Showing papers in "Molecular Breeding in 1996"
TL;DR: A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated and wild soybean accessions, estimates based on RFLPs, RAPD, AFLPs and SSRs are highly correlated, indicating congruence between these assays.
Abstract: The utility of RFLP (restriction fragment length polymorphism), RAPD (random-amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat, microsatellite) markers in soybean germplasm analysis was determined by evaluating information content (expected heterozygosity), number of loci simultaneously analyzed per experiment (multiplex ratio) and effectiveness in assessing relationships between accessions. SSR markers have the highest expected heterozygosity (0.60), while AFLP markers have the highest effective multiplex ratio (19). A single parameter, defined as the marker index, which is the product of expected heterozygosity and multiplex ratio, may be used to evaluate overall utility of a marker system. A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated (Glycine max) and wild soybean (Glycine soja) accessions, estimates based on RFLPs, AFLPs and SSRs are highly correlated, indicating congruence between these assays. However, correlations of RAPD marker data with those obtained using other marker systems were lower. This is because RAPDs produce higher estimates of interspecific similarities. If the comparisons involvedG. max only, then overall correlations between marker systems are significantly lower. WithinG. max, RAPD and AFLP similarity estimates are more closely correlated than those involving other marker systems.
2,521 citations
TL;DR: Randon-amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to map submergence tolerance in 169 F2 plants and the resulting F3 families of a cross between a tolerant indica rice line, IR40931-26, and a susceptible japonica line, PI543851.
Abstract: Submergence stress is a widespread problem in rice-growing environments where drainage is impeded. A few cultivars can tolerate more than 10 days of submergence, but the genes conferring this tolerance have not been identified. We used randon-amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers to map submergence tolerance in 169 F2 plants and the resulting F3 families of a cross between a tolerant indica rice line, IR40931-26, and a susceptible japonica line, PI543851. IR40931-26 inherited strong submergence tolerance from the unimproved cultivar FR13A. Eight-day old F3 seedlings were submerged for 14–16 days in 55-cm deep tanks, and tolerance was scored after 7 days recovery on a scale of 1 (tolerant) to 9 (susceptible). The tolerant and susceptible parents scored 1.5 and 8.4, respectively, and the F3 means ranged from 1.6 to 8.9. Two bulks were formed with DNA from F2 plants corresponding to the nine most tolerant and the nine most susceptible F3 families. Of 624 RAPD primers used to screen the bulks, five produced bands associated with either tolerance or susceptibility. These markers were mapped to a region of chromosome 9 by linkage to RFLP markers. A submergence tolerance quantitative trait locus (QTL), here designatedSub1, was located ca. 4 cM from the RFLP marker C1232 and accounted for 69% of the phenotypic variance for the trait.
334 citations
TL;DR: Small-scale field tests showed that the transgenic rice plants expressing the CpTi gene had significantly increased resistance to two species of rice stem borers, which are major rice insect pests.
Abstract: The gene encoding a cowpea trypsin inhibitor (CpTI), which confers insect resistance in trangenic tobacco, was introduced into rice. Expression of the CpTi gene driven by the constitutively active promoter of the rice actin 1 gene (Act1) leads to high-level accumulation of the CpTI protein in transgenic rice plants. Protein extracts from transgenic rice plants exhibit a strong inhibitory activity against bovine trypsin, suggesting that the proteinase inhibitor produced in transgenic rice is functionally active. Small-scale field tests showed that the transgenic rice plants expressing the CpTi gene had significantly increased resistance to two species of rice stem borers, which are major rice insect pests. Our results suggest that the cowpea trypsin inhibitor may be useful for the control of rice insect pests.
196 citations
TL;DR: The high efficiencies of gene transfer, selection and cotransformation in the optimised system, coupled with the simple initiation and regeneration of embryogenic callus, provide an effective tool for practical genetic transformation of sugarcane.
Abstract: Transient expression of the maize anthocyanin regulatory elements,R andC1, was used to optimise parameters for microprojectile-mediated delivery of DNA into sugarcane embryogenic callus. Osmotic treatment of target tissues and particle acceleration in a high-pressure helium pulse increased the frequency of transient expression to 5–8×103 cells per bombardment, with minimal tissue damage. An average of 0.34% of transiently expressing cells developed into stably transformed, anthocyanin-pigmented proembryoids which subsequently regenerated into plantlets. However, constitutive expression ofR andC1 proved deleterious, and no anthocyanin-pigmented plant survived beyond 3 cm in height. We also compared selective subculture of callus portions showing luciferase activity with antibiotic selection on medium containing G418 or phosphinothricin, upon bombardment of callus with constructs driving strong expression ofluc, aphA orbar genes. Selective subculture based on luciferase activity enabled recovery of 1.4±0.5 independent transgenic plants per bombardment, compared to 19.8±3.7 independent transgenic plants per bombardment from an optimised G418 selection regimen, and no transformed plants from phosphinothricin selection. Whenluc andaphA on separate plasmids were coprecipitated onto microprojectiles before bombardment, 67–79% of callus lines selected for G418 resistance also showed luciferase activity detectable under a low-light camera. Southern analysis confirmed a very high cotransformation frequency, with variable copy numbers of introduced genes. The high efficiencies of gene transfer, selection and cotransformation in the optimised system, coupled with the simple initiation and regeneration of embryogenic callus, provide an effective tool for practical genetic transformation of sugarcane.
