Applications and potential of genome editing in crop improvement

Genome-editing tools provide advanced biotechnological techniques that enable the precise and efficient targeted modification of an organism’s genome. Genome-editing systems have been utilized in a wide variety of plant species to characterize gene functions and improve agricultural traits. We describe the current applications of genome editing in plants, focusing on its potential for crop improvement in terms of adaptation, resilience, and end-use. In addition, we review novel breakthroughs that are extending the potential of genome-edited crops and the possibilities of their commercialization. Future prospects for integrating this revolutionary technology with conventional and new-age crop breeding strategies are also discussed.     

Organ specificity and transcriptional control of metabolic routes revealed by expression QTL profiling of source-sink tissues in a segregating potato population

Background  

With the completion of genome sequences belonging to some of the major crop plants, new challenges arise to utilize this data for crop improvement and increased food security. The field of genetical genomics has the potential to identify genes displaying heritable differential expression associated to important phenotypic traits. Here we describe the identification of expression QTLs (eQTLs) in two different potato tissues of a segregating potato population and query the potato genome sequence to differentiate between cis- and trans-acting eQTLs in relation to gene subfunctionalization.     

360度群体遗传变异扫描——大豆泛基因组研究

大豆(Glycine max)是重要的油料和蛋白作物, 其丰富的遗传变异为生物学性状挖掘和育种改良提供了重要的资源基础。然而, 单个基因组信息无法全面揭示种质资源的遗传变异, 泛基因组研究为解决这一不足提供了新方案。近日, 中国科学院遗传与发育生物学研究所田志喜和梁承志研究团队从2 898份大豆种质中选取26份代表性材料, 并整合已有的3个基因组, 构建了包含野生和栽培大豆的泛基因组和图基因组(graph-based genome), 鉴定了整个群体的绝大多数结构变异数据集, 确定了大豆种质的核心、非必需和个体特异的基因集。利用这些数据系统地揭示了生育期位点E3的等位基因变异和基因融合事件、种皮颜色基因I的单体型和演化关系以及结构变异对铁离子转运基因表达和地区适应性选择的影响。该研究为作物基因组学研究提供了一个新的模式, 同时将加速推动大豆遗传变异的鉴定、性状解析和种质创新。     

Base editing in crops: current advances,limitations and future implications

Targeted mutagenesis via genome‐editing technologies holds great promise in developing improved crop varieties to meet future demands. Point mutations or single nucleotide polymorphisms often determine important agronomic traits of crops. Genome‐editing‐based single‐base changes could generate elite trait variants in crop plants which help in accelerating crop improvement. Among the genome‐editing technologies, base editing has emerged as a novel and efficient genome‐editing approach which enables direct and irreversible conversion of one target base into another in a programmable manner. A base editor is a fusion of catalytically inactive CRISPR–Cas9 domain (Cas9 variants) and cytosine or adenosine deaminase domain that introduces desired point mutations in the target region enabling precise editing of genomes. In the present review, we have summarized the development of different base‐editing platforms. Then, we have focussed on the current advances and the potential applications of this precise technology in crop improvement. The review also sheds light on the limitations associated with this technology. Finally, the future perspectives of this emerging technology towards crop improvement have been highlighted.     

Progress of chromosome engineering mediated by asymmetric somatic hybridization

Plant somatic hybridization has progressed steadily over the past 35 years. Many hybrid plants have been generated from fusion combinations of different phylogenetic species, some of which have been utilized in crop breeding programs. Among them, asymmetric hybrid, which usually contains a fraction of alien genome, has received more attention because of its importance in crop improvement. However, few studies have dealt with the heredity of the genome of somatic hybrid for a long time, which has limited the progress of this approach. Over recent ten years, along with the development of an effective cytogenetical tool ＂in situ hybridization （ISH）＂, asymmetric fusion of common wheat （Triticum aestivum L.） with different grasses or cereals has been greatly developed. Genetics, genomes, functional genes and agricultt, ral traits of wheat asymmetric hybrids have been subject to systematic investigations using gene cloning, genomic in situ hybridization （GISH） and molecular makers. The future goal is to fully elucidate the functional relationships among improved agronomic traits, the genes and underlying molecular mechanisms, and the genome dynamics of somatic introgression lines. This will accelerate the development of elite germplasms via somatic hybridization and the application of these materials in the molecular improvement of crop plants.     

