Anther culture and chromosome reduction in wheat × Thinopyrum wide crosses

Callus cultures of Ambrosia tenuifolia were established from sub-apical leaves. The explants were grown on basal media MS (Murashige and Skoog) supplemented with 10 μM kinetin, 1 μM 2-4 dichlorophenoxyacetic, ascorbic acid and cysteine and cultivated either in light or darkness. To determine the effect of each individual salt component on the growing rate of the callus and the psilostachyinolides and altamisine production in the culture, the concentrations of each nutrient was tested at different levels, ranging from 50% above to 50% below the standard medium. Interestingly, increased concentration of boron in the medium, resulted in a four fold increase in the production of sesquiterpene lactones by the callus. However, positive production of psilostachynolides and altamisine was only detected when the basal concentrations of the others components of the basal media were used. In addition, production of altamisine was highly sensitive to the variations of nutrients. No statiscally effect on terpenoide production by the callus was detected by varying light exposure. These results indicates that conditions for the optimal production of these terpenoids can be enhanced by modifying nutrients of the MS basal media. This revised version was published online in June 2006 with corrections to the Cover Date.     

Advanced backcross QTL analysis of a hard winter wheat × synthetic wheat population

Advanced backcross quantitative trait locus (AB-QTL) analysis was used to identify QTLs for yield and yield components in a backcross population developed from a cross between hard red winter wheat (Triticum aestivum L.) variety Karl 92 and the synthetic wheat line TA 4152-4. Phenotypic data were collected for agronomic traits including heading date, plant height, kernels per spike, kernel weight, tiller number, biomass, harvest index, test weight, grain yield, protein content, and kernel hardness on 190 BC₂F_2:4 lines grown in three replications in two Kansas environments. Severity of wheat soilborne mosaic virus (WSBMV) reaction was evaluated at one location. The population was genotyped using 151 microsatellite markers. Of the ten putative QTLs identified, seven were located on homoeologous group 2 and group 3 chromosomes. The favorable allele was contributed by cultivated parent Karl 92 at seven QTLs including a major one for WSBMV resistance, and by the synthetic parent at three QTLs: for grain hardness, kernels per spike, and tiller number. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

Expression and secretion of wheat α-amylase in Kluyveromyces lactis

The plasmid pCR1 has been constructed to express a wheat -amylase enzyme in Kluyveromyces lactis strains. The contruct is based on the vector pCXJ-kan1, which has been derived from pDK1, a native plasmid of K. lactis var. drosophilarum containing the essential regions for plasmid replication and stability. Contruct pCR1 produces an -amylase by DNA isolated from a wheat cDNA clone and is controlled by a Saccharomyces cerevisia PGK promoter. Correspondence to: C. Russell     

Molecular characterization of the genome composition of partial amphiploids derived from Triticum aestivum×Thinopyrum ponticum and T. aestivum×Th. intermedium as sources of resistance to wheat streak mosaic virus and its vector, Aceria tosichella

 Wheat streak mosaic virus (WSMV), vectored by the wheat curl mite (WCM), is one of the most important viral diseases of wheat (Triticum aestivum) in the world. Genetic resistance to WSMV and the WCM does not exist in wheat. Resistance to WSMV and the WCM was evaluated in five different partial amphiploids namely Agrotana, OK7211542, ORRPX, Zhong 5 and TAF 46, which were derived from hybrids of wheat with decaploid Thinopyrum ponticum or with hexaploid Th. intermedium. Agrotana was shown to be immune to WSMV and the WCM; the other four partial amphiploids were susceptible to the WCM. Genomic in situ hybridization (GISH) using genomic DNA probes from Th. elongatum (EE, 2n=14), Th. bessarabicum (JJ, 2n=14), Pseudoroegneria strigosa (SS, 2n=14) and T. aestivum (AABBDD, 2n=42) demonstrated that three of the partial amphiploids, Agrotana, OK7211542 and ORRPX, have almost identical alien genome constitutions: all have 16 alien chromosomes, with 8 chromosomes being closely related to the J^s genome and 8 chromosomes belonging to the E or J genomes and no evidence of any S-genome chromosomes. GISH confirmed that the alien genomes of Agrotana and OK7211542, like ORRPX, were all derived from Th. ponticum, and not from Th. intermedium. However, in contrast to Agrotana, ORRPX and OK7211542 were susceptible to the WCM and WSMV. The partial amphiploid Zhong 5 had a reconstituted alien genome composed of 4 S-and 4 J^s-genome chromosomes of Th. intermedium with 6 translocated chromosomes between the S and J^s genomes. This line was highly resistant to WSMV, but was susceptible to the WCM. TAF 46, which contained a synthetic genome consisting of 3 pairs of S-genome chromosomes and 4 pairs of E- or J-genome chromosomes in addition to the 21 pairs of wheat chromosomes, was susceptible to the WCM, but moderately resistant to WSMV. Agrotana offers great potential for transferring WSMV and WCM resistance into wheat. Received: 27 January 1998 / Accepted: 10 February 1998     

