Advanced backcross QTL mapping of resistance to Fusarium head blight and plant morphological traits in a <Emphasis Type="Italic">Triticum macha</Emphasis> × <Emphasis Type="Italic">T. aestivum</Emphasis> population

While many reports on genetic analysis of Fusarium head blight (FHB) resistance in bread wheat have been published during the past decade, only limited information is available on FHB resistance derived from wheat relatives. In this contribution, we report on the genetic analysis of FHB resistance derived from Triticum macha (Georgian spelt wheat). As the origin of T. macha is in the Caucasian region, it is supposed that its FHB resistance differs from other well-investigated resistance sources. To introduce valuable alleles from the landrace T. macha into a modern genetic background, we adopted an advanced backcross QTL mapping scheme. A backcross-derived recombinant-inbred line population of 321 BC₂F₃ lines was developed from a cross of T. macha with the Austrian winter wheat cultivar Furore. The population was evaluated for Fusarium resistance in seven field experiments during four seasons using artificial inoculations. A total of 300 lines of the population were genetically fingerprinted using SSR and AFLP markers. The resulting linkage map covered 33 linkage groups with 560 markers. Five novel FHB-resistance QTL, all descending from T. macha, were found on four chromosomes (2A, 2B, 5A, 5B). Several QTL for morphological and developmental traits were mapped in the same population, which partly overlapped with FHB-resistance QTL. Only the 2BL FHB-resistance QTL co-located with a plant height QTL. The largest-effect FHB-resistance QTL in this population mapped at the spelt-type locus on chromosome 5A and was associated with the wild-type allele q, but it is unclear whether q has a pleiotropic effect on FHB resistance or is closely linked to a nearby resistance QTL.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Karyotype reshufflings of <Emphasis Type="Italic">Festuca pratensis</Emphasis> × <Emphasis Type="Italic">Lolium perenne</Emphasis> hybrids

Many different processes have an impact on the shape of plant karyotype. Recently, cytogenetic examination of Lolium species has revealed the occurrence of spontaneous fragile sites (FSs) associated with 35S rDNA regions. The FSs are defined as the chromosomal regions that are sensitive to forming gaps or breaks on chromosomes. The shape of karyotype can also be determined by interstitial telomeric sequences (ITSs), what was recognized for the first time in this paper in chromosomes of Festuca pratensis × Lolium perenne hybrids. Both FSs and ITSs can contribute to genome instabilities and chromosome rearrangements. To evaluate whether these cytogenetic phenomena have an impact on karyotype reshuffling observed in Festuca × Lolium hybrids, we examined F₁ F. pratensis × L. perenne plants and generated F₂-F₉ progeny by fluorescent in situ hybridization (FISH) using rDNA sequences, telomere and centromere probes, as well as by genomic in situ hybridization (GISH). Analyses using a combination of FISH and GISH revealed that intergenomic rearrangements did not correspond to FSs but overlapped with ITSs for several analyzed genotypes. It suggests that internal telomeric repeats can affect the shape of F. pratensis × L. perenne karyotypes. However, other factors that are involved in rearrangements and have a more crucial impact could exist, but they are still unknown.     

Assessment of antifungal effects of a novel compound from <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> against <Emphasis Type="Italic">Fusarium solani</Emphasis> by fluorescent staining

A novel compound CF66I produced by Burkholeria cepacia was investigated for its antifungal effects against Fusarium solani by three different fluorescent dyes. Dual staining with propidium iodide (PI) and fluorescein diacetate (FDA) demonstrated high doses of CF66I (120.0 μg ml⁻¹) killed the fungi by acting primarily on the cell membrane. However, at fungistatic concentration (20.0 μg ml⁻¹) of this compound, microscopic observations revealed swelling hyphae with abnormal chitin deposition, as determined by Calcofluor white (CFW) staining, which was indicative of the alterations in cell wall structure. In addition, inhibition of intracellular esterases activity was observed. These results led us to conclude that low doses of CF66I probably inhibited the fungal growth by interfering with the cell metabolic pathways.     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.     

