Expression of hydrogenase activity in barley (Hordeum vulgare L.) after anaerobic stress

Germinated seeds of barley, after anaerobic treatment, produce considerable amounts of hydrogen. The anaerobic stress induces hydrogenase activity, as measured by the ability to evolve hydrogen from reduced methyl viologen. Several pieces of evidence, including bacteriological controls and induction in axenic calluses, indicate that the hydrogenase activity is induced by the plant itself and not by contaminant microorganisms. The hydrogenase is selectively induced in roots and to a lower extent in hypocotyls, but no hydrogenase activity can be detected in leaves.     

Studies of soil moisture stress in barley (Hordeum vulgare L.)

Summary The objective was to find the optimum range of water contents for inducing better growth, physiological efficiency and yield potential of barley plants (Hordeum vulgare L. var. K18). A pot culture experiment was conducted in the Division of Crop Physiology and Biochemistry Kanpur-2. The plants were subjected to various soil moisture stresses,i.e., 0.15, 0.30, 0.45, 0.60 and 0.75 atm tension throughout the crop growth period measured by irrometers.Plants maintained at 0.45 soil moisture tension required 19.07 litre of water and had the best water use efficiency (1765 mg dm/litre of water) which favourably influenced the leaf water balance (85.9%), plant growth as measured by plant height (85.4 cm) and tiller production (35.6) per hill, photosynthetic efficiency (2.185 mg CO₂/g dm/h), grain number (722) and grain yield (33.7 g) per hill while plants irrigated at a tension greater than 0.45 SMT did not develop as well. However, protein and gluten percentage increased gradually with the subsequent increase in soil moisture tension. On the other hand respiration rate (2.090 mg CO₂/g dm/hr) and leaf area (4375 cm²) were recorded to be the highest at 0.60 and 0.30 atm SMT respectively.Thus it is suggested that for reaping high harvest of barley crop, the physiological need of water (19.07 litre) in total of plant life should be made available through scheduled irrigation based on maintenance of plant at 0.45 SMT from seeding to maturity.     

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.     

Distorted segregation of the esterase isozyme genotypes in barley (Hordeum vulgare L.)

Distorted segregation of the esterase isozyme genotypes was observed in F2 population which was produced from a cross combination between 'Ko A' and 'Mokusekko 3' of barley varieties. After examining the segregation of esterase isozyme genotypes in B1F1 hybrids derived from the reciprocal backcrosses, it was made clear that the distorted segregation was caused by certation between pollens of different genotypes, independent of the female genotypes. Furthermore, the certation was controlled by a newly designated gene, Ga2, at the locus which was linked with the multiocus, Est1, Est2 and Est4, for esterase isozymes at the long arm of chromosome 3. The distorted segregation ratios did not significantly vary among F2 populations derived from F1 hybrids of the same combination which were grown in different years. Mechanically mixed pollens of the parents, however, could not induce the certation.     

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.     

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     

The barley (Hordeum vulgare L.) dehydrin multigene family: sequences, allele types, chromosome assignments, and expression characteristics of 11 Dhn genes of cv Dicktoo

 Dehydrins (LEA D11 proteins) have been identified in both higher and lower plants, and are associated with tolerance to, or response to the onset of, low temperature or dehydration. Several studies have suggested that specific alleles of Dhn genes may contribute to a number of phenotypic traits, including the emergence of seedlings in cool or saline soils and the frost tolerance of more-mature plants. However, an incomplete collection of the Dhn multigene family in any system and nucleic acid cross-hybridization between Dhn gene-family members have limited the precision of these studies. We attempted to overcome these impediments by determining the nucleotide sequences of the entire Dhn multigene family in barley and by developing gene-specific probes. We identified 11 unique Dicktoo Dhn genes. Seven appear to be alleles of Dhn genes identified previously in other barley cultivars. Another, Dhn9, appears to be orthologous to a Triticum durum Dhn gene. A statistical analysis of the total collection of genomic clones brings the estimated size of the barley Dhn gene family to 13. Allelic differences in the protein-coding regions appear to result principally from duplications of entire Φ-segments or single amino-acid substitutions, suggesting that polypeptide structural constraints have been a strong force in the evolution of Dhn alleles. Chromosome mapping by PCR with wheat-barley addition lines established the presence of Dhn genes in four barley chromosomes (3H, 4H, 5H, 6H). RT-PCR demonstrated that the Dhn genes are differentially regulated under dehydration, low temperature and ABA treatment, consistent with putative regulatory elements located upstream of the respective Dhn coding regions. This whole-genome, gene-specific study unifies what previously seemed to be disparate-mapping, expression, and genetic-variation data for Dhn genes in the Triticeae and other plant systems. Received: 23 September 1998 / Accepted: 13 October 1998     

AB-QTL analysis reveals new alleles associated to proline accumulation and leaf wilting under drought stress conditions in barley (Hordeum vulgare L.)

