Breeding Tools Research Articles

Assessing inbreeding in perennial ryegrass (Lolium perenne) as a step towards F1 hybrid breeding

AbstractPerennial ryegrass is an important turf and forage grass in temperate regions worldwide. Limited genetic gains have been made with current breeding strategies compared with other grass crops, such as rice and maize, which benefit from Filial 1 (F1) hybrid breeding. One of the largest constraints on hybrid breeding in ryegrass is self‐incompatibility preventing inbreeding, as homozygous parental lines are required to develop hybrids with maximal hybrid vigour. Obligate outcrossing in ryegrass has resulted in cultivars with high levels of heterozygosity, lacking trait uniformity across the population. A naturally occurring self‐fertile (SF) locus that overcomes the self‐incompatibility system has been identified in a European perennial ryegrass population. This study crossed the SF locus into an elite cultivar, producing a self‐compatible population that was inbred for several generations. Genotyping‐by‐sequencing (GBS) was used to assess the population structure and degree of inbreeding in the self‐compatible population. Phenotypic analysis indicated that increased homozygosity did not necessarily affect growth and performance. This study concludes that self‐compatible ryegrass is a promising tool for hybrid breeding and agronomic improvement of perennial ryegrass.

Development and validation of a genome-wide InDel marker set discriminating the alleles between the BB-genome Oryza species and rice (O. sativa)

Wild relatives of cultivated rice (Oryza sativa) belonging to the genus Oryza are regarded as ‘genetic reservoirs’ for rice improvement. Their genes are transferable to cultivated rice through crossing and they have been contributing significantly to rice variety improvement by introgression of their diverse valuable traits, especially biotic stress resistance. Moreover, active use of wild rice resources might be one of the ideal solutions to seek novel or superior alleles/genes to cope with climate change and stable high-yield rice production. DNA markers are essential tools for genetic analysis and breeding. However, to date, there are no suitable DNA marker sets for BB-genome species (O. punctata). In this study, we developed a genome genome-wide InDel marker set evenly distributed (∼2 Mb intervals) across the 12 chromosomes for BB-genome species. The markers were validated by PCR-agarose gel analysis with BB-genome containing species: four accessions each of O. punctata (BB) and O. minuta (BBCC). Out of the 191 InDel markers designed, 184 (96.3%) and 138 (72.2%) were able to differentiate the alleles between O. sativa and O. punctata and between O. sativa and O. minuta respectively. The same marker sets were also tested in other genome types species (CC, CCDD, EE, FF, GG, HHJJ, and KKLL) and one accession of Leersia perrieri (a sister genus to Oryza). The number of polymorphic markers (O. sativa vs other genome types) was drastically reduced in other genome types. In contrast, the number of markers showing no PCR amplification increased, especially in FF, GG, HHJJ, and KKLL species. This suggests that the development of genome type-specific marker sets would be more efficient rather than testing random InDel markers. The newly developed InDel markers maybe be useful for the identification of valuable genetic factors from the BB or BBCC-genome species and also for transferring the identified genes/QTLs into elite variety backgrounds.

ClPSY1 gene-based SNP markers identified from whole-genome resequencing for the determination of orange flesh color and carotenoid content in watermelon

Watermelon (Citrullus lanatus) is an economically important crop worldwide. The color of watermelon flesh is affected by the type and content of carotenoids. Carotenoids are high in antioxidants, which are associated with lower risk of various cancers and cardiovascular diseases and support visual function. Accordingly, the color of watermelon flesh is considered an important characteristic as it not only provides color diversity but also serves as the main motivator of consumer choice in terms of nutrition. Molecular markers have emerged as a powerful tool in plant breeding and the development of markers for selection of important traits and highly desirable. We previously identified genome-wide single nucleotide polymorphism (SNP) by performing whole-genome resequencing (WGRS) of 24 inbred watermelon lines with different flesh color traits (PRJNA516776). In this study, we proposed candidate genes for orange flesh color based on SNPs unique to orange flesh genotypes. One SNP in the phytoene synthase (ClPSY1) allele, which mediates the first step in carotenoid biosynthesis, was found to be associated with the orange color of watermelon flesh. Based on the polymorphism present in the ClPSY1 gene, we developed a molecular marker for predicting watermelon flesh color and confirmed the association of the SNP with orange flesh. This marker and markers derived from several other previously reported genes could be useful for the selective breeding of watermelons with a specific flesh color.

