Diversity changes in an intensively bred wheat germplasm during the 20th century

Plant breeding may lead to narrowing genetic diversity of cultivatedcrops, thereby affecting sustained selection gains in crop improvement. A totalof 47 microsatellite primer pairs (mapped to the 21 wheat genetic linkagegroups) were assessed in 75 Nordic spring wheat cultivars bred during the20^th century to determine the variation of genetic diversity in thisgermplasm throughout this period. The number of alleles ranged from one toseven, with an average of 3.6 alleles per microsatellite marker. A dendrogramresulting from analysis of the matrix of dissimilarities using the unweightedpair-group method with arithmetic average discriminated all cultivars andrevealed clusters of accessions released both from some geographical area inthe Nordic Region and the breeding era, i.e. before and after World War II. Geneticdiversity in this wheat material increased from 1900 to 1940 and again from1960 onwards. In between these two periods there was a loss of diversity, whichcould not be explained by changes in a single genome or in one or few chromosomesets. Effects of different selection within countries are revealed by cleardifferences in frequency of some microsatellite alleles. In adition somemicrosatellite alleles were lost during the first quarter of the century whileseveral new alleles were introduced in the Nordic spring wheat material duringthe second half of the century. These results suggest that genetic diversity inNordic spring wheat was enhanced by plant breeding in the first quarter of the20^th century and following a decrease during the second quarter wasincreased again by plant breeding.     

Chromosome substitutions of Triticum timopheevii in common wheat and some observations on the evolution of polyploid wheat species

Whether the two tetraploid wheat species, the well known Triticum turgidum L. (macaroni wheat, AABB genomes) and the obscure T. timopheevii Zhuk. (A^tA^tGG), have monophyletic or diphyletic origin from the same or different diploid species presents an interesting evolutionary problem. Moreover, T. timopheevii and its wild form T. araraticum are an important genetic resource for macaroni and bread-wheat improvement. To study these objectives, the substitution and genetic compensation abilities of individual T. timopheevii chromosomes for missing chromosomes of T. aestivum Chinese Spring (AABBDD) were analyzed. Chinese Spring aneuploids (nullisomic-tetrasomics) were crossed with a T. timopheevii x Aegilops tauschii amphiploid to isolate T. timopheevii chromosomes in a monosomic condition. The F₁ hybrids were backcrossed one to four times to Chinese Spring aneuploids without selection for the T. timopheevii chromosome of interest. While spontaneous substitutions involving all A^t- and G-genome chromosomes were identified, the targeted T. timopheevii chromosome was not always recovered. Lines with spontaneous substitutions from T. timopheevii were chosen for further backcrossing. Six T. timopheevii chromosome substitutions were isolated: 6A^t (6A), 2G (2B), 3G (3B), 4G (4B), 5G (5B) and 6G (6B). The substitution lines had normal morphology and fertility. The 6A^t of T. timopheevii was involved in a translocation with chromosome 1G, resulting in the transfer of the group-1 gliadin locus to 6A^t. Chromosome 2G substituted for 2B at a frequency higher than expected and may carry putative homoeoalleles of gametocidal genes present on group-2 chromosomes of several alien species. Our data indicate a common origin for tetraploid wheat species, but from separate hybridization events because of the presence of a different spectrum of intergenomic translocations.     

Identification of the G-genome donor to Triticum timopheevii by DNA:DNA hybridizations

In vitro DNA:DNA hybridizations and hydroxyapatite thermal-elution chromatography were employed to identify the diploid Triticum species ancestral to the G genome of Triticum timopheevii. Total genomic, unique-sequence, and repeated-sequence fractions of ³H-T. timopheevii DNA were hybridized to the corresponding fractions of unlabeled DNAs of T. searsii, T. speltoides, T. sharonensis, T. longissimum, and T. bicorne. The heteroduplex thermal stabilities indicated that, of the five species examined, T. speltoides was the most closely related to the G genome of T. timopheevii. Thus, T. spelotides appears to be the G-genome donor to T. timopheevii. The thermal stability profiles further indicated that the repeated DNA fractions from the five diploid species and the tetraploid T. timopheevii are more similar than the unique DNA fractions. This indicates that all of these species are closely related and that the sequences which comprise the current repeated fractions in the various species have not undergone any significant change since the formation of various species.Published with the approval of the Director of the West Virginia Agriculture and Forestry Experiment Station as Scientific Paper No. 1850.     

