Molecular marker assisted genetic analysis of head shattering in six-rowed barley

Head shattering in barley (Hordeum vulgare L.) has two forms; brittle rachis and weak rachis. Brittle rachis is not observed in cultivated barley since all cultivars carry non-brittle alleles at one of the two complementary brittle rachis loci (Btr1;Btr2). Weak rachis causes head shattering in barley cultivars and may be confused with brittle rachis. Brittle rachis has been mapped to the chromosome 3 (3H) short arm while map position(s) of the weak rachis is unknown. Two major and a putative minor QTL for head shattering were mapped using the Steptoe × Morex doubled haploid line population. The largest QTL, designated Hst-3, located on the chromosome 3 (3H) centromeric region, is associated with a major yield QTL. The Steptoe Hst-3 region, when transferred into Morex, resulted in a substantial decrease in head shattering. High-resolution mapping of Hst-3 was achieved using isogenic lines. Brittle rachis was mapped with molecular markers and shown to be located in a different position from that of Hst-3. The second major QTL, designated Hst-2 S, is located on chromosome 2 S. This locus is associated with an environmentally sensitive yield QTL.     

AFLP analysis to assess genomic stability in Solanum regenerants derived from wild and cultivated species

The cultivated potato as well as its tuber-bearing relatives are considered model plants for cell and tissue culture, and therefore for exploiting the genetic variation induced by in vitro culture. The association between molecular stability and tissue culture in different genetic backgrounds and ploidy levels has already been explored. However, it still remains to be ascertained whether somaclonal variation differs between callus-derived chromosome-doubled and undoubled regenerants. Our research aimed at investigating, through amplified fragment length polymorphism (AFLP) markers, the genetic changes in marker-banding patterns of diploid and tetraploid regenerants obtained from one clone each of Solanum bulbocastanum Dunal and S. cardiophyllum Lindl (both 2n = 2x = 24) and tetraploids from cultivated S. tuberosum L. (2n = 4x = 48). Pairwise comparisons between the banding patterns of regenerants and parents allowed detecting considerable changes associated to in vitro culture both at diploid and tetraploid level. The percentages of polymorphic bands between diploid and tetraploid regenerants were, respectively, 57 and 69% in S. bulbocastanum and 58 and 63% in S. cardiophyllum. On average, the frequencies of lost parental fragments in regenerants were significantly higher than novel bands both in S. bulbocastanum (48 vs. 22%) and S. tuberosum (36 vs. 18%) regenerants. By contrast, in S. cardiophyllum, a similar incidence of the two events was detected (32 vs. 29%). Our results revealed that structural changes after tissue culture process strongly affected the genome of the species studied, but diploid and tetraploids regenerated plants responded equally.     

Molecular marker analyses of powdery mildew resistance in barley

Powdery mildew, caused byEryisphe graminis f. sp.hordei, is one of the most important diseases of barley (Hordeum vulgare). A number of loci conditioning resistance to this disease have been reported previously. The objective of this study was to use molecular markers to identify chromosomal regions containing genes for powdery mildew resistance and to estimate the resistance effect of each locus. A set of 28 F₁ hybrids and eight parental lines from a barley diallel study was inoculated with each of five isolates ofE. graminis. The parents were surveyed for restriction fragment length polymorphisms (RFLPs) at 84 marker loci that cover about 1100 cM of the barley genome. The RFLP genotypes of the F₁s were deduced from those of the parents. A total of 27 loci, distributed on six of the seven barley chromosomes, detected significant resistance effects to at least one of the five isolates. Almost all the chromosomal regions previously reported to carry genes for powdery mildew resistance were detected, plus the possible existence of 1 additional locus on chromosome 7. The analysis indicated that additive genetic effects are the most important component in conditioning powdery mildew resistance. However, there is also a considerable amount of dominance effects at most loci, and even overdominance is likely to be present at a number of loci. These results suggest that quantitative differences are likely to exist among alleles even at loci which are considered to carry major genes for resistance, and minor effects may be prevalent in cultivars that are not known to carry major genes for resistance.     