179 citations
TL;DR: Agrobacterium-mediated transformation of Javanica cultivars Gulfmont and Jefferson that are widely used or about to enter commercial cultivation in the southern USA is reported, confirming the suitability of this approach for biotechnological improvement of elite rice cultivars.
Abstract: Difficulties frequently encountered using direct DNA transfer methods for transformation of Javanica varieties of rice (Oryza sativa L.) have limited the application of biotechnology to these varieties. We now reportAgrobacterium-mediated transformation of Javanica cultivars Gulfmont and Jefferson that are, respectively, widely used or about to enter commercial cultivation in the southern USA. Vigorous, phenotypically normal, fertile plants expressing both the selectable marker and the gene of interest were obtained. Southern analysis showed that only one or two copies of the T-DNA insert were present. Sequence analysis of right border fragments of one line confirmed that insertion was into a coding region of rice nuclear DNA. This analysis also revealed the presence of relatively short regions of permuted T-DNA border sequences, similar to those found afterAgrobacterium-mediated transformation of dicots. Progeny analysis of lines bearing two copies showed co-segregation, indicating that they were located relatively closely on the same chromosome. The introduced genes were transmitted to the R1 and R2 generations in a Mendelian fashion, confirming the suitability of this approach for biotechnological improvement of elite rice cultivars.
166 citations
TL;DR: In this paper, the authors constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs).
Abstract: Rice is a leading grain crop and the staple food for over half of the “world” population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14208 clones with an average insert size of 130 kb, which represents 4.4. rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.
157 citations
TL;DR: Different results show that it is possible to manipulate lignin profiles through targeted genetic transformation of plants and that lignins exhibit a relative flexibility of their chemical structure.
Abstract: Although lignins play important roles in plants, they often represent an obstacle to the utilization of plant biomass in different areas: pulp industry, forage digestibility. The recent characterization of different lignification genes has stimulated research programmes aimed at modifying the lignin profiles of plants through genetic engineering (antisense and sense suppression of gene expression). The first transgenic plants with a modification of monomeric composition of lignins and lignin content have been recently obtained. Down regulation of the OMT gene induces dramatic reduction of syringyl units. CAD down regulated plants exhibit a unusual red phenotype associated with the developing xylem and several chemical modifications of their lignins including an increase in cinnamaldehydes in the polymer structure. Interestingly this novel lignin is removed more easily during the pulping process. In both OMT and CAD down regulated plants no changes in phenotypic characteristics such as growth architecture and morphology were observed. More recent experiments have shown that a reduction of CCR activity determines specific changes in the coloration of the xylem area suggesting significant chemical modifications which are currently being studied. These different results show that it is possible to manipulate lignins through targeted genetic transformation of plants and that lignins exhibit a relative flexibility of their chemical structure. Future developments should probe the impact of down regulating the expression of other recently characterized lignification genes such as F5H and CCoAOMT and also of a combination of genes in order to tailor lignins more adapted to specific purposes. In addition to biotechnological applications which should provide important economical benefits for utilization of wood in the pulp industry, genetic engineering of lignins offer very promising perspectives for the understanding of lignin synthesis, structure and properties.
124 citations
TL;DR: Herbicide-resistant transgenic cotton plants carrying mutant forms of a native acetohydroxyacid synthase (AHAS) gene have been obtained by Agrobacterium and biolistic transformation, and transgenic Acala plants resistant to imidazolinone herbicides were obtained.