Expression of α-amylase and other gibberellin-regulated genes in aleurone tissue of developing wheat grains

Aleurone tissue from freshly harvested immature wheat grains (Triticum aestivum L. cv. Sappo) which is normally unresponsive to gibberellic acid can be made responsive by subjecting the tissue to a pre-incubation treatment in a simple buffered medium prior to the addition of the growth substance. The effectiveness of this treatment is dependent on grain age, with grains less than 15–20 days post anthesis failing to become converted to a responsive state whilst tissue from grains older than this become increasingly susceptible. Tissue from grains of a certain age (approx. 25–28 days post anthesis) produce small amounts of -amylase following this treatment even in the absence of exogenously applied growth substance. Using different ³²-labelled complementary-DNA probes for -amylase in wheat it was demonstrated that the failure of freshly harvested tissue to produce -amylase was correlated with the absence of the appropriate mRNA species. Inability to accumulate -amylase mRNA in response to gibberellic acid was removed by the pre-iccubation treatment and also by enforced drying. The gibberellin-regulated expression of other unidentified genes also responds to pre-incubation or drying. Induction of gibberellin-responsiveness in immature aleurone cells did not extend to the secretion of acid phosphatase, protease and ribonuclease.Abbreviations cDNA complementary DNA - dpa days post anthesis - GA gibberellin - GA₃ gibberellic acid     

The assignment of a Thinopyrum distichum (Thunb.) Löve-derived translocation to the long arm of wheat chromosome 7D using endopeptidase polymorphisms

Summary Endopeptidase zymograms of the translocation line Indis revealed the presence of several major and minor bands that had differential expression in coleoptile and seed tissues. While Indis lacks Ep-D1a, which is present in the parental cultivar Inia 66, it also may not express any of the Th. distichum bands. The Indis zymogram was found to be identical to that of an isogenic line of Inia 66 possessing Lr19. Since the absence of an Ep-D1a product appears to be linked to the 7DL translocation, it is possible to use the null condition as a marker for both the Lr19 or Indis translocations. The Indis translocation also did not show recombination with the cn-D1 chlorophyl mutant on 7DL, confirming that a part of 7D was involved. The results of a telocentric mapping experiment involving the 7D telosomes indicated that in Indis a chromosome segment from Th. distichum replaced a large section of 7DL of Inia 66.     

Mitotic instability in wheat×Thinopyrum ponticum derivatives revealed by chromosome counting, nuclear DNA content and histone H3 phosphorylation pattern

To evaluate the mitotic stability of Triticum aestivum×Thinopyrum ponticum derivatives (BC₂F₇ and BC₂F₅ doubled haploids), chromosome counting by both conventional and immunostaining techniques, and measurement of DNA content were performed. The wheat progenitor line, PF 839197, the wheat recurrent parent CEP 19 and the control Chinese Spring were also investigated. In the hybrid derivatives, chromosome number ranged from 2n=36 to 60, with a predominance of chromosome numbers higher than 2n=42, that was confirmed by determination of nuclear DNA content. Chinese Spring and PF 839197 were stable, but CEP 19 showed chromosome number variation (20%). Analyses of non-pretreated cells revealed the presence of anaphase bridges, lagging chromatids, chromosome fragments and micronuclei. Immunostaining with an antibody recognizing histone H3 phosphorylated showed dicentric chromatids forming anaphase bridges and pericentromeric phosphorylation at centric chromosome fragments but not at lagging chromatids. The possible causes of the observed mitotic instability are discussed.     

