Showing papers in "Molecular Breeding in 1997"
TL;DR: This article describes a network experiment involving several European laboratories, in which the reproducibility of three popular molecular marker techniques was examined: random-amplified fragment length polymorphism (RAPD), amplified fragment length SNP (AFLP) and sequence-tagged microsatellites (SSR).
Abstract: A number of PCR-based techniques can be used to detect polymorphisms in plants. For their wide-scale usage in germplasm characterisation and breeding it is important that these marker technologies can be exchanged between laboratories, which in turn requires that they can be standardised to yield reproducible results, so that direct collation and comparison of the data are possible. This article describes a network experiment involving several European laboratories, in which the reproducibility of three popular molecular marker techniques was examined: random-amplified fragment length polymorphism (RAPD), amplified fragment length polymorphism (AFLP) and sequence-tagged microsatellites (SSR). For each technique, an optimal system was chosen, which had been standardised and routinely used by one laboratory. This system (genetic screening package) was distributed to different participating laboratories in the network and the results obtained compared with those of the original sender. Different experiences were gained in this exchange experiment with the different techniques. RAPDs proved difficult to reproduce. For AFLPs, a single-band difference was observed in one track, whilst SSR alleles were amplified by all laboratories, but small differences in their sizing were obtained.
895 citations
TL;DR: Applications of genome mapping and marker-assisted selection in crop improvement are reviewed and the use of MAS in breeding for disease and pest resistance is considered.
Abstract: Applications of genome mapping and marker-assisted selection (MAS) in crop improvement are reviewed. The following aspects are considered: a comparison of the choice of markers available for the generation of linkage maps (including amplified fragment length polymorphisms (AFLP); restriction fragment length polymorphisms (RFLP); randomly amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR)); quantitative trait loci (QTL) analysis; use of molecular markers in the exploitation of hybrid vigour; physical genome mapping; map-based cloning and transposon tagging of agriculturally important genes; synteny in cereal genomes; and the use of MAS in breeding for disease and pest resistance.
738 citations
TL;DR: A software application embodying two design principles: conventional reduction of raw genetic marker data to numerical summary statistics, and fast, interactive graphical display of both data and statistics.
Abstract: Efficient use of DNA markers for genomic research and crop improvement will depend as much on computational tools as on laboratory technology. The large size and multidimensional character of marker datasets invite novel approaches to data visualization. Described here is a software application embodying two design principles: conventional reduction of raw genetic marker data to numerical summary statistics, and fast, interactive graphical display of both data and statistics. The program performs various analyses for mapping quantitative-trait loci in real or simulated datasets and other analyses in aid of phenotypic and marker-assisted breeding. Functionality is described and some output is illustrated.
622 citations
TL;DR: The application of AFLPs, RAPDs and SSRs to examine genetic relationships in the primary northwestern European cultivated potato gene pool was investigated and marker index, the product of DI and EMR, was used to evaluate the overall utility of each marker system.
Abstract: The application of AFLPs, RAPDs and SSRs to examine genetic relationships in the primary northwestern European cultivated potato gene pool was investigated. Sixteen potato cultivars were genotyped using five AFLP primer combinations, 14 RAPD primers, and 17 database-derived SSR primer pairs. All three approaches successfully discriminated between the 16 cultivars using a minimum of one assay. Similarity matrices produced for each marker type on the basis of Nei and Li coefficients showed low correlations when compared with different statistical tests. Dendrograms were produced from these data for each marker system. The usefulness of each system was examined in terms of number of loci revealed (effective multiplex ratio, or EMR) and the amount of polymorphism detected (diversity index, or DI). AFLPs had the highest EMR, and SSRs the highest DI. A single parameter, marker index (MI), which is the product of DI and EMR, was used to evaluate the overall utility of each marker system. The use of these PCR-based marker systems in potato improvement and statutory applications is discussed. Abbreviations: PCR, polymerase chain reaction; AFLP, amplified fragment length polymorphism; RAPD, randomly amplified polymorphic DNA; DNA, deoxyribonucleic acid; EMR, effective multiplex ratio; DI, diversity index; MI, marker index; RFLP, restriction fragment length polymorphism.
436 citations
TL;DR: In this article, a transformant showing high-level expression of avidin was selected, and the protein was purified to greater than 90% purity by affinity chromatography after extraction from ground mature maize seed, showing that the N-terminal amino acid sequence and biotin binding characteristics are identical to the native protein with near identical molecular weight and glycosylation.
