Classical Plant Breeding Research Articles

Exploring Pan-Genomes: An Overview of Resources and Tools for Unraveling Structure, Function, and Evolution of Crop Genes and Genomes.

The availability of multiple sequenced genomes from a single species made it possible to explore intra- and inter-specific genomic comparisons at higher resolution and build clade-specific pan-genomes of several crops. The pan-genomes of crops constructed from various cultivars, accessions, landraces, and wild ancestral species represent a compendium of genes and structural variations and allow researchers to search for the novel genes and alleles that were inadvertently lost in domesticated crops during the historical process of crop domestication or in the process of extensive plant breeding. Fortunately, many valuable genes and alleles associated with desirable traits like disease resistance, abiotic stress tolerance, plant architecture, and nutrition qualities exist in landraces, ancestral species, and crop wild relatives. The novel genes from the wild ancestors and landraces can be introduced back to high-yielding varieties of modern crops by implementing classical plant breeding, genomic selection, and transgenic/gene editing approaches. Thus, pan-genomic represents a great leap in plant research and offers new avenues for targeted breeding to mitigate the impact of global climate change. Here, we summarize the tools used for pan-genome assembly and annotations, web-portals hosting plant pan-genomes, etc. Furthermore, we highlight a few discoveries made in crops using the pan-genomic approach and future potential of this emerging field of study.

Furanocoumarins from Heracleum persicum L.: Unveiling their biosynthesis and gene expression

Due to the high value of Persian angelica (Heracleum persicum L.) and the limited genetic information regarding this plant, the investigation of the main secondary metabolites and corresponding biosynthesis pathway is of great importance. In this study, sequencing and transcriptome analysis were performed for both leaf and fruit tissues of H. persicum. Functional annotation and gene ontology analysis for 165,597 assembled transcripts was carried out. The most abundant categories belonged to proteins involved in cellular, metabolic, binding, and biosynthetic processes class. After comprehensive transcript annotation, the genes involved in the important metabolites biosynthesis of this plant were identified and the possible pathway for the biosynthesis of furanocoumarins and terpenoids were proposed. Finally, the relative expression of the genes involved in the biosynthesis of furanocoumarin metabolites as important compounds of this plant and their amount were investigated using real-time PCR and HPLC methods, respectively. In general, according to the results of the study, the expression of most genes in fruit tissue was higher than in leaf tissue. Angelicin synthase, bergaptol methyltransferase, and psoralen synthase in fruit tissue expressed 3.3, 2.4, and 1.9 times more than that in leaf tissue, respectively. The amount of bergapten, psoralen, xanthotoxol, and isopimpinellin in fruit was measured as 94.06, 4.6, 17.9, and 0.1 mg per gram of dry weight, respectively. Our findings will be valuable in designing metabolite engineering strategies based on genetic engineering and classical plant breeding with the aim of genetic manipulation and metabolite engineering in this plant.

Recent Advances and Achievements in Mutation Breeding of Fruit Crops: A Review

Genetic variation is essential for crop breeding. In classical plant breeding programme, variation is generated by hybridization and selections are made from the resulting segregating generations. Induced mutagenesis can supplement hybridization or able to replace as a source of variability. Since, mutations bring about variation, they provide the ultimate basis for evolution of new forms, varieties or species. Induced and spontaneous mutations have played an important role in developing improved cultivars of various fruit crops as a supplementary method to conventional breeding. But, induced mutations also have well defined limitations in fruit breeding applications, but their possibilities may be expanded by the use of in vitro mutation techniques.