Comparative analysis of the meiotic effects of wheat ph1b and ph2b mutations in wheat×rye hybrids

 Wheat-wheat and wheat-rye homoeologous pairing at metaphase I and wheat-rye recombination at anaphase I were examined by genomic in situ hybridization (GISH) in wild-type (Ph1Ph2) and mutant ph1b and ph2b wheat×rye hybrids. The metaphase-I analysis revealed that the relative contribution of wheat-rye chromosome associations in ph2b wheat×rye was similar to that of the wild-type hybrid genotype but differed from the effect of the ph1b mutation. The greater pairing promotion effect of the ph1b mutation appears to be relatively more on distant homoeologous partner metaphase-I associations, whereas the lower promoting effect of ph2b is evenly distributed among all types of homoeologous associations. This finding reveals that distinct mechanisms are involved in the control of wheat homoeologous pairing by the two Ph genes. The frequency of wheat-rye recombination calculated from anaphase-I analysis was lower than expected from the metaphase-I data. A greater discrepancy was found in ph2b than in ph1b wheat×rye hybrids, which may suggest a more distal chiasma localization in the former hybrid genotype. Received: 20 June 1997 / Accepted: 9 December 1997     

Expression of TNF-α and IL-1β in splenic dendritic cells and their serum levels in mouse sparganosis

Sparganosis is a tissue invading helminthiasis infecting intermediate hosts, including humans. Strong immune responses are expected to occur in early phases of infection. Thus, we investigated cytokine expressions in splenic dendritic cells and in sera after experimental infection of mice. In splenic dendritic cells, TNF-α and IL-1β expression peaked at week 1 and week 3 post-infection (PI), respectively, and also early phase (week 2 PI) depressed cytokine expression was noticed. Serum IL-1β concentration increased significantly at week 2 PI and peaked at week 6 PI, and that of TNF-α peaked at week 6 PI. These results showed that pro-inflammatory cytokines, TNF-α and IL-1β, are chronologically regulated in mouse sparganosis.     

Plant regeneration from interspecific hybrid and backcross progeny of Helianthus eggertii × Helianthus annuus

An efficient protocol for plant regeneration from leaves of the interspecific hybrid Helianthus eggertii Small. × Helianthus annuus L. was developed. The regeneration capacity of the first backcross progeny is reported. Leaves from the F1 interspecific hybrid were cultured on Murashige and Skoog basal media (MS) supplemented with -naphthalenacetic acid (NAA), N ⁶-benzyladenine (BA), AgNO₃, KNO₃, casein hydrolysate and adenine sulfate. Embryo-like structures and/or shoots regeneration were observed on most of the tested media. The best results were obtained on media with a higher concentration of cytokinin (8.8 M BA) and lower concentration of auxin (1.08 M NAA). The addition of casein hydrolysate in the media increased the regeneration efficiency. Plant regeneration was achieved via somatic embryogenesis and direct organogenesis. The regeneration potential of leaf, stem and root explants of eighteen first backcross lines was studied. Most of the tested lines were highly regenerable and some of them had DNA content closely related to that of Helianthus annuus L.     

Genomic constitution and variation in five partial amphiploids of wheat–<Emphasis Type="Italic">Thinopyrum intermedium</Emphasis> as revealed by GISH,multicolor GISH and seed storage protein analysis