Abstract: Herbicide-resistant transgenic cotton (Gossypium hirsutum L.) plants carrying mutant forms of a native aceto- hydroxyacid synthase (AHAS) gene have been obtained by Agrobacterium and biolistic transformation. The native gene, A19, was mutated in vitro to create amino acid substitutions at residue 563 or residue 642 of the precursor polypeptide. Transformation with the mutated forms of the Al9 gene produced resistance to imidazolinone and sulfonylurea herbicides (563 substitution), or imidazolinones only (642 substitution). The herbicide-resistant phen- otype of transformants was also manifested in their in vitro AHAS activity. Seedling explants of both Coker and Acala cotton varieties were transformed with the mutated forms of the Al9 gene using Agrobacterium. In these experiments, hundreds of transformation events were obtained with the Coker varieties, while the Acala varieties were transformed with an efficiency about one-tenth that of Coker. Herbicide-resistant Coker and Acala plants were regenerated from a subset of transformation events. Embryonic cell suspension cultures of both Coker and Acala varieties were biolistically transformed at high frequencies using cloned cotton DNA fragments carrying the mutated forms of the Al9 gene. In these transformation experiments the mutated Al9 gene served as the selectable marker, and the efficiency of selection was comparable to that obtained with the NPT II gene marker of vector Bin 19. Using this method, transgenic Acala plants resistant to imidazolinone herbicides were obtained. Southern blot analyses indicated the presence of two copies of the mutated A 19 transgene in one of the biolistically transformed Ra plants, and a single copy in one of the Ro plants transformed with Agrobacterium. As expected, progeny seedlings derived from outcrosses involving the Ro plant transformed with Agrobacterium segregated in a 1: 1 ratio with respect to herbicide resistance. The resistant progeny grew normally after irrigation with 175 pg/l of the imidazolinone herbicide imazaquin, which is five times the field application rate. In contrast, untransformed sibling plants were severely stunted.
110 citations
TL;DR: Results demonstrate that the bar gene was effective in conferring field-level resistance to glufosinate in rice, and showed the necessity to generate several independent transgenic lines of each cultivar.
Abstract: The commerical cultivars ‘Gulfmont’, ‘IR72’ and ‘Koshihikari’ were genetically engineered using electric discharge particle bombardment to express the bar gene which confers resistance to the broad-range herbicide glufosinate. Southern and northern blot analyses of transgenics material revealed stable integration and expression of introduced transgenes in the lines evaluated. In a few plants, silencing of the uidA marker gene was detected at the transcriptional level. Field studies were conducted in 1993 and 1994 at the Rice Research Station near Crowley, LA. This report summarizes results from the first two years of field trial for transgenic Gulfmont and Koshihikari. Transgenic cultivar IR72 was tested in 1995 and preliminary results are similar to those reported for transgenic Gulfmont. All 11 independently derived transgenic lines produced fertile, normal looking seed at maturity. Significant differences were observed in the absence of the herbicide between parental cultivars and transgenic Gulfmont-and Koshihikari-derived lines for days to 50% heading (20% of transgenic lines), plant height (13%), and grain yield (7%). Foliar application of glufosinate had little or no effect on agronomic performance of all transgenic Gulfmont and IR72 lines, while herbicide applications affected grain, yield and plant height of some transgenic Koshhikari. Non-transgenic plants of all three cultivars at the 4-leaf stage were killed within 7 days after 1.12 or 2.24 kg/ha glufosinate applications. Significant differences among certain transgenic lines were observed for agronomic traits after herbicide applications. These results demonstrate that the bar gene was effective in conferring field-level resistance to glufosinate in rice. Variation among transgenic lines required traditional breeding selection procedures to identify superior agronomic types with high levels of herbicide resistance and showed the necessity to generate several independent transgenic lines of each cultivar.
107 citations
TL;DR: DNA markers linked to a root-knot nematode resistance gene derived from wild peanut species have been identified and different distributions of resistance in the BC5F2 progeny and TxAG-7 suggest the presence of additional resistance genes in TxAg-7.
Abstract: DNA markers linked to a root-knot nematode resistance gene derived from wild peanut species have been identified. The wild diploid peanut accessions K9484 (Arachis batizocoi Krapov. & W. C. Gregory), GKP10017, (A. cardenasii Krapov & W. C. Gregory), and GKP10602 (A. diogoi Hoehne) possess genes for ressitance to Meloidogyne arenaria. These three accessions and A. hypogaea cv. Florunner were crossed to generate the hybrid resistant breeding line TxAg-7. This line was used as donor parent to develop a BC4F2 population segregating for resistance. Three RAPD markers associated with nematode resistance were identified in this population by bulked segregant analysis. Linkage was confirmed by screening 21 segregatingh BC4F2 and 63 BC5F2 single plants. Recombination between marker RKN410 and resistance, and between marker RKN440 and resistance, was estimated to be 5.4±1.9% and 5.8±2.1%, respectively, on a per-generation basis. These two markers identified a resistance gene derived from either A. cardenasii or A. diogoi, and were closely linked to each other. Recombination between a third marker, RKN229, inherited from A. cardenasii or A. diogoi, and resistance was 9.0±3.2% per generation. Markers RKN410 and RKN229 appeared to be linked genetically and flank the same resistance gene. All markers were confirmed by hybridization of cloned or gel-purified marker DNA to blots of PCR-amplified DNA. Pooled data on the segregation of BC5F2 plants was consistent with the presence of one resistance gene in the advanced breeding lines. Different distributions of resistance in the BC5F2 progeny and TxAG-7 suggest the presence of additional resistance genes in TxAG-7.
101 citations
TL;DR: It is concluded that cultivated roses display a high level of genetic variability despite the fact that single morphological and physiological characters may be less polymorphic within rose groups, which contrasts with the widely accepted opinion of a lack of Genetic variability in roses.
