Showing papers in "Molecular Breeding in 1999"

A cautiously optimistic vision for marker-assisted breeding.

Abstract: Even though marker-assisted selection now plays a prominent role in the field of plant breeding, examples of successful, practical outcomes are rare. It is clear that DNA markers hold great promise, but realizing that promise remains elusive. Despite innovations like better marker systems and improved genetic mapping strategies, most marker associations are not sufficiently robust for successful marker-assisted selection. In large part this is due to inadequate experimental design. Molecular breeders must reassess their research programs so that DNA marker work leads to useful selection tools and valuable germplasm. As molecular breeders adopt more rigorous experimental guidelines and ambitious goals, they also need to integrate the growing body of knowledge from genomics and bioinformatics.

Expression of the insecticidal lectin from snowdrop (Galanthus nivalis agglutinin; GNA) in transgenic wheat plants: effects on predation by the grain aphid Sitobion avenae.

Abstract: Transgenic wheat plants containing the gene encoding snowdrop lectin (Galanthus nivalis agglutinin; GNA) under the control of constitutive and phloem-specific promoters were generated through the particle bombardment method. Thirty-two independently derived plants were subjected to molecular and biochemical analyses. Transgene integration varied from one to twelve estimated copies per haploid genome, and levels of GNA expression from 0 to ca. 0.2% of total soluble protein were observed in different transgenic plants. Seven transgenic plants were selected for further study. Progeny plants from these parental transformants were selected for transgene expression, and tested for enhanced resistance to the grain aphid (Sitobion avenae) by exposing the plants to nymphal insects under glasshouse conditions. Bioassay results show that transgenic wheat plants from lines expressing GNA at levels greater than ca. 0.04% of total soluble protein decrease the fecundity, but not the survival, of grain aphids. We propose that transgenic approaches using insecticidal genes such as gna in combination with integrated pest management present promising opportunities for the control of damaging wheat pests.

QTL for field resistance to late blight in potato are strongly correlated with maturity and vigour

Abstract: Field resistance to Phytophthora infestans, the causal agent of foliage and tuber blight in cultivated potatoes, earliness (maturity) and vigour, were examined in a diploid segregating potato population grown in replicated trials over three consecutive growing seasons A genetic linkage map of this population was constructed in parallel using PCR-based SSR, AFLP and CAPS markers Analysis of the trait scores alongside the marker segregation data allowed the identification of regions of the genome which were significantly correlated with components of the respective characters The most significant associations for all four traits were with marker alleles on potato linkage group V originating from the male (susceptible) parent In the case of foliage resistance to late blight, the positions of the majority of the effects, which were located on eleven of the twelve potato linkage groups, have been detected in previous [16] and parallel studies [21] The absence of Solanum demissum-derived R genes for hypersensitive response to late blight and the co-localisation of QTL for resistance, vigour and earliness suggest that developmental and/or physiological factors play a major role in determining the level of foliage resistance in this population In contrast with previous findings, a negative correlation was found between foliage and tuber blight resistance

Identification of quantitative trait loci for nitrogen use efficiency in maize

Abstract: Intensively managed crop systems are normally dependent on nitrogen input to maximize yield potential. Improvements in nitrogen- use efficiency (NUE) in crop plants may support the development of cropping systems that are more economically efficient and environment friendly. The objective of this study was to map and characterize quantitative trait loci (QTL) for NUE in a maize population. In preliminary experiments, inbred lines contrasting for NUE were identified and were used to generate populations of F2:3 families for genetic study. A total of 214 F2:3 families were evaluated in replicated trials under high nitrogen (280 kg/ha) and low nitrogen (30 kg/ha) conditions in 1996 and 1997. Analysis of ear-leaf area, plant height, grain yield, ears per plant, kernels number per ear, and kernel weight indicated significant genetic variation among F2:3 families. The heritability of these traits was found to be high (h2=0.57–0.81). The mapping population were genotyped using a set of 99 restriction fragment length polymorphism (RFLP) markers. A linkage map of these markers was developed and used to identify QTL. Between two and six loci were found to be associated with each trait. The correspondence of several genomic regions with traits measured under nitrogen limited conditions suggests the presence of QTL associated with NUE. QTLs will help breeders to improve their maize ideotype of a low-nitrogen efficiency by identifying those constitutive and adaptive traits involved in the expression of traits significantly correlated with yield, such as ear leaf area and number of ears per plant.

Transgenic approaches to increase dehydration-stress tolerance in plants.