Genomic in situ hybridization (GISH) and multicolor GISH (mcGISH) methodology were used to establish the cytogenetic constitution of five partial amphiploid lines obtained from wheat × Thinopyrum intermedium hybridizations. Line Zhong 1, 2n=52, contained 14 chromosomes from each of the wheat genomes plus ten Th. intermedium chromosomes, with one pair of A-genome chromosomes having a Th. intermedium chromosomal segment translocated to the short arm. Line Zhong 2, 2n=54, had intact ABD wheat genome chromosomes plus 12 Th. intermedium chromosomes. The multicolor GISH results, using different fluorochrome labeled Th. intermedium and the various diploid wheat genomic DNAs as probes, indicated that both Zhong 1 and Zhong 2 contained one pair of Th. intermedium chromosomes with a significant homology to the wheat D genome. High-molecular-weight (HMW) glutenin and gliadin analysis revealed that Zhong 1 and Zhong 2 had identical banding patterns that contained all of the wheat bands and a specific HMW band from Th. intermedium. Zhong 1 and Zhong 2 had good HMW subunits for wheat breeding. Zhong 3 and Zhong 5, both 2n=56, possessed no gross chromosomal aberrations or translocations that were detectable at the GISH level. Zhong 4 also had a chromosome number of 2n=56 and contained the complete wheat ABD-genome chromosomes plus 14 Th. intermedium chromosomes, with one pair of Th. intermedium chromosomes being markedly smaller. Multicolor GISH results indicated that Zhong 4 also contained two pairs of reciprocally translocated chromosomes involving the A and D genomes. Zhong 3, Zhong 4 and Zhong 5 contained a specific gliadin band from Th. intermedium. Based on the above data, it was concluded that inter-genomic transfer of chromosomal segments and/or sequence introgression had occurred in these newly synthesized partial amphiploids despite their diploid-like meiotic behavior and disomic inheritance.     

Expression and localization of VCX／Y proteins and their possible in-volvement in regulation of ribosome assembly during spermatogenesis

Variable Charge X/Y (VCX/Y) is a human testis-specific gene family that localized on X and Y chromo-somes. In this study, VCY protein was expressed in E. coli in the form of glutathione-S-transferase (GST)fusion protein. With the purified fusion protein as antigen, the anti-GST-VCY antibody was generated andthe localization of VCY protein in human testis was determined by immunohistochemistry. In the testisseminiferous epithelium, VCY proteins were highly expressed in nuclei of germ cells. Using propidium io-dide staining and green fluorescent protein (GFP) tag technologies, VCY and VCX-8r proteins were mainlylocalized in the nucleoli of COS7 cells. In addition, the colocalization for VCY and VCX-8r in COS7 cellswas also observed. With VCY cDNA as bait, a cDNA fragment of acidic ribosomal protein PO was obtainedusing yeast two-hybrid system. All the information above indicates that VCX/Y protein family might beinvolved in the regulation of ribosome assembly during spermatogenesis.     

Expression of regulatory receptors on γδ T Cells and their cytokine production in Behcet's disease

Introduction

Behcet''s disease (BD) is a multi-systemic disorder with muco-cutaneous, ocular, arthritic, vascular or central nervous system involvement. The role of γδ T cells is implicated in BD. The activation status of γδ T cells and their cytokine secretion against phosphoantigens are evaluated in BD.

Methods

NKG2A, NKG2C, NKG2D, CD16 and CCR7 molecules on γδ T cells were analyzed in 70 BD, 27 tuberculosis (TB) patients and 26 healthy controls (HC). Peripheral γδ T cells were expanded with a phosphoantigen (BrHPP) and IL-2, restimulated with BrHPP and a TLR3 ligand, and cytokine production was measured.

Results

γδ T cells were not increased in both BD and TB patients, but the proportions of TCRVδ2⁺ T cells were lower (58.9 and 50.7 vs. 71.7%, P = 0.04 and P = 0.005) compared to HC. Higher proportion of TCRVδ2⁺ T cells were CD16⁺ (26.2 and 33.9 vs. 16.6%, P = 0.02 and P = 0.001) and CCR7^- (32.2 and 27.9 vs. 17.7%, P < 0.0001 and P = 0.014) in BD and TB patients compared to HC. NKG2C⁺ γδ⁺ T cells were relatively increased (0.5 and 0.6 vs. 0.3%, P = 0.008 and 0.018), whereas NKG2D positivity was decreased in patients with BD and TB (77.7 and 75.8 vs. 87.5%, P = 0.001 and 0.004). Expansion capacity of γδ T cells in BD and TB as well as production of IL-13, IFN-γ, granulocyte monocyte colony stimulating factor (GM-CSF), TNF-α, CCL4 and CCL5 in BD was lower compared to HC, when restimulated by TLR3 ligand and BrHPP.