Editing of TOM1 gene in tobacco using CRISPR/Cas9 confers resistance to Tobacco mosaic virus

Genome editing technology has become one of the excellent tools for precise plant breeding to develop novel plant germplasm. The Tobacco mosaic virus (TMV) is the most prominent pathogen that infects several Solanaceae plants, such as tobacco, tomato, and capsicum, which requires critical host factors for infection and replication of its genomic RNA in the host. The Tobamovirus multiplication (TOM) genes, such as TOM1, TOM2A, TOM2B, and TOM3, are involved in the multiplication of Tobamoviruses. TOM1 is a transmembrane protein necessary for efficient TMV multiplication in several plant species. The TOM genes are crucial recessive resistance genes that act against the tobamoviruses in various plant species. The single guided RNA (sgRNA) was designed to target the first exon of the NtTOM1 gene and cloned into the pHSE401 vector. The pHSE401-NtTOM1 vector was introduced into Agrobacterium tumefaciens strain LBA4404 and then transformed into tobacco plants. The analysis on T0 transgenic plants showed the presence of the hptII and Cas9 transgenes. The sequence analysis of the NtTOM1 from T0 plants showed the indels. Genotypic evaluation of the NtTOM1 mutant lines displayed the stable inheritance of the mutations in the subsequent generations of tobacco plants. The NtTOM1 mutant lines successfully conferred resistance to TMV. CRISPR/Cas genome editing is a reliable tool for investigating gene function and precision breeding across different plant species, especially the species in the Solanaceae family.

Alternative Splicing of NAC Transcription Factor Gene CmNST1 Is Associated with Naked Seed Mutation in Pumpkin, Cucurbita moschata.

In pumpkin (Cucurbita moschata), the naked or hull-less seed phenotype has great benefits for breeding this crop for oil or snack use. We previously identified a naked seed mutant in this crop. In this study, we report genetic mapping, identification, and characterization of a candidate gene for this mutation. We showed that the naked seed phenotype is controlled by a single recessive gene (N). The bulked segregant analysis identified a 2.4 Mb region on Chromosome 17 with 15 predicted genes. Multiple lines of evidence suggested that CmoCh17G004790 is the most probable candidate gene for the N locus which encodes a NAC transcription factor WALL THICKENING PROMOTING FACTOR 1 (CmNST1). No nucleotide polymorphism or structural variation was found in the genomic DNA sequences of CmNST1 between the mutant and the wildtype inbred line (hulled seed). However, the cDNA sequence cloned from developing seed coat samples of the naked seed mutant was 112 bp shorter than that from the wildtype which is due to seed coat-specific alternative splicing in the second exon of the mutant CmNST1 transcript. The expression level of CmNST1 in the developing seed coat was higher in the mutant than in the wildtype during early seed coat development which was reversed later. Transcriptomic profiling with RNA-Seq at different stages of seed development in the mutant and wildtype revealed a critical role of CmNST1 as a master regulator for the lignin biosynthesis pathway during seed coat development while other NAC and MYB transcription factors were also involved in forming a regulatory network for the building of secondary cell walls. This work provides a novel mechanism for the well-characterized NST1 transcription factor gene in regulating secondary cell wall development. The cloned gene also provides a useful tool for marker-assisted breeding of hull-less C. moschata varieties.