A Review On Citrus tristeza virus (CTV) And Its Management Approaches

Citrus tristeza virus (CTV), one of the casual agents of citrus decline, is responsible for the death of millions of citrus trees and reduced production and productivity of citrus orchards worldwide. CTV epidemics has been recorded from several parts of the world where mainly steam pitting (SP) and quick decline (QD) strains of CTV have induced severe disease reactions. Identification and characterization of CTV isolates primarily has been focused on the biological assaying in indicator plants, serology-based ELISA and molecular PCR tests. Controlling the presence and spread of CTV where it is absent or establishment is limited heavily relies upon preventive measures, quarantine and legislations. Cross protection is an appealing technique especially for controlling CTV – Stem Pitting strains and use of CTV resistant rootstocks largely prevent infection by CTV – Quick Decline strains. More reliable and effective way to control CTV is breeding for resistant or tolerant cultivars. Advances in molecular biology have lead scientists to find out genes and map genetic loci of CTV resistant citrus and related species that could be exploited in breeding. However incorporation of resistant ability offered by a handful of citrus and its related species into the susceptible cultivars containing other desirable agronomical traits is challenging through classical plant breeding approaches. The following review work is based on Citrus tristza virus and its management practices.

Genetic Improvement of Camelina sativa (L.) Crantz: Opportunities and Challenges.

In recent years, a renewed interest in novel crops has been developing due to the environmental issues associated with the sustainability of agricultural practices. In particular, a cover crop, Camelina sativa (L.) Crantz, belonging to the Brassicaceae family, is attracting the scientific community's interest for several desirable features. It is related to the model species Arabidopsis thaliana, and its oil extracted from the seeds can be used either for food and feed, or for industrial uses such as biofuel production. From an agronomic point of view, it can grow in marginal lands with little or no inputs, and is practically resistant to the most important pathogens of Brassicaceae. Although cultivated in the past, particularly in northern Europe and Italy, in the last century, it was abandoned. For this reason, little breeding work has been conducted to improve this plant, also because of the low genetic variability present in this hexaploid species. In this review, we summarize the main works on this crop, focused on genetic improvement with three main objectives: yield, seed oil content and quality, and reduction in glucosinolates content in the seed, which are the main anti-nutritional substances present in camelina. We also report the latest advances in utilising classical plant breeding, transgenic approaches, and CRISPR-Cas9 genome-editing.

Multiplex PCR assay for the simultaneous identification of race specific and non-specific leaf resistance genes in wheat (Triticum aestivum L.)

Race-nonspecific resistance is a key to sustainable management of pathogens in bread wheat (Triticum aestivum L.) breeding. It is more durable compared to race-specific immunity, conferred by the major genes (R), which are often overcome by pathogens. The accumulation of the genes, which provide the resistance to a specific race of a pathogen, together with the introduction of race-non-specific resistance genes is the most effective strategy aimed at preventing the breakdown of genetically conditioned immunity. PCR markers improved the productivity and accuracy of classical plant breeding by means of marker-assisted selection (MAS). Multiplexing assays provide increased throughput, reduced reaction cost, and conservation of limited sample material, which are beneficial for breeding purposes. Here, we described the process of customizing multiplex PCR assay for the simultaneous identification of the major leaf rust resistance genes Lr19, Lr24, Lr26, and Lr38, as well as the slow rusting, race-nonspecific resistance genes: Lr34 and Lr68, in thirteen combinations. The adaptation of PCR markers for multiplex assays relied on: (1) selection of primers with an appropriate length; (2) selection of common annealing/extension temperature for given primers; and (3) PCR mixture modifications consisting of increased concentration of primers for the scanty band signals or decreased concentration of primers for the strong bands. These multiplex PCR protocols can be integrated into a marker-assisted selection of the leaf rust-resistant wheat genotypes.

Understanding tobamovirus-plant interactions: implications for breeding resistance to tomato brown rugose fruit virus