Abstract: The genetic variability based on random-amplified polymorphic DNA markers was analysed among 10 cultivated rose varieties and 9 wild species from three different series of the genus Rosa. Using 13 different RAPD primers, 104 polymorphic DNA fragments with a high potential to differentiate rose genotypes could be produced. A dendrogram displaying the relative genetic similarities among the genotypes shows the existence of large genetic diversity among the cultivated roses as compared to the wild species. Furthermore, the main clusters found here are in agreement with known pedigrees and the classical taxonomy. However, the relationships between cultivated roses as inferred by RAPD markers do not correlate with the classical rose classification system. From the present data it is concluded that cultivated roses display a high level of genetic variability despite the fact that single morphological and physiological characters may be less polymorphic within rose groups. This contrasts with the widely accepted opinion of a lack of genetic variability in roses. This is also in accordance with the reported history of rose breeding which makes it highly probable that rose genomes comprise mosaics of different species genomes. As a consequence, it may be possible to utilize the high genetic variability of all genetic traits not under actual selection by breeders for future breeding programmes.
TL;DR: Pollen dispersal was low in both years, but increased with an increase in the size of the source plot in the second year, and results indicate that 20 m buffer zones would serve to limit dispersal of transgenic pollen from small-scale field tests.
Abstract: The testing of transgenic crops in the field is a necessary part of the validation of genetically engineered cultivars, but in the early stages of testing, biosafety procedures must be carefully monitored to ensure that the modified plants do not have deleterious effects on the environment. This study was carried out over two seasons to determine the effectiveness of containment procedures under australian environmental conditions by measuring the dispersal of pollen amay from a test plot of transgenic cotton into a surrounding buffer field of non-transgenic cotton plants whose function was to act as a sink for pollen carried by nectar feeding and pollen-gathering insects. Dispersal was estimated by measuring the frequency of the dominant selectable marker transgene, neomycin phosphotransferase (NptII) in the progeny of the buffer plants. The presence of nptii was determined by a sensitive radioactive enzyme assay. Pollen dispersal was low in both years, but increased with an increase in the size of the source plot in the second year. In the first year outcrossing averaged from 0.15% of progeny at 1 m to below 0.08% at 4 m from the test plot. Outcrossing was highest within the central test plot where progeny from non-transgenic control plants, immediately adjacent to transgenic plants, had transgenic progeny at frequencies of up to 1.7%. In the second year, with a bigger source of transgenic plants, outcrossing declined on average from 0.4% at 1 m to below 0.03% at 16 m into the buffer zone. These results indicate that 20 m buffer zones would serve to limit dispersal of transgenic pollen from small-scale field tests.
TL;DR: A population of diploid potato was used for the genetic analysis and mapping of a locus for resistance to the potato cyst nematode Globodera rostochiensis, introgressed from the wild potato species Solanum vernei.
Abstract: A population of diploid potato (Solanum tuberosum) was used for the genetic analysis and mapping of a locus for resistance to the potato cyst nematode Globodera rostochiensis, introgressed from the wild potato species Solanum vernei. Resistance tests of 108 genotypes of a F1 population revealed the presence of a single locus with a dominant allele for resistance to G. rostochiensis pathotype Ro1. This locus, designated GroV1, was located on chromosome 5 with RFLP markers. Fine-mapping was performed with RAPD and SCAR markers. The GroV1 locus was found in the same region of the potato genome as the S. tuberosum ssp. andigena H1 nematode resistance locus. Both resistance loci could not excluded to be allelic. The identification of markers flanking the GroV1 locus offers a valuable strategy for marker-assisted selection for introgression of this nematode resistance.
TL;DR: The method presented enables the transformation of this particular cassava genotype, thus allowing the genetic improvement of this important tropical crop by transgenesis.
Abstract: Cassava embroids derived from friable embryogenic callus of the genotype TMS60444 were bombarded with DNA of the constructs pJIT100 or pJIT64. Both constructs contain the non-invasive reporter gene luciferase from firefly driven by the CaMV 35S promoter. The influence of several particle gun machine parameters and pretreatment of plant material on transient luciferase activity were studied to determine the most essential conditions for stable transformation. Two weeks after bombardment pieces of friable calli with luciferase activity were selected. In total, 67 independent selected calli with luciferase activity (spots), derived from five different experiments, were further cultured either in liquid or on solid medium. Per plate or flask one spot was cultured. In subsequent selection rounds all spots of one individual plate or flask were cultured as one individual group. In this way different transformation events were separated and multiplied. Eight weeks after bombardment 34 cultures still contained luciferase activity. The mean number of luciferase spots per culture had increased from 1 to 4.6 spots in liquid and to 2.5 spots on solid medium. After two more months of subsequent culture and luciferase selection presence of the construct in these cultures was confirmed at the molecular level using the polymerase chain reaction assay and Southern analysis. Friable embryos derived from four transformation events were cultured for maturation. Between 3% and 21% of the mature embryos of the different transformation events were luciferase-positive. After multiplication of the luciferase-positive mature embryos by secondary somatic embryogenesis they were germinated. The plantlets analysed contained one to several copies of the inserted DNA. The method presented enables the transformation of this particular cassava genotype, thus allowing the genetic improvement of this important tropical crop by transgenesis.