Morphological and cytological diversity of regenerants derived from half-anther cultures of anthurium

Anthurium anther culture was successfully established using half-anthers as explants. Explants were cultured on Winarto–Teixeira basal medium (WT-1) containing 0.01 mg/l α-naphthalene acetic acid (NAA), 0.5 mg/l thidiazuron (TDZ), and 1.0 mg/l 6-benzylaminopurine (BAP), or on New Winarto–Teixeira basal medium (NWT-3) supplemented with 0.02 mg/l NAA, 1.5 mg/l TDZ, and 0.75 mg/l BAP for callus initiation. Regenerated calli produced multiple shoots on WT-1, which were then rooted in NWT-3 supplemented with 1% activated charcoal. Plantlets were acclimatized ex vitro using a mixture of burned rice husk, rice husk, and bamboo peat (1:1:1, v/v/v) as the potting medium. There was considerable morphological and cytological diversity of regenerants derived from anther culture, which are described in detail in this study. The callus cluster color ranged from green to light green and had a high regeneration capacity (7.3 and 4.8 shoots/callus cluster), light reddish-yellow callus showed moderate regeneration (2.6 shoots/callus cluster), while reddish-yellow callus had the lowest regeneration capacity (1.5 shoots/callus cluster). Morphological variations clearly observed in regenerants derived from this technique included alterations in plant size, peduncle length, spathe position compared to leaves, the type and number of buds, spathe and spadix color, and spadix length. There were also cytological variations in both in vitro and ex vitro regenerants of anther culture with 23–29% haploids, 5–10% aneuploids, 56–69% diploids, and 3–4% triploids. The results strengthen other studies in which the development of anther cultures, especially via callus formation, resulted in morphological and cytological alterations. These variations have been discussed to great length in this paper.     

Molecular evolution of the seed storage proteins of barley, rye and wheat

The major storage proteins (prolamins) of barley, rye and wheat are characterized by the presence of two or more unrelated structural domains, one of which contains repeated sequences. Because of this repetitive structure and their restricted distribution (only in grasses), it has been suggested that the prolamins are of recent origin. Contrary to this hypothesis, we show that parts of the non-repetitive domain of one group of prolamins are homologous with sequences present in a large group of seed proteins from monocotyledonous and dicotyledonous plants; including Bowman-Birk protease inhibitors, cereal inhibitors of alpha-amylase and trypsin, and 2 S globulin storage proteins of castor bean and oil seed rape. This implies an ancient origin for these non-repetitive domains. The origins of the repetitive domains are not known but may lie within the grasses.     

Molecular analysis of mitochondrial DNA from rye (Secale cereale L.)

Summary Molecular characterization of mitochondrial (mt) DNA of rye (Secale cereale L.), free of significant amounts of contaminating chloroplast (cp) DNA, was initiated using the open-pollinated cultivar Halo as a source of mtDNA. Based on the compilation of data from restriction patterns, the molecular size of the mtDNA was estimated to be 410 Kb and its buoyant density was determined as 1.705 g/ml. Southern hybridization, using labelled cp genes (P700 and ribulosebiphosphate-carboxylase large subunit), indicated the presence of cpDNA-homologous regions on putative mtDNA fragments. Mt DNAs of inbred lines with fertile and cytoplasmic male sterile (CMS) Pampa cytoplasm were also analysed. Whereas the restriction patterns of mtDNAs of Halors and the fertile line turned out to be identical, Pampa mtDNA showed a unique restriction pattern, indicating (as in most other CMS systems) the involvement of mtDNA rearrangements in the expression of male sterility in rye. All 3 mtDNAs investigated contain regions homologous to the plasmid S1 of the CMS-S cytoplasm of Maize (Zea mays), as indicated by hybridization experiments. In Pampa cytoplasm the S-homologous sequence is located within a rearranged region of mtDNA.     

Molecular detection of an importation of type 3 wild poliovirus into Canada from The Netherlands in 1993.