The genus Tobamovirus comprises a group of single-stranded RNA viruses that affect a wide variety of vegetables of economic importance. Tobamoviruses express a series of proteins that interact with the plant’s cellular machinery, allowing viral infection; during incompatible interactions, active defense is mediated by host proteins encoded by resistance genes. The genes conferring viral resistance and tolerance in non-susceptible hosts have been studied for their ability to transfer desired resistance traits to different crops. The N gene from Nicotiana spp., the repertoire of Tm genes in Solanum spp., the L locus from Capsicum spp., and TOM genes are the most studied genetic sequences for understanding resistance to tobamoviruses. Through classical plant breeding and genetic engineering techniques, it has been possible to introgress these resistance genes (R) into new species. However, new reports highlight the ability of tobamoviruses to overcome R-mediated defense. One of the most notorious recent cases is the tomato brown rugose fruit virus (ToBRFV). The main characteristic of ToBRFV is its capacity to overcome the resistance mediated by the Tm-22 gene, resulting in a limited repertoire of options to combat the virus. To defeat emerging viruses, it is necessary to apply the knowledge from other tobamoviruses-host relationships and use new technologies such as genome-wide association studies (GWAS) to understand and associate the architecture of resistance genes present in the Solanaceae family for the benefit of plant breeding. Although new genomic tools such as CRISPR systems open the possibility of coping with viral diseases, there are no commercial ToBRFV-resistant tomato varieties. Hence, the world’s leading seed suppliers compete to develop and bring these varieties to market.

Site-Directed Mutagenesis – A Chance to Meet Environmental Challenges and Provide Healthy Food for People or an Unacceptable Hazard to Humans, Animals, and the Environment. Consequences of the European Court of Justice Judgment in Case C-528/16

Abstract One of the EU's strategic goals is to reduce the environmental and climate footprint of the EU food system and strengthen its resilience, while ensuring food security for European citizens. Thus, the EU Farm to Fork strategy, which is one of the central pillars of the European Green Deal, set ambitious targets for 2030 to reduce pesticide use in agriculture by 50% and fertilizers use by 20%, with a concomitant 50% reduction of nutrient leakage to surface and groundwater. Additionally, it is recommended that at least 25% of the EU agricultural land shall be kept under organic farming. These goals are far-reaching, but several recent studies indicate that implementing them without significant progress in research and innovation (R & I) may result in a yield decrease by up to 30%, depending on the crop, and an increase in the price of agricultural commodities by up to 18%. Especially affected would be horticulture due to its high dependence on plant protection against pests and diseases. Therefore, the studies recommend accelerating plant breeding in order to produce new plant cultivars genetically resistant to pests and diseases and better equipped to cope with abiotic stresses like limited nutrition and water deficit. The progress in classical plant breeding is a lengthy process. It is especially slow in the case of woody species, like most fruit plants, due to their long juvenile periods and limited genetic variance. Recent advances in functional genomics, bioinformatics, and molecular methods provided tools that speed up the breeding process significantly. Several site-directed mutation technologies allow modifying a specific gene at a predefined site, by deletion or insertion of single or multiple nucleotides, without affecting off-target genes. Several valuable cultivars have been bred so far using these methods, and a large number of others are under trials. However, their release will be severely impeded by the decision of the Court of Justice of the European Union, dated 25 July 2018, that the release of organisms obtained by site-specific mutations, as opposed to organisms obtained by induced random mutation, is controlled by Directive 2001/18/EC2 on genetically modified organisms. This paper reviews the new generation breeding techniques, especially site-directed mutagenesis, and their benefits as well as potential hazards to consumers and the environment.

Progress and prospects of C4 trait engineering in plants.

Incorporating C4 photosynthetic traits into C3 crops is a rational approach for sustaining future demands for crop productivity. Using classical plant breeding, engineering this complex trait is unlikely to achieve its target. Therefore, it is critical and timely to implement novel biotechnological crop improvement strategies to accomplish this goal. However, a fundamental understanding of C3 , C4 , and C3 -C4 intermediate metabolism is crucial for the targeted use of biotechnological tools. This review assesses recent progress towards engineering C4 photosynthetic traits in C3 crops. We also discuss lessons learned from successes and failures of recent genetic engineering attempts in C3 crops, highlighting the pros and cons of using rice as a model plant for short-, medium- and long-term goals of genetic engineering. This review provides an integrated approach towards engineering improved photosynthetic efficiency in C3 crops for sustaining food, fibre and fuel production around the globe.