Abstract: We have produced in transgenic maize seed the glycoprotein, avidin, which is native to avian, reptilian, and amphibian egg white. A transformant showing high-level expression of avidin was selected. Southern blot data revealed that four copies of the gene are present in this transformant. The foreign protein represents >2% of aqueous soluble extracted protein from populations of dry seed, a level higher than any heterologous protein previously reported for maize. In seed, greater than 55% of the extractable transgenic protein is present in the embryo, an organ representing only 12% of the dry weight of the seed. This indicates that the ubiquitin promoter which is generally considered to be constitutive, in this case may be showing a strong tissue preference in the seed. The mature protein is primarily localized to the intercellular spaces. An interesting trait of the transgenic plants expressing avidin is that the presence of the gene correlates with partial or total male sterility. Seed populations from transgenic plants were maintained by outcrossing and segregate 1:1 for the trait. In generations T2–T4, avidin expression remained high at 2.3% (230 mg/kg seed) of extractable protein from seed, though it varied from 1.5 to 3.0%. However, levels of expression did not appear to depend on pollen parent or growing location. Cracked and flaked kernels stored at −29°C or 10 °C for up to three months showed no significant loss of avidin activity. Commercial processing of harvested seed also generated no apparent loss of activity. The protein was purified to greater than 90% purity by affinity chromatography after extraction from ground mature maize seed. Physical characterization of purified maize-derived avidin demonstrated that the N-terminal amino acid sequence and biotin binding characteristics are identical to the native protein with near identical molecular weight and glycosylation. This study shows that producing avidin from maize is not only possible but has practical advantages over current methods.
395 citations
TL;DR: Quantitative trait loci (QTL) analysis identified 13 regions of the genome associated with one or more measures of post-flowering drought tolerance and many loci that were associated with both rate and duration of grain development.
Abstract: Drought is a serious agronomic problem and the single greatest factor contributing to crop yield loss in the world today. This problem may be alleviated by developing crops that are well adapted to dry-land environments. Sorghum (Sorghum bicolor (L.) Moench) is one of the most drought-tolerant grain crops and is an excellent crop model for evaluating mechanisms of drought tolerance. In this study, a set of 98 recombinant inbred (RI) sorghum lines was developed from a cross between two genotypes with contrasting drought reactions, TX7078 (pre-flowering-tolerant, post-flowering susceptible) and B35 (pre-flowering susceptible, post-flowering-tolerant). The RI population was characterized under drought and non-drought conditions for the inheritance of traits associated with post-flowering drought tolerance and for potentially related components of grain development. Quantitative trait loci (QTL) analysis identified 13 regions of the genome associated with one or more measures of post-flowering drought tolerance. Two QTL were identified with major effects on yield and 'staygreen’ under post-flowering drought. These loci were also associated with yield under fully irrigated conditions suggesting that these tolerance loci have pleiotropic effects on yield under non-drought conditions. Loci associated with rate and/or duration of grain development were also identified. QTL analysis indicated many loci that were associated with both rate and duration of grain development. High rate and short duration of grain development were generally associated with larger seed size, but only two of these loci were associated with differences in stability of performance under drought.
238 citations
TL;DR: The hypothesis that GNA has a significant antifeedant effect on insects is supported, and expression of CpTI in transgenic potatoes had similar effects to expression of GNA on total insect biomass and survival, but did not afford protection against insect damage to the plant.
Abstract: Insecticidal effects of three plant-derived genes, those encoding snowdrop lectin (GNA), bean (Phaseolus vulgaris) chitinase (BCH) and wheat α-amylase (WAI), were investigated and compared with effects of the cowpea trypsin inhibitor gene (CpTI) Transgenic potato plants containing each of the three genes singly, and in pairwise combinations were produced All the introduced genes were driven by the CaMV 35S promoter; expression was readily detectable at the RNA level in transformants, but not detectable accumulation of WAI could be detected in transgenic potatoes containing its encoding gene GNA and BCH were accumulated at levels up to 20% of total soluble protein; both proteins were expressed in a functional form, and GNA was shown to undergo 'correct' N-terminal processing Accumulation levels of individual proteins were higher in plants containing a single foreign gene than in plants containing two foreign genes
220 citations
TL;DR: RFLP framework map presented here will be useful for mapping other genes segregating in this doubled haploid population and rapid generation of doubled haploids lines and their unbiased segregation make it very attractive for gene mapping.
Abstract: We have developed an RFLP framework map with 146 RFLP markers based on a doubled haploid population derived from a cross between an indica variety IR64 and a japonica variety Azucena. The population carries 50.2% of IR64 loci and 49.8% of Azucena loci, indicating an equal amount of genetic materials from each parent has been transmitted to the progenies through anther culture. However, some markers show segregation distortion. These distorted marker loci are located on 10 chromosomal segments. Using this map we were able to place 8 isozymes, 14 RAPDs, 12 cloned genes, 1 gene for brown planthopper (BPH) resistance, and 12 QTLs for grain length, grain width and length/width ratio onto rice chromosomes. The major gene for BPH resistance was mapped on chromosome 12 near RG463 and isozyme Sdh-1. Most of the QTLs identified for the three grain characters were closely linked on chromosomes 1, 2, 3 and 10. We concluded that the RFLP framework map presented here will be useful for mapping other genes segregating in this doubled haploid population. Thus rapid generation of doubled haploid lines and their unbiased segregation make it very attractive for gene mapping.
189 citations
TL;DR: The maize-expressed CryIA(b) protein was found to have the expected size and to be immunoreactive with antibodies prepared against crystals from Bacillus thuringiensis subsp.