Nuclear genomic composition of asymmetric fusion products between irradiated transgenic Solanum brevidens and S. tuberosum: limited elimination of donor chromosomes and polyploidization of the recipient genome

Summary The production of asymmetric somatic hybrid calli after fusion between gamma-irradiated protoplasts from transgenic Solanum brevidens and protoplasts from S. tuberosum are reported. Transgenic (kanamycin-resistant, GUS-positive) S. brevidens plants and hairy root clones were obtained after transformation with Agrobacterium tumefaciens LBA 1060 (pRi1855) (pBI121) and LBA 4404 (pRAL4404) (pBI121), and A. rhizogenes LBA 9402 (pRi1855) (pBI121), respectively. Leaf protoplasts isolated from the transgenic plants or root protoplasts from the hairy root clones were fused with S. tuberosum leaf protoplasts, and several calli were selected on kanamycin-containing medium. The relative nuclear DNA content of the hybrid calli was measured by flow cytometry (FCM), and the percentages of DNA of the S. brevidens and S. tuberosum genomes in the calli were determined by dot blot analysis using species-specific DNA probes. Chromosome-specific restriction fragment length polymorphism (RFLP) markers were used to investigate the elimination of specific S. brevidens chromosomes in the hybrids. The combined data on FCM, dot blot and RFLP analysis revealed that 18–62% of the S. brevidens DNA was eliminated in the hybrid calli and that the RFLP marker for chromosome 7 was absent in seven out of ten calli. The absence of RFLP markers for chromosomes 5 and 11 hardly ever occurred. In most of the hybrids the ploidy level of the S. tuberosum genome had increased considerably.     

Cytogenetic investigation of Triticum timopheevii (Zhuk.) Zhuk. and related species using the C-banding technique

Triticum timopheevii and related species T. militinae (2n=28, A^tG) and T. zhukovskyi (2n=42, A^mA^tG), hybrids T. kiharae, T. miguschovae, the amphidiploid T. timopheevii x T. tauschii (all 2n=42, A^tGD), T. fungicidum (ABA^tG) and T. timonovum (2n=56, A^tA^tGG) were analyzed using the C-banding technique. Chromosomes of the A^m and A^t genomes in the karyotype of T. zhukovskyi differed in their C-banding pattern. Partial substitutions of A^t-genome chromosomes and a complete substitution of the G-genome chromosomes by homoeologous chromosomes of an unidentified tetraploid wheat species with an AB genome composition were found in the T. timonovum karyotype. A^t- and G-genome chromosomes in the karyotypes of all studied species had similar C-banding patterns and were characterized by a low level of polymorphism. The comparative stability of the A^t and G genomes is determined by the origin and specifity of cultivation of studied species.     

Synaptic behaviour of the tetraploid wheat Triticum timopheevii

Triticum timopheevii (2n=4x=A^tA^tGG) is an allotetraploid wheat which shows a diploid-like behaviour at metaphase-I. The synaptic process was analyzed in fully traced spread nuclei at mid-zygotene, late-zygotene and pachytene. The length and type of synaptonemal complexes, as well as the number of bivalent and multivalent associations, were determined in each nucleus. A high number of bivalents per nucleus was detected at all three stages. Nuclei at pachytene showed a lower frequency of multivalents than did zygotene nuclei, which suggests the existence of a pairing correction mechanism. At metaphase-I only homologous bivalents and, rarely, one pair of univalents were observed. Similarities between the diploidization mechanism of T. timopheevii and that of allohexaploid wheat, controlled by chromosome 5B, are discussed.     