TL;DR: The commonly used expression vector pRT100 and several pGPTV binary vectors are modified by replacing 6 bp restriction sites with 8 bp sequences recognized by rare-cutter restriction enzymes to facilitate cloning and transfer of chimeric expression cassettes.
Abstract: Cloning of long open reading frames (ORFs) into plant gene expression vectors and transfer of the chimeric expression cassettes into binary vectors is often hampered by the presence of restriction enzyme cleavage sites internal to the open reading frame (ORF) to be expressed. We therefore modified the commonly used expression vector pRT100 [7] and several pGPTV binary vectors [2] by replacing 6 bp restriction sites with 8 bp sequences recognized by rare-cutter restriction enzymes.
TL;DR: Results conclusively demonstrate that the gene from L. douglasii encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase which can function in rape and incorporate erucic acid at the sn-2 position of triacylglycerols in seed.
Abstract: A cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii was introduced into oil seed rape (Brassica napus) under the control of a napin promoter. Seed triacylglycerols from transgenic plants were analysed by reversed-phase HPLC and trierucin was detected at a level of 0.4% and 2.8% in two transgenic plants but was not found in untransformed rape seed. Total fatty acid composition analysis of seeds from these selected plants revealed that the erucic acid content was no higher than the maximum found in the starting population. Analysis of fatty acids at the sn-2 position showed no erucic acid in untransformed rape but in the selected transgenic plants 9% (mol/mol) and 28.3% (mol/mol) erucic acid was present. These results conclusively demonstrate that the gene from L. douglasii encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase which can function in rape and incorporate erucic acid at the sn-2 position of triacylglycerols in seed. Additional modifications may further increase levels of trierucin.
TL;DR: In the unevenly coloured inflorescences detected among anti-gchs1 transformants during their growth, relaxation of the antisense effect was here shown to start from the most distant member of the gene family, further demonstrating the influence of sequence homology in the stability of antisense inhibition.
Abstract: Suppression of gene expression using antisense technology has been successful in various applications. In this paper we report differential inhibition of gene expression of the chalcone synthase (chs) gene superfamily members in transgenic Gerbera hybrida (Asteraceae) plants. We have transformed two different cDNAs of the chs gene family, gchs 1 [4] and gchs2, in antisense orientation under control of the CaMV 35S promoter into gerbera. Gchs1 codes for an enzyme with chalcone synthase activity while gchs2 is a more diverged member of the gene family having distinct structure and expression pattern. Furthermore, gchs2 is evidently not involved in anthocyanin synthesis and encodes an enzyme with novel catalytic properties. In both cases effective blocking of the resident sense gene expression was detected. In addition, the transformation affected differentially the expression of other members of the chs gene family. The degree of inhibition appeared to depend on the sequence homology between the antisense and the target genes. In the unevenly coloured inflorescences detected among anti-gchs1 transformants during their growth, relaxation of the antisense effect was here shown to start from the most distant member of the gene family, further demonstrating the influence of sequence homology in the stability of antisense inhibition.
TL;DR: Quantitative trait loci involved in the resistance of maize to Setosphaeria turcica, the causal agent of northern leaf blight, were located by interval mapping analysis of 121 F2:3 lines derived from a cross between Mo17 (moderately resistant) and B52 (susceptible).
Abstract: Quantitative trait loci (QTL) involved in the resistance of maize to Setosphaeria turcica, the causal agent of northern leaf blight, were located by interval mapping analysis of 121 F2:3 lines derived from a cross between Mo17 (moderately resistant) and B52 (susceptible) A linkage map spanning 112 RFLP loci with 15 cM mean interval length was constructed, based on marker data recorded in a previous study Field tests with artificial inoculation were conducted at three sites in tropical mid- to high-altitude regions of Kenya, East Africa Host-plant response was measured in terms of incubation period, disease severity (five scoring dates), and the area under the disease progress curve (AUDPC) Heritability of all traits was high (around 075) QTL associated with the incubation period were located on chromosomes 2S and 8L For disease severity and AUDPC, significant QTL were detected in the putative centromeric region of chromosome 1 and on 2S, 3L, 5S, 6L, 7L, 8L and 9S On 2S the same marker interval which carried a gene enhancing latent period was also associated with reduced disease severity of juvenile plants QTL on chromosomes 3L, 5S, 7L and 8L were significant across environments but all other QTL were affected by a large genotype x environment interaction Partially dominant gene action for resistance as well as for susceptibility was prevailing Single QTL explained 10 to 38% of the phenotypic variation of the traits All but the QTL on chromosomes 1, 6 and 9 were contributed by the resistant parent Mo17 On chromosome 8L a QTL mapped to the same region as the major race-specific gene Ht2, supporting the hypothesis that some qualitative and quantitative resistance genes may be allelic
TL;DR: The research experience on transformation of Brassica oleracea is collated to highlight the problems encountered and methods that enhance bacterial virulence and increase the proportion of cells susceptible to transformation and competent for regeneration are discussed.