Mapping QTL associated with resistance to <Emphasis Type="Italic">Fusarium</Emphasis> head blight in the Nanda2419 × Wangshuibai population. I. Type II resistance

Scab disease caused by Fusarium spp. has been a major concern for both wheat producers and consumers. Deployment of scab-resistant varieties is the major strategy to curb this disease. To identify the scab resistance genes in wheat cv. Wangshuibai, we produced a F_6:7 recombinant inbred line (RIL) population by crossing Wangshuibai with the scab-susceptible cultivar Nanda2419. The RILs were evaluated for scab resistance in the field by single floret inoculation in two replicates in 2002 and one replicate in 2003. The number of diseased spikelets (NDS) and the length of diseased rachides (LDR) were investigated to reflect the Type II resistance. Among 654 simple sequence repeat (SSR) markers surveyed, 326 were found to be polymorphic between the parents. A partial molecular map was constructed with these markers that covered over 2,210 cM of the wheat genome. Six chromosome regions showed association with both NDS and LDR in a one-way anova analysis, even though the variation explained by them varied between the two traits. Eight intervals were detected for their association with Type II resistance through interval mapping, five of which were not identified in single-point analysis. The quantitative trait loci (QTL) with large effects were the ones in the interval of Xgwm533-3–Xgwm533-1 on chromosome 3B and in the interval of Xwmc539–Xbarc024 on chromosome 6B, whose alleles favoring resistance originate from Wangshuibai. In addition, a QTL whose resistance allele originated from Nanda2419 was consistently detected in the interval of Xs1021m–Xgwm47-1 on chromosome 2B. These results suggest that Wangshuibai is the major source for Type II resistance in this population. The markers associated with these QTL would facilitate the use of scab-resistant genes of Wangshuibai in scab resistance breeding programs of wheat.F. Lin and Z.X. Kong have equally contributed to this work.     

Mapping and characterization of wheat stem rust resistance genes <Emphasis Type="Italic">SrTm5</Emphasis> and <Emphasis Type="Italic">Sr60</Emphasis> from <Emphasis Type="Italic">Triticum monococcum</Emphasis>

Key message

The new stem rust resistance gene Sr60 was fine-mapped to the distal region of chromosome arm 5A^mS, and the TTKSK-effective gene SrTm5 could be a new allele of Sr22.

Abstract

The emergence and spread of new virulent races of the wheat stem rust pathogen (Puccinia graminis f. sp. tritici; Pgt), including the Ug99 race group, is a serious threat to global wheat production. In this study, we mapped and characterized two stem rust resistance genes from diploid wheat Triticum monococcum accession PI 306540. We mapped SrTm5, a previously postulated gene effective to Ug99, on chromosome arm 7A^mL, completely linked to Sr22. SrTm5 displayed a different race specificity compared to Sr22 indicating that they are distinct. Sequencing of the Sr22 homolog in PI 306540 revealed a novel haplotype. Characterization of the segregating populations with Pgt race QFCSC revealed an additional resistance gene on chromosome arm 5A^mS that was assigned the official name Sr60. This gene was also effective against races QTHJC and SCCSC but not against TTKSK (a Ug99 group race). Using two large mapping populations (4046 gametes), we mapped Sr60 within a 0.44 cM interval flanked by sequenced-based markers GH724575 and CJ942731. These two markers delimit a 54.6-kb region in Brachypodium distachyon chromosome 4 and a 430-kb region in the Chinese Spring reference genome. Both regions include a leucine-rich repeat protein kinase (LRRK123.1) that represents a potential candidate gene. Three CC–NBS–LRR genes were found in the colinear Brachypodium region but not in the wheat genome. We are currently developing a Bacterial Artificial Chromosome library of PI 306540 to determine which of these candidate genes are present in the T. monococcum genome and to complete the cloning of Sr60.

     

Somatic hybridization between the diploids of <Emphasis Type="Italic">S.</Emphasis> × <Emphasis Type="Italic">michoacanum</Emphasis> and <Emphasis Type="Italic">S. tuberosum</Emphasis>

Interspecific somatic hybrids between a diploid potato clone DG 81-68 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuber-bearing species Solanum × michoacanum were generated and analyzed. About 30 regenerants displaying an intermediate morphology were obtained as a result of three separate PEG-mediated fusion experiments. The RAPD analysis confirmed the hybridity of all the regenerants. About 50% of the hybrid plants exhibited vigorous growth and were stable in culture, while the rest of them rooted poorly and grew slowly in vitro. Most of the hybrid clones were at the tetraploid level (70%), while 30% of the clones examined were at the hexaploid level. The S. × michoacanum (+) DG 81-68 hybrids with growth anomalies were aneuploid. The variation in late blight resistance of the hybrid clones was found in detached leaflet tests, with enhanced resistance characteristic for three tetraploid hybrids.     

An integrated molecular linkage map of diploid wheat based on a <Emphasis Type="Italic">Triticum boeoticum</Emphasis> × <Emphasis Type="Italic">T. monococcum</Emphasis> RIL population

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum × Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A^m. With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A^m of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.     