Raising Climate-Resilient Crops: Journey From the Conventional Breeding to New Breeding Approaches

BackgroundIn order to meet the demands of the ever-increasing human population, it has become necessary to raise climate-resilient crops. Plant breeding, which involves crossing and selecting superior gene pools, has contributed tremendously towards achieving this goal during the past few decades. The relatively newer methods of crop improvement based on genetic engineering are relatively simple, and targets can be achieved in an expeditious manner. More recently emerged genome editing technique using CRISPR has raised strong hopes among plant scientists for precise integration of valuable traits and removal of undesirable ones.ConclusionGenome editing using Site-Specific Nucleases (SSNs) is a good alternative to the plant breeding and genetic engineering approaches as it can modify the genomes specifically and precisely at the target site in the host genome. Another added advantage of the genome editing approach is the simpler biosafety regulations that have been adopted by many countries for commercialization of the products thus generated. This review provides a critical assessment of the available methods for improving the stress tolerance in crop plants. Special emphasis has been given on genome editing approach in light of the diversity of tools, which are being discovered on an everyday basis and the practical applications of the same. This information will serve as a beginner’s guide to initiate the crop improvement programs as well as giving technical insight to the expert to plan the research strategically to tackle even multigenic traits in crop plants.     

Shaping polyploid wheat for success: Origins,domestication, and the genetic improvement of agronomic traits

Bread wheat (Triticum aestivum L., AABBDD, 2n = 6x = 42), which accounts for most of the cultivated wheat crop worldwide, is a typical allohexaploid with a genome derived from three diploid wild ancestors. Bread wheat arose and evolved via two sequential allopolyploidization events and was further polished through multiple steps of domestication. Today, cultivated allohexaploid bread wheat has numerous advantageous traits, including adaptive plasticity, favorable yield traits, and extended end-use quality, which have enabled its cultivation well beyond the ranges of its tetraploid and diploid progenitors to become a global staple food crop. In the past decade, rapid advances in wheat genomic research have considerably accelerated our understanding of the bases for the shaping of complex agronomic traits in this polyploid crop. Here, we summarize recent advances in characterizing major genetic factors underlying the origin, evolution, and improvement of polyploid wheats. We end with a brief discussion of the future prospects for the design of gene cloning strategies and modern wheat breeding.     

Current status and future possibilities of molecular genetics techniques in <Emphasis Type="Italic">Brassica napus</Emphasis>

As PCR methods have improved over the last 15 years, there has been an upsurge in the number of new DNA marker tools, which has allowed the generation of high-density molecular maps for all the key Brassica crop types. Biotechnology and molecular plant breeding have emerged as a significant tool for molecular understanding that led to a significant crop improvement in the Brassica napus species. Brassica napus possess a very complicated polyploidy-based genomics. The quantitative trait locus (QTL) is not sufficient to develop effective markers for trait introgression. In the coming years, the molecular marker techniques will be more effective to determine the whole genome impairing desired traits. Available genetic markers using the single-nucleotide sequence (SNP) technique and high-throughput sequencing are effective in determining the maps and genome polymorphisms amongst candidate genes and allele interactions. High-throughput sequencing and gene mapping techniques are involved in discovering new alleles and gene pairs, serving as a bridge between the gene map and genome evaluation. The decreasing cost for DNA sequencing will help in discovering full genome sequences with less resources and time. This review describes (1) the current use of integrated approaches, such as molecular marker technologies, to determine genome arrangements and interspecific outcomes combined with cost-effective genomes to increase the efficiency in prognostic breeding efforts. (2) It also focused on functional genomics, proteomics and field-based breeding practices to achieve insight into the genetics underlying both simple and complex traits in canola.     

The Regulatory Status of Genome‐edited Crops

Genome editing with engineered nucleases (GEEN) represents a highly specific and efficient tool for crop improvement with the potential to rapidly generate useful novel phenotypes/traits. Genome editing techniques initiate specifically targeted double strand breaks facilitating DNA‐repair pathways that lead to base additions or deletions by non‐homologous end joining as well as targeted gene replacements or transgene insertions involving homology‐directed repair mechanisms. Many of these techniques and the ancillary processes they employ generate phenotypic variation that is indistinguishable from that obtained through natural means or conventional mutagenesis; and therefore, they do not readily fit current definitions of genetically engineered or genetically modified used within most regulatory regimes. Addressing ambiguities regarding the regulatory status of genome editing techniques is critical to their application for development of economically useful crop traits. Continued regulatory focus on the process used, rather than the nature of the novel phenotype developed, results in confusion on the part of regulators, product developers, and the public alike and creates uncertainty as of the use of genome engineering tools for crop improvement.     

