This document discusses cisgenesis and intragenesis, which involve genetically modifying a crop plant using genes isolated from a crossable donor plant or from the same plant species, respectively. It defines cisgenic and intragenic plants and outlines their similarities and differences. It describes the prerequisites and various methods for constructing intragenic vectors and producing marker-free cisgenic/intragenic plants. The document presents several case studies demonstrating the development and evaluation of cisgenic plants with improved disease resistance. It discusses regulations around cisgenic/intragenic crops in different countries and potential benefits compared to transgenic and conventional breeding approaches.
it cover almost all content in cis/intragesis, right from introduction definition, explanation, production of marker free transgenic, intragenic vector construction, regulatory guide lines, current and future status, limitation, advantage over existing technique, swot analysis etc
its very useful for your seminar and presentations. it contain lot of picture, table, figure for your easy understanding
thank you
Mahesh
This document presents a seminar on cisgenesis and intragenesis as new tools in crop improvement. It begins with introductions to cisgenesis and intragenesis, noting they allow for the introduction of isolated genes from crossable species or the crop itself. It then discusses why cisgenesis/intragenesis are important alternatives to issues with transgenesis, traditional breeding, and translocation breeding. Methods for developing cisgenic/intragenic plants including vector design and transformation techniques are covered. Examples of crops modified with cisgenesis/intragenesis including late blight resistant potato and apple scab resistant apple are provided. A case study on stacking two late blight resistance genes in potato cisgenically is also summarized
The use of the term cisgenesis is an attempt to distinguish GM plants or other organisms produced in this way from transgenics that is GM plants that contain DNA from unrelated organisms. Schouten et al. (2006) introduced the term cisgenesis and defined cisgenesis as the modification in the genetic background of a recipient plant by a naturally derived gene from a cross compatible species including its introns and its native promoter and terminator flanked in the normal sense orientation. Since cisgenes shared a common gene pool available for traditional breeding the final cisgenic plant should be devoid of any kind of foreign DNA viz., selection markers and vector- backbone sequences. Sometimes the word cisgenesis is also referred to as Agrobacterium-mediated gene transfer from a sexually compatible plant where only the T-DNA borders may be present in the recipient organism after transformation (EFSA, 2012). The cisgenesis precludes linkage drag, and hence, prevents hazards from unidentified hitch hiking genes (Schouten, and Jacobsen, 2008). Compared to transgenesis, one of the disadvantages shared by cisgenesis is that characters outside the sexually compatible gene pool cannot be introduced. Furthermore, development of cisgenic crops involves extraordinary proficiency and time compared to transgenic crops. Therefore, the required genes or fragments of genes may not be readily accessible but have to be isolated from the sexually compatible gene pool (Holme et al., 2013).
On 16 February 2012, European Food Safety Authority (EFSA, 2012) reported the detail study concerning the safety aspects of cisgenic plants and validated that cisgenic plants are secure to be used in terms of environment, food and feed, similar to the traditionally bred plants. However, the present GMO regulation keeps the cisgenic micro-organisms out from its supervision. The first scientific statement of bringing forth a true plant obtained by cisgenic approach was reported in apple through the insertion of the internal scab resistance gene HcrVf2 influenced by their own regulatory genes into the cultivar Gala, a scab susceptible cultivar (Vanblaere et al., 2011). Barley with improved phytase activity was produced successfully by Holme et al. 2011, through cisgenic approach. Late blight resistant potatoes have developed by cisgene stacking of R- gene (jo et al., 2014).
The document describes the development of a QTL map for Egyptian durum wheat using an F2 mapping population derived from a cross between two parental varieties, Baniswif-1 and Souhag-2. A variety of DNA markers including SSRs, RAPDs, AFLPs, ESTs and SCoTs were used to genotype the mapping population. Traits related to yield and drought tolerance such as root length, plant height, spike characteristics, and biomass were measured. Linkage analysis was performed to construct a genetic linkage map, which was then used to detect QTLs associated with the traits of interest.
This document discusses speed breeding, a technique to accelerate crop breeding cycles. Traditional breeding can take many years to develop new varieties while meeting future food demands poses challenges. Speed breeding uses controlled environmental conditions like extended photoperiod and supplemental lighting to complete multiple generations in a year. Case studies show this approach led wheat and barley to flower in half the time and generated 5 soybean generations per year. Speed breeding holds potential to rapidly develop climate-resilient varieties on a smaller scale while combining with genomics and other innovations.
Molecular Markers, their application in crop improvementMrinali Mandape
Molecular markers such as SNPs, SSRs, RAPDs, AFLPs, and RFLPs can be used for crop improvement through applications like marker-assisted selection, linkage mapping, and trait-based selection. Molecular markers are DNA sequences that can identify specific locations in the genome and are linked to important agronomic traits. They are useful because they are selectively neutral, co-segregate with traits of interest, and follow Mendelian inheritance patterns.
This document discusses allele mining as a technique for improving crops. It defines allele mining as identifying allelic variation within genetic resources collections to find superior alleles. There are two main approaches - TILLING based allele mining which uses mutagenized populations and enzymatic cleavage to find mutations, and sequencing-based allele mining which uses PCR and sequencing to identify natural variation. Both have benefits and limitations. Applications of allele mining include finding alleles for resistance, abiotic stress tolerance, and improved yield and quality. Overall, allele mining is a promising approach for utilizing genetic resources to discover variants that can aid crop breeding.
Diversity array technology (DArT) is a high-throughput marker system that does not require sequence information. DArT arrays have been developed for chickpea, pigeonpea, and groundnut comprising 15,360 clones each. DArT markers showed 35% and 9% polymorphism in chickpea and pigeonpea mapping populations, but are not cost-effective for detecting variation in cultivated germplasm. DArT may be useful for introgressing segments from wild species into elite varieties, as seen with introgressing resistance genes from C. platycarpus into pigeonpea. Next-generation sequencing has also been used to develop SSR markers for trait mapping in these
it cover almost all content in cis/intragesis, right from introduction definition, explanation, production of marker free transgenic, intragenic vector construction, regulatory guide lines, current and future status, limitation, advantage over existing technique, swot analysis etc
its very useful for your seminar and presentations. it contain lot of picture, table, figure for your easy understanding
thank you
Mahesh
This document presents a seminar on cisgenesis and intragenesis as new tools in crop improvement. It begins with introductions to cisgenesis and intragenesis, noting they allow for the introduction of isolated genes from crossable species or the crop itself. It then discusses why cisgenesis/intragenesis are important alternatives to issues with transgenesis, traditional breeding, and translocation breeding. Methods for developing cisgenic/intragenic plants including vector design and transformation techniques are covered. Examples of crops modified with cisgenesis/intragenesis including late blight resistant potato and apple scab resistant apple are provided. A case study on stacking two late blight resistance genes in potato cisgenically is also summarized
The use of the term cisgenesis is an attempt to distinguish GM plants or other organisms produced in this way from transgenics that is GM plants that contain DNA from unrelated organisms. Schouten et al. (2006) introduced the term cisgenesis and defined cisgenesis as the modification in the genetic background of a recipient plant by a naturally derived gene from a cross compatible species including its introns and its native promoter and terminator flanked in the normal sense orientation. Since cisgenes shared a common gene pool available for traditional breeding the final cisgenic plant should be devoid of any kind of foreign DNA viz., selection markers and vector- backbone sequences. Sometimes the word cisgenesis is also referred to as Agrobacterium-mediated gene transfer from a sexually compatible plant where only the T-DNA borders may be present in the recipient organism after transformation (EFSA, 2012). The cisgenesis precludes linkage drag, and hence, prevents hazards from unidentified hitch hiking genes (Schouten, and Jacobsen, 2008). Compared to transgenesis, one of the disadvantages shared by cisgenesis is that characters outside the sexually compatible gene pool cannot be introduced. Furthermore, development of cisgenic crops involves extraordinary proficiency and time compared to transgenic crops. Therefore, the required genes or fragments of genes may not be readily accessible but have to be isolated from the sexually compatible gene pool (Holme et al., 2013).
On 16 February 2012, European Food Safety Authority (EFSA, 2012) reported the detail study concerning the safety aspects of cisgenic plants and validated that cisgenic plants are secure to be used in terms of environment, food and feed, similar to the traditionally bred plants. However, the present GMO regulation keeps the cisgenic micro-organisms out from its supervision. The first scientific statement of bringing forth a true plant obtained by cisgenic approach was reported in apple through the insertion of the internal scab resistance gene HcrVf2 influenced by their own regulatory genes into the cultivar Gala, a scab susceptible cultivar (Vanblaere et al., 2011). Barley with improved phytase activity was produced successfully by Holme et al. 2011, through cisgenic approach. Late blight resistant potatoes have developed by cisgene stacking of R- gene (jo et al., 2014).