During the fall and winter of 1992-1993 an outbreak of wild poliovirus type 3-associated poliomyelitis involving 71 patients occurred in The Netherlands. Almost all of the individuals involved in the outbreak belonged to an orthodox religious denomination that prohibits vaccination. A surveillance was initiated to determine if there had been an importation of this same strain of wild poliovirus into a southern Alberta community with a similar religious affiliation. Viral culture of stool samples from consenting individuals in the community resulted in viral isolates which typed as poliovirus type 3. Sequencing of amplicons generated from both the 5' nontranslated region and the VP1/2A portion of the genomes from representative poliovirus isolates indicated a greater than 99% genetic similarity to the strain from The Netherlands. The results of this study show that the utilization of PCR-based diagnostics offers an important molecular tool for the concise and rapid surveillance of possible cases of wild poliovirus importation into communities with individuals at risk for infection.     

AB-QTL analysis in spring barley. I. Detection of resistance genes against powdery mildew, leaf rust and scald introgressed from wild barley

The objective of this study was to map new resistance genes against powdery mildew (Blumeria graminis f. sp. hordei L.), leaf rust (Puccinia hordei L.) and scald [Rhynchosporium secalis (Oud.) J. Davis] in the advanced backcross doubled haploid (BC₂DH) population S42 derived from a cross between the spring barley cultivar Scarlett and the wild barley accession ISR42-8 (Hordeum vulgare ssp. spontaneum). Using field data of disease severity recorded in eight environments under natural infestation and genotype data of 98 SSR loci, we detected nine QTL for powdery mildew, six QTL for leaf rust resistance and three QTL for scald resistance. The presence of the exotic QTL alleles reduced disease symptoms by a maximum of 51.5, 37.6 and 16.5% for powdery mildew, leaf rust and scald, respectively. Some of the detected QTL may correspond to previously identified qualitative (i.e. Mla) and to quantitative resistance genes. Others may be newly identified resistance genes. For the majority of resistance QTL (61.0%) the wild barley contributed the favourable allele demonstrating the usefulness of wild barley in the quest for resistant cultivars.     

Biochemical and physiological estimation of barley regenerants obtained in selective systems

Callus lines of barley (Hordeum vulgare L.) resistant to aluminum toxicity and osmotic stress from which plants regenerated were obtained in selective systems. Comparative estimation of the intensity of lipid peroxidation, the content of anthocyans and ascorbic acid in leaves, the parameters of CO₂/H₂O gas exchange in leaves of regenerant plants obtained in selective mediums with gradual addition of aluminum ions and polyethylene glycol and in mediums without selective agents was performed in conditions of an artificial climate. The obtained data were compared with biochemical and physiological parameters of barley plants of the initial genotypes grown in the same conditions. Significant changes in the intensity of apparent photosynthesis, transpiration, and efficacy of water use by barley leaves were revealed as a result of callus culturing and especially due to the use of selective systems in vitro.     

High yield isolation of mesophyll protoplasts from wheat, barley and rye

Efficient procedures are described for high-yield isolation of mesophyll protoplasts from spring wheat ( Triticum aestivum L. cv. Glenlea), winter wheat ( Triticum aestivum L. cv. Frederick), barley ( Hordeum vulgare L. cv. Bruce) and rye ( Secale cereale L. cv. Puma). Factors such as plant age, composition of the incubation medium during isolation, purification procedures and culture medium affect protoplast yield, viability and metabolic competence, as measured by light-dependent CO₂ fixation. Optimal osmolarity of the isolation medium was equivalent to 1.8 times that measured in the leaves of all plant material used. The presence of 2 m M ascorbic acid in the preincubation and isolation medium increased the yield by 50% and conserved viability and metabolic competence. The protoplasts were stable for up to 48 h without loss of either viability or of original activity of CO₂ fixation, which was in the order of 100 μmol CO₂ (mg chl)⁻¹h⁻¹.
In our MC-56 liquid medium these protoplasts regenerated cell walls within 72 h and a few divided.     

Composition of guttation fluid from rye, wheat, and barley seedlings

This work was undertaken to determine the kinds and amount of substances that would account for the previously demonstrated differential growth of Claviceps purpurea on guttation fluids from Rosen rye, Genesee wheat, and Traill barley seedlings. Chromatographic methods were used for determining amino acids and sugars, spot tests and spectrometric methods for inorganic materials, and microbiological methods for vitamins.