Abstract: The range and stability of expression of the transgenic CryIA(b) protein was examined in Ciba Seeds Bt maize plants derived from Event 176. Specifically, CryIA(b) levels were determined for: (1) various plant tissues and developmental stages in three maize lines from 1993 field tests; (2) pollen and leaves from plants representing four backcross generations of two genotypes; (3) leaves of 6 precommercial hybrids; and (4) silage from one Bt maize hybrid. Significant levels were found only in pollen and leaves. Genetic background did not greatly impact the level seen in either tissue. CryIA(b) expression in maize plants derived from transformation Event 176 was stable over at least four successive generations. On a per acre basis, the highest amount of CryIA(b) protein (estimated to be 2-4 g CryIA(b) protein/acre) was found to occur at anthesis, consistent with the stage at which maximum plant vegetative biomass is reached. CryIA(b) was not detected in silage prepared from CryIA(b)-expression plants. The maize-expressed CryIA(b) protein was found to have the expected size and to be immunoreactive with antibodies prepared against crystals from Bacillus thuringiensis subsp. kurstaki.
180 citations
TL;DR: The enhancement of stem borer resistance in cv.
Abstract: Rice cultivars of isozyme group V include high-quality, aromatic rices that are difficult to improve by traditional methods because of the loss of quality characters upon sexual hybridization. Their low-tillering plant type predisposes them to economic loss from attack by stem borers, a group of insects to which they are susceptible. We report here the enhancement of stem borer resistance in cv. Tarom Molaii through transformation by microprojectile bombardment. Embryogenic calli derived from mature seeds were bombarded with gold particles coated with plasmid pCIB4421, carrying a synthetic truncated toxin gene based on the cryIA(b) gene from Bacillus thuringiensis, and plasmid pHygII, carrying the hygromycin phosphotransferase (hpt) selectable marker gene. Inclusion of 50 mg/l hygromycin B in culture media from bombardment through to rooting of plantlets eliminated escapes. The procedure generated three independent hpt transformants of which two also contained the cryIA(b) gene. One such line (No. 827) produced truncated (67 kDa) CryIA(b) protein equivalent to about 0.1% of total soluble protein. The cryIA(b) gene was controlled by the promoter of the maize C4 PEP carboxylase gene and was expressed in leaf blades but was not expressed to a detectable level in dehulled mature grain. Line 827 contained about 3 copies of the cryIA(b) gene which segregated as a single dominant Mendelian locus in the second (T1) and third (T2) generations and co-segregated with enhanced resistance to first-instar larvae of striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas). T2 line 827-6 homozygous for the cryIA(b) gene showed no dead hearts or whiteheads after infestation with stem borers, whereas T2 line 827-25 lacking the gene averaged 7 dead hearts per plant and 2.25 whiteheads per plant. These results establish that transformation of high-quality rices of group V is a feasible alternative to sexual hybridization.
167 citations
TL;DR: A truncated cryIA(b) gene encoding the active region of the Bacillus thuringiensis δ-endotoxin was expressed in transgenic sugarcane plants under the control of the CaMV 35S promoter and showed significant larvicidal activity despite the low expression of CryIA( b).
Abstract: A truncated cryIA(b) gene encoding the active region of the Bacillus thuringiensis δ-endotoxin was expressed in transgenic sugarcane plants (Saccharum officinarum L.) under the control of the CaMV 35S promoter. Genetic transformation was accomplished by electroporation of intact cells. The levels of recombinant toxin were established and biological activity tests were performed against neonate sugarcane borer (Diatraea saccharalis F.) larvae. Transgenic sugarcane plants showed significant larvicidal activity despite the low expression of CryIA(b).
TL;DR: Investigations of the relationships revealed between rice groups using the two types of PCR-based marker led to investigations of their map positions using an intraspecific doubled haploid mapping population.
Abstract: Genetic variation between samples of Oryza sativafrom 19 localities in Bangladesh and Bhutan was assessed using two PCR-based molecular marker systems: RAPD (random amplification of polymorphic DNA) and ISSR-PCR (inter-simple sequence repeat polymerase chain reaction). Employing RAPD, a set of 14 decanucleotides of arbitrary sequence directed the amplification of 94 reproducible marker bands, 47 (50%) of which were polymorphic. In addition, a set of 9 ISSR primers were used to direct amplification of 71 PCR products, 40 (56%) of which were polymorphic. Multivariate analyses of the two PCR-based molecular marker data sets provided evidence that the patterns of variation correspond with the classification described by Glaszmann [9] using isozyme analysis. Subtle differences in the relationships revealed between rice groups using the two types of PCR-based marker led to investigations of their map positions using an intraspecific doubled haploid mapping population. The observation that the chromosomal locations of markers can influence diversity assessments is presented and the significance of this is discussed.
TL;DR: Results indicate that pathogen-derived resistance can provide effective protection against a viral disease over a significant portion of the crop cycle of a perennial species.