The document describes the development of a QTL map for Egyptian durum wheat using an F2 mapping population derived from a cross between two parental varieties, Baniswif-1 and Souhag-2. A variety of DNA markers including SSRs, RAPDs, AFLPs, ESTs and SCoTs were used to genotype the mapping population. Traits related to yield and drought tolerance such as root length, plant height, spike characteristics, and biomass were measured. Linkage analysis was performed to construct a genetic linkage map, which was then used to detect QTLs associated with the traits of interest.
This document discusses speed breeding, a technique to accelerate crop breeding cycles. Traditional breeding can take many years to develop new varieties while meeting future food demands poses challenges. Speed breeding uses controlled environmental conditions like extended photoperiod and supplemental lighting to complete multiple generations in a year. Case studies show this approach led wheat and barley to flower in half the time and generated 5 soybean generations per year. Speed breeding holds potential to rapidly develop climate-resilient varieties on a smaller scale while combining with genomics and other innovations.
Molecular Markers, their application in crop improvementMrinali Mandape
Molecular markers such as SNPs, SSRs, RAPDs, AFLPs, and RFLPs can be used for crop improvement through applications like marker-assisted selection, linkage mapping, and trait-based selection. Molecular markers are DNA sequences that can identify specific locations in the genome and are linked to important agronomic traits. They are useful because they are selectively neutral, co-segregate with traits of interest, and follow Mendelian inheritance patterns.
This document discusses allele mining as a technique for improving crops. It defines allele mining as identifying allelic variation within genetic resources collections to find superior alleles. There are two main approaches - TILLING based allele mining which uses mutagenized populations and enzymatic cleavage to find mutations, and sequencing-based allele mining which uses PCR and sequencing to identify natural variation. Both have benefits and limitations. Applications of allele mining include finding alleles for resistance, abiotic stress tolerance, and improved yield and quality. Overall, allele mining is a promising approach for utilizing genetic resources to discover variants that can aid crop breeding.
Diversity array technology (DArT) is a high-throughput marker system that does not require sequence information. DArT arrays have been developed for chickpea, pigeonpea, and groundnut comprising 15,360 clones each. DArT markers showed 35% and 9% polymorphism in chickpea and pigeonpea mapping populations, but are not cost-effective for detecting variation in cultivated germplasm. DArT may be useful for introgressing segments from wild species into elite varieties, as seen with introgressing resistance genes from C. platycarpus into pigeonpea. Next-generation sequencing has also been used to develop SSR markers for trait mapping in these
This document discusses marker-assisted backcrossing (MAB) for introgressing traits from a donor parent into a recipient line. MAB uses DNA markers linked to target genes/QTLs to aid in selection. Markers can be used for foreground selection of target genes, background selection to recover the recipient genome, and recombinant selection to minimize linkage drag. A case study is described where MAB was used over multiple generations to introgress 5 drought resistance QTLs from a donor rice variety into a recipient variety. Through foreground, background, and recombinant selection using DNA markers, lines were developed with the target QTLs and most of the recipient genetic background.
A new era of genomics for plant science research has opened due the complete genome sequencing projects of Arabidopsis thaliana and rice. The sequence information available in public database has highlighted the need to develop genome scale reverse genetic strategies for functional analysis (Till et al., 2003). As most of the phenotypes are obscure, the forward genetics can hardly meet the demand of a high throughput and large-scale survey of gene functions. Targeting Induced Local Lesions in Genome TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identity point mutations in regions of interest (McCallum et al., 2000). This strategy works with a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms in genes of interest. A newly developed general reverse genetic strategy helps to locate an allelic series of induced point mutations in genes of interest. It allows the rapid and inexpensive detection of induced point mutations in populations of physically or chemically mutagenized individuals. To create an induced population with the use of physical/chemical mutagens is the first prerequisite for TILLING approach. Most of the plant species are compatible with this technique due to their self-fertilized nature and the seeds produced by these plants can be stored for long periods of time (Borevitz et al., 2003). The seeds are treated with mutagens and raised to harvest M1 plants, which are consequently, self-fertilized to raise the M2 population. DNA extracted from M2 plants is used in mutational screening (Colbert et al., 2001). To avoid mixing of the same mutation only one M2 plant from each M1 is used for DNA extraction (Till et al., 2007). The M3 seeds produce by selfing the M2 progeny can be well preserved for long term storage. Ethyl methane sulfonate (EMS) has been extensively used as a chemical mutagen in TILLING studies in plants to generate mutant populations, although other mutagens can be effective. EMS produces transitional mutations (G/C, A/T) by alkylating G residues which pairs with T instead of the conservative base pairing with C (Nagy et al., 2003). It is a constructive approach for users to attempt a range of chemical mutagens to assess the lethality and sterility on germinal tissue before creating large mutant populations.
This document discusses genome editing in fruit crops using CRISPR/Cas9 technology. It provides examples of using CRISPR to edit genes involved in fruit ripening, pigmentation, and flowering time regulation in strawberry, banana, apple, and kiwifruit. Specifically, it describes using CRISPR to increase beta-carotene levels in banana, induce early flowering in apple and pear, and generate dwarf kiwifruit plants. The document concludes that integrating biotechnology like CRISPR with conventional breeding is a promising strategy for fruit crop improvement.
Introduction:
Proposed by Meuwissen et al. (2001)
GS is a specialized form of MAS, in which information from genotype data on marker alleles covering the entire genome forms the basis of selection.
The effects associated with all the marker loci, irrespective of whether the effects are significant or not, covering the entire genome are estimated.
The marker effect estimates are used to calculate the genomic estimated breeding values (GEBVs) of different individuals/lines, which form the basis of selection.
Why to go for genomic selection:
Marker-assisted selection (MAS) is well-suited for handling oligogenes and quantitative trait loci (QTLs) with large effects but not for minor QTLs.
MARS attempts to take into account small effect QTLs by combining trait phenotype data with marker genotype data into a combined selection index.
Based on markers showing significant association with the trait(s) and for this reason has been criticized as inefficient
The genomic selection (GS) scheme was to rectify the deficiency of MAS and MARS schemes. The GS scheme utilizes information from genome-wide marker data whether or not their associations with the concerned trait(s) are significant.
GEBV: GenomicEstimated Breeding Values-
The sum total of effects associated with all the marker alleles present in the individual and included in the GS model applied to the population under selection
Calculated on a single individual basis
Gene-assisted genomic selection:
A GS model that uses information about prior known QTLs, the targeted QTLs were accumulated in much higher frequencies than when the standard ridge regression was used
The sum total of effects associated with all the marker alleles present in the individual and included in the GS model applied to the population under selection
Calculated on a single individual basis
Population used:
Training population: used for training of the GS model and for obtaining estimates of the marker-associated effects needed for estimation of GEBVs of individuals/lines in the breeding population.
Breeding population: the population subjected to GS for achieving the desired improvement and isolation of superior lines for use as new varieties/parents of new improved hybrids.
Training population-
large enough: must be representative of the breeding population: max. trait variance with marker : by cluster analysis
should have either equal or comparable LD, LD decay rates with breeding populations
Updated by including individuals/lines from the breeding population
Training more than one generation
Low colinearity between markers is needed since high colinearity tends to reduce prediction accuracy of certain GS models. (colinearity disturbed by recombination)
Gene introgression from wild relatives to cultivated plantsManjappa Ganiger
This document summarizes a seminar on using crop wild relatives to introduce beneficial genes into cultivated crops. It discusses how crop wild relatives contain genetic diversity that can provide traits like pest and disease resistance, abiotic stress tolerance, and improved yields. Specific examples are given of introducing disease resistance genes from wild relatives into tomatoes and rust resistance genes into wheat. The use of wild rice species to develop rice varieties with improved resistance to various diseases and insects is also described.
Association genetics‟ or ‟association studies,” or ‟linkage disequilibrium mapping”.
Tool to resolve complex trait variation down to the sequence level by exploiting historical and evolutionary recombination events at the population level.
Natural population surveyed to determine MTA using LD.
Advances in Vegetable Improvement through Biotechnological ApproachAditika Sharma
The document discusses various biotechnological approaches that can be used to improve vegetables, including genetic engineering, molecular markers, and tissue culture techniques. It provides examples of how transgenic crops have been developed with traits like virus resistance, herbicide tolerance, and improved nutrition. Molecular markers can be used for marker-assisted selection, genetic mapping, and introgressing traits from wild relatives. The global adoption of biotech crops is also summarized. Genome sequencing of various crops is helping with marker development and gene discovery.
Genome-wide association mapping identifies genomic regions associated with phenotypes by analyzing phenotypic and genotypic data. Phenotypic data includes traits like flowering time and yield, while genotypic data consists of genetic markers spanning the genome. Single nucleotide polymorphisms (SNPs) are commonly used markers. Association mapping fits statistical models to test for association between each SNP and the phenotype. Accounting for population structure and relatedness through mixed models reduces false positives. Significant associations between SNPs and traits suggest the SNP directly affects the trait or is linked to a causal variant. Results are visualized through Manhattan plots and QQ-plots.