Modification of the salinity response of wheat by the genome ofElytrigia elongatum

Summary Triticum aestivum cv. Chinese Spring wheat,Elytrigia elongatum (tall wheatgrass), and theTriticum-Elytrigia amphiploid were grown in complete nutrient culture containing, in addition, 0, 40, 80 and 120 mM NaCl. The 3 genotypes responded quite differently to increasing salinity; the Na concentration of wheat shoots increased in direct proportion to the increase in salinity of the external medium whereas the Elytrigia response was interpreted as showing high affinity for Na at low external Na (40 mM) but comparative exclusion of Na at high salinities (120 mM). In contrast, Na levels of the amphiploid were less than those of either wheat or Elytrigia under both low and high salinities. Thus the amphiploid behaved like wheat at 40 mM NaCl but more like Elytrigia at 120 mM NaCl because Na transport to the amphiploid shoot was restricted over the whole salinity range. The K concentration of the amphiploid shoot at high salinities was significantly greater than the K concentrations of either wheat or Elytrigia.     

Effect of Heat Stress During Grain Filling on Phosphoenolpyruvate Carboxylase and Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Activities of Various Green Organs in Winter Wheat

In two winter wheat (Triticum aestivum L.) cultivars differing in their response to high temperature, JD8 (tolerant) and J411 (sensitive) we studied the effect of heat stress on the activities of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase (RuBPC) in green organs during grain-filling. There were significantly higher PEPC activities and lower RuBPC activities in each of the non-leaf organs (awn, glume, lemma, peduncle, and sheath) than in the flag leaf blade. Under heat stress for 12 d, the activity of RuBPC quickly declined and the activity of PEPC first increased and later declined in all organs, resulting in a great increase of the PEPC/RuBPC ratios in the organs, particularly in non-leaf organs which had a higher PEPC/RuBPC than the flag leaf blade in all times. The PEPC activity and PEPC/RuBPC ratio in every organ of JD8 were higher than those in the same organ of J411. Thus the differences in PEPC activities and PEPC/RuBPC may be associated with the differences in photosynthetic heat tolerance among the organs of the same plant or between the two cultivars.     

Effects of Water Deficit during Germination of Wheat Seeds

Germinating seeds of spring wheat (Triticum aestivum L.) were tolerant to dehydration up to the 4^th day following imbibition and from the 5^th day the seedling survival decreased. Dehydration also inhibited the rate of seed dry mass depletion and seedling dry matter accumulation and increased the content of soluble sugars both in grain and seedlings. Glucose supplied either to dry seeds or to 4-d-old seedlings increased survival of dehydrated seedlings. In contrast, exogenously supplied non-readily metabolizable sorbose and mannose suppressed seedling survival.     

Microsatellite tagging of the leaf rust resistance gene <Emphasis Type="Italic">Lr16</Emphasis> on wheat chromosome 2BSc

Leaf rust, caused by Puccinia triticina, is one of the most damaging diseases of wheat worldwide. Lr16 is a widely deployed leaf rust resistance gene effective at the seedling stage. Although virulence to Lr16 exists in the Canadian P. triticina population, Lr16 provides a level of partial resistance in the field. The primary objective of this study was to identify markers linked to Lr16 that are suitable for marker-assisted selection (MAS). Lr16 was tagged with microsatellite markers on the distal end of chromosome 2BS in three mapping populations. Seven microsatellite loci mapped within 10 cM of Lr16, with the map distances varying among populations. Xwmc764 was the closest microsatellite locus to Lr16, and mapped 1, 9, and 3 cM away in the RL4452/AC Domain, BW278/AC Foremost, and HY644/McKenzie mapping populations, respectively. Lr16 was the terminal locus mapped in all three populations. Xwmc764, Xgwm210, and Xwmc661 were the most suitable markers for selection of Lr16 because they had simple PCR profiles, numerous alleles, high polymorphism information content (PIC), and were tightly linked to Lr16. Twenty-eight spring wheat lines were evaluated for leaf rust reaction with the P. triticina virulence phenotypes MBDS, MBRJ, and MGBJ, and analyzed with five microsatellite markers tightly linked to Lr16. There was good agreement between leaf rust infection type (IT) data and the microsatellite allele data. Microsatellite markers were useful for postulating Lr16 in wheat lines with multiple leaf rust resistance genes.     