Abstract: Transgenic Carica papaya plants (cv. Sunset, R0 clone 55-1) carrying the coat protein gene of papaya ringspot virus (strain HA 5-1) remained symptomless and ELISA-negative for 24 months after inoculation with Hawaiian strains of papaya ringspot virus under field conditions. Non-transgenic and transgenic control plants lacking the coat protein gene developed disease symptoms within one month after manual inoculation or within four months when natural aphid populations were the inoculum vectors. Mean trunk diameter was significantly greater in cloned 55-1 plants compared with virus-infected controls (14.7 cm versus 9.3 cm after 18 months). Fruit brix, plant morphology, and fertility of 55-1 plants were all normal, and no pleiotropic effects of the coat protein gene were observed. These results indicate that pathogen-derived resistance can provide effective protection against a viral disease over a significant portion of the crop cycle of a perennial species.
TL;DR: Assessment of the effectiveness of molecular marker assisted selection for malting quality traits found tandem genotypic and phenotypic selection was more effective than phenotypes selection, but for QTL2 was not as effective as phenotyping selection due to a lack of QTL1 effects in the selection population.
Abstract: Selection for malting quality in breeding programs by micromalting and micromashing is time-consuming, and resource-intensive. More efficient and feasible approaches for identifying genotypes with good malting quality would be highly desirable. With the advent of molecular markers, it is possible to map and tag the loci affecting malting quality. The objective of this study was to assess the effectiveness of molecular marker assisted selection for malting quality traits. Two major quantitative trait loci (QTL) regions in six-row barley for malt extract percentage, α-amylase activity, diastatic power, and malt β-glucan content on chromosomes 1 (QTL1) and 4 (QTL2) have been previously identified. The flanking markers, Brz and Amy2, and WG622 and BCD402B, for these two major QTL regions were used in marker-assisted selection. Four alternative selection strategies; phenotypic selection, genotypic selection, tandem genotypic and phenotypic selection, and combined phenotypic and genotypic selection, were compared for both single and multiple trait selection in a population consisting of 92 doubled haploid lines derived from ‘Steptoe’ × ‘Morex’ crosses. Marker assisted selection for QTL1 (tandem genotypic and phenotypic selection, and combined phenotypic and genotypic selection) was more effective than phenotypic selection, but for QTL2 was not as effective as phenotypic selection due to a lack of QTL2 effects in the selection population. The effectiveness of tandem genotypic and phenotypic selection makes marker assisted selection practical for traits which are extremely difficult or expensive to measure such as most malting quality traits. It can substantially eliminate undesirable genotypes by early genotyping and keeping only desirable genotypes for later phenotypic selection.
TL;DR: Evaluation of QTL associated with several fruit number determinants of early, first-harvest yield demonstrating additive genetic variance suggests that marker-assisted selection may have utility for the development of determinate, multiple lateral branching germplasm suited for once-over mechanical harvesting in this population.
Abstract: An 80-point genetic map [77 random-amplified polymorphic DNAs (RAPD), F (female sex expression), de (determinate), and ll (little leaf)] was constructed from a narrow cross in cucumber using the determinate, gynoecious, standard-sized leaf line G421 and the indeterminate, monoecious, little leaf line H-19. The map defined nine linkage groups and spanned ca. 600 cM with an average distance between markers of 8.4 ± 9.4 cM. The RAPD loci BC-551 and BC-592 were found to flank ll at 3.4 and 12.2 cM, respectively. The locus OP-L18-2 was linked (16 cM) to de, and the F locus was flanked by markers at 44 and 31 cM. One-hundred F3 families were used to identify quantitative trait loci (QTL) for sex expression, main stem length, number of lateral branches, days to anthesis, fruit number and weight, fruit length and diameter, and fruit length: diameter ratio in two replicated test locations (Wisconsin and Georgia). QTL on linkage group B explained major portions (R2 = ca. 2 to 74%) of the variation observed for sex expression, main stem length, lateral branch number, and fruit diameter (LOD = 2.1 to 29.8). Although ca. 62 to 74% of the variation for sex expression was associated with a putative QTL spanning the F locus (OP-AJ-2 to F and F to de), other regions (three) of the genome were important for the determination of sex in the F3 families examined depending upon environment. The number of genomic regions affecting main stem length (five) and number of lateral branches (three) coincided with expectations as determined by calculations of minimum number of genes in previous studies. Evaluation of QTL associated with several fruit number determinants of early, first-harvest yield demonstrating additive genetic variance (i.e., sex expression, main stem length, and number of laterals) suggests that marker-assisted selection may have utility for the development of determinate, multiple lateral branching germplasm suited for once-over mechanical harvesting in this population.
TL;DR: Principal co-ordinate (PCO) analysis of genetic similarities (gs) confirmed the marked contrast in the cultivars used in the 1970s and 1980s and provided insight into the effects of selection for disease resistance and the antagonism between malting quality and particular resistance genes.