Protein structure determination as a powerful tool for the sustainable development of agriculture field (and its potential relevance in Indonesia)

Traditional agricultural practices, such as classical plant breeding and pest management, have been utilized for many years to increase crop yield and improve its properties. However, more effective and sustainable practices are needed to meet the increasing global demands for agricultural-based products. In the past decades, DNA recombinant technology, and protein structure determination have been employed as a tool for the development of the agricultural field. Protein structure determination enables us to understand the structure-function relationship of a given protein, such as plant hormone, receptor, or growth regulators, thus introducing modifications to improve the properties of the crop. This review introduces the latest development of protein structure determination techniques ranging from X-ray crystallography, protein NMR, and cryo-electron microscopy. Notable examples of how these techniques can be utilized as a powerful tool for the sustainable development of the agricultural field, including their potential relevance in Indonesia, are introduced.

Transcriptomic and volatile signatures associated with maize defense against corn leaf aphid

BackgroundMaize (Zea mays L.) is a major cereal crop, with the United States accounting for over 40% of the worldwide production. Corn leaf aphid [CLA; Rhopalosiphum maidis (Fitch)] is an economically important pest of maize and several other monocot crops. In addition to feeding damage, CLA acts as a vector for viruses that cause devastating diseases in maize. We have shown previously that the maize inbred line Mp708, which was developed by classical plant breeding, provides heightened resistance to CLA. However, the transcriptomic variation conferring CLA resistance to Mp708 has not been investigated.ResultsIn this study, we contrasted the defense responses of the resistant Mp708 genotype to those of the susceptible Tx601 genotype at the transcriptomic (mRNA-seq) and volatile blend levels. Our results suggest that there was a greater transcriptomic remodeling in Mp708 plants in response to CLA infestation compared to the Tx601 plants. These transcriptomic signatures indicated an activation of hormonal pathways, and regulation of sesquiterpenes and terpenoid synthases in a constitutive and inducible manner. Transcriptomic analysis also revealed that the resistant Mp708 genotype possessed distinct regulation of ethylene and jasmonic acid pathways before and after aphid infestation. Finally, our results also highlight the significance of constitutive production of volatile organic compounds (VOCs) in Mp708 and Tx601 plants that may contribute to maize direct and/or indirect defense responses.ConclusionsThis study provided further insights to understand the role of defense signaling networks in Mp708’s resistance to CLA.

Concept and rationale of evolutionary plant breeding and its status in Nepal

Nepal has released and registered a total of 623 genetically uniform (mono genotyped) varieties. These varieties were developed by both conventional and classical plant breeding, biotech-assisted plant breeding, and participatory plant breeding methods. However, these varieties have been shown to vary in their yield performance over the years and locations. Smallholder farmers dominate agriculture with 53% of the land-owning households with their land holding size of less than 0.5 ha in Nepal. Farmers are increasingly losing their own saved seeds. There have been impacts of weather variability, often modern crop varieties are not available to suit with these changing conditions. Farmers are looking for crop varieties that can better adapt to these changing conditions, and seeds of which can be saved for the next season planting. Evolutionary Plant Breeding (EPB), which creates and maintains a high degree of genetic diversity (i.e. polymorphic population), is a choice for breeders and farmers for accelerating the development of climate resilient and sustainably high-performance crop varieties. In 2015, the National Gene Bank in Nepal started an EPB program for the local rice variety, Jumli Marshi with the objective of enhancing genetic conservation through creating a dynamic gene pool. An evolutionary population can be compared to a living gene bank, not only in line with bringing greater yield stability, but also greater diversity in aroma, nutritional value and quality. Evolutionary populations have the potential to produce higher yields and perform better than their local or improved counterparts in adverse, or stress conditions. Under stress conditions, evolutionary populations have also been shown to be more resistant to weeds, diseases and pests damage than homogenous crop populations. Based on the source of diversity used in EPB, two different types of populations- Composite Cross population, and Composite Mixtures, population are developed. With the exception of Europe, and only for some crops, existing seed policies do not favor such populations. Therefore, there is a need to revise seed regulations in order to allow the cultivation of a higher degree of genetic diversity.