ABSTRACT: BACKGROUND: Land plants have evolved several measures to maintain their life against abiotic stresses. The accumulation of proline is the most generalized response of plants under drought, heat or salt stress conditions. It is known as an osmoprotectant which also acts as an instant source of energy during drought recovery process. But, both its role and genetic inheritance are poorly understood in agriculture crops. In the present work, advanced backcross quantitative trait locus (AB-QTL) analysis was performed to elucidate genetic mechanisms controlling proline accumulation and leaf wilting in barley under drought stress conditions. RESULTS: The analysis revealed eight QTL associated to proline content (PC) and leaf wilting (WS). QTL for PC were localized on chromosome 3 H, 4 H, 5 H and 6 H. The strongest QTL effect QPC.S42.5 H was detected on chromosome 5 H where drought inducible exotic allele was associated to increase PC by 54%. QTL effects QPC.S42.3 H, QPC.S42.4 H and QPC.S42.6 H were responsible to heighten PC due to the preeminence of elite alleles over the exotic alleles which ranged from 26% to 43%. For WS, QTL have been localized on chromosome 1 H, 2 H, 3 H and 4 H. Among these, QWS.S42.1 H and QWS.S42.4 H were associated to decrease in WS due to the introgression of exotic alleles. In addition, two digenic epistatic interaction effects were detected for WS where the additive effect of exotic alleles imparted a favorable increase in the trait value. CONCLUSIONS: The present data represents a first report on whole-genome mapping of proline accumulation and leaf wilting in barley. The detected QTL are linked to new alleles from both cultivated and wild accessions which bring out an initial insight on the genetic inheritance of PC and WS. These QTL alleles are fixed in the isogenic background of Scarlett, which will allow for positional cloning of underlying genes and to develop drought resilient barley cultivars.     

Proteomics analysis of sensitive and tolerant barley genotypes under drought stress

Drought is a severe environmental constraint to plant productivity and an important factor limiting barley yield. To investigate the initial response of barley to drought stress, changes in protein profile were analyzed using a proteomics technique. Three-day-old barley seedlings of sensitive genotype 004186 and tolerant genotype 004223 were given two treatments, one with 20 % polyethylene glycol and the second with drought induced by withholding water. After 3 days of treatments, proteins were extracted from shoots and separated by 2-dimensional polyacrylamide gel electrophoresis. Metabolism related proteins were decreased in the sensitive genotype under drought; however, they were increased in the tolerant genotype. Photosynthetic related proteins were decreased and increased among the three sensitive and three tolerant genotypes, respectively. In addition, amino acid synthesis and degradation related proteins were increased and decreased among the three tolerant genotypes. These results suggest that chloroplastic metabolism and energy related proteins might play a significant role in the adaptation process of barley seedlings under drought stress.     

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.     

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.     

Superoxide dismutase and peroxidase activities in drought sensitive and resistant barley (Hordeum vulgare L.) varieties

The effects of water deficit on relative water content (RWC), on the activity superoxide dismutase (SOD) and peroxidase (POX) from leaves of two drought-resistant barley strains (Hordeum vulgare L.) varieties (TOKAK-157/37 and 56000/MISC-233) and one sensitive (ERGINEL-90) were studied. In 21 day old seedlings, drought stress was initiated by withholding water and lasted for 12 days. Activity of SOD increased by the effect of drought treatments in the leaves of drought-resistant varieties TOKAK-157/37 and 56000/MISC-233 as compared to sensitive variety ERGINEL-90. The drought treatment resulted in a 418 % and 59 % increase in SOD activity in resistant varieties at the end of the 12^th day of experimental period. However, an increase in activity of SOD was not accompanied by an increase in activity of POX in drought-resistant TOKAK-157/37 and 56000/MISC-233 except on the 6^th day of drought treatment in 56000/MISC-233. In drought-sensitive variety, ERGINEL-90, POX activity did not change throughout drought period.     