Abstract: Brassica oleracea is a highly polymorphic species encompassing a wide range of important vegetable and fodder crops. Gene transfer into cultivated forms of this species requires reproducible and efficient methods for genetic transformation and plant regeneration. In this review, we have collated the research experience on transformation ofB. oleracea to highlight the problems encountered. Most research effort has been directed at developingAgrobacterium-mediated transformation methods with relatively little emphasis to date on direct gene transfer techniques. Common procedures for the transformation ofB. oleracea have not emerged, due to the inherent variability between and amongst genotypes. Future progress would be facilitated by the use of genetically fixed material, such as double-haploid or inbred lines, to reduce variation of response within genotypes and would avoid the need for cultivar-specific transformation protocols if responsive lines amenable to crossing with cultivated forms could be identified. The principal difficulties relate to combining efficient plant regeneration with gene transfer. Methods that enhance bacterial virulence and increase the proportion of cells susceptible to transformation and competent for regeneration are discussed. Inefficient selection is a major cause of poor transformation frequencies inB. oleracea and has resulted in the regeneration of chimeric plants uponAgrobacterium tumefaciens-mediated transformation. Promising results have been obtained withAgrobacterium rhizogenes-mediated transformation but the impact of therol genes on flowering of primary transformants has not yet been fully assessed. Strategies to reduce the deleterious effects of therol genes on flowering are discussed. Few agronomically useful characters have been introduced, the majority of research having been confined to the introduction of marker and reporter genes; possible candidate genes are discussed.
TL;DR: In order to help conventional breeding, RAPD markers were used to map the two genes involved in determining the erucic acid content in a doubled haploid progeny derived from a ‘low x high’ erucing acid F1 hybrid, and it was shown that theTwo genes do not contribute uniformly to the C22:1 level.
Abstract: The recent development of the industrial use of rapeseed oil rich in erucic acid has led to increased interest in the improvement of the high-erucic-acid (50–60%) varieties and to research towards genotypes containing a very high erucic acid content. This trait is controlled by two genes with additive effects. The low-erucic-acid trait was relatively easily introduced through backcrosses into various backgrounds because the zero-erucic-acid homozygotes were clearly identified in the segregating populations. To select for high erucic acid level is more difficult because of the partial overlap of the high-erucic-acid homozygous class and the intermediate one, containing heterozygotes. In order to help conventional breeding, RAPD markers were used to map the two genes involved in determining the erucic acid content in a doubled haploid progeny derived from a ‘low x high’ erucic acid F1 hybrid. The two genes were successfully localized in two independent linkage group, through a QTL approach. A close association was found between individual plant genotypes and the erucic acid content of the doubled haploid progeny, and it was shown that the two genes do not contribute uniformly to the C22:1 level. The value of molecular gene mapping of such a trait in a conventional breeding programme is discussed.
TL;DR: A highly significant correlation was found between fruit shape and ovary shape indicating that thefs8.1 gene product acts early in ovary development (preanthesis).
Abstract: In two previous quantitative trait locus (QTL) mapping studies conducted inLycopersicon esculentum x L. pimpinellifolium BC1 and BC2 populations we had localized a major QTL for fruit shape,fs8.1, to a ca. 20 cM interval on the short arm of chromosome 8, flanked by markers TG176 and CT92. At this QTL the allele from the wild species reduces the length of fruit, giving round-shaped fruit. In order to define more precisely the location offs8.1, near-isogenic lines (NILs) segregating for the region of interest were developed. The results from substitution mapping show that no recombination occurred betweenfs8.1 and the marker CD40 in 322 meioses. The gene action forfs8.1 was determined in a BC4F3 population to be partial dominance. The main effect offs8.1 is exerted on fruit length while fruit diameter is not significantly affected. A highly significant correlation (r=0.89;P<0.01) was found between fruit shape and ovary shape indicating that thefs8.1 gene product acts early in ovary development (preanthesis). Implications for the evolution of fruit shape and the feasibility of map-based cloning of this QTL are discussed.
TL;DR: The most polymorphic primer pairs were used successfully to fingerprint a range of closely related varieties of kiwifruit (A. deliciosa) and the highest number of bands and the largest size variation were observed with a complex microsatellite harbouring four different repeat motifs.