Wheat storage proteins: diversity of HMW glutenin subunits in wild emmer from Israel

Summary The diversity of HMW glutenin subunits in the tetraploid wild progenitor of wheat, Triticum turgidum var. dicoccoides was studied electrophoretically in 231 individuals representing 11 populations of wild emmer from Israel. The results show that (a) The two HMW glutenin loci, Glu-A1 and Glu-B1, are rich in variation, having 11 and 15 alleles, respectively, (b) Genetic variation in HMW glutenin subunits is often severely restricted in individual populations, supporting an island population genetic model, (c) Significant correlations were found between glutenin diversity and the frequencies of specific glutenin alleles and physical (climate and soil) and biotic (vegetation) variables. Our results suggest that: (a) at least part of the glutenin polymorphisms in wild emmer can be accounted for by environmental factors and (b) the endosperm of wild emmer contains many allelic variants of glutenin storage proteins that are not present in bread wheat and could be utilized in breeding varieties with improved bread-making qualities.     

小麦Fe超氧歧化酶基因的原核表达分析

采用RT-PCR技术分离小麦Fe超氧歧化酶基因(FeSOD)的ORF全长cDNA,然后构建其原核表达载体,并对其表达的诱导时间、IPTG浓度、温度进行优化,以期获得较大量的重组蛋白。结果表明:实验获得了小麦FeSOD基因的ORF全长（600 bp）,ORF全长与原核表达载体pET-Dute1相连接构建了原核表达载体pET-FeSOD,将pET-FeSOD导入宿主菌Rosetta(DE3)中,经SDS-PAGE电泳结果显示,可以高效表达融合蛋白且表达的蛋白均主要以包涵体的形式存在;重组质粒表达出25.8 kD的融合蛋白,除去载体pET-Duet1自身表达的3.0 kD蛋白后,与FeSOD编码的约为22.8 kD蛋白的大小一致;对诱导表达条件的优化结果显示,融合蛋白 pET-FeSOD最佳的诱导表达条件为:0.5 mmol/L的IPTG浓度,37 ℃诱导5 h。该研究结果为进一步深入研究该基因的特性与功能奠定了基础。     

Wheat phylogeny determined by RFLP analysis of nuclear DNA. 2. Wild tetraploid wheats

Intra- and inter-specific variations in the nuclear DNA of Triticum dicoccoides Körn. (2n = 28, genome constitution AABB) and T. araraticum Jakubz. (2n = 28, AAGG), wild species, respectively, of the Emmer and Timopheevi group, were studied by restriction fragment length polymorphism (RFLP) analysis. Total DNAs of 32 T. dicoccoides and 24 T. araraticum accessions, collected from throughout the distribution areas of these species, were treated with two 6-bp cutters and hybridized with 30 nuclear DNA clones as probes to detect RFLPs. A total of 167 hybrid bands were observed per accession. All the enzyme-probe combinations showed RFLPs between accessions. The average genetic distance between the T. dicoccoides accessions was 0.0135 ± 0.0031 and that between the T. araraticum accessions 0.0036 ± 0.0015, indicative of about a four-fold intraspecific variation in T. dicoccoides as compared to T. araraticum in terms of genetic distance. No significant genetic differentiation was found for the geographical populations of these species, the genetic distance between the two species being 0.0482 ± 0.0022. The interspecific divergence corrected for intraspecific divergence was 0.0395, about three times that for T. dicoccoides and 11 times that for T. araraticum. The results show that in the wild state the Emmer and Timopheevi groups are clearly differentiated and that T. dicoccoides has much greater variation than T. araraticum, suggesting a relatively recent origin for the latter and therefore a diphyletic origin for these species.Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, Japan, No. 535     

Comparative analysis of whole genome structure of Streptococcus suis using whole genome PCR scanning

An outbreak associated with Streptococcus suis infection in humans emerged in Sichuan province, China in 2005. The outbreak is atypical for the apparent large number of human cases, high fatality rate and geographical spread. To determine whether the bacterium has changed, we compared both human and animal isolates from the Sichuan outbreak with those collected previously within China and in other countries using whole genome PCR scanning (WGPScaning) comparative sequencing of several known virulence factor genes and multilocus sequence typing (MLST) analysis. WGPScanning analysis showed that all primer pairs yielded PCR products of the expected sizes in all four strains tested. The nucleotide sequences of all the detected virulence factor genes are identical in the four strains and MLST results showed that the four isolates studied and reference strain all belonged to the ST1 complex. No new genetic changes were found in the genome structure of the isolates from this Sichuan outbreak. Contributed equally to this work Supported by the National Key Technologies Research and Development Program (Grant No. 2005BA711A09) from the Ministry of Science and Technology of China     