Abstract: Plant productivity is strongly influenced by abiotic stress conditions induced by drought, high salt and low temperature. Plants respond to these conditions with an array of biochemical and physiological adaptations, at least some of which are the result of changes in gene expression. Transgenic approaches offer a powerful means of gaining valuable information to better understand the mechanisms governing stress tolerance. They also offer new opportunities to improve dehydration-stress tolerance in crops by incorporating a gene involved in stress protection into species that lack them. In this review, we discuss progress made towards understanding the molecular elements involved in dehydration-stress responses that have been used to improve salt or drought tolerance following several transgenic approaches. Further, we discuss various strategies being used to produce transgenic plants with increased tolerance to dehydration stress. These include the overproduction of enzymes responsible for biosynthesis of osmolytes, late-embryogenesis-abundant proteins and detoxification enzymes. At this time, there is a need for a careful appraisal of the genes to be selected and promoter elements to be used, because constitutive expression of these genes may not be desirable in all applications. In this context, the advantages and limitations of transgenic approaches currently being used are discussed together with the importance of using stress-inducible promoters and the introduction of multiple genes for the improvement of dehydration-stress tolerance.

Tri-trophic interactions involving pest aphids, predatory 2-spot ladybirds and transgenic potatoes expressing snowdrop lectin for aphid resistance

Abstract: Transgenic crops genetically engineered for enhanced insect resistance should be compatible with other components of IPM for the pest resistance to be durable and effective. An experimental potato line was genetically engineered to express an anti-aphid plant protein (snowdrop lectin, GNA), and assessed for possible interactions of the insect resistance gene with a beneficial pest predator. These extended laboratory studies are the first to demonstrate adverse tri-trophic interactions involving a lectin- expressing transgenic crop, a target pest aphid and a beneficial aphidophagous predator. When adult 2-spot ladybirds (Adalia bipunctata[L.]) were fed for 12 days on peach-potato aphids (Myzus persicae Sulzer) colonising transgenic potatoes expressing GNA in leaves, ladybird fecundity, egg viability and longevity significantly decreased over the following 2–3 weeks. No acute toxicity due to the transgenic plants was observed, although female ladybird longevity was reduced by up to 51%. Adverse effects on ladybird reproduction, caused by eating peach-potato aphids from transgenic potatoes, were reversed after switching ladybirds to feeding on pea aphids from non-transgenic bean plants. These results demonstrate that expression of a lectin gene for insect resistance in a transgenic potato line can cause adverse effects to a predatory ladybird via aphids in its food chain. The significance of these potential ecological risks under field conditions need to be further evaluated.

A genetic analysis of quantitative resistance to late blight in potato: towards marker-assisted selection

Abstract: Late blight caused by the oomycete Phytophthora infestans is the most important fungal disease in potato cultivation worldwide Resistance to late blight is controlled by a few major genes (R genes) which can be easily overcome by new races of P infestans and/or by an unknown number of genes expressing a quantitative type of resistance which may be more durable Quantitative resistance of foliage to late blight was evaluated in five F1 hybrid families originating from crosses among seven different diploid potato clones Tuber resistance was evaluated in four of the families Two of the families were scored for both foliage maturity and vigour The five families were genotyped with DNA-based markers and tested for linkage with the traits analysed QTL (quantitative trait locus) analysis identified at least twelve segments on ten chromosomes of potato having genes that affect reproducibly foliage resistance Two of those segments also have major R genes for resistance to late blight The segments are tagged by 21 markers that can be analyzed based on PCR (polymerase chain reaction) with specific oligonucleotide primers One QTL was detected for tuber resistance and one for foliage vigour Two QTLs were mapped for foliage maturity Major QTL effects on foliage and tuber resistance to late blight and on foliage maturity and vigour were all linked with marker GP179 on linkage group V of potato Plants having alleles at this QTL, which increased foliage resistance, exhibited decreased tuber resistance, later maturity and more vigour

Commercial production of aprotinin in transgenic maize seeds

Abstract: The development of genetic transformation technology for plants has stimulated an interest in using transgenic plants as a novel manufacturing system for producing different classes of proteins of industrial and pharmaceutical value. In this regard, we report the generation and characterization of transgenic maize lines producing recombinant aprotinin. The transgenic aprotinin lines recovered were transformed with the aprotinin gene using the bar gene as a selectable marker. The bar and aprotinin genes were introduced into immature maize embryos via particle bombardment. Aprotinin gene expression was driven by the maize ubiquitin promoter and protein accumulation was targeted to the extracellular matrix. One line that showed a high level of aprotinin expression was characterized in detail. The protein accumulates primarily in the embryo of the seed. Southern blot analysis showed that the line had at least 20 copies of the bar and aprotinin genes. Further genetic analysis revealed that numerous plants derived from this transgenic line had a large range of levels of expression of the aprotinin gene (0–0.069%) of water-soluble protein in T2 seeds. One plant lineage that showed stable expression after 4 selfing generations was recovered from the parental transgenic line. This line showed an accumulation of the protein in seeds that was comparable to the best T2 lines, and the recombinant aprotinin could be effectively recovered and purified from seeds. Biochemical analysis of the purified aprotinin from seeds revealed that the recombinant aprotinin had the same molecular weight, N-terminal amino acid sequence, isoelectric point, and trypsin inhibition activity as native aprotinin. The demonstration that the recombinant aprotinin protein purified from transgenic maize seeds has biochemical and functional properties identical to its native counterpart provides a proof-of-concept example for producing new generation products for the pharmaceutical industry.

