24 research outputs found

    Genetic manipulation of self-incompatibility in diploid potato species

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    Many of the wild and some cultivated species of potato are true diploids and are therefore more amenable for genetic studies than the majority of tetraploid cultivars. However, the use of these diploid Solanum species is complicated by almost universal self-incompatibility (SI). In Solanum, SI is gametophytic and pistil specificity is controlled by a polymorphic ribonuclease (S-RNase), as found in other members of the Solanaceae. The genetic engineering of self-compatible (SC) diploid potato lines would benefit potato breeding in general and allow inbred lines to be established for the first time. This would facilitate genetic analyses including that of complex traits such as drought resistance or yield. The aim of this thesis is to down regulate the expression of S-RNases in diploid potatoes using the RNAi technique and established procedures for Agrobacterium-mediated transformation. This approach to engineering self-compatibility has already been successfully demonstrated in SI Petunia inflata (Lee et al., 1994) and other species of the Solanaceae. To date just a handful of S-RNase sequences are available for potato species. The characterization of S-RNases in targeted diploid Solanum species was an initial requirement for our approach. To develop the tools, S-alleles have initially been characterized in both Petunia inflata and P. hybrida cv Mitchell both phenotypically (by pollination tests using a diallel cross) and/or genotypically (by RT-PCR). This approach was then transferred to three diploid potato species, specifically accessions of Solanum stenotomum, Solanum phureja and Solanum okadae. These wild species are important sources of new traits studied by The James Hutton Institute (formally the Scottish Crop Research Institute). The approach taken to amplify partialS-RNase sequences from pistil RNA was RT-PCR using a degenerate primer. PCR products were cloned using a TA vector (Invitrogen) and sequenced. For two alleles full length sequences were obtained by 5'RACE. Database searches with these sequences, revealed sixteen S-RNases several of which are novel. Phylogenetic analYSis was carried out with the cloned S-RNases together with selected published S-RNase and S-like RNase sequences of solanaceous species. The S-RNases revealed extensive trans-generic evolution and are clearly distinct from and distantly related to S-like RNases. For two alleles (501 and 502), S-RNase gene expression profiling was performed to check the developmental expression of the S-RNase gene, tissue-specific expression and also test whether these S-RNases (e.g. Sor and S02-RNases) are expressed at a similar level. Wide variation in S-RNase gene expression levels have been reported in the literature. An RNAi construct has been designed to down-regulate two specific 5-RNases in an 501/502 heterozygote of S. okadae. To increase the chance of Silencing, the RNAi construct has been designed to use a chimeric 5-RNase gene involving the 5' end of the SorRNase and the 3' end of the S02-RNase. The correct chimeric S-RNase construct (SOl/S02-RNase) has now been identified and inserted into an RNAi vector (pHelisgate8) using Gateway® technology. This RNAi construct (pHG8-S01/S02) is now a valuable resource for use in S-RNase gene silencing in potato leading to the development of self-compatible diploid potato lines and ultimately the development of the first inbred lines of S. okadae

    Genetic manipulation of self-incompatibility in diploid potato species

    Get PDF
    Many of the wild and some cultivated species of potato are true diploids and are therefore more amenable for genetic studies than the majority of tetraploid cultivars. However, the use of these diploid Solanum species is complicated by almost universal self-incompatibility (SI). In Solanum, SI is gametophytic and pistil specificity is controlled by a polymorphic ribonuclease (S-RNase), as found in other members of the Solanaceae. The genetic engineering of self-compatible (SC) diploid potato lines would benefit potato breeding in general and allow inbred lines to be established for the first time. This would facilitate genetic analyses including that of complex traits such as drought resistance or yield. The aim of this thesis is to down regulate the expression of S-RNases in diploid potatoes using the RNAi technique and established procedures for Agrobacterium-mediated transformation. This approach to engineering self-compatibility has already been successfully demonstrated in SI Petunia inflata (Lee et al., 1994) and other species of the Solanaceae. To date just a handful of S-RNase sequences are available for potato species. The characterization of S-RNases in targeted diploid Solanum species was an initial requirement for our approach. To develop the tools, S-alleles have initially been characterized in both Petunia inflata and P. hybrida cv Mitchell both phenotypically (by pollination tests using a diallel cross) and/or genotypically (by RT-PCR). This approach was then transferred to three diploid potato species, specifically accessions of Solanum stenotomum, Solanum phureja and Solanum okadae. These wild species are important sources of new traits studied by The James Hutton Institute (formally the Scottish Crop Research Institute). The approach taken to amplify partialS-RNase sequences from pistil RNA was RT-PCR using a degenerate primer. PCR products were cloned using a TA vector (Invitrogen) and sequenced. For two alleles full length sequences were obtained by 5'RACE. Database searches with these sequences, revealed sixteen S-RNases several of which are novel. Phylogenetic analYSis was carried out with the cloned S-RNases together with selected published S-RNase and S-like RNase sequences of solanaceous species. The S-RNases revealed extensive trans-generic evolution and are clearly distinct from and distantly related to S-like RNases. For two alleles (501 and 502), S-RNase gene expression profiling was performed to check the developmental expression of the S-RNase gene, tissue-specific expression and also test whether these S-RNases (e.g. Sor and S02-RNases) are expressed at a similar level. Wide variation in S-RNase gene expression levels have been reported in the literature. An RNAi construct has been designed to down-regulate two specific 5-RNases in an 501/502 heterozygote of S. okadae. To increase the chance of Silencing, the RNAi construct has been designed to use a chimeric 5-RNase gene involving the 5' end of the SorRNase and the 3' end of the S02-RNase. The correct chimeric S-RNase construct (SOl/S02-RNase) has now been identified and inserted into an RNAi vector (pHelisgate8) using Gateway® technology. This RNAi construct (pHG8-S01/S02) is now a valuable resource for use in S-RNase gene silencing in potato leading to the development of self-compatible diploid potato lines and ultimately the development of the first inbred lines of S. okadae