An Enhancing Effect of Exogenous Mannitol on the Antioxidant Enzyme Activities in Roots of Wheat Under Salt Stress

The role of mannitol as an osmoprotectant, a radical scavenger, a stabilizer of protein and membrane structure, and protector of photosynthesis under abiotic stress has already been well described. In this article we show that mannitol applied exogenously to salt-stressed wheat, which normally cannot synthesize mannitol, improved their salt tolerance by enhancing activities of antioxidant enzymes. Wheat seedlings (3 days old) grown in 100 mM mannitol (corresponding to −0.224 MPa) for 24 h were subjected to 100 mM NaCl treatment for 5 days. The effect of exogenously applied mannitol on the salt tolerance of plants in view of growth, lipid peroxidation levels, and activities of antioxidant enzymes in the roots of salt-sensitive wheat (Triticum aestivum L. cv. Kızıltan-91) plants with or without mannitol was studied. Although root growth decreased under salt stress, this effect could be alleviated by mannitol pretreatment. Peroxidase (POX) and ascorbate peroxidase (APX) activities increased, whereas superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GR) activities decreased in Kızıltan-91 under salt stress. However, activities of antioxidant enzymes such as SOD, POX, CAT, APX, and GR increased with mannitol pretreatment under salt stress. Although root tissue extracts of salt-stressed wheat plants exhibited only nine different SOD isozyme bands of which two were identified as Cu/Zn-SOD and Mn-SOD, mannitol treatment caused the appearance of 11 different SOD activity bands. On the other hand, five different POX isozyme bands were determined in all treatments. Enhanced peroxidation of lipid membranes under salt stress conditions was reduced by pretreatment with mannitol. We suggest that exogenous application of mannitol could alleviate salt-induced oxidative damage by enhancing antioxidant enzyme activities in the roots of salt-sensitive Kızıltan-91.     

Effect of Polyamines on Chlorophyll and Protein Contents, Photochemical Activity, and Energy Transfer in Detached Wheat Leaves during Dark Incubation

Spermine as compared to putrescine or spermidine retarded the loss of chlorophyll and protein contents to a greater extent in wheat primary leaves during dark incubation. Activities of whole chain electron transport, photosystem (PS) 1 and PS2, and absorbed excitation energy distribution in favour of PS 1 were protected by these amines in valency dependent manner during 72-h dark incubation.     

Wheat cells accumulate a syringyl-rich lignin during the hypersensitive resistance response

The stem rust fungus Puccinia graminis f.sp. tritici is an obligately biotrophic pathogen attacking wheat (Triticum aestivum). In compatible host/pathogen-interactions, the fungus participates in the host's metabolism by establishing functional haustoria in the susceptible plant cells. In highly resistant wheat cultivars, fungal attack is stopped by a hypersensitive response of penetrated host cells. This mechanism of programmed cell death of single plant cells is accompanied by the intracellular accumulation of material with UV-fluorescence typical of phenolic compounds. A similar reaction can be induced in healthy wheat leaves by the application of a rust-derived elicitor. We analysed the biochemical composition of this defense-induced phenolic material. Contents of total soluble and cell wall esterified and etherified phenolic acids were determined in rust-inoculated and elicitor-treated leaves of the fully susceptible wheat cultivar Prelude and its highly resistant, near-isogenic line Prelude-Sr5. While no resistance-related changes occured in any of these fractions, the lignin content as determined by the thioglycolic acid and the acetyl bromide methods increased after elicitor treatment. Nitrobenzene oxidation revealed that the entire increase can be explained by an increase in syringyl units only. These biochemical data were confirmed by fluorescence emission spectra analyses which indicated a defense-induced enrichment of syringyl lignin for cell wall samples both from elicitor-treated wheat leaves and single host cells undergoing a hypersensitive response upon fungal penetration.     