Multiple traits of agronomic importance in transgenic indica rice plants: analysis of transgene integration patterns, expression levels and stability

Abstract: We cotransformed indica rice (Oryza sativa L. cvs. Basmati 370 and M7) with three plasmids, carrying a total of four genes, by particle bombardment. The Bacillus thuringiensis (Bt) δ-endotoxin genes cry1Ac and cry2A were carried on separate vectors, while the gna (snowdrop lectin) and hpt (hygromycin phosphotransferase) genes were linked on the same, cointegrate vector. We evaluated the molecular and expression profiles of 29 independently derived transgenic lines over two generations. The gna and hpt genes cointegrated with a frequency of 100% as expected. Furthermore, 60% of the transgenic plants carried all four genes. Southern blot analysis showed that transgene copy number ranged from 1 to 15. We used western blots to determine protein expression levels in R0 and R1 plants. We observed wide variation in the expression levels of the nonselected transgenes among independently-derived lines, but expression levels within lines derived from the same clone were similar. Consistent with previous reports, we observed no correlation between transgene copy number and the level or stability of protein expression. We show that the introduction of multiple agronomically valuable genes into the rice genome by cotransformation is a practical strategy for engineering elite rice varieties.

Heterologous expression of the Arabidopsis etr1-1 allele inhibits the senescence of carnation flowers

Abstract: The Arabidopsis thaliana etr1-1 allele, capable of conferring ethylene insensitivity in a heterologous host, was introduced into transgenic carnation plants. This gene was expressed under control of either its own promoter, the constitutive CaMV 35S promoter or the flower-specific petunia FBP1 promoter. In about half of the transgenic plants obtained flower senescence was delayed by at least 6 days relative to control flowers, with a maximum delay of 16 days, a 3-fold increase in vase life. These flowers did not show the petal inrolling phenotype typical of ethylene-dependent carnation flower senescence. Instead, petals remained firm and finally started to rot and decolorize.

Increased insect resistance in transgenic wheat stably expressing trypsin inhibitor CMe

Abstract: Proteinase inhibitors have been proposed to function as plant defence agents against herbivorous pests We have introduced the barley trypsin inhibitor CMe (BTI-CMe) into wheat (Triticum aestivum L) by biolistic bombardment of cultured immature embryos Of the 30 independent transgenic wheat lines selected, 16 expressed BTI-CMe BTI-CMe was properly transcribed and translated as indicated by northern and western blot, with a level of expression in transgenic wheat seeds up to 11% of total extracted protein No expression was detected in untransformed wheat seeds Functional integrity of BTI-CMe was confirmed by trypsin inhibitor activity assay The significant reduction of the survival rate of the Angoumois grain moth (Sitotroga cerealella, Lepidoptera: Gelechiidae), reared on transgenic wheat seeds expressing the trypsin inhibitor BTI-CMe, compared to the untransformed control confirmed the potential of BTI-CMe for the increase of insect resistance However, only early-instar larvae were inhibited in transgenic seeds and expression of BTI-CMe protein in transgenic leaves did not have a significant protective effect against leaf-feeding insects

Development of SCARs by direct sequencing of RAPD products: a practical tool for the introgression and marker-assisted selection of wheat

Abstract: RAPD markers generated by mixtures of two different primers were developed for octoploid × Tritordeum (amphiploid Hordeum chilense × Triticum aestivum) and its parents Addition lines were used to identify 21 specific RAPD markers for the H chilense chromosomes detectable in a wheat background Ten RAPD bands were selected and eight of them were converted into dominant SCAR markers by direct sequencing of the RAPD products, avoiding the costly and time-consuming cloning step The methodology overcomes some of the pitfalls associated with the election of the right clones when developing SCARs from RAPD markers The SCARs generated have maintained both the chromosome specificity and the possibility of detection in a wheat background This strategy provides a rapid method for the characterization of RAPD markers and for the development of PCR-based markers for both the characterization of the introgression of H chilense in bread and durum wheat, as well as the efficient and reliable screening of tritordeum lines

Transformation of pasta wheat (Triticum turgidum L. var. durum) with high-molecular-weight glutenin subunit genes and modification of dough functionality