Conclusion

The changes on γδ T cells of BD as well as TB patients implicate that γδ T cells have already been exposed to regulatory effects, which changed their activity. Lower cytokine response of γδ T cells implicates down modulation of these cells in BD.     

Expression of cystathionine β-synthase and cystathionine γ-lyase in human pregnant myometrium and their roles in the control of uterine contractility

Background

Human uterus undergoes distinct molecular and functional changes during pregnancy and parturition. Hydrogen sulfide (H₂S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H₂S produced locally in the control of uterine contractility during labour is unknown.

Methodology/Principal Findings

Human myometrium biopsies were obtained from pregnant women undergoing cesarean section at term. Immunohistochemistry analysis showed that cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS), the principle enzymes responsible for H₂S generation, were mainly localized to smooth muscle cells of human pregnant myometrium. The mRNA and protein expression of CBS as well as H₂S production rate were down-regulated in labouring tissues compared to nonlabouring tissues. Cumulative administration of L-cysteine (10⁻⁷–10⁻² mol/L), a precursor of H₂S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10⁻³ mol/L) increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (K_ATP) channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips.

Conclusion/Significance

H₂S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H₂S synthetic enzymes is down-regulated during labour, suggesting that H₂S is one of the factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition.     

Production of trigeneric (barley × wheat) × rye hybrids

Summary Rye (Secale cereale cv. Prolific 2n=14 and 2n =14 + 2B was crossed onto hybrids between barley (Hordeum vulgare 2n = 14) and wheat (Triticum aestivum 2n= 42). Pollinated florets were injected with GA₃ to promote fertilization and hybrid embryo development. At 16 days after pollination the watery caryopses were removed, embryos dissected and cultured on a modified B₅ medium. Approximately 20% of the cultured embryos produced both roots and coleoptile and developed into viable seedlings. Viable seeds were also obtained at a low frequency from the same cross combinations. The hybrids were wheat-like except for the hairy neck characteristic of rye. There were 35 chromosomes in somatic tissue; 21 wheat, 7 barley and 7 rye. The rye chromosomes were distinguishable by their larger size and terminal C-bands. A lower seed set was obtained using pollen from rye plants with 2n=14 + 2B chromosomes than from plants without B chromosomes.Contribution No. 577, Ottawa Research Station     

Molecular study and C-banding of chromosomes in common wheat alloplasmic lines obtained from the backcross progeny of barley–wheat hybrids Hordeum vulgare L. (2n = 14) × Triticum aestivum L. (2n = 42) and differing in fertility

We studied common wheat alloplasmic lines differing in fertility traits, which had been obtained from the backcross progeny of barley—wheat hybrids Hordeum vulgare L. (2n = 14) × Triticum aestivum L. (2n = 42), using molecular analysis and chromosome C-banding. It was found that the nuclei of all alloplasmic lines studied, regardless of their fertility traits, contained only the common wheat chromosomes (2n = 42). The formation of line L-79(10)(3)F₆, stable for self-fertility, from line L-79(10)F₃ was accompanied by changes of the proportions of simple sequence repeats of the parental common wheat varieties in the nuclear genome. The presence of barley genome fragments in line accessions with incomplete self-fertility was shown by RAPD. Heteroplasmy for mitochondrial genome loci was detected in these lines with the use of primers specific to the tMet-18S-5S repeat of mitochondrial ribosomal genes.Translated from Genetika, Vol. 40, No. 12, 2004, pp. 1668–1677.Original Russian Text Copyright © 2004 by Bildanova, Badaeva, Pershina, Salina.     