Machine Learning for Plant Breeding and Biotechnology

Classical univariate and multivariate statistics are the most common methods used for data analysis in plant breeding and biotechnology studies. Evaluation of genetic diversity, classification of plant genotypes, analysis of yield components, yield stability analysis, assessment of biotic and abiotic stresses, prediction of parental combinations in hybrid breeding programs, and analysis of in vitro-based biotechnological experiments are mainly performed by classical statistical methods. Despite successful applications, these classical statistical methods have low efficiency in analyzing data obtained from plant studies, as the genotype, environment, and their interaction (G × E) result in nondeterministic and nonlinear nature of plant characteristics. Large-scale data flow, including phenomics, metabolomics, genomics, and big data, must be analyzed for efficient interpretation of results affected by G × E. Nonlinear nonparametric machine learning techniques are more efficient than classical statistical models in handling large amounts of complex and nondeterministic information with “multiple-independent variables versus multiple-dependent variables” nature. Neural networks, partial least square regression, random forest, and support vector machines are some of the most fascinating machine learning models that have been widely applied to analyze nonlinear and complex data in both classical plant breeding and in vitro-based biotechnological studies. High interpretive power of machine learning algorithms has made them popular in the analysis of plant complex multifactorial characteristics. The classification of different plant genotypes with morphological and molecular markers, modeling and predicting important quantitative characteristics of plants, the interpretation of complex and nonlinear relationships of plant characteristics, and predicting and optimizing of in vitro breeding methods are the examples of applications of machine learning in conventional plant breeding and in vitro-based biotechnological studies. Precision agriculture is possible through accurate measurement of plant characteristics using imaging techniques and then efficient analysis of reliable extracted data using machine learning algorithms. Perfect interpretation of high-throughput phenotyping data is applicable through coupled machine learning-image processing. Some applied and potentially applicable capabilities of machine learning techniques in conventional and in vitro-based plant breeding studies have been discussed in this overview. Discussions are of great value for future studies and could inspire researchers to apply machine learning in new layers of plant breeding.

Selection signatures across seven decades of hard winter wheat breeding in the Great Plains of the United States

Classical plant breeding has been instrumental in changing the genetic makeup of crop plants for better ecological adaptation and improved quality. This paper provides insights of the genomic changes effected in hard winter wheat (Triticum aestivum L.) through decades of breeding and selection in the Great Plains of the United States. Population structure and differentiation analyses were conducted on 185 wheat cultivars released from 1943 to 2013. Cultivars were grouped into four distinct clusters using discriminant analysis of principal components (DAPC). One of the clusters was unique in that 15 out of the 18 individuals were recent releases (2000-2010), while 12 of the 18 shared the cultivar 'Jagger' in their genetic background. Jagger carries a 2NS/2AS translocation segment from Aegilops ventricosa, an important segment for resistance to several foliar diseases. Using the outlier approach, Wright's population fixation index (Fst) identified 450 loci that were directionally selected. The largest signature of selection was found on chromosome 2A. Genetic diversity was high while the inbreeding coefficient was low, indicating extensive hybridization and germplasm exchange among breeding programs within the region. Foliar disease pressure and selection for resistance helped shape the microevolution of wheat in the southern Great Plains. The results showed that high genetic diversity remains in hard winter wheat cultivars adapted to the Great Plains of the USA, and modern plant breeding did not cause any sizable reduction in genetic diversity of the crop in this region.