This document discusses genomic selection in plants. It begins with an introduction to genomic selection and its history. Genomic selection uses dense genetic markers and phenotypic data from a reference population to develop prediction equations that can then be applied to other populations to estimate genomic breeding values without additional phenotyping. The document outlines the steps involved, including preparing phenotypic and genotypic data, constructing prediction models, fitting and evaluating models, and applying genomic selection in breeding programs. It provides examples of software used and factors that affect prediction accuracy. The document concludes with two case studies, one on genomic selection for hybrid rice and another on genomic selection to improve wheat grain quality.
Speed Breeding and its implications in crop improvementANILKUMARDASH2
Introduction
History of speed breeding
Methods of speed breeding
Advantages over conventional breeding
Integration with various technologies
Case studies
Opportunities and challenges
Conclusions
Genomics, proteomics and metabolomics are the three core omics technologies, which respectively deal with the analysis of genome, proteome and metabolome of cells and tissues of an organism.
This document discusses different types of mapping populations that can be used for genetic mapping and molecular breeding programs. It describes F2, F2:F3, backcross, doubled haploid (DH), recombinant inbred line (RIL), and near-isogenic line (NIL) populations. For each type, it provides details on how they are developed, their characteristics, and merits and demerits relative to mapping objectives. The document emphasizes that the choice of mapping population depends on the research goals, availability of markers, and existing molecular maps. RILs and DHs allow for replication over environments but require more time and resources to develop.
The document discusses gene stacking in crop plants. It begins by explaining the importance of genetic variation and how plant breeders take advantage of genetic variants to improve crops. It then discusses various sources of genetic variability that can be used for transgenic development, including local germplasms, obsolete varieties, wild species, and interspecies or intergenera crosses. The document goes on to define gene stacking as combining two or more genes of interest in the host plant genome. It provides examples of early gene stacked crops and discusses different strategies for achieving gene stacking, including iterative procedures, re-transformation, and co-transformation. It also covers polycistronic transgenes, polyprotein expression systems, and selection methods for gene stacked crops.
The document discusses MAGIC (Multi-parent Advanced Generation Inter-Cross) populations, which are created by intercrossing multiple parent lines over several generations. This increases recombination and genetic diversity. Key points:
- MAGIC populations allow more precise mapping of QTLs controlling quantitative traits compared to biparental populations.
- Two case studies describe the development of MAGIC populations in rice with 8 founders each, and tomato with 8 founders. Traits like yield, disease resistance, and abiotic stress tolerance were evaluated.
- Advantages include exploiting more genetic variation, developing varieties with favorable trait combinations, and more accurate gene mapping. Limitations include requiring more time, resources for phenotyping and breeding.
This document discusses precision breeding techniques in plants, including their objectives, advantages, and challenges. It covers molecular marker-assisted selection, mutagenesis using chemicals or radiation, and newer gene editing tools like CRISPR/Cas9. CRISPR allows more precise genetic modifications than traditional techniques by targeting specific genes. However, increasing the efficiency of the homology directed repair pathway is still an area of research, as non-homologous end joining is the dominant repair pathway in plants. Overall, precision breeding holds promise for accelerating crop improvement but developing methods for high-efficiency homology directed repair remains important.
Reverse breeding is a novel plant breeding technique that allows the development of parental lines directly from any superior heterozygous plant. It involves suppressing meiotic recombination to produce gametes with whole parental chromosome sets, followed by doubling of haploids to generate parental lines. Two case studies demonstrate using RNAi to silence meiotic genes in Arabidopsis thaliana, producing parental lines that reconstitute the original hybrid when crossed. A second technique, marker-assisted reverse breeding, uses high-density SNP genotyping instead of gene silencing to select maize lines similar to original parents within one year. Reverse breeding techniques accelerate breeding and facilitate hybrid improvement without prior knowledge of parental lines.
Quantitative trait loci (QTL) analysis and its applications in plant breedingPGS
Abstract
Many agriculturally important traits such as grain yield, protein content and relative disease resistance are controlled by many genes and are known as quantitative traits (also polygenic or complex traits). A quantitative trait depends on the cumulative actions of many genes and the environment. The genomic regions that contain genes associated with a quantitative trait are known as quantitative trait loci (QTLs). Thus, a QTL could be defined as a genomic region responsible for a part of the observed phenotypic variation for a quantitative trait. A QTL can be a single gene or a cluster of linked genes that affect the trait. The effects of individual QTLs may differ from each other and change from environment to environment. The genetics of a quantitative trait can often be deduced from the statistical analysis of several segregating populations. Recently, by using molecular markers, it is feasible to analyze quantitative traits and identify individual QTLs or genes controlling the traits of interest in breeding programs.
The document discusses allele mining, which aims to identify allelic variations in genetic resources collections that are relevant for traits of interest. It describes how allele mining works to unlock hidden genetic variation by identifying single nucleotide polymorphisms and new haplotypes. The document then provides details on a case study of allele mining focused on three genes - calmodulin, LEA3, and SalT - important for abiotic stress tolerance in rice and related species. Primers were developed to amplify regions of these three genes from 64 accessions representing rice and other grasses.
This case study describes the development of cisgenic barley with improved phytase activity through Agrobacterium-mediated transformation. 72 transgenic T0 barley plants were produced by co-transforming with two vectors, one containing the HvPAPhy_a gene and the other a selectable marker. 19 plants were found to contain only the HvPAPhy_a insert through PCR analysis. Two of these plants, PAPhy05 and PAPhy07, showed increased phytase activity and a 3:1 segregation ratio in progeny, indicating single insertions of HvPAPhy_a without the selectable marker. This demonstrates the successful production and analysis of cisgenic barley
This document provides an overview of cisgenesis, which involves genetically modifying a plant with a natural gene from a sexually compatible plant species. It defines cisgenesis and provides examples of cisgenic crops. It discusses the limitations of traditional breeding and transgenesis, and how cisgenesis addresses some of these limitations. The document outlines the process for developing cisgenic plants, including gene isolation, vector construction, transformation and selection methods. It summarizes a case study on developing cisgenic apple with scab resistance. Finally, it discusses opportunities and challenges for the future of cisgenesis.
This document discusses marker-assisted backcrossing (MAB) for introgressing traits from a donor parent into a recipient line. MAB uses DNA markers linked to target genes/QTLs to aid in selection. Markers can be used for foreground selection of target genes, background selection to recover the recipient genome, and recombinant selection to minimize linkage drag. A case study is described where MAB was used over multiple generations to introgress 5 drought resistance QTLs from a donor rice variety into a recipient variety. Through foreground, background, and recombinant selection using DNA markers, lines were developed with the target QTLs and most of the recipient genetic background.
A new era of genomics for plant science research has opened due the complete genome sequencing projects of Arabidopsis thaliana and rice. The sequence information available in public database has highlighted the need to develop genome scale reverse genetic strategies for functional analysis (Till et al., 2003). As most of the phenotypes are obscure, the forward genetics can hardly meet the demand of a high throughput and large-scale survey of gene functions. Targeting Induced Local Lesions in Genome TILLING is a general reverse genetic technique that combines chemical mutagenesis with PCR based screening to identity point mutations in regions of interest (McCallum et al., 2000). This strategy works with a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms in genes of interest. A newly developed general reverse genetic strategy helps to locate an allelic series of induced point mutations in genes of interest. It allows the rapid and inexpensive detection of induced point mutations in populations of physically or chemically mutagenized individuals. To create an induced population with the use of physical/chemical mutagens is the first prerequisite for TILLING approach. Most of the plant species are compatible with this technique due to their self-fertilized nature and the seeds produced by these plants can be stored for long periods of time (Borevitz et al., 2003). The seeds are treated with mutagens and raised to harvest M1 plants, which are consequently, self-fertilized to raise the M2 population. DNA extracted from M2 plants is used in mutational screening (Colbert et al., 2001). To avoid mixing of the same mutation only one M2 plant from each M1 is used for DNA extraction (Till et al., 2007). The M3 seeds produce by selfing the M2 progeny can be well preserved for long term storage. Ethyl methane sulfonate (EMS) has been extensively used as a chemical mutagen in TILLING studies in plants to generate mutant populations, although other mutagens can be effective. EMS produces transitional mutations (G/C, A/T) by alkylating G residues which pairs with T instead of the conservative base pairing with C (Nagy et al., 2003). It is a constructive approach for users to attempt a range of chemical mutagens to assess the lethality and sterility on germinal tissue before creating large mutant populations.