Abstract: Particle bombardment has been used to transform three cultivars (L35, Ofanto, Svevo) and one breeding line (Latino × Lira) of durum wheat (Triticum turgidum L. var. durum). These varieties were co-transformed with plasmids containing selectable and scorable marker genes (bar and uidA) and plasmids containing one of two high-molecular-weight (HMW) glutenin subunit genes (encoding subunits 1Ax1 or 1Dx5). Ten independent transgenic lines were recovered from 1683 bombarded scutella (transformation efficiency thus 0.6%). Five lines expressed either subunit 1Dx5 or 1Ax1 at levels similar to those of endogenous subunits encoded on chromosome 1B. To identify the effects of the transgenes on the functional properties of grain, three lines showing segregation for transgene expression were used to isolate sibling T2 plants which were null or positive for the transgene product. Analysis of these plants using a small-scale mixograph showed that expression of the additional subunits resulted in increased dough strength and stability, demonstrating that transformation can be used to modify the quality of durum wheat for bread and pasta making.

Single large-scale marker-assisted selection (SLS-MAS)

Abstract: This paper presents a new approach for plant improvement that interactively combines the use of DNA markers and conventional breeding. This approach involves selecting plants at early generation with a fixed, favorable genetic background at specific loci, conducting a single large-scale marker-assisted selection (SLS-MAS) while maintaining as much as possible the allelic segregation in the rest of the genome. First, the identification of elite lines presenting high allelic complementarity and being outstanding for traits of interest is required to capture favorable alleles from different parental lines. Second, after identification of the most favorable genomic regions for each selected parental line, those lines are intercrossed to develop segregating populations from which plants homozygous for favorable alleles at target loci are selected. One objective of the scheme is to conduct the marker-assisted selection only once, and it requires the selection of a minimum number of plants to maintain sufficient allelic variability at the unselected loci. Therefore, the selection pressure exerted on the segregating population is quite high and the screening of large populations is required to achieve the objectives of the scheme. No selection is applied outside the target genomic regions, to maintain as much as possible the Mendelian allelic segregation among the selected genotypes. After selection with DNA markers, the genetic diversity at un-selected loci may allow breeders to generate new varieties and hybrids through conventional breeding in response to various local needs. Although the single large-scale MAS scheme described here is oriented toward maize and large-scale breeding programs with substantial resources, the flexibility of this scheme would allow breeding programs to develop options compatible with local resources.

Soybean Kunitz trypsin inhibitor (SKTI) confers resistance to the brown planthopper (Nilaparvata lugens Stål) in transgenic rice

Abstract: Proteinase inhibitors are widely distributed in animals, plants and microorganisms and their roles in plants are associated with defense against pests The utilization of proteinase inhibitors for crop protection has been actively investigated with a variety of proteinase inhibitors Soybean Kunitz trypsin inhibitor (SKTI), one of the major seed storage protein, is synthesized for a short period during seed development To investigate the role of SKTI in a plant's defense system against insect predation, a recombinant plasmid containing the full-length cDNA of SKTI under control of the CaMV 35S promoter was introduced into rice protoplasts by using the PEG direct gene transfer method and a large number of transgenic rice plants were regenerated The integration, expression, and inheritance of this gene was demonstrated in R1 and R2 generations by Southern, northern, and western analyses Accumulation levels (005–25% of soluble proteins) of SKTI protein were detected in R1 and R2 plants Bioassay with R1 and R2 transgenic plants revealed that transgenic plants are more resistant to destructive insect pest of rice, brown planthopper (Nilaparvata lugens Stal), than the control plants Thus, introduction of SKTI into rice plants can be used to control insect pests

Genetic engineering of parthenocarpic fruit development in tomato.

Abstract: Parthenocarpy was engineered in two genotypes of Lycopersicon esculentum Mill. by using the DefH9-iaaM chimeric gene. The parthenocarpic trait consists of fruit set and growth in the absence of fertilization. Seedless parthenocarpic fruits were obtained from emasculated flowers, and fruits with seeds from pollinated flowers. All parthenocarpic tomato plants analysed expressed the DefH9-iaaM gene during flower development. The fruit set percentage of emasculated transgenic flowers was similar to that of control plants. In 7 out of 8 independent transgenic plants, the fresh weight of fruits derived from pollinated or emasculated flowers did not significantly differ from that of fruits obtained by pollination of the control plants. The pH of the parthenocarpic fruit was generally unaffected and the soluble solid concentration was either unchanged or increased. Thus, the DefH9-iaaM gene is a genetic tool that might be used to improve tomato productivity.

