.