TWENTY-TWO-YEAR PAPAYA BREEDING PROGRAM: FROM BREEDING STRATEGY ESTABLISHMENT TO CULTIVAR DEVELOPMENT

The papaya crop occupies 32 thousand hectares of planted area in Brazil, with a total annual production of 1.6 million tons (12.5% of the world supply). The country stands out in this scenario as the second biggest producer of the fruit worldwide, only after India. The narrow genetic base of the crop once limited its variability, but the use of classical and molecular plant breeding techniques has enabled the development of a number of higher-yielding cultivars with different levels of resistance to fungal diseases. However, many studies still ought to be undertaken to investigate the papaya crop, given the constant search for higher-yielding cultivars with quality and flavor attributes and the wide range of pathogens affecting the crop, which has not yet shown fully resistant genotypes. Advances in the genomics of papaya provide tools that may improve cultivar production and development systems. This article describes studies conducted by the genetics and breeding group at UENF, in a partnership with Caliman Agricola S.A., using conventional breeding, diallel cross, and topcross, among other techniques, for the development of 21 hybrids, which were registered at MAPA, in addition to studies with DNA-based markers for sex determination and for the generation of resistant and productive cultivars. This review focuses on the 22 years of conventional breeding for the most recent molecular progress in papaya growing. The information reported here is extremely useful for breeders to develop resistant, productive, and high-quality varieties through assisted selection.

The pathogen Moniliophthora perniciosa promotes differential proteomic modulation of cacao genotypes with contrasting resistance to witches\xb4 broom disease

BackgroundWitches’ broom disease (WBD) of cacao (Theobroma cacao L.), caused by Moniliophthora perniciosa, is the most important limiting factor for the cacao production in Brazil. Hence, the development of cacao genotypes with durable resistance is the key challenge for control the disease. Proteomic methods are often used to study the interactions between hosts and pathogens, therefore helping classical plant breeding projects on the development of resistant genotypes. The present study compared the proteomic alterations between two cacao genotypes standard for WBD resistance and susceptibility, in response to M. perniciosa infection at 72 h and 45 days post-inoculation; respectively the very early stages of the biotrophic and necrotrophic stages of the cacao x M. perniciosa interaction.ResultsA total of 554 proteins were identified, being 246 in the susceptible Catongo and 308 in the resistant TSH1188 genotypes. The identified proteins were involved mainly in metabolism, energy, defense and oxidative stress. The resistant genotype showed more expressed proteins with more variability associated with stress and defense, while the susceptible genotype exhibited more repressed proteins. Among these proteins, stand out pathogenesis related proteins (PRs), oxidative stress regulation related proteins, and trypsin inhibitors. Interaction networks were predicted, and a complex protein-protein interaction was observed. Some proteins showed a high number of interactions, suggesting that those proteins may function as cross-talkers between these biological functions.ConclusionsWe present the first study reporting the proteomic alterations of resistant and susceptible genotypes in the T. cacao x M. perniciosa pathosystem. The important altered proteins identified in the present study are related to key biologic functions in resistance, such as oxidative stress, especially in the resistant genotype TSH1188, that showed a strong mechanism of detoxification. Also, the positive regulation of defense and stress proteins were more evident in this genotype. Proteins with significant roles against fungal plant pathogens, such as chitinases, trypsin inhibitors and PR 5 were also identified, and they may be good resistance markers. Finally, important biological functions, such as stress and defense, photosynthesis, oxidative stress and carbohydrate metabolism were differentially impacted with M. perniciosa infection in each genotype.

Evolution of plant mutagenesis tools: a shifting paradigm from random to targeted genome editing

Mutations are the basis of all genetic variation, and classical plant breeding has exploited the power of natural mutations in the development of high-yielding varieties. Since the discovery of chemical and radiation mutagens, various mutagenesis methods have been employed effectively in molecular plant breeding to study gene functions, to identify crucial genetic mutations for conferring novel traits to plants. Here, we review the historical development and use of plant mutagenesis tools, mainly physical and chemical mutagens, PCR-based methods, T-DNA insertions, transposon insertions, RNA interference, and meganucleases. The unique feature of meganucleases such as the CRISPR/Cas system is the control of site-directed DNA changes. We highlight the recent advancements in CRISPR/Cas-based mutagenesis tools that enable various applications of genetic manipulations, including gene knock-out, gene replacement, targeted base substitutions, and nucleotide diversification of user-defined sites. In addition, we review the use of CRISPR/Cas-based mutagenesis tools in agriculture.

