Mapping of chromosome regions conferring boron toxicity tolerance in barley (Hordeum vulgare L.)

 Boron toxicity has been recognised as an important problem limiting production in the low-rainfall regions of southern Australia, West Asia and North Africa. Genetic variation for boron toxicity tolerance in barley has been characterised but the mode of inheritance and the location of genes controlling tolerance were not previously known. A population of 150 doubled-haploid lines from a cross between a boron toxicity tolerant Algerian landrace, Sahara 3771, and the intolerant Australian cultivar Clipper was screened in four tolerance assays. An RFLP linkage map of the Clipper×Sahara population was used to identify chromosomal regions associated with boron tolerance in barley. Interval regression-mapping allowed the detection of four chromosomal regions involved in the boron tolerance traits measured. A region on chromosome 2H was associated with leaf-symptom expression, a region on chromosome 3H was associated with a reduction of the affect of boron toxicity on root growth suppression, a region on chromosome 6H was associated with reduced boron uptake, and a region on chromosome 4H was also associated with the control of boron uptake as well as being associated with root-length response, dry matter production and symptom expression. The benefits and potential of marker-assisted selection for boron toxicity tolerance are discussed. Received: 18 December 1997 / Accepted: 28 November 1998     

Salinity tolerance in different cultivars of barley (Hordeum vulgare L.)

Seven barley(Hordeum vulgäre L.) cultivars tested varied greatly in their responses to root medium salinity (electrical conductivity of 3, 5, 10, 15 and 20 dS nr^-1)-lant growth was relatively more adversely affected than seed germination. Dry/fresh mass ratio increased at higher salinity levels in all barley cultivars indicating reduced water uptake. Higher K/Na ratio in plant shoots compared to that in the root medium solution indicated selective uptake of K that seems to be among processes involved in tolerance of cultivars to salinity stress.     

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.     

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.     

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.     

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.     

Accumulation of phenolics and growth rate of barley seedlings (Hordeum vulgare L.)

Accumulation of phenolics in barley seedlings was examined in relation to elongation; the seedlings were cultivated at 5 °C or 26 °C in light or in darkness. It was found that a higher accumulation of phenolics (mainly saponarin) was accompanied by slower elongation. This relation was repeatedly observed regardless of whether growth retardation or stimulation was obtained by light and temperature conditions of growth or treatment with CCO orp-fluorophenylalanine (p-FPA), respectively. It is proposed that PAL and peroxidase activities are responsible for maintaining the level of phenolics in seedlings. These enzyme activities are differently influenced by temperature conditions of growth. It is also suggested that accumulation of saponarin may lead to slowing down the growth by stimulating IAA oxidase and lowering the auxin level in the tissues. Thus, phenolics may belong to the factors through which environmental conditions influence elongation of seedlings.     

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.     

Salt tolerance of barley (Hordeum vulgare L.) cultivars at the germination stage: Analysis of the response functions

Two models, initially proposed by Van Genuchten (1983) for evaluating salinity-yield response curves at the adult stage, were applied to study the salinity response of 24 barley cultivars at the germination stage. According to the calculated salinity threshold, EC_t (the solution electrical conductivity, EC, at which germination starts to decrease), and EC₅₀ (the solution EC at which germination is reduced by 50%) parameters, both models give similar results, although model 2, a sigmoid-form curve, fits the observed data slightly better than model 1, a piecewise response function. Also, the results suggest that, for model 1, EC_t seems to be the most reliable parameter for screening barley germplasm because it clearly discriminates the relative salt-tolerance of the studied cultivars and, furthermore, it basically determines their salinity response for the 100 to 50% germination interval. On the other hand, the model 1 s parameter — percent germination decrease per unit salinity increase bove EC_t—is less relevant because of its smaller variation interval and lack of correlation with EC₅₀, indicating that the salinity response of the studied cultivars for the 50% germination value is independent of this parameter.     

The allene oxide cyclase of barley (Hordeum vulgare L.)--cloning and organ-specific expression

The naturally occurring enantiomer of the various octadecanoids and jasmonates is established in a biosynthetic step catalyzed by the allene oxide cyclase (AOC). The AOC converts an allene oxide formed by an allene oxide synthase (AOS). Here, we show cloning and characterization of cDNAs encoding the AOC and a third AOS, respectively, in addition to the two AOSs previously published (Plant J. 21, 199-213, 2000). The ORF of the AOC-cDNA of 717 bp codes for a protein of 238 amino acid residues carrying a putative chloroplast target sequence. Overexpression without chloroplast target sequence revealed AOC activity. The AOC was found to be a single copy gene which mapped on chromosome 6H. AOC mRNA accumulation appeared in leaf segments upon treatment with various jasmonates, octadecanoids and ABA or during stress such as treatment with sorbitol or glucose solutions. Infection with powdery mildew activated AOC expression in susceptible and resistant lines of barley which correlated with PR1b expression. Among different tissues of barley seedlings, the scutellar node and leaf base accumulated AOC mRNA preferentially which correlated with accumulation of mRNAs for other biosynthetic enzymes (lipoxygenases, AOSs). AOC mRNA accumulation appeared also abundantly in parts of the root containing the tip and correlated with elevated levels of jasmonates. The data suggest a link of AOC expression and JA formation and support role of JA in stress responses and development of barley.     

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.     

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.     

Callus formation and plant regeneration in standard and microexplants from seedlings of barley (Hordeum vulgare L.)

Callus was induced in standard (1 mm) leaf base explants and in cross sections (microexplants) through the seedling axis from seedlings of Hordeum vulgare L.. Reduced callus formation was observed with increasing distance from the leaf base, and explants from the first and second leaves gave the best response. In serial hand sections of the seedling axis frequency of callus formation decreased from 100% in the apical region to 5% in the basal region. Callus formed from all tissues outside the central vascular elements, except for the coleoptile and the scutellum. Plants were regenerated from callus induced from both types of explants.     

Assessment of paraquat genotoxicity on barley (Hordeum vulgare L.) seedlings using molecular and biochemical parameters

The effects of exposure of barley seedlings to different concentration (10^?6 to 10^?3 mol/l) of paraquat on seed germination, root length, antioxidant enzyme activities and randomly amplified polymorphic DNA (RAPD) profiles were investigated. The results revealed that malondialdehyde content significantly increased by exposing paraquat in a concentration-dependent manner (p < 0.05). A significant increase in peroxidase and catalase activities in seedlings was observed with increased concentration of paraquat, and then decreased when the value reached 10^?3 mol/l, whereas the activities of superoxide dismutase gradually increased with increasing paraquat concentration. Germination and root elongation also decreased with the increase of paraquat concentration. On the other hand, alterations of DNA in barley seedlings were detected using RAPD technique. The changes occurring in RAPD profiles of seedlings following paraquat treatment included loss of bands found in DNA of control seedlings and appearance of new bands. The results of this study showed that paraquat induced DNA damage in a dose-dependent manner and the root cells of barley showed a defense against paraquat-induced oxidative stress by enhancing their antioxidant activities.