Abstract: The generation of AFLPs in spring barley cultivars provided genetic information relating to the development of the crop in the UK since 1953. Principal co-ordinate (PCO) analysis of genetic similarities (gs) confirmed the marked contrast in the cultivars used in the 1970s and 1980s. The earliest cultivars, many derived from Proctor, were succeeded by tall-strawed, disease-resistant types with high yield but poor malting potential. In the 1980s they were in turn replaced by short-strawed cultivars with excellent yield and good malting quality, which originated from Triumph. A PCO plot of gs provided insight into the effects of selection for disease resistance and the antagonism between malting quality and particular resistance genes. The analysis of gs was more useful than pedigrees and estimates of kinship in revealing the genetic relationship between cultivars. Theoretical considerations for maximising the efficiency of an AFLP genotyping programme are discussed in the context of the number of primer pairs required to distinguish genotypes at varying levels of similarity.
TL;DR: These data demonstrate that the markers derived from sequences of Lrk10 are highly specific for the Lr10 gene in breeding material of very diverse genetic origin and will contribute to the elucidation of the role of L r10 in polygenic resistances against leaf rust.
Abstract: We recently showed that the Lr10 wheat leaf rust resistance gene cosegregated with the candidate resistance gene Lrk10 which encodes a putative receptor-like kinase. The aim of this study was to develop Lrk10-derived molecular markers for the detection of the Lr10 gene in breeding material. Different subfragments of Lrk10 were tested as RFLP markers for the Lr10 resistance gene. The most specific fragment (Lrk10-6) was converted into the PCR-based STS marker STSLrk10-6. Both the RFLP and the STS marker did not give a signal with near isogenic lines containing a different Lr gene. The applicability of these markers for the detection of Lr10 in genetically diverse material was tested with 62 wheat and spelt breeding lines, mostly from European breeding programmes. Twelve varieties known to have Lr10 showed the same alleles as the originally characterized line ThatcherLr10. Most of the lines with unknown composition at the Lr10 locus had a null allele with both the RFLP marker Lrk10-6 and the marker STSLrk10-6 whereas 20% of the lines had a different allele. For six lines, including a traditional spelt variety derived from a landrace, both markers showed the same allele as Thatcher Lr10. Artificial infections of these lines with an isolate avirulent on Lr10 resulted in a hypersensitive reaction of all these lines, indicating also the presence of the Lr10 resistance gene. These data demonstrate that the markers derived from sequences of Lrk10 are highly specific for the Lr10 gene in breeding material of very diverse genetic origin. The markers will allow the defined deployment of Lr10 in wheat breeding programmes and will contribute to the elucidation of the role of Lr10 in polygenic resistances against leaf rust.
TL;DR: Repeated feeding trials with Helicoverpa armigera larvae fed on clonally derived T0 and T1 plants expressing GTPI demonstrated that, relative to those fed on control plants, some growth inhibition occurs, but there was no increase in larval mortality.
Abstract: A giant taro proteinase inhibitor (GTPI) cDNA was expressed in transgenic tobacco using three different gene constructs. The highest expression level obtained was ca. 0.3% of total soluble protein when the cDNA was driven by the Arabidopsis rbcS ats1 promoter. Repeated feeding trials with Helicoverpa armigera larvae fed on clonally derived T0 and T1 plants expressing GTPI demonstrated that, relative to those fed on control plants, some growth inhibition (22–40%) occurs, but there was no increase in larval mortality. Proteinase activities of larvae fed on GTPI-expressing tobacco or GTPI-containing diet were examined to monitor the spectrum of digestive proteinases in the midgut. Total proteinase activity was reduced by 13%, but GTPI-insensitive proteinase activity was increased by up to 17%. Trypsin was inhibited by 58%, but chymotrypsin and elastase were increased by 26% and 16% respectively. These results point to an adaptive mechanism in this insect that elevates the levels of other classes of proteinases to compensate for the trypsin activity inhibited by dietary proteinase inhibitors.
TL;DR: It is confirmed that one major gene is involved in segregation for iron chlorosis deficiency in the Anoka x A7 population, supporting a previous hypothesis that two separate genetic mechanisms control iron deficiency in soybean.
Abstract: The objective of this study was to map genes controlling iron deficiency chlorosis in two intraspecific soybean [Glycine max (L.) Merrill] populations. Chlorosis symptoms were evaluated by visual scores and spectrometric chlorophyll determinations at the V4 stage (third trifoliolate leaf fully developed) in the field in 1993, and at V2 (first trifoliolate leaf fully developed) and V4 stages in 1994. A total of 89 RFLP and 10 SSR markers in the Pride B216 x A15 population, and 82 RFLP, 14 SSR and 1 morphological I (hilum color) markers in the Anoka x A7 population were used to map quantitative trait loci (QTL) affecting iron deficiency chlorosis. QTL with minor effects were detected on six linkage groups of the Pride B216 x A15 population, suggesting a typical polygene mechanism. In contrast, in the Anoka x A7 population, one QTL contributed an average of 72.7% of the visual score variation and 68.8% of the chlorophyll concentration variation and was mapped on linkage group N. Another QTL for visual score variation, and one for chlorophyll concentration variation were detected on linkage groups A1 and I, respectively. Due to the large LOD score and major genetic effect of the QTL on linkage group N, the quantitative data was reclassified into qualitative data fitting a one major gene model according to the means of the QTL genotypic classes. The major gene was mapped in the same interval of linkage group N using both visual scores and chlorophyll concentrations, thus verifying that one major gene is involved in segregation for iron chlorosis deficiency in the Anoka x A7 population. This study supported a previous hypothesis that two separate genetic mechanisms control iron deficiency in soybean.