Total sugar content is about equal in rye and barley fluids, but lower in wheat. Glucose is the principal sugar component of the rye and barley fluids and galactose highest in wheat. Most of the amino acid in all 3 fluids is aspartic acid or asparagine. Barley fluid is far higher than the other 2 in total amino acids, with wheat the lowest. Most inorganic elements are found to be highest in barley and lowest in wheat, with the exception of iron where rye is highest and barley lowest. Barley fluid is highest in choline, p-aminobenzoic acid, thiamine, and uracil, while rye is highest in inositol and pyridoxine. Wheat is much lower than the other 2 in choline and inositol.

     

Molecular cytogenetic analysis of durum wheat x tritordeum hybrids.

Southern and in situ hybridization were used to examine the chromosome constitution, genomic relationships, repetitive DNA sequences, and nuclear architecture in durum wheat x tritordeum hybrids (2n = 5x = 35), where tritordeum is the fertile amphiploid (2n = 6x = 42) between Hordeum chilense and durum wheat. Using in situ hybridization, H. chilense total genomic DNA hybridized strongly to the H. chilense chromosomes and weakly to the wheat chromosomes, which showed some strongly labelled bands. pHcKB6, a cloned repetitive sequence isolated from H. chilense, enabled the unequivocal identification of each H. chilense chromosome at metaphase. Analysis of chromosome disposition in prophase nuclei, using the same probes, showed that the chromosomes of H. chilense origin were in individual domains with only limited intermixing with chromosomes of wheat origin. Six major sites of 18S-26S rDNA genes were detected on the chromosomes of the hybrids. Hybridization to Southern transfers of restriction enzyme digests using genomic DNA showed some variants of tandem repeats, perhaps owing to methylation. Both techniques gave complementary information, extending that available from phenotypic, chromosome morphology, or isozyme analysis, and perhaps are useful for following chromosomes or chromosome segments during further crossing of the lines in plant breeding programs.     

The isolation of high molecular weight DNA from wheat,barley and rye for analysis by pulse-field gel electrophoresis

A method is presented for the preparation of large DNA molecules from protoplasts embedded in agarose blocks of three different cereals-hexaploid bread wheat (Triticum aestivum), barley (Hordeum vulgare) and rye (Secale cereale). Pulse-field gel electrophoresis (PFGE) analysis of these DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA molecules was greater than 6 Mb. DNA samples prepared by this method were shown to be useful for restriction analysis using both frequent and rare cutting enzymes.     

Molecular marker assisted broadening of the Central European heterotic groups in rye with Eastern European germplasm

Broadening the genetic base of heterotic pools is a key to ensure continued genetic gains in hybrid breeding and extend hybrid cultivation to new areas. In the present study, two Central European heterotic pools (Carsten and Petkus) and five Eastern European open-pollinated varieties (OPVs, Pop-1 to Pop-5) were studied with the objectives to (1) investigate the genetic diversity in OPVs and the heterotic pools using molecular and field data, (2) evaluate the molecular diversity among OPVs, (3) examine the combining ability for grain yield of the OPVs when crossed with testers in field trials, and (4) develop a strategy for targeted introgression of OPV germplasm into the heterotic pools. In total, 610 S₀ plants, 347 from OPVs and 263 from heterotic pools, were developed. Clones of the S₀ plants of OPVs were crossed with two testers belonging to each heterotic pool, while clones of heterotic pools were crossed with only the opposite tester. Testcrosses were evaluated for grain yield in multi-location trials. In addition, 589 S₀ plants were fingerprinted with 30 SSR markers. The data revealed that the Carsten pool has a narrow genetic base and should be the primary target for broadening the established heterotic pattern. Mean and genetic variance suggested that Pop-2 and Pop-4 are good candidates for introgression in Petkus pool and Pop-5 in Carsten pool. Nevertheless, introgression of Pop-5 in Carsten could reduce the genetic diversity between heterotic pools. Therefore, we suggest that either selected plants of Pop-5 should be introgressed or more Eastern European germplasm should be fingerprinted and field evaluated to identify promising germplasm for broadening the established heterotic pattern.     