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.     

Uzu mutation in barley (Hordeum vulgare L.) reduces the leaf unrolling response to brassinolide

A sensitive method to examine the brassinolide (BL) response of barley (Hordeum vulgare L.) using dark-grown leaf segments was established based on the known method for wheat. BL responses of 53 dwarf isogenic lines of barley were examined, and two lines were found having a uzu gene that doesn't respond significantly. These results indicate that uzu dwarfism may be caused by the non-responding character to BL.     

Genetic analysis of the components of winterhardiness in barley (Hordeum vulgare L.)

Winterhardiness in cereals is the consequence of a number of complex and interacting component characters: cold tolerance, vernalization requirement, and photoperiod sensitivity. An understanding of the genetic basis of these component traits should allow for more-effective selection. Genome map-based analyses hold considerable promise for dissecting complex phenotypes. A 74-point linkage map was developed from 100 doubled haploid lines derived from a winter x spring barley cross and used as the basis for quantitative trait locus (QTL) analyses to determine the chromosome location of genes controlling components of winterhardiness. Despite the greater genome coverage provided by the current map, a previously-reported interval on chromosome 7 remains the only region where significant QTL effects for winter survival were detected in this population. QTLs for growth habit and heading date, under 16 h and 24 h light, map to the same region. A QTL for heading date under these photoperiod regimes also maps to chromosome 2. Contrasting alleles at these loci interact in an epistatic fashion. A distinct set of QTLs mapping to chromosomes 1, 2, 3, and 5 determined heading date under 8 h of light. Under field conditions, all QTLs identified under controlled environment conditions were determinants of heading date. Patterns of differential QTL expression, coupled with additive and additive x additive QTL effects, underscore the complexity of winterhardiness. The presence of unique phenotype combinations in the mapping population suggests that coincident QTLs for heading date and winter survival represent the effects of linkage rather than pleiotropy.     

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.     

Microarray analysis of gene expression in germinating barley embryos (Hordeum vulgare L.)

A cDNA library containing approximately 5,000 clones from germinating barley embryos was constructed and used to examine the variation in gene expression patterns during the first 4 days postimbibition. The expression profiles of embryos (including scutellum) from 4 to 96 h postimbibition were compared to a reference profile from 24 h postimbibition using microarray analysis. A subset of clones exhibiting tenfold or greater differential expression patterns was sequenced to elucidate function. All of the sequenced clones could be identified to at least EST level with 64% exhibiting homology to published protein sequences. Almost 95% of the library exhibited similar expression levels at the 4 h time point as at the 24 h reference point. From 24 to 96 h, however, considerable fluctuations in gene expression occurred. The observed patterns of gene expression for the classified genes are consistent with the expected genetic changes required to prepare an embryo for germinative development. A replicate set of clones for the 23-kDa jasmonate-induced protein was identified. The current data not only provides conclusive evidence for the expression patterns of this abundant stress-response protein in germinating embryos, but also serves to validate previous research into JIP-23 isoforms, function and the relationship between timing of mRNA upregulation and protein abundance.     

Trisomic analysis of ribosomal RNA cistron multiplicity in barley (Hordeum vulgare L.)

Ribosomal RNA cistron numbers in all the seven primary trisomics of diploid barley (Hordeum vulgare L.) were determined by DNA-rRNA filter hybridisation. Trisomies for the nucleolus organiser (NO) chromosomes 6 and 7 showed the highest levels of rDNA (DNA complementary to rRNA) indicating the localisation of rRNA cistrons on the NOs. Chromosomes 6 and 7 possessed 1,580 and 2,690 rRNA (18S + 5.8S + 26S) cistrons respectively. Trisomics for the other chromosomes (except for 3) also displayed levels of rDNA significantly higher (22–32%) than the diploid controls although the dosage of NOs was not altered. These non-specific increases were also present in trisomics for 6 and 7 (NOs) which showed further increases equivalent to their respective contributions. The nonspecific increases due to trisomy is indicative of rDNA compensation. Such increases did not persist in diploid sibs of the trisomics, demonstrating the nonheritable nature of the compensation.