Consolidating 23 years of historical data from an irrigated subtropical rice breeding program in Uruguay

AbstractBreeding programs generate vast amount of data which are often scattered in separate files. This hinders the application of modern breeding tools such as multi‐environment analyses and genomic selection. This research work describes the process of consolidating 23 years of phenotypic, pedigree, and genomic records from the Uruguayan national rice (Oryza sativa L.) breeding program, and the features and structure of the resulting database. Using a custom‐made R code, we gathered all the available data from 1997 to 2020 corresponding to field trials, blast disease evaluation nurseries, laboratory analyses of milling and cooking quality, pedigree information, and genomic information for selected advanced breeding lines, and organized it into a relational database. Records of 996 trials in 12 locations over a span of 23 years, 91,636 field plots with information on 14 phenotypic variables, pedigree for 19,447 genotypes, and genomic information regarding 61,260 single nucleotide polymorphism (SNP) markers for 965 genotypes were recovered. The dataset is structured in trials, phenotypes, lines, genomic information, and SNP tables in an easy‐to‐access relational database, which will be a valuable resource for rice breeding.

Role of Molecular Breeding Tools in Enhancing the Breeding of Drought-Resilient Cotton Genotypes: An Updated Review

Drought stress is an inevitable factor that disturbs the production of plants by altering morphological, physiological, biochemical, and molecular functions. Breeding for drought tolerance requires a complete understanding of the molecular factors controlling stress-responsive pathways. The plant responds to drought stress by adopting four mechanisms: avoidance, escape, tolerance, and recovery. Traditional plant-breeding tools have been employed to increase tolerance in cotton, but the complexity of drought tolerance has limited the use of these breeding methods. The plant adopts several key strategies against drought stress, such as activating the signaling network and activating molecular factors. Cotton breeders have been engaged in elucidating the molecular mechanisms of drought tolerance in cotton using significant molecular tools such as quantitative trait loci (QTL) mapping, transcription factor (TFs) analysis, transcriptome analysis, genome-wide association studies (GWAS), genetic engineering, and CRISPR/Cas9. Breeders have studied the functional description of genes and the interacting pathways accountable for controlling drought tolerance in cotton. Hundreds of genes/QTL have been identified, and many have been cloned for drought tolerance in cotton; however, a complete understanding of these traits still needs more study. This review presents a detailed overview of molecular tools, their application for improving drought tolerance in cotton, and their prospects. This review will help future researchers to conduct further studies to develop drought-tolerant cotton genotypes that can thrive under conditions of water scarcity.

Improving the cultivation of fish planting material in Central Federal district

The article outlines the issues of introducing a factory method of growing fish planting material in individual fish farms of the Central Federal District on the basis of creating a network of specialized farms (reproducers), which will solve the problem of rapid introduction of existing and newly created fish species into production. At the same time, it is proposed to integrate production on the basis of cooperation and specialization, including vertical production links of various sectors of commercial fish farming (breeding, reproducers, etc.), which will create prerequisites for the efficiency of pond farms. Correlation analysis was used as a research tool for breeding and commodity farms of the Moscow region, on the basis of which the main factors affecting the cultivation of commercial fish were identified.

Potential of Molecular Plant Breeding for Sustaining the Global Food Security

New alleles are continuously added to bring novel traits which are selected using genetic manipulations. Understanding the genes responsible for a particular phenotype involves recent genomic approaches which are to be integrated with conventional breeding programs for the crop improvement. Use of biotechnological tools merged with the conventional breeding practices has resulted molecular plant breeding which has significant contributions to food production, despite the presence of narrow genetic base in current materials used for breeding purposes. Use of molecular markers along with high-throughput genome sequencing efforts, have dramatically augmented our information to characterize the elite germplasm for the production of hybrids and improved populations. This review focuses on possibilities for the need and application of molecular breeding tools in the genetic improvement of the crop plants which can ensure sustainable food production for the increasing global population.

Genomic prediction and selection response for grain yield in safflower.

In plant breeding programs, multiple traits are recorded in each trial, and the traits are often correlated. Correlated traits can be incorporated into genomic selection models, especially for traits with low heritability, to improve prediction accuracy. In this study, we investigated the genetic correlation between important agronomic traits in safflower. We observed the moderate genetic correlations between grain yield (GY) and plant height (PH, 0.272-0.531), and low correlations between grain yield and days to flowering (DF, -0.157-0.201). A 4%-20% prediction accuracy improvement for grain yield was achieved when plant height was included in both training and validation sets with multivariate models. We further explored the selection responses for grain yield by selecting the top 20% of lines based on different selection indices. Selection responses for grain yield varied across sites. Simultaneous selection for grain yield and seed oil content (OL) showed positive gains across all sites with equal weights for both grain yield and oil content. Combining g×E interaction into genomic selection (GS) led to more balanced selection responses across sites. In conclusion, genomic selection is a valuable breeding tool for breeding high grain yield, oil content, and highly adaptable safflower varieties.