Molecular marker linked to a chromosome region regulating seed Zn accumulation in barley

Zinc deficiency is a critical nutritional problem in soils, restricting yield and nutritional quality of barley (Hordeum vulgare L.). Some genotypes (Zn-efficient) can produce greater yield and accumulate more Zn in seed under Zn deficiency than standard (Zn-inefficient) genotypes. However, there is little information regarding the genetics of Zn uptake/accumulation and location of genes conferring Zn efficiency in barley. Selection through molecular markers for seed Zn accumulation might be an efficient complementary breeding tool in barley. With the aim of developing molecular markers for increased accumulation of Zn in seed, a population of 150 DH lines derived from a cross between Clipper (low-Zn-accumulator) and Sahara 3771 (high-Zn-accumulator) was screened in the field and glasshouse for seed Zn concentration and content. One dominant DNA polymorphism was detected using the microsatellite-anchored fragment length polymorphism (MFLP) technique. The candidate MFLP marker was isolated from the MFLP gel, re-amplified by PCR, cloned, sequenced, and converted into simple sequence-specific and PCR-based marker. This marker, located on the short arm of chromosome 2H, might be useful for the improvement of barley nutritional quality and productivity programs in Zn-deficient environments. However, high seed Zn alone can not replace the need for Zn fertilization.     

A comparative analysis of genetic polymorphism in wild and cultivated barley from Tibet using isozyme and ribosomal DNA markers.

This study was conducted to address some of the issues concerning the possible significance of Tibet in the origin and evolution of cultivated barley. A total of 1757 barley accessions from Tibet, including 1496 entries of Hordeum vulgare ssp. vulgare (HV), 229 entries of the six-rowed wild barley H. vulgare ssp. agriocrithon (HA), and 32 entries of the two-rowed wild barley H. vulgare ssp. spontaneum (HS), were assayed for allozymes at four esterase loci. A subsample of 491 accessions was surveyed for spacer-length polymorphism at two ribosomal DNA loci. Genetic variation is extensive in these barley groups, and the amount of genetic diversity in cultivated barley of this region is comparable with that of cultivated barley worldwide. The level of genetic variation of HA is significantly lower than the other two barley groups, and there is also substantial heterogeneity in the level of polymorphism among different agrigeographical subregions. However, little genetic differentiation was detected among the three barley groups (HV, HA, and HS), as well as among different agrigeographical subregions. Comparison of the results from this and previous studies indicated a strong differentiation between Oriental and Occidental barley, thus favoring the hypothesis of a diphyletic origin of cultivated barley.     

Prevalence of spindle cell sarcomas among wild Canada geese from southern Illinois.

Over a 6-yr period, two cases of neoplastic disease were diagnosed among 1,272 wild Canada geese (Branta canadensis interior) collected in Illinois (USA), Wisconsin (USA), and Ontario, Canada. One juvenile goose of each sex was involved. In both cases > 35 discrete masses were observed subdermally, intramuscularly, and within the body cavity. The tumors, diagnosed as spindle cell sarcomas, are among the most commonly observed neoplasms in free-living North American waterfowl. Based on our data, we suggest that these neoplasms occur with extremely low prevalence (0.002%) in free-living Canada geese.     

普通小麦-纤毛鹅观草染色体异附加系的分子标记鉴定

随机选取定位于小麦和大麦7个部分同源群上的135对EST、27对STS和253对SSR引物对24个可能的普通小麦-纤毛鹅观草二体异附加系的基因组DNA进行扩增。结果表明, 55对引物在亲本普通小麦中国春、Inayama Komugi、纤毛鹅观草和Inayama Komugi-纤毛鹅观草双二倍体间有多态性扩增, 其中31对引物可以在异附加系中扩增到纤毛鹅观草特异条带。根据PCR扩增结果, 异附加系07K02、07K06、07K39、07K201、07K202、07K255和07K256所添加的纤毛鹅观草染色体归属小麦第1部分同源群; 07K07、07K08、07K09、07K11、07K14和07K17所添加的纤毛鹅观草染色体归属小麦第2部分同源群; 07K15、07K16、07K21和07K47所添加的纤毛鹅观草染色体归属小麦第6部分同源群。