Abstract: We have identified a set of informative microsatellite markers for genome analysis in kiwifruit and related Actinidia species. A small-insert genomic library was constructed from Actinidia chinensis DNA, and screened for microsatellites. About 1.2% of the total colonies hybridised to a (GA)8 probe, 0.4% to (GT)8, and 0.1% to a mixture of three different trinucleotide repeat probes, (CAA)5, (GAA)5 and (CTA)5. From the DNA sequences of 35 hybridising clones, 18 primer pairs were designed, and used to amplify genomic DNA from 38 individual plants, representing 30 different accessions of ten Actinidia species. The banding patterns for most of the dinucleotide repeats showed a high degree of polymorphism in the diploid and tetraploid A. chinensis, and in the hexaploid A. deliciosa (kiwifruit). Heterozygosity levels of up to 100% were found among eight diploid accessions of A. chinensis examined, and the number of different-sized bands among all the species varied from 3 to 36 for each microsatellite. One simple CT microsatellite gave 21 bands with sizes suggesting that the number of repeats ranged from 9 to 37. The highest number of bands (36) and the largest size variation (>100 bp) were observed with a complex microsatellite harbouring four different repeat motifs. The majority of primer pairs amplified bands from most of the ten Actinidia species tested. The most polymorphic primer pairs were used successfully to fingerprint a range of closely related varieties of kiwifruit (A. deliciosa).
TL;DR: Northern and western blot analysis showed that the tachyplesin I gene was expressed in transgenic plants, and small tubers of 17 transgenic clones were screened twice for soft rot resistance to Erwinia carotovora ssp.
Abstract: Tachyplesin I is a 2.3 kDa antimicrobial peptide isolated from Southeast Asian horseshoe crabs. Bacterial suspensions containing 1×106 colony-forming units/ml of six isolates of pectolytic Erwinia spp., the causal pathogens of potato soft rot and blackleg, were killed in vitro by 1.4 to 11.1 μg/ml of tachyplesin I. In an attempt to enhance resistance to Erwinia spp., each of the potato cultivars Bintje, Karnico and Kondor were transformed with two gene constructs encoding different precursor tachyplesin I proteins under the control of a cauliflower mosaic virus 35S promotor. Northern and western blot analysis showed that the tachyplesin I gene was expressed in transgenic plants. Small tubers of 17 transgenic clones were screened twice for soft rot resistance to Erwinia carotovora ssp. atroseptica. Under aerobic or anaerobic conditions, transgenic clones showed slightly less rot than control tubers.
TL;DR: Compared to other types of pathogens, efforts in genetic engineering allocated to the development of resistance to phytopathogenic bacteria in transgenic plants is quite small, and work has focused mostly on potato or tobacco.
Abstract: Bacterial diseases are of high economic importance in many crop plant species including potato, different vegetable species, fruit trees, rice, cotton and grapevine. Conventional breeding efforts in potato has not resulted in the generation of varieties resistant to soft rot and black leg due to the lack of known resistance traits in lines or species which are sexually compatible with potato. Resistance traits known to be present in wild species often cannot be used for classical breeding programmes as these are genetically too distant from today’s cultivars. Similarly, in other plant species susceptible to bacterial pathogens often no valuable resistance or tolerance traits are known. Furthermore, chemical plant protection is not as evolved as for other pathogens. Actually, the most prominent means of prevention of bacterial infections are phytosanitary practices and highly developed harvesting techniques minimizing wounding as well as storage conditions reducing temperature and humidity. Genetic engineering allows a new approach to an old problem. Foreign proteins or peptides can be expressed in transgenic plants and might introduce new resistance factors or complement the natural mechanisms. An excellent model host-pathogen system of high economic importance is the interaction between Solanurn tuberosum and Etwinia carotovora. This bacterial species is the causative agent of soft rot and black leg in potato. Especially in tropical and subtropical climates the pathogen provokes severe damage and losses of harvest both in the field and during storage. Compared to other types of pathogens, efforts in genetic engineering allocated to the development of resistance to phytopathogenic bacteria in transgenic plants is quite small. In general, this work has focused mostly on potato or tobacco. This mini-review will summarize the different research approaches published so far.
TL;DR: Apical segments of embryonic axes of sunflower embryos submitted to co-culture experiments with a disarmed strain of Agrobacterium tumefaciens, harbouring a plasmid coding for the marker enzyme β-glucuronidase are consistent with the hypothesis that transformed shoots originate from transformation events that have occurred within the existing meristems.
Abstract: Apical segments of embryonic axes of sunflower (Helianthus annuus L.) embryos were submitted to co-culture experiments with a disarmed strain of Agrobacterium tumefaciens, harbouring a plasmid coding for the marker enzyme β-glucuronidase. The expression patterns of this marker were analysed at different developmental stages of the regenerated shoots. The results are consistent with the hypothesis that transformed shoots originate from transformation events that have occurred within the existing meristems. Two of the resulting chimaeric plants have been analysed in detail, and some representative gene integration patterns are presented.