CRISPR/Cas9 establishment-mediated targeted mutagenesis in Macrobrachium nipponense.

Introduction: CRISPR/Cas9 is a gene-editing technology which could specifically cleave dsDNA and induce target gene mutation. CRISPR/Cas9 has been widely used in gene functional studies in many fields, such as medicine, biology, and agriculture due to its simple design, low cost, and high efficiency. Although it has been well developed in model fish and freshwater fish for gene function analysis, it is still novel in the studies dealing with economic crustacean species. Methods: In this study, we established a CRISPR/Cas9 system based on microinjection for M. nipponense, an important economic crustacean aquaculture species. The vitellogenin (Vg) gene and the eyeless (Ey) gene were selected as the targeted genes for mutation. Two sgRNAs were designed for Mn-Vg and Mn-Ey gene editing, respectively. Results and Discussion: For sg-Vg-1, the gastrula survival ratio was 8.69%, and the final hatching ratio was 4.83%. The blastula mutant ratio was 10%, and the hatching individual mutant ratio was 30%. For sg-Vg-2, the gastrula survival ratio was 5.85%, and the final hatching ratio was 3.89%. The blastula mutant ratio was 16.67%, and no mutant sequences were detected in hatching individuals. For sg-Ey-1, the gastrula survival ratio was 6.25%, and the final hatching ratio was 2.34%. The blastula mutant ratio was 10.00%, and the hatching individual mutant ratio was 66.67%. For sg-Ey-2, the gastrula survival ratio was 6.00%, and the final hatching ratio was 2.67%. No mutant sequence was detected in both blastula stage and hatching individuals. There were no significant morphological changes observed in the Mn-Vg group. Two deformed types were detected in sg-Ey-1-injected embryos. An evident developmental delay of the compound eye was detected in Ey-sg1-H1 in the zoea stage. The compound eyes of the Ey-sg1-H2 embryo could not form well-defined spheres, and the whole compound eye appeared to diffuse at the end of the late zoea stage. The establishment of a gene-editing platform based on CRISPR/Cas9 will not only provide an efficient and convenient method for gene function analysis but also provide a powerful tool for molecular-assisted breeding of Macrobrachium nipponense.

Rice3K56 is a high-quality SNP array for genome-based genetic studies and breeding in rice (Oryza sativa L.)

Single nucleotide polymorphism (SNP) genotyping arrays provide an optimal high-throughput platform for genetic research and molecular breeding programs in both animals and plants. In this study, a high-quality and custom-designed Rice3K56 SNP array was developed with the resequencing data of 3024 rice accessions worldwide, which was then tested extensively in 192 representative rice samples. Printed on the GeneTitan chips of Affymetrix Axiom each containing 56,606 SNP markers, the Rice3K56 array has a high genotyping reliability (99.6%), high and uniform genome coverage (an average of 6.7-kb between adjacent SNPs), abundant polymorphic information and easy automation, compared with previously developed rice SNP arrays. When applied in rice varietal differentiation, population diversity analysis, gene mapping of 13 complex traits by a genome-wide association study analysis (GWAS), and genome selection experiments in a recombinant inbred line and a multi-parent advanced generation inter-cross populations, these properties of the Rice3K56 array were well demonstrated for its power and great potential to be a highly efficient tool for rice genetic research and genomic breeding.

Novel QTL for chilling tolerance at germination and early seedling stages in sorghum.