TL;DR: The results confirmed the previous suggestion that salt tolerance during germination in tomato is polygenically controlled and suggests the likelihood of recovering transgressive segregants in progeny derived from these parental genotypes.
Abstract: This study was conducted to identify genomic regions (quantitative trait loci, QTLs) affecting salt tolerance during germination in tomato Germination response of an F2 population of a cross between UCT5 (Lycopersicon esculentum, salt-sensitive) and LA716 (L pennellii, salt-tolerant) was evaluated at a salt-stress level of 175 mM NaCl + 175 mM CaCl2 (water potential ca −950 kPa) Germination was scored visually as radicle protrusion at 6 h intervals for 30 consecutive days Individuals at both extremes of the response distribution (ie, salt-tolerant and salt-sensitive individuals) were selected The selected individuals were genotyped at 84 genetic markers including 16 isozymes and 68 restriction fragment length polymorphisms (RFLPs) Trait-based marker analysis (TBA) which measures changes (differences) in marker allele frequencies in selected lines was used to identify marker-linked QTLs Eight genomic regions were identified on seven tomato chromosomes bearing genes (QTLs) with significant effects on this trait The results confirmed our previous suggestion that salt tolerance during germination in tomato is polygenically controlled The salt-tolerant parent contributed favorable QTL alleles on chromosomes 1, 3, 9 and 12 whereas the salt sensitive parent contributed favorable QTL alleles on chromosomes 2, 7 and 8 The identification of favorable alleles in both parents suggests the likelihood of recovering transgressive segregants in progeny derived from these parental genotypes The results can be used for marker-assisted selection and breeding of salt-tolerant tomatoes
TL;DR: For a number of QTL the identification of linked markers provided suitable opportunities for marker-assisted selection and improvement of barley and reference markers with which to analyse the homoeologous chromosome regions of wheat and other cereals.
Abstract: An RFLP map constructed from 99 doubled haploid lines of a cross between two spring barley varieties (Blenheim × Kym) was used to localize quantitative trait loci (QTL) controlling grain yield and yield components by marker regression and single-marker analysis. Trials were conducted over three years. Genotype-by-year interaction was detected for plant grain weight and ear grain weight so they were analysed separately for each year. None was detected for thousand-grain weight and ear grain number so data were pooled over years. A total of eleven QTL were detected for plant grain weight over two years and fourteen for ear grain weight over three years. Seven QTL were detected for plot yield. The locus with the largest effect was on chromosome 2(2H)L and accounted for 19% of the variation in the progeny. Eight QTL were detected for thousand-grain weight and five for ear grain number. Many of the QTL detected were in comparable positions in each year. Yield and yield components were only partly correlated. Comparisons based on common RFLP markers showed that some QTL were found in positions similar to those identified in other studies. For a number of QTL the identification of linked markers provided suitable opportunities for marker-assisted selection and improvement of barley and reference markers with which to analyse the homoeologous chromosome regions of wheat and other cereals.
TL;DR: The effectiveness of different promoters for use in Indica rice transformation was compared in this paper, where plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 and Ubi1 promoters were delivered into cell suspension cultures by particle bombardment.
Abstract: The effectiveness of different promoters for use in Indica rice transformation was compared Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1 Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbi1-gas plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes These genes were stably inherited by the T1 generation
TL;DR: The recovery of acylsugar accumulation in progeny of the intermated BC3F1 plants supports the involvement of at least some of the 5 target regions in acylSugar biosynthesis, however, it is likely that another, as of yet unidentified, region is necessary for accumulation of higher levels of acylsugars.
Abstract: Acylsugars exuded from type IV trichomes mediate the multiple pest resistance found in the wild tomato species, Lycopersicon pennellii. A marker-assisted selection breeding program was used to attempt the transfer of the ability to accumulate acylsugars to cultivated tomato. RFLP and PCR-based markers were used through three backcross generations to select plants containing 5 target regions associated by QTL analysis with acylsugar accumulation. The BC1F1 plant selected possessed all 5 target regions and accumulated acylsugars at a moderate level similar to that of the interspecific F1 control. The BC2F1 and BC3F1 selections contained complementary subsets of the 5 target regions and did not accumulate acylsugars. BC3F1 plants with complementary subsets of the 5 target regions were intermated to produce populations segregating for the 5 target regions. From 1000 BC3F1-intermated plants, three plants were found which accumulated acylsugars at low levels and contained 3 to 5 of the target regions. The recovery of acylsugar accumulation in progeny of the intermated BC3F1 plants supports the involvement of at least some of the 5 target regions in acylsugar biosynthesis. However, since the levels of acylsugars accumulated by these plants were lower than that of the interspecific F1, it is likely that another, as of yet unidentified, region is necessary for accumulation of higher levels of acylsugars.