Subcellular targeting of green fluorescent protein to plastids in transgenic rice plants provides a high-level expression system

Abstract: In order to develop a high-level expression system in transgenic rice, we inserted a synthetic gene (sgfp) encoding a modified form of the green fluorescent protein (GFP) into two expression vectors, Act1-sgfp for an untargeted and rbcS-Tp-sgfp for a chloroplast targeted expression. Several fertile transgenic rice plants were produced by the Agrobacterium-mediated method. Confocal microscopic analyses demonstrated that, in cells expressing the Act1-sgfp, GFP fluorescence was localized within the cytoplasm and nucleoplasm whereas, in cells expressing the rbcS-Tp-sgfp fusion gene, the fluorescence was specifically targeted to chloroplasts and non-green plastids. The levels of sgfp expression were about 0.5% of the total soluble protein in mature leaf tissues of the Act1-sgfp transformed lines. In contrast, expression levels were markedly increased in mature leaf tissues of the rbcS-Tp-sgfp transformed lines, yielding about 10% of the total soluble protein. N-terminal sequencing of the localized GFPs revealed that the Tp-GFP fusion protein was correctly processed during import to non-green plastids, as well as to chloroplasts. Thus, our results demonstrate that GFP can be produced at high levels and localized in specific subcellular spaces of transgenic plants, providing a high-level expression system for general use in rice, an agronomically important cereal.

Stable genetic transformation of white pine (Pinus strobus L.) after cocultivation of embryogenic tissues with Agrobacterium tumefaciens.

Abstract: A genetic transformation procedure for white pine has been developed after cocultivation of embryogenic tissues with Agrobacterium tumefaciens This efficient transformation procedure led to an average of four independent transformed lines per gram of cocultivated embryogenic tissue and up to 50 transformed lines can be obtained in a routine experiment Constructs bearing the uidA gene or the green fluorescent protein (GFP) gene were introduced and β-glucuronidase (GUS) activity was followed over time The expression of the uidA gene was lowest with a 35S-gus-intron construct and was 20-fold higher with a 35S-35S-AMVgus::nptII construct The addition of scaffold attachment region (SAR) sequences surrounding the gus::nptII fusion did not significantly enhance the GUS activity Transformed mature somatic embryos have been germinated and plantlets are presently being acclimatized

Identification of molecular markers for the detection of the yellow rust resistance gene Yr17 in wheat

Abstract: The Yr17 gene, which is present in many European wheat cultivars, displays yellow rust resistance at the seedling stage. The gene introduced into chromosome 2A from Aegilops ventricosa was previously found to be closely linked (0.5 cM) to leaf and stem rust resistance genes Lr37 and Sr38, respectively. The objective of this study was to identify molecular markers linked to the Yr17 gene. We screened with RAPD primers, for polymorphism, the DNAs of cv. Thatcher and the leaf rust-resistant near-isogenic line (NIL) RL 6081 of cv. Thatcher carrying the Lr37 gene. Using a F2 progeny of the cross between VPM1 (resistant) and Thesee (susceptible), the RAPD marker OP-Y15580 was found to be closely linked to the Yr17 gene. We converted the OP- Y15580 RAPD marker into a sequence characterized amplified region (SCAR). This SCAR marker (SC-Y15) was linked at 0.8 ± 0.7 cM to the Yr17 resistance gene. We tested the SC-Y15 marker over a survey of 37 wheat cultivars in order to verify its consistency in different genetic backgrounds and to explain the resistance of some cultivars against yellow rust. Moreover, we showed that the Xpsr150-2Mv locus marker of Lr gene described by Bonhomme et al. [6] which possesses A. ventricosa introgression on the 2A chromosome was also closely linked to the Yr17 gene. Both the SCAR SC-Y15 and Xpsr150-2Mv markers should be used in breeding programmes in order to detect the cluster of the three genes Yr17, Lr37 and Sr38 in cross progenies.

Concanavalin A inhibits development of tomato moth (Lacanobia oleracea) and peach-potato aphid (Myzus persicae) when expressed in transgenic potato plants

Abstract: The effects of concanavalin A (ConA), a glucose/mannose-specific lectin from jackbean (Canavalia ensiformis), on insect crop pests from two different orders, Lepidoptera and Homoptera, were investigated. When fed to larvae of tomato moth (Lacanobia oleracea) at a range of concentrations (0.02–2.0% of total protein) in artificial diet, ConA decreased survival, with up to 90% mortality observed at the highest dose level, and retarded development, but had only a small effect on larval weight. When fed to peach-potato aphids (Myzus persicae) at a range of concentrations (1–9μM) in liquid artificial diet, ConA reduced aphid size by up to 30%, retarded development to maturity, and reduced fecundity (production of offspring) by >35%, but had little effect on survival. With both insects, there was a poor correlation between lectin dose and the quantitative effect. Constitutive expression of ConA in transgenic potatoes driven by the CaMV 35S promoter resulted in the protein accumulating to levels lower than predicted, possibly due to potato not being able to adequately reproduce the post-translational processing of this lectin which occurs in jackbean. However, the expressed lectin was functionally active as a haemagglutinin. Bioassay of L. oleracea larvae on ConA-expressing potato plants showed that the lectin retarded larval development, and decreased larval weights by >45%, but had no significant effect on survival. It also decreased consumption of plant tissue by the larvae. In agreement with the diet bioassay results, ConA-expressing potatoes decreased the fecundity of M. persicae by up to 45%. ConA thus has potential as a protective agent against insect pests in transgenic crops.