Sorghum (Sorghum bicolor L.) a drought tolerant staple crop for half a billion people in Africa and Asia, an important source of animal feed throughout the world and a biofuel feedstock of growing importanceorghum's originated from tropical regions rendering the crop to be cold sensitive. Low temperature stresses such as chilling and frost greatly affect the agronomic performance of sorghum and limit its geographical distribution, posing a major problem in temperate environments when sorghum is planted early. Understanding the genetic basis of wide adaptability and of sorghum would facilitate molecular breeding programs and studies of other C4 crops. The objective of this study is to conduct quantitative trait loci analysis using genotying by sequencing for early seed germination and seedling cold tolerance in two sorghum recombinant inbred lines populations. To accomplish that, we used two populations of recombinant inbred lines (RIL) developed from crosses between cold-tolerant (CT19, ICSV700) and cold-sensitive (TX430, M81E) parents. The derived RIL populations were evaluated for single nucleotide polymorphism (SNP) using genotype-by-sequencing (GBS) in the field and under controlled environments for their response to chilling stress. Linkage maps were constructed with 464 and 875 SNPs for the CT19 X TX430 (C1) and ICSV700 X M81 E (C2) populations respectively. Using quantitative trait loci (QTL) mapping, we identified QTL conferring tolerance to chilling tolerance at the seedling stage. A total of 16 and 39 total QTL were identified in the C1 and C2 populations, respectively. Two major QTL were identified in the C1 population, and three major QTL were mapped in the C2 population. Comparisons between the two populations and with previously identified QTL show a high degree of similarity in QTL locations. Given the amount of co-localization of QTL across traits and the direction of allelic effect supports that these regions have a pleiotropic effect. These QTL regions were also identified to be highly enriched for genes encoding chilling stress and hormonal response genes. This identified QTL can be useful in developing tools for molecular breeding of sorghums with improved low-temperature germinability.

BnIR: A multi-omics database with various tools for Brassica napus research and breeding

In the post-genome-wide association study era, multi-omics techniques have shown great power and potential for candidate gene mining and functional genomics research. However, due to the lack of effective data integration and multi-omics analysis platforms, such techniques have not still been applied widely in rapeseed, an important oil crop worldwide. Here, we report a rapeseed multi-omics database (BnIR; http://yanglab.hzau.edu.cn/BnIR), which provides datasets of six omics including genomics, transcriptomics, variomics, epigenetics, phenomics, and metabolomics, as well as numerous “variation–gene expression–phenotype” associations by using multiple statistical methods. In addition, a series of multi-omics search and analysis tools are integrated to facilitate the browsing and application of these datasets. BnIR is the most comprehensive multi-omics database for rapeseed so far, and two case studies demonstrated its power to mine candidate genes associated with specific traits and analyze their potential regulatory mechanisms.

Recent advances in the crosstalk between adipose, muscle and bone tissues in fish.

Control of tissue metabolism and growth involves interactions between organs, tissues, and cell types, mediated by cytokines or direct communication through cellular exchanges. Indeed, over the past decades, many peptides produced by adipose tissue, skeletal muscle and bone named adipokines, myokines and osteokines respectively, have been identified in mammals playing key roles in organ/tissue development and function. Some of them are released into the circulation acting as classical hormones, but they can also act locally showing autocrine/paracrine effects. In recent years, some of these cytokines have been identified in fish models of biomedical or agronomic interest. In this review, we will present their state of the art focusing on local actions and inter-tissue effects. Adipokines reported in fish adipocytes include adiponectin and leptin among others. We will focus on their structure characteristics, gene expression, receptors, and effects, in the adipose tissue itself, mainly regulating cell differentiation and metabolism, but in muscle and bone as target tissues too. Moreover, lipid metabolites, named lipokines, can also act as signaling molecules regulating metabolic homeostasis. Regarding myokines, the best documented in fish are myostatin and the insulin-like growth factors. This review summarizes their characteristics at a molecular level, and describes both, autocrine effects and interactions with adipose tissue and bone. Nonetheless, our understanding of the functions and mechanisms of action of many of these cytokines is still largely incomplete in fish, especially concerning osteokines (i.e., osteocalcin), whose potential cross talking roles remain to be elucidated. Furthermore, by using selective breeding or genetic tools, the formation of a specific tissue can be altered, highlighting the consequences on other tissues, and allowing the identification of communication signals. The specific effects of identified cytokines validated through in vitro models or in vivo trials will be described. Moreover, future scientific fronts (i.e., exosomes) and tools (i.e., co-cultures, organoids) for a better understanding of inter-organ crosstalk in fish will also be presented. As a final consideration, further identification of molecules involved in inter-tissue communication will open new avenues of knowledge in the control of fish homeostasis, as well as possible strategies to be applied in aquaculture or biomedicine.