TL;DR: It was shown that the enzyme immobilized on oil-bodies could be recycled by flotation several times without loss of activity and the functioning of the xylanase as a fusion protein.
Abstract: Canola seed oil-bodies were investigated as a production vehicle and immobilization matrix for xylanases. A recombinant xynC gene from Neocallimastix patriciarum encoding a xylanase (XynC) was fused to an oleosin coding sequence suitable for targeting the xylanase to the oil-body membrane. This fusion gene was introduced into Brassica napus using Agrobacterium-mediated transformation. Transgenic Canola plants were obtained expressing xylanase which was targeted to the oil-bodies of seeds as shown by analysis with XynC-specific antibodies. Oil-bodies extracted from transgenic seeds exhibited xylanase activity, indicating the immobilization of XynC on the surface of oil bodies and the functioning of the xylanase as a fusion protein. The immobilized XynC retained its optimal temperature, Km value and specificity. However, it exhibited reduced sensitivity to pH. Furthermore, it was shown that the enzyme immobilized on oil-bodies could be recycled by flotation several times without loss of activity.
TL;DR: In this paper, the rootstock Rosa hybrida L. cv. was produced via a two-step procedure, in which kanamycin resistant roots were generated on stem slices from micropropagated shoots, which were cocultivated with Agrobacterium tumefaciens containing the neomycin phosphotransferase II (NPTII) gene, together with individual ROL genes from A. rhizogenes, and these roots were used to regenerate transgenic plants via somatic embryogenesis.
Abstract: Transgenic plants of the rootstock Rosa hybrida L. cv. Moneyway were produced via a two-step procedure. First, kanamycin-resistant roots were generated on stem slices from micropropagated shoots, which were cocultivated with Agrobacterium tumefaciens containing the neomycin phosphotransferase II (NPTII) gene for conferring kanamycin resistance, together with individual ROL genes from A. rhizogenes. Root formation was quite efficient and up to two kanamycin-resistant roots per stem slice were produced. In the second step, these roots were used to regenerate transgenic plants via somatic embryogenesis. Although regeneration lasted up to 12 months, production of several transformants was successfully accomplished. Untransformed escapes were not found, indicating that the initial selection on kanamycin resistance was reliable. The presence of a combination of ROLA, B and C genes enhanced adventitious root formation on micropropagated shoots and explants of stems and leaves. It appears that the auxin sensitivity was increased to such a degree that cells were able to respond even to endogenous auxins present in shoots and leaves. Rooting experiments in greenhouse demonstrated that adventitious root formation on cuttings was improved threefold upon introduction of these ROL genes. It is concluded that a method was developed for the production of ROL gene transformed roses with improved rooting characteristics.
TL;DR: It is demonstrated that Agrobacterium-mediated gene transfer to preorganised meristematic tissue combined with axillary regeneration can form the basis of a routine transformation system for legume crop species which are difficult to regenerate from other explants.
Abstract: Transgenic plants of Lupinus angustifolius L. (cvs. Unicrop and Merrit) were routinely generated using Agrobacterium-mediated gene transfer to shoot apices. The bar gene for resistance to phosphinothricin (PPT, the active ingredient of the herbicide Basta) was used as the selectable marker. After co-cultivation, the shoot apex explants were transferred onto a PPT-free regeneration medium and their tops were thoroughly wetted with PPT solution (2 mg/ml). The multiple axillary shoots developing from the shoot apices were excised onto a medium containing 20 mg/l PPT. The surviving shoots were transferred every second week onto fresh medium containing 20 mg/l PPT. At each transfer, the number of surviving shoots decreased, until it stabilized. Indeed, some of these chimeric shoots surviving the PPT selection, eventually produced new green healthier axillary shoots which could be transferred to soil. This whole process took from 5 to 9 months after co-cultivation. Average transformation frequencies of 2.8% for cv. Unicrop and of 0.4% for the commercial cultivar Merrit were achieved. Molecular analysis of T0, T1, and T2 generations demonstrated stable integration of the foreign gene into the plant genome and expression of the integrated gene. Transformed plants of the T1 and T2 generations were resistant in glasshouse trials where the herbicide Basta (0.1 mg/ml) was sprayed onto whole plants. These results demonstrate that Agrobacterium-mediated gene transfer to preorganised meristematic tissue combined with axillary regeneration can form the basis of a routine transformation system for legume crop species which are difficult to regenerate from other explants.
TL;DR: This is the first report on extensive field trials designed to assess the resistance to mixed infection by CMV, ZYMV, and WMV-2, and to evaluate the yield of commercial quality cantaloupes that are genetically engineered.