Chromosome structure of Triticum longissimum relative to wheat

Homoeologous pairing at meiotic metaphase I was analyzed in T. longissimum x T. aestivum hybrids in order to reconfirm the homoeologous relationships of T. longissimum chromosomes to wheat. Hybrids between T. longissimum and Chinese Spring carrying the Ph1 gene or theph1b mutation, which showed low and high pairing levels, respectively, were used. Chromosome arms associated at metaphase I were identified by C-banding. The homoeology of chromosomes 1S ^l , 2S ^l , 3S ^l , 5S ^l and 6S ^l to wheat group 1,2, 3, 5, and 6 chromosomes, respectively, was confirmed. Chromsome arms 4S ^l S and 7S ^l S showed normal homoeologous relationships to wheat. The 4S ^l L arm carries a translocated segment from 7S ^l L relative to wheat. The 7S ^l L arm seldom paired, likely because this arm lost a relatively long segment and received a very short segment in the interchange with 4S ^l L. Available data suggest that translocation 4S ^l L/7S ^l L arose in the evolution of T. longissimum, which implies that this species was not the donor of the B genome of wheat.     

Wheat tissues freeze-etched during exposure to extracellular freezing: distribution of ice

Pieces excised from leaf bases and laminae of seedlings of Triticum aestivum L. cv. Lennox were slowly frozen, using a specially designed apparatus, to temperatures between 2° and 14° C. These treatments ranged from non-damaging to damaging, based on ion-leakage tests to be found in the accompanying report (Pearce and Willison 1985, Planta 163, 304–316). The frozen tissue pieces were then freeze-fixed by rapidly cooling them, via melting Freon, to liquid-nitrogen temperature. The tissue was subsequently prepared for electron microscopy by freeze-etching. Ice crystals formed during slow freezing would tend to be much larger than those formed during subsequent freeze-fixation. Ice crystals surrounding the excised tissues were much larger in the frozen than in the control tissues (the latter rapidly freeze-fixed from room temperature). Large ice crystals were present between cells of frozen laminae and absent from controls. Intercellular spaces were infrequent in control leaf bases and no ice-filled intercellular spaces were found in frozen leaf bases. Intracellular ice crystals were smaller in frozen tissues than in controls. It is concluded that all ice formation before freeze-fixation was extracellular. This extracellular ice was either only extra-tissue (leaf bases), or extra-tissue and intercellular (laminae). Periplasmic ice was sometimes present, in control as well as slowly frozen tissues, and the crystals were always small; thus they were probably formed during freeze-fixation rather than during slow freezing. The plasma membrane sometimes showed imprints of cell-wall microfibrils. These were less abundant in leaf bases at 8° C than in controls, and were present on only a minority of plasma membranes from laminae. Therefore, extracellular ice probably did not compress the cells substantially, and changes in cell size and shape were possibly primarily a result of freezing-induced dehydration. Fine-scale distortions (wrinkles) in the plasma membrane, while absent from controls, were present, although only rarely, in both damaged and non-damaged tissues; they were therefore ice-induced but not directly related to the process of damage.     

The chromosomal location of peroxidase isozymes of the wheat kernel

Summary The analysis of the individual parts of the Triticum aestivum L. kernel yields a total of 11 peroxidase isozymes: m, n, a, c, d₁, d, d₂, e, f, g and h (in order from faster to slower migration). Isozymes a, c and d are found in the endosperm (Ed) and seed coats (C), while m, n, d₁, d₂, e, f, g and h are peculiar to the embryo and scutellum (E + S). The use of the nullitetrasomic and ditellosomic series of Chinese Spring wheat allows peroxidase isozymes to be associated with specific chromosome arms. Isozymes a, c and d (Ed) are associated with chromosome arms 7D^S, 4B^L and 7A^S; whereas isozymes m, d₂, e and f are associated with chromosome arms 3D^S, 3B^L, 3D^L and 3D^L, respecitvely. Thus, the E + S isozymes are associated with homoeology group 3 and the Ed isozymes with homoeology groups 7 (a and d isozymes) or 4 (c isozymes).