TILLING,high-resolution melting (HRM), and next-generation sequencing (NGS) techniques in plant mutation breeding

Induced mutations have been used effectively for plant improvement. Physical and chemical mutagens induce a high frequency of genome variation. Recently, developed screening methods have allowed the detection of single nucleotide polymorphisms (SNPs) and the identification of traits that are difficult to identify at the molecular level by conventional breeding. With the assistance of reverse genetic techniques, sequence variation information can be linked to traits to investigate gene function. Targeting induced local lesions in genomes (TILLING) is a high-throughput technique to identify single nucleotide mutations in a specific region of a gene of interest with a powerful detection method resulted from chemical-induced mutagenesis. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of genome size and ploidy level. However, TILLING requires laborious and time-consuming steps, and a lack of complete genome sequence information for many crop species has slowed the development of suitable TILLING targets. Another method, high-resolution melting (HRM), which has assisted TILLING in mutation detection, is faster, simpler and less expensive with non-enzymatic screening system. Currently, the sequencing of crop genomes has completely changed our vision and interpretation of genome organization and evolution. Impressive progress in next-generation sequencing (NGS) technologies has paved the way for the detection and exploitation of genetic variation in a given DNA or RNA molecule. This review discusses the applications of TILLING in combination with HRM and NGS technologies for screening of induced mutations and discovering SNPs in mutation breeding programs.     

Sugarcane improvement: how far can we go?

In recent years, efforts to improve sugarcane have focused on the development of biotechnology for this crop. It has become clear that sugarcane lacks tools for the biotechnological route of improvement and that the initial efforts in sequencing ESTs had limited impact for breeding. Until recently, the models used by breeders in statistical genetics approaches have been developed for diploid organisms, which are not ideal for a polyploid genome such as that of sugarcane. Breeding programs are dealing with decreasing yield gains. The contribution of multiple alleles to complex traits such as yield is a basic question underlining the breeding efforts that could only be addressed by the development of specific tools for this grass. However, functional genomics has progressed and gene expression profiling is leading to the definition of gene networks. The sequencing of the sugarcane genome, which is underway, will greatly contribute to numerous aspects of research on grasses. We expect that both the transgenic and the marker-assisted route for sugarcane improvement will contribute to increased sugar, stress tolerance, and higher yield and that the industry for years to come will be able to rely on sugarcane as the most productive energy crop.     

Recombinase-directed plant transformation for the post-genomic era

Plant genomics promises to accelerate genetic discoveries for plant improvements. Machine-driven technologies are ushering in gene structural and expressional data at an unprecedented rate. Potential bottlenecks in this crop improvement process are steps involving plant transformation. With few exceptions, genetic transformation is an obligatory final step by which useful traits are engineered into plants. In addition, transgenesis is most often needed to confirm gene function, after deductions made through comparative genomics, expression profiles, and mutation analysis. This article reviews the use of recombinase systems to deliver DNA more efficiently into the plant genome.     

New traits in crops produced by genome editing techniques based on deletions

One of the most promising New Plant Breeding Techniques is genome editing (also called gene editing) with the help of a programmable site-directed nuclease (SDN). In this review, we focus on SDN-1, which is the generation of small deletions or insertions (indels) at a precisely defined location in the genome with zinc finger nucleases (ZFN), TALENs, or CRISPR-Cas9. The programmable nuclease is used to induce a double-strand break in the DNA, while the repair is left to the plant cell itself, and mistakes are introduced, while the cell is repairing the double-strand break using the relatively error-prone NHEJ pathway. From a biological point of view, it could be considered as a form of targeted mutagenesis. We first discuss improvements and new technical variants for SDN-1, in particular employing CRISPR-Cas, and subsequently explore the effectiveness of targeted deletions that eliminate the function of a gene, as an approach to generate novel traits useful for improving agricultural sustainability, including disease resistances. We compare them with examples of deletions that resulted in novel functionality as known from crop domestication and classical mutation breeding (both using radiation and chemical mutagens). Finally, we touch upon regulatory and access and benefit sharing issues regarding the plants produced.     

Establishment of a soybean (Glycine max Merr. L) transposon-based mutagenesis repository

Soybean is a major crop species providing valuable feedstock for food, feed and biofuel. In recent years, considerable progress has been made in developing genomic resources for soybean, including on-going efforts to sequence the genome. These efforts have identified a large number of soybean genes, most with unknown function. Therefore, a major research priority is determining the function of these genes, especially those involved in agronomic performance and seed traits. One means to study gene function is through mutagenesis and the study of the resulting phenotypes. Transposon-tagging has been used successfully in both model and crop plants to support studies of gene function. In this report, we describe efforts to generate a transposon-based mutant collection of soybean. The Ds transposon system was used to create activation-tagging, gene and enhancer trap elements. Currently, the repository houses approximately 900 soybean events, with flanking sequence data derived from 200 of these events. Analysis of the insertions revealed approximately 70% disrupted known genes, with the majority matching sequences derived from either Glycine max or Medicago truncatula sequences. Among the mutants generated, one resulted in male-sterility and was shown to disrupt the strictosidine synthase gene. This example clearly demonstrates that it is possible to disrupt soybean gene function by insertional mutagenesis and to derive useful mutants by this approach in spite of the tetraploid nature of the soybean genome.     