Advanced backcross quantitative trait locus analysis in winter wheat: Dissection of stripe rust seedling resistance and identification of favorable exotic alleles originated from a primary hexaploid wheat (Triticum turgidum ssp. dicoccoides?×?Aegilops tauschii)

Stripe rust (Puccinia striiformis W.) causes a range of disease symptoms in hexaploid wheat. We have utilized the AB-QTL (advanced backcross quantitative trait locus) strategy for the genetic dissection of complex disease resistance against stripe rust. An advanced backcross population designated Z86 was made by crossing the winter wheat cultivar Zentos (Triticum aestivum L.) and the primary (exotic) synthetic wheat accession Syn86L (T. turgidum ssp. dicoccoides?×?Aegilops tauschii). The population Z86, containing 150 BC2F3 lines, was inoculated with the stripe rust isolate R108E141. The disease symptoms were subjected to QTL analysis by using a genetic map based on 118 simple sequence repeat markers. This analysis revealed six QTL effects that were located on chromosomes 1B, 2B, 6B, 7B, 1D and 4D. At four loci, the exotic alleles were associated to increased resistance against stripe rust. The strongest effect, QYrs.Z86-1B, was detected on the short arm of chromosome 1B. Here, the introgression of the exotic allele resulted in 86% enhancement of resistance which explained 37.2% of the genetic variance (R ²). The second favorable effect of an exotic allele was detected on chromosome 1D at QYrs.Z86-1D, which accounted for 72% increase in resistance and explained 18.4% of the R ². Each of the exotic allele at QTL QYrs.Z86-6B and QYrs.Z86-7B accounted for around 60% enhancement of resistance against stripe rust. At QTL QYrs.Z86-2B and QYrs.Z86-4D, the relative performance of the exotic alleles was inferior due to the pre-eminence of the elite alleles which ranged from 67 to 72%. In addition, QTL analysis revealed four QTL by marker interaction effects. In most cases, the interaction between the elite and exotic alleles brought up resistance in the mixed background of BC2F3 lines. The data presented here provide valuable new genetic resources to be used for stripe rust resistance breeding as well as to isolate new alleles of exotic origin.     

High-density genetic map of durum wheat × wild emmer wheat based on SSR and DArT markers

A genetic linkage map of tetraploid wheat was constructed based on a cross between durum wheat [Triticum turgidum ssp. durum (Desf.) MacKey] cultivar Langdon and wild emmer wheat [T. turgidum ssp. dicoccoides (K?rn.) Thell.] accession G18-16. One hundred and fifty-two single-seed descent derived F(6) recombinant inbred lines (RILs) were analyzed with a total of 690 loci, including 197 microsatellite and 493 DArT markers. Linkage analysis defined 14 linkage groups. Most markers were mapped to the B-genome (60%), with an average of 57 markers per chromosome and the remaining 40% mapped to the A-genome, with an average of 39 markers per chromosome. To construct a stabilized (skeleton) map, markers interfering with map stability were removed. The skeleton map consisted of 307 markers with a total length of 2,317 cM and average distance of 7.5 cM between adjacent markers. The length of individual chromosomes ranged between 112 cM for chromosome 4B to 217 cM for chromosome 3B. A fraction (30.1%) of the markers deviated significantly from the expected Mendelian ratios; clusters of loci showing distorted segregation were found on chromosomes 1A, 1BL, 2BS, 3B, and 4B. DArT markers showed high proportion of clustering, which may be indicative of gene-rich regions. Three hundred and fifty-two new DArT markers were mapped for the first time on the current map. This map provides a useful groundwork for further genetic analyses of important quantitative traits, positional cloning, and marker-assisted selection, as well as for genome comparative genomics and genome organization studies in wheat and other cereals.     

Location of β-amylase sequences in wheat and its relatives

Summary A -amylase cDNA clone isolated from barley has been used to locate -amylase encoding sequences on wheat, rye, and Aegilops umbellulata chromosomes by hybridisation to restriction endonuclease digested DNA obtained from wheat aneuploid and wheat-alien addition lines. Structural genes were identified on homoeologous group 4 and 5 chromosomes, confirming the results of isozyme studies. In addition, a further set of structural genes was found on homoeologous group 2 chromosomes. It is proposed that there are two homoeoallelic series, -Amy-1 on group 4 or 5 chromosomes, and -Amy-2 on group 2 chromosomes. Evidence is presented that each locus contains one or two -amylase structural genes, and it is suggested that the large number of isozymes seen upon IEF are due to post-translational modifications.