Verification of yield QTL through realized molecular marker- assisted selection responses in a barley cross

Abstract: Verification of putative quantitative trait loci (QTL) is an essential step towards implementing the use of marker-assisted selection (MAS) in cultivar improvement In a previous study with 150 doubled haploid lines derived from the 6-row cross Steptoe/Morex (S/M), four regions (QTL1–4) of the barley genome were associated with differential genotypic expression for grain yield across environments The objectives of this study were to verify the value of these four QTL for selection and to compare the efficiency of alternative MAS strategies using these QTL vs conventional phenotypic selection for grain yield A total of 92 DHLs derived from the S/M cross that were not used in the original mapping efforts were used for QTL verification Confirmation of QTL effects was first accomplished by assessing yield differences between individuals carrying alternative alleles at each putative locus in three environments QTL1 on chromosome 3 was confirmed as the most important and consistent locus to determine yield across sites, with the S allele being favorable The M allele at QTL3 on chromosome 6 was beneficial for grain yield across sites, but to a lesser degree than QTL1 Magnitudes of allele effects at QTL2 (chromosome 2) and QTL4 (chromosome 7) were highly influenced by the environment where the genotypes were grown Verification of QTL effects was best achieved by comparing realized selection response Genotypic (MAS) and tandem genotypic and phenotypic selection were at least as good as phenotypic selection Consistent selection responses were detected for QTL1 alone and together with QTL3 Genotypic selection for lines carrying the S allele at QTL1 resulted in the identification of high-yielding genotypes Selection responses increased when the M allele at QTL3 was combined with the S allele at QTL1 Significant qualitative QTL × environment interactions for QTL2 and QTL4 were detected through differential realized selection responses at different sites Without a thorough understanding of the physiological and agronomic particulars of any QTL and the target environment, MAS for QTL showing qualitative interactions should be minimized

Transgenic broccoli with high levels of Bacillus thuringiensis Cry1C protein control diamondback moth larvae resistant to Cry1A or Cry1C

Abstract: A synthetic Bacillus thuringiensis (Bt) cry1C gene was introduced into broccoli (Brassica oleracea ssp. italica) by Agrobacterium-mediated transformation. Twenty-one Cry1C transgenic plants were regenerated from 400 hypocotyl and petiole explants. Variable amounts of stable steady- state cry1C mRNA accumulated in different transgenic plants. Cry1C protein (up to 0.4% of total soluble protein) was produced in correlation with the cry1C mRNA levels. Leaf section and whole-plant bioassays were done using diamondback moth (DBM) larvae from lines susceptible to Bt or resistant to Cry1A or Cry1C proteins (Cry1AR or Cry1CR, respectively). Plants with high levels of Cry1C protein caused rapid and complete mortality of all three types of DBM larvae with no defoliation. Plants with lower levels of Cry1C protein showed an increasing differential between control of susceptible of Cry1AR DBM. This study demonstrated that high production of Cry1C protein can protect transgenic broccoli not only from susceptible or Cry1AR DBM larvae but also from DBM selected for moderate levels of resistance of Cry1C. The Cry1C- transgenic broccoli were also resistant to two other lepidopteran pests of crucifers (cabbage looper and imported cabbage worm). These plants will be useful in studies of resistance management strategies involving multiple transgenes.

Expression of bacterial cellulase genes in transgenic alfalfa (Medicago sativa L.), potato (Solanum tuberosum L.) and tobacco (Nicotiana tabacum L.)