Haplotype hitchhiking promotes trait coselection in Brassica napus

Local haplotype patterns surrounding densely spaced DNA markers with significant trait associations can reveal information on selective sweeps and genome diversity associated with important crop traits. Relationships between haplotype and phenotype diversity, coupled with analysis of gene content in conserved haplotype blocks, can provide insight into coselection for nonrelated traits. We performed genome‐wide analysis of haplotypes associated with the important physiological and agronomic traits leaf chlorophyll and seed glucosinolate content, respectively, in the major oilseed crop species Brassica napus. A locus on chromosome A01 showed opposite effects on leaf chlorophyll content and seed glucosinolate content, attributed to strong linkage disequilibrium (LD) between orthologues of the chlorophyll biosynthesis genes EARLY LIGHT‐INDUCED PROTEIN and CHLOROPHYLL SYNTHASE, and the glucosinolate synthesis gene ATP SULFURYLASE 1. Another conserved haplotype block, on chromosome A02, contained a number of chlorophyll‐related genes in LD with orthologues of the key glucosinolate biosynthesis genes METHYLTHIOALKYMALATE SYNTHASE‐LIKE 1 and 3. Multigene haplogroups were found to have a significantly greater contribution to variation for chlorophyll content than haplotypes for any single gene, suggesting positive effects of additive locus accumulation. Detailed reanalysis of population substructure revealed a clade of ten related accessions exhibiting high leaf chlorophyll and low seed glucosinolate content. These accessions each carried one of the above‐mentioned haplotypes from A01 or A02, generally in combination with further chlorophyll‐associated haplotypes from chromosomes A05 and/or C05. The phenotypic rather than pleiotropic correlations between leaf chlorophyll content index and seed GSL suggest that LD may have led to inadvertent coselection for these two traits.     

Exploring the potential of CRISPR/Cas genome editing for vegetable crop improvement: An overview of challenges and approaches

Vegetables provide many nutrients in the form of fiber, vitamins, and minerals, which make them an important part of our diet. Numerous biotic and abiotic stresses can affect crop growth, quality, and yield. Traditional and modern breeding strategies to improve plant traits are slow and resource intensive. Therefore, it is necessary to find new approaches for crop improvement. Clustered regularly interspaced short palindromic repeats/CRISPR associated 9 (CRISPR/Cas9) is a genome editing tool that can be used to modify targeted genes for desirable traits with greater efficiency and accuracy. By using CRISPR/Cas9 editing to precisely mutate key genes, it is possible to rapidly generate new germplasm resources for the promotion of important agronomic traits. This is made possible by the availability of whole genome sequencing data and information on the function of genes responsible for important traits. In addition, CRISPR/Cas9 systems have revolutionized agriculture, making genome editing more versatile. Currently, genome editing of vegetable crops is limited to a few vegetable varieties (tomato, sweet potato, potato, carrot, squash, eggplant, etc.) due to lack of regeneration protocols and sufficient genome sequencing data. In this article, we summarize recent studies on the application of CRISPR/Cas9 in improving vegetable trait development and the potential for future improvement.     

Mapping of yield influencing QTL in <Emphasis Type="Italic">Brassica juncea</Emphasis>: implications for breeding of a major oilseed crop of dryland areas

Quantitative trait loci (QTL) analysis of yield influencing traits was carried out in Brassica juncea (AABB) using a doubled haploid (DH) mapping population of 123 lines derived from a cross between Varuna (a line representing the Indian gene pool) and Heera (representing the east European gene pool) to identify potentially useful alleles from both the parents. The existing AFLP based map of B. juncea was further saturated with RFLP and SSR markers which led to the identification of the linkage groups belonging to the A (B. rapa) and B (B. nigra) genome components of B. juncea. For QTL dissection, the DH lines were evaluated at three different environments and phenotyped for 12 quantitative traits. A total of 65 QTL spread over 13 linkage groups (LG) were identified from the three environments. QTL analysis showed that the A genome has contributed more than the B genome to productivity (68% of the total QTL detected) suggesting a more prominent role of the A genome towards domestication of this crop. The east European line, Heera, carried favorable alleles for 42% of the detected QTL and the remaining 58% were in the Indian gene pool line, Varuna. We observed clustering of major QTL in a few linkage groups, particularly in J7 and J10 of the A genome, with QTL of different traits having agronomically antagonistic allelic effects co-mapping to the same genetic interval. QTL analysis also identified some well-separated QTL which could be readily transferred between the two pools. Based on the QTL analysis, we propose that improvement in yield could be achieved more readily by heterosis breeding rather than by pure line breeding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.