Fertility and precocity of <Emphasis Type="Italic">Osmunda</Emphasis> × <Emphasis Type="Italic">intermedia</Emphasis> offspring in culture

The feasibility of later-generation hybrid production in ferns has not been previously studied, although it is a significant factor in relation to reproductive isolation. Osmunda × intermedia, a hybrid between O. japonica and O. lancea, is semifertile and has moderate spore germination rates. Under the artificial conditions of this study, F2 and F3 offspring were formed. Some of the F2 offspring showed precocity, and some of the F3 offspring also showed precocity. This fertility suggests that introgressive hybridization might be ongoing in nature. This also indicates a currently unknown genetic control over the timing of fertile frond production in Osmunda.     

<Emphasis Type="Italic">Fusarium solani</Emphasis>, Tax-3, a new endophytic taxol-producing fungus from <Emphasis Type="Italic">Taxus chinensis</Emphasis>

An endophytic fungus, Tax-3, was isolated from barks of Taxus chinensis grown in the Qinba mountains, China. The strain was classified into Fusarium solani based on the morphological characteristics and the molecular phylogenetics inferred from the nuclear ribosomal DNA ITS sequences with the sequence similarity values of 100%. High performance liquid chromatography (HPLC) assay showed the F. solani, Tax-3, produced taxol with a higher yield of 163.35 μg/L in the reformative potato dextrose liquid medium (d), revealing its potential applications for taxol production. Bai Wan Deng and Kai Hui Liu contributed equally to this work.     

AB-QTL analysis in winter wheat: I. Synthetic hexaploid wheat (<Emphasis Type="Italic">T. turgidum</Emphasis> ssp. <Emphasis Type="Italic">dicoccoides </Emphasis> × <Emphasis Type="Italic">T. tauschii</Emphasis>) as a source of favourable alleles for milling and baking quality traits

The advanced backcross QTL (AB-QTL) strategy was utilised to locate quantitative trait loci (QTLs) for baking quality traits in two BC₂F₃ populations of winter wheat. The backcrosses are derived from two German winter wheat cultivars, Batis and Zentos, and two synthetic, hexaploid wheat accessions, Syn022 and Syn086. The synthetics originate from hybridisations of wild emmer (T. turgidum spp. dicoccoides) and T. tauschii, rather than from durum wheat and T. tauschii and thus allowed for the first time to test for exotic QTL effects on wheat genomes A and B in addition to genome D. The investigated quality traits comprised hectolitre weight, grain hardness, flour yield Type 550, falling number, grain protein content, sedimentation volume and baking volume. One hundred and forty-nine SSR markers were applied to genotype a total of 400 BC₂F₃ lines. For QTL detection, a mixed-model ANOVA was conducted, including the effects DNA marker, BC₂F₃ line, environment and marker × environment interaction. Overall 38 QTLs significant for a marker main effect were detected. The exotic allele improved trait performance at 14 QTLs (36.8%), while the elite genotype contributed the favourable effect at 24 QTLs (63.2%). The favourable exotic alleles were mainly associated with grain protein content, though the greatest improvement of trait performance due to the exotic alleles was achieved for the traits falling number and sedimentation volume. At the QTL on chromosome 4B the exotic allele increased the falling number by 19.6% and at the QTL on chromosome 6D the exotic allele led to an increase of the sedimentation volume by 21.7%. The results indicate that synthetic wheat derived from wild emmer × T. tauschii carries favourable QTL alleles for baking quality traits, which might be useful for breeding improved wheat varieties by marker-assisted selection.     

Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.     

<Emphasis Type="Italic">TNF</Emphasis><Emphasis Type="Italic">α</Emphasis> and <Emphasis Type="Italic">LT</Emphasis><Emphasis Type="Italic">α</Emphasis> gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.     

Regeneration and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of mature woody species <Emphasis Type="Italic">Photinia</Emphasis> × <Emphasis Type="Italic">fraseri</Emphasis> “Red Robin”

An efficient regeneration protocol for genetic transformation was developed from the leaves of an 11-year-old Phtinia × fraseri “Red Robin” tree. A high frequency of adventitious buds (88.63 ± 1.38%) and the highest maximum mean number of adventitious buds per explant (4.65 ± 0.48) were obtained in light conditions on Murashige–Skoog (MS) medium containing 2 mg l⁻¹ benzyladenine (BA) and 0.2 mg l⁻¹ α-Naphthaleneacetic acid (NAA). After preculturing for 6 days, over 95% of the shoots successfully rooted on 1/2 MS medium supplemented with 0.3 mg l⁻¹ indole-3-butyric acid (IBA) within 3 weeks. For genetic transformation, two crucial parameters (30 mg l⁻¹ kanamycin for selection and 30-min suspension time) were optimized. Taken together, a reliable transformation system with an efficiency of more than 5% was established. This genetic transformation protocol can be utilized for further genetic manipulation of the Photinia tree.