Abstract: The genes encoding thermostable cellulases E2 and E3 of Thermomonospora fusca were expressed in plants under the control of the constitutive, hybrid Mac promoter For both E2 and E3, the genes were modified so as to remove the sequence encoding the bacterial leader peptide Western blot analysis indicated that expression levels of recombinant cellulase in tobacco lines ranged up to about 01% (E2) and 002% of soluble protein (E3) No phenotypic effect of cellulase expression was noted Recombinant E2 expressed in either tobacco or alfalfa was active and retained heat stability These findings are an important first step in the development of crop plants as a production system for cellulases

Using microsatellites, isozymes and AFLPs to evaluate genetic diversity and redundancy in the cassava core collection and to assess the usefulness of DNA-based markers to maintain germplasm collections

Abstract: The cassava core collection was selected to represent, with minimum repetitiveness, the potential genetic diversity of the crop. The core (630 accessions) was chosen from the base collection (over 5500 accessions) on the basis of diversity of origin (country and geographic), morphology, isozyme patterns and specific agronomic criteria. To asses the genetic diversity of the core, 521 accessions were typed with four microsatellite loci. Allele diversity and frequency, and size variance of dinucleotide repeats (Rst statistic) were estimated. Microsatellite allele numbers and frequencies varied among countries: Colombia and Brazil had the largest number of different alleles across all loci. Mexico also had a high number, ranking fifth after Peru, Costa Rica and Venezuela (which tied). Unique alleles were present in accessions from Brazil, Colombia, Guatemala, Venezuela and Paraguay. A small number (1.34%) of potential duplicates were identified through isozyme and AFLP profiles. Thus, the present results indicated that traditional markers have been highly effective at selecting unique genotypes for the core. Future selections of cassava germplasm sets can be aided by DNA-based markers to ensure genetically representative, non-redundant samples.

Mapping of QTLs for lycopene and other fruit traits in a Lycopersicon esculentum × L. pimpinellifolium cross and comparison of QTLs across tomato species

Abstract: Quantitative trait loci (QTLs) for several fruit traits in tomato were mapped and characterized in a backcross population of an interspecific cross between Lycopersicon esculentum fresh-marker breeding line NC84173 and L. pimpinellifolium accession LA722. A molecular linkage map of this cross that was previously constructed based on 119 BC1 individuals and 151 RFLP markers was used for the QTL mapping. The parental lines and 119 BC1S1 families (self-pollinated progeny of BC1 individuals) were grown under field conditions at two locations, Rock Spring, PA, and Davis, CA, and fruits were scored for weight (FW), polar (PD) and equatorial diameters (ED), shape (FS), total soluble solids content (SSC), pH and lycopene content (LYC). For each trait, between 4 and 10 QTLs were identified with individual effects ranging between 4.4% and 32.9% and multilocus QTL effects ranging between 39% and 75% of the total phenotypic variation. Most QTL effects were predictable from the parental phenotypes, and several QTLs were identified that affected more than one trait. A few pairwise epistatic interactions were detected between QTL-linked and QTL-unlinked markers. Despite great differences between PA and CA growing conditions, the majority of FW QTLs (78%) and SSC QTLs (75%) in the two locations shared similar genomic positions. Almost all of the QTLs that were identified in the present study for FW and SSC were previously identified in six other studies that used different interspecific crosses of tomato; this indicates conservation of QTLs for fruit traits across tomato species. Altogether, the seven studies identified at least 28 QTLs for FW and 32 QTLs for SSC on the 12 tomato chromosomes. However, for each trait a few major QTLs were commonly identified in 4 or more studies; such ‘popular’ QTLs should be of considerable interest for breeding purposes as well as basic research towards cloning of QTLs. Notably, a majority of QTLs for increased SSC also contributed to decreased fruit size. Therefore, to significantly increase SSC of the cultivated tomato, some compromise in fruit size may be unavoidable.

Detection of linked QTL for soybean brown stem rot resistance in ‘BSR 101’ as expressed in a growth chamber environment*

Abstract: The objective of this study was to map the gene(s) conferring resistance to brown stem rot in the soybean cultivar BSR 101. A population of 320 recombinant inbred lines (RIL) was derived from a cross of BSR 101 and PI 437.654. Seedlings of each RIL and parent were inoculated by injecting stems with a suspension of spores and mycelia of Phialophora gregata, incubated in a growth chamber at 17 °C, and assessed for resistance by monitoring the development of foliar and stem symptoms. The population also was evaluated with 146 RFLPs, 760 AFLPs, and 4 probes for resistance gene analogs (RGAs). Regression analysis identified a significant association between resistance and several markers on Linkage Group J of the USDA-ARS molecular marker linkage map. Interval analysis with Mapmaker QTL identified a major peak between marker RGA2V-1 and AFLP marker AAGATG152M on Linkage Group J. A second peak, associated only with stem symptoms, was identified between the RFLP B122I-1 and RGA2V-1, also on Linkage Group J. When composite interval mapping with QTL Cartographer was used, two linked QTL were identified with both foliar and stem disease assessment methods: a major QTL between AFLP markers AAGATG152E and ACAAGT260, and a minor QTL between RGA3I-3 and RGA3I-2. These results demonstrate that composite interval mapping gives increased precision over interval mapping and is capable of distinguishing two linked QTL. The minor QTL associated with the cluster of RGA3I loci is of special interest because it is the first example of a disease resistance QTL associated with a resistance gene analog.