TL;DR: This work investigated the possibilities of transgenic plants as bioreactors for the production of industrial enzymes using cell wall-hydrolysing enzymes as first examples and found XYLD-C accumulated in the intercellular space and was one of the most prominent bands in protein gels.
Abstract: We are investigating the possibilities of transgenic plants as bioreactors for the production of industrial enzymes using cell wall-hydrolysing enzymes as first examples. Within the frame work of this work two distinct domains of the xynD gene from Ruminococcus flavefaciens encoding a xylanase (XYLD-A) and a β(1–3, 1–4)glucanase (XYLD-C) were separately cloned into a plant expression vector which would target the proteins into the apoplast. Transgenic tobacco plants were obtained expressing xylan-hydrolysing as well as lichenan-hydrolysing activities. Despite similar steady-state levels of the respective mRNAs xylan hydrolysis rates were between 40 and 170 μmol min−1 m−2 leaf area depending on the transgenic plant while β(1–3, 1–4)glucan degradation was much more effective ranging between 200 and 2000 μmol min−1 m−2. The high activity levels of the XYLD-C expressing plants were reflected on the protein level. XYLD-C accumulated in the intercellular space and was one of the most prominent bands in protein gels. Despite their apoplastic location as confirmed by activity measurements using intercellular fluids the transgenic plants had not undergone any phenotypic alteration.
TL;DR: The use of dot blot hybridization to analyse PCR products well decrease the cost and time requirements of marker-assisted selection and facilitate the rapid application of PCR-based maps.
Abstract: Scoring of the results of RAPD analysis using gel electrophoresis imposes a constraint on throughput. To circumvent this barrier, dot-blot hybridization was substituted for electrophoresis. Arbitrarily amplified fragments from barley and wheat genomic DNA were labelled and used as probes for the identification of identical fragments in subsequent amplification reactions. None of the twelve fragments used as probes exhibited significant levels of croos-hybridization to other fragments amplified by the same arbitrary primer. The strength of the hybridization signal facilitates more accurate and more sensitive detection of diagnostic fragments than gel electrophoresis. In addition, the defined spatial orientation (microtitre dish format) of the ± results provide an excellent format for automated data collection. The use of dot blot hybridization to analyse PCR products well decrease the cost and time requirements of marker-assisted selection. This technique will also facilitate the rapid application of PCR-based maps.
TL;DR: A method for using alkali treated intact plant tissue as a DNA source for the polymerase chain reaction (PCR) was applied to barley and was shown to work with several genotypes and different primers.
Abstract: A method for using alkali treated intact plant tissue as a DNA source for the polymerase chain reaction (PCR) was applied to barley. This method saves up to two days and more than USD 50 per 40 samples by eliminating the need for DNA extraction to produce template for PCR. The conditions were optimized for various barley tissues. Fresh leaves, freeze-dried leaves, and anthers worked well as templates while root, embryo, and endosperm tissues did not. The method was shown to work with several genotypes and different primers. The resulting PCR product could be cut with restriction enzyme to produce clear polymorphism without any interference. This method can be a practical breeding tool by providing a fast, inexpensive method for screening large populations.
TL;DR: Changes in RAPD marker frequencies with recurrent selection are demonstrated and linkage of RAPD markers to genes controlling pigment in red beet is suggested.
Abstract: In the past twenty years, betalain pigments found in red beet (Beta vulgaris L.) have been adopted for use as natural red food colorings. Utility and extractability of these compounds is dependent upon native concentrations of both pigment and total dissolved solids. In an effort to develop red beet populations with elevated levels of betalain pigment, recurrent half-sib family selection for high pigment and both high and low solids was practiced for seven cycles. This scheme resulted in the development of a high pigment/high solids and a high pigment/low solids population. A total of 200 randomly chosen oligonucleotide decamer primers were used to amplify genomic DNA of individual plants in the selection scheme for repeatability and clarity of amplification products. Thirty-one primers were chosen on this basis and used to assess random-amplified polymorphic DNA (RAPD) marker frequencies on genomic DNA samples isolated from 47 randomly-chosen individual plants in each of cycles 1, 3, and 6 in both high pigment/high solids and high pigment/low solids. Number of scorable Polymerase Chain Reaction products ranged from 1–10, resulting in a total of 161 RAPD markers. Chi-square and regression analyses were performed to determine the presence/absence of a linear trend in marker frequencies during the selection scheme. Comparisons were made among cycles within high pigment/high solids and high pigment/low solids populations. Significant linear trends were detected in both cases for certain RAPD markers. Formulae were used to test whether observed linear marker frequency trends were due to the effects of selection or random genetic drift. Chi-square tests revealed a subset of markers which exhibited significant frequency changes across cycles changed due to selection. These data demonstrate changes in RAPD marker frequencies with recurrent selection and suggest linkage of RAPD markers to genes controlling pigment in red beet.