    Assessing Mating Designs Utilized in Cassava Population Improvement

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    Cassava breeders are curious about appropriate breeding strategies utilized to generate elite genotypes with desired complimentary traits or genes from parents used in crossing. Use of appropriate mating design is influenced by a good understanding of the flower biology of the putative parent plants, type of pollination, crossing technique, pollen dissemination, the presence of male-sterility system, the purpose of the project (that is either breeding or genetic studies), and the size of population needed. The objective of this book chapter is to assess the current knowledge on mating designs, their applications and limitations in cassava improvement. This book chapter discusses the floral biology, genetic improvement, breeding procedures and mating designs in cassava. The information utilized in this study were obtained from various sources including documentary search of the journals, books and websites of relevant stakeholder organizations. Empirical findings of selected mating designs in cassava and their impacts were discussed. Findings serve as a good guide for selection of appropriate mating arrangement to obtain useful information on parents and progenies. Findings are relevant to scientists, researchers, scholars, lecturers and other relevant stakeholders

    Brown midrib 6 and 12 Genes introgression in two nigerien and one malian sorghum varieties: A practical guide to young scientists with limited molecular facility

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    Introgression of bmr genes from less adapted donor parent to well adapted high yielding biomass varieties with poor nutritional value is very important for sustainable cattle feeding during pasture scare time in the Sahel. The main objective of this work was to introgress bmr6 and bmr12 genes in Nigerien and Malian sorghum varieties background for dual purpose grain and biomass potential. The plant material was composed of two improved sorghum varieties (Sepon82 and Kalla Kene) and El mota a farmer preferred variety as recurrent parents. bmr donor parents were redlan bmr6, Tx630 bmr12 and Wheatland bmr12. The hand emasculation technique was used to introgress bmr genes in recurrent parents to produce F3 and BC1F3 populations at Sotuba research Station in Mali from January 2016 to June 2017. Anthocyanin pigment and heterosis effects were key phenotypic traits to identify F1 and BC1F1 plants during the population development. Anthocyanin allowed the identification of F1 plants in a cross involving anthocyanin (purple plant) and tan plants, while for both tan plants cross, heterosis effect was major key to discriminate F1 from parental lines and bmr segregation in F2 to ascertain successful crosses. The χ2 test was used to analyze bmr segregation ration. Segregation ratios of bmr plants in F2 and BC1F2 showed a good fit of a single recessive gene (3:1). bmr 6 and 12 genes were successfully transferred to three recurrent parents varieties which are at F4 and BC1F3 generation for grain and biomass yields potential tests in Niger during the 2017 cropping season

    Cross-species gene expression analysis of species specific differences in the preclinical assessment of pharmaceutical compounds

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    Animals are frequently used as model systems for determination of safety and efficacy in pharmaceutical research and development. However, significant quantitative and qualitative differences exist between humans and the animal models used in research. This is as a result of genetic variation between human and the laboratory animal. Therefore the development of a system that would allow the assessment of all molecular differences between species after drug exposure would have a significant impact on drug evaluation for toxicity and efficacy. Here we describe a cross-species microarray methodology that identifies and selects orthologous probes after cross-species sequence comparison to develop an orthologous cross-species gene expression analysis tool. The assumptions made by the use of this orthologous gene expression strategy for cross-species extrapolation is that; conserved changes in gene expression equate to conserved pharmacodynamic endpoints. This assumption is supported by the fact that evolution and selection have maintained the structure and function of many biochemical pathways over time, resulting in the conservation of many important processes. We demonstrate this cross-species methodology by investigating species specific differences of the peroxisome proliferatoractivator receptor (PPAR) a response in rat and human