Comparison of PCR-based molecular marker analyses of Musa breeding populations

Abstract: Progress in the breeding of plantain and banana has been restricted by the complex genetic structure and behaviour of cultivated polyploid Musa. Genetic improvement has been hindered due to the large amount of space required for growth and maintenance of plant populations, in addition to the long growth cycle and the low levels of fertility and seed viability characteristic of cultivated genotypes. Molecular marker assisted breeding has the potential to dramatically enhance the pace and efficiency of genetic improvement in Musa. This study was conducted to compare different PCR-based marker systems (RAPD, VNTR and AFLP) for the analysis of breeding populations generated from two diverse Musa breeding schemes. All three assays detected a high level of polymorphism between parental genotypes and within progeny populations. As expected, AFLP assays had by far the highest multiplex ratio while VNTR analysis detected the highest levels of polymorphism. AFLP analysis of a full-sib tetraploid hybrid population confirmed previous reports based on VNTR analysis, of a high frequency of recombination during 2n (3x) gamete formation by a triploid plantain landrace. In addition, both VNTR and RAPD analyses of a full-sib triploid hybrid population suggested a high frequency of homoeologous recombination during n (2x) gamete formation by tetraploid hybrids. In general, there was a poor correlation between estimates of genetic similarity based on different types of marker. The implications of these findings for the molecular breeding of Musa crops are discussed.

Rice plant (Oryza sativa L.) containing rice tungro spherical virus (RTSV) coat protein transgenes are resistant to virus infection.

Abstract: The coat protein (CP) genes CP1, CP2 and CP3 of Rice tungro spherical virus (RTSV) were introduced individually or together to indica and/or japonica rice cells by particle bombardment and transgenic plants were produced. Plants derived from selfed progeny of the primary transformants were subjected to virus inoculation via leafhoppers, the natural vector of the virus. Sixteen out of the nineteen selected transgenic plant lines, as well as their R1, R2 and/or R3 progeny that contained the target gene, accumulated transcripts of the chimeric CP gene(s) by RNA blot analysis. We obtained evidence of moderate levels of protection to RTSV infection, ranging from 17% to 73% of seedlings that escaped infection and a significant delay of virus replication under greenhouse conditions in plant lines that expressed the RTSV-CP1, CP2 and CP3 genes singly or together. There was not an additive effect on resistance when more than one CP gene is expressed. This study is the first to report pathogen-derived resistance to infection by RTSV, one of the two viruses that are involved in rice tungro disease. It is also the first example of CP-mediated protection against a virus that contains more than one CP gene from the same virus.

Transgenic tobacco and peas expressing a proteinase inhibitor from Nicotiana alata have increased insect resistance

Abstract: Proteinase inhibitors have been used to increase resistance to insect pests in transgenic plants A cDNA clone encoding a multi-domain proteinase inhibitor precursor from Nicotiana alata (Na-PI) was transferred into tobacco and peas under the control of a promoter from a ribulose-1, 5-bisphosphate carboxylase small subunit gene The Na-PI precursor was cleaved in the leaves of transgenic tobacco and peas, and Mr 6000 polypeptides accumulated to levels of 03% and 01%, respectively, of the total soluble protein The Na-PI cDNA segregated as a dominant Mendelian trait and was stably transmitted for at least two generations of both species Helicoverpa armigera larvae that ingested tobacco or pea leaves containing Na-PI exhibited higher mortality or were delayed in growth and development relative to control larvae

Expression of the Talaromyces flavus glucose oxidase gene in cotton and tobacco reduces fungal infection, but is also phytotoxic

Abstract: Glucose oxidase secreted by the fungus Talaromyces flavus generates, in the presence of glucose, hydrogen peroxide that is toxic to phytopathogenic fungi responsible for economically important diseases in many crops. A glucose oxidase gene from T. flavus, was modified with a carrot extensin signal peptide and fused to either a constitutive or root-specific plant promoter. T1 tobacco plants expressing the enzyme constitutively were protected against infection by the seedling pathogen Rhizoctonia solani. Constitutive expression in tobacco was associated with reduced root growth, and slow germination on culture medium, and with reduced seed set in glasshouse conditions. Several independent transformed cotton plants with a root-specific construct expressed high glucose oxidase activity in the roots, excluding the root tip. Selected T3 homozygous lines showed some protection against the root pathogen, Verticillium dahliae, but not against Fusarium oxysporum. High levels of glucose oxidase expression in cotton roots were associated with reduced height, seed set and seedling germination and reduced lateral root formation. If this gene is to be of value for crop protection against pathogens it will require precise control of its expression to remove the deleterious phenotypes.