New Possibilities on the Horizon: Genome Editing Makes the Whole Genome Accessible for Changes.

The emergence of new genome editing techniques, such as the site-directed nucleases, clustered regulatory interspaced short palindromic repeats (CRISPRs)/Cas9, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs), has greatly increased the feasibility of introducing any desired changes into the genome of a target organism. The ability to target a Cas nuclease to DNA sequences with a single-guide RNA (sgRNA) has provided a dynamic tool for genome editing and is naturally derived from an adaptive immune system in bacteria and archaea. CRISPR/Cas systems are being rapidly improved and refined, thereby opening up even more possibilities. Classical plant breeding is based on genetic variations that occur naturally and is used to select plants with improved traits. Induced mutagenesis is used to enhance mutational frequency and accelerate this process. Plants have evolved cellular processes, including certain repair mechanisms that ensure DNA integrity and the maintenance of distinct DNA loci. The focus of this review is on the characterization of new potentials in plant breeding through the use of CRISPR/Cas systems that eliminate natural limitations in order to induce thus far unachievable genomic changes.

Precision breeding with genomic tools: A decade long journey of molecular breeding in rice

Plant breeding helps in recombining and fixation of desirable alleles which enables the development of improved varieties with better productivity. Though classical plant breeding has contributed enormously in achieving genetic gains in different crops including rice, the challenges imposed by resource constraints as well as greater anticipated demand need better tools to meet the expectations. Molecular breeding which uses modern genomic tools integrated with plant breeding offers enormous potential for improvement of crop varieties. The present review focusses on the achievements of ICAR-IARI in molecular breeding in rice wherein as many as eight rice varieties were incorporated with genes governing resistance/tolerance to different traits and released for commercial cultivation in India. The various factors which enabled the successful integration of molecular breeding including development of infrastructure, human resources and the genetic and genomic resources have been discussed in detail. All the examples of translational research are from the marker assisted backcross breeding strategy, which limits the scope for achieving yield gains. In the foregoing discussion, the challenges and the opportunities for implementation of molecular breeding in rice to realize the full potential of these tools in plant breeding are discussed based on the decade long experience, which may be valuable for other crops as well.

Efficient Targeted Mutagenesis in Apple and First Time Edition of Pear Using the CRISPR-Cas9 System.

Targeted genome engineering has emerged as an alternative to classical plant breeding and transgenic methods to improve crop plants. Among other methods (zinc finger nucleases or TAL effector nucleases) the CRISPR-Cas system proved to be the most effective, convenient and least expensive method. In this study, we optimized the conditions of application of this system on apple and explored its feasibility on pear. As a proof of concept, we chose to knock-out the Phytoene Desaturase (PDS) and Terminal Flower 1 (TFL1) genes. To improve the edition efficiency, two different single guide RNAs (gRNAs) were associated to the Cas9 nuclease for each target gene. These gRNAs were placed under the control of the U3 and U6 apple promoters. Characteristic albino phenotype was obtained for 85% of the apple transgenic lines targeted in MdPDS gene. Early flowering was observed in 93% of the apple transgenic lines targeted in MdTFL1.1 gene and 9% of the pear transgenic lines targeted in PcTFL1.1. Sequencing of the target zones in apple and pear CRISPR-PDS and CRISPR-TFL1.1 transgenic lines showed that the two gRNAs induced mutations but at variable frequencies. In most cases, Cas9 nuclease cut the DNA in the twenty targeted base pairs near the protospacer adjacent motif and insertions were more frequent than deletions or substitutions. The most frequent edition profile of PDS as well as TFL1.1 genes was chimeric biallelic. Analysis of a sample of potential off-target sequences of the CRISPR-TFL1.1 construct indicated the absence of edition in cases of three mismatches. In addition, transient transformation with the CRISPR-PDS construct produced two T-DNA free edited apple lines. Our overall results indicate that, despite the frequent occurrence of chimerism, the CRISPR-Cas 9 system is a powerful and precise method to induce targeted mutagenesis in the first generation of apple and pear transgenic lines.