Polyploid Induction and Karyotype Analysis of Dendrobium officinale

Dendrobium officinale Kimura et Migo is an orchid with both medicinal and edible values and a high economic value. The wild resources of D. officinale are in an endangered state. Compared with the wild D. officinale, cultivated D. officinale exhibits inferior quality and a low content of medicinal components. Polyploid induction is a conventional breeding tool for genome doubling of species, which can effectively increase the total amount of plant components to improve the medicinal efficacy of D. officinale. In this study, D. officinale tetraploids were generated by treating the protocorms with colchicine. Morphological observations showed that tetraploids exhibited decreased plant size and leaf shape index and increased stem diameter. Cytological observations showed that the polyploid plants had larger stomata and a lower number of stomata per unit area compared with normal plants. The highest stomata variation of 30.00% was observed when the plant was treated with 0.3% colchicine for 24 h. Chromosomal observations showed that treatment of plants with 0.2% colchicine for 48 h resulted in the highest tetraploid induction rate of 10.00%. A total of 10 tetraploids were successfully obtained by inducing plant protoplasts with colchicine. The number of diploid D. officinale chromosomes was 38 with a base number of 19, and the karyotype formula was 2n = 2x = 38 = 24m + 14sm with a karyotype asymmetry coefficient of 60.59%, belonging to type 2B. The number of tetraploid D. officinale chromosomes was 76 with a base number of 19, and the karyotype formula was 2n = 4x = 76 = 58m + 18sm with a karyotype asymmetry coefficient of 60.04%, belonging to type 2B. This study determined the optimal mutagenesis treatment based on the chromosome observation results, investigated the relationship between the phenotype and ploidy level, and generated the polyploid germplasm of D. officinale, thereby laying the foundation for the breeding of new D. officinale cultivars enriched with compounds of medicinal value.

THE RICH PICTURE IN CONTEMPORARY POET POET COMPANION JAWAQ MODEL

Folmen that the spinning is one of the oldest hair types that have been on the throne of the poetic topics on the ages - from the Jahili to the modern era - which took a large space of poetic wealth, both on the level of poetry or at the level Lovers and tasty, and no doubt that every era of its own features, which has been shaded by the spirit of public, and the invasion, especially, but the forthcoming poetry in the modern era was characterized by the representation of the nature of the Arabs away from the innovation and all the Svih In the period of stagnant periods of the Abbasid Age as a result of the mixing of dear. This research is considered as saying: [The rich picture in contemporary poet poet companion Jawaq model]. The portrait of the poet Rafiq al-Jawash, who revealed several major results First, the poet Rafiq al-Jawash from poets representing the image of the general poetry, and the pork is particularly, as well as that he described the distant virgin all the dimension of instincts, lusts and excitement, adhered to its spinning images in the vulnerable image AlAfifi without mentioned for alleys of the beloved or the alumni in the spinning and is a feature of the times The poet employed internal music employment of his own breeding tools, and there has been a rhino-quarreling, metaphor and a metaphor and this is indicating that the jaws can be able to hire it.

Cost-effective duplex Kompetitive Allele Specific PCR markers for homologous genes facilitating wheat breeding