    Scalable Sparse Testing Genomic Selection Strategy for Early Yield Testing Stage

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    To enable a scalable sparse testing genomic selection (GS) strategy at preliminary yield trials in the CIMMYT maize breeding program, optimal approaches to incorporate genotype by environment interaction (GEI) in genomic prediction models are explored. Two cross-validation schemes were evaluated: CV1, predicting the genetic merit of new bi-parental populations that have been evaluated in some environments and not others, and CV2, predicting the genetic merit of half of a bi-parental population that has been phenotyped in some environments and not others using the coefficient of determination (CDmean) to determine optimized subsets of a full-sib family to be evaluated in each environment. We report similar prediction accuracies in CV1 and CV2, however, CV2 has an intuitive appeal in that all bi-parental populations have representation across environments, allowing efficient use of information across environments. It is also ideal for building robust historical data because all individuals of a full-sib family have phenotypic data, albeit in different environments. Results show that grouping of environments according to similar growing/management conditions improved prediction accuracy and reduced computational requirements, providing a scalable, parsimonious approach to multi-environmental trials and GS in early testing stages. We further demonstrate that complementing the full-sib calibration set with optimized historical data results in improved prediction accuracy for the cross-validation schemes

    Allelic diversity of S‑RNase alleles in diploid potato species

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    S-ribonucleases (S-RNases) control the pistil specificity of the self-incompatibility (SI) response in the genus Solanum and several other members of the Solanaceae. The nucleotide sequences of S-RNases corresponding to a large number of S-alleles or S-haplotypes have been characterised. However, surprisingly few S-RNase sequences are available for potato species. The identification of new S-alleles in diploid potato species is desirable as these stocks are important sources of traits such as biotic and abiotic resistance. S-RNase sequences are reported here from three distinct diploid types of potato: cultivated Solanum tuberosum Group Phureja, S. tuberosum Group Stenotomum, and the wild species Solanum okadae. Partial S-RNase sequences were obtained from pistil RNA by RT-PCR or 3’RACE (Rapid Amplification of cDNA Ends) using a degenerate primer. Full length sequences were obtained for two alleles by 5’RACE. Database searches with these sequences, identified sixteen S-RNases in total, all of which are novel. The sequence analysis revealed all the expected features of functional S-RNases. Phylogenetic analysis with selected published S-RNase and S-like-RNase sequences from the Solanaceae revealed extensive trans-generic evolution of the S-RNases and a clear distinction from S-like-RNases. Pollination tests were used to confirm the self-incompatibility status and cross-compatibility relationships of the S. okadae accessions. All the S. okadae accessions were found to be self-incompatible as expected with crosses amongst them exhibiting both cross-compatibility and semi-compatibility consistent with the S-genotypes determined from the S-RNase sequence data. The progeny analysis of four semi-compatible crosses examined by allele-specific PCR provided further confirmation that these are functional S-RNases