This document discusses genome editing in fruit crops using CRISPR/Cas9 technology. It provides examples of using CRISPR to edit genes involved in fruit ripening, pigmentation, and flowering time regulation in strawberry, banana, apple, and kiwifruit. Specifically, it describes using CRISPR to increase beta-carotene levels in banana, induce early flowering in apple and pear, and generate dwarf kiwifruit plants. The document concludes that integrating biotechnology like CRISPR with conventional breeding is a promising strategy for fruit crop improvement.
Introduction:
Proposed by Meuwissen et al. (2001)
GS is a specialized form of MAS, in which information from genotype data on marker alleles covering the entire genome forms the basis of selection.
The effects associated with all the marker loci, irrespective of whether the effects are significant or not, covering the entire genome are estimated.
The marker effect estimates are used to calculate the genomic estimated breeding values (GEBVs) of different individuals/lines, which form the basis of selection.
Why to go for genomic selection:
Marker-assisted selection (MAS) is well-suited for handling oligogenes and quantitative trait loci (QTLs) with large effects but not for minor QTLs.
MARS attempts to take into account small effect QTLs by combining trait phenotype data with marker genotype data into a combined selection index.
Based on markers showing significant association with the trait(s) and for this reason has been criticized as inefficient
The genomic selection (GS) scheme was to rectify the deficiency of MAS and MARS schemes. The GS scheme utilizes information from genome-wide marker data whether or not their associations with the concerned trait(s) are significant.
GEBV: GenomicEstimated Breeding Values-
The sum total of effects associated with all the marker alleles present in the individual and included in the GS model applied to the population under selection
Calculated on a single individual basis
Gene-assisted genomic selection:
A GS model that uses information about prior known QTLs, the targeted QTLs were accumulated in much higher frequencies than when the standard ridge regression was used
The sum total of effects associated with all the marker alleles present in the individual and included in the GS model applied to the population under selection
Calculated on a single individual basis
Population used:
Training population: used for training of the GS model and for obtaining estimates of the marker-associated effects needed for estimation of GEBVs of individuals/lines in the breeding population.
Breeding population: the population subjected to GS for achieving the desired improvement and isolation of superior lines for use as new varieties/parents of new improved hybrids.
Training population-
large enough: must be representative of the breeding population: max. trait variance with marker : by cluster analysis
should have either equal or comparable LD, LD decay rates with breeding populations
Updated by including individuals/lines from the breeding population
Training more than one generation
Low colinearity between markers is needed since high colinearity tends to reduce prediction accuracy of certain GS models. (colinearity disturbed by recombination)
Gene introgression from wild relatives to cultivated plantsManjappa Ganiger
This document summarizes a seminar on using crop wild relatives to introduce beneficial genes into cultivated crops. It discusses how crop wild relatives contain genetic diversity that can provide traits like pest and disease resistance, abiotic stress tolerance, and improved yields. Specific examples are given of introducing disease resistance genes from wild relatives into tomatoes and rust resistance genes into wheat. The use of wild rice species to develop rice varieties with improved resistance to various diseases and insects is also described.
Association genetics‟ or ‟association studies,” or ‟linkage disequilibrium mapping”.
Tool to resolve complex trait variation down to the sequence level by exploiting historical and evolutionary recombination events at the population level.
Natural population surveyed to determine MTA using LD.
Advances in Vegetable Improvement through Biotechnological ApproachAditika Sharma
The document discusses various biotechnological approaches that can be used to improve vegetables, including genetic engineering, molecular markers, and tissue culture techniques. It provides examples of how transgenic crops have been developed with traits like virus resistance, herbicide tolerance, and improved nutrition. Molecular markers can be used for marker-assisted selection, genetic mapping, and introgressing traits from wild relatives. The global adoption of biotech crops is also summarized. Genome sequencing of various crops is helping with marker development and gene discovery.
Genome-wide association mapping identifies genomic regions associated with phenotypes by analyzing phenotypic and genotypic data. Phenotypic data includes traits like flowering time and yield, while genotypic data consists of genetic markers spanning the genome. Single nucleotide polymorphisms (SNPs) are commonly used markers. Association mapping fits statistical models to test for association between each SNP and the phenotype. Accounting for population structure and relatedness through mixed models reduces false positives. Significant associations between SNPs and traits suggest the SNP directly affects the trait or is linked to a causal variant. Results are visualized through Manhattan plots and QQ-plots.
This document discusses genomic selection in plants. It begins with an introduction to genomic selection and its history. Genomic selection uses dense genetic markers and phenotypic data from a reference population to develop prediction equations that can then be applied to other populations to estimate genomic breeding values without additional phenotyping. The document outlines the steps involved, including preparing phenotypic and genotypic data, constructing prediction models, fitting and evaluating models, and applying genomic selection in breeding programs. It provides examples of software used and factors that affect prediction accuracy. The document concludes with two case studies, one on genomic selection for hybrid rice and another on genomic selection to improve wheat grain quality.
Speed Breeding and its implications in crop improvementANILKUMARDASH2
Introduction
History of speed breeding
Methods of speed breeding
Advantages over conventional breeding
Integration with various technologies
Case studies
Opportunities and challenges
Conclusions
Genomics, proteomics and metabolomics are the three core omics technologies, which respectively deal with the analysis of genome, proteome and metabolome of cells and tissues of an organism.
This document discusses different types of mapping populations that can be used for genetic mapping and molecular breeding programs. It describes F2, F2:F3, backcross, doubled haploid (DH), recombinant inbred line (RIL), and near-isogenic line (NIL) populations. For each type, it provides details on how they are developed, their characteristics, and merits and demerits relative to mapping objectives. The document emphasizes that the choice of mapping population depends on the research goals, availability of markers, and existing molecular maps. RILs and DHs allow for replication over environments but require more time and resources to develop.
The document discusses gene stacking in crop plants. It begins by explaining the importance of genetic variation and how plant breeders take advantage of genetic variants to improve crops. It then discusses various sources of genetic variability that can be used for transgenic development, including local germplasms, obsolete varieties, wild species, and interspecies or intergenera crosses. The document goes on to define gene stacking as combining two or more genes of interest in the host plant genome. It provides examples of early gene stacked crops and discusses different strategies for achieving gene stacking, including iterative procedures, re-transformation, and co-transformation. It also covers polycistronic transgenes, polyprotein expression systems, and selection methods for gene stacked crops.
The document discusses MAGIC (Multi-parent Advanced Generation Inter-Cross) populations, which are created by intercrossing multiple parent lines over several generations. This increases recombination and genetic diversity. Key points:
- MAGIC populations allow more precise mapping of QTLs controlling quantitative traits compared to biparental populations.
- Two case studies describe the development of MAGIC populations in rice with 8 founders each, and tomato with 8 founders. Traits like yield, disease resistance, and abiotic stress tolerance were evaluated.
- Advantages include exploiting more genetic variation, developing varieties with favorable trait combinations, and more accurate gene mapping. Limitations include requiring more time, resources for phenotyping and breeding.
This document discusses precision breeding techniques in plants, including their objectives, advantages, and challenges. It covers molecular marker-assisted selection, mutagenesis using chemicals or radiation, and newer gene editing tools like CRISPR/Cas9. CRISPR allows more precise genetic modifications than traditional techniques by targeting specific genes. However, increasing the efficiency of the homology directed repair pathway is still an area of research, as non-homologous end joining is the dominant repair pathway in plants. Overall, precision breeding holds promise for accelerating crop improvement but developing methods for high-efficiency homology directed repair remains important.
Reverse breeding is a novel plant breeding technique that allows the development of parental lines directly from any superior heterozygous plant. It involves suppressing meiotic recombination to produce gametes with whole parental chromosome sets, followed by doubling of haploids to generate parental lines. Two case studies demonstrate using RNAi to silence meiotic genes in Arabidopsis thaliana, producing parental lines that reconstitute the original hybrid when crossed. A second technique, marker-assisted reverse breeding, uses high-density SNP genotyping instead of gene silencing to select maize lines similar to original parents within one year. Reverse breeding techniques accelerate breeding and facilitate hybrid improvement without prior knowledge of parental lines.
Quantitative trait loci (QTL) analysis and its applications in plant breedingPGS
Abstract
Many agriculturally important traits such as grain yield, protein content and relative disease resistance are controlled by many genes and are known as quantitative traits (also polygenic or complex traits). A quantitative trait depends on the cumulative actions of many genes and the environment. The genomic regions that contain genes associated with a quantitative trait are known as quantitative trait loci (QTLs). Thus, a QTL could be defined as a genomic region responsible for a part of the observed phenotypic variation for a quantitative trait. A QTL can be a single gene or a cluster of linked genes that affect the trait. The effects of individual QTLs may differ from each other and change from environment to environment. The genetics of a quantitative trait can often be deduced from the statistical analysis of several segregating populations. Recently, by using molecular markers, it is feasible to analyze quantitative traits and identify individual QTLs or genes controlling the traits of interest in breeding programs.