BackgroundOwing to successful cloning of wheat functional genes in recent years, more traits can be selected by diagnostic markers, and consequently, effective molecular markers will be powerful tools in wheat breeding programs.ResultsThe present study proposed a cost-effective duplex Kompetitive Allele Specific PCR (dKASP) marker system that combined multiplex PCR and KASP™ technology to yield twice the efficiency at half the cost compared with the common KASP™ markers and provide great assistance in breeding selection. Three dKASP markers for the major genes controlling plant height (Rht-B1/Rht-D1), grain hardness (Pina-D1/Pinb-D1), and high-molecular-weight glutenin subunits (Glu-A1/Glu-D1) were successfully developed and applied in approved wheat varieties growing in the middle and lower reaches of the Yangtze River and advanced lines from our breeding program. Three markers were used to test six loci with high efficiency. In the approved wheat varieties, Rht-B1b was the most important dwarfing allele, and the number of accessions carrying Pinb-D1b was much greater than that of the accessions carrying Pina-D1b. Moreover, the number of accessions carrying favorable alleles for weak-gluten wheat (Null/Dx2) was much greater than that of the accessions carrying favorable alleles for strong-gluten wheat (Ax1 or Ax2*/Dx5). In the advanced lines, Rht-B1b and Pinb-D1b showed a significant increase compared with the approved varieties, and the strong-gluten (Ax1 or Ax2*/Dx5) and weak-gluten (Null/Dx2) types also increased.ConclusionA cost-effective dKASP marker system that combined multiplex PCR and KASP™ technology was proposed to achieve double the efficiency at half the cost compared with the common KASP™ markers. Three dKASP markers for the major genes controlling PH (Rht-B1/Rht-D1), GH (Pina-D1/Pinb-D1), and HMW-GS (Glu-A1/Glu-D1) were successfully developed, which would greatly improve the efficiency of marker-assisted selection of wheat.

Effects of Different Gene Editing Modes of CRISPR/Cas9 on Soybean Fatty Acid Anabolic Metabolism Based on GmFAD2 Family

Δ12-fatty acid dehydrogenase (FAD2) is the essential enzyme responsible for catalyzing the formation of linoleic acid from oleic acid. CRISPR/Cas9 gene editing technology has been an essential tool for molecular breeding in soybeans. To evaluate the most suitable type of gene editing in soybean fatty acid synthesis metabolism, this study selected five crucial enzyme genes of the soybean FAD2 gene family-GmFAD2-1A, GmFAD2-1B, GmFAD2-2A, GmFAD2-2B, and GmFAD2-2C-and created a CRISPR/Cas9-mediated single gene editing vector system. The results of Sanger sequencing showed that 72 transformed plants positive for T1 generation were obtained using Agrobacterium-mediated transformation, of which 43 were correctly edited plants, with the highest editing efficiency of 88% for GmFAD2-2A. The phenotypic analysis revealed that the oleic acid content of the progeny of GmFAD2-1A gene-edited plants had a higher increase of 91.49% when compared to the control JN18, and the rest of the gene-edited plants in order were GmFAD2-2A, GmFAD2-1B, GmFAD2-2C, and GmFAD2-2B. The analysis of gene editing type has indicated that base deletions greater than 2bp were the predominant editing type in all editing events. This study provides ideas for the optimization of CRISPR/Cas9 gene editing technology and the development of new tools for precise base editing in the future.

A comparison of methods for training population optimization in genomic selection

Key messageMaximizing CDmean and Avg_GRM_self were the best criteria for training set optimization. A training set size of 50–55% (targeted) or 65–85% (untargeted) is needed to obtain 95% of the accuracy. With the advent of genomic selection (GS) as a widespread breeding tool, mechanisms to efficiently design an optimal training set for GS models became more relevant, since they allow maximizing the accuracy while minimizing the phenotyping costs. The literature described many training set optimization methods, but there is a lack of a comprehensive comparison among them. This work aimed to provide an extensive benchmark among optimization methods and optimal training set size by testing a wide range of them in seven datasets, six different species, different genetic architectures, population structure, heritabilities, and with several GS models to provide some guidelines about their application in breeding programs. Our results showed that targeted optimization (uses information from the test set) performed better than untargeted (does not use test set data), especially when heritability was low. The mean coefficient of determination was the best targeted method, although it was computationally intensive. Minimizing the average relationship within the training set was the best strategy for untargeted optimization. Regarding the optimal training set size, maximum accuracy was obtained when the training set was the entire candidate set. Nevertheless, a 50–55% of the candidate set was enough to reach 95–100% of the maximum accuracy in the targeted scenario, while we needed a 65–85% for untargeted optimization. Our results also suggested that a diverse training set makes GS robust against population structure, while including clustering information was less effective. The choice of the GS model did not have a significant influence on the prediction accuracies.