    Genetic manipulation of self-incompatibility in diploid potato species

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    Many of the wild and some cultivated species of potato are true diploids and are therefore more amenable for genetic studies than the majority of tetra ploid cultivars. However, the use of these diploid Solanum species is complicated by almost universal self-incompatibility (51). In Solanum, 51 is gametophytic and pistil specificity is controlled by a polymorphic ribonuclease (S-RNase), as found in other members of the Solanaceae. The genetic engineering of self-compatible (SC) diploid potato lines would benefit potato breeding in genera l and allow inbred lines to be established for the first time. This would facilitate genetic analyses including that of complex traits such as drought resistance or yield. The aim of this thesis is to downregulate the expression of S-RNases in diploid potatoes using the RNAi technique and established procedures for Agrobacterium-mediated transformation. This approach to engineering self-compatibility has already been successfully demonstrated in SI Petunia infiata (Lee et al., 1994) and other species of the Solanaceae. To date just a handful of S-RNase sequences are available for potato species. The characterization of S-RNases in targeted diploid Solanum species was an initial requirement for our approach, To develop the tools, S-alleles have initially been characterized in both Petunia infiata and p, hybrida cv Mitchell both phenotypically (by pollination tests using a diallel cross) and/or genotypically (by RT-PCR). This approach was then transferred to three diploid potato species, specifically accessions of Solanum stenotomum, Solanum phureja and Solanum okadae. These wild species are important sources of new traits studied by The James Hutton Institute (formally the Scottish Crop Research Institute). The approach taken to amplify partial S-RNase sequences from pistil RNA was RT-PCR using a degenerate primer. PCR products were cloned using a TA vector (Invitrogen) and sequenced . For two alleles full length sequences were obtained by 5'RACE . Database searches with t hese sequences, revealed sixteen S-RNases several of which are novel. Phylogenetic analysis was carried out with the cloned S-RNases together with selected published S-RNase and S- like RNase sequences of solanaceous species. The S-RNases revealed extensive trans-generic evolution and are clearly distinct from and distantly related to S-like RNases. For two alleles (501 and 502), S-RNase gene expression profiling was performed to check the developmental expression of the S-RNase gene, t issue-specific expression and also test whether t hese S-RNases (e.g. Sor and SorRNases) are expressed at a similar level. Wide variation in S-RNase gene expression levels have been reported in the literature. An RNAi construct has been designed to down-regulate two speCific 5- RNases in an SOl/S02 heterozygote of S. okadae. To increase the chance of silencing, the RNAi construct has been designed to use a chimeric 5- RNase gene involving the 5' end of the SorRNase and the 3' end of the SoyRNase. The correct chimeric S-RNase construct (Sol/SorRNase) has now been identified and inserted into an RNAi vector (pHellsgate8) using Gateway~ technology. This RNAi construct (pHG8-S01 / So.2) is now a valuable resource for use in S-RNase gene silencing in potato leading to the development of self-compatible diploid potato lines and ultimately the development of the first inbred lines of S. okadae.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

    Genetic manipulation of self-incompatibility in diploid potato species

    Get PDF
    Many of the wild and some cultivated species of potato are true diploids and are therefore more amenable for genetic studies than the majority of tetra ploid cultivars. However, the use of these diploid Solanum species is complicated by almost universal self-incompatibility (51). In Solanum, 51 is gametophytic and pistil specificity is controlled by a polymorphic ribonuclease (S-RNase), as found in other members of the Solanaceae. The genetic engineering of self-compatible (SC) diploid potato lines would benefit potato breeding in genera l and allow inbred lines to be established for the first time. This would facilitate genetic analyses including that of complex traits such as drought resistance or yield. The aim of this thesis is to downregulate the expression of S-RNases in diploid potatoes using the RNAi technique and established procedures for Agrobacterium-mediated transformation. This approach to engineering self-compatibility has already been successfully demonstrated in SI Petunia infiata (Lee et al., 1994) and other species of the Solanaceae. To date just a handful of S-RNase sequences are available for potato species. The characterization of S-RNases in targeted diploid Solanum species was an initial requirement for our approach, To develop the tools, S-alleles have initially been characterized in both Petunia infiata and p, hybrida cv Mitchell both phenotypically (by pollination tests using a diallel cross) and/or genotypically (by RT-PCR). This approach was then transferred to three diploid potato species, specifically accessions of Solanum stenotomum, Solanum phureja and Solanum okadae. These wild species are important sources of new traits studied by The James Hutton Institute (formally the Scottish Crop Research Institute). The approach taken to amplify partial S-RNase sequences from pistil RNA was RT-PCR using a degenerate primer. PCR products were cloned using a TA vector (Invitrogen) and sequenced . For two alleles full length sequences were obtained by 5'RACE . Database searches with t hese sequences, revealed sixteen S-RNases several of which are novel. Phylogenetic analysis was carried out with the cloned S-RNases together with selected published S-RNase and S- like RNase sequences of solanaceous species. The S-RNases revealed extensive trans-generic evolution and are clearly distinct from and distantly related to S-like RNases. For two alleles (501 and 502), S-RNase gene expression profiling was performed to check the developmental expression of the S-RNase gene, t issue-specific expression and also test whether t hese S-RNases (e.g. Sor and SorRNases) are expressed at a similar level. Wide variation in S-RNase gene expression levels have been reported in the literature. An RNAi construct has been designed to down-regulate two speCific 5- RNases in an SOl/S02 heterozygote of S. okadae. To increase the chance of silencing, the RNAi construct has been designed to use a chimeric 5- RNase gene involving the 5' end of the SorRNase and the 3' end of the SoyRNase. The correct chimeric S-RNase construct (Sol/SorRNase) has now been identified and inserted into an RNAi vector (pHellsgate8) using Gateway~ technology. This RNAi construct (pHG8-S01 / So.2) is now a valuable resource for use in S-RNase gene silencing in potato leading to the development of self-compatible diploid potato lines and ultimately the development of the first inbred lines of S. okadae.EThOS - Electronic Theses Online ServiceGBUnited Kingdo
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