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.     

Aldehyde oxidase in roots, leaves and seeds of barley (Hordeum vulgare L.)

Aldehyde oxidase (AO, EC 1.2.3.1) proteins in leaves, roots and seeds of barley (Hordeum vulgare L.) plants were studied. Differences in substrate specificity and mobility in native PAGE between AO proteins extracted from roots, leaves and seeds have been observed. Four clear bands of AO reacting proteins were detected in barley plants capable of oxidizing a number of aliphatic and aromatic aldehydes such as indole-3-aldehyde, acetaldehyde, heptaldehyde, and benzaldehyde. Mouse polyclonal antibodies raised against purified maize AO cross-reacted with barley AO proteins extracted from roots, leaves and seeds. At least three different AO proteins were detected in roots on the basis of their mobility during PAGE after native Western blot analysis while in leaves and seeds only one polypeptide cross-reacted with the antibody. SDS-immunoblot analysis showed marked differences in molecular weight between subunits of the AO bands extracted from roots, leaves and seeds. Two distinct subunit bands were observed in roots, with relatively close molecular weights (160 kDa and 145 kDa), while a single subunit with a molecular weight of 150 kDa was observed in leaf and seed extracts.Menadione, a specific and potent inhibitor of animal AO did not affect barley AO proteins. Root and leaf AO differed in their thermostability and susceptibility to exogenous tungstate. The AO proteins in plants may be a group of enzymes with different substrate specificity, tissue distribution and presumably fulfilling different metabolic roles in each plant organ.     

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.     

Effect of anther orientation on microspore-callus production in barley (Hordeum vulgare L.)

Barley anthers from cold pretreated spikes produced no or few calluses when plated with both loculi in contact with the medium (flat). When anthers were plated with only one loculus in contact with the medium (up), a high proportion of the anthers produced calluses. The top loculus of the up anthers was most productive. Flat anthers, when compared with up anthers, were not only slower to produce multicellular pollen grains (MCPs) and microcalluses, but also produced fewer of them and ceased production earlier. The MCPs and microcalluses in flat anthers grew more slowly and few developed beyond the 30 cell stage. These results establish the importance of anther orientation for barley anther culture.     

Involvement of ABA in induction of secondary dormancy in barley (Hordeum vulgare L.) seeds

At harvest, barley seeds are dormant because their germination is difficult above 20 degrees C. Incubation of primary dormant seeds at 30 degrees C, a temperature at which they do not germinate, results in a loss of their ability to germinate at 20 degrees C. This phenomenon which corresponds to an induction of a secondary dormancy is already observed after a pre-treatment at 30 degrees C as short as 4-6 h, and is optimal after 24-48 h. It is associated with maintenance of a high level of embryo ABA content during seed incubation at 30 degrees C, and after seed transfer at 20 degrees C, while ABA content decreases rapidly in embryos of primary dormant seeds placed directly at 20 degrees C. Induction of secondary dormancy also results in an increase in embryo responsiveness to ABA at 20 degrees C. Application of ABA during seed treatment at 30 degrees C has no significant additive effect on the further germination at 20 degrees C. In contrast, incubation of primary dormant seeds at 20 degrees C for 48 and 72 h in the presence of ABA inhibits further germination on water similarly to 24-48 h incubation at 30 degrees C. However fluridone, an inhibitor of ABA synthesis, applied during incubation of the grains at 30 degrees C has only a slight effect on ABA content and secondary dormancy. Expression of genes involved in ABA metabolism (HvABA8'OH-1, HvNCED1 and HvNCED2) was studied in relation to the expression of primary and secondary dormancies. The results presented suggest a specific role for HvNCED1 and HvNCED2 in regulation of ABA synthesis in secondary seed dormancy.     

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.     

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.     

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.     

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.     

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.     

High resolution analysis of meiotic chromosome structure and behaviour in barley (Hordeum vulgare L.)

Reciprocal crossing over and independent assortment of chromosomes during meiosis generate most of the genetic variation in sexually reproducing organisms. In barley, crossovers are confined primarily to distal regions of the chromosomes, which means that a substantial proportion of the genes of this crop rarely, if ever, engage in recombination events. There is potentially much to be gained by redistributing crossovers to more proximal regions, but our ability to achieve this is dependent upon a far better understanding of meiosis in this species. This study explores the meiotic process by describing with unprecedented resolution the early behaviour of chromosomal domains, the progression of synapsis and the structure of the synaptonemal complex (SC). Using a combination of molecular cytogenetics and advanced fluorescence imaging, we show for the first time in this species that non-homologous centromeres are coupled prior to synapsis. We demonstrate that at early meiotic prophase the loading of the SC-associated structural protein ASY1, the cluster of telomeres, and distal synaptic initiation sites occupy the same polarised region of the nucleus. Through the use of advanced 3D image analysis, we show that synapsis is driven predominantly from the telomeres, and that new synaptic initiation sites arise during zygotene. In addition, we identified two different SC configurations through the use of super-resolution 3D structured illumination microscopy (3D-SIM).     

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.     

Root contribution to nitrate reduction in barley seedlings (Hordeum vulgare L.)

Summary The leaf and root nitrate reductase activities were measured in 7 day-old barley seedlings by anoxic nitrite accumulation in darkness, during 48h after the transfer from a N-starved medium to a 1.5 mM K¹⁵NO₃ medium. Thisin situ nitrate reduction was compared with the¹⁵N incorporation in the reduced N fraction of the whole seedlings.The nitrate reduction integrated fromin situ measurements was lower than the reduced¹⁵N accumulation. The rootin situ nitrate reductase activity seemed to account for only the third of the real root nitrate reduction, which may have been responsible for the overall underestimation. This discrepancy was partly explained by the ability of the root to reduce nitrite in an anoxic environment.These results suggest that, after correction of thein situ estimation of the nitrate reduction. the roots contribute to about 50% of the total assimilation.     

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.     

5-n-Alkylresorcinols from grains of winter barley (Hordeum vulgare L.)

The resorcinolic lipid content and homologue composition of winter barley grains harvested at two field locations were evaluated. Depending on the crop location, the predominant alkylresorcinols identified were 1,3-dihydroxy-5-n-heneicosylbenzene or 1,3-dihydroxy-5-n-pentacosylbenzene. Both resorcinol concentration and their homologue profiles were diverse in samples harvested at different fields indicating a prevailing role of the environment upon the alkylresorcinol biosynthesis in cereals.     

Identification of a phytase gene in barley (Hordeum vulgare L.)

Background

Endogenous phytase plays a crucial role in phytate degradation and is thus closely related to nutrient efficiency in barley products. The understanding of genetic information of phytase in barley can provide a useful tool for breeding new barley varieties with high phytase activity.

Methodology/Principal Findings

Quantitative trait loci (QTL) analysis for phytase activity was conducted using a doubled haploid population. Phytase protein was purified and identified by the LC-ESI MS/MS Shotgun method. Purple acid phosphatase (PAP) gene was sequenced and the position was compared with the QTL controlling phytase activity. A major QTL for phytase activity was mapped to chromosome 5 H in barley. The gene controlling phytase activity in the region was named as mqPhy. The gene HvPAP a was mapped to the same position as mqPhy, supporting the colinearity between HvPAP a and mqPhy.

Conclusions/Significance

It is the first report on QTLs for phytase activity and the results showed that HvPAP a, which shares a same position with the QTL, is a major phytase gene in barley grains.