Abstract: Cantaloupe line CZW-30 containing coat protein gene constructs of cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus 2 potyvirus (WMV-2) was investigated in the field over two consecutive years for resistance to infections by CMV, ZYMV, and/or WMV-2. Resistance was evaluated under high disease pressure achieved by mechanical inoculations and/or natural challenge inoculations by indigenous aphid vectors. Across five different trials, homozygous plants were highly resistant in that they never developed systemic symptoms as did the nontransformed plants but showed few symptomatic leaves confined close to the vine tips. Hemizygous plants exhibited a significant delay (2–3 weeks) in the onset of disease compared to control plants but had systemic symptoms 9–10 weeks after transplanting to the field. Importantly, ELISA data revealed that transgenic plants reduced the incidence of mixed infections. Only 8% of the homozygous and 33% of the hemizygous plants were infected by two or three viruses while 99% of the nontransformed plants were mixed infected. This performance is of epidemiological significance. In addition, control plants were severely stunted (44% reduction in shoot length) and had poor fruit yield (62% loss) compared to transgenic plants, and most of their fruits (60%) were unmarketable. Remarkably, hemizygous plants yielded 7.4 times more marketable fruits than control plants, thus suggesting a potential commercial performance. This is the first report on extensive field trials designed to assess the resistance to mixed infection by CMV, ZYMV, and WMV-2, and to evaluate the yield of commercial quality cantaloupes that are genetically engineered.
TL;DR: Near-isogenic lines in conjunction with bulked segregant analysis were used to identify a DNA marker in wheat associated with the H21 gene conferring resistance to biotype L of Hessian fly larvae.
Abstract: Near-isogenic lines in conjunction with bulked segregant analysis were used to identify a DNA marker in wheat (Triticum aestivum L.) associated with the H21 gene conferring resistance to biotype L of Hessian fly [Mayetiola destructor (Say)] larvae. Near-isogenic lines were developed by backcross introgression BC3F3:4 (‘Coker 797’ * 4 / ‘Hamlet’) and differed by the presence or absence of H21 (on 2RL) derived from ‘Chaupon’ rye (Secale cereale L.). Bulked DNA samples were prepared from near-isogenic lines and BC3F2 population individuals segregating for reaction to Hessian fly biotype L and screened for random amplified polymorphic DNA markers using 46 10mer primers. Random-amplified polymorphic DNA markers from resistant and susceptible individuals and parental lines were scored and these data were used to identify a 3 kb DNA fragment that was related to the occurrence of H21. This fragment was amplified from DNA isolated from Hamlet, a near-isogenic line carrying 2RL, and bulked DNA from resistant BC3F2 individuals, but not from the recurrent parent Coker 797 or DNA bulks from susceptible BC3F2 plants. Analysis of 111 BC3F2 segregating individuals and BC3F2:3 segregants confirmed the co-segregation of the 3 kb DNA marker with the H21 resistance gene to Hessian fly. Use of this marker could facilitate more rapid screening of plant populations for Hessian fly resistance and monitoring the introgression of H21.
TL;DR: A previously uncharacterized deletion mutation is found in which segments of both the intron and exon have been deleted and replaced by other sequences in the active wild-type COMT gene.
Abstract: The caffeic acid O-methyltransferase (COMT) gene plays an important role in the synthesis of lignin We have used the polymerase chain reaction in conjuction with genomic analysis to characterize deletion mutations of this gene in maize In addition, we have analyzed and compared regions of the COMT gene from three distinct heterotic groups Both PCR and Southern analysis indicate that the active wild-type COMT gene can be polymorphic We suggest that the intron domain of at least one heterotic inbred can contribute to the alteration of the wild-type gene In addition, multiple deletion mutations have occurred at this locus We have found a previously uncharacterized deletion mutation in which segments of both the intron and exon have been deleted and replaced by other sequences Precise knowledge of its sequence has allowed us to develop an assay by which we can follow this mutation in a breeding program
TL;DR: In marker-assisted selection with coupling-repulsion-phase markers, SCAR F6 can be used in combination with N9, or together with any other RAPD marker linked in repulsion to the Rrl allele, in a group of sugar beet elite lines containing the ‘Holly’ type of rhizomania resistance.
Abstract: In sugar beet genotypes with the ‘Holly’ type of resistance to rhizomania, a disease due to infection of the beet necrotic yellow vein virus (BNYVV), the major gene rrl is responsible for resistance. Twelve RAPD markers linked to rrl were selected by BSA and mapped on linkage group IV using a segregating population previously analysed by the same group. Markers F61050 and N9600 were tightly linked, respectively in coupling and repulsion, to the Rrl allele (recombination values of 1.4 cM for both markers). After sequencing the products amplified by F61050 and N9600, new PCR primers were used to generate the two SCAR markers F6 and N9. The simultaneous use of these markers in a PCR reaction allows the correct fingerprinting of rrl rrl, Rrl rrl and Rrl Rrl sugar beet plants in populations segregating for the ‘Holly’ resistance. In a group of sugar beet elite lines containing the ‘Holly’ type of rhizomania resistance, SCAR F6 is always present whereas the SCAR N9 fragment is absent. Thus, in marker-assisted selection with coupling-repulsion-phase markers, SCAR F6 can be used in combination with N9, or together with any other RAPD marker linked in repulsion to the Rrl allele.