The document discusses allele mining, which aims to identify allelic variations in genetic resources collections that are relevant for traits of interest. It describes how allele mining works to unlock hidden genetic variation by identifying single nucleotide polymorphisms and new haplotypes. The document then provides details on a case study of allele mining focused on three genes - calmodulin, LEA3, and SalT - important for abiotic stress tolerance in rice and related species. Primers were developed to amplify regions of these three genes from 64 accessions representing rice and other grasses.
This case study describes the development of cisgenic barley with improved phytase activity through Agrobacterium-mediated transformation. 72 transgenic T0 barley plants were produced by co-transforming with two vectors, one containing the HvPAPhy_a gene and the other a selectable marker. 19 plants were found to contain only the HvPAPhy_a insert through PCR analysis. Two of these plants, PAPhy05 and PAPhy07, showed increased phytase activity and a 3:1 segregation ratio in progeny, indicating single insertions of HvPAPhy_a without the selectable marker. This demonstrates the successful production and analysis of cisgenic barley
This document provides an overview of cisgenesis, which involves genetically modifying a plant with a natural gene from a sexually compatible plant species. It defines cisgenesis and provides examples of cisgenic crops. It discusses the limitations of traditional breeding and transgenesis, and how cisgenesis addresses some of these limitations. The document outlines the process for developing cisgenic plants, including gene isolation, vector construction, transformation and selection methods. It summarizes a case study on developing cisgenic apple with scab resistance. Finally, it discusses opportunities and challenges for the future of cisgenesis.
Chloroplast transformation allows for the integration of foreign genes into the chloroplast genome. This is beneficial as it provides high levels of transgene expression without epigenetic effects or position effects. Chloroplast transformation requires a chloroplast specific expression vector, a method for DNA delivery such as biolistics, and an efficient selection marker such as spectinomycin resistance. Successful transformation is confirmed by PCR and Southern blot analysis showing integration of the transgene into the chloroplast genome. Applications of chloroplast transformation include production of biopharmaceuticals, vaccines, industrial enzymes, and biomaterials as well as phytoremediation.
Marker free transgenic crops can be generated using several strategies to avoid issues with marker genes. Selectable marker genes allow selection of transformed cells but their products may be undesirable in food. Strategies include co-transformation using two plasmids without linking marker and trait genes, replacing selectable markers with screenable markers, and excising the selectable marker from the genome after selection using site-specific recombination, transposition, or homologous recombination. Case studies demonstrate applying these strategies successfully in tobacco and mustard to generate marker free transgenic plants.
Transgenic plants can be engineered for insect resistance by introducing genes from bacteria, plants or animals. This study generated transgenic broccoli expressing a Cry1Aa gene from Bacillus thuringiensis for resistance against diamondback moth. A second study transformed two cabbage varieties with a sporamin gene, conferring resistance against diamondback moth. Transgene insertion was confirmed, and resistant transgenic lines were selected and shown to inherit resistance stably to subsequent generations. Transgenic crops hold promise for reduced pesticide use and increased sustainable food production.
How transgenic plant is used in agricultural fieldFOODCROPS
The document summarizes the work of the Biotechnology Research Institute, CAAS. It has over 150 staff members working in departments focused on plant biotechnology and molecular biology and molecular microbiology. The institute has made notable achievements including developing insect-resistant Bt cotton and producing transgenic corn that expresses phytase to increase phosphorus availability. The document also provides an overview of plant biotechnology techniques, including defining biotechnology, cloning genes, and developing transgenic plants by introducing transformation cassettes containing genes of interest and selectable markers via Agrobacterium or particle bombardment.
The document presents on gene stacking, pathway engineering, and marker-free transgenic development strategies. It discusses various methods for combining multiple genes/traits into plants, including gene stacking, gene pyramiding, sexual hybridization, re-transformation, co-transformation, and marker-free techniques. The goal is to develop crops with improved agronomic traits through plant genome engineering approaches.
Crisper cas-9 is a powerful gene editing technology and helps to solve many problems in shorter time and more precisely. The two main molecules is an enzyme CAS-9 and a piece of RNA known as GUIDE RNA, that introduce a change into the DNA.
Advanced genetic tools for plant biotechnology muhammad shoaib
This document discusses advanced genetic tools that are being developed for plant biotechnology. It begins by outlining the need for new tools to address challenges in improving crop traits and developing biosensing abilities. Recent tools described include synthetic promoters and transcription factors for precise spatiotemporal gene expression control, and genome editing tools like CRISPR/Cas9 that allow for precise genetic modifications. Methods for assembling and transforming large DNA constructs, like entire pathways or synthetic chromosomes, are also discussed. Examples of applications around the world include engineering crops for improved biofuel production. Overall, the development of these new genetic tools is poised to greatly enhance the precision and capabilities of plant biotechnology.
This document summarizes a presentation on using CRISPR-Cas9 for crop improvement. It begins with an introduction to CRISPR-Cas9 and how it is used to edit genomes by removing, adding, or altering DNA sequences. It then discusses the mechanism of the CRISPR-Cas9 complex and how it creates breaks in DNA that are repaired. The document reviews several case studies where CRISPR was used to modify crops, including creating low-gluten wheat and improving rice. It finds that CRISPR can efficiently edit multiple genes simultaneously with few off-target effects. The conclusion states that CRISPR is revolutionizing agriculture by enabling the creation of higher yielding, more resistant crop varieties without transgenes.
CRISPR/Cas9 is a new genome editing tool that allows geneticists to precisely edit DNA sequences. It uses a bacterial immune system to cut DNA at a specific location so that parts of the genome can be removed, replaced, or added. The system involves a Cas9 enzyme guided by RNA to a targeted DNA site, where it cuts both strands of the DNA. This allows desired modifications to the genome. The summary discusses how CRISPR can be used to create genetic variability, develop disease resistance in crops, and potentially edit human organs for transplantation with less immune rejection risk. It also provides an example of using CRISPR to enhance blast resistance in rice.
Genetic Transformation of Maize: Conventional Methods and Precision Geno...Ananya Sinha
Genetic Transformation of Maize: Conventional Methods and Precision Genome Modification
This is a summary of the above chapter mentioned in "Biotechnology of Major Cereals".
The precision genome modification is defined majorly focused on comparing the conventional genome modification methods with the modern ones.
This document discusses clean gene technology for developing transgenic plants without selectable marker genes. It presents 5 methods for producing marker-free transgenic plants: 1) co-transformation, 2) site-specific recombination-mediated marker deletion using the Cre/loxP system, 3) transposon-based marker methods, 4) intrachromosomal recombination, and 5) removal of chloroplast marker genes using homologous recombination. Each method is described briefly along with their advantages and limitations. The document concludes with a list of references on clean gene technology and selectable marker genes.
Genome walking – a new strategy for identification of nucleotide sequence in ...Dr. Mukesh Chavan
Identification of unknown nucleotide sequences flanking already characterized DNA regions can be pursued by number of different PCR- based methods commonly known as Genome walking (GW)
GW methods have been developed in the last 20 years, with continuous improvements added to the first basic strategies
First reported by Hengen in 1995 in comparison with other technologies
Hui et al., in 1998 reviewed in detail
The extreme flexibility of GW strategies makes its application possible in every standardly equipped research laboratory. In addition, the possibility of merging GW strategies to next generation sequencing approaches will undoubtedly extend the future application of this by now basic technique of molecular biology.
Gene stacking and its materiality in crop improvementShamlyGupta
Gene stacking involves combining two or more transgenes into a host plant genome. It can be achieved through iterative crossing of transgenic plants, re-transformation of transgenic plants with additional genes, or co-transformation of multiple genes simultaneously. Co-transformation allows multiple genes to be introduced together but risks silencing effects if the same promoter is used. Iterative crossing is time-consuming but avoids this issue. Gene stacking holds promise for improving crop traits like disease resistance and nutrition but careful selection is needed to maintain expression levels of all genes. Recent examples demonstrate progress in stacking drought tolerance, yield, and nutrition genes into elite crop varieties.
Chloroplast transformation allows the integration of genes into the chloroplast genome. Genes integrated into chloroplasts are highly expressed due to high polyploidy of chloroplasts and proper folding of proteins. Genes integrated at the trnA/trnI site are most highly expressed. Selection of transformants is done using spectinomycin resistance encoded by the aadA gene. High-level expression of vaccines, biomaterials, and therapeutic proteins such as human serum albumin is achieved through chloroplast transformation, offering potential for low-cost production. Genes conferring herbicide and insect resistance have also been successfully expressed via the chloroplast genome.
This paper reviews recent advances in engineering Streptomyces bacteria for the production of medicines and bioproducts. Key approaches discussed include using genome editing tools and laboratory automation to rapidly engineer Streptomyces metabolism. The review highlights how integrating these methods within a design-build-test-learn framework is accelerating the discovery of new bioactive compounds and engineered Streptomyces cell factories. It provides an overview of this iterative engineering cycle and how each stage contributes to continual improvements in metabolic engineering outcomes for these industrially important bacteria.
TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics technique that uses chemical mutagenesis and screening to identify point mutations in genes of interest. It involves mutagenizing an organism's genome with chemicals like EMS, pooling DNA from mutagenized individuals, amplifying target genes via PCR, treating products with enzymes like CEL1 to detect mutations, and analyzing cleavage products on gels to find mutations. TILLING has been used to identify mutations in many crops to determine gene function and discover traits like disease resistance. It provides an efficient way to study gene function without transgenic approaches.
This document summarizes information about two herbicide resistance genes - bar and pat genes. The bar gene codes for the enzyme phosphinothricin acetyl transferase (PAT) which inactivates the herbicide phosphinothricin (PPT). Plants containing the bar gene are tolerant to PPT. The bar gene has been introduced into crops like rapeseed oil and maize. The document also discusses imidazolinone herbicides and tolerance achieved by mutating the acetolactate synthase gene in maize. Environmental impacts of herbicide tolerant crops include concerns about reducing biodiversity and development of herbicide resistant weeds.
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
Unlocking the mysteries of reproduction: Exploring fecundity and gonadosomati...AbdullaAlAsif1
The pygmy halfbeak Dermogenys colletei, is known for its viviparous nature, this presents an intriguing case of relatively low fecundity, raising questions about potential compensatory reproductive strategies employed by this species. Our study delves into the examination of fecundity and the Gonadosomatic Index (GSI) in the Pygmy Halfbeak, D. colletei (Meisner, 2001), an intriguing viviparous fish indigenous to Sarawak, Borneo. We hypothesize that the Pygmy halfbeak, D. colletei, may exhibit unique reproductive adaptations to offset its low fecundity, thus enhancing its survival and fitness. To address this, we conducted a comprehensive study utilizing 28 mature female specimens of D. colletei, carefully measuring fecundity and GSI to shed light on the reproductive adaptations of this species. Our findings reveal that D. colletei indeed exhibits low fecundity, with a mean of 16.76 ± 2.01, and a mean GSI of 12.83 ± 1.27, providing crucial insights into the reproductive mechanisms at play in this species. These results underscore the existence of unique reproductive strategies in D. colletei, enabling its adaptation and persistence in Borneo's diverse aquatic ecosystems, and call for further ecological research to elucidate these mechanisms. This study lends to a better understanding of viviparous fish in Borneo and contributes to the broader field of aquatic ecology, enhancing our knowledge of species adaptations to unique ecological challenges.
Immersive Learning That Works: Research Grounding and Paths ForwardLeonel Morgado
We will metaverse into the essence of immersive learning, into its three dimensions and conceptual models. This approach encompasses elements from teaching methodologies to social involvement, through organizational concerns and technologies. Challenging the perception of learning as knowledge transfer, we introduce a 'Uses, Practices & Strategies' model operationalized by the 'Immersive Learning Brain' and ‘Immersion Cube’ frameworks. This approach offers a comprehensive guide through the intricacies of immersive educational experiences and spotlighting research frontiers, along the immersion dimensions of system, narrative, and agency. Our discourse extends to stakeholders beyond the academic sphere, addressing the interests of technologists, instructional designers, and policymakers. We span various contexts, from formal education to organizational transformation to the new horizon of an AI-pervasive society. This keynote aims to unite the iLRN community in a collaborative journey towards a future where immersive learning research and practice coalesce, paving the way for innovative educational research and practice landscapes.
Current Ms word generated power point presentation covers major details about the micronuclei test. It's significance and assays to conduct it. It is used to detect the micronuclei formation inside the cells of nearly every multicellular organism. It's formation takes place during chromosomal sepration at metaphase.
ESR spectroscopy in liquid food and beverages.pptxPRIYANKA PATEL
With increasing population, people need to rely on packaged food stuffs. Packaging of food materials requires the preservation of food. There are various methods for the treatment of food to preserve them and irradiation treatment of food is one of them. It is the most common and the most harmless method for the food preservation as it does not alter the necessary micronutrients of food materials. Although irradiated food doesn’t cause any harm to the human health but still the quality assessment of food is required to provide consumers with necessary information about the food. ESR spectroscopy is the most sophisticated way to investigate the quality of the food and the free radicals induced during the processing of the food. ESR spin trapping technique is useful for the detection of highly unstable radicals in the food. The antioxidant capability of liquid food and beverages in mainly performed by spin trapping technique.
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
The binding of cosmological structures by massless topological defectsSérgio Sacani
Assuming spherical symmetry and weak field, it is shown that if one solves the Poisson equation or the Einstein field
equations sourced by a topological defect, i.e. a singularity of a very specific form, the result is a localized gravitational
field capable of driving flat rotation (i.e. Keplerian circular orbits at a constant speed for all radii) of test masses on a thin
spherical shell without any underlying mass. Moreover, a large-scale structure which exploits this solution by assembling
concentrically a number of such topological defects can establish a flat stellar or galactic rotation curve, and can also deflect
light in the same manner as an equipotential (isothermal) sphere. Thus, the need for dark matter or modified gravity theory is
mitigated, at least in part.
Remote Sensing and Computational, Evolutionary, Supercomputing, and Intellige...University of Maribor
Slides from talk:
Aleš Zamuda: Remote Sensing and Computational, Evolutionary, Supercomputing, and Intelligent Systems.
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Inter-Society Networking Panel GRSS/MTT-S/CIS Panel Session: Promoting Connection and Cooperation
https://www.etran.rs/2024/en/home-english/
hematic appreciation test is a psychological assessment tool used to measure an individual's appreciation and understanding of specific themes or topics. This test helps to evaluate an individual's ability to connect different ideas and concepts within a given theme, as well as their overall comprehension and interpretation skills. The results of the test can provide valuable insights into an individual's cognitive abilities, creativity, and critical thinking skills
Authoring a personal GPT for your research and practice: How we created the Q...Leonel Morgado
Thematic analysis in qualitative research is a time-consuming and systematic task, typically done using teams. Team members must ground their activities on common understandings of the major concepts underlying the thematic analysis, and define criteria for its development. However, conceptual misunderstandings, equivocations, and lack of adherence to criteria are challenges to the quality and speed of this process. Given the distributed and uncertain nature of this process, we wondered if the tasks in thematic analysis could be supported by readily available artificial intelligence chatbots. Our early efforts point to potential benefits: not just saving time in the coding process but better adherence to criteria and grounding, by increasing triangulation between humans and artificial intelligence. This tutorial will provide a description and demonstration of the process we followed, as two academic researchers, to develop a custom ChatGPT to assist with qualitative coding in the thematic data analysis process of immersive learning accounts in a survey of the academic literature: QUAL-E Immersive Learning Thematic Analysis Helper. In the hands-on time, participants will try out QUAL-E and develop their ideas for their own qualitative coding ChatGPT. Participants that have the paid ChatGPT Plus subscription can create a draft of their assistants. The organizers will provide course materials and slide deck that participants will be able to utilize to continue development of their custom GPT. The paid subscription to ChatGPT Plus is not required to participate in this workshop, just for trying out personal GPTs during it.
The use of Nauplii and metanauplii artemia in aquaculture (brine shrimp).pptxMAGOTI ERNEST
Although Artemia has been known to man for centuries, its use as a food for the culture of larval organisms apparently began only in the 1930s, when several investigators found that it made an excellent food for newly hatched fish larvae (Litvinenko et al., 2023). As aquaculture developed in the 1960s and ‘70s, the use of Artemia also became more widespread, due both to its convenience and to its nutritional value for larval organisms (Arenas-Pardo et al., 2024). The fact that Artemia dormant cysts can be stored for long periods in cans, and then used as an off-the-shelf food requiring only 24 h of incubation makes them the most convenient, least labor-intensive, live food available for aquaculture (Sorgeloos & Roubach, 2021). The nutritional value of Artemia, especially for marine organisms, is not constant, but varies both geographically and temporally. During the last decade, however, both the causes of Artemia nutritional variability and methods to improve poorquality Artemia have been identified (Loufi et al., 2024).
Brine shrimp (Artemia spp.) are used in marine aquaculture worldwide. Annually, more than 2,000 metric tons of dry cysts are used for cultivation of fish, crustacean, and shellfish larva. Brine shrimp are important to aquaculture because newly hatched brine shrimp nauplii (larvae) provide a food source for many fish fry (Mozanzadeh et al., 2021). Culture and harvesting of brine shrimp eggs represents another aspect of the aquaculture industry. Nauplii and metanauplii of Artemia, commonly known as brine shrimp, play a crucial role in aquaculture due to their nutritional value and suitability as live feed for many aquatic species, particularly in larval stages (Sorgeloos & Roubach, 2021).
8. CISGENIC PLANT:
“A crop plant that has been
genetically modified with one or
more genes isolated from a crossable
donor plant” (Schouten et al., 2006).
INTRAGENIC PLANT:
The intragenic concept as the
isolation of specific genetic elements
from a plant, recombination of these
elements in vitro and insertion of the
resulting expression cassettes into a
plant belonging to the same sexual
compatibility group (Rommens et al.,
2004).
8
10. Similarity and Differences
• Similarity:
• Differences:
Genes transferred are either within the species or
from related species.
Intragenesis
• P-DNA borders are
used.
• In-vitro rearrangements
are made.
Cisgenesis
• No use of P-DNA
borders
• No in-vitro
rearrangement
10
11. To appreciate CISGENESIS and
INTRAGENESIS…..
we need to understand the problems related to…..
Traditional breeding
Transgenic approach
11
12. P = Promoter
C = Coding sequence
T = Terminator
Source: Vivek., 2016
12
13. Pre-requisites of cis/intragenic plant
Complete genome sequence information of the plant.
The isolation and characterization of genes of interest
from crossable relatives.
Transformation technique
Marker free transformation/ Clean vector technology
Intragenic vectors development
13
14. 1. Website address for data base
information
Crop plant Web addresses
Grass species http://www.gramene.org/qtl/index.html
Potato http://www.scri.ac.uk/research/genetics/GeneticsAndBreeding/p
otatoes/mappingqtls
Tomato http://164.107.85.47:8004/cgi-bin/_information.pl
http://zamir.sgn.cornell.edu/Qtl/Html/home.htm
Cucurbitaceous www.icugi.org
14
15. 2. Selection, confirmation and introduction of alleles from
the breeder’s gene pool
• Examples of
traits that can be
incorporated into
a plant by either
transferring or
modifying the
expression of
native genes.
(Rommens, 2004)
15
16. 3.Methods of plant transformation
16
• INDIRECT METHOD : Agrobacterium mediated gene transfer
• DIRECT METHOD :
Particle gun method
Polyethylene glycol method
Microinjection
Macroinjection
Electroporation
Lipofection
17. 4.Clean vector technology
Aims to produce GM plants with only the gene-of-
interest .
The goal is to avoid the use or the continued presence of
antibiotic resistance genes as selectable markers.
This can be done by the following approaches:
17
18. Methods to produce marker free cis/intragenic
plant
1. Transformation without marker gene
2. Co- transformation
3. Site-specific recombination
4. Transposon based expelling system
18
19. Transformation without marker gene
Without the use of a selectable agent.
Depending on the regeneration and gene transfer frequencies,
some plantlets will be transformed.
Potato and cassava – 1-5% ( via Agrobacterium mediated
transformation)
Tobacco – 18% (via protoplast fusion)
Limited to model species and a low number of specific crop
cultivars, e.g. potato.
19
20. Co-transformation
It involves co-integration of gene of interest and a selectable
marker delivered by two separate DNA molecules and thereafter,
segregation of both in the progeny
It is based on Agrobacterium/ biolistic mediated transformation.
20
24. Methods used to produce marker-free
intragenic/cisgenic plants
24
25. Intragenic vector development
Components of intragenic vector:
1.a plant derived T-DNA like region that should contain 2 or
at least 1 T-DNA border like sequences in the correct
orientation.
2.an origin of replication(ori)
3.a selectable antibiotic resistant gene
25
26. P-DNA Approach
Functional plant analogs of the Agrobacterium T-DNA
borders.
Putative transfer DNAs from pooled DNA’s of 66 potato
accessions were isolated and PCR is done using border
specific degenerate primers.
Amplified fragment were sequenced.
Inverse PCR with nested primers to confirm the sequence of
the border like regions.
Identified sequence similarity with Agrobacterium T-DNA.
(Rommens et al., 2005)
27. Putative P-DNA both the inverted repeats flanking a 2.6-kb rice DNA
fragment share homology with T-DNA borders, especially with the
highlighted left border of Agrobacterium nopaline strains and right
border of octopine strains (shaded).
27
28. Limitations of P-DNA
• Found in some species only.
• finding such features on a single relatively short fragment in a
plant genome is rare.
• Reduced frequencies of gene transfer.
(Rommens et al., 2005)
Left border
1-2 kb apart Restriction
sites
Right border
28
29. Combines benefits of traditional breeding and genetic
engineering.
Identifying and assembling T-DNA like fragments with
functional equivalents of T-DNA border sequences.
single step without linkage drag
Without the incorporation of “foreign DNA”
29
33. )
• Objective : Improve the existing variety (Gala) for apple scab resistance
• Material : ‘Gala’ cultivar (susceptible variety) for apple scab caused
by Venturia inaequalis
33
34. Source of natural resistance
Source of natural resistance to scab diseases is known
Classical breeding - Vf resistance gene from Malus floribunda 821
(Lespinasse, 1989; MacHardy, 1996).
In this study HcrVf2 gene (homologue of the Cladosporium
fulvum resistance genes of the Vf region) has taken from ‘Florina’
cultivar.
34
36. Methodology
Transformation with stable
integration using positive
selection, e.g. on kanamycin
(nptII)
↓
Removal of marker by
chemical induction of
Recombinase R activity
(Dexamethasone treatment)
↓
Selection for marker-free
plants using negative selection
(cod A) on
5-FluoroCytosine
36
37. Results
• 6 Independent transformation experiments were performed
resulting in regeneration of 10 transgenic lines out of 1635
explants.
• 2 experiments failed to regenerate transgenic shoots.
37
38. • Eight transgenic lines (T7.1, T7.2, T7.3, T7.4, T8.1, T8.2, T8.3,
T11.2) showed nptIII insertion, confirming backbone integration.
Backbone integration: PCR analysis using primers specific for nptIII.
38
41. Conclusion
Cisgenic ‘Gala’ lines - HcrVf2.
DEX treatment is not impairing apple explant regeneration.
Elimination of fusion marker gene and the R recombinase gene,
occurred as expected.
Delayed negative selection with 5-FC: better recovery of cisgenic
shoots.
Overall no negative effects on the growth of the shoots.
High degree of backbone integration (80%) pose considerable
problem.
41
43. Materials used:
• Potato varieties –Atlantic (America) , Bintje (Dutch) , potae9 (Korea)
• Tested with five Phytophthora infestans isolates with varied virulence
• IPO-C
90128
• EC1
Pic9189
• DHD11 Korean
European
American
43
44. Vector
• Resistance governing genes
i. Rpi-vnt 1.1- S . Venturii
ii. Rpi-sto 1 - S. Stoloniferum
• pBINPLUS – binary vector
• pBINPLUS:Rpi-vnt1.1
• pBINPLUS:Rpi-sto1
• pBINAW2- modified form of
pBINPLUS
44
constructs
45. Detached leaf assays of transgenic
potatoes with single R gene constructs
• Non transformed Atlantic or Bintje were
susceptible to four P. infestans isolates.
45
47. Results
• PCR - Rpi-sto1, Rpi-vnt1.1, or both genes
• bbf - number of vector backbone free events;
• % bbf - percentage of vector backbone c carrying both Rpi-sto1 and Rpi-vnt1.1 47
48. Functional validation of cisgenic
transformants by agroinfiltration and
resistance assays
• A. Avrvnt1.1- and Avrsto1-induced hypersensitive responses in
cisgenic transformant H43-7.
• B. Detached leaf assays for cisgenic transformant H43-7.
48
50. 50
Conclusion
• The narrow late blight resistance spectra of the selected varieties were
upgraded to broad spectrum resistance after the successful introduction of
two cisgenic late blight R genes.
• Cisgenic upgrades of local potato varieties might even ensure food
security.
51. • Objective :
• Improving the viral resistance using RNAi-mediated
transcriptional regulation in marker free intragenic potato
without affecting plant phenotype and productivity.
51
52. Materials and methods
• Potato variety-‘Pirol NN’
• Vector- pMF1
(T-DNA was removed by VspI sites, and resulted expression
cassette was cut at AscI and SbfI sites to insert the hairpin
sequence- pWS-E1-Lhca3).
52
53. Construction of the intron-spliced-hairpin
construct for RNAi–mediated silencing
• pWS-E1-Lhca3 : vector
• Lhca3 gene promoter from
cv“Manhattan”
• rbcs1-ribulose-(1.5)-bisphosphate
carboxylase gene
• ColE1- E. coli origin of replication
• oriV - A. tumefaciens ori site
• trfA- replication protein,
• nptIII- neomycin phosphotransferase III
53
54. Molecular characterization of intragenic plants
• PCR analysis- pws 4.6, pws 10.1
• Southern blot analysis
Southern blot analysis
of EcoRV-digested DNA
isolated from
PCR-positive potato lines
using the probes specific
for the sequence
of Lhca3 gene promoter
(a) and the sequence of
eIF4E1 translation
initiation factor (b)
54
60. BIOSAFETY AND REGULATIONS
• Genetically modified
organisms (GMOs) and
the products are
regulated under the
“Rules for the
manufacture, use,
import, export &
storage of hazardous
microorganisms,
genetically engineered
organisms or cells,
1989”.
60
61. • These Rules are implemented by the Ministry of Environment, Forest and
Climate Change, Department of Biotechnology and State Governments
through six competent authorities.
61
62. Definitions in Rules, 1989
“Genome Edited cells/organisms”(GEd) living cells and/or organisms with
targeted genetic change(s) in genome.
It is used to create a wide range of genome modifications that includes:
• production of ‘nature-identical’ traits,
• production of cisgenic and intragenic plants and animals, and
• introduction of exogenous genes with minimum change in the
cell’s/organism’s genome.
“Genetic engineering” means the technique by which heritable material, which
does not usually occur or will not occur naturally in the organism or cell
concerned, generated outside the organism or the cell is inserted into said cell or
organism.
62
63. Why we need biosafety assessment for GEd ?
Currently available nucleases used for
genome editing experiments are not
completely error-free.
Therefore, biosafety assessment of
GEd organisms/Cells takes into
account :
1) Modified/introduced trait
efficacy as well as
2) The off-target effects leading to
undesirable genetic changes in
the genome and/or phenotype.
63
Point
mutations
and
deletions
Long
sequences
inserted
64. Tiered approach for the risk assessment of GEd
products / organisms
Regulatory scrutiny will be determined by:
• the kind of the genome editing tools/process used like CRISPR/Cas9
• extent of the resulting genetic change (edit),
• the characteristics of GEd organism/cells,
• un-intended changes if any
Risk categories:
• Group I - chemical/radiation mutagenesis, targeted editing using SDN-1(Site
Targeted Nucleases),ODM (Oligo Directed Mutagenesis)
• Group II - targeted editing using SDN-2
• Group III - targeted editing using SDN-3
64
65. • Group I -trait
performance data
using greenhouse or
nethouse.
• Group II - the trait
efficacy data would
be assessed on a
case-by-case basis
depending on the
crop-trait
combination.
• Group III - besides
green house/ net
house data, trait
efficacy data from
limited confined
field trials would be
required.
Draft Document on Genome Edited Organisms,DBT, 2020
65
66. • Regarding regulation of genome engineering technologies, there is still a
debate in our country.
• The definition of GE technology is very broad based and include
“modification, deletion or removal of parts of heritable material”.
• This implies that all new technologies (cis/intragenesis) will be subject to
regulation under provision of Rules, 1989.
• It is still under examination of regulatory agencies and appropriate
Guidelines and Standard Operating Procedures(SOPs) to be drafted.
66
67. ITALY:
• On May 18, 2018, MIPAAF (Italian Ministry of Agricultural, Food, and
Forestry Policies) approved a three-year sustainable agriculture research plan
‘BIOTECH’.
• This research focuses on genome editing and cisgenesis for grapevine, olive,
apple, citrus fruit, apricot, peach, cherry, strawberry, kiwifruit, eggplant, tomato,
basil, artichoke, wheat, rice, and poplar trees.
• In 2021, after the completion of the project, a decision will be taken about the
plant generated by BIOTECH, based on the legislation on force . 67
68. CANADA:
• Follows unique approach based on “Novel traits”
• Canadian Food Inspection Agency (CFIA)
• Novelty is the major trigger for regulatory oversight in Canada and not the
method used to introduce it.
• A herbicide-tolerant variety of canola was developed based on genome
editing oligonucleotides by Cibus Global, a San Diego based company -
approved by the CFIA, is in market from 2016.
• Gen-1 potato with reduced bruising and black spots and reduced asparagine
produced by RNAi silencing has been approved by CFIA for release in
2016.
• Cisgenic plants with ‘Novel Traits’ have to demonstrate that they are
environmentally safe and do not cause any deleterious effect on human and
animal health.
68
69. Cisgenic cultivar registration:
• Establishment of the superiority of the cisgenic cultivar over
the non-cisgenic based on field trials for crop production
traits
• Its impact on food/feed in regard to health effects should be
tested.
• Results are then submitted to the Variety Registration
Office (VRO) of the CFIA.
Ajjamada et al. 2016
69
71. Current status of cis/intragenic crops
• In most countries, the release of cisgenic or intragenic crops
currently falls under the same regulatory guidelines as
transgenic crops.
• The greatest expression of interest for less stringent
regulations of these crops has been within the USA and New
Zealand.
71
Apple production highly impaired by apple scab disease caused by v.en .
Many researches have come up with many resistant varieties but the incorporated genes were exogenous coupled with marker genes.
Inorder to find a solution vanblaere and his coworkers came out with the development of cisgenic apple.
As apple cultivars are self incompatible and highly heterozygous – breeding produces genotypes with new and distict characters
Monoculturing- susceptibility
Established cultivars are susceptible and resistant cultivars developed could not meet up the quality characters of already established caultivars
Schematic representation of the pMF1 vector containing the HcrVf2 gene controlled by its native regulatory sequences.
The segment between the left (LB)and the right border (RB) is transferred into the plant cell, and
the segment between the recombination sites (RS) is then removed on recombinase-mediated deletion,
with the exception of one of the RS.
Rec LBD – LIGAND BINDING DOMAIN inactivates the R recombinase
The gene hcrvf2 will be isolated from florina cultivar and ligated into the vector at pacl and ascl sites of the binary vector pMF1
To verify the correct insertion the gene is isolated and sequenced
Top 4 youngest leaves from 4 week old invitro shoots from gala cv were cut and cocultivated with agrobacterium for 3 days.
Transferred into regeneration medium with kanamycin
For callus induction it was kept at 25 degree for 2 weeks in dark
Resulting transgenic lines were transferred into fresh media after every 4-5 week
Slide
By PCR results they confirmed the presence of hcrvf2 & fusion marker gene in all the 10 transgenic lines.
For marker free transformation all 10 transgenic lines were subjected to recombination and subsequent regeneration through an induction step of R recombinase activity by culturing explts on dexamethasone containing medium.
Later for identification of the marker free transformants 2 diff strategies were applied
1.Normal neg selection
2.Delayed neg selection – prevent the plants containing cod A gene
From the putative cisgenic shoots which was confirmed by neg selection 3 lines selected
Earlier breeding for late blight resistance was done by introgressing R genes from S.demissum – there was rapid breakdown of the resistance.
Therefore combination of R genes from diff spectra came into the thought of researchers
Sarpomira a variety with 4 R genes with durable resistance was released but it failed bcz of no market value as farmers prefer already established varieties.
Transgenic atlantic and bintje with rpivnt1.1 were suscept to EC1 While that of Potae9 was resist to EC1
Those with rpisto1 were sucptible to 99189 but they were resistant to the other strains
So these 2 genes were combined as a cisgenic R gene stack in selected varieties.
19 lines were transferred to greenhouse for phenotypic characterization of the disease
After 3 weeks 5 showed abnormalities
14 lines were tested for their response to Avrvnt1 and avrsto1 after agroinfiltration
AGROINFILTRATION: it’s a methid to induce transient expression of genes in the transformed plant/leaf in order to produce a desired protein
PVY is most harmful viral disease
Severe cases leads to 50-100%yield loss
Classical control practices for the vector control + use of certified high grade potatoes provided only some amount of resistance
Therefore Breeding for viral resistance at genetic level was a promising approach
100 nodal explants
224 plantlets – were collected
For simplifying the search 8plantlets were pooled into I culture vessel
DNA isolation from pooling the samples from each culture vessel
PCR Analysis-Analysis of samples through amplification of chimeric union of left arm of hairpin construct with rbcs intron
Presence of intragenic plts in 4th and 10th vessel
DNA isolation from each individual plants in 4th and 10t vessel and PCR
2 independent events pws4.6, pws10.1 found positive and confirmed the presence of hairpin construct in both the lines
To verify the expression level of the 2 genes
To assess whether the reduction in m RNA of these 2 genes was because of RNAi silencing , the accumulation of hairpin derived si RNA was analysed by Northern blotting
Genome editing is a precise molecular method of mutation leading to deletion or addition or substitution of target base pair(s) in the native genes/ nucleic acid sequences. On the contrary, GE organisms (also known as GMOs/LMOs) typically contain foreign genes or DNA (with/ without prior knowledge of genome structure and function) derived from related or unrelated organisms to modify an existing trait or introduce a new trait. In addition, genome editing also facilitates the introduction of a foreign gene(s) to introduce a new trait(s), which is similar to GE organisms, but the site of integration is predetermined in GEd organisms unlike in GE organisms where site of